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Alnaimat SM, Abushattal S, Dmour SM, Al-Awaida WJ, Ayyash AM, Goh KW. Genomic insights into the taxonomic status and bioactive gene cluster profiling of Bacillus velezensis RVMD2 isolated from desert rock varnish in Ma'an, Jordan. PLoS One 2025; 20:e0319345. [PMID: 40273114 PMCID: PMC12021177 DOI: 10.1371/journal.pone.0319345] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2024] [Accepted: 01/30/2025] [Indexed: 04/26/2025] Open
Abstract
Extreme environments like arid and semi-arid deserts harbor unique microbial diversity, offering rich sources of specialized microbial metabolites. This study explores Bacillus velezensis RVMD2, a strain isolated from rock varnish in the Ma'an Desert, Jordan. The genome was sequenced using the Illumina NextSeq 2000 platform, resulting in a 4,212,579 bp assembly with a GC content of 45.94%. The assembled genome comprises 112 contigs and encodes 4,250 proteins, 77 tRNA genes, and 4 rRNA genes. Phylogenetic analysis of the 16S rRNA gene indicated a 99.84% similarity to previously identified B. velezensis strains. Whole-genome phylogeny using EzBiome, MiGA, and TYGS confirmed its classification as B. velezensis. Functional annotation identified genes involved in carbohydrate metabolism, including 324 carbohydrate-active enzyme (CAZyme) genes, stress response, and secondary metabolite biosynthesis. The genome also contains 50 genes associated with heavy metal resistance and plant growth promotion. Analysis using AntiSMASH identified 12 biosynthetic gene clusters involved in the production of secondary metabolites, including fengycin, surfactin, polyketides, terpenes, and bacteriocins. Notably, several clusters did not match any known sequences, suggesting the presence of potentially novel antimicrobial compounds. The genomic features of RVMD2 highlight its adaptability to extreme environments and its potential for biotechnological applications, including bioremediation and the discovery of novel bioactive metabolites.
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Affiliation(s)
- Sulaiman M. Alnaimat
- Department of Medical Analysis, Princess Aisha Bint Al-Hussein College of Nursing and Health Sciences, Al-Hussein Bin Talal University, Ma’an, Jordan
| | - Saqr Abushattal
- Department of Medical Analysis, Princess Aisha Bint Al-Hussein College of Nursing and Health Sciences, Al-Hussein Bin Talal University, Ma’an, Jordan
| | - Saif M. Dmour
- Department of Medical Analysis, Princess Aisha Bint Al-Hussein College of Nursing and Health Sciences, Al-Hussein Bin Talal University, Ma’an, Jordan
| | - Wajdy J. Al-Awaida
- Department of Biology and Biotechnology, Faculty of Science, American University of Madaba, Madaba, Jordan
| | - Amani M. Ayyash
- Department of Pharmacy, Faculty of Health Sciences, American University of Madaba, Madaba, Jordan
| | - Khang Wen Goh
- Faculty of Data Science and Information Technology, INTI International University, Nilai, Malaysia
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Sari RF, Fadilah F, Maladan Y, Sarassari R, Khoeri MM, Harimurti K, Alimsardjono L, Safari D. An insight of Streptococcus pneumoniae serotype 3 genomic profile in Indonesia. JOURNAL OF MICROBIOLOGY, IMMUNOLOGY, AND INFECTION = WEI MIAN YU GAN RAN ZA ZHI 2025:S1684-1182(25)00031-3. [PMID: 39956730 DOI: 10.1016/j.jmii.2025.01.007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/10/2024] [Revised: 01/17/2025] [Accepted: 01/23/2025] [Indexed: 02/18/2025]
Abstract
BACKGROUND Streptococcus pneumoniae Serotype 3 (SPN3) remains a significant cause of morbidity and mortality worldwide despite of pneumococcal conjugate vaccine (PCV) implementation. We explored genomic profile of SPN3 from children and adult groups to understand population structure and evolution dynamics of SPN3 in Indonesia. METHODS We undertook whole genome sequencing (WGS) from 19 isolates of SPN3 in Indonesia between 2017 and 2021 prior to PCV introduction. This study assessed sequence types (STs), global pneumococcal sequence cluster (GPSC), genome prediction of antimicrobial resistance (AMR) profile, pangenome analysis, phylogenetic tree, and genome comparative of capsular polysaccharide (cps) locus. RESULTS We identified ST451-GPSC234 (n = 5) and ST180-GPSC12 (n = 4), ST458-GPSC51 (n = 2), ST3805-GPSC12 (n = 2), ST4909-GPSC363 (n = 2), ST700-GPSC10 (n = 1), ST5292-GPSC309 (n = 1), ST505-GPSC12 (n = 1), and ST4233 (n = 1). Genome prediction of AMR discover isolates were resistant to tetracycline (n = 5); co-resistant of chloramphenicol and tetracycline (n = 2); co-trimoxazole and tetracycline (n = 1). We observed SPN3 possess closed pangenome characteristic, indicates more stable genetic repertoire. We found 5 absent genes in cps locus including cpsABCD and tnp in ST700-GPSC10 lineage. CONCLUSIONS SPN3 has potential genomic profile to enhance the ability of this strain to endure selective pressure such as PCV introduction.
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Affiliation(s)
- Ratna Fathma Sari
- Biomedical Sciences, Faculty of Medicine, University of Indonesia, Jl. Salemba Raya No. 6, Jakarta Pusat, DKI Jakarta, 10430, Indonesia; Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Jl. Raya Bogor KM 46, Cibinong, Jawa Barat, Indonesia
| | - Fadilah Fadilah
- Medical Chemistry Department, Faculty of Medicine, University of Indonesia, Jl. Salemba Raya No. 6, Jakarta Pusat, DKI Jakarta, 10430, Indonesia
| | - Yustinus Maladan
- Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Jl. Raya Bogor KM 46, Cibinong, Jawa Barat, Indonesia
| | - Rosantia Sarassari
- Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Jl. Raya Bogor KM 46, Cibinong, Jawa Barat, Indonesia
| | - Miftahuddin Majid Khoeri
- Infectious Disease and Immunology Research Center, Indonesia Medical and Education Research Institute (IMERI), Faculty of Medicine, University of Indonesia, Indonesia
| | - Kuntjoro Harimurti
- Department of Internal Medicine, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia
| | - Lindawati Alimsardjono
- Department of Clinical Microbiology, Dr. Soetomo General Academic Hospital, Surabaya, East Java, Indonesia
| | - Dodi Safari
- Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Jl. Raya Bogor KM 46, Cibinong, Jawa Barat, Indonesia.
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Fritzenwanker M, Falgenhauer J, Hain T, Imirzalioglu C, Chakraborty T, Yao Y. The Detection of Extensively Drug-Resistant Proteus mirabilis Strains Harboring Both VIM-4 and VIM-75 Metallo-β-Lactamases from Patients in Germany. Microorganisms 2025; 13:266. [PMID: 40005633 PMCID: PMC11857796 DOI: 10.3390/microorganisms13020266] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2024] [Revised: 01/13/2025] [Accepted: 01/22/2025] [Indexed: 02/27/2025] Open
Abstract
Proteus mirabilis is a well-known opportunistic pathogen predominantly associated with urinary tract infections. It exhibits natural resistance to multiple antibiotics, including last-resort options like colistin. The emergence and spread of multidrug-resistant P. mirabilis isolates, including those producing ESBLs, AmpC cephalosporinases, and carbapenemases, are now more frequently reported. The most common carbapenemase types found in P. mirabilis are KPC-2, IMP, VIM, NDM, and OXA-48. We sequenced the genomes of three carbapenem-resistant P. mirabilis isolates harboring both blaVIM-4 and blaVIM-75 from Germany using both short-read and long-read sequencing techniques. We found that the isolates were only distantly related genetically. Both blaVIM-4 and blaVIM-75 genes were located on a class I integron, which in two cases was located on the chromosome and in one case on a plasmid. This is the first report on the complete genomes of P. mirabilis strains harboring a rare genetic element encoding both blaVIM-4 and blaVIM-75. Our results emphasize a key role for class 1 integrons in the transmission of VIM carbapenemases in P. mirabilis.
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Affiliation(s)
- Moritz Fritzenwanker
- Institute of Medical Microbiology, Justus Liebig University Giessen, Schubertstrasse 81, 35392 Giessen, Germany; (M.F.); (J.F.); (T.H.); (C.I.); (T.C.)
- German Center for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Justus-Liebig University Giessen, 35392 Giessen, Germany
| | - Jane Falgenhauer
- Institute of Medical Microbiology, Justus Liebig University Giessen, Schubertstrasse 81, 35392 Giessen, Germany; (M.F.); (J.F.); (T.H.); (C.I.); (T.C.)
- Institute for Hygiene and Environmental Medicine, Justus Liebig University Giessen, Schubertstrasse 81, 35392 Giessen, Germany
| | - Torsten Hain
- Institute of Medical Microbiology, Justus Liebig University Giessen, Schubertstrasse 81, 35392 Giessen, Germany; (M.F.); (J.F.); (T.H.); (C.I.); (T.C.)
- German Center for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Justus-Liebig University Giessen, 35392 Giessen, Germany
| | - Can Imirzalioglu
- Institute of Medical Microbiology, Justus Liebig University Giessen, Schubertstrasse 81, 35392 Giessen, Germany; (M.F.); (J.F.); (T.H.); (C.I.); (T.C.)
- German Center for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Justus-Liebig University Giessen, 35392 Giessen, Germany
| | - Trinad Chakraborty
- Institute of Medical Microbiology, Justus Liebig University Giessen, Schubertstrasse 81, 35392 Giessen, Germany; (M.F.); (J.F.); (T.H.); (C.I.); (T.C.)
- German Center for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Justus-Liebig University Giessen, 35392 Giessen, Germany
| | - Yancheng Yao
- Institute of Medical Microbiology, Justus Liebig University Giessen, Schubertstrasse 81, 35392 Giessen, Germany; (M.F.); (J.F.); (T.H.); (C.I.); (T.C.)
- German Center for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Justus-Liebig University Giessen, 35392 Giessen, Germany
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Saratto T, Visuri K, Lehtinen J, Ortega-Sanz I, Steenwyk JL, Sihvonen S. Solu: a cloud platform for real-time genomic pathogen surveillance. BMC Bioinformatics 2025; 26:12. [PMID: 39806295 PMCID: PMC11731562 DOI: 10.1186/s12859-024-06005-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2024] [Accepted: 12/05/2024] [Indexed: 01/16/2025] Open
Abstract
BACKGROUND Genomic surveillance is extensively used for tracking public health outbreaks and healthcare-associated pathogens. Despite advancements in bioinformatics pipelines, there are still significant challenges in terms of infrastructure, expertise, and security when it comes to continuous surveillance. The existing pipelines often require the user to set up and manage their own infrastructure and are not designed for continuous surveillance that demands integration of new and regularly generated sequencing data with previous analyses. Additionally, academic projects often do not meet the privacy requirements of healthcare providers. RESULTS We present Solu, a cloud-based platform that integrates genomic data into a real-time, privacy-focused surveillance system. EVALUATION Solu's accuracy for taxonomy assignment, antimicrobial resistance genes, and phylogenetics was comparable to established pathogen surveillance pipelines. In some cases, Solu identified antimicrobial resistance genes that were previously undetected. Together, these findings demonstrate the efficacy of our platform. CONCLUSIONS By enabling reliable, user-friendly, and privacy-focused genomic surveillance, Solu has the potential to bridge the gap between cutting-edge research and practical, widespread application in healthcare settings. The platform is available for free academic use at https://platform.solugenomics.com .
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Affiliation(s)
- Timo Saratto
- Solu Healthcare Oy, Kalevankatu 31 A 13, 00100, Helsinki, Finland.
| | - Kerkko Visuri
- Solu Healthcare Oy, Kalevankatu 31 A 13, 00100, Helsinki, Finland
| | - Jonatan Lehtinen
- Solu Healthcare Oy, Kalevankatu 31 A 13, 00100, Helsinki, Finland
| | - Irene Ortega-Sanz
- Department of Food Technology, Safety and Health, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000, Ghent, Belgium
| | - Jacob L Steenwyk
- Howards Hughes Medical Institute and the Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA
| | - Samuel Sihvonen
- Solu Healthcare Oy, Kalevankatu 31 A 13, 00100, Helsinki, Finland
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Maladan Y, Retnaningrum E, Daryono BS, Sarassari R, Sari RF, Balqis SA, Wahid GA, Safari D. A New Serotyping Method of Streptococcus pneumoniae Based on CRISPR/Cas9-Targeted Sequencing. J Mol Diagn 2024; 26:1045-1054. [PMID: 39603754 DOI: 10.1016/j.jmoldx.2024.08.009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2024] [Revised: 08/05/2024] [Accepted: 08/15/2024] [Indexed: 11/29/2024] Open
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) application for targeted sequencing has made a breakthrough in the genomic research era. High diversity in the capsular polysaccharide (cps) locus of Streptococcus pneumoniae has hampered identification of the serotype. This study developed a new serotyping method for S. pneumoniae using CRISPR/Cas9-targeted sequencing with the Oxford Nanopore Technologies platform. A probe was designed at the position of the cps locus using an excision approach on two sides flanking genes between the dexB and aliA genes with approximately 20 kb. A native barcoding method was used for multiplexing. The probe will attach to a specific side followed by attachment of CRISPR/Cas9 to cut the recognition area. The study used de novo assembly to reconstruct sequence reads, which were analyzed using PneumoCRISPR, a new serotyping pipeline for Oxford Nanopore Technologies sequencing data output. Four CRISPR/Cas9 probes have been designed and recognize the cps locus of S. pneumoniae. Serotyping results align precisely with serotyping data from whole-genome sequencing. This serotyping method also allows researchers to use multiple samples in a single run. The new serotyping method based on CRISPR/Cas9-targeted sequencing holds immense promise for serotype identification of S. pneumoniae.
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Affiliation(s)
- Yustinus Maladan
- Doctorate Program of Biology, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia
| | - Endah Retnaningrum
- Microbiology Laboratory, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia.
| | - Budi Setiadi Daryono
- Genetics Laboratory, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia
| | - Rosantia Sarassari
- Eijkman Research Center for Molecular Biology, Cibinong Science Center, Bogor, Indonesia
| | - Ratna Fathma Sari
- Eijkman Research Center for Molecular Biology, Cibinong Science Center, Bogor, Indonesia
| | - Sarah Azhari Balqis
- Eijkman Research Center for Molecular Biology, Cibinong Science Center, Bogor, Indonesia
| | - Ghina Athyah Wahid
- Eijkman Research Center for Molecular Biology, Cibinong Science Center, Bogor, Indonesia
| | - Dodi Safari
- Eijkman Research Center for Molecular Biology, Cibinong Science Center, Bogor, Indonesia.
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Alnaimat SM, Abushattal S, Dmour SM. Comparative genomic characterization of Cellulosimicrobium funkei isolate RVMD1 from Ma'an desert rock varnish challenges Cellulosimicrobium systematics. Front Microbiol 2024; 15:1445943. [PMID: 39575183 PMCID: PMC11579708 DOI: 10.3389/fmicb.2024.1445943] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2024] [Accepted: 10/22/2024] [Indexed: 11/24/2024] Open
Abstract
Desert environments harbor unique microbial communities. This study focuses on Cellulosimicrobium funkei isolate RVMD1, isolated from rock varnish in the Ma'an Desert. Initial identification was achieved using 16S rRNA gene sequencing, followed by whole-genome sequencing (WGS) for comprehensive characterization. The genome comprises 4,264,015 base pairs (857 contigs) with a high G + C content of 74.59%. A total of 4,449 proteins were predicted. Comparative analysis utilizing OrthoANI, ANI, AAI, and dDDH metrics suggests that RVMD1 belongs to the C. cellulans group, with the highest similarity to C. funkei (97.71% ANI). Phylogenomic analysis of 43 Cellulosimicrobium genomes revealed significant heterogeneity within the genus. Our results challenge current systematics, with C. cellulans potentially representing up to 9 distinct genomospecies. Isolate RVMD1 shows genetic adaptations to its desert environment, including genes for denitrification, oxygen and sulfur cycling, and diverse hydrogen metabolism. Pangenomic analysis uncovered a considerable number of unique genes within RVMD1, highlighting its genetic distinctiveness. Gene family expansions suggest evolution in response to stressors like UV radiation and nutrient limitation. This study represents the first whole-genome analysis of a bacterium isolated from Jordanian rock varnish, emphasizing the value of WGS in understanding microbial diversity and adaptation in extreme environments.
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Affiliation(s)
- Sulaiman M. Alnaimat
- Department of Medical Analysis, Princess Aisha Bint Al-Hussein College of Nursing and Health Sciences, Al-Hussein Bin Talal University, Ma’an, Jordan
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Maladan Y, Retnaningrum E, Daryono BS, Salsabila K, Sarassari R, Khoeri MM, Sari RF, Balqis SA, Wahid GA, Safari D. Pneumococcal transposon profiling associated with macrolide, tetracycline, and chloramphenicol resistance from carriage isolates of serotype 19F in Indonesia. INFECTION, GENETICS AND EVOLUTION : JOURNAL OF MOLECULAR EPIDEMIOLOGY AND EVOLUTIONARY GENETICS IN INFECTIOUS DISEASES 2024; 125:105672. [PMID: 39313084 DOI: 10.1016/j.meegid.2024.105672] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/26/2024] [Revised: 08/22/2024] [Accepted: 09/20/2024] [Indexed: 09/25/2024]
Abstract
Genetic evolution of resistance due to mutations and transposon insertions is the primary cause of antimicrobial resistance in Streptococcus pneumoniae. Resistance to macrolide, tetracycline, and chloramphenicol is caused by the insertion of specific genes that carried by transposon (Tn). This study aims to analyze transposon profiling associated with macrolide, tetracycline, and chloramphenicol resistance from carriage isolates of S. pneumoniae serotype 19F in Indonesia. S. pneumoniae serotype 19F isolates were collected from nasopharyngeal swab specimens from different regions in Indonesia. Genomic DNA was extracted from sixteen isolates and whole genome sequencing was performed on Illumina platform. Raw sequence data were analyzed using de novo assembly by ASA3P and Microscope server. The presence of transposons was identified with detection of int and xis genes and visualized by pyGenomeViz. The genome size of S. pneumoniae ranges from 2,040,117 bp to 2,437,939 bp, with a GC content of around 39 %. ST1464 (4/16) and ST271 (3/16) were found as the predominant sequence type among isolates. Tn2010 was the most common transposon among S. pneumoniae serotype 19F isolates (7/16) followed by Tn2009 (4/16), and Tn5253 (3/16). We identified two deletion sites within the tetM gene (2 bp and 58 bp) that confer tetracycline susceptibility from one isolate. This study suggests that genomic analysis can be employed for the detection and surveillance of antimicrobial resistance genes among S. pneumoniae strains isolated from various regions in Indonesia.
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Affiliation(s)
- Yustinus Maladan
- Biology Doctoral Students, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia; Eijkman Research Center for Molecular Biology, The National Research and Innovation Agency, Cibinong Science Center, Bogor, Indonesia
| | - Endah Retnaningrum
- Microbiology Laboratory, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia.
| | - Budi Setiadi Daryono
- Genetics Laboratory, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia
| | - Korrie Salsabila
- Eijkman Research Center for Molecular Biology, The National Research and Innovation Agency, Cibinong Science Center, Bogor, Indonesia
| | - Rosantia Sarassari
- Eijkman Research Center for Molecular Biology, The National Research and Innovation Agency, Cibinong Science Center, Bogor, Indonesia
| | - Miftahuddin Majid Khoeri
- Eijkman Research Center for Molecular Biology, The National Research and Innovation Agency, Cibinong Science Center, Bogor, Indonesia; Doctoral program in Biomedical, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia
| | - Ratna Fathma Sari
- Eijkman Research Center for Molecular Biology, The National Research and Innovation Agency, Cibinong Science Center, Bogor, Indonesia
| | - Sarah Azhari Balqis
- Eijkman Research Center for Molecular Biology, The National Research and Innovation Agency, Cibinong Science Center, Bogor, Indonesia
| | - Ghina Athyah Wahid
- Eijkman Research Center for Molecular Biology, The National Research and Innovation Agency, Cibinong Science Center, Bogor, Indonesia
| | - Dodi Safari
- Eijkman Research Center for Molecular Biology, The National Research and Innovation Agency, Cibinong Science Center, Bogor, Indonesia.
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Rahman MH, El Zowalaty ME, Falgenhauer L, Khan MFR, Alam J, Popy NN, Rahman MB. Draft genome sequences of clinical mastitis-associated Enterococcus faecalis and Enterococcus faecium carrying multiple antimicrobial resistance genes isolated from dairy cows. J Glob Antimicrob Resist 2024; 38:111-115. [PMID: 38795772 DOI: 10.1016/j.jgar.2024.05.011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2023] [Revised: 03/19/2024] [Accepted: 05/15/2024] [Indexed: 05/28/2024] Open
Abstract
OBJECTIVES The emergence of antimicrobial-resistant and mastitis-associated Enterococcus faecalis and Enterococcus faecium is of great concern due to the huge economic losses associated with enterococcal infections. Here we report the draft genome sequences of E. faecalis and E. faecium strains that were isolated from raw milk samples obtained from mastitis-infected cows in Bangladesh. METHODS The two strains were isolated, identified, and genomic DNA was sequenced using the Illumina NextSeq 550 platform. The assembled contigs were analysed for virulence, antimicrobial resistance genes, and multilocus sequence type. The genomes were compared to previously reported E. faecalis and E. faecium genomes to generate core genome phylogenetic trees. RESULTS E. faecalis strain BR-MHR218Efa and E. faecium strain BR-MHR268Efe belonged to multilocus sequence types ST-190 and ST-22, respectively, both of which appear to represent relatively rare sequence types. BR-MHR268Efe harboured only one antibiotic resistance gene encoding resistance towards macrolides (lsa(A)), while BR-MHR218Efa harboured ten different antibiotic resistance genes encoding resistance to aminoglycosides (ant[6]-Ia, aph(3')-III), sulphonamides (aac(6')-II), lincosamides (lnu(B)), macrolides (erm(B)), MLSB antibiotics (msr(C)), tetracyclines (tet(M), tet(L)), trimethoprim (dfrG), and pleuromutilin-lincosamide-streptogramin A (lsa(E)). Virulence gene composition was different between the two isolates. BR-MHR218Efa harboured only two virulence genes involved in adherence (acm and scm). BR-MHR268Efe harboured eight complete virulence operons including three operons involved in adherence (Ace, Ebp pili, and EfaA), two operons involved in biofilm formation (BopD and Fsr), and three exoenzymes (gelatinase, hyaluronidase, SprE). CONCLUSIONS The genome sequences of the strains BR-MHR268Efe and BR-MHR218Efa will serve as a reference point for molecular epidemiological studies of mastitis-associated E. faecalis and E. faecium. Additionally, the findings will help understand the complex antimicrobial-resistance in livestock-assoiated Enterococci.
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Affiliation(s)
- Mohammad H Rahman
- Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
| | - Mohamed E El Zowalaty
- Department of Microbiology and Immunology, Faculty of Pharmacy, Ahram Canadian University, Giza, Egypt.
| | - Linda Falgenhauer
- Institute of Hygiene and Environmental Medicine, Justus Liebig University Giessen, Biomedical Research Center Seltersberg, Schubertstrasse 81, 35392 Giessen, Germany
| | | | - Jahangir Alam
- National Institute of Biotechnology, Savar, Dhaka, Bangladesh
| | - Najmun Nahar Popy
- Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
| | - Md Bahanur Rahman
- Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.
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Marquis B, Pillonel T, Carrara A, Bertelli C. zDB: bacterial comparative genomics made easy. mSystems 2024; 9:e0047324. [PMID: 38940522 PMCID: PMC11264898 DOI: 10.1128/msystems.00473-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2024] [Accepted: 05/31/2024] [Indexed: 06/29/2024] Open
Abstract
The analysis and comparison of genomes rely on different tools for tasks such as annotation, orthology prediction, and phylogenetic inference. Most tools are specialized for a single task, and additional efforts are necessary to integrate and visualize the results. To fill this gap, we developed zDB, an application integrating a Nextflow analysis pipeline and a Python visualization platform built on the Django framework. The application is available on GitHub (https://github.com/metagenlab/zDB) and from the bioconda channel. Starting from annotated Genbank files, zDB identifies orthologs and infers a phylogeny for each orthogroup. A species phylogeny is also constructed from shared single-copy orthologs. The results can be enriched with Pfam protein domain prediction, Cluster of Orthologs Genes and Kyoto Encyclopedia of Genes and Genomes annotations, and Swissprot homologs. The web application allows searching for specific genes or annotations, running Blast queries, and comparing genomic regions and whole genomes. The metabolic capacities of organisms can be compared at either the module or pathway levels. Finally, users can run queries to examine the conservation of specific genes or annotations across a chosen subset of genomes and display the results as a list of genes, Venn diagram, or heatmaps. Those features make zDB useful for both bioinformaticians and researchers more accustomed to laboratory research.IMPORTANCEGenome comparison and analysis rely on many independent tools, leaving to scientists the burden to integrate and visualize their results for interpretation. To alleviate this burden, we have built zDB, a comparative genomics tool that includes both an analysis pipeline and a visualization platform. The analysis pipeline automates gene annotation, orthology prediction, and phylogenetic inference, while the visualization platform allows scientists to easily explore the results in a web browser. Among other features, the interface allows users to visually compare whole genomes and targeted regions, assess the conservation of genes or metabolic pathways, perform Blast searches, or look for specific annotations. Altogether, this tool will be useful for a broad range of applications in comparative studies between two and hundred genomes. Furthermore, it is designed to allow sharing of data sets easily at a local or international scale, thereby supporting exploratory analyses for non-bioinformaticians on the genome of their favorite organisms.
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Affiliation(s)
- Bastian Marquis
- Lausanne University Hospital and University of Lausanne, Institute of Microbiology, Lausanne, Switzerland
| | - Trestan Pillonel
- Lausanne University Hospital and University of Lausanne, Institute of Microbiology, Lausanne, Switzerland
| | - Alessia Carrara
- Lausanne University Hospital and University of Lausanne, Institute of Microbiology, Lausanne, Switzerland
| | - Claire Bertelli
- Lausanne University Hospital and University of Lausanne, Institute of Microbiology, Lausanne, Switzerland
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Le DQ, Nguyen TT, Nguyen CH, Ho TH, Vo NS, Nguyen T, Nguyen HA, Vinh LS, Dang TH, Cao MD, Nguyen SH. AMRomics: a scalable workflow to analyze large microbial genome collections. BMC Genomics 2024; 25:709. [PMID: 39039439 PMCID: PMC11264974 DOI: 10.1186/s12864-024-10620-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2024] [Accepted: 07/15/2024] [Indexed: 07/24/2024] Open
Abstract
Whole genome analysis for microbial genomics is critical to studying and monitoring antimicrobial resistance strains. The exponential growth of microbial sequencing data necessitates a fast and scalable computational pipeline to generate the desired outputs in a timely and cost-effective manner. Recent methods have been implemented to integrate individual genomes into large collections of specific bacterial populations and are widely employed for systematic genomic surveillance. However, they do not scale well when the population expands and turnaround time remains the main issue for this type of analysis. Here, we introduce AMRomics, an optimized microbial genomics pipeline that can work efficiently with big datasets. We use different bacterial data collections to compare AMRomics against competitive tools and show that our pipeline can generate similar results of interest but with better performance. The software is open source and is publicly available at https://github.com/amromics/amromics under an MIT license.
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Affiliation(s)
- Duc Quang Le
- AMROMICS JSC, Nghe An, Vietnam.
- Faculty of Information Technology, VNU University of Engineering and Technology, Hanoi, Vietnam.
- Faculty of IT, Hanoi University of Civil Engineering, Hanoi, Vietnam.
| | - Tam Thi Nguyen
- Oxford University Clinical Research Unit, Hanoi, Vietnam
| | - Canh Hao Nguyen
- Bioinformatics Center, Institute for Chemical Research, Kyoto University, Kyoto, Japan
| | - Tho Huu Ho
- Department of Medical Microbiology, The 103 Military Hospital, Vietnam Military Medical University, Hanoi, Vietnam
- Department of Genomics & Cytogenetics, Institute of Biomedicine & Pharmacy, Vietnam Military Medical University, Hanoi, Vietnam
| | - Nam S Vo
- Center for Biomedical Informatics, Vingroup Big Data Institute, Hanoi, Vietnam
| | | | | | - Le Sy Vinh
- Faculty of Information Technology, VNU University of Engineering and Technology, Hanoi, Vietnam
| | - Thanh Hai Dang
- Faculty of Information Technology, VNU University of Engineering and Technology, Hanoi, Vietnam
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11
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Nuhamunada M, Mohite OS, Phaneuf P, Palsson B, Weber T. BGCFlow: systematic pangenome workflow for the analysis of biosynthetic gene clusters across large genomic datasets. Nucleic Acids Res 2024; 52:5478-5495. [PMID: 38686794 PMCID: PMC11162802 DOI: 10.1093/nar/gkae314] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2023] [Revised: 03/22/2024] [Accepted: 04/11/2024] [Indexed: 05/02/2024] Open
Abstract
Genome mining is revolutionizing natural products discovery efforts. The rapid increase in available genomes demands comprehensive computational platforms to effectively extract biosynthetic knowledge encoded across bacterial pangenomes. Here, we present BGCFlow, a novel systematic workflow integrating analytics for large-scale genome mining of bacterial pangenomes. BGCFlow incorporates several genome analytics and mining tools grouped into five common stages of analysis such as: (i) data selection, (ii) functional annotation, (iii) phylogenetic analysis, (iv) genome mining, and (v) comparative analysis. Furthermore, BGCFlow provides easy configuration of different projects, parallel distribution, scheduled job monitoring, an interactive database to visualize tables, exploratory Jupyter Notebooks, and customized reports. Here, we demonstrate the application of BGCFlow by investigating the phylogenetic distribution of various biosynthetic gene clusters detected across 42 genomes of the Saccharopolyspora genus, known to produce industrially important secondary/specialized metabolites. The BGCFlow-guided analysis predicted more accurate dereplication of BGCs and guided the targeted comparative analysis of selected RiPPs. The scalable, interoperable, adaptable, re-entrant, and reproducible nature of the BGCFlow will provide an effective novel way to extract the biosynthetic knowledge from the ever-growing genomic datasets of biotechnologically relevant bacterial species.
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Affiliation(s)
- Matin Nuhamunada
- The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby 2800, Denmark
| | - Omkar S Mohite
- The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby 2800, Denmark
| | - Patrick V Phaneuf
- The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby 2800, Denmark
| | - Bernhard O Palsson
- The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby 2800, Denmark
- Department of Bioengineering, University of California San Diego, La Jolla, CA 92093, USA
| | - Tilmann Weber
- The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby 2800, Denmark
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12
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Le DQ, Nguyen SH, Nguyen TT, Nguyen CH, Ho TH, Vo NS, Nguyen T, Nguyen HA, Cao MD. AMRViz enables seamless genomics analysis and visualization of antimicrobial resistance. BMC Bioinformatics 2024; 25:193. [PMID: 38755527 PMCID: PMC11100100 DOI: 10.1186/s12859-024-05792-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2023] [Accepted: 04/18/2024] [Indexed: 05/18/2024] Open
Abstract
We have developed AMRViz, a toolkit for analyzing, visualizing, and managing bacterial genomics samples. The toolkit is bundled with the current best practice analysis pipeline allowing researchers to perform comprehensive analysis of a collection of samples directly from raw sequencing data with a single command line. The analysis results in a report showing the genome structure, genome annotations, antibiotic resistance and virulence profile for each sample. The pan-genome of all samples of the collection is analyzed to identify core- and accessory-genes. Phylogenies of the whole genome as well as all gene clusters are also generated. The toolkit provides a web-based visualization dashboard allowing researchers to interactively examine various aspects of the analysis results. Availability: AMRViz is implemented in Python and NodeJS, and is publicly available under open source MIT license at https://github.com/amromics/amrviz .
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Affiliation(s)
- Duc Quang Le
- AMROMICS JSC, Nghe An, Vietnam.
- Faculty of IT, Hanoi University of Civil Engineering, Hanoi, Vietnam.
| | | | - Tam Thi Nguyen
- Oxford University Clinical Research Unit, Hanoi, Vietnam
| | - Canh Hao Nguyen
- Bioinformatics Center, Institute for Chemical Research, Kyoto University, Kyoto, Japan
| | - Tho Huu Ho
- Department of Medical Microbiology, The 103 Military Hospital, Vietnam Military Medical University, Hanoi, Vietnam
- Department of Genomics and Cytogenetics, Institute of Biomedicine and Pharmacy, Vietnam Military Medical University, Hanoi, Vietnam
| | - Nam S Vo
- Center for Biomedical Informatics, Vingroup Big Data Institute, Hanoi, Vietnam
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13
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Bouras G, Houtak G, Wick RR, Mallawaarachchi V, Roach MJ, Papudeshi B, Judd LM, Sheppard AE, Edwards RA, Vreugde S. Hybracter: enabling scalable, automated, complete and accurate bacterial genome assemblies. Microb Genom 2024; 10:001244. [PMID: 38717808 PMCID: PMC11165638 DOI: 10.1099/mgen.0.001244] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2024] [Accepted: 04/16/2024] [Indexed: 05/21/2024] Open
Abstract
Improvements in the accuracy and availability of long-read sequencing mean that complete bacterial genomes are now routinely reconstructed using hybrid (i.e. short- and long-reads) assembly approaches. Complete genomes allow a deeper understanding of bacterial evolution and genomic variation beyond single nucleotide variants. They are also crucial for identifying plasmids, which often carry medically significant antimicrobial resistance genes. However, small plasmids are often missed or misassembled by long-read assembly algorithms. Here, we present Hybracter which allows for the fast, automatic and scalable recovery of near-perfect complete bacterial genomes using a long-read first assembly approach. Hybracter can be run either as a hybrid assembler or as a long-read only assembler. We compared Hybracter to existing automated hybrid and long-read only assembly tools using a diverse panel of samples of varying levels of long-read accuracy with manually curated ground truth reference genomes. We demonstrate that Hybracter as a hybrid assembler is more accurate and faster than the existing gold standard automated hybrid assembler Unicycler. We also show that Hybracter with long-reads only is the most accurate long-read only assembler and is comparable to hybrid methods in accurately recovering small plasmids.
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Affiliation(s)
- George Bouras
- Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, Australia
- The Department of Surgery – Otolaryngology Head and Neck Surgery, University of Adelaide and the Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, Adelaide, South Australia, Australia
| | - Ghais Houtak
- Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, Australia
- The Department of Surgery – Otolaryngology Head and Neck Surgery, University of Adelaide and the Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, Adelaide, South Australia, Australia
| | - Ryan R. Wick
- Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
| | - Vijini Mallawaarachchi
- Flinders Accelerator for Microbiome Exploration, College of Science and Engineering, Flinders University, Adelaide, Australia
| | - Michael J. Roach
- Flinders Accelerator for Microbiome Exploration, College of Science and Engineering, Flinders University, Adelaide, Australia
- Adelaide Centre for Epigenetics and South Australian Immunogenomics Cancer Institute, The University of Adelaide, Adelaide, Australia
| | - Bhavya Papudeshi
- Flinders Accelerator for Microbiome Exploration, College of Science and Engineering, Flinders University, Adelaide, Australia
| | - Lousie M. Judd
- Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
| | - Anna E. Sheppard
- School of Biological Sciences, The University of Adelaide, Adelaide, Australia
| | - Robert A. Edwards
- Flinders Accelerator for Microbiome Exploration, College of Science and Engineering, Flinders University, Adelaide, Australia
| | - Sarah Vreugde
- Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, Australia
- The Department of Surgery – Otolaryngology Head and Neck Surgery, University of Adelaide and the Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, Adelaide, South Australia, Australia
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14
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Bouras G, Houtak G, Wick RR, Mallawaarachchi V, Roach MJ, Papudeshi B, Judd LM, Sheppard AE, Edwards RA, Vreugde S. Hybracter: Enabling Scalable, Automated, Complete and Accurate Bacterial Genome Assemblies. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2023.12.12.571215. [PMID: 38168369 PMCID: PMC10760025 DOI: 10.1101/2023.12.12.571215] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2024]
Abstract
Improvements in the accuracy and availability of long-read sequencing mean that complete bacterial genomes are now routinely reconstructed using hybrid (i.e. short- and long-reads) assembly approaches. Complete genomes allow a deeper understanding of bacterial evolution and genomic variation beyond single nucleotide variants (SNVs). They are also crucial for identifying plasmids, which often carry medically significant antimicrobial resistance (AMR) genes. However, small plasmids are often missed or misassembled by long-read assembly algorithms. Here, we present Hybracter which allows for the fast, automatic, and scalable recovery of near-perfect complete bacterial genomes using a long-read first assembly approach. Hybracter can be run either as a hybrid assembler or as a long-read only assembler. We compared Hybracter to existing automated hybrid and long-read only assembly tools using a diverse panel of samples of varying levels of long-read accuracy with manually curated ground truth reference genomes. We demonstrate that Hybracter as a hybrid assembler is more accurate and faster than the existing gold standard automated hybrid assembler Unicycler. We also show that Hybracter with long-reads only is the most accurate long-read only assembler and is comparable to hybrid methods in accurately recovering small plasmids.
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Affiliation(s)
- George Bouras
- Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, Australia
- The Department of Surgery - Otolaryngology Head and Neck Surgery, University of Adelaide and the Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, South Australia, Australia
| | - Ghais Houtak
- Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, Australia
- The Department of Surgery - Otolaryngology Head and Neck Surgery, University of Adelaide and the Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, South Australia, Australia
| | - Ryan R. Wick
- Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
| | - Vijini Mallawaarachchi
- Flinders Accelerator for Microbiome Exploration, College of Science and Engineering, Flinders University, Adelaide, Australia
| | - Michael J. Roach
- Flinders Accelerator for Microbiome Exploration, College of Science and Engineering, Flinders University, Adelaide, Australia
- Adelaide Centre for Epigenetics and South Australian Immunogenomics Cancer Institute, The University of Adelaide, Adelaide, Australia
| | - Bhavya Papudeshi
- Flinders Accelerator for Microbiome Exploration, College of Science and Engineering, Flinders University, Adelaide, Australia
| | - Lousie M. Judd
- Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
| | - Anna E. Sheppard
- School of Biological Sciences, The University of Adelaide, Adelaide, Australia
| | - Robert A. Edwards
- Flinders Accelerator for Microbiome Exploration, College of Science and Engineering, Flinders University, Adelaide, Australia
| | - Sarah Vreugde
- Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, Australia
- The Department of Surgery - Otolaryngology Head and Neck Surgery, University of Adelaide and the Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, South Australia, Australia
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15
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Khoeri MM, Maladan Y, Salsabila K, Alimsardjono L, Vermasari N, Puspitasari I, Yunita R, Tafroji W, Sarassari R, Sari RF, Balqis SA, Wahid GA, Purwanto DS, Harimurti K, Soebandrio A, Safari D. Whole genome sequencing data of Streptococcus pneumoniae isolated from Indonesian population. Data Brief 2024; 53:110251. [PMID: 38533114 PMCID: PMC10964051 DOI: 10.1016/j.dib.2024.110251] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2023] [Revised: 12/21/2023] [Accepted: 02/20/2024] [Indexed: 03/28/2024] Open
Abstract
Streptococcus pneumoniae is the leading cause of bacterial pneumonia, bacteremia, and meningitis. Indonesia introduced the pneumococcal conjugate vaccine (PCV) nationwide in 2022. In this study, we present whole genome sequence (WGS) data of 94 S. pneumoniae isolates that were obtained from hospitalized patients, healthy children, and adult groups from different regions prior to PCV program in Indonesia. DNA sequences of S. pneumoniae were obtained using the TruSeq Nano DNA kit (Illumina NovaSeq6000 Platform). The genome data of S. pneumoniae features a 1,969,562 bp to 2,741,371 bp circular chromosome with 39-40% G+C content. The genome includes 1935-3319 coding sequences (CDS), 2 to 5 rRNA genes, 43 to 49 tRNA genes, and 56 to 71 ncRNA. These data will be useful for analyzing the serotype, sequence type, virulence genes, antimicrobial resistance genes, and the impact of pneumococcal vaccination in Indonesia. The FASTQ raw files of these sequences are available under BioProject accession number PRJNA995903 and Sequence Read Archive accession numbers SRR25316461-SRR25316554.
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Affiliation(s)
- Miftahuddin Majid Khoeri
- Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Cibinong, West Java, Indonesia
- Doctoral program in Biomedical, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia
| | - Yustinus Maladan
- Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Cibinong, West Java, Indonesia
| | - Korrie Salsabila
- Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Cibinong, West Java, Indonesia
| | | | | | | | - Rina Yunita
- Haji Adam Malik Hospital, Medan, North Sumatera, Indonesia
- Department of Microbiology, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia
| | - Wisnu Tafroji
- Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Cibinong, West Java, Indonesia
| | - Rosantia Sarassari
- Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Cibinong, West Java, Indonesia
| | - Ratna Fathma Sari
- Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Cibinong, West Java, Indonesia
| | - Sarah Azhari Balqis
- Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Cibinong, West Java, Indonesia
| | - Ghina Athyah Wahid
- Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Cibinong, West Java, Indonesia
| | - Diana Shinta Purwanto
- Department of Biochemistry, Faculty of Medicine, Sam Ratulangi University, Manado, North Sulawesi, Indonesia
| | - Kuntjoro Harimurti
- Departement of Internal Medicine, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia
| | - Amin Soebandrio
- Departement of Clinical Microbiology, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia
| | - Dodi Safari
- Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Cibinong, West Java, Indonesia
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16
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El Zowalaty ME, Falgenhauer L, Ashour HM, Zishiri OT, Forsythe S, Helmy YA. Draft genome sequences of Bacillus licheniformis strains MEZBL63 and MEZBL64 harboring the lichenysin toxin operon isolated from livestock in South Africa. Microbiol Resour Announc 2024; 13:e0096723. [PMID: 38323846 DOI: 10.1128/mra.00967-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2023] [Accepted: 01/05/2024] [Indexed: 02/08/2024] Open
Abstract
Here, we report the draft genome sequences of two Bacillus licheniformis strains harboring the lichenysin operon that were isolated from healthy goat and horse in South Africa. The genomes were sequenced using Illumina MiSeq and had a length of 4,152,826 and 4,110,075 bp, respectively, with a G + C content of 46%.
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Affiliation(s)
- Mohamed E El Zowalaty
- Veterinary Medicine and Food Security Research Group, Medical Laboratory Sciences Program, Faculty of Health Sciences, Abu Dhabi Women's Campus, Higher Colleges of Technology, Abu Dhabi, United Arab Emirates
| | - Linda Falgenhauer
- Institute of Hygiene and Environmental Medicine, German Center for Infection Research, Site Giessen-Marburg-Langen and Hessian University Competence Center for Hospital Hygiene (HuKKH), Justus Liebig University Giessen, Giessen, Germany
| | - Hossam M Ashour
- Department of Integrative Biology, College of Arts and Sciences, University of South Florida, St. Petersburg, Florida, USA
| | - Oliver T Zishiri
- Discipline of Genetics, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Durban, South Africa
| | - Stephen Forsythe
- Foodmicrobe.com Ltd, Adams Hill, Keyworth, Nottingham, United Kingdom
| | - Yosra A Helmy
- Department of Veterinary Science, Martin-Gatton College of Agriculture, Food, and Environment, University of Kentucky, Lexington, Kentucky, USA
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17
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Rahman MH, El Zowalaty ME, Falgenhauer L, Khan MFR, Alam J, Popy NN, Ashour HM, Rahman MB. Molecular identification and whole genome sequence analyses of methicillin-resistant and mastitis-associated Staphylococcus aureus sequence types 6 and 2454 isolated from dairy cows. J Genomics 2024; 12:19-25. [PMID: 38321997 PMCID: PMC10845239 DOI: 10.7150/jgen.90833] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2023] [Accepted: 12/19/2023] [Indexed: 02/08/2024] Open
Abstract
The emergence of antimicrobial-resistant and mastitis-associated Staphylococcus aureus is of great concern due to the huge economic losses worldwide. Here, we report draft genome sequences of two Staphylococcus aureus strains which were isolated from raw milk samples obtained from mastitis-infected cows in Bangladesh. The strains were isolated and identified using conventional microbiological and molecular polymerase chain reaction (PCR) methods. Antibiotic susceptibility testing was performed. Genomic DNA of the two strains was extracted and the strains were sequenced using the Illumina NextSeq 550 platform. The assembled contigs were analyzed for virulence determinants, antimicrobial resistance genes, extra-chromosomal plasmids, and multi-locus sequence type (MLST). The genomes of the two strains were compared with other publicly available genome sequences of Staphylococcus aureus strains. The raw read sequences were downloaded and all sequence files were analyzed identically to generate core genome phylogenetic trees. The genome of BR-MHR281strain did not harbour any antibiotic resistance determinants, however BR-MHR220 strain harbored mecA and blaZ genes. Analysis of BR-MHR220 strain revealed that it was assigned to sequence type (ST-6), clonal complex (CC) 5 and spa type t304, while BR-MHR281 strain belonged to ST-2454, CC8, and harbored the spa type t7867. The findings of the present study and the genome sequences of BR-MHR220 and BR-MHR281 strains will provide data on the detection and genomic analysis and characterization of mastitis-associated Staphylococcus aureus in Bangladesh. In addition, the findings of the present study will serve as reference genomes for future molecular epidemiological studies and will provide significant data which help understand the prevalence, pathogenesis and antimicrobial resistance of mastitis-associated Staphylococcus aureus.
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Affiliation(s)
- Mohammad H. Rahman
- Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
| | - Mohamed E. El Zowalaty
- Veterinary Medicine and Food Security Research Group, Medical Laboratory Sciences Program, Faculty of Health Sciences, Abu Dhabi Women's Campus, Higher Colleges of Technology, Abu Dhabi, UAE
| | - Linda Falgenhauer
- Institute of Hygiene and Environmental Medicine, Justus Liebig University Giessen, Biomedical research center Seltersberg, Schubertstrasse 81, 35392 Giessen, Germany
| | - Mohammad F. R. Khan
- Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
| | - Jahangir Alam
- National Institute of Biotechnology, Savar, Dhaka, Bangladesh
| | - Najmun N. Popy
- Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
| | - Hossam M. Ashour
- Department of Integrative Biology, College of Arts and Sciences, University of South Florida, St. Petersburg, Florida, USA
| | - Md. Bahanur Rahman
- Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
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18
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El Zowalaty ME, Lamichhane B, Falgenhauer L, Mowlaboccus S, Zishiri OT, Forsythe S, Helmy YA. Antimicrobial resistance and whole genome sequencing of novel sequence types of Enterococcus faecalis, Enterococcus faecium, and Enterococcus durans isolated from livestock. Sci Rep 2023; 13:18609. [PMID: 37903806 PMCID: PMC10616195 DOI: 10.1038/s41598-023-42838-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2023] [Accepted: 09/15/2023] [Indexed: 11/01/2023] Open
Abstract
The emergence of antimicrobial-resistant, livestock-associated Enterococcus faecalis represents a public health concern. Here, we report the isolation, molecular detection of virulence and antimicrobial resistance determinants, in addition to the phylogenetic analyses of 20 Enterococcus species using whole genome sequencing analysis of 15 Enterococcus faecalis strains including six strains of three novel sequence types, three Enterococcus faecium and two Enterococcus durans. All strains were isolated from food chain animals in South Africa. Enterococcus strains were isolated on bile aesculin azide agar, followed by identification using MALDI-TOF MS analysis. Antibiotic susceptibility testing was performed using the Kirby-Bauer disk diffusion method. The genomic DNA of the isolates was extracted and sequencing was performed using the Illumina MiSeq platform. Sequence reads were trimmed and de novo assembled. The assembled contigs were analyzed for antimicrobial resistance genes and chromosomal mutations, extra-chromosomal plasmids, and multi-locus sequence type (MLST). Multidrug antimicrobial resistance genes conferring resistance to aminoglycosides (ant(6)-Ia, aph(3')-IIIa, sat4, and spw), lincosamides (lnu(B), lsa(A), and lsa(E)), macrolides (erm(B)), trimethoprim (dfrG) and tetracyclines (tet(L) and tet(M)) were identified. Plasmid replicons were detected in seven E. faecalis and three E. faecium isolates. The sequence type (ST) of each isolate was determined using the Enterococcus PubMLST database. Ten STs were identified in the collection, three of which (ST1240, ST1241, and ST1242) have not been previously reported and are described in the present study for the first time. To compare the sequenced strains to other previously sequenced E. faecalis strains, assembled sequences of E. faecalis from livestock were downloaded from the PubMLST database. Core genome-based phylogenetic analysis was performed using ParSNP. The detection of multiple drug-resistance in Enterococcus including E. faecalis and E. faecium highlights the significance of genomic surveillance to monitor the spread of antimicrobial resistance in food chain animals. In addition, the genome sequences of Enterococcus strains reported in the present study will serve as a reference point for future molecular epidemiological studies of livestock-associated and antibiotic-resistant E. faecalis in Africa. In addition, this study enables the in-depth analysis of E. faecalis genomic structure, as well as provides valuable information on the phenotypic and genotypic antimicrobial resistance, and the pathogenesis of livestock-associated E. faecalis and E. faecium.
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Affiliation(s)
- Mohamed E El Zowalaty
- Veterinary Medicine and Food Security Research Group, Medical Laboratory Sciences Program, Faculty of Health Sciences, Abu Dhabi Women's Campus, Higher Colleges of Technology, Abu Dhabi, 41012, UAE.
| | - Bibek Lamichhane
- Department of Veterinary Science, Martin-Gatton College of Agriculture, Food, and Environment, University of Kentucky, Lexington, KY, 40546, USA
| | - Linda Falgenhauer
- Institute of Hygiene and Environmental Medicine, Justus Liebig University Giessen, Biomedical Research Center Seltersberg, Schubertstrasse 81, 35392, Giessen, Germany
| | - Shakeel Mowlaboccus
- Antimicrobial Resistance and Infectious Diseases Research Laboratory, College of Science, Health, Engineering and Education, Murdoch University, Murdoch, WA, Australia
- Department of Microbiology, PathWest Laboratory Medicine-WA, Fiona Stanley Hospital, Murdoch, WA, Australia
| | - Oliver T Zishiri
- Discipline of Genetics, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Private Bag X54001, Westville, Durban, 4000, South Africa
| | - Stephen Forsythe
- Foodmicrobe.com Ltd., Adams Hill, Keyworth, Nottingham, NG12 5GY, UK
| | - Yosra A Helmy
- Department of Veterinary Science, Martin-Gatton College of Agriculture, Food, and Environment, University of Kentucky, Lexington, KY, 40546, USA.
- Department of Zoonoses, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, 41522, Egypt.
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19
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de Almeida FM, de Campos TA, Pappas Jr GJ. Scalable and versatile container-based pipelines for de novo genome assembly and bacterial annotation. F1000Res 2023; 12:1205. [PMID: 37970066 PMCID: PMC10646344 DOI: 10.12688/f1000research.139488.1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 08/16/2023] [Indexed: 11/17/2023] Open
Abstract
Background: Advancements in DNA sequencing technology have transformed the field of bacterial genomics, allowing for faster and more cost effective chromosome level assemblies compared to a decade ago. However, transforming raw reads into a complete genome model is a significant computational challenge due to the varying quality and quantity of data obtained from different sequencing instruments, as well as intrinsic characteristics of the genome and desired analyses. To address this issue, we have developed a set of container-based pipelines using Nextflow, offering both common workflows for inexperienced users and high levels of customization for experienced ones. Their processing strategies are adaptable based on the sequencing data type, and their modularity enables the incorporation of new components to address the community's evolving needs. Methods: These pipelines consist of three parts: quality control, de novo genome assembly, and bacterial genome annotation. In particular, the genome annotation pipeline provides a comprehensive overview of the genome, including standard gene prediction and functional inference, as well as predictions relevant to clinical applications such as virulence and resistance gene annotation, secondary metabolite detection, prophage and plasmid prediction, and more. Results: The annotation results are presented in reports, genome browsers, and a web-based application that enables users to explore and interact with the genome annotation results. Conclusions: Overall, our user-friendly pipelines offer a seamless integration of computational tools to facilitate routine bacterial genomics research. The effectiveness of these is illustrated by examining the sequencing data of a clinical sample of Klebsiella pneumoniae.
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Affiliation(s)
- Felipe Marques de Almeida
- Programa de Pós-graduação em Biologia Molecular, Universidade de Brasilia, Brasília, FD, 70910-900, Brazil
- Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, 70910-900, Brazil
| | - Tatiana Amabile de Campos
- Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, 70910-900, Brazil
- Programa de Pós-graduação em Biologia Microbiana, Universidade de Brasília, Brasília, DF, 70910-900, Brazil
| | - Georgios Joannis Pappas Jr
- Programa de Pós-graduação em Biologia Molecular, Universidade de Brasilia, Brasília, FD, 70910-900, Brazil
- Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, 70910-900, Brazil
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Maladan Y, Safari D, Parikesit AA. Structural dynamics insights into the M306L, M306V, and D1024N mutations in Mycobacterium tuberculosis inducing resistance to ethambutol. Genomics Inform 2023; 21:e32. [PMID: 37813628 PMCID: PMC10584647 DOI: 10.5808/gi.23019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2023] [Revised: 05/18/2023] [Accepted: 08/07/2023] [Indexed: 10/11/2023] Open
Abstract
Resistance to anti-tuberculosis drugs, especially ethambutol (EMB), has been widely reported worldwide. EMB resistance is caused by mutations in the embB gene, which encodes the arabinosyl transferase enzyme. This study aimed to detect mutations in the embB gene of Mycobacterium tuberculosis from Papua and to evaluate their impact on the effectiveness of EMB. We analyzed 20 samples of M. tuberculosis culture that had undergone whole-genome sequencing, of which 19 samples were of sufficient quality for further bioinformatics analysis. Mutation analysis was performed using TBProfiler, which identified M306L, M306V, D1024N, and E378A mutations. In sample TB035, the M306L mutation was present along with E378A. The binding affinity of EMB to arabinosyl transferase was calculated using AutoDock Vina. The molecular docking results revealed that all mutants demonstrated an increased binding affinity to EMB compared to the native protein (-0.948 kcal/mol). The presence of the M306L mutation, when coexisting with E378A, resulted in a slight increase in binding affinity compared to the M306L mutation alone. The molecular dynamics simulation results indicated that the M306L, M306L + E378A, M306V, and E378A mutants decreased protein stability. Conversely, the D1024N mutant exhibited stability comparable to the native protein. In conclusion, this study suggests that the M306L, M306L + E378A, M306V, and E378A mutations may contribute to EMB resistance, while the D1024N mutation may be consistent with continued susceptibility to EMB.
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Affiliation(s)
- Yustinus Maladan
- Eijkman Research Center for Molecular Biology, The National Research and Innovation Agency, Cibinong, Bogor 16911, Indonesia
| | - Dodi Safari
- Eijkman Research Center for Molecular Biology, The National Research and Innovation Agency, Cibinong, Bogor 16911, Indonesia
| | - Arli Aditya Parikesit
- Department of Bioinformatics, School of Life Sciences, Indonesia International Institute for Life Sciences (I3L), Jakarta 13210, Indonesia
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Rahman MH, El Zowalaty ME, Falgenhauer L, Khan MFR, Alam J, Popy NN, Rahman MB. Draft Genome Sequences of Two Clinical Mastitis-Associated Escherichia coli Strains, of Sequence Type 101 and Novel Sequence Type 13054, Isolated from Dairy Cows in Bangladesh. Microbiol Resour Announc 2023; 12:e0016623. [PMID: 37428031 PMCID: PMC10443403 DOI: 10.1128/mra.00166-23] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2023] [Accepted: 05/13/2023] [Indexed: 07/11/2023] Open
Abstract
Here, we report the draft genome sequences of two Escherichia coli strains that were isolated from raw milk samples obtained from lactating cows with mastitis in Bangladesh. One strain was assigned to a novel sequence type 13054, and the other strain belonged to sequence type 101.
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Affiliation(s)
- Mohammad H. Rahman
- Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, Bangladesh
| | - Mohamed E. El Zowalaty
- Veterinary Medicine and Food Security Research Group, Medical Laboratory Sciences Program, Faculty of Health Sciences, Abu Dhabi Women's Campus, Higher Colleges of Technology, Abu Dhabi, United Arab Emirates
| | - Linda Falgenhauer
- German Center for Infection Research, Site Giessen-Marburg-Langen, Giessen, Germany
- Institute of Hygiene and Environmental Medicine, Justus Liebig University Giessen, Giessen, Germany
- Hessian University Competence Center for Hospital Hygiene, Justus Liebig University Giessen, Giessen, Germany
| | | | - Jahangir Alam
- National Institute of Biotechnology, Savar, Dhaka, Bangladesh
| | - Najmun Nahar Popy
- Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, Bangladesh
| | - M. Bahanur Rahman
- Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, Bangladesh
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22
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Ortega-Sanz I, Barbero-Aparicio JA, Canepa-Oneto A, Rovira J, Melero B. CamPype: an open-source workflow for automated bacterial whole-genome sequencing analysis focused on Campylobacter. BMC Bioinformatics 2023; 24:291. [PMID: 37474912 PMCID: PMC10357626 DOI: 10.1186/s12859-023-05414-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2023] [Accepted: 07/14/2023] [Indexed: 07/22/2023] Open
Abstract
BACKGROUND The rapid expansion of Whole-Genome Sequencing has revolutionized the fields of clinical and food microbiology. However, its implementation as a routine laboratory technique remains challenging due to the growth of data at a faster rate than can be effectively analyzed and critical gaps in bioinformatics knowledge. RESULTS To address both issues, CamPype was developed as a new bioinformatics workflow for the genomics analysis of sequencing data of bacteria, especially Campylobacter, which is the main cause of gastroenteritis worldwide making a negative impact on the economy of the public health systems. CamPype allows fully customization of stages to run and tools to use, including read quality control filtering, read contamination, reads extension and assembly, bacterial typing, genome annotation, searching for antibiotic resistance genes, virulence genes and plasmids, pangenome construction and identification of nucleotide variants. All results are processed and resumed in an interactive HTML report for best data visualization and interpretation. CONCLUSIONS The minimal user intervention of CamPype makes of this workflow an attractive resource for microbiology laboratories with no expertise in bioinformatics as a first line method for bacterial typing and epidemiological analyses, that would help to reduce the costs of disease outbreaks, or for comparative genomic analyses. CamPype is publicly available at https://github.com/JoseBarbero/CamPype .
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Affiliation(s)
- Irene Ortega-Sanz
- Department of Biotechnology and Food Science, University of Burgos, 09006, Burgos, Spain
| | | | | | - Jordi Rovira
- Department of Biotechnology and Food Science, University of Burgos, 09006, Burgos, Spain
| | - Beatriz Melero
- Department of Biotechnology and Food Science, University of Burgos, 09006, Burgos, Spain.
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23
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Elek CKA, Brown TL, Le Viet T, Evans R, Baker DJ, Telatin A, Tiwari SK, Al-Khanaq H, Thilliez G, Kingsley RA, Hall LJ, Webber MA, Adriaenssens EM. A hybrid and poly-polish workflow for the complete and accurate assembly of phage genomes: a case study of ten przondoviruses. Microb Genom 2023; 9:mgen001065. [PMID: 37463032 PMCID: PMC10438801 DOI: 10.1099/mgen.0.001065] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2023] [Accepted: 06/17/2023] [Indexed: 07/21/2023] Open
Abstract
Bacteriophages (phages) within the genus Przondovirus are T7-like podoviruses belonging to the subfamily Studiervirinae, within the family Autographiviridae, and have a highly conserved genome organisation. The genomes of these phages range from 37 to 42 kb in size, encode 50-60 genes and are characterised by the presence of direct terminal repeats (DTRs) flanking the linear chromosome. These DTRs are often deleted during short-read-only and hybrid assemblies. Moreover, long-read-only assemblies are often littered with sequencing and/or assembly errors and require additional curation. Here, we present the isolation and characterisation of ten novel przondoviruses targeting Klebsiella spp. We describe HYPPA, a HYbrid and Poly-polish Phage Assembly workflow, which utilises long-read assemblies in combination with short-read sequencing to resolve phage DTRs and correcting errors, negating the need for laborious primer walking and Sanger sequencing validation. Our assembly workflow utilised Oxford Nanopore Technologies for long-read sequencing for its accessibility, making it the more relevant long-read sequencing technology at this time, and Illumina DNA Prep for short-read sequencing, representing the most commonly used technologies globally. Our data demonstrate the importance of careful curation of phage assemblies before publication, and prior to using them for comparative genomics.
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Affiliation(s)
- Claire K. A. Elek
- Quadram Institute Bioscience, Rosalind Franklin Road, Norwich Research Park, Norwich, UK
- University of East Anglia, Norwich Research Park, Norwich, UK
| | - Teagan L. Brown
- Quadram Institute Bioscience, Rosalind Franklin Road, Norwich Research Park, Norwich, UK
| | - Thanh Le Viet
- Quadram Institute Bioscience, Rosalind Franklin Road, Norwich Research Park, Norwich, UK
| | - Rhiannon Evans
- Quadram Institute Bioscience, Rosalind Franklin Road, Norwich Research Park, Norwich, UK
| | - David J. Baker
- Quadram Institute Bioscience, Rosalind Franklin Road, Norwich Research Park, Norwich, UK
| | - Andrea Telatin
- Quadram Institute Bioscience, Rosalind Franklin Road, Norwich Research Park, Norwich, UK
| | - Sumeet K. Tiwari
- Quadram Institute Bioscience, Rosalind Franklin Road, Norwich Research Park, Norwich, UK
| | - Haider Al-Khanaq
- Quadram Institute Bioscience, Rosalind Franklin Road, Norwich Research Park, Norwich, UK
| | - Gaëtan Thilliez
- Quadram Institute Bioscience, Rosalind Franklin Road, Norwich Research Park, Norwich, UK
| | - Robert A. Kingsley
- Quadram Institute Bioscience, Rosalind Franklin Road, Norwich Research Park, Norwich, UK
- University of East Anglia, Norwich Research Park, Norwich, UK
| | - Lindsay J. Hall
- Quadram Institute Bioscience, Rosalind Franklin Road, Norwich Research Park, Norwich, UK
- University of East Anglia, Norwich Research Park, Norwich, UK
- Chair of Intestinal Microbiome, ZIEL—Institute for Food and Health, School of Life Sciences, Technical University of Munich, Freising, Germany
| | - Mark A. Webber
- Quadram Institute Bioscience, Rosalind Franklin Road, Norwich Research Park, Norwich, UK
- University of East Anglia, Norwich Research Park, Norwich, UK
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24
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Neidhöfer C, Sib E, Neuenhoff M, Schwengers O, Dummin T, Buechler C, Klein N, Balks J, Axtmann K, Schwab K, Holderried TAW, Feldmann G, Brossart P, Engelhart S, Mutters NT, Bierbaum G, Parčina M. Hospital sanitary facilities on wards with high antibiotic exposure play an important role in maintaining a reservoir of resistant pathogens, even over many years. Antimicrob Resist Infect Control 2023; 12:33. [PMID: 37061726 PMCID: PMC10105422 DOI: 10.1186/s13756-023-01236-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2022] [Accepted: 03/29/2023] [Indexed: 04/17/2023] Open
Abstract
BACKGROUND Hospitals with their high antimicrobial selection pressure represent the presumably most important reservoir of multidrug-resistant human pathogens. Antibiotics administered in the course of treatment are excreted and discharged into the wastewater system. Not only in patients, but also in the sewers, antimicrobial substances exert selection pressure on existing bacteria and promote the emergence and dissemination of multidrug-resistant clones. In previous studies, two main clusters were identified in all sections of the hospital wastewater network that was investigated, one K. pneumoniae ST147 cluster encoding NDM- and OXA-48 carbapenemases and one VIM-encoding P. aeruginosa ST823 cluster. In the current study, we investigated if NDM- and OXA-48-encoding K. pneumoniae and VIM-encoding P. aeruginosa isolates recovered between 2014 and 2021 from oncological patients belonged to those same clusters. METHODS The 32 isolates were re-cultured, whole-genome sequenced, phenotypically tested for their antimicrobial susceptibility, and analyzed for clonality and resistance genes in silico. RESULTS Among these strains, 25 belonged to the two clusters that had been predominant in the wastewater, while two others belonged to a sequence-type less prominently detected in the drains of the patient rooms. CONCLUSION Patients constantly exposed to antibiotics can, in interaction with their persistently antibiotic-exposed sanitary facilities, form a niche that might be supportive for the emergence, the development, the dissemination, and the maintenance of certain nosocomial pathogen populations in the hospital, due to antibiotic-induced selection pressure. Technical and infection control solutions might help preventing transmission of microorganisms from the wastewater system to the patient and vice versa, particularly concerning the shower and toilet drainage. However, a major driving force might also be antibiotic induced selection pressure and parallel antimicrobial stewardship efforts could be essential.
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Affiliation(s)
- Claudio Neidhöfer
- Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Venusberg Campus 1, 53127, Bonn, Germany.
| | - Esther Sib
- Institute for Hygiene and Public Health, University Hospital Bonn, Bonn, Germany
| | - Marcel Neuenhoff
- Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Venusberg Campus 1, 53127, Bonn, Germany
- Bioinformatics and Systems Biology, Justus Liebig University Giessen, Giessen, Germany
| | - Oliver Schwengers
- Bioinformatics and Systems Biology, Justus Liebig University Giessen, Giessen, Germany
| | - Tobias Dummin
- Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Venusberg Campus 1, 53127, Bonn, Germany
| | - Christian Buechler
- Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Venusberg Campus 1, 53127, Bonn, Germany
| | - Niklas Klein
- Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Venusberg Campus 1, 53127, Bonn, Germany
- Department of Microbiology and Hospital Hygiene, Bundeswehr Central Hospital Koblenz, Koblenz, Germany
| | - Julian Balks
- Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Venusberg Campus 1, 53127, Bonn, Germany
| | - Katharina Axtmann
- Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Venusberg Campus 1, 53127, Bonn, Germany
| | - Katjana Schwab
- Department of Oncology, Hematology, and Rheumatology, University Hospital Bonn, Bonn, Germany
| | - Tobias A W Holderried
- Department of Oncology, Hematology, and Rheumatology, University Hospital Bonn, Bonn, Germany
| | - Georg Feldmann
- Department of Oncology, Hematology, and Rheumatology, University Hospital Bonn, Bonn, Germany
| | - Peter Brossart
- Department of Oncology, Hematology, and Rheumatology, University Hospital Bonn, Bonn, Germany
| | - Steffen Engelhart
- Institute for Hygiene and Public Health, University Hospital Bonn, Bonn, Germany
| | - Nico T Mutters
- Institute for Hygiene and Public Health, University Hospital Bonn, Bonn, Germany
| | - Gabriele Bierbaum
- Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Venusberg Campus 1, 53127, Bonn, Germany
| | - Marijo Parčina
- Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Venusberg Campus 1, 53127, Bonn, Germany
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Kumar R, Yadav G, Kuddus M, Ashraf GM, Singh R. Unlocking the microbial studies through computational approaches: how far have we reached? ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH INTERNATIONAL 2023; 30:48929-48947. [PMID: 36920617 PMCID: PMC10016191 DOI: 10.1007/s11356-023-26220-0] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 03/23/2022] [Accepted: 02/24/2023] [Indexed: 04/16/2023]
Abstract
The metagenomics approach accelerated the study of genetic information from uncultured microbes and complex microbial communities. In silico research also facilitated an understanding of protein-DNA interactions, protein-protein interactions, docking between proteins and phyto/biochemicals for drug design, and modeling of the 3D structure of proteins. These in silico approaches provided insight into analyzing pathogenic and nonpathogenic strains that helped in the identification of probable genes for vaccines and antimicrobial agents and comparing whole-genome sequences to microbial evolution. Artificial intelligence, more precisely machine learning (ML) and deep learning (DL), has proven to be a promising approach in the field of microbiology to handle, analyze, and utilize large data that are generated through nucleic acid sequencing and proteomics. This enabled the understanding of the functional and taxonomic diversity of microorganisms. ML and DL have been used in the prediction and forecasting of diseases and applied to trace environmental contaminants and environmental quality. This review presents an in-depth analysis of the recent application of silico approaches in microbial genomics, proteomics, functional diversity, vaccine development, and drug design.
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Affiliation(s)
- Rajnish Kumar
- Amity Institute of Biotechnology, Amity University Uttar Pradesh Lucknow Campus, Lucknow, Uttar Pradesh, India
- Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA
| | - Garima Yadav
- Amity Institute of Biotechnology, Amity University Uttar Pradesh Lucknow Campus, Lucknow, Uttar Pradesh, India
| | - Mohammed Kuddus
- Department of Biochemistry, College of Medicine, University of Hail, Hail, Saudi Arabia
| | - Ghulam Md Ashraf
- Department of Medical Laboratory Sciences, College of Health Sciences, and Sharjah Institute for Medical Research, University of Sharjah, Sharjah , 27272, United Arab Emirates
| | - Rachana Singh
- Amity Institute of Biotechnology, Amity University Uttar Pradesh Lucknow Campus, Lucknow, Uttar Pradesh, India.
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Maechler F, Weber A, Schwengers O, Schwab F, Denkel L, Behnke M, Gastmeier P, Kola A. Split k-mer analysis compared to cgMLST and SNP-based core genome analysis for detecting transmission of vancomycin-resistant enterococci: results from routine outbreak analyses across different hospitals and hospitals networks in Berlin, Germany. Microb Genom 2023; 9:mgen000937. [PMID: 36748706 PMCID: PMC9973845 DOI: 10.1099/mgen.0.000937] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2022] [Accepted: 11/22/2022] [Indexed: 01/31/2023] Open
Abstract
The increase of Vancomycin-resistant Enterococcus faecium (VREfm) in recent years has been partially attributed to the rise of specific clonal lineages, which have been identified throughout Germany. To date, there is no gold standard for the interpretation of genomic data for outbreak analyses. New genomic approaches such as split k-mer analysis (SKA) could support cluster attribution for routine outbreak investigation. The aim of this project was to investigate frequent clonal lineages of VREfm identified during suspected outbreaks across different hospitals, and to compare genomic approaches including SKA in routine outbreak investigation. We used routine outbreak laboratory data from seven hospitals and three different hospital networks in Berlin, Germany. Short-read libraries were sequenced on the Illumina MiSeq system. We determined clusters using the published Enterococcus faecium-cgMLST scheme (threshold ≤20 alleles), and assigned sequence and complex types (ST, CT), using the Ridom SeqSphere+ software. For each cluster as determined by cgMLST, we used pairwise core-genome SNP-analysis and SKA at thresholds of ten and seven SNPs, respectively, to further distinguish cgMLST clusters. In order to investigate clinical relevance, we analysed to what extent epidemiological linkage backed the clusters determined with different genomic approaches. Between 2014 and 2021, we sequenced 693 VREfm strains, and 644 (93 %) were associated within cgMLST clusters. More than 74 % (n=475) of the strains belonged to the six largest cgMLST clusters, comprising ST117, ST78 and ST80. All six clusters were detected across several years and hospitals without apparent epidemiological links. Core SNP analysis identified 44 clusters with a median cluster size of three isolates (IQR 2-7, min-max 2-63), as well as 197 singletons (41.4 % of 475 isolates). SKA identified 67 clusters with a median cluster size of two isolates (IQR 2-4, min-max 2-19), and 261 singletons (54.9 % of 475 isolates). Of the isolate pairs attributed to clusters, 7 % (n=3064/45 596) of pairs in clusters determined by standard cgMLST, 15 % (n=1222/8500) of pairs in core SNP-clusters and 51 % (n=942/1880) of pairs in SKA-clusters showed epidemiological linkage. The proportion of epidemiological linkage differed between sequence types. For VREfm, the discriminative ability of the widely used cgMLST based approach at ≤20 alleles difference was insufficient to rule out hospital outbreaks without further analytical methods. Cluster assignment guided by core genome SNP analysis and the reference free SKA was more discriminative and correlated better with obvious epidemiological linkage, at least recently published thresholds (ten and seven SNPs, respectively) and for frequent STs. Besides higher overall discriminative power, the whole-genome approach implemented in SKA is also easier and faster to conduct and requires less computational resources.
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Affiliation(s)
- Friederike Maechler
- Institute of Hygiene and Environmental Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany
| | - Anna Weber
- Institute of Hygiene and Environmental Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany
| | - Oliver Schwengers
- Bioinformatics and Systems Biology, Justus Liebig University Giessen, Giessen, Germany
| | - Frank Schwab
- Institute of Hygiene and Environmental Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany
| | - Luisa Denkel
- Institute of Hygiene and Environmental Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany
| | - Michael Behnke
- Institute of Hygiene and Environmental Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany
| | - Petra Gastmeier
- Institute of Hygiene and Environmental Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany
| | - Axel Kola
- Institute of Hygiene and Environmental Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany
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Draft genome sequence of Cronobacter sakazakii strain MEZCS99 sequence type 3 isolated from chicken in South Africa. J Glob Antimicrob Resist 2022; 31:292-294. [PMID: 36049732 DOI: 10.1016/j.jgar.2022.08.016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2022] [Revised: 08/18/2022] [Accepted: 08/19/2022] [Indexed: 12/30/2022] Open
Abstract
OBJECTIVES Cronobacter sakazakii is an emerging opportunistic foodborne pathogen that is frequently associated with life-threatening infections such as infantile septicemia, meningitis, and necrotizing enterocolitis. The emergence of antimicrobial-resistant, livestock-associated C. sakazakii is a great public health concern. Here, we report on the first draft genome sequence of C. sakazakii strain MEZCS99 sequence type 3 (ST3) isolated from feces from a healthy chicken in KwaZulu-Natal Province, South Africa. METHODS The genomic DNA of C. sakazakii was sequenced using an Illumina MiSeq platform (Illumina Inc., San Diego, CA). Generated reads were trimmed and de novo assembled. The assembled contigs were analyzed for virulence and antimicrobial resistance genes, extra-chromosomal plasmids, and multilocus sequence type (MLST). To compare the sequenced strains to other previously sequenced C. sakazakii strains, available raw read sequences of C. sakazakii were downloaded and all sequence files were treated identically to generate a core genome phylogenetic tree. RESULTS Intrinsic beta-lactam resistance gene blaCSA-1 was detected in MEZCS99. No colistin or other antibiotic resistance genes were detected. MEZCS99 belonged to ST3 and harbored an extra-chromosomal plasmid (IncFIB (pCTU3)). The genome of MEZCS99 strain showed two CRISPR/Cas cluster arrays of I-E (n = 1) and I-F (n = 1) type. CONCLUSION The genome sequence of strain MEZCS99 will serve as a reference point for molecular epidemiological studies of livestock-associated C. sakazakii in Africa. In addition, this study allows in-depth analysis of the genomic structure and will provide valuable information that helps understand the pathogenesis and antimicrobial resistance of livestock-associated C. sakazakii.
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A Practical Bioinformatics Workflow for Routine Analysis of Bacterial WGS Data. Microorganisms 2022; 10:microorganisms10122364. [PMID: 36557617 PMCID: PMC9781918 DOI: 10.3390/microorganisms10122364] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2022] [Revised: 11/18/2022] [Accepted: 11/25/2022] [Indexed: 12/03/2022] Open
Abstract
The use of whole-genome sequencing (WGS) for bacterial characterisation has increased substantially in the last decade. Its high throughput and decreasing cost have led to significant changes in outbreak investigations and surveillance of a wide variety of microbial pathogens. Despite the innumerable advantages of WGS, several drawbacks concerning data analysis and management, as well as a general lack of standardisation, hinder its integration in routine use. In this work, a bioinformatics workflow for (Illumina) WGS data is presented for bacterial characterisation including genome annotation, species identification, serotype prediction, antimicrobial resistance prediction, virulence-related genes and plasmid replicon detection, core-genome-based or single nucleotide polymorphism (SNP)-based phylogenetic clustering and sequence typing. Workflow was tested using a collection of 22 in-house sequences of Salmonella enterica isolates belonging to a local outbreak, coupled with a collection of 182 Salmonella genomes publicly available. No errors were reported during the execution period, and all genomes were analysed. The bioinformatics workflow can be tailored to other pathogens of interest and is freely available for academic and non-profit use as an uploadable file to the Galaxy platform.
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29
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Chitale P, Lemenze AD, Fogarty EC, Shah A, Grady C, Odom-Mabey AR, Johnson WE, Yang JH, Eren AM, Brosch R, Kumar P, Alland D. A comprehensive update to the Mycobacterium tuberculosis H37Rv reference genome. Nat Commun 2022; 13:7068. [PMID: 36400796 PMCID: PMC9673877 DOI: 10.1038/s41467-022-34853-x] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2022] [Accepted: 11/08/2022] [Indexed: 11/19/2022] Open
Abstract
H37Rv is the most widely used Mycobacterium tuberculosis strain, and its genome is globally used as the M. tuberculosis reference sequence. Here, we present Bact-Builder, a pipeline that uses consensus building to generate complete and accurate bacterial genome sequences and apply it to three independently cultured and sequenced H37Rv aliquots of a single laboratory stock. Two of the 4,417,942 base-pair long H37Rv assemblies are 100% identical, with the third differing by a single nucleotide. Compared to the existing H37Rv reference, the new sequence contains ~6.4 kb additional base pairs, encoding ten new regions that include insertions in PE/PPE genes and new paralogs of esxN and esxJ, which are differentially expressed compared to the reference genes. New sequencing and de novo assemblies with Bact-Builder confirm that all 10 regions, plus small additional polymorphisms, are also present in the commonly used H37Rv strains NR123, TMC102, and H37Rv1998. Thus, Bact-Builder shows promise as an improved method to perform accurate and reproducible de novo assemblies of bacterial genomes, and our work provides important updates to the primary M. tuberculosis reference genome.
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Affiliation(s)
- Poonam Chitale
- Ray V. Lourenco Center for the Study of Emerging and Re-emerging Pathogens, Rutgers University - New Jersey Medical School, Newark, NJ, USA
- Public Health Research Institute, Rutgers University - New Jersey Medical School, Newark, NJ, USA
| | - Alexander D Lemenze
- Department of Pathology, Immunology and Laboratory Medicine, New Jersey Medical School, Rutgers-The State University of New Jersey, Newark, NJ, USA
| | - Emily C Fogarty
- Department of Medicine, University of Chicago, Chicago, IL, USA
- Committee on Microbiology, University of Chicago, Chicago, IL, USA
| | - Avi Shah
- Ray V. Lourenco Center for the Study of Emerging and Re-emerging Pathogens, Rutgers University - New Jersey Medical School, Newark, NJ, USA
- Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers University- New Jersey Medical School, Newark, NJ, USA
| | - Courtney Grady
- Ray V. Lourenco Center for the Study of Emerging and Re-emerging Pathogens, Rutgers University - New Jersey Medical School, Newark, NJ, USA
- Public Health Research Institute, Rutgers University - New Jersey Medical School, Newark, NJ, USA
| | - Aubrey R Odom-Mabey
- Division of Computational Biomedicine, Boston University School of Medicine and Bioinformatics Program, Boston University, Boston, MA, USA
- Bioinformatics Program, Boston University, Boston, MA, USA
| | - W Evan Johnson
- Ray V. Lourenco Center for the Study of Emerging and Re-emerging Pathogens, Rutgers University - New Jersey Medical School, Newark, NJ, USA
- Public Health Research Institute, Rutgers University - New Jersey Medical School, Newark, NJ, USA
- Center for Data Science, Rutgers University - New Jersey Medical School, Newark, NJ, USA
| | - Jason H Yang
- Ray V. Lourenco Center for the Study of Emerging and Re-emerging Pathogens, Rutgers University - New Jersey Medical School, Newark, NJ, USA
- Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers University- New Jersey Medical School, Newark, NJ, USA
| | - A Murat Eren
- Helmholtz Institute for Functional Marine Biodiversity (HIFMB), Oldenburg, Germany
- Bay Paul Center, Marine Biological Laboratory, Woods Hole, MA, USA
| | - Roland Brosch
- Institut Pasteur, Université Paris Cité, Unit for Integrated Mycobacterial Pathogenomics, CNRS UMR 6047, Paris, France
| | - Pradeep Kumar
- Ray V. Lourenco Center for the Study of Emerging and Re-emerging Pathogens, Rutgers University - New Jersey Medical School, Newark, NJ, USA
- Public Health Research Institute, Rutgers University - New Jersey Medical School, Newark, NJ, USA
| | - David Alland
- Ray V. Lourenco Center for the Study of Emerging and Re-emerging Pathogens, Rutgers University - New Jersey Medical School, Newark, NJ, USA.
- Public Health Research Institute, Rutgers University - New Jersey Medical School, Newark, NJ, USA.
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30
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Guzman J, Poehlein A, Glaeser SP, Schwengers O, Blom J, Hollensteiner J, Kämpfer P, Vilcinskas A. Pseudocitrobacter corydidari sp. nov., isolated from the Asian emerald cockroach Corydidarum magnifica. Int J Syst Evol Microbiol 2022; 72. [DOI: 10.1099/ijsem.0.005497] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
A Gram-negative bacterial strain, G163CMT, was isolated from the gut of the Asian emerald cockroach Corydidarum magnifica. The 16S rRNA gene sequence (1416 bp) of strain G163CMT showed 99.22% similarity to
Pseudocitrobacter faecalis
CCM 8479T and
Pseudocitrobacter vendiensis
CPO20170097T. The average nucleotide identity, digital DNA–DNA hybridization and average amino acid identity values of strain G163CMT were 92.4, 48.8 and 95.7% to
P. faecalis
CCM 8479T, and 93.3, 52.4 and 95.7% to
P. vendiensis
CPO20170097T. This strongly supports the designation of G163CMT as representing a new species in the genus
Pseudocitrobacter
. Phylogenetic trees based on the alignment of 16S rRNA, multilocus sequence analysis of six single-copy genes (fusA, pyrG, leuS, rpoB, recN and mnmE) and 107 core protein sequences consistently showed G163CMT to be a member of the genus
Pseudocitrobacter
, closely related to
P. vendiensis
CPO20170097T. In contrast to
P. faecalis
CCM 8479T and
P. vendiensis
CPO20170097T, the genome of G163CMT did not encode for proteins conferring resistance to antibiotics. However, all three genomes encoded a similar number of virulence factors and specialized metabolite biosynthetic proteins. The major fatty acids of strain G163CMT were C16:0 (31.5 %), C18:1 ω7c (22.6 %), C17:0 cyclo (15.3 %) and C14:0 (6.5 %). Based on the polyphasic results, we conclude that strain G163CMT represents a novel species of the genus
Pseudocitrobacter
and we propose the name Pseudocitrobacter corydidari sp. nov. with the type strain G163CMT (=DSM 112648T=CCM 9160T).
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Affiliation(s)
- Juan Guzman
- Department of Bioresources, Fraunhofer Institute for Molecular Biology and Applied Ecology, Giessen, Germany
- Institute for Insect Biotechnology, Justus-Liebig-University of Giessen, Giessen, Germany
| | - Anja Poehlein
- Genomic and Applied Microbiology and Göttingen Genomics Laboratory, Institute of Microbiology and Genetics, University of Göttingen, Göttingen, Germany
| | - Stefanie P. Glaeser
- Institute for Applied Microbiology, Justus-Liebig-University of Giessen, Giessen, Germany
| | - Oliver Schwengers
- Bioinformatics and Systems Biology, Justus Liebig University Giessen, Giessen, Germany
| | - Jochen Blom
- Bioinformatics and Systems Biology, Justus Liebig University Giessen, Giessen, Germany
| | - Jacqueline Hollensteiner
- Genomic and Applied Microbiology and Göttingen Genomics Laboratory, Institute of Microbiology and Genetics, University of Göttingen, Göttingen, Germany
| | - Peter Kämpfer
- Institute for Applied Microbiology, Justus-Liebig-University of Giessen, Giessen, Germany
| | - Andreas Vilcinskas
- Institute for Insect Biotechnology, Justus-Liebig-University of Giessen, Giessen, Germany
- Department of Bioresources, Fraunhofer Institute for Molecular Biology and Applied Ecology, Giessen, Germany
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31
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Sváb D, Falgenhauer L, Mag T, Chakraborty T, Tóth I. Genomic Diversity, Virulence Gene, and Prophage Arrays of Bovine and Human Shiga Toxigenic and Enteropathogenic Escherichia coli Strains Isolated in Hungary. Front Microbiol 2022; 13:896296. [PMID: 35865933 PMCID: PMC9294531 DOI: 10.3389/fmicb.2022.896296] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2022] [Accepted: 06/13/2022] [Indexed: 11/23/2022] Open
Abstract
Escherichia coli belonging to the enterohemorrhagic (EHEC), Shiga toxin-producing (STEC) and atypical enteropathogenic (aEPEC) pathotypes are significant foodborne zoonotic pathogens posing serious health risks, with healthy cattle as their main reservoir. A representative sampling of Hungarian cattle farms during 2017-2018 yielded a prevalence of 6.5 and 5.8% for STEC and aEPEC out of 309 samples. The draft genomes of twelve STEC (of them 9 EHEC) and four aEPEC of bovine origin were determined. For comparative purposes, we also included 3 EHEC and 2 aEPEC strains of human origin, as well four commensal isolates and one extraintestinal pathogenic E. coli (ExPEC) obtained from animals in a final set of 26 strains for a WGS-based analysis. Apart from key virulence genes, these isolates harbored several additional virulence genes with arrays characteristic for the site of isolation. The most frequent insertion site of Shiga toxin (stx) encoding prophages was yehV for the Stx1 prophage and wrbA and sbcB for Stx2. For O157:H7 strains, the locus of enterocyte effacement pathogenicity island was present at the selC site, with integration at pheV for other serotypes, and pheU in the case of O26:H11 strains. Several LEE-negative STEC and aEPEC as well as commensal isolates carried additional prophages, with an average of ten prophage regions per isolate. Comparative phylogenomic analysis showed no clear separation between bovine and human lineages among the isolates characterized in the current study. Similarities in virulence gene arrays and close phylogenetic relations of bovine and human isolates underline the zoonotic potential of bovine aEPEC and STEC and emphasize the need for frequent monitoring of these pathogens in livestock.
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Affiliation(s)
- Domonkos Sváb
- Veterinary Medical Research Institute, Budapest, Hungary
| | - Linda Falgenhauer
- Institute of Hygiene and Environmental Medicine and German Center for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Justus Liebig University Giessen, Giessen, Germany
| | - Tünde Mag
- National Public Health Center, Budapest, Hungary
| | - Trinad Chakraborty
- Institute of Medical Microbiology, German Center for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Justus Liebig University Giessen, Giessen, Germany
| | - István Tóth
- Veterinary Medical Research Institute, Budapest, Hungary
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32
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CleanSeq: A Pipeline for Contamination Detection, Cleanup, and Mutation Verifications from Microbial Genome Sequencing Data. APPLIED SCIENCES-BASEL 2022. [DOI: 10.3390/app12126209] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Contaminations frequently occur in bacterial cultures, which significantly affect the reproducibility and reliability of the results from whole-genome sequencing (WGS). Decontaminated WGS data with clean reads is the only desirable source for detecting possible variants correctly. Improvements in bioinformatics are essential to analyze the contaminated WGS dataset. Existing pipelines usually contain contamination detection, decontamination, and variant calling separately. The efficiency and results from existing pipelines fluctuate since distinctive computational models and parameters are applied. It is then promising to develop a bioinformatical tool containing functions to discriminate and remove contaminated reads and improve variant calling from clean reads. In this study, we established a Python-based pipeline named CleanSeq for automatic detection and removal of contaminating reads, analyzing possible genome variants with proper verifications via local re-alignments. The application and reproducibility are proven in either simulated, publicly available datasets or actual genome sequencing reads from our experimental evolution study in Escherichia coli. We successfully obtained decontaminated reads, called out all seven consistent mutations from the contaminated bacterial sample, and derived five colonies. Collectively, the results demonstrated that CleanSeq could effectively process the contaminated samples to achieve decontaminated reads, based on which reliable results (i.e., variant calling) could be obtained.
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33
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Yao Y, Falgenhauer L, Falgenhauer J, Hauri AM, Heinmüller P, Domann E, Chakraborty T, Imirzalioglu C. Carbapenem-Resistant Citrobacter spp. as an Emerging Concern in the Hospital-Setting: Results From a Genome-Based Regional Surveillance Study. Front Cell Infect Microbiol 2021; 11:744431. [PMID: 34858870 PMCID: PMC8632029 DOI: 10.3389/fcimb.2021.744431] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2021] [Accepted: 10/26/2021] [Indexed: 11/13/2022] Open
Abstract
The rise of Carbapenem-resistant Enterobacterales (CRE) represents an increasing threat to patient safety and healthcare systems worldwide. Citrobacter spp., long considered not to be a classical nosocomial pathogen, in contrast to Klebsiella pneumoniae and Escherichia coli, is fast gaining importance as a clinical multidrug-resistant pathogen. We analyzed the genomes of 512 isolates of 21 CRE species obtained from 61 hospitals within a three-year-period and found that Citrobacter spp. (C. freundii, C. portucalensis, C. europaeus, C. koseri and C. braakii) were increasingly detected (n=56) within the study period. The carbapenemase-groups detected in Citrobacter spp. were KPC, OXA-48/-like and MBL (VIM, NDM) accounting for 42%, 31% and 27% respectively, which is comparable to those of K. pneumoniae in the same study. They accounted for 10%, 17% and 14% of all carbapenemase-producing CRE detected in 2017, 2018 and 2019, respectively. The carbapenemase genes were almost exclusively located on plasmids. The high genomic diversity of C. freundii is represented by 22 ST-types. KPC-2 was the predominantly detected carbapenemase (n=19) and was located in 95% of cases on a highly-conserved multiple-drug-resistance-gene-carrying pMLST15 IncN plasmid. KPC-3 was rarely detected and was confined to a clonal outbreak of C. freundii ST18. OXA-48 carbapenemases were located on plasmids of the IncL/M (pOXA-48) type. About 50% of VIM-1 was located on different IncN plasmids (pMLST7, pMLST5). These results underline the increasing importance of the Citrobacter species as emerging carriers of carbapenemases and therefore as potential disseminators of Carbapenem- and multidrug-resistance in the hospital setting.
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Affiliation(s)
- Yancheng Yao
- Institute of Medical Microbiology, Justus Liebig University Giessen, Giessen, Germany.,German Center for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Justus-Liebig University Giessen, Giessen, Germany
| | - Linda Falgenhauer
- German Center for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Justus-Liebig University Giessen, Giessen, Germany.,Institute for Hygiene and Environmental Medicine, Justus Liebig University Giessen, Giessen, Germany
| | - Jane Falgenhauer
- Institute of Medical Microbiology, Justus Liebig University Giessen, Giessen, Germany.,German Center for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Justus-Liebig University Giessen, Giessen, Germany
| | - Anja M Hauri
- Department of Epimeiology, Hessisches Landesprüfungs- und Untersuchungsamt im Gesundheitswesen (HLPUG), Dillenburg, Germany.,Department of Epimeiology, Helmholtz Centre for Infection Research, Braunschweig, Germany
| | - Petra Heinmüller
- Department of Epimeiology, Hessisches Landesprüfungs- und Untersuchungsamt im Gesundheitswesen (HLPUG), Dillenburg, Germany
| | - Eugen Domann
- Institute of Medical Microbiology, Justus Liebig University Giessen, Giessen, Germany.,German Center for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Justus-Liebig University Giessen, Giessen, Germany.,Institute for Hygiene and Environmental Medicine, Justus Liebig University Giessen, Giessen, Germany
| | - Trinad Chakraborty
- Institute of Medical Microbiology, Justus Liebig University Giessen, Giessen, Germany.,German Center for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Justus-Liebig University Giessen, Giessen, Germany
| | - Can Imirzalioglu
- Institute of Medical Microbiology, Justus Liebig University Giessen, Giessen, Germany.,German Center for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Justus-Liebig University Giessen, Giessen, Germany
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34
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Schönborn JW, Stewart FA, Enriquez KM, Akhtar I, Droste A, Waschina S, Beller M. Modeling Drosophila gut microbe interactions reveals metabolic interconnectivity. iScience 2021; 24:103216. [PMID: 34712918 PMCID: PMC8528732 DOI: 10.1016/j.isci.2021.103216] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2021] [Revised: 07/08/2021] [Accepted: 09/30/2021] [Indexed: 11/19/2022] Open
Abstract
We know a lot about varying gut microbiome compositions. Yet, how the bacteria affect each other remains elusive. In mammals, this is largely based on the sheer complexity of the microbiome with at least hundreds of different species. Thus, model organisms such as Drosophila melanogaster are commonly used to investigate mechanistic questions as the microbiome consists of only about 10 leading bacterial species. Here, we isolated gut bacteria from laboratory-reared Drosophila, sequenced their respective genomes, and used this information to reconstruct genome-scale metabolic models. With these, we simulated growth in mono- and co-culture conditions and different media including a synthetic diet designed to grow Drosophila melanogaster. Our simulations reveal a synergistic growth of some but not all gut microbiome members, which stems on the exchange of distinct metabolites including tricarboxylic acid cycle intermediates. Culturing experiments confirmed our predictions. Our study thus demonstrates the possibility to predict microbiome-derived growth-promoting cross-feeding.
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Affiliation(s)
- Jürgen W. Schönborn
- Institut für Mathematische Modellierung Biologischer Systeme, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany
- Systembiologie des Fettstoffwechsels, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany
| | - Fiona A. Stewart
- Institut für Mathematische Modellierung Biologischer Systeme, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany
- Systembiologie des Fettstoffwechsels, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany
| | - Kerstin Maas Enriquez
- Institut für Mathematische Modellierung Biologischer Systeme, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany
- Systembiologie des Fettstoffwechsels, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany
| | - Irfan Akhtar
- Institut für Mathematische Modellierung Biologischer Systeme, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany
- Systembiologie des Fettstoffwechsels, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany
| | - Andrea Droste
- Institut für Mathematische Modellierung Biologischer Systeme, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany
- Systembiologie des Fettstoffwechsels, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany
| | - Silvio Waschina
- Christian-Albrechts-University Kiel, Institute of Human Nutrition and Food Science, Nutriinformatics, Heinrich-Hecht-Platz 10, 24118 Kiel, Germany
| | - Mathias Beller
- Institut für Mathematische Modellierung Biologischer Systeme, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany
- Systembiologie des Fettstoffwechsels, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany
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35
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Schwengers O, Jelonek L, Dieckmann MA, Beyvers S, Blom J, Goesmann A. Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identification. Microb Genom 2021; 7:000685. [PMID: 34739369 PMCID: PMC8743544 DOI: 10.1099/mgen.0.000685] [Citation(s) in RCA: 323] [Impact Index Per Article: 80.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2021] [Accepted: 09/08/2021] [Indexed: 12/21/2022] Open
Abstract
Command-line annotation software tools have continuously gained popularity compared to centralized online services due to the worldwide increase of sequenced bacterial genomes. However, results of existing command-line software pipelines heavily depend on taxon-specific databases or sufficiently well annotated reference genomes. Here, we introduce Bakta, a new command-line software tool for the robust, taxon-independent, thorough and, nonetheless, fast annotation of bacterial genomes. Bakta conducts a comprehensive annotation workflow including the detection of small proteins taking into account replicon metadata. The annotation of coding sequences is accelerated via an alignment-free sequence identification approach that in addition facilitates the precise assignment of public database cross-references. Annotation results are exported in GFF3 and International Nucleotide Sequence Database Collaboration (INSDC)-compliant flat files, as well as comprehensive JSON files, facilitating automated downstream analysis. We compared Bakta to other rapid contemporary command-line annotation software tools in both targeted and taxonomically broad benchmarks including isolates and metagenomic-assembled genomes. We demonstrated that Bakta outperforms other tools in terms of functional annotations, the assignment of functional categories and database cross-references, whilst providing comparable wall-clock runtimes. Bakta is implemented in Python 3 and runs on MacOS and Linux systems. It is freely available under a GPLv3 license at https://github.com/oschwengers/bakta. An accompanying web version is available at https://bakta.computational.bio.
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Affiliation(s)
- Oliver Schwengers
- Bioinformatics and Systems Biology, Justus Liebig University Giessen, Giessen 35392, Germany
| | - Lukas Jelonek
- Bioinformatics and Systems Biology, Justus Liebig University Giessen, Giessen 35392, Germany
| | - Marius Alfred Dieckmann
- Bioinformatics and Systems Biology, Justus Liebig University Giessen, Giessen 35392, Germany
| | - Sebastian Beyvers
- Bioinformatics and Systems Biology, Justus Liebig University Giessen, Giessen 35392, Germany
| | - Jochen Blom
- Bioinformatics and Systems Biology, Justus Liebig University Giessen, Giessen 35392, Germany
| | - Alexander Goesmann
- Bioinformatics and Systems Biology, Justus Liebig University Giessen, Giessen 35392, Germany
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36
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Dieckmann MA, Beyvers S, Nkouamedjo-Fankep RC, Hanel PHG, Jelonek L, Blom J, Goesmann A. EDGAR3.0: comparative genomics and phylogenomics on a scalable infrastructure. Nucleic Acids Res 2021; 49:W185-W192. [PMID: 33988716 PMCID: PMC8262741 DOI: 10.1093/nar/gkab341] [Citation(s) in RCA: 80] [Impact Index Per Article: 20.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2021] [Revised: 04/11/2021] [Accepted: 04/21/2021] [Indexed: 01/29/2023] Open
Abstract
The EDGAR platform, a web server providing databases of precomputed orthology data for thousands of microbial genomes, is one of the most established tools in the field of comparative genomics and phylogenomics. Based on precomputed gene alignments, EDGAR allows quick identification of the differential gene content, i.e. the pan genome, the core genome, or singleton genes. Furthermore, EDGAR features a wide range of analyses and visualizations like Venn diagrams, synteny plots, phylogenetic trees, as well as Amino Acid Identity (AAI) and Average Nucleotide Identity (ANI) matrices. During the last few years, the average number of genomes analyzed in an EDGAR project increased by two orders of magnitude. To handle this massive increase, a completely new technical backend infrastructure for the EDGAR platform was designed and launched as EDGAR3.0. For the calculation of new EDGAR3.0 projects, we are now using a scalable Kubernetes cluster running in a cloud environment. A new storage infrastructure was developed using a file-based high-performance storage backend which ensures timely data handling and efficient access. The new data backend guarantees a memory efficient calculation of orthologs, and parallelization has led to drastically reduced processing times. Based on the advanced technical infrastructure new analysis features could be implemented including POCP and FastANI genomes similarity indices, UpSet intersecting set visualization, and circular genome plots. Also the public database section of EDGAR was largely updated and now offers access to 24,317 genomes in 749 free-to-use projects. In summary, EDGAR 3.0 provides a new, scalable infrastructure for comprehensive microbial comparative gene content analysis. The web server is accessible at http://edgar3.computational.bio.
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Affiliation(s)
- Marius Alfred Dieckmann
- Bioinformatics & Systems Biology, Justus Liebig University Gießen, Heinrich-Buff-Ring 58, 35390 Gießen, Hesse, Germany
| | - Sebastian Beyvers
- Bioinformatics & Systems Biology, Justus Liebig University Gießen, Heinrich-Buff-Ring 58, 35390 Gießen, Hesse, Germany
| | | | - Patrick Harald Georg Hanel
- Institute of Computational Biology, Helmholtz Center Munich, German Research Center for Environmental Health, Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany
| | - Lukas Jelonek
- Bioinformatics & Systems Biology, Justus Liebig University Gießen, Heinrich-Buff-Ring 58, 35390 Gießen, Hesse, Germany
| | - Jochen Blom
- Bioinformatics & Systems Biology, Justus Liebig University Gießen, Heinrich-Buff-Ring 58, 35390 Gießen, Hesse, Germany
| | - Alexander Goesmann
- Bioinformatics & Systems Biology, Justus Liebig University Gießen, Heinrich-Buff-Ring 58, 35390 Gießen, Hesse, Germany
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37
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Murigneux V, Roberts LW, Forde BM, Phan MD, Nhu NTK, Irwin AD, Harris PNA, Paterson DL, Schembri MA, Whiley DM, Beatson SA. MicroPIPE: validating an end-to-end workflow for high-quality complete bacterial genome construction. BMC Genomics 2021; 22:474. [PMID: 34172000 PMCID: PMC8235852 DOI: 10.1186/s12864-021-07767-z] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2021] [Accepted: 06/03/2021] [Indexed: 11/23/2022] Open
Abstract
Background Oxford Nanopore Technology (ONT) long-read sequencing has become a popular platform for microbial researchers due to the accessibility and affordability of its devices. However, easy and automated construction of high-quality bacterial genomes using nanopore reads remains challenging. Here we aimed to create a reproducible end-to-end bacterial genome assembly pipeline using ONT in combination with Illumina sequencing. Results We evaluated the performance of several popular tools used during genome reconstruction, including base-calling, filtering, assembly, and polishing. We also assessed overall genome accuracy using ONT both natively and with Illumina. All steps were validated using the high-quality complete reference genome for the Escherichia coli sequence type (ST)131 strain EC958. Software chosen at each stage were incorporated into our final pipeline, MicroPIPE. Further validation of MicroPIPE was carried out using 11 additional ST131 E. coli isolates, which demonstrated that complete circularised chromosomes and plasmids could be achieved without manual intervention. Twelve publicly available Gram-negative and Gram-positive bacterial genomes (with available raw ONT data and matched complete genomes) were also assembled using MicroPIPE. We found that revised basecalling and updated assembly of the majority of these genomes resulted in improved accuracy compared to the current publicly available complete genomes. Conclusions MicroPIPE is built in modules using Singularity container images and the bioinformatics workflow manager Nextflow, allowing changes and adjustments to be made in response to future tool development. Overall, MicroPIPE provides an easy-access, end-to-end solution for attaining high-quality bacterial genomes. MicroPIPE is available at https://github.com/BeatsonLab-MicrobialGenomics/micropipe. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-021-07767-z.
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Affiliation(s)
- Valentine Murigneux
- QCIF Facility for Advanced Bioinformatics, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia
| | - Leah W Roberts
- University of Queensland Centre for Clinical Research, Brisbane, Queensland, Australia. .,Queensland Children's Hospital, Brisbane, Queensland, Australia. .,European Bioinformatics Institute, European Molecular Biology Laboratory (EMBL), Hinxton, Cambridge, UK.
| | - Brian M Forde
- University of Queensland Centre for Clinical Research, Brisbane, Queensland, Australia
| | - Minh-Duy Phan
- School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia
| | - Nguyen Thi Khanh Nhu
- School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia
| | - Adam D Irwin
- University of Queensland Centre for Clinical Research, Brisbane, Queensland, Australia.,Queensland Children's Hospital, Brisbane, Queensland, Australia
| | - Patrick N A Harris
- University of Queensland Centre for Clinical Research, Brisbane, Queensland, Australia.,Central Microbiology, Pathology Queensland, Royal Brisbane & Women's Hospital, Brisbane, Queensland, Australia
| | - David L Paterson
- University of Queensland Centre for Clinical Research, Brisbane, Queensland, Australia
| | - Mark A Schembri
- School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia
| | - David M Whiley
- University of Queensland Centre for Clinical Research, Brisbane, Queensland, Australia.,Queensland Children's Hospital, Brisbane, Queensland, Australia
| | - Scott A Beatson
- School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia. .,Australian Centre for Ecogenomics, The University of Queensland, Brisbane, Queensland, Australia.
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38
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Sserwadda I, Mboowa G. rMAP: the Rapid Microbial Analysis Pipeline for ESKAPE bacterial group whole-genome sequence data. Microb Genom 2021; 7:000583. [PMID: 34110280 PMCID: PMC8461470 DOI: 10.1099/mgen.0.000583] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2020] [Accepted: 04/13/2021] [Indexed: 12/13/2022] Open
Abstract
The recent re-emergence of multidrug-resistant pathogens has exacerbated their threat to worldwide public health. The evolution of the genomics era has led to the generation of huge volumes of sequencing data at an unprecedented rate due to the ever-reducing costs of whole-genome sequencing (WGS). We have developed the Rapid Microbial Analysis Pipeline (rMAP), a user-friendly pipeline capable of profiling the resistomes of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) using WGS data generated from Illumina's sequencing platforms. rMAP is designed for individuals with little bioinformatics expertise, and automates the steps required for WGS analysis directly from the raw genomic sequence data, including adapter and low-quality sequence read trimming, de novo genome assembly, genome annotation, single-nucleotide polymorphism (SNP) variant calling, phylogenetic inference by maximum likelihood, antimicrobial resistance (AMR) profiling, plasmid profiling, virulence factor determination, multi-locus sequence typing (MLST), pangenome analysis and insertion sequence characterization (IS). Once the analysis is finished, rMAP generates an interactive web-like html report. rMAP installation is very simple, it can be run using very simple commands. It represents a rapid and easy way to perform comprehensive bacterial WGS analysis using a personal laptop in low-income settings where high-performance computing infrastructure is limited.
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Affiliation(s)
- Ivan Sserwadda
- Department of Immunology and Molecular Biology, College of Health Sciences, School of Biomedical Sciences, Makerere University, Kampala, Uganda
- Department of Biochemistry and Bioinformatics, School of Pure and Applied Sciences, Pwani University, Kilifi, Kenya
| | - Gerald Mboowa
- Department of Immunology and Molecular Biology, College of Health Sciences, School of Biomedical Sciences, Makerere University, Kampala, Uganda
- The African Center of Excellence in Bioinformatics and Data-Intensive Sciences, the Infectious Diseases Institute, College of Health Sciences, Makerere University, Kampala, Uganda
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39
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Pavlovikj N, Gomes-Neto JC, Deogun JS, Benson AK. ProkEvo: an automated, reproducible, and scalable framework for high-throughput bacterial population genomics analyses. PeerJ 2021; 9:e11376. [PMID: 34055480 PMCID: PMC8142932 DOI: 10.7717/peerj.11376] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2020] [Accepted: 04/08/2021] [Indexed: 12/28/2022] Open
Abstract
Whole Genome Sequence (WGS) data from bacterial species is used for a variety of applications ranging from basic microbiological research, diagnostics, and epidemiological surveillance. The availability of WGS data from hundreds of thousands of individual isolates of individual microbial species poses a tremendous opportunity for discovery and hypothesis-generating research into ecology and evolution of these microorganisms. Flexibility, scalability, and user-friendliness of existing pipelines for population-scale inquiry, however, limit applications of systematic, population-scale approaches. Here, we present ProkEvo, an automated, scalable, reproducible, and open-source framework for bacterial population genomics analyses using WGS data. ProkEvo was specifically developed to achieve the following goals: (1) Automation and scaling of complex combinations of computational analyses for many thousands of bacterial genomes from inputs of raw Illumina paired-end sequence reads; (2) Use of workflow management systems (WMS) such as Pegasus WMS to ensure reproducibility, scalability, modularity, fault-tolerance, and robust file management throughout the process; (3) Use of high-performance and high-throughput computational platforms; (4) Generation of hierarchical-based population structure analysis based on combinations of multi-locus and Bayesian statistical approaches for classification for ecological and epidemiological inquiries; (5) Association of antimicrobial resistance (AMR) genes, putative virulence factors, and plasmids from curated databases with the hierarchically-related genotypic classifications; and (6) Production of pan-genome annotations and data compilation that can be utilized for downstream analysis such as identification of population-specific genomic signatures. The scalability of ProkEvo was measured with two datasets comprising significantly different numbers of input genomes (one with ~2,400 genomes, and the second with ~23,000 genomes). Depending on the dataset and the computational platform used, the running time of ProkEvo varied from ~3-26 days. ProkEvo can be used with virtually any bacterial species, and the Pegasus WMS uniquely facilitates addition or removal of programs from the workflow or modification of options within them. To demonstrate versatility of the ProkEvo platform, we performed a hierarchical-based population structure analyses from available genomes of three distinct pathogenic bacterial species as individual case studies. The specific case studies illustrate how hierarchical analyses of population structures, genotype frequencies, and distribution of specific gene functions can be integrated into an analysis. Collectively, our study shows that ProkEvo presents a practical viable option for scalable, automated analyses of bacterial populations with direct applications for basic microbiology research, clinical microbiological diagnostics, and epidemiological surveillance.
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Affiliation(s)
- Natasha Pavlovikj
- Department of Computer Science and Engineering, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America
| | - Joao Carlos Gomes-Neto
- Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.,Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America
| | - Jitender S Deogun
- Department of Computer Science and Engineering, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America
| | - Andrew K Benson
- Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.,Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America
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40
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Deneke C, Brendebach H, Uelze L, Borowiak M, Malorny B, Tausch SH. Species-Specific Quality Control, Assembly and Contamination Detection in Microbial Isolate Sequences with AQUAMIS. Genes (Basel) 2021; 12:644. [PMID: 33926025 PMCID: PMC8145556 DOI: 10.3390/genes12050644] [Citation(s) in RCA: 50] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2021] [Revised: 04/23/2021] [Accepted: 04/24/2021] [Indexed: 01/13/2023] Open
Abstract
Sequencing of whole microbial genomes has become a standard procedure for cluster detection, source tracking, outbreak investigation and surveillance of many microorganisms. An increasing number of laboratories are currently in a transition phase from classical methods towards next generation sequencing, generating unprecedented amounts of data. Since the precision of downstream analyses depends significantly on the quality of raw data generated on the sequencing instrument, a comprehensive, meaningful primary quality control is indispensable. Here, we present AQUAMIS, a Snakemake workflow for an extensive quality control and assembly of raw Illumina sequencing data, allowing laboratories to automatize the initial analysis of their microbial whole-genome sequencing data. AQUAMIS performs all steps of primary sequence analysis, consisting of read trimming, read quality control (QC), taxonomic classification, de-novo assembly, reference identification, assembly QC and contamination detection, both on the read and assembly level. The results are visualized in an interactive HTML report including species-specific QC thresholds, allowing non-bioinformaticians to assess the quality of sequencing experiments at a glance. All results are also available as a standard-compliant JSON file, facilitating easy downstream analyses and data exchange. We have applied AQUAMIS to analyze ~13,000 microbial isolates as well as ~1000 in-silico contaminated datasets, proving the workflow's ability to perform in high throughput routine sequencing environments and reliably predict contaminations. We found that intergenus and intragenus contaminations can be detected most accurately using a combination of different QC metrics available within AQUAMIS.
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Affiliation(s)
| | | | | | | | | | - Simon H. Tausch
- Department Biological Safety, German Federal Institute for Risk Assessment, 10589 Berlin, Germany; (C.D.); (H.B.); (L.U.); (M.B.); (B.M.)
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41
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Falgenhauer L, Zur Nieden A, Harpel S, Falgenhauer J, Domann E. Clonal CTX-M-15-Producing Escherichia coli ST-949 Are Present in German Surface Water. Front Microbiol 2021; 12:617349. [PMID: 33912141 PMCID: PMC8072356 DOI: 10.3389/fmicb.2021.617349] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2020] [Accepted: 03/24/2021] [Indexed: 11/13/2022] Open
Abstract
Extended-spectrum beta-lactamase (ESBL)-producing bacterial isolates are emerging within the last years. To understand this emergence, a thorough genome-based analysis of ESBL isolates from different sources (One Health approach) is needed. Among these, analysis of surface water is underrepresented. Therefore, we performed a genome-based analysis of ESBL-producing Escherichia coli isolates from surface water samples. Water samples were collected from eleven different surface water sites (lakes, river). ESBL-producing E. coli were recovered from these samples using filters and chromogenic media. Whole-genome sequencing of ESBL-producing E. coli was performed followed by determination of the multilocus sequence type (ST), ESBL-type, and virulence genes. Phylogenetic analysis was done using single nucleotide analysis. From all water samples taken, nineteen ESBL-producing E. coli were recovered. All of them harbored an ESBL gene. Nine different multilocus STs were determined, among which ST-949 was the ST detected most frequently. Phylogenetic analysis of ST-949 isolates revealed that all those isolates were closely related. In addition, they harbored an identical chromosomal insertion of bla CTX-M-15 , indicating a clonal relationship among these isolates. Genetic comparison with isolates from all over the world revealed that these isolates were closely related to human clinical isolates derived from New Zealand and Sweden. An ESBL-producing E. coli ST-949 clone was detected in German surface waters. Its close relationship to human clinical isolates suggests its ability to colonize or even infect humans. Our findings reveal that water sources indeed may play a hitherto underreported role in spread of ESBL-producing isolates.
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Affiliation(s)
- Linda Falgenhauer
- Institute of Hygiene and Environmental Medicine, Justus Liebig University Giessen, Giessen, Germany.,German Center for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Justus Liebig University Giessen, Giessen, Germany
| | - Anja Zur Nieden
- Institute of Hygiene and Environmental Medicine, Justus Liebig University Giessen, Giessen, Germany
| | - Susanne Harpel
- Institute of Hygiene and Environmental Medicine, Justus Liebig University Giessen, Giessen, Germany
| | - Jane Falgenhauer
- German Center for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Justus Liebig University Giessen, Giessen, Germany.,Institute of Medical Microbiology, Justus Liebig University Giessen, Giessen, Germany
| | - Eugen Domann
- Institute of Hygiene and Environmental Medicine, Justus Liebig University Giessen, Giessen, Germany.,German Center for Infection Research (DZIF), Partner Site Giessen-Marburg-Langen, Justus Liebig University Giessen, Giessen, Germany.,Institute of Medical Microbiology, Justus Liebig University Giessen, Giessen, Germany
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42
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Falgenhauer L, Preuser I, Imirzalioglu C, Falgenhauer J, Fritzenwanker M, Mack D, Best C, Heudorf U, Chakraborty T. Changing epidemiology of vancomycin-resistant Enterococcus faecium: Results of a genome-based study at a regional neurological acute hospital with intensive care and early rehabilitation treatment. Infect Prev Pract 2021; 3:100138. [PMID: 34368749 PMCID: PMC8335922 DOI: 10.1016/j.infpip.2021.100138] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2021] [Accepted: 03/22/2021] [Indexed: 12/04/2022] Open
Abstract
Background Vancomycin-resistant Enterococcus faecium (VREfm) are an emerging threat worldwide. In Germany, a VRE-belt with higher VREfm prevalences transversing its central east-west axis and including the state of Hesse was previously described. Recently, we detected a predominant VREfm clone in hospitals throughout the Rhine-Main metropolitan area of Hesse. Aim Here we expanded our study on VREfm to a regional neurological acute hospital outside of the metropolitan area with patient referrals from throughout Hesse and the neighboring federal state of Rhineland-Palatinate. Material/Methods VREfm isolates obtained between 2016-2018 from a regional neurological acute hospital with intensive care and early rehabilitation units were investigated (n=55). Patient data was collected and analyzed together with whole-genome sequencing data to investigate antibiotic resistance and virulence determinants of the VREfm. The population structure of VREfm was investigated using the Core genome-based multilocus sequence typing (cgMLST). Findings The average age of the patients was 67.1 years. For 96% of the patients, a previous hospital stay was reported. 64% of the patients were treated with antibiotics. All VREfm harbored the vanB vancomycin resistance gene. The multilocus sequence types (STs) detected changed abruptly from four different STs in 2016 to a predominant ST in 2017 and 2018 (ST117). Most of the ST117 isolates were members of the cgMLST type CT71. Conclusion The results indicate a sudden shift of the VREfm population structure from a semi-heterogeneous population to a pre-dominant clone within an interval of two years. Further investigations are warranted to understand the epidemiology and emergence of this clone.
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Affiliation(s)
- Linda Falgenhauer
- Institute of Hygiene and Environmental Medicine, Justus Liebig University Giessen, Schubertstrasse 81, Giessen, 35392, Germany.,German Center for Infection Research (DZIF), Site Giessen-Marburg-Langen, Justus Liebig University Giessen, Schubertstrasse 81, Giessen, 35392, Germany
| | - Ingeborg Preuser
- Department of Neurology, Vitos-Weil-Lahn, Weilstrasse 10, Weilmünster, 35789, Germany
| | - Can Imirzalioglu
- German Center for Infection Research (DZIF), Site Giessen-Marburg-Langen, Justus Liebig University Giessen, Schubertstrasse 81, Giessen, 35392, Germany.,Institute of Medical Microbiology, Justus Liebig University Giessen, Schubertstrasse 81, Giessen, 35392, Germany
| | - Jane Falgenhauer
- German Center for Infection Research (DZIF), Site Giessen-Marburg-Langen, Justus Liebig University Giessen, Schubertstrasse 81, Giessen, 35392, Germany.,Institute of Medical Microbiology, Justus Liebig University Giessen, Schubertstrasse 81, Giessen, 35392, Germany
| | - Moritz Fritzenwanker
- German Center for Infection Research (DZIF), Site Giessen-Marburg-Langen, Justus Liebig University Giessen, Schubertstrasse 81, Giessen, 35392, Germany.,Institute of Medical Microbiology, Justus Liebig University Giessen, Schubertstrasse 81, Giessen, 35392, Germany
| | - Dietrich Mack
- Institut für Medizinische Diagnostik GmbH, Bioscientia Labor Ingelheim, Konrad-Adenauer-Straße 17, Ingelheim am Rhein, 55218, Germany
| | - Christoph Best
- Department of Neurology, Vitos-Weil-Lahn, Weilstrasse 10, Weilmünster, 35789, Germany
| | - Ursel Heudorf
- Network on MDRO Rhine-Main, Breite Gasse 28, Frankfurt/Main, 60313, Germany
| | - Trinad Chakraborty
- German Center for Infection Research (DZIF), Site Giessen-Marburg-Langen, Justus Liebig University Giessen, Schubertstrasse 81, Giessen, 35392, Germany.,Institute of Medical Microbiology, Justus Liebig University Giessen, Schubertstrasse 81, Giessen, 35392, Germany
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43
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Dong Y, Li C, Kim K, Cui L, Liu X. Genome annotation of disease-causing microorganisms. Brief Bioinform 2021; 22:845-854. [PMID: 33537706 PMCID: PMC7986607 DOI: 10.1093/bib/bbab004] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2020] [Revised: 12/23/2020] [Accepted: 01/03/2021] [Indexed: 11/13/2022] Open
Abstract
Humans have coexisted with pathogenic microorganisms throughout its history of evolution. We have never halted the exploration of pathogenic microorganisms. With the improvement of genome-sequencing technology and the continuous reduction of sequencing costs, an increasing number of complete genome sequences of pathogenic microorganisms have become available. Genome annotation of this massive sequence information has become a daunting task in biological research. This paper summarizes the approaches to the genome annotation of pathogenic microorganisms and the available popular genome annotation tools for prokaryotes, eukaryotes and viruses. Furthermore, real-world comparisons of different annotation tools using 12 genomes from prokaryotes, eukaryotes and viruses were conducted. Current challenges and problems were also discussed.
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Affiliation(s)
- Yibo Dong
- College of Public Health, University of South Florida, Tampa, FL, USA
| | - Chang Li
- College of Public Health, University of South Florida, Tampa, FL, USA
| | - Kami Kim
- Division of Infectious Disease and International Medicine, Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
| | - Liwang Cui
- Division of Infectious Disease and International Medicine, Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
| | - Xiaoming Liu
- College of Public Health, University of South Florida, Tampa, FL, USA
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44
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Chakraborty T, Barbuddhe SB. Enabling One Health solutions through genomics. Indian J Med Res 2021; 153:273-279. [PMID: 33906989 PMCID: PMC8204826 DOI: 10.4103/ijmr.ijmr_576_21] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2021] [Indexed: 12/02/2022] Open
Affiliation(s)
- Trinad Chakraborty
- Institute for Medical Microbiology, Faculty of Medicine, Justus-Liebig University Giessen, Schubertstraße 81, 35392 Giessen, Germany
| | - Sukhadeo B. Barbuddhe
- Department of Meat Safety, ICAR-National Research Centre on Meat, Chengicherla, Boduppal P.O., Hyderabad 500 092, Telangana, India
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45
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Cross-Border Emergence of Escherichia coli Producing the Carbapenemase NDM-5 in Switzerland and Germany. J Clin Microbiol 2021; 59:JCM.02238-20. [PMID: 33361340 DOI: 10.1128/jcm.02238-20] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2020] [Accepted: 12/04/2020] [Indexed: 01/05/2023] Open
Abstract
A series of clinical NDM-5-producing Escherichia coli isolates obtained from two surveillance networks for carbapenem-producing Enterobacterales from 2018 to 2019, namely, Switzerland (NARA) and Germany (SurvCARE), were analyzed. The 33 NDM-5-producing E. coli isolates were highly resistant to β-lactams, including novel β-lactam/β-lactamase inhibitor combinations (ceftazidime-avibactam, imipenem-relebactam, and meropenem-vaborbactam), and remained susceptible to fosfomycin, colistin, and tigecycline. These isolates were assigned to different sequence types (STs) and indicated a predominance of isolates exhibiting ST167 in Switzerland and Germany (n = 10) (phylogenetic group C), followed by ST405 (n = 4) (phylogenetic group E), ST1284 (n = 4) (phylogenetic group C), and ST361 (n = 4) (phylogenetic group C). The bla NDM-5 gene was predominantly present on an IncF-type plasmid (n = 29) and, to a lesser extent, on the narrow-host-range IncX3 plasmid (n = 4). Sequence analyses of eight NDM-5 plasmids indicated that NDM-5-encoding F-type plasmids varied in size between 86 and 132 kb. The two IncX3 plasmids pCH8NDM5 and pD12NDM5 were 46 and 45 kb in size, respectively. The highly conserved bla NDM-5 genetic surrounding structures (ΔISAba125-bla NDM-5-ble MBL-trpT-dsbD-IS26) of both the F-type and IncX3 plasmids suggested a common genetic origin. The emergence of the NDM-5 carbapenemase was evidenced in particular for the E. coli ST167 clone, which is a successful epidemic clone known to be associated with both multiresistance and virulence traits and is therefore of high public health concern. The occurrence of clonally related NDM-5-producing E. coli isolates in Switzerland and Germany further indicates the international spread of this multidrug-resistant superbug at least throughout Europe.
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46
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Sváb D, Falgenhauer L, Horváth B, Maróti G, Falgenhauer J, Chakraborty T, Tóth I. Genome Analysis of a Historical Shigella dysenteriae Serotype 1 Strain Carrying a Conserved Stx Prophage Region. Front Microbiol 2021; 11:614793. [PMID: 33488558 PMCID: PMC7819885 DOI: 10.3389/fmicb.2020.614793] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Accepted: 12/09/2020] [Indexed: 11/13/2022] Open
Abstract
Shigella dysenteriae are significant agents of bacillary dysentery, accounting for a considerable number of illnesses with high morbidity worldwide. The Shiga toxin (Stx) encoded by a defective prophage is the key virulence factor of S. dysenteriae type 1 (SD1) strains. Here we present the full genome sequence of an SD1 strain HNCMB 20080 isolated in 1954, compare it to other sequenced SD1 genomes, and assess the diversity of Stx-prophages harbored by previously sequenced SD1 strains. The genome of HNCMB 20080 consists of a chromosome sized 4,393,622 bp containing 5,183 CDSs, as well as two small plasmids. Comparative genomic analysis revealed a high degree of uniformity among SD1 genomes, including the structure of Stx prophage regions, which we found to form two subgroups termed PT-I and PT-II. All PT-I strains are members of the sequence type (ST) 146 or ST260, while the only PT-II harboring strain, Sd1617 proved to be ST untypeable. In accordance with data from previous reports, the Stx1 prophage could not be induced from HNCMB 20080. Our cumulative data do not support the notion that stx-harboring phages in STEC are derived from historical SD1 isolates.
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Affiliation(s)
- Domonkos Sváb
- Institue for Veterinary Medical Research, Centre for Agricultural Research, Martonvásár, Hungary
| | - Linda Falgenhauer
- Institute of Hygiene and Environmental Medicine, Justus Liebig University Giessen, Giessen, Germany.,German Centre for Infection Research, Site Giessen-Marburg-Langen, Giessen, Germany
| | | | - Gergely Maróti
- Institute of Plant Biology, Biological Research Centre, Szeged, Hungary.,Faculty of Water Sciences, University of Public Service, Baja, Hungary
| | - Jane Falgenhauer
- German Centre for Infection Research, Site Giessen-Marburg-Langen, Giessen, Germany.,Institute for Medical Microbiology, Justus Liebig University Giessen, Giessen, Germany
| | - Trinad Chakraborty
- Institute of Hygiene and Environmental Medicine, Justus Liebig University Giessen, Giessen, Germany.,Institute for Medical Microbiology, Justus Liebig University Giessen, Giessen, Germany
| | - István Tóth
- Institue for Veterinary Medical Research, Centre for Agricultural Research, Martonvásár, Hungary
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47
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Complete Genome Sequences of Novel Bovine T4, rv5-Like, and Dhillonviruses Effective against Escherichia coli O157. Microbiol Resour Announc 2021; 10:10/1/e01261-20. [PMID: 33414348 PMCID: PMC8407724 DOI: 10.1128/mra.01261-20] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Enterohemorrhagic Escherichia coli (EHEC) isolates of serotype O157:H7 are serious foodborne zoonotic pathogens and prime targets for biocontrol using bacteriophages. We report on the complete genome sequences of 11 novel lytic bacteriophages, representing three viral genera, isolated from cattle in Hungary that target E. coli O157 strains. Enterohemorrhagic Escherichia coli (EHEC) isolates of serotype O157:H7 are serious foodborne zoonotic pathogens and prime targets for biocontrol using bacteriophages. We report on the complete genome sequences of 11 novel lytic bacteriophages, representing three viral genera, isolated from cattle in Hungary that target E. coli O157 strains.
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48
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Falgenhauer L, Nordmann P, Imirzalioglu C, Yao Y, Falgenhauer J, Hauri AM, Heinmüller P, Chakraborty T. Cross-border emergence of clonal lineages of ST38 Escherichia coli producing the OXA-48-like carbapenemase OXA-244 in Germany and Switzerland. Int J Antimicrob Agents 2020; 56:106157. [DOI: 10.1016/j.ijantimicag.2020.106157] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Revised: 08/31/2020] [Accepted: 09/06/2020] [Indexed: 10/23/2022]
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49
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Buckley SJ, Davies MR, McMillan DJ. In silico characterisation of stand-alone response regulators of Streptococcus pyogenes. PLoS One 2020; 15:e0240834. [PMID: 33075055 PMCID: PMC7571705 DOI: 10.1371/journal.pone.0240834] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2020] [Accepted: 10/02/2020] [Indexed: 12/20/2022] Open
Abstract
Bacterial “stand-alone” response regulators (RRs) are pivotal to the control of gene transcription in response to changing cytosolic and extracellular microenvironments during infection. The genome of group A Streptococcus (GAS) encodes more than 30 stand-alone RRs that orchestrate the expression of virulence factors involved in infecting multiple tissues, so causing an array of potentially lethal human diseases. Here, we analysed the molecular epidemiology and biological associations in the coding sequences (CDSs) and upstream intergenic regions (IGRs) of 35 stand-alone RRs from a collection of global GAS genomes. Of the 944 genomes analysed, 97% encoded 32 or more of the 35 tested RRs. The length of RR CDSs ranged from 297 to 1587 nucleotides with an average nucleotide diversity (π) of 0.012, while the IGRs ranged from 51 to 666 nucleotides with average π of 0.017. We present new evidence of recombination in multiple RRs including mga, leading to mga-2 switching, emm-switching and emm-like gene chimerization, and the first instance of an isolate that encodes both mga-1 and mga-2. Recombination was also evident in rofA/nra and msmR loci with 15 emm-types represented in multiple FCT (fibronectin-binding, collagen-binding, T-antigen)-types, including novel emm-type/FCT-type pairings. Strong associations were observed between concatenated RR allele types, and emm-type, MLST-type, core genome phylogroup, and country of sampling. No strong associations were observed between individual loci and disease outcome. We propose that 11 RRs may form part of future refinement of GAS typing systems that reflect core genome evolutionary associations. This subgenomic analysis revealed allelic traits that were informative to the biological function, GAS strain definition, and regional outbreak detection.
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Affiliation(s)
- Sean J. Buckley
- School of Health and Sports Sciences, University of the Sunshine Coast, Sippy Downs, Queensland, Australia
- * E-mail:
| | - Mark R. Davies
- Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
| | - David J. McMillan
- School of Health and Sports Sciences, University of the Sunshine Coast, Sippy Downs, Queensland, Australia
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50
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Schwengers O, Barth P, Falgenhauer L, Hain T, Chakraborty T, Goesmann A. Platon: identification and characterization of bacterial plasmid contigs in short-read draft assemblies exploiting protein sequence-based replicon distribution scores. Microb Genom 2020; 6:mgen000398. [PMID: 32579097 PMCID: PMC7660248 DOI: 10.1099/mgen.0.000398] [Citation(s) in RCA: 94] [Impact Index Per Article: 18.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2019] [Accepted: 06/02/2020] [Indexed: 12/22/2022] Open
Abstract
Plasmids are extrachromosomal genetic elements that replicate independently of the chromosome and play a vital role in the environmental adaptation of bacteria. Due to potential mobilization or conjugation capabilities, plasmids are important genetic vehicles for antimicrobial resistance genes and virulence factors with huge and increasing clinical implications. They are therefore subject to large genomic studies within the scientific community worldwide. As a result of rapidly improving next-generation sequencing methods, the quantity of sequenced bacterial genomes is constantly increasing, in turn raising the need for specialized tools to (i) extract plasmid sequences from draft assemblies, (ii) derive their origin and distribution, and (iii) further investigate their genetic repertoire. Recently, several bioinformatic methods and tools have emerged to tackle this issue; however, a combination of high sensitivity and specificity in plasmid sequence identification is rarely achieved in a taxon-independent manner. In addition, many software tools are not appropriate for large high-throughput analyses or cannot be included in existing software pipelines due to their technical design or software implementation. In this study, we investigated differences in the replicon distributions of protein-coding genes on a large scale as a new approach to distinguish plasmid-borne from chromosome-borne contigs. We defined and computed statistical discrimination thresholds for a new metric: the replicon distribution score (RDS), which achieved an accuracy of 96.6 %. The final performance was further improved by the combination of the RDS metric with heuristics exploiting several plasmid-specific higher-level contig characterizations. We implemented this workflow in a new high-throughput taxon-independent bioinformatics software tool called Platon for the recruitment and characterization of plasmid-borne contigs from short-read draft assemblies. Compared to PlasFlow, Platon achieved a higher accuracy (97.5 %) and more balanced predictions (F1=82.6 %) tested on a broad range of bacterial taxa and better or equal performance against the targeted tools PlasmidFinder and PlaScope on sequenced Escherichia coli isolates. Platon is available at: http://platon.computational.bio/.
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Affiliation(s)
- Oliver Schwengers
- Bioinformatics and Systems Biology, Justus Liebig University Giessen, Giessen, Germany
- Institute of Medical Microbiology, Justus Liebig University Giessen, Giessen, Germany
- German Center for Infection Research (DZIF), partner site Giessen-Marburg-Langen, Giessen, Germany
| | - Patrick Barth
- Bioinformatics and Systems Biology, Justus Liebig University Giessen, Giessen, Germany
| | - Linda Falgenhauer
- Institute of Medical Microbiology, Justus Liebig University Giessen, Giessen, Germany
- German Center for Infection Research (DZIF), partner site Giessen-Marburg-Langen, Giessen, Germany
- Present address: Institute of Hygiene and Environmental Health, Justus Liebig University, Giessen, Germany
| | - Torsten Hain
- Institute of Medical Microbiology, Justus Liebig University Giessen, Giessen, Germany
- German Center for Infection Research (DZIF), partner site Giessen-Marburg-Langen, Giessen, Germany
| | - Trinad Chakraborty
- Institute of Medical Microbiology, Justus Liebig University Giessen, Giessen, Germany
- German Center for Infection Research (DZIF), partner site Giessen-Marburg-Langen, Giessen, Germany
| | - Alexander Goesmann
- Bioinformatics and Systems Biology, Justus Liebig University Giessen, Giessen, Germany
- German Center for Infection Research (DZIF), partner site Giessen-Marburg-Langen, Giessen, Germany
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