|
1
|
Lotfian Z, Nave HH, Khalili R, Saffari F. Contribution of efflux activity in resistance to antibiotics in Escherichia coli clinical isolates. J Infect Chemother 2025; 31:102725. [PMID: 40318805 DOI: 10.1016/j.jiac.2025.102725] [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: 01/05/2025] [Accepted: 04/30/2025] [Indexed: 05/07/2025]
Abstract
BACKGROUND In an attempt to find new therapeutic strategies for multidrug-resistant infections, understanding the contribution of efflux pumps is of particular importance. This study aimed to determine the contribution of efflux pumps in development of resistance to different classes of antibiotics in clinical isolates of E. coli in order to adopt more effective strategies in the treatment of related infections. METHODS Two methods, i.e. measurement the expression level of some efflux related genes and assessment of efflux activity by H33342 accumulation assay using CCCP as an efflux inhibitor, were applied. The relationship of efflux activity with antibiotic resistance, was evaluated using three criteria including HA (dye accumulation in the absence of CCCP), HAC (dye accumulation in the presence of CCCP) and HAR (HAC/HA). RESULTS Expression of tolC in ceftazidime, cefotaxime and azithromycin resistant groups and expression of acrA and mdfA in tetracycline resistant isolates were significantly higher than susceptible ones (p < 0.05). By investigating the simultaneous meeting of three proposed criteria for any studied antibiotics, no significant association was found between efflux activity and antibiotic resistance. CONCLUSION Finding a relation between the overexpression of some efflux genes and resistance to tetracycline, azithromycin and beta-lactams, confirmed the role of these pumps in resistance to mentioned antibiotics and highlighted the neglected role of efflux pumps in resistance to beta-lactams. Meeting two criteria in >70% of investigated antibiotics, confirmed the contribution of efflux as a partial resistance mechanism in these isolates.
Collapse
Affiliation(s)
- Zahra Lotfian
- Student Research Committee Centre, Kerman University of Medical Sciences, Kerman, Iran
| | - Hossein Hosseini Nave
- Medical Mycology and Bacteriology Research Centre, Kerman University of Medical Sciences, Kerman, Iran
| | - Razieh Khalili
- Student Research Committee Centre, Kerman University of Medical Sciences, Kerman, Iran
| | - Fereshteh Saffari
- Medical Mycology and Bacteriology Research Centre, Kerman University of Medical Sciences, Kerman, Iran.
| |
Collapse
|
|
2
|
Feye KM, Rasmussen MA, Yeater KM, Anderson RC, Crippen TL, Harvey RB, Poole TL, Ricke SC. Chlorophyllin Supplementation of Medicated or Unmedicated Swine Diets Impact on Fecal Escherichia coli and Enterococci. Animals (Basel) 2024; 14:1955. [PMID: 38998066 PMCID: PMC11240447 DOI: 10.3390/ani14131955] [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: 05/20/2024] [Revised: 06/12/2024] [Accepted: 06/26/2024] [Indexed: 07/14/2024] Open
Abstract
Considering that certain catabolic products of anaerobic chlorophyll degradation inhibit efflux pump activity, this study was conducted to test if feeding pigs a water-soluble chlorophyllin product could affect the antibiotic resistance profiles of select wild-type populations of fecal bacteria. Trial 1 evaluated the effects of chlorophyllin supplementation (300 mg/meal) on fecal E. coli and enterococcal populations in pigs fed twice daily diets supplemented without or with ASP 250 (containing chlortetracycline, sulfamethazine and penicillin at 100, 100 and 50 g/ton, respectively). Trial 2, conducted similarly, evaluated chlorophyllin supplementation in pigs fed diets supplemented with or without 100 g tylosin/ton. Each trial lasted 12 days, and fecal samples were collected and selectively cultured at 4-day intervals to enumerate the total numbers of E. coli and enterococci as well as populations of these bacteria phenotypically capable of growing in the presence of the fed antibiotics. Performance results from both studies revealed no adverse effect (p > 0.05) of chlorophyllin, antibiotic or their combined supplementation on average daily feed intake or average daily gain, although the daily fed intake tended to be lower (p = 0.053) for pigs fed diets supplemented with tylosin than those fed diets without tylosin. The results from trial 1 showed that the ASP 250-medicated diets, whether without or with chlorophyllin supplementation, supported higher (p < 0.05) fecal E. coli populations than the non-medicated diets. Enterococcal populations, however, were lower, albeit marginally and not necessarily significantly, in feces from pigs fed the ASP 250-medicated diet than those fed the non-medicated diet. Results from trial 2 likewise revealed an increase (p < 0.05) in E. coli and, to a lesser extent, enterococcal populations in feces collected from pigs fed the tylosin-medicated diet compared with those fed the non-medicated diet. Evidence indicated that the E. coli and enterococcal populations in trial 1 were generally insensitive to penicillin or chlortetracycline, as there were no differences between populations recovered without or with antibiotic selection. Conversely, a treatment x day of treatment interaction observed in trial 2 (p < 0.05) provided evidence, albeit slight, of an enrichment of tylosin-insensitive enterococci in feces from the pigs fed the tylosin-medicated but not the non-medicated diet. Under the conditions of the present study, it is unlikely that chlorophyllin-derived efflux pump inhibitors potentially present in the chlorophyllin-fed pigs were able to enhance the efficacy of the available antibiotics. However, further research specifically designed to optimize chlorophyll administration could potentially lead to practical applications for the swine industry.
Collapse
Affiliation(s)
- Kristina M. Feye
- Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR 72701, USA
| | - Mark A. Rasmussen
- Department of Animal Science, Iowa State University, Ames, IA 50011, USA;
| | - Kathleen M. Yeater
- United States Department of Agriculture/Agricultural Research Service, Plains Area Office of the Director, Fort Collins, CO 80521, USA;
| | - Robin C. Anderson
- Southern Plains Agricultural Research Center, United States Department of Agriculture/Agricultural Research Service, College Station, TX 77845, USA; (R.C.A.); (T.L.C.); (R.B.H.); (T.L.P.)
| | - Tawni L. Crippen
- Southern Plains Agricultural Research Center, United States Department of Agriculture/Agricultural Research Service, College Station, TX 77845, USA; (R.C.A.); (T.L.C.); (R.B.H.); (T.L.P.)
| | - Roger B. Harvey
- Southern Plains Agricultural Research Center, United States Department of Agriculture/Agricultural Research Service, College Station, TX 77845, USA; (R.C.A.); (T.L.C.); (R.B.H.); (T.L.P.)
| | - Toni L. Poole
- Southern Plains Agricultural Research Center, United States Department of Agriculture/Agricultural Research Service, College Station, TX 77845, USA; (R.C.A.); (T.L.C.); (R.B.H.); (T.L.P.)
| | - Steven C. Ricke
- Meat Science and Animal Biologics Discovery Program, Department of Animal and Dairy Sciences, University of Wisconsin, Madison, WI 53706, USA
| |
Collapse
|
|
3
|
Lin MM, Yang SS, Huang QY, Cui GH, Jia XF, Yang Y, Shi ZM, Ye H, Zhang XZ. Effect and mechanism of Qingre Huashi decoction on drug-resistant Helicobacter pylori. World J Gastroenterol 2024; 30:3086-3105. [PMID: 38983958 PMCID: PMC11230061 DOI: 10.3748/wjg.v30.i24.3086] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/25/2024] [Revised: 05/05/2024] [Accepted: 05/29/2024] [Indexed: 06/25/2024] Open
Abstract
BACKGROUND Helicobacter pylori (HP), the most common pathogenic microorganism in the stomach, can induce inflammatory reactions in the gastric mucosa, causing chronic gastritis and even gastric cancer. HP infection affects over 4.4 billion people globally, with a worldwide infection rate of up to 50%. The multidrug resistance of HP poses a serious challenge to eradication. It has been de-monstrated that compared to bismuth quadruple therapy, Qingre Huashi decoction (QHD) combined with triple therapy exhibits comparable eradication rates but with a lower incidence of adverse reactions; in addition, QHD can directly inhibit and kill HP in vitro. AIM To explore the effect and mechanism of QHD on clinically multidrug-resistant and strong biofilm-forming HP. METHODS In this study, 12 HP strains were isolated in vitro after biopsy during gastroscopy of HP-infected patients. In vitro, the minimum inhibitory concentration (MIC) values for clinical HP strains and biofilm quantification were determined through the E-test method and crystal violet staining, respectively. The most robust biofilm-forming strain of HP was selected, and QHD was evaluated for its inhibitory and bactericidal effects on the strain with strong biofilm formation. This assessment was performed using agar dilution, E-test, killing dynamics, and transmission electron microscopy (TEM). The study also explored the impact of QHD on antibiotic resistance in these HP strains with strong biofilm formation. Crystalline violet method, scanning electron microscopy, laser confocal scanning microscopy, and (p)ppGpp chromatographic identification were employed to evaluate the effect of QHD on biofilm in strong biofilm-forming HP strains. The effect of QHD on biofilm and efflux pump-related gene expression was evaluated by quantitative polymerase chain reaction. Non-targeted metabolomics with UHPLC-MS/MS was used to identify potential metabolic pathways and biomarkers which were different between the NC and QHD groups. RESULTS HP could form biofilms of different degrees in vitro, and the intensity of formation was associated with the drug resistance of the strain. QHD had strong bacteriostatic and bactericidal effects on HP, with MICs of 32-64 mg/mL. QHD could inhibit the biofilm formation of the strong biofilm-forming HP strains, disrupt the biofilm structure, lower the accumulation of (p)ppGpp, decrease the expression of biofilm-related genes including LuxS, Spot, glup (HP1174), NapA, and CagE, and reduce the expression of efflux pump-related genes such as HP0605, HP0971, HP1327, and HP1489. Based on metabolomic analysis, QHD induced oxidative stress in HP, enhanced metabolism, and potentially inhibited relevant signaling pathways by upregulating adenosine monophosphate (AMP), thereby affecting HP growth, metabolism, and protein synthesis. CONCLUSION QHD exerts bacteriostatic and bactericidal effects on HP, and reduces HP drug resistance by inhibiting HP biofilm formation, destroying its biofilm structure, inhibiting the expression of biofilm-related genes and efflux pump-related genes, enhancing HP metabolism, and activating AMP in HP.
Collapse
Affiliation(s)
- Miao-Miao Lin
- Department of Integrated Traditional Chinese and Western Medicine, Peking University First Hospital, Beijing 100034, China
- Institute of Integrated Traditional Chinese and Western Medicine, Peking University, Beijing 100034, China
| | - Shan-Shan Yang
- Department of Integrated Traditional Chinese and Western Medicine, Peking University First Hospital, Beijing 100034, China
- Institute of Integrated Traditional Chinese and Western Medicine, Peking University, Beijing 100034, China
| | - Qiu-Yue Huang
- Department of Integrated Traditional Chinese and Western Medicine, Peking University First Hospital, Beijing 100034, China
- Institute of Integrated Traditional Chinese and Western Medicine, Peking University, Beijing 100034, China
| | - Guang-Hui Cui
- Department of Integrated Traditional Chinese and Western Medicine, Peking University First Hospital, Beijing 100034, China
- Institute of Integrated Traditional Chinese and Western Medicine, Peking University, Beijing 100034, China
| | - Xiao-Fen Jia
- Department of Integrated Traditional Chinese and Western Medicine, Peking University First Hospital, Beijing 100034, China
- Institute of Integrated Traditional Chinese and Western Medicine, Peking University, Beijing 100034, China
| | - Yao Yang
- Department of Integrated Traditional Chinese and Western Medicine, Peking University First Hospital, Beijing 100034, China
- Institute of Integrated Traditional Chinese and Western Medicine, Peking University, Beijing 100034, China
| | - Zong-Ming Shi
- Department of Integrated Traditional Chinese and Western Medicine, Peking University First Hospital, Beijing 100034, China
- Institute of Integrated Traditional Chinese and Western Medicine, Peking University, Beijing 100034, China
| | - Hui Ye
- Department of Integrated Traditional Chinese and Western Medicine, Peking University First Hospital, Beijing 100034, China
- Institute of Integrated Traditional Chinese and Western Medicine, Peking University, Beijing 100034, China
| | - Xue-Zhi Zhang
- Department of Integrated Traditional Chinese and Western Medicine, Peking University First Hospital, Beijing 100034, China
- Institute of Integrated Traditional Chinese and Western Medicine, Peking University, Beijing 100034, China
| |
Collapse
|
|
4
|
Novelli M, Bolla JM. RND Efflux Pump Induction: A Crucial Network Unveiling Adaptive Antibiotic Resistance Mechanisms of Gram-Negative Bacteria. Antibiotics (Basel) 2024; 13:501. [PMID: 38927168 PMCID: PMC11200565 DOI: 10.3390/antibiotics13060501] [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: 04/26/2024] [Revised: 05/22/2024] [Accepted: 05/27/2024] [Indexed: 06/28/2024] Open
Abstract
The rise of multi-drug-resistant (MDR) pathogenic bacteria presents a grave challenge to global public health, with antimicrobial resistance ranking as the third leading cause of mortality worldwide. Understanding the mechanisms underlying antibiotic resistance is crucial for developing effective treatments. Efflux pumps, particularly those of the resistance-nodulation-cell division (RND) superfamily, play a significant role in expelling molecules from bacterial cells, contributing to the emergence of multi-drug resistance. These are transmembrane transporters naturally produced by Gram-negative bacteria. This review provides comprehensive insights into the modulation of RND efflux pump expression in bacterial pathogens by numerous and common molecules (bile, biocides, pharmaceuticals, additives, plant extracts, etc.). The interplay between these molecules and efflux pump regulators underscores the complexity of antibiotic resistance mechanisms. The clinical implications of efflux pump induction by non-antibiotic compounds highlight the challenges posed to public health and the urgent need for further investigation. By addressing antibiotic resistance from multiple angles, we can mitigate its impact and preserve the efficacy of antimicrobial therapies.
Collapse
Affiliation(s)
- Marine Novelli
- Aix Marseille Univ, INSERM, SSA, MCT, 13385 Marseille, France;
- Université Paris Cité, CNRS, Biochimie des Protéines Membranaires, F-75005 Paris, France
| | | |
Collapse
|
|
5
|
Wilhelm J, Pos KM. Molecular insights into the determinants of substrate specificity and efflux inhibition of the RND efflux pumps AcrB and AdeB. MICROBIOLOGY (READING, ENGLAND) 2024; 170:001438. [PMID: 38358391 PMCID: PMC10924465 DOI: 10.1099/mic.0.001438] [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: 11/01/2022] [Accepted: 01/30/2024] [Indexed: 02/16/2024]
Abstract
Gram-negative bacterial members of the Resistance Nodulation and cell Division (RND) superfamily form tripartite efflux pump systems that span the cell envelope. One of the intriguing features of the multiple drug efflux members of this superfamily is their ability to recognize different classes of antibiotics, dyes, solvents, bile salts, and detergents. This review provides an overview of the molecular mechanisms of multiple drug efflux catalysed by the tripartite RND efflux system AcrAB-TolC from Eschericha coli. The determinants for sequential or simultaneous multiple substrate binding and efflux pump inhibitor binding are discussed. A comparison is made with the determinants for substrate binding of AdeB from Acinetobacter baumannii, which acts within the AdeABC multidrug efflux system. There is an apparent general similarity between the structures of AcrB and AdeB and their substrate specificity. However, the presence of distinct conformational states and different drug efflux capacities as revealed by single-particle cryo-EM and mutational analysis suggest that the drug binding and transport features exhibited by AcrB may not be directly extrapolated to the homolog AdeB efflux pump.
Collapse
Affiliation(s)
- Julia Wilhelm
- Institute of Biochemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany
| | - Klaas Martinus Pos
- Institute of Biochemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany
| |
Collapse
|
|
6
|
Abstract
Gram-negative bacteria are intrinsically resistant to many antibiotics, due in large part to the permeability barrier formed by their cell envelope. The complex and synergistic interplay of the two Gram-negative membranes and active efflux prevents the accumulation of a diverse range of compounds that are effective against Gram-positive bacteria. A lack of detailed information on how components of the cell envelope contribute to this has been identified as a key barrier to the rational development of new antibiotics with efficacy against Gram-negative species. This review describes the current understanding of the role of the different components of the Gram-negative cell envelope in preventing compound accumulation and the state of efforts to describe properties that allow compounds to overcome this barrier and apply them to the development of new broad-spectrum antibiotics.
Collapse
Affiliation(s)
- Claire Maher
- College of Engineering, Science and Environment, University of Newcastle, Newcastle, Australia
- ARC Centre of Excellence in Synthetic Biology, Macquarie University, Sydney, Australia
| | - Karl A. Hassan
- College of Engineering, Science and Environment, University of Newcastle, Newcastle, Australia
- ARC Centre of Excellence in Synthetic Biology, Macquarie University, Sydney, Australia
| |
Collapse
|
|
7
|
Kavanaugh LG, Mahoney AR, Dey D, Wuest WM, Conn GL. Di-berberine conjugates as chemical probes of Pseudomonas aeruginosa MexXY-OprM efflux function and inhibition. NPJ ANTIMICROBIALS AND RESISTANCE 2023; 1:12. [PMID: 39843773 PMCID: PMC11721654 DOI: 10.1038/s44259-023-00013-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/24/2023] [Accepted: 08/17/2023] [Indexed: 01/24/2025]
Abstract
The Resistance-Nodulation-Division (RND) efflux pump superfamily is pervasive among Gram-negative pathogens and contributes extensively to clinical antibiotic resistance. The opportunistic pathogen Pseudomonas aeruginosa contains 12 RND-type efflux systems, with four contributing to resistance including MexXY-OprM which is uniquely able to export aminoglycosides. At the site of initial substrate recognition, small molecule probes of the inner membrane transporter (e.g., MexY) have potential as important functional tools to understand substrate selectivity and a foundation for developing adjuvant efflux pump inhibitors (EPIs). Here, we optimized the scaffold of berberine, a known but weak MexY EPI, using an in-silico high-throughput screen to identify di-berberine conjugates with enhanced synergistic action with aminoglycosides. Further, docking and molecular dynamics simulations of di-berberine conjugates reveal unique contact residues and thus sensitivities of MexY from distinct P. aeruginosa strains. This work thereby reveals di-berberine conjugates to be useful probes of MexY transporter function and potential leads for EPI development.
Collapse
Affiliation(s)
- Logan G Kavanaugh
- Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA
| | | | - Debayan Dey
- Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA
| | - William M Wuest
- Department of Chemistry, Emory University, Atlanta, GA, USA.
- Emory Antibiotic Resistance Center, Emory University, Atlanta, GA, USA.
| | - Graeme L Conn
- Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA.
- Emory Antibiotic Resistance Center, Emory University, Atlanta, GA, USA.
| |
Collapse
|
|
8
|
Kavanaugh LG, Mahoney AR, Dey D, Wuest WM, Conn GL. Di-berberine conjugates as chemical probes of Pseudomonas aeruginosa MexXY-OprM efflux function and inhibition. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.03.24.533986. [PMID: 37425949 PMCID: PMC10327050 DOI: 10.1101/2023.03.24.533986] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/11/2023]
Abstract
The Resistance-Nodulation-Division (RND) efflux pump superfamily is pervasive among Gram-negative pathogens and contributes extensively to clinical antibiotic resistance. The opportunistic pathogen Pseudomonas aeruginosa contains 12 RND-type efflux systems, with four contributing to resistance including MexXY-OprM which is uniquely able to export aminoglycosides. At the site of initial substrate recognition, small molecule probes of the inner membrane transporter (e.g., MexY) have potential as important functional tools to understand substrate selectivity and a foundation for developing adjuvant efflux pump inhibitors (EPIs). Here, we optimized the scaffold of berberine, a known but weak MexY EPI, using an in-silico high-throughput screen to identify di-berberine conjugates with enhanced synergistic action with aminoglycosides. Further, docking and molecular dynamics simulations of di-berberine conjugates reveal unique contact residues and thus sensitivities of MexY from distinct P. aeruginosa strains. This work thereby reveals di-berberine conjugates to be useful probes of MexY transporter function and potential leads for EPI development.
Collapse
Affiliation(s)
- Logan G. Kavanaugh
- Department of Biochemistry, Emory University School of Medicine, Atlanta, GA
| | | | - Debayan Dey
- Department of Biochemistry, Emory University School of Medicine, Atlanta, GA
| | - William M. Wuest
- Department of Chemistry, Emory University, Atlanta, GA
- Emory Antibiotic Resistance Center, Emory University, Atlanta, GA
| | - Graeme L. Conn
- Department of Biochemistry, Emory University School of Medicine, Atlanta, GA
- Emory Antibiotic Resistance Center, Emory University, Atlanta, GA
| |
Collapse
|
|
9
|
Kumawat M, Nabi B, Daswani M, Viquar I, Pal N, Sharma P, Tiwari S, Sarma DK, Shubham S, Kumar M. Role of bacterial efflux pump proteins in antibiotic resistance across microbial species. Microb Pathog 2023:106182. [PMID: 37263448 DOI: 10.1016/j.micpath.2023.106182] [Citation(s) in RCA: 21] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2023] [Revised: 05/24/2023] [Accepted: 05/30/2023] [Indexed: 06/03/2023]
Abstract
Efflux proteins are transporter molecules that actively pump out a variety of substrates, including antibiotics, from cells to the environment. They are found in both Gram-positive and Gram-negative bacteria and eukaryotic cells. Based on their protein sequence homology, energy source, and overall structure, efflux proteins can be divided into seven groups. Multidrug efflux pumps are transmembrane proteins produced by microbes to enhance their survival in harsh environments and contribute to antibiotic resistance. These pumps are present in all bacterial genomes studied, indicating their ancestral origins. Many bacterial genes encoding efflux pumps are involved in transport, a significant contributor to antibiotic resistance in microbes. Efflux pumps are widely implicated in the extrusion of clinically relevant antibiotics from cells to the extracellular environment and, as such, represent a significant challenge to antimicrobial therapy. This review aims to provide an overview of the structures and mechanisms of action, substrate profiles, regulation, and possible inhibition of clinically relevant efflux pumps. Additionally, recent advances in research and the pharmacological exploitation of efflux pump inhibitors as a promising intervention for combating drug resistance will be discussed.
Collapse
Affiliation(s)
- Manoj Kumawat
- Department of Microbiology, ICMR- National Institute for Research in Environmental Health, Bhopal, 462030, India
| | - Bilkees Nabi
- Department of Biochemistry & Biochemical Engineering, SHUATS, Allahabad, 211007, India
| | - Muskan Daswani
- Department of Biotechnology, SantHirdaram Girls College, Bhopal, 462030, India
| | - Iqra Viquar
- Department of Biotechnology, SantHirdaram Girls College, Bhopal, 462030, India
| | - Namrata Pal
- Department of Microbiology, ICMR- National Institute for Research in Environmental Health, Bhopal, 462030, India
| | - Poonam Sharma
- Department of Microbiology, ICMR- National Institute for Research in Environmental Health, Bhopal, 462030, India
| | - Shikha Tiwari
- Department of Microbiology, ICMR- National Institute for Research in Environmental Health, Bhopal, 462030, India
| | - Devojit Kumar Sarma
- Department of Microbiology, ICMR- National Institute for Research in Environmental Health, Bhopal, 462030, India
| | - Swasti Shubham
- Department of Microbiology, ICMR- National Institute for Research in Environmental Health, Bhopal, 462030, India
| | - Manoj Kumar
- Department of Microbiology, ICMR- National Institute for Research in Environmental Health, Bhopal, 462030, India.
| |
Collapse
|
|
10
|
Omollo C, Singh V, Kigondu E, Wasuna A, Agarwal P, Moosa A, Ioerger TR, Mizrahi V, Chibale K, Warner DF. Developing synergistic drug combinations to restore antibiotic sensitivity in drug-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother 2023; 65:AAC.02554-20. [PMID: 33619062 PMCID: PMC8092878 DOI: 10.1128/aac.02554-20] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2020] [Accepted: 02/14/2021] [Indexed: 12/17/2022] Open
Abstract
Tuberculosis (TB) is a leading global cause of mortality owing to an infectious agent, accounting for almost one-third of antimicrobial resistance (AMR) deaths annually. We aimed to identify synergistic anti-TB drug combinations with the capacity to restore therapeutic efficacy against drug-resistant mutants of the causative agent, Mycobacterium tuberculosis We investigated combinations containing the known translational inhibitors, spectinomycin (SPT) and fusidic acid (FA), or the phenothiazine, chlorpromazine (CPZ), which disrupts mycobacterial energy metabolism. Potentiation of whole-cell drug efficacy was observed in SPT-CPZ combinations. This effect was lost against an M. tuberculosis mutant lacking the major facilitator superfamily (MFS) efflux pump, Rv1258c. Notably, the SPT-CPZ combination partially restored SPT efficacy against an SPT-resistant mutant carrying a g1379t point mutation in rrs, encoding the mycobacterial 16S ribosomal RNA. Combinations of SPT with FA, which targets the mycobacterial elongation factor G, exhibited potentiating activity against wild-type M. tuberculosis Moreover, this combination produced a modest potentiating effect against both FA-monoresistant and SPT-monoresistant mutants. Finally, combining SPT with the frontline anti-TB agents, rifampicin (RIF) and isoniazid, resulted in enhanced activity in vitro and ex vivo against both drug-susceptible M. tuberculosis and a RIF-monoresistant rpoB S531L mutant.These results support the utility of novel potentiating drug combinations in restoring antibiotic susceptibility of M. tuberculosis strains carrying genetic resistance to any one of the partner compounds.
Collapse
Affiliation(s)
- Charles Omollo
- Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
- South African Medical Research Council Drug Discovery and Development Research Unit, University of Cape Town, Rondebosch 7701, South Africa
- SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DSI/NRF Centre of Excellence for Biomedical Tuberculosis Research, Department of Pathology, University of Cape Town, Rondebosch 7701, South Africa
- Institute of Infectious Disease & Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
| | - Vinayak Singh
- Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
- South African Medical Research Council Drug Discovery and Development Research Unit, University of Cape Town, Rondebosch 7701, South Africa
- SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DSI/NRF Centre of Excellence for Biomedical Tuberculosis Research, Department of Pathology, University of Cape Town, Rondebosch 7701, South Africa
- Institute of Infectious Disease & Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
| | - Elizabeth Kigondu
- Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
- South African Medical Research Council Drug Discovery and Development Research Unit, University of Cape Town, Rondebosch 7701, South Africa
- SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DSI/NRF Centre of Excellence for Biomedical Tuberculosis Research, Department of Pathology, University of Cape Town, Rondebosch 7701, South Africa
| | - Antonina Wasuna
- Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
- South African Medical Research Council Drug Discovery and Development Research Unit, University of Cape Town, Rondebosch 7701, South Africa
- SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DSI/NRF Centre of Excellence for Biomedical Tuberculosis Research, Department of Pathology, University of Cape Town, Rondebosch 7701, South Africa
| | - Pooja Agarwal
- SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DSI/NRF Centre of Excellence for Biomedical Tuberculosis Research, Department of Pathology, University of Cape Town, Rondebosch 7701, South Africa
- Institute of Infectious Disease & Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
| | - Atica Moosa
- SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DSI/NRF Centre of Excellence for Biomedical Tuberculosis Research, Department of Pathology, University of Cape Town, Rondebosch 7701, South Africa
- Institute of Infectious Disease & Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
| | - Thomas R Ioerger
- Texas A&M University, Department of Computer Science, College Station, TX, 77843, USA
| | - Valerie Mizrahi
- SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DSI/NRF Centre of Excellence for Biomedical Tuberculosis Research, Department of Pathology, University of Cape Town, Rondebosch 7701, South Africa
- Institute of Infectious Disease & Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
- Wellcome Centre for Infectious Diseases Research in Africa, University of Cape Town, Rondebosch 7701, South Africa
| | - Kelly Chibale
- Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
- South African Medical Research Council Drug Discovery and Development Research Unit, University of Cape Town, Rondebosch 7701, South Africa
- Institute of Infectious Disease & Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
| | - Digby F Warner
- SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DSI/NRF Centre of Excellence for Biomedical Tuberculosis Research, Department of Pathology, University of Cape Town, Rondebosch 7701, South Africa
- Institute of Infectious Disease & Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
- Wellcome Centre for Infectious Diseases Research in Africa, University of Cape Town, Rondebosch 7701, South Africa
| |
Collapse
|
|
11
|
Moore-Machacek A, Gloe A, O'Leary N, Reen FJ. Efflux, Signaling and Warfare in a Polymicrobial World. Antibiotics (Basel) 2023; 12:antibiotics12040731. [PMID: 37107093 PMCID: PMC10135244 DOI: 10.3390/antibiotics12040731] [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: 02/24/2023] [Revised: 03/30/2023] [Accepted: 04/04/2023] [Indexed: 04/29/2023] Open
Abstract
The discovery void of antimicrobial development has occurred at a time when the world has seen a rapid emergence and spread of antimicrobial resistance, the 'perfect storm' as it has often been described. While the discovery and development of new antibiotics has continued in the research sphere, the pipeline to clinic has largely been fed by derivatives of existing classes of antibiotics, each prone to pre-existing resistance mechanisms. A novel approach to infection management has come from the ecological perspective whereby microbial networks and evolved communities already possess small molecular capabilities for pathogen control. The spatiotemporal nature of microbial interactions is such that mutualism and parasitism are often two ends of the same stick. Small molecule efflux inhibitors can directly target antibiotic efflux, a primary resistance mechanism adopted by many species of bacteria and fungi. However, a much broader anti-infective capability resides within the action of these inhibitors, borne from the role of efflux in key physiological and virulence processes, including biofilm formation, toxin efflux, and stress management. Understanding how these behaviors manifest within complex polymicrobial communities is key to unlocking the full potential of the advanced repertoires of efflux inhibitors.
Collapse
Affiliation(s)
| | - Antje Gloe
- School of Microbiology, University College Cork, T12 K8AF Cork, Ireland
- Institute for Pharmaceutical Microbiology, University of Bonn, D-53113 Bonn, Germany
| | - Niall O'Leary
- School of Microbiology, University College Cork, T12 K8AF Cork, Ireland
| | - F Jerry Reen
- School of Microbiology, University College Cork, T12 K8AF Cork, Ireland
- Synthesis and Solid-State Pharmaceutical Centre, University College Cork, T12 K8AF Cork, Ireland
| |
Collapse
|
|
12
|
Chauviat A, Meyer T, Favre-Bonté S. Versatility of Stenotrophomonas maltophilia: Ecological roles of RND efflux pumps. Heliyon 2023; 9:e14639. [PMID: 37089375 PMCID: PMC10113797 DOI: 10.1016/j.heliyon.2023.e14639] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2023] [Revised: 03/13/2023] [Accepted: 03/14/2023] [Indexed: 03/30/2023] Open
Abstract
S. maltophilia is a widely distributed bacterium found in natural, anthropized and clinical environments. The genome of this opportunistic pathogen of environmental origin includes a large number of genes encoding RND efflux pumps independently of the clinical or environmental origin of the strains. These pumps have been historically associated with the uptake of antibiotics and clinically relevant molecules because they confer resistance to many antibiotics. However, considering the environmental origin of S. maltophilia, the ecological role of these pumps needs to be clarified. RND efflux systems are highly conserved within bacteria and encountered both in pathogenic and non-pathogenic species. Moreover, their evolutionary origin, conservation and multiple copies in bacterial genomes suggest a primordial role in cellular functions and environmental adaptation. This review is aimed at elucidating the ecological role of S. maltophilia RND efflux pumps in the environmental context and providing an exhaustive description of the environmental niches of S. maltophilia. By looking at the substrates and functions of the pumps, we propose different involvements and roles according to the adaptation of the bacterium to various niches. We highlight that i°) regulatory mechanisms and inducer molecules help to understand the conditions leading to their expression, and ii°) association and functional redundancy of RND pumps and other efflux systems demonstrate their complex role within S. maltophilia cells. These observations emphasize that RND efflux pumps play a role in the versatility of S. maltophilia.
Collapse
|
|
13
|
Manrique PD, López CA, Gnanakaran S, Rybenkov VV, Zgurskaya HI. New understanding of multidrug efflux and permeation in antibiotic resistance, persistence, and heteroresistance. Ann N Y Acad Sci 2023; 1519:46-62. [PMID: 36344198 PMCID: PMC9839546 DOI: 10.1111/nyas.14921] [Citation(s) in RCA: 21] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Antibiotics effective against Gram-negative ESKAPE pathogens are a critical area of unmet need. Infections caused by these pathogens are not only difficult to treat but finding new therapies to overcome Gram-negative resistance is also a challenge. There are not enough antibiotics in development that target the most dangerous pathogens and there are not enough novel drugs in the pipeline. The major obstacle in the antibiotic discovery pipeline is the lack of understanding of how to breach antibiotic permeability barriers of Gram-negative pathogens. These barriers are created by active efflux pumps acting across both the inner and the outer membranes. Overproduction of efflux pumps alone or together with either modification of the outer membrane or antibiotic-inactivating enzymes and target mutations contribute to clinical levels of antibiotics resistance. Recent efforts have generated significant advances in the rationalization of compound efflux and permeation across the cell envelopes of Gram-negative pathogens. Combined with earlier studies and novel mathematical models, these efforts have led to a multilevel understanding of how antibiotics permeate these barriers and how multidrug efflux and permeation contribute to the development of antibiotic resistance and heteroresistance. Here, we discuss the new developments in this area.
Collapse
Affiliation(s)
- Pedro D. Manrique
- Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM 87545, United States
- Present address: Physics Department, George Washington University, Washington D.C. 20052, United States
| | - Cesar A. López
- Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM 87545, United States
| | - S. Gnanakaran
- Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM 87545, United States
| | - Valentin V. Rybenkov
- Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, United States
| | - Helen I. Zgurskaya
- Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, United States
| |
Collapse
|
|
14
|
Goetz JA, Kuehfuss NM, Botschner AJ, Zhu S, Thompson LK, Cox G. Exploring functional interplay amongst Escherichia coli efflux pumps. MICROBIOLOGY (READING, ENGLAND) 2022; 168. [PMID: 36318669 DOI: 10.1099/mic.0.001261] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Bacterial efflux pumps exhibit functional interplay that can translate to additive or multiplicative effects on resistance to antimicrobial compounds. In diderm bacteria, two different efflux pump structural types - single-component inner membrane efflux pumps and cell envelope-spanning multicomponent systems - cooperatively export antimicrobials with cytoplasmic targets from the cell. Harnessing our recently developed efflux platform, which is built upon an extensively efflux-deficient strain of Escherichia coli, here we explore interplay amongst a panel of diverse E. coli efflux pumps. Specifically, we assessed the effect of simultaneously expressing two efflux pump-encoding genes on drug resistance, including single-component inner membrane efflux pumps (MdfA, MdtK and EmrE), tripartite complexes (AcrAB, AcrAD, MdtEF and AcrEF), and the acquired TetA(C) tetracycline resistance pump. Overall, the expression of two efflux pump-encoding genes from the same structural type did not enhance resistance levels regardless of the antimicrobial compound or efflux pump under investigation. In contrast, a combination of the tripartite efflux systems with single-component pumps sharing common substrates provided multiplicative increases to antimicrobial resistance levels. In some instances, resistance was increased beyond the product of resistance provided by the two pumps individually. In summary, the developed efflux platform enables the isolation of efflux pump function, facilitating the identification of interactions between efflux pumps.
Collapse
Affiliation(s)
- James A Goetz
- College of Biological Sciences, Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Rd E, Guelph, Ontario, N1G 2W1, Canada
| | - Noah M Kuehfuss
- College of Biological Sciences, Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Rd E, Guelph, Ontario, N1G 2W1, Canada
| | - Alexander J Botschner
- College of Biological Sciences, Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Rd E, Guelph, Ontario, N1G 2W1, Canada
| | - Shawna Zhu
- College of Biological Sciences, Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Rd E, Guelph, Ontario, N1G 2W1, Canada
| | - Laura K Thompson
- College of Biological Sciences, Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Rd E, Guelph, Ontario, N1G 2W1, Canada
| | - Georgina Cox
- College of Biological Sciences, Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Rd E, Guelph, Ontario, N1G 2W1, Canada
| |
Collapse
|
|
15
|
A genetic platform to investigate the functions of bacterial drug efflux pumps. Nat Chem Biol 2022; 18:1399-1409. [PMID: 36065018 DOI: 10.1038/s41589-022-01119-y] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2021] [Accepted: 07/21/2022] [Indexed: 11/08/2022]
Abstract
Efflux pumps are a serious challenge for the development of antibacterial agents. Overcoming efflux requires an in-depth understanding of efflux pump functions, specificities and the development of inhibitors. However, the complexities of efflux networks have limited such studies. To address these challenges, we generated Efflux KnockOut-35 (EKO-35), a highly susceptible Escherichia coli strain lacking 35 efflux pumps. We demonstrate the use of this strain by constructing an efflux platform comprising EKO-35 strains individually producing efflux pumps forming tripartite complexes with TolC. This platform was profiled against a curated diverse compound collection, which enabled us to define physicochemical properties that contribute to transport. We also show the E. coli drug efflux network is conditionally essential for growth, and that the platform can be used to investigate efflux pump inhibitor specificities and efflux pump interplay. We believe EKO-35 and the efflux platform will have widespread application for the study of drug efflux.
Collapse
|
|
16
|
MDR Pumps as Crossroads of Resistance: Antibiotics and Bacteriophages. Antibiotics (Basel) 2022; 11:antibiotics11060734. [PMID: 35740141 PMCID: PMC9220107 DOI: 10.3390/antibiotics11060734] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2022] [Revised: 05/21/2022] [Accepted: 05/26/2022] [Indexed: 01/27/2023] Open
Abstract
At present, antibiotic resistance represents a global problem in modern medicine. In the near future, humanity may face a situation where medicine will be powerless against resistant bacteria and a post-antibiotic era will come. The development of new antibiotics is either very expensive or ineffective due to rapidly developing bacterial resistance. The need to develop alternative approaches to the treatment of bacterial infections, such as phage therapy, is beyond doubt. The cornerstone of bacterial defense against antibiotics are multidrug resistance (MDR) pumps, which are involved in antibiotic resistance, toxin export, biofilm, and persister cell formation. MDR pumps are the primary non-specific defense of bacteria against antibiotics, while drug target modification, drug inactivation, target switching, and target sequestration are the second, specific line of their defense. All bacteria have MDR pumps, and bacteriophages have evolved along with them and use the bacteria’s need for MDR pumps to bind and penetrate into bacterial cells. The study and understanding of the mechanisms of the pumps and their contribution to the overall resistance and to the sensitivity to bacteriophages will allow us to either seriously delay the onset of the post-antibiotic era or even prevent it altogether due to phage-antibiotic synergy.
Collapse
|
|
17
|
Rattanapanadda P, Kuo HC, Chang SK, Tell LA, Shia WY, Chou CC. Effect of Carbonyl Cyanide Chlorophenylhydrazone on Intrabacterial Concentration and Antimicrobial Activity of Amphenicols against Swine Resistant Actinobacillus pleuropneumoniae and Pasteurella multocida. Vet Res Commun 2022; 46:903-916. [PMID: 35322371 DOI: 10.1007/s11259-022-09917-4] [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: 10/23/2021] [Accepted: 03/15/2022] [Indexed: 12/13/2022]
Abstract
Effects and mechanism of carbonyl cyanide chlorophenylhydrazone (CCCP) on antimicrobial activity of florfenicol (FF) and thiamphenicol (TAP) were investigated against amphenicol-resistant Actinobacillus pleuropneumoniae and Pasteurella multocida isolated from diseased swine. Broth microdilution and time-kill assays indicated that CCCP dose-dependently and substantially (4-32 fold MIC reduction) improved amphenicol antimicrobial activity. When combined with CCCP at the lowest literature reported dose (2-5 μg/mL), 85% FF resistant A. pleuropneumoniae and 92% resistant P. multocida showed significantly reduced FF MICs (≥ 4-fold). In contrast, none or few of the susceptible A. pleuropneumoniae and P. multocida had FF MICs reduction ≥ 4-fold. 90% FF resistant A. pleuropneumoniae and 96% resistant P. multocida carried the floR gene, indicating strong association with the FloR efflux pump. With CCCP, the intracellular FF concentration increased by 71% in floR+ resistant A. pleuropneumoniae and 156% in floR+ resistant P. multocida strains but not the susceptible strains. The degree of reduction in TAP MICs was found consistently in parallel to FF for both bacteria. Taken together, partially attributed to blockage of drug-efflux, the combination of FF or TAP with CCCP at sub-cytotoxic concentrations was demonstrated and showed feasibility to combat amphenicol-resistant A. pleuropneumoniae and P. multocida isolated from diseased swine.
Collapse
Affiliation(s)
- Porjai Rattanapanadda
- Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan, 402.,Department of Livestock Development, Ministry of Agriculture and Cooperatives, Bangkok, 10400, Thailand
| | - Hung-Chih Kuo
- Department of Veterinary Medicine, College of Veterinary Medicine, National Chiayi University, Chiayi, Taiwan, 600
| | - Shao-Kuang Chang
- Department of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan, 106
| | - Lisa Ann Tell
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA, 95616, USA
| | - Wei-Yau Shia
- Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan, 402
| | - Chi-Chung Chou
- Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan, 402.
| |
Collapse
|
|
18
|
Zhang W, Yuan Y, Li S, Deng B, Zhang J, Li Z. Comparative transcription analysis of resistant mutants against four different antibiotics in Pseudomonas aeruginosa. Microb Pathog 2021; 160:105166. [PMID: 34480983 DOI: 10.1016/j.micpath.2021.105166] [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: 06/10/2021] [Revised: 08/26/2021] [Accepted: 08/29/2021] [Indexed: 11/28/2022]
Abstract
The emergence of antibiotic resistance has severely impaired the treatment of infections caused by Pseudomonas aeruginosa. There are few studies related to comparing the antibiotics resistance mechanisms of P. aeruginosa against different antibiotics. In this study, RNA sequencing was used to investigate the differences of transcriptome between wild strain and four antibiotics resistant strains of P. aeruginosa PAO1 (polymyxin B, ciprofloxacin, doxycycline, and ceftriaxone). Compared to the wild strain, 1907, 495, 2402, and 116 differentially expressed genes (DEGs) were identified in polymyxin B, ciprofloxacin, doxycycline, and ceftriaxone resistant PAO1, respectively. After analysis of genes related to antimicrobial resistance, we found genes implicated in biofilm formation (pelB, pelC, pelD, pelE, pelF, pelG, algA, algF, and alg44) were significantly upregulated in polymyxin B-resistant PAO1, efflux pump genes (mexA, mexB, oprM) and biofilm formation genes (pslJ, pslK and pslN) were upregulated in ciprofloxacin-resistant PAO1; other efflux pump genes (mexC, mexD, oprJ) were upregulated in doxycycline-resistant PAO1; ampC were upregulated in ceftriaxone-resistant PAO1. As a consequence of antibiotic resistance, genes related to virulence factors such as type Ⅱ secretion system (lasA, lasB and piv) were significantly upregulated in polymyxin B-resistant PAO1, and type Ⅲ secretion system (exoS, exoT, exoY, exsA, exsB, exsC, exsD, pcrV, popB, popD, pscC, pscE, pscG, and pscJ) were upregulated in doxycycline-resistant PAO1. While, ampC were upregulated in ceftriaxone-resistant PAO1. In addition, variants were obtained in wild type and four antibiotics resistant PAO1. Our findings provide a comparative transcriptome analysis of antibiotic resistant mutants selected by different antibiotics, and might assist in identifying potential therapeutic strategies for P. aeruginosa infection.
Collapse
Affiliation(s)
- Wenlu Zhang
- School of Basic Medical Sciences, Henan University of Science and Technology, Luoyang, 471003, China
| | - Yaping Yuan
- School of Basic Medical Sciences, Henan University of Science and Technology, Luoyang, 471003, China
| | - Shasha Li
- School of Basic Medical Sciences, Henan University of Science and Technology, Luoyang, 471003, China
| | - Bo Deng
- School of Basic Medical Sciences, Henan University of Science and Technology, Luoyang, 471003, China
| | - Jiaming Zhang
- School of Basic Medical Sciences, Henan University of Science and Technology, Luoyang, 471003, China
| | - Zhongjie Li
- School of Basic Medical Sciences, Henan University of Science and Technology, Luoyang, 471003, China.
| |
Collapse
|
|
19
|
Foong WE, Wilhelm J, Tam HK, Pos KM. Tigecycline efflux in Acinetobacter baumannii is mediated by TetA in synergy with RND-type efflux transporters. J Antimicrob Chemother 2021; 75:1135-1139. [PMID: 32049277 DOI: 10.1093/jac/dkaa015] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2019] [Accepted: 01/08/2020] [Indexed: 01/05/2023] Open
Abstract
OBJECTIVES To investigate the role of Major Facilitator Superfamily (MFS)-type transporters from Acinetobacter baumannii AYE in tigecycline efflux. METHODS Two putative tetracycline transporter genes of A. baumannii AYE (tetA and tetG) were heterologously expressed in Escherichia coli and drug susceptibility assays were conducted with tigecycline and three other tetracycline derivatives. The importance of TetA in tigecycline transport in A. baumannii was determined by complementation of tetA in WT and Resistance Nodulation cell Division (RND) gene knockout strains of A. baumannii ATCC 19606. Gene expression of the MFS-type tetA gene and RND efflux pump genes adeB, adeG and adeJ in A. baumannii AYE in the presence of tigecycline was analysed by quantitative real-time RT-PCR. RESULTS Overproduction of TetA or TetG conferred resistance to doxycycline, minocycline and tetracycline in E. coli. Cells expressing tetA, but not those expressing tetG, conferred resistance to tigecycline, implying that TetA is a determinant for tigecycline transport. A. baumannii WT and RND-knockout strains complemented with plasmid-encoded tetA are significantly less susceptible to tigecycline compared with non-complemented strains. Efflux pump genes tetA and adeG are up-regulated in A. baumannii AYE in the presence of subinhibitory tigecycline concentrations. CONCLUSIONS TetA plays an important role in tigecycline efflux of A. baumannii by removing the drug from cytoplasm to periplasm and, subsequently, the RND-type transporters AdeABC and AdeIJK extrude tigecycline across the outer membrane. When challenged with tigecycline, tetA is up-regulated in A. baumannii AYE. Synergy between TetA and the RND-type transporters AdeABC and/or AdeIJK appears necessary for A. baumannii to confer higher tigecycline resistance via drug efflux.
Collapse
Affiliation(s)
- Wuen Ee Foong
- Institute of Biochemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany
| | - Jochen Wilhelm
- Excellence Cluster Cardio-Pulmonary Institute (CPI), Universities of Giessen & Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Justus-Liebig University Giessen, Gaffkystrasse 11, 35392 Giessen, Germany
| | - Heng-Keat Tam
- Institute of Biochemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany
| | - Klaas M Pos
- Institute of Biochemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany
| |
Collapse
|
|
20
|
Klenotic PA, Moseng MA, Morgan CE, Yu EW. Structural and Functional Diversity of Resistance-Nodulation-Cell Division Transporters. Chem Rev 2021; 121:5378-5416. [PMID: 33211490 PMCID: PMC8119314 DOI: 10.1021/acs.chemrev.0c00621] [Citation(s) in RCA: 55] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Multidrug resistant (MDR) bacteria are a global threat with many common infections becoming increasingly difficult to eliminate. While significant effort has gone into the development of potent biocides, the effectiveness of many first-line antibiotics has been diminished due to adaptive resistance mechanisms. Bacterial membrane proteins belonging to the resistance-nodulation-cell division (RND) superfamily play significant roles in mediating bacterial resistance to antimicrobials. They participate in multidrug efflux and cell wall biogenesis to transform bacterial pathogens into "superbugs" that are resistant even to last resort antibiotics. In this review, we summarize the RND superfamily of efflux transporters with a primary focus on the assembly and function of the inner membrane pumps. These pumps are critical for extrusion of antibiotics from the cell as well as the transport of lipid moieties to the outer membrane to establish membrane rigidity and stability. We analyze recently solved structures of bacterial inner membrane efflux pumps as to how they bind and transport their substrates. Our cumulative data indicate that these RND membrane proteins are able to utilize different oligomerization states to achieve particular activities, including forming MDR pumps and cell wall remodeling machineries, to ensure bacterial survival. This mechanistic insight, combined with simulated docking techniques, allows for the design and optimization of new efflux pump inhibitors to more effectively treat infections that today are difficult or impossible to cure.
Collapse
Affiliation(s)
- Philip A. Klenotic
- Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland OH 44106, USA
| | - Mitchell A. Moseng
- Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland OH 44106, USA
| | - Christopher E. Morgan
- Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland OH 44106, USA
| | - Edward W. Yu
- Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland OH 44106, USA
| |
Collapse
|
|
21
|
Rybenkov VV, Zgurskaya HI, Ganguly C, Leus IV, Zhang Z, Moniruzzaman M. The Whole Is Bigger than the Sum of Its Parts: Drug Transport in the Context of Two Membranes with Active Efflux. Chem Rev 2021; 121:5597-5631. [PMID: 33596653 PMCID: PMC8369882 DOI: 10.1021/acs.chemrev.0c01137] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Cell envelope plays a dual role in the life of bacteria by simultaneously protecting it from a hostile environment and facilitating access to beneficial molecules. At the heart of this ability lie the restrictive properties of the cellular membrane augmented by efflux transporters, which preclude intracellular penetration of most molecules except with the help of specialized uptake mediators. Recently, kinetic properties of the cell envelope came into focus driven on one hand by the urgent need in new antibiotics and, on the other hand, by experimental and theoretical advances in studies of transmembrane transport. A notable result from these studies is the development of a kinetic formalism that integrates the Michaelis-Menten behavior of individual transporters with transmembrane diffusion and offers a quantitative basis for the analysis of intracellular penetration of bioactive compounds. This review surveys key experimental and computational approaches to the investigation of transport by individual translocators and in whole cells, summarizes key findings from these studies and outlines implications for antibiotic discovery. Special emphasis is placed on Gram-negative bacteria, whose envelope contains two separate membranes. This feature sets these organisms apart from Gram-positive bacteria and eukaryotic cells by providing them with full benefits of the synergy between slow transmembrane diffusion and active efflux.
Collapse
Affiliation(s)
- Valentin V Rybenkov
- Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019, United States
| | - Helen I Zgurskaya
- Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019, United States
| | - Chhandosee Ganguly
- Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019, United States
| | - Inga V Leus
- Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019, United States
| | - Zhen Zhang
- Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019, United States
| | - Mohammad Moniruzzaman
- Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019, United States
| |
Collapse
|
|
22
|
Ahmed MS, Lauersen KJ, Ikram S, Li C. Efflux Transporters' Engineering and Their Application in Microbial Production of Heterologous Metabolites. ACS Synth Biol 2021; 10:646-669. [PMID: 33751883 DOI: 10.1021/acssynbio.0c00507] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Metabolic engineering of microbial hosts for the production of heterologous metabolites and biochemicals is an enabling technology to generate meaningful quantities of desired products that may be otherwise difficult to produce by traditional means. Heterologous metabolite production can be restricted by the accumulation of toxic products within the cell. Efflux transport proteins (transporters) provide a potential solution to facilitate the export of these products, mitigate toxic effects, and enhance production. Recent investigations using knockout lines, heterologous expression, and expression profiling of transporters have revealed candidates that can enhance the export of heterologous metabolites from microbial cell systems. Transporter engineering efforts have revealed that some exhibit flexible substrate specificity and may have broader application potentials. In this Review, the major superfamilies of efflux transporters, their mechanistic modes of action, selection of appropriate efflux transporters for desired compounds, and potential transporter engineering strategies are described for potential applications in enhancing engineered microbial metabolite production. Future studies in substrate recognition, heterologous expression, and combinatorial engineering of efflux transporters will assist efforts to enhance heterologous metabolite production in microbial hosts.
Collapse
Affiliation(s)
- Muhammad Saad Ahmed
- Institute for Synthetic Biosystem/Department of Biochemical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology (BIT), Beijing 100081, P. R. China
- Department of Biological Sciences, National University of Medical Sciences (NUMS), Abid Majeed Road, The Mall, Rawalpindi 46000, Pakistan
| | - Kyle J. Lauersen
- Biological and Environmental Sciences and Engineering Division (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal 23955, Kingdom of Saudi Arabia
| | - Sana Ikram
- Beijing Higher Institution Engineering Research Center for Food Additives and Ingredients, Beijing Technology & Business University (BTBU), Beijing 100048, P. R. China
| | - Chun Li
- Institute for Synthetic Biosystem/Department of Biochemical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology (BIT), Beijing 100081, P. R. China
- SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, Key Laboratory of Systems Bioengineering, Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, P. R. China
- Key Laboratory for Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, P. R. China
| |
Collapse
|
|
23
|
Luo YH, Lai YS, Zheng C, Ilhan ZE, Ontiveros-Valencia A, Long X, Krajmalnik-Brown R, Rittmann BE. Increased expression of antibiotic-resistance genes in biofilm communities upon exposure to cetyltrimethylammonium bromide (CTAB) and other stress conditions. THE SCIENCE OF THE TOTAL ENVIRONMENT 2021; 765:144264. [PMID: 33418325 DOI: 10.1016/j.scitotenv.2020.144264] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/02/2020] [Revised: 11/12/2020] [Accepted: 11/28/2020] [Indexed: 06/12/2023]
Abstract
Quaternary ammonium compounds (QAC, e.g., cetyltrimethylammonium bromide, (CTAB)) are widely used as surfactants and disinfectants. QAC already are commonly found in wastewaters, and their concentration could increase, since QAC are recommended to inactivate the SARS-CoV-2 (COVID-19) virus. Exposure of bacteria to QAC can lead to proliferation of antibiotic resistance genes (ARG). In particular, O2-based membrane biofilm reactors (O2-MBfRs) achieved excellent CTAB biodegradation, but ARG increased in their biofilms. Here, we applied meta-transcriptomic analyses to assess the impacts of CTAB exposure and operating conditions on microbial community's composition and ARG expression in the O2-MBfRs. Two opportunistic pathogens, Pseudomonas aeruginosa and Stenotrophomonas maltophilia, dominated the microbial communities and were associated with the presence of ARG. Operating conditions that imposed stress on the biofilms, i.e., limited supplies of O2 and nitrogen or a high loading of CTAB, led to large increases in ARG expression, particularly for genes conferring antibiotic-target protection. Important within the efflux pumps was the Resistance-Nodulation-Division (RND) family, which may have been active in exporting CTAB from cells. Oxidative stress appeared to be the key factor that triggered ARG proliferation by selecting intrinsically resistant species and accentuating the expression of ARG. Our findings suggest that means to mitigate the spread of ARG, such as shown here in a O2-based membrane biofilm reactor, need to consider the impacts of stressors, including QAC exposure and stressful operating conditions.
Collapse
Affiliation(s)
- Yi-Hao Luo
- Biodesign Swette Center for Environmental Biotechnology, Arizona State University, P.O. Box 875701, Tempe, AZ 85287-5701, USA
| | - YenJung Sean Lai
- Biodesign Swette Center for Environmental Biotechnology, Arizona State University, P.O. Box 875701, Tempe, AZ 85287-5701, USA.
| | - Chenwei Zheng
- Biodesign Swette Center for Environmental Biotechnology, Arizona State University, P.O. Box 875701, Tempe, AZ 85287-5701, USA
| | - Zehra Esra Ilhan
- Biodesign Swette Center for Environmental Biotechnology, Arizona State University, P.O. Box 875701, Tempe, AZ 85287-5701, USA; INRAE, Micalis Institute, Université Paris-Saclay, AgroParisTech, 78350 Jouy-en-Josas, France
| | - Aura Ontiveros-Valencia
- Biodesign Swette Center for Environmental Biotechnology, Arizona State University, P.O. Box 875701, Tempe, AZ 85287-5701, USA; Division de Ciencias Ambientales, Instituto Potosino de Investigación Científica y Tecnológica, Camino a la Presa de San José 2055, ZC 78216 San Luis Potosí, Mexico
| | - Xiangxing Long
- Biodesign Swette Center for Environmental Biotechnology, Arizona State University, P.O. Box 875701, Tempe, AZ 85287-5701, USA; Nanosystems Engineering Research Center for Nanotechnology-Enabled Water Treatment, School of Sustainable Engineering and the Built Environment, Arizona State University, Tempe, AZ 85287-5306, USA
| | - Rosa Krajmalnik-Brown
- Biodesign Swette Center for Environmental Biotechnology, Arizona State University, P.O. Box 875701, Tempe, AZ 85287-5701, USA
| | - Bruce E Rittmann
- Biodesign Swette Center for Environmental Biotechnology, Arizona State University, P.O. Box 875701, Tempe, AZ 85287-5701, USA
| |
Collapse
|
|
24
|
Henderson PJF, Maher C, Elbourne LDH, Eijkelkamp BA, Paulsen IT, Hassan KA. Physiological Functions of Bacterial "Multidrug" Efflux Pumps. Chem Rev 2021; 121:5417-5478. [PMID: 33761243 DOI: 10.1021/acs.chemrev.0c01226] [Citation(s) in RCA: 89] [Impact Index Per Article: 22.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Bacterial multidrug efflux pumps have come to prominence in human and veterinary pathogenesis because they help bacteria protect themselves against the antimicrobials used to overcome their infections. However, it is increasingly realized that many, probably most, such pumps have physiological roles that are distinct from protection of bacteria against antimicrobials administered by humans. Here we undertake a broad survey of the proteins involved, allied to detailed examples of their evolution, energetics, structures, chemical recognition, and molecular mechanisms, together with the experimental strategies that enable rapid and economical progress in understanding their true physiological roles. Once these roles are established, the knowledge can be harnessed to design more effective drugs, improve existing microbial production of drugs for clinical practice and of feedstocks for commercial exploitation, and even develop more sustainable biological processes that avoid, for example, utilization of petroleum.
Collapse
Affiliation(s)
- Peter J F Henderson
- School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom
| | - Claire Maher
- School of Environmental and Life Sciences, University of Newcastle, Callaghan 2308, New South Wales, Australia
| | - Liam D H Elbourne
- Department of Biomolecular Sciences, Macquarie University, Sydney 2109, New South Wales, Australia.,ARC Centre of Excellence in Synthetic Biology, Macquarie University, Sydney 2019, New South Wales, Australia
| | - Bart A Eijkelkamp
- College of Science and Engineering, Flinders University, Bedford Park 5042, South Australia, Australia
| | - Ian T Paulsen
- Department of Biomolecular Sciences, Macquarie University, Sydney 2109, New South Wales, Australia.,ARC Centre of Excellence in Synthetic Biology, Macquarie University, Sydney 2019, New South Wales, Australia
| | - Karl A Hassan
- School of Environmental and Life Sciences, University of Newcastle, Callaghan 2308, New South Wales, Australia.,ARC Centre of Excellence in Synthetic Biology, Macquarie University, Sydney 2019, New South Wales, Australia
| |
Collapse
|
|
25
|
Bagelman S, Zvigule-Neidere G. Insight into Kytococcus schroeteri Infection Management: A Case Report and Review. Infect Dis Rep 2021; 13:230-238. [PMID: 33799382 PMCID: PMC8005950 DOI: 10.3390/idr13010026] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/20/2020] [Revised: 02/25/2021] [Accepted: 03/10/2021] [Indexed: 01/14/2023] Open
Abstract
Background: Kytococcus schroeteri is a member of normal skin microflora, which can cause lethal infections in immunosuppressed hosts. In this review we attempted to draw patterns of its pathogenicity, which seem to vary regarding host immune status and the presence of implantable devices. Evidence suggests this pathogen houses many resistance-forming proteins, which serve to exacerbate the challenge in curing it. Available information on K. schroeteri antibacterial susceptibility is scarce. In this situation, a novel, genome-based antibiotic resistance analysis model, previously suggested by Su et al., could aid clinicians dealing with unknown infections. In this study we merged data from observed antibiotic resistance patterns with resistance data demonstrated by DNA sequences. Methods: We reviewed all available articles and reports on K. schroeteri, from peer-reviewed online databases (ClinicalKey, PMC, Scopus and WebOfScience). Information on patients was then subdivided into patient profiles and tabulated independently. We later performed K. schroeteri genome sequence analysis for resistance proteins to understand the trends K. schroeteri exhibits. Results: K. schroeteri is resistant to beta-lactams, macrolides and clindamycin. It is susceptible to aminoglycosides, tetracyclines and rifampicin. We combined data from the literature review and sequence analysis and found evidence for the existence of PBP, PBP-2A and efflux pumps as likely determinants of K. schroeteri. Conclusions: Reviewing the data permits the speculation that baseline immune status plays a role in the outcome of a Kytococcal infection. Nonetheless, our case report demonstrates that the outcome of a lower baseline immunity could still be favorable, possibly using rifampicin in first-line treatment of infection caused by K. schroeteri.
Collapse
Affiliation(s)
- Shelly Bagelman
- International Students Department, Riga Stradins University, LV-1007 Riga, Latvia
- Correspondence: ; Tel.: +371-972-549-066-373
| | | |
Collapse
|
|
26
|
Burkholderia ubonensis High-Level Tetracycline Resistance Is Due to Efflux Pump Synergy Involving a Novel TetA(64) Resistance Determinant. Antimicrob Agents Chemother 2021; 65:AAC.01767-20. [PMID: 33318011 DOI: 10.1128/aac.01767-20] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2020] [Accepted: 12/07/2020] [Indexed: 02/06/2023] Open
Abstract
Burkholderia ubonensis, a nonpathogenic soil bacterium belonging to the Burkholderia cepacia complex (Bcc), is highly resistant to some clinically significant antibiotics. The concern is that B. ubonensis may serve as a resistance reservoir for Bcc or B. pseudomallei complex (Bpc) organisms that are opportunistic human pathogens. Using a B. ubonensis strain highly resistant to tetracycline (MIC, ≥256 µg/ml), we identified and characterized tetA(64) that encodes a novel tetracycline-specific efflux pump of the major facilitator superfamily. TetA(64) and associated TetR(64) regulator expression are induced by tetracyclines. Although TetA(64) is the primary tetracycline and doxycycline resistance determinant, maximum tetracycline and doxycycline resistance requires synergy between TetA(64) and the nonspecific AmrAB-OprA resistance nodulation cell division efflux pump. TetA(64) does not efflux minocycline, tigecycline, and eravacycline. Comprehensive screening of genome sequences showed that TetA(64) is unequally distributed in the Bcc and absent from the Bpc. It is present in some major cystic fibrosis pathogens, like Burkholderia cenocepacia, but absent from others like Burkholderia multivorans The tetR(64)-tetA(64) genes are located in a region of chromosome 1 that is highly conserved in Burkholderia sp. Because there is no evidence for transposition, the tetR(64)-tetA(64) genes may have been acquired by homologous recombination after horizontal gene transfer. Although Burkholderia species contain a resident multicomponent efflux pump that allows them to respond to tetracyclines up to a certain concentration, the acquisition of the single-component TetA(64) by some species likely provides the synergy that these bacteria need to defend against high tetracycline concentrations in niche environments.
Collapse
|
|
27
|
Verma P, Tiwari M, Tiwari V. Efflux pumps in multidrug-resistant Acinetobacter baumannii: Current status and challenges in the discovery of efflux pumps inhibitors. Microb Pathog 2021; 152:104766. [PMID: 33545327 DOI: 10.1016/j.micpath.2021.104766] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2020] [Revised: 01/13/2021] [Accepted: 01/22/2021] [Indexed: 12/14/2022]
Abstract
Acinetobacter baumannii is an ESKAPE pathogen known to cause fatal nosocomial infections. With the surge of multidrug resistance (MDR) in the bacterial system, effective treatment measures have become very limited. The MDR in A. baumannii is contributed by various factors out of which efflux pumps have gained major attention due to their broad substrate specificity and wide distribution among bacterial species. The efflux pumps are involved in the MDR as well as contribute to other physiological processes in bacteria, therefore, it is critically important to inhibit efflux pumps in order to combat emerging resistance. The present review provides insight about the different efflux pump systems in A. baumannii and their role in multidrug resistance. A major focus has been put on the different strategies and alternate therapeutics to inhibit the efflux system. This includes use of different efflux pump inhibitors-natural, synthetic or combinatorial therapy. The use of phage therapy and nanoparticles for inhibiting efflux pumps have also been discussed here. Moreover, the present review provides the knowledge of barriers in development of efflux pump inhibitors (EPIs) and their approval for commercialization. Here, different prospectives have been discussed to improve the therapeutic development process and make it more compatible for clinical use.
Collapse
Affiliation(s)
- Privita Verma
- Department of Biochemistry, Central University of Rajasthan, Bandarsindri, Ajmer, 305817, India
| | - Monalisa Tiwari
- Department of Biochemistry, Central University of Rajasthan, Bandarsindri, Ajmer, 305817, India
| | - Vishvanath Tiwari
- Department of Biochemistry, Central University of Rajasthan, Bandarsindri, Ajmer, 305817, India.
| |
Collapse
|
|
28
|
Leus IV, Adamiak J, Trinh AN, Smith RD, Smith L, Richardson S, Ernst RK, Zgurskaya HI. Inactivation of AdeABC and AdeIJK efflux pumps elicits specific nonoverlapping transcriptional and phenotypic responses in Acinetobacter baumannii. Mol Microbiol 2020; 114:1049-1065. [PMID: 32858760 DOI: 10.1111/mmi.14594] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2020] [Revised: 08/17/2020] [Indexed: 12/24/2022]
Abstract
Multidrug resistant (MDR) strains of Acinetobacter baumannii present a serious clinical challenge. The development of antibiotic resistance in this species is enabled by efflux pumps of the Resistance-Nodulation-Division (RND) superfamily of proteins creating an efficient permeability barrier for antibiotics. At least three RND pumps, AdeABC, AdeIJK, and AdeFGH are encoded in the A. baumannii genome and are reported to contribute to antibiotic resistance in clinical isolates. In this study, we analyzed the contributions of AdeABC and AdeIJK in antibiotic resistance and growth physiology of the two MDR strains, AYE and AB5075. We found that not only the two pumps have nonoverlapping substrate specificities, their inactivation leads to specific nonoverlapping changes in gene expression as determined by RNA sequencing and confirmed by gene knockouts and growth phenotypes. Our results suggest that inactivation of AdeIJK elicits broader changes in the abundances of mRNAs and this response is modified in the absence of AdeB. In contrast, inactivation of AdeB leads to a focused cellular response, which is not sensitive to the activity of AdeIJK. We identified additional efflux pumps and transcriptional regulators that contribute to MDR phenotype of clinical A. baumannii isolates.
Collapse
Affiliation(s)
- Inga V Leus
- Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA
| | - Justyna Adamiak
- Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA
| | - Anhthu N Trinh
- Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA
| | - Richard D Smith
- Department of Microbial Pathogenesis, University of Maryland, Baltimore, Baltimore, MD, USA
| | - Lauren Smith
- Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA
| | - Sophie Richardson
- Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA
| | - Robert K Ernst
- Department of Microbial Pathogenesis, University of Maryland, Baltimore, Baltimore, MD, USA
| | - Helen I Zgurskaya
- Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA
| |
Collapse
|
|
29
|
Teelucksingh T, Thompson LK, Cox G. The Evolutionary Conservation of Escherichia coli Drug Efflux Pumps Supports Physiological Functions. J Bacteriol 2020; 202:e00367-20. [PMID: 32839176 PMCID: PMC7585057 DOI: 10.1128/jb.00367-20] [Citation(s) in RCA: 56] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Bacteria harness an impressive repertoire of resistance mechanisms to evade the inhibitory action of antibiotics. One such mechanism involves efflux pump-mediated extrusion of drugs from the bacterial cell, which significantly contributes to multidrug resistance. Intriguingly, most drug efflux pumps are chromosomally encoded components of the intrinsic antibiotic resistome. In addition, in terms of xenobiotic detoxification, bacterial efflux systems often exhibit significant levels of functional redundancy. Efflux pumps are also considered to be highly conserved; however, the extent of conservation in many bacterial species has not been reported and the majority of genes that encode efflux pumps appear to be dispensable for growth. These observations, in combination with an increasing body of experimental evidence, imply alternative roles in bacterial physiology. Indeed, the ability of efflux pumps to facilitate antibiotic resistance could be a fortuitous by-product of ancient physiological functions. Using Escherichia coli as a model organism, we here evaluated the evolutionary conservation of drug efflux pumps and we provide phylogenetic analysis of the major efflux families. We show the E. coli drug efflux system has remained relatively stable and the majority (∼80%) of pumps are encoded in the core genome. This analysis further supports the importance of drug efflux pumps in E. coli physiology. In this review, we also provide an update on the roles of drug efflux pumps in the detoxification of endogenously synthesized substrates and pH homeostasis. Overall, gaining insight into drug efflux pump conservation, common evolutionary ancestors, and physiological functions could enable strategies to combat these intrinsic and ancient elements.
Collapse
Affiliation(s)
- Tanisha Teelucksingh
- College of Biological Sciences, Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
| | - Laura K Thompson
- College of Biological Sciences, Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
| | - Georgina Cox
- College of Biological Sciences, Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
| |
Collapse
|
|
30
|
Li P, Zhu T, Zhou D, Lu W, Liu H, Sun Z, Ying J, Lu J, Lin X, Li K, Ying J, Bao Q, Xu T. Analysis of Resistance to Florfenicol and the Related Mechanism of Dissemination in Different Animal-Derived Bacteria. Front Cell Infect Microbiol 2020; 10:369. [PMID: 32903722 PMCID: PMC7438884 DOI: 10.3389/fcimb.2020.00369] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2020] [Accepted: 06/16/2020] [Indexed: 12/26/2022] Open
Abstract
Bacterial resistance to antibiotics has become an important concern for public health. This study was aimed to investigate the characteristics and the distribution of the florfenicol-related resistance genes in bacteria isolated from four farms. A total of 106 florfenicol-resistant Gram-negative bacilli were examined for florfenicol-related resistance genes, and the positive isolates were further characterized. The antimicrobial sensitivity results showed that most of them (100, 94.33%) belonged to multidrug resistance Enterobacteriaceae. About 91.51% of the strains carried floR gene, while 4.72% carried cfr gene. According to the pulsed-field gel electrophoresis results, 34 Escherichia coli were subdivided into 22 profiles, the genetic similarity coefficient of which ranged from 80.3 to 98.0%. The multilocus sequence typing (MLST) results revealed 17 sequence types (STs), with ST10 being the most prevalent. The genome sequencing result showed that the Proteus vulgaris G32 genome consists of a 4.06-Mb chromosome, a 177,911-bp plasmid (pG32-177), and a 51,686-bp plasmid (pG32-51). A floR located in a drug-resistant region on the chromosome of P. vulgaris G32 was with IS91 family transposase, and the other floR gene on the plasmid pG32-177 was with an ISCR2 insertion sequence. The cfr gene was located on the pG32-51 flanked by IS26 element and TnpA26. This study suggested that the mobile genetic elements played an important role in the replication of resistance genes and the horizontal resistance gene transfer.
Collapse
Affiliation(s)
- Peizhen Li
- Key Laboratory of Medical Genetics of Zhejiang Province, Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, China.,School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China.,Institute of Biomedical Informatics, Wenzhou Medical University, Wenzhou, China
| | - Tingyuan Zhu
- Key Laboratory of Medical Genetics of Zhejiang Province, Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, China.,School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China.,Institute of Biomedical Informatics, Wenzhou Medical University, Wenzhou, China
| | - Danying Zhou
- Key Laboratory of Medical Genetics of Zhejiang Province, Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, China.,School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China
| | - Wei Lu
- Key Laboratory of Medical Genetics of Zhejiang Province, Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, China.,School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China
| | - Hongmao Liu
- Key Laboratory of Medical Genetics of Zhejiang Province, Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, China.,School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China
| | - Zhewei Sun
- Key Laboratory of Medical Genetics of Zhejiang Province, Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, China.,School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China
| | - Jun Ying
- Key Laboratory of Medical Genetics of Zhejiang Province, Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, China.,School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China
| | - Junwan Lu
- Key Laboratory of Medical Genetics of Zhejiang Province, Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, China.,School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China
| | - Xi Lin
- Key Laboratory of Medical Genetics of Zhejiang Province, Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, China.,School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China.,Institute of Biomedical Informatics, Wenzhou Medical University, Wenzhou, China
| | - Kewei Li
- Key Laboratory of Medical Genetics of Zhejiang Province, Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, China.,School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China.,Institute of Biomedical Informatics, Wenzhou Medical University, Wenzhou, China
| | - Jianchao Ying
- Key Laboratory of Medical Genetics of Zhejiang Province, Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, China.,School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China.,Institute of Biomedical Informatics, Wenzhou Medical University, Wenzhou, China
| | - Qiyu Bao
- Key Laboratory of Medical Genetics of Zhejiang Province, Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, China.,School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China.,Institute of Biomedical Informatics, Wenzhou Medical University, Wenzhou, China
| | - Teng Xu
- Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China.,Institute of Translational Medicine, Baotou Central Hospital, Baotou, China
| |
Collapse
|
|
31
|
Dolatabadi A, Noorbazargan H, Khayam N, Moulavi P, Zamani N, Asghari Lalami Z, Ashrafi F. Ecofriendly Biomolecule-Capped Bifidobacterium bifidum-Manufactured Silver Nanoparticles and Efflux Pump Genes Expression Alteration in Klebsiella pneumoniae. Microb Drug Resist 2020; 27:247-257. [PMID: 32635796 DOI: 10.1089/mdr.2019.0366] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Background: Klebsiella pneumoniae is currently considered as an immediate threat to human health due to its various multidrug efflux pumps. Microbially synthesized silver nanoparticles (AgNPs) are an attractive and eco-friendly approach to prevent antibiotic resistance in bacteria. In the present study, we compared the inhibitory effect of both commercial and green AgNPs by Bifidobacterium bifidum on OxqAB efflux pump genes in ciprofloxacin-resistant strains of K. pneumoniae. Materials and Methods: AgNPs were characterized by ultraviolet-visible spectrophotometer, Fourier transform infrared spectroscopy, X-ray diffraction, zeta potential, transmission electron microscopy, and scanning electron microscopy. Antibiogram was used to identify resistant isolates and the effect of the biosynthesized AgNPs against OxqAB efflux pump strains was assessed by the minimum inhibitory concentration (MIC) method. The expression levels of oxqAB genes were evaluated using real-time polymerase chain reaction (PCR) followed by exposure to subMICs of the AgNPs. Results: PCR results showed that 25 strains had OxqAB efflux pump and the MIC method indicated that AgNPs had an inhibitory effect on all resistant strains with OxqAB efflux pump. The efficacy of the synthetic nanoparticles was assessed by comparing the antiefflux pump activity with commercial AgNPs. In ciprofloxacin-resistant isolates, the oxqAB genes expression levels reduced in the subMIC of both AgNPs, whereas biosynthesized AgNPs had greater bactericidal effects compared with the commercial AgNPs. Conclusions: Efflux pumps could be an attractive target for our biosynthesized AgNPs. The oxqAB genes expression levels reduced in subMIC of both AgNPs, whereas biosynthesized AgNPs had greater bactericidal effects than the commercial AgNPs.
Collapse
Affiliation(s)
- Aghigh Dolatabadi
- Department of Biology, Tehran North Branch, Islamic Azad University, Tehran, Iran
| | - Hassan Noorbazargan
- Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Nazanin Khayam
- Department of Biology, Tehran North Branch, Islamic Azad University, Tehran, Iran
| | - Pooria Moulavi
- Department of Biology, Tehran North Branch, Islamic Azad University, Tehran, Iran
| | - Naghmeh Zamani
- Department of Biology, Tehran North Branch, Islamic Azad University, Tehran, Iran
| | | | - Fatemeh Ashrafi
- Department of Biology, Tehran North Branch, Islamic Azad University, Tehran, Iran
| |
Collapse
|
|
32
|
Meng L, Liu H, Lan T, Dong L, Hu H, Zhao S, Zhang Y, Zheng N, Wang J. Antibiotic Resistance Patterns of Pseudomonas spp. Isolated From Raw Milk Revealed by Whole Genome Sequencing. Front Microbiol 2020; 11:1005. [PMID: 32655503 PMCID: PMC7326020 DOI: 10.3389/fmicb.2020.01005] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2019] [Accepted: 04/24/2020] [Indexed: 12/11/2022] Open
Abstract
Psychrotrophic bacteria in raw milk are most well known for their spoilage potential and the economic losses they cause to the dairy industry. Food-related psychrotrophic bacteria are increasingly reported to have antibiotic resistance features. The aim of this study was to evaluate the resistance patterns of Pseudomonas spp. isolated from bulk-tank milk. In total, we investigated the antibiotic susceptibility profiles of 86 Pseudomonas spp. isolates from raw milk. All strains were tested against 15 antimicrobial agents. Pseudomonas isolates were most highly resistant to imipenem (95.3%), followed by trimethoprim-sulfamethoxazole (69.8%), aztreonam (60.5%), chloramphenicol (45.3%), and meropenem (27.9%). Their multiple antibiotic resistance (MAR) index values ranged from 0.0 to 0.8. Whole-genome sequencing revealed the presence of intrinsic resistance determinants, such as BcI, ampC-09, blaCTX-M, oprD, sul1, dfrE, catA1, catB3, catI, floR, and cmlV. Moreover, resistance-nodulation-cell division (RND) and ATP-binding cassette (ABC) antibiotic efflux pumps were also found. This study provides further knowledge of the antibiotic resistance patterns of Pseudomonas spp. in milk, which may advance our understanding of resistance in Pseudomonas and suggests that antibiotic resistance of Pseudomonas spp. in raw milk should be a concern.
Collapse
Affiliation(s)
- Lu Meng
- Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.,Key Laboratory of Quality & Safety Control for Milk and Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Huimin Liu
- Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.,Key Laboratory of Quality & Safety Control for Milk and Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Tu Lan
- Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.,Key Laboratory of Quality & Safety Control for Milk and Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Lei Dong
- Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.,Key Laboratory of Quality & Safety Control for Milk and Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Haiyan Hu
- Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.,Key Laboratory of Quality & Safety Control for Milk and Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.,College of Animal Science and Technology, Anhui Agricultural University, Hefei, China
| | - Shengguo Zhao
- Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.,Key Laboratory of Quality & Safety Control for Milk and Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Yangdong Zhang
- Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.,Key Laboratory of Quality & Safety Control for Milk and Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Nan Zheng
- Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.,Key Laboratory of Quality & Safety Control for Milk and Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Jiaqi Wang
- Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.,Key Laboratory of Quality & Safety Control for Milk and Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| |
Collapse
|
|
33
|
High Level of Resistance to Antimicrobials and Heavy Metals in Multidrug-Resistant Pseudomonas sp. Isolated from Water Sources. Curr Microbiol 2020; 77:2694-2701. [DOI: 10.1007/s00284-020-02052-w] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2020] [Accepted: 05/23/2020] [Indexed: 01/11/2023]
|
|
34
|
Saha P, Sikdar S, Krishnamoorthy G, Zgurskaya HI, Rybenkov VV. Drug Permeation against Efflux by Two Transporters. ACS Infect Dis 2020; 6:747-758. [PMID: 32039579 DOI: 10.1021/acsinfecdis.9b00510] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
The development of new antibiotics against Gram-negative bacteria is hampered by the powerful protective properties of their cell envelope. This envelope consists of two membranes augmented by efflux transporters, which act in synergy to restrict cellular access to a broad range of chemical compounds. Recently, a kinetic model of this system has been constructed. The model revealed a complex, nonlinear behavior of the system, complete with a bifurcation, and matched very well to experimental uptake data. Here, we expand the model to include multiple transporters and apply it to an experimental analysis of antibiotic accumulation in wild-type and efflux-deficient Escherichia coli. We show that transporters acting across the inner and outer membranes have synergistic effects with each other. In contrast, transporters acting across the same membrane are additive as a rule but can be synergistic under special circumstances owing to a bifurcation controlled by the barrier constant. With respect to ethidium bromide, the inner membrane transporter MdfA was synergistic to the TolC-dependent efflux across the outer membrane. The agreement between the model and drug accumulation was very good across a range of tested drug concentrations and strains. However, antibiotic susceptibilities related only qualitatively to the accumulation of the drugs or predictions of the model and could be fit to the model only if additional assumptions were made about the physiological consequences of prolonged cell exposure to the drugs. Thus, the constructed model correctly predicts transmembrane permeation of various compounds and potentially their intracellular activity.
Collapse
Affiliation(s)
- Paramita Saha
- Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019, United States
| | - Samapan Sikdar
- Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019, United States
| | - Ganesh Krishnamoorthy
- Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019, United States
| | - Helen I. Zgurskaya
- Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019, United States
| | - Valentin V. Rybenkov
- Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019, United States
| |
Collapse
|
|
35
|
Fanelli F, Chieffi D, Di Pinto A, Mottola A, Baruzzi F, Fusco V. Phenotype and genomic background of Arcobacter butzleri strains and taxogenomic assessment of the species. Food Microbiol 2020; 89:103416. [PMID: 32138986 DOI: 10.1016/j.fm.2020.103416] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2019] [Revised: 11/08/2019] [Accepted: 01/08/2020] [Indexed: 11/29/2022]
Abstract
In this study the phenotypic and genomic characterization of two Arcobacter butzleri (Ab) strains (Ab 34_O and Ab 39_O) isolated from pre-cut ready-to-eat vegetables were performed. Results provided useful data about their taxonomy and their overall virulence potential with particular reference to the antibiotic and heavy metal susceptibility. These features were moreover compared with those of two Ab strains isolated from shellfish and a genotaxonomic assessment of the Ab species was performed. The two Ab isolated from vegetables were confirmed to belong to the Aliarcobacter butzleri species by 16S rRNA gene sequence analysis, MLST and genomic analyses. The genome-based taxonomic assessment of the Ab species brought to the light the possibility to define different subspecies reflecting the source of isolation, even though further genomes from different sources should be available to support this hypothesis. The strains isolated from vegetables in the same geographic area shared the same distribution of COGs with a prevalence of the cluster "inorganic ion transport and metabolism", consistent with the lithotrophic nature of Arcobacter spp. None of the Ab strains (from shellfish and from vegetables) metabolized carbohydrates but utilized organic acids and amino acids as carbon sources. The metabolic fingerprinting of Ab resulted less discriminatory than the genome-based approach. The Ab strains isolated from vegetables and those isolated from shellfish endowed multiple resistance to several antibiotics and heavy metals.
Collapse
Affiliation(s)
- Francesca Fanelli
- Institute of Sciences of Food Production of the National Research Council of Italy (CNR-ISPA), Bari, 70126, Italy
| | - Daniele Chieffi
- Institute of Sciences of Food Production of the National Research Council of Italy (CNR-ISPA), Bari, 70126, Italy
| | - Angela Di Pinto
- Department of Veterinary Medicine, University of Bari Aldo Moro, Valenzano, Bari, 70010, Italy
| | - Anna Mottola
- Department of Veterinary Medicine, University of Bari Aldo Moro, Valenzano, Bari, 70010, Italy
| | - Federico Baruzzi
- Institute of Sciences of Food Production of the National Research Council of Italy (CNR-ISPA), Bari, 70126, Italy
| | - Vincenzina Fusco
- Institute of Sciences of Food Production of the National Research Council of Italy (CNR-ISPA), Bari, 70126, Italy.
| |
Collapse
|
|
36
|
Guest RL, Court EA, Waldon JL, Schock KA, Raivio TL. Impaired Efflux of the Siderophore Enterobactin Induces Envelope Stress in Escherichia coli. Front Microbiol 2019; 10:2776. [PMID: 31866967 PMCID: PMC6908949 DOI: 10.3389/fmicb.2019.02776] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2019] [Accepted: 11/14/2019] [Indexed: 01/04/2023] Open
Abstract
The Cpx response is one of several envelope stress responses that monitor and maintain the integrity of the gram-negative bacterial envelope. While several conditions that are known or predicted to generate misfolded inner membrane proteins activate the Cpx response, the molecular nature of the Cpx inducing cue is not yet known. Studies have demonstrated that mutation of multidrug efflux pumps activates the Cpx response in many gram-negative bacteria. In Vibrio cholerae, pathway activation is due to accumulation of the catechol siderophore vibriobactin. However, the mechanism by which the Cpx response is activated by mutation of efflux pumps in Escherichia coli remains unknown. Here we show that inhibition of efflux by deletion of tolC, the outer membrane channel of several multidrug efflux pumps, activates the Cpx response in E. coli as a result of impaired efflux of the siderophore enterobactin. Enterobactin accumulation in the tolC mutant reduces activity of the nicotinamide adenine dinucleotide (NADH) oxidation arm of the aerobic respiratory chain. However, the Cpx pathway remains active in the tolC mutant when either NADH dehydrogenase I, NADH dehydrogenase II, or cytochrome bo3 is absent. Finally, we show that the Cpx response down-regulates transcription of the enterobactin biosynthesis operon. These results suggest that the Cpx response promotes adaptation to envelope stress in enteric bacteria that are exposed to iron-limited environments, which are rich in envelope-damaging compounds and conditions.
Collapse
Affiliation(s)
- Randi L Guest
- Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada
| | - Emily A Court
- Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada
| | - Jayne L Waldon
- Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada
| | - Kiersten A Schock
- Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada
| | - Tracy L Raivio
- Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada
| |
Collapse
|
|
37
|
Genome-Wide Transposon Screen of a Pseudomonas syringae mexB Mutant Reveals the Substrates of Efflux Transporters. mBio 2019; 10:mBio.02614-19. [PMID: 31662463 PMCID: PMC6819667 DOI: 10.1128/mbio.02614-19] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Bacteria express numerous efflux transporters that confer resistance to diverse toxicants present in their environment. Due to a high level of functional redundancy of these transporters, it is difficult to identify those that are of most importance in conferring resistance to specific compounds. The resistance-nodulation-division (RND) protein family is one such example of redundant transporters that are widespread among Gram-negative bacteria. Within this family, the MexAB-OprM protein complex is highly expressed and conserved among Pseudomonas species. We exposed barcoded transposon mutant libraries in isogenic wild-type and ΔmexB backgrounds in P. syringae B728a to diverse toxic compounds in vitro to identify mutants with increased susceptibility to these compounds. Mutants with mutations in genes encoding both known and novel redundant transporters but with partially overlapping substrate specificities were observed in a ΔmexB background. Psyr_0228, an uncharacterized member of the major facilitator superfamily of transporters, preferentially contributes to tolerance of acridine orange and acriflavine. Another transporter located in the inner membrane, Psyr_0541, contributes to tolerance of acriflavine and berberine. The presence of multiple redundant, genomically encoded efflux transporters appears to enable bacterial strains to tolerate a diversity of environmental toxins. This genome-wide screen performed in a hypersusceptible mutant strain revealed numerous transporters that would otherwise be dispensable under these conditions. Bacterial strains such as P. syringae that likely encounter diverse toxins in their environment, such as in association with many different plant species, probably benefit from possessing multiple redundant transporters that enable versatility with respect to toleration of novel toxicants.IMPORTANCE Bacteria use protein pumps to remove toxic compounds from the cell interior, enabling survival in diverse environments. These protein pumps can be highly redundant, making their targeted examination difficult. In this study, we exposed mutant populations of Pseudomonas syringae to diverse toxicants to identify pumps that contributed to survival in those conditions. In parallel, we examined pump redundancy by testing mutants of a population lacking the primary efflux transporter responsible for toxin tolerance. We identified partial substrate overlap for redundant transporters, as well as several pumps that appeared more substrate specific. For bacteria that are found in diverse environments, having multiple, partially redundant efflux pumps likely allows flexibility in habitat colonization.
Collapse
|
|
38
|
Helmann TC, Ongsarte CL, Lam J, Deutschbauer AM, Lindow SE. Genome-Wide Transposon Screen of a Pseudomonas syringae mexB Mutant Reveals the Substrates of Efflux Transporters. mBio 2019. [PMID: 31662463 DOI: 10.1101/684605] [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] [Indexed: 05/12/2023] Open
Abstract
Bacteria express numerous efflux transporters that confer resistance to diverse toxicants present in their environment. Due to a high level of functional redundancy of these transporters, it is difficult to identify those that are of most importance in conferring resistance to specific compounds. The resistance-nodulation-division (RND) protein family is one such example of redundant transporters that are widespread among Gram-negative bacteria. Within this family, the MexAB-OprM protein complex is highly expressed and conserved among Pseudomonas species. We exposed barcoded transposon mutant libraries in isogenic wild-type and ΔmexB backgrounds in P. syringae B728a to diverse toxic compounds in vitro to identify mutants with increased susceptibility to these compounds. Mutants with mutations in genes encoding both known and novel redundant transporters but with partially overlapping substrate specificities were observed in a ΔmexB background. Psyr_0228, an uncharacterized member of the major facilitator superfamily of transporters, preferentially contributes to tolerance of acridine orange and acriflavine. Another transporter located in the inner membrane, Psyr_0541, contributes to tolerance of acriflavine and berberine. The presence of multiple redundant, genomically encoded efflux transporters appears to enable bacterial strains to tolerate a diversity of environmental toxins. This genome-wide screen performed in a hypersusceptible mutant strain revealed numerous transporters that would otherwise be dispensable under these conditions. Bacterial strains such as P. syringae that likely encounter diverse toxins in their environment, such as in association with many different plant species, probably benefit from possessing multiple redundant transporters that enable versatility with respect to toleration of novel toxicants.IMPORTANCE Bacteria use protein pumps to remove toxic compounds from the cell interior, enabling survival in diverse environments. These protein pumps can be highly redundant, making their targeted examination difficult. In this study, we exposed mutant populations of Pseudomonas syringae to diverse toxicants to identify pumps that contributed to survival in those conditions. In parallel, we examined pump redundancy by testing mutants of a population lacking the primary efflux transporter responsible for toxin tolerance. We identified partial substrate overlap for redundant transporters, as well as several pumps that appeared more substrate specific. For bacteria that are found in diverse environments, having multiple, partially redundant efflux pumps likely allows flexibility in habitat colonization.
Collapse
Affiliation(s)
- Tyler C Helmann
- Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, USA
| | - Caitlin L Ongsarte
- Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, USA
| | - Jennifer Lam
- Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, USA
| | - Adam M Deutschbauer
- Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, USA
- Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA
| | - Steven E Lindow
- Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, USA
| |
Collapse
|
|
39
|
Kobylka J, Kuth MS, Müller RT, Geertsma ER, Pos KM. AcrB: a mean, keen, drug efflux machine. Ann N Y Acad Sci 2019; 1459:38-68. [PMID: 31588569 DOI: 10.1111/nyas.14239] [Citation(s) in RCA: 111] [Impact Index Per Article: 18.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2019] [Revised: 08/21/2019] [Accepted: 09/02/2019] [Indexed: 12/23/2022]
Abstract
Gram-negative bacteria are intrinsically resistant against cytotoxic substances by means of their outer membrane and a network of multidrug efflux systems, acting in synergy. Efflux pumps from various superfamilies with broad substrate preferences sequester and pump drugs across the inner membrane to supply the highly polyspecific and powerful tripartite resistance-nodulation-cell division (RND) efflux pumps with compounds to be extruded across the outer membrane barrier. In Escherichia coli, the tripartite efflux system AcrAB-TolC is the archetype RND multiple drug efflux pump complex. The homotrimeric inner membrane component acriflavine resistance B (AcrB) is the drug specificity and energy transduction center for the drug/proton antiport process. Drugs are bound and expelled via a cycle of mainly three consecutive states in every protomer, constituting a flexible alternating access channel system. This review recapitulates the molecular basis of drug and inhibitor binding, including mechanistic insights into drug efflux by AcrB. It also summarizes 17 years of mutational analysis of the gene acrB, reporting the effect of every substitution on the ability of E. coli to confer resistance toward antibiotics (http://goethe.link/AcrBsubstitutions). We emphasize the functional robustness of AcrB toward single-site substitutions and highlight regions that are more sensitive to perturbation.
Collapse
Affiliation(s)
- Jessica Kobylka
- Institute of Biochemistry, Goethe-University Frankfurt, Frankfurt am Main, Germany
| | - Miriam S Kuth
- Institute of Biochemistry, Goethe-University Frankfurt, Frankfurt am Main, Germany
| | - Reinke T Müller
- Institute of Biochemistry, Goethe-University Frankfurt, Frankfurt am Main, Germany
| | - Eric R Geertsma
- Institute of Biochemistry, Goethe-University Frankfurt, Frankfurt am Main, Germany
| | - Klaas M Pos
- Institute of Biochemistry, Goethe-University Frankfurt, Frankfurt am Main, Germany
| |
Collapse
|
|
40
|
Efflux Pumps of Burkholderia thailandensis Control the Permeability Barrier of the Outer Membrane. Antimicrob Agents Chemother 2019; 63:AAC.00956-19. [PMID: 31383661 DOI: 10.1128/aac.00956-19] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2019] [Accepted: 07/28/2019] [Indexed: 01/27/2023] Open
Abstract
Burkholderia comprises species that are significant biothreat agents and common contaminants of pharmaceutical production facilities. Their extreme antibiotic resistance affects all classes of antibiotics, including polycationic polymyxins and aminoglycosides. The major underlying mechanism is the presence of two permeability barriers, the outer membrane with modified lipid A moieties and active drug efflux pumps. The two barriers are thought to be mechanistically independent and act synergistically to reduce the intracellular concentrations of antibiotics. In this study, we analyzed the interplay between active efflux pumps and the permeability barrier of the outer membrane in Burkholderia thailandensis We found that three efflux pumps, AmrAB-OprA, BpeEF-OprC, and BpeAB-OprB, of B. thailandensis are expressed under standard laboratory conditions and provide protection against multiple antibiotics, including polycationic polymyxins. Our results further suggest that the inactivation of AmrAB-OprA or BpeAB-OprB potentiates the antibacterial activities of antibiotics not only by reducing their efflux, but also by increasing their uptake into cells. Mass spectrometry analyses showed that in efflux-deficient B. thailandensis cells, lipid A species modified with 4-amino-4-deoxy-l-aminoarabinose are significantly less abundant than in the parent strain. Taken together, our results suggest that changes in the outer membrane permeability due to alterations in lipid A structure could be contributing factors in antibiotic hypersusceptibilities of B. thailandensis cells lacking AmrAB-OprA and BpeAB-OprB efflux pumps.
Collapse
|
|
41
|
Abstract
Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this Review, we describe recent advances that have increased our understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria. Clinical and laboratory data indicate that efflux pumps function not only in the drug extrusion process but also in virulence and the adaptive responses that contribute to antimicrobial resistance during infection. The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities.
Collapse
|
|
42
|
Deletion of the major Escherichia coli multidrug transporter AcrB reveals transporter plasticity and redundancy in bacterial cells. PLoS One 2019; 14:e0218828. [PMID: 31251753 PMCID: PMC6599122 DOI: 10.1371/journal.pone.0218828] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2019] [Accepted: 06/11/2019] [Indexed: 12/28/2022] Open
Abstract
Multidrug Transporters (MDTs) are major contributors to the acquisition and maintenance of Antimicrobial Resistance (AMR), a growing public health threat of broad concern. Despite the large number of MDTs, the overwhelming majority of the studies performed thus far in Gram-negative bacteria emphasize the supremacy of the AcrAB-TolC complex. To unveil the potential role of other MDTs we studied the behavior of a null AcrB Escherichia coli strain when challenged with chloramphenicol, a bacteriostatic antibiotic. We found that such a strain developed an extremely high-level of resistance to chloramphenicol, cross resistance to quinolones and erythromycin and displayed high levels of expression of the single component MFS transporter MdfA and multiple TolC-dependent transporters. The results suggest that the high versatility of the whole ensemble of transporters, the bacterial Effluxome, is an essential part of a strategy of survival in everchanging, at times noxious, environments. The concept of a functional Effluxome presents an alternative to the existing paradigms in the field and provides novel targets for the search for inhibitors of transporters as adjuvants of existing antibiotics.
Collapse
|
|
43
|
Chetri S, Bhowmik D, Dhar D, Chakravarty A, Bhattacharjee A. Effect of concentration gradient carbapenem exposure on expression of bla NDM-1 and acrA in carbapenem resistant Escherichia coli. INFECTION GENETICS AND EVOLUTION 2019; 73:332-336. [PMID: 31170528 DOI: 10.1016/j.meegid.2019.05.024] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/25/2019] [Revised: 05/28/2019] [Accepted: 05/31/2019] [Indexed: 10/26/2022]
Abstract
Escherichia coli, one of the major pathogens, frequently exhibits carbapenem resistance. It would be of interest to investigate which of the mechanisms responds when a strain that carries both blaNDM-1 and over expressed AcrAB-TolC efflux pump system is exposed against carbapenem under differential concentration gradient stress. Four different sets of strains were used in the study; (i) Strain that have blaNDM-1 and over expressed AcrAB-TolC system (ii) Strain that harbour blaNDM-1 and express AcrAB-TolC at basal level (iii) the strain that is devoid of blaNDM-1 but having over expressed AcrAB-TolC systems and (iv) E. coli AG100A (ΔAcrAB) and E. coli HUE 1 (ΔAcrAB-TolC) where blaNDM-1was cloned. The Quantitative Real time PCR showed blaNDM-1 was over expressed under meropenem and imipenem stress irrespective of concentration gradient. In case of ertapenem, at lower concentration AcrA were over expressed whereas, at higher concentration blaNDM-1 showed elevated expression. A consistent elevated expression of AcrA and AcrB was observed against all carbapenems in the strains devoid of blaNDM-1 where as in case of the strain with basal level expression of AcrA, no significant over expression could be observed for blaNDM-1. In case of clones in group IV, expression of blaNDM-1 was elevated in the presence of carbapenem stress.
Collapse
Affiliation(s)
- Shiela Chetri
- Department of Microbiology, Assam University, Silchar, India
| | | | - Debadatta Dhar
- Department of Microbiology, Silchar Medical College and Hospital, Silchar, India
| | - Atanu Chakravarty
- Department of Microbiology, Silchar Medical College and Hospital, Silchar, India
| | | |
Collapse
|
|
44
|
Foong WE, Tam HK, Crames JJ, Averhoff B, Pos KM. The chloramphenicol/H+ antiporter CraA of Acinetobacter baumannii AYE reveals a broad substrate specificity. J Antimicrob Chemother 2019; 74:1192-1201. [DOI: 10.1093/jac/dkz024] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2018] [Revised: 11/23/2018] [Accepted: 01/08/2019] [Indexed: 12/15/2022] Open
Affiliation(s)
- Wuen Ee Foong
- Institute of Biochemistry, Goethe‐University Frankfurt, Max‐von‐Laue‐Str. 9, Frankfurt am Main, Germany
| | - Heng-Keat Tam
- Institute of Biochemistry, Goethe‐University Frankfurt, Max‐von‐Laue‐Str. 9, Frankfurt am Main, Germany
| | - Jan J Crames
- Institute of Biochemistry, Goethe‐University Frankfurt, Max‐von‐Laue‐Str. 9, Frankfurt am Main, Germany
| | - Beate Averhoff
- Department of Molecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, Goethe University Frankfurt, Frankfurt am Main, Germany
| | - Klaas M Pos
- Institute of Biochemistry, Goethe‐University Frankfurt, Max‐von‐Laue‐Str. 9, Frankfurt am Main, Germany
| |
Collapse
|
|
45
|
Türkel İ, Yıldırım T, Yazgan B, Bilgin M, Başbulut E. Relationship between antibiotic resistance, efflux pumps, and biofilm formation in extended-spectrum β-lactamase producing Klebsiella pneumoniae. J Chemother 2019; 30:354-363. [DOI: 10.1080/1120009x.2018.1521773] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Affiliation(s)
- İbrahim Türkel
- Department of Biology, Faculty of Arts and Sciences, Amasya University, Amasya, Turkey,
| | - Tuba Yıldırım
- Department of Biology, Faculty of Arts and Sciences, Amasya University, Amasya, Turkey,
- Central Research Laboratory, Amasya University, Amasya, Turkey,
| | - Burak Yazgan
- Central Research Laboratory, Amasya University, Amasya, Turkey,
- Department of Medical Sevices and Techniques, Amasya University Sabuncuoğlu Serefeddin Vocational Higher School of Health Services, Amasya, Turkey,
| | - Melek Bilgin
- Microbiology Laboratory, Education and Research Hospital, Samsun, Turkey
| | - Eşe Başbulut
- Microbiology Laboratory, Education and Research Hospital, Samsun, Turkey
| |
Collapse
|
|
46
|
Peterson E, Kaur P. Antibiotic Resistance Mechanisms in Bacteria: Relationships Between Resistance Determinants of Antibiotic Producers, Environmental Bacteria, and Clinical Pathogens. Front Microbiol 2018; 9:2928. [PMID: 30555448 PMCID: PMC6283892 DOI: 10.3389/fmicb.2018.02928] [Citation(s) in RCA: 503] [Impact Index Per Article: 71.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2018] [Accepted: 11/14/2018] [Indexed: 11/13/2022] Open
Abstract
Emergence of antibiotic resistant pathogenic bacteria poses a serious public health challenge worldwide. However, antibiotic resistance genes are not confined to the clinic; instead they are widely prevalent in different bacterial populations in the environment. Therefore, to understand development of antibiotic resistance in pathogens, we need to consider important reservoirs of resistance genes, which may include determinants that confer self-resistance in antibiotic producing soil bacteria and genes encoding intrinsic resistance mechanisms present in all or most non-producer environmental bacteria. While the presence of resistance determinants in soil and environmental bacteria does not pose a threat to human health, their mobilization to new hosts and their expression under different contexts, for example their transfer to plasmids and integrons in pathogenic bacteria, can translate into a problem of huge proportions, as discussed in this review. Selective pressure brought about by human activities further results in enrichment of such determinants in bacterial populations. Thus, there is an urgent need to understand distribution of resistance determinants in bacterial populations, elucidate resistance mechanisms, and determine environmental factors that promote their dissemination. This comprehensive review describes the major known self-resistance mechanisms found in producer soil bacteria of the genus Streptomyces and explores the relationships between resistance determinants found in producer soil bacteria, non-producer environmental bacteria, and clinical isolates. Specific examples highlighting potential pathways by which pathogenic clinical isolates might acquire these resistance determinants from soil and environmental bacteria are also discussed. Overall, this article provides a conceptual framework for understanding the complexity of the problem of emergence of antibiotic resistance in the clinic. Availability of such knowledge will allow researchers to build models for dissemination of resistance genes and for developing interventions to prevent recruitment of additional or novel genes into pathogens.
Collapse
Affiliation(s)
- Elizabeth Peterson
- Department of Biology, Georgia State University, Atlanta, GA, United States
| | - Parjit Kaur
- Department of Biology, Georgia State University, Atlanta, GA, United States
| |
Collapse
|
|
47
|
Multidrug-Resistant Acinetobacter baumannii Chloramphenicol Resistance Requires an Inner Membrane Permease. Antimicrob Agents Chemother 2018; 62:AAC.00513-18. [PMID: 29891596 DOI: 10.1128/aac.00513-18] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2018] [Accepted: 06/04/2018] [Indexed: 11/20/2022] Open
Abstract
Acinetobacter baumannii is a Gram-negative organism that is a cause of hospital-acquired multidrug-resistant (MDR) infections. A. baumannii has a unique cell surface compared to those of many other Gram-negative pathogens in that it can live without lipopolysaccharide (LPS) and it has a high content of cardiolipin in the outer membrane. Therefore, to better understand the cell envelope and mechanisms of MDR A. baumannii, we screened a transposon library for mutants with defective permeability barrier function, defined as a deficiency in the ability to exclude the phosphatase chromogenic substrate 5-bromo-4-chloro-3-indolylphosphate (XP). We identified multiple mutants with mutations in the ABUW_0982 gene, predicted to encode a permease broadly present in A. baumannii isolates with increased susceptibility to the ribosome-targeting antibiotic chloramphenicol (CHL). Moreover, compared to other known CHL resistance genes, such as chloramphenicol acyltransferase genes, we found that ABUW_0982 is the primary determinant of intrinsic CHL resistance in A. baumannii strain 5075 (Ab5075), an important isolate responsible for severe MDR infections in humans. Finally, studies measuring the efflux of chloramphenicol and expression of ABUW_0982 in CHL-susceptible Escherichia coli support the conclusion that ABUW_0982 encodes a single-component efflux protein with specificity for small, hydrophobic molecules, including CHL.
Collapse
|
|
48
|
Ramaswamy VK, Vargiu AV, Malloci G, Dreier J, Ruggerone P. Molecular Determinants of the Promiscuity of MexB and MexY Multidrug Transporters of Pseudomonas aeruginosa. Front Microbiol 2018; 9:1144. [PMID: 29910784 PMCID: PMC5992780 DOI: 10.3389/fmicb.2018.01144] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2018] [Accepted: 05/14/2018] [Indexed: 12/14/2022] Open
Abstract
Secondary multidrug transporters of the resistance-nodulation-cell division (RND) superfamily contribute crucially to antibiotic resistance in Gram-negative bacteria. Compared to the most studied transporter AcrB of Escherichia coli, little is known about the molecular determinants of distinct polyspecificities of the most important RND transporters MexB and MexY of Pseudomonas aeruginosa. In an effort to add knowledge on this topic, we performed an exhaustive atomic-level comparison of the main putative recognition sites (access and deep binding pockets) in these two Mex transporters. We identified an underlying link between some structural, chemical and dynamical features of the binding pockets and the physicochemical nature of the corresponding substrates recognized by either one or both pumps. In particular, mosaic-like lipophilic and electrostatic surfaces of the binding pockets provide for both proteins several multifunctional sites for diffuse binding of diverse substrates. Specific lipophilicity signatures of the weakly conserved deep pocket suggest a key role of this site as a selectivity filter as in Acr transporters. Finally, the different dynamics of the bottom-loop in MexB and MexY support its possible role in binding of large substrates. Our work represents the first comparative study of the major RND transporters in P. aeruginosa and also the first structure-based study of MexY, for which no experimental structure is available yet.
Collapse
Affiliation(s)
| | - Attilio V Vargiu
- Department of Physics, University of Cagliari, Monserrato, Italy
| | - Giuliano Malloci
- Department of Physics, University of Cagliari, Monserrato, Italy
| | - Jürg Dreier
- Basilea Pharmaceutica International Ltd., Basel, Switzerland
| | - Paolo Ruggerone
- Department of Physics, University of Cagliari, Monserrato, Italy
| |
Collapse
|
|
49
|
Neuberger A, Du D, Luisi BF. Structure and mechanism of bacterial tripartite efflux pumps. Res Microbiol 2018; 169:401-413. [PMID: 29787834 DOI: 10.1016/j.resmic.2018.05.003] [Citation(s) in RCA: 38] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2017] [Revised: 02/20/2018] [Accepted: 05/14/2018] [Indexed: 12/22/2022]
Abstract
Efflux pumps are membrane proteins which contribute to multi-drug resistance. In Gram-negative bacteria, some of these pumps form complex tripartite assemblies in association with an outer membrane channel and a periplasmic membrane fusion protein. These tripartite machineries span both membranes and the periplasmic space, and they extrude from the bacterium chemically diverse toxic substrates. In this chapter, we summarise current understanding of the structural architecture, functionality, and regulation of tripartite multi-drug efflux assemblies.
Collapse
Affiliation(s)
- Arthur Neuberger
- Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK
| | - Dijun Du
- Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK
| | - Ben F Luisi
- Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.
| |
Collapse
|
|
50
|
Lu WJ, Lin HJ, Janganan TK, Li CY, Chin WC, Bavro VN, Lin HTV. ATP-Binding Cassette Transporter VcaM from Vibrio cholerae is Dependent on the Outer Membrane Factor Family for Its Function. Int J Mol Sci 2018; 19:ijms19041000. [PMID: 29584668 PMCID: PMC5979437 DOI: 10.3390/ijms19041000] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2018] [Revised: 03/23/2018] [Accepted: 03/25/2018] [Indexed: 11/16/2022] Open
Abstract
Vibrio cholerae ATP-binding cassette transporter VcaM (V. cholerae ABC multidrug resistance pump) has previously been shown to confer resistance to a variety of medically important drugs. In this study, we set to analyse its properties both in vitro in detergent-solubilised state and in vivo to differentiate its dependency on auxiliary proteins for its function. We report the first detailed kinetic parameters of purified VcaM and the rate of phosphate (Pi) production. To determine the possible functional dependencies of VcaM on the tripartite efflux pumps we then utilized different E. coli strains lacking the principal secondary transporter AcrB (Acriflavine resistance protein), as well as cells lacking the outer membrane factor (OMF) TolC (Tolerance to colicins). Consistent with the ATPase function of VcaM we found it to be susceptible to sodium orthovanadate (NaOV), however, we also found a clear dependency of VcaM function on TolC. Inhibitors targeting secondary active transporters had no effects on either VcaM-conferred resistance or Hoechst 33342 accumulation, suggesting that VcaM might be capable of engaging with the TolC-channel without periplasmic mediation by additional transporters. Our findings are indicative of VcaM being capable of a one-step substrate translocation from cytosol to extracellular space utilising the TolC-channel, making it the only multidrug ABC-transporter outside of the MacB-family with demonstrable TolC-dependency.
Collapse
Affiliation(s)
- Wen-Jung Lu
- Department of Food Science, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung 202, Taiwan.
| | - Hsuan-Ju Lin
- Department of Food Science, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung 202, Taiwan.
| | - Thamarai K Janganan
- School of Life Sciences, University of Bedfordshire, University Square, Luton LU1 3JU, UK.
| | - Cheng-Yi Li
- Department of Food Science, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung 202, Taiwan.
| | - Wei-Chiang Chin
- Department of Food Science, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung 202, Taiwan.
| | - Vassiliy N Bavro
- School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK.
| | - Hong-Ting Victor Lin
- Department of Food Science, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung 202, Taiwan.
- Center of Excellence for the Oceans, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung 202, Taiwan.
| |
Collapse
|