• ARGs
• Escherichia coli
• antibiotic resistance genes
• antibiotics
• resistance mechanisms

• antibiotic resistance
• antibiotics
• bacteria
• crystal structure
• enzyme catalysis
• enzyme inhibitor
• enzyme kinetics
• molecular dynamics
• molecular evolution

• Humans
• Amino Acids/genetics
• Anti-Bacterial Agents/pharmacology
• Anti-Bacterial Agents/therapeutic use
• Bacterial Proteins/chemistry
• Bacterial Proteins/genetics
• Bacterial Proteins/metabolism
• beta-Lactamases/chemistry
• beta-Lactamases/genetics
• beta-Lactamases/metabolism
• Ceftazidime/pharmacology
• Klebsiella Infections/drug therapy
• Klebsiella Infections/microbiology
• Klebsiella pneumoniae/drug effects
• Klebsiella pneumoniae/genetics
• Drug Resistance, Bacterial/genetics
• Crystallography, X-Ray
• Catalytic Domain/genetics
• Protein Structure, Tertiary
• Models, Molecular

• P30 GM124169 NIGMS NIH HHS
• R01 AI032956 NIAID NIH HHS
• R37 AI032956 NIAID NIH HHS
• R56 AI032956 NIAID NIH HHS

• UTI
• antibiotic resistance genes
• antimicrobial resistance genes
• bacterial genetics
• molecular diagnostics
• multiplex PCR
• polymicrobial infections
• urinary tract infection

• bloodstream infection
• carbapenem-resistant Enterobacteriaceae
• carbapenemase-producing Enterobacteriaceae
• mortality

Infections with multidrug-resistant organisms among patients in intensive care units (ICUs) are associated with high mortality. Among multidrug-resistant organisms, carbapenem-resistant Enterobacterales (CRE) harbor important pathogens for healthcare-associated infections, including pneumonia, bacteremia, and urinary tract infections. Risk factors for CRE colonization include underlying comorbid conditions, prior antibiotics exposure, prior use of healthcare facilities, device use, and longer ICU stay. The mortality rate due to invasive CRE infection is 22%–49%, and CRE colonization is associated with an approximately 10-fold increased risk of CRE infection. Infection control measures include hand hygiene, contact precautions, minimizing the use of devices, and environmental control. Additionally, implementing active surveillance of CRE carriage should be considered in ICU settings.

• carbapenem
• carbapenem-resistant enterobacteriaceae
• drug resistance
• infection control
• intensive care units

The rapidly increasing prevalence of Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing bacteria has become a serious challenge to public health. Currently, the bla_KPC–2 gene is mainly disseminated through plasmids of different sizes and replicon types. However, the plasmids carrying the bla_KPC–2 gene have not been fully characterized. In this study, we report the complete genome sequences of two novel bla_KPC–2-harboring incompatibility group U (IncU) plasmids, pEC2341-KPC and pEC2547-KPC, from international high-risk clones of Escherichia coli isolated from Zhejiang, China. Two KPC-2-producing E. coli isolates (EC2341 and EC2547) were collected from clinical samples. Whole-genome sequencing (WGS) analysis indicated that EC2341 and EC2547 belonged to the ST410 and ST131 clones, respectively. S1-nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blot and conjugation experiments confirmed the presence of the bla_KPC–2 gene on the pEC2341-KPC plasmid and that this was a conjugative plasmid, while the bla_KPC–2 gene on the pEC2547-KPC plasmid was a non-conjugative plasmid. In addition, plasmid analysis further revealed that the two bla_KPC–2-harboring plasmids have a close evolutionary relationship. To the best of our knowledge, this is the first report of E. coli strains carrying the bla_KPC–2 gene on IncU plasmids. The emergence of the IncU-type bla_KPC–2-positive plasmid highlights further dissemination of bla_KPC–2 in Enterobacteriaceae. Therefore, effective measures should be taken immediately to prevent the spread of these bla_KPC–2–positive plasmids.

• E. coli
• IncU plasmid
• KPC-2
• high-risk clones
• whole genome sequencing

Plasmids can provide a selective advantage for microorganisms to survive and adapt to new environmental conditions. Plasmid-encoded traits, such as antimicrobial resistance (AMR) or virulence, impact the ecology and evolution of bacteria and can significantly influence the burden of infectious diseases. Insight about the identity and functions encoded on plasmids on the global scale are largely lacking. Here, we investigate the plasmidome of 24 samples (22 countries, 5 continents) from the global sewage surveillance project. We obtained 105-Gbp Oxford Nanopore and 167-Gbp Illumina NextSeq DNA sequences from plasmid DNA preparations and assembled 165,302 contigs (159,322 circular). Of these, 58,429 carried genes encoding for plasmid-related and 11,222 for virus/phage-related proteins. About 90% of the circular DNA elements did not have any similarity to known plasmids. Those that exhibited similarity had similarity to plasmids whose hosts were previously detected in these sewage samples (e.g., Acinetobacter, Escherichia, Moraxella, Enterobacter, Bacteroides, and Klebsiella). Some AMR classes were detected at a higher abundance in plasmidomes (e.g., macrolide-lincosamide-streptogramin B, macrolide, and quinolone) compared to the respective complex sewage samples. In addition to AMR genes, a range of functions were encoded on the candidate plasmids, including plasmid replication and maintenance, mobilization, and conjugation. In summary, we describe a laboratory and bioinformatics workflow for the recovery of plasmids and other potential extrachromosomal DNA elements from complex microbiomes. Moreover, the obtained data could provide further valuable insight into the ecology and evolution of microbiomes, knowledge about AMR transmission, and the discovery of novel functions.

IMPORTANCE This is, to the best of our knowledge, the first study to investigate plasmidomes at a global scale using long read sequencing from complex untreated domestic sewage. Previous metagenomic surveys have detected AMR genes in a variety of environments, including sewage. However, it is unknown whether the AMR genes were present on the microbial chromosome or located on extrachromosomal elements, such as plasmids. Using our approach, we recovered a large number of plasmids, of which most appear novel. We identified distinct AMR genes that were preferentially located on plasmids, potentially contributing to their transmissibility. Overall, plasmids are of great importance for the biology of microorganisms in their natural environments (free-living and host-associated), as well as for molecular biology and biotechnology. Plasmidome collections may therefore be valuable resources for the discovery of fundamental biological mechanisms and novel functions useful in a variety of contexts.

• Illumina NextSeq
• Oxford Nanopore
• Oxford Nanopore sequencing
• animals
• human
• microbiome
• plasmid DNA isolation
• plasmid reconstruction
• plasmids
• wastewater

• Klebsiella species
• antibiotics
• capsular polysaccharide
• complement system
• exopolysaccharide
• lipopolysaccharide
• multidrug resistance
• vaccine

• Amino Acid Sequence
• Animals
• Anti-Bacterial Agents/chemistry
• Anti-Bacterial Agents/pharmacology
• Biological Transport
• Complement System Proteins/immunology
• Drug Resistance, Bacterial/drug effects
• Extracellular Space/metabolism
• Host-Pathogen Interactions/immunology
• Humans
• Klebsiella/drug effects
• Klebsiella/immunology
• Klebsiella/metabolism
• Klebsiella Infections/metabolism
• Klebsiella Infections/microbiology
• Lipopolysaccharides/chemistry
• Lipopolysaccharides/metabolism
• Models, Molecular
• Molecular Conformation
• Molecular Structure
• Protein Binding
• Sugars/chemistry
• Sugars/metabolism
• Vaccines/immunology

Treatment for nosocomial bloodstream infections (BSI) caused by multidrug-resistant (MDR) Gram-negative bacteria (GNB) is challenging. Rising antimicrobial resistance, especially in extended spectrum beta-lactamase production, inadvertently increases empiric carbapenem consumption. Three antipseudomonal carbapenems (imipenem, meropenem [MER], and doripenem [DOR]) are available commercially against MDR GNB in Singapore. The study aims to determine the most optimal empiric carbapenem dosing regimens (CDR) and evaluate their cost-effectiveness for GNB-BSI in the face of increasing MDR GNB.

Carbapenem minimum inhibitory concentrations (MICs) were generated for non-repeat GNB-BSI obtained in 2013–2014 from two hospitals. Monte Carlo simulations were used to assess the cumulative fraction of response (CFR) of various CDRs using the percentage of time above MIC for 40% (%T > MIC of 40%) as the pharmacokinetic (PK)–pharmacodynamic (PD) parameter for efficacy. Carbapenem costs were based on patient antibiotic costs. Antibiotic cost-effectiveness was calculated as total daily drug cost/CFR.

A total of 1,140 bloodstream isolates were collected. They comprised 116 Acinetobacter baumannii, 237 Pseudomonas aeruginosa, and 787 Enterobacteriaceae. All CDRs achieved ~40, ~80, and ≥90% CFRs against A. baumannii, P. aeruginosa, and Enterobacteriaceae, respectively. Against P. aeruginosa, MER 2 g every 8 h infused over 3 h and DOR 1 g every 8 h infused over 4 h achieved CFRs 84 and 81%, respectively. Against Enterobacteriaceae, the cost of MER 2 g every 8 h infused over 3 h was the lowest among the three carbapenems at $0.40/percentage of CFR.

This study demonstrates the utility of PK–PD modeling to formulate the optimal selection of a cost-effective empiric CDR in antibiotics guidelines and formulary inclusion. The findings support the selection of high MER doses of prolonged infusions as empiric coverage for GNB-BSI in our institutions.

• Gram-negative bacteria
• bloodstream infections
• empiric carbapenem regimens
• multidrug resistant

• antibiotic resistance
• epidemic plasmids
• extremely drug resistant (XDR)
• high-risk clones
• mobile genetic elements (MGEs)
• resistome

• Enterobacteriaceae
• ISKpn8
• ST131
• antimicrobial resistance
• blaKPC
• dissemination
• genetic environment

• Avibactam
• Ceftazidime
• Cephalosporin
• Pharmacology

• CRKP
• Carbapenemases
• Colistin
• K. pneumoniae
• KPC-KP

Carbapenemase-producing Enterobacteriaceae have been steadily spreading worldwide during the last decade. Nine patients were identified prospectively and were followed during their hospitalization course to identify the epidemiology, clinical profiles and outcomes. These patients had one or more cultures positive for a CRE isolate, contributing to a total of eleven positive cultures from various sites without including duplicates of isolates obtained from the same site. Isolates from these patients included five Klebseilla pneumoniae, three Escherichia coli, and one Enterobacter aerogenes. Five isolates were grown from blood cultures, three from wound cultures, one from urine cultures, one from respiratory cultures and one from an abscess collection. Five survived the hospital course. The other five patients died due to severe sepsis, septic shock or multi-organ failure. Of the nine isolates of CRE identified for which molecular analysis were available, four K. pneumonia were confirmed as blaNDM and one as OXA-48. For the purpose of controlling the spread of CRE in our institution, we recommend considering active surveillance cultures and screening patients transferred from other hospitals or coming from highly endemic settings at admission for these organisms.

• Carbapenem-resistant
• Enterobacteriaceae

• Carbapenem resistance
• Carbapenemase
• Escherichia coli
• Klebsiella pneumoniae carbapenemase
• OXA-48

• bacteriuria
• carbapenem-resistant Enterobacteriaceae
• carbapenem-resistant Klebsiella pneumoniae
• kidney transplantation
• multidrug resistance

• carbapenem-resistant Enterobacteriaceae
• carbapenemases
• carbapenems
• resistant infections
• treatment

Carbapenems are potent β-lactams with activity against extended-spectrum cephalosporinases and β-lactamases. These antibiotics, derived from thienamycn, a carbapenem produced by the environmental bacterium Streptomyces cattleya, were initially used as last-resort treatments for severe Gram-negative bacterial infections presenting resistance to most β-lactams but have become an empirical option in countries with high prevalence of Extended Spectrum β-lactamase-producing bacterial infections. Imipenem, the first commercially available carbapenem, was approved for clinical use in 1985. Since then, a wide variety of carbapenem-resistant bacteria has appeared, primarily Enterobacteriaceae such as Escherichia coli or Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa and Acinetobacter baumannii, presenting different resistance mechanisms. The most relevant mechanism is the production of carbapenem-hydrolyzing β-lactamases, also known as carbapenemases. These enzymes also inactivate all known β-lactams, and some of these enzymes can be acquired through horizontal gene transfer. Moreover, plasmids, transposons and integrons harboring these genes typically carry other resistance determinants, rendering the recipient bacteria resistant to almost all currently used antimicrobials, as is the case for K. pneumoniae carbapenemase - or New Delhi metallo-β-lactamases-type enzymes. The recent advent of these enzymes in the health landscape presents a serious challenge. First, the emergence of carbapenemases limits the currently available treatment options; second, these enzymes pose a risk to patients, as some studies have demonstrated high mortality associated with carbapenemase-producing bacterial infections; and third, these circumstances require an extra cost to sanitary systems, which are particularly cumbersome in developing countries. Therefore, emphasis should be placed on the early detection of these enzymes, the prevention of the spread of carbapenemase-producing bacteria and the development of new drugs resistant to carbapenemase hydrolysis.

• Carbapenemases
• Origin
• Epidemiology
• Evolution
• Multi-Drug-resistant bacteria

• Antimicrobial resistance
• Carbapenem-resistant Klebsiella pneumoniae
• Intensive care unit
• Renal disease

• IncFIIK
• carbapenemases
• resistance mechanisms

The emergence of Klebsiella pneumoniae carbapenemases (KPCs) producing bacteria has become a significant global public health challenge while the optimal treatment remains undefined. We performed a systematic review of published studies and reports of treatment outcomes of KPC infections using MEDLINE (2001–2011). Articles or cases were excluded if one of the following was fulfilled: no individual patient data provided, no treatment regimen specified, no treatment outcome specified, report of colonization, or greater than three antibiotics were used to treat the KPC infection. Data extracted included patient demographics, site of infection, organism, KPC subtype, antimicrobial therapy directed at KPC-infection, and treatment outcome. Statistical analysis was performed in an exploratory manner. A total of 38 articles comprising 105 cases were included in the analysis. The majority of infections were due to K. pneumoniae (89%). The most common site of infection was blood (52%), followed by respiratory (30%), and urine (10%). Forty-nine (47%) cases received monotherapy and 56 (53%) cases received combination therapy directed at the KPC-infection. Significantly more treatment failures were seen in cases that received monotherapy compared to cases who received combination therapy (49% vs 25%; p= 0.01). Respiratory infections were associated with higher rates of treatment failure with monotherapy compared to combination therapy (67% vs 29% p= 0.03). Polymyxin monotherapy was associated with higher treatment failure rates compared to polymyxin-based combination therapy (73% vs 29%; p= 0.02); similarly, higher treatment failure rates were seen with carbapenem monotherapy compared to carbapenem-based combination therapy (60% vs 26%; p= 0.03). Overall treatment failure rates were not significantly different in the three most common antibiotic-class combinations: polymyxin plus carbapenem, polymyxin plus tigecycline, polymyxin plus aminoglycoside (30%, 29%, and 25% respectively; p=0.6). In conclusion, combination therapy is recommended for the treatment of KPC infections; however, which combination of antimicrobial agents needs to be established in future prospective clinical trials.

Klebsiella pneumoniae carbapenemases (KPCs) confer resistance to nearly all β-lactams. This broad-spectrum drug resistance mechanism has rapidly spread in the United States and is reportedly increasing elsewhere in the world. Thus, the emergence of KPC resistance is a major threat to global health. This article reviews the epidemiology and provides an overview of the dissemination of KPC-producing organisms.

• beta-lactam resistance
• carbapenemase
• drug resistance
• epidemiology
• treatment failure

Carbapenem resistant Enterobacteriaceae is a worldwide threat, with increasing prevalence in many countries. Restricted usage of higher end antibiotics, especially carbapenem is of great importance in tackling these super bugs. Purpose of this retrospective study was to analyse the impact of antibiotic stewardship activities on the prevalence of carbapenem resistant Enterobacteriaceae in our hospital.

In the first Quarter of 2009, average usage of carbapenem group of antibiotics was 955 vials a month while in 2010, the usage dropped to 745 vials per month. Carbapenem resistant E.coli rate dropped from 3.7% in 2009 to 1.6% in 2010 and Klebsiella rate reduced from 6% in 2009 to 3.6% in 2010.

Strict antibiotic stewardship strategies in conjunction with good infection control practices are useful in restricting higher end antibiotic usage and reducing the prevalence of carbapenem resistant Enterobacteriaceae.

• Aged
• Aged, 80 and over
• Anti-Bacterial Agents/pharmacology
• Bacterial Proteins/biosynthesis
• Brazil
• Female
• Humans
• Klebsiella Infections/microbiology
• Klebsiella pneumoniae/drug effects
• Klebsiella pneumoniae/enzymology
• Klebsiella pneumoniae/genetics
• Male
• Microbial Sensitivity Tests
• Middle Aged
• beta-Lactamases/biosynthesis