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Background: Carbapenemase-producing, carbapenem-resistant Pseudomonas aeruginosa (CP-CRPA) are extensively drug-resistant bacteria. We investigated the source of a multistate CP-CRPA outbreak., Methods: Cases were defined as a US patient's first isolation of P. aeruginosa sequence type 1203 with carbapenemase gene blaVIM-80 and cephalosporinase gene blaGES-9 from any specimen source collected and reported to the Centers for Disease Control and Prevention during 1 January 2022-15 May 2023. We conducted a 1:1 matched case-control study at the post-acute care facility with the most cases, assessed exposures associated with case status for all case-patients, and tested products for bacterial contamination., Results: We identified 81 case-patients from 18 states, 27 of whom were identified through surveillance cultures. Four (7%) of 54 case-patients with clinical cultures died within 30 days of culture collection, and 4 (22%) of 18 with eye infections underwent enucleation. In the case-control study, case-patients had increased odds of receiving artificial tears versus controls (crude matched OR, 5.0; 95% CI, 1.1-22.8). Overall, artificial tears use was reported by 61 (87%) of 70 case-patients with information; 43 (77%) of 56 case-patients with brand information reported use of Brand A, an imported, preservative-free, over-the-counter (OTC) product. Bacteria isolated from opened and unopened bottles of Brand A were genetically related to patient isolates. Food and Drug Administration inspection of the manufacturing plant identified likely sources of contamination., Conclusions: A manufactured medical product serving as the vehicle for carbapenemase-producing organisms is unprecedented in the United States. The clinical impacts from this outbreak underscore the need for improved requirements for US OTC product importers., Competing Interests: Potential conflicts of interest. M. M. and L. E. S. report support for attending meetings or travel from the APHL CSTE. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2024.)

Background: Bacteroides fragilis, an anaerobic gut bacterium and opportunistic pathogen, comprises two genetically divergent groups (or divisions) at the species level. Differences exist both in the core and accessory genomes and the beta-lactamase genes, with the cephalosporinase gene cepA represented only in division I and the carbapenemase gene cfiA only in division II., Methods: Multidrug resistance in a clinical B. fragilis strain was examined by whole-genome sequencing., Results: Strain CNM20200260 carried the antimicrobial resistance genes cepA, cfiA2, ant(6'), erm(F), mef(En2), est(T), tet(Q) and cat(A), along with 82-Phe mutation in gyrA (together with 47 amino acid changes in gyrA/B and parC/parE). bexA/B and other efflux pump genes were also observed. None of the detected insertion sequences was located upstream of cfiA2. The genome-based taxonomy coefficients (average nucleotide identity, DNA-DNA hybridization similarity and difference in genomic G + C%) with respect to genomes of the strains of B. fragilis division II and the novel species Bacteroides hominis (both cfiA-positive) met the criteria for CNM20200260 to belong to either species (>95%, >70% and <1%, respectively). No such similarity was seen with type strain NCTC 9343 or the representative genome FDAARGOS 1225 of B. fragilis (division I, cfiA-negative). Strain CNM20200260 harboured four out of nine Kyoto Encyclopedia of Genes and Genomes orthologues defined for division I and one of two defined for division II., Conclusions: This is the first description of the co-occurrence of cepA and cfiA in a Bacteroides strain, confirming the complexity of the taxonomy of this species., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Enterobacter cloacae starred different pioneer studies that enabled the development of a widely accepted model for the peptidoglycan metabolism-linked regulation of intrinsic class C cephalosporinases, highly conserved in different Gram-negatives. However, some mechanistic and fitness/virulence-related aspects of E. cloacae choromosomal AmpC-dependent resistance are not completely understood. The present study including knockout mutants, β-lactamase cloning, gene expression analysis, characterization of resistance phenotypes, and the Galleria mellonella infection model fills these gaps demonstrating that: (i) AmpC enzyme does not show any collateral activity impacting fitness/virulence; (ii) AmpC hyperproduction mediated by ampD inactivation does not entail any biological cost; (iii) alteration of peptidoglycan recycling alone or combined with AmpC hyperproduction causes no attenuation of E. cloacae virulence in contrast to other species; (iv) derepression of E. cloacae AmpC does not follow a stepwise dynamics linked to the sequential inactivation of AmpD amidase homologues as happens in Pseudomonas aeruginosa; (v) the enigmatic additional putative AmpC-type β-lactamase generally present in E. cloacae does not contribute to the classical cephalosporinase hyperproduction-based resistance, having a negligible impact on phenotypes even when hyperproduced from multicopy vector. This study reveals interesting particularities in the chromosomal AmpC-related behavior of E. cloacae that complete the knowledge on this top resistance mechanism., (© 2024. The Author(s).)

Class C Acinetobacter -derived cephalosporinases (ADCs) represent an important target for inhibition in the multidrug-resistant pathogen Acinetobacter baumannii . Many ADC variants have emerged, and characterization of their structural and functional differences is essential. Equally as important is the development of compounds that inhibit all prevalent ADCs despite these differences. The boronic acid transition state inhibitor, MB076 , a novel heterocyclic triazole with improved plasma stability, was synthesized and inhibits seven different ADC β-lactamase variants with K i values <1 μM. MB076 acted synergistically in combination with multiple cephalosporins to restore susceptibility. ADC variants containing an alanine duplication in the Ω-loop, specifically ADC-33, exhibited increased activity for larger cephalosporins, such as ceftazidime, cefiderocol, and ceftolozane. X-ray crystal structures of ADC variants in this study provide a structural context for substrate profile differences and show that the inhibitor adopts a similar conformation in all ADC variants, despite small changes near their active sites.

Traditional studies on the evolution of antibiotic resistance development use approaches that can range from laboratory-based experimental studies, to epidemiological surveillance, to sequencing of clinical isolates. However, evolutionary trajectories also depend on the environment in which selection takes place, compelling the need to more deeply investigate the impact of environmental complexities and their dynamics over time. Herein, we explored the within-patient adaptive long-term evolution of a Pseudomonas aeruginosa hypermutator lineage in the airways of a cystic fibrosis (CF) patient by performing a chronological tracking of mutations that occurred in different subpopulations; our results demonstrated parallel evolution events in the chromosomally encoded class C β-lactamase ( bla PDC ). These multiple mutations within bla PDC shaped diverse coexisting alleles, whose frequency dynamics responded to the changing antibiotic selective pressures for more than 26 years of chronic infection. Importantly, the combination of the cumulative mutations in bla PDC provided structural and functional protein changes that resulted in a continuous enhancement of its catalytic efficiency and high level of cephalosporin resistance. This evolution was linked to the persistent treatment with ceftazidime, which we demonstrated selected for variants with robust catalytic activity against this expanded-spectrum cephalosporin. A "gain of function" of collateral resistance toward ceftolozane, a more recently introduced cephalosporin that was not prescribed to this patient, was also observed, and the biochemical basis of this cross-resistance phenomenon was elucidated. This work unveils the evolutionary trajectories paved by bacteria toward a multidrug-resistant phenotype, driven by decades of antibiotic treatment in the natural CF environmental setting. IMPORTANCE Antibiotics are becoming increasingly ineffective to treat bacterial infections. It has been consequently predicted that infectious diseases will become the biggest challenge to human health in the near future. Pseudomonas aeruginosa is considered a paradigm in antimicrobial resistance as it exploits intrinsic and acquired resistance mechanisms to resist virtually all antibiotics known. AmpC β-lactamase is the main mechanism driving resistance in this notorious pathogen to β-lactams, one of the most widely used classes of antibiotics for cystic fibrosis infections. Here, we focus on the β-lactamase gene as a model resistance determinant and unveil the trajectory P. aeruginosa undertakes on the path toward a multidrug-resistant phenotype during the course of two and a half decades of chronic infection in the airways of a cystic fibrosis patient. Integrating genetic and biochemical studies in the natural environment where evolution occurs, we provide a unique perspective on this challenging landscape, addressing fundamental molecular mechanisms of resistance.

Resistance to antipseudomonal penicillins and cephalosporins is often driven by the overproduction of the intrinsic β-lactamase AmpC. However, OXA-10-family β-lactamases are a rich source of resistance in Pseudomonas aeruginosa. OXA β-lactamases have a propensity for mutation that leads to extended spectrum cephalosporinase and carbapenemase activity. In this study, we identified isolates from a subclade of the multidrug-resistant (MDR) high risk P. aeruginosa clonal complex CC446 with a resistance to ceftazidime. A genomic analysis revealed that these isolates harbored a plasmid containing a novel allele of bla OXA-10 , named bla OXA-935 , which was predicted to produce an OXA-10 variant with two amino acid substitutions: an aspartic acid instead of a glycine at position 157 and a serine instead of a phenylalanine at position 153. The G157D mutation, present in OXA-14, is associated with the resistance of P. aeruginosa to ceftazidime. Compared to OXA-14, OXA-935 showed increased catalytic efficiency for ceftazidime. The deletion of bla OXA-935 restored the sensitivity to ceftazidime, and susceptibility profiling of P. aeruginosa laboratory strains expressing bla OXA-935 revealed that OXA-935 conferred ceftazidime resistance. To better understand the impacts of the variant amino acids, we determined the crystal structures of OXA-14 and OXA-935. Compared to OXA-14, the F153S mutation in OXA-935 conferred increased flexibility in the omega (Ω) loop. Amino acid changes that confer extended spectrum cephalosporinase activity to OXA-10-family β-lactamases are concerning, given the rising reliance on novel β-lactam/β-lactamase inhibitor combinations, such as ceftolozane-tazobactam and ceftazidime-avibactam, to treat MDR P. aeruginosa infections.

Wildlife play a role in the acquisition, maintenance, and dissemination of antimicrobial resistance (AMR). This is especially true at the human-domestic animal-wildlife interface, like urbanized areas, where interactions occur that can promote the cross-over of AMR bacteria and genes. We conducted a 2-year fecal surveillance ( n  = 783) of a white-tailed deer (WTD) herd from an urban park system in Ohio to identify and characterize cephalosporin-resistant and carbapenemase-producing bacteria using selective enrichment. Using generalized linear mixed models we found that older (OR = 2.3, P  < 0.001), male (OR = 1.8, P  = 0.001) deer from urbanized habitats (OR = 1.4, P  = 0.001) were more likely to harbor extended-spectrum cephalosporin-resistant Enterobacterales. In addition, we isolated two carbapenemase-producing Enterobacterales (CPE), a Klebsiella quasipneumoniae harboring bla KPC-2 and an Escherichia coli harboring bla NDM-5 , from two deer from urban habitats. The genetic landscape of the plasmid carrying bla KPC-2 was unique, not clustering with other reported plasmids encoding KPC-2, and only sharing 78% of its sequence with its nearest match. While the plasmid carrying bla NDM-5 shared sequence similarity with other reported plasmids encoding NDM-5, the intact IS26 mobile genetic elements surrounding multiple drug resistant regions, including the bla NDM-5 , has been reported infrequently. Both carbapenemase genes were successfully conjugated to a J53 recipient conferring a carbapenem-resistant phenotype. Our findings highlight that urban environments play a significant role on the transmission of AMR bacteria and genes to wildlife and suggest WTD may play a role in the dissemination of clinically and epidemiologically relevant antimicrobial resistant bacteria. IMPORTANCE The role of wildlife in the spread of antimicrobial resistance is not fully characterized. Some wildlife, including white-tailed deer (WTD), can thrive in suburban and urban environments. This may result in the exchange of antimicrobial resistant bacteria and resistance genes between humans and wildlife, and lead to their spread in the environment. We found that WTD living in an urban park system carried antimicrobial resistant bacteria that were important to human health and resistant to antibiotics used to treat serious bacterial infections. This included two deer that carried bacteria resistant to carbapenem antibiotics which are critically important for treatment of life-threatening infections. These two bacteria had the ability to transfer their AMR resistance genes to other bacteria, making them a threat to public health. Our results suggest that WTD may contribute to the spread of antimicrobial resistant bacteria in the environment.

Aims: Polymyxin-based combination therapy is often used to treat carbapenem-resistant Acinetobacter baumannii (A. baumannii) infections. Although sulbactam is intrinsically active against A. baumannii, few studies have investigated colistin/sulbactam combinations against carbapenem-resistant A. baumannii., Methods: Whole genome sequencing was undertaken on eight carbapenem-resistant (colistin-susceptible) isolates of A. baumannii from Chinese patients. Bacterial killing of colistin and sulbactam, alone and in combination, was examined with checkerboard (all isolates) and static and dynamic time-kill studies (three isolates). In the dynamic studies, antibiotics were administered in various clinically-relevant dosing regimens that mimicked patient pharmacokinetics., Results: The eight isolates consisted of ST195, ST191 and ST208 belonging to clonal complex 208, which is the most epidemic clonal type of A. baumannii globally. All isolates possessed Acinetobacter-derived cephalosporinase (ADC-61 or ADC-78) and seven of eight isolates contained the carbapenem-resistance gene bla OXA-23 . The colistin/sulbactam combination was synergistic against two of eight isolates in checkerboard studies. In time-kill studies, rapid bacterial killing of ca. 3-6 log 10 CFU/mL was observed with colistin monotherapy, followed by steady regrowth. Sulbactam monotherapy was generally ineffective. Substantially enhanced bacterial killing was observed with colistin/sulbactam combinations in both static and dynamic models, especially with the higher sulbactam concentration (2 g) and/or longer sulbactam infusion time (2 hours) in the dynamic model., Conclusions: This study was the first to use a pharmacokinetics/pharmacodynamics model to investigate synergistic activity of colistin/sulbactam combinations against A. baumannii. It showed that clinically-relevant dosing regimens of colistin combined with sulbactam may substantially improve bacterial killing of multidrug-resistant and carbapenem-resistant A. baumannii., (Copyright © 2021 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Boronic acid transition state inhibitors (BATSIs) are known reversible covalent inhibitors of serine β-lactamases. The selectivity and high potency of specific BATSIs bearing an amide side chain mimicking the β-lactam's amide side chain are an established and recognized synthetic strategy. Herein, we describe a new class of BATSIs where the amide group is replaced by a bioisostere triazole; these compounds were designed as molecular probes. To this end, a library of 26 α-triazolylmethaneboronic acids was synthesized and tested against the clinically concerning Acinetobacter -derived cephalosporinase, ADC-7. In steady state analyses, these compounds demonstrated K i values ranging from 90 nM to 38 μM (±10%). Five compounds were crystallized in complex with ADC-7 β-lactamase, and all the crystal structures reveal the triazole is in the putative amide binding site, thus confirming the triazole-amide bioisosterism. The easy synthetic access of these new inhibitors as prototype scaffolds allows the insertion of a wide range of chemical groups able to explore the enzyme binding site and provides insights on the importance of specific residues in recognition and catalysis. The best inhibitor identified, compound 6q ( K i 90 nM), places a tolyl group near Arg340, making favorable cation-π interactions. Notably, the structure of 6q does not resemble the natural substrate of the β-lactamase yet displays a pronounced inhibition activity, in addition to lowering the minimum inhibitory concentration (MIC) of ceftazidime against three bacterial strains expressing class C β-lactamases. In summary, these observations validate the α-triazolylboronic acids as a promising template for further inhibitor design.

Yersinia enterocolitica is an enteric bacterium which can cause severe gastroenteritis. Beta-lactams are the most widely used antibiotics against Y. enterocolitica. Y. enterocolitica produces two chromosomal β-lactamases, BlaA and BlaB. BlaB is an Ambler Class C inducible broad spectrum cephlaosporinase which showed differential enzyme activity in different biotypes of Y. enterocolitica. The expression of blaB is mainly regulated by ampR- the transcriptional regulator and, ampD - which helps in peptidoglycan recycling. The aim of this study was to identify and characterize genetic determinants underlying differential enzyme activity of BlaB in Y. enterocolitica biotypes 1 A, IB, 2 and 4. Thus, ampR, blaB and ampD were PCR-amplified and modeled in silico. The intercistronic region containing promoters of ampR and blaB was also investigated. Our results indicated that blaB was more inducible in biotypes 2 and 4, than in biotypes 1 A and 1B. Superimposition of in silico modeled proteins suggested that variations in amino acid sequences of AmpR, BlaB and AmpD were not responsible for hyper-production of BlaB in biotypes 2 and 4. Analysis of promoter regions of ampR and blaB revealed variations at -30, -37 and -58 positions from blaB transcription start site. Studies on relative expression levels of blaB in different biotypes by qRT-PCR indicated that nucleotide variations at these positions might contribute to a higher enzyme activity of BlaB in biotypes 2 and 4. However, this is a preliminary study and further studies including more strains of each biotype are required to strengthen our findings. Nevertheless, to the best of our knowledge, this is the first study which has investigated the genetic determinants underlying differential inducible production of BlaB in different biotypes of Y. enterocolitica.

A wide range of intrinsic Acinetobacter-derived cephalosporinases (ADC) along with other carbapenemases has now been detected in Acinetobacter baumannii leaving clinicians with few treatment options. The present study reports the spread of ADC-30 co-producing KPC-2 along with other β-lactamases among carbapenem resistant A. baumannii strains obtained from ICU patients in two Indian hospitals. Primer extension analysis revealed higher transcript level of the ADC gene when induced with cefoxitin at 8 μg/ml (170 fold), ceftriaxone at 8 μg/ml (136 fold), ceftazidime at 4 μg/ml (65 fold), cefepime at 8 μg/ml (77 fold) and aztreonam at 8 μg/ml (21 fold) when compared with the basal level without antibiotic pressure. Slight increase in expression of bla ADC-30 when induced with imipenem and meropenem at 0.25 μg/ml (3 and 6 fold) was observed and may help in conferring resistance to carbapenem. MLST analysis revealed the circulation of A. baumannii sequence types ST188, ST386, ST583 and ST390 in these hospitals., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Objectives: This study investigates the frequency and characteristics of carbapenemase-producing Escherichia coli/Klebsiella pneumoniae (CPE/K) and extended-spectrum cephalosporinase-producing E. coli/K. pneumoniae (ESCE/K) in healthy humans and livestock in rural Cambodia. Additionally, household practices as risk factors for faecal carriage of ESCE/K are identified., Methods: Faecal samples were obtained from 307 humans and 285 livestock including large ruminants, pigs and poultry living in 100 households in rural Cambodia in 2011. Each household was interviewed, and multilevel logistic model determined associations between household practices/meat consumption and faecal carriage of ESCE/K. CPE and ESCE/K were detected and further screened for colistin resistance genes., Results: CPE/K isolates harbouring bla OXA-48 were identified in two humans. The community carriage of ESCE/K was 20% in humans and 23% in livestock. The same ESBL genes: bla CTX-M-15 , bla CTX-M-14 , bla CTX-M-27 , bla CTX-M-55 , bla SHV-2 , bla SHV-12 , bla SHV-28 ; AmpC genes: bla CMY-2 , bla CMY-42, bla DHA-1 ; and colistin resistance genes: mcr-1-like and mcr-3-like were detected in humans and livestock. ESCE/K was frequently detected in women, young children, pigs and poultry, which are groups in close contact. The practice of burning or burying meat waste and not collecting animal manure indoors and outdoors daily were identified as risk factors for faecal carriage of ESCE/K., Conclusions: Faecal carriage of E. coli and K. pneumoniae harbouring extended-spectrum cephalosporinase genes are common in the Cambodian community, especially in women and young children. Exposure to animal manure and slaughter products are risk factors for intestinal colonization of ESCE/K in humans., (© 2019 The Authors. Zoonoses and Public Health Published by Blackwell Verlag GmbH.)

Yersinia enterocolitica biotype 1A strains are emerging pathogens, frequently isolated from clinical samples across the globe. Y. enterocolitica strains produce two chromosomal β-lactamases, BlaA and BlaB. BlaA is a constitutively expressed, Ambler class A, penicillinase, whereas BlaB is Ambler class C-type, inducible cephalosporinase. An earlier study from our laboratory indicated that instead of BlaB, Y. enterocolitica biotype 1A produced a "BlaB-like" enzyme. The objective of this work was to study the molecular characteristics of "Bla-B like" β-lactamases produced by biotype 1A strains to discern their similarity with AmpC-type β-lactamases and the basis of varied levels of minimum inhibitory concentration (MIC) to cefotaxime. Thus, the promoters and blaB genes were investigated in four strains of biotype 1A. Three-dimensional structures of the "BlaB-like" enzymes were modeled, and docked in silico with cefotaxime to understand how specific substitutions in gene sequences affect antibiotic susceptibilities. Our results indicated that all the reported key catalytic residues were present in variants of "Bla-B-like" enzymes, discerned in biotype 1A strains, but at different positions. Molecular docking of enzyme variants with cefotaxime revealed that lesser was the number of the H-binding residues with cefotaxime in a strain, lower was the MIC of cefotaxime in that strain. To the best of our knowledge, this is the first study in which the molecular characteristics and enzymatic interactions of "BlaB-like" cephalosporinases of Y. enterocolitica biotype 1A strains have been reported.

Yersinia enterocolitica strains produce two chromosomal β‑lactamases, BlaA - a constitutively produced penicillinase, and BlaB - an inducible "AmpC-type" cephalosporinase. As in other members of Enterobacteriaceae, expression of ampC in Y. enterocolitica is regulated by the genes - ampR and ampD. The ampR encodes a transcriptional regulator which represses the expression of ampC and, ampD encodes a cytoplasmic N‑acetyl‑anhydromuramyl‑l‑alanine amidase which participates in recycling of peptidoglycan. Exposure of bacteria to antibiotics like imipenem and cefoxitin results in generation and accumulation of large quantities of muropeptides in cytoplasm which is beyond the recycling capability of AmpD. These muropeptides bind to AmpR, converting it into an activator of ampC expression (ampC de-repression). Earlier studies from our laboratory indicated that instead of BlaB, Y. enterocolitica biotype 1A strains produced a "BlaB-like" enzyme which was non-heterogeneous and showed a differential expression when induced with imipenem. The detection of "BlaB-like" cephalosporinase which was also induced differentially in Y. enterocolitica biotype 1A strains presented an opportunity to discern newer mechanisms, if any, which may underlie inducible expression of "AmpC-type" cephalosporinases. Thus, the objective of the present study was to understand the role of ampR and ampD in regulating differential expression of "BlaB-like" cephalosporinases in biotype 1A strains. Analysis of promoters and amino acid sequences of AmpR revealed that these were conserved in all strains of biotype 1A. Analysis of AmpD amino acid sequences revealed that five variants of AmpD were present which did not contribute to hyper-inducible production of "BlaB-like" enzyme. In-silico prediction of the mRNA secondary structures of ampD revealed significant differences, which might have affected the rate of translation of ampD and accumulation of un-recycled muropeptides inside the cell leading to hyper production of "BlaB-like" cephalosporinases in some Y. enterocolitica biotype 1A strains. The findings provide newer insights to our understanding of the mechanisms underlying regulation of expression of "AmpC-type" β‑lactamases., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In Enterobacter cloacae complex (ECC), the overproduction of the chromosome-encoded cephalosporinase (cAmpC) associated with decreased outer membrane permeability may result in carbapenem resistance. In this study, we have characterized ACT-28, a cAmpC with weak carbapenemase activity, from a single Enterobacter kobei lineage. ECC clinical isolates were characterized by whole-genome sequencing (WGS), susceptibility testing, and MIC, and carbapenemase activity was monitored using diverse carbapenem hydrolysis methods. ACT-28 steady-state kinetic parameters were determined. Among 1,039 non-carbapenemase-producing ECC isolates with decreased susceptibility to carbapenems received in 2016-2017 at the French National Reference Center for antibiotic resistance, only 8 had a positive carbapenemase detection test (Carba NP). These eight ECC isolates were resistant to broad-spectrum cephalosporins due to AmpC derepression, showed decreased susceptibility to carbapenems, and were categorized as carbapenemase-producing Enterobacteriaceae (CPE) according to several carbapenemase detection assays. WGS identified a single clone of E. kobei ST125 expressing only its cAmpC, ACT-28. The bla ACT-28 gene was expressed in a wild-type and in a porin-deficient Escherichia coli background and compared to the bla ACT-1 gene. Detection of carbapenemase activity was positive only for E. coli expressing the bla ACT-28 gene. Kinetic parameters of purified ACT-28 revealed a slightly increased imipenem hydrolysis compared to that of ACT-1. In silico porin analysis revealed the presence of a peculiar OmpC-like protein specific to E. kobei ST125 that could impair carbapenem influx into the periplasm and thus enhance carbapenem-resistance caused by ACT-28. We described a widespread lineage of E. kobei ST125 producing ACT-28, with weak carbapenemase activity that can lead to false-positive detection by several biochemical and phenotypic diagnostic tests., (Copyright © 2019 American Society for Microbiology.)

The chromogenic βLACTA™ test was evaluated to detect ceftazidime resistance in P. aeruginosa isolates from patients with cystic fibrosis. Best results were obtained after one hour of incubation with low sensitivity (64.1%), high specificity (98.3%), and negative and positive predictive values of 80.3% and 96.2%, respectively., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A clinical isolate of Citrobacter freundii (JA99) obtained from a bile culture of a Taiwanese patient was found to produce a plasmid-encoded β-lactamase conferring resistance to oxyimino-cephalosporins and cephamycins. Resistance arising from production of the β-lactamase could be transferred by conjugation with an IncW plasmid (pJA99) into Escherichia coli J53. The substrate and inhibition profiles of this enzyme resembled that of an AmpC β-lactamase. The resistance gene of pJA99, cloned and expressed in E. coli DH5α, was shown to contain an open reading frame showing 92% amino acid identity with the plasmid-encoded enzyme CFE-1 of E. coli KU6400. DNA sequence analysis also identified a gene upstream of ampC in pJA99 whose sequence was 95.0% identical to the ampR gene from E. coli KU6400. In addition, orf1, the fumarate operon (frdABCD), blc, lolB and repB surrounding the ampR-ampC genes in C. freundii were identified. This DNA fragment was absent in other Citrobacter spp. Therefore, we describe a new plasmid-encoded AmpC β-lactamase, named CFE-2. This study highlights the emergence of broad-spectrum cephalosporin resistance in C. freundii owing to a new type of AmpC β-lactamase., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Pseudomonas aeruginosa is responsible for chronic respiratory tract colonisation and acute exacerbations in cystic fibrosis (CF) patients. This Gram-negative bacterium often develops multidrug resistance, which represents a therapeutic challenge. The objective of this study was to characterise the phenotypic and genetic β-lactam resistance traits of P. aeruginosa strains isolated from CF patients at Grenoble Alpes University Hospital (Grenoble, France). The susceptibility to β-lactam compounds of 123 P. aeruginosa strains collected from the lower respiratory tract of 45 CF patients between 2010-2014 was evaluated. Genetic analyses focused on characterisation of the presence of carbapenemase- and extended-spectrum β-lactamases (ESBL)-encoding genes as well as alterations in the oprD gene encoding the OprD porin. Among the 123 P. aeruginosa strains evaluated, 25 were susceptible to both ceftazidime (CAZ) and imipenem (IPM), 9 only to IPM and 36 only to CAZ; 53 strains were resistant to both drugs. CAZ resistance could be reverted by cloxacillin in 29 strains, indicating overproduction of cephalosporinase. Genetic analyses performed for 79 P. aeruginosa strains revealed no ESBL- or carbapenemases-encoding genes. Among the 74 IPM-resistant strains, 42 (56.8%) displayed major alterations in the OprD protein sequence. This study shows that in this CF patient cohort, cephalosporinase overproduction and OprD alterations were the main resistance mechanisms of P. aeruginosa to CAZ and IPM, respectively. No genes coding for ESBLs or carbapenemases were detected, but monitoring of the emergence of such resistance genes in CF patients is warranted owing to their ability to rapidly spread by horizontal gene transfer., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Introduction: The wide distribution of extended-spectrum β-lactamase (ESBL) producing Shigella spp., along with the emergence of fluoroquinolone resistant isolates, is a serious threat to public health, posing a new challenge for the effective treatment of shigellosis. The purpose of this study was to determine the level of antimicrobial resistance, the presence of genes encoding resistance to cephalosporins, and plasmid-mediated quinolone resistance (PMQR) among the clinical isolates of Shigella spp. in Iran., Materials and Methods: A total of 142 Shigella isolates were collected from different parts of Iran. All of the cephalosporin resistant Shigella strains were selected based on ESBL and AmpC production. The presence of PMQR regions was assessed in ciprofloxacin-resistant isolates, and genetic relatedness in the isolates was determined., Results: Seventy-eight Shigella isolates were found to be resistant to extended-spectrum cephalosporin (ESC). The bla CTX-M15 was the most prevalent cephalosporinase. Four ESBL-producing isolates were also resistant to ciprofloxacin. Among the PMQR regions, aac(6')-lb-cr gene was the most prevalent, as it was seen in 83.3% of the ciprofloxacin resistant isolates, while qnrA was positive in 16.7%. Clonal relatedness showed a limited variety of clones was responsible for Shigella infection in the region studied., Conclusion: Overall, our findings indicated that a large number of ESBL producing Shigella spp. were mediated mainly by bla CTX-M15 . This study is the first report on ciprofloxacin-resistant ESBL-producing Shigella isolates from patients in Iran.

Recent research has demonstrated that selection for antibiotic resistance occurs at very low antibiotic concentrations in single-species experiments, but the relevance of these findings when species are embedded in complex microbial communities is unclear. We show that the strength of selection for naturally occurring resistance alleles in a complex community remains constant from low subinhibitory to above clinically relevant concentrations. Selection increases with antibiotic concentration before reaching a plateau where selection remains constant over a 2-order-magnitude concentration range. This is likely to be due to cross protection of the susceptible bacteria in the community following rapid extracellular antibiotic degradation by the resistant population, shown experimentally through a combination of chemical quantification and bacterial growth experiments. Metagenome and 16S rRNA analyses of sewage-derived bacterial communities evolved under cefotaxime exposure show preferential enrichment for bla CTX-M genes over all other beta-lactamase genes, as well as positive selection and co-selection for antibiotic resistant, opportunistic pathogens. These findings have far-reaching implications for our understanding of the evolution of antibiotic resistance, by challenging the long-standing assumption that selection occurs in a dose-dependent manner. IMPORTANCE Antibiotic resistance is one of the greatest global issues facing society. Still, comparatively little is known about selection for resistance at very low antibiotic concentrations. We show that the strength of selection for clinically important resistance genes within a complex bacterial community can remain constant across a large antibiotic concentration range (wide selective space). Therefore, largely understudied ecological compartments could be just as important as clinical environments for selection of antibiotic resistance., (Copyright © 2018 Murray et al.)

The aim of this review is to provide an update on the plasmids mediating DHA-1 cephalosporinase in Klebsiella pneumoniae. These plasmids have been mainly found in this bacterium but not only. The first was isolated from Salmonella sp. in France in the early 1990s. They are currently reported worldwide. Bla DHA-1 beta-lactamase gene is usually co-expressed with many other antibiotic resistance genes such as extended-spectrum β-lactamases (bla CTX-M -, bla SHV -types), oxacillinases (bla OXA-1, bla OXA-30 ), penicillinases (bla TEM -type), carbapenemases (bla OXA48 , bla KPC-2 ), aminoglycosides (aacA, aadA, armA), fluoroquinolones (qnrB4, aac6'-1b-cr), and sulfonamide (sul1) resistance genes. Plasmids carrying DHA-1 cephalosporinase have different sizes (22 to 313 kb), belong to diverse groups of incompatibility (R, L/M, FII(k), FIB, A/C2, HI2, HIB), and are self-transferable or not. The multidrug resistance region consists of a mosaic structure composed of resistance genes, insertion sequences, composite transposon, and integrons.

Escherichia coli PBP5, a DD-carboxypeptidase (DD-CPase), helps in maintaining cell shape and intrinsic β-lactam resistance. Though PBP5 does not have β-lactamase activity under physiological pH, it has a common but shorter Ω-like loop resembling class A β-lactamases. However, such Ω-like loop lacks the key glutamic acid residue that is present in β-lactamases. It is speculated that β-lactamases and DD-CPases might have undergone divergent evolution leading to distinct enzymes with different substrate specificities and functions indicating the versatility of the Ω-loops. Nonetheless, direct experimental evidence favoring the idea is insufficient. Here, aiming to investigate the effect of introducing a glutamic acid residue in the PBP5 Ω-like loop, we substituted A184 to E to create PBP5&#95;A184E. Expression of PBP5&#95;A184E in E. coli ∆PBP5 mutant elevates the β-lactam resistance, especially for cephalosporins. However, like PBP5, PBP5&#95;A184E has the ability to complement the aberrantly shaped E. coli septuple PBP mutant indicating an unaffected in vivo DD-CPase activity. Biochemical and bioinformatics analyses have substantiated the dual enzyme nature of the mutated enzyme possessing both DD-CPase and β-lactamase activities. Therefore, substitution of A184 to E of Ω-like loop alone can introduce the cephalosporinase activity in E. coli PBP5 supporting the phenomenon of a single amino acid polymorphism.

Metallo-β-lactamases (MBLs) are the major group of carbapenemases produced by bacterial pathogens. The design of MBL inhibitors has been limited by, among other issues, incomplete knowledge about how these enzymes modulate substrate recognition. While most MBLs are broad-spectrum enzymes, B2 MBLs are exclusive carbapenemases. This narrower substrate profile has been attributed to a sequence insertion present in B2 enzymes that limits accessibility to the active site. In this work, we evaluate the role of sequence insertions naturally occurring in the B2 enzyme Sfh-I and in the broad-spectrum B1 enzyme SPM-1. We engineered a chimeric protein in which the sequence insertion of SPM-1 was replaced by the one present in Sfh-I. The chimeric variant is a selective cephalosporinase, revealing that the substrate profile of MBLs can be further tuned depending on the protein context. These results also show that the stable scaffold of MBLs allows a modular engineering much richer than the one observed in nature., (Copyright © 2018 American Society for Microbiology.)

The exchange of mobile genomic islands (MGIs) between microorganisms is often mediated by phages, which may provide benefits to the phage's host. The present study started with the identification of Enterobacter cloacae, Klebsiella pneumoniae and Escherichia coli isolates with exceptional cephalosporin and carbapenem resistance phenotypes from patients in a neonatal ward. To identify possible molecular connections between these isolates and their β-lactam resistance phenotypes, the respective bacterial genome sequences were compared. This unveiled the existence of a family of ancient MGIs that were probably exchanged before the species E. cloacae, K. pneumoniae and E. coli emerged from their common ancestry. A representative MGI from E. cloacae was named MIR17-GI, because it harbors the novel β-lactamase gene variant bla MIR17 . Importantly, our observations show that the MIR17-GI-like MGIs harbor genes associated with high-level resistance to cephalosporins. Among them, MIR17-GI stands out because MIR17 also displays carbapenemase activity. As shown by mass spectrometry, the MIR17 carbapenemase is among the most abundantly expressed proteins of the respective E. cloacae isolate. Further, we show that MIR17-GI-like islands are associated with integrated P4-like prophages. This implicates phages in the spread of cephalosporin and carbapenem resistance amongst Enterobacteriaceae. The discovery of an ancient family of MGIs, mediating the spread of cephalosporinase and carbapenemase genes, is of high clinical relevance, because high-level cephalosporin and carbapenem resistance have serious implications for the treatment of patients with enterobacteriaceal infections.

Cephalosporinases, which are naturally present in some enterobacterial species, can be mobilized by transposons, migrate to plasmids, and spread into other species such as Escherichia coli. The aim of this study was to characterize genes responsible for the production of extended-spectrum β-lactamases (ESBL) in E. coli isolates from urinary origin isolated in two hospitals in Senegal. Thus, a fortuitous discovery of plasmidic cephalosporinase in two isolates was noted. One of the isolates produced dha-1 associated with ESBL CTX-M-14, the other produced cmy-2, ESBL CTXM-15, tem-1 penicillinase, and oxa-1. This confirms the circulation of multidrug-resistant bacteria producing plasmidic cephalosporinase in Senegal. However, a large study is needed to better understand the prevalence and the nature of the genes involved.

The molecular recognition and binding interaction of beta-lactamase II from Bacillus cereus (Bc II) with penicillin V (PV) and sulbactam (Sul) at 277 K were studied by spectroscopic analysis and molecular docking. The results showed that a non-fluorescence static complex was separately formed between Bc II and two ligands, the molecular ratio of Bc II to PV or Sul was both 1:1 in the binding and the binding constants were 2.00 × 10 6 and 3.98 × 10 5 (L/mol), respectively. The negative free energy changes and apparent activation energies indicated that both the binding processes were spontaneous. Molecular docking showed that in the binding process, the whole Sul molecule entered into the binding pocket of Bc II while only part of the whole PV molecule entered into the pocket due to a long side chain, and electrostatic interactions were the major contribution to the binding processes. In addition, a weak conformational change of Bc II was also observed in the molecular recognition and binding process of Bc II with PV or Sul. This study may provide some valuable information for exploring the recognition and binding of proteins with ligands in the binding process and for the design of novel super-antibiotics., (Copyright © 2017 John Wiley & Sons, Ltd.)

Background: Treatment alternatives for DHA-1 harboring strains are challenging as it confers resistance to broad spectrum cephalosporins and may further limit treatment option when expressed at higher levels. Therefore, this study was designed to know the prevalence of DHA genes and analyse the transcription level of DHA-1 against different β-lactam stress., Methods: Screening of AmpC β-lactamase phenotypically by modified three dimensional extract method followed by Antimicrobial Susceptibility and MIC determination. Genotyping screening of β-lactamase genes was performed by PCR assay followed by their sequencing. The bla DHA-1 transcriptional response was evaluated under different cephalosporin stress by RT PCR. Transferability of bla DHA gene was performed by transformation and conjugation and plasmid incompatibility typing, DNA fingerprinting by enterobacterial repetitive intergenic consensus sequences PCR., Results: 16 DHA-1 genes were screened positive from 176 Escherichia coli isolates and primer extension analysis showed a significant increase in DHA-1 mRNA transcription in response to cefotaxime at 8 µg/ml (6.99 × 10 2 fold), ceftriaxone at 2 µg/ml (2.63 × 10 3 fold), ceftazidime at 8 µg/ml (7.06 × 10 3 fold) and cefoxitin at 4 µg/ml (3.60 × 10 4 fold) when compared with untreated strain. These transcription data were found significant when analyzed statistically using one way ANOVA. Four different ESBL genes were detected in 10 isolates which include CTX-M (n = 6), SHV (n = 4), TEM (n = 3) and OXA-10 (n = 1), whereas, carbapenemase gene (NDM) was detected only in one isolate. Other plasmid mediated AmpC β-lactamases CIT (n = 9), EBC (n = 2) were detected in nine isolates. All DHA-1 genes detected were encoded in plasmid and incompatibility typing from the transformants indicated that the plasmid encoding bla DHA-1 was carried mostly by the FIA and L/M Inc group., Conclusion: This study demonstrates the prevalence of DHA-1 gene in this region and highlights high transcription of DHA-1 when induced with different β-lactam antibiotics. Therefore, cephalosporin treatment must be restricted for the patients infected with pathogen expressing this resistance determinant.

Very little is known about the occurrence and various types of extended-spectrum β-lactamase (ESBL), AmpC and carbapenemase in Iran. The aims of this study were to determine the prevalence of ESBLs, AmpCs and carbapenemase genes among Enterobacteriaceae in Azerbaijan and to characterize the genetic composition of the detected genes. A total of 307 Enterobacteriaceae isolates, recovered from five medical centres, were screened for ESBL, AmpC and carbapenemase activities by the disc diffusion method and phenotypic confirmatory tests. The 162 selected strains (third-generation cephalosporins, cefoxitin- or carbapenem-resistant strains with positive or negative phenotypic confirmatory tests) were selected for multiplex PCR screening for β-lactamase genes, and detected genes were confirmed by sequencing. Of 162 isolates, 156 harboured 1 to 6 β-lactamase genes of 41 types. The most prevalent genes were blaTEM-1 (29.9 %), followed by blaCTX-M-15 (25.7 %). Plasmid-mediated AmpC was detected in 66 strains (21.5 %) alone or in combination with other genes. Carbapenemase-encoding genes were detected in 18 strains (5.8 %) of 27 carbapenem-non-susceptible isolates including 11, 7, 3 and 1 cases of blaOXA-48, blaNDM-1, blaKPC-2 and blaKPC-3 genes, respectively. Interestingly, 148 (94.8 %) of 156 strains with any β-lactamase gene were found to have a multidrug-resistant pattern. The rate of resistance to β-lactams and multidrug-resistant Enterobacteriaceae is high in Azerbaijan. All positive strains for carbapenemase genes were resistant to all β-lactams. The present study reveals the high occurrence of CTX-M-type ESBLs followed by TEM and SHV variants among Enterobacteriaceae isolates. East Azerbaijan seems to be an alarming focus for OXA-48, NDM-1 and KPC dissemination.

Extended-spectrum-cephalosporin-resistant Enterobacteriaceae are a public health concern due to limited treatment options. Here, we report on the occurrence and the molecular characteristics of extended-spectrum-cephalosporin-resistant Enterobacteriaceae recovered from wild birds (kelp gulls). Our results revealed kelp gulls as a reservoir of various extended-spectrum cephalosporinase genes associated with different genetic platforms. In addition, we report for the first time the presence of a known epidemic clone of Salmonella enterica serotype Heidelberg (JF6X01.0326/XbaI.1966) among wild birds., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Resistance to β-lactams is considered one of the major global problems and recently it became the most frequently studied topic in the area of antimicrobial resistance. The study was focused on phenotypic and genetic characterisation of commensal Escherichia coli (E. coli), including those producing cephalosporinases, isolated from gut flora of healthy slaughter animals. E. coli were cultured simultaneously on MacConkey agar (MCA) and cefotaxime supplemented MCA. The isolates were confirmed with ONPG and indol tube tests as well as PCR targeting uspA gene. Microbroth dilution method was applied for determination of Minimal Inhibitory Concentrations and interpreted according to EUCAST epidemiological cut-off values. Cephalosporin resistance phenotypes were defined by E-tests (BioMerieux) and relevant gene amplicons from selected strains were sequenced. A total of 298 E. coli isolates with cephalosporin resistance (ESC) found in 99 ones, were obtained from 318 cloacal or rectal swabs deriving from broilers, layers, turkeys, pigs and cattle. Both extended spectrum β-lactamase (ESBL) and ampC-cephalosporinase resistance phenotypes were noted in all tested animal species but cattle. At least one of the analysed genes was identified in 90 out of 99 cephalosporin-resistant isolates: bla TEM (n=44), bla CMY (n=38), bla CTX-M (n=33) and bla SHV (n=12). None of the phenotypes was identified in nine isolates. Sequencing of PCR products showed occurrence of ESBL-genes: bla CTX-M-1/-61 , bla SHV-12 , bla TEM-1,-52/-92,-135 and ampC-gene bla CMY-2 . They were located on numerous and diverse plasmids and resistance transferability was proved by electroporation of bla SHV-12 and bla CTX-M-1/-61 located on X1 plasmids. Detection of cephalosporin resistant E. coli confirms the existence of resistance genes reservoir in farm animals and their possible spread (i.e. via IncX1 plasmids) to other bacteria including human and animal pathogens. The identified genetic background indicates on ecological aspects of selection and dissemination of cephalosporin resistance in E. coli isolated from food-producing animals rather than its potential role for public health threats., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Klebsiella pneumoniae is responsible for a wide range of infections, including urinary tract infections, pneumonia, bacteremia, and liver abscesses. In addition to susceptible clinical isolates involved in nosocomial infections, multidrug-resistant (MDR) and hypervirulent (hvKP) strains have evolved separately in distinct clonal groups. The rapid geographic spread of these isolates is of particular concern. However, we still know little about the virulence of K. pneumoniae except for hvKP, whose secrets are beginning to be revealed. The treatment of K. pneumoniae infections is threatened by the emergence of antimicrobial resistance. The dissemination of resistance is associated with genetic mobile elements, such as plasmids that may also carry virulence determinants. A proficient pathogen should be virulent, resistant to antibiotics, and epidemic. However, the interplay between resistance and virulence is poorly understood. Here, we review current knowledge on the topic.

OXA-48-like carbapenemases have only recently emerged in Europe. OXA-162 is a rare OXA-48 variant usually coexpressed with extended-spectrum β-lactamases. Here, we report the identification of the first OXA-162 carbapenemase-producing Klebsiella pneumoniae isolates, which coexpressed an AmpC cephalosporinase (DHA-1), retrieved from a patient in Greece. They belonged to a single sequence type (ST11) and caused the first documented community-onset urinary tract infections attributable to an OXA-48-like-producing Enterobacteriaceae strain., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The emergence of carbapenem resistance among Pseudomonas aeruginosa is an increasing problem in many parts of the world. In particular, metallo-β-lactamases (MBLs) and AmpC β- lactamases are responsible for high-level resistance to carbapenem and cephalosporin. We studied the diversity and frequency of β-lactamases and characterized chromosomal AmpC β- lactamase from carbapenem-resistant P. aeruginosa isolates. Sixty-one carbapenem-resistant P. aeruginosa isolates were collected from patients in a tertiary hospital in Daejeon, Korea, from January 2011 to June 2014. Minimum inhibitory concentrations (MICs) of four antimicrobial agents were determined using the agar-dilution method. Polymerase chain reaction and sequencing were used to identify the various β-lactamase genes, class 1 integrons, and chromosomally encoded and plasmid-mediated ampC genes. In addition, the epidemiological relationship was investigated by multilocus sequence typing. Among 61 carbapenem-resistant P. aeruginosa isolates, 25 isolates (41.0%) were MBL producers. Additionally, 30 isolates producing PDC (Pseudomonas-derived cephalosporinase)-2 were highly resistant to ceftazidime (MIC50 = 256 μg/ml) and cefepime (MIC50 = 256 μg/ml). Of all the PDC variants, 25 isolates harboring MBL genes showed high levels of cephalosporin and carbapenem resistance, whereas 36 isolates that did not harbor MBL genes revealed relatively low-level resistance (ceftazidime, p < 0.001; cefepime, p < 0.001; imipenem, p = 0.003; meropenem, p < 0.001). The coexistence of MBLs and AmpC β-lactamases suggests that these may be important contributing factors for cephalosporin and carbapenem resistance. Therefore, efficient detection and intervention to control drug resistance are necessary to prevent the emergence of P. aeruginosa possessing this combination of β-lactamases.

Extended-spectrum cephalosporinase resistance is currently the fastest emerging antimicrobial resistance problem worldwide; however, evidence documenting the effect of potential risk factors is limited. The main objective of this study was to investigate the effect of using third and fourth generation cephalosporins on the occurrence of extended-spectrum cephalosporinase-producing Escherichia coli (ESC-Ec) in Danish pig herds. Conventional, integrated, medium to large herds were selected based on information from the Danish Central Husbandry Register and two groups were formed based on the use of third and fourth generation cephalosporins within a specified period, namely, 20 herds with no cephalosporin use (non-exposed) and 19 herds with frequent use (exposed). Data on prescribed antimicrobials were obtained from the National database (VetStat). Management data were obtained through a questionnaire. At the herd level, three pooled faecal samples were collected from sows with their piglets (farrowing pens), weaners, and finishers. ESC-Ec were then identified using selective enrichment. Because several of the herds only had a low number of weaners and/or finishers, analysis was only performed on samples from the farrowing pens. Logistic regression showed a significant effect of using cephalosporins-III/IV on the occurrence of ESC-Ec in the farrowing pens, even when adjusted for use of other antimicrobials 1 year prior to sampling. No confounding effect was identified in relation to management data. The relative risk ESC-Ec in exposed compared to non-exposed was 4.7 (95% confidence interval 2.0-11.5), confirming that regular use of cephalosporins-III/IV was a significant risk factor for the occurrence of ESC-Ec., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objectives: To identify β-lactamase genes in gut commensal Bacteroides species and to assess the impact of these enzymes, when carried by outer membrane vesicles (OMVs), in protecting enteric pathogens and commensals., Methods: A deletion mutant of the putative class A β-lactamase gene (locus tag BT&#95;4507) found in the genome of the human commensal Bacteroides thetaiotaomicron was constructed and a phenotypic analysis performed. A phylogenetic tree was built from an alignment of nine Bacteroides cephalosporinase protein sequences, using the maximum likelihood method. The rate of cefotaxime degradation after incubation with OMVs produced by different Bacteroides species was quantified using a disc susceptibility test. The resistance of Salmonella Typhimurium and Bifidobacterium breve to cefotaxime in liquid culture in the presence of B. thetaiotaomicron OMVs was evaluated by measuring bacterial growth., Results: The B. thetaiotaomicron BT&#95;4507 gene encodes a β-lactamase related to the CepA cephalosporinase of Bacteroides fragilis. OMVs produced by B. thetaiotaomicron and several other Bacteroides species, except Bacteroides ovatus, carried surface-associated β-lactamases that could degrade cefotaxime. β-Lactamase-harbouring OMVs from B. thetaiotaomicron protected Salmonella Typhimurium and B. breve from an otherwise lethal dose of cefotaxime., Conclusions: The production of membrane vesicles carrying surface-associated β-lactamases by Bacteroides species, which constitute a major part of the human colonic microbiota, may protect commensal bacteria and enteric pathogens, such as Salmonella Typhimurium, against β-lactam antibiotics., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Antimicrobial resistance in Acinetobacter baumannii is a growing public health concern and an important pathogen in nosocomial infections. We investigated the genes involved in resistance to carbapenems and cephalosporins in clinical A. baumannii isolates from a tertiary medical centre in Malaysia. A. baumannii was isolated from 167 clinical specimens and identified by sequencing of the 16S rRNA and rpoB genes. The MIC for imipenem, meropenem, ceftazidime and cefepime were determined by the E-test method. The presence of carbapenemase and cephalosporinase genes was investigated by PCR. The isolates were predominantly nonsusceptible to carbapenems and cephalosporins (>70 %) with high MIC values. ISAba1 was detected in all carbapenem-nonsusceptible A. baumannii harbouring the blaOXA-23-like gene. The presence of blaOXA-51-like and ISAba1 upstream of blaOXA-51 was not associated with nonsusceptibility to carbapenems. A. baumannii isolates harbouring ISAba1-blaADC (85.8 %) were significantly associated with nonsusceptibility to cephalosporins (P<0.0001). However, ISAba1-blaADC was not detected in a minority (<10 %) of the isolates which were nonsusceptible to cephalosporins. The acquired OXA-23 enzymes were responsible for nonsusceptibility to carbapenems in our clinical A. baumannii isolates and warrant continuous surveillance to prevent further dissemination of this antibiotic resistance gene. The presence of ISAba1 upstream of the blaADC was a determinant for cephalosporin resistance. However, the absence of this ISAba1-blaADC in some of the isolates may suggest other resistance mechanisms and need further investigation., (© 2015 The Authors.)

β-Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. Much of this resistance to cephalosporins derives from the expression of the class C β-lactamase enzymes, known as Acinetobacter-derived cephalosporinases (ADCs). Currently, β-lactamase inhibitors are structurally similar to β-lactam substrates and are not effective inactivators of this class C cephalosporinase. Herein, two boronic acid transition state inhibitors (BATSIs S02030 and SM23) that are chemically distinct from β-lactams were designed and tested for inhibition of ADC enzymes. BATSIs SM23 and S02030 bind with high affinity to ADC-7, a chromosomal cephalosporinase from Acinetobacter baumannii (Ki = 21.1 ± 1.9 nM and 44.5 ± 2.2 nM, respectively). The X-ray crystal structures of ADC-7 were determined in both the apo form (1.73 Å resolution) and in complex with S02030 (2.0 Å resolution). In the complex, S02030 makes several canonical interactions: the O1 oxygen of S02030 is bound in the oxyanion hole, and the R1 amide group makes key interactions with conserved residues Asn152 and Gln120. In addition, the carboxylate group of the inhibitor is meant to mimic the C3/C4 carboxylate found in β-lactams. The C3/C4 carboxylate recognition site in class C enzymes is comprised of Asn346 and Arg349 (AmpC numbering), and these residues are conserved in ADC-7. Interestingly, in the ADC-7/S02030 complex, the inhibitor carboxylate group is observed to interact with Arg340, a residue that distinguishes ADC-7 from the related class C enzyme AmpC. A thermodynamic analysis suggests that ΔH driven compounds may be optimized to generate new lead agents. The ADC-7/BATSI complex provides insight into recognition of non-β-lactam inhibitors by ADC enzymes and offers a starting point for the structure-based optimization of this class of novel β-lactamase inhibitors against a key resistance target.

A population analysis of 103 multidrug-resistant Pseudomonas aeruginosa isolates from Croatian hospitals was performed. Twelve sequence types (STs) were identified, with a predominance of international clones ST235 (serotype O11 [41%]), ST111 (serotype O12 [15%]), and ST132 (serotype O6 [11%]). Overexpression of the natural AmpC cephalosporinase was common (42%), but only a few ST235 or ST111 isolates produced VIM-1 or VIM-2 metallo-β-lactamases or PER-1 or GES-7 extended-spectrum β-lactamases., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Seventeen Klebsiella pneumoniae isolates producing DHA-1 β-lactamase were collected in an intensive care unit between 2006 and 2010. Molecular analysis revealed the predominance of ST48 and ST1263 clones of K. pneumoniae and the spread of DHA-1-encoding plasmids belonging to incompatibility group IncL/M or IncHI2., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The presence of multidrug-resistant bacterial pathogens in the environment poses a serious threat to public health. The opportunistic Acinetobacter spp. are among the most prevalent causes of nosocomial infections. Here, we performed complete genome sequencing of the Acinetobacter calcoaceticus strain XM1570, which was originally cultivated from the sputum of a patient diagnosed with pneumonia in Xiamen in 2010. We identified carbapenem resistance associated gene bla(NDM-1) located on a 47.3-kb plasmid. Three methods--natural reproduction, sodium dodecyl sulfate treatment and nalidixic acid treatment--were used to eliminate the bla(NDM-1)-encoding plasmid, which achieved elimination rates of 3.32% (10/301), 83.78% (278/332), and 84.17% (298/354), respectively. Plasmid elimination dramatically increased antibiotic sensitivity, reducing the minimum bacteriostatic concentration of meropenem from 256 µg/ml in the clinical strain to 0.125 µg/ml in the plasmid-eliminated strain. Conjugation transfer assays showed that the bla(NDM-1)-containing plasmid could be transferred into Escherichia coli DH5α:pBR322 in vitro as well as in vivo in mice. The bla(NDM-1) genetic environment was in accordance with that of other bla(NDM-1) genes identified from India, Japan, and Hong-Kong. The multilocus sequence type of the isolate was identified as ST-70. Two novel genes encoding intrinsic OXA and ADC were identified and named as OXA-417 and ADC-72. The finding of bla(NDM-1) in species like A. calcoaceticus demonstrates the wide spread of this gene in gram-negative bacteria which is possible by conjugative plasmid transfer. The results of this study may help in the development of a treatment strategy for controlling NDM-1 bacterial infection and transmission.

In the community, close contacts between humans and dogs may promote the transfer of extended-spectrum beta-lactamase/plasmidic AmpC cephalosporinase (ESBL/pAmpC) genes. Large-scale prevalence studies on ESBL/pAmpC carriage in dogs are rare, and data on ESBL/pAmpC plasmids are even more limited. Here, a considerable rate of 18.5% ESBL/pAmpC carriers was found among 368 unrelated healthy dogs in Paris, France. This prevalence is much higher than the one found in healthy humans in the same city (6%) but close to that recently reported in dogs in China (24.5%). All isolates were identified as Escherichia coli, except one Salmonella enterica and one Klebsiella pneumoniae isolate. The sequence type 131 (ST131) clone was rare (2/73 isolates). Interestingly, two plasmids (blaCTX-M-1/IncI1/ST3 and blaCMY-2/IncI1/ST2) were unexpectedly highly predominant, raising the question of their successful spread. Considering that CTX-M-1 was recently found to be equally as abundant as CTX-M-15 in healthy Parisian subjects, the question of dogs being a CTX-M-1 reservoir for humans is open. Such a high prevalence of the blaCMY-2/IncI1/ST2 plasmid may result from the use of cephalexin in veterinary medicine, as previously demonstrated experimentally. In all, our study points out healthy urban dogs as a potential source of ESBL/pAmpC genes that can further disseminate to the human community., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Objectives: This study investigated the occurrence of extended spectrum cephalosporinase (ESC)-producing Escherichia coli in a broiler production with no cephalosporin use and a low use of antimicrobials in general. Furthermore, it investigated whether the current consumption of aminopenicillins selects for ESC-producing E. coli and whether certain clones or plasmids spread from imported parent flocks to the meat., Materials and Methods: ESC-producing E. coli was isolated using MacConkey broth with 1 mg/L of ceftriaxone. ESC genes were identified using polymerase chain reaction and sequencing. Isolates with blaCMY-2 were subtyped by pulsed-field gel electrophoresis (PFGE), phylotyping, and antimicrobial susceptibility testing. Selected isolates were used as donors in filter-mating experiments, multilocus sequence typing (MLST), and plasmid replicons were typed. Aminopenicillin use at the farm (not flock) level was obtained from VetStat, a database for mandatory registration of veterinary prescriptions in Denmark., Results: ESC-producing E. coli occurred in 93% (27/29) of broiler parent farms in 2011, 27% (53/197) of broiler flocks in 2010, and 3.3% (4/121) of Danish retail broiler meat in 2009 and 8.6% (16/187) in 2010. The ESC producing E. coli contained blaCMY-2, blaSHV-2 or blaCTX-M-1. Isolates with blaCMY-2 represented 35 PFGE groups. One group dominated (39 isolates) and included isolates with indistinguishable PFGE patterns from parents, broilers, and meat. Most blaCMY-2 isolates were susceptible to non-β-lactams, and blaCMY-2 was mostly present on horizontally transferable incI1 or incK plasmids. Phylogroup D was most common and E. coli MLST types previously found in humans were observed. Broiler farms with registered aminopenicillin use had significantly higher occurrence of ESC E. coli., Conclusions: ESC-producing E. coli from flocks of imported broiler parents spread clonally and horizontally to broiler meat (including potentially human pathogenic types) even in a country with no cephalosporin use. Use of aminopenicillins may influence the persistence of ESC-producing E. coli in the broiler production, but other factors should be investigated.

Background: Pseudomonas aeruginosa commonly causes nosocomial bloodstream infections and the emergence of a variety of β-lactamases (BLs) is worrying. In 5 hospitals in Belo Horizonte, Brazil, the presence of phenotypes encoding BL genes was established and the genetic diversity of the P. aeruginosa strains recovered from bloodstream infections was analyzed., Materials and Methods: The isolates were investigated using a disk diffusion (DD) method and the Etest, for encoding metallo-β-lactamases (MBLs), oxacillinases and cephalosporinases. Genes and genetic diversity were evaluated by random amplified polymorphic DNA (RAPD) genotyping and enterobacterial repetitive intergenic consensus (ERIC)-PCR., Results: Twelve strains (30%) were positive for MBLs by Etest and DD, 15 were cephalosporinase-positive and 87.5% were positive for blaSPM-1 and blaVIM-1. Twenty-three strains (57.5%) were grouped into profile A, 32.5% into profile B and 10% into profile C by RAPD genotyping. ERIC-PCR revealed a varying degree of similarity between strains, ranging from 45 to 100%., Conclusions: The results suggest distinct clonal populations in the 5 hospitals studied, indicating a potentially problematic epidemiological situation in Belo Horizonte, Brazil.

Whole-genome sequencing of a collection of 103 Acinetobacter strains belonging to 22 validly named species and another 16 putative species allowed detection of genes for 50 new class D β-lactamases and 65 new Acinetobacter-derived cephalosporinases (ADC). All oxacillinases (OXA) contained the three typical motifs of class D β-lactamases, STFK, (F/Y)GN, and K(S/T)G. The phylogenetic tree drawn from the OXA sequences led to an increase in the number of OXA groups from 7 to 18. The topologies of the OXA and RpoB phylogenetic trees were similar, supporting the ancient acquisition of blaOXA genes by Acinetobacter species. The class D β-lactamase genes appeared to be intrinsic to several species, such as Acinetobacter baumannii, Acinetobacter pittii, Acinetobacter calcoaceticus, and Acinetobacter lwoffii. Neither blaOXA-40/143- nor blaOXA-58-like genes were detected, and their origin remains therefore unknown. The phylogenetic tree analysis based on the alignment of the sequences deduced from blaADC revealed five main clusters, one containing ADC belonging to species closely related to A. baumannii and the others composed of cephalosporinases from the remaining species. No indication of blaOXA or blaADC transfer was observed between distantly related species, except for blaOXA-279, possibly transferred from Acinetobacter genomic species 6 to Acinetobacter parvus. Analysis of β-lactam susceptibility of seven strains harboring new oxacillinases and cloning of the corresponding genes in Escherichia coli and in a susceptible A. baumannii strain indicated very weak hydrolysis of carbapenems. Overall, this study reveals a large pool of β-lactamases in different Acinetobacter spp., potentially transferable to pathogenic strains of the genus.

Aeromonas dhakensis, a recently described Aeromonas sp. formerly called Aeromonas aquariorum, is associated with human infections. In this study, a chromosomal gene, blaAQU-1, was identified in A. dhakensis AAK1 that constitutes a 1143-bp open reading frame and is 87% identical to the gene encoding CepH in Aeromonas hydrophila. An Escherichia coli TOP10 cell transformant harbouring blaAQU-1 was resistant to cefotaxime but not to cefepime. mRNA expression of blaAQU-1 in the cefotaxime-resistant mutant strain AAK1m was 70-fold higher than in the wild strain AAK1. In all 16 A. dhakensis isolates (the major species of 51 consecutive Aeromonas blood isolates collected from June 1999 to June 2001) as well as in A. aquariorum MDC47(T) and A. hydrophila subsp. dhakensis LMG 19562(T), but not in the reference strains or clinical isolates of other A. hydrophila subspecies, Aeromonas caviae, Aeromonas veronii or Aeromonas enteropelogenes, blaAQU-1-related genes were detected by PCR. Overall, 13 (81%) of the 16 A. dhakensis blood isolates exhibited either cefotaxime resistance or the in vitro emergence of derepressed cefotaxime-resistant mutants. In vivo selection of an A. dhakensis resistant mutant was noted in a burn patient undergoing cefotaxime monotherapy. These observations suggest that AQU-1 is a chromosomal cephalosporinase in A. dhakensis. Cefotaxime monotherapy for severe A. dhakensis infections should be used cautiously., (Copyright © 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

We examined the prevalence of various cephalosporins' resistance mechanisms in Acinetobacter baumannii clinical isolates. Phenotypic and molecular detection of Ambler classes A, B and D β-lactamases was performed on 75 isolates. Clonal relatedness was defined using Repetitive Extragenic Palindromic PCR. PCR mapping was used to examine the linkage of insertion sequences and the ampC gene, and ampC expression was analyzed by TaqMan reverse transcriptase-PCR. Twenty-six (37%) isolates carried at least one of the blaPER-1 or blaTEM-1. Sixty-nine (98.5%) out of 70 cephalosporin-resistant isolates had insertions upstream of the ampC gene, of which 48 (69%) and 6 (8%) were identified as ISAba1and ISAba125, respectively. Higher level of expression was obtained in resistant isolates lacking ISAba1/ampC combination in comparison with that in positive ones. The ability to up-regulate the expression of ampC gene in association with different insertion elements has become an important factor in A. baumannii resistance to cephalosporins., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objectives: To measure the effect of a voluntary ban on cephalosporin usage in the Danish pig production on the prevalence of extended-spectrum cephalosporinase (ESC)-producing Escherichia coli in pigs and pork., Methods: Data on cephalosporin consumption were obtained from the VetStat database. For detection of ESC-producing E. coli, three sampling types were included: at slaughter, caecal samples were collected from pigs in 2009 and 2010 (June) before and in two periods (2010 and 2011) after a voluntary ban on cephalosporins was effected (July 2010); at farm level, pools of five stool samples from different pigsties were collected in 2010 and in 2011; and samples from pork were collected randomly at retail stores and outlets from 2009 to 2011. ESC-producing E. coli was isolated after selective enrichment in MacConkey broth with 1 mg/L ceftriaxone. ESC genes were detected using PCR, microtube array and sequencing., Results: From July 2010 the consumption of cephalosporins approximated zero. The occurrence of ESC-producing E. coli in pigs at slaughter was not significantly different (P=0.7) between 2009 [10.8% (85/786)] and 2010 [11.8% (48/407)], but in 2011 the occurrence [3.6% (28/777)] decreased significantly (P<0.001). A significant decrease (P=0.002) in occurrence of ESC-producing E. coli at pig farm level from 2010 [11% (11/99)] to 2011 (0/78) was also observed. The bla(CTX-M-1) gene was most often detected (63%), but bla(CTX-M-14) and bla(CTX-M-15) were also found. Occurrence in pork was between 1.3% and 0.9%., Conclusions: The discontinuation of an already low use of cephalosporins in pig production has significantly reduced the occurrence of ESC-producing E. coli.