CTX-M Extended-spectrum Β-lactamases Research Articles

Nanopore R10.4 metagenomic detection of blaCTX-M/blaDHA antimicrobial resistance genes and their genetic environments in stool

The increasing prevalence of gut colonization with CTX-M extended-spectrum β-lactamase- and/or DHA plasmid-mediated AmpC-producing Escherichia coli is a concern. Here, we evaluate Nanopore-shotgun metagenomic sequencing (Nanopore-SMS) latest V14 chemistry to detect blaCTX-M and blaDHA genes from healthy stools. We test 25 paired samples characterized with culture-based methods (native and pre-enriched). Antimicrobial resistant genes (ARGs) are detected from reads and meta-assembled genomes (MAGs) to determine their associated genetic environments (AGEs). Sensitivity and specificity of native Nanopore-SMS are 61.1% and 100%, compared to 81.5% and 75% for pre-enriched Nanopore-SMS, respectively. Native Nanopore-SMS identifies only one sample with an AGE, whereas pre-enriched Nanopore-SMS recognizes 9/18 plasmids and 5/9 E. coli chromosomes. Pre-enriched Nanopore-SMS identifies more ARGs than native Nanopore-SMS (p < 0.001). Notably, blaCTX-Ms and blaDHAs AGEs (plasmid and chromosomes) are identified within 1 hour of sequencing. Furthermore, microbiota analyses show that pre-enriched Nanopore-SMS results in more E. coli classified reads (47% vs. 3.1%), higher differential abundance (5.69 log2 fold) and lower Shannon diversity index (p < 0.0001). Nanopore-SMS has the potential to be used for intestinal colonization screening. However, sample pre-enrichment is necessary to increase sensitivity. Further computational improvements are needed to reduce the turnaround time for clinical applications.

Molecular characterization of cefepime and aztreonam nonsusceptibility in Haemophilus influenzae.

Cefepime and aztreonam are highly efficacious against H. influenzae, and resistant strains are rare. In this study, we isolated cefepime- and aztreonam-nonsusceptible H. influenzae strains and addressed the molecular basis of their resistance to cefepime and aztreonam. Two hundred and 28 specimens containing H. influenzae were screened, of which 32 isolates were enrolled and applied to antimicrobial susceptibility testing and whole-genome sequencing. Genetic variations that were detected in all nonsusceptible isolates with statistical significance by Fisher's exact tests were identified as cefepime or aztreonam nonsusceptibility related. Functional complementation assays were conducted to assess the in vitro effects of proteins with sequence substitutions on drug susceptibility. Three H. influenzae isolates were nonsusceptible to cefepime, one of which was also nonsusceptible to aztreonam. Genes encoding TEM, SHV and CTX-M extended-spectrum β-lactamases were not detected in the cefepime- and aztreonam-nonsusceptible isolates. Five genetic variations in four genes and 10 genetic variations in five genes were associated with cefepime and aztreonam nonsusceptibility, respectively. Phylogenetic analyses revealed that changes in FtsI were correlated strongly with the MIC of cefepime and moderately with aztreonam. FtsI Thr532Ser-Tyr557His cosubstitution linked to cefepime nonsusceptibility and Asn305Lys-Ser385Asn-Glu416Asp cosubstitution to aztreonam nonsusceptibility. Functional complementation assays revealed that these cosubstitutions increased MICs of cefepime and aztreonam in susceptible H. influenzae isolates, respectively. Genetic variations relevant to resistant phenotypes of cefepime and aztreonam nonsusceptibility in H. influenzae were identified. Moreover, the effects of FtsI cosubstitutions on increasing MICs of cefepime and aztreonam in H. influenzae were demonstrated.

OXA-48-Like β-Lactamases: Global Epidemiology, Treatment Options, and Development Pipeline.

Modern medicine is threatened by the rising tide of antimicrobial resistance, especially among Gram-negative bacteria, where resistance to β-lactams is most often mediated by β-lactamases. The penicillin and cephalosporin ascendancies were, in their turn, ended by the proliferation of TEM penicillinases and CTX-M extended-spectrum β-lactamases. These class A β-lactamases have long been considered the most important. For carbapenems, however, the threat is increasingly from the insidious rise of a class D carbapenemase, OXA-48, and its close relatives. Over the past 20 years, OXA-48 and "OXA-48-like" enzymes have proliferated to become the most prevalent enterobacterial carbapenemases across much of Europe, Northern Africa, and the Middle East. OXA-48-like enzymes are notoriously difficult to detect because they often cause only low-level in vitro resistance to carbapenems, meaning that the true burden is likely underestimated. Despite this, they are associated with carbapenem treatment failures. A highly conserved incompatibility complex IncL plasmid scaffold often carries blaOXA-48 and may carry other antimicrobial resistance genes, leaving limited treatment options. High conjugation efficiency means that this plasmid is sometimes carried by multiple Enterobacterales in a single patient. Producers evade most β-lactam-β-lactamase inhibitor combinations, though promising agents have recently been licensed, notably ceftazidime-avibactam and cefiderocol. The molecular machinery enabling global spread, current treatment options, and the development pipeline of potential new therapies for Enterobacterales that produce OXA-48-like β-lactamases form the focus of this review.

Evaluation of a diagnostic algorithm for rapid identification of Gram-negative species and detection of extended-spectrum β-lactamase and carbapenemase directly from blood cultures.

To evaluate a rapid diagnostic algorithm based on MALDI-TOF MS, lateral flow immunoassays (LFIAs) and molecular testing performed directly from positive blood cultures (BCs) for Gram-negative species identification and detection of CTX-M extended-spectrum β-lactamases and main carbapenemases. Non-duplicate BCs positive to Gram-negative bacteria at microscope examination were subjected to species identification by direct MALDI-TOF MS following recovery of bacterial pellet by Rapid MBT Sepsityper® kit. Subsequently, NG-Test® CARBA 5 and NG-Test® CTX-M MULTI LFIAs were performed according to identified microbial species. Eazyplex® SuperBug CRE molecular assay was performed in cases of NG-Test® CARBA 5 negative results in patients with documented carbapenemase-producers carriage. Results of rapid diagnostic workflow were compared with those obtained by conventional diagnostic routine. Overall, the direct MALDI-TOF MS protocol allowed reliable identification to the species level of 92.1% of the 2133 monomicrobial BCs. Rate of matched identification was significantly higher for Enterobacterales (97.3%) in comparison to non-fermenting Gram-negative species (80.2%), obligate anaerobic bacteria (42.1%) and fastidious Gram-negative species (41.5%). The overall sensitivity of NG-Test® CARBA 5 and NG-Test® CTX-M MULTI was 92.2% and 91.6%, respectively. Integration of Easyplex® SuperBug CRE allowed the detection of blaKPC mutants associated with ceftazidime/avibactam resistance, reaching 100% sensitivity in carbapenemase detection. Both LFIAs and molecular testing showed no false-positive results. Algorithms based on MALDI-TOF MS, LFIAs and molecular testing may represent a cost-effective tool to timely identify Gram-negative species and detect resistance markers directly from BCs. According to local epidemiology, these results may allow antimicrobial stewardship interventions including prompt use of new approved drugs.

Evaluation of a lateral flow immunoassay to detect CTX-M extended-spectrum β-lactamases (ESBL) directly from positive blood cultures for its potential use in Antimicrobial Stewardship programs.

BackgroundBloodstream infections (BSI) caused by extended-spectrum beta-lactamases Enterobacteriaceae (ESBL-E) are associated with high rates of treatment failure and increased mortality, especially when appropriate antimicrobial therapy is delayed. Our aim was to evaluate the anticipation of ESBLs detection and the potential improvement of the time response of the Vitek 2 System (BioMérieux; France).MethodsWe compared this lateral flow immunoassay when used directly on fluid from positive blood cultures to the VITEK2 AST system. We evaluated 80 isolates, 61 tested directly on fluid from positive blood cultures and 19 tested on fluid from blood cultures spiked with known ESBL positive and negative organisms.ResultsThe concordance between the CTX-LFIA and the reference method (Vitek 2) had a Cohen´s Kappa coefficient of 0.97, indicating a particularly good correlation between both compared methods.ConclusionThis lateral flow immunoassay can be more rapid than the Vitek 2 for earlier presumptive identification of CTX- M ESBLs and can be useful to anticipate results and the adjustment of antimicrobial therapy.

In Vivo Evolution of CTX-M-215, a Novel Narrow-Spectrum β-Lactamase in an Escherichia coli Clinical Isolate Conferring Resistance to Mecillinam.

Here, we report a novel narrow-spectrum β-lactamase CTX-M-215 identified in an Escherichia coli clinical isolate in China and conferring high-level resistance to mecillinam but not to cefotaxime. CTX-M-215 differed from CTX-M-125, a CTX-M extended-spectrum β-lactamase (ESBL), by an N132D substitution, which decreased hydrolytic activities toward penicillins and cephalosporins except for mecillinam. High similarity was observed between CTX-M-215- and CTX-M-125-bearing plasmids, carried by different isolates in the same patient, indicating in vivo evolution of CTX-M-215 from CTX-M-125.

Structural Basis and Binding Kinetics of Vaborbactam in Class A β-Lactamase Inhibition.

Class A β-lactamases are a major cause of β-lactam resistance in Gram-negative bacteria. The recently FDA-approved cyclic boronate vaborbactam is a reversible covalent inhibitor of class A β-lactamases, including CTX-M extended-spectrum β-lactamase and KPC carbapenemase, both frequently observed in the clinic. Intriguingly, vaborbactam displayed different binding kinetics and cell-based activity for these two enzymes, despite their similarity. A 1.0-Å crystal structure of CTX-M-14 demonstrated that two catalytic residues, K73 and E166, are positively charged and neutral, respectively. Meanwhile, a 1.25-Å crystal structure of KPC-2 revealed a more compact binding mode of vaborbactam versus CTX-M-14, as well as alternative conformations of W105. Together with kinetic analysis of W105 mutants, the structures demonstrate the influence of this residue and the unusual conformation of the β3 strand on the inactivation rate, as well as the stability of the reversible covalent bond with S70. Furthermore, studies of KPC-2 S130G mutant shed light on the different impacts of S130 in the binding of vaborbactam versus avibactam, another recently approved β-lactamase inhibitor. Taken together, these new data provide valuable insights into the inhibition mechanism of vaborbactam and future development of cyclic boronate inhibitors.

Advantage of the F2:A1:B- IncF Pandemic Plasmid over IncC Plasmids in In Vitro Acquisition and Evolution of blaCTX-M Gene-Bearing Plasmids in Escherichia coli.

Despite a fitness cost imposed on bacterial hosts, large conjugative plasmids play a key role in the diffusion of resistance determinants, such as CTX-M extended-spectrum β-lactamases. Among the large conjugative plasmids, IncF plasmids are the most predominant group, and an F2:A1:B- IncF-type plasmid encoding a CTX-M-15 variant was recently described as being strongly associated with the emerging worldwide Escherichia coli sequence type 131 (ST131)-O25b:H4 H30Rx/C2 sublineage. In this context, we investigated the fitness cost of narrow-range F-type plasmids, including the F2:A1:B- IncF-type CTX-M-15 plasmid, and of broad-range C-type plasmids in the K-12-like J53-2 E. coli strain. Although all plasmids imposed a significant fitness cost to the bacterial host immediately after conjugation, we show, using an experimental-evolution approach, that a negative impact on the fitness of the host strain was maintained throughout 1,120 generations with the IncC-IncR plasmid, regardless of the presence or absence of cefotaxime, in contrast to the F2:A1:B- IncF plasmid, whose cost was alleviated. Many chromosomal and plasmid rearrangements were detected after conjugation in transconjugants carrying the IncC plasmids but not in transconjugants carrying the F2:A1:B- IncF plasmid, except for insertion sequence (IS) mobilization from the fliM gene leading to the restoration of motility of the recipient strains. Only a few mutations occurred on the chromosome of each transconjugant throughout the experimental-evolution assay. Our findings indicate that the F2:A1:B- IncF CTX-M-15 plasmid is well adapted to the E. coli strain studied, contrary to the IncC-IncR CTX-M-15 plasmid, and that such plasmid-host adaptation could participate in the evolutionary success of the CTX-M-15-producing pandemic E. coli ST131-O25b:H4 lineage.

Direct use of eazyplex® SuperBug CRE assay from positive blood cultures in conjunction with inpatient infectious disease consulting for timely appropriate antimicrobial therapy in Escherichia coli and Klebsiella pneumoniae bloodstream infections.

Objectives: To describe a rapid workflow based on the direct detection of Escherichia coli (Ec) and Klebsiella pneumoniae (Kp) producing CTX-M extended-spectrum β-lactamase (ESBL) and/or carbapenemases (eg, KPC, VIM) from blood cultures (BCs) and the infectious disease (ID) consulting for timely appropriate antimicrobial therapy.Methods: This observational, retrospective study included adult patients with a first episode of Ec or Kp bloodstream infection (BSI) in a large Italian university hospital, where an inpatient ID consultation team (IDCT) has been operational. Results from the BCs tested for detecting blaCTX-M, blaKPC, blaNDM, blaOXA-48-like, and blaVIM genes by the eazyplex® SuperBug CRE assay in Ec and Kp organisms had been notified for antimicrobial therapy consulting.Results: In 321 BSI episodes studied, we found that 151 (47.0%) of Ec or Kp organisms harbored blaCTX-M and/or blaKPC and/or blaVIM (meantime from BC collection: 18.5 h). Empirical antimicrobial treatment was appropriate in 21.8% (33/151) of BSIs, namely 5.9% (3/51) of BSIs caused by KPC/VIM producers and 30.0% (30/100) of BSIs caused by CTX-M producers. After notification of results, the IDCT modified antimicrobial therapy (mean time from BC collection: 20 h) such that the proportion of appropriate treatments increased to 84.8% (128/151) of BSIs, namely 70.6% (36/51) of BSIs caused by KPC/VIM producers and 92.0% (92/100) of BSIs caused by CTX-M producers.Conclusion: Our study shows that a rapid diagnostic-driven clinical strategy allowed for early prescription of potentially effective antimicrobial therapy in BSIs caused by CTX-M ESBL- and/or KPC/VIM carbapenemase-producing Ec and Kp organisms.

Differences in Antimicrobial Resistance Phenotypes by the Group of CTX-M Extended-Spectrum β-Lactamase

Differences in Antimicrobial Resistance Phenotypes by the Group of CTX-M Extended-Spectrum β-Lactamase

Rapid in vitro detection of CTX-M groups 1, 2, 8, 9 resistance genes by LAMP assays

BackgroundThe prevalence of bacteria producing CTX-M Extended-Spectrum β-lactamases (ESBLs) has increased around the world and some of them became a major cause of infections such as bloodstream or urinary tract infections (UTI). We developed a loop-mediated isothermal amplification (LAMP) assay for a simple, rapid and sensitive detection of the four most common CTX-M groups, namely CTX-M groups 1, 2, 8 and 9.MethodsLAMP primers targeting the four ESBLs CTX-M groups were designed using the Primer Explorer V4 software. The detection limit of the method was tested by serial dilution of reference DNAs. The primer specificity of the LAMP reaction was tested on DNA extracted from six strains producing various group of CTX-M and validated using DNA extracted from CTX-M-resistant clinical isolates (isolated from pus, urine, or blood). Results were compared with those of conventional PCR.ResultsWe were able to detect down to 0.1 pg/ul of DNA using the newly developed LAMP assays whereas the minimal amount detectable for conventional PCR was 50 to 100pg/ul, indicating that the LAMP assay was found to have a detection limit at least 500 to 1000 times lower than the PCR. Additionally, representative genes from the CTX-M groups 1, 2, 8 and 9 were amplified using the designed assay and no cross amplification was observed between the four CTX-M groups, demonstrating the specificity of the LAMP assay. Of the 37 clinical strains tested, the four LAMP assays showed 100% sensitivity and 87%, 97%, 100%, 100% specificity for the CTX-M groups 1, 2, 8 and 9 respectively.ConclusionBeing sensitive, specific, rapid and standard methods, the LAMP assays developed in this study have a potential to be beneficial tools in molecular epidemiology and surveillance studies of the four prevalent EBSLs CTX-M groups even in low cost laboratory.

Diversity of CTX-M Extended-Spectrum β-Lactamases in Escherichia coli Isolates from Retail Raw Ground Beef: First Report of CTX-M-24 and CTX-M-32 in Algeria.

The aim of this study was to investigate the prevalence and molecular features of extended-spectrum cephalosporin resistance in Escherichia coli isolates contaminating ground beef at retail in Algeria. Of 371 ground beef samples, 27.5% were found to contain cefotaxime-resistant E. coli isolates distributed into A (24.5%), B1 (60.8%), and D (14.7%) phylogroups. A rate of 88.2% of isolates had a multidrug-resistance phenotype. All strains were producers of CTX-M type extended-spectrum β-lactamases (ESBLs): CTX-M-1, CTX-M-3, CTX-M-14, CTX-M-15, CTX-M-24, or CTX-M-32. Conjugation assays allowed the transfer of blaCTX-M-1 in association with IncI1 plasmids, blaCTX-M-15 with IncI1 and IncK+B/O plasmids, blaCTX-M-3 with IncK plasmids, and blaCTX-M-14 with IncF1B or IncK plasmids. Sequence analysis of gyrA and parC genes showed mutations in 98.6% of ciprofloxacin-resistant isolates. The patterns "GyrA: S83L+D87N, ParC: S80I" (46.5%) and "ParC: S80I" (42.3%) were predominant. qnrS1, qnrB, and aac(6')-Ib-cr were detected in 18.7% of isolates. The tet genes, tetA, tetB, and tetA+tetB, were present in 95.7% of tetracycline-resistant isolates. The sul genes (sul1, sul2, sul3, sul1+sul2, sul2+sul3, and sul1+sul3) and the dfr gene clusters (dfrA1, dfrA5, dfrA7, dfrA8, dfrA12, dfrA5+dfrA12, dfrA1+dfrA5, dfrA7+dfrA12, dfrA5+dfrA7, and dfrA1+dfrA5+dfrA7) were found in 96.4% and 85.5% of sulfamethoxazole/trimethoprim-resistant isolates, respectively. Classes 1 and 2 integrons were detected in 67.6% and 9.8% of isolates, respectively. This study highlighted the significant presence of resistance genes, in particular those of CTXM ESBLs, in the beef meat, with the risk of their transmission to humans through food chain.

CTX-M-15 Type β-lactamases From Clinical Isolates of Escherichia coli by Polymerase Chain Reaction and DNA Sequencing

Background: E. coli is a common cause of urinary tract infection and bacteremia in humans. That is frequently resistant to β-lactam antibiotics. Objectives: The aim of this study was to determine the antibiotic resistance and CTX-M extended-spectrum β-lactamases (ESBL) carrying E. coli isolated in hospitals from two different areas in Iran. Methods: In this descriptive cross-sectional study, 93 clinical isolates of E. coli were collected from urine culture of inpatients in Mashhad and Qom hospitals from August 2013 to February 2014. Bacteria were re-confirmed by Microgen kit. Antimicrobial and ESBL tests were performed using by standard disc diffusion method. Polymerase chain reaction (PCR) was used for detection of plasmid mediated CTX-M-1 and CTX-M-2 ESBLs. Four of the blaCTX-Mgenes were sequenced. Statistical analysis carried out using SPSS (version 16) software and Chi-square test. Results: High prevalent isolates were resistant to ampicillin, cephalothin, nalidixic acid and cotrimoxazole, but sensitive to imipenem, nitrofurantoin and amikacin. The ESBL phenotype was detected in 48 (51.6%) isolates, 42 of which were carrying blaCTX-Mgene. There was no significant difference between the β-lactamase-producing bacteria from the two studied cities. 81% and 85% of blaCTX-Mcarriers from hospitals in Qom and Mashhad were respectively found to have CTX-M-1 ESBL, whereas none of them showed CTX-M-2 ESBL cluster. BlaCTX-M-15was found in four selected bacteria by sequencing. Conclusions: This study showed a high rate of CTX-M-1 cluster and also presence of CTX-M-15 ESBL in two different areas of Iran, which emphasize the need for employing an excellent management program in antibiotic therapy.

Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate.

ABSTRACTThe monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A β-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed.

Phylogenetic Analysis of Enteroaggregative Escherichia coli (EAEC) Isolates from Japan Reveals Emergence of CTX-M-14-Producing EAEC O25:H4 Clones Related to Sequence Type 131.

Enteroaggregative Escherichia coli (EAEC) causes acute or persistent diarrhea. The aggR gene is widely used as a marker for typical EAEC. The heterogeneity of EAEC is well known; however, there are few reports on the phylogenetic relationships of EAEC. Recently, CTX-M extended-spectrum β-lactamase (ESBL)-producing EAEC strains have been reported worldwide. To characterize EAEC strains in Japan, we investigated the population structure of EAEC. A total of 167 aggR-positive strains isolated from stool specimens from diarrheal patients in Kagoshima (139 strains) and Osaka (28 strains), Japan, between 1992 and 2010 were examined for the prevalence of EAEC virulence markers, the blaCTX-M gene, and the capacity to form biofilms. Multilocus sequence typing was also conducted. EAEC strains were widely distributed across four major E. coli phylogroups. Strains of O111:H21/clonal group 40 (CG40) (30 strains), O126:H27/CG200 (13 strains), and O86a:H27/CG3570 (11 strains) in phylogroup B1 are the historical EAEC clones in Japan, and they exhibited strong biofilm formation. Twenty-nine strains of EAEC O25:H4/CG131 were identified in phylogroup B2, 79% of which produced CTX-M-14. This clone has emerged since 2003. The clone harbored plasmid-encoded EAEC virulence genes but not chromosomal virulence genes and had lower biofilm-forming capacity than historical EAEC strains. This clone most likely emerged from a pandemic uropathogenic O25:H4/sequence type 131 clone by acquiring an EAEC virulence plasmid from canonical EAEC. Surveillance of the horizontal transfer of both virulence and ESBL genes among E. coli strains is important for preventing a worldwide increase in antimicrobial drug resistance.

Incidence of SHV and CTX-M Extended spectrum β-lactamases producing gram negative bacterial isolates from antenatal mother with asymptomatic bacteriuria

Incidence of SHV and CTX-M Extended spectrum β-lactamases producing gram negative bacterial isolates from antenatal mother with asymptomatic bacteriuria

Translational Inhibition of CTX-M Extended Spectrum β-Lactamase in Clinical Strains of Escherichia coli by Synthetic Antisense Oligonucleotides Partially Restores Sensitivity to Cefotaxime.

Synthetic antisense oligomers are DNA mimics that can specifically inhibit gene expression at the translational level by ribosomal steric hindrance. They bind to their mRNA targets by Watson-Crick base pairing and are resistant to degradation by both nucleases and proteases. A 25-mer phosphorodiamidate morpholino oligomer (PMO) and a 13-mer polyamide (peptide) nucleic acid (PNA) were designed to target mRNA (positions -4 to +21, and –17 to –5, respectively) close to the translational initiation site of the extended-spectrum β-lactamase resistance genes of CTX-M group 1. These antisense oligonucleotides were found to inhibit β-lactamase activity by up to 96% in a cell-free translation-transcription coupled system using an expression vector carrying a blaCTX-M-15 gene cloned from a clinical isolate. Despite evidence for up-regulation of CTX-M gene expression, they were both found to significantly restore sensitivity to cefotaxime (CTX) in E. coli AS19, an atypical cell wall permeable mutant, in a dose dependant manner (0-40 nM). The PMO and PNA were covalently bound to the cell penetrating peptide (CPP; (KFF)3K) and both significantly (P < 0.05) increased sensitivity to CTX in a dose dependent manner (0-40 nM) in field and clinical isolates harboring CTX-M group 1 β-lactamases. Antisense oligonucleotides targeted to the translational initiation site and Shine-Dalgarno region of blaCTX-M-15 inhibited gene expression, and when conjugated to a cell penetrating delivery vehicle, partially restored antibiotic sensitivity to both field and clinical isolates.

Inhibitory activity of avibactam against selected β-lactamases expressed in an isogenic Escherichia coli strain

Avibactam restored the in-vitro antibacterial activity of ceftazidime, ceftaroline, and aztreonam against isogenic Escherichia coli expressing class A, class C, and class D β-lactamases. The enzymes included TEM and CTX-M extended spectrum β-lactamases, ACT, CMY and FOX AmpC-type enzymes, and carbapenemases including rarer KPC variants and OXA-139.

High Diversity of CTX-M Extended-Spectrum β-Lactamases in Municipal Wastewater and Urban Wetlands.

The CTX-M-type extended-spectrum β-lactamases (ESBLs) present a serious public health threat as they have become nearly ubiquitous among clinical gram-negative pathogens, particularly the enterobacteria. To aid in the understanding and eventual control of the spread of such resistance genes, we sought to determine the diversity of CTX-M ESBLs not among clinical isolates, but in the environment, where weaker and more diverse selective pressures may allow greater enzyme diversification. This was done by examining the CTX-M diversity in municipal wastewater and urban coastal wetlands in southern California, United States, by Sanger sequencing of polymerase chain reaction amplicons. Of the five known CTX-M phylogroups (1, 2, 8, 9, and 25), only genes from groups 1 and 2 were detected in both wastewater treatment plants (WWTPs), and group 1 genes were also detected in one of the two wetlands after a winter rain. The highest relative abundance of blaCTX-M group 1 genes was in the sludge of one WWTP (2.1 × 10(-4) blaCTX-M copies/16S rRNA gene copy). Gene libraries revealed surprisingly high nucleotide sequence diversity, with 157 new variants not found in GenBank, representing 99 novel amino acid sequences. Our results indicate that the resistomes of WWTPs and urban wetlands contain diverse blaCTX-M ESBLs, which may constitute a mobile reservoir of clinically relevant resistance genes.

Characterization of CTX-M-Type Extend-Spectrum β-Lactamase Producing Klebsiella spp. in Kashan, Iran.

Context:The CTX-M family consists of more than 50 β-lactamases, which are grouped on the basis of sequences into five subtypes including CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25.Objectives:The current study aimed to detect subtypes of CTX-M extended-spectrum β-lactamases (ESBLs) among ESBL positive Klebsiella isolates from patients in Kashan, Iran.Materials and Methods:A total of 100 clinical isolates of Klebsiella were collected and the isolates, which showed resistance or reduced susceptibility to cefotaxime, ceftazidime and/or aztreonam by the disk diffusion method were selected. These isolates were identified as ESBL-producing isolates by double disk synergy tests using clavulanic acid, cefotaxime, ceftazidime and aztreonam. The blaCTX-M type determinants were identified by the Polymerase Chain Reaction (PCR) method followed by DNA sequencing.Results:Of the 100 Klebsiella isolates, 41 (41%) demonstrated resistance or reduced susceptibility to ceftazidime and/or aztreonam and 35% (n = 35) were ESBL-producers. Twenty-eight (8o%) of the ESBL-producing isolates carried the blaCTX-M type genes. Based on PCR assays and sequencing of blaCTX-M genes, CTX-M-1, CTX-M-2 and CTX-M-9 were identified in 21 (60%), 15 (42%) and nine (34%) of these isolates, respectively (GenBank accession numbers KJ803828-KJ803829).Conclusions:Our study showed that the frequency of blaCTX-M genes among Klebsiella isolates in our region is at an alarming rate. Also, we found a high prevalence of blaCTX-M-1 β-lactamase in Klebsiella isolates in Kashan.