Project description:CTX-M enzymes, the plasmid-mediated cefotaximases, constitute a rapidly growing family of extended-spectrum β-lactamases (ESBLs) with significant clinical impact. CTX-Ms are found in at least 26 bacterial species, particularly in Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. At least 109 members in CTX-M family are identified and can be divided into seven clusters based on their phylogeny. CTX-M-15 and CTX-M-14 are the most dominant variants. Chromosome-encoded intrinsic cefotaximases in Kluyvera spp. are proposed to be the progenitors of CTX-Ms, while ISEcp1, ISCR1 and plasmid are closely associated with their mobilization and dissemination.
Project description:CTX-M-25 is a novel extended-spectrum beta-lactamase isolated from a single Canadian Escherichia coli isolate. Susceptibility testing demonstrated that this enzyme confers resistance to both cefotaxime and ceftazidime, but the level of resistance was reduced with the addition of beta-lactamase inhibitors. The bla(CTX-M-25) gene was detected on a 111-kb plasmid. It is a member of the CTX-M-8 group and has the closest amino acid identity (99%; three amino acid substitutions) with CTX-M-26. The bla(CTX-M-26) gene was detected on a 100-kb plasmid isolated from a Klebsiella pneumoniae strain from the United Kingdom, and plasmid profiling revealed that it showed some homology to the bla(CTX-M-25)-harboring plasmid. Both CTX-M genes were located downstream of ISEcp1, although the copy upstream of bla(CTX-M-25) was disrupted by IS50-A. Comparative kinetic studies of recombinant CTX-M-25 and CTX-M-26 enzymes showed that CTX-M-25 has a higher level of ceftazidime hydrolysis (kcat values, 33 and 0.005 s(-1) for CTX-M-25 and CTX-M-26, respectively).
Project description:Denaturing high-performance liquid chromatography (dHPLC) is a powerful technique which has been used extensively to detect genetic variation. This is the first report of the application of dHPLC for rapid genotyping of bacterial beta-lactamase genes. The technique was specifically developed to genotype members of all blaCTX-M DNA homology groups. Thirteen well-defined blaCTX-M extended-spectrum beta-lactamase (ESBL)-producing strains were used to develop and optimize the dHPLC genotyping assay. Further evaluation was carried out with a blinded panel of 62 clinical isolates. The results of blaCTX-M genotyping achieved by dHPLC were comparable to the typing results obtained by DNA sequencing. Applying the newly developed dHPLC-based genotyping method, we successfully genotyped all 73 blaCTX-M ESBL-producing strains from the 4-month survey study. Furthermore, we found the first reported cases in the United Kingdom of clinically significant disease caused by CTX-M-14- and CTX-M-1-producing Escherichia coli strains. We conclude that the novel dHPLC assay is highly accurate, rapid, and cost-effective for the genotyping of blaCTX-M-producing ESBLs and has great potential for determining the clinical relevance of different and new blaCTX-M genotypes, as well as for epidemiological studies and surveillance programs.
Project description:Among 603 isolates of Enterobacteriaceae collected between June and November 2003 from three university hospitals within Korea, bla(CTX-M-3), bla(CTX-M-15), bla(CTX-M-14), and bla(CTX-M-9) were detected in 41 isolates of species from five different genera of Enterobacteriaceae, Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter spp., and Serratia marcescens.
Project description:The presence of antimicrobial-resistant bacteria and resistance genes in aquatic environments is a serious public health concern. This study focused on Escherichia coli possessing blaCTX-M genes in wastewater inflows. Twelve crude inflow water samples from wastewater treatment plant (WWTP) A and two samples each from three other WWTPs were collected in 2017 and 2018. A total of 73 E. coli isolates with 31 different sequence types (STs) harboring distinctive blaCTX-M gene repertoires were detected. In WWTP A influents, blaCTX-M-14 (14 isolates) was dominant, followed by blaCTX-M-15 (12 isolates) and blaCTX-M-27 (10 isolates). The chimeric blaCTX-M-64 and blaCTX-M-123 genes were each identified in one of the E. coli isolates from the same WWTP A inflow port. The blaCTX-M-27 gene was associated with five of seven B2-ST131 isolates, including three isolates of the B2-O25b-ST131-H30R/non-Rx lineage. One of the remaining two isolates belonged to the B2-O25b-ST131-H30R/Rx lineage harboring the blaCTX-M-15 gene. As for the B2-O25b-ST131-H30R/non-Rx lineage, two isolates with blaCTX-M-27 were recovered from each of the WWTP B and D influents, and one isolate with blaCTX-M-174 was also recovered from WWTP B influent. Whole-genome sequencing of chimeric blaCTX-M-harboring E. coli isolates revealed that the blaCTX-M-64 gene was integrated into the chromosome of ST10 E. coli B22 via ISEcp1-mediated transposition of a 9,467-bp sequence. The blaCTX-M-123-carrying IncI1 plasmid pB64 was 109,169 bp in length with pST108. The overall findings suggest that wastewater may act as a probable reservoir of clinically significant clonal lineages mediating antimicrobial resistance genes and chimeric genes that have not yet been identified from human isolates of domestic origin in Japan.IMPORTANCE Global spread of CTX-M-type extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is a critical concern in both clinical and community settings. This dominance of CTX-M-type ESBL producers may be largely due to the successful international spread of epidemic clones, as represented by the extraintestinal pathogenic Escherichia coli (ExPEC) ST131. Our findings highlight the worrisome presence of diverse E. coli clones associated with humans, including ExPEC lineages harboring the most common blaCTX-M variants in untreated wastewater samples. Moreover, the chimeric genes blaCTX-M-64 and blaCTX-M-123, which have not yet been identified from human isolates of domestic origin in Japan, were identified. Exposure to untreated wastewater through combined sewer overflow caused by heavy rains derived from abnormal weather change could pose a risk for human health due to ingesting those antimicrobial-resistant bacteria.
Project description:A huge variety of extended-spectrum beta-lactamases (ESBLs) have been detected during the last 20 years. The majority of these have been of the TEM or SHV lineage. We have assessed ESBLs occurring among a collection of 455 bloodstream isolates of Klebsiella pneumoniae, collected from 12 hospitals in seven countries. Multiple beta-lactamases were produced by isolates with phenotypic evidence of ESBL production (mean of 2.7 beta-lactamases per isolate; range, 1 to 5). SHV-type ESBLs were the most common ESBL, occurring in 67.1% (49 of 73) of isolates with phenotypic evidence of ESBL production. In contrast, TEM-type ESBLs (TEM-10 type, -12 type, -26 type, and -63 type) were found in just 16.4% (12 of 73) of isolates. The finding of TEM-10 type and TEM-12 type represents the first detection of a TEM-type ESBL in South America. PER (for Pseudomonas extended resistance)-type beta-lactamases were detected in five of the nine isolates from Turkey and were found with SHV-2-type and SHV-5-type ESBLs in two of the isolates. CTX-M-type ESBLs (bla(CTX-M-2) type and bla(CTX-M-3) type) were found in 23.3% (17 of 73) of isolates and were found in all study countries except for the United States. We also detected CTX-M-type ESBLs in four countries where they have previously not been described-Australia, Belgium, Turkey, and South Africa. The widespread emergence and proliferation of CTX-M-type ESBLs is particularly noteworthy and may have important implications for clinical microbiology laboratories and for physicians treating patients with serious K. pneumoniae infections.
Project description:CTX-M beta-lactamases, which show a high cefotaxime hydrolytic activity, constitute the most prevalent extended-spectrum beta-lactamase (ESBL) type found among clinical isolates. The recent explosive diversification of CTX-M enzymes seems to have taken place due to the appearance of more efficient enzymes which are capable of hydrolyzing both cefotaxime and ceftazidime, especially among the CTX-M-1 cluster. A combined strategy of in vitro stepwise evolution experiments using bla(CTX-M-1), bla(CTX-M-3), and bla(CTX-M-10) genes and site-directed mutagenesis has been used to evaluate the role of ceftazidime and other beta-lactam antibiotics in triggering the diversity found among enzymes belonging to this cluster. Two types of mutants, P167S and D240G, displaying high ceftazidime MICs but reduced resistance to cefotaxime and/or cefepime, respectively, were identified. Such an antagonistic pleiotropic effect was particularly evident with P167S/T mutations. The incompatibility between P167S and D240G changes was demonstrated, since double mutants reduced susceptibility to both ceftazidime and cefotaxime-cefepime; this may explain the absence of strains containing both mutations in the clinical environment. The role of A77V and N106S mutations, which are frequently associated with P167S/T and/or D240G, respectively, in natural strains, was investigated. The presence of A77V and N106S contributes to restore a high-level cefotaxime resistance phenotype, but only when associated with mutations P167S and D240G, respectively. However, A77V mutation increases resistance to both cefotaxime and ceftazidime when associated with CTX-M-10. This suggests that in this context this mutation might be considered a primary site involved in resistance to broad-spectrum cephalosporins.
Project description:BackgroundPatients with faecal carriage of extended-spectrum beta-lactamases (ESBL)-producing Enterobacterales serve as reservoirs and sources of dissemination and infection.ObjectiveThis report examined immunocompetent patients for faecal carriage of ESBL-producing Enterobacterales in a district care hospital setting in Ghana.MethodsBetween March 2019 and May 2020, cross-sectional sampling was performed to enrol patients and conduct questionnaire-structured interviews for factors that predispose patients to ESBL faecal carriage. Faecal samples from study patients were quantified for ESBL-producing Enterobacterales. The ESBL genes were characterised by polymerase chain reaction and sequencing.ResultsThe overall proportion of ESBL faecal carriage was 35.5% (n = 38/107). The blaCTX-M gene, mostly CTX-M-15, was detected in 89.5% (n = 34/38) of the ESBL-producing isolates. The other ESBL types included blaSHV (n = 3) and blaOXA (n = 1). The CTX-M-15-positive isolates, when present in a faecal sample compared to the non-ESBL-CTX-M-15 isolates, constituted the predominant faecal Enterobacterales, with significantly higher colony counts than all other enterobacteria in that sample. In multivariate regression, independent risk factors for faecal carriage of ESBL-producing Enterobacterales were hospitalisation in the past year, infections since admission, use of antibiotics in the past 6 weeks, and admission from another hospital.ConclusionThe study found that CTX-M-15-producing isolates were the predominant faecal Enterobacterales, and that further investigations are needed to determine the reasons behind this dominance.What this study addsThe CTX-M-15-producing isolates dominance in this study shows the misuse and abuse of antibiotics in an African medical facility and indicates the potential role of immunity in controlling ESBL spread, which is to be investigated further.