Plasmid-mediated AmpC Beta-lactamases Research Articles

Survival of highly related ESBL- and pAmpC- producing Escherichia coli in broiler farms identified before and after cleaning and disinfection using cgMLST

BackgroundBroiler chickens are frequently colonized with Extended-Spectrum Beta-Lactamase- (ESBL-) and plasmid mediated AmpC Beta-Lactamase- (pAmpC-) producing Enterobacterales, and we are confronted with the potential spread of these resistant bacteria in the food chain, in the environment, and to humans. Research focused on identifying of transmission routes and investigating potential intervention measures against ESBL- and pAmpC- producing bacteria in the broiler production chain. However, few data are available on the effects of cleaning and disinfection (C&D) procedures in broiler stables on ESBL- and pAmpC- producing bacteria.ResultsWe systematically investigated five broiler stables before and after C&D and identified potential ESBL- and pAmpC- colonization sites after C&D in the broiler stables, including the anteroom and the nearby surrounding environment of the broiler stables. Phenotypically resistant E. coli isolates grown on MacConkey agar with cefotaxime were further analyzed for their beta-lactam resistance genes and phylogenetic groups, as well as the relation of isolates from the investigated stables before and after C&D by whole genome sequencing. Survival of ESBL- and pAmpC- producing E. coli is highly likely at sites where C&D was not performed or where insufficient cleaning was performed prior to disinfection. For the first time, we showed highly related ESBL-/pAmpC- producing E. coli isolates detected before and after C&D in four of five broiler stables examined with cgMLST. Survival of resistant isolates in investigated broiler stables as well as transmission of resistant isolates from broiler stables to the anteroom and surrounding environment and between broiler farms was shown. In addition, enterococci (frequently utilized to detect fecal contamination and for C&D control) can be used as an indicator bacterium for the detection of ESBL-/pAmpC- E. coli after C&D.ConclusionWe conclude that C&D can reduce ESBL-/pAmpC- producing E. coli in conventional broiler stables, but complete ESBL- and pAmpC- elimination does not seem to be possible in practice as several factors influence the C&D outcome (e.g. broiler stable condition, ESBL-/pAmpC- status prior to C&D, C&D procedures used, and biosecurity measures on the farm). A multifactorial approach, combining various hygiene- and management measures, is needed to reduce ESBL-/pAmpC- E. coli in broiler farms.

Rare Plasmid-Mediated AmpC Beta-Lactamase DHA-1 Located on Easy Mobilized IS26-Related Genetic Element Detected in Escherichia coli from Livestock and Food in Germany.

AmpC beta-lactamases cause resistance to third-generation cephalosporins, including beta-lactamase inhibitors. In Escherichia coli from the German food production chain, the majority of AmpC beta-lactamase activity can be attributed to plasmid-mediated CMY-2 or overproduction of chromosomal AmpC beta-lactamase, but occasionally other enzymes like DHA-1 are involved. This study investigated the prevalence of the AmpC beta-lactamase DHA-1 in ESBL/AmpC-producing E. coli (n = 4706) collected between 2016 and 2021 as part of a German antimicrobial resistance monitoring program along the food chain. Eight isolates (prevalence < 0.2%) were detected and further characterized by PFGE, transformation and conjugation experiments as well as short-read and long-read sequencing. All eight strains harbored blaDHA-1 together with qnrB4, sul1 and mph(A) resistance genes on an IS26 composite transposon on self-transferable IncFII or IncFIA/FIB/II plasmids. During laboratory experiments, activation of the translocatable unit of IS26-bound structures was observed. This was shown by the variability of plasmid sizes in original isolates, transconjugants or transferred plasmids, and correspondingly, duplications of resistance fragments were found in long-read sequencing. This activation could be artificial due to laboratory handling or naturally occurring. Nevertheless, DHA-1 is a rare AmpC beta-lactamase in livestock and food in Germany, and its dissemination will be monitored in the future.

AmpC β-Lactamases in Enterobacteriaceae - A Mini Review

Beta-lactamases are enzymes that confer resistance to β-lactam antibiotics. Both Gram-positive and Gram-negative bacteria produces these enzymes. There are about 3000 enzymes that initially emerged from bacteria found in the environment to protect themselves from the natural β-lactam. After the 1980s, many transmissible enzymes were detected periodically, resistant to cephalosporins, monobactam, and carbapenems. These enzymes were classified based on function and molecular structure. Among them AmpC β-lactamases were found to be resistant to β-lactams and β-lactamases inhibitors. They are class C cephalosporinases that confer resistance to the first, second, third generation cephalosporins and cephamycin, and also resistance to beta-lactamases inhibitors such as sulbactam, tazobactam, and clavulanic acid. Family Enterobacteriaceae comprises many organisms that cause community and nosocomial infections, such as Escherichia coli, Klebsiella pneumoniae, Citrobacter spp, Enterobacter aerogenes, and Salmonella species. Beta-lactamases are produced by Enterobacteriaceae, where AmpC beta-lactamases are found to be one of the mechanisms. Different types of AmpC beta-lactamases: mutation/attenuation in the chromosome, induced plasmid-mediated AmpC beta-lactamases. Some Enterobacteriaceae, like Enterobacter, carry it on their chromosome, and some other Enterobacteriaceae has plasmid-mediated AmpC beta-lactamases. This type of resistance has led to increased mortality and morbidity. It is challenging to detect these AmpC beta-lactamases in diagnostic settings. Still, the detection of AmpC β- lactamases is cumbersome, and no approved methods are found in CLSI guidelines. But the prevalence of AmpC beta-lactamases has increased drastically in Asia. The review aims to give an overview of AmpC β-lactamases. The objective of this review is to review the evolution, types, detection methods, recent world epidemiology, treatment options, and current updates of the AmpC beta-lactamases.

Characterisation of ESBL/AmpC-Producing Enterobacteriaceae isolated from poultry farms in Peninsular Malaysia.

Extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases (AmpCs)-producing Enterobacteriaceae have been increasingly reported and imposing significant threat to public. Livestock production industry might be the important source for clinically important ESBL-producing Enterobacteriaceae. This study aims to investigate the resistance profile, phenotypic ESBL production, beta-lactamase genes, virulence factors, and plasmid replicon types among 59 Enterobacteriaceae strains isolated from poultry faecal samples in Malaysia's commercial poultry farm. There were 38.7% and 32.3% of Escherichia coli resistant to cefotaxime and cefoxitin, respectively, while Klebsiellaspp. demonstrated resistance rate of 52.6% to both mentioned antimicrobials. Majority of the E. coli isolates carried blaTEM and blaCMY-2 group. blaSHV was the most prevalent gene detected in Klebsiellaspp., followed by blaDHA and blaTEM. Resistance to extended spectrum cephalosporin in our isolates was primarily mediated by plasmid mediated AmpC beta-lactamase such as CMY-2 group and DHA enzyme. The CTX-M genes were found in two ESBL-producing E. coli. IncF, IncI1, and IncN plasmids were most frequently detected in E. coli and Klebsiellaspp. The virulence factor, including EAST1 and pAA were identified at low frequency. This study highlights the poultry as a reservoir of resistance and virulence determinants and prevalence of plasmids in Enterobacteriaceae might drive their dissemination.

Rapid identification of carbapenemases by CarbAcineto NP test and the rate of beta-lactamases among Acinetobacter baumannii from a teaching hospital.

Background and Objectives:Acinetobacter baumannii has emerged as a major organism accounting for hospital acquired infections particularly in intensive care units. Due to production of different kinds of beta lactamases these bacteria have developed drug resistance rendering the treatment of such infections very difficult and expensive. Rapid identification of A. baumannii producing such beta-lactamases is the need of the hour in reducing morbidity and mortality associated with A. baumannii infections.Materials and Methods:A. baumannii was isolated from clinical samples like endotracheal aspirates, sputum, urine, exudates using standard culture techniques. Identification and drug sensitivity was done using Vitek 2 system. All the isolates were subjected to detection of ESBLs using phenotypic confirmatory test, plasmid mediated AmpC beta-lactamase by AmpC disc test, Carbapenemase production by CarbAcineto NP Test and Modified hodge method.Results:149 A. baumannii isolates were analysed for antimicrobial susceptibility and various beta-lactamase production. Results were evaluated for statistical significance using Chi-Square and P value. 81.8% of isolates were from male patients with majority of them above 50 years of age. 88.5% of samples were from ventilator associated pneumonia patients. 83.8% of isolates were sensitive to tigecycline. Only 10% to 12% of isolates were sensitive to carbapenems. 23.4% of isolates were ESBL producers and 46.9% of them were AmpC producers. Modified Hodge test method identified 63.7% of A. baumannii as carbapenemase producers where as CarbAcineto NP test identified 63% and exibiting 94.74% sensitivity, 93.22% specificity when compared to Modified Hodge test.Conclusion:Multidrug resistant Acinetobacter spp. is on the rise. Present study showed that high percentage of drug resistance in A. baumannii could be due to production of ESBLs, AmpC and carbapenemases. Among all beta lactamases carbapenemase producers are more and quickly raising in A. baumannii. Rapid, cost effective assay which can be adopted in all clinical laboratories is critical to prevent their further transmission particularly in hospital environment.

Effect of the Different Plasmid-Mediated AmpC Beta-Lactamase Genotypes on the Phenotypic Detection of ESBL in Enterobacteriaceae Isolates.

Increasingly, Enterobacteriaceae isolates that are positive for the extended-spectrum-beta-lactamase (ESBL) by phenotypic screening tests yield a negative result when tested with the reference Clinical and Laboratory Standards Institute (CLSI) ESBL confirmatory test. The aim is to determine to what extent the different plasmid AmpC (pAmpC) genotypes could affect the CLSI ESBL confirmatory test for detection of the ESBL phenotype in ESBL/pAmpC co-producers. A total of 253 Enterobacteriaceae isolates were screened for ESBL and AmpC production according to CLSI guidelines. Out of 186 ESBL and AmpC-screen-positive isolates, 96 isolates were selected for ESBL confirmation by the combined disc diffusion test (CDDT) as well as for detection of the most common ESBL and pAmpC encoding-genes by multiplex PCR. Out of the 96 ESBL/AmpC-screen-positive isolates, all (100%) were positive for at least one of the investigated ESBL genes, and 88 (91.7%) were positive for any of the investigated pAmpC genes. CDDT correctly identified ESBL phenotype more frequently in non-pAmpC carriers than pAmpC carriers (75% vs. 52.3%). CIT alone-containing isolates were associated more with non-confirmed ESBL phenotype rather than confirmed ESBL phenotype (76.2% vs. 30.4%, p < 0.001), especially when co-harbored blaCTXM alone (76.9% vs. 33.3%, p < 0.001) or both blaCTXM/blaSHV genes (100% vs. 0%). On the other hand, DHA-carrying isolates were more associated with confirmed ESBL phenotype than with non-confirmed ESBL phenotype when co-harboring either blaCTXM alone (47.6% vs. 0%, p < 0.001) or blaCTXM/blaSHV genes (100% vs. 0%, p = 0.022) or blaCTX-M/blaTEM genes (100% vs. 0%, p = 0.03). For ESBL/pAmpC co-producers of Enterobacteriaceae, CDDT results vary with the type of pAmpC genes and with different ESBL/pAmpC genotype combinations. Therefore, the ESBL-screening test is more sensitive than the CDDT in detecting ESBL phenotype among ESBL/pAmpC coproducers of Enterobacteriaceae.

Phenotypic and genotypic characterization of plasmid-mediated AmpC beta-lactamases in enteric Gram-negative bacteria from patients with lower respiratory tract infections in a tertiary hospital, southwest Nigeria

Background: AmpC or class C or group 1 beta lactamases are class C cephalosporinases that hydrolyse a wide variety of beta-lactam antibiotics including alpha methoxy beta-lactams (cefoxitin), narrow and broad spectrum cephalosporins. This study was conducted to characterize plasmid-mediated AmpC producing enteric Gram- negative bacteria from patients with lower respiratory tract infections in Obafemi Awolowo University Teaching Hospital Complex (OAUTHC) Ile Ife, Osun State, NigeriaMethodology: A total of 149 patients with clinical features of lower respiratory tract infections (LRTI) were selected by simple random sampling for the study. All Gram-negative isolates recovered from standard microbiological cultures of respiratory specimens of these patients were tested against cefoxitin, third generation cephalosporins (3GCs), and other antibiotics using the disc diffusion AST method, and also screened for production of AmpC beta-lactamases phenotypically by the CLSI method. Plasmid DNA extraction was carried out on twenty-nine cefoxitin-resistant selected isolates using the Kado and Lin method, while genotypic detection of plasmid-mediated AmpC gene was carried out by the polymerase chain reaction (PCR) assay.Results: The results showed that 204 (43.3%) of 471 isolates recovered from the 149 selected patients were resistant to 3GC in the AST assay, among which 121 (59.3%) were resistant to cefoxitin, and 189 of the 471 isolates (40.1%) were AmpC producers. The AmpC producers concurrently showed multiple resistance pattern to other antibiotics tested in this study. Ninety six percent of the 29 selected isolates for plasmid analysis contained plasmids, 45% of which amplified positive on PCR for CMY, 38% for FOX, and 31% for ACC types of AmpC genes.Conclusion: This study showed a high degree of antibiotic resistance among enteric Gram-negative bacteria recovered from patients with LRTIs, as well as high degree of plasmid-encoded AmpC genes responsible for this high antibiotic resistance among the isolates. Proper antibiotic policy and regulation are required to limit the spread of plasmid mediated AmpC β-lactamase producing organisms because they can lead to therapeutic failure in infected patients in the nearest future.&#x0D; &#x0D; French title: Caractérisation phénotypique et génotypique des bêta-lactamases AmpC à médiation plasmidique dans les bactéries entériques Gram-négatives de patients atteints d'infections des voies respiratoires inférieures dans un hôpital tertiaire, sud-ouest du Nigéria&#x0D; Contexte: Les bêta-lactamases AmpC ou de classe C ou de groupe 1 sont des céphalosporinases de classe C qui hydrolysent une grande variété d'antibiotiques bêta-lactamines, y compris les alpha-méthoxy bêta-lactamines (céfoxitine), les céphalosporines à spectre étroit et large. Cette étude a été menée pour caractériser les bactéries à Gram négatif entériques produisant de l'AmpC à médiation plasmidique chez des patients atteints d'infections des voies respiratoires inférieures du complexe hospitalier universitaire d'Obafemi Awolowo (OAUTHC) Ile Ife, État d'Osun, NigériaMéthodologie: Un total de 149 patients présentant des caractéristiques cliniques d'infections des voies respiratoires inférieures (LRTI) ont été sélectionnés par échantillonnage aléatoire simple pour l'étude. Tous les isolats à Gram négatif récupérés à partir de cultures microbiologique standard d'échantillons respiratoires de ces patients ont été testés contre la céfoxitine, les céphalosporines de troisième génération (3GC) et d'autres antibiotiques en utilisant la méthode AST de diffusion sur disque, et également criblés pour la production de bêtalactamases AmpC phénotypiquement par le Méthode CLSI. L'extraction de l'ADN plasmidique a été réalisée sur 29 isolats sélectionnés résistants à la céfoxitine en utilisant la méthode Kado et Lin, tandis que la détection génotypique du gène AmpC à médiation plasmidique a été réalisée par le test de réaction en chaîne par polymérase (PCR).Résultats: Les résultats ont montré que 204 (43,3%) des 471 isolats récupérés des 149 patients sélectionnés étaient résistants à la 3GC dans le test AST, parmi lesquels 121 (59,3%) étaient résistants à la céfoxitine et 189 des 471 isolats (40,1%) étaient des producteurs d'AmpC. Les producteurs d'AmpC ont montré simultanément plusieurs profils de résistance à d'autres antibiotiques testés dans cette étude. Quatre-vingt-seize pour cent des 29 isolats sélectionnés pour l'analyse des plasmides contenaient des plasmides, dont 45% amplifiés positifs par PCR pour CMY, 38% pour FOX et 31% pour les types ACC des gènes AmpC.Conclusion: Cette étude a montré un degré élevé de résistance aux antibiotiques parmi les bactéries entériques Gram-négatives récupérées chez des patients atteints de LRTI, ainsi qu'un degré élevé de gènes AmpC codés par plasmide responsable de cette résistance élevée aux antibiotiques parmi les isolats. Une politique et une réglementation appropriées en matière d'antibiotiques sont nécessaires pour limiter la propagation des organismes producteurs β-lactamase d'AmpC à médiation plasmidique car ils peuvent conduire à un échec thérapeutique chez les patients infectés dans un avenir proche.&#x0D;

Prevalence of plasmid-mediated AmpC beta-lactamases in Enterobacteria isolated from urban and rural folks in Uganda

Background: AmpC beta-lactamase-producing bacteria are associated with increased resistance to third-generation cephalosporins. Here, we describe plasmid-mediated AmpC beta-lactamase-producing enterobacteria isolated from urban and rural dwellers in Uganda.Methods: Stool and urine from 1,448 individuals attending outpatient clinics in Kampala and two rural districts in central Uganda were processed for isolation of Escherichia coli and Klebsiella. Following antibiotic susceptibility testing, cefoxitin resistant isolates, and amoxicillin/clavulanate resistant but cefoxitin susceptible isolates, were tested for AmpC beta-lactamase production using the cefoxitin-cloxacillin double-disc synergy test. Carriage of plasmid-mediated AmpC beta-lactamase-encoding genes (pAmpC) and extended spectrum beta-lactamase (ESBL) encoding genes was determined by PCR.Results: Nine hundred and thirty E. coli and 55 Klebsiella were recovered from the cultured samples, yielding 985 isolates investigated (one per participant). One hundred and twenty-nine isolates (13.1%, 129/985) were AmpC beta-lactamase producers, of which 111 were molecularly characterized for pAmpC and ESBL gene carriage. pAmpC genes were detected in 60% (67/111) of the AmpC beta-lactamase producers; pAmpC genes were also detected in 18 AmpC beta-lactamase non-producers and in 13 isolates with reduced susceptibility to third-generation cephalosporins, yielding a total of 98 isolates that carried pAmpC genes. Overall, the prevalence of pAmpC genes in cefoxitin resistant and/or amoxicillin/clavulanate resistant E. coli and Klebsiella was 59% (93/157) and 26.1% (5/23), respectively. The overall prevalence of pAmpC-positive enterobacteria was 10% (98/985); 16.4% (45/274) in Kampala, 6.2% (25/406) Kayunga, and 9.2% (28/305) Mpigi. Ciprofloxacin use was associated with carriage of pAmpC-positive bacteria while residing in a rural district was associated with protection from carriage of pAmpC-positive bacteria.Conclusion: pAmpC beta-lactamase producing enterobacteria are prevalent in urban and rural dwellers in Uganda; therefore, cefoxitn should be considered during routine susceptibility testing in this setting.

Plasmid-Encoded AmpC and Extended-Spectrum Beta-Lactamases in Multidrug-Resistant Escherichia coli Isolated from Piglets in Portugal.

Considering the concept of "One Health," the aim of this study was to determine susceptibility profiles of Escherichia coli in piglets' intestinal microbiota from different farms in Portugal. Beyond antimicrobial susceptibility, the occurrence of multiple antibiotic resistance and detection of phenotypic/genotypic extended-spectrum beta-lactamases (ESBLs) and plasmid mediated AmpC beta-lactamases (pAmpC) were done. From 10 different pig farms, 340 E. coli isolates were obtained from 75 feces samples. Susceptibility to amoxicillin-clavulanic acid (AMC), piperacillin (PIP), cefoxitin (FOX), ceftazidime (CAZ), cefepime (FEP), aztreonam (AZT), imipenem (IP), amikacin (AK), ciprofloxacin (CIP), and trimethoprim-sulfamethoxazole (SXT) was determined. Five-gene panel for amplification of bla genes was used for ESBL (TEM, SHV, CTX-M) and pAmpC (CMY-2, ACC). Among E. coli isolates, 209 were distributed in three resistance profiles: 57.7% MDR, 3.5% extensively drug-resistance (XDR) (resistant to CIP, SXT, and beta-lactams, except IP, with variability to AK) and 0.3% pandrug-resistance (PDR) (resistant to all antibiotics used). pAmpC and/or ESBLs genes were presented in 65% of the isolates. Presence of different associations of bla genes in the same isolate was the most observed (31%), and the most common were an ESBL (TEM) and a pAmpC (CMY-2). Presence of three or four bla genes in various associations were detected. These isolates were very resistant, especially those with four genes, which were resistant to beta-lactams (except IP), CIP, and SXT. This study showed a surprisingly high rate of MDR E. coli isolated in Portuguese piglets, with enzymes that impair activity of the most used antibiotics in human therapeutic.

Relating the prevalence of plasmid-mediated AmpC beta-lactamase genes to aquatic environmental factors

It is important that environmental parameters that may affect the prevalence of AmpC beta-lactamase genes are investigated to devise frameworks for their surveillance, management and prevention. The aim of this study was thus to determine which environmental parameters are associated with the prevalence of clinically relevant AmpC beta-lactamase genes in aquatic systems. River water was sampled from seven sites in the Crocodile West River, South Africa. Physical-chemical parameters, metal levels and beta-lactam levels were measured. Environmental DNA was extracted from the water samples and six AmpC beta-lactamase gene groups (ACC, ACT/MIR, BIL/LAT/CMY, DHA, FOX, MOX/CMY) were quantified using quantitative PCR. Additionally, 16S rRNA gene metabarcoding analyses were performed on eDNA for each site and metabolic pathways were predicted using PICRUST2. Network analysis was performed to establish co-occurrences of AmpC genes with environmental factors. Quantification results indicated that AmpC gene copy numbers were significantly high (Kruskal Wallis H Test, p < 0.05) at Sites 1–3 of the Crocodile West River. In contrast, no significant changes regarding environmental factors were observed across the seven sites. Results of network analysis indicated that the AmpC gene groups had limited associations with all the environmental parameters, except for some key bacterial families, specifically Pseudomonadaceae, Aeromonadaceae and Enterobacteriaceae. A significant positive correlation between population density and AmpC genes suggested that in more densely populated areas more faecal pollution will be prevalent which is associated with high AmpC gene levels. Areas such as these are also likely to be linked with more antibiotic use which supports the notion that pre-selection of AmpC genes occurs before entering the aquatic environment. Moreover, it was demonstrated that prevalent selectors of AmpC genes do not ensure that continuous selection occurs in an aquatic environment. This information could be vital in future detection and management of AmpC genes in aquatic systems.

Prevalence of plasmid-mediated AmpC beta-lactamases in Enterobacteria isolated from urban and rural folks in Uganda.

Background: AmpC beta-lactamase-producing bacteria are associated with increased resistance to third-generation cephalosporins. Here, we describe plasmid-mediated AmpC beta-lactamase-producing enterobacteria isolated from urban and rural dwellers in Uganda. Methods: Stool and urine from 1,448 individuals attending outpatient clinics in Kampala and two rural districts in central Uganda were processed for isolation of Escherichia coli and Klebsiella. Following antibiotic susceptibility testing, cefoxitin resistant isolates, and amoxicillin/clavulanate resistant but cefoxitin susceptible isolates, were tested for AmpC beta-lactamase production using the cefoxitin-cloxacillin double-disc synergy test. Carriage of plasmid-mediated AmpC beta-lactamase-encoding genes (pAmpC) and extended spectrum beta-lactamase (ESBL) encoding genes was determined by PCR. Results: Nine hundred and thirty E. coli and 55 Klebsiella were recovered from the cultured samples, yielding 985 isolates investigated (one per participant). One hundred and twenty-nine isolates (13.1%, 129/985) were AmpC beta-lactamase producers, of which 111 were molecularly characterized for pAmpC and ESBL gene carriage. pAmpC genes were detected in 60% (67/111) of the AmpC beta-lactamase producers; pAmpC genes were also detected in 18 AmpC beta-lactamase non-producers and in 13 isolates with reduced susceptibility to third-generation cephalosporins, yielding a total of 98 isolates that carried pAmpC genes. Overall, the prevalence of pAmpC genes in cefoxitin resistant and/or amoxicillin/clavulanate resistant E. coli and Klebsiella was 59% (93/157) and 26.1% (5/23), respectively. The overall prevalence of pAmpC-positive enterobacteria was 10% (98/985); 16.4% (45/274) in Kampala, 6.2% (25/406) Kayunga, and 9.2% (28/305) Mpigi. Ciprofloxacin use was associated with carriage of pAmpC-positive bacteria while residing in a rural district was associated with protection from carriage of pAmpC-positive bacteria. Conclusion: pAmpC beta-lactamase producing enterobacteria are prevalent in urban and rural dwellers in Uganda; therefore, cefoxitn should be considered during routine susceptibility testing in this setting.

Detection and quantification of clinically relevant plasmid-mediated AmpC beta-lactamase genes in aquatic systems

Abstract The extent and impact of plasmid-mediated AmpC beta-lactamase genes (pAmpCs) prevalence in aquatic environments is poorly understood. The aim of this study was to detect and quantify pAmpCs from the aquatic environment. The following pAmpCs were analysed with clinical TaqMan assays from isolated plasmids: ACC, ACT/MIR, BIL/LAT/CMY, DHA, FOX and MOX/CMY. Quantification was conducted using quantitative PCR (qPCR) and 3D chip-based digital PCR. The results of qPCR yielded 4,875.27 copies/ng DNA and dPCR, 1,640.58 copies/ng (Mann–Whitney U Test, p= 0.868). Redundancy analysis indicated that land coverage explains 90.49% (ANOVA, p= 0.601) of pAmpC variance. There was a correlation between the frequency and quantities of pAmpCs detected in each river and this could be related to anthropogenic influence. Frequencies of detection for pAmpCs were 25/36 for the Crocodile West River and 13/36 for the Marico River. Quantification resulted in higher copy numbers for the Crocodile West River and high copies in only two sites of the Marico River, thus reflecting degrees of anthropogenic influences on both rivers. The presence of these clinically relevant pAmpCs in aquatic systems are cause for concern, considering their potential impact if these genes are harboured by pathogens and become dispersed to human populations.

Genetic Diversity of CMY Beta-Lactamase Genes in Clinical Isolates of Escherichia coli in Myanmar: Identification of Three Novel Types and Updated Phylogenetic Classification of blaCMY.

The dissemination of CMY-type enzymes, one of the plasmid-mediated AmpC beta-lactamases, among Enterobacteriaceae has become an important public health concern. In this study, genetic diversity of CMY beta-lactamase genes was investigated for 50 blaCMY-positive isolates detected from 426 clinical isolates of Escherichia coli in Yangon, Myanmar. CMY genes were differentiated into 9 types, with blaCMY-42 being predominant (22 isolates, 44%), followed by blaCMY-2, blaCMY-6, blaCMY-146, and included three novel types (CMY-156, CMY-158, CMY-159). Among E. coli harboring blaCMY, phylogenetic group D-sequence type (ST)405 and A-ST410 were the most common genotypes, and blaCTX-M-15 was detected in 72% (36/50) of isolates. blaCMY-42 was distributed to phylogenetic groups A, B1, and D E. coli with 11 STs, which included 10 isolates harboring carbapenemase genes (blaNDM-4, blaNDM-5, or blaNDM-7). Phylogenetic analysis of all the blaCMY genes reported to date, including the three novel types in the present study, revealed the presence of at least four distinct genetic groups, that is, CMY-1, CMY-2, CMY-70, and CMY-98 group, showing less than 91% nucleotide sequence identities among different groups. CMY-2 group beta-lactamase genes, which contained by far the largest number of CMY types (89.7%) with extensive diversity, were divided into two clusters (I and II). While eight CMY types identified in the present study were classified into CMY-2 group cluster I, novel type CMY-159 was assigned into CMY-98 group with a Citrobacter freundii strain in Thailand.

Off-label use of ceftiofur in one-day chicks triggers a short-term increase of ESBL-producing E. coli in the gut.

This trial was designed to evaluate the off-label use of ceftiofur with Marek’s vaccine in one-day-old broiler chicks, a prophylactic treatment that has been done in some commercial hatcheries, on the emergence of extended-spectrum beta-lactamase producing Escherichia coli (ESBL-E. coli). A total of 168 chicks (Cobb500®) were used in a completely randomized design. Birds were assigned to two treatments (Marek’s vaccine plus saline vs Marek’s vaccine plus ceftiofur) and six repetitions, with 14 animals each. Cloacal swabs were collected from 1 to 14 days post-hatch. The majority (86%; p<0.0001) of the ESBL-producing isolates harboring blaCTX-M and blaSHV genes originated from animals receiving the antimicrobial. None of the isolates were positive for plasmid-mediated AmpC betalactamase genes (blaACC, blaCMY-2, blaDHA, blaFOX, blaMOX and blaMIR). These findings indicate that the off-label use of ceftiofur with Marek’s vaccine is associated with the short-term increase in ESBL-producing Escherichia coli in the gut of chicks.

Detection of Plasmid-Mediated AmpC Beta-lactamase Enzyme among Escherichia coli Isolates in Livestock, South Nigeria

Detection of Plasmid-Mediated AmpC Beta-lactamase Enzyme among Escherichia coli Isolates in Livestock, South Nigeria

Convergence of plasmid architectures drives emergence of multi-drug resistance in a clonally diverse Escherichia coli population from a veterinary clinical care setting

The purpose of this study was to determine the plasmid architecture and context of resistance genes in multi-drug resistant (MDR) Escherichia coli strains isolated from urinary tract infections in dogs. Illumina and single-molecule real-time (SMRT) sequencing were applied to assemble the complete genomes of E. coli strains associated with clinical urinary tract infections, which were either phenotypically MDR or drug susceptible. This revealed that multiple distinct families of plasmids were associated with building an MDR phenotype. Plasmid-mediated AmpC (CMY-2) beta-lactamase resistance was associated with a clonal group of IncI1 plasmids that has remained stable in isolates collected up to a decade apart. Other plasmids, in particular those with an IncF replicon type, contained other resistance gene markers, so that the emergence of these MDR strains was driven by the accumulation of multiple plasmids, up to 5 replicons in specific cases. This study indicates that vulnerable patients, often with complex clinical histories provide a setting leading to the emergence of MDR E. coli strains in clonally distinct commensal backgrounds. While it is known that horizontally-transferred resistance supplements uropathogenic strains of E. coli such as ST131, our study demonstrates that the selection of an MDR phenotype in commensal E. coli strains can result in opportunistic infections in vulnerable patient populations. These strains provide a reservoir for the onward transfer of resistance alleles into more typically pathogenic strains and provide opportunities for the coalition of resistance and virulence determinants on plasmids as evidenced by the IncF replicons characterised in this study.

Plasmid-mediated resistance to cephalosporins and quinolones in Escherichia coli from American crows in the USA.

American crow (Corvus brachyrhynchos) faeces were tested for Escherichia coli with plasmid-mediated quinolone resistance (PMQR), extended-spectrum beta-lactamases (ESBL) and AmpC beta-lactamases. A total of 590 faecal samples were collected at four roosting sites in the USA and cultivated on selective media. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were performed to assess clonality. Transferability of resistance genes was studied using conjugation and transformation bioassays. In total, 78 (13%, n = 590) cefotaxime-resistant isolates were obtained, of which 66 and 12 displayed AmpC and ESBL phenotypes, respectively. Fifty-four AmpC-producing isolates carried blaCMY-2 . Isolates producing ESBLs contained genes blaCTX-M-27 (5 isolates), blaCTX-M-15 (4), blaCTX-M-14 (2) and blaCTX-M-1 (1). Ninety isolates (15%, n = 590) with reduced susceptibility to ciprofloxacin were obtained, among which 14 harboured PMQR genes aac(6')-Ib-cr (4 isolates), qnrB19 (3), qnrS1 (2), qnrA1 (2), qnrB2 (1), qnrB6 (1) and qnrD3 (1). High genetic diversity was revealed by PFGE and MLST. Epidemiologically important E. coli clones (e.g., ST131, ST405) were identified. Plasmids carrying blaCMY-2 were assigned predominantly to IncA/C (8 plasmids), IncI1/ST23 (5) and IncI1/ST12 (3). The study demonstrates a widespread occurrence of E. coli with ESBL, AmpC and PMQR genes associated with clinically important multidrug-resistant clones and epidemic plasmids, in American crows.

Emergence of serotype K1 Klebsiella pneumoniae ST23 strains co-producing the plasmid-mediated AmpC beta-lactamase DHA-1 and an extended-spectrum beta-lactamase in Korea.

BackgroundSerotype K1 Klebsiella pneumoniae has emerged as an important community pathogen causing various infections, including liver abscesses. Although serotype K1 K. pneumoniae community isolates have been reported as susceptible to most classes of antimicrobial agents, a few cases of infection caused by extended-spectrum beta-lactamase (ESBL)-producing serotype K1 K. pneumoniae have recently been reported in Asian countries. We identified three ESBL-producing strains of serotype K1 K. pneumoniae and conducted a molecular characterization of their drug resistance.MethodsThree ESBL-producing serotype K1 K. pneumoniae ST23 strains were identified from strains in the Asian Bacterial Bank. Antimicrobial susceptibility testing was performed using the broth microdilution method, and ESBL production was tested by the double-disk synergy test and a confirmatory test. PCR was performed to detect the genes for plasmid-mediated ESBL and AmpC beta-lactamases.ResultsAll three strains were resistant to cefotaxime, ceftazidime, and piperacillin/tazobactam, and all were determined to be ESBL-producers. No known ESBL genes, including bla SHV, bla TEM, bla CTX-M, bla GES, bla PER, and bla VEB, were detected among the three strains. Of all plasmid-mediated AmpC beta-lactamase (PAB) genes, including bla DHA-1, bla CMY, bla FOX, and bla MOX, the bla DHA-1 gene was detected in two of the strains. The PFGE patterns revealed that the two isolates carrying bla DHA-1 were closely related (84% similarity).ConclusionsNo ESBL genes were detected among three ESBL-producing serotype K1 K. pneumoniae ST23 strains. Two strains contained the PAB gene bla DHA-1. The emergence of resistant strains of community-origin serotype K1 K. pneumoniae has important implications for effective treatment and infection control practices.

Emergence of co-production of plasmid-mediated AmpC beta-lactamase and ESBL in cefoxitin-resistant uropathogenic Escherichia coli.

Plasmid-mediated AmpC (pAmpC) and ESBL co-production was detected in Escherichia coli a major etiologic agent of urinary tract infection. Isolates resistant to cefoxitin by CLSI methodology were tested for pAmpC beta-lactamase using phenylboronic acid and ESBLs by combined disk diffusion method. pAmpC/ESBL genes were characterized by PCR and sequencing. Transconjugation experiments were done to study the transfer of pAmpC and ESBL production from clinical isolates as donor to E. coli J53 AziR as recipient. Incompatibility groups of transmissible plasmids were classified by PCR-based replicon typing (PBRT). Among 148 urine culture positive isolates, E. coli was reported in 39.86% (59/148), with 93.22% (55/59) of cefoxitin resistance. pAmpC production was detected in 25, with varied distribution of blaCMY-2 and blaDHA-1type genes alone (n = 13 and 7 respectively) or in combination (n = 5). ESBL co-production was observed in 88% (22/25) of pAmpC producing isolates with predominance of blaTEM (n = 20). Twenty-three transconjugants showed transmission of pAmpC-and ESBL-resistant genes with co-carriage of blaCMY-2 and blaTEM (n = 15) in plasmids of IncF type (n = 9) being predominant, followed by IncI1 (n = 4) and IncH1 (n = 2) in combination. All clinical isolates were clonally diverse. Resistance against different beta-lactams in uropathogenic E. coli has been an emerging concern in resource- poor countries such as India. Knowledge on the occurrence of AmpC beta-lactamases and ESBL amongst this pathogen and its transmission dynamics may aid in hospital infection control.

Characteristics of extended-spectrum β-lactamase (ESBL)- and pAmpC beta-lactamase-producing Enterobacteriaceae of water samples in Tunisia

The presence of extended-spectrum beta-lactamase and plasmid-mediated AmpC beta-lactamase producing Enterobacteriaceae (ESBL-Eb and pAmpC-Eb, respectively) was analyzed in 57 wastewater and 57 surface-water samples in Tunisia. Twenty-four of the 57 wastewater samples (42.1%) and one of the 57 surface-water samples (1.7%, a river that received effluents of a wastewater-treatment-plant) contained ESBL-Eb or pAmpC-Eb; one ESBL/pAmpC-Eb per positive sample was further characterized. Beta-lactamase genes detected were as follows: blaCTX-M-1 (10 Escherichia coli),blaCTX-M-15 (eight E. coli, one Klebsiella pneumoniae, one Citrobacter freundii), blaCTX-M-14 (one E. coli) and blaCMY-2 (four E. coli). The blaTEM-1, blaOXA-1 or blaSHV-1 genes were also found in 72% of these isolates. The ISEcp1, orf477 or IS903 sequences were found upstream or downstream of blaCTX-M genes. Class 1 integrons were present in 16 of the 25 ESBL-Eb/pAmpC-Eb strains (64%), and contained five different gene-cassette arrays. Most of the strains (76%) showed a multiresistant phenotype and qnr genes were identified in four strains. Molecular typing of ESBL/CMY-2-producing E. coli isolates showed 23 different PFGE-patterns and 15 different sequence-types (ST10, ST46, ST48, ST58, ST69, ST101, ST117, ST131, ST141, ST288, ST359, ST399, ST405, ST617, and the new ST4530); these strains were ascribed to phylogroups A (11 isolates), B1 (3 isolates), D (6 isolates) and B2 (3 isolates). From one to five plasmids were detected in each strain (size from 30kb to >240kb) and ESBL or pAmpC genes were transferred by conjugation in 69.5% of the E. coli strains. In conclusion, ESBL-Eb and pAmpC-Eb strains are frequently detected in wastewater samples and they might be a source for dissemination in other environments with repercussion in public health.