pAmpC Genes Research Articles

Extended-Spectrum Beta-Lactamase- and Plasmidic AmpC-Producing Enterobacterales among the Faecal Samples in the Bulgarian Community.

The aim of the present work was to genetically characterise cefotaxime-resistant enterobacteria isolated from community carriers in Bulgaria. In total, 717 faecal samples from children and adults in five medical centres in Sofia, Pleven and Burgas were examined. Antimicrobial susceptibility was evaluated by the disk diffusion method. blaESBL or plasmidic AmpC (pAmpC) genes were detected by PCR and sequencing. MLST and ERIC-PCR were used to detect clonal relatedness. Among the faecal samples, 140 cefotaxime-resistant enterobacteria were found. The most frequently detected species was Escherichia coli (77.9%, 109/140 samples), followed by Klebsiella pneumoniae (7.9%, 11/140). Among the isolates, blaCTX-M-15 (37.1%) was predominant, followed by blaCTX-M-3 (19.2%), blaCTX-M-14 (10%), and blaCTX-M-27 (4.3 %). Genes encoding pAmpC were observed in 11.4% (blaDHA-1, 16/140) and in 1.4% (blaCMY-2, 2/140). The frequency of ESBL and pAmpC producers among the subjects was 14.6% and 2.5%, respectively. No carbapenem-resistant isolates were found. Four main clonal complexes (CC131, CC10, CC38, and CC155) were detected among E. coli isolates. The most common type was ST131, phylogroup B2 (16.5%). The increased frequency of ESBL- and pAmpC-producing enterobacteria in the community is a prerequisite for treatment failures of the associated infections and a good background for further studies.

Occurrence and characteristics of extended-spectrum-β-lactamase- and pAmpC-producing Klebsiella pneumoniae isolated from companion animals with urinary tract infections.

This study examined 70 Klebsiella pneumoniae isolates derived from companion animals with urinary tract infections in Taiwan. Overall, 81% (57/70) of the isolates carried extended-spectrum β-lactamase (ESBL) and/or plasmid-encoded AmpC (pAmpC) genes. ESBL genes were detected in 19 samples, with blaCTX-M-1, blaCTX-M-9, and blaSHV being the predominant groups. pAmpC genes were detected in 56 isolates, with blaCIT and blaDHA being the predominant groups. Multilocus sequence typing revealed that sequence types (ST)11, ST15, and ST655 were prevalent. wabG, uge, entB, mrkD, and fimH were identified as primary virulence genes. Two isolates demonstrated a hypermucoviscosity phenotype in the string test. Antimicrobial susceptibility testing exhibited high resistance to β-lactams and fluoroquinolones in ESBL-positive isolates but low resistance to aminoglycosides, sulfonamides, and carbapenems. Isolates carrying pAmpC genes exhibited resistance to penicillin-class β-lactams. These findings provide valuable insights into the role of K. pneumoniae in the context of the concept of One Health.

Plasmid-mediated fosfomycin resistance in Escherichia coli isolates of worldwide origin

Fosfomycin is a first-line treatment for uncomplicated urinary tract infections (UTIs) in several European countries, and it is increasingly becoming the treatment of choice globally. Resistance to fosfomycin in Escherichia coli can be exerted through several mechanisms, including the acquisition of fosfomycin-modifying enzymes, of which the FosA-type enzymes are the most common. This study analysed, both phenotypically and genotypically, an international collection of E. coli strains harbouring acquired fosA genes. Thirty-one fosA-positive E. coli isolates were obtained from both clinical and environmental sources, from seven countries (Portugal (n=12), Switzerland (n=9), China (n=3), France (n=2), Nepal (n=2), South Africa (n=2), Kuwait (n=1)). MICs were determined according to EUCAST guidelines. Whole genome sequencing (WGS) was performed on 23 isolates, and complete fosA plasmid sequences were determined for 12. Conjugation assays were performed on seven isolates. All isolates exhibited high-level resistance to fosfomycin (64 to >256 mg/L). WGS of 23 isolates identified 17 sequence types (STs), and 16 harboured fosA3, four fosA4, two fosA8, and one fosA10. ESBLs, pAmpC, or carbapenemase genes were present in 15, four, and three isolates, respectively. The fosA plasmids of 12 isolates were determined and were diverse in size (∼67 kb to ∼235 kb), resistance gene carriage, and replicon types. Six fosA plasmids additionally carried ESBL or carbapenemase genes. Conjugation assays, performed on seven isolates harbouring diverse plasmids, identified that all were capable of being transmitted. This study highlights the necessity of the surveillance and close monitoring of fosfomycin resistance in E. coli, essential to maintain the optimal use of this treatment option.

Detection of plasmid-mediated AmpC β-lactamases in Klebsiella pneumoniae clinical isolates from Bushehr province, Iran.

Plasmid-mediated AmpC producers are considered an increasing concern. The aim of this study was to investigate the prevalence of plasmid-mediated AmpC β-lactamases (pAmpCs) in Klebsiella pneumoniae isolates. A total of 228 clinical isolates of K. pneumoniae were collected in Bushehr province, Iran, from December 2017 to February 2019. Cefoxitin disks were applied for screening AmpC-producing isolates. Furthermore, 3 phenotypic confirmatory tests including combine disk test (CDT), double disk synergy test (DDST) and modified three dimensional test (M3DT) were used. Finally, the presence of pAmpC genes was tested by multiplex PCR. We identified 18 pAmpC-KP isolates among the 228 isolates (7.9%): 12 DHA (66.6%) and 6 CMY (33.3%). In the present study only 47% of cefoxitin-resistant isolates were pAmpC producers. The sensitivity of CDT, DDST, and M3DT was 89%, 67% and 100% and the specificity was 90%, 90% and 85%, respectively. In addition, M3DT displayed a higher rate of efficiency (92%) than CDT (89%) and DDST (79%) in detecting plasmid-meditated AmpC producers. DHA was the most prevalent pAmpC beta-lactamase in this study. DDST and CDT tests proved inefficient to detect two and six pAmpC producers, respectively, while M3DT represented the best overall performance.

Plasmid-mediated AmpC in Klebsiella pneumoniae and Escherichia coli from septicaemic neonates: diversity, transmission and phenotypic detection

Presence and dissemination of plasmid-mediated AmpC genes (pAmpCs) have made bacteria cephalosporin-resistant and assessment of their prevalence and diversity is essential. Coexistence of pAmpCs with New Delhi metallo-β-lactamase (blaNDM) has facilitated their spread and NDM interferes with correct pAmpC phenotypic identification. Assessment of pAmpCs in different species and sequence types (STs), co-transmission with blaNDM and phenotypic detection were analysed among Klebsiella pneumoniae (n=256) and Escherichia coli (n=92) isolated from septicaemic neonates over 13 years. pAmpCs were present in 9% (30/348) of strains, 5% in K. pneumoniae and 18% in E. coli. pAmpC genes (blaCMY and blaDHA) were detected, blaCMY-42 and blaDHA-1 variants being predominant. Strains were resistant to most antimicrobials tested. blaCMY and blaDHA were dominant among E. coli (14/17) and K. pneumoniae (9/13), respectively. pAmpC-bearing strains belonged to diverse STs, including epidemic K. pneumoniae ST11 and ST147. Some strains co-harboured carbapenemase genes, blaNDM (17/30) and blaOXA-48 (5/30). In 40% (12/30) of strains, pAmpC genes were transferred by conjugation, of which 8/12 exhibited co-transfer with blaNDM. pAmpCs were frequently found in replicons as follows: blaDHA-1 with IncHIB-M, blaCMY-4 with IncA/C, blaCMY-6 with IncA/C, and blaCMY-42 with IncFII. The combination disk-diffusion test correctly detected pAmpC in 77% (23/30) of pAmpC-bearing strains. However, correct detection of pAmpC was higher in strains that did not harbour blaNDM vs. those with blaNDM (85% vs. 71%). Presence of pAmpCs along with carbapenemases, linkage with multiple STs, and replicon types indicated their potential for spread. pAmpCs can go undetected in the presence of blaNDM; hence, regular surveillance is required.

Development of a Method for the Fast Detection of Extended-Spectrum β-Lactamase- and Plasmid-Mediated AmpC β-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae from Dogs and Cats in the USA.

Antibiotic resistance, such as resistance to beta-lactams and the development of resistance mechanisms, is associated with multifactorial phenomena and not only with the use of third-generation cephalosporins. Many methods have been recommended for the detection of ESBL and pAmpC β-lactamase production but they are very subjective and the appropriate facilities are not available in most laboratories, especially not in clinics. Therefore, for fast clinical antimicrobial selection, we need to rapidly detect ESBL- and pAmpC β-lactamase-producing bacteria using a simple method with samples containing large amounts of bacteria. For the detection of ESBL- and pAmpC phenotypes and genes, the disk diffusion test, DDST and multiplex PCR were conducted. Of the 109 samples, 99 (90.8%) samples were grown in MacConkey broth containing cephalothin, and 71 samples were grown on MacConkey agar containing ceftiofur. Of the 71 samples grown on MacConkey agar containing ceftiofur, 58 Escherichia coli and 19 Klebsiella pneumoniae isolates, in particular, harbored β-lactamase genes. Of the 38 samples that did not grow in MacConkey broth containing cephalothin or on MacConkey agar containing ceftiofur, 32 isolates were identified as E. coli, and 10 isolates were identified as K. pneumoniae; β-lactamase genes were not detected in these E. coli and K. pneumoniae isolates. Of the 78 ESBL- and pAmpC β-lactamase-producing E. coli and K. pneumoniae, 55 (70.5%) isolates carried one or more ESBL genes and 56 (71.8%) isolates carried one or more pAmpC β-lactamase genes. Our method is a fast, and low-cost tool for the screening of frequently encountered ESBL- and pAmpC β-lactamase-producing bacteria and it would assist in diagnosis and improve therapeutic treatment in animal hospitals.

Multidrug-Resistant ESBL-Producing E. coli in Clinical Samples from the UK.

Globally, cephalosporin therapy failure is a serious problem for infection control. One causative agent of cephalosporin-resistant infections is multidrug-resistant (MDR) E. coli producing extended-spectrum β-lactamases (ESBLs) and/or plasmid-encoded AmpC (pAmpC) β-lactamases. We evaluated the occurrence of ESBL/pAmpC genetic determinants in phenotypically MDR E. coli isolated from clinical samples of blood, faeces, ear effusion, urine and sputum from a UK hospital. Phenotypic resistance profiling for 18 antibiotics (from seven classes) showed that 32/35 isolates were MDR, with resistance to 4-16 of the tested antibiotics. Of the isolates, 97.1% showed resistance to ampicillin, 71.4% showed resistance to co-amoxiclav, cefotaxime, ceftazidime and ceftiofur, and 68.5% showed resistance to cefquinome. blaCTX-M, blaTEM and blaOXA-1 genes were detected in 23, 13 and 12 strains, respectively, and Intl1 was detected in 17 isolates. The most common subtypes among the definite sequence types were CTX-M-15 (40%) and TEM-1 (75%). No E. coli isolates carried pAmpC genes. Significant correlations were seen between CTX-M carriage and cefotaxime, ceftiofur, aztreonam, ceftazidime and cefquinome resistance; between blaCTX-M, blaTEM and blaOXA-1 carriage and ciprofloxacin resistance; and between Intl1 carriage and trimethoprim/sulfamethoxazole resistance. Thus, MDR phenotypes may be conferred by a relatively small number of genes. The level and pattern of antibiotic resistance highlight the need for better antibiotic therapy guidelines, including reduced use and improved surveillance.

Rapid Detection of Plasmid AmpC Beta-Lactamases by a Flow Cytometry Assay.

Plasmidic AmpC (pAmpC) enzymes are responsible for the hydrolysis of extended-spectrum cephalosporins but they are not routinely investigated in many clinical laboratories. Phenotypic assays, currently the reference methods, are cumbersome and culture dependent. These methods compare the activity of cephalosporins with and without class C inhibitors and the results are provided in 24–48 h. Detection by molecular methods is quicker, but several genes should be investigated. A new assay for the rapid phenotypic detection of pAmpC enzymes of the Enterobacterales group-I (not usually AmpC producers) based on flow cytometry technology was developed and validated. The technology was evaluated in two sites: FASTinov, a spin-off of Porto University (Portugal) where the technology was developed, and the Microbiology Department of Ramón y Cajal University Hospital in Madrid (Spain). A total of 100 strains were phenotypically screened by disk diffusion for the pAmpC with the new 2 h assay. Molecular detection of the pAmpC genes was also performed on discrepant results. Forty-two percent of the strains were phenotypically classified as pAmpC producers using disk diffusion. The percentage of agreement of the flow cytometric assay was 93.0%, with 95.5% sensitivity and 91.1% specificity. Our proposed rapid assay based on flow cytometry technology can, in two hours, accurately detect pAmpC enzymes.

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.

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.

Phenotypic and Genotypic Characteristics of Uropathogenic Escherichia coli Isolates from Kenya.

Introduction: Uropathogenic Escherichia coli (UPECs) are a significant cause of urinary tract infections (UTIs). In Kenya, UTIs are typically treated with β-lactam antibiotics without antibiotic susceptibility testing, which could accelerate antibiotic resistance among UPEC strains.Aim: This study determined the occurrence of UPEC producing extended-spectrum β-lactamases (ESBLs), the genes conferring resistance to β-lactams, and the phylogenetic groups associated with ESBLs in Kenyan UPECs.Methodology: Ninety-five UPEC isolates from six Kenyan hospitals were tested for ESBL and plasmid-mediated AmpC β-lactamase (pAmpC) production by combined disk diffusion and disk approximation tests, respectively. Real-time and conventional polymerase chain reactions (PCRs) were used to detect three ESBL and six pAmpC genes, respectively, and phylogenetic groups were assigned by a quadruplex PCR method.Results: Twenty-four percent UPEC isolates were ESBL producers with blaCTX-M (95.6%), blaTEM (95.6%), and blaSHV (21.7%) genes detected. Sixteen isolates had blaCTX-M/TEM, whereas five had blaTEM/CTX-M/SHV. A total of 5/23 ESBLs were cefoxitin resistant, but no AmpC genes were detected. The UPECs belonged predominantly to phylogenetic groups B2 (31/95; 32.6%) and D (30/95; 31.6%), while groups B2 and A had the most ESBL producers.Conclusions: β-Lactam antibiotics have reduced utility for treating UTIs as a quarter of UPECs were ESBL producing. Single or multiple ESBL genes were present in UPECs, belonging primarily to phylogenetic groups B2 and A.

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.

Occurrence, identification, and antibiogram signatures of selected Enterobacteriaceae from Tsomo and Tyhume rivers in the Eastern Cape Province, Republic of South Africa.

The increasing occurrence of multidrug-resistant Enterobacteriaceae in clinical and environmental settings has been seen globally as a complex public health challenge, mostly in the developing nations where they significantly impact freshwater used for a variety of domestic purposes and irrigation. This paper detail the occurrence and antibiogram signatures of the Enterobacteriaceae community in Tsomo and Tyhume rivers within the Eastern Cape Province, the Republic of South Africa, using standard methods. The average distribution of the presumptive Enterobacteriaceae in the rivers ranged from 1 × 102 CFU/100ml to 1.95 × 104 CFU/100ml. We confirmed 56 (70.8%) out of 79 presumptive Enterobacteriaceae isolated being species within the family Enterobacteriaceae through the Matrix-Assisted Laser Desorption Ionization Time of Flight technique. Citrobacter-, Enterobacter-, Klebsiella species, and Escherichia coli were selected (n = 40) due to their pathogenic potentials for antibiogram profiling. The results of the antibiotic susceptibility testing gave a revelation that all the isolates were phenotypically multidrug-resistant. The resistance against ampicillin (95%), tetracycline and doxycycline (88%), and trimethoprim-sulfamethoxazole (85%) antibiotics were most prevalent. The Multiple Antibiotic Resistance indices stretched from 0.22 to 0.94, with the highest index observed in a C. freundii isolate. Molecular characterisation using the PCR technique revealed the dominance of blaTEM (30%; 12/40) among the eight groups of β-lactams resistance determinants assayed. The prevalence of others was blaCTX-M genes including group 1, 2 and 9 (27.5%), blaSHV (20%), blaOXA-1-like (10%), blaPER (2.5%), and blaVEB (0%). The frequencies of the resistance determinants for the carbapenems were blaKPC (17.6%), blaGES (11.8%), blaIMP (11.8%), blaVIM (11.8%), and blaOXA-48-like (5.9%). Out of the six plasmid-mediated AmpC (pAmpC) genes investigated blaACC, blaEBC, blaFOX, blaCIT, blaDHA, and blaMOX, only the first four were detected. In this category, the most dominant was blaEBC, with 18.4% (7/38). The prevalence of the non-β-lactamases include tetA (33.3%), tetB (30.5%), tetC (2.8%), tetD (11.1%), tetK (0%), tetM (13.9%), catI (12%), catII (68%), sulI (14.3%), sulII (22.9%) and aadA (8.3%). Notably, a C. koseri harboured 42.8% (12/28) of the genes assayed for which includes five of the ESBL genes (including the only blaPER detected in this study), two of the pAmpC resistance genes (blaACC and blaCIT), and five of the non-β-lactamase genes. This study gives the first report on C. koseri exhibiting the co-occurrence of ESBL/AmpC β-lactamase genes from the environment to the best of our knowledge. The detection of a blaPER producing Citrobacter spp. in this study is remarkable. These findings provide evidence that freshwater serves as reservoirs of antimicrobial resistance determinants, which can then be easily transferred to human beings via the food chain and water.

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.

Microbiological risk assessment of Turkey and chicken meat for consumer: Significant differences regarding multidrug resistance, mcr or presence of hybrid aEPEC/ExPEC pathotypes of E. coli

To assess the microbiological risk for consumers, we propose a lab workflow based on six virulence/antimicrobial resistance (AMR) traits, and including a duplex PCR for the screening of extraintestinal pathogenic E. coli (ExPEC). This protocol was tested in 100 poultry meat products. The characterization of 323 isolates revealed that poultry meat is a rich phylogenetic source of E. coli phylogroups (A to G) and Escherichia clade I. Non-susceptible E. coli isolates to monobactams, 3rd-generation cephalosporins and/or fluoroquinolones, were present in 71% of the samples. Besides, 47% carried ≥2 different E. coli positive for ESBL, pAmpC or mcr genes. Isolates from 78% of the poultry meat exhibited ExPEC status, and 53% were carriers of isolates positive for the uropathogenic (UPEC) status. The sequence types (STs) identified in 86% of the samples belonged to the so-called ExPEC high-risk lineages, being 73% carriers of clonal groups identified in human infections of the same Health Area. Moreover, different human-associated clones co-occurred in same meat sample: ST131-B2 (CH40-22), ST648-F (CH4-58), ST93-A (CH11-neg) or ST95-B2 (CH38-27), ST354-F (CH88-58), ST155-B1 (CH4-neg). Globally, 84% of the meat samples posed ≥3 risks, including resistance genes, successful clones and virulence traits. Turkey meat showed significant higher rates concerning mcr-carriage or multidrug resistance; while the ExPEC status rate, or the presence of hybrid pathotypes such as the aEPEC/ExPEC O153:H10-A-ST10 (CH11-54), were associated with chicken origin (P < 0.05). In a “Farm to Fork Strategy”, ExPEC should be clearly included in food surveillance.

β-lactam and fluoroquinolone resistance in Enterobacteriaceae from imported and locally-produced chicken in Mozambique.

Plasmid-mediated resistance to β-lactam and fluoroquinolone antibiotics was investigated in Enterobacteriaceae isolated from retailed frozen chickens from Brazil, South Africa and Mozambique. Carcass swabs and the liquid thaw of 33 chickens from each of the three countries constituted the total sample size of 198. Isolates were identified by biochemical tests, antibiotic susceptibility was ascertained by the disc diffusion assay and β-lactamases were detected using the double-disk synergy test. PCR was used to detect the presence of blaCTX-M, blaSHV, blaTEM, blaCMY, blaMOX, blaFOX, blaDHA, qnrB, qnrD, qnrS and qepA genes. A random selection of CTX-M genes was sequenced. The 198 samples yielded 27 (13.6%) putative extended-spectrum β-lactamase (ESBL)-positive isolates, 15 from carcass swabs and 12 from the liquid thaw from 22 chickens with 19, 5 and 3 isolates from South African, Mozambican and Brazilian chicken, respectively. Isolates exhibited the following resistance: ampicillin 100%, ceftriaxone 89%, trimethoprim-sulfamethoxazole 78%, cefotaxime 74%, ciprofloxacin 70%, ceftazidime 67%, cefoxitin 22% and gentamicin 8%. The predominant putative ESBL gene was blaSHV (85%), followed by blaCTX-M (62.9%) and blaTEM (44.4%) whilst blaMOX and blaDHA were the most common pAmpC genes at 33.3%. The predominant plasmid-mediated fluoroquinolone-resistance gene was qepA (22.2%). DNA sequencing identified blaCTX-M-55/-79/-101/-164. ERIC-PCR profiles did not show strong evidence of clonality. The Mozambican population is exposed to a reservoir of plasmid-mediated, and hence mobile β-lactam and quinolone resistance genes via imported, and to a lesser extent, locally produced poultry. This presents a food safety concern.

Molecular characteristics of antimicrobial resistance determinants and integrons in Salmonella isolated from chicken meat in Korea

Summary The increase and spread of antimicrobial resistance, particularly multidrug resistance (MDR), in Salmonella isolated from food animal pose a serious public health concern worldwide. Therefore, the objective of this study was to characterize antimicrobial resistance and analyze the transferability of integrons and antimicrobial resistance genes in MDR Salmonella isolates from retail chicken meat in Korea. Among the 57 Salmonella isolates, 43 (75.4%) showed resistance to at least 1 antimicrobial agent and 29 (50.9%) exhibited MDR. The highest resistance was to nalidixic acid (73.7%), ampicillin (50.9%), tetracycline (42.1%), chloramphenicol (33.3%), and trimethoprim/sulfamethoxazole (33.3%). β-lactamase–encoding genes were detected in 9 (31.0%) of 29 β-lactam–resistant isolates. These genes were as follows: blaCMY-2 (1 S. Albany), blaCTX-M-79 (1 S. Virchow), and blaCTX-M-15 (6 S. Virchow and 1 S. Enteritidis). Seventeen (70.8%) of the 24 tetracycline-resistant isolates harbored only tetA. Among the 19 trimethoprim/sulfamethoxazole-resistant isolates, sul1 was only identified in 3 (68.4%) isolates. cmlA was detected in 4 (21.1%) of the 19 chloramphenicol-resistant isolates. Both aminoglycoside-resistant isolates were positive for ant(2″)-I. Class 1 integrons were observed in 28 (49.1%) isolates belonging to S. Albany (21 isolates), S. Virchow (6 isolate), and S. Enteritidis (1 isolate). All 29 MDR isolates carried one or more integrons and antimicrobial resistance genes. All isolates harboring the ESBL and pAmpC genes exhibited resistance to at least 4 different classes of antimicrobials, including cephalosporins and noncephalosporins. Furthermore, most (79.3%) of the MDR isolates carried 1 or 2 of conjugable plasmids, and the transfer of integrons and resistance genes via plasmids was confirmed in transconjugants, which showed resistance patterns similar to those of the donor strains.

Prevalence of AmpC and Extended-Spectrum Beta-Lactamase Genes in Klebsiella pneumoniae and Escherichia coli Isolates

Background: In recent years, the prevalence of antibiotic resistance has steadily increased and also the antibiotic-resistant strains producing extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases have emerged among the Enterobacteriaceae, predominantly in Escherichia coli and Klebsiella pneumoniae species. Objectives: This prospective study aimed at determining the production of ESBL or AmpC, phenotypically and also at the molecular level, in E. coli and K. pneumoniae isolates collected from various clinical specimens. Methods: In total, 78 K. pneumoniae and 92 E. coli isolates were collected from various clinical infectious sources available in different wards of the Imam Reza Hospital, Tabriz, Iran, from July 2017 to December 2018. All isolates were subjected to antimicrobial susceptibility testing. MAST 4-disc test and polymerase chain reaction (PCR) were applied for phenotypic and genotypic detection of ESBLs and plasmid-encoded AmpCs (pAmpCs) among isolates, respectively. Results: Overall, 78 K. pneumoniae and 92 E. coli isolates were evaluated, of which 46 K. pneumoniae (58.9%) and 51 E. coli (55.4%) isolates were resistant to cefotaxime/ceftazidime and included in the study. Among the K. pneumoniae and E. coli isolates resistant to cefotaxime/ceftazidime, 40 (86.9%) and 40 (78.4%) isolates were ESBL producers and 8 (17.3%) and 2 (3.9%) isolates were pAmpC producers, respectively. In addition, 40 E. coli (78.4%) isolates were positive for both CTX-M-14 and CTX-M-15 genes. Regarding K. pneumoniae isolates, 40 isolates (86.9%) were positive for CTX-M-15 gene and 18 isolates (39.1%) for CTX-M-14 gene. Among 51 ceftazidime/cefotaxime-resistant E. coli isolates, 32 isolates (62.7%) were positive for DHA-1 gene and 33 isolates (64.7%) isolates for CMY-2 gene. Also, among 46 ceftazidime/cefotaxime-resistant K. pneumoniae isolates, 15 isolates (32.6%) had DHA-1 gene and 27 isolates (58.7%) had CMY-2 gene in the genome. Conclusions: The high prevalence of ESBL and AmpC production among E. coil and K. pneumoniae isolates was a serious concern in the studied region. Therefore, a simple and rapid PCR-based technique is essential to detect and distinguish various pAmpC and ESBL genes to discriminate other resistance determinants

CTX-M-producing Escherichia coli Isolated from urban pigeons (Columba livia domestica) in Brazil.

Worldwide urban pigeons (Columba livia domestica) are an important reservoir of pathogenic and multidrug-resistant bacteria (MDR). Plasmids are key genetic elements in the dissemination of antimicrobial drug resistance in bacteria, including beta-lactams and quinolones, which are the most important classes of drugs for treatment of Enterobacteriaceae infections in human and veterinary medicine. The aim of this study was to determine the presence of Escherichia coli (E. coli) harboring plasmids containing extend-spectrum (ESBL) and pAmpC beta-lactamases, also plasmid-mediated quinolone resistance (PMQR) genes in urban pigeons from São Paulo State, Brazil. A collection of 107 isolates of E. coli from urban pigeons from four cities was screened by antimicrobial resistance phenotypic and PCR for genes encoding ESBL, pAmpC and PMQR genes. Clonality was evaluated by ERIC-PCR. We found three strains positive for blaCTX-M genes. In two clonally related CTX-M-8-producing strains, the gene was associated with IncI1 plasmids. An MDR strain harboring blaCTX-M-2, the plasmid could not be transferred. No strain was positive for PMQR genes. These results indicate that CTX-M-2 and CTX-M-8-producing E. coli are present in urban pigeons, which could serve as a reservoir for ESBL-producing E. coli in Brazil.

CTX-M- and pAmpC-Encoding Genes Are Associated with Similar Mobile Genetic Elements in Escherichia coli Isolated from Different Brands of Brazilian Chicken Meat.

In this study we characterized the genetic environment of blaCTX-M and blaCMY-2 genes carried by 46 Escherichia coli isolates obtained from 20 chicken carcasses produced by five different brands in Brazil, including exporters and antibiotic-free-certified producers, purchased between 2010 and 2014. Similar plasmids characterized according to size and incompatibility group (Inc) were identified in E. coli belonging to different MLST-ST collected, regardless of carcass brand or production system. Hybridization assays with transconjugant strains revealed that blaCMY-2 gene (n = 19) was located on 85 kb plasmids of IncB/O, IncI1, IncFIB, or nontypeable groups. blaCTX-M-8 (n = 9) was located on 90 kb IncI1 plasmids. blaCTX-M-2 (n = 14) was inserted in class 1 integrons and conjugated only by one isolate in a 125 kb IncP plasmid. blaCTX-M-15 (n = 1), rarely described in isolates from food-producing animals in South America, was characterized by whole genome sequencing of transconjugant; the gene was carried in a 49.3 kb IncX1 plasmid. Sequencing of bla gene-flanking regions indicated the association of these genes with previously described insertion sequences. These results suggest that conserved genetic environments are related to ESBL and pAmpC genes in the Brazilian chicken production chain.