Plasmid-mediated AmpC Β-lactamase Research Articles

Rapid detection of plasmid-mediated AmpC-producers by eazyplex® SuperBug AmpC assay compared to whole-genome sequencing

Current methods for plasmid-mediated AmpC β-lactamase (pAmpC) detection in routine microbiological laboratories are based on various phenotypic tests. Eazyplex®SuperBug AmpC assay is a molecular assay based on isothermal amplification for rapid detection of the most common pAmpC types from bacterial culture: CMY-2 group, DHA, ACC and MOX. Our aim was to evaluate the diagnostic performance of this assay. The assay was evaluated on 64 clinical isolates of Enterobacterales without chromosomal inducible AmpC, and with phenotypically confirmed AmpC production. The results were confirmed, and isolates further characterized by whole-genome sequencing (WGS). eazyplex®SuperBug AmpC assay correctly detected the two most common pAmpC types CMY-2 group (16/16) and DHA (19/19). Detection of ACC and MOX could not be evaluated on our set of isolates since there was only one isolate harbouring ACC and none with MOX. pAmpC encoding genes could be detected in only eight of 36 investigated Escherichia coli isolates. The remaining 28 E. coli isolates harboured previously described mutations in the blaEC promoter, leading to the overexpression of chromosomally encoded E. coli specific AmpC β-lactamase. All results were 100% concordant with the results of WGS. eazyplex®SuperBug AmpC assay enabled rapid and reliable detection of pAmpC-encoding genes in Enterobacterales like Klebsiella spp. and Proteus spp. and the distinction between plasmid-mediated and chromosomally encoded AmpC in E. coli.

Global transmission of extended-spectrum cephalosporin resistance in Escherichia coli driven by epidemic plasmids

Extended-spectrum cephalosporins (ESCs) are third and fourth generation cephalosporin antimicrobials used in humans and animals to treat infections due to multidrug-resistant (MDR) bacteria. Resistance to ESCs (ESC-R) in Enterobacterales is predominantly due to the production of extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases (AmpCs). The dynamics of ESBLs and AmpCs are changing across countries and host species, the result of global transmission of ESC-R genes. Plasmids are known to play a key role in this dissemination, but the relative importance of different types of plasmids is not fully understood. In this study, Escherichia coli with the major ESC-R genes blaCTX-M-1, blaCTX-M-15, blaCTX-M-14 (ESBLs) and blaCMY-2 (AmpC), were selected from diverse host species and other sources across Canada, France and Germany, collected between 2003 and 2017. To examine in detail the vehicles of transmission of the ESC-R genes, long- and short-read sequences were generated to obtain complete contiguous chromosome and plasmid sequences (n=192 ESC-R E.coli). The types, gene composition and genetic relatedness of these plasmids were investigated, along with association with isolate year, source and geographical origin, and put in context with publicly available plasmid sequences. We identified five epidemic resistance plasmid subtypes with distinct genetic properties that are associated with the global dissemination of ESC-R genes across multiple E.coli lineages and host species. The IncI1 pST3 blaCTX-M-1 plasmid subtype was found in more diverse sources than the other main plasmid subtypes, whereas IncI1 pST12 blaCMY-2 was more frequent in Canadian and German human and chicken isolates. Clonal expansion also contributed to the dissemination of the IncI1 pST12 blaCMY-2 plasmid in ST131 and ST117 E.coli harbouring this plasmid. The IncI1 pST2 blaCMY-2 subtype was predominant in isolates from humans in France, while the IncF F31:A4:B1 blaCTX-M-15 and F2:A-:B- blaCTX-M-14 plasmid subtypes were frequent in human and cattle isolates across multiple countries. Beyond their epidemic nature with respect to ESC-R genes, in our collection almost all IncI1 pST3 blaCTX-M-1 and IncF F31:A4:B1 blaCTX-M-15 epidemic plasmids also carried multiple antimicrobial resistance (AMR) genes conferring resistance to other antimicrobial classes. Finally, we found genetic signatures in the regions surrounding specific ESC-R genes, identifying the predominant mechanisms of ESC-R gene movement, and using publicly available databases, we identified these epidemic plasmids from widespread bacterial species, host species, countries and continents. We provide evidence that epidemic resistance plasmid subtypes contribute to the global dissemination of ESC-R genes, and in addition, some of these epidemic plasmids confer resistance to multiple other antimicrobial classes. The success of these plasmids suggests that they may have a fitness advantage over other plasmid types and subtypes. Identification and understanding of the vehicles of AMR transmission are crucial to develop and target strategies and interventions to reduce the spread of AMR. This project was supported by the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR), through the Medical Research Council (MRC, MR/R000948/1), the Canadian Institutes of Health Research (CFC-150770), and the Genomics Research and Development Initiative (Government of Canada), the German Federal Ministry of Education and Research (BMBF) grant no. 01KI1709, the French Agency for food environmental and occupational health & safety (Anses), and the French National Reference Center (CNR) for antimicrobial resistance. Support was also provided by the Biotechnology and Biological Sciences Research Council (BBSRC) through the BBSRC Institute Strategic Programme Microbes in the Food ChainBB/R012504/1 and its constituent project BBS/E/F/000PR10348 (Theme 1, Epidemiology and Evolution of Pathogens in the Food Chain).

The causes of bacterial bloodstream infections and antimicrobial resistance patterns in children attending a secondary care hospital in Bhaktapur, Nepal, 2017-2022: a retrospective study.

Data on antimicrobial resistance (AMR) among children in Nepal are limited. Here we have characterized the causes of bacterial bloodstream infections (BSIs), antimicrobial resistance patterns and the mechanisms of β-lactamase production in Enterobacterales among children attending outpatient and inpatient departments of a secondary care paediatric hospital in Nepal. We retrospectively collected demographic and clinical data of culture-proven bacterial BSIs between January 2017 and December 2022 among children <18 years attending a 50-bedded paediatric hospital. Stored isolates were subcultured for antimicrobial susceptibility testing against commonly used antimicrobials. Enterobacterales displaying non-susceptibility to β-lactams were phenotypically and genotypically investigated for ESBLs, plasmid-mediated AmpC (pAmpC) β-lactamases and carbapenemases. A total of 377 significant bacteria were isolated from 27 366 blood cultures. Among 91 neonates with a BSI, Klebsiella pneumoniae (n = 39, 42.4%), Pseudomonas aeruginosa (n = 15, 16.3%) and Acinetobacter baumannii complex (n = 13, 14.1%) were most common. In the non-neonates, 275/285 (96.5%) infections were community-acquired including Staphylococcus aureus (n = 89, 32.4%), Salmonella Typhi (n = 54, 19.6%) and Streptococcus pneumoniae (n = 32, 11.6%). Among the 98 S. aureus, 29 (29.6%) were methicillin-resistant Staphylococcus aureus. K. pneumoniae and Escherichia coli demonstrated non-susceptibility to extended-spectrum cephalosporins and carbapenems in both community and hospital-acquired cases. For E. coli and K. pneumoniae, blaCTX-M (45/46), blaEBC (7/10) and blaOXA-48 (5/6) were common among their respective groups. We determined significant levels of AMR among children attending a secondary care paediatric hospital with BSI in Nepal. Nationwide surveillance and implementation of antimicrobial stewardship policies are needed to combat the challenge imposed by AMR.

Spatial diarrheal disease risks and antibiogram diversity of diarrheagenic Escherichia coli in selected access points of the Buffalo River, South Africa.

Freshwater sources, often used for domestic and agricultural purposes in low- and middle-income countries are repositories of clinically significant bacterial pathogens. These pathogens are usually diversified in their antibiogram profiles posing public health threats. This study evaluated the spatial diarrhoeal disease risk and antibiogram diversity of diarrheagenic Escherichia coli (DEC) in four access points of the Buffalo River, Eastern Cape Province, South Africa using standard epidemiological, culture, and molecular methods. The diarrhoeal disease risk was characterised using the Monte Carlo simulation, while the antibiogram diversity was assessed using the species observed Whittaker's single alpha-diversity modelling. E. coli mean count was highest in King William's Town dam [16.0 × 102 CFU/100ml (SD: 100.0, 95% CI: 13.5 × 102 to 18.5 × 102)]. Enterohemorrhagic E. coli (stx1/stx2) was the most prevalent DEC pathotype across the study sites. A high diarrhoeal disease risk of 25.0 ×10-2 exceeding the World Health Organization's standard was recorded across the study sites. The average single and multiple antimicrobial resistance indices of the DEC to test antimicrobials were highest in the Eluxolzweni dam [0.52 (SD: 0.25, 95% CI: 0.37 to 0.67)] and King William's Town dam [0.42 (SD: 0.25, 95% CI: 0.27 to 0.57)] respectively. The prevalent antibiotic resistance genes detected were tetA, blaFOX and blaMOX plasmid-mediated AmpC, blaTEM and blaSHV extended-spectrum β-lactamases, which co-occurred across the study sites on network analysis. The phenotypic and genotypic resistance characteristics of the DEC in Maden dam (r = 0.93, p<0.00), Rooikrantz dam (r = 0.91, p<0.00), King William's Town dam (r = 0.83, p = 0.0), and Eluxolzweni dam (r = 0.91, p<0.00) were strongly correlated. At least, three phylogenetic clades of the DEC with initial steep descent alpha-diversity curves for most of the test antimicrobials were observed across the study sites, indicating high diversity. The occurrence of diversified multi drug resistant DEC with diarrhoeal disease risks in the Buffalo River substantiates the role surface water bodies play in the dissemination of drug-resistant bacterial pathogens with public health implications.

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.

Genetic relatedness of third-generation cephalosporin-resistant Escherichia coli among livestock, farmers, and patients in Japan

ObjectivesThe third-generation cephalosporin (3GC)-resistant E. coli strains have been detected worldwide in humans and animals. Hence, in this study, we evaluated the prevalence and genetic characteristics of 3GC-resistant E. coli in livestock, farmers, and patients to further analyse if livestock serves as a potential reservoir of antimicrobial-resistant bacteria. MethodsFaecal samples were collected from 330 healthy livestock (216 cattle and 114 swine), 61 healthy livestock farmers (52 cattle farmers and 9 swine farmers), and 68 non-duplicate 3GC-resistant E. coli isolates were also obtained from the clinical specimens of patients in Japan between 2013 and 2015. Genes associated with resistance in 3GC-resistant E. coli were identified using polymerase chain reaction (PCR) and DNA sequencing. Genotypic diversity was determined by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). ResultsWe obtained 39 and 17 non-duplicated 3GC-resistant E. coli strains from healthy livestock (33 cattle and six swine) and livestock farmers, respectively. All isolates carried either CTX-M-type extended-spectrum β-lactamase or plasmid-mediated AmpC β-lactamase genes, with CTX-M-14 being the most frequent. CTX-M producers from livestock and patients belonged to 22 and 19 different sequence types (STs), respectively, and only three STs were the same. Among the 3GC-resistant E. coli from livestock and farmers, three types of CTX-M producers have shown similar characteristics (CTX-M genotype, ST, PFGE patterns, and antimicrobial susceptibilities) and were identified as clonal isolates shared among their farms. ConclusionsOur study findings indicate that CTX-M-14 is predominant in Japan. No distinct relationship was observed between the 3GC-resistant E. coli isolated from livestock and patients; however, some clonal relatedness was observed between the isolates from livestock and farmers due to their close contact.

Biofilm Formation Ability of ESBL/pAmpC-Producing Escherichia coli Isolated from the Broiler Production Pyramid.

Escherichia coli able to produce extended spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases (pAmpCs) represents a serious threat to public health, since these genes confer resistance to critically important antimicrobials (i.e., third generation cephalosporins) and can be transferred to non-resistant bacteria via plasmids. E. coli are known to be able to form a biofilm, which represents a favorable environment for the exchange of resistance determinants. Here, we assessed the ability of 102 ESBL/pAmpC-producing E. coli isolated from the broiler production pyramid to form a biofilm and to identify genetic factors involved in biofilm formation. All but one of the ESBL/pAmpC-producing E. coli were able to form a biofilm, and this represents a great concern to public health. E. coli belonging to phylogroups D, E, and F, as well as strains harboring the blaCTX-M-type gene, seem to be associated with an increased biofilm capability (p &lt; 0.05). Furthermore, virulence genes involved in adherence and invasion (i.e., csgBAC, csgDEFG, matABCDEF, and sfaX) seem to enhance biofilm formation in E. coli. Efforts should be made to reduce the presence of ESBL/pAmpC- and biofilm-producing E. coli in the broiler production pyramid and, therefore, the risk of dissemination of resistant bacteria and genes.

A Novel Single-Tube Eicosaplex/Octaplex PCR System for the Detection of Extended-Spectrum β-Lactamases, Plasmid-Mediated AmpC β-Lactamases, and Integrons in Gram-Negative Bacteria.

We developed two multiplex polymerase chain reactions (PCRs) for the detection of extended-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC β-lactamases, aac(6')-Ib gene, and integrase genes (intI1, intI2, and intI3) in class 1, 2, and 3 integrons in Gram-negative bacteria. We evaluated the PCRs using 109 Gram-negative isolates from non-organic (ANO) and organic (AO) vegetables and fruits. Screening of ANO substances identified five SHV, one TEM-1, one CTX-M, 20 AmpC-CS, and two intI1 positives. DNA sequencing revealed CTX-M in Pantoea spp. was blaRANH-2, a plasmid-mediated CTX-M related ESBL gene only found in Rahnella spp. Of the 20 AmpC-CS positives, 10 were CMY/MIR/ACT/EC (3 new variants), eight were ACT, one was AZECL, and one was new Pseudomonas-related AmpC family. Screening of AO substances identified 11 SHV, two TEM-1, three CTX-M (one OXY-2, two CTX-M-14/-15), two OXA-9, 13 AmpC-CS and one intI1 positives. The 13 AmpC-CS positives were five CMY/MIR/ACT/EC, three ACT, one MOX-12 variant, and four ADC (one ADC-25 and three new variants). We developed a rapid, easy-to-perform, low-cost, and reliable multiplex PCR system for screening clinically relevant β-lactamases and integrons in Gram-negative bacteria. We showed the prevalence of ESBLs and AmpC β-lactamases among our panel of ampicillin-resistant Gram-negative strains and detection of NDM and OXA carbapenemases.

Distribution of β-lactamases and emergence of carbapenemases co-occurring Enterobacterales isolates with high-level antibiotic resistance identified from patients with intra-abdominal infection in the Asia-Pacific region, 2015-2018.

In this study, we aimed to assess the geographic distribution and molecular characteristics of β-lactamases among Enterobacterales isolates causing intra-abdominal infections (IAIs) from 2015 to 2018 in the Asia-Pacific region. Isolates were investigated for extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases, and carbapenemases using multiplex PCR assays and full-gene DNA sequencing. A total of 832 Enterobacterales isolates from 8 different countries with β-lactamase genes were analysed. Plasmid-mediated ESBLs and AmpC β-lactamases were encoded in 598 (71.9%) and 314 (37.7%) isolates, respectively. In 710 (85.3%) carbapenemase-negative isolates, positivity for both AmpC β-lactamases and ESBLs was identified in 51 (8.5%) Escherichia coli and 24 (3.4%) Klebsiella pneumoniae isolates. The most prevalent countries were Taiwan and Vietnam, and the co-occurrence of CMY/CTX-M in E. coli and DHA-1/ESBLs in K. pneumoniae was predominant. All isolates showed high susceptibility to colistin, but susceptibility to carbapenems varied among different resistance mechanism combinations. Among 122 (14.7%) isolates encoding carbapenemase, NDM (n=67, including 64.2% NDM-1) was the most common, followed by the OXA-48-type (n=49), KPC (n=24) and IMP (n=4). The most prevalent country was Thailand (n=44), followed by Vietnam (n=35) and the Philippines (n=21). Twenty-two isolates were found to encode multiple carbapenemases, 16 of which were collected from Thailand and harbored NDM-1, OXA-232 and CTX-M-15. Despite high susceptibility to amikacin, susceptibility to colistin was only 56%. The emergence of carbapenem-non-susceptible AmpC/ESBL co-occurring Enterobacterales and colistin non-susceptible carbapenemases co-occurring K. pneumoniae highlights potential therapeutic challenges in the Asia-Pacific region.

Evolution of Beta-Lactamases in Urinary Klebsiella pneumoniae Isolates from Croatia; from Extended-Spectrum Beta-Lactamases to Carbapenemases and Colistin Resistance.

K. pneumoniae isolates often harbor various antibiotic resistance determinants including extended-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC β-lactamases (p-Amp-C) and carbapenemases. In this study we analyzed 65K. pneumoniae isolates obtained from urinary tract infections in the outpatients setting, with regard to antibiotic susceptibility, β-lactamase production, virulence traits and plasmid content.Antibiotic susceptibility was determined by broth microdilution method. PCR was applied to detect genes encoding ESBLs, p-Amp-C and carbapenemases and plasmid incompatibility groups. Phenotypic methods were applied to characterize virulence determinants. Increasing resistance trend was observed for amoxicillin/clavulanate, imipenem, meropenem and ciprofloxacin. The study showed that ESBLs belonging to the CTX-M family, conferring high level of resistance to expanded-spectrum cephalosporins (ESC) were the dominant resistance trait among early isolates (2013 to 2016) whereas OXA-48 carbapenemase, belonging to class D, emerged in significant numbers after 2017. OXA-48 producing organisms coharbored ESBLs. KPC-2 was dominant among isolates from Dubrovnik in the recent years. Colistin resistance was reported in three isolates. Inc L/M was the dominant plasmid in the later period, encoding OXA-48. Hyperviscosity was linked to KPC positivity and emerged in the later period. This report describes evolution of antibiotic resistance in K. pneumoniae from ESBLs to carbapenemases and colistin resistance. The study demonstrated the ability of K. pneumoniae to acquire various resistance determinants, over time. The striking diversity of the UTI isolates could result from introduction of the isolates from the hospitals, transfer of plasmids and multidirectional evolution.

Fecal Carriage of Extended-Spectrum β-Lactamases and pAmpC Producing Enterobacterales in an Iranian Community: Prevalence, Risk Factors, Molecular Epidemiology, and Antibiotic Resistance.

This study aimed to determine the prevalence and risk factors associated with intestinal carriage of extended-spectrum β-lactamases producing Enterobacterales (ESBL-PE) and plasmid-mediated AmpC β-lactamase producing Enterobacterales (AmpC-PE) in healthy children in Ardabil, Iran. A total of 305 fecal samples were collected. Isolates underwent antimicrobial susceptibility testing, phenotypic and genotypic identification of β-lactamase production, and epidemiologic molecular typing. In total, 21.5%, 1.5%, and 1.2% of volunteers were extended-spectrum β-lactamase (ESBL)-, AmpC-, and simultaneous ESBL/AmpC-PE carriers, respectively. Escherichia coli was the predominant ESBL producing bacterium (70.2%) found in ESBL-PE colonized subjects. Beyond ESBL positive isolates, bla CTX-M group genes were the most common type (75.6%) and bla TEM (non-bla TEM-1 and non- bla TEM-2) were in the second place (25.6%). Among bla CTX-M genes, bla CTX-M-1 (55.3%) and bla CTX-M-15 (55.3%) were the most predominant types with equal prevalence. Some isolates were multi-enzyme producers. bla CIT and bla DHA genes were common AmpC type enzyme encoding genes found in AmpC-PE isolates. Most isolates produced both enzymes at the same time. The number of students in the classes was statistically associated with ESBL-PE intestinal carriage (p < 0.05). Moreover, 46 (65.7%), 3 (60%), 4 (100%), and 98 (39.8%) ESBL-, AmpC-, ESBL/AmpC, and non-ESBL/AmpC-PE isolates were multidrug-resistant, respectively. Overall, regardless of β-lactam antibiotics, 62% and 59.5% of isolates were resistant to co-trimoxazole and tetracycline, respectively. The majority of ESBL producing E. coli isolates (69.2%) belonged to phylogroup A. According to Enterobacterial repetitive intergenic consensus polymerase chain reaction, there was no clonal relatedness between isolates. This study showed a high rate of multi-resistant ESBL-PE intestinal carriage among healthy individuals in Iran.

Changes in the characteristics of community-onset fluoroquinolone-resistant Escherichia coli isolates causing community-acquired acute pyelonephritis in South Korea.

This study aimed to examine the changes in the characteristics of community-onset fluoroquinolone-resistant (FQ-R) Escherichia coli isolates causing community-acquired acute pyelonephritis (APN) in South Korea. Blood or urine samples were prospectively collected from patients aged ≥15 years with community-acquired APN who were admitted to one of the eight Korean hospitals included in this study between September 2017 and August 2018. Phylogenetic typing, multilocus sequence typing, and molecular characterization of β-lactamase resistance and plasmid-mediated quinolone resistance (PMQR) determinants were performed. The data were compared with those from a previous study with the same design conducted in 2010-2011. A total of 300 and 346 isolates were identified in 2010-2011 and 2017-2018, respectively. Among them, 76 (22.0%) and 77 (25.7%) FQ-R isolates were identified in 2010-2011 and 2017-2018, respectively. A significantly higher antimicrobial resistance against third-to fourth-generation cephalosporins, including cefotaxime (23.9% vs. 77.9%, P<0.001), were observed among FQ-R isolates in 2017-2018 than among those in 2010-2011. A higher proportion of ST131 isolates (27.6% vs. 66.2%, P<0.001), as well as isolates that had extended-spectrum β-lactamase (ESBL)/plasmid-mediated AmpC β-lactamase (PABL) (23.7% vs. 79.2%, P<0.001), was observed in 2017-2018 than in 2010-2011. Further, more PMQR determinants (11.8% vs. 40.8%, P<0.001) were observed in 2017-2018 than in 2010-2011. Among uropathogenic FQ-R E. coli isolates in South Korea, the prevalence of ST131 and the proportion of isolates containing ESBL and/or PMQR determinants have increased.

Evaluation of NG-Test CARBA 5 for the detection of carbapenemase-producing Gram-negative bacilli.

Carbapenemase-producing Enterobacterales (CPE) pose one of the most serious antimicrobial resistance threats to public health worldwide. The outcome of CPE infection differs depending on the resistance mechanism. Therefore, rapid detection of CPE infection is essential for optimizing patient management. The carbapenem inactivation method (CIM) and modified CIM (mCIM) are standard methods for detecting CPE, but they usually require 24 h to generate results. Recently, an immunochromatographic assay, NG-Test CARBA 5, has become commercially available. It detects the five most common carbapenemase producers (KPC, IMP, NDM, VIM, and OXA-48) rapidly and accurately. We aimed to evaluate the diagnostic accuracy of NG-Test CARBA 5 for detecting carbapenemase-producing Gram-negative bacilli (CPGNB). We used 116 carbapenemase-producing strains and 48 non-carbapenemase-producing strains. Of the 116 carbapenemase-producing strains, 107 harboured genes for at least one of the five most common carbapenemases, KPC, IMP, NDM, VIM, and OXA-48-like. Forty-eight non-carbapenemase-producing strains, including carbapenem-resistant Enterobacterales, harboured genes for extended-spectrum β-lactamases (CTX-M groups [n=25] and SHV groups [n=2]) or plasmid-mediated AmpC β-lactamases (DHA [n=3], CMY-2 [n=2], and CFE-1 [n=1]). Antimicrobial susceptibility was tested using the agar dilution method, according to the Clinical and Laboratory Standards Institute guidelines. Of the 116 carbapenemase-producing strains, 79 were resistant to at least meropenem or imipenem. The sensitivity and specificity of the NG-Test CARBA 5 for the strains were 99.1 % (106 strains positive for 107 strains of the five most common carbapenemase producers) and 100 % (60 strains negative for other types of CPGNB [n=10] and non-CPGNB strains [n=48]), respectively. The carbapenemase-producing strain with a false-negative result produced IMP-66. The NG-Test CARBA 5 had high sensitivity and specificity for detecting carbapenemase-producing strains.

Presence of the Extended-Spectrum-β-Lactamase and Plasmid-Mediated AmpC-Encoding Genes in Escherichia coli from Companion Animals-A Study from a University-Based Veterinary Hospital in Taipei, Taiwan.

Extended-spectrum-β-lactamase (ESBL) and AmpC β-lactamase are two enzymes commonly found in Enterobacteriaceae that confer resistance to major antibiotics, such as third-generation cephalosporins that are widely prescribed for both human and animals. We screened for Escherichia coli producing ESBL and plasmid-mediated AmpC β-lactamase (pAmpC) from dogs and cats brought to National Taiwan University Veterinary Hospital, Taipei, Taiwan from 29 June 2020, to 31 December 2020. The genotypes and phylogenetic relatedness of these E. coli were also analyzed. Fifty samples of E. coli obtained from 249 bacterial isolates were included in this study. Among them, eight isolates had ESBL, seven had pAmpC, and one had both. Thirty-two percent (16/50) of E. coli isolates were resistant to third-generation cephalosporins. The detected ESBL genes included the blaCTX-M-1 and blaCTX-M-9 groups, and the blaCMY-2 group was the only gene type found in pAmpC. ESBL-producing E. coli belonged to the pathogenic phylogroup B2, and the sequence types (STs) were ST131 and ST1193. Three isolates were determined to be ST131-O25b, a highly virulent epidemic clone. The pAmpC-producing E. coli were distributed in multiple phylogroups, primarily the commensal phylogroup B1. The STs of the pAmpC-producing E. coli included ST155, ST315, ST617, ST457, ST767, ST372, and ST93; all of these have been reported in humans and animals. Imipenem was active against all the ESBL/pAmpC-producing E. coli; however, since in humans it is a last-resort antimicrobial, its use in companion animals should be restricted.

Detection and characterisation of extended-spectrum and plasmid-mediated AmpC β-lactamase produced by Escherichia coli isolates found at poultry farms in Bosnia and Herzegovina.

Extended-spectrum β-lactamases (ESBLs) hydrolyse extended-spectrum cephalosporins (ESC) and aztreonam. As ESBL-producing organisms have been identified in food producing animals, the aim of our study was to detect and analyse such Escherichia coli isolates from poultry. Antibiotic susceptibility of the isolates was determined with disk-diffusion and broth microdilution methods. ESBLs were detected with the double-disk synergy and inhibitor-based test with clavulanic acid. The transferability of cefotaxime resistance was determined with conjugation experiments, and genes encoding ESBLs, plasmid-mediated AmpC β-lactamases, and quinolone resistance determinants identified by polymerase chain reaction. The study included 108 faecal samples (cloacal swabs) from 25 different poultry farms in the Zenica-Doboj Canton, Bosnia and Herzegovina. Of these, 75 (69.4 %) were positive for E. coli, of which 27 were resistant to cefotaxime, amoxicillin, cefazoline, and cefriaxone, and susceptible to imipenem, meropenem, ertapenem, and amikacin. All 27 cefotaxime-resistant isolates were positive in double-disk synergy and combined disk tests. Eighteen isolates transferred cefotaxime resistance to E. coli recipient. Twenty-one isolates were positive for the blaCTX-M-1 cluster genes and seven for blaCTX-M-15. Fourteen were positive for the blaTEM genes. The most frequent plasmid incompatibility group was IncFIB, whereas IncFIA and Inc HI1 were present in only a few isolates. Two different sequence types (STs) were identified: ST117 and ST155. The emergence of ESBL-producing E. coli in farm animals presents a public health threat, as they can colonise the intestine and cause infections in humans.

Prevalence of plasmid-mediated AmpC β-lactamases among uropathogenic Escherichia coli isolates in southwestern Iran.

ObjectivesThis study was undertaken to evaluate AmpC β-lactamase-producing Escherichia coli urine isolates and to characterize the frequency of plasmid-mediated AmpC (pAmpC)-encoding genes.MethodsAntimicrobial susceptibility tests were performed using the disk diffusion technique. AmpC β-lactamase production was assessed with a phenotypic inhibitor-based method. The presence of 6 pAmpC-encoding cluster genes was detected by multiplex polymerase chain reaction (PCR).ResultsThe proportion of antibiotic resistance of E. coli isolates ranged from 7.4% to 90.5%, and more than half (51.6%) of the total isolates were multidrug-resistant (MDR). Among the 95 E. coli isolates, 60 (63.2%) were found to be cefoxitin-resistant, but only 14 (14.7%) isolates were confirmed as AmpC β-lactamase-producers. In the PCR assay, pAmpC-encoding genes were found in 15 (15.8%) isolates, and blaDHA was the most prevalent type. However, blaFOX, blaMOX, and blaACC genes were not detected in the isolates.ConclusionOur findings contributed valuable information concerning antibiotic resistance, confirmatory phenotypic testing for AmpC production, and pAmpC β-lactamase gene content in E. coli isolates in southwestern Iran. The level of MDR recorded in AmpC-producing strains of this study was worrying; therefore, implementing strong infection control approaches to reduce the MDR burden is recommended.

Third-generation cephalosporin (3GC) resistance and its association with Extra-intestinal pathogenic Escherichia coli (ExPEC). Focus on broiler carcasses

The worldwide spread of Extra-intestinal Pathogenic Escherichia coli (ExPEC), together with the antimicrobial resistance linked with extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases (pAmpCs) are pressing threats for public health. This study aimed to investigate the presence of ExPEC genes in third-generation cephalosporin (3 GC)-resistant E. coli and to study their distribution in broiler carcasses at the slaughterhouse after the chilling process. To this purpose, isolates from a collection of 3 GC-resistant E. coli from carcasses of broilers originating from twelve broiler farms and three production chains were investigated. Several multivariate statistical approaches were adopted to elucidate the relationships among features. Phylogroup F was predominant in all broiler batches and was mainly associated with blaTEM and ESBL genes but less correlated to ExPEC genes. Another remarkable finding was the predominance of ExPEC strains assigned to uncommon phylogroups, such as B2, D, E and Clade I, commonly found into the environment. This study represents a first step for a comprehensive characterization of ExPEC genes harboured by 3 GC-resistant E. coli. These findings may be valuable for the identification of potential risks associated to broiler carcasses as source of uncommon E. coli phylogroups.

Prevalence and Characterization of ESBL/AmpC Producing Escherichia coli from Fresh Meat in Portugal.

The present study aimed to characterize the extended-spectrum β-lactamases and plasmid-mediated AmpC β-lactamases (ESBL/PMAβ) among Escherichia coli producers isolated from beef, pork, and poultry meat collected at retail, in Portugal. A total of 638 meat samples were collected and inoculated on selective medium for the search of E. coli resistant to 3rd generation cephalosporins. Isolates were characterized by antimicrobial susceptibility testing, molecular assays targeting ESBL/AmpC, plasmid-mediated quinolone resistance (PMQR), and plasmid-mediated colistin resistance (PMCR) encoding genes. The highest frequency of E. coli non-wild type to 3rd generation cephalosporins and fluoroquinolones was observed in broiler meat (30.3% and 93.3%, respectively). Overall, a diversity of acquired resistance mechanisms, were detected: blaESBL [blaCTX-M-1 (n = 19), blaCTX-M-15 (n = 4), blaCTX-M-32 (n = 12), blaCTX-M-55 (n = 8), blaCTX-M-65 (n = 4), blaCTX-M-27 (n = 2), blaCTX-M-9 (n = 1), blaCTX-M-14 (n = 11), blaSHV-12 (n = 27), blaTEM-52 (n = 1)], blaPMAβ [blaCMY-2 (n = 8)], PMQR [qnrB (n = 27), qnrS (n = 21) and aac(6’)-Ib-type (n = 4)] and PMCR [mcr-1 (n = 8)]. Our study highlights that consumers may be exposed through the food chain to multidrug-resistant E. coli carrying diverse plasmid-mediated antimicrobial resistance genes, posing a great hazard to food safety and a public health risk.

Occurrence of the Colistin Resistance Gene mcr-1 and Additional Antibiotic Resistance Genes in ESBL/AmpC-Producing Escherichia coli from Poultry in Lebanon: A Nationwide Survey.

ABSTRACTThe objective of the present study was to determine genomic characteristics of expanded-spectrum cephalosporin (ESC)-resistant Escherichia coli spreading in healthy broilers in Lebanon in 2018. Rectal swabs (n = 280) from 56 farms were screened for the presence of ESC-resistant E. coli isolates. Antimicrobial susceptibility and extended-spectrum β-lactamase (ESBL)/AmpC production were determined by the disk diffusion method. Whole-genome sequencing (WGS) of 102 representative isolates of E. coli was performed to determine their phylogenetic diversity, serotypes, sequence types (ST), acquired resistance genes, and virulence-associated genes. Fifty-two out of 56 farms housed broilers carrying ESC-resistant E. coli isolates. These farms had large and recurrent antimicrobial practices, using, for some of them, critically important antibiotics for prophylactic and therapeutic purposes. Among the 102 sequenced multidrug-resistant (MDR) E. coli isolates, the proportion of ESBL, plasmid-mediated AmpC β-lactamase (pAmpC) producers, and ESBL/pAmpC coproducers was 60%, 27.6%, and 12.4%, respectively. The most prevalent ESBL/pAmpC genes were blaCMY-2, blaCTX-M-3, blaCTX-M-15, blaCTX-M-27, and blaCTX-M-14b (n = 42, n = 31, n =15, n = 9, and n = 7, respectively). These ESBL/pAmpC producers were distributed in different STs, most being well-known avian-associated and sometimes pathogenic STs (ST-10, ST-48, ST-93, ST-115, ST-117, and ST-457). Phylogenetic single nucleotide polymorphism (SNP) analysis confirmed their genetic diversity and wide dispersion across the Lebanese territory. Most isolates were also resistant to ciprofloxacin (101/102 with 3 QRDR mutations), and 19/102 isolates from 11 unrelated STs also carried the mobile resistance gene mcr-1. This survey illustrates the alarming prevalence of MDR E. coli resistant to medically important antibiotics in broilers in Lebanon. This advocates the need for surveillance programs of antimicrobial resistance in Lebanon and the reduction of excessive use of antibiotics to limit the spread of MDR E. coli in food-producing animals.IMPORTANCE Poultry production is a main contributor of the global trend of antimicrobial resistance arising from food-producing animals worldwide. In Lebanon, inappropriate use of antibiotics is frequent in chickens for prophylactic reasons and to improve productivity, resulting in an alarming prevalence of extended-spectrum β-lactamase (ESBL)/AmpC-producing Escherichia coli, also resistant to other medically important antibiotics (i.e., colistin and ciprofloxacin). Their complex genomic epidemiology highlighted by an important genetic diversity suggests that these resistance determinants are largely spreading in enteric bacteria in Lebanese poultry. Further molecular surveillance is needed to understand the country-specific epidemiology of ESBL/AmpC and mcr-1 genes in Lebanese poultry production. In addition, decisive interventions are urgently needed in order to ban the use of critically important antibiotics for human medicine in food-producing animals and limit the spread of antibiotic resistance in Lebanon.

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.