Prevalence and Characterization of ESBL/AmpC Producing Escherichia coli from Fresh Meat in Portugal.
ABSTRACT: 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.
Project description:Animals are considered important sources of ESBL/AmpC-producing bacteria in humans. We analyzed indications of transfer of ESBL/AmpC genes between pigs and pig farmers in Vietnam by analyzing whole genome sequences of 114 ESBL/AmpC-producing <i>E. coli</i> isolated from the two hosts, and performed conjugation experiments and plasmid profiling to confirm that such transfer could have happened. ESBL-encoding genes detected in pigs and pig farmers included <i>bla</i> <sub>CTX-M-55</sub>, <i>bla</i> <sub>CTX-M-27</sub>, <i>bla</i> <sub>CTX-M-65</sub>, <i>bla</i> <sub>CTX-M-15</sub>, <i>bla</i> <sub>CTX-M-14</sub>, <i>bla</i> <sub>CTX-M-3</sub>, <i>bla</i> <sub>CTX-M-24</sub>, and <i>bla</i> <sub>CARB-2</sub>, and AmpC β-lactamases included <i>bla</i> <sub>CMY-2</sub>, <i>bla</i> <sub>DHA-1</sub>, and <i>bla</i> <sub>CMY-42</sub>. The most frequent ESBL gene, <i>bla</i> <sub>CTX-M-55</sub>, was carried on plasmid with replicons types IncF, IncX, IncH, IncN, IncR, and IncP. The insertion transposases downstream of the <i>bla</i> <sub>CTX-M-55</sub> gene were different in plasmids carried by different strains. The second most detected gene, <i>bla</i> <sub>CTX-M-27</sub>, is found in a stable genetic arrangement with the same flanking transposons seen across strains, and the gene was located on similar conjugal IncF plasmid types, suggesting a horizontal spread of these plasmids. In three strains, we observed a novel <i>bla</i> <sub>CTX-M-27</sub> harboring IncF type of plasmid which had not been reported before. Its closest reference in NCBI was the non-ESBL <i>Salmonella</i> Typhimurium plasmid pB71 that might have experienced an insertion of <i>bla</i> <sub>CTX-M-27</sub>. Our data also point to an emergence of plasmids co-carrying ESBL genes, <i>mcr</i> genes, quinolones and other antimicrobials resistance determinants, and such plasmids require special attention. Plasmids phylogeny confirmed that the <i>bla</i> <sub>CTX-M-55</sub> encoding plasmids varied considerably, while those encoding <i>bla</i> <sub>CTX-M-27</sub> were closely related. Plasmids harboring both ESBL genes were confirmed to be conjugative and not to differ in transfer efficacy. The isolates carrying the plasmids, even those with plasmids of similar types, showed wide genetic variation with high number of SNPs, suggesting horizontal spread of plasmids into different clonal lines. Their virulence profiles did not confirm to known pathotypes, suggesting that unrelated commensals are a main reservoir for ESBL and AmpC β-lactamases in both humans and pigs. Overall, despite evidence of transferability of plasmids in the analyzed strains, our findings do not support that ESBL-producing <i>E. coli</i> from pigs or their ESBL/AmpC encoding plasmids are commonly spread to workers in close contact with the animals.
Project description:The emergence and spread of multidrug-resistant <i>Salmonella enterica</i> (<i>S. enterica</i>) to humans through food of animal origin are considered a major global public health concern. Currently, little is known about the prevalence of important antimicrobial resistance genes in <i>S. enterica</i> from retail food in Africa. Therefore, the screening and characterization of the extended-spectrum β-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes in <i>S. enterica</i> isolated from retail meats and slaughterhouses in Egypt were done by using PCR and DNA sequencing techniques. Twenty-eight out of thirty-four (82.4%) non-duplicate <i>S. enterica</i> isolates showed multidrug-resistance phenotypes to at least three classes of antimicrobials, and fourteen (41.2%) exhibited an ESBL-resistance phenotype and harbored at least one ESBL-encoding gene. The identified β-lactamase-encoding genes included <i>bla</i><sub>CTX-M-1</sub>, <i>bla</i><sub>CTX-M-3</sub>, <i>bla</i><sub>CTX-M-13</sub>, <i>bla</i><sub>CTX-M-14</sub>, <i>bla</i><sub>CTX-M-15</sub>, and <i>bla</i><sub>SHV-12</sub> (ESBL types); <i>bla</i><sub>CMY-2</sub> (AmpC type); and <i>bla</i><sub>TEM-1</sub> and <i>bla</i><sub>OXA-1</sub> (narrow-spectrum types). PMQR genes (included <i>qnrA</i>, <i>qnrB</i>, <i>qnrS</i>, and <i>aac(6')-Ib-cr</i>) were identified in 23 (67.6%) isolates. The presence of ESBL- and PMQR-producing <i>S. enterica</i> with a high prevalence rate in retail meats and slaughterhouses is considered a major threat to public health as these strains with resistance genes could be transmitted to humans through the food chain.
Project description:Plasmid-mediated extended-spectrum beta-lactamase (ESBL), AmpC, and carbapenemase producing Enterobacteriaceae, in particular <i>Escherichia coli</i> and <i>Klebsiella pneumoniae</i>, with potential zoonotic transmission routes, are one of the greatest threats to global health. The aim of this study was to investigate global food products as potential vehicles for ESBL/AmpC-producing bacteria and identify plasmids harboring resistance genes. We sampled 200 food products purchased from Finland capital region during fall 2018. Products originated from 35 countries from six continents and represented four food categories: vegetables (<i>n</i> = 60), fruits and berries (<i>n</i> = 50), meat (<i>n</i> = 60), and seafood (<i>n</i> = 30). Additionally, subsamples (<i>n</i> = 40) were taken from broiler meat. Samples were screened for ESBL/AmpC-producing Enterobacteriaceae and whole genome sequenced to identify resistance and virulence genes and sequence types (STs). To accurately identify plasmids harboring resistance and virulence genes, a hybrid sequence analysis combining long- and short-read sequencing was employed. Sequences were compared to previously published plasmids to identify potential epidemic plasmid types. Altogether, 14 out of 200 samples were positive for ESBL/AmpC-producing <i>E. coli</i> and/or <i>K. pneumoniae</i>. Positive samples were recovered from meat (18%; 11/60) and vegetables (5%; 3/60) but were not found from seafood or fruit. ESBL/AmpC-producing <i>E. coli</i> and/or <i>K. pneumoniae</i> was found in 90% (36/40) of broiler meat subsamples. Whole genome sequencing of selected isolates (<i>n</i> = 21) revealed a wide collection of STs, plasmid replicons, and genes conferring multidrug resistance. <i>bla</i> <sub>CTX-M-15</sub>-producing <i>K. pneumoniae</i> ST307 was identified in vegetable (<i>n</i> = 1) and meat (<i>n</i> = 1) samples. Successful IncFII plasmid type was recovered from vegetable and both IncFII and IncI1-I? types from meat samples. Hybrid sequence analysis also revealed chromosomally located beta-lactamase genes in two of the isolates and indicated similarity of food-derived plasmids to other livestock-associated sources and also to plasmids obtained from human clinical samples from various countries, such as IncI type plasmid harboring <i>bla</i> <sub>TEM-52C</sub> from a human urine sample obtained in the Netherlands which was highly similar to a plasmid obtained from broiler meat in this study. Results indicate certain foods contain bacteria with multidrug resistance and pose a possible risk to public health, emphasizing the importance of surveillance and the need for further studies on epidemiology of epidemic plasmids.
Project description:Extended-spectrum ?-lactamase (ESBL) and plasmid-mediated AmpC ?-lactamase (pAmpC) genes confer resistance to extended spectrum cephalosporin's. The spread of these genes is mostly facilitated by plasmid-mediated horizontal transfer. National surveillance activities to detect ESBL/pAmpC-producers in commensal bacteria from livestock are in place in the Netherlands since several years. This study aimed at reporting gene and plasmid diversity of commensal ESBL/pAmpC-producing <i>Escherichia coli</i> isolated from healthy animals during surveillance activities between 2007 and 2017. A collection of 2304 extended-spectrum cephalosporin-resistant (ESC-R) <i>E. coli</i> isolated from feces of broilers, dairy cattle, slaughter pigs, turkeys, ducks, and veal calves was investigated and ESBL/pAmpC genes were determined. Gene location of a selection of 473 <i>E. coli</i> isolates was determined and typing of plasmids linked to the ESBL/pAmpC genes was performed. Twenty-two different ESBL/pAmpC genes were identified with <i>bla</i> <sub>CTX-M-1</sub> being the most prevalent gene in livestock (43.7%), followed by <i>bla</i> <sub>CMY -2</sub> and <i>bla</i> <sub>SHV -12</sub>, independent of the animal source. Prevalence of typically human associated <i>bla</i> <sub>CTX-M-15</sub> was highest in cattle. Less than 10% <i>E. coli</i> isolates owed their ESC-R phenotype to promoter mutations of the chromosomal <i>ampC</i> gene. Majority (92%) of ESBL/pAmpC genes analyzed were plasmid located, with IncI1? being the most represented plasmid family in isolates from all animals, followed by IncF (veal calves, dairy cattle and slaughter pigs), IncK (broilers and laying hens), IncX1 in broilers, and emerging IncX3 in broilers and dairy cattle. Prevalence and molecular diversity of ESC-R <i>E. coli</i> isolated from livestock over an 11-year period revealed a composite scenario of gene-plasmid combinations.
Project description:Antimicrobial resistance (AMR) is a major health threat for public and animal health in the twenty-first century. In Ecuador, antibiotics have been used by the poultry industry for decades resulting in the presence of multi-drug resistant (MDR) bacteria in the poultry meat production chain, with the consequent risk for public health. This study evaluated the prevalence of ESBL/AmpC and <i>mcr</i> genes in third-generation cephalosporin-resistant <i>Escherichia coli</i> (3GC-R <i>E. coli</i>) isolated from broiler farms (animal component), broiler carcasses (food component), and human enteritis (human component) in Quito-Ecuador. Samples were collected weekly from November 2017 to November 2018. For the animal, food, and human components, 133, 335, and 302 samples were analyzed, respectively. Profiles of antimicrobial resistance were analyzed by an automated microdilution system. Resistance genes were studied by PCR and Sanger sequencing. From all samples, 122 (91.7%), 258 (77%), and 146 (48.3%) samples were positive for 3GC-R <i>E. coli</i> in the animal, food, and human components, respectively. Most of the isolates (472/526, 89.7%) presented MDR phenotypes. The ESBL <i>bla</i> <sub>CTX-<i>M-</i>55</sub>, <i>bla</i> <sub>CTX-<i>M-</i>3</sub>, <i>bla</i> <sub>CTX-<i>M-</i>15</sub>, <i>bla</i> <sub>CTX-<i>M-</i>65</sub>, <i>bla</i> <sub>CTX-<i>M-</i>27</sub>, and <i>bla</i> <sub>CTX-<i>M-</i>14</sub> were the most prevalent ESBL genes while <i>bla</i> <sub>CMY-2</sub> was the only AmpC detected gene. The <i>mcr</i>-1 gene was found in 20 (16.4%), 26 (10.1%), and 3 (2.1%) of isolates from animal, food, and human components, respectively. The implication of poultry products in the prevalence of ESBL/AmpC and <i>mcr</i> genes in 3GC-R must be considered in the surveillance of antimicrobial resistance.
Project description:In order to estimate herd-level prevalence of extended-spectrum ?-lactamase/AmpC ?-lactamase (ESBL/AmpC)- and carbapenemase-producing commensal <i>Escherichia coli</i> in ruminants in the Basque Country (northern Spain), a cross-sectional survey was conducted in 2014 to 2016 in 300 herds using selective isolation. ESBL-/AmpC-producing <i>E. coli</i> was isolated in 32.9% of dairy cattle herds, 9.6% of beef cattle herds, and 7.0% of sheep flocks. No carbapenemase-producing <i>E. coli</i> was isolated. Phenotypic antimicrobial susceptibility determined by broth microdilution using EUCAST epidemiological cutoff values identified widespread coresistance to extended-spectrum cephalosporins and other antimicrobials (110/135 isolates), particularly tetracycline, sulfamethoxazole, trimethoprim, and ciprofloxacin. All isolates were susceptible to tigecycline, imipenem, meropenem, and colistin. The genomes of 66 isolates were sequenced using an Illumina NovaSeq 6000 and screened for antimicrobial resistance determinants against ResFinder and PointFinder. The plasmid/chromosomal locations of resistance genes were predicted with PlasFlow, and plasmid replicons were identified using PlasmidFinder. Fifty-two acquired resistance genes and point mutations in another four genes that coded for resistance to 11 antimicrobial classes were identified. Fifty-five genomes carried ESBL-encoding genes, <i>bla</i> <sub>CTX-M-14</sub> being the most common, and 11 carried determinants of the AmpC phenotype, mostly the <i>bla</i> <sub>CMY-2</sub> gene. Additionally, genes coding for ?-lactamases of the CTX-M group 9 were detected as well as the sporadic presence of <i>bla</i> <sub>SHV-12</sub>, <i>bla</i> <sub>CMY-4</sub>, and a point mutation in the <i>ampC</i> promoter. Only a bovine isolate coharbored more than one ESBL/AmpC genetic determinant (<i>bla</i> <sub>CTX-M-14</sub> and a mutation in the <i>ampC</i> promoter), confirming its ESBL- and AmpC ?-lactamase-producing phenotype. Most ESBL/AmpC genes were located in IncI1 plasmids, which also carried a great variety of other antimicrobial resistance genes.<b>IMPORTANCE</b> Extended-spectrum ?-lactamase (ESBL)- and AmpC ?-lactamase (AmpC)-producing <i>E. coli</i> isolates have emerged in recent years as some of the fastest spreading antimicrobial resistance determinants in humans and food-producing animals, becoming a concern for animal and public health. This study provided insight into the prevalence of cefotaxime-resistant <i>E. coli</i> in cattle and sheep in the Basque Country and the associated genetic determinants of antimicrobial resistance. These constituted an important contribution to the limited repository of such data for cattle in the region and for sheep worldwide. Antimicrobial susceptibility testing by phenotypic and molecular methods is key in surveillance programs to enhance early detection of resistance development, monitor resistance trends, and provide guidance to clinicians in selecting the adequate therapy.
Project description:ESC-resistant <i>E. coli</i> isolates were collected from broiler chickens, a slaughterhouse, and retail meat to assess their dispersion and their involvement in cross-contamination. ESBL-/AmpC-producing <i>E. coli</i> were isolated during the slaughter process of all six investigated chicken flocks from scalding, feather removal, first conveyor, evisceration, second washing, third conveyor, and third washing areas, and from handling workers in the slaughterhouse. ESC-resistant <i>E. coli</i> isolates with the same pulsed-field gel electrophoresis type were found in the same site (scalding) on different sampling days. ESBL/AmpC-producing <i>E. coli</i> isolates were absent in the lairage area in the slaughterhouse, but present in the retail markets in 36.8% (7/19) of the chicken flocks. The <i>bla</i><sub>CTX-M</sub> genes and <i>bla</i><sub>CMY-2</sub> were conjugated to recipient <i>E. coli</i> J53 in 67.5% (27/40) and 56.1% (23/41) of ESBL-producing and AmpC-producing <i>E. coli</i> isolates, respectively. The presence of the same conjugative plasmids was found in genetic diversity ESC-resistant <i>E. coli</i> colonies collected on different sampling days. Our study emphasizes that cross-contamination of ESBL/AmpC-producing <i>E. coli</i> in slaughterhouse has a crucial impact on the occurrence of ESC resistance in retail chicken meat.
Project description:Draft genomes of seven extended-spectrum β-lactamase (ESBL)-producing <i>Escherichia coli</i> strains recovered from New Zealand waterways are described. The mean genome size was 5.1 Mb, with 4,724 coding sequences. All genomes contained the ESBL gene <i>bla</i> <sub>CTX-M</sub>, and one carried a plasmid-mediated AmpC gene, <i>bla</i> <sub>CMY-2</sub> A multidrug-resistant genotype was detected in three isolates.
Project description:A total of 2,283 <i>Salmonella</i> isolates were recovered from 18,334 samples, including samples from patients with diarrhea, food of animal origin, and pets, across 5 provinces of China. The highest prevalence of <i>Salmonella</i> spp. was detected in chicken meats (39.3%, 486/1,237). Fifteen serogroups and 66 serovars were identified, with <i>Salmonella enterica</i> serovars Typhimurium and Enteritidis being the most dominant. Most (85.5%, 1,952/2,283) isolates exhibited resistance to??1 antimicrobial, and 56.4% were multidrug resistant (MDR). A total of 222 isolates harbored extended-spectrum ?-lactamases (ESBLs), and 200 of these were of the CTX-M type and were mostly detected in isolates from chicken meat and turtle fecal samples. Overall, eight <i>bla</i> <sub>CTX-M</sub> genes were identified, with <i>bla</i> <sub>CTX-M-65</sub>, <i>bla</i> <sub>CTX-M-123</sub>, <i>bla</i> <sub>CTX-M-14</sub>, <i>bla</i> <sub>CTX-M-79</sub>, and <i>bla</i> <sub>CTX-M-130</sub> being the most prevalent. In total, 166 of the 222 ESBL-producing isolates had amino acid substitutions in GyrA (S83Y, S83F, D87G, D87N, and D87Y) and ParC (S80I), while the plasmid-mediated quinolone resistance (PMQR)-encoding genes <i>oqxA</i>, <i>oqxB</i>, <i>qepA</i>, <i>qnrB</i>, and <i>qnrS</i> were detected in almost all isolates. Of the 15 sequence types (STs) identified in the 222 ESBLs, ST17, ST11, ST34, and ST26 ranked among the top 5 in number of isolates. Our study revealed considerable serovar diversity and a high prevalence of the co-occurrence of MDR determinants, including CTX-M-type ESBLs, quinolone resistance-determining region (QRDR) mutations, and PMQR genes. This is the first report of CTX-M-130 <i>Salmonella</i> spp. from patients with diarrhea and QRDR mutations from turtle fecal samples. Our study emphasizes the importance of actions, both in health care settings and in the veterinary medicine sector, to control the dissemination of MDR, especially the CTX-M-type ESBL-harboring <i>Salmonella</i> isolates.
Project description:This study aimed to investigate the phylogenetic diversity and epidemiology of extended-spectrum beta-lactamase (ESBL)-producing <i>Escherichia coli</i> and <i>Klebsiella pneumoniae</i> from chicken, chicken meat, and human clinical isolates in Sao Paolo, Brazil, and characterize their respective ESBL-encoding plasmids. Three hundred samples from chicken cloaca, chicken meat, and clinical isolates were phenotypically and genotypically assessed for ESBL resistance. Isolates were identified by MALDI TOF-MS and further characterized by MLST analysis and phylogenetic grouping. ESBL genes were characterized and their location was determined by I-Ceu-I-PFGE and Southern blot, conjugation, transformation, and PCR-based replicon typing experiments. Thirty-seven ESBL-producing isolates (28 <i>E. coli</i> and 9 <i>K. pneumoniae</i>) that were positive for the <i>bla</i> <sub><i>CTX-M</i>-1</sub> or <i>bla</i> <sub><i>CTX-M</i>-2</sub> gene groups were obtained. Two isolates were negative in the transformation assay, and the chromosomal location of the genes was deduced by Southern blot. The <i>bla</i> <sub><i>CTX-M</i></sub> genes identified were carried on plasmid replicon-types X1, HI2, N, FII-variants, I1 and R. The <i>E. coli</i> isolates belonged to nine sequence types, while the <i>K. pneumoniae</i> isolates belonged to four sequence types. The <i>E. coli</i> isolates belonged to phylotype classification groups A, B1, D, and F. This study demonstrated that isolates from cloacal swabs, chicken meat, and human feces had genetic diversity, with a high frequency of <i>bla</i> <sub><i>CTX-M</i>-15</sub> among chickens, chicken meat, and human feces. Thus, this reinforces the hypothesis that chickens, as well as their by-products, could be an important source of transmission for ESBL-producing pathogens to humans in South America.