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

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

Traditional marketed meats as a reservoir of multidrug-resistant Escherichia coli.

This study aimed to analyze Escherichia coli from marketed meat samples in Peru. Sixty-six E. coli isolates were recovered from 21 meat samples (14 chicken, 7 beef), and antimicrobial resistance levels and the presence of mechanisms of antibiotic resistance, as well as clonal relationships and phylogeny of colistin-resistant isolates, were established. High levels of antimicrobial resistance were detected, with 93.9% of isolates being multi-drug resistant (MDR) and 76.2% of samples possessing colistin-resistant E. coli; of these, 6 samples from 6 chicken samples presenting mcr-1-producer E. coli. Colistin-resistant isolates were classified into 22 clonal groups, while phylogroup A (15 isolates) was the most common. Extended-spectrum ß-lactamase- and pAmpC-producing E. coli were found in 18 and 8 samples respectively, with blaCTX-M-55 (28 isolates; 16 samples) and blaCIT (8 isolates; 7 samples) being the most common of each type. Additionally, blaCTX-M-15, blaCTX-M-65, blaSHV-27, blaOXA-5/10-like, blaDHA, blaEBC and narrow-spectrum blaTEM were detected. In addition, 5 blaCTX-M remained unidentified, and no sought ESBL-encoding gene was detected in other 6 ESBL-producer isolates. The tetA, tetE and tetX genes were found in tigecycline-resistant isolates. This study highlights the presence of MDR E. coli in Peruvian food-chain. The high relevance of CTX-M-55, the dissemination through the food-chain of pAmpC, as well as the high frequency of unrelated colistin-resistant isolates is reported.

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 ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-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.

Antimicrobial Resistance in Salmonella enterica Serovar Paratyphi B Variant Java in Poultry from Europe and Latin America.

Salmonella enterica serovar Paratyphi B variant Java sequence type 28 is prevalent in poultry and poultry meat. We investigated the evolutionary relatedness between sequence type 28 strains from Europe and Latin America using time-resolved phylogeny and principal component analysis. We sequenced isolates from Colombia, Guatemala, Costa Rica, and the Netherlands and complemented them with publicly available genomes from Europe, Africa, and the Middle East. Phylogenetic time trees and effective population sizes (Ne) showed separate clustering of strains from Latin America and Europe. The separation is estimated to have occurred during the 1980s. Ne of strains increased sharply in Europe around 1995 and in Latin America around 2005. Principal component analysis on noncore genes showed a clear distinction between strains from Europe and Latin America, whereas the plasmid gene content was similar. Regardless of the evolutionary separation, similar features of resistance to ß-lactams and quinolones/fluoroquinolones indicated parallel evolution of antimicrobial resistance in both regions.

A Cross-Sectional Cohort Study of Extended-Spectrum-Beta-Lactamase-Producing Enterobacterales in Patients with Traveler's Diarrhea.

Patients with traveler's diarrhea (TD) can acquire extended-spectrum-beta-lactamase (ESBL)-producing members of the Enterobacterales (EPE) during travel to areas of endemicity. The aim of the present study was to investigate the prevalence and characteristics of EPE carriage in travelers from southern Sweden who were sampled for bacterial diagnostics of TD compared to those of EPE carriage 10 years ago. Clinical samples sent for culture of common causes of bacterial enterocolitis, if the referral stated foreign travel, were included in the study. Antimicrobial susceptibility testing was done according to the EUCAST disk diffusion test method. EPE strains were subjected to whole-genome sequencing (WGS). Eighty-four of 303 patients carried a total of 92 ESBL-producing members of the Enterobacterales The overall prevalence of EPE in tested samples was thus 28%, compared to 24% 10 years earlier (P = 0.33). Among 86 strains available for WGS, 47 different sequence types (STs) were identified, and there were only 5 ST131 strains. Of the 79 Escherichia coli isolates, 76% carried at least one fim (type 1 fimbria) gene, 29% carried at least one pap (p-fimbriae) gene, and 43% were extraintestinal pathogenic E. coli (ExPEC) or uropathogenic E. coli (UPEC). Over half of the E. coli strains (57%) were intestinal pathogenic E. coli, most commonly enteroaggregative E. coli (EAEC) (33%), and enteroinvasive E. coli EIEC (22%). A relatively high proportion of patients with traveler's diarrhea carry EPE, but there was no significant increase compared to 10 years ago. Most E. coli strains were intestinal pathogenic strains. A comparatively high proportion of the strains were ExPEC/UPEC, many expressing the virulence genes pap and/or fim (This project was assigned ClinicalTrials.gov number NCT03866291.).

High occurrence of CMY-2-type beta-lactamase-producing Escherichia coli among broiler flocks in Turkey.

In this study, the prevalence of ESBL/pAmpC-producing Escherichia coli and their molecular characterization from cloacal swab samples were investigated. All samples were obtained from broiler flocks that are located in Hatay, Adana, and Mersin provinces of Turkey. Antimicrobial susceptibilities of the isolates were determined by disk diffusion method following the CLSI criteria. Genetic mechanisms mediating resistance in ESBL/pAmpC-producing E. coli isolates were identified by polymerase chain reaction (PCR) and followed by DNA sequencing. Phylogenetic groups and plasmid replicon types of the isolates were also investigated by PCR. The clonal relationship of selected isolates was investigated by enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus sequence typing (MLST) method. Of 430 cloacal swab samples, 154 (35.8%) were positive for ESBL/pAmpC-producing E. coli. The ESBL/pAmpC type beta-lactamases were as follows: CMY-2 (n = 46), CMY-2 + TEM-1b (n = 63), SHV-12 (n = 5), SHV-12 + TEM-1b (n = 12), CTX-M-3 (n = 14), CTX-M-3 + TEM-1b (n = 1), CTX-M-15 (n = 4), CTX-M-15 + TEM-1b (n = 4), and CTX-M-1 (n = 3). Moreover, various rates of resistance to different antimicrobials were determined such as nalidixic acid (92.9%), ciprofloxacin (76%), sulfamethoxazole-trimethoprim (78.6%), tetracycline (73.4%), streptomycin (52.6%), chloramphenicol (44.2%), kanamycin (27.9%), tobramycin (24.7%), gentamicin (19.5%), and amikacin (0.6%). Furthermore, 148 (96.1%) isolates were found to be MDR. The ESBL/pAmpC-producing isolates were distributed into the following phylogroups: E (n = 61), B1 (n = 30), F (n = 20), A (n = 19), B2 (n = 11), D (n = 10), and C (n = 3). ERIC-PCR analysis showed 51 unrelated patterns. Out of the 28 selected isolates, the following sequence types (STs) were detected: ST354 (n = 3), ST114 (n = 3), ST5696 (n = 2), ST156 (n = 2), ST174 (n = 2), ST362 (n = 2), ST157 (n = 2), ST5114 (n = 2), ST6635, ST539, ST457, ST1640, ST95, ST5843, ST1158, ST10, ST648, and ST4248. The results of the current study revealed that broilers in Turkey are important reservoir of ESBL/pAmpC-producing E. coli, which suggest that these agents have a great potential of transmission to humans by food chain or direct contact.

Faecal colonization of E. coli and Klebsiella spp. producing extended-spectrum beta-lactamases and plasmid-mediated AmpC in Mozambican university students.

BACKGROUND: In recent years, the world has seen a surge in Enterobacteriaceae resistant to broad-spectrum beta-lactam antibiotics due to the production of extended-spectrum beta-lactamases (ESBLs) or plasmid-mediated AmpC (pAmpC) enzymes. Data on the epidemiology of cephalosporin-resistant Enterobacteriaceae in Sub-Saharan Africa are still limited. METHODS: Two hundred seventy-five non-repetitive stool samples were collected from Mozambican university students of both sexes. Samples were cultured on MacConkey agar with and without ceftriaxone (1 mg/L) for selection of third-generation cephalosporin-resistant isolates, which were subjected to antimicrobial susceptibility testing by disc diffusion, characterization of resistance genes by PCR and ERIC-PCR analysis for strain clonality. RESULTS: Among the 275 students, 55 (20%) carried a total of 56 E. coli (n = 35) and Klebsiella spp. (n = 21) isolates resistant to ceftriaxone and phenotypically positive for ESBL- and/or pAmpC-production. Forty-three percent of the isolates (24/56) contained only ESBL genes, 11% (6/56) only pAmpC genes, and 36% (20/56) both ESBL and pAmpC genes. The remaining six isolates were negative for the CTX-M/pAmpC genes included in the test panel. E. coli and Klebsiella spp. combined demonstrated 70% resistance to tetracycline and co-trimoxazole, 63% to ceftazidime and 34% to ciprofloxacin. In total, 89% of ESBL/pAmpC-positive isolates were defined as multi-resistant by being resistant to three or more antibiotic classes. ERIC-PCR fingerprinting demonstrated low similarity among isolates. None of the participants reported recent hospitalization and just 12.5% had taken antibiotics 3 months prior to the study. CONCLUSION: This study demonstrated 20% colonization with multi-resistant E. coli and Klebsiella spp. among Mozambican students with a diversity of ESBL and pAmpC genes. Colonization was not related to prior hospitalization or antimicrobial consumption.

Extended-Spectrum-Beta-Lactamase- and Plasmid-Encoded Cephamycinase-Producing Enterobacteria in the Broiler Hatchery as a Potential Mode of Pseudo-Vertical Transmission.

Antimicrobial resistance through extended-spectrum beta-lactamases (ESBLs) and transferable (plasmid-encoded) cephamycinases (pAmpCs) represents an increasing problem in human and veterinary medicine. The presence of ESBL-/pAmpC-producing commensal enterobacteria in farm animals, such as broiler chickens, is considered one possible source of food contamination and could therefore also be relevant for human colonization. Studies on transmission routes along the broiler production chain showed that 1-day-old hatchlings are already affected. In this study, ESBL-/pAmpC-positive broiler parent flocks and their corresponding eggs, as well as various environmental and air samples from the hatchery, were analyzed. The eggs were investigated concerning ESBL-/pAmpC-producing enterobacteria on the outer eggshell surface (before/after disinfection), the inner eggshell surface, and the egg content. Isolates were analyzed concerning their species, their phylogroup in the case of Escherichia coli strains, the respective resistance genes, and the phenotypical antibiotic resistance. Of the tested eggs, 0.9% (n = 560) were contaminated on their outer shell surface. Further analyses using pulsed-field gel electrophoresis showed a relationship of these strains to those isolated from the corresponding parent flocks, which demonstrates a pseudo-vertical transfer of ESBL-/pAmpC-producing enterobacteria into the hatchery. Resistant enterobacteria were also found in environmental samples from the hatchery, such as dust or surfaces which could pose as a possible contamination source for the hatchlings. All 1-day-old chicks tested negative directly after hatching. The results show a possible entry of ESBL-/pAmpC-producing enterobacteria from the parent flocks into the hatchery; however, the impact of the hatchery on colonization of the hatchlings seems to be low. IMPORTANCE: ESBL-/pAmpC-producing enterobacteria occur frequently in broiler-fattening farms. Recent studies investigated the prevalence and possible transmission route of these bacteria in the broiler production chain. It seemed very likely that the hatcheries play an important role in transmission and/or contamination events. There are only few data on transmission investigations from a grandparent or parent flock to their offspring. However, reliable data on direct or indirect vertical transmission events in the hatchery are not available. Therefore, we conducted our study and intensively investigated the broiler hatching eggs from ESBL-/pAmpC-positive broiler parent flocks as well as the hatchlings and the environment of the hatchery.

Limited Dissemination of Extended-Spectrum ß-Lactamase- and Plasmid-Encoded AmpC-Producing Escherichia coli from Food and Farm Animals, Sweden.

Extended-spectrum ß-lactamase (ESBL)- and plasmid-encoded ampC (pAmpC)-producing Enterobacteriaceae might spread from farm animals to humans through food. However, most studies have been limited in number of isolates tested and areas studied. We examined genetic relatedness of 716 isolates from 4,854 samples collected from humans, farm animals, and foods in Sweden to determine whether foods and farm animals might act as reservoirs and dissemination routes for ESBL/pAmpC-producing Escherichia coli. Results showed that clonal spread to humans appears unlikely. However, we found limited dissemination of genes encoding ESBL/pAmpC and plasmids carrying these genes from foods and farm animals to healthy humans and patients. Poultry and chicken meat might be a reservoir and dissemination route to humans. Although we found no evidence of clonal spread of ESBL/pAmpC-producing E. coli from farm animals or foods to humans, ESBL/pAmpC-producing E. coli with identical genes and plasmids were present in farm animals, foods, and humans.

Phenotypic and Genotypic Identification of Four Cases of Plasmid-Mediated AmpC ß-Lactamases-Producing Escherichia coli Admitted to a Tertiary Centre.

We describe four patients with a positive culture of AmpC ß-lactamases-producing Escherichia coli (E. coli), despite the fact that our understanding of plasmid-mediated AmpC ß-lactamases (pAmpC) is currently limited. Three out of four cases of AmpC ß-lactamases-producing Escherichia coli were isolated from a urine sample, and one was from a peritoneal fluid sample. All four isolates are resistant to cefoxitin disc and were subjected to a confirmatory AmpC phenotypic test (AmpC induction test) and monoplex polymerase chain reaction (PCR) for the determination of six pAmpC genotypes (blaDHA, blaEBC, blaMOX, blaFOX, blaACC, and blaCIT). All four E. coli isolates tested negative for the AmpC induction test, while monoplex PCR analysis was positive only for the blaDHA pAmpC genotype and negative for all five other genotypes (blaEBC, blaMOX, blaFOX, blaACC, and blaCIT). A common clinical characteristic across all patients was fever. One patient was treated for perforated sigmoid diverticulitis, while the other three patients were treated for acute pyelonephritis or urinary tract infections (UTIs). Each patient improved significantly and was successfully discharged.

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

Current methods for plasmid-mediated AmpC ß-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 ß-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.

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

OBJECTIVES: 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-ß-lactamase (blaNDM) has facilitated their spread and NDM interferes with correct pAmpC phenotypic identification. METHODS: 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. RESULTS: 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%). CONCLUSION: 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.

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

Escherichia coli able to produce extended spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-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 < 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.

World Health Organization critical priority Escherichia coli clone ST648 in magnificent frigatebird (Fregata magnificens) of an uninhabited insular environment.

Antimicrobial resistance is an ancient natural phenomenon increasingly pressured by anthropogenic activities. Escherichia coli has been used as markers of environmental contamination and human-related activity. Seabirds may be bioindicators of clinically relevant bacterial pathogens and their antimicrobial resistance genes, including extended-spectrum-beta-lactamase (ESBL) and/or plasmid-encoded AmpC (pAmpC), in anthropized and remote areas. We evaluated cloacal swabs of 20 wild magnificent frigatebirds (Fregata magnificens) of the Alcatrazes Archipelago, the biggest breeding colony of magnificent frigatebirds in the southern Atlantic and a natural protected area with no history of human occupation, located in the anthropized southeastern Brazilian coast. We characterized a highly virulent multidrug-resistant ST648 (O153:H9) pandemic clone, harboring bla CTX-M-2, bla CMY-2, qnrB, tetB, sul1, sul2, aadA1, aac(3)-VIa and mdfA, and virulence genes characteristic of avian pathogenic (APEC) (hlyF, iroN, iss, iutA, and ompT) and other extraintestinal E. coli (ExPEC) (chuA, kpsMII, and papC). To our knowledge, this is the first report of ST648 E. coli co-producing ESBL and pAmpC in wild birds inhabiting insular environments. We suggest this potentially zoonotic and pathogenic lineage was likely acquired through indirect anthropogenic contamination of the marine environment, ingestion of contaminated seafood, or by intra and/or interspecific contact. Our findings reinforce the role of wild birds as anthropization sentinels in insular environments and the importance of wildlife surveillance studies on pathogens of critical priority classified by the World Health Organization.

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.

Lineages, Virulence Gene Associated and Integrons among Extended Spectrum ß-Lactamase (ESBL) and CMY-2 Producing Enterobacteriaceae from Bovine Mastitis, in Tunisia.

Extended Spectrum Beta-Lactamase (ESBL) Enterobacteriaceae are becoming widespread enzymes in food-producing animals worldwide. Escherichia coli and Klebseilla pneumoniae are two of the most significant pathogens causing mastitis. Our study focused on the characterization of the genetic support of ESBL/pAmpC and antibiotic resistance mechanisms in cefotaxime-resistant (CTXR) and susceptible (CTXS) Enterobacteriaceae isolates, recovered from bovine mastitis in Tunisia, as well as the analyses of their clonal lineage and virulence-associated genes. The study was carried out on 17 ESBL/pAmpC E. coli and K. pneumoniae and 50 CTXS E. coli. Detection of resistance genes and clonal diversity was performed by PCR amplification and sequencing. The following ß-lactamase genes were detected: blaCTX-M-15 (n = 6), blaCTX-M-15 + blaOXA-1 (2), bla CTX-M-15 + blaOXA-1 + blaTEM-1b (2), blaCTX-M-15 + blaTEM-1b (4), blaCMY-2 (3). The MLST showed the following STs: ST405 (n = 4 strains); ST58 (n = 3); ST155 (n = 3); ST471 (n = 2); and ST101 (n = 2). ST399 (n = 1) and ST617 (n = 1) were identified in p(AmpC) E. coli producer strains. The phylogroups A and B1 were the most detected ones, followed by the pathogenic phylogroup B2 that harbored the shigatoxin genes stx1/stx2, associated with the cnf, fimA, and aer virulence factors. The qnrA/qnrB, aac(6')-Ib-cr genes and integrons class 1 with different gene cassettes were detected amongst these CTXR/S isolated strains. The presence of different genetic lineages, associated with resistance and virulence genes in pathogenic bacteria in dairy farms, may complicate antibiotic therapies and pose a potential risk to public health.

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 ß-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 ß-lactamases (ESBLs), the genes conferring resistance to ß-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 ß-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: ß-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.

Genetic characterization of ESBL/pAmpC-producing Escherichia coli isolated from forest, urban park and cereal culture soils.

Little is known about the role of forestland and non-fertilized agriculture soils as reservoirs of extended-spectrum beta-lactamase (ESBL) and plasmid-borne AmpC (pAmpC)-producing Escherichia coli isolates. Thus, in the present study, 210 soil samples from various origins (forest of Oued Zen (Ain Drahem), non-agriculture soils from different park gardens in Tunis City, cereal culture soils and home gardens) were investigated to characterize cefotaxime-resistant E. coli isolates. A total of 22 ESBL/pAmpC-producing E. coli were collected, and all harbored variants of the blaCTX-M gene (15 blaCTX-M-1, 5 blaCTX-M-55 and 2 blaCTX-M-15). A total of seven and two isolates harbored also blaEBC and blaDHA-like genes, respectively. Resistances to tetracycline, sulfonamides and fluoroquinolones were encoded by tetA (n = 4)/tetB (n = 12), sul1 (n = 17)/sul2 (n = 19) and aac(6')-Ib-cr (n = 2)/qnrA (n = 1)/qnrS (n = 1) genes, respectively. A total of seven isolates were able to transfer by conjugation cefotaxime-resistance in association or not with other resistance markers. PFGE showed that ten and two isolates were clonally related (pulsotypes P1 and P2). The 10 P1 isolates had been collected from forestland, cereal culture soils and an urban park garden in Tunis City, arguing for a large spread of clonal strains. Our findings highlight the occurrence of ESBL/pAmpC-E. coli isolates in soils under limited anthropogenic activities and the predominance of CTX-M enzymes that are largely disseminated in E. coli from humans and animals in Tunisia.

Multiple sequence types, virulence determinants and antimicrobial resistance genes in multidrug- and colistin-resistant Escherichia coli from agricultural and non-agricultural soils.

In soils, the presence of clinically relevant bacteria carrying ARGs, including extended-spectrum ß-lactamase- and plasmid-mediated AmpC ß-lactamase-encoding genes, is an underestimated public health problem that requires more attention. For this investigation, 300 samples from agricultural and non-agricultural soils were used to obtain 41 MDR E. coli isolates, standing out the resistance to ß-lactams, fluoroquinolones and colistin. Virulence genes related to diarrheagenic E. coli and extraintestinal pathogenic E. coli were detected. Several ARGs were found, highlighting the presence of at least one ß-lactamase-encoding gene (blaTEM, blaCMY, blaSHV, blaOXA-1-like, blaCTX-M-2, and/or blaCTX-M-15) in each isolate. Among the fluoroquinolone-resistant E. coli isolates, the plasmid-mediated quinolone resistance genes (qnrB and oqxA) and substitutions in the quinolone resistance-determining regions were detected. Some isolates were resistant to colistin (MICs of 4-8 mg/L) and, although no mcr-like gene was detected, substitutions in the two-component systems involving PhoP/PhoQ and PmrA/PmrB were found. Furthermore, the E. coli isolates presented plasmids and class 1 integrons, the last one detected in all isolates. The ARGs blaTEM, aadA and dfrA and the lpfA virulence-associated gene presented statistically significant differences (P < 0.05) in agricultural soils, while the blaOXA-1-like gene presented a statistically significant difference in non-agricultural soils. Thirty-eight sequence types (STs) were identified among the isolates, spotlighting the 20 different STs that carried blaCMY and blaCTX-M-type genes and those commonly reported in infections worldwide. The occurrence of virulent, multidrug- and colistin-resistant E. coli isolates in soils could lead to contamination of surrounding environments and food, increasing the risk of human and animal exposure. Therefore, this study contributes to a better understanding of E. coli in soils and reinforces the importance of the One Health approach to antimicrobial resistance surveillance.

First Detection of Human ST131-CTX-M-15-O25-B2 Clone and High-Risk Clonal Lineages of ESBL/pAmpC-Producing E. coli Isolates from Diarrheic Poultry in Tunisia.

Circulation of a multi-resistance clone of bacteria associated with genetic elements in diseased animals constitutes a global public health problem. Our study focused on the characterization of the support of ESBL in cefotaxime resistant E. coli (CTXR) isolates recovered from poultry with diarrhea, analysis of their clonal lineage, and virulence-associated genes. The study was carried out on 130 samples of chickens with diarrhea, collected in 2015 from poultry farms in Tunisia. Isolates of 20 CTXR E. coli strains were identified as ESBL and AmpC ß- lactamase producers. The following ß-lactamase genes (number of isolates) were detected: blaCTX-M-15+ blaOXA1 (4), blaCTX-M-15 + blaOXA1 + blaTEM-1b (2), blaCTX-M-1 + blaTEM-1b (9), blaCTX-M-1 (2), blaCMY2 + blaTEM-1b (3). Six E. coli harboring blaCTXM-15 were allocated to ST131-B2-O25b-; six and three blaCTX-M-1 were grouped in ST155, ST10, and ST58, respectively, related to the phylogroup D and A. The qnrB gene, the variant aac(6')-Ib-cr, and the class 1 integrons with different gene cassettes, were detected amongst our 20 isolated strains, which were classified as ExPEC and aEPEC. Our findings highlighted the emergence of the human pandemic ST131-CTX-M-15-O25-B2 clone and the high risk of such clonal lineage strains in diarrheic poultry, in Tunisia, which could constitute a risk of their transfer to healthy animals and humans.