Unexpected role of pig nostrils in the clonal and plasmidic dissemination of extended-spectrum beta-lactamase-producing Escherichia coli at farm level.

The presence of methicillin-resistant or -susceptible S. aureus in pig nostrils has been known for a long time, but the occurrence of extended-spectrum beta-lactamase (ESBL)-producing E. coli has hardly been investigated. Here, we collected 25 E. coli recovered from nasal samples of 40 pigs/10 farmers of four farms. Nine ESBL-producing isolates belonging to ST48, ST117, ST847, ST5440, ST14914 and ST10 were retrieved from seven pigs. All blaESBL genes (blaCTX-M-32,blaCTX-M-14,blaCTX-M-1,blaCTX-M-65, and blaSHV-12) were horizontally transferable by conjugation through plasmids belonging to IncI1 (n=3), IncX1 (n=3) and IncHI2 (n=1) types. IncI1-plasmids displayed different genetic environments: i) IS26-blaSHV-12-deoR-IS26, ii) wbuC-blaCTX-M-32-ISKpn26 (IS5), and iii) IS930-blaCTX-M-14-IS26. The IncHI2-plasmid contained the genetic environment IS903-blaCTX-M-65-fipA with multiple resistance genes associated either to: a) Tn21-like transposon harbouring genes conferring aminoglycosides/beta-lactams/chloramphenicol/macrolides resistance located on two atypical class 1 integrons with an embedded ΔTn5393; or b) Tn1721-derived transposon displaying an atypical class 1 integron harbouring aadA2-arr3-cmlA5-blaOXA-10-aadA24-dfrA14, preceding the genetic platform IS26-blaTEM-95-tet(A)-lysR-floR-virD2-ISVsa3-IS3075-IS26-qnrS1, as well as the tellurite resistance module. Other plasmids harbouring clinically relevant genes were detected, such as a ColE-type plasmid carrying the mcr-4.5 gene. Chromosomally encoded genes (fosA7) or integrons (intI1-dfrA1-aadA1-qacE-sul1/intI1-IS15-dfrA1-aadA2) were also identified. Finally, an IncY plasmid harbouring a class 2 integron (intI2-dfrA1-sat2-aadA1-qacL-IS406-sul3) was detected but not associated with a blaESBL gene. Our results evidence that pig nostrils might favour the spread of ESBL-E. coli and mcr-mediated colistin-resistance. Therefore, enhanced monitoring should be considered, especially in a sector where close contact between animals in intensive farming increases the risk of spreading antimicrobial resistance.

Enterobacterales high-risk clones and plasmids spreading blaESBL/AmpC and blaOXA-48 genes within and between hospitalized dogs and their environment.

BACKGROUND: Compared with healthcare settings, the role of veterinary hospitals in the spread of extended-spectrum cephalosporin- and carbapenem-resistant (ESC-R/CP-R) bacteria has been overlooked. OBJECTIVES: To investigate using genome-based approaches the dynamics of ESC-R and CP-R Enterobacterales among 125 dogs admitted to the same veterinary hospital over a 4 month period. METHODS: Dogs (n = 125) were sampled within 48 h of admission and at discharge. ESC-R/CP-R were phenotypically characterized and whole-genome sequenced using short- and long-read technologies. Phylogenetic analyses were performed using appropriate pipelines. RESULTS: ESC-R/CP-R prevalence in dogs was 4.8% (6/125) upon admission and reached 24.8% (31/125) at discharge, reflecting multiple acquisitions of ESBL/AmpC and OXA-48-positive Enterobacterales during hospitalization. Indistinguishable or closely related isolates were found within dogs, shared between dogs, and shared between dogs and their environment, suggesting numerous clonal and plasmid spreads. Even though carbapenems are not licensed for use in companion animals, a wide distribution of the blaOXA-48/IncL plasmid was evidenced across different bacterial species and dogs. CONCLUSIONS: This study highlights nosocomial acquisitions of ESBL/AmpC and carbapenemase-producing Enterobacterales by companion animals and the risk of further transmission within the community in a One Health perspective. Reinforced infection prevention and control measures and screening procedures are urgently needed in small animal veterinary settings where advanced therapeutics and intensive care is provided.

Interplay between Bacterial Clones and Plasmids in the Spread of Antibiotic Resistance Genes in the Gut: Lessons from a Temporal Study in Veal Calves.

Intestinal carriage of extended spectrum ß-lactamase (ESBL)-producing Escherichia coli is a frequent, increasing, and worrying phenomenon, but little is known about the molecular scenario and the evolutionary forces at play. We screened 45 veal calves, known to have high prevalence of carriage, for ESBL-producing E. coli on 514 rectal swabs (one randomly selected colony per sample) collected over 6 months. We characterized the bacterial clones and plasmids carrying blaESBL genes with a combination of genotyping methods, whole genome sequencing, and conjugation assays. One hundred and seventy-three ESBL-producing E. coli isolates [blaCTX-M-1 (64.7%), blaCTX-M-14 (33.5%), or blaCTX-M-15 (1.8%)] were detected, belonging to 32 bacterial clones, mostly of phylogroup A. Calves were colonized successively by different clones with a trend in decreasing carriage. The persistence of a clone in a farm was significantly associated with the number of calves colonized. Despite a high diversity of E. coli clones and blaCTX-M-carrying plasmids, few blaCTX-M gene/plasmid/chromosomal background combinations dominated, due to (i) efficient colonization of bacterial clones and/or (ii) successful plasmid spread in various bacterial clones. The scenario "clone versus plasmid spread" depended on the farm. Thus, epistatic interactions between resistance genes, plasmids, and bacterial clones contribute to optimize fitness in specific environments. IMPORTANCE The gut microbiota is the epicenter of the emergence of resistance. Considerable amount of knowledge on the molecular mechanisms of resistance has been accumulated, but the ecological and evolutionary forces at play in nature are less studied. In this context, we performed a field work on temporal intestinal carriage of extended spectrum ß-lactamase (ESBL)-producing Escherichia coli in veal farms. Veal calves are animals with one of the highest levels of ESBL producing E. coli fecal carriage, due to early high antibiotic exposure. We were able to show that calves were colonized successively by different ESBL-producing E. coli clones, and that two main scenarios were at play in the spread of blaCTX-M genes among calves: efficient colonization of several calves by a few bacterial clones and successful plasmid spread in various bacterial clones. Such knowledge should help develop new strategies to fight the emergence of antibiotic-resistance.

Identification and detection of three new F17 fimbrial variants in Escherichia coli strains isolated from cattle.

F17 fimbriae are produced by pathogenic Escherichia coli involved in diarrhea and septicemia outbreaks in calves and lambs. These proteins result from the expression of four different clustered genes, namely f17A, f17D, f17C and f17G, encoding a pilin protein, a periplasmic protein, an anchor protein and an adhesin protein, respectively. Several variants of f17A and f17G genes have been reported and found genetically associated with typical virulence factors of bovine pathogenic E. coli strains. In this study, a new F17e-A variant, closely related to F17b-A, was identified from a collection of 58 E. coli isolates from diarrheic calves in Iran. While highly prevalent in Iranian F17-producing clinical isolates from calves, this variant was rare among E. coli from a French healthy adult bovine population, suggesting a possible association with virulence. The f17Ae gene was also found in the genome of the Shiga-like toxin variant Stx1d-producing bovine E. coli strain MHI813, and belonged to a gene cluster also encoding a new F17-G3 variant, which greatly differed from F17-G1 and F17-G2. This gene cluster was located on a pathogenicity island integrated in the tRNA pheV gene. The gene coding for a third new F17f-A variant corresponding to a combination of F17c-A and F17d-A was also identified on the pVir68 plasmid in the bovine pathogenic E. coli strain 6.0900. In conclusion, we identified three new F17-A and F17-G variants in cattle E. coli, which may also have significant impact on the development of new diagnostics and vaccination tools.

Occurrence and persistence of multidrug-resistant Enterobacterales isolated from urban, industrial and surface water in Monastir, Tunisia.

The One Health approach of antimicrobial resistance highlighted the role of the aquatic environment as a reservoir and dissemination source of resistance genes and resistant bacteria, especially due to anthropogenic activities. Resistance to extended-spectrum cephalosporins (ESC) conferred by extended-spectrum beta-lactamases (ESBLs) in E. coli has been proposed as the major marker of the AMR burden in cross-sectoral approaches. In this study, we investigated wastewater, surface water and seawater that are subjected to official water quality monitoring in Monastir, Tunisia. While all but one sample were declared compliant according to the official tests, ESC-resistant bacteria were detected in 31 (19.1 %) samples. Thirty-nine isolates, coming from urban, industrial and surface water in Monastir, were collected and characterized using antibiograms and whole-genome sequencing. These isolates were identified as 27 Escherichia coli (69.3 %) belonging to 13 STs, 10 Klebsiella pneumoniae (25.6 %) belonging to six STs, and two Citrobacter freundii (5.1 %). We observed the persistence and dissemination of clones over time and in different sampling sites, and no typically human-associated pathogens could be identified apart from one ST131. All isolates presented a blaCTX-M gene - blaCTX-M-15 (n = 22) and blaCTX-M-55 (n = 8) being the most frequent variants - which were identified on plasmids (n = 20) or on the chromosome (n = 19). In conclusion, we observed ESC resistance in rather ubiquitous bacteria that are capable of surviving in the water environment. This suggests that including the total coliform count and the ESBL count as determined by bacterial growth on selective plates in the official monitoring would greatly improve water quality control in Tunisia.

CTX-M-15/27-positive Escherichia coli and VIM-2-producing Pseudomonas putida in free-living pigeons (Columba livia) in Tunisia.

OBJECTIVES: Wild birds are vectors of antimicrobial resistance. Birds living in close contact with humans or other animals, like feral pigeons (Columba livia), might be especially prone to acquire resistance genes such as those encoding extended-spectrum beta-lactamases (ESBLs) and carbapenemases. METHODS: Cloacal samples (n = 206) of free-living feral pigeons (C. livia) were collected in Sousse and Monastir, Tunisia. Antimicrobial susceptibility profiles were determined by disc-diffusion, and resistant isolates were short- and long-read whole-genome sequenced. Sequence analysis was performed using tools of the Centre for Genomic Epidemiology, and Phylogenetic analysis was performed based on the core-genome MLST. RESULTS: Fourteen (14/206, 6.8%) pigeons harboured Enterobacterales resistant to last-generations cephalosporins, of which 10 were CTX-M-15- or CTX-M-27-producers, while two (1.0%) carried a VIM-2-producing Pseudomonas putida. Positive pigeons lived on four different livestock farms. Three STs (ST206, ST5584, ST8149) were identified among E. coli, of which ST5584 and ST8149 were found in two different farms. Genetic diversity was also observed in Enterobacter cloacae and P. putida isolates. The blaCTX-M-27 genes were chromosomally encoded, while the blaCTX-M-15 genes were carried on highly similar IncF/F-:A-:B53 plasmids. The blaVIM-2 gene was located on a class 1 integron co-harbouring several resistance genes. CONCLUSION: Pigeons living on livestock farms carried clinically important resistance genes encoding ESBLs and carbapenemases. Our results evidenced that both clonal (ST8149 and ST5584) and plasmidic (IncF/F-:A-:B53) transfers played a role in the spread of resistance genes among pigeons. Further studies are needed to identify factors favouring the transfer and persistence of resistance genes within the pigeon communities.

Characterization of blaCTX-M IncFII plasmids and clones of Escherichia coli from pets in France.

OBJECTIVES: To characterize bla(CTX-M) IncFII plasmids and clones of Escherichia coli from cats and dogs and to compare them with bla(CTX-M) IncFII plasmids reported in humans. METHODS: From December 2006 to April 2010, 518 E. coli isolates from clinical infections in cats and dogs were screened for extended-spectrum ß-lactamase (ESBL) production. Antimicrobial susceptibility was performed by disc diffusion and resistance genes were identified by PCR and sequencing. Plasmids were characterized using PCR-based replicon typing and sub-typing schemes, restriction fragment length polymorphism analysis, S1-PFGE and Southern hybridization. Isolates were characterized by PFGE, phylogenetic grouping, O25b typing and multilocus sequence typing. RESULTS: Nineteen E. coli isolates (3.7%) produced ESBLs, of which 14 (74%) carried bla(CTX-M) IncFII plasmids. The bla(CTX-M) gene was predominant and located on F31:A4:B1, F36:A4:B1 or F36:A1:B20 plasmids, abundantly reported in humans. The bla(CTX-M) F22:A1:B20 or F2:A2:B20 plasmids were also found. Different sequence types (STs) were identified, such as ST10, ST410, ST359, ST617 and ST224. Only one E. coli isolate belonged to the ST131 E. coli clone and carried a bla(CTX-M) F2:A2:B20 plasmid. CONCLUSIONS: This is the first known extensive study on ESBL-producing E. coli isolates from pets in France. The ST131 clone was rare. However, the predominance of human-like bla(CTX-M) IncFII plasmids suggests exchanges of these plasmids with the human reservoir.

Detection and genetic characterization of blaESBL-carrying plasmids of cloacal Escherichia coli isolates from white stork nestlings (Ciconia ciconia) in Spain.

OBJECTIVES: This study aimed to characterize Escherichia coli isolates from cloacal samples of white stork nestlings, with a special focus on extended-spectrum ß-lactamases (ESBLs)-producing E. coli isolates and their plasmid content. METHODS: Cloacal samples of 88 animals were seeded on MacConkey-agar and chromogenic-ESBL plates to recover E. coli and ESBL-producing E. coli. Antimicrobial susceptibility was screened using the disc diffusion method, and the genotypic characterization was performed by polymerase chain reaction (PCR) and subsequent sequencing. S1 nuclease Pulsed-Field-Gel-Electrophoresis (PFGE), Southern blotting, and conjugation essays were performed on ESBL-producing E. coli, as well as whole-genome sequencing by short- and long-reads. The four blaESBL-carrying plasmids were completely sequenced. RESULTS: A total of 113 non-ESBL-producing E. coli isolates were collected on antibiotic-free MacConkey-agar, of which 27 (23.9%) showed a multidrug-resistance (MDR) phenotype, mainly associated with ß-lactam-phenicol-sulfonamide resistance (blaTEM/cmlA/floR/sul1/sul2/sul3). Moreover, four white stork nestlings carried ESBL-producing E. coli (4.5%) with the following characteristics: blaSHV-12/ST38-D, blaSHV-12/ST58-B1, blaCTX-M-1/ST162-B1, and blaCTX-M-32/ST155-B1. Whole-genome sequencing followed by Southern blot hybridizations on S1-PFGE gels in ESBL-positive isolates proved that the blaCTX-M-1 gene and one of the blaSHV-12 genes were carried by IncI1/pST3 plasmids, while the second blaSHV-12 gene and the blaCTX-M-32 gene were located on IncF plasmids. The two blaSHV-12 genes and the two blaCTX-M genes had similar but non-identical close genetic environments, as all four genes were flanked by a variety of insertion sequences. CONCLUSION: The role played by several genetic platforms in the mobility of ESBL genes allows for interchangeability on a remarkably small scale (gene-plasmid-clones), which may support the spread of ESBL genes.

Molecular characterization of highly prevalent Escherichia coli and Escherichia marmotae resistant to extended-spectrum cephalosporins in European starlings (Sturnus vulgaris) in Tunisia.

European starlings are widespread migratory birds that have already been described as carrying bacteria resistant to extended-spectrum cephalosporins (ESC-R). These birds are well known in Tunisia because they spend the wintertime in this country and are hunted for human consumption. The goal of our study was to estimate the proportion of ESC-R in these birds and to characterize the collected isolates using whole-genome sequencing. Results showed that 21.5% (42/200) of the birds carried either an extended-spectrum beta-lactamase (ESBL) or an acquired AmpC gene. Diverse bla CTX-M genes were responsible for the ESBL phenotype, bla CTX-M-14 being the most prevalent, while only bla CMY-2 and one bla CMY-62 were found in AmpC-positive isolates. Likewise, different genetic determinants carried these resistance genes, including IncHI2, and IncF plasmids for bla CTX-M genes and IncI1 plasmids for bla CMY-2 genes. Three chromosomally encoded bla CTX-M-15 genes were also identified. Surprisingly, species identification revealed a large proportion (32.7%) of Escherichia marmotae isolates. This species is phenotypically indistinguishable from Escherichia coli and has obviously the same capacity to acquire ESC-R genes. Our data also strongly suggest that at least the IncHI2/pST3 plasmid can spread equally between E. coli and E. marmotae. Given the potential transmission routes between humans and animals, either by direct contact with dejections or through meat preparation, it is important to closely monitor antimicrobial resistance in European starlings in Tunisia and to set up further studies to identify the sources of contamination of these birds. IMPORTANCE The One Health concept highlighted knowledge gaps in the understanding of the transmission routes of resistant bacteria. A major interest was shown in wild migratory birds since they might spread resistant bacteria over long distances. Our study brings further evidence that wild birds, even though they are not directly submitted to antibiotic treatments, can be heavily contaminated by resistant bacteria. Our results identified numerous combinations of resistance genes, genetic supports, and bacterial clones that can spread vertically or horizontally and maintain a high level of resistance in the bird population. Some of these determinants are widespread in humans or animals (IncHI2/pST3 plasmids and pandemic clones), while some others are less frequent (atypical IncI1 plasmid and minor clones). Consequently, it is essential to be aware of the risks of transmission and to take all necessary measures to prevent the proportions of resistant isolates from increasing uncontrollably.

A USA300 variant and other human-related methicillin-resistant Staphylococcus aureus strains infecting cats and dogs in France.

OBJECTIVES: To characterize methicillin-resistant Staphylococcus aureus (MRSA) clinical strains from cats and dogs in France, and to compare the clones identified with the distribution of French human MRSA. METHODS: Susceptibilities to antimicrobials were assessed by disc diffusion. Resistance and virulence genes were screened using a microarray-based assay. Isolates were additionally characterized by SmaI macrorestriction analysis and spa typing. RESULTS: From 2006 to 2010, the proportion of MRSA infections in pets in France was low (1.8%), but most isolates (87.0%, 20/23) belonged to human clones. The most common clones were the Lyon clone (69.6%, 16/23), the livestock-associated CC398 (13.0%, 3/23) and the Geraldine clone (8.7%, 2/23). Interestingly, we report the first USA300 clone infecting a European dog, which was probably imported by a US patient. CONCLUSIONS: Over a 5 year period, the proportion of MRSA infections in pets appears low (<2%) in France, but the distribution of the clones mostly mirrors the epidemiology of human invasive clones. These data highlight the role of pets as both victims and reservoirs of endemic, epidemic and/or invasive MRSA.

Spread of the mcr-1 colistin-resistance gene in Escherichia coli through plasmid transmission and chromosomal transposition in French goats.

Introduction: Colistin-resistance widely disseminated in food-producing animals due to decades of colistin use to treat diarrhea. The plasmid-borne mcr-1 gene has been extensively reported from bovine, swine and chicken worldwide, but smaller productions such as the goat farming sector were much less surveyed. Methods: We looked for colistin-resistant isolates presenting plasmid-borne genes of the mcr family in both breeding (n=80) and fattening farms (n=5). Localization of the mcr-1 gene was performed using Southern blot analysis coupled to short-read and long-read sequencing. Results: Only the mcr-1 gene was identified in 10% (8/80) of the breeding farms and four over the five fattening farms. In total, 4.2% (65/1561) of the animals tested in breeding farms and 60.0% (84/140) of those tested in fattening farms presented a mcr-1-positive E. coli. The mcr-1 gene was located either on the chromosome (32.2%) or on IncX4 (38.9%) and IncHI2 (26.8%) plasmids. As expected, both clonal expansion and plasmidic transfers were observed in farms where the mcr-1 gene was carried by plasmids. Tn6330 transposition was observed in the chromosome of diverse E. coli sequence types within the same farm. Discussion: Our results show that the mcr-1 gene is circulating in goat production and is located either on plasmids or on the chromosome. Evidence of Tn6330 transposition highlighted the fact that chromosomal insertion does not impair the transmission capability of the mcr-1 gene. Only strict hygiene and biosecurity procedures in breeding farms, as well as a prudent use of antibiotics in fattening farms, can avoid such complex contamination pathways.

Escherichia coli ST224 and IncF/blaCTX-M-55 plasmids drive resistance to extended-spectrum cephalosporins in poultry flocks in Parana, Brazil.

Resistant Enterobacterales of avian intestinal origin can contaminate carcasses during broiler processing and thereby spread through the human food chain. This study aimed at assessing the prevalence, diversity and genomic characteristics of ESBL/AmpC Enterobacterales in poultry flocks from different farms and cities in the state of Paraná, Brazil. Enterobacterales isolated from cloacal samples were subjected to antimicrobial susceptibility testing (AST). ESBL/AmpC isolates were whole-genome sequenced and subjected to S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) followed by Southern blotting to determine the location of resistant genes on plasmids. A surprisingly high proportion of E. coli (40.6 %) collected on non-selective plates presented an ESBL/AmpC phenotype. Multidrug resistance was statistically not higher in ESBL/AmpC E. coli having the potential to be Avian Pathogenic (APEC-like) compared to non-APEC-like ESBL/AmpC E. coli isolates. Resistance to antibiotics not authorized for use in poultry in the State of Paraná was observed, suggesting that antimicrobial resistance (AMR) is co-selected by the use of veterinary-licensed antibiotics. Phylogenetic analyzes revealed the presence of identical or highly similar ESBL/AmpC E. coli clones on farms distant up to 100 km of each other; this strongly suggests that the centralization and verticalization of the poultry industry can facilitate the spread of resistant bacteria among different farms, companies, and cities. The molecular characterization of clones and plasmids proved the dominance of the ST224 E. coli lineage and the IncF/blaCTX-M-55 plasmid, possibly indicating the emergence of successful clones and plasmids adapted to the chicken host. Our data contribute to the epidemiological tracking of resistance mechanisms in Enterobacterales from poultry and to knowledge for further One Health studies to control the spread of resistant bacteria from food animals to humans.

Prevalence and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from cattle between 2002 and 2006 in France.

Feces from 2,255 cattle (calves, young beef cattle, and culled cows) were collected at slaughter from nine departments across France. Campylobacter was recovered from 16.5% of the 2,255 samples (C. jejuni from 12.8% and C. coli from 3.7%), predominantly from calves. Antimicrobial resistance to six antibiotics of medical and/or veterinary interest was tested with the E-test. Resistance to tetracycline was found in most isolates (52.8% of C. jejuni isolates and 88.1% of C. coli isolates) in contrast to low but consistent resistance to ampicillin and erythromycin. Only two C. coli isolates were resistant to gentamicin. Multiple resistance was frequently detected in C. jejuni and C. coli isolates, and 0.8% (3 of 372) of the isolates were resistant to five of the six antimicrobials. An upward trend in the resistance to quinolones and fluoroquinolones in C. jejuni from calves was found; resistance to nalidixic acid reached 70.4% in 2006 and fluoroquinolone resistance increased from 29.7 to 70.4% during 2002 through 2006. All data were analyzed in parallel using clinical breakpoints or epidemiological cutoff values, and the results overlapped largely, except those for gentamicin. This 5-year survey (2002 through 2006) gives the first overview of the prevalence and antimicrobial resistance of C. jejuni and C. coli in cattle in France and documents to what extent cattle may contribute to the environmental reservoir of Campylobacter in France in the context of recurrent reports on links between human campylobacterioses and livestock. The results underline a notable increase in the resistance to fluoroquinolones in C. jejuni from cattle that may be of significant importance for public health.

Dynamics and molecular features of OXA-48-like-producing Klebsiella pneumoniae lineages in a Tunisian hospital.

OBJECTIVES: The aim of this study was to elucidate the molecular features of genes, plasmids and clones of OXA-48-like producingKlebsiella pneumoniae isolates recovered in Sahloul Hospital (Sousse, Tunisia) in the period 2012-2014. METHODS: In vitro antimicrobial susceptibility testing, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blotting and PCR-based replicon typing (PBRT) were performed. Extended-spectrum ß-lactamase (ESBL) and carbapenemases genes were detected by PCR and sequencing. The clonality of isolates was assessed by PFGE and multilocus sequence typing (MLST). RESULTS: Klebsiella pneumoniae accounted for 26.8% (1095/4083) of clinical Enterobacterales isolates identified during 2012-2014, of which 21.9% (240/1095) were resistant to carbapenems, mostly harbouring blaOXA-48-like genes (196/240; 81.7%). Plasmid analysis showed that blaOXA-204 and blaOXA-48 were mostly carried by IncA/C and IncL plasmids, respectively. The current data highlight the dominance of two ST101 and ST147 lineages spreading OXA-48 and OXA-204, respectively, through successive clonal spreads at this hospital. In addition, a large diversity of other K. pneumoniae lineages was also identified, such as ST15, ST36 and ST525 spreading OXA-48 as well as ST340, ST2032, ST301, ST199 and ST1561 spreading OXA-48 or OXA-204, constituting a reservoir of possible dominant clones in the future. CONCLUSION: This study reports the full molecular characterisation of carbapenem resistance in K. pneumoniae and the predominance of a few clones responsible for the dissemination of OXA-48 and OXA-204 enzymes in a Tunisian hospital.

vanA in Enterococcus faecium, Enterococcus faecalis, and Enterococcus casseliflavus detected in French cattle.

The goal of this study was to assess the presence of enterococci species presenting van-mediated glycopeptide resistance in French cattle. Fecal samples were collected from healthy and sick animals, and enterococci were screened for vancomycin resistance. Vancomycin resistance was principally encountered in Enterococcus gallinarum and Enterococcus casseliflavus strains. However, glycopeptide resistance was detected in three different species of enterococci (E. faecalis, E. faecium, and E. casseliflavus). Molecular characterization of the genetic support proved that they all presented the prototypic VanA element. Interestingly, the E. casseliflavus strain displayed a remarkable VanB phenotype/vanA-vanC genotype. Transferability, associated resistances, and factors of vanA cotransfer were sought. This study proved that acquired vanA genes can still be detected in food-producing animals more than a decade after the avoparcin ban. Indeed, calves, which are recurrently exposed to antibiotics in France, may allow the re-emergence of glycopeptide resistance through coselection factors, and this might potentially be concerning for human health.

Diversity of CTX-M Extended-Spectrum ß-Lactamases in Escherichia coli Isolates from Retail Raw Ground Beef: First Report of CTX-M-24 and CTX-M-32 in Algeria.

The aim of this study was to investigate the prevalence and molecular features of extended-spectrum cephalosporin resistance in Escherichia coli isolates contaminating ground beef at retail in Algeria. Of 371 ground beef samples, 27.5% were found to contain cefotaxime-resistant E. coli isolates distributed into A (24.5%), B1 (60.8%), and D (14.7%) phylogroups. A rate of 88.2% of isolates had a multidrug-resistance phenotype. All strains were producers of CTX-M type extended-spectrum ß-lactamases (ESBLs): CTX-M-1, CTX-M-3, CTX-M-14, CTX-M-15, CTX-M-24, or CTX-M-32. Conjugation assays allowed the transfer of blaCTX-M-1 in association with IncI1 plasmids, blaCTX-M-15 with IncI1 and IncK+B/O plasmids, blaCTX-M-3 with IncK plasmids, and blaCTX-M-14 with IncF1B or IncK plasmids. Sequence analysis of gyrA and parC genes showed mutations in 98.6% of ciprofloxacin-resistant isolates. The patterns "GyrA: S83L+D87N, ParC: S80I" (46.5%) and "ParC: S80I" (42.3%) were predominant. qnrS1, qnrB, and aac(6')-Ib-cr were detected in 18.7% of isolates. The tet genes, tetA, tetB, and tetA+tetB, were present in 95.7% of tetracycline-resistant isolates. The sul genes (sul1, sul2, sul3, sul1+sul2, sul2+sul3, and sul1+sul3) and the dfr gene clusters (dfrA1, dfrA5, dfrA7, dfrA8, dfrA12, dfrA5+dfrA12, dfrA1+dfrA5, dfrA7+dfrA12, dfrA5+dfrA7, and dfrA1+dfrA5+dfrA7) were found in 96.4% and 85.5% of sulfamethoxazole/trimethoprim-resistant isolates, respectively. Classes 1 and 2 integrons were detected in 67.6% and 9.8% of isolates, respectively. This study highlighted the significant presence of resistance genes, in particular those of CTXM ESBLs, in the beef meat, with the risk of their transmission to humans through food chain.