Estelle Saras

Co-occurrence of extended spectrum β lactamase and MCR-1 encoding genes on plasmids

Findings reported by Yi-Yun Liu and colleagues identified the plasmidborne gene mcr-1 encoding resistance to colistin with a high prevalence in Escherichia coli isolates from animals, foodstuff , and human beings in China. The same gene was then reported in Europe (Denmark) among extendedspectrum β lactamase (ESBL) and AmpC-producing E coli isolates from chicken meat and human infections, but at a very low prevalence. We screened ESBL-positive E coli isolates collected in France for colistin resistance. Isolates were collected between 2005 and mid-2014 from faeces of diarrhoeic veal calves at farms, as part of a survey in the context of the French antimicrobial resistance Resapath surveillance network for animal pathogens. We screened these isolates for colistin resistance using disk diff usion and minimum inhibitory concentration determination by broth microdilution. We analysed plasmids bearing the mcr-1 gene by conjugation, S1-pulsed-fi eld gel electrophoresis, PCRbased replicon typing, and Southern blot. We analysed clonal relationship of all isolates by enterobacterial repetitive intergenic consensus PCR and pulsedfi eld gel electrophoresis. Of 517 ESBL-producing E coli isolates collected, 106 (21%) were mcr-1 positive. Notably, the oldest mcr-1positive E coli isolate had been collected in 2005. The 106 mcr-1-positive E coli isolates originated from diff erent individuals located in 94 widely distant farms, and they were clonally unrelated. Sequencing of the whole mcr-1 gene in 75 mcr-1-positive isolates revealed a 100% identity compared with the original sequence. Co-occurrence of the mcr-1 and ESBL genes was identifi ed in a subset of seven isolates, with mcr-1 and blaCTX-M-1 being found on a large and conjugative IncHI2type plasmid together with genes conferring resistance to sulfonamides and tetracyclines, two antibiotics widely used in veterinary medicine. These findings demonstrate a colocation of the mcr-1 gene along with an ESBL gene on a single plasmid, and additional studies are needed to clarify the diversity of the plasmid backbones spreading these two genes within our collection. Noticeably, the prevalence of the mcr-1 gene among ESBL producers in veal calves was much higher than that found in ESBL-positive E coli isolates in human beings and chicken meat reported in Denmark. This diff erence may refl ect a major spread of the mcr-1 gene in European live animals. We showed that the dissemination of mcr-1, at least in France, had already occurred more than a decade ago, with one E coli isolate collected in 2005 identifi ed as mcr-1 positive. Altogether, available data reveal the occurrence of mcr-1 among diff erent animals and human contexts over time. Worryingly, we show that selection pressure with broadspectrum cephalosporins may select for colistin resistance and vice-versa, further highlighting the likelihood of a pandemic spread of mcr-1. Of note, the substantial use of tetracyclines and sulfonamides in animals might also substantially contribute to the dissemination of mcr-1 plasmids. In a one-health perspective, and considering the renewed importance of colistin in human medicine, our data and those from others underscore the urgent need to limit the spread of mcr-1-positive plasmids by reconsidering the massive use of colistin in veterinary medicine worldwide.

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.

Diversity and Mobility of Integrative and Conjugative Elements in Bovine Isolates of Streptococcus agalactiae, S. dysgalactiae subsp. dysgalactiae, and S. uberis

ABSTRACT Bovine isolates of Streptococcusagalactiae (n = 76), Streptococcusdysgalactiae subsp. dysgalactiae (n = 32), and Streptococcusuberis (n = 101) were analyzed for the presence of different integrative and conjugative elements (ICEs) and their association with macrolide, lincosamide, and tetracycline resistance. The diversity of the isolates included in this study was demonstrated by multilocus sequence typing for S. agalactiae and pulsed-field gel electrophoresis for S. dysgalactiae and S. uberis. Most of the erythromycin-resistant strains carry an ermB gene. Five strains of S. uberis that are resistant to lincomycin but susceptible to erythromycin carry the lin(B) gene, and one has both linB and lnuD genes. In contrast to S. uberis, most of the S. agalactiae and S. dysgalactiae tetracycline-resistant isolates carry a tet(M) gene. A tet(S) gene was also detected in the three species. A Tn916-related element was detected in 30 to 50% of the tetracycline-resistant strains in the three species. Tetracycline resistance was successfully transferred by conjugation to an S. agalactiae strain. Most of the isolates carry an ICE integrated in the rplL gene. In addition, half of the S. agalactiae isolates have an ICE integrated in a tRNA lysine (tRNALys) gene. Such an element is also present in 20% of the isolates of S. dysgalactiae and S. uberis. A circular form of these ICEs was detected in all of the isolates tested, indicating that these genetic elements are mobile. These ICEs could thus also be a vehicle for horizontal gene transfer between streptococci of animal and/or human origin.

High Prevalence of blaCTX-M-1/IncI1/ST3 and blaCMY-2/IncI1/ST2 Plasmids in Healthy Urban Dogs in France

ABSTRACT In the community, close contacts between humans and dogs may promote the transfer of extended-spectrum beta-lactamase/plasmidic AmpC cephalosporinase (ESBL/pAmpC) genes. Large-scale prevalence studies on ESBL/pAmpC carriage in dogs are rare, and data on ESBL/pAmpC plasmids are even more limited. Here, a considerable rate of 18.5% ESBL/pAmpC carriers was found among 368 unrelated healthy dogs in Paris, France. This prevalence is much higher than the one found in healthy humans in the same city (6%) but close to that recently reported in dogs in China (24.5%). All isolates were identified as Escherichia coli, except one Salmonella enterica and one Klebsiella pneumoniae isolate. The sequence type 131 (ST131) clone was rare (2/73 isolates). Interestingly, two plasmids (blaCTX-M-1/IncI1/ST3 and blaCMY-2/IncI1/ST2) were unexpectedly highly predominant, raising the question of their successful spread. Considering that CTX-M-1 was recently found to be equally as abundant as CTX-M-15 in healthy Parisian subjects, the question of dogs being a CTX-M-1 reservoir for humans is open. Such a high prevalence of the blaCMY-2/IncI1/ST2 plasmid may result from the use of cephalexin in veterinary medicine, as previously demonstrated experimentally. In all, our study points out healthy urban dogs as a potential source of ESBL/pAmpC genes that can further disseminate to the human community.

CTX-M-15 extended-spectrum β-lactamase in a shiga toxin-producing Escherichia coli isolate of serotype O111:H8.

ABSTRACT We report the discovery of a CTX-M-15-producing Escherichia coli (STEC) of serogroup O111:H8, a major serotype responsible for human enterohemorrhagic Escherichia coli (EHEC) infections. In line with the recent CTX-M-15/O104:H4 E. coli outbreak, these data may reflect an accelerating spread of resistance to expanded-spectrum cephalosporins within the E. coli population, including STEC isolates.

High Prevalence of the Animal-Associated blaCTX-M-1 IncI1/ST3 Plasmid in Human Escherichia coli Isolates

In humans and animals, extended-spectrum beta-lactamases (ESBLs) are widespread enzymes conferring resistance to broad-spectrum cephalosporins. Consumption of ESBL-contaminated foodstuffs or contacts with ESBL-colonized/infected animals enhance the risk of human spread of ESBL genes from nonhuman

ermB-mediated erythromycin resistance in Streptococcus uberis from bovine mastitis

The ermB gene was identified in 111 erythromycin resistant isolates of Streptococcus uberis from cases of bovine mastitis associated either with a constitutive (47/111) or an inducible (64/111) phenotype, as well as a phenotypic resistance to all macrolides tested. Resistance to lincosamides was identified in 14 other isolates of S. uberis from bovine mastitis cases and was mainly mediated by the linB gene; resistance conferred by a combination of two genes (linB-lnuD, ermB-linB) was also detected.

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.

ST147 NDM-1-producing Klebsiella pneumoniae spread in two Tunisian hospitals

Unité de Recherche: Résistances Bactériennes Émergentes et Sécurité des Soins ‘UR12SP37’, Laboratoire de Microbiologie, Hôpital Universitaire Sahloul, Sousse, Tunisia; French Agency for Food, Environmental and Occupational Health Safety (ANSES), Unité Antibiorésistance et Virulence Bactériennes, Lyon, France; Institut Supérieur des Sciences Appliquées et de Technologie de Mahdia, Université de Monastir, Monastir, Tunisia; Laboratoire de Microbiologie, Hopital Tahar Sfar, Mahdia, Tunisia; Faculté de Pharmacie de Monastir, Université de Monastir, Monastir, Tunisia; Faculté de Médecine de Sousse, Université de Sousse, Sousse, Tunisia

High prevalence of international ESBL CTX-M-15-producing Enterobacter cloacae ST114 clone in animals

OBJECTIVES The objective of this study was to characterize ESBL-producing Enterobacter cloacae isolated from animals and to compare their clonal distribution with that of human-related isolates. METHODS Among 635 clinical E. cloacae from horses, dogs and cats collected in France between 2010 and 2013, 36 were resistant to ceftiofur as determined by disc diffusion. ESBL genes were identified by sequencing. Plasmids carrying ESBL-encoding genes were characterized by PCR-based replicon typing, S1-PFGE and Southern blotting. IncHI2 plasmids were subtyped using the plasmid double-locus sequence typing scheme and multiplex amplification of the hipA, smr0092 and smr0183 genes. All E. cloacae were typed by PFGE and MLST. ST clustering was analysed by eBURST. RESULTS All 36 ceftiofur-resistant E. cloacae produced an ESBL. Their PFGE patterns formed 23 clusters of high similarity and 13 STs and were isolated from epidemiologically unrelated animals (14 horses, 11 dogs and 11 cats) distributed throughout France. ST114, the most prevalent clone in humans, was over-represented in animals (16/36) compared with other human-related clones detected here. The blaCTX-M-15 gene was dominant (66.7%) and mostly carried on IncHI2 plasmids (ST1 subtype). ST114 isolates always produced CTX-M-15. CONCLUSIONS Most ESBL-producing E. cloacae from animals studied here (69.4%) belonged to potentially high-risk clones in humans, in particular ST114 (44.4%). These data raise questions and potential concerns about the transfer of E. cloacae between animals and humans.