Pierre Châtre

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.

Prevalence of Fecal Carriage of Acquired Expanded-Spectrum Cephalosporin Resistance in Enterobacteriaceae Strains from Cattle in France

In gram-negative pathogens, extended-spectrum beta-lactamases (ESBLs) confer resistance to penicillins, cephalosporins (including extended-spectrum cephalosporins), and aztreonam, but not to cephamycins and carbapenems, and are inhibited by beta-lactamase inhibitors ([8][1]). Recently, cefotaximases

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 β-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.

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.

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.

Comparative prevalence and characterization of ESBL-producing Enterobacteriaceae in dominant versus subdominant enteric flora in veal calves at slaughterhouse, France.

Food-producing animals have become a growing reservoir of Extended-Spectrum Beta-Lactamase (ESBL)-producing bacteria. In cattle, veal calves are exposed to high amounts of antibiotics but ESBL prevalence data are still limited compared to other food sectors such as poultry production. Based on the investigation of 491 veal calves from different slaughtering batches at 12 abattoirs, this study shows a prevalence of 29.4% of ESBL producers in the faecal flora of veal calves in France in 2012. A variety of blaCTX-M genes was found, reflecting possible diverse pathways of dissemination in cattle. Another major conclusion is the comparison of the ESBL prevalence in the dominant versus sub-dominant Escherichia coli population of the same calves (1% and 29.4%, respectively). Also, the ESBL E. coli clones in the sub-dominant flora mostly differed from the non-ESBL dominant E. coli clones of the same calves. Of note, the distribution of blaCTX-M genes and E. coli phylogroups were similar to the ones previously found in ESBL E. coli clones from diseased calves. The hypothesis that ESBL genes may distribute more abundantly in certain backgrounds of E. coli was also discussed. In all, as recently reported in the Netherlands, these results strongly suggest a recent increase in the prevalence of ESBL carriage in French veal calves, which should be considered one of the major ESBL reservoirs in food animals.

Staphylococcal nasal carriage in calves: multiresistant Staphylococcus sciuri and immune evasion cluster (IEC) genes in methicillin-resistant Staphylococcus aureus ST398

Sir, Methicillin-resistant Staphylococcus aureus (MRSA) are increasingly associated with non-clinical settings or with companion and food-producing animals. This has raised questions about staphylococcal host specificity and the potential transmissibility from animals to humans or vice versa. Several clonal complexes (CCs) were reported as specifically deriving from animals (CC151 in cows) or humans (CC15, CC25 and CC45), whereas others are not restricted to particular hosts. In particular, the ST398 clone, originally described in pigs, has since been identified in humans, horses, cattle and poultry, and has become a public health concern, especially for persons in contact with animals. Conversely, coagulase-negative staphylococci (CoNS) are also responsible for severe infections in both animals and humans, and constitute a less-documented reservoir of mecA-carrying cassettes. In this context, our objective was to document the presence of MRSA and methicillin-resistant CoNS (MRCoNS) in veal calves at the slaughterhouse, and to characterize the isolates to gain insight into the potential risks for human health. Between April and June 2009, nasal swabs of 123 veal calves coming from 14 different feeding farms were sampled at the slaughterhouse in Lyon, France. Fifty-three (43.1%) methicillinresistant staphylococci, including 8 MRSA (6.5%) and 45 MRCoNS (36.6%), were isolated on selective plates after a pre-enrichment step. MRSA were sampled from eight different calves coming from four geographically unrelated farms. All belonged to the t899 spa-type ST398 clone, as confirmed by PCR, displayed an SCCmec type IV cassette and the agr1 allele, and shared identical resistance and genetic profiles by microarray (S. aureus genotyping; Identibac, Alere) and Cfr9I PFGE (see Figure S1, available as Supplementary data at JAC Online). Since ST398 can rapidly spread at slaughterhouses, a unique clone might have disseminated within this environment or during transport to it—possibly originating from the only calf fed on a pig–veal mixed farm. Nevertheless, the presence of a specific clone in the four farms cannot be excluded. Antimicrobial susceptibility tests (disc diffusion using clinical breakpoints recommended by the Antimicrobial Committee of the French Society of Microbiology, www.sfm-microbiologie.org) revealed phenotypic resistances to penicillin, tetracycline and tobramycin, as confirmed by the detection of blaZ, tet(M) and aadD genes on the microarray. This multidrug pattern was completed by the presence of the vga(A) and dfrA genes, and by the phenotypic detection of additional resistances to fusidic acid, ciprofloxacin, lincomycin and macrolides. Interestingly, while devoid of toxin genes, all isolates carried immune evasion cluster (IEC) type B genes (sak, chp and scn, but not sea), which are known to play an important role in human colonization but are usually not found in ST398 or animal staphylococci. The link with human adaptation remains to be elucidated, especially since MRSA or methicillin-sensitive S. aureus (MSSA) ST398 have rarely caused hospitalizations in France until now, even in the main pig-farming area (F. Laurent, National Reference Centre for Staphylococci, personal communication). Yet, severe infections have been reported in The Netherlands, and the acquisition of IEC genes in ST398 should be surveyed, especially regarding the transmission capacities and resistance patterns of this clone. In parallel, MRCoNS were identified by matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (Bruker Daltonics, Bremen, Germany) as Staphylococcus haemolyticus (n1⁄41) and Staphylococcus sciuri (n1⁄444), and confirmed by sequencing of the tuf gene. In addition to cefoxitin and penicillin resistance, susceptibility tests revealed resistances to antibiotics of human and veterinary interest, including fusidic acid, tetracycline, macrolides or aminoglycosides (Table 1), but not to the molecules prescribed in human medicine (vancomycin, teicoplanin, pristinamycin, linezolid, tigecycline and rifampicin). Results revealed a wide variety of 24 different susceptibility patterns. Overall, the number of associated resistances was considerably higher than what has recently been reported in human S. sciuri isolates, but the potential interspecific transmission of these resistance determinants remains to be investigated. The SCCmec cassette was nontypeable for 16 isolates (mecA, mecI and IS431, but no ccr gene) and 28 S. sciuri presented an SCCmec type III element (mecA, mecI and ccrAB3). Since all MRSA presented a type IV cassette (mecA, ccrAB2 and IS1272), there was no indication for a direct genetic exchange between S. sciuri and S. aureus, as recently reported for human staphylococci. Lastly, three veal calves coming from different farms carried both an MRCoNS and an MRSA, but, in each case, both bacteria shared no common resistance pattern or SCCmec cassette.

Emergence of Escherichia coli producing extended-spectrum AmpC β-lactamases (ESAC) in animals

In both humans and animals, the spread of Extended-Spectrum β-Lactamases (ESBL)/AmpC producers has become a major issue, particularly due to the plasmidic dissemination of most of these genes. Besides, over-expression of the chromosomal ampC gene was largely reported in human and animal Enterobacteriaceae and, more recently, modifications within the coding region of the ampC gene [encoding Extended-spectrum AmpC β-lactamases (ESACs)] were shown to be responsible for an hydrolysis spectrum expanded to oxyiminocephalosporins in humans. In this study, among 6765 cattle E. coli isolates, 28 (0.37%) isolates harboring a reduced susceptibility to cefepime (MICs ranging from 0.5 to 12 μg/ml) were investigated as presumptive ESACs producers. Highly conserved mutations in the promoter/attenuator region were identified at positions −88, −82, −42, −18, −1, and +58. Using sequencing and cloning experiments, amino acid substitutions of the AmpC beta-lactamase were characterized at positions 287 (mostly S287N, but also S287C), 292 (A292V) and 296 (H296P), similarly to data reported in humans. Interestingly, those cattle ESAC-producing E. coli isolates predominantly belonged to the Clonal Complex (CC) 23, thus mirroring what has been described in humans. The driving forces for the selection of ESACs in animals are unknown, and their prevalence needs to be further investigated in the different animal sectors. Considering the over-representation of ESAC-producing E. coli belonging to CC23 in both humans and animals, exchanges of ESAC producers between the two populations may have occurred as well. To our best knowledge, this study is the first report of ESACs in animals worldwide, which should be considered an emerging mechanism contributing to the resistance to extended-spectrum cephalosporins in the animal population.

Dissemination of multidrug-resistant blaCTX-M-15/IncFIIk plasmids in Klebsiella pneumoniae isolates from hospital- and community-acquired human infections in Tunisia

This study investigated the molecular features of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae from hospital- and community-acquired (HA/CA) infections in the region of Mahdia, Tunisia. Among 336 K. pneumoniae isolates recovered from both clinical contexts between July 2009 and December 2011, 49 and 15 were ESBL producers and originated from clinical and community sources, respectively. All isolates produced the CTX-M-15 enzyme. As shown by Southern blot on S1 nuclease treatment followed by pulsed-field gel electrophoresis (PFGE) gels, the blaCTX-M-15 gene was carried on IncFII (n=4), IncFIIk (n=25), IncL/M (n=4), IncK (n=1), or untypeable (n=15) plasmids in HA isolates. In CA isolates, the blaCTX-M-15 gene was carried on IncFIIk (n=6), IncFII (n=1), IncHI1 (n=1), or untypeable (n=7) plasmids. In all, 23 and 11 PFGE types were found among the HA and CA isolates. Multilocus sequence typing on representative isolates shows diverse sequence types (STs), such as ST307, ST101, ST39, ST4, ST140, ST15, and ST307 in HA isolates and ST101, ST664, and ST323 in CA isolates. This study is the first comprehensive report of ESBL plasmids in K. pneumoniae from HA and CA infections in Tunisia.

Characterisation of clinical canine meticillin-resistant and meticillin-susceptible Staphylococcus pseudintermedius in France

Staphylococcus pseudintermedius is a frequent pathogen in dogs. The emergence of meticillin-resistant S. pseudintermedius (MRSP), which is concomitantly resistant to nearly all veterinary licensed antibiotics used for systemic treatment in dogs, is a major problem for veterinarians. In France, 16.9% (41/243) of the S. pseudintermedius collected in 2010 were MRSP. They mainly belonged to the multiresistant MLST sequence type ST71, spa type t02, SCCmec type II-III (ST71-t02-II-III) European clone. Moreover, we also report the emergence of multiresistant meticillin-susceptible S. pseudintermedius isolates presenting atypical and/or new spa types. This study highlights the need for surveillance, optimised treatment guidelines and new therapeutic alternatives.