Safia Dahmen

Characterization of extended-spectrum beta-lactamase (ESBL)-carrying plasmids and clones of Enterobacteriaceae causing cattle mastitis in France.

Extended-spectrum beta-lactamases (ESBLs) have become widespread enzymes in food-producing and companion animals worldwide. However, in cattle mastitis, a major cause of economic loss in the dairy industry, ESBL-producers were rarely described. In this study, from a collection of 1427 Escherichia coli and Klebsiella pneumoniae isolates causing clinical mastitis in France, we report 0.4% (6/1427) of the isolates carrying an ESBL gene. These six isolates were genetically unrelated and recovered over a 3-year period of time. The bla(CTX-M-14) gene was found in 4/6 isolates, and was predominantly located on F2:A-:B- IncFII plasmids. The bla(CTX-M-1) IncI1/ST3, which is widespread in various animal species in France, was found as well. Interestingly, among the five E. coli isolates, the ST23 and ST58 clones were found twice, together with the ST10 clone, all of which were previously found as ESBL-carriers in humans. Despite the very limited number of ESBL-producers recovered, this study shows a surprisingly low molecular diversity of the strains causing mastitis in France with respect to ESBL genes, plasmids and clones. Further work is needed to understand the major driving forces of the ESBL epidemiology in animals, including for different infections within the same animal species.

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.

IncI1/ST3 plasmids contribute to the dissemination of the blaCTX-M-1 gene in Escherichia coli from several animal species in France

Sir, In Gram-negative bacteria, extended-spectrum b-lactamases (ESBLs) are widespread enzymes conferring resistance to most b-lactams, including third-generation cephalosporins but with the exception of carbapenems and cephamycins. In animals, the blaCTX-M-1 gene is one of the most frequently reported ESBL-encoding genes; in contrast, in humans the blaCTX-M-15 gene is highly prevalent. Plasmids ascribed to different incompatibility (Inc) groups play a key role in the spread of ESBL-encoding genes. However, certain combinations display epidemiological success, such as the blaCTX-M-15 gene on IncFII plasmids in humans, often associated with the B2-O25b:H4-ST131 E. coli clone. Interestingly, in France, the same blaCTX-M-1-carrying IncI1/ST3 plasmid has been reported in various serovars of Salmonella enterica isolated from humans, poultry and cattle, and in Escherichia coli from healthy poultry. – 5 Here, we investigate eight ESBL E. coli isolates recovered from 2006 to 2010 from non-food-producing animals, a cat, four dogs, two horses (one a pony) and a goat, that were confirmed to harbour the blaCTX-M-1 gene by PCR and sequencing. Those isolates were collected through the RESAPATH network, which carries out surveillance of antimicrobial resistance in animals in France (www.resapath.anses.fr). As determined by disc diffusion according to the CA-SFM guidelines (www.sfm-microbiologie.fr), all strains were resistant to ceftiofur but susceptible to cefoxitin, with a typical double-disc synergy. The blaCTX-M-1 gene was preceded by the ISEcp1 element. Three isolates additionally produced the b-lactamase TEM-1 and one presented an OXA-1 (Table 1). Resistance to non-b-lactams varied depending on the isolates, but resistance to tetracyclines and sulphonamides was constant (Table 1). All isolates were unrelated, as proved by distinct PFGE profiles of BlnI-digested genomic DNA (data not shown), and belonged to the phylogenetic groups A (n1⁄42), B1 (n1⁄42), B2 (n1⁄41) or D (n1⁄43) (Table 1). The B2 isolate did not belong to the B2-O25b:H4-ST131 E. coli clone, which has spread worldwide among humans and was also recovered from livestock and companion animals. Resistance to ceftiofur was transferable by conjugation and the blaCTX-M-1 gene was identified in all recipients, together with a single IncI1 replicon (Table 1). As determined on S1-PFGE gels, IncI1 plasmids ranged between 112 and 120 kb in size (Table 1). Southern blot on S1-PFGE gels with blaCTX-M and IncI1 probes demonstrated that the blaCTX-M-1 gene was carried on the IncI1 plasmid. Subtypes of the IncI1 plasmids were determined using the plasmid multilocus sequence typing (‘pMLST’) scheme and all blaCTX-M-1/IncI1 plasmids were of sequence type ST3. Restriction fragment length polymorphism (RFLP) on PstI-digested plasmid DNA from transconjugants showed that the blaCTX-M-1/IncI1/ST3 plasmids were indistinguishable or highly similar (see Figure S1, available as Supplementary data at JAC Online). Indeed, RFLP profiles of the blaCTX-M-1/IncI1/ST3 plasmids from pets were identical to that found previously in S. enterica in humans, poultry and cattle. –5 Those from horses and the goat were indistinguishable as well, albeit slightly different from those from pets. Southern blot on the RFLP gel revealed the same PstI fragment in all plasmids from pets, also of the same size as that previously found in S. enterica (Figure S1; lanes 9 and 10). – 5 For horses and the goat, Southern blot with a blaCTX-M probe confirmed the differences observed on RFLP gels. Hybridization with a blaTEM probe suggested that the slight differences among blaCTX-M-1/IncI1/ ST3 plasmids were probably due to additional resistance (i.e. blaTEM) genes and not to major variations of the plasmid scaffold (Figure S2, available as Supplementary data at JAC Online). In this study, we report indistinguishable or highly similar blaCTX-M-1/IncI1/ST3 plasmids in different E. coli isolates from a wide range of animal species in France. All animals were unrelated, had different owners and originated from highly distant areas. They were also sampled at various periods of time, from 2006 to 2010. Consequently, we demonstrate the spread of the blaCTX-M-1/IncI1/ST3 plasmid in the animal population in France, irrespective of the E. coli backgrounds and animal species. To our best knowledge, this is also the very first report of an ESBL in a goat. In a previous work, we suggested that blaCTX-M-1/IncI1/ST3 plasmids could have transferred to cattle from poultry, a recognized reservoir of IncI1 plasmids carrying ESBL genes. In fact, this ESBL plasmid may have spread more extensively than previously thought into the animal population. Alternately, IncI1 plasmids, which are highly prevalent in animals, may have acquired the blaCTX-M-1 gene independently within different hosts. Equally worrying is the detection of an ESBL producer in small ruminants, farming of which is relatively spared from excessive antibiotic usage. Interestingly, blaCTX-M-1/IncI1/ST3 plasmids successfully expanded in animals in France, whereas most blaCTX-M-1/IncI1 plasmids reported from food-producing animals in the Netherlands were of the ST7 subgroup. Taken together, the differential expansion among countries of different ESBL plasmid subtypes

Characterization and Molecular Epidemiology of Extended-Spectrum β-Lactamases in Clinical Isolates of Enterobacteriaceae in a Tunisian University Hospital

One hundred extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae were recovered from the intensive care unit and the urology ward of the University Hospital of Sahloul in Tunisia between May 2005 and May 2006. The majority of strains showed a high level of resistance to cefotaxime and ceftazidime. Double-disk synergy test and E-test strips were used to confirm production of ESBLs. The molecular analysis revealed that the majority of strains (91%) carried genes encoding CTX-M-15. SHV-12 and SHV-2a were produced, respectively, by 9% and 3% of the strains. Pulsed-field gel electrophoresis of ESBL-producing Klebsiella pneumoniae isolates revealed four different clonal groups and three for Escherichia coli, showing the absence of spread of any epidemic clone. The CTX-M-15 ESBL-producing E. coli of the major clonal group belong to the B2 phylogenetic group, to the sequence type 131, and has a high virulence potential. In conclusion, CTX-M-15 ESBLs accounted for the overwhelming majority of ESBL types among Enterobacteriaceae from our hospital. This study confirms the high rate of ESBLs in Tunisia and further demonstrates the worldwide spread of genes coding for CTX-M-15 enzymes in clinical isolates.

The blaCTX-M-1 IncI1/ST3 plasmid is dominant in chickens and pets in Tunisia

Sir, Extended-spectrum b-lactamases (ESBLs) of the CTX-M group have become widespread enzymes conferring resistance to broadspectrum cephalosporins in humans and animals, and plasmids play a key role in the horizontal transfer of the corresponding genes. Contrary to the situation in humans, the blaCTX-M-1 gene is abundant in animals. It is also frequently located on plasmids of specific incompatibility (Inc) groups and subtypes, such as IncI1/ST3 plasmids, which can consequently be considered as dominant plasmids. Indeed, indistinguishable blaCTX-M-1 IncI1/ ST3 plasmids have been found in Escherichia coli from cattle, goats, poultry, horses and pets and in Salmonella enterica from cattle, poultry and humans. – 5 In Tunisia, the blaCTX-M-1 gene was also reported in pets, foodstuff and food animals, particularly in chickens. – 8 Moreover, 7.3% of Tunisian healthy humans proved to carry CTX-M-1-producing E. coli, suggesting that foodstuff of poultry origin may contribute to the transfer of the blaCTX-M-1 gene from animals to humans. The aims of this study were to characterize blaCTX-M-carrying plasmids from E. coli isolates from chickens and pets in Tunisia and to compare them with previously reported blaCTX-M-carrying plasmids. Between December 2011 and April 2012, faecal swabs from 193 chickens, 41 dogs and 4 cats were collected in Tunisia and plated onto cefotaxime-supplemented (4 mg/L) MacConkey agar. One presumptive E. coli colony was selected per plate and identification was confirmed using API20E galleries. All E. coli isolates were then tested for antimicrobial susceptibility by disc diffusion according to the Antibiogram Committee of the French Society for Microbiology guidelines (www.sfm-microbiologie.fr) and ESBL production was confirmed by the double-disc synergy test. Chickens originated from 12 unrelated farms (each with 1000–5000 animals) in the Sousse (7), Mahdia (1), Kairouan (2) and Monastir (2) governorates, and were either diseased or dead (colibacillosis). Dogs and cats were sampled at a single clinic during routine examination (Sousse), and all but three animals were healthy. Seven of the 12 chicken farms (58.3%) were found positive for ESBL producers (each governorate was represented), and eight unrelated ESBL E. coli isolates (using PFGE, not shown) were recovered from the 193 samples (8/193, 4.1%). Seven additional unrelated ESBL E. coli isolates were recovered from the 45 pets (7/45, 15.6%). No isolate belonged to the pandemic human O25b-ST131 E. coli clone. All but one isolate displayed multiple co-resistances (Table 1). ESBL-carrying plasmids were transferred into electrocompetent E. coli TOP10 cells, and the blaCTX-M-1 gene was confirmed by PCR and sequencing in 13/15 (87%) transformants. The two last transformants harboured the blaCTX-M-9 and the blaCTX-M-15 genes, respectively. Southern blots on S1-PFGE gels with blaCTX-M, IncI1 and IncF probes demonstrated that all blaCTX-M-1 and the blaCTX-M-9 genes were carried on IncI1 plasmids, whereas the blaCTX-M-15 gene was located on an IncFII plasmid (not shown). The IncI1 plasmids sizes ranged from 100 to 120 kb, whereas the size of the blaCTX-M-15 IncFII plasmid was 160 kb (Table 1). In chicken isolates, the blaCTX-M-1 IncI1 plasmids belonged to the ST3 subtype (allelic profile 2/1/4/1/2) or to the closely related ST87 (8/1/4/1/2) subtype (Table 1), which only differs from ST3 by four base pairs at the end of a single locus (repA gene; http ://pubmlst.org/plasmid). The blaCTX-M-9 gene was also located on an IncI1/ST87 plasmid. In pet isolates, all plasmids but one blaCTX-M-1 IncI1 plasmid belonged to the ST3 subtype, whereas one isolate belonged to the unrelated ST25 (1/4/5/4/1) subtype (Table 1). Restriction fragment length polymorphism (RFLP) profiles of the IncI1/ST3 and IncI1/ST87 plasmids after digestion with PstI or EcoRI were either indistinguishable or highly similar, whatever the animal origin (Figure S1, available as Supplementary data at JAC Online). Hence, this study shows that the blaCTX-M-1 IncI1/ST3 plasmid is broadly disseminated in chickens and pets in Tunisia. Interestingly enough, the blaCTX-M-1 IncI1/ST3 plasmids described here were identical or highly similar to blaCTX-M-1 IncI1/ ST3 plasmids recently reported in France and Belgium in several animal species and humans (Figure S1, available as Supplementary data at JAC Online ). These plasmids were reported not only in E. coli but also in S. enterica, in particular of serovars Llandoff, London, Newport and Typhimurium of chicken and human origin. In conclusion, the blaCTX-M-1 IncI1/ST3 plasmid, which is dominant invarious animal species in Europe and has alsobeen reported in humans infected with S. enterica, appears to be dominant in foodand non-food-producing Tunisian animals. These data underline the international role of the blaCTX-M-1 IncI1/ST3 plasmid in ESBLepidemiology in animals. The risk of dissemination of the blaCTX-M-1 IncI1/ST3 plasmid from E. coli to S. enterica in Tunisia, as reported in Europe, is also a major concern. Furthermore, this study suggests that the surprisingly high prevalence of CTX-M-1 producers in the Tunisian community might result from the abundance of the blaCTX-M-1 IncI1/ST3 plasmid in animals. There is an urgent need to set up surveillance systems for antimicrobial resistance and antibiotic usage in animals (particularly in the food chain) in Tunisia.

Emergence of SHV-2a Extended-Spectrum β-Lactamases in Clinical Isolates of Pseudomonas aeruginosa in a University Hospital in Tunisia

Extended-spectrum beta-lactamases (ESBLs) in Pseudomonas aeruginosa are increasingly reported worldwide. In our study, a total of 70 clinical isolates of multidrug-resistant P. aeruginosa were studied. Isoelectric focusing electrophoresis, PCR, and PCR product sequencing were designed to characterize the contained ESBLs. The Double Disk Synergy Test in Cloxacillin (250 microg/ml)-containing Mueller-Hinton agar plates with a 20 mm distance between disks was the most reliable ESBL-screening method. Seven out of 70 multidrug-resistant P. aeruginosa clinical isolates were positive for ESBL and have the bla(SHV-2a) ESBL gene. The bla(SHV-2a)-positive isolates were clonally related according to Enterobacterial Repetetive Intergenic Consensus-PCR (ERIC-PCR) results. The bla(SHV-2a) gene was found to be chromosomally located, and the flanking IS26 sequence in the immediate upstream region of the bla(SHV-2a) gene was detected in all SHV-2a-producing isolates. This is the first report of SHV-2a-producing P. aeruginosa isolates from Tunisia.

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.

blaCTX-M-15-Carrying F2:A-:B- Plasmid in Escherichia coli from Cattle Milk in Tunisia

Extended-spectrum β-lactamases (ESBL) are widespread enzymes in animals, and the risk of transmission of ESBL genes to humans has become a major issue. In Tunisia, recent data showed a high prevalence of ESBL-producing Escherichia coli isolates in healthy animals, mostly in chickens. In this study, we report the first data on ESBL in diseased Tunisian animals (chickens and cattle), highlighting a major difference in ESBL prevalence in the infectious versus noninfectious E. coli flora. Interestingly, the only ESBL producer was an ST10 E. coli from a cattle, and not from chicken. Moreover, this E. coli isolate harbored the bla(CTX-M-15) gene on an F2:A-:B- plasmid, a combination frequently found in humans. This plasmid was also highly similar to a bla(CTX-M-15) F2:A-:B- plasmid recently reported in cattle in France. Altogether, this study is also the first report of the bla(CTX-M-15) gene in food animals in Tunisia, and, to our best knowledge, the first report of an ESBL producer in cattle in Africa. Since this plasmid was recognized in cattle in France and worldwide in humans, the question of its origin in Tunisian cattle is open. The detection of ESBL producers in milk in Tunisia may also constitute a risk of ESBL transmission from animals to humans through food consumption.

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.

F2:A-:B- plasmid carrying the extended-spectrum β-lactamase blaCTX-M-55/57 gene in Proteus mirabilis isolated from a primate

Didier Raoult a,b,∗ a Aix Marseille Université, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE, UM63), CNRS 7278, IRD 198, INSERM 1095, 13005 Marseille, France b AP-HM, CHU Timone, Pôle Infectieux, 13005 Marseille, France c Service de Bactériologie, Hospices Civils de Lyon, Lyon, France d Service de Chirurgie Cardio-vasculaire, CHU Dijon, Dijon, France e Service de Cardiologie, CHU Dijon, Dijon, France