In veterinary medicine, beta-lactam antibiotics are arguably

MICROBIAL DRUG RESISTANCE Volume 22, Number 4, 2016 ª Mary Ann Liebert, Inc. DOI: 10.1089/mdr.2015.0196 Emergence of Carbapenemase-Producing Escherichia coli Isolated from Companion Animals in Algeria Massilia Yousfi, 1 Abdelaziz Touati, 1 Assia Mairi, 1 Lucien Brasme, 2,3 Alima Gharout-Sait, 1 Thomas Guillard, 2,3 and Christophe De Champs 2,3 The emergence and worldwide spread of carbapenemase-producing Enterobacteriaceae is of great concern to public health. The aim of this study was to investigate the occurrence of carbapenemase-producing Escherichia coli in companion animals in Algeria. Two hundred fecal samples were obtained from healthy and diseased s and cats in one veterinary office and private owners in Bejaia city, Algeria, during November 2014 to March 2015. Isolates were screened by polymerase chain reaction for the presence of carbapenemase, acquired plasmidic AmpC (pampc) and extended-spectrum beta-lactamase genes. Five carbapenemase-producing E. coli isolates were detected including four OXA-48-producing isolates and one isolate producing NDM-5. Coexpression of ESBL and pampc genes was observed in these isolates. Phylogenetic grouping revealed that these isolates belonged to A and D phylogroups. The results of this study show that carbapenemase-producing E. coli spread to the companion animals in Algeria. Introduction In veterinary medicine, beta-lactam antibiotics are arguably the most important and widely used antimicrobial class for treating bacterial infections, including those caused by Enterobacteriaceae. 23 A large variety of beta-lactams is currently licensed for use in veterinary medicine and thus provides opportunity for selection pressure in the development of beta-lactam resistance. Indeed, beta-lactam resistance, including resistance to extended-spectrum beta-lactams, has now been increasingly observed in bacteria of animal origin and of human health concern. 16 During the last decade, the emergence of carbapenemaseproducing strains among Gram negative bacilli is remarkable. In Enterobacteriaceae, the carbapenemases belong to three types, the metallo-b-lactamases (MbLs), Klebsiella pneumoniae carbapenemases (KPC)-type, and the OXA-48 type enzymes. 27 The MBLs of Ambler class B are active against penicillins, older and newer cephalosporins, and carbapenems, but not aztreonam. 27 Recently, the emergence and dissemination of NDM-1 (New Delhi metallo b-lactamase) from India has garnered much attention. 23 The NDM-type b-lactamase was first isolated in 2009 from a Swedish patient returning from India. bla NDM has now spread to all inhabited continents and is carried by multiple Gram-negative species. 28 Although OXA-48 hydrolyses penicillins at a high level, it hydrolyses carbapenems only at a low level. In addition, it shows very weak activity against extended-spectrum cephalosporins. The OXA-48 type carbapenemases are found most commonly in Escherichia coli and K. pneumoniae, and are endemic in Turkey, northern Africa, and India. 21 The KPC exhibit activity against a wide spectrum of b-lactams, including penicillins, older and newer cephalosporins, aztreonam, and carbapenems. 27 Reports on carbapenemase-producing bacteria in animals are rare, but are increasing. Isolates of E. coli and Salmonella producing carbapenemase VIM-1 were recovered from pigs in Germany. 8,9 Poirel et al., reported one Acinetobacter baumannii isolate expressing carbapenemase OXA-23 recovered from cattle in France 20 and NDM-1-producing A. baumannii and Acinetobacter lwoffii isolates were reported from porcine and chicken sources in south China. 30 Concerning carbapenem resistance among Gram-negative bacteria isolated from companion animals, only two reports were published, including isolates of E. coli producing NDM- 1 reported from USA 25 and OXA-48 producing E. coli and K. pneumoniae identified in Germany. 26 These indicate that 1 Laboratoire d Ecologie Microbienne, FSNV, Université de Bejaia, Bejaia, Algérie. 2 Laboratoire de Bactériologie Virologie-Hygiène Hospitalière, CHU Reims, Hôpital Robert DEBRE, Reims, France. 3 EA4687 SFR CAP-Santé (FED 4231), Université de Reims-Champagne-Ardenne, Reims, France. 342

NDM-5 AND OXA-48 IN ESCHERICHIA COLI FROM PETS 343 carbapenemase-producing isolates are an emerging problem and can constitute a serious threat for public health. In Algeria, no studies have been published on antimicrobial resistance in companion animals and no data were available. This study aimed to determine the prevalence of colonization by carbapenemase-producing E. coli recovered from companion animals in Algeria. Materials and Methods Bacterial strains A total of 200 rectal swabs were obtained from healthy (i.e., pets admitted to veterinary office for vaccination) and diseased s and cats (for diagnostic investigation) in one veterinary office and private owners in Bejaia city, Algeria, during November 2014 to March 2015. Swabs were analyzed for the presence of carbapenemase-producing E. coli within 4 hr of collection. Swabs were enriched in 10 ml nutrient broth (Fluka, St Louis, MO) containing ½ disc of 10 mg ertapenem (BioRad, Marnes La Coquette, France). After incubation at 37 C/ 24 hr, 100 ml of the culture were streaked on MacConkey agar (Fluka) plates containing imipenem (0.5 mg/l) and vancomycin (16 mg/l) to inhibit Gram-positive bacteria and incubated under aerobic conditions during 24 hr at 37 C. E. coli isolates were identified using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (IVD MALDI Biotyper; Bruker Biospin SAS, Wissembourg, France). Antimicrobial susceptibility testing Antibiotic susceptibility was determined on Mueller Hinton agar using the standard disc diffusion procedure as described by the European Committee on Antimicrobial Susceptibility Testing. 7 The following antibiotics were tested: ticarcillin, ticarcillin clavulanic acid, piperacillin, piperacillin tazobactam, amoxicillin clavulanic acid, ceftazidime, cefotaxime, cefepime, cefoxitin, imipenem, ertapenem, meropenem, aztreonam, temocillin, amikacin, tobramycin, ciprofloxacin, chloramphenicol, tigecycline, and cotrimoxazole (BioRad). Determination of minimum inhibitory concentrations Isolates classified as resistant or intermediate to ertapenem by disk diffusion were further analyzed by determining the minimum inhibitory concentrations ( MICs) of imipenem, ertapenem, meropenem, doripenem, ceftazidime, cefotaxime, aztreonam, and piperacillin tazobactam by Etest (AB biomérieux, Marcy l Etoile, France). MICs were interpreted according to the breakpoints recommended by EUCAST. 7 Phenotypic carbapenemase detection The Modified Hodge Test (MHT) was performed on a Mueller Hinton agar (biomérieux, Marcy l Etoile, France) plate with both imipenem and meropenem (BioRad) with and without EDTA 0.5M (5 ml on the ertapenem disc) and E. coli ATCC 25922 as the indicator organism for detection of carbapenemases as described previously. 6,7 In addition, a double-disc synergy test was designed for detecting MbLs as described previously. 29 K. pneumoniae ATCC BAA-1705 was used as a positive control and K. pneumoniae ATCC 8291T was used as a negative control. Detection of antibiotic resistance genes Carbapenemase encoding genes, bla KPC, bla VIM, bla IMP, bla NDM-1, bla OXA-23-like, bla OXA-58-like, and bla OXA-48-like, were screened as previously described. 10 Polymerase chain reaction (PCR) assays for the presence of genes encoding for b-lactamases (bla TEM, bla SHV, and bla CTX-M ) and for plasmid-mediated AmpC-type genes (bla ACC, bla FOX, bla MOX, bla DHA, bla CMY, and bla MIR ) were performed as described previously. 15 A multiplex real-time PCR assay was used to screen the presence of qnra, qnrb, qnrs, qnrc, qnrd, oqxab, and qepa genes as described previously. 11,13 Pyrosequencing method was used for the rapid detection of aac(6 )-Ib-cr determinant. 12 PCR products were sequenced and the sequencing results were compared to reported sequences available in GenBank. Phylogenetic groups and virulence genotyping of E. coli PCRs were performed to determine the phylogenetic groups (A, B1, B2, C, D, E, F, and clade I) of the E. coli isolates, using the newly revised Clermont method. 5 The presence of 11 virulence factors was investigated by PCR. These factors included sfa/foc (S and F1C fimbriae), papg alleles (G adhesin classes of P fimbriae), papc (C adhesin classes of P fimbriae), hlya (alpha-hemolysin A), cnf (cytotoxic necrotizing factor 1), fyua (genes of yersiniabactin), iuta (aerobactin receptor), ibea (invasion protein IbeA), stx1, 22 stx2 4, and eae. 19 Results During a 5-month period, we collected 200 sample swabs taken from 200 pets from a veterinary practice (93 s and 81 cats) and private owners (23 s and 3 cats). A total of 173 of companion animals were healthy and 27 were diseased. We isolated five E. coli isolate-producing carbapenemase from five animals. The overall prevalence of carbapenemase isolates in the 200 animals was 2.5%. The prevalence of E. coli-producing carbapenemase in s was 2.58% and in cats was 2.83%. Four isolates were from healthy pets (prevalence of 2.3%; n = 173) and one isolate was obtained from a diseased cat admitted to a veterinary office for a respiratory infection (prevalence of 3.7%; n = 27). All isolates were recovered from the veterinary office. These E. coli isolates were suspected to produce carbapenemases by Hodge test, which are not inhibited by EDTA, except for one isolate (S174). The antibiotic susceptibility profiles of the carbapenemaseproducing E. coli strains isolated in this study are shown in Table 1. E. coli S174 isolate showed a synergy between aztreonam and co-amoxiclav when tested by DD-test (Fig. 1). Concerning nonbeta-lactam antibiotics, isolates showed different susceptibility pattern (Table 1), but all isolates were susceptible to colistin, tigecycline, and amikacin. The antibiotics MICs are shown in Table 1. Except E. coli S174 isolate, which showed high carbapenem MICs (24 mg/ L for doripenem and >32 mg/l for ertapenem, meropenem,

344 YOUSFI ET AL. Table 1. Characteristics of Escherichia coli Strains Producing Carbapenemase Isolated from Pets MIC (mg/l) ETP MEM DOR IMP ATM CAZ CTX TZP b-lactamases and PMQR identified Resistance phenotype Virulence genes Phylogenetic group Isolate Origin OXA-48, TEM-1 1.5 0.38 0.25 0.38 0.125 0.25 1 192 D fyua ERT, TIC, TCC, AMC, PIP, TZP, TEM, TOB, GEN, SXT S100 Healthy 2 0.5 0.25 0.75 6 24 12 192 OXA-48, TEM-1, CMY-2 ERT, TIC, TCC, AMC, PIP, TZP, TEM, CAZ, FOX, CIP, NAL, SXT U fyua, iuta S132 Diseased cat >32 >32 24 >32 >256 >256 >32 >256 NDM-5, CTX-M-15, CMY-42, AAC-(6 )- Ib-cr ERT, TIC, TCC, AMC, PIP, TZP, FEP, FOX, CTX, CAZ, ATM, IPM, TEM, TOB, CIP, NAL A fyua, iuta S174 Healthy OXA-48 2 0.38 0.25 0.5 0.125 0.38 0.75 192 A fyua ERT, TIC, TCC, AMC, PIP, TZP, TEM, CIP, NAL, SXT S192 Healthy OXA-48, TEM-1 2 0.38 0.19 0.5 0.94 0.38 1 192 D fyua ERT, TIC, TCC, AMC, PIP, TZP, TEM, TOB, GEN, SXT S99 Healthy cat AMC, amoxicillin clavulanate; ATM, aztreonam; CAZ, ceftazidime; CIP, ciprofloxacin; CTX, cefotaxime; DOR, doripenem; ERT, ertapenem; FOX, cefoxitin; GEN, gentamicin; IPM, imipenem; MIC, minimum inhibitory concentration; NAL, nalidixic acid; PIP, piperacillin; PMQR, plasmid-mediated quinolone resistance; SXT, co-trimoxazole; TCC, ticarcillin clavulanate; TEM, temocillin; TIC, ticarcillin; TOB, tobramycin; TZP, piperacillin tazobactam; U, unknown. FIG. 1. Result of susceptibility testing of the Escherichia coli isolate S174. From the top and from left to right: TIC, ticarcillin; PIP, piperacillin; CTX, cefotaxime clavulanate; CCAZ, ceftazidime clavulanate; AMC, amoxicillin clavulanate; TCC, ticarcillin clavulanate; CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; CPO, cefpirome; ATM, aztreonam; IPM, imipenem; TZP, piperacillin tazobactam; FOX, cefoxitin; CFEP, cefepime clavulanate; ERT, ertapenem. and imipenem), the other isolates showed very low MICs to carbapenems ranging from 0.19 mg/l to doripenem to 2 mg/l to ertapenem. Genotypic analysis of antibiotic resistance genes The presence of the bla OXA-48 gene in four isolates and bla NDM-5 in one isolate (S174) were confirmed with PCR amplification and sequencing. In addition to carbapenemase determinants, four isolates produced other beta-lactamases (Table 1). The unique detected plasmid-mediated quinolone resistance (PMQR) gene was aac(6 )-Ibcr identified in E. coli S174 producing NDM-5, CMY-42, and CTX-M-15. No isolates were found to carry qnr, qepa,oroqxab determinants. E. coli phylogenetic groups and virulence factors Analysis of E. coli phylogroups showed that these isolates belong to phylogroup A (S174 and S192), phylogroup D (S99 and S100), and unknown phylogroup (S132) (Table 1). All isolates carried fyua gene encoding yersiniabactin. Two isolates, S174 and S132, were also found to carry iuta encoding aerobactin receptor. Discussion Although carbapenems are not registered for use in animals in any major jurisdiction, a case of use of these molecules has been reported in s for the treatment of urinary tract infection and postoperative infection caused by multidrugresistant E. coli. 1 Indeed, the emergence of carbapenemaseproducing bacteria is a major public health problem, mainly in

NDM-5 AND OXA-48 IN ESCHERICHIA COLI FROM PETS 345 the community setting. In our study, we report for the first time the emergence of carbapenemase-producing E. coli in companion animals in Algeria and in Africa. Until now, only two reports on carbapenemase-producing Gram-negative bacteria were reported from companion animals, NDM-1-producing E. coli isolates reported in the USA 25 and OXA-48 in E. coli and K. pneumoniae isolates in Germany. 26 In the Shaheen study, 944 canine and feline clinical E. coli isolates from different veterinary laboratories recovered between May 2008 and 2009 were analyzed. Of these, six different isolates were found to produce bla NDM-1 genes. 25 Reports on NDM-producing isolates in Algeria are limited and concern essentially isolates of A. baumannii recovered from clinical specimens. 3,17 NDM-5 was first reported by Hornsey et al. 14 NDM-5 differed from existing enzymes due to substitutions at positions 88 (Val3Leu) and 154 (Met3Leu). 14. Sassi et al. reported NDM-5-producing E. coli recovered from urine and blood specimens of three patients admitted to the University Hospital of Annaba, Algeria. These isolates coexpressed bla CTX-M-15 and bla TEM-1. 24 In our study, besides NDM-5, the E. coli S174 isolate coexpressed CTX-M-15 and CMY-42 allowing the isolate to resist all beta-lactams available. Zhang et al. reported two clinical isolates of E. coli harboring either bla NDM-1, bla CTX-M-14, and bla CMY-42 or bla NDM-1, bla SHV-12, bla CTX-M-14,andbla CMY-42. 31 Detection of plasmidic AmpC (pampc) in these types of isolates is only possible with molecular screening by PCR, because isolates harboring MbL are resistant to all betalactams, except aztreonam. So the production of pampc is masked by MbL. Thus, we recommend using PCR multiplex to detect the coproduction of pampc in isolates producing MbL. In addition a DD-test with aztreonam and co-amoxiclav should be used to screen the coexpression of ESBL. OXA-48 was first described in 2001 in a K. pneumoniae isolate from a patient in Istanbul, Turkey. It is suggested that the main reservoirs of OXA-48-harbouring K. pneumoniae and E. coli are in North African countries and Turkey. 18 OXA-48 E. coli and K. pneumoniae producers were isolated from s for the first time in 2012 in Germany. 26 In this study, the authors reported eight carbapenem-resistant K. pneumoniae (n = 5) and E. coli (n = 3) isolated from six s admitted to a veterinary clinic producing the bla OXA-48 gene. These E. coli isolates also coproduced bla CMY-2, bla SHV-12,andbla CTX-M-1. 26 In Algeria, there is only one report on Enterobacteriaceae isolates producing OXA-48 in a military hospital. 2 In this study, the authors reported one isolate of E. coli belonging to phylogroup B1 producing OXA-48. 2 In our study, in three of the four isolates producing OXA-48, coproduction of additional beta-lactamases conferred resistance to extendedspectrum beta-lactams, cephamycins, and aztreonam. We used two steps for the isolation of E. coli-producing carbapenemases. These methods allowed obtaining both types of carbapenemase NDM and OXA-48. When we analyze the carbapenem MICs obtained, we note that isolates harboring only bla OXA-48 showed very low carbapenem MICs ranging from 1.5 to 2 mg/l for ertapenem and from 0.38 to 0.75 mg/l for imipenem. Thus, to select Enterobacteriaceae-producing OXA-48, we recommend using, in enrichment and isolation steps, ertapenem at a final concentration of 0.5 mg/l. In terms of international spread, there are three major routes of NDM acquisition, nosocomial, personal travel, and community acquisition. 28 Unfortunately, information regarding the foreign travel history of the owners were not collected by the veterinary office at the time of the medical visit. Thus, the origin of these carbapenemase-producing isolates was uncertain. Phylogenetic analyses have shown that E. coli strains fall into four main phylogroups (A, B1, B2, and D), in which groups A and B1 typically contain commensal isolates and isolates of groups B2 and D are considered to be more likely carrying pathogenicity-associated genes. 5 In our study, isolates were assigned to phylogroup A and D. One limitation of our study is the small size of our samples. Therefore, we agree that a potential caveat of our study could be the extrapolation of our results to the general cat and population at the national level. We should be cautious. Nonetheless, only one veterinary practice was involved because there is only veterinary practice for companion animals in Bejaia city. It should be pointed out that the adoption of companion animals by private owners in Algeria is a phenomenon that has appeared in recent years. In general, these owners belong to a high socioeconomic class. Even though our study was conducted in one veterinary practice in Bejaia city, a surveillance program for screening of carbapenemase-producing isolates among healthy companion animals should be established. In conclusion, our study demonstrates that the prevalence of carbapenemase-producing E. coli in companion animals in Algeria is 2.5%. Our findings strongly suggest that isolates producing carbapenemase are currently circulating among companion animals and can act as a reservoir of gene transmission between humans and animals. Further studies are needed to survey the dissemination of carbapenemaseproducing strains in the population. 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Antimicrob. Chemother. 68:1681 1682. 31. Zhang, X., D. Lou, Y. Xu, Y. Shang, D. Li, X. Huang, Y. Li, L. Hu, L. Wang, and F. Yu. 2013. First identification of coexistence of bla NDM-1 and bla CMY-42 among Escherichia coli ST167 clinical isolates. BMC Microbiol. 13:282. Address correspondence to: Abdelaziz Touati, PhD Laboratoire d Ecologie Microbienne FSNV Université de Bejaia Bejaia 06000 Algérie E-mail: ziz1999@yahoo.fr