Emergence of Escherichia coli ST131 H30/H30-Rx subclones in companion animals

Sir, The Escherichia coli O25b-ST131, with its fluoroquinolone-resistant H30 subclone and its nested ESBL CTX-M-15-associated H30-Rx subclone, is the most disseminated MDR and virulent E. coli clonal group worldwide.1 In previous work, we have reported the first detection of this O25b-ST131 clone causing urinary tract infection (UTI) in a dog.2 Additionally, later on, we found within-lineage variability of ST131 E. coli UTI isolates from humans and companion animals by PFGE analysis.3 The detection of human high-risk pandemic E. coli clones causing UTI in companion animals is a great public health concern.4 Between 1999 and 2015, 342 uropathogenic E. coli were isolated at the Laboratory of Antimicrobial Resistance, Faculty of Veterinary Medicine, University of Lisbon from companion animals (dogs and cats) with UTI. Significant bacteriuria was determined based on a quantitative urine culture according to the urine collection method used (puncture, catheter or free catch). Urine samples were collected from companion animals at the teaching hospital of the Faculty of Veterinary Medicine and at private veterinary hospitals in Lisbon, Portugal. All B2 phylogroup isolates were studied by PCR for the ST131-associated SNP in the mdh and gyrB genes;5 14.5% (n = 25/172) were the pandemic O25b:H4-B2-ST131 clone. The E. coli O25b:H4-B2-ST131 H30 and H30-Rx subclones were screened by PCR as previously described.6 Seven (n = 7/25; 28%) of the O25b:H4-B2-ST131 clone companion animal isolates were the H30 subclone and three out of these seven were the H30-Rx subclone (Table 1). To the best of our knowledge this is the first report of E. coli ST131 H30/H30-Rx subclones causing UTI in companion animals in Europe. Characterization of E. coli O25b:H4-B2-ST131 H30 and H30-Rx subclones isolated from companion animals AMC, amoxicillin/clavulanate; AMP, ampicillin; AMK, amikacin; CAZ, ceftazidime; CIP, ciprofloxacin; GEN, gentamicin; CTX, cefotaxime; ENR, enrofloxacin; FOX, cefoxitin; SXT, trimethoprim/sulfamethoxazole; TET, tetracycline; TOB, tobramycin; CEF, cefalotin; pAmpC, plasma-mediated AmpC. Susceptibility was accessed according to CLSI M100-S27. Classification of the virotype was in accordance with Nicolas-Chanoine et al.1 Characterization of E. coli O25b:H4-B2-ST131 H30 and H30-Rx subclones isolated from companion animals AMC, amoxicillin/clavulanate; AMP, ampicillin; AMK, amikacin; CAZ, ceftazidime; CIP, ciprofloxacin; GEN, gentamicin; CTX, cefotaxime; ENR, enrofloxacin; FOX, cefoxitin; SXT, trimethoprim/sulfamethoxazole; TET, tetracycline; TOB, tobramycin; CEF, cefalotin; pAmpC, plasma-mediated AmpC. Susceptibility was accessed according to CLSI M100-S27. Classification of the virotype was in accordance with Nicolas-Chanoine et al.1 The presence of uropathogenic E. coli (UPEC) virotype markers was assessed, i.e. Pap fimbriae (papEF operon segment), Sfa fimbriae and Afa afimbrial adhesin (sfa and afa genes, respectively), the hlyA gene from the α-haemolysin operon, cytotoxic necrotizing factor 1 (cnf1 gene), aerobactin siderophore (iucD gene), E. coli common pilus (ecpA gene) and the uropathogenic specific protein (usp gene).7,8 The distribution of alleles I, II and III of the P adhesin gene papG was studied.9 Additionally, pathogenicity-associated islands (PAIs) (PAIIV536, PAII536, PAIII536, PAIIII536, PAIIJ96, PAIIIJ96, PAIICFT073, PAIIICFT073) were identified.10 The E. coli O25b:H4-B2-ST131-H30/H30-Rx most common pathogenicity and virulence-associated gene profiles were PAIICFT073-PAIIV536-PAIIICFT073 (42.9%, n = 3/7) and ecpA-hlyA-papEF-iucD (28.6%, n = 2/7), respectively. The full pathogenicity and virulence genotype of E. coli O25b:H4-B2-ST131-H30/H30-Rx is shown in Table 1. All H30/H30-Rx subclones causing UTI in companion animals belonged to virotype D, which confirms their virulent characteristics.1 Resistance to third-generation cephalosporins was detected only in the three isolates of the H30-Rx subclone. β-Lactamase genes were screened as reported elsewhere.2 As expected from other studies, O25b:H4-B2-ST131 H30 and H30-Rx isolates were fluoroquinolone resistant,1 yet one O25b:H4-B2-ST131-H30-Rx E. coli did not carry the ESBL blaCTX-M-15, but instead carried the frequent blaCTX-M-1 gene, which is associated with E. coli isolates of farm animal origin and recently described in humans in Turkey with UTI.1,11 The mechanism of resistance to other antimicrobial classes was characterized by PCR and nucleotide sequencing for sul1, sul2, sul3, dfrA1, dfraA12 and the plasmid-mediated quinolone resistance (PMQR) genes [qnrA, qnrB, qnrS, qnrC, qnrD, qepA, aac(6′)-Ib and the MDR oqxAB genes of the efflux pump].12 The aac(6′)-Ib-cr gene was the most common PMQR gene detected (57.1%, n = 4/7) and was more frequently found in ST131 H30-Rx E. coli isolates than in ST131 H30 isolates. The qnrB2 and aac(6′)-Ib-cr genes were detected in the FMV5825/04 dog isolate as expected from previous data (Table 1).2 In Europe, carbadox and olaquindox, which are quinoxaline derivatives with antibacterial properties, were used for the prevention of dysentery and as growth promotors in pigs since 1974 and 1976, respectively. Fortunately, their use has been banned in farm animals in Europe for decades, but not in China.13 The efflux pump OqxAB also extrudes antibiotics such as chloramphenicol and fluoroquinolones. Isolate FMV5695/09 O25b:H4-B2-ST131-H30 harboured both oqxA and oqxB efflux pump genes, which could potentially be related to the reduced fluoroquinolone susceptibility. To the best of our knowledge, this is the first description of an ST131 E. coli harbouring the oqxAB efflux pump. Further studies will be necessary to elucidate the importance of this resistance mechanism in the ST131 pandemic E. coli clone. This study reports the detection and frequency of the E. coli O25b:H4-B2-ST131 H30/H30-Rx subclones in companion animals with UTI in Portugal and, to the best of our knowledge, in Europe. In conclusion, the findings presented in this study are relevant and, to the best of our knowledge, represent the first detection of ST131 H30/H30-Rx E. coli isolates associated with UTI in companion animals in Europe. To the best of our knowledge, this is the first report in Europe of the disseminated E. coli O25b:H4-B2-ST131-H30/H30-Rx, MDR, fluoroquinolone-resistant human high-risk clone and its CTX-M-15-H30-Rx and CTX-M-1-H30-Rx subsets in companion animals with UTI. Studies of ST131 H30/H30-Rx in humans in Portugal are scarce, yet H30/H30-Rx subclones have been described in faecal samples in healthy humans.14 These results raise public health concerns since these subclones may have an impact on human health through the close and direct contact between companion animals and owners. Moreover, the close contact between companion animals and humans creates opportunities for interspecies transmission of resistant bacteria. We are grateful to Vincent Perreten for the gift of the sul3-harbouring E. coli strain. This work was supported by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the Programa Operacional Factores de Competitividade (COMPETE) and by National funds through the Fundação para a Ciência e a Tecnologia (FCT) [CIISA Project UID/CVT/00276/2013 and PET-Risk Consortium Project Joint Programming Initiative on Antimicrobial Resistance (JPIAMR/0002/2016)]. Adriana Belas (SFRH/BD/113142/2015) and Cátia Marques (SFRH/BD/77886/2011) hold FCT PhD grants. None to declare.