Characterization of CTX-M-14- and CTX-M-15-producing Escherichia coli of porcine origin

Abstract

Sir, The emergence of extended-spectrum b-lactamase (ESBL)producing Escherichia coli among human patients is largely due to the spread of CTX-M, especially the clonal spread of sequence type (ST) 131 E. coli producing CTX-M-15. In recent years ESBLproducing E. coli have also been detected in food and animals and ST131 has been detected in E. coli of animal origin. To our knowledge, E. coli ST131 producing CTX-M-15 has been detected in companion animals, but not in other animals or in meat. Major questions include whether animals can be a reservoir of the STs that have been associated with ESBL-producing E. coli causing infections in humans, and whether animals and meat can be a reservoir of ESBL genes that can be transferred to some of the pathogenic E. coli STs (e.g. ST131) in the human intestine. Recently Agerso et al. reported on the prevalence of cephalosporinase-producing E. coli from Danish pig (n1⁄4786), Danish pork (n1⁄4153) and imported pork (n1⁄4173) samples taken in 2009. CTX-M-1-producing E. coli were most common among the ESBL-producing E. coli from pigs and pork, whereas a few isolates were producers of CTX-M-14 and CTX-M-15; whether these types are established in pigs is unknown. In the present study, one CTX-M-14-producing E. coli isolate from imported pork, six CTX-M-14-producing E. coli from Danish pigs and two CTX-M-15-producing E. coli from Danish pigs originating from the cephalosporinase prevalence study mentioned above were characterized for PFGE profile, multilocus sequence typing (MLST) ST, resistance profile, phylotype, clonal group A status, virulence genes and transfer of blaCTX-M genes (Table 1). PFGE with XbaI was performed on the nine E. coli isolates. MLST was performed using seven conserved housekeeping genes (adk, fumC, gyrB, icd, mdh, purA and recA) (http://mlst. ucc.ie/mlst/dbs/Ecoli). The MICs of chloramphenicol, ciprofloxacin, gentamicin, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline and trimethoprim for the isolates were determined (Trek Diagnostic, East Grinstead, UK). Results were interpreted according to the EUCAST (www.EUCAST.org) clinical breakpoint except for results for nalidixic acid, tetracycline, streptomycin and sulfamethoxazole, which were interpreted according to CLSI. E. coli ATCC 25922 was used for quality control. The phylogenetic background (A, B1, B2 or D) of the isolates was determined by PCR. Results obtained allowed classification of the isolates into the four major E. coli phylogenetic lineages or non-typeable isolates. Isolates classified as phylogroup D were tested for the presence of single-nucleotide polymorphisms in fumB and gyrB, which has been associated with a clonal group of extraintestinal pathogenic E. coli (ExPEC), designated clonal group A. Furthermore, the isolates were investigated for the ExPEC-related virulence genes kpsM II, papA, papC, iutA, sfaS, focG, afa and dra. In vitro matings were performed with a rifampicin-resistant mutant of an ST131 B2 E. coli of Danish pork origin as recipient. Three selected transconjugants from each mating were examined for the presence of the transferred blaCTX-M gene by PCR. Furthermore, PFGE was performed on the three selected transconjugants, the donor and the recipient. The isolates had different PFGE types (≤80% identical) and were not clonally related to any of the 80 ESBL-producing E. coli blood isolates of human origin from 2009 obtained in our BioNumerics database (data not shown). The nine CTX-M-producing E. coli isolates in the present study had nine different STs, of which ST48, ST101, ST117 and ST167 had previously been detected in human patients (Table 1). – 8 Six of the nine isolates were resistant to other classes of antimicrobial agents beside the b-lactams (Table 1). Urinary tract infections are most often associated with phylogroup B2 and to some extent with phylogroup D. None of the nine isolates belonged to phylogroup B2 (Table 1). However, blaCTX-M-14and blaCTX-M-15-producing E. coli isolates from patients with infections belonging to phylogroup A, B1 and D have been detected. – 5 Two of the tested E. coli belonged to phylogroup D, but not to the clonal group A. A previous Danish study of phylogroup D E. coli of porcine origin likewise failed to identify any clonal group A isolates. Isolate 2700-1 (CTX-M-14) was positive for focG (F1C fimbriae) and iutA (aerobactin siderophore) and isolate 2469-1 (CTX-M-14) was positive for papA/papC (P fimbriae) and iutA, which defined both isolates as ExPEC isolates according to the molecular definition.