Coexistence of CTX-M-2, CTX-M-55, CMY-2, FosA3, and QnrB19 in Extraintestinal Pathogenic Escherichia coli from Poultry in Brazil

Abstract

E xtraintestinal pathogenic Escherichia coli (ExPEC) is a pathogen that causes a wide range of diseases worldwide (1). Based on molecular and phenotypic analysis, many reports suggest that poultry is a reservoir of ExPEC (1, 2). In addition to having pathogenic potential, many ExPEC lineages are multidrug resistant, leaving treatment options scarce (1). In this study, we report the coexistence of plasmid-mediated resistance to cephalosporins, quinolones, and fosfomycin in virulent lineages of ExPEC from poultry. Between 2013 and 2016, during a surveillance study conducted to monitor the prevalence of ExPEC isolates in poultry farms, six strains exhibiting a multidrug-resistant profile were recovered from cloacal swabs of healthy broilers in 3 farms located in Paraná State, southern Brazil. Isolates displayed resistance to cephalosporins, fosfomycin, and fluoroquinolones, as evaluated by disk diffusion and agar dilution methods (3, 4). The presence of genes encoding ␤-lactamases, plasmid-mediated quinolone resistance (PMQR), and fosfomycin resistance was assessed by PCR and sequencing (5-7). Plasmids were characterized by conjugation in E. coli C600 STR or transformation in E. coli TOP10, PCR-based replicon typing (PBRT) (8), plasmid multilocus sequence typing (pMLST) for IncI1 plasmids (9), replicon sequence typing (RST) for IncF plasmids (10), and plasmid sizing determination. Plasmid analysis revealed that all strains bearing an ϳ70-kb conjugative F33:A-:Bplasmid harbored bla CTX-M-55 , bla TEM-1b , and fosA3 genes (see Table S1 in the supplemental material), whereas four strains additionally harbored the qnrB19 gene in ColElike plasmids (ϳ3 kb). Identification of bla CTXM-2 and bla CMY-2 was confirmed in two and three E. coli strains, respectively (Table 1). bla CMY-2 was related to 80-kb IncI1/ST12 plasmids. Virulence genotypes were elucidated using PCR to target 27 virulence markers (Table 1) (11-13). In this concern, all six strains were classified as an ExPEC pathotype based on the Johnson criterion (the presence of at least two of five virulence markers, namely, afa, kpsMTII, iutA, papC, and sfa/foc) (14), and the presence of genes encoding adhesins, siderophores, and serum resistance was confirmed in all strains. Phylogenetic analyses was determined by MLST (http://mlst.ucc.ie/mlst/mlst/dbs/ Ecoli), using the Clermont method (15), and single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis. Four strains belonging to sequence types ST117, ST453, ST457, and ST1706 were clonally unrelated. In this regard, ST117 has been recognized as a lineage of avian-pathogenic E. coli (APEC) and human ExPEC (2). Furthermore, in a study conducted in China, ST117 was associated with an F33:A-:Bplasmid harboring bla CTX-M-65 , fosA3, and rmtB (16). In this study, ExPEC strain FA46

Identification and characterization of plasmid-mediated quinolone resistance determinants in Enterobacteriaceae isolated from healthy poultry in Brazil

“…There is few data about PMQR determinants either in humans or in animals in Brazil, therefore the prevalence of these genes is poorly understood. Another study in Brazil, also reported the gene qnrB19 carried by ColElike plasmids in four ExPEC isolates from poultry (Cunha et al, 2017).…”

Antimicrobial Resistance in Farm Animals in Brazil: An Update Overview

“…Part of isolates characterized by Fernandes and coworkers was not able to transfer bla CTX-M-2 to E. coli, suggesting a possible chromosomal location of this gene [145]. In contrast, in E. coli, most studies suggest a chromosomal location of bla CTX-M-2 with a few reports of this gene in plasmids belonging to IncK, IncHI2, IncP incompatibility groups ranging from 35 to 280 kb [137,139,146,147,157]. Always when investigated, both in Salmonella sp.…”

“…Studies indicate that these genes are mainly carried by E. coli and nontyphoid Salmonella, but also by Escherichia fergusonii, Klebsiella pneumoniae, Citrobacter diversus, and Proteus mirabilis [136][137][138][139][140]. Reports include animals on farms [36,[136][137][138][141][142][143][144][145][146][147], whole carcasses, or meat pieces available in Brazilian retail markets [69,136,138,140,145,148,149], and exported chicken meat [150][151][152]. The same genes were detected in turkeys with clinical signs of colibacillosis [153].…”

“…In contrast, this genetic environment is more diverse in E. coli. The mobilization of the gene seems to be associated with ISEcp1, and with IncI, IncFIB, IncK, IncB/O, IncAC, and non-typable plasmids, most of them 80-90 kb long [143,146,147,157].…”

ESBL-producing <i>Escherichia coli</i> and Its Rapid Rise among Healthy People

“…UTIs would be found to harbor fosA3-mediating plasmids in high frequency in China 145) . Moreover, FosA3 producers have also been spreading outside of Asia [146][147][148][149][150] . Therefore, further prevalence of fosA3 among the ESBL-producing E. coli showing resistance to fluoroquinolones, which are mostly E. coli ST131 subclones, H30 151,152) and H30Rx 153) clades, should be more carefully monitored in days to come within the community, especially in UTI patients 154) .…”