A new multidrug-resistant enterotoxigenic Escherichia coli pulsed-field gel electrophoresis cluster associated with enrofloxacin non-susceptibility in diseased pigs

Abstract

Aims: To describe the temporal trends in Escherichia coli pathotypes and antimicrobial resistance detected in isolates from diseased-pig cases submitted to the EcL from 2008 to 2016, in Quebec, Canada, and to investigate the presence of spatiotemporal and phylogenetic clusters.

Methods and results: Detection of 12 genes coding for virulence factors in pathogenic E. coli in pigs by PCR and antimicrobial resistance standard disc diffusion assay were performed. Demographic and clinical data were entered in the Animal Pathogenic and Zoonotic E. coli (APZEC) database. ETEC:F4 was the most prevalent pathovirotype among the 3773 cases submitted. The LT:STb:F4 virotype was predominant until 2014, then was overtaken by the LT:STb:STa:F4 virotype. More than 90% of the ETEC:F4 isolates were multidrug resistant. A spatiotemporal cluster of LT:STb:STa:F4 isolates non-susceptible to enrofloxacin was detected between 4/2015 and 9/2016. Pulsed-field gel electrophoresis analysis of 137 ETEC:F4 isolates revealed the presence of a cluster composed mainly of LT:STb:STa:F4 isolates non-susceptible to enrofloxacin.

Conclusions: The APZEC database was useful to highlight temporal trends in E. coli pathotypes. A high-risk ETEC:F4 clone might disseminate in the pig population in Quebec since 2015.

Significance and impact of the study: Surveillance is crucial to identify new clones and develop control strategies.

Keywords: Escherichia coli; ETEC; antimicrobial resistance; clonal complex; fluoroquinolones non-susceptibility; pigs.

Figure 1

Outline of the EcL approach for the detection of the virulence genes which define the Escherichia coli pathotypes commonly found in pigs. LB = Luria–Bertani broth. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Percentage of positive cases per pathovirotype in diseased pigs in Quebec among all cases submitted to the EcL from 2008 to 2016. The pathovirotypes were classified as pathogenic agents if they carried a colonization factor (F4, F18 or F5) and as possible pathogenic agents if they carried only enterotoxins, shiga toxin or the eae gene. Cases are defined as a gathering of samples coming from the same farm on the same day, but not necessarily from the same animal. Undetermined: the sample possesses only fimbrial genes (and could represent a vaccinal strain). During the study period there were 3773 cases and 1870 positive cases, including 89 mixed cases (i.e. several pathovirotypes per case) (Pathovirotype: a: ETEC:F4; STEC:F18; ETEC:F5; ETEC:F18; ETEC:STEC:F18; b: ETEC; undetermined; EPEC; STEC; EPEC:STEC; ETEC:STEC; ExPEC). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Number of positive cases per virotype in diseased pigs in Quebec among ETEC:F4 cases submitted to the EcL from 2008 to 2016. During the study period there were 611 ETEC:F4 cases, including 81 mixed cases (i.e. several virotypes per case). Miscellaneous ETEC:F4 is a group of isolates carrying other combinations of enterotoxin genes (virotype ETEC:F4: LT:STb:STa; LT:STb; STa:STb; miscellaneous). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

Susceptibility to enrofloxacin in ETEC:F4 isolates from positive cases, classified by virotype, detected in samples from diseased pigs of Quebec submitted to the EcL from 2008 to 2016. Miscellaneous ETEC:F4 is a group of isolates carrying other combinations of enterotoxin genes. During the study period there were 692 ETEC:F4 isolates from 611 cases, including 81 cases with more than one isolate. Only one isolate per virotype was kept for each case ( susceptible; intermediate; resistant). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5

Number of multidrug resistant ETEC:F4 isolates, classified by virotype, detected in samples from diseased pigs of Quebec submitted to the EcL from 2008 to 2016. Miscellaneous ETEC:F4 is a group of isolates carrying all other combination of enterotoxin genes. During the study period there were 692 ETEC:F4 isolates from 611 cases, including 81 cases with more than one isolate. Only one isolate per virotype was kept for each case ( 0–2 classes; 3–4 classes; 5–6 classes; 7–8 classes). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6

Representation of the spatio‐temporal cluster. Geographical distribution of cases (in red) and control (in blue) in the province of Quebec, Canada, before (a), during (b) and after (c) the spatiotemporal cluster. On each map, the graphic in the upper right indicates the temporal distribution of cases and controls sampled per month during the study period. The highlighted period is the period illustrated on the corresponding map. On the map b, the striped circle indicates the spatial cluster, and the striped bars represent the cases sampled in the spatial cluster, whereas the unstriped bars represent the cases sampled out of the spatial cluster. The size of the circles indicates the number of cases at this location. In this analysis, a case is defined as an APZEC case positive for an ETEC:F4:LT:STa:STb isolate non‐susceptible to enrofloxacin and a control is any other APZEC case (a: case; control; 1 case; 4 cases: b: case; control; spatial cluster; 1 case; 4 cases: c: case; control; 1 case; 2 cases).

Figure 7

Phylogenetic additive tree based on the PFGE profiles of ETEC:F4 isolates collected from 2013 to 2016 by the EcL in diseased pigs in Quebec. D = Diarrhoea, SD = Sudden Death, O = Other, Ne = Neonates, Nu = Nursery, G = Grower, ID = Identification, A = Apramycin, N = Neomycin, S = Spectinomycin, G = Gentamicin, TM = Trimethoprim Sulfisoxazole, AM = Ampicillin, T = Tetracycline, F = Florfenicol, E = Enrofloxacin (the squares are coloured in red), C = Ceftiofur, MDR = Multidrug resistance.

Figure 8

Geographical distribution of isolates belonging to the PFGE clusters over the Quebec territory in 2013–2016 according to the EcL database. The size of the circles is proportional to the number of isolates identified in the 6‐digit zip code region ( 1 isolate; 5 isolates; cluster 0; cluster 1; cluster 2; cluster 3; cluster 4; cluster 5; cluster 6; cluster 7; cluster 8; cluster 9; cluster 10; cluster 11; cluster 12; cluster 13).