Additional file 1: of Antimicrobial resistance profiles of Shiga toxin-producing Escherichia coli O157 and Non-O157 recovered from domestic farm animals in rural communities in Northwestern Mexico

Antimicrobial agents used in the present study. (DOCX 14 kb

Genotypic Analyses of Shiga Toxin-Producing <em>Escherichia coli</em> O157 and Non-O157 Recovered from Feces of Domestic Animals on Rural Farms in Mexico

<div><p>Shiga toxin-producing <em>Escherichia coli</em> (STEC) are zoonotic enteric pathogens associated with human gastroenteritis worldwide. Cattle and small ruminants are important animal reservoirs of STEC. The present study investigated animal reservoirs for STEC in small rural farms in the Culiacan Valley, an important agricultural region located in Northwest Mexico. A total of 240 fecal samples from domestic animals were collected from five sampling sites in the Culiacan Valley and were subjected to an enrichment protocol followed by either direct plating or immunomagnetic separation before plating on selective media. Serotype O157:H7 isolates with the virulence genes <em>stx2</em>, <em>eae</em>, and <em>ehxA</em> were identified in 40% (26/65) of the recovered isolates from cattle, sheep and chicken feces. Pulse-field gel electrophoresis (PFGE) analysis grouped most O157:H7 isolates into two clusters with 98.6% homology. The use of multiple-locus variable-number tandem repeat analysis (MLVA) differentiated isolates that were indistinguishable by PFGE. Analysis of the allelic diversity of MLVA loci suggested that the O157:H7 isolates from this region were highly related. In contrast to O157:H7 isolates, a greater genotypic diversity was observed in the non-O157 isolates, resulting in 23 PFGE types and 14 MLVA types. The relevant non-O157 serotypes O8:H19, O75:H8, O111:H8 and O146:H21 represented 35.4% (23/65) of the recovered isolates. In particular, 18.5% (12/65) of all the isolates were serotype O75:H8, which was the most variable serotype by both PFGE and MLVA. The non-O157 isolates were predominantly recovered from sheep and were identified to harbor either one or two <em>stx</em> genes. Most non-O157 isolates were <em>ehxA</em>-positive (86.5%, 32/37) but only 10.8% (4/37) harbored <em>eae</em>. These findings indicate that zoonotic STEC with genotypes associated with human illness are present in animals on small farms within rural communities in the Culiacan Valley and emphasize the need for the development of control measures to decrease risks associated with zoonotic STEC.</p> </div

Characterization of 11 variable-number tandem repeat loci in the O157:H7 STEC isolates used in this study.

1<p>Nei’s diversity index for each locus was calculated as 1-∑(allele frequency)²<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051565#pone.0051565-Nei1" target="_blank">[67]</a>.</p

Proportion of fecal samples positive for presumptive STEC.

1<p>Enrichment broths were subjected to direct plating from July to December 2008 or to immunomagnetic separation (IMS) from January to June 2009.</p>2<p>Sampling sites correspond to regions in the Culiacan Valley, Sinaloa, Mexico, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051565#pone-0051565-g001" target="_blank">Figure 1</a>.</p>3<p>NA, None available.</p

Analysis of the genetic relatedness among the non-O157 STEC isolates by PFGE and MLVA methods.

<p>Dendograms of the combined PFGE types with <i>Xba</i>I and <i>Bnl</i>I restriction enzymes were constructed using Bionumerics software v6.1 with the Dice coefficient and the UPGMA method. MLVA types were designated to each unique profile after analysis of tandem repeats in 10 genomic loci using Bionumerics software.</p

Phylogenetic relationships of MLVA types for the 26 O157:H7 STEC isolates.

<p>A minimum-spanning tree of MLVA types was generated with Bionumerics software v6.1 and the Manhattan distance algorithm. Each circle in the tree represents a different MLVA type, and the number in the circle indicates the MLVA type number. The circle size corresponds to the number of isolates with an identical MLVA type. Colors represent the sampling sites (A) or the sampling source (B) for each MLVA type.</p

List of <i>E. coli</i> non-O157 isolates used in this study.

1<p>NT, H-antigen non-typeable; ONT, O-antigen non-typeable.</p>2<p>Enrichment broths subjected to direct plating from July to December 2008 or to immunomagnetic separation from January to June 2009.</p>3<p>Sampling sites correspond to regions in the Culiacan Valley, Sinaloa, Mexico, as show in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051565#pone-0051565-g001" target="_blank">Figure 1</a>.</p

List of <i>E. coli</i> O157 isolates used in this study.

1<p>NT, H-antigen non-typeable; ONT, O-antigen non-typeable.</p>2<p>Enrichment broths subjected to direct plating from July to December 2008 or to immunomagnetic separation from January to June 2009.</p>3<p>Sampling sites correspond to regions in the Culiacan Valley, Sinaloa, Mexico, as show in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051565#pone-0051565-g001" target="_blank">Figure 1</a>.</p

Phylogenetic relationships of MLVA types for the 37 non-O157 STEC isolates.

<p>A minimum-spanning tree was generated with Bionumerics software v61 and the Manhattan distance algorithm. Each circle in the tree represents a different MLVA type, and the number in the circle indicates the MLVA type number. The circle size corresponds to the number of isolates with an identical MLVA type. Colors represent the sampling sites (A) or the sampling source (B) for each MLVA type.</p

Sampling sites in the Culiacan Valley in Northwest Mexico.

<p>Map of the five sampling sites, Jotagua (A), Agua Caliente (B), Cofradia de Navolato (C), Iraguato (D), and El Castillo (E) that were selected to be 14–38 km apart and to represent the study area in the Culiacan Valley, Sinaloa, Mexico. Dark areas in the map indicate urban zones with more than 20,000 inhabitants. Scale bar corresponds to 50 km.</p