Frecuencia, caracterización y análisis genotípico de Escherichia coli productor de toxina Shiga en frigoríficos de carne bovina de Argentina / Frequency, characterization and genotypic analysis of Shiga toxin-producing Escherichia coli in beef slaughterhouses of Argentina

The objectives of this study were: (1) to estimate STEC frequency in hide and carcass samples taken from beef slaughterhouses supplying the domestic market in Argentina, (2) to establish the pheno-genotypic characteristics of STEC and non-toxigenic Escherichia coli of serogroups O26, O45, O103, O121, O111, O145 or O157 isolated from the analyzed samples and, (3) to study their clonal relatedness. Sixty hides and 60 carcasses were analyzed. At the screening step, 48% of hide and 80% of carcass samples tested positive for the stx gene by endpoint PCR. The STEC isolation rate was 5% for hides and 8% for carcasses. The isolation rate of STEC-positive for O26, O45, O103, O111, O145 or O157 serogroups was 0% for hides and 2% for carcasses. With the purpose of studying the clonal relatedness of isolates, macrorestriction fragment analysis by pulsed-field gel electrophoresis was performed. The results indicated cross-contamination between hides and between carcasses of animals in the same lot and, that the origin of carcass contamination was their own hide, or the hides of other animals in the same lot. The high detection rate at the screening step, especially in carcasses, and the evidence of cross-contamination show the need to apply additional in-plant intervention strategies aimed at preventing carcass contamination.

Los objetivos del presente estudio fueron tres: 1) estimar la frecuencia de Escherichia coli productor de toxina Shiga (STEC) en muestras de cuero y carcasa de bovinos en frigoríficos de consumo interno de Argentina; 2) realizar la caracterización feno-genotípica de las cepas STEC y de Escherichia coli no toxigénicas pertenecientes a los serogrupos O26, O45, 0103, O121, O145 u O157 aisladas a partir de las muestras analizadas; 3) establecer la relación clonal de ese conjunto de cepas. Se analizaron 60 cueros y 60 carcasas. En la etapa de tamizaje, el gen stx se detectó en el 48% de las muestras de cuero y en el 80% de las muestras de carcasa por una PCR de punto final. La frecuencia de recuperación de cepas STEC fue del 5% en cueros y del 8% en carcasas, y la de cepas STEC positivas para los serogrupos O26, O45, O103, O121, O111, O145 u O157 fue del 0% en los cueros y del 2% en las carcasas. La relación clonal de las cepas aisladas se investigó a través de electroforesis de campo pulsado y análisis de los patrones de macrorrestricción generados. Los resultados demostraron la existencia de contaminación cruzada entre cueros y carcasas de animales pertenecientes a un mismo lote, y también que el origen de la contaminación fue el propio cuero del animal o el cuero de otros animales pertenecientes al mismo lote. Los altos porcentajes de detección en la etapa de tamizaje, especialmente en carcasas, y la evidencia de contaminación cruzada ponen de manifiesto la necesidad de evaluar la implementación de estrategias de intervención tendientes a evitar la contaminación de carcasas.

Frequency, characterization and genotypic analysis of Shiga toxin-producing Escherichia coli in beef slaughterhouses of Argentina.

The objectives of this study were: (1) to estimate STEC frequency in hide and carcass samples taken from beef slaughterhouses supplying the domestic market in Argentina, (2) to establish the pheno-genotypic characteristics of STEC and non-toxigenic Escherichia coli of serogroups O26, O45, O103, O121, O111, O145 or O157 isolated from the analyzed samples and, (3) to study their clonal relatedness. Sixty hides and 60 carcasses were analyzed. At the screening step, 48% of hide and 80% of carcass samples tested positive for the stx gene by endpoint PCR. The STEC isolation rate was 5% for hides and 8% for carcasses. The isolation rate of STEC-positive for O26, O45, O103, O111, O145 or O157 serogroups was 0% for hides and 2% for carcasses. With the purpose of studying the clonal relatedness of isolates, macrorestriction fragment analysis by pulsed-field gel electrophoresis was performed. The results indicated cross-contamination between hides and between carcasses of animals in the same lot and, that the origin of carcass contamination was their own hide, or the hides of other animals in the same lot. The high detection rate at the screening step, especially in carcasses, and the evidence of cross-contamination show the need to apply additional in-plant intervention strategies aimed at preventing carcass contamination.

Geographically distinct Escherichia coli O157 isolates differ by lineage, Shiga toxin genotype, and total shiga toxin production.

While the differential association of Escherichia coli O157 genotypes with animal and human hosts has recently been well documented, little is known about their distribution between countries and how this might affect regional disease rates. Here, we used a 48-plex single nucleotide polymorphism (SNP) assay to segregate 148 E. coli O157 isolates from Australia, Argentina, and the United States into 11 SNP lineages. We also investigated the relationship between SNP lineages, Shiga toxin (Stx) gene profiles, and total Stx production. E. coli O157 isolates clearly segregated into SNP lineages that were differentially associated with each country. Of the 11 SNP lineages, seven were detected among isolates from a single country, two were detected among isolates from all three countries, and another two were detected only among U.S. and Argentinean isolates. A number of Australian (30%) and Argentinean (14%) isolates were associated with novel, previously undescribed SNP lineages that were unique to each country. Isolates within SNP lineages that were strongly associated with the carriage of stx2a produced comparatively more Stx on average than did those lacking the stx2a subtype. Furthermore, the proportion of isolates in stx2a-associated SNP lineages was significantly higher in Argentina and the United States than Australia (P < 0.05). This study provides evidence for the geographic divergence of E. coli O157 and for a prominent role of stx2a in total Stx production. These results also highlight the need for more comprehensive studies of the global distribution of E. coli O157 lineages and the impacts of regionally predominant E. coli O157 lineages on the prevalence and severity of disease.

Phylogenetically related Argentinean and Australian Escherichia coli O157 isolates are distinguished by virulence clades and alternative Shiga toxin 1 and 2 prophages.

Shiga toxigenic Escherichia coli O157 is the leading cause of hemolytic uremic syndrome (HUS) worldwide. The frequencies of stx genotypes and the incidences of O157-related illness and HUS vary significantly between Argentina and Australia. Locus-specific polymorphism analysis revealed that lineage I/II (LI/II) E. coli O157 isolates were most prevalent in Argentina (90%) and Australia (88%). Argentinean LI/II isolates were shown to belong to clades 4 (28%) and 8 (72%), while Australian LI/II isolates were identified as clades 6 (15%), 7 (83%), and 8 (2%). Clade 8 was significantly associated with Shiga toxin bacteriophage insertion (SBI) type stx(2) (locus of insertion, argW) in Argentinean isolates (P < 0.0001). In Argentinean LI/II strains, stx(2) is carried by a prophage inserted at argW, whereas in Australian LI/II strains the argW locus is occupied by the novel stx(1) prophage. In both Argentinean and Australian LI/II strains, stx(2c) is almost exclusively carried by a prophage inserted at sbcB. However, alternative q(933)- or q(21)-related alleles were identified in the Australian stx(2c) prophage. Argentinean LI/II isolates were also distinguished from Australian isolates by the presence of the putative virulence determinant ECSP_3286 and the predominance of motile O157:H7 strains. Characteristics common to both Argentinean and Australian LI/II O157 strains included the presence of putative virulence determinants (ECSP_3620, ECSP_0242, ECSP_2687, ECSP_2870, and ECSP_2872) and the predominance of the tir255T allele. These data support further understanding of O157 phylogeny and may foster greater insight into the differential virulence of O157 lineages.

Subtyping of Escherichia coli O157:H7 strains isolated from human infections and healthy cattle in Argentina.

Shiga toxin-producing Escherichia coli (STEC) cause nonbloody (NBD) and bloody diarrhea (BD), and hemolytic uremic syndrome (HUS). Cattle have been described as their main reservoir. STEC O157:H7 is recognized as the predominant serotype in clinical infections, but much less is known about the dominant subtypes in humans and animals or their genetic relatedness. The aims of this study were to compare the STEC O157 subtypes found in sporadic human infections with those in the bovine reservoir using stx-genotyping, phage typing, and XbaI-pulsed-field gel electrophoresis (PFGE), and correlate the subtypes with the severity of clinical manifestations. The 280 STEC O157:H7 strains collected included in this study were isolated from HUS (n=122), BD (n=69), and NBD (n=30) cases, and healthy carriers (n=5), and from bovines (n=54) in the abattoirs. The stx-genotyping showed that stx2/stx(2c(vh-a)) was predominant in human (76.1%) and in bovine strains (55.5%), whereas the second more important genotype was stx2 (20.8%) in human and stx(2c(vh-a)) (16.7%) in cattle strains. In human strains, PT4 (37.6%), PT49 (24.3%), and PT2 (18.6%) were the most frequent PTs (80.5%). In bovine isolates, PT2 (26%), PT39 (16.7%), and PT4 and PT49 (11.1% each) were predominant. By XbaI-PFGE, all 280 strains yielded 148 patterns with 75% similarity, and 169 strains were grouped in 37 clusters. Identical PT-PFGE-stx profile combinations were detected in strains of both origins: PT4-AREXH01.0011-stx2/stx(2c(vh-a)) (12 humans and one bovine), PT4-AREXH01.0543-stx2/stx(2c(vh-a)) (one human and four bovines), PT2-AREXH01.0076-stx2/stx(2c(vh-a)) (one human and four bovines), PT49-AREXH01.0175-stx2/stx(2c(vh-a)) (seven humans and one bovine), and PT49-AREXH01.0022-stx2/stx(2c(vh-a)) (seven humans and one bovine). No correlation was found among the stx-genotypes, the phage type, and the clinical symptoms.