Nicole Cernela

Rapid species specific identification and subtyping of Yersinia enterocolitica by MALDI-TOF mass spectrometry.

Yersinia enterocolitica are Gram-negative pathogens and known as important causes of foodborne infections. Rapid and reliable identification of strains of the species Y. enterocolitica within the genus Yersinia and the differentiation of the pathogenic from the non-pathogenic biotypes has become increasingly important. We evaluated here the application of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid species identification and subtyping of Y. enterocolitica. To this end, we developed a reference MS database library including 19 Y. enterocolitica (non-pathogenic biotype 1A and pathogenic biotypes 2 and 4) as well as 24 non-Y. enterocolitica strains, belonging to eleven different other Yersinia spp. The strains provided reproducible and unique mass spectra profiles covering a wide molecular mass range (2000 to 30,000 Da). Species-specific and biotype-specific biomarker protein mass patterns were determined for Y. enterocolitica. The defined biomarker mass patterns (SARAMIS SuperSpectrum™) were validated using 117 strains from various Y. enterocolitica bioserotypes in a blind-test. All strains were correctly identified and for all strains the mass spectrometry-based identification scheme yielded identical results compared to a characterization by a combination of biotyping and serotyping. Our study demonstrates that MALDI-TOF-MS is a reliable and powerful tool for the rapid identification of Y. enterocolitica strains to the species level and allows subtyping of strains to the biotype level.

Detection of the Emerging Shiga Toxin-Producing Escherichia coli O26:H11/H− Sequence Type 29 (ST29) Clone in Human Patients and Healthy Cattle in Switzerland

ABSTRACT Shiga toxin-producing Escherichia coli O26:H11/H− strains showing the characteristics of the emerging human-pathogenic ST29 clone (stx 2a + only, eae +, plasmid gene profile hlyA + etpD +) were detected from human patients and healthy cattle, indicating a possible reservoir. Sheep also appear to shed strains related to the new pathogenic clone O26:H11/H− (ST29, stx 1a + only, eae +, plasmid gene profile hlyA + etpD +).

Shiga Toxin Subtypes Associated with Shiga Toxin–Producing Escherichia coli Strains Isolated from Red Deer, Roe Deer, Chamois, and Ibex

A total of 52 Shiga toxin-producing Escherichia coli (STEC) strains, isolated from fecal samples of six ibex, 12 chamois, 15 roe deer, and 19 red deer were further characterized by subtyping the stx genes, examining strains for the top nine serogroups and testing for the presence of eae and ehxA. Eleven of the 52 strains belonged to one of the top nine STEC O groups (O26, O45, O91, O103, O111, O113, O121, O145, and O157). Eight STEC strains were of serogroup O145, two strains of serogroup O113, and one strain of serogroup O157. None of the strains harbored stx2a, stx2e, or stx2f. Stx2b (24 strains) and stx1c (21 strains) were the most frequently detected stx subtypes, occurring alone or in combination with another stx subtype. Eight strains harbored stx2g, five strains stx2d, three strains stx1a, two strains stx2c, and one strain stx1d. Stx2g and stx1d were detected in strains not harboring any other stx subtype. The eae and ehxA genes were detected in two and 24 STEC strains, respectively. Considering both, the serogroups and the virulence factors, the majority of the STEC strains isolated from red deer, roe deer, chamois, and ibex do not show the typical patterns of highly pathogenic STEC strains. To assess the potential pathogenicity of STEC for humans, strain isolation and characterization is therefore of central importance.

Development and Validation of a PulseNet Standardized Protocol for Subtyping Isolates of Cronobacter Species

Cronobacter (formerly known as Enterobacter sakazakii) is a genus comprising seven species regarded as opportunistic pathogens that can be found in a wide variety of environments and foods, including powdered infant formula (PIF). Cronobacter sakazakii, the major species of this genus, has been epidemiologically linked to cases of bacteremia, meningitis in neonates, and necrotizing enterocolitis, and contaminated PIF has been identified as an important source of infection. Robust and reproducible subtyping methods are required to aid in the detection and investigation, of foodborne outbreaks. In this study, a pulsed-field gel electrophoresis (PFGE) protocol was developed and validated for subtyping Cronobacter species. It was derived from an existing modified PulseNet protocol, wherein XbaI and SpeI were the primary and secondary restriction enzymes used, generating an average of 14.7 and 20.3 bands, respectively. The PFGE method developed was both reproducible and discriminatory for subtyping Cronobacter species.

Hemolytic uremic syndrome in a 65 year-old male linked to a very unusual type of stx2e and eae harboring O51:H49 Shiga-toxin producing Escherichia coli

ABSTRACT We report on a 65-year-old male patient with a Shiga toxin-producing Escherichia coli O51:H49 gastrointestinal infection and sepsis associated with hemolytic uremic syndrome (HUS) with a fatal outcome. The strains isolated harbored stx 2e and eae, a very unusual and new virulence profile for an HUS-associated enterohemorrhagic E. coli.

Evaluation of Seven Different Commercially Available Real-Time PCR Assays for Detection of Shiga Toxin 1 and 2 Gene Subtypes

Following the recent outbreak of Shiga toxin-producing Escherichia coli (STEC) O104:H4 infection in Germany, the demand for fast detection of STEC has again increased. Various real-time PCR-based methods enabling detection of Shiga toxin genes (stx) have been developed and can be used for applications in food microbiology. The present study was conducted to evaluate the reliability of seven commercially available real-time PCR systems for detection of stx1 and stx2 subtypes. For this purpose, pure cultures of 18 STEC strains harboring all known stx1 and/or stx2 subtypes were tested. Only one of the seven real-time PCR systems detected all known stx1 and stx2 subtypes. Six systems failed to detect the stx2f subtype. One system missed stx2 subtypes reported in association with severe human disease. Because the presence of certain stx genes (subtypes) is considered an important indicator of STEC virulence, systems differentiating between the stx1 and stx2 gene groups provide added value. Reliable and fast detection of stx genes is of major importance for both diagnostic laboratories and the food industry.

Pathogenic Yersinia enterocolitica O:3 isolated from a hunted wild alpine ibex.

Occurrence of Yersinia spp. in wild ruminants was studied and the strains were characterized to get more information on the epidemiology of enteropathogenic Yersinia in the wildlife. In total, faecal samples of 77 red deer, 60 chamois, 55 roe deer and 27 alpine ibex were collected during 3 months of the hunting season in 2011. The most frequently identified species was Y. enterocolitica found in 13%, 10%, 4% and 2% of roe deer, red deer, alpine ibex and chamois, respectively. Interestingly, one Y. enterocolitica O:3 strain, isolated from an alpine ibex, carried the important virulence genes located on the virulence plasmid (yadA and virF) and in the chromosome (ail, hreP, myfA and ystA). Most of the Y. enterocolitica strains belonged to biotype 1A of which 14 were ystB positive. Further studies are needed to clarify the importance of alpine ibex as a reservoir of pathogenic Y. enterocolitica.

Prevalence of subtilase cytotoxin-encoding subAB variants among Shiga toxin-producing Escherichia coli strains isolated from wild ruminants and sheep differs from that of cattle and pigs and is predominated by the new allelic variant subAB2-2

Subtilase cytotoxin (SubAB) is an AB5 toxin produced by Shiga toxin (Stx)-producing Escherichia coli (STEC) strains usually lacking the eae gene product intimin. Three allelic variants of SubAB encoding genes have been described: subAB1, located on a plasmid, subAB2-1, located on the pathogenicity island SE-PAI and subAB2-2 located in an outer membrane efflux protein (OEP) region. SubAB is becoming increasingly recognized as a toxin potentially involved in human pathogenesis. Ruminants and cattle have been identified as reservoirs of subAB-positive STEC. The presence of the three subAB allelic variants was investigated by PCR for 152 STEC strains originating from chamois, ibex, red deer, roe deer, cattle, sheep and pigs. Overall, subAB genes were detected in 45.5% of the strains. Prevalence was highest for STEC originating from ibex (100%), chamois (92%) and sheep (65%). None of the STEC of bovine or of porcine origin tested positive for subAB. None of the strains tested positive for subAB1. The allelic variant subAB2-2 was detected the most commonly, with 51.4% possessing subAb2-1 together with subAB2-2. STEC of ovine origin, serotypes O91:H- and O128:H2, the saa gene, which encodes for the autoagglutinating adhesin and stx2b were significantly associated with subAB-positive STEC. Our results suggest that subAB2-1 and subAB2-2 is widespread among STEC from wild ruminants and sheep and may be important as virulence markers in STEC pathogenic to humans.

Antimicrobial resistance patterns and genotypes of Salmonella enterica serovar Hadar strains associated with human infections in Switzerland, 2005-2010.

Salmonella Hadar ranks in the top ten serovars reported from humans in Switzerland. In this study, all 64 S. Hadar strains isolated from different patients from 2005 to 2010 in Switzerland were characterized by (i) assessing phenotypic antimicrobial resistance profiles using the disk diffusion method and (ii) by genotyping using pulsed-field gel electrophoresis (PFGE) in order to evaluate the relationship of the strains. The annual incidences varied between 0.32/100,000 in 2005 (highest incidence) and 0.065/100,000 in 2007 (lowest incidence). In total 71.8% of the isolates were resistant to nalidixic acid. Although 40.6% of the strains were resistant to the β-lactam antibiotic ampicillin, they remained susceptible to the third-generation cephalosporin cefotaxime. Genotyping revealed a primary cluster consisting of 42 strains, sharing a similarity of >92%, with a subcluster of 18 strains with indistinguishable patterns. Resistance profiles allowed further differentiation within this subcluster providing a link of two strains to an outbreak in Spain.

Characteristics of Shigatoxin-Producing Escherichia coli Strains Isolated during 2010–2014 from Human Infections in Switzerland

Objectives: The aim of this study was to characterize a collection of 95 Shigatoxin-producing E.coli (STEC) isolated from human patients in Switzerland during 2010–2014. Methods: We performed O and H serotyping and molecular subtyping. Results: The five most common serogroups were O157, O145, O26, O103, and O146. Of the 95 strains, 35 (36.8%) carried stx1 genes only, 43 strains (45.2%) carried stx2 and 17 (17.9%) harbored combinations of stx1 and stx2 genes. Stx1a (42 strains) and stx2a (32 strains) were the most frequently detected stx subtypes. Genes for intimin (eae), hemolysin (hly), iron-regulated adhesion (iha), and the subtilase cytotoxin subtypes subAB1, subAB2-1, subAB2-2, or subAB2-3 were detected in 70.5, 83.2, 74.7, and 20% of the strains, respectively. Multilocus sequence typing assigned the majority (58.9%) of the isolates to five different clonal complexes (CC), 11, 32, 29, 20, and 165, respectively. CC11 included all O157:[H7] and O55:[H7] isolates. CC32 comprised O145:[H28] isolates, and O145:[H25] belonged to sequence type (ST) 342. CC29 contained isolates of the O26:[H11], O111:[H8] and O118:[Hnt] serogroups, and CC20 encompassed isolates of O51:H49/[Hnt] and O103:[H2]. CC165 included isolates typed O80:[H2]-ST301, all harboring stx2d, eae-ξ, hly, and 66.7% additionally harboring iha. All O80:[H2]-ST301 strains harbored at least 7 genes carried by pS88, a plasmid associated with extraintestinal virulence. Compared to data from Switzerland from the years 2000–2009, an increase of the proportion of non-O157 STEC infections was observed as well as an increase of infections due to STEC O146. By contrast, the prevalence of the highly virulent German clone STEC O26:[H11]-ST29 decreased from 11.3% during 2000–2009 to 1.1% for the time span 2010–2014. The detection of O80:[H2]-ST301 harboring stx2d, eae-ξ, hly, iha, and pS88 related genes suggests an ongoing emergence in Switzerland of an unusual, highly pathogenic STEC serotype. Conclusions: Serotyping and molecular subtyping of clinical STEC demonstrate that although STEC O157 predominates among STEC isolated from diseased humans, non-O157 STEC infections are increasing in Switzerland, including those due to STEC O146:[H2/H21/H28]-ST442/ST738 harboring subAB variants, and the recently emerged STEC O80:[H2]-ST301 harboring eae-ξ and pS88 associated extraintestinal pathogenic virulence genes.