Stefan Hertwig

Shiga Toxin-Producing Escherichia coli O104:H4: a New Challenge for Microbiology

ABSTRACT In 2011, Germany experienced the largest outbreak with a Shiga toxin-producing Escherichia coli (STEC) strain ever recorded. A series of environmental and trace-back and trace-forward investigations linked sprout consumption with the disease, but fecal-oral transmission was also documented. The genome sequences of the pathogen revealed a clonal outbreak with enteroaggregative E. coli (EAEC). Some EAEC virulence factors are carried on the virulence plasmid pAA. From an unknown source, the epidemic strains acquired a lambdoid prophage carrying the gene for the Shiga toxin. The resulting strains therefore possess two different mobile elements, a phage and a plasmid, contributing essential virulence genes. Shiga toxin is released by decaying bacteria in the gut, migrates through the intestinal barrier, and is transported via the blood to target organs, like the kidney. In a mouse model, probiotic bifidobacteria interfered with transport of the toxin through the gut mucosa. Researchers explored bacteriophages, bacteriocins, and low-molecular-weight inhibitors against STEC. Randomized controlled clinical trials of enterohemorrhagic E. coli (EHEC)-associated hemolytic uremic syndrome (HUS) patients found none of the interventions superior to supportive therapy alone. Antibodies against one subtype of Shiga toxin protected pigs against fatal neurological infection, while treatment with a toxin receptor decoy showed no effect in a clinical trial. Likewise, a monoclonal antibody directed against a complement protein led to mixed results. Plasma exchange and IgG immunoadsoprtion ameliorated the condition in small uncontrolled trials. The epidemic O104:H4 strains were resistant to all penicillins and cephalosporins but susceptible to carbapenems, which were recommended for treatment.

Virulence and resistance determinants of German Staphylococcus aureus ST398 isolates from nonhuman sources.

ABSTRACT A series of 100 Staphylococcus aureus isolates ascribed to sequence type 398 (ST398) and recovered from different sources (healthy carrier and diseased pigs, dust from pig farms, milk, and meat) in Germany were investigated for their virulence and antimicrobial resistance genetic background. Antimicrobial resistance was determined by the disk diffusion method. Virulence and resistance determinants (37 and 31 genes, respectively) were tested by PCR. Only two virulence profiles, including the accessory gene regulator agrI and three or four hemolysin-encoding genes, were detected. In contrast, 33 resistance profiles were distinguished (only 11 were shown by more than one isolate). Fifty-nine isolates were multiresistant (four or more antimicrobial classes), and 98 were methicillin resistant (mecA positive). All of the ST398 isolates showed resistance to tetracycline [encoded by tet(M) alone or together with tet(K) and/or tet(L)]. In addition, 98% were resistant to other antimicrobials, including macrolide-lincosamine-streptogramin B (70%, encoded by ermA, ermB, and ermC, alone or in combination), trimethoprim (65%, mostly due to dfrK and dfrG), kanamycin and gentamicin [29% and 14%, respectively, mainly related to aac(6′)-Ie-aph(2″)-Ia and/or ant(4′)-Ia but also to aph(3′)-IIIa], chloramphenicol (9%, fexA or cfr), quinupristin-dalfopristin (9%), ciprofloxacin (8%), and trimethoprim-sulfamethoxazole (4%). The heterogeneity of the resistance profiles underlines the ability of the ST398 clone to acquire multiple antimicrobial resistance genes. However, the virulence gene content of the tested isolates was low. Continuous surveillance is needed to clarify whether its pathogenicity potential for animals and humans will increase over time.

Characterization of enterocoliticin, a phage tail-like bacteriocin, and its effect on pathogenic Yersinia enterocolitica strains.

ABSTRACT Yersinia enterocolitica 29930 (biogroup 1A; serogroup O:7,8) produces a bacteriocin, designated enterocoliticin, that shows inhibitory activity against enteropathogenic strains of Y. enterocolitica belonging to serogroups O:3, O:5,27 and O:9. Enterocoliticin was purified, and electron micrographs of enterocoliticin preparations revealed the presence of phage tail-like particles. The particles did not contain nucleic acids and showed contraction upon contact with susceptible bacteria. Enterocoliticin addition to logarithmic-phase cultures of susceptible bacterial strains led to a rapid dose-dependent reduction in CFU. Calorimetric measurements of the heat output of cultures of sensitive bacteria showed a complete loss of cellular metabolic activity immediately upon addition of enterocoliticin. Furthermore, a dose-dependent efflux of K+ ions into the medium was determined, indicating that enterocoliticin has channel-forming activity.

Isolation of a Lysogenic Bacteriophage Carrying the stx1OX3 Gene, Which Is Closely Associated with Shiga Toxin-Producing Escherichia coli Strains from Sheep and Humans

ABSTRACT A specific PCR for the detection of a variant of the gene encoding Shiga toxin 1 (stx1) calledstx1OX3 (GenBank accession no. Z36901 ) was developed. The PCR was used to investigate 148 Stx1-producing Escherichia colistrains from human patients (n = 72), cattle (n = 27), sheep (n = 48), and a goat (n = 1) for the presence of thestx1OX3 gene. Thestx1OX3 gene was present in 38 Shiga toxin-producing E. coli (STEC) strains from sheep belonging to serogroups O5, O125, O128, O146, and OX3 but was absent from Stx1-positive ovine STEC O91 strains. Thestx1OX3 gene was also detected in 22 STEC strains from humans with nonbloody diarrhea and from asymptomatic excreters. Serotypes O146:H21 and O128:H2 were most frequently associated withstx1OX3-carrying STEC from sheep and humans. In contrast, Stx1-producing STEC strains from cattle and goats and 50 STEC strains from humans were all negative for the stx1OX3 gene. Thestx1OX3-negative strains belonged to 13 serotypes which were different from those of thestx1OX3-positive STEC strains. Moreover, the stx1OX3gene was not associated with STEC belonging to enterohemorrhagicE. coli (EHEC) serogroups O26, O103, O111, O118, O145, and O157. A bacteriophage carrying thestx1OX3 gene (phage 6220) was isolated from a human STEC O146:H21 strain. The phage was able to lysogenize laboratory E. coli K-12 strain C600. Phage 6220 shared a similar morphology and a high degree of DNA homology with Stx2-encoding phage 933W, which originates from EHEC O157. In contrast, few similarities were found between phage 6220 and Stx1-encoding bacteriophage H-19B from EHEC O26.

Prevalence of MRSA types in slaughter pigs in different German abattoirs.

To investigate the prevalence of types of meticillinresistant Staphylococcus aureus (MRSA) in slaughter pigs in German abattoirs, nasal swabs were collected from a total of 1026 pigs in five abattoirs after stunning in the course of two studies, and examined for MRSA. Study 1 included four abattoirs; study 2 was carried out in one large abattoir. Isolates were tested for antimicrobial susceptibility and characterised using spa-typing, multilocus sequence typing (MLST) and typing of the staphylococcal cassette chromosome, SCCmec. Overall, MRSA was isolated from 70·8 per cent of 520 samples in study 1 and from 49·0 per cent of 506 samples in study 2. The proportion of positive samples varied substantially between the abattoirs in study 1. Most isolates belonged to spa-types t011 and t034 and SCCmec types III and V. MLST of selected isolates revealed that they were all MLST ST398. Besides β-lactams, 100 per cent of the isolates were resistant to tetracycline, 80·5 per cent were resistant to erythromycin and 80·7 per cent were resistant to clindamycin. Less than 5 per cent of the isolates were resistant to other antimicrobials.

Simultaneous Identification of DNA and RNA Viruses Present in Pig Faeces Using Process-Controlled Deep Sequencing

Background Animal faeces comprise a community of many different microorganisms including bacteria and viruses. Only scarce information is available about the diversity of viruses present in the faeces of pigs. Here we describe a protocol, which was optimized for the purification of the total fraction of viral particles from pig faeces. The genomes of the purified DNA and RNA viruses were simultaneously amplified by PCR and subjected to deep sequencing followed by bioinformatic analyses. The efficiency of the method was monitored using a process control consisting of three bacteriophages (T4, M13 and MS2) with different morphology and genome types. Defined amounts of the bacteriophages were added to the sample and their abundance was assessed by quantitative PCR during the preparation procedure. Results The procedure was applied to a pooled faecal sample of five pigs. From this sample, 69,613 sequence reads were generated. All of the added bacteriophages were identified by sequence analysis of the reads. In total, 7.7% of the reads showed significant sequence identities with published viral sequences. They mainly originated from bacteriophages (73.9%) and mammalian viruses (23.9%); 0.8% of the sequences showed identities to plant viruses. The most abundant detected porcine viruses were kobuvirus, rotavirus C, astrovirus, enterovirus B, sapovirus and picobirnavirus. In addition, sequences with identities to the chimpanzee stool-associated circular ssDNA virus were identified. Whole genome analysis indicates that this virus, tentatively designated as pig stool-associated circular ssDNA virus (PigSCV), represents a novel pig virus. Conclusion The established protocol enables the simultaneous detection of DNA and RNA viruses in pig faeces including the identification of so far unknown viruses. It may be applied in studies investigating aetiology, epidemiology and ecology of diseases. The implemented process control serves as quality control, ensures comparability of the method and may be used for further method optimization.

Livestock Associated Methicillin-Resistant Staphylococcus aureus (LaMRSA) Isolated from Lesions of Pigs at Necropsy in Northwest Germany Between 2004 and 2007

An increasing number of reported detections of methicillin‐resistant Staphylococcus aureus (MRSA) in food animals since 2007 has led to the assumption that there is an emerging zoonotic problem with livestock associated (la)MRSA potentially aggravating the MRSA problem in humans. It was the objective of the study to investigate, whether MRSA was present in clinical specimens of pigs collected at post‐mortem since 2004 and to further characterize these isolates. We studied 138 isolates of S. aureus collected between 2004 and 2007 from various pathological lesions of pigs at necropsy. Potential MRSA were identified by growth on selective chromogenic media. Isolates were confirmed as MRSA using multiplex PCR. Confirmed isolates were spa‐ and SCCmec‐typed and were tested for antimicrobial resistance. Overall, 60 (43%) S. aureus isolates were identified as MRSA. The majority (57/60) of the MRSA isolates found in the altered porcine tissues were spa‐types associated with MRSA ST398. Three MRSA were ST97 isolates, a type that has not been described as an MRSA in pigs before. Other clonal complexes (ST9, ST30) dominated among the methicillin‐sensitive S. aureus. MRSA were found in similar frequency in all 4 years. We assume that MRSA in pigs may have occurred earlier than 2004 and might be not really ‘emerging’, but rather have been overlooked until recently. The potentially causative role of the MRSA in the lesions warrants further investigation.

High Heterogeneity within Methicillin-Resistant Staphylococcus aureus ST398 Isolates, Defined by Cfr9I Macrorestriction-Pulsed-Field Gel Electrophoresis Profiles and spa and SCCmec Types

ABSTRACT During recent years, the animal-associated methicillin-resistant Staphylococcus aureus clone ST398 has extensively been studied. The DNA of these isolates turned out to be refractory to SmaI restriction, and consequently, SmaI is unsuitable for subtyping this clone by standard pulsed-field gel electrophoresis (PFGE). Very recently, ST398 DNA was shown to be digested by Cfr9I, a neoschizomer of SmaI. In the present study, we employed Cfr9I PFGE on 100 German and 5 Dutch ST398 isolates and compared their PFGE profiles, protein A gene variable repeat regions (spa types), and types of the staphylococcal cassette chromosome mec (SCCmec). The isolates (from healthy carrier pigs, clinical samples from pigs, dust from farms, milk, and meat) were assigned to 35 profiles, which were correlated to the SCCmec type. A dendrogram with the Cfr9I patterns assigned all profiles to two clusters. Cluster A grouped nearly all isolates with SCCmec type V, and cluster B comprised all SCCmec type IVa and V* (a type V variant first identified as III) carriers plus one isolate with SCCmec type V. Both clusters also grouped methicillin-susceptible S. aureus isolates. The association of the majority of isolates with SCCmec type V in one large cluster indicated the presence of a successful subclone within the clonal complex CC398 from pigs, which has diversified. In general, the combination of Cfr9I PFGE with spa and SCCmec typing demonstrated the heterogeneity of the series analyzed and can be further used for outbreak investigations and traceability studies of the MRSA ST398 emerging clone.

A suggested classification for two groups of Campylobacter myoviruses

Most Campylobacter bacteriophages isolated to date have long contractile tails and belong to the family Myoviridae. Based on their morphology, genome size and endonuclease restriction profile, Campylobacter phages were originally divided into three groups. The recent genome sequencing of seven virulent campylophages reveal further details of the relationships between these phages at the genome organization level. This article details the morphological and genomic features among the campylophages, investigates their taxonomic position, and proposes the creation of two new genera, the “Cp220likevirus” and “Cp8unalikevirus” within a proposed subfamily, the “Eucampyvirinae”

Factors associated with the occurrence of MRSA CC398 in herds of fattening pigs in Germany

BackgroundThe purpose of this study was to investigate the prevalence of MRSA in herds of fattening pigs in different regions of Germany, and to determine factors associated with the occurrence of this pathogen. For this purpose pooled dust samples were collected, and a questionnaire covered information regarding herd characteristics and management practices. Samples were pre-enriched in high-salt medium followed by selective enrichment containing cefoxitin/aztreonam, and culturing. Presumptive colonies were confirmed by multiplex-PCR targeting nuc-, mecA- and 16S rRNA-genes. Isolates were spa- and SCCmec-, and in selected cases, multilocus sequence-typed. Susceptibilities to 13 antimicrobials were determined by broth microdilution. Statistical analysis was carried out using backward stepwise logistic regression to calculate odds ratios with the MRSA test result as the outcome and herd characteristics as categorical covariates.ResultsOverall, 152 of 290 (52%) fattening pig farms tested positive for MRSA. The prevalence in the east, north- and south-west of Germany ranged from 39 to 59%.t011 (66%) and t034 (23%) were the most commonly identified spa-types, and 85% of isolates carried SCCmec Type V. Identified spa-types were all associated with clonal complex CC398. Susceptibility testing revealed that all isolates were resistant to tetracycline. High resistance rates were also found for sulfamethoxazole/trimethoprim (40%), and quinupristin/dalfopristin (32%). In addition, 83% of strains displayed multidrug resistant (> 3 substance classes) phenotypes.Logistic regression revealed herd size (large farms OR: 5.4; CI: 2.7-11.2; p < 0.05), and production type (wean-to-finish OR: 4.0; CI: 1.6-10.4; p < 0.05) as risk factors associated with a positive MRSA finding in fattening pig operations.ConclusionsMRSA CC398 is widely distributed among herds of fattening pigs in Germany. Farm management plays a crucial role in the dissemination of MRSA with herd size, and production type representing potential major indicators.