[Show abstract][Hide abstract] ABSTRACT: The mutS-rpoS intergenic region in E. coli displays a mosaic structure which revealed pathotype specific patterns. To assess the importance of this region as a surrogate marker for the identification of highly virulent extraintestinal pathogenic E. coli (ExPEC) strains we aimed to: (i) characterize the genetic diversity of the mutS gene and the o454-nlpD genomic region among 510 E. coli strains from animals and humans; (ii) delineate associations between the polymorphism of this region and features such as phylogenetic background of E. coli, pathotype, host species, clinical condition, serogroup and virulence associated genes (VAG)s; and (iii) identify the most important VAGs for classification of the o454-nlpD region.
[Show abstract][Hide abstract] 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 stx2e and eae, a very unusual and new virulence profile for a HUS-associated enterohemorrhagic E. coli.
Journal of clinical microbiology 02/2014; · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Extraintestinal pathogenic and intestinal pathogenic (diarrheagenic) Escherichia coli differ phylogenetically and by virulence profiles. Classic theory teaches simple linear descent in this species, where non-pathogens acquire virulence traits and emerge as pathogens. However, diarrheagenic Shiga toxin-producing E. coli (STEC) O2:H6 not only possess and express virulence factors associated with diarrheagenic and uropathogenic E. coli but also cause diarrhea and urinary tract infections. These organisms are phylogenetically positioned between members of an intestinal pathogenic group (STEC) and extraintestinal pathogenic E. coli. STEC O2:H6 is, therefore, a 'heteropathogen,' and the first such hybrid virulent E. coli identified. The phylogeny of these E. coli and the repertoire of virulence traits they possess compel consideration of an alternate view of pathogen emergence, whereby one pathogroup of E. coli undergoes phased metamorphosis into another. By understanding the evolutionary mechanisms of bacterial pathogens, rational strategies for counteracting their detrimental effects on humans can be developed.
EMBO Molecular Medicine 01/2014; · 7.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To discern the relevance of ST648 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli as a putative new group of multiresistant and extraintestinal pathogenic strains in animals, its frequency, ESBL types, antimicrobial resistance patterns and virulence gene (VG) profiles should be determined and compared with ST131 strains from the same collection of strains.
ESBL-producing E. coli isolates (n = 1152), consecutively sampled from predominantly dogs, cats and horses between 2008 and 2011, were assigned to a phylogenetic group by PCR. Partial multilocus sequence typing was performed for group D and B2 strains and strains presumed to be D-ST648 and B2-ST131 were fully typed. ESBL genes and extraintestinal pathogenic E. coli (ExPEC)-like VGs were characterized by PCR and sequence analysis and antimicrobial resistance was determined by broth dilution. Clonal analysis was done by PFGE.
Forty (3.5%) ESBL-producing E. coli were determined as D-ST648, whereas B2-ST131 isolates occurred less frequently (2.8%). Although the predominant ESBL type in both groups was CTX-M-15 (72.5% versus 46.9%), ST648 strains from companion animals and horses displayed a lower variety of ESBL types (CTX-M-1, -3, -14, -15 and -61 versus CTX-M-1,-2,-14,-15,-27 and -55 and SHV-12). In contrast to ST131 strains, a higher proportion of ST648 strains showed resistance to most non-β-lactam antibiotics. Overall, VGs were less abundant in ST648 strains, although some strains had VG profiles comparable to those of ST131 strains. ExPEC-associated serotype O1:H6 was predominant (46.8%) among the ST648 strains. Some PFGE clusters comprised ST648 isolates from pets, horses and wild birds and humans included from previous studies.
Our findings demonstrate that certain subgroups of E. coli D-ST648-CTX-M may represent a novel genotype that combines multiresistance, extraintestinal virulence and zoonotic potential.
Journal of Antimicrobial Chemotherapy 01/2014; · 5.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Salmonella enterica serovar Kottbus has been continuously isolated from poultry and poultry meat, especially from turkey. We investigated by comparative molecular typing 95 S. Kottbus isolates obtained in Germany between 2000 and 2011 from poultry/poultry meat, pig/pork, cattle, reptiles, the environment as well as from human cases to identify potential infection sources for humans, especially the role of poultry and poultry products as vehicle in transmission of S. Kottbus isolates to humans. Multilocus sequence typing analysis detected three main genetic lineages. Most human isolates belonged to lineage 1 represented by sequence types ST212 and ST808. Part of the isolates isolated from cattle and pork were also linked to this lineage. Nevertheless, human isolates and especially isolates from poultry/poultry meat, and with less extend from other livestock, grouped in lineage 2 represented by ST582. Four additional isolates from reptiles and humans belonging to ST1669 represented the third lineage. The three lineages were also reflected by pulsed-field gel electrophoresis typing data and DNA microarray analysis of 102 pathogenicity genes. Antimicrobial resistance especially to nalidixic acid and ciprofloxacin was predominantly observed in isolates assigned to lineage 2, which contains predominantly resistant isolates compared to lineage 1 and 3. Sequencing of the quinolone resistance-determining region of gyrA revealed a point mutation in codon 83 or 87 responsible for nalidixic acid resistance and MIC values for ciprofloxacin between 0.125 and 0.25 mg/l. Overall, this study showed that in Germany a specific S. Kottbus lineage (ST582), which is well-established in poultry, can be transmitted to humans by poultry meat and, consequently, poses a risk for human health.
[Show abstract][Hide abstract] ABSTRACT: The so far highest number of life-threatening hemolytic uremic syndrome was associated with a food-borne outbreak in 2011 in Germany which was caused by an enterohemorrhagic Escherichia coli (EHEC) of the rare serotype O104:H4. Most importantly, the outbreak strain harbored genes characteristic of both EHEC and enteroaggregative E. coli (EAEC). Such strains have been described seldom but due to the combination of virulence genes show a high pathogenicity potential. To evaluate the importance of EHEC/EAEC hybrid strains in human disease, we analyzed the EHEC strain collection of the German National Reference Centre for Salmonella and other Bacterial Enteric Pathogens (NRC). After exclusion of O104:H4 EHEC/EAEC strains, out of about 2400 EHEC strains sent to NRC between 2008 and 2012, two strains exhibited both EHEC and EAEC marker genes, specifically were stx2 and aatA positive. Like the 2011 outbreak strain, one of the novel EHEC/EAEC harbored the Shiga toxin gene type stx2a. The strain was isolated from a patient with bloody diarrhea in 2010, was serotyped as O59:H-, belonged to MLST ST1136, and exhibited genes for type IV aggregative adherence fimbriae (AAF). The second strain was isolated from a patient with diarrhea in 2012, harbored stx2b, was typed as Orough:H-, and belonged to MLST ST26. Although the strain conferred the aggregative adherence phenotype, no known AAF genes corresponding to fimbrial types I to V were detected. In summary, EHEC/EAEC hybrid strains are currently rarely isolated from human disease cases in Germany and two novel EHEC/EAEC of rare serovars/MLST sequence types were characterized.
PLoS ONE 01/2014; 9(4):e95379. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Uropathogenic Escherichia coli (UPEC) are the most common cause of community and hospital-acquired urinary tract infections (UTIs). Isolates from community-acquired, uncomplicated UTIs express a variety of virulence traits which promote efficient colonization of the urinary tract. In contrast, nosocomial UTIs can be caused by E.coli strains which differ in their virulence traits from community-acquired UTI isolates. UPEC virulence markers are used to distinguish these facultative extraintestinal pathogens, which belong to the intestinal flora of many healthy individuals, from intestinal pathogenic E.coli (IPEC). IPEC are diarrheagenic pathogens with characteristic virulence gene sets which are absent in UPEC. Here, we characterized 265 isolates from patients with UTI during inpatient or outpatient treatment at a hospital regarding phylogeny and IPEC or UPEC virulence traits. Interestingly, 28 of these isolates (10.6%) carried typical IPEC virulence genes characteristic for enteroaggregative E.coli (EAEC), Shiga toxin-producing E.coli (STEC), and atypical enteropathogenic E.coli (aEPEC), although IPEC are not considered uropathogens. Twenty-three isolates harbored the astA gene coding for the EAEC heat-stable enterotoxin 1 (EAST1), and most of them carried characteristic virulence genes of UPEC and/or EAEC. Our results indicate that UPEC from hospital patients can differ from archetypal community-acquired isolates of uncomplicated UTI by the spectrum of virulence traits. They represent a diverse group, including EAEC, as well as other IPEC pathotypes, which, in addition, contain typical UPEC virulence genes. The combination of typical ExPEC and IPEC virulence determinants in some isolates demonstrates the marked E.coli genome plasticity and calls for re-evaluating the strict pathotype classification of EAEC.
Journal of clinical microbiology 11/2013; · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Yersinia enterocolitica is a food-borne, gastro-intestinal pathogen with world-wide distribution. Only 11 serotypes have been isolated from patients, with O:3, O:9, O:8 and O:5,27 being the serotypes most commonly associated with human yersiniosis. Serotype is an important characteristic of Y. enterocolitica strains, allowing differentiation for epidemiology, diagnosis and phylogeny studies. Conventional serotyping, performed by slide agglutination, is a tedious and laborious procedure whose interpretation tends to be subjective, leading to poor reproducibility. Here we present a PCR-based typing scheme for molecular identification and patho-serotyping of Y. enterocolitica. Genome-wide comparison of Y. enterocolitica sequences allowed analysis of the O-antigen gene clusters of different serotypes, uncovering their formerly unknown genomic locations, and selection of targets for serotype-specific amplification. Two multiplex PCRs and one additional PCR were designed and tested on various reference strains and isolates from different origins. Our genotypic assay proved to be highly specific for identification of Y. enterocolitica species, discrimination between virulent and non-virulent strains, distinguishing the main human-related serotypes, and typing of conventionally untypeable strains. This genotyping scheme could be applied in microbiology laboratories as an alternative or complementary method to the traditional phenotypic assays, providing data for epidemiological studies.
International journal of medical microbiology: IJMM 10/2013; · 4.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Campylobacter spp., Salmonella enterica, and Yersinia enterocolitica are common causes of foodborne infections in humans with pork as a potential source. Monitoring programs at farm level are, to date, only implemented for S. enterica, while epidemiological knowledge of the other two pathogens is still lacking. This study aimed to assess the pathogen load (in the pigs' environment) in fattening pig herds, their simultaneous occurrence, and the occurrence of Campylobacter spp. and Y. enterocolitica in herds in different Salmonella risk categories. In 50 fattening pig herds in northern Germany, four pooled fecal samples and 10 swab samples from the pigs' direct environment (pen walls, nipple drinkers), indirect environment (hallways, drive boards), and flies and rodent droppings were collected from each herd and submitted for cultural examination. Campylobacter spp. were detected in 38.1% of fecal, 32.7% of direct environment, 5.3% of indirect environment, and 4.6% of flies/pests samples collected, and Y. enterocolitica in 17.1, 8.1, 1.2, and 3.1% and S. enterica in 11.2, 7.7, 4.1, and 1.5%, respectively. For Campylobacter spp., Y. enterocolitica, and S. enterica, 80, 48, and 32% of herds were positive, respectively; 22 herds were positive for both Campylobacter spp. and Y. enterocolitica, 12 for Campylobacter spp. and S. enterica, and 7 for Y. enterocolitica and S. enterica. There was no significant association between the pathogens at herd level. Campylobacter spp. and Y. enterocolitica were found more often in samples from the low Salmonella risk category (odds ratio, 0.51; confidence interval, 0.36 to 0.73, and 0.3, 0.17 to 0.57), and this was also the case for Y. enterocolitica at herd level (odds ratio, 0.08; confidence interval, 0.02 to 0.3). This study provides evidence that the pigs' environment should be accounted for when implementing control measures on farms against Campylobacter spp. and Y. enterocolitica. An extrapolation from the current Salmonella monitoring to the other two pathogens does not seem feasible.
Journal of food protection 10/2013; 76(10):1704-1711. · 1.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Yersinia enterocolitica is a human pathogen that is ubiquitous in livestock, especially pigs. The bacteria are able to colonize the intestinal tract of a variety of mammalian hosts, but the severity of induced gut-associated diseases (Yersiniosis) differs significantly between hosts. To gain more information about the individual virulence determinants that contribute to colonization and induction of immune responses in different hosts, we analyzed and compared the interaction of different human- and animal-derived isolates of the serotypes O:3, O:5,27, O:8 and O:9 with murine, porcine and human intestinal cells and macrophages. Examined strains exhibited significant serotype-specific cell binding and entry characteristics, but adhesion and uptake into the different host cells were not host-specific and independent of the source of the isolate. In contrast, survival and replication within macrophages and the induced proinflammatory response differed between murine, porcine and human macrophages suggesting a host-specific immune response. In fact, similar levels of the proinflammatory cytokine MIP-2 were secreted by murine bone marrow derived macrophages with all tested isolates, but the equivalent IL-8 response of porcine bone marrow derived macrophages was strongly serotype-specific and considerably lower in O:3 compared to O:8 strains. In addition, all tested Y. enterocolitica strains caused a considerably higher secretion of the anti-inflammatory cytokine IL-10 by porcine compared to murine macrophages. This could contribute to limit the severity of the infection (in particular of serotype O:3 strains) in pigs which are the primary reservoir of Y. enterocolitica strains pathogen to humans.
Infection and immunity 08/2013; · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report three patients with terminal ileitis and positive fecal cultures with Yersinia pseudotuberculosis. From one patient, a virulence plasmid (pYV)-negative Y. pseudotuberculosis was isolated, which represents the second finding of a pYV-negative isolate associated with human disease. All patients were treated with ciprofloxacin and fully recovered. Since conventional culture methods for yersiniosis are gradually replaced with molecular tests not recognizing Y. pseudotuberculosis, we recommend to include a specific culture medium or to apply a specific polymerase chain reaction (PCR) assay on fecal samples from patients suspected of terminal ileitis.
European Journal of Clinical Microbiology 08/2013; · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Shiga toxin (Stx)-producing Escherichia coli (STEC) of serogroup O174 are human pathogenic intimin gene (eae)-negative STEC. To facilitate diagnosis and subtyping, we genotypically and phenotypically characterized 25 STEC O174 isolates from humans with different clinical outcomes and from animals and the environment. fliC genotyping resulted in four different genotypes (fliCH2 : n = 5; fliCH8 : n = 8; fliCH21 : n = 11; fliCH46 : n = 1). Twenty-three strains were motile expressing the corresponding H antigen; two non-motile isolates possessed fliCH8 . The stx genotypes and non-stx virulence loci, including toxins, serine-proteases and adhesins correlated well with serotypes but showed no differences with respect to the isolates' origins. Multilocus sequence typing identified seven sequence types that correlated with serotypes. Core gene typing further specified the four serotypes, including a previously unknown O174:H46 combination, and revealed distant relationships of the different serotypes within serogroup O174 and in relation to other haemolytic uremic syndrome (HUS)-associated STEC. Only serotype O174:H21 was associated with HUS. Differences in virulence factors and in the adherence capacity of STEC O174 corroborated this separation into four distinct groups. Our study provides a basis for O174 subtyping, unravels considerable genotypic and phenotypic heterogeneity and sheds light to potential environmental and animal reservoirs.
[Show abstract][Hide abstract] ABSTRACT: With the intention to deepen the knowledge of the vertical transmission of particular subtypes of Salmonella enterica from "the stable to the table" a case1-case2 analysis in Lower Saxony, Germany, was conducted. The data collection was based on standardised telephone interviews with 1741 Salmonella case persons. Single-factor-analyses revealed statistically significant associations between S. Typhimurium infections and animal keeping (odds ratio (OR): 1.4; 95%-Confidence-interval (CI): 1.2-1.7), especially rodents (OR 1.5; CI 1.2-2.1), and with consumption of meat (OR 1.9; CI 1.3-2.8), raw ground pork (OR 3.0; CI 2.1-4.2) and uncooked pork sausage (OR 2.1; CI 1.6-2.9). The S. Typhimurium phage type DT 104 was associated most with consumption of uncooked pork sausage (OR 3.6; CI 1.3-8.5). Multiple logistic regression analyses confirmed the associations between S. Typhimurium infection and consumption of raw ground pork and with animal contact. The results circumstantiate the assumption of raw pork products still being a relevant source for S. Typhimurium infections in Germany. Therefore, it is recommended to intensify efforts to reduce salmonella infections caused by raw pork products. S. Enteritidis infection was associated statistically significantly with travelling abroad (OR 2.1; CI 1.6-3.3), consumption of raw tomatoes (OR 1.8; CI 1.5-2.1), dried herbs (OR 2.1; CI 1.0-1.8), and undercooked eggs (OR 1.3; CI 1.1-1.6) compared with other serovars. These results were confirmed in multiple logistic regression analyses, as well.
International journal of hygiene and environmental health 07/2013; 216(4):428-434. · 2.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Salmonella enterica serovar 4,,12:b:- is a monophasic serovar not able to express the second phase flagellar antigen (H2-antigen). In Germany, the serovar is occasionally isolated from poultry, reptiles, fish, food and humans. In this study a selection of 67 epidemiologically unrelated Salmonella enterica serovar 4,,12:b:- strains isolated in Germany between 2000 and 2011 from the environment, animal, food, and humans was investigated by phenotypic and genotypic methods to better understand the population structure and to identify potential sources of human infections. Strains of this monophasic serovar were highly diverse. Within the 67 strains analyzed we identified 52 different pulsed-field gel electrophoresis XbaI profiles, twelve different multilocus sequence types and 18 different pathogenicity array types. The relatedness of strains based on the pathogenicity gene repertoire (102 markers tested) was in good agreement with grouping by MLST. S. enterica serovar 4,,12:b:- is distributed across multiple unrelated eBurst groups and consequently highly polyphyletic. Two sequence types (ST88 and ST127) were linked to S. enterica serovar Paratyphi B (D-tartrate +), two single locus variants of ST1583 were linked to S. enterica serovar Abony and one sequence type (ST1484) was associated with S. enterica serovar Mygdal, a recently defined new serovar. From the characterization of clinical isolates and those of non-human origin it can be concluded that the potential sources of sporadic human infections with S. enterica serovar 4,,12:b:- most likely are mushrooms, shellfish/fish or poultry.
Applied and Environmental Microbiology 06/2013; · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Escherichia coli is one of the major causative agents of bovine mastitis, a disease affecting dairy herds. The lipopolysaccharide (LPS) of E. coli was plays a prominent role during infection. Here, we report on the O-antigen chemical structure of the LPS from Escherichia coli strain 2188 (serotype O174:H28) isolated from a mastitis-diseased cow. The structure of the OPS was analyzed by 1 and 2D NMR spectroscopy, and methylation analysis, which identified the branched repeating tetrasaccharide biological unit.
Carbohydrate research 03/2013; 373C:18-21. · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Seventy-five food-associated Shiga toxin-producing Escherichia coli (STEC) were analyzed by molecular and phylogenetic methods to describe their pathogenic potential. The presence of the locus of proteolysis (LPA), the chromosomal island PAI I(CL3), and the autotransporter encoding gene sabA, were examined by PCR. Furthermore, the occupation of the chromosomal integration sites of the locus of enterocyte effacement (LEE), selC, pheU and pheV as well as the Stx-phage integration sites yehV, yecE, wrbA, z2577 and ssrA were analyzed. Moreover, the antibiotic resistance phenotypes of all STEC were determined.Multilocus sequence typing (MLST) was performed and sequence types (ST) as well as sequence type complexes (STC) were compared with 42 HUS-associated enterohemorrhagic E. coli (HUSEC) strains. Besides 59 STs and 4 STCs, three larger clusters were defined in this strain collection. Clusters A and C mostly consist of highly pathogenic eae-positive HUSEC strains and some related food-borne STEC. A member of a new O26 HUS-associated clone and the 2011 outbreak strain E. coli O104:H4 was found in group A. Cluster B comprises only eae-negative food-borne STEC as well as mainly eae-negative HUSEC. Although food-borne strains of cluster B were not clearly associated with disease, serotypes of important pathogens such as O91:H21 and O113:H21 were in this group and closely related to the food-borne strains.Clonal analysis demonstrated eight closely related genetic groups of food-borne STEC and HUSEC that shared the same ST and were similar in their virulence gene composition. These groups should be considered with respect to their potential for human infection.
Applied and Environmental Microbiology 02/2013; · 3.95 Impact Factor