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Background Plasmid-encoded extended-spectrum beta-lactamase (ESBL)-enzymes are frequently produced by Escherichia coli. Several ESBL-plasmids contain genes for toxin-antitoxin (TA) systems, which assure the maintenance of plasmids in bacteria and prevent the cells from “post-segregational killing”. These systems limit options to “cure” plasmids of ESBL-wild-type strains due to the death of the bacterial cells. A helpful tool to understand the role of ESBL-plasmids in the dissemination of pandemic multi-resistant E. coli are ESBL-plasmid-“cured”-variants (PCVs) and their comparison to ESBL-wild-type strains. The purpose of this study was to construct PCVs of ESBL-wild-type E. coli strains despite the presence of genes for TA systems. Findings Using enhanced temperatures and brain-heart-infusion broth it was possible to construct viable PCVs of wild-type ESBL-E. coli strains. The occurrence of TA system-genes including hok/sok, srnB/C, vagC/D, pemI/K on ESBL-plasmids of replicon types FIA or FIB was demonstrated by bioinformatic analyses. The loss of the plasmid and the genetic identity of PCV and corresponding wild-type strain was confirmed via different methods including plasmid-profile-analysis, pulsed-field gel electrophoresis and bioinformatics using generated whole genome data of the strains. Conclusions This short report describes the successful construction of viable PCVs of ESBL-wild-type E. coli strains. The results are hence surprising due to the fact that all “cured” ESBL-plasmids contained at least one complete toxin-antitoxin system, whose loss would normally mean the death of bacterial cells.

Over the past decades the use of probiotics in food has increased largely due to the manufacturer’s interest in placing “healthy” food on the market based on the consumer’s ambitions to live healthy. Due to this trend, health benefits of products containing probiotic strains such as lactobacilli are promoted and probiotic strains have been established in many different products with their numbers increasing steadily. Probiotics are used as starter cultures in dairy products such as cheese or yoghurts and in addition they are also utilized in non-dairy products such as fermented vegetables, fermented meat and pharmaceuticals, thereby, covering a large variety of products. To assure quality management, several pheno-, physico- and genotyping methods have been established to unambiguously identify probiotic lactobacilli. These methods are often specific enough to identify the probiotic strains at genus and species levels. However, the probiotic ability is often strain dependent and it is impossible to distinguish strains by basic microbiological methods. Therefore, this review aims to critically summarize and evaluate conventional identification methods for the genus Lactobacillus, complemented by techniques that are currently being developed.

Naturally occurring enterohemolysin negative variants were observed during studies on bovine Shiga-like toxin-producing E. coli (SLTEC). Examination of three strains (413/89-1 and 332, 026:H-, and 570/89, O111:H-) and their isogenic variants (413/89-6, 332-I and 570/89-I, respectively) showed, that in each strain loss of the enterohemolytic phenotype correlated with the loss of a large plasmid ranging from 94 to 104 kb in size. The hemolysin determinant present on the 94 kb plasmid of strain 413/89-1 was cloned and discovered by DNA and N-terminal aminoacid sequence analysis to be highly homologous to the recently published EHEC-hemolysin (HlyEHEC; Schmidt et al., 1994; 1995). When a recombinant plasmid harboring this determinant was reintroduced into the enterohemolysin negative isogenic mutant 413/89-6, the enterohemolytic phenotype was restored. Southern blot hybridization analysis was used to demonstrate that the HlyEHEC is plasmid-borne in SLTEC-strains. Our cumulative data suggest that the enterohemolytic phenotype of SLTEC is encoded by the plasmid-borne HlyEHEC. These results further demonstrate the close similarity between SLTEC-isolates from bovine and human.

Shiga-like toxin (SLT)-producing Escherichia coli (SLTEC) O101 has recently been associated with hemorrhagic colitis and hemolytic-uremic syndrome in humans. In this study, SLTEC O101 strains from humans and pigs were characterized for clonal relatedness by nucleotide sequence analysis of their slt genes, DNA finger-printing of genomic DNA, and determination of virulence factors. The slt genes of five E. coli O101 strains were cloned and sequenced. For all strains, the deduced amino acid sequences of the B subunits were identical to those of the SLT-IIe present in the classical SLTEC O139 strains that cause edema disease in pigs. The A subunit revealed more than 99% homology to that of SLT-IIe. DNA fingerprinting revealed a high degree of genetic relatedness between the human and porcine O101 isolates. None of the O101 strains investigated had virulence factors frequently found in porcine (F107 fimbriae or heat-stable or heat-labile enterotoxins) or human SLTEC strains (eaeA or enterohemorrhagic E. coli hemolysin). The absence of virulence factors typical of SLT-I- and SLT-II-producing E. Coli together with the presence of SLT-IIe, a toxin previously seen only in porcine E. coli, suggests a new pathogenic mechanism for E. coli O101 infection of humans. For diagnostic purposes, we recommend the use of PCR primers and DNA probes complementary to slt-IIe to correctly identify such strains and to further evaluate their role in human diseases.

Previous or present infection with Shiga-like toxin producing E. coli (SLTEC) was detected by an indirect neutralization assay of antibody titer. Bovine colostra and sera blocked the cytotoxic effects of Shiga-like toxin on Vero cell monolayers. SLT neutralizing antibodies were present in 84.0% (189/225) of the colostrum samples from randomly chosen cows in Bavaria, Germany. While all of the colostra with neutralizing activity reacted with SLT-I, only 14.7% neutralized both SLT-I and -II. Approximately 93.0% (37/40) of sera from heifers had SLT neutralizing activity. To quantify the neutralizing antibodies, colostra were tested in the Vero cell assay for their capability to reduce the 50% cytotoxic dose (CD50) of SLT standards, where the neutralizing units/ml (nu/ml) equal the log10 of CD50 reduction. Almost half of reactive colostra (48.7%) reduced the CD50 of the SLT-I standard by 10(4) to 10(5) (4-5 nu/ml). Higher reactivity (5-7 nu/ml) was found in 46.5% of positive colostra. The remaining colostra samples had over 7 nu/ml. To determine if the colostra were blocking receptors for SLT on Vero cells, cells were preincubated with colostra, and SLT was later added. No neutralizing activity was detected, indicating the reactivity of colostra was directed against SLT. When the colostra were subjected to ammonium sulphate precipitation and DEAE anion exchange chromatography, high levels of neutralizing activity were found in the IgG1 containing fractions. Colostrum fractions were tested for SLT-I binding antibodies in a capture ELISA, based on the binding of SLT-I to the toxin receptor analogue P1-glycoprotein. Only fractions from colostra with over 5 nu/ml were reactive in this assay, indicating the ELISA was less sensitive than the Vero cell assay. The results support the theory that SLTEC exposure of cows in Germany is more widespread than expected from epidemiological studies based on bacterial isolation. This possibly indicates a higher risk of human SLTEC infection via beef and milk products.

Background Shiga toxin producing Escherichia coli (STEC) are an important cause of human gastro-enteritis and extraintestinal sequelae, with ruminants, especially cattle, as the major source of infection and reservoir. In this study, the fecal STEC shedding of 133 dairy cows was analyzed over a period of twelve months by monthly sampling with the aim to investigate shedding patterns and risk factors. Results Overall, 24.7% (in total 407) of 1,646 fecal samples were tested positive for stx by PCR with inner-herd prevalences on the different farms of 11.1% to 32.3%. At individual levels, cows were stx-positive on zero to eight consecutive samplings. According to a strictly longitudinal definition of Super-Shedding, in the present study 14 cows were identified as Super-Shedders of non-O157 serotypes. Significant risk factors for the shedding of STEC were the month of sampling, the number of lactations and days in lactation, the nutritional condition, the somatic cell count and the content of protein in milk. Most notably, the presence of STEC Super-Shedding cows in the herd was a significant risk factor, revealing that STEC Super-Shedding is not restricted to STEC O157:H7 alone. Conclusions These data have implications for possible interventions, as removing single non-O157:H7 STEC Super-Shedding cattle from farms would significantly reduce STEC burden.

The extraintestinal pathogen, avian pathogenic E. coli (APEC), known to cause systemic infections in chickens, is responsible for large economic losses in the poultry industry worldwide. In order to identify genes involved in the early essential stages of pathogenesis, namely adhesion and colonization, Signature-tagged mutagenesis (STM) was applied to a previously established lung colonization model of infection by generating and screening a total of 1,800 mutants of an APEC strain IMT5155 (O2:K1:H5; Sequence type complex 95). The study led to the identification of new genes of interest, including two adhesins, one of which coded for a novel APEC fimbrial adhesin (Yqi) not described for its role in APEC pathogenesis to date. Its gene product has been temporarily designated ExPEC Adhesin I (EA/I) until the adhesin-specific receptor is identified. Deletion of the ExPEC adhesin I gene resulted in reduced colonization ability by APEC strain IMT5155 both in vitro and in vivo. Furthermore, complementation of the adhesin gene restored its ability to colonize epithelial cells in vitro. The ExPEC adhesin I protein was successfully expressed in vitro. Electron microscopy of an afimbriate strain E. coli AAEC189 over-expressed with the putative EA/I gene cluster revealed short fimbrial-like appendages protruding out of the bacterial outer membrane. We observed that this adhesin coding gene yqi is prevalent among extraintestinal pathogenic E. coli (ExPEC) isolates, including APEC (54.4%), uropathogenic E. coli (UPEC) (65.9%) and newborn meningitic E. coli (NMEC) (60.0%), and absent in all of the 153 intestinal pathogenic E. coli strains tested, thereby validating the designation of the adhesin as ExPEC Adhesin I. In addition, prevalence of EA/I was most frequently associated with the B2 group of the EcoR classification and ST95 complex of the multi locus sequence typing (MLST) scheme, with evidence of a positive selection within this highly pathogenic complex. This is the first report of the newly identified and functionally characterized ExPEC adhesin I and its significant role during APEC infection in chickens.

Background Although antimicrobial resistance and persistence of resistant bacteria in humans and animals are major health concerns worldwide, the impact of antimicrobial resistance on bacterial intestinal colonization in healthy domestic animals has only been rarely studied. We carried out a retrospective analysis of the antimicrobial susceptibility status and the presence of resistance genes in intestinal commensal E. coli clones from clinically healthy pigs from one production unit with particular focus on effects of pheno- and/or genotypic resistance on different nominal and numerical intestinal colonization parameters. In addition, we compared the occurrence of antimicrobial resistance phenotypes and genotypes with the occurrence of virulence associated genes typical for extraintestinal pathogenic E. coli. Results In general, up to 72.1% of all E. coli clones were resistant to ampicillin, chloramphenicol, kanamycin, streptomycin, sulfamethoxazole or tetracycline with a variety of different resistance genes involved. There was no significant correlation between one of the nominal or numerical colonization parameters and the absence or presence of antimicrobial resistance properties or resistance genes. However, there were several statistically significant associations between the occurrence of single resistance genes and single virulence associated genes. Conclusion The demonstrated resistance to the tested antibiotics might not play a dominant role for an intestinal colonization success in pigs in the absence of antimicrobial drugs, or cross-selection of other colonization factors e.g. virulence associated genes might compensate "the cost of antibiotic resistance". Nevertheless, resistant strains are not outcompeted by susceptible bacteria in the porcine intestine. Trial Registration The study was approved by the local animal welfare committee of the "Landesamt für Arbeitsschutz, Gesundheitsschutz und technische Sicherheit" Berlin, Germany (No. G0037/02).

Among coagulase-positive staphylococci of animal origin, the members of the Staphylococcus intermedius-group (SIG: S. intermedius, Staphylococcus pseudintermedius and Staphylococcus delphini ) are important opportunistic pathogens in different animal hosts and occasionally in humans. However, the unambiguous species diagnosis of SIG is often challenging. Therefore, matrix-assisted laser desorption ionization—time of flight mass spectrometry (MALDI-TOF MS) -based SIG-identification with Bruker Microflex LT in combination with Biotyper 3.0 software (Bruker Daltonics, Bremen, Germany) was evaluated using (i) the original database content and (ii) the database after extension with distinct hierarchical clustered reference spectra for 60 SIG. A convenience sample comprising 200 isolates was used to compare both database performances. As a result, 17 isolates initially diagnosed as S. intermedius with the current content of the Bruker database were identified as S. pseudintermedius by applying the in-house reference spectra extended version. Furthermore, a significant improvement (average rise of log score value: 0.24) of the SIG identification score values was achieved, emphasizing that further sequence-based refinement of the Bruker database content allows improvement of MALDI-TOF MS-based identification.

Streptococcus canis is an animal pathogen that occasionally causes human infections. Isolates recovered from infections of animals ( n = 78, recovered from 2000 to 2010 in three European countries, mainly from house pets) and humans ( n = 7, recovered from 2006 to 2010 in Portugal) were identified by phenotypic and genotypic methods and characterized by antimicrobial susceptibility testing, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and emm typing. S. canis isolates presented considerable variability in biochemical profiles and 16S rRNA. Resistance to antimicrobial agents was low, with the most significant being tet (M)- and tet (O)-mediated tetracycline resistance. MLST analysis revealed a polyclonal structure of the S. canis population causing infections, where the same genetic lineages were found infecting house pets and humans and were disseminated in distinct geographic locations. Phylogenetic analysis indicated that S. canis was a divergent taxon of the sister species Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis and found evidence of acquisition of genetic material by S. canis from S. dysgalactiae subsp. equisimilis . PFGE confirmed the MLST findings, further strengthening the similarity between animal and human isolates. The presence of emm -like genes was restricted to a few isolates and correlated with some MLST-based genetic lineages, but none of the human isolates could be emm typed. Our data show that S. canis isolates recovered from house pets and humans constitute a single population and demonstrate that isolates belonging to the main genetic lineages identified have the ability to infect the human host, providing strong evidence for the zoonotic nature of S. canis infection.

The possible zoonotic spread of antimicrobial-resistant bacteria is controversial. This review discusses global molecular epidemiological data combining both analyses of the chromosomal background, using multilocus sequence typing (MLST), and analyses of plasmid (episomal) extended-spectrum β-lactamase (ESBL)/AmpC genes in Escherichia coli present in humans and animals. For consideration of major epidemiological differences, animals were separated into livestock and companion animals. MLST revealed the existence of ESBL-producing isolates thoughout the E. coli population, with no obvious association with any ancestral EcoR group. A similar distribution of major ESBL/AmpC types was apparent only in human isolates, regardless of their geographical origin from Europe, Asia, or the Americas, whereas in animals this varied extensively between animal groups and across different geographical areas. In contrast to the diversity of episomal ESBL/AmpC types, isolates from human and animals mainly shared identical sequence types (STs), suggesting transmission or parallel micro-evolution. In conclusion, the opinion that animal ESBL-producing E. coli is a major source of human infections is oversimplified, and neglects a highly complex scenario.

We have isolated Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from the feces of feral pigeons which contained a new Stx2 variant gene designated stx 2f . This gene is most similar to sltIIva of patient E. coli O128:B12 isolate H.I.8. Stx2f reacted only weakly with commercial immunoassays. The prevalence of STEC organisms carrying the stx 2f gene in pigeon droppings was 12.5%. The occurrence of a new Stx2 variant in STEC from pigeons enlarges the pool of Stx2 variants and raises the question whether horizontal gene transfer to E. coli pathogenic to humans may occur.

The 1-kb BamHI-SalI fragment from plasmid pMAR2 termed the enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) probe was cloned in pUC19 and pK18. The nucleotide sequence of this fragment was determined, and a set of primers was designed to amplify a 397-bp region associated with pMAR2 by PCR. An analysis of the whole EAF sequence with database libraries indicated no significant homology to any known genes. However, between bases 701 and 787 of the fragment, an 82.8% homology between the EAF and the insertion sequence IS630 of Shigella sonnei exists. The results of PCR with primers of the EAF sequence demonstrated that all of the 151 EAF probe-positive EPEC strains with localized adherence to HEp-2 cells yielded positive EAF PCR results. In contrast, none of the 277 EAF probe-negative strains reacted to the EAF PCR. In addition, the PCR assay was successfully used to generate vector-free digoxigenin-labeled EAF fragments that gave valid results in colony blot hybridization assays. The EAF PCR appears to be a specific and efficient method for the detection of EPEC strains carrying the EAF plasmids.

Out of 174 bovine Shiga toxin-producing Escherichia coli (STEC) strains isolated from diarrheic calves in Germany and Belgium, 122 strains (70.1%) were selected because of their reactivity with the eae (E. coli attaching and effacing gene) probe ECW1-ECW2. One hundred seven of these eae-positive strains (87.7%) harbored stx1 genes, 13 strains (10.7%) had stx2 genes, and 2 strains (1.6%) had both stx genes. The strains displayed 17 different O types, the majority (97 strains) [79.5%]) belonging to O5 (5 strains), O26 (21 strains), O111 (13 strains) O118 (36 strains), O145 (9 strains), and O157 (13 strains). In the HEp-2 cell adhesion assay, 99 strains (81.1%) showed a localized adhesion, and 80 strains (65.6%) stimulated actin accumulation, as determined in the fluorescence actin staining test. None of the strains harbored genes coding for bundle-forming pili (bfpA), clearly differentiating them from enteropathogenic. E. cole. espB gene sequences were only detectable in 23 (18.9%) of the eae-positive bovine STEC strains. Three different PCRs were established, differentiating between eae sequences of enteropathogenic E. coli strain E2348/69 (O127:H6) and STEC strain EDL933 (O157: H7). Primers matching in the more heterologous downstream eae sequences gave amplicons in only 8 of the 17 O types (O84:H-, O103:H2, O111:H-, O111:H2, O119:H25, O128:H-, O145:H28, and O157:H-). Only 15 STEC strains, belonging to serotypes O111H:-, O111H:2, O145:H28, and O157:H-, gave amplicons in all three eae-specific PCRs. These data demonstrate that bovine STEC strains are a heterogeneous group of pathogenic bacteria, a lot of which share virulence markers with STEC strains causing infections in humans. However, in contrast to human STEC strains, bovine eae-positive STEC strains are mainly restricted to the stx1 genotype. The observation that espB sequences are not highly conserved might have consequences for the serological recognition of the ESPB protein in patients. Like in human STEC strains, eae-related sequences are closely associated with certain E. coli O groups; however, they are not serotype specific.