Anti-Shiga toxin 2 antibodies in enterohemorrhagic Escherichia coli O104:H4 infected patients may predict hemolytic uremic syndrome.

BACKGROUND AND AIM: An outbreak of Shiga toxin 2 (Stx2) producing enterohemorrhagic and enteroaggregative Escherichia coli O104:H4 infection in May 2011 in Germany caused enterocolitis and an unprecedented high 22% rate of hemolytic uremic syndrome (HUS). We hypothesized that anti-Stx2 IgM or IgG titers might predict HUS development. METHODS: Thirty-two patients infected with enterohemorrhagic Escherichia coli O104:H4 (HUS: n = 23; non-HUS: n = 9) were retrospectively screened for anti-Stx2 IgM/IgG and matched with clinical data regarding HUS development, fever, superinfection, dialysis, neurological symptoms, intensive care, antibiotic treatment, and plasmapheresis. RESULTS: Only HUS patients showed a prominent Stx2-specific humoral response in the early acute phase. Despite a strong trend towards prediction of HUS development, statistical analysis revealed no significant correlation between high IgM/IgG titers and further key clinical parameters such as fever, superinfection, neurological symptoms, antibiotic treatment, and plasmapheresis. CONCLUSIONS: Anti-Stx2 antibodies seem to accompany or even precede HUS development.

Heteropathogenic virulence and phylogeny reveal phased pathogenic metamorphosis in Escherichia coli O2:H6.

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.

Mobilisation and remobilisation of a large archetypal pathogenicity island of uropathogenic Escherichia coli in vitro support the role of conjugation for horizontal transfer of genomic islands.

BACKGROUND: A substantial amount of data has been accumulated supporting the important role of genomic islands (GEIs)--including pathogenicity islands (PAIs)--in bacterial genome plasticity and the evolution of bacterial pathogens. Their instability and the high level sequence similarity of different (partial) islands suggest an exchange of PAIs between strains of the same or even different bacterial species by horizontal gene transfer (HGT). Transfer events of archetypal large genomic islands of enterobacteria which often lack genes required for mobilisation or transfer have been rarely investigated so far. RESULTS: To study mobilisation of such large genomic regions in prototypic uropathogenic E. coli (UPEC) strain 536, PAI II536 was supplemented with the mobRP4 region, an origin of replication (oriVR6K), an origin of transfer (oriTRP4) and a chloramphenicol resistance selection marker. In the presence of helper plasmid RP4, conjugative transfer of the 107-kb PAI II536 construct occured from strain 536 into an E. coli K-12 recipient. In transconjugants, PAI II536 existed either as a cytoplasmic circular intermediate (CI) or integrated site-specifically into the recipient's chromosome at the leuX tRNA gene. This locus is the chromosomal integration site of PAI II536 in UPEC strain 536. From the E. coli K-12 recipient, the chromosomal PAI II536 construct as well as the CIs could be successfully remobilised and inserted into leuX in a PAI II536 deletion mutant of E. coli 536. CONCLUSIONS: Our results corroborate that mobilisation and conjugal transfer may contribute to evolution of bacterial pathogens through horizontal transfer of large chromosomal regions such as PAIs. Stabilisation of these mobile genetic elements in the bacterial chromosome result from selective loss of mobilisation and transfer functions of genomic islands.

Chromosomal instability in enterohaemorrhagic Escherichia coli O157:H7: impact on adherence, tellurite resistance and colony phenotype.

Tellurite (Tel) resistant enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a global pathogen. In strain EDL933 Tel resistance (Tel(R) ) is encoded by duplicate ter cluster in O islands (OI) 43 and 48, which also harbour iha, encoding the adhesin and siderophore receptor Iha. We identified five EHEC O157:H7 strains that differentiate into large (L) colonies and small (S) colonies with high and low Tel minimal inhibitory concentrations (MICs) respectively. S colonies (Tel-MICs ≤ 4 µg ml⁻¹) sustained large internal deletions within the Tel(R) OIs via homologous recombination between IS elements and lost ter and iha. Moreover, complete excision of the islands occurred by site-specific recombination between flanking direct repeats. Complete excision of OI 43 and OI 48 occurred in 1.81 × 10⁻³ and 1.97 × 10⁻4 cells in culture, respectively; internal deletion of OI 48 was more frequent (9.7 × 10⁻¹ cells). Under iron limitation that promotes iha transcription, iha-negative derivatives adhered less well to human intestinal epithelial cells and grew slower than did their iha-positive counterparts. Experiments utilizing iha deletion and complementation mutants identified Iha as the major factor responsible for these phenotypic differences. Spontaneous deletions affecting Tel(R) OIs contribute to EHEC O157 genome plasticity and might impair virulence and/or fitness.

Evolutionary analysis and distribution of type III effector genes in pathogenic Escherichia coli from human, animal and food sources.

Molecular analysis of Shiga toxin-producing Escherichia coli (STEC) from different sources is considered as a major approach to assess their risk potential. However, only limited data are available about the correlation of evolutionary relationship, the presence of major virulence factor genes and the putative risk of an STEC strain for human infection. In this study, we analysed the evolutionary relationship of 136 pathogenic E. coli strains from human, animal and food sources by multi-locus sequence typing (MLST) and molecular subtyping of their Shiga toxin (stx) and intimin (eae) genes. Moreover, the distribution of three type III effector genes, encoded within the locus of enterocyte effacement (LEE), and 16 effector genes, which are encoded outside the LEE, was analysed. One hundred and five strains from different sources harboured 5-15 of the analysed non-LEE-encoded effector genes. In 101 of these strains, the LEE genes eae, map, espF and espG were present simultaneously. Thirty-one isolates deriving mainly from food and patients suffering from haemolytic uraemic syndrome (HUS) were eae-negative and did not carry any of the analysed effector genes. By combination of MLST and virulence gene data, we defined five genetic clusters. Within these clusters a clear-cut affiliation of particular sequence types and the occurrence of certain effector genes was observed. However, in contrast to other studies, a significant correlation between the amount and type of effector genes and the risk to cause HUS could not be demonstrated.

Distribution and phylogeny of immunoglobulin-binding protein G in Shiga toxin-producing Escherichia coli and its association with adherence phenotypes.

eibG in Shiga toxin-producing Escherichia coli (STEC) O91 encodes a protein (EibG) which binds human immunoglobulins G and A and contributes to bacterial chain-like adherence to human epithelial cells. We investigated the prevalence of eibG among STEC, the phylogeny of eibG, and eibG allelic variations and their impact on the adherence phenotype. eibG was found in 15.0% of 240 eae-negative STEC strains but in none of 157 eae-positive STEC strains. The 36 eibG-positive STEC strains belonged to 14 serotypes and to eight multilocus sequence types (STs), with serotype O91:H14/H(-) and ST33 being the most common. Sequences of the complete eibG gene (1,527 bp in size) from eibG-positive STEC resulted in 21 different alleles with 88.11% to 100% identity to the previously reported eibG sequence; they clustered into three eibG subtypes (eibG-alpha, eibG-beta, and eibG-gamma). Strains expressing EibG-alpha and EibG-beta displayed a mostly typical chain-like adherence pattern (CLAP), with formation of long chains on both human and bovine intestinal epithelial cells, whereas strains with EibG-gamma adhered in short chains, a pattern we termed atypical CLAP. The same adherence phenotypes were displayed by E. coli BL21(DE3) clones containing the respective eibG-alpha, eibG-beta, and eibG-gamma subtypes. We propose two possible evolutionary scenarios for eibG in STEC: a clonal development of eibG in strains with the same phylogenetic background or horizontal transfer of eibG between phylogenetically unrelated STEC strains.

Phylogeny and disease association of Shiga toxin-producing Escherichia coli O91.

The diversity and relatedness of 100 Shiga toxin-producing Escherichia coli O91 isolates from different patients were examined by multilocus sequence typing. We identified 10 specific sequence types (ST) and 4 distinct clonal groups. ST442 was significantly associated with hemolytic uremic syndrome.

Analysis of collection of hemolytic uremic syndrome-associated enterohemorrhagic Escherichia coli.

Multilocus sequence typing of 169 non-O157 enterohemorrhagic Escherichia coli (EHEC) isolated from patients with hemolytic uremic syndrome (HUS) demonstrated 29 different sequence types (STs); 78.1% of these strains clustered in 5 STs. From all STs and serotypes identified, we established a reference panel of EHEC associated with HUS (HUSEC collection).

Shiga toxin-negative attaching and effacing Escherichia coli: distinct clinical associations with bacterial phylogeny and virulence traits and inferred in-host pathogen evolution.

BACKGROUND: Attaching and effacing Escherichia coli (AEEC) that lack Shiga toxin genes (stx) and the enteropathogenic E. coli adherence factor (EAF) plasmid (stx-/EAF-) are classified as atypical enteropathogenic E. coli and cause diarrhea worldwide. However, it is unknown whether there are bacterial lineage-specific human disease phenotypes. We compared stx-/EAF- AEEC recovered from patients (mostly children) with bloody and nonbloody diarrhea. METHODS: stx-/EAF- AEEC were isolated using eae colony blot hybridization and were serotyped, tested (by polymerase chain reaction) for putative virulence genes, and analyzed for phylogenetic relationships by use of multilocus sequence typing. RESULTS: During the period 1995-2007, stx-/EAF- AEEC were isolated as the only bacterial pathogens from stool specimens obtained from 18 (15.3%) of 118 patients with bloody diarrhea and from 141 (1.3%) of 10,550 patients with nonbloody diarrhea (P<.001). All but 1 of 18 strains recovered from patients with bloody diarrhea resembled enterohemorrhagic E. coli (EHEC) on the basis of serotypes, non-stx virulence profiles, and multilocus sequence types. In contrast, most (75.9%) of 141 stx-/EAF- AEEC recovered from patients with nonbloody diarrhea belonged to other serotypes and differed from the former strains phylogenetically and with regard to virulence genes. Three of 18 patients with bloody diarrhea and none of 141 patients with nonbloody diarrhea who shedded stx-/EAF- AEEC developed hemolytic uremic syndrome. CONCLUSIONS: Most stx-/EAF- AEEC associated with bloody diarrhea are plausibly EHEC that lost Shiga toxin during infection (EHEC-LST). To prevent serious complications of such infections, Shiga toxin-independent diagnostic strategies to accurately and rapidly identify such patients should be developed and applied. Multilocus sequence typing has potential to distinguish EHEC-LST from less pathogenic stx-/EAF- AEEC.

Delineation of the recombination sites necessary for integration of pathogenicity islands II and III into the Escherichia coli 536 chromosome.

In uropathogenic Escherichia coli strain 536, six pathogenicity islands (PAIs) encode key virulence factors. All PAIs except PAI IV(536) are flanked by direct repeats and four of them encode integrases responsible for their chromosomal excision. To study recombination sites used for the integration by PAI II(536) and III(536) integrases, we measured site-specific recombination between the chromosomal integration site attB, and the PAI-specific attachment site attP. We show that PAI III(536) IntB, but not IntA, mediates PAI III(536) integration. Studies of integrative recombination sites of both PAIs show that, when using a large cognate attP site (839 bp for PAI II(536) and 268 bp for PAI III(536)), PAI II(536) and III(536) attB sites could be reduced to 16 bp and 20 bp, respectively, without affecting recombination. Further reduction to 14 bp for PAI II(536) and 13 bp for PAI III(536) diminished recombination efficiency. Surprisingly, attP sites could also be reduced to 14 bp (PAI II(536)) and 20 bp (PAI III(536)). The integration host factor (IHF) and the DNA-bending HU protein do not influence PAI II(536) recombination, but IHF enhances PAI-III(536) excision and negatively affects its integration. These data suggest that PAI intasomes differ from those of lambda and P4 integrase paradigms.

Role of pathogenicity island-associated integrases in the genome plasticity of uropathogenic Escherichia coli strain 536.

The genome of uropathogenic Escherichia coli isolate 536 contains five well-characterized pathogenicity islands (PAIs) encoding key virulence factors of this strain. Except PAI IV(536), the four other PAIs of strain 536 are flanked by direct repeats (DRs), carry intact integrase genes and are able to excise site-specifically from the chromosome. Genome screening of strain 536 identified a sixth putative asnW-associated PAI. Despite the presence of DRs and an intact integrase gene, excision of this island was not detected. To investigate the role of PAI-encoded integrases for the recombination process the int genes of each unstable island of strain 536 were inactivated. For PAI I(536) and PAI II(536), their respective P4-like integrase was required for their excision. PAI III(536) carries two integrase genes, intA, encoding an SfX-like integrase, and intB, coding for an integrase with weak similarity to P4-like integrases. Only intB was required for site-specific excision of this island. For PAI V(536), excision could not be abolished after deleting its P4-like integrase gene but additional deletion of the PAI II(536)-specific integrase gene was required. Therefore, although all mediated by P4-like integrases, the activity of the PAI excision machinery is most often restricted to its cognate island. This work also demonstrates for the first time the existence of a cross-talk between integrases of different PAIs and shows that this cross-talk is unidirectional.

Phg, a novel member of the autotransporter family present in Bordetella species.

Several proteins encoded in the genomes of Bordetella species show significant sequence similarity to the autotransporter domains of surface exposed or secreted virulence factors of bordetellae such as pertactin, tracheal colonization factor or Vag8. One of these putative autotransporters, provisionally termed Phg, is encoded by the pertactin homologous gene (phg), which is highly conserved in Bordetella pertussis, B. bronchiseptica and B. parapertussis, but absent in B. avium and B. petrii. In contrast to homologues with documented functions in host interaction and virulence, several key amino acids probably involved in proteolytic processing of the autotransporter domain are not conserved in Phg. The transcription start site of phg was identified by primer extension analysis, but differential transcription of phg could not be detected in B. bronchiseptica strains under conditions that lead to enhanced expression of other known Bordetella autotransporter proteins. A mutant of B. pertussis was constructed in which major parts of phg are substituted by a kanamycin resistance cassette. Virulence testing of this mutant in a mouse respiratory infection model showed the same colonization properties as the wild-type strain.

Pathogenomics of mobile genetic elements of toxigenic bacteria.

The growing knowledge of genetic diversity and whole genome organization in bacteria shows that pathogenicity islands (PAIs) represent a subtype of a more general genetic element, termed genomic island (GEI), which is widespread among pathogenic and non-pathogenic microbes. These findings mirror the importance of horizontal gene transfer, genome reduction and recombination events as fundamental mechanisms involved in evolution of bacterial variants. GEIs are part of the flexible gene pool and carry selfish genes, but also determinants which may be beneficial under certain conditions thus increasing bacterial fitness and consequently their survival or transmission. In this review, we focus on the role of mobile genetic elements that may also contain toxin-encoding genes for genome variability and evolution of bacteria.

Instability of pathogenicity islands in uropathogenic Escherichia coli 536.

The uropathogenic Escherichia coli strain 536 carries at least five genetic elements on its chromosome that meet all criteria characteristic of pathogenicity islands (PAIs). One main feature of these distinct DNA regions is their instability. We applied the so-called island-probing approach and individually labeled all five PAIs of E. coli 536 with the counterselectable marker sacB to evaluate the frequency of PAI-negative colonies under the influence of different environmental conditions. Furthermore, we investigated the boundaries of these PAIs. According to our experiments, PAI II536 and PAI III536 were the most unstable islands followed by PAI I536 and PAI V536, whereas PAI IV536 was stable. In addition, we found that deletion of PAI II536 and PAI III536 was induced by several environmental stimuli. Whereas excision of PAI I536, PAI II536, and PAI V536 was based on site-specific recombination between short direct repeat sequences at their boundaries, PAI III536 was deleted either by site-specific recombination or by homologous recombination between two IS100-specific sequences. In all cases, deletion is thought to lead to the formation of nonreplicative circular intermediates. Such extrachromosomal derivatives of PAI II536 and PAI III536 were detected by a specific PCR assay. Our data indicate that the genome content of uropathogenic E. coli can be modulated by deletion of PAIs.