Suppression subtractive hybridization identifies an autotransporter adhesin gene of E. coli IMT5155 specifically associated with avian pathogenic Escherichia coli (APEC).

BACKGROUND: Extraintestinal pathogenic E. coli (ExPEC) represent a phylogenetically diverse group of bacteria which are implicated in a large range of infections in humans and animals. Although subgroups of different ExPEC pathotypes, including uropathogenic, newborn meningitis causing, and avian pathogenic E. coli (APEC) share a number of virulence features, there still might be factors specifically contributing to the pathogenesis of a certain subset of strains or a distinct pathotype. Thus, we made use of suppression subtractive hybridization and compared APEC strain IMT5155 (O2:K1:H5; sequence type complex 95) with human uropathogenic E. coli strain CFT073 (O6:K2:H5; sequence type complex 73) to identify factors which may complete the currently existing model of APEC pathogenicity and further elucidate the position of this avian pathotype within the whole ExPEC group. RESULTS: Twenty-eight different genomic loci were identified, which are present in IMT5155 but not in CFT073. One of these loci contained a gene encoding a putative autotransporter adhesin. The open reading frame of the gene spans a 3,498 bp region leading to a putative 124-kDa adhesive protein. A specific antibody was raised against this protein and expression of the adhesin was shown under laboratory conditions. Adherence and adherence inhibition assays demonstrated a role for the corresponding protein in adhesion to DF-1 chicken fibroblasts. Sequence analyses revealed that the flanking regions of the chromosomally located gene contained sequences of mobile genetic elements, indicating a probable spread among different strains by horizontal gene transfer. In accordance with this hypothesis, the adhesin was found to be present not only in different phylogenetic groups of extraintestinal pathogenic but also of commensal E. coli strains, yielding a significant association with strains of avian origin. CONCLUSIONS: We identified a chromosomally located autotransporter gene in a highly virulent APEC strain which confers increased adherence of a non-fimbriated E. coli K-12 strain to a chicken fibroblast cell line. Even though flanked by mobile genetic elements and three different genetic regions upstream of the gene, most probably indicating horizontal gene transfer events, the adhesin gene was significantly linked with strains of avian origin. Due to the nucleotide sequence similarity of 98% to a recently published adhesin-related gene, located on plasmid pAPEC-O1-ColBM, the name aatA (APEC autotransporter adhesin A) was adopted from that study.Our data substantiate that AatA might not only be of relevance in APEC pathogenicity but also in facilitating their reservoir life style in the chicken intestine, which might pave the way for future intestinal preventive strategies.

The chicken as a natural model for extraintestinal infections caused by avian pathogenic Escherichia coli (APEC).

E. coli infections in avian species have become an economic threat to the poultry industry worldwide. Several factors have been associated with the virulence of E. coli in avian hosts, but no specific virulence gene has been identified as being entirely responsible for the pathogenicity of avian pathogenic E. coli (APEC). Needless to say, the chicken would serve as the best model organism for unravelling the pathogenic mechanisms of APEC, an extraintestinal pathogen. Five-week-old white leghorn SPF chickens were infected intra-tracheally with a well characterized APEC field strain IMT5155 (O2:K1:H5) using different doses corresponding to the respective models of infection established, that is, the lung colonization model allowing re-isolation of bacteria only from the lung but not from other internal organs, and the systemic infection model. These two models represent the crucial steps in the pathogenesis of APEC infections, including the colonization of the lung epithelium and the spread of bacteria throughout the bloodstream. The read-out system includes a clinical score, pathomorphological changes and bacterial load determination. The lung colonization model has been established and described for the first time in this study, in addition to a comprehensive account of a systemic infection model which enables the study of severe extraintestinal pathogenic E. coli (ExPEC) infections. These in vivo models enable the application of various molecular approaches to study host-pathogen interactions more closely. The most important application of such genetic manipulation techniques is the identification of genes required for extraintestinal virulence, as well as host genes involved in immunity in vivo. The knowledge obtained from these studies serves the dual purpose of shedding light on the nature of virulence itself, as well as providing a route for rational attenuation of the pathogen for vaccine construction, a measure by which extraintestinal infections, including those caused by APEC, could eventually be controlled and prevented in the field.

Characterization of a yjjQ mutant of avian pathogenic Escherichia coli (APEC).

Infections with extraintestinal avian pathogenic Escherichia coli (APEC) cause significant economic losses in the poultry industry worldwide. In a previous study we applied signature-tagged transposon mutagenesis and identified 28 virulence-associated genes in APEC strain IMT5155 (O2 : H5 : K1). One of them, yjjQ, encodes a putative transcriptional regulator whose function and role in pathogenesis are still unknown. In the present study, this mutant has been characterized. The yjjQ-defective mutant of IMT5155 (M18E10) was out-competed by the wild-type strain in vivo, and infection of chickens with this yjjQ mutant led to strongly reduced bacterial loads in several organs. Expression studies showed that transcription of yjjQ was significantly upregulated in M9 minimal medium. Correspondingly, the yjjQ mutant showed significantly reduced growth in M9 medium. Although the mutation could not be complemented, a yjjQ deletion mutant showed phenotypes similar to the transposon-generated mutant M18E10, whereas deletion and overexpression of the downstream gene bglJ did not cause a growth defect in M9. To identify virulence genes regulated by YjjQ, one- and two-dimensional protein gel electrophoresis was performed. The proteomic analysis revealed that in the yjjQ mutant M18E10 the expression of several genes involved in iron uptake was downregulated and some other genes were upregulated. The regulation of genes involved in iron uptake was shown to occur at the transcription level using real-time RT-PCR. Taking the results together, this functional analysis strongly suggests that YjjQ is a regulator involved in virulence of APEC by affecting iron uptake.

Application of Campylobacter molecular classification and typing techniques in veterinary medicine: old-established methods and new perspectives.

In this review the application and usefulness of Campylobacter genotypical classification and typing in veterinary medicine will be discussed.While there is a large area of overlapping applications between the veterinary and the medical field, several differences exist, as the spectrum of veterinary pathogens is different from the human and contaminated food of healthy animal origin may cause disease in man. In general, genotyping in the veterinary field can be applied in three different areas: (a) purely diagnostic purposes for classification of Campylobacter species and subspecies, (b) typing methods useful for monitoring or surveillance of animals as well as food products of animal origin, and (c) typing methods that can be applied during outbreaks and for source tracing. In addition, typing methods applied in areas (b) and (c) should be distinguished in regard to local short-term and global long-term epidemiology, respectively. While a whole plethora of discriminative typing methods are available, classification tools of certain species and subspecies are still missing. Perspectively, as the genomes of many relevant Campylobacter species have now been sequenced, this will help to identify several species specific loci, the products of which should be available to develop easy and fast applicable diagnostic tools. Global cooperation, sharing of strains and databases should close the currently existing gaps in Campylobacter identification tools.

Avian pathogenic, uropathogenic, and newborn meningitis-causing Escherichia coli: how closely related are they?

Avian pathogenic Escherichia coli (APEC), uropathogenic E. coli (UPEC), and newborn meningitis-causing E. coli (NMEC) establish infections in extraintestinal habitats (extraintestinal pathogenic E. coli; ExPEC) of different hosts. As diversity, epidemiological sources, and evolutionary origins of ExPEC are so far only partially defined, we screened a collection of 526 strains of medical and veterinary origin of various O-types for assignment to E. coli reference collection (ECOR) group and virulence gene patterns. Results of ECOR typing confirmed that human ExPEC strains mostly belong to groups B2, followed by group D. Although a considerable portion of APEC strains did also fell into ECOR group B2 (35.1%), a higher amount (46.1%) belonged to group A, which has previously been described to also harbour strains with a high pathogenic potential for humans. The number of virulence-associated genes of single strains ranged from 5 to 26 among 33 genes tested and high numbers were rather related to K1-positive and ECOR B2 strains than to a certain pathotype. With a few exceptions (iha, afa/draB, sfa/foc, and hlyA), which were rarely present in APEC strains, most chromosomally located genes were widely distributed among all ExPEC strains irrespective of host and pathotype. However, prevalence of invasion genes (ibeA and gimB) and K1 capsule-encoding gene neuC indicated a closer relationship between APEC and NMEC strains. Genes associated with ColV plasmids (tsh, iss, and the episomal sit locus) were in general more prevalent in APEC than in UPEC and NMEC strains, indicating that APEC could be a source of ColV-located genes or complete plasmids for other ExPEC strains. Our data support the hypothesis that (a) poultry may be a vehicle or even a reservoir for human ExPEC strains, (b) APEC potentially serve as a reservoir of virulence-associated genes for UPEC and NMEC, (c) some ExPEC strains, although of different pathotypes, may share common ancestors, and (d) as a conclusion certain APEC subgroups have to be considered potential zoonotic agents. The finding of different evolutionary clusters within these three pathotypes implicates an independently and parallel evolution, which should be resolved in the future by thorough phylogenetic typing.

Identification of genes required for avian Escherichia coli septicemia by signature-tagged mutagenesis.

Infections with avian pathogenic Escherichia coli (APEC) cause colibacillosis, an acute and largely systemic disease resulting in significant economic losses in poultry industry worldwide. Although various virulence-associated genes have been identified in APEC, their actual role in pathogenesis is still not fully understood, and, furthermore, certain steps of the infection process have not been related to previously identified factors. Here we describe the application of a signature-tagged transposon mutagenesis (STM) approach to identify critical genes required for APEC infections in vivo. Twenty pools of about 1,800 IMT5155 (O2:H5) mutants were screened in an infection model using 5-week-old chickens, and potentially attenuated mutants were subjected to a secondary screen and in vivo competition assays to confirm their attenuation. A total of 28 genes required for E. coli septicemia in chickens were identified as candidates for further characterization. Among these disrupted genes, six encode proteins involved in biosynthesis of extracellular polysaccharides and lipopolysaccharides; two encode iron transporters that have not been previously characterized in APEC in in vivo studies, and four showed similarity to membrane or periplasmic proteins. In addition, several metabolic enzymes, putative proteins with unknown function, and open reading frames with no similarity to other database entries were identified. This genome-wide analysis has identified both novel and previously known factors potentially involved in pathogenesis of APEC infection.

Long-term clonal lineages within Campylobacter jejuni O:2 strains from different geographical regions and hosts.

The genomic diversity of 11 epidemiologically unrelated strains of Campylobacter jejuni (serotype O:2) isolated from different hosts and different geographical regions in Germany over a period of 15 years was studied and results were compared with the reference strain NCTC11168. By flagellin PCR-RFLP typing six fla types were identified, while macrorestriction analysis with three different restriction enzymes revealed almost identical patterns for two human and one bovine strain, even though they were isolated between 1984 and 1996. Interestingly, the PFGE and fla profiles from strain NCTC11168, which was originally isolated in 1977 from an outbreak case in Worcester (UK), were highly similar or even identical to the profiles of these strains. Besides this, mapping of selective genetic markers to the obtained restriction fragments by Southern blot hybridization showed a conserved localization of the investigated sequences in several strains. Our data confirmed considerable degrees of genomic conservation and the occurrence of long-term O:2 serotype-associated clonal lineages in C. jejuni in different geographical regions and hosts.

Description of a 111-kb pathogenicity island (PAI) encoding various virulence features in the enterohemorrhagic E. coli (EHEC) strain RW1374 (O103:H2) and detection of a similar PAI in other EHEC strains of serotype 0103:H2.

Human infections with enterohemorrhagic E. coli (EHEC) strains of serotype O103:H2 are of increasing importance in Germany. As bovines are the principal EHEC reservoir behind the occurrence of human infections, we analyzed a pathogenicity island (PAI I(RW1374)) of bovine O103:H2 strain RW1374 to identify putative virulence features. This PAI I(RW1374) harbors a functional 34-kb locus of enterocyte effacement (LEE) core region and has a total length of 111 kb. About 43 kb upstream of the LEE core a gene cassette consisting of efa1/lifA gene and flanking IS elements suggests another putative transposon within the PAI(IRW1374). In addition, the ent gene, encoding a Shigella ShET-2 enterotoxin homologue, is present about 57 kb upstream of the LEE core. This PAI is therefore a complex assembly of various virulence determinants including the efa1/lifA and the ent gene resembling O157:H7 PAI OI-122/SpLE3 as well as the LEE core region. An integrase gene on the very left end of PAI I(Rw1374) is disrupted by an IS629 homologue. In an attempt to mobilize the LEE core we performed conjugation, transformation and transduction experiments. We were, however, unable to mobilize the whole or even single regions of PAI I(RW1374). Comparative studies with other strains of serotype O103:H2 isolated from humans, bovines and food showed that they all harbored a similar phe V-inserted PAI including the virulence genes ent and lifA/efa1 as well as the large virulence-associated plasmid encoding the EHEC hemolysin. This combination of several virulence factors confirms the complex virulence of O103:H2 EHEC and may at least partly explain the high virulence of this EHEC serotype in humans.