Isolation of recombinant antibodies against EspA and intimin of Escherichia coli O157:H7

Intimin, Tir, and EspA proteins are expressed by attaching-effacing Escherichia coli, which include enteropathogenic and enterohemorrhagic E. coli pathotypes. EspA proteins are part of the type three secretion system needle complex that delivers Tir to the host epithelial cell, while surface arrayed intimin docks the bacterium to the translocated Tir. This intimate attachment leads to attaching and effacing lesions. Recombinant forms of these effector proteins from enterohemorrhagic E. coli O157:H7 were produced by using E. coli expression vectors. Binding of intimin and Tir fragments in enzyme-linked immunosorbent assay (ELISAs) demonstrated the interaction of intimin fragments containing the C-terminal 282 or 188 amino acids to a Tir fragment containing amino acid residues 258 to 361. Recombinant intimin and EspA proteins were used to elicit immune responses in rabbits and immune phage-display antibody libraries were produced. Screening of these immune libraries by conventional phage-antibody panning and colony filter screening produced a panel of antibodies with specificity for EspA or intimin. Antibodies recognizing different C-terminal epitopes on intimin bound specifically to the gamma intimin of O157:H7 and not to other classes of intimin. Antibodies recognizing EspA from E. coli O157 also recognized the protein from the eae-deficient O157 mutant DM3 and from E. coli O111. Anti-intimin antibodies were also produced as fusion proteins coupled to the reporter molecule alkaline phosphatase, allowing the one-step detection of gamma intimin. The isolated recombinant monoclonal antibodies were functional in a range of assay formats, including ELISA, Western blotting, and dot blots, thus demonstrating their diagnostic potential.

Antibiotic resistance genes, integrons and multiple antibiotic resistance in thirty-five serotypes of Salmonella enterica isolated from humans and animals in the UK

Objectives: To examine 397 strains of Salmonella enterica of human and animal origin comprising 35 serotypes for the presence of aadB, aphAI-IAB, aadA1, aadA2, bla(Carb(2)) or pse1, bla(Tem), cat1, cat2, dhfr1, floR, strA, sul1, sul2, tetA(A), tetA(B) and tetA(G) genes, the presence of class 1 integrons and the relationship of resistance genes to integrons and antibiotic resistance. Results: Some strains were resistant to ampicillin (91), chloramphenicol (85), gentamicin (2), kanamycin (14), spectinomycin (81), streptomycin (119), sulfadiazine (127), tetracycline (108) and trimethoprim (45); 219 strains were susceptible to all antibiotics. bla(Carb(2)), floR and tetA(G) genes were found in S. Typhimurium isolates and one strain of S. Emek only. Class 1 integrons were found in S. Emek, Haifa, Heidelberg, Mbandaka, Newport, Ohio, Stanley, Virchow and in Typhimurium, mainly phage types DT104 and U302. These strains were generally multi-resistant to up to seven antibiotics. Resistance to between three and six antibiotics was also associated with class 1 integron-negative strains of S. Binza, Dublin, Enteritidis, Hadar, Manhattan, Mbandaka, Montevideo, Newport, Typhimurium DT193 and Virchow. Conclusion: The results illustrate specificity of some resistance genes to S. Typhimurium or non- S. Typhimurium serotypes and the involvement of both class 1 integron and non-class 1 integron associated multi-resistance in several serotypes. These data also indicate that the bla(Carb(2)), floR and tetA(G) genes reported in the SG1 region of S. Typhimurium DT104, U302 and some other serotypes are still predominantly limited to S. Typhimurium strains.

Identification of core and variable components of the Salmonella enterica subspecies I genome by microarray

We have performed microarray hybridization studies on 40 clinical isolates from 12 common serovars within Salmonella enterica subspecies I to identify the conserved chromosomal gene pool. We were able to separate the core invariant portion of the genome by a novel mathematical approach using a decision tree based on genes ranked by increasing variance. All genes within the core component were confirmed using available sequence and microarray information for S. enterica subspecies I strains. The majority of genes within the core component had conserved homologues in Escherichia coli K-12 strain MG1655. However, many genes present in the conserved set which were absent or highly divergent in K-12 had close homologues in pathogenic bacteria such as Shigella flexneri and Pseudomonas aeruginosa. Genes within previously established virulence determinants such as SPI1 to SPI5 were conserved. In addition several genes within SPI6, all of SPI9, and three fimbrial operons (fim, bcf, and stb) were conserved within all S. enterica strains included in this study. Although many phage and insertion sequence elements were missing from the core component, approximately half the pseudogenes present in S. enterica serovar Typhi were conserved. Furthermore, approximately half the genes conserved in the core set encoded hypothetical proteins. Separation of the core and variant gene sets within S. enterica subspecies I has offered fundamental biological insight into the genetic basis of phenotypic similarity and diversity across S. enterica subspecies I and shown how the core genome of these pathogens differs from the closely related E. coli K-12 laboratory strain.

Attaching-effacing bacteria in animals

Enteric bacteria with a demonstrable or potential ability to form attaching-effacing lesions, so-called attaching-effacing (AE) bacteria, have been found in the intestinal tracts of a wide variety of warm-blooded animal species, including man. In some host species, for example cattle, pigs, rabbits and human beings, attaching-effacing Escherichia coli (AEEC) have an established role as enteropathogens. In other host species, AE bacteria are of less certain significance. With continuing advances in the detection and typing of AE strains, the importance of these bacteria for many hosts is likely to become clearer. The pathogenic effects of AE bacteria result from adhesion to the intestinal mucosa by a variety of mechanisms, culminating in the formation of the characteristic intimate adhesion of the AE lesion. The ability to induce AE lesions is mediated by the co-ordinated expression of some 40 bacterial genes organized within a so-called pathogenicity island, known as the "Locus for Enterocyte Effacement". It is also believed that the production of bacterial toxins, principally Vero toxins, is a significant virulence factor for some A-EEC strains. Recent areas of research into AE bacteria include: the use of Citrobacter rodentium to model human AEEC disease; quorum-sensing mechanisms used by AEEC to modulate virulence gene expression; and the potential role of adhesion in the persistent colonization of the intestine by AE bacteria. This review of AE bacteria covers their molecular biology, their occurrence in various animal species, and the diagnosis, pathology and clinical aspects of animal diseases with which they are associated. Reference is made to human pathogens where appropriate. The focus is mainly on natural colonization and disease, but complementary experimental data are also included. (C) 2004 Elsevier Ltd. All rights reserved.

Use of miniaturized protein arrays for Escherichia coli O serotyping

Serological typing of Escherichia coli O antigens is a well-established method used for differentiation and identification of O serotypes commonly associated with disease. In this feasibility study, we have developed a novel somatic antibody-based miniaturized microarray chip, using 17 antisera, which can be used to detect bound whole-cell E. coli antigen with its corresponding immobilized antibody, to assess the feasibility of this approach. The chip was tested using the related 17 control strains, and the O types found by the microarray chip showed 100% correlation with the O types found by conventional typing. A blind trial was performed in which 100 E. coli isolates that had been O serotyped previously by the conventional assay were tested by the array approach. Overall, the O serotypes of 88% of isolates were correctly identified by the microarray method. For several isolates, ambiguity of O-type designation by microarray arose due to increased sensitivity of this method, allowing signal intensities of cross-reactions to be quantified. Investigation of discrepancies between conventional and microarray O serotyping indicated that some isolates upon storage had become untypeable and, therefore, gave poor signal intensity when tested by the microarray or retested by conventional means. For all 20 serotype O26 and O157 isolates, the apparent discrepancy in O serotyping was analyzed further by a third independent test, which confirmed the microarray results. Therefore, the use of miniaturized protein arrays increases the speed and efficiency of O serotyping in a cost-effective manner, and these preliminary findings suggest the microarray approach may have a higher accuracy than those of traditional O-serotyping methods.

Sources and spread of thermophilic Campylobacter spp. during partial depopulation of broiler chicken flocks

The practice of partial depopulation or ‘thinning’, i.e. early removal of a proportion of birds from a commercial broiler flock, is a reported risk factor for Campylobacter colonization of residual birds because of the difficulty in maintaining biosecurity during the process. Therefore, the effect of this practice was studied in detail for 51 target flocks, each at a different growing farm belonging to one of seven major poultry companies throughout the United Kingdom. On 21 of these farms, the target flock was already colonized by Campylobacter and at slaughter all cecal samples examined were positive, with a mean of log10 8 cfu / g. A further 27 flocks became positive within 2 – 6 days of the start of thinning and had similarly high levels of cecal carriage at slaughter. Just prior to the thinning process, Campylobacter could be isolated frequently from the farm driveways, transport vehicles, equipment and personnel. Strains from seven such farms on which flocks became colonized after thinning were examined by PFGE typing. The study demonstrated an association between strains occurring at specific sampling sites and those isolated subsequently from the thinned flocks. There were also indications that particular strains had spread from one farm to another, when the farms were jointly company-owned and served by the same bird-catching teams and / or vehicles. The results highlighted the need for better hygiene control in relation to catching equipment and personnel, and more effective cleaning and disinfection of vehicles, and bird-transport crates.

Hepatitis C virus NS5A inhibits mixed lineage kinase 3 to block apoptosis

Hepatitis C virus (HCV) infection results in the activation of numerous stress responses including oxidative stress, with the potential to induce an apoptotic state. Previously we have shown that HCV attenuates the stress-induced, p38MAPK-mediated up-regulation of the K+ channel Kv2.1, to maintain the survival of infected cells in the face of cellular stress. We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1. In this study, we investigated the host cell proteins targeted by NS5A in order to mediate Kv2.1 inhibition. We screened a phage-display library expressing the entire complement of human SH3 domains for novel NS5A-host cell interactions. This analysis identified mixed lineage kinase 3 (MLK3) as a putative NS5A interacting partner. MLK3 is a serine/threonine protein kinase that is a member of the MAPK kinase kinase (MAP3K) family and activates p38MAPK. An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and western blot analysis. We further demonstrate a novel role of MLK3 in the modulation of Kv2.1 activity, whereby MLK3 overexpression leads to the up-regulation of channel activity. Accordingly, coexpression of NS5A suppressed this stimulation. Additionally we demonstrate that overexpression of MLK3 induced apoptosis which was also counteracted by NS5A. We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K+ homeostasis.

Comparative genomics of Salmonella enterica serovars Derby and Mbandaka, two prevalent serovars associated with different livestock species in the UK

Background Despite the frequent isolation of Salmonella enterica sub. enterica serovars Derby and Mbandaka from livestock in the UK and USA little is known about the biological processes maintaining their prevalence. Statistics for Salmonella isolations from livestock production in the UK show that S. Derby is most commonly associated with pigs and turkeys and S. Mbandaka with cattle and chickens. Here we compare the first sequenced genomes of S. Derby and S. Mbandaka as a basis for further analysis of the potential host adaptations that contribute to their distinct host species distributions. Results Comparative functional genomics using the RAST annotation system showed that predominantly mechanisms that relate to metabolite utilisation, in vivo and ex vivo persistence and pathogenesis distinguish S. Derby from S. Mbandaka. Alignment of the genome nucleotide sequences of S. Derby D1 and D2 and S. Mbandaka M1 and M2 with Salmonella pathogenicity islands (SPI) identified unique complements of genes associated with host adaptation. We also describe a new genomic island with a putative role in pathogenesis, SPI-23. SPI-23 is present in several S. enterica serovars, including S. Agona, S. Dublin and S. Gallinarum, it is absent in its entirety from S. Mbandaka. Conclusions We discovered a new 37 Kb genomic island, SPI-23, in the chromosome sequence of S. Derby, encoding 42 ORFS, ten of which are putative TTSS effector proteins. We infer from full-genome synonymous SNP analysis that these two serovars diverged, between 182kya and 625kya coinciding with the divergence of domestic pigs. The differences between the genomes of these serovars suggest they have been exposed to different stresses including, phage, transposons and prolonged externalisation. The two serovars possess distinct complements of metabolic genes; many of which cluster into pathways for catabolism of carbon sources.

Comparative Analysis of ESBL-Positive Escherichia coli isolates from Animals and Humans from the UK, the Netherlands and Germany

The putative virulence and antimicrobial resistance gene contents of extended spectrum β-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the suitability of this approach with regard to determining their similarities. A selection of isolates (n=313) were also analysed by multilocus sequence typing (MLST). Isolates harbouring blaCTX-M-group-1 dominated (66%, n=418) and originated from both animals and cases of human infections in all three countries; 23% (n=144) of all isolates contained both blaCTX-M-group-1 and blaOXA-1-like genes, predominantly from humans (n=127) and UK cattle (n=15). The antimicrobial resistance and virulence gene profiles of this collection of isolates were highly diverse. A substantial number of human isolates (32%, n=87) did not share more than 40% similarity (based on the Jaccard coefficient) with animal isolates. A further 43% of human isolates from the three countries (n=117) were at least 40% similar to each other and to five isolates from UK cattle and one each from Dutch chicken meat and a German dog; the members of this group usually harboured genes such as mph(A), mrx, aac(6’)-Ib, catB3, blaOXA-1-like and blaCTX-M-group-1. forty-four per cent of the MLST-typed isolates in this group belonged to ST131 (n=18) and 22% to ST405 (n=9), all from humans. Among animal isolates subjected to MLST (n=258), only 1.2% (n=3) were more than 70% similar to human isolates in gene profiles and shared the same MLST clonal complex with the corresponding human isolates. The results suggest that minimising human-to-human transmission is essential to control the spread of ESBL-positive E. coli in humans.

Bacterial motility confers fitness advantage in the presence of phages

Dispersal provides the opportunity to escape harm and colonize new patches, enabling populations to expand and persist. However, the benefits of dispersal associated with escaping harm will be dependent on the structure of the environment and the likelihood of escape. Here, we empirically investigate how the spatial distribution of a parasite influences the evolution of host dispersal. Bacteriophages are a strong and common threat for bacteria in natural environments and offer a good system with which to explore parasite-mediated selection on host dispersal. We used two transposon mutants of the opportunistic bacteria, Pseudomonas aeruginosa, which varied in their motility (a disperser and a nondisperser), and the lytic bacteriophage ФKZ. The phage was distributed either in the central point of colony inoculation only, thus offering an escape route for the dispersing bacteria; or, present throughout the agar, where benefits of dispersal might be lost. Surprisingly, we found dispersal to be equally advantageous under both phage conditions relative to when phages were absent. A general explanation is that dispersal decreased the spatial structuring of host population, reducing opportunities for parasite transmission, but other more idiosyncratic mechanisms may also have contributed. This study highlights the crucial role the parasites can play on the evolution of dispersal and, more specifically, that bacteriophages, which are ubiquitous, are likely to select for bacterial motility.

Using experimental evolution to explore natural patterns between bacterial motility and resistance to bacteriophages

Resistance of bacteria to phages may be gained by alteration of surface proteins to which phages bind, a mechanism that is likely to be costly as these molecules typically have critical functions such as movement or nutrient uptake. To address this potential trade-off, we combine a systematic study of natural bacteria and phage populations with an experimental evolution approach. We compare motility, growth rate and susceptibility to local phages for 80 bacteria isolated from horse chestnut leaves and, contrary to expectation, find no negative association between resistance to phages and bacterial motility or growth rate. However, because correlational patterns (and their absence) are open to numerous interpretations, we test for any causal association between resistance to phages and bacterial motility using experimental evolution of a subset of bacteria in both the presence and absence of naturally associated phages. Again, we find no clear link between the acquisition of resistance and bacterial motility, suggesting that for these natural bacterial populations, phage-mediated selection is unlikely to shape bacterial motility, a key fitness trait for many bacteria in the phyllosphere. The agreement between the observed natural pattern and the experimental evolution results presented here demonstrates the power of this combined approach for testing evolutionary trade-offs.

Klebsiella pneumoniae subsp. pneumoniae–bacteriophage combination from the caecal effluent of a healthy woman

A sample of caecal effluent was obtained from a female patient who had undergone a routine colonoscopic examination. Bacteria were isolated anaerobically from the sample, and screened against the remaining filtered caecal effluent in an attempt to isolate bacteriophages (phages). A lytic phage, named KLPN1, was isolated on a strain identified as Klebsiella pneumoniae subsp. pneumoniae (capsular type K2, rmpA+). This Siphoviridae phage presents a rosette-like tail tip and exhibits depolymerase activity, as demonstrated by the formation of plaque-surrounding haloes that increased in size over the course of incubation. When screened against a panel of clinical isolates of K. pneumoniae subsp. pneumoniae, phage KLPN1 was shown to infect and lyse capsular type K2 strains, though it did not exhibit depolymerase activity on such hosts. The genome of KLPN1 was determined to be 49,037 bp (50.53 %GC) in length, encompassing 73 predicted ORFs, of which 23 represented genes associated with structure, host recognition, packaging, DNA replication and cell lysis. On the basis of sequence analyses, phages KLPN1 (GenBank: KR262148) and 1513 (a member of the family Siphoviridae, GenBank: KP658157) were found to be two new members of the genus “Kp36likevirus”.

Diversity of STs, plasmids and ESBL genes among Escherichia coli from humans, animals and food in Germany, the Netherlands and the UK

This study aimed to compare ESBL-producing Escherichia coli causing infections in humans with infecting or commensal isolates from animals and isolates from food of animal origin in terms of the strain types, the ESBL gene present and the plasmids that carry the respective ESBL genes. A collection of 353 ESBL-positive E. coli isolates from the UK, the Netherlands and Germany were studied by MLST and ESBL genes were identified. Characterization of ESBL gene-carrying plasmids was performed using PCR-based replicon typing. Moreover, IncI1-Iγ and IncN plasmids were characterized by plasmid MLST. The ESBL-producing E. coli represented 158 different STs with ST131, ST10 and ST88 being the most common. Overall, blaCTX-M-1 was the most frequently detected ESBL gene, followed by blaCTX-M-15, which was the most common ESBL gene in the human isolates. The most common plasmid replicon type overall was IncI1-Iγ followed by multiple IncF replicons. ESBL genes were present in a wide variety of E. coli STs. IncI1-Iγ plasmids that carried the blaCTX-M-1 gene were widely disseminated amongst STs in isolates from animals and humans, whereas other plasmids and STs appeared to be more restricted to isolates from specific hosts.

Regional atmospheric circulation over Europe during the Last Glacial Maximum and its links to precipitation

The Last Glacial Maximum (LGM) exhibits different large-scale atmospheric conditions compared to present-day climate due to altered boundary conditions. The regional atmospheric circulation and associated precipitation patterns over Europe are characterized for the first time with a weather typing approach (circulation weather types, CWT) for LGM paleoclimate simulations. The CWT approach is applied to four representative regions across Europe. While the CWTs over Western Europe are prevailing westerly for both present-day and LGM conditions, considerable differences are identified elsewhere: Southern Europe experienced more frequent westerly and cyclonic CWTs under LGM conditions, while Central and Eastern Europe was predominantly affected by southerly and easterly flow patterns. Under LGM conditions, rainfall is enhanced over Western Europe but is reduced over most of Central and Eastern Europe. These differences are explained by changing CWT frequencies and evaporation patterns over the North Atlantic Ocean. The regional differences of the CWTs and precipitation patterns are linked to the North Atlantic storm track, which was stronger over Europe in all considered models during the LGM, explaining the overall increase of the cyclonic CWT. Enhanced evaporation over the North Atlantic leads to higher moisture availability over the ocean. Despite the overall cooling during the LGM, this explains the enhanced precipitation over southwestern Europe, particularly Iberia. This study links large-scale atmospheric dynamics to the regional circulation and associated precipitation patterns and provides an improved regional assessment of the European climate under LGM conditions.