Phenotype MicroArray (TM) in the metabolic characterisation of Salmonella serotypes Agona, Enteritidis, Give, Hvittingfoss, Infantis, Newport and Typhimurium

The Phenotype MicroArray (TM) (PM) technology was used to study the metabolic characteristics of 29 Salmonella strains belonging to seven serotypes of S. enterica spp. enterica. Strains of serotypes Typhimurium (six strains among definite phage types DTs 1, 40 and 104) and Agona (two strains) were tested for 949 substrates, Enteritidis (six strains of phage type PT1), Give, Hvittingfoss, Infantis and Newport strains (two of each) were tested for 190 substrates and seven other Agona strains for 95 substrates. The strains represented 18 genotypes in pulsed-field gel electrophoresis (PFGE). Among 949 substrates, 18 were identified that could be used to differentiate between the strains of those seven serotypes or within a single serotype. Unique metabolic differences between the Finnish endemic Typhimurium DT1 and Agona strains were detected, for example, in the metabolism of d-tagatose, d-galactonic acid gamma-lactone and l-proline as a carbon source. Thus, the PM technique is a useful tool for identifying potential differential markers on a metabolic basis that could be used for epidemiological surveillance.

Molecular typing of Salmonella serotypes prevalent in animals in England: assessment of methodology

Salmonella enterica serotypes Derby, Mbandaka, Montevideo, Livingstone, and Senftenberg were among the 10 most prevalent serotypes isolated from farm animals in England and Wales in 1999. These serotypes are of potential zoonotic relevance; however, there is currently no "gold standard" fingerprinting method for them. A collection of isolates representing the former serotypes and serotype Gold Coast were analyzed using plasmid profiling, pulsed-field gel electrophoresis (PFGE), and ribotyping. The success of the molecular methods in identifying DNA polymorphisms was different for each serotype. Plasmid profiling was particularly useful for serotype Derby isolates, and it also provided a good level of discrimination for serotype Senftenberg. For most serotypes, we observed a number of nontypeable plasmid-free strains, which represents a limitation of this technique. Fingerprinting of genomic DNA by ribotyping and PFGE produced a significant variation in results, depending on the serotype of the strain. Both PstI/SphI ribotyping and XbaI-PFGE provided a similar degree of strain differentiation for serotype Derby and serotype Senftenberg, only marginally lower than that achieved by plasmid profiling. Ribotyping was less sensitive than PFGE when applied to serotype Mbandaka or serotype Montevideo. Serotype Gold Coast isolates were found to be nontypeable by XbaI-PFGE, and a significant proportion of them were found to be plasmid free. A similar situation applies to a number of serotype Livingstone isolates which were nontypeable by plasmid profiling and/or PFGE. In summary, the serotype of the isolates has a considerable influence in deciding the best typing strategy; a single method cannot be relied upon for discriminating between strains, and a combination of typing methods allows further discrimination.

Virulence factors of Escherichia coli serotypes associated with avian colisepticaemia

The current understanding of the pathogenesis of avian pathogenic Escherichia coli (APEC) in colisepticaemia is limited. This review discusses putative virulence determinants per se, such as a number of surface organelles including fimbriae and flagella; together with other factors such as iron sequestering mechanisms, which are involved in the survival of E. coli in the host rather than initiation of infection. It is concluded that avian colisepticaemia is a multi-factorial disease and that to date only a limited number of virulence factors of APEC have been thoroughly elucidated. Crown Copyright (C) 2002 Published by Elsevier Science Ltd. All rights reserved.

Interaction between attaching and effacing Escherichia coli serotypes O157:H7 and O26:K60 in cell culture

Ruminants harbour both O157:H7 and non-O157 Attaching Effacing Escherichia coli (AEEC) strains but to date only nonO157 AEEC have been shown to induce attaching effacing lesions in naturally infected animals. However, O157 may induce lesions in deliberate oral inoculation studies and persistence is considered dependent upon the bacterially encoded locus for enterocyte effacement. In concurrent infections in ruminants it is unclear whether non-O157 AEEC contribute either positively or negatively to the persistence of E. coli O157:H7. To investigate this, and prior to animal studies, E. coli O157:H7 NCTC 12900, a non-toxigenic strain that persists in conventionally reared sheep, and non-toxigenic AEEC O26:K60 isolates of sheep origin were tested for adherence to Hep-2 tissue culture alone and in competition one with another. Applied together, both strains adhered in similar numbers but lower than when either was applied separately. Pre-incubation of tissue culture with either one strain reduced significantly (P < 0.05) the extent of adherence of the strain that was applied second. It was particularly noticeable that AEEC O26 when applied first reduced adherence and inhibited microcolony formation, as demonstrated by confocal microscopy, of E. coli 01 57:H7. The possibility that prior colonisation of a ruminant by non-O157 AEEC such as O26 may antagonise O157 colonisation and persistence in ruminants is discussed. (C) 2004 Elsevier B.V. All rights reserved.

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.

Restriction fragment length polymorphism of polymerase chain reaction products applied to the differentiation of poultry campylobacters for epidemiological investigations

A technique for subtyping Camplobacter jejuni isolates has been developed by using the restriction fragment length polymorphism (Rnp) of polymerase chain reaction (PCR) products of the fluA and flaB genes. The technique was validated by using strains representing 28 serotypes of C jejuni and it may also be applied to C coli. From these strains 12 distinct RFLP profiles were observed but there was no direct relationship between the RFLP profile and the serotype. One hundred and thirty-five campylobacter isolates from 15 geographically distinct broiler flocks were investigated. All the isolates could be subtyped by using the RFLP method. Isolates from most of the flocks had a single RFLP profile despite data indicating that several serotypes were involved. Although it is possible that further restriction analysis may have demonstrated profile variations in these strains, it is more likely that antigenic variation can occur within genotypically related campylobacters. As a result, serotyping may give conflicting information for veterinary epidemiological purposes. This RFLP typing scheme appears to provide a suitable tool for the investigation of the sources and routes of transmission of campylobacters in chickens.

Distribution, gene sequence and expression in vivo of the plasmid encoded fimbrial antigen of Salmonella serotype Enteritidis

The pefA gene which encoded the serotype associated plasmid (SAP) mediated fimbrial major subunit antigen of Salmonella enterica serotype Typhimurium shared genetic identity with 128 of 706 salmonella isolates as demonstrated by dot (colony) hybridization. Seventy-seven of 113 isolates of Typhimurium and individual isolates of serotypes Bovis-morbificans, Cholerae-suis and Enteritidis phage type 9b hybridized pefA strongly, whereas 48 isolates of Enteritidis hybridized pefA weakly and one Enteritidis isolate of phage type 14b failed to hybridize. Individual isolates of 294 serotypes and 247 individual isolates of serotype Dublin did not hybridize pefA. Southern hybridization of plasmids extracted from Enteritidis demonstrated that the pefA gene probe hybridized strongly an atypical SAP of 80 kb in size harboured by one Enteritidis isolate of phage-type 9b, whereas the typical SAP of 58 kb in size harboured by 48 Enteritidis isolates hybridized weakly. One Enteritidis isolate of phage type 14b which failed to hybridize pefA in dot (colony) hybridization experiments was demonstrated to be plasmid free. A cosmid library of Enteritidis phage type 4 expressed in Escherichia coli K12 was screened by hybridization for the presence of pef sequences. Recombinant clones which were deduced to harbour the entire pef operon elaborated a PEF-like fimbrial structure at the cell surface. The PEF-like fimbrial antigen was purified from one cosmid clone and used in western blot experiments with sera from chickens infected with Enteritidis phage-type 4. Seroconversion to the fimbrial antigen was observed which indicated that the Enteritidis PEF-like fimbrial structure was expressed at some stage during infection. Nucleotide sequence analysis demonstrated that the pefA alleles of Typhimurium and Enteritidis phage-type 4 shared 76% DNA nucleotide and 82% deduced amino acid sequence identity.

Sequence analysis and distribution of an IS3-like insertion element isolated from Salmonella enteritidis

The nucleotide sequence of a 3 kb region immediately upstream of the sef operon operon of Salmonella enteritidis was determined. A 1230 base pair insertion sequence which shared sequence identity (> 75%) with members of the IS3 family was revealed. This element, designated IS1230, had almost identical (90% identity) terminal inverted repeats to Escherichia coli IS3 but unlike other IS3-like sequences lacked the two characteristic open reading frames which encode the putative transposase. S. enteritidis possessed only one copy of this insertion sequence although Southern hybridisation analysis of restriction digests of genomic DNA revealed another fragment located in a region different from the sef operon which hybridised weakly which suggested the presence of an IS1230 homologue. The distribution of IS1230 and IS1230-like elements was shown to be widespread amongst salmonellas and the patterns of restriction fragments which hybridised differed significantly between Salmonella serotypes and it is suggested that IS1230 has potential for development as a differential diagnostic tool.

Detection of Chlamydia psittaci DNA in avian clinical samples by polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed to detect Chlamydia psittaci DNA in faeces and tissue samples from avian species. Primers were designed to amplify a 264 bp product derived from part of the 5' non-translated region and part of the coding region of the ompA gene which encodes the major outer membrane protein. Amplified sequences were confirmed by Southern hybridization using an internal probe. The sensitivity of the combined assay was found to be between 60 to 600 fg of chlamydial DNA (approximately 6 to 60 genome copies). The specificity of the assay was confirmed since PCR product was not obtained from samples containing several serotypes of C. trachomatis, strains of C. pneumoniae, the type strain of C. pecorum, nor from samples containing microorganisms commonly found in the avian gut flora. In this study, 404 avian faeces and 141 avian tissue samples received by the Central Veterinary Laboratory over a 6 month period were analysed by PCR, antigen detection ELISA and where possible, cell culture isolation. PCR performed favourably compared with ELISA and cell culture, or with ELISA alone. The PCR assay was especially suited to the detection of C. psittaci DNA in avian faeces samples. The test was also useful when applied to tissue samples from small contact birds associated with a case of human psittacosis where ELISA results were negative and chlamydial isolation was a less favourable method due to the need for rapid diagnosis.

Association between cyclohexane resistance in Salmonella of different serovars and increased, resistance to multiple antibiotics, disinfectants and dyes

A panel of 388 salmonellas of animal and human origin, comprising 35 serotypes, was tested for resistance to cyclohexane and to a range of antibiotics, disinfectants and dyes. Cyclohexane resistance was detected in 41 isolates (10.6%): these comprised members of the serovars Binza (1 of 15), Dublin (1 of 24), Enteritidis (1 of 61), Fischerkietz (4 of 5), Livingstone (9 of 11), Montevideo (1 of 32), Newport (4 of 23), Saint-paul (1 of 3), Senftenberg (10 of 24) and Typhimurium (9 of 93). Most (39 of 41) of the cyclohexane-resistant isolates were from poultry. Statistical analysis showed that the cyclohexane-resistant strains were significantly more resistant than the cyclohexane-susceptible strains to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, nalidixic acid, tetracycline, trimethoprim, cetrimide and triclosan. The multiresistance patterns seen,were typical of those caused by efflux pumps, such as AcrAB. The emergence of such resistance may play an important role in the overall antibiotic resistance picture of Salmonella, with particular effect on ciprofloxacin.

Membrane ruffling and invasion of human and avian cell lines is reduced for aflagellate mutants of Salmonella enterica serotype Enteritidis

Independent studies have demonstrated that flagella are associated with the invasive process of Salmonella enterica serotypes, and aflagellate derivatives of Salmonella enterica serotype Enteritidis are attenuated in murine and avian models of infection. One widely held view is that the motility afforded by flagella, probably aided by chemotactic responses, mediates the initial interaction between bacterium and host cell. The adherence and invasion properties of two S. Enteritidis wild-type strains and isogenic aflagellate mutants were assessed on HEp-2 and Div-1 cells that are of human and avian epithelial origin, respectively. Both aflagellate derivatives showed a significant reduction of invasion compared with wild type over the three hours of the assays. Complementation of the defective fliC allele recovered partially the wild-type phenotype. Examination of the bacterium-host cell interaction by electron and confocal microscopy approaches showed that wild-type bacteria induced ruffle formation and significant cytoskeletal rearrangements on HEp-2 cells within 5 minutes of contact. The aflagellate derivatives induced fewer ruffles than wild type. Ruffle formation on the Div-1 cell line was less pronounced than for HEp-2 cells for wild-type S. Enteritidis. Collectively, these data support the hypothesis that flagella play an active role in the early events of the invasive process.

Detection of mutations in Salmonella enterica gyrA, gyrB, parC and parE genes by denaturing high performance liquid chromatography (DHPLC) using standard HPLC instrumentation

Aims: Quinolone antibiotics are the agents of choice for treating systemic Salmonella infections. Resistance to quinolones is usually mediated by mutations in the DNA gyrase gene gyrA. Here we report the evaluation of standard HPLC equipment for the detection of mutations (single nucleotide polymorphisms; SNPs) in gyrA, gyrB, parC and parE by denaturing high performance liquid chromatography (DHPLC). Methods: A panel of Salmonella strains was assembled which comprised those with known different mutations in gyrA (n = 8) and fluoroquinolone-susceptible and -resistant strains (n = 50) that had not been tested for mutations in gyrA. Additionally, antibiotic-susceptible strains of serotypes other than Salmonella enterica serovar Typhimurium strains were examined for serotype-specific mutations in gyrB (n = 4), parC (n = 6) and parE (n = 1). Wild-type (WT) control DNA was prepared from Salmonella Typhimurium NCTC 74. The DNA of respective strains was amplified by PCR using Optimase (R) proofreading DNA polymerase. Duplex DNA samples were analysed using an Agilent A1100 HPLC system with a Varian Helix (TM) DNA column. Sequencing was used to validate mutations detected by DHPLC in the strains with unknown mutations. Results: Using this HPLC system, mutations in gyrA, gyrB, parC and parE were readily detected by comparison with control chromatograms. Sequencing confirmed the gyrA predicted mutations as detected by DHPLC in the unknown strains and also confirmed serotype-associated sequence changes in non-Typhimurium serotypes. Conclusions: The results demonstrated that a non-specialist standard HPLC machine fitted with a generally available column can be used to detect SNPs in gyrA, gyrB, parC and parE genes by DHPLC. Wider applications should be possible.

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.

Recombinant anti-EspA antibodies block Escherichia coli O157:H7-induced attaching and effacing lesions in vitro

Intimin and EspA proteins are virulence factors expressed by attaching and effacing Escherichia coli (AEEC) such as enteropathogenic and enterohaemorrhagic E. coli. The EspA protein makes up a filament structure forming part of the type III secretion system (TTSS) that delivers effector proteins to the host epithelial cell. Bacterial surface displayed intimin interacts with translocated intimin receptor in the host cell membrane leading to intimate attachment of the bacterium and subsequent attaching and effacing lesions. Here, we have assessed the use of recombinant monoclonal antibodies against E. coli O157:147 EspA and intimin for the disruption of AEEC interaction with the host cell. Anti-gamma intimin antibodies did not reduce either adhesion of E. coli O157:H7 to host cell mono-layers or subsequent host cell actin rearrangement. Anti-EspA antibodies similarly had no effect on bacterial adhesion however they had a marked effect upon E. coli O157:H7-induced host cell actin rearrangement, where both monoclonal and polyclonal antibodies completely blocked cytoskeletal changes within the host cell. Furthermore, these anti-EspA antibodies were shown to reduce actin rearrangement induced by some but not all other AEEC serotypes tested. Both polyclonal and monoclonal antibodies could be used to label E. coli O157 EspA filaments and these immunoreagents did not inhibit the formation of such filaments. This is the first report of monoclonal antibodies to EspA capable of disrupting the TTSS function of E. coli O157:H7. (c) 2005 Elsevier SAS. All rights reserved.

Structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design

Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.