Biomedical subjects

Stephen L W On

Publications and source records attributed to Stephen L W On.

18 recordsLinked to original sources

PubMed2006

Numerical analysis of DNA microarray data of Campylobacter jejuni strains correlated with survival, cytolethal distending toxin and haemolysin analyses.

Molecular epidemiological studies of the enteric pathogen Campylobacter jejuni have suggested that not all animal isolates are equally pathogenic to humans. We examined the use of numerical analysis of whole-genomotype data as a potential tool for evaluating C. jejuni virulence potential. Whole-genome microarray analysis was used to determine the gene-level complementarity of 12 Danish strains to the pathogenic, genome-sequenced strain NCTC 11168. Cytolethal distending toxin (CDT) and haemolysin activities, and survival characteristics under aerobic conditions at room temperature were also determined. Among the strains examined, 439 genes were polymorphic. Numerical analysis of these data by use of the squared Euclidean distance coefficient and Ward's clustering method clearly delineated strains into two clusters. CDT and haemolysin activities of cluster 1 strains were not statistically significantly different from cluster 2 strains. However, viability during aerobic incubation of cluster 1 strains was statistically significantly lower than corresponding estimates of cluster 2 strains. The number of missing or highly divergent genes in cluster 1 strains with respect to NCTC 11168 was also statistically significantly greater compared with those of cluster 2 strains. Sixty-seven genes present in NCTC 11168 were characteristically missing or divergent among cluster 1 strains. Of these, 53 genes were localised within 11 major gene clusters, of which eight were associated with surface structures and included flagellar, lipo-oligosaccharide, and membrane transport proteins. Our data indicate a correlation between C. jejuni genomic content, particularly in surface-coding regions, and its capacity for environmental survival, and may help explain why certain serotypes are more commonly reported in human disease.

Animals↗

PubMed2006

Genotyping of human and porcine Yersinia enterocolitica, Yersinia intermedia, and Yersinia bercovieri strains from Switzerland by amplified fragment length polymorphism analysis.

In this study, 231 strains of Yersinia enterocolitica, 25 strains of Y. intermedia, and 10 strains of Y. bercovieri from human and porcine sources (including reference strains) were analyzed using amplified fragment length polymorphism (AFLP), a whole-genome fingerprinting method for subtyping bacterial isolates. AFLP typing distinguished the different Yersinia species examined. Representatives of Y. enterocolitica biotypes 1A, 1B, 2, 3, and 4 belonged to biotype-related AFLP clusters and were clearly distinguished from each other. Y. enterocolitica biotypes 2, 3, and 4 appeared to be more closely related to each other (83% similarity) than to biotypes 1A (11%) and 1B (47%). Biotype 1A strains exhibited the greatest genetic heterogeneity of the biotypes studied. The biotype 1A genotypes were distributed among four major clusters, each containing strains from both human and porcine sources, confirming the zoonotic potential of this organism. The AFLP technique is a valuable genotypic method for identification and typing of Y. enterocolitica and other Yersinia spp.

Animals↗

PubMed2006

Helicobacter pullorum in chickens, Belgium.

A total of 110 broilers from 11 flocks were tested for Helicobacter pullorum by polymerase chain reaction; positive samples were reexamined with a conventional isolation method. H. pullorum isolates were examined by amplified fragment length polymorphism (AFLP) fingerprinting for interstrain genetic diversity and relatedness. Sixteen isolates from cecal samples from 2 different flocks were obtained. AFLP analysis showed that these isolates and 4 additional isolates from a different flock clustered according to their origin, which indicates that H. pullorum colonization may occur with a single strain that disseminates throughout the flock. Strains isolated from different hosts or geographic sources displayed a distinctive pattern. H. pullorum is present in approximately one third of live chickens in Belgium and may represent a risk to human health.

Abattoirs↗

PubMed2005

Arcobacter cibarius sp. nov., isolated from broiler carcasses.

Twenty Gram-negative, rod-shaped, slightly curved, non-spore-forming bacteria that gave a negative result in Arcobacter species-specific PCR tests but that yielded an amplicon in an Arcobacter genus-specific PCR test were isolated from 13 unrelated broiler carcasses. Numerical analysis of the profiles obtained by SDS-PAGE of whole-cell proteins clustered all isolates in a single group distinct from the other Arcobacter species. DNA-DNA hybridization among four representative strains exhibited DNA binding values above 91 %. DNA-DNA hybridization with reference strains of the current four Arcobacter species revealed binding levels below 47 %. The G+C contents ranged between 26.8 and 27.3 mol%. Pairwise comparison of 16S rRNA gene sequences revealed the mean values for similarity to the type strain of Arcobacter cryaerophilus (97.5 %), Arcobacter butzleri (96.5 %), Arcobacter skirrowii (96.0 %) and Arcobacter nitrofigilis (95.0 %). The levels of similarity to Campylobacter and Helicobacter species were below 88 and 87 %, respectively. The isolates could be distinguished from other Arcobacter species by the following biochemical tests: catalase, oxidase and urease activities; reduction of nitrate; growth at 25 and 37 degrees C under aerobic conditions; growth on 2-4 % (w/v) NaCl media; and susceptibility to cephalothin. These data demonstrate that the 20 isolates represent a single novel Arcobacter species, for which the name Arcobacter cibarius sp. nov. is proposed, with LMG 21996(T) (=CCUG 48482(T)) as the type strain.

Animals↗

PubMed2005

Mucispirillum schaedleri gen. nov., sp. nov., a spiral-shaped bacterium colonizing the mucus layer of the gastrointestinal tract of laboratory rodents.

The mammalian gastrointestinal tract is covered by a layer of mucus that can harbour a range of bacterial species specifically adapted to colonize this ecological niche. Examination of 110 bacterial isolates cultivated from the gastrointestinal tract of 23 mice revealed the presence of a subgroup of 30 isolates that did not correspond genetically with genera commonly associated with this site, i.e. members of the epsilon-Proteobacteria such as Helicobacter and Campylobacter species. Instead this group of isolates was found to lie within the phylum Deferribacteres, a completely distinct lineage in the domain Bacteria. There was a high level of consensus in results obtained from the phenotypic and genotypic characterization of a number of the isolates, which showed they were distinct from other members of the Deferribacteres. As such, they are proposed to constitute a new genus and species, Mucispirillum schaedleri gen. nov., sp. nov. These organisms are anaerobic, Gram-negative, spiral-shaped rods with bipolar flagella. The type strain is HRI I17(T) (= ATCC BAA-1009(T) = ACM 5223(T)).

Anaerobiosis↗

PubMed2005

Arcobacter halophilus sp. nov., the first obligate halophile in the genus Arcobacter.

A Gram-negative bacterium, designated LA31B(T), was isolated from water collected from a hypersaline lagoon on Laysan Atoll in the north-western Hawaiian Islands. Single cells of LA31B(T) were slightly curved but became helical as their length increased. Preliminary characterization based on 16S rRNA gene sequence analysis showed that LA31B(T) shared 96.0 % identity with an Arcobacter sp. isolated from a cyanobacterial mat in hypersaline Lake Sinai, and 94 % identity with Arcobacter nitrofigilis, the type species of the genus Arcobacter. A polyphasic taxonomic study was conducted and confirmed the phylogenetic affiliation of strain LA31B(T) to the genus Arcobacter. However, LA31B(T) was found to be distinct from all recognized Arcobacter species, by a comprehensive biochemical test analysis, whole-cell fatty acid profiling, DNA G + C content (35 mol% in LA31B(T)) and degree of DNA-DNA reassociation. Most notably, LA31B(T) was found to be an obligate halophile, a hitherto undescribed feature among recognized Arcobacter species. These data indicate that LA31B(T) should be considered to represent a novel species in the genus Arcobacter, for which the name Arcobacter halophilus sp. nov. is proposed. This is the first obligately halophilic member of the genus. The type strain is LA31B(T) (=ATCC BAA-1022(T) = CIP 108450(T)).

Arcobacter↗

PubMed2005

Application of fluorescent amplified fragment length polymorphism for comparison of human and animal isolates of Yersinia enterocolitica.

An amplified fragment length polymorphism (AFLP) method, developed to genotype Yersinia enterocolitica, has been used to investigate 70 representative strains isolated from humans, pigs, sheep, and cattle in the United Kingdom. AFLP primarily distinguished Y. enterocolitica strains according to their biotype, with strains dividing into two distinct clusters: cluster A, comprising largely the putatively pathogenic biotypes (BT2 to -4), and cluster B, comprising the putatively nonpathogenic biotype 1A strains and a single BT1B isolate. Within these two clusters, subclusters formed largely on the basis of serotype. However, AFLP profiles also allowed differentiation of strains within these serotype-related subclusters, indicating the high discriminatory power of the technique for Y. enterocolitica. Investigation of the relationship between strain AFLP profile and host confirmed that pigs are, and provides further proof that sheep may be, potential sources of human infection with putatively pathogenic strains. However, the results suggest that some strains causing human disease do not come from veterinary sources identifiable at this time. The distribution of some BT1A isolates within cluster A raises questions about the relationship between virulence potential and biotype.

Abattoirs↗

PubMed2005

Delineation of Campylobacter concisus genomospecies by amplified fragment length polymorphism analysis and correlation of results with clinical data.

Campylobacter concisus has been as frequently isolated from human diarrhea as the important enteropathogen Campylobacter jejuni, but it also occurs in the feces of healthy individuals. The role of C. concisus in human disease has been difficult to determine, since the species comprises at least two phenotypically indistinguishable but genetically distinct taxa (i.e., genomospecies) that may vary in pathogenicity. We examined 62 C. concisus strains by amplified fragment length polymorphism (AFLP) profiling and correlated the results with clinical data. All C. concisus strains gave unique AFLP profiles, and numerical analysis of these data distributed the strains among four clusters. The clustering was of taxonomic significance: two clusters contained, respectively, the type strain (of oral origin) and a reference strain (from diarrhea) of each of the known genomospecies. Genomospecies 2 strains were more frequently isolated from immunocompetent patients and/or patients without concomitant infections that presented with fever, chronic diarrhea, and gut inflammation than was genomospecies 1, clustering with the type strain of oral origin. Bloody diarrhea was recorded only with C. concisus genomospecies 2 infections. We identified two additional C. concisus genomospecies: genomospecies 3 comprised a single strain from an immunocompetent patient, and genomospecies 4 contained five isolates from severely immunodeficient patients, i.e., organ transplantation recipients or those with hematological malignancies. All genomospecies 4 strains were of the same protein profile group and failed to react with a C. concisus species-specific PCR assay based on 23S rRNA gene sequences: the taxonomic position of this group requires closer investigation. Campylobacter concisus is genetically and taxonomically diverse and contains at least four distinct genomospecies that may exhibit differences in their spectra of virulence potential.

Adolescent↗

PubMed2005

Extended multilocus sequence typing system for Campylobacter coli, C. lari, C. upsaliensis, and C. helveticus.

A multilocus sequence typing (MLST) system has been reported previously for Campylobacter jejuni to both differentiate strains and identify clonal lineages. However, sequence variation at the MLST loci prevents its use for closely related Campylobacter species. We describe herein an expanded MLST method to include three clinically relevant Campylobacter species, C. coli, C. lari, and C. upsaliensis, and a fourth Campylobacter species, C. helveticus. The C. coli and C. helveticus methods use the same seven C. jejuni loci (aspA, atpA, glnA, gltA, glyA, pgm, and tkt); however, adk and pgi were substituted for aspA and gltA in C. lari and for gltA and pgm in C. upsaliensis. Multiple C. coli (n = 57), C. lari (n = 20), C. upsaliensis (n = 78), and C. helveticus (n = 9) isolates, representing both clinical and environmental sources, were typed. All four species were genetically diverse: the majority (> 80%) of the isolates had unique sequence types (STs). Using this method, mixed C. lari, C. upsaliensis, and C. helveticus isolates were identified; upon separation, each isolate was shown to contain two strains of the same species with distinct STs. Additionally, the expanded MLST method was able to detect potential lateral transfer events between C. jejuni and either C. coli or C. lari and between C. upsaliensis and C. helveticus. Thus, the expanded MLST method will prove useful in differentiating strains of five Campylobacter species, identifying mixed Campylobacter cultures, and detecting genetic exchange within the genus.

Base Sequence↗

PubMed2004

Identification and phylogeny of Enterobacter sakazakii relative to Enterobacter and Citrobacter Species.

The phylogenetic relationships of Enterobacter sakazakii strains were investigated using 16S ribosomal DNA (rDNA) and hsp60 sequencing. Each analysis distributed E. sakazakii strains among four clusters, indicating substantial taxonomic heterogeneity. The E. sakazakii type strain 16S rDNA sequence was 97.8% similar to that of Citrobacter koseri but 97.0% similar to that of Enterobacter cloacae.

Chaperonin 60↗

PubMed2003

RpoD promoters in Campylobacter jejuni exhibit a strong periodic signal instead of a -35 box.

We have used a hidden Markov model (HMM) to identify the consensus sequence of the RpoD promoters in the genome of Campylobacter jejuni. The identified promoter consensus sequence is unusual compared to other bacteria, in that the region upstream of the TATA-box does not contain a conserved -35 region, but shows a very strong periodic variation in the AT-content and semi-conserved T-stretches, with a period of 10-11 nucleotides. The TATA-box is in some, but not all cases, preceded by a TGx, similar to an extended -10 promoter. We predicted a total of 764 presumed RpoD promoters in the C.jejuni genome, of which 654 were located upstream of annotated genes. A similar promoter was identified in Helicobacter pylori, a close phylogenetic relative of Campylobacter, but not in Escherichia coli, Vibrio cholerae, or six other Proteobacterial genomes, or in Staphylococcus aureus. We used upstream regions of high confidence genes as training data (n=529, for the C.jejuni genome). We found it necessary to limit the training set to genes that are preceded by an intergenic region of >100bp or by a gene oriented in the opposite direction to be able to identify a conserved sequence motif, and ended up with a training set of 175 genes. This leads to the conclusion that the remaining genes (354) are more rarely preceded by a (RpoD) promoter, and consequently that operon structure may be more widespread in C.jejuni than has been assumed by others. Structural predictions of the regions upstream of the TATA-box indicates a region of highly curved DNA, and we assume that this facilitates the wrapping of the DNA around the RNA polymerase holoenzyme, and offsets the absence of a conserved -35 binding motif.

Bacterial Proteins↗

PubMed2003

Assessment of PCR-DGGE for the identification of diverse Helicobacter species, and application to faecal samples from zoo animals to determine Helicobacter prevalence.

Helicobacter species are fastidious bacterial pathogens that are difficult to culture by standard methods. A PCR-denaturing gradient gel electrophoresis (PCR-DGGE) technique for detection and identification of different Helicobacter species was developed and evaluated. The method involves PCR detection of Helicobacter DNA by genus-specific primers that target 16S rDNA and subsequent differentiation of Helicobacter PCR products by use of DGGE. Strains are identified by comparing mobilities of unknown samples to those determined for reference strains; sequence analysis can also be performed on purified amplicons. Sixteen DGGE profiles were derived from 44 type and reference strains of 20 Helicobacter species, indicating the potential of this approach for resolving infection of a single host by multiple Helicobacter species. Some more highly related species were not differentiated whereas in highly heterogeneous species, sequence divergence was observed and more than one PCR-DGGE profile was obtained. Application of the PCR-DGGE method to DNA extracted from faeces of zoo animals revealed the presence of Helicobacter DNA in 13 of 16 samples; a correlation was seen between the mobility of PCR products in DGGE analysis and DNA sequencing. In combination, this indicated that zoo animals are colonized by a wide range of different Helicobacter species; seven animals appeared to be colonized by multiple Helicobacter species. By this approach, presumptive identifications were made of Helicobacter bilis and Helicobacter hepaticus in a Nile crocodile, Helicobacter cinaedi in a baboon and a red panda, and Helicobacter felis in a wolf and a Taiwan beauty snake. All of these PCR products ( approximately 400 bp) showed 100 % sequence similarity to 16S rDNA sequences of the mentioned species. These results demonstrate the potential of PCR-DGGE-based analysis for identification of Helicobacter species in complex ecosystems, such as the gastrointestinal tract, and could contribute to a better understanding of the ecology of helicobacters and other pathogens with a complex aetiology.

Animals↗

PubMed2003

Avian reservoirs and zoonotic potential of the emerging human pathogen Helicobacter canadensis.

A polyphasic identification approach was used to investigate the taxonomic position of Campylobacter-like isolates recovered from barnacle geese (Branta leucopsis) and Canada geese (Branta candensis). Seven strains were selected from a collection of 21 isolates and analyzed by extensive phenotypic testing; four strains were characterized by 16S rRNA gene sequence analysis. The results clearly identified the bird isolates as Helicobacter canadensis, recently described as an emerging human pathogen. This is the first report of an animal reservoir for this organism and of its presence in Europe and confirms the zoonotic potential of H. canadensis.

Animals↗

PubMed2003

Evaluation of 11 PCR assays for species-level identification of Campylobacter jejuni and Campylobacter coli.

We examined the sensitivity and specificity of 11 PCR assays described for the species identification of Campylobacter jejuni and Campylobacter coli by using 111 type, reference, and field strains of C. jejuni, C. coli, and Campylobacter lari. For six assays, an additional 21 type strains representing related Campylobacter, Arcobacter, and Helicobacter species were also included. PCR tests were initially established in the laboratory by optimizing conditions with respect to five type and reference strains of C. jejuni, C. coli, and C. lari. One PCR test for C. coli failed to give appropriate results during this initial setup phase and was not evaluated further. The remaining 10 assays were used to examine heated lysate and purified DNA templates as appropriate of well-characterized type, reference, and field strains of C. jejuni (n = 62), C. coli (n = 34), and C. lari (n = 15). The tests varied considerably in their sensitivity and specificity for their respective target species. No assay was found to be 100% sensitive and/or specific for all C. jejuni strains tested, but four assays for C. coli gave appropriate responses for all strains examined. Between one and six strains of C. jejuni gave amplicons in four of seven C. jejuni PCR tests only where purified DNA was used as the template; corresponding results were seen with one strain of C. coli in each of three assays for the latter species. Our findings indicate that a polyphasic strategy for PCR-based identification should be used to identify C. jejuni and C. coli strains. The data may assist laboratories in selecting assays suited for their needs and in designing evaluations of future PCR tests aimed to identify these species.

Campylobacter coli↗

PubMed2003

Molecular typing of Campylobacter jejuni isolates involved in a neonatal outbreak indicates nosocomial transmission.

Genotypic typing by restriction fragment length polymorphism and pulsed-field gel electrophoresis showed that two neonates in a neonatal ward were infected with the same Campylobacter jejuni strain. Isolates from the mother and brother of the index patient were identical to each other but distinct from the neonatal type. Genotyping results therefore suggested that the neonatal C. jejuni infection was nosocomial in origin.

Anti-Bacterial Agents↗

PubMed2002

Prevalence and diversity of Arcobacter spp. isolated from the internal organs of spontaneous porcine abortions in Denmark.

A study was conducted to determine the prevalence and possible significance of campylobacteria in pig abortions in Denmark. Surface-cauterised liver and kidney samples from 55 aborted pig fetuses submitted to the Danish Veterinary Laboratory were taken and a sensitive isolation procedure used to examine pooled tissue samples for Campylobacter, Arcobacter and Helicobacter spp. Routine microbiological, immunological, and histopathological examinations were also performed to identify concurrent infections or histopathological changes. The abortions tested negative for established abortifacient pathogens (Brucella, Leptospira, PPV, PRRSV), but Arcobacter spp. were recovered from 23/55 abortions. Co-infections with Streptococcus suis, Escherichia coli, and haemolytic streptococci were observed in 7/23 Arcobacter-positive fetuses, and in 4/32 Arcobacter-negative fetuses. Histopathological analyses identified placentitis, pneumonia, hepatitis and encephalitis among the study group. However, no obvious pathologic features were solely associated with Arcobacter-positive cases, nor were Arcobacter-like bacteria observed in tissue samples. Protein profile analyses of the 27 Arcobacter isolates identified 11 as A. cryaerophilus and 10 as A. skirrowii. Six strains could not be classified into any existing species and were phenotypically distinct, thus, potentially representing at least one new species. The identification results showed that multiple taxa could be found in a single fetus, and in distinct aborted fetuses from a single sow. The high prevalence of arcobacters in Danish pig abortions may account for at least some of the >90% of cases in which no established abortifacient agent is detected, but further studies are needed to define the role of each species, especially where co-infections with other bacteria are present.

Abortion, Veterinary↗

PubMed2002

Extragastric Helicobacter species.

The genus Helicobacter has expanded at a rapid pace and no fewer than 31 species have been named since the proposal of the genus in 1989. Of these 31 species, 22 are principally associated with extragastric niches and there is increasing interest in the role of these taxa in diseases of humans and animals. Substantial evidence attests to certain species playing a role in the pathogenesis of enteric, hepatic and biliary disorders and some taxa demonstrate zoonotic potential. The importance of extragastric Helicobacters is likely to be an important topic for research in the near future. Here, important papers published in the field this last year are reviewed.

Animals↗

PubMed2002

Prevalence of Campylobacter concisus in diarrhoea of immunocompromised patients.

The importance of Campylobacter species other than C. jejuni/coli in diarrhoeal disease is largely unknown. We wished to determine the prevalence and clinical presentation of C. concisus infection in patients with enteric disease in a tertiary hospital. Stool specimens were routinely tested for the presence of Campylobacter species, by use of the filter isolation method. The medical records of the C. concisus-positive patients were reviewed. Of 224 Campylobacter isolates obtained, 110 were identified as C. concisus. Concomitant infection occurred in only 27% of cases. By means of protein profiling we assigned C. concisus into 2 groups. The predominant C. concisus group 2 was the only strain to infect immunocompetent patients and children: 71% of the infected patients were immunocompromised, the majority being adults (84%). C. concisus may be a frequent cause of diarrhoea in immunocompromised patients. Two groups of C. concisus were found and these groups possibly vary in their pathogenic potential.

Age Distribution↗