Cyril J. Smyth

Fine-structure molecular epidemiological analysis of Staphylococcus aureus recovered from cows.

Sixty-three Staphylococcus aureus isolates recovered from bovine sources in the USA and the Republic of Ireland were characterized by multilocus enzyme electrophoresis (MLEE), ribotyping, and random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) typing at two separate laboratories. The S. aureus isolates were assigned by MLEE to 10 electrophoretic types (ETs) (Index of Discrimination, D = 0.779). In contrast, the same isolates were assigned to 13 ribotypes (D = 0.888), and to 12 RAPD types (D = 0.898). A common clone, ET3, of worldwide distribution, was represented by six distinct combinations of ribotypes and RAPD types. S. aureus clones recovered from cows in Ireland were also associated with mastitis in dairy cows in the USA. These findings are consistent with the hypothesis that only a few specialized clones of S. aureus are responsible for the majority of cases of bovine mastitis, and that these clones have a broad geographic distribution.

Evolutionary Genomics of Staphylococcus aureus Reveals Insights into the Origin and Molecular Basis of Ruminant Host Adaptation

Phenotypic biotyping has traditionally been used to differentiate bacteria occupying distinct ecological niches such as host species. For example, the capacity of Staphylococcus aureus from sheep to coagulate ruminant plasma, reported over 60 years ago, led to the description of small ruminant and bovine S. aureus ecovars. The great majority of small ruminant isolates are represented by a single, widespread clonal complex (CC133) of S. aureus, but its evolutionary origin and the molecular basis for its host tropism remain unknown. Here, we provide evidence that the CC133 clone evolved as the result of a human to ruminant host jump followed by adaptive genome diversification. Comparative whole-genome sequencing revealed molecular evidence for host adaptation including gene decay and diversification of proteins involved in host–pathogen interactions. Importantly, several novel mobile genetic elements encoding virulence proteins with attenuated or enhanced activity in ruminants were widely distributed in CC133 isolates, suggesting a key role in its host-specific interactions. To investigate this further, we examined the activity of a novel staphylococcal pathogenicity island (SaPIov2) found in the great majority of CC133 isolates which encodes a variant of the chromosomally encoded von Willebrand-binding protein (vWbpSov2), previously demonstrated to have coagulase activity for human plasma. Remarkably, we discovered that SaPIov2 confers the ability to coagulate ruminant plasma suggesting an important role in ruminant disease pathogenesis and revealing the origin of a defining phenotype of the classical S. aureus biotyping scheme. Taken together, these data provide broad new insights into the origin and molecular basis of S. aureus ruminant host specificity.

Molecular genetic typing reveals further insights into the diversity of animal-associated Staphylococcus aureus.

Staphylococcus aureus is an important pathogen of man, but is also able to colonize and cause disease in a wide variety of mammals and birds. An extended multilocus sequencing approach, involving multilocus sequence typing (MLST), sas typing, spa typing and agr typing, was used to examine the molecular diversity of 118 S. aureus isolates recovered from a range of host species and to compare these data with the known diversity of human-derived isolates. MLST revealed that the commonest animal-associated MLST types were ST133, ST5, ST71, ST97, ST126 and ST151. ST133 appears to be an ungulate-animal-specific genotype, as no evidence of ST133 associating with humans has yet been found in the literature. Novel and unique sas alleles were identified in the animal-associated strains that may represent animal-associated sas alleles. However, sas typing exhibited a lower typeability than MLST for the animal strains (91.3 %). Phylogenetic analyses using neighbour-joining and maximum-parsimony trees localized ruminant-associated MLST lineages to both previously identified S. aureus subspecies aureus subgroups, thus explaining the finding of all four agr types within the ruminant-associated strains. S. aureus isolates recovered from chickens and rabbits were genotypically more similar to known human genotypes than the ruminant-associated lineages.

Molecular population and virulence factor analysis of Staphylococcus aureus from bovine intramammary infection

Staphylococcus aureus isolates from cows in Ireland (n = 102) and the USA (n = 42) were characterized by RAPD‐PCR and analysed for the production of a number of putative virulence factors. Of these strains 63 representative isolates were screened for the corresponding virulence factor genes by PCR or Southern hybridization or both. The isolates were divided into 12 distinct clonal types on the basis of their RAPD fingerprint profiles. Of the isolates, 107 (74·3%) tested positive for clumping factor in a slide agglutination test, all 24 RAPD type 7 isolates being negative for clumping factor. PCR analysis of region R, a repeat region of the clfA gene, revealed eight region‐R sizes. There was a strong association between RAPD type and the clfA region‐R genotype among Irish isolates. Of the RAPD type 7 isolates, 21 (87·5%) coproduced toxic shock syndrome toxin‐1 (TSST‐1) and staphylococcal enterotoxin C (SEC). Over 90% of isolates demonstrated haemolytic activity on sheep or rabbit red blood cells and all isolates harboured the gamma‐haemolysin (hlg) locus. Of the Irish isolates, all those of RAPD type 7 were sensitive to penicillin G, whereas 86% of RAPD types 4 and 5 strains were resistant. Furthermore, RAPD types 5 and 7 were more likely to be associated with clinical mastitis whereas RAPD type 4 isolates were more often associated with a latent infection. The current study identifies some of the putative virulence factors produced by the predominant clonal types of bovine Staph. aureus that may be considered as components of a vaccine.

Recrudescence of Helicobacter pylori after apparently successful eradication: novel application of randomly amplified polymorphic DNA fingerprinting.

The aim of this study was to find out if reinfection or recrudescence accounted for the recurrence of Helicobacter pylori infections after apparent eradication of the bacterium. Three hundred and twenty patients were treated with colloidal bismuth subcitrate (120 mg four times daily for four weeks), metronidazole and tetracycline (400 mg and 500 mg, respectively, thrice daily for the first week). H pylori was eradicated four weeks after the end of treatment as assessed by the rapid urease test, histological examination, Gram staining, and culture. However, the infection recurred in 29 (9.1%) of the patients one year after apparent eradication. Pre and posteradication isolates from five patients were available. DNA was extracted and used for restriction endonuclease analysis with Hind III and Hae III, and for polymerase chain reaction (PCR) based randomly amplified polymorphic DNA fingerprinting with a combination of two 10 nucleotide primers. Sodium dodecyl sulphate polyacrylamide gel electrophoretic analysis was performed also. Randomly amplified polymorphic DNA fingerprinting was unique in that it yielded highly discriminatory fingerprints, which showed that the pretreatment and recurrent isolates obtained from each of the five patients were indistinguishable from one another. This shows that recurrence of H pylori infection is probably caused by recrudescence and that the discriminatory power of randomly amplified polymorphic DNA fingerprinting is a practicable and discriminatory typing scheme for H pylori.

Pathogenomic Analysis of the Common Bovine Staphylococcus aureus Clone (ET3): Emergence of a Virulent Subtype with Potential Risk to Public Health

A common clone (ET3) of Staphylococcus aureus is responsible for a large proportion of cases of bovine mastitis and occasionally causes zoonotic infections of humans. In the present study, we report the identification of a virulent clonal subtype (ST151) of ET3, which resulted in increased tissue damage and mortality in a mouse model of mastitis. ST151 has undergone extensive diversification in virulence and regulatory-gene content, including the acquisition of genetic elements encoding toxins not made by other ET3 strains. Furthermore, ST151 had elevated levels of RNAIII and cytolytic toxin-gene expression, consistent with the enhanced virulence observed during experimental infection. Previously, the ST151 clone was shown to be hypersusceptible to the acquisition of vancomycin-resistance genes from Enterococcus spp. Taken together, these data indicate the emergence of a virulent subtype of the common ET3 clone, which could present an enhanced risk to public health.

Molecular typing of nasal carriage isolates of Staphylococcus aureus from an Irish university student population based on toxin gene PCR, agr locus types and multiple locus, variable number tandem repeat analysis

Forty-eight Staphylococcus aureus isolates collected from a young, healthy, Irish university student population from 1995 to 2004 were screened for 16 enterotoxin (SE) and enterotoxin-like (SEl) genes (sea-see, seg-sei, selj-selo, selq, selu), and for the toxic shock toxin syndrome toxin-1 gene, tst. All of the isolates harboured at least one SE or SEl gene and 66.7 % possessed a classical SE gene (sea, seb, sec), the commonest being the seb gene. Most of the isolates (85.4 %) had a complete egc locus (selo, selm, sei, seln, seg). The intergenic sei-seln region of the egc locus was typed by PCR-RFLP in 34 isolates, 15 possessing pseudogenes psient1 and psient2 and 19 having the selu gene. The seh and sell genes, the selk-selq gene combination, and the tst gene were each found in <15 % of isolates. The agr genotype distribution was agr type III, 37.5 %; agr type I, 35.4 %; agr type II, 25 %; and agr type IV, 2.1 %. There was no association between SE-SEl genotype and agr type. All tst gene-positive isolates were of agr type III and harboured a classical SE gene. Multiple locus, variable number tandem repeat analysis (MLVA) produced 47 different patterns. While the sdr locus was present in all isolates, half of them lacked one or two of the sdr gene amplimers. Twenty isolates harboured the bbp gene, its presence being associated with agr type III, but not with the SE-SEl gene profile. The agr types of isolates were associated with MLVA subclusters. Selective MLST analysis revealed seven novel sequence types and a new aroE allele. Five clonal clusters (CCs), including CCs comprising major pandemic clones CC30, CC5 and CC22 and minor lineages CC6 and CC9, and three singletons were identified.

Molecular epidemiology of group B streptococci in Ireland: associations between serotype, invasive status and presence of genes encoding putative virulence factors

Group B streptococcal isolates (n = 159) from the three Dublin maternity hospitals, were serotyped and analysed for the bac, bca, hylB, pepB, and rib genes. The serotype distribution of the isolates was Ia, 19.5%; Ib, 18.9%; II, 10.7%; III, 29.5%; IV, 1.9%; V, 15.1%; non-typeable, 4.4%. There was a statistically significant association between the serotype and invasive status (carriage or infection) of isolates (P < 0.005), but no significant association between serotype and degree of invasiveness was demonstrated. The presence or absence of each analysed gene was not associated with the invasive status of isolates. Statistically significant associations were revealed between bca and hylB (IS1548) (P = 0.0004) and between bac and bca (P=0.014). The bac, bca, hylB (IS1548) and rib genes and the numbers of tandem repeats in the bca gene showed significant associations with serotype. Almost 50% of serotype III isolates possessed at least one of the bac and bca genes and 55-65% of strains of serotypes Ia, Ib and II possessed the rib gene. Most serotype III isolates had IS1548 in their hylB genes. Serotype Ib was the only serotype in which more than half of the strains contained more tandem repeats in the bca gene than the overall mean for the GBS population studied of 7.4 repeats. These findings indicate that some previously reported associations between putative virulence factors and GBS disease require further study and clarification.

Associations between enterotoxin gene cluster types egc1, egc2 and egc3, agr types, enterotoxin and enterotoxin-like gene profiles, and molecular typing characteristics of human nasal carriage and animal isolates of Staphylococcus aureus

Twenty genes encoding enterotoxin and enterotoxin-like proteins have been described in Staphylococcus aureus strains. Five of these occur commonly in the enterotoxin gene cluster (egc: selo, selm, sei, seln and seg). In the sei-seln intergenic region, two pseudogenes, psient1 and psient2, can be present or an additional gene designated selu or a variant selu(v). Whilst frequencies of loci bearing pseudogenes (egc1) or the selu gene (egc2) have been reported, the distinction between selu-bearing and selu(v)-bearing (egc3) loci has rarely been made. A PCR-RFLP procedure involving cleavage of the sei-seln intergenic region by restriction endonuclease BbvI or TseI was developed that allowed differentiation of selu(+) and selu(v)(+) loci. In addition, PCR primers were designed to yield a 203 bp amplimer for sequencing of a selu or selu(v) intragenic region, which encompassed ten signature nucleotide differences. A total of 43 egc(+) human nasal isolates and 53 egc(+) bovine, ovine, caprine, leporine and gallinaceous isolates were egc typed and agr typed. None of the animal isolates was of agr type III. A total of 12 out of 17 egc3(+) human nasal isolates were of agr type III, the other 5 being agr type I. On the basis of representative multilocus sequence typing, agr type III/egc3(+) strains belonged to CC30. Human nasal isolates bearing an egc1 locus were distributed evenly across agr types I, II and III. Only two nasal isolates had an egc2 locus. All 14 agr type IV isolates, only 1 of which was of human origin, possessed an egc2 locus. The agr IV nasal isolate was fusidic acid sensitive and was found to be ST123 (CC121). There were strong associations between bovine, leporine and gallinaceous S. aureus clonal types and egc locus types. The PCR-RFLP procedure was used to screen an additional 45 S. aureus isolates from dogs, cats, rats, pigs and horses for egc locus types. Of these, 33 were egc(-). Six equine isolates were selu(+). One canine and three porcine isolates possessed pseudogenes psient1 and psient2. One porcine and one canine isolate each had the selu(v) gene. Putative relationships between disease-causing propensity and egc type need (re-)evaluation.

Fine-Structure Molecular Typing of Irish Helicobacter pylori Isolates and Their Genetic Relatedness to Strains from Four Different Continents

ABSTRACT Genotyping of 74 Irish Helicobacter pylori isolates was performed at four different loci (vacA signal sequence and mid-region, insertion-deletion polymorphisms at the 3′ end of the cag pathogenicity island, and cagA). The predominant vacA alleles and insertion-deletion motifs suggest an ancestral relationship between Irish isolates and either specific East Asian or Northern European strains. In addition, fluorescent amplified fragment length polymorphism-PCR genotyping and phylogenetic analysis of 32 representative Irish H. pylori isolates and 22 isolates from four different continents demonstrated that the Irish H. pylori isolates examined were weakly clonal and showed some association with both European and Asian isolates. These three genotyping techniques show that Irish H. pylori isolates have distinctive features that may have evolved in this insular European population.