Previous studies have demonstrated that a proportion of Staphylococcus aureus isolates from bovine mastitis coproduce toxic shock syndrome toxin (TSST) and staphylococcal enterotoxin C (SEC). In this study, molecular genetic analysis of one such strain, RF122, revealed the presence of a 15,891-bp putative pathogenicity island (SaPIbov) encoding the genes for TSST (tst), the SEC bovine variant (sec-bovine), and a gene ( A closely related strain, RF120, of the same multilocus enzyme electrophoretic type, random amplified polymorphic DNA type, and ribotype, does not contain the island, implying that the element is mobile and that a recent insertion/deletion event has taken place. TSST and TSST/SEC-deficient mutants of S. aureus strain RF122 were constructed by allele replacement. In vitro bovine V-specific lymphocyte expansion analysis by culture supernatants of wild-type strains and of tst and sec-bovine allele replacement mutants revealed that TSST stimulates BTB13-specific T cells whereas SEC-bovine stimulates BTB93-specific T cells. This suggests that the presence of SaPIbov may contribute to modulation of the bovine immune response.
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.
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.
Porcine enteropathogenic Escherichia coli strains possessing or lacking K88 antigen were studied by using hydrophobic interaction chromatography on crosslinked agarose gels with alkyl or aryl substituents (amphiphilic gels) to determine whether or not they possessed surface-associated hydrophobic properties. Strains with K88ab or K88ac antigen adsorbed to phenyl and octyl Sepharose gels in the presence of 4 M sodium chloride. This property correlated with phenotypic expression of K88 antigen. Cells grown at 37°C but not those grown at 18°C possessed hydrophobic adsorptive characteristics in addition to the property of mannose-resistant hemagglutination of guinea pig erythrocytes. Adsorption of K88-positive strains to gels with hydrophobic ligands was independent of 0 group and enterotoxicity. Strains lacking K88 antigen did not adsorb to the hydrophobically substituted derivatives of Sepharose and lacked mannose-resistant hemagglutinating characteristics. Neither the presence of additional polysaccharide K antigens nor nonhemagglutinating pili conferred the property of hydrophobic interaction on the strains. K88-positive bacteria had a lower electrophoretic migration rate than did K88-negative bacteria of the same serotype in free-zone electrophoresis. K88-positive bacteria also adsorbed strongly to hydrophobic ligands in the presence of 1 M ammonium sulfate, whereas K88-negative strains did not. These observations provide evidence for the suspected role of hydrophobic interaction in the adhesive properties of certain enteropathogenic strains of E. coli. Moreover, hydrophobic interaction chromatography provides convenient and rapid alternative means of screening strains for a property potentially associated with adhesiveness.
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 ruminantassociated 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.
Enterotoxigenic Escherichia coli of serotype 0 6 : K 15 : H16 or Hhave been shown to cause MRHA of bovine erythrocytes (MRHAbov), a characteristic associated with possession of socalled CFA (CFA/II) which facilitates adhesion to the intestinal mucosa. Using putative anti-CFA/II antisera, raised to whole cell vaccines of MRHAL,, prototype strains of different biotypes with subsequent absorption with their MRHAco, variants, three immunologically distinct CS antigens were identified on enterotoxigenic strains of this serotype. CS1 antigen was found only on MRHA,+,, isolates of biotype A (rhamnose-negative). Specific anti-CS 1 antigen serum agglutinated rhamnose-negative strains, gave a single immunoprecipitate in double immunodiffusion against cell surface extracts of these bacteria, and only inhibited the MRHA,,, activity of biotype A strains. The presence of CSl serologically correlated with the presence of a 16.3 kDal polypeptide band upon SDS-PAGE of extracts. CS2 antigen was identified on MRHAk,, strains of biotypes B, C and F (rhamnose-positive). Specific anti-CS2 antigen serum agglutinated only the latter strains, gave a single immunoprecipitate against cell surface extracts of only rhamnose fermenting strains, and only inhibited MRHAbov activity of strains of these biotypes. The presence of CS2 antigen serologically was associated with the presence of a 15.3 kDal polypeptide band in SDS-PAGE patterns of extracts. The CS3 antigen was identified by immunodiffusion tests with extracts of most 0 6 : K 15 : H 16 or H-strains possessing CS 1 or CS2 antigen, i.e. independently of biotype, and was associated with the presence of a 14-8 kDal polypeptide band by SDS-PAGE. Anti-CS3 antigen antibodies did not agglutinate nor did they possess haemagglutination inhibition activity. CS3 antigen was also found on an enterotoxigenic isolate series of serotype 0 8 : K40 : H9, which was MRHA;,, and lacked the CS1 and CS2 antigens. A number of CS2-only 0-serogroup 6 strains were identified. Expression of MRHAbov activity and the production of CS1, CS2 and CS3 antigens were phenotypically suppressed by growth at 18 "C. Loss of ST and also, in most instances, of LT production accompanied loss of MRHAb,, activity and loss of production of the CS1, CS2 and CS3 antigens. Thus, the findings demonstrate the presence of two serologically distinct, biotype-associated haemagglutinins with different molecular weight properties on MRHA;,,, enterotoxigenic E. coli of serotype 0 6 : K15 : H 16 or Hof human origin and of a third non-haemagglutinating surface-associated antigen common to most strains which may also facilitate adhesion.
In recent years several new staphylococcal enterotoxins (SEs) have been described, which currently have largely unknown frequencies of occurrence and roles in human or animal disease. One hundred and ninety-one Staphylococcus aureus isolates from cows (99), goats (39), sheep (23), rabbits (15), chickens (15) and a cat (1) were screened for SE genes sea-see, seg-seo and seq and for the tst gene encoding staphylococcal toxic shock syndrome toxin-1 using multiplex PCRs and individual PCRs for the seb and sek genes. One hundred and ten isolates tested positive for at least one of these 16 superantigen (SAg)-encoding genes. There were statistically significant differences in the frequencies of some of these SAg genes between isolates from different animals. No strain possessed either the sea or see gene. The sec gene was present in 51 isolates, the sed gene in eight and the seb gene in one. The seh gene was found in four strains and the sek and seq genes together in one isolate. The most common combinations of genes were the egc cluster, bearing the seg, sei, sem, sen and seo genes, in 47 isolates, the sec, sel and tst gene combination typical of the SaPIbov pathogenicity island in 44 isolates, the egc cluster lacking the seg gene in 11 isolates, the sed and sej genes in nine isolates, and the sec and tst genes without the sel gene in seven isolates. The higher frequencies of the sec and tst genes together and the lower frequencies of the egc gene cluster among the SAg gene-positive sheep or goat isolates compared to bovine isolates were statistically significant. Of 36 bovine isolates that were mitogenic for human T lymphocytes, four were negative for the 16 SAg genes tested for, while a further 14 gave borderline results in the mitogenicity assay, 12 of which were SAg gene-negative. Twenty-nine strains lacking all the SAg genes did not induce T-cell proliferation. This survey indicates that novel SE genes seg, sei, sel, sem, sen and seo along with the sec and tst genes predominate in S. aureus from animal hosts. The mitogenicity assays indicate that further uncharacterized SAgs may be present in bovine isolates. INTRODUCTIONStaphylococcus aureus is a major human pathogen that causes a wide variety of diseases ranging in severity from food poisoning (McCormick et al., 2001;Le Loir et al., 2003) and life-threatening toxic shock syndrome (Llewelyn & Cohen, 2002;Proft & Fraser, 2003) to less serious infections, e.g. boils (Stulberg et al., 2002). S. aureus can also cause a number of infections in animals, such as tick-associated pyaemia in lambs (Webster & Mitchell, 1989), staphylococcosis in rabbits (Hermans et al., 2003), oedematous and necrotic dermatitis, septicaemia, abcesses and chondronecrosis in chickens McNamee et al., 1998;Takeuchi et al., 2002) S. aureus is the most frequent cause of bovine mastitis, a disease that is of economic importance worldwide (Beck et al., 1992;Miles et al., 1992). Typically staphylococcal mastitis is chronic in nature, with subclinical mastitis being the most common form (Gruet et a...
Enterotoxigenic Escherichia coli (ETEC) of serotype 06:H16, biotype A, bearing colonization factor antigen II (CFA/II) possesses two distinct coli surface antigens, CS1 and CS3, whereas CFA/II-positive ETEC of serotype 08:H9 manifests only CS3. CS1 has been shown to be fimbrial in nature, but heretofore the morphology of CS3 has not been described. Accordingly, by immune electron microscopy we investigated the morphological characteristics of CS3 on bacterial cells and after purification. CS3 was found to consist of thin (2-nm), flexible, wiry, "fibrillar" fimbriae, visible both on bacteria (06:H16, biotype A, and 08:H9 strains) and in the pure state. In contrast, CS1 exists as wider (6-nm), rigid fimbriae on the surface of 06:H16, biotype A, strains. By the use of antisera to CS1 and CS3 in immune electron microscopy, immunodiffusion in gel, and immunoblotting techniques, CS1 and CS3 were found to be immunologically as well as morphologically distinct. Six of nine volunteers who developed diarrhea after challenge with an 0139:H28 ETEC strain bearing CS1 and CS3 had significant serological rises to purified CS1 and CS3 antigens, suggesting that both antigens are elaborated in vivo, play a role in pathogenesis, and stimulate an immune response.
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