Agalactiae Strains Research Articles

Genetic diversity of rRNA operons of unrelated Streptococcus agalactiae strains isolated from cerebrospinal fluid of neonates suffering from meningitis.

The genetic diversity of a collection of 54 unrelated Streptococcus agalactiae strains isolated from the cerebrospinal fluid of neonates and of 60 unrelated carrier strains was evaluated by investigating the restriction fragment length polymorphism of the rRNA gene region. Three restriction enzymes were selected for use: PstI, HindIII, and CfoI. Clustering analysis revealed two phylogenetic groups of strains with 40% divergence. Group I contained two clusters, A and B, and group II contained three clusters, C, D, and E. Strains of serotype Ia were mostly distributed in cluster A, and strains of serotype Ib were mostly distributed in cluster E. Serotype III isolates did not cluster. Nevertheless, 37 of 39 isolates belonging to cluster B were serotype III. With HindIII, two rRNA gene banding patterns characterized 38 of the 39 strains of cluster B, which represents a high-virulence group. In addition, two rRNA gene banding patterns with each enzyme and/or a pair of CfoI fragments of 905 and 990 bp identified 81% of the invasive strains. On account of the genetic homogeneity of the cerebrospinal fluid strains, ribotyping is a powerful typing method for investigation of nosocomial or epidemic invasive infections only when all three enzymes are used or when PstI and HindIII or PstI and CfoI are combined with serotyping (index of discrimination, > 0.95).

A species-specific DNA probe for the detection of Mycoplasma agalactiae

A 4.5 kb M. agalactiae DNA fragment present in strains from different areas of Sardinia was cloned and used to detect M. agalactiae DNA in sheep milk samples by dot blot hybridization. The probe recognized only M. agalactiae strains and did not cross-hybridize with other mycoplasmas ( M. capricolum, M. mycoides subsp. capri, M. mycoides subsp. mycoides, M. putrefaciens and M. arginini) or bacteria ( E. coli, P. hemolytica, S. aureus, S. epidermis, L. casei, S. durans, S. lactis and S. thermophilus which may also be found in sheep milk.

Use of Modified Rambach Agar to Differentiate Streptococcus uberis from Other Mastitis Streptococci

The medium of Rambach was modified to permit differentiation of mastitis streptococci. A total of 377 streptococci isolated from bovine IMI was used in the study. Of the 159 strains identified as Streptococcus uberis, 151 strains (94.9%) were β-galactosidase-positive and yielded blue colonies on modified Rambach agar. In comparison, Streptococcus agalactiae, Streptococcus dysgalactiae, Enterococcus saccharolyticus, or Enterococcus faecalis strains were negative for propylene glycol utilization (red colonies) or β-galactosidase production; all strains yielded colorless colonies on modified Rambach agar. However, 5 of 35 (14.3%) Streptococcus equinus strains were also positive for β-galactosidase. Results indicate that modified Rambach agar is a convenient medium for the differentiation of the Strep. uberis from the other mastitis streptococci. Furthermore, modified Rambach agar could be easily incorporated as a screening medium for Strep. uberis in mastitis bacteriology laboratories.

Screening of type Ia and Ib Streptococcus agalactiae strains with high sialic acid levels by determination of susceptibility to tetracyclines.

The type-specific capsular polysaccharide antigen of Streptococcus agalactiae is recognized to be an antiphagocytic factor in strains having large amounts of it. In the present study, it was indicated that vaginal isolates of types Ia and Ib could be classified into two groups on the basis of both their levels of the sialic acid, which occupies the terminal side chains of the polysaccharide, and their susceptibility to tetracyclines: one group comprised strains with low sialic acid levels (less than 9 micrograms/mg of cell dry weight) as well as with susceptibility to tetracyclines (MIC, less than or equal to 0.5 micrograms/ml), and the other comprised strains with higher sialic acid levels (greater than or equal to 9 micrograms/mg) and resistance to tetracyclines (MIC, greater than or equal to 8 micrograms/ml). A few isolates were found to have low levels of sialic acid and to be resistant to tetracyclines, but no isolates that were both relatively high in sialic acid and susceptible to tetracyclines were ever detected. Among strains of those serotypes, the MICs of tetracyclines were not in proportion to the sialic acid levels and were not affected when the sialic acid levels of each strain were altered by using Todd-Hewitt broth with various concentrations of Na2HPO4 and glucose. It was, therefore, apparent that the correlation of sialic acid levels with susceptibility to tetracyclines was not related directly to the sialic acid content or to the amount of the capsular polysaccharide. Since no plasmid DNAs were detected among representative strains that were tetracycline resistant, it was apparent that at least for the strains tested, resistance was chromosomal gene associated. In strains of S. agalactiae of types of Ia and Ib, the determination of susceptibility to tetracyclines was considered to be useful for screening strains with higher sialic acid levels.

Evaluation of the Minitek Gram-Positive Set for identification of streptococci isolated from bovine mammary glands.

A total of 127 isolates were used to evaluate the Minitek Gram-Positive Set for identification of streptococci cultured from bovine mammary glands. The overall accuracy of the Minitek Gram-Positive Set was 34.6%. Of 12 Streptococcus agalactiae strains, 4 (33.3%) were correctly identified. Of 43 Streptococcus dysgalactiae strains, 32 (74.4%) were identified correctly. Of 44 Streptococcus uberis strains, 42 (95.5%) were identified as Enterococcus spp. Poor performance was attributed to the limited number of veterinary strains in the data base. Incorporation of large numbers of veterinary isolates into the data base is needed for further development of this system.

Adherence of group B streptococci to human buccal epithelial cells, its dependence on the biological state of the culture

The adherence activity of Streptococcus agalactiae (group B) strains is directly dependent on the biological state of cultures. The aim of the present paper was to consider the effect of repeated transfers of cultures alternately on solid and liquid media and the effect of the growth phase. The strains, adhering weakly, strongly and variably to epithelial cells were employed in studies on the effect of repeated transfer. The percentage of adherent epithelial cells differed significantly after the first or the second transfer. On storage of the strains after the 3rd transfer at 4 degrees C for 4 d, the adherence activity decreased to the level of non-transferred strains. Ultrastructural analysis revealed in all strains the presence of capsular material, its character being similar both in strongly and in weakly adhering cultures. In weakly adherent strains, the structure of the capsular layer has not changed during transfer whereas the adherence of the strain increased considerably. The effect of the growth phase was studied during 3-48 h of incubation. The growth phase did not influence the adherence activity in strains that had been allowed to multiply for 3-24 h. After a long-term multiplication beyond 24 h, the adherence activity gradually decreased.

The cross reaction Between two Agalactiae strains Lorestan and Aik2 in sera of vaccinated sheep and Goats Inoculated with live Vaccine

The cross reaction Between two Agalactiae strains Lorestan and Aik2 in sera of vaccinated sheep and Goats Inoculated with live Vaccine

Identification of a Streptococcus agalactiae protein antigen associated with bovine mastitis isolates.

Immunoblotting was used to analyze the immune response of cows to Streptococcus agalactiae. Antibody from the milk of cows immunized (via the superficial inguinal lymph node) with formalinized S. agalactiae cells or from the milk of cows with S. agalactiae mastitis reacted strongly with a group of high-molecular-weight proteinaceous antigens. The two most predominant antigenic polypeptides in this group had apparent molecular weights of 97,000 and 104,000. Because the data indicated that these two antigens, as well as several minor antigens sometimes observed in the 70- to 100-kilodalton size range, seemed to be different forms of the same protein, we refer to the entire group as Sas97/104. A monoclonal antibody that was reactive with Sas97/104 was derived and was used to purify the antigen by affinity chromatography. Whole-cell and colony blot enzyme-linked immunoassays with either the monoclonal antibody or a polyclonal serum sample raised against the affinity-purified antigen indicated that this antigen (or cross-reactive proteins with higher molecular weights) is present on the S. agalactiae strains that were freshly isolated from mastitic cows. However, the antigen was not detected in S. agalactiae of human origin, bovine strains of S. agalactiae maintained for a prolonged period in the laboratory, or other streptococci. The data are consistent with the notion that Sas97/104 is a surface antigen and does not correspond to previously described type-specific antigens of group B streptococci.

Specific Agglutination of Streptococcus agalactiae from Bovine Mastitis by Casein Components of Bovine Milk

Streptococcus agalactiae strains freshly isolated from bovine mastitis cases clumped within 15min of addition of small amounts of bovine milk to a broth culture. This reaction was not observed with isolates from human infections or bovine strains that had been maintained in the laboratory for extensive periods. Intensity of the clumping response as measured by a microtiter dilution assay was highly variable. Analysis of several single colony isolates derived from one strain indicated that the clumping phenotype was genetically unstable. The clumping reaction took place in the presence of rifampicin or chloramphenicol. Milk components that caused aggregation were heat stable, relatively insensitive to proteases, and were larger than 10,000 daltons. Purified casein also induced clumping in these strains.

A note on bile tolerance in Streptococcus agalactiae.

Streptococcus agalactiae strains isolated from humans were found to be able to grow in the presence of high levels of bile but not in media containing levels of bile salts or surfactants commonly considered equivalent to bile in selective media.

Lack of virulence of bovine type IIIStreptococcus agalactiae strains for mice correlates with reduced in vitro production of extracellular type-specific antigen

Six strains of type IIIStreptococcus agalactiae isolated from milk samples from cases of bovine mastitis were examined for in vitro production of three potential extracellular virulence factors: neuraminidase, protease, and extracellular type-specific antigen. Virulence in mice, expressed as LD50 values, was examined for these six strains to determine if a relationship existed between the in vitro production of any of the three extracellular products and mouse lethality. Only in vitro production of extracellular type-specific antigen showed a correlation with virulence of these organisms in the mouse model. All six bovine strains were relatively avirulent in the mouse while producing reduced levels in vitro of extracellular typespecific antigen as compared to nine human isolates. The bovineS. agalactiae strains were an average of 538-fold less virulent for the mouse than were the high type-specific antigen producers isolated from human sources.

Homology among tet determinants in conjugative elements of streptococci.

A mutation to tetracycline sensitivity in a resistant strain of Streptococcus pneumoniae was shown by several criteria to be due to a point mutation in the conjugative omega (cat-tet) element found in the chromosomes of strains derived from BM6001, a clinical strain resistant to tetracycline and chloramphenicol. Strains carrying the mutation were transformed back to tetracycline resistance with the high efficiency of a point marker by donor deoxyribonucleic acids from its ancestral strain and from nine other clinical isolates of pneumococcus and by deoxyribonucleic acids from group D Streptococcus faecalis and group B Streptococcus agalactiae strains that also carry conjugative tet elements in their chromosomes. It was not transformed to resistance by tet plasmid deoxyribonucleic acids from either gram-negative or gram-positive species, except for one that carried transposon Tn916, the conjugative tet element present in the chromosomes of some S. faecalis strains. The results showed that the tet determinants in conjugative elements of several streptococcal species share a high degree of deoxyribonucleic acid sequence homology and suggested that they differ from other tet genes.

Differentiation of Mycoplasma agalactiae from other mycoplasmas of sheep and goats

Mycoplasma agalactiae strains consistently produced slight acidity in glucose broth and characteristic lytic patterns on solid media containing sheep, horse and chicken blood, enabling them to be differentiated from other mycoplasmas of sheep and goats in Turkey. With these and additional observations on cultural, morphological and serological characteristics, 67 of 128 Mycoplasma strains were shown to be M. agalactiae. A further 58 strains, some isolated from the nose and eyes of healthy sheep and goats and others from animals affected with contagious agalactiae, were sufficiently homogeneous to be classed as another group, type N. The remaining strains for a third group, type C.

Identification of Streptococcus Agalactiae by the Fluorescent Antibody

A fluorescent antibody technic for identification of Streptococcus agalactiae is described. Smears from colonies on blood agar plates were tested. A pool of conjugates to four different Streptococcus agalactiae antisera stained all the 80 Streptococcus agalactiae strains investigated. The pool proved superior to individual conjugates. Also strains of groups C and G were stained by the Streptococcus agalactiae conjugates. These, however, could be differentiated from Streptococcus agalactiae strains by examination of the controls because the conjugates of antisera to some group C strains stained group C and G strains but not Streptococcus agalactiae strains.

The detection of agglutinins to Str. Agalactiae strains using a milk ring test

A milk ring test, using a haematoxylin-stained antigen of Str. agalactiae strain S 13 is described. The test detected agglutinins in the milk of five cows experimentally infected with strain S 13, and was not inferior to a whey slide agglutination test for this purpose. When compared with standard Brucella ring antigen the streptococcal antigen appeared to the species-specific.