Streptococcus Agalactiae Strains Research Articles

Identification of lactoferrin-binding proteins in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae isolated from cows with mastitis

Three strains of Streptococcus dysgalactiae subsp. dysgalactiae ( S. dysgalactiae) and five strains of Streptococcus agalactiae were used to identify lactoferrin-binding proteins (LBPs). LBPs from extracted surface proteins were detected by polyacrylamide gel electrophoresis and Western blotting. All strains of S. dysgalactiae evaluated had 52- and 74-kDa protein bands. All strains of S. agalactiae evaluated had 52-, 70- and 110-kDa protein bands. In addition, a 45-kDa band was detected in two of five S. agalactiae strains evaluated. This study demonstrated that S. dysgalactiae and S. agalactiae of bovine origin contain two and three major LBPs, respectively

Identification of lactoferrin-binding proteins in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae isolated from cows with mastitis.

Three strains of Streptococcus dysgalactiae subsp. dysgalactiae (S. dysgalactiae) and five strains of Streptococcus agalactiae were used to identify lactoferrin-binding proteins (LBPs). LBPs from extracted surface proteins were detected by polyacrylamide gel electrophoresis and Western blotting. All strains of S. dysgalactiae evaluated had 52- and 74-kDa protein bands. All strains of S. agalactiae evaluated had 52-, 70- and 110-kDa protein bands. In addition, a 45-kDa band was detected in two of five S. agalactiae strains evaluated. This study demonstrated that S. dysgalactiae and S. agalactiae of bovine origin contain two and three major LBPs, respectively.

Different cytokine expression in cord blood mononuclear cells after stimulation with neonatal sepsis or colonizing strains of Streptococcus agalactiae.

Streptococcus agalactiae (group B streptococcus, GBS) is the major pathogen of neonatal sepsis. In some newborns, GBS sepsis may have a severe course, including septic shock with a high mortality rate, whereas other newborns are colonized with GBS on their surfaces without any clinical signs of bacterial infection. The reason for this discrepancy is far from clear. We sought, in this study, to compare cytokine expression in cord blood mononuclear cells after stimulation with GBS strains isolated from newborns with sepsis, and strains isolated from newborns without any symptoms of invasive infection. Cord blood mononuclear cells were incubated with either heat-killed bacteria of different strains or lipopolysaccharide, respectively. After 6 and 24 h, cells were harvested and cytokine mRNA-expression was analyzed by reverse-transcriptase PCR. Likewise, supernatants were tested for IL-6 and tumor necrosis factor-alpha concentrations by enzyme immunoassay. When comparing IL-6 and tumor necrosis factor-alpha secretion, there were significantly higher IL-6 levels after stimulation with sepsis than with colonizing isolates. Likewise, mRNA expression of IL-6, IL-1 beta, and IL-12p40 was significantly higher after stimulation with sepsis isolates. This was also true when normalizing to cytokine expression after stimulation with lipopolysaccharide. These findings indicate that the different clinical pictures in response to GBS, either septic infection or colonization, might reflect strain-specific properties. If the respective characteristics can be defined, it might become possible to distinguish by molecular methods potentially "dangerous" from "harmless" strains. Moreover, our findings underline the essential role of these cytokines in the pathogenesis of neonatal GBS sepsis.

The major bovine mastitis pathogens have different cell tropisms in cultures of bovine mammary gland cells

We previously showed that Staphylococcus aureus cells adhered mainly to an elongated cell type, present in cultures of bovine mammary gland cells. Moreover, we showed that this adhesion was mediated by binding to fibronectin. The same in vitro model was used here, to study adhesion of other important mastitis pathogens. Like the S. aureus strains, the Streptococcus dysgalactiae strains adhered mainly to elongated cells, which seemed to be mediated by fibronectin binding. In contrast, Streptococcus uberis strains adhered mainly to cubic cells. Since the cubic cells did not express fibronectin and S. uberis cells bound fibronectin less efficiently, the adhesion of S. uberis cells was independent of fibronectin binding. Streptococcus agalactiae strains adhered poorly to both cell types. The specificity and efficiency of adhesion of Escherichia coli strains was strongly strain dependent. None of the S. agalactiae and E. coli strains tested was able to bind fibronectin efficiently. The results suggest that the different mastitis pathogens have different target cell specificities and use different mechanisms to adhere to cells of the bovine mammary gland.

Antimicrobial activity of everninomicin against clinical isolates of Enterococcus spp., Staphylococcus spp., and Streptococcus spp. tested by Etest.

The in-vitro antimicrobial activity of everninomicin, a novel oligosaccharide antibiotic, was tested against clinical isolates of 11 methicillin-susceptible Staphylococcus aureus (MSSA) strains, 11 methicillin-resistant S. aureus (MRSA) strains, 22 Staphylococcus epidermidis strains, 23 Enterococcus faecalis strains, 23 Enterococcus faecium strains, 23 Enterococcus avium strains, 27 Streptococcus pneumoniae strains, 22 Streptococcus pyogenes strains, and 20 Streptococcus agalactiae strains. The minimum inhibitory concentrations (MICs) of everninomicin were determined by Etest and compared with those of vancomycin, teicoplanin, and minocycline. The Etest showed that everninomicin exhibited excellent activity (MIC, <0.016 to 1.5 microg/ml) against the gram-positive cocci tested. The antimicrobial activity of everninomicin against S. aureus and S. epidermidis was stronger than that of vancomycin, teicoplanin, and minocycline, in particular against MRSA, with an MIC50 of 0.38 microg/ml and an MIC90 of 0.5 microg/ml. Against strains of enterococci and streptococci, everninomicin was more active than vancomycin, but as active as teicoplanin.

In vitro and in vivo antibacterial activities of a new injectable carbapenem, S-4661, against gynaecological pathogens.

Carbapenems are often used to treat infections in obstetrics and gynaecology because of their activity against anaerobes. S-4661, a new injectable carbapenem antibiotic, has favourable pharmacokinetic properties and is not hydrolysed by dehydropeptidase I. We investigated the in vitro and in vivo antibacterial activities of S-4661 against strains of Streptococcus agalactiae, Escherichia coli, Peptostreptococcus magnus, Bacteroides fragilis and Prevotella bivia, which are major pathogens in the fields of obstetrics and gynaecology. The MIC(50) and MIC(90) of S-4661 for these strains were 0.25 and 1 mg/L, respectively. The in vivo efficacy of S-4661 was evaluated in a rat model of intrauterine infection, namely pyometra caused by E. coli and B. fragilis. The accumulation of neutrophils in the uterus in the S-4661-treated group was less marked and the number of bacteria significantly lower than those in the untreated group. These results suggest that S-4661 may be useful for treating polymicrobial infections in the fields of obstetrics and gynaecology.

Investigation of the translation-initiation factor IF2 gene, infB, as a tool to study the population structure of Streptococcus agalactiae.

The sequence of infB, encoding the prokaryotic translation-initiation factor 2 (IF2), was determined in eight strains of Streptococcus agalactiae (group B streptococcus) and an alignment revealed limited intraspecies diversity within S. agalactiae. The amino acid sequence of IF2 from S. agalactiae and from related species were aligned and revealed an interspecies conserved central and C-terminal part, and an N-terminal part that is highly variable in length and amino acid sequence. The diversity and relationships in a collection of 58 genetically distinct strains of S. agalactiae were evaluated by comparing a partial sequence of infB. A total of six alleles were detected for the region of infB analysed. The alleles correlated with the separation of the same strains of S. agalactiae into major evolutionary lineages, as shown in previous work. The partial sequences of infB were furthermore used in phylogenetic analyses of species closely related to S. agalactiae, yielding an evolutionary tree which had a topology similar to a tree constructed using 16S rRNA sequences from the same species.

Bca, beta gene, and gene product divergency in reference and prototype strains of streptococcus agalactiae.

Reference and prototype strains of Streptococcus agalactiae (GBS) were originally selected on the basis of phenotypic traits which, however, do not always mirror genotypic traits. A total of 14 reference and prototype GBS strains were examined by PCR designed to detect the bca and beta genes, encoding the c proteins c(alpha) and c(beta), respectively. The cognate proteins were detected by whole-cell-based fluorescent antibody testing and Western blotting. The PCR for beta gene detection and the antibody-based c(beta) protein detection showed concordant results with all of the isolates, whereas 7 of 14 strains which did not express c(alpha) protein at detectable levels contained bca gene elements, consistent with bca gene and gene product divergency in these strains. The results emphasize the importance of genetic characterization of reference and prototype strains of GBS which, in the past, have been selected on the basis of phenotypic traits.

Characteristics in adherence of streptococci and Staphylococcus aureus isolated from various infective skin lesions: serum IgA decreases adherence of Streptococcus pyogenes but not Staphylococcus aureus

We characterized adherence of streptococci and Staphylococcus aureus strains isolated from various infective skin lesions in terms of hydrophobicity, negative charge, tube adherence, slime production, and influence on adherence to coverslips by plasma and serum immunoglobulins. High hydrophobicity was more frequently observed in Streptococcus pyogenes strains than in Streptococcus agalactiae strains ( P<0.01) and S. aureus strains ( P<0.001) and slime production was more frequently observed in S. agalactiae strains than in S. pyogenes strains ( P<0.05). Serum IgA decreased adherence to coverslips of S. pyogenes strains but not that of S. aureus strains.

Physical and genetic chromosomal maps of Streptococcus agalactiae, serotypes II and III; rRNA operon organization

A detailed analysis of two Streptococcus agalactiae (group B streptococcus, GBS) strains was performed by pulsed field gel electrophoresis (PFGE). Digestion of the chromosomal DNA with SmaI and SgrAI endonucleases, followed by separation and analysis of fragments by PFGE was carried out. Physical chromosomal maps of serotype II/(α+β) and III/α strains of S. agalactiae were constructed. The GBS genome size was estimated to be 2200 kb. Sixteen GBS genes were used as probes and were located on the restriction maps of both strains by DNA-DNA hybridization. Six copies of ribosomal operons were found in the genome of the analyzed strains. Significant differences in the restriction patterns of chromosomal DNA and DNA-DNA hybridization between the two analyzed strains were detected so that DNA restriction patterns may be used to trace outbreaks of disease. The overall GBS chromosomal organization as determined is fairly conserved.

Physical and genetic chromosomal maps of Streptococcus agalactiae, serotypes II and III; rRNA operon organization.

A detailed analysis of two Streptococcus agalactiae (group B streptococcus, GBS) strains was performed by pulsed field gel electrophoresis (PFGE). Digestion of the chromosomal DNA with SmaI and SgrAI endonucleases, followed by separation and analysis of fragments by PFGE was carried out. Physical chromosomal maps of serotype II/(alpha + beta) and III/alpha strains of S. agalactiae were constructed. The GBS genome size was estimated to be 2200 kb. Sixteen GBS genes were used as probes and were located on the restriction maps of both strains by DNA-DNA hybridization. Six copies of ribosomal operons were found in the genome of the analyzed strains. Significant differences in the restriction patterns of chromosomal DNA and DNA-DNA hybridization between the two analyzed strains were detected so that DNA restriction patterns may be used to trace outbreaks of disease. The overall GBS chromosomal organization as determined is fairly conserved.

A new ketolide, HMR 3004, active against streptococci inducibly resistant to erythromycin.

HMR 3004 is a new hydrazono ketolide characterized by a 3-keto function instead of the cladinose moiety. The effect of this antimicrobial agent on inducible and constitutive macrolide-lincosamide-streptogramin B (MLSB) resistance was tested in a lacZ reporter system under control of several ermAM-like attenuator variants. For one constitutively resistant Streptococcus agalactiae strain, three inducibly resistant Streptococcus pneumoniae strains, and one inducibly resistant Enterococcus faecalis strain, the attenuators fused with lacZ were cloned into the shuttle plasmid pJIM2246 and the plasmid was introduced into Staphylococcus aureus RN4220. For the wild-type attenuators, HMR 3004 was a very weak inducer, unlike its cladinose counterpart RU 6652 and erythromycin. As expected, for the fusion originating from the constitutively resistant S. agalactiae strain, the level of uninduced beta-galactosidase synthesis was high. For one S. pneumoniae attenuator, mutations in the 3' end of the attenuator that weakened the stem-loop structure that sequesters the ribosome-binding site and start codon for ermAM methylase could explain the high level of uninduced beta-galactosidase produced. For streptococci, the activity of HMR 3004 correlated with the basal level of beta-galactosidase synthesized. The weak inducer activity of HMR 3004 explained its activity against inducibly MLSB-resistant S. pneumoniae but did not correlate with the moderate activity of the antibiotic against inducibly resistant E. faecalis.

The Streptococcus agalactiae hylB gene encoding hyaluronate lyase: completion of the sequence and expression analysis

We report the cloning, sequencing and expression analysis of the Streptococcus agalactiae strain 4755 hylB 4755 allele, the first chromosomally-encoded streptococcal hyaluronate lyase gene to be cloned and sequenced completely. This gene lies in a region homologous to that found in S. mutans, between the mutX and rmlB genes, a region involved in the synthesis of the serotype c-specific polysaccharide antigen of this organism. Sequencing of hylB 4755 revealed a 3216-bp open reading frame that encodes a 121.2-kDa polypeptide possessing a 30-amino acid signal sequence which was theoretically predicted and experimentally confirmed. A recombinant plasmid, pHYB100, containing hylB 4755 together with its promoter and terminator was constructed and used to analyze the expression of the gene in Escherichia coli. In Northern hybridization experiments, hylB 4755 was found to be transcribed as 3.3-kb monocistronic mRNA from its own promoter which exhibits an extended, σ 70-like 10 consensus sequence. Transcript mapping by primer extension analysis placed the major transcription initiation site leading to the longest transcript 38 bp upstream of the translational initiation codon, ATG. E. coli TG1(pHYB100) efficiently synthesized hyaluronan-cleaving enzyme activity at ∼7000 working units/10 9 cells, with lyase activity detectable in all principle cellular locations. Zymography and Western analysis identified functional activity in TG1(pHYB100) to be associated with ∼118, 110 and 94-kDa polypeptides, with the two low molecular weight species constituting the major components of the enzyme purified from the culture supernatant fluid of S. agalactiae 4755. The 118-kDa form was shown to represent the undegraded mature enzyme, whereas the smaller species are likely to arise from proteolytic cleavage in the N-terminal part of the mature protein. The HylB 4755 protein showed extensive sequence identity to the homologous enzymes from S. agalactiae 3502 and S. pneumoniae characterized by others but sequence comparisons clearly show that incomplete genes truncated at their 5′ ends had been isolated from these two organisms.

Sociability and cognitive processing: Linkages between temperament, habituation and sequential memory

Conjugative plasmid transfer is one of the major mechanisms responsible for the spread of antibiotic resistance and virulence genes. The incompatibility (Inc) 18 group of plasmids is a family of plasmids replicating by the theta-mechanism, whose members have been detected frequently in enterococci and streptococci. Inc18 plasmids encode a variety of antibiotic resistances, including resistance to vancomycin, chloramphenicol and the macrolide-lincosamide-streptogramine (MLS) group of antibiotics. These plasmids comprising insertions of Tn1546 were demonstrated to be responsible for the transfer of vancomycin resistance encoded by the vanA gene from vancomycin resistant enterococci (VRE) to methicillin resistant Staphylococcus aureus (MRSA). Thereby vancomycin resistant S. aureus (VRSA) were generated, which are serious multi-resistant pathogens challenging the health care system. Inc18 plasmids are widespread in the clinic and frequently have been detected in the environment, especially in domestic animals and wastewater. pIP501 is one of the best-characterized conjugative Inc18 plasmids. It was originally isolated from a clinical Streptococcus agalactiae strain and is, due to its small size and simplicity, a model to study conjugative plasmid transfer in Gram-positive bacteria. Here, we report on the occurrence and spread of Inc18-type plasmids in the clinic and in different environments as well as on the exchange of the plasmids among them. In addition, we discuss molecular details on the transfer mechanism of Inc18 plasmids and its regulation, as exemplified by the model plasmid pIP501. We finish with an outlook on promising approaches on how to reduce the emerging spread of antibiotic resistances.

Effect of C5a and tumor necrosis factor-alpha on phagocytosis of Streptococcus agalactiae NT/X and IV/X by bovine neutrophils.

Neutrophils can be primed by inflammatory mediators to have enhanced functional activity, so that when they reach the site of an infection, these cells are no longer in a quiescent state as they are in the blood compartment but are ready to exert their phagocytic and bactericidal potential. Previous studies had shown (3, 4) that purified antibodies (Ab) to group B polysaccharide (GBP) or to X-protein are poorly efficient opsonins for mastitis-causing strains of Streptococcus agalactiae in the absence of a source of complement. The fact that Ab to X-protein alone were poorly efficient but promoted phagocytosis by bovine neutrophils in the setting of heat-treated (56°C, 30 min) immune serum (4), suggested that serum may contain components that activate neutrophils and allow them to ingest IgG-coated bacteria. Here we show that C5adesArg and TNF-α, inflammatory mediators commonly encountered at inflammation sites, were able to enhance the in vitro phagocytosis of S. agalactiae opsonized with either normal bovine serum (NBS), anti-GBP or anti-X IgG.KeywordsOxidative BurstMean Fluorescence IntensityUnstimulated CellImmune SerumBlood CompartmentThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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).

Population structure of Streptococcus agalactiae reveals an association between specific evolutionary lineages and putative virulence factors but not disease

To evaluate the genetic diversity and relationships in a collection of 85 Danish strains of Streptococcus agalactiae (group B streptococcus) we have performed restriction fragment length polymorphism analysis on EcoRI- and MspI-digested whole-cell DNA using as probes rRNA, DNA fragments representing the genes encoding hyaluronidase, C5a-peptidase, alpha-antigen, and beta-antigen as well as two randomly selected genomic DNA fragments for which the coding potential is unknown. In addition, we have assayed for expression of hyaluronidase activity and beta-antigen. Combined analyses of our data and those previously obtained by multilocus enzyme electrophoresis and serotyping revealed a population separating into six major lineages that correlate with individual serotypes. The significant linkage disequilibrium of alleles indicates that the S. agalactiae population examined is predominantly clonal. Notably, strains expressing the serotype III capsule divide into two distant evolutionary lineages, of which one lacks expression of hyaluronidase activity. Six North American isolates of serotype III clustered together with multiple Danish serotype III strains, showing that the combinations of characters on which the phylogenetic tree was based are conserved worldwide. Occurrence of beta-antigen correlated with a specific version of the alpha-antigen gene and was exclusively associated with a single major phylogenetic lineage. Comparisons with the clinical history of the strains revealed no evidence of differences in pathogenic potential among the six major genetic divisions.

A fluorometric β-glucuronidase assay for analysis of bacterial growth in milk

The growth of common mastitis-causing bacteria in milk was followed by a fluorometric technique based on the release of fluorescent 4-methylumbelliferone (4-MU) from 4-methylumbelliferyl-β-D-glucuronide by the β-glucuronidase of bacterial or milk origin. Three of four Escherichia coli strains, all four strains of Streptococcus uberis ( 4 4 ) and Streptococcus agalactiae ( 4 4 ) produced β-glucuronidase. Four Staphylococcus aureus strains ( 4 4 ) and one E. coli strain, though unable to produce the enzyme, activated the milk β-glucuronidase most probably by lowering the pH of bacterial cultures in milk for optimum activity of the indigenous enzyme. The β-glucuronidase of milk, Str. uberis and Str. agalactiae origin had similar optimum pH ranges (5.3–6.6) while E. coli β-glucuronidase was more active at neutral or slightly alkaline pH (6.8–7.7). The increase of β-glucuronidase activity in milk cultures of E. coli, Str. uberis, Str. agalactiae and S. aureus seemed to parallel the increase of colony forming units and were dependent on the inoculum size. The time to reach a predetermined enzyme threshold in E. coli-milk cultures showed excellent linear relationship with the inoculum size.

Purification and characterization of hyaluronidase from Streptococcus agalactiae

Hyaluronidase from two different strains of Streptococcus agalactiae was purified and characterized. The purification was performed successively by chromatography and rechromatography on phenylsepharose, gel filtration with FPLC on Superdex G 200 and isoelectric focusing. The purified hyaluronidase had an isoelectric point of 8.75 and a molecular weight of approximately 116,000 D. It showed maximal enzyme activity at pH 6.30 and 40 degrees C. The Michaelis constant was estimated to be 8.17 x 10(-2) mg/ml. Hyaluronidase was stimulated only by Mg++ and inhibited by Zn++, Al , Cu++ and Fe++ at a final concentration of 10 mmol/l, respectively. The enzyme splitted hyaluronic acid and in low amounts dermatan sulphate and chondroitin sulphate A. Additionally, synthetic polyanions (like polymers of gentisic acid with formaldehyde and hydroxy sulphonic acid with formaldehyde) turned out to be also potent inhibitors of the enzyme.

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.