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Multiplex polymerase chain reaction as a mastitis screening test for Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis in bulk milk samples.

Effective diagnostic tools for screening herds for mastitis pathogens are important in development and monitoring of mastitis control programmes. A multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis was used in preliminary studies to assess its applicability as an alternative method for monitoring mastitis caused by these organisms at the herd level. PCR was used to detect the presence of these organisms in bulk milk samples. Correlations with bulk milk somatic cell counts (BMCC), total bacteria counts and thermoduric bacteria counts were evaluated. A total of 176 bulk milk samples were collected from 42 herds on five consecutive occasions at approx. 10-d intervals. Str. uberis was the most common organism in these bulk milk samples. There was no relationship between presence of either Staph. aureus, Str. dysgalactiae or Str. uberis and BMCC, total bacteria counts or thermoduric bacteria counts. However, presence of Str. agalactiae was associated with high BMCC and total bacteria counts. The results of this study show that regular analysis of bulk milk using this multiplex PCR assay may be a useful tool for monitoring herd status with respect to Str. agalactiae, but is of less value for monitoring occurrence of Staph. aureus, Str. dysgalactiae and Str. uberis. Further investigations are needed to clarify the relationship between positive PCR results and the prevalence of infected cows in the herd.

Animals↗

Recommended conservation of the names Streptococcus sanguis, Streptococcus rattus, Streptococcus cricetus, and seven other names included in the Approved Lists of Bacterial Names. Request for an opinion.

With reference to the first Principle of the International Code of Nomenclature of Bacteria, which emphasizes stability of names, it is proposed that the original names Streptococcus sanguis, Streptococcus rattus, Streptococcus cricetus, Erwinia ananas, Eubacterium tarantellus, Lactobacillus sake, Nitrosococcus oceanus, Pseudomonas betle, Rickettsia canada and Streptomyces rangoon, all included in the Approved Lists of Bacterial Names, be conserved. Request for an Opinion.

Streptococcus↗

Immunological relationships between glucosyltransferases synthesizing insoluble glucan from Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei.

The Mr values and isoelectric points of glucosyltransferases synthesizing insoluble glucan (GTF-Is) were determined, and the immunological relationships between them studied. The GTF-I enzymes were from Streptococcus cricetus (mutans group serotype a), Streptococcus sobrinus (mutans group serotypes d and g) and Streptococcus downei (mutans group serotype h). By double immunodiffusion tests, the GTF-I enzymes from the three species possessed a common antigenic determinant; in addition, the GTF-I enzymes of serotypes d, g and h shared a further determinant. The S. sobrinus serotypes d and g GTF-I enzymes were immunologically identical. The GTF-I enzymes of S. sobrinus serotypes d and g, and of S. downei, had an Mr of 161,000 and isoelectric points of 4.8-4.9, while S. cricetus GTF-I had a lower Mr (150,000) and a higher isoelectric point (5.2). This suggests that the S. cricetus GTF-I enzyme may lack a sequence of amino acids which include the determinant shared by S. sobrinus and S. downei GTF-I enzymes. Antibodies specific to the determinant shared by all four serotypes inhibited the homologous and heterologous enzymes by 94-100%.

Bacterial Proteins↗

Resistance mechanism of chloramphenicol in Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis.

The chloramphenicol resistance of Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis isolated from clinical materials was proved to be due to an inactivating enzyme produced by these bacteria. The inactivated products of chloramphenicol were identified as 1-acetoxy, 3-acetoxy and 1,3-diacetoxy derivatives by thin-layer chromatography and infrared spectroscopy. The responsible enzyme was thus confirmed to be chloramphenicol acetyltransferase. The enzyme was inducible. It was partially purified by ammonium sulfate precipitation, DEAE-cellulose chromatography and gel filtration on Sephadex G-150. The enzymes obtained from S. haemolyticus, S. pneumoniae and S. faecalis have been compared with the conclusion that they are identical with respect to molecular weight (approximately 75,000-80,000), optimum pH and heat stability.

Acetyltransferases↗

[Evaluation of rapid diagnostic kits for the detection of group A streptococcus to Streptococcus pyogenes and Streptococcus spp. with Lancefield's group A antigen].

We studied the basic performance of eight rapid diagnostic kits for the detection of Group A streptococcus by immunochromatography under the same conditions. Kits were the; QuickVue Dipstick Strep A (Sumitomo Seiyaku Biomedical Co., Ltd.), TESTPACK Plus STREP A (ABBOTT JAPAN Co., Ltd), CLEAVIEW STREP A (Nihon Schering K. K.), QuickVue STREP A (Wako Pure Chemical Industries, Ltd), ImmunoCard STAT! STREP A (TFB, INC.), DIPSTICK 'Eiken' STREP A (Eiken Chemical Co., Ltd.), Rapid Testa Strep A (Daiichi Pure Chemical Co., Ltd.), and StatCheck Strep A (KAINOS Laboratories, Inc.). Four of these kits, i.e. QuickVue Dipstick Strep A, TESTPACK Plus STREP A, Rapid Testa Strep A, and StatCheck Strep A showed sensitivity at 1.0 x 10(5) CFU/mL (1.0 x 10(4)CFU/test) with all of S. pyogenes tested, while the Anginosus group and S. dysgalactiae subsp. equisimilis with Lancefield' s group A antigen showed sensitivity very similar to S. pyogenes. Of these strains, S. dysgalactiae subsp. equisimilis formed a beta-hemolytic colony resembling that of S. pyogenes on sheep blood agar, and was sensitive to bacitracin. It is thus indispensable to identify the colony using biochemical tests such as the PYR (pyrrolidonylarylamidase production) test. In using rapid diagnostic kits for the detection of Group A streptococcus, it is important to rule out the possibility of Group A streptococcus other than S. pyogenes in throats. Severe invasive group-G streptococcal infections are increasing recently. Concerning S. dysgalactiae subsp. equisimilis, it is especially important to conduct these identification tests.

Antigens, Bacterial↗

The cell wall polysaccharide of Streptococcus gordonii 38: structure and immunochemical comparison with the receptor polysaccharides of Streptococcus oralis 34 and Streptococcus mitis J22.

As part of our ongoing investigations involving lectin-mediated adhesion among oral bacteria, the receptor polysaccharide from Streptococcus gordonii 38 was isolated and characterized. Carbohydrate analysis of the hydrolysed S. gordonii 38 polysaccharide by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) showed galactose (Gal) (2 mol), N-acetylgalactosamine (GalNAc) (1 mol), rhamnose (Rha) (2 mol), glucose (Glc) (1 mol) and galactosamine-6-phosphate (1 mol). Mild acid hydrolysis of the polysaccharide yielded a heptasaccharide repeating unit. The structure of the heptasaccharide repeating unit was determined by high-resolution NMR spectroscopy which includes various homonuclear (DQF-COSY, TQF-COSY, NOESY and HOHAHA) and heteronuclear experiments (HMQC), including linkage assignments by 1H-13C long-range correlation (HMBC). Complete 1H and 13C NMR assignments for the intact polysaccharide yielded the covalent structure of a heptasaccharide repeating unit: [Formula: see text] The structure of the strain 38 polysaccharide is closely related to those of Streptococcus mitis J22 and Streptococcus oralis 34. Thus, the difference between the strain 38 and J22 heptasaccharides was at their reducing ends, with GaLNAc beta-(1-->3)-Gal in the former and Gal beta-(1-->3)-GalNAc in the latter, while the difference between the 38 heptasaccharide and 34 hexasaccharide was at the non-reducing ends, where a rhamnose branch occurred in the former but not the latter structure. When compared by their quantitative precipitin curves with rabbit antibodies against each streptococcal strain, the strain 38 polysaccharide reacted more like the polysaccharide of strain J22 than that of strain 34. In contrast, each strain was recognized by the Gal- and GalNAc-reactive lectins of Actinomyces spp., but only strains 38 and 34 were recognized by GalNAc-sensitive lectins of other streptococci. These findings strongly support the hypothesis that the immunogenic features of these polysaccharides are distinct from those detected by lectin binding.

Animals↗

Streptococcus peroris sp. nov. and Streptococcus infantis sp. nov., new members of the Streptococcus mitis group, isolated from human clinical specimens.

Taxonomic studies were performed on eight strains of alpha-haemolytic streptococci that showed very low DNA-DNA hybridization similarity values with all established members of the mitis group of the genus Streptococcus. These strains were isolated from the tooth surface and pharynx of humans. 16S rRNA gene sequence analysis showed that these strains belonged to the mitis group, but that they fell into two new branches. DNA-DNA hybridization demonstrated two new similarity groups. From the results of the present study, the names Streptococcus peroris sp. nov. and Streptococcus infantis sp. nov. are proposed for these new groups. The type strains are O-66T (= GTC 848T = JCM 10158T) and O-122T (= GTC 849T = JCM 10157T), respectively.

Base Composition↗

Streptococcus infantarius sp. nov., Streptococcus infantarius subsp. infantarius subsp. nov. and Streptococcus infantarius subsp. coli subsp. nov., isolated from humans and food.

Eighteen strains isolated from human specimens or from food products were characterized as atypical variants of mannitol-negative Streptococcus bovis. They were tested for extended biochemical criteria, ribotyping and DNA-DNA hybridization in order to define their taxonomic status. These strains were demonstrated to constitute a DNA relatedness group that includes strains of DNA group 4 of Farrow et al. (1984). Comparative analysis of 16S rRNA sequences demonstrated that these strains represent a new species which belongs to the Streptococcus bovis/Streptococcus equinus complex and which has been provisionally named S. infantarius by Bouvet et al. (1997). Biotyping and ribotyping allowed differentiation of these strains from the aesculin-positive strains of S. bovis belonging to the previously described biotypes I, II.1 and II.2. The results of the ribotyping and hybridization assays demonstrated the presence of two different DNA subgroups within the 18 strains. On the basis of these data, the names S. infantarius subsp. infantarius (aesculin-negative for five strains out of seven, including the type strain HDP 90056T = NCDO 599T) and S. infantarius subsp. coli (aesculin-positive, reference strain HDP 90248 = NCDO 2620) are proposed as the names for these two subspecies within the S. infantarius species.

Animals↗

Physico-chemical and structural properties of the surfaces of Peptostreptococcus micros and Streptococcus mitis as compared to those of mutans streptococci, Streptococcus sanguis and Streptococcus salivarius.

The surface properties of nine Streptococcus mitis and four Peptostreptococcus micros strains from the oral cavity were examined and compared with a large group of oral streptococci. Zeta potential and contact angle measurements were employed to determine physico-chemical cell surface properties. In addition, elemental surface concentration ratios were obtained via X-ray photoelectron spectroscopy, and surface structures were examined with transmission electron microscopy. The S. mitis and P. micros strains were found to have higher isoelectric points, higher hydrophobicities and higher N/C surface concentration ratios than some other oral streptococci. The combined data suggest that both species possess large amounts of surface protein. All the S. mitis strains displayed abundant surface fibrils in negative staining, but the P. micros strains were devoid of surface appendages indicating that surface protein is present in different forms in the two species. The surfaces of S. mitis and P. micros type strains differed significantly from the other strains examined.

Bacterial Adhesion↗

Relatedness among penicillin-binding protein 2b genes of Streptococcus mitis, Streptococcus oralis, and Streptococcus pneumoniae.

Penicillin-binding protein (PBP) 2b similarities among Streptococcus mitis, S. oralis, and S. pneumoniae using DNA fingerprinting and sequencing were investigated. The polymerase chain reaction (PCR) was performed on 41 penicillin-susceptible and -resistant clinical isolates of S. mitis and S. oralis using the susceptible S. pneumoniae R6 PBP 2b primers. PCR products were then analyzed using Hinf I and Sty I restriction enzymes. Of 41 S. mitis/S. oralis isolates studied 15 strains produced a PCR product of a similar size to that of S. pneumoniae R6. On fingerprinting these 15 strains, 11 different patterns were seen using Sty I restriction enzyme and 12 different patterns with Hinf I. The PBP 2b genes of the S. mitis and S. oralis isolates studied were found to be very heterogenous. The PBP 2b genes of two S. mitis isolates, MICs 0.5 and 2 micrograms/ml, were sequenced. These PBP 2b genes were found to possess a mosaic structure when compared to those of other S. pneumoniae and viridans streptococcal species. Analysis of these mosaic blocks indicates that both S. mitis strains contain areas that originated from S. pneumoniae as well as regions of unknown origin. PBP 2b sequence comparisons of a susceptible S. oralis with reported sequences of S. pneumoniae R6 and S. mitis NCTC 10712 revealed what appears at this stage to be nucleotide regions unique to S. oralis. A penicillin-resistant S. oralis strain contained a pneumococcal region of 272 bp that was flanked by S. oralis sequences. These specific S. oralis regions have been located in PBP 2b genes of penicillin-resistant S. oralis and S. pneumoniae isolates described from Europe and South Africa.

Bacterial Proteins↗

Genetic transformation in Streptococcus sanguis. Effects on genetic transformation by culture filtrates of Streptococcus sanguis (serogroups H and W) and streptococcus mitis (mitior) with reference to identification.

Streptococcus sanguis (serogroups H and W) strains are frequently competent in genetic transformation. In a collection of 18 strains of S. sanguis isolated from blood cultures, 12 strains were spontaneously competent in transformation by streptomycin-resistant DNA, and three strains were induced to competence by the use of culture filtrates containing competence factor (CF). Culture filtrates from spontaneously competent S. sanguis strains only induced competence in transformation of strains of the same species. In addition to the three "constitutional" types of S. sanguis with regard to specificity of transformation previously reported (originally represented by the strain Challis, 13b and NCTC 7863), a possible fourth type, (represented by strain 21452) was found. Further studies are required to ascertain the role of these types. The "constitutional" types are characterized by the range of activity of their CF's. These specificities seem useful in identification of S. sanguis and its separation from S. mitis (mitior). Tests of spontaneously competent strains with culture filtrates from other competent strains showed variable effects on the transformation frequencies in different strains.

Bacterial Proteins↗

Mixed continuous cultures of Streptococcus mutans with Streptococcus sanguis or with Streptococcus oralis as a model to study the ecological effects of the lactoperoxidase system.

Mixed continuous cultures of Streptococcus species were obtained, using complex carbohydrate (mucin) as a source of nutrients, to study the ecological effects of oxygen and the lactoperoxidase system. S. mutans NCTC 10449 was unable to grow as a pure culture on mucin, but attained a significant population size in the presence of S. oralis and S. sanguis strains. The cell densities of the anaerobic mixed cultures decreased when oxygen was supplied, and S. mutans was more suppressed by oxygen than were S. sanguis and S. oralis. However, the concentrations of hydrogen peroxide (30 mumol/l in the mixed culture of S. mutans with S. sanguis and 640 mumol/l in the culture with S. oralis) indicated a certain resistance of the organisms to hydrogen peroxide. Addition of lactoperoxidase and thiocyanate to the oxygen-supplied cultures had a differential effect on the streptococcal populations. While S. mutans was inhibited, and even disappeared in the culture with S. oralis, the growth of S. sanguis and S. oralis was unaffected. This latter observation was in accordance with the OSCN- reductase activities of these organisms. When hydrogen peroxide was also added together with lactoperoxidase and thiocyanate, a further inhibition of S. mutans in the culture with S. sanguis was observed. Under these conditions, S. oralis was also inhibited, perhaps by the strong accumulation of OSCN-, exceeding the capacity of the OSCN- reductase. The effects of lactoperoxidase on mixed cultures may reflect the situation in the mouth.(ABSTRACT TRUNCATED AT 250 WORDS)

Colony Count, Microbial↗

Physiological characteristics of Streptococcus dysgalactiae and Streptococcus uberis and the effect of the lactoperoxidase complex on their growth in a chemically-defined medium and milk.

Aerobic or anaerobic degradation of glucose by Streptococcus dysgalactiae and Streptococcus uberis yielded products qualitatively similar to those observed previously for Streptococcus agalactiae. There were, however, quantitative differences. Though acetoin was formed during aerobic growth of Streptococcus uberis, there was none with Streptococcus dysgalactiae. Differences between Streptococcus dysgalactiae and Streptococcus uberis in their aerobic metabolism of glucose was in lower oxygen consumption (.5 mol/mol of glucose), greater conversion of glucose to lactic acid, and lower molar growth yields with Streptococcus uberis. Cell suspensions of Streptococcus uberis had strong peroxidase activity, and no hydrogen peroxide accumulated during the respiration on glucose. With Streptococcus dysgalactiae, there was more oxygen consumed during growth (1.5 mol/mol of glucose used), greater conversion of glucose to acetic and formic acids and carbon dioxide, and a cell yield of about 6 g of dry cells more per mole of glucose than with Streptococcus uberis. This increase in molar growth yield with Streptococcus dysgalactiae over Streptococcus uberis could be nearly all accounted for by differences in the amount of substrate level adenosine triphosphate generated. Cell suspensions oxidizing glucose accumulated hydrogen peroxide and showed no peroxidase activity. Streptococcus dysgalactiae showed the same growth relationships in three milk media as Streptococcus agalactiae, although growth and acid formation values were much lower. Growth inhibition by the lactoperoxidase complex was reversed with cystine. Acid formation by Streptococcus uberis was decreased by the lactoperoxidase complex and increased by the addition of cystine; however, neither appeared to affect the growth of the organism.

Animals↗

[The effect of zinc chloride mouthwashes on caries-inducing plaque streptococci. 1. In vitro research on the antimicrobial efficacy of zinc chloride on reference strains of Streptococcus mutans (BHT), Streptococcus sanguis (HKop) and Streptococcus salivarius (NCTN 8618)].

The antimicrobial efficiency of zinc chloride solution in concentration from 0.1% to 2.0% and 0.2% chlorhexidine solution has been examined on the strains of reference in a comparative study. Three test methods were applied: the agar diffusion test, the dilution test and the suspension test. As was to be expected the antibacterial effect of the zinc chloride solution was dependent on concentration. Between 0.1% and 2.0% the zinc chloride solution showed a bacteriostatic effect on the strains of reference only; the application of higher concentrated solutions is not indicated intraorally. 0.2% chlorhexidine solution showed a bactericidal effect on the test germs.

Chlorhexidine↗