Pulsed-Field Gel Electrophoresis Is More Discriminating Than Multilocus Enzyme Electrophoresis and Random Amplified Polymorphic DNA Analysis for Typing Pyogenic Streptococci

The SmaI restriction endonuclease digestion patterns of chromosomal DNAs from 99 pyogenic streptococci belonging to Lancefield group A (41 Streptococcus pyogenes), group C (seven S. dysgalactiae, 11 \QS. equisimilis\W, three S. equi, eight S. zooepidemicus) and group G (25 human group G Streptococcus, four S. canis) were analyzed by pulsed-field gel electrophoresis (PFGE), and the results were compared with those previously obtained by multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA analysis (RAPD). PFGE revealed 93 distinct types among the 99 strains, and no patterns were common to strains of different species. The discriminatory power of PFGE was greater than that of MLEE and RAPD for groups A and G streptococci. The polymorphism among group C streptococci was similar with the three techniques. PFGE is, therefore, the most efficacious method for epidemiological typing of pyogenic streptococci. Received: 31 July 1996 / Accepted: 17 August 1996     

The incidence of inflammation among patients suffering from cervix cancer with positive beta haemolytic streptococci cultures from genital tract

AimThe main goal of this investigation was to evaluate the influence of positive beta haemolytic streptococci culture from the genital tract on patients receiving radiation therapy who suffer from cervical cancer. The other aim was to observe radiation therapy complications.BackgroundGroup B streptococci (GBS), group C streptococci (GCS) and group G streptococci (GGS) have been described as frequent invasive pathogens in elderly patients, often in association with underlying medical conditions including immunodeficiency and cancer.Materials and methodsIn the years 2006–2015, vaginal swabs from 452 patients were examined. A total of 118 women with positive beta haemolytic streptococci (BHS) groups A, B, C, F, G cultures were analysed, of whom 111 were diagnosed with cervix cancer of IB to IVA degree according to the FIGO 1988 clinical classification.ResultsOf the 452 patients suffering from cervix cancer 26.1% were positive for A, B, C, F or G group BHS isolated from the genital tract. All of the 114 examined strains were sensitive to beta-lactam antibiotics. The antimicrobials for which resistance was noted were erythromycin, clindamycin, ciprofloxacin and tetracycline.ConclusionsPositive cultures of BHS from the genital tract were demonstrated to occur in patients with cervix cancer. Complications were found during radiotherapy in 30 (27%) of these patients, including 20 (18%) patients suffering from clinical symptoms of inflammation. When beta-lactam antibiotics are not recommended because of allergy, sensitivity tests to other drugs are necessary.     

Allelic variation of the streptokinase gene in β-hemolytic streptococci group C and G isolates of human origin

Abstract Genetic diversity of the streptokinase gene ( sk ) from 36 strains of S. equisimilis and 54 strains of group G streptococci was examined. The strains were isolated from patients with various streptococcal disease manifestations and healthy carriers. The region of the gene that corresponds to amino acid residues 174–244, was PCR amplified. The amplified product was subjected to Mlu I, Pvu II, Dra I and Dde I digestion. Based on the restriction enzyme digestion patterns nine sk alleles were recognized. There was no correlation between the various sk gene alleles and streptococcal disease manifestations. Three of the nine sk gene alleles, sk 4, sk 7, and sk 8, were detected earlier among group A streptococci. The other six alleles were unique to S. equisimilis and group G streptococci. The most common alleles were sk 5, found in 21/90 (23%) and sk 10 detected in 43/90 (47%) of the strains. Alleles sk 1 and sk 2, the most frequent among group A streptococci, were not found among the strains in the present investigation. Thus, it appears that the sk gene has been evolving in line with other species distinguishing features of the streptococci.     

Effects of medium composition on penicillin-induced hydrolysis and loss of RNA and culture turbidity in group A streptococci.

Exposure to penicillin G of exponentially growing cultures of group A streptococci growing in chemically defined medium (CDM) can lead to extensive loss of culture turbidity. Significant reductions in culture turbidity did not accompany comparable treatments of group A streptococci growing in Todd-Hewitt broth (THB). Studies with THB and a high-molecular-weight (greater than 12,000) fraction of THB demonstrated that components in this complex medium inhibited the efflux of RNA hydrolysis products from otherwise intact cells. Hydrolysis products accumulated intracellularly and inhibited the extensive hydrolysis of RNA and consequently the loss of culture turbidity. Results of survival studies with cultures of group A streptococci exposed to penicillin G in THB demonstrated that this treatment protocol produces conditions of phenotypic tolerance relative to exposure in CDM. In combination, these findings provide further support for the hypothesis of RNA hydrolysis as the bactericidal mechanism of penicillin G action in this nonlytic death phenotype.     

Characterization of clinical isolates of group C and G streptococci on the basis of protein G gene

Treatment of human group C and G streptococci with cyanogen bromide results in solubilization of surface protein G molecules. Strain-to-strain variation in the quanity, molar mass and functional activity of protein G extracted from representative group C and G isolates led to the identification of three structurally and functionally distinct forms of the protein. Using different molecular biological approaches it was possible to determine the group of streptococci (C or G), or the quantity of IgG and HSA domains. Presented at theInternational Conference on Recent Problems in Microbiology and Immunology, Košice (Slovakia), 13–15 October 1999.     

Localization of group A streptococcal nontype-specific antigens and their occurrence among streptococci of different groups]

It was shown by immunodiffusion methods that nontypespecific antigens revealed in the HCl extracts of streptococcus, group A, were localized in the cell wall. In B, E, H, K, L, M, P, S, T streptococci groups there was revealed only one, and in C and G streptococci groups--two antigens identical to the HTC antigens of streptococci, group A. Besides, an antigen, which was apparently specific specific for group A streptococcus only, was detected. The data obtained should be taken into consideration in the elaboration of improved method of grouping and typing group A streptococcus.     

Some observations about the camp reaction and its application to human β haemolytic streptococci

Summary Detailed information concerning the performing and the reading of theCamp reaction is given in this study. ComparableCamp reactions were carried out on 1223 human streptococcals strains. Group B strains reacted positive in 99.5%++94%,+in 5% and ±in 0.5%. Group A strains were positive in about 80%; ++ in 30%, +in 28% and ±in 22%. The reactions in the A group were often incomplete. There are reasons to suppose that the responsible agents in the two groups of streptococci are not quite identical. C and G group streptococci both reacted positive in only 0.5%; all tested F and ungrouped strains were negative. By adding theCamp reaction to simple bacteriological methods it is possible to recognize with certainty human group B streptococci. Some observations were made over the interaction of both components of the reaction, the β toxin and the lytic agent produced by B group streptococci (Camp factor). Preliminary chemical studies make it likely that the Camp factor has a polypeptidic structure. Aided by a grant from the National Health Research Councils T.N.O.     

NAD+-glycohydrolase productivity of haemolytic streptococci assayed by a simple fluorescent method and its relation to T serotype

Abstract The ability of haemolytic streptococci to produce NAD⁺-glycohydrolase was investigated by a fluorescent assay. Enzyme production was found in 31 (91%) of 34 group A, 17 (61%) of 28 group C and eight (27%) of 30 group G isolates. The high producers were found in 22 (65%) of group A, one (4%) of group C and none of group G isolates. The high producers of the group A isolates belonged to T-1, T-3, T-4 or T-12 serotype. These results suggest that NAD+-glycohydrolase productivity of streptococci is closely related to specific Lancefield's groups or T serotypes.     

Composition and properties of a group A streptococcal teichoic acid

Teichoic acid-like material extracted by cold trichloroacetic acid from lyophilized whole cells of streptococci from groups A,D,E,O, and T was shown to give a positive precipitin reaction with group antisera. Similar material from cells of groups B,C,F,G,H,K,L,M,N,P,Q,R, and S did not give a positive reaction with group antisera. The group A material also reacted with anti-E serum; however, the opposite did not occur. A similar result was also obtained on the group T material and anti-O serum. The group A teichoic acid was purified by Sephadex column chromatography, and was shown to be free of cell wall peptidoglycan and polysaccharide, and ribitol teichoic acid. It was composed of glycerol, phosphate, alanine, and glucosamine. Alkaline hydrolysis showed the presence of ester-linked alanine and glucosaminylglycerol. Phosphorus was released from ester linkage by alkaline phosphatase. N-acetylglucosamine produced a 72% inhibition of the precipitin test at a level of 10 mumoles, and d-alanine methyl ester was significantly stronger than the l-alanine ester. A single precipitin band was seen with group A serum. The data indicate that teichoic acid of group A streptococci is a polymer composed of glycerol phosphate and containing N-acetylglucosamine and alanine. Antisera to these streptococci contain antibodies specific for the alanine and the glucosamine linkages. The use of serum containing antibodies to alanine-polyglycerophosphate shows that the occurrence of this type of teichoic acid is widespread among the streptococci.     

Presumptive Identification of Group A, B, and D Streptococci

A battery of five tests was used for presumptive identification of the pathogenic streptococci. The non-serological methods included determination of hemolysis for all strains, bacitracin susceptibility for group A streptococci, hippurate hydrolysis by group B streptococci, and bile-esculin reaction for group D streptococci. Enterococcal group D streptococci were differentiated from non-enterococcal group D streptococci by 6.5% NaCl tolerance. Two other categories of streptococci resulted: beta-hemolytic streptococci non-groups A, B, or D; and alpha- or nonhemolytic streptococci, not enterococci, not further identified (viridans streptococci). The tests were used as a battery and not as single entities. In this manner more than 99% of the group A, 99% of the group B, 81% of the beta-hemolytic streptococci non-group A, B, or D, 99% of the group D enterococci, 97% of the group D non-enterococci, and 94% of the viridans streptococci were correctly identified.     

Identification of beta-hemolytic streptococci isolated from hospitalized children in Baghdad.

Two hundred and twenty four hospitalized children in Baghdad aged between 1 month and 10 years were examined for Streptococcal infections. Thirty-four percent of the throat and saliva specimens were positive for beta-hemolytic streptococci. Males were more susceptible to infection with group A streptococci than females. Streptococcus of group A was isolated from 39.5% of the positive cases while group G was 47.4%. The etiological significance of the latter group in tonsillitis and otitis media is to be further investigated. Ninety six percent of the isolated streptococci were T typable and 13.3% of the strains were M typable. A high frequency of type T-11 was found in streptococcal infections. T type 3875 was found to be a new provisional type. All isolates were M untypable, and antiopacity factor negative except for two isolates of T type 4 which were positive in both typings.     

Contribution to the taxonomy of haemolytic corynebacteria

In an attempt to assess the taxonomic relationships among human (Corynebacterium haemolyticum), animal (Corynebacterium pyogenes bovis) haemolytio corynebacteria, typical corynebacteria (Corynebacterium diphteriae mitis, C. ovis, C. ulcerans) and group A and G streptococci, a number of biochemical parameters were established: the DNA content of G + C, the presence of the cytochrome system, composition of fatty acids in free lipids and production of carboxylic acids as end products of fermentation. It was found that according to the above criteria, streptococci differed significantly from the corynebacteria studied. In addition, it was possible to differentiate a subgroup of typically aerobic haemolytic corynebacteria (different from both human and animal corynebacteria), possessing a complete cytochrome system, producing propionic acid and having a different composition of fatty acids.     

Monoclonal antibodies against the common polysaccharide ofStreptococcus agalactiœ

A monoclonal antibody giving a dominant reaction with the group-specific polysaccharide of streptococcus group B in an ELISA test has been developed. The purified polysaccharide exhibited a high positivity with reference anti B streptococcal antiserum in the ELISA test. Cross-tests of antibodies with other groups of streptococci provided a minimum cross-reaction only in the case of G streptococci. Monoclonal antibodies were prepared usingStreptococcus agalactiœ S 589 MT strain isolated from a case of bovine mastitis which does not express Ia, Ib, II, III, IV and V type antigens, nor C, R and X protein antigens.     

Demonstration of a Bactericidal Substance Against β-Hemolytic Streptococci in Supernatant Fluids of Staphylococcal Cultures

Staphylococcal skin isolates belonging to phage type 71 were found to produce a bactericidal substance against some streptococci, pneumococci, and corynebacteria. Fifteen strains of group A streptococci belonging to 13 different M types, group C streptococci, and group D streptococci were uniformly inhibited on solid media and in broth by membrane-filtered supernatant fluids of the staphylococcal broth cultures. Inhibition of group G streptococci and other staphyloccoci was variable, and no inhibition of group B streptococci or of a variety of gram-negative rods was demonstrable. A quantitative variation observed to exist among susceptible organisms was a function of the inoculum size of the inhibited strains. The bactericidal substance could be detected best from 24 to 48 hr after inoculation of the staphylococci in tryptic soy broth or in a dialysate of tryptic soy broth. Little or no bactericidal activity was noted when the organisms were grown in several other liquid media. The bactericidal substance was nondialyzable and could be precipitated with ammonium sulfate. It was heat-stable and its activity was not altered within a pH range of 4.0 to 8.5. Pronase and three times crystallized trypsin totally abolished its activity. The concentrated ammonium sulfate precipitate could be fractionated on a Sephadex G-100 column into several peaks, with the bactericidal activity localized to a single peak.     

Electrophoretic patterns of extracellular deoxyribonuclease (DNase) and their correlation with T-type in group A streptococci

Molecular heterogeneity of the extracellular deoxyribonuclease (DNase) in group A streptococci was demonstrated in 42 clinical isolates. Although polyacrylamide gel electrophoretic patterns of the extracellular DNase of all the isolates were heterogeneous, they could be divided into five main patterns with respect to the presence or absence of three DNase components including DNase B. By comparing the electrophoretic patterns of DNase in all the isolates with their T-types, we found that the patterns were quite characteristic for their T-types, especially in the prevalent T-types 12 and 1, and that the isolates of T-types 12 and 1 produced DNase B as their major extracellular DNase. Relative DNase B activity in the total extracellular DNase activity of group A, B, and G isolates was determined by the rapid method of neutralization with anti-DNase B antibody. The results showed neutralization of DNase activity in all the isolates of group A streptococci, largely corresponding to their T-types, but not of the isolates of groups B and G. These results indicate that the electrophoretic patterns of the extracellular DNase of group A streptococci are closely correlated with their T-types, suggesting the physicochemical taxonomic value of these properties.     

Distribution and serological specificity of sialidase produced by various groups of streptococci

The occurrence of a streptococcal sialidase (designated St-sialidase) in culture fluids of various streptococci was investigated. St-sialidase was found to occur in strains belonging to groups A, B, C, E, G, H, and L, and the unclassified strains, Streptococcus sanguis and Streptococcus uberis. St-sialidase of group A was confined predominantly to types 4 and 22. St-sialidases, extracted from the culture fluids of some selected strains, were antigenic, eliciting the formation of antibody which effectively neutralized the enzymatic activity of the enzyme. Antisera to the St-sialidases of groups A, B, C, E, G, and L, and Streptococcus sanguis were produced in rabbits. The St-sialidases of groups A, B, and E streptococci were serologically distinct and group-specific. The St-sialidases from groups C, G, and L were serologically homologous, but distinct from St-sialidases of the other groups. Antiserum to the enzyme of strain 10557 (S. sanguis) cross-reacted with the St-sialidase of strain 9927 (S. uberis).     

A Three-Year Survey of the Antibacterial Spectra of the More Commonly Used Chemotherapeutic Agents

Eight major bacterial groups (25,000 strains) of gram-positive and gram-negative organisms which were isolated from a variety of clinical specimens were tested by the disc-plate and tube dilution procedure. The in vitro antibacterial spectra of 17 commonly used chemotherapeutic agents were recorded and evaluated statistically during a three-year period. Penicillin G, erythromycin and chloramphenicol were very effective against members of the diplococci and streptococci genera. The synthetic penicillins inhibited 99% of Staph. aureus whereas penicillin G was effective against only 45% of these strains. There was a significant increase in the number of tetracycline-resistant strains of both D. pneumoniae and the Lancefield Group A streptococci. A yearly increase in gram-negative pathogens was noted. These organisms (i.e. Escherichia, Aerobacter, Proteus, Pseudomonas) showed greater resistance to the majority of chemotherapeutic agents than did the gram-positive organisms. The percentage of susceptible strains for each bacterial group appears in the text.     

Sialidase-like Enzymes Produced by Group A, B, C, G, and L Streptococci and by Streptococcus sanguis

A group of enzymes were prepared from the culture fluids of streptococci belonging to groups A, B, C, G, and L, and from a strain of Streptococcus sanguis. These streptococcal enzymes (designated St-sialidases) released a substance shown to belong to the sialic acid group from the specific substrate BSM-St, a sialomucoid prepared from bovine submaxillary gland. They were inactive on N-acetylneuramin lactose prepared from bovine colustrum and on a sialomucoid prepared from bovine submaxillary mucin, whereas these substances are susceptible to sialidases produced by group K streptococci and by Vibrio cholerae. Some of the St-sialidases were markedly activated by divalent cations, but others showed little response. The heat stability of the enzymes produced by the different strains varied. The optimal pH was between 5.5 and 6.5 with acetate buffer and was about 7 with phosphate buffer. K(m) values were determined for the St-sialidases with BSM-St as substrate.     

A 28-kilodalton fibronectin-binding protein of group a streptococci

Lipoteichoic acid (LTA) has been implicated as a major adhesin of group A streptococci that interacts with fibronectin (Fn). It has been suggested that protein adhesins may also be involved in the binding of Fn to streptococci. We searched for such a protein by transblotting membrane preparations from M types 5, 19, and 24 group A streptococci to nitrocellulose and reacting the blot with¹²⁵I-Fn. The Fn reacted with a 28-kDa polypeptide from all three serotypes of streptococci. Using affinity-purified antibodies to the 28-kDa protein in immunoblots of membrane preparations from various streptococci, we demonstrated that the 28-kDa protein is present in all 17 strains tested. Affinity-purified antibodies to the 28-kDa protein also reacted in varying degrees with intact streptococci, demonstrating that the antigen is exposed on the surface of intact organisms. Our results suggest that, in addition to LTA, group A streptococci contain a common Fn-binding moiety that is expressed as a major component of membrane preparations and that is accessible on the surface of streptococci for interactions with Fn.     

Lysis and Lysogenization of Groups A, C, and G Streptococci by a Transducing Bacteriophage Induced from a Group G Streptococcus

A temperate bacteriophage, designated GT-234, was isolated from a group G Streptococcus after ultraviolet irradiation. After several single-plaque passages in a group G indicator strain, this phage formed plaques in 3 of 14 group A strains, in 3 of 15 group C strains, and in 4 of 13 group G strains-but not in some representatives of several other serogroups. After propagation in each of the sensitive strains, the progeny from each was shown to be the same phage by (i) adsorption and plaque formation in each of the other groups, (ii) lysogenization in each of the other groups, (iii) high titers on infection of each serogroup, regardless of the group of propagating strain, and (iv) neutralization of infection in each of the other groups by antiserum against the phage propagated in group G. Phage GT-234 is serologically related to virulent group A phage A25, from which it is morphologically indistinguishable. Like A25, it is a transducing phage. Other studies showed that A25, as well as a group A temperate transducing phage (AT-298), could also infect strains of group C and G. These results indicate a need for reassessment of group specificity and phage receptors among streptococci of groups A, C, and G and raise possibilities for intergroup transduction.