Identification of Group B Streptococci by Immunofluorescence Staining

Gamma globulin fractions of rabbit antisera prepared with whole cell vaccines of group B types Ia, Ib, II, and III and labeled with fluorescein isothiocyanate stained group B streptococci type specifically. Type Ic cells, which contain the Ia polysaccharide antigen of type Ia and the Ic protein antigen of type Ib, were specifically stained by both Ia and Ib conjugates. A group B conjugate pool (B pool) that contained one conjugate specific for each group B type at its predetermined titer gave positive fluorescent-antibody (FA) reactions (4+ intensity) with group B stock strains and negative FA reactions (less than 2+ intensity) with stock strains of streptococcal groups A, C through H, and K through U, viridans streptococci, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria gonorrhoeae, and representative Enterobacteriaceae. Examination of 883 clinical isolates submitted to the Streptococcus Laboratory (Center for Disease Control, Atlanta, Ga.) for identification revealed a 99.1% agreement between FA and culture-precipitin methods. All 305 group B streptococci identified by culture-precipitin and six nonhemolytic group B streptococci missed initially by culture tests were identified correctly by FA. Results of cultural and FA methods in a double-blind study of 99 vaginal swabs agreed on 96 of 99 strains. Three nonhemolytic group B streptococci were identified first by FA and later confirmed by culture-precipitin tests.     

Comparison of Several Laboratory Media for Presumptive Identification of Enterococci and Group D Streptococci

Bile-esculin (Difco), modified bile-esculin (Difco), selective enterococcus (Pfizer Co.), and eosin-methylene blue agar media were evaluated for accuracy in identifying group D streptococci. The regular and modified bile-esculin media performed equally well, but the selective enterococcus and eosin-methylene blue agars did not accurately differentiate the group D from non-group D streptococci. A modified 6.5% NaCl broth was compared with unmodified 6.5% NaCl broth and Streptococcus faecalis (SF; Difco) broth for accuracy in differentiating enterococci from non-enterococci. The modified and unmodified broths worked equally well in the salt tolerance test, but the lot-to-lot variability of SF broth made this medium unusable as an indicator for enterococci. With all seven media, the number of strains giving positive tests decreased when the tests were incubated at 45 C as compared with 35 C, and the number of strains giving negative tests increased. Thus, the number of false-positive identifications decreased, but the number of false-negative identifications increased. Variability in the susceptibility of group D non-enterococcal streptococci to oxacillin and methicillin sensitivity disks limited the usefulness of these tests for presumptive identification of either enterococci or group D streptococci.     

Cores, Microbial Organelles Possibly Specific to Group D Streptococci

A long, thin, approximately cylindrical core spans the interior of cells of 24-hr cultures of all group D streptococci that were examined, five strains of Streptococcus faecalis, single strains of S. faecalis subsp. zymogenes and S. durans, and three strains of Streptococcus spp. In one strain of S. faecalis, serial section electron microscopy showed that most cells possess a core. The core is 0.10 to 0.16 mum thick and consists of a matrix and an axial array of ribosomelike particles. It resembles one of two types of cores present in a stable protoplast form of one of the S. faecalis strains. Cores were not present in single strains of S. pyogenes (beta-hemolytic group A), S. agalactiae (group B), S. dysgalactiae (group C), S. equisimilis (group C), and S. mitis (viridans group) that were examined; nor were cores observed in single strains of Staphylococcus aureus, Escherichia coli, and Bacillus megaterium. Cores may be useful, therefore, in identification of group D streptococci. For preservation and rapid recognition of cores, a glutaraldehyde-osmium tetroxide sequence of fixation appears superior to the osmium tetroxide method often employed in processing bacteria for electron microscopy.     

Rapid Extraction Method with Pronase B for Grouping Beta-Hemolytic Streptococci

The enzyme, Pronase B, was used to extract the C carbohydrates of betahemolytic streptococci for serological grouping. Reference strains of streptococci, groups A through N, were accurately grouped by using this extraction method. Of the beta-hemolytic streptococci isolated from patients' cultures, 1,197 were classified as either group A or not group A by this method. There was 100% correlation with the reference Lancefield method. In contrast, false reactions occurred with the presumptive bacitracin disc method in 4.1% of the group A and 12.7% of the non-group A cultures. The Pronase method proved simple, accurate, and readily adaptable to the clinical laboratory routine.     

Antibiotic sensitivity of beta-hemolytic streptococci isolated from throat swabs and purulent material

The aim of this study was to evaluate the prevalence and susceptibility of beta-hemolytic streptococci isolated from throat swabs (142--29.9%) and purulent material (333--70.1%) taken from patients treated at University Hospital dr. A. Jurasz in Bydgoszcz Collegium Medicum. L. Rydygier in Bydgoszcz, Nicolaus Copernicus University in Torun in 2005-2009. Of the 475 tested strains, 156 (32.8%) were identified as S. pyogenes. This species accounted for 38.8% of strains isolated from purulent material and 19.0% of swabs from the throat. Among the strains isolated from throat swabs of 62 (43.7%) were identified as Streptococcus group C. Only 5.1% strains were identified as Streptococcus group F. All strains of beta-hemolytic streptococci were susceptible to ampicillin or penicillin, fluoroquinolones, vancomycin and linezolid. Erythromycin-susceptible strains was 83.8%, and 89.1% for clindamycin. A total of 51.3% of erythromycin resistance strains had the cMLS(B) phenotype (63.3% for strains from throat swabs and 46.3% of the purulent materials). Sensitivity to tetracycline was characterized by 51.2% of strains of beta-hemolytic streptococci. The percentage of strains susceptible to this antibiotic among isolates from throat swabs was 63.1%, and purulent material--48.0%. The lowest percentage of strains susceptible to tetracycline (14.1%) were found among S. agalactiae and Streptococcus group G (33.6%) strains. During the study time, saw an increase in the percentage of strains susceptible to tetracycline and erythromycin.     

Time, Cost, and Efficacy Study of Identifying Group A Streptococci with Commercially Available Reagents

During the 12-month period primary throat, wound, and skin cultures, tentatively identified as B streptococci, were submitted by 10 different clinical laboratories for evaluation. A total of 692 beta-hemolytic streptococci were isolated from cultures submitted and examined in parallel by the fluorescent-antibody, precipitin, and bacitracin techniques. An evaluation of the specificity and sensitivity in conjunction with basic and personnel costs was determined for each method. The standard Lancefield precipitin method was established as the standard by which the bacitracin and flourescent antibody techniques were compared. With some variation depending on the commerical source of the disc, approximately 7% of the strains examined produced false reactions with the bacitracin disc. False-negative reactions were rarely noted by the group A fluorescent antibody technique (0.5%), but an appreciable number of other Lancefield groups (B, C, and G) were nonreactive with homologous conjugates.     

The pharyngeal beta-hemolytic streptococcal carrier state among blood donors

459 blood donors aged 18-50 years were examined in 1987-1988 in Moscow. Among them, carrier state with respect to beta-hemolytic streptococci was detected in 107 donors (23.3%). The number of carriers gradually decreased with the increase of age of the examined donors. Group C streptococci occurred least of all (6.9%). Group A beta-hemolytic streptococci were isolated in 16.7% of the carriers. The isolation rate of streptococci from blood achieved its maximum in autumn and winter months and did not depend on preceding diseases, unhealthy working conditions, the rhesus factor and, with the exception of group A streptococci, the blood group. Among tonsillectomized donors carrier state with respect to beta-hemolytic streptococci occurred 2.2 times less frequently than among donors who had not undergone tonsillectomy. Carrier state with respect to beta-hemolytic streptococci was accompanied by higher levels of salivary sIgA antibodies to polysaccharide A, serum antibodies to polysaccharide A and circulating polysaccharide A. All beta-hemolytic streptococci were sensitive to erythromycin. All groups of streptococci showed the highest percentage of cultures resistant to gentamicin and tetracycline. In 100% of cases group A streptococci were sensitive to benzylpenicillin, methicillin, ampicillin, erythromycin and lincomycin.     

Antibiotic sensitivity of the microflora isolated in ENT diseases]

Sensitivity of beta-hemolytic streptococci of group A, Streptococcus viridans, Staphylococcus aureus, epidermal staphylococci, pneumococci, Proteus and Ps. aeruginosa isolated in 1975-1978 from patients with tonsillitis, otitis, sinusitis and other otorhinolaryngological diseases was studied with respect to 19 antibiotics. Data on comparison of the antibiotic sensitivity of the microflora isolated from the patients with otorhinolaryngological diseases in 1964-1974 and 1975-1978 are presented. It was shown that beta-hemolytic streptococci were highly sensitive to all the antibiotics tested except tetracycline. Among Streptococcus viridans the strains resistant to many antibiotics were more frequent than among beta-hemolytic streptococci. Most of the Staphylococcus aureus strains were sensitive to gentamycin, cephaloridin, oxacillin and resistant to the other antibiotics. The epidermal staphylococci were characterized by approximately the same antibiotic sensitivity as Staphylococcus aureus. Resistance of the predominating majority of the Pneumococcus strains to tetracycline was noted. Proteus and Ps. aeruginosa were resistant to all the antibiotics except aminoglycosides. The microflora isolated from the cases with otorhinolaryngological diseases in 1975-1978 were mainly characterized by lower antibiotic sensitivity than that isolated from the cases with the same diseases in 1964-1974. It is possible to suppose that the microorganisms isolated from the patients with otorhinolaringological diseases had no significant differences with respect to their antibiotic sensitivity from those isolated from the patients with other pathological processes.     

Detection and Enumeration of Fecal Indicator Organisms in Frozen Sea Foods: II. Enterococci

Consistently high recoveries of enterococci as compared to the low numbers of coliforms obtained from the same samples of frozen sea foods are indirect evidence that enterococci are better indicators of contamination in such foods.

The use of azide dextrose broth, modified by the incorporation of bromthymol blue, and of ethyl violet azide broth as presumptive and confirmation tests, respectively, were found to be highly specific for the detection and enumeration of enterococci in these samples. Tetrazolium agar medium, when used as a third step after the confirmation test, provides a reliable differentiation of Streplococcus faecalis types from other group D streptococci. A simple procedure is described for further identification of S. faecalis varieties and other enterococcal species.

Incidence of biotypes within certain species is noted and relationships of these subgroups to the organisms described by other workers is discussed.

The striking resistance of all group D streptococci to dihydrostreptomycin and polymyxin B seems to offer promise for evolving a new selective medium for these organisms.

     

Periurethral aerobic microflora of pregnant and non-pregnant women.

Seventy-two pregnant and 88 non-pregnant women were examined to see whether the periurethral region had been colonised with group B streptococci (Streptococcus agalactiae), enterococci, and Gram-negative rods belonging to the Enterobacteriaeceae. A semi-quantitative method was used for periurethral sampling, and paired urethral swabs were also collected to compare the isolation rates of group B streptococci from the two sites and with the two sampling methods. A higher isolation rate was found with periurethral sampling. Most specimens showed no or scanty growth of Gram-negative rods. Pregnancy was often associated with heavy growth of enterococci. Sampling performed during menstruation and while oral contraceptives were being used produced high isolation rates of group B streptococci. These results seem to suggest that the periurethral area might protect against genital colonisation with group B streptococci as it does against urinary tract infection and that hormonal factors influence the carriage of these organisms.     

Broad-host-range shuttle vectors for screening of regulated promoter activity in viridans group streptococci: isolation of a pH-regulated promoter

Viridans group streptococci are major constituents of the normal human oral flora and are also identified as the predominant pathogenic bacteria in native valve infective endocarditis. Little information is available regarding the regulation of gene expression in viridans group streptococci, either in response to changes in the oral environment or during development of endocarditis. We therefore constructed a set of broad-host-range vectors for the isolation of promoters from viridans group streptococci that are activated by specific environmental stimuli in vitro or in vivo. A genomic library of Streptococcus gordonii strain CH1 was constructed in one of the new vectors, and this library was introduced into a homologous bacterium by using an optimized electroporation protocol for viridans group streptococci. Because viridans group streptococci entering the bloodstream from the oral cavity encounter an increase in pH, we selected promoters upregulated by this specific stimulus. One of the selected promoter sequences showed homology to the promoter region of the hydA gene from Clostridium acetobutylicum, the expression of which is known to be regulated by the environmental pH. The isolation of this pH-regulated promoter shows that S. gordonii can sense an increase in the environmental pH, which serves as a signal for bacterial gene activation. Furthermore, this demonstrates the usefulness of these new selection vectors in research on adaptive gene expression of viridans group streptococci and possibly also of other gram-positive bacteria.     

Characteristics of a Bacteriocin Derived from Streptococcus faecalis var. zymogenes Antagonistic to Diplococcus peumoniae

A bacteriocin-producing strain of Streptococcus faecalis var. zymogenes (E-1) was isolated from clinical material (conjunctiva). The active substance differed from bacteriocins described by other investigators primarily in its spectrum of antibacterial activity, especially by its marked inhibition of Diplococcus pneumoniae. The E-1 bacteriocin also inhibited nonhemolytic strains of enterococci as well as one-third of the Viridans group of streptococcal strains investigated. The degree of inhibition, however, as indicated by the size of the zones against the latter organisms, was significantly reduced. No activity was detected against any of the strains belonging to the following groups of bacteria: hemolytic enterococci, beta-hemolytic streptococci, nonhemolytic streptococci, staphylococci, and various gram-negative species. Similarly, three strains each of Bacillus cereus and Listeria monocytogenes and one strain of Erysipelothrix insidiosa were not inhibited. The bacteriocin was able to diffuse through bacterial membranes as well as cellulose dialyzer tubing. It was inactivated by heating to 80 C for 20 min but resisted inactivation by either trypsin or chloroform.     

Evaluation of the usefulness of selected commercial systems for identification of Streptococcus species

The usefulness was assessed of three commercially available systems for rapid identification of streptococcal strains. The studied material comprised 68 strains of streptococci and enterococci (including 24 standard strains) belonging to serological groups: A (14 strains), B (10), C (11), D (10), F (3) and G (10), as well as 10 S. pneumoniae strains. The strains were isolated from throat, nasal, wound swabs, blood, pus of inpatients and throat and nasal swabs of outpatients. For the identification of streptococci 3 commercially available systems were used: API 20 STREP (bioMerieux, France), rapid ID 32 Strep (bioMerieux, France), Streptoplast PPL 18 (HTL, Poland). The determinations were done according to producer's instructions. The highest percent of correctly identified strains was obtained with the rapid ID 32 Strep--80.9%, with the API 20 STREP--76.4% strains were identified correctly and with the PPL 18--61.8%. The study showed that the API 20 STREP and rapid ID 32 STREP are suitable for the identification of streptococcal strains from groups: A, B, C, D, F and enterococci--group D. The proportions of correctly identified strains from these groups with the Streptoplast PPL 18 were lower than those determined with the bioMerieux systems. Using of three identification systems streptococci from group G and S. pneumoniae strains cannot be identified.     

Broad-Host-Range Shuttle Vectors for Screening of Regulated Promoter Activity in Viridans Group Streptococci: Isolation of a pH-Regulated Promoter

Viridans group streptococci are major constituents of the normal human oral flora and are also identified as the predominant pathogenic bacteria in native valve infective endocarditis. Little information is available regarding the regulation of gene expression in viridans group streptococci, either in response to changes in the oral environment or during development of endocarditis. We therefore constructed a set of broad-host-range vectors for the isolation of promoters from viridans group streptococci that are activated by specific environmental stimuli in vitro or in vivo. A genomic library of Streptococcus gordonii strain CH1 was constructed in one of the new vectors, and this library was introduced into a homologous bacterium by using an optimized electroporation protocol for viridans group streptococci. Because viridans group streptococci entering the bloodstream from the oral cavity encounter an increase in pH, we selected promoters upregulated by this specific stimulus. One of the selected promoter sequences showed homology to the promoter region of the hydA gene from Clostridium acetobutylicum, the expression of which is known to be regulated by the environmental pH. The isolation of this pH-regulated promoter shows that S. gordonii can sense an increase in the environmental pH, which serves as a signal for bacterial gene activation. Furthermore, this demonstrates the usefulness of these new selection vectors in research on adaptive gene expression of viridans group streptococci and possibly also of other gram-positive bacteria.     

New Method of Grouping Beta-Hemolytic Streptococci Directly on Sheep Blood Agar Plates by Coagglutination of Specifically Sensitized Protein A-Containing Staphylococci

A technique is described that allows the grouping of beta-hemolytic streptococci directly upon the primary colony. This was accomplished by applying a small drop of specifically sensitized protein A-containing Staphylococcus aureus over a colony of streptococci, rocking the plate to allow mixing of the particles with the soluble group-specific polysaccharide, which in the case of beta-hemolytic streptocicci was produced in abundance during colony formation, and observing for agglutination of the sensitized particles. Such a simple test for group A beta-hemolytic streptococci should allow accurate identification of group A streptococci in small laboratories, such as in clinics or physicians' offices, as well as in the larger public health and private laboratories.     

Occurrence and drug-resistance of beta-hemolytic streptococci

The aim of this study was the analysis of drug-resistance and frequency appearance of beta-hemolytic streptococci strains which were isolated in 2003-2005 in the University Hospital at the L. Rydygier Collegium Medicum in Bydgoszcz University of Nicolaus Copernicus in Toruń. Among investigeted beta-hemolytic streptococci the most frequency isolated species was S. agalactiae. All isolates examined in our study were susceptible to penicillin, the higest rate of resistance was found for tetracycline. The rates of resistence to macrolide-lincosamide-streptogramin B (phenotyp MLS(B)) were as follows: S. agalactiae (18.7%), S. pyogenes (10.1%), group G streptococci (10.6%) and group C streptococci (8.0%). In our study we presented also a special case patient from which in investigeted period S. agalactiae was isolated twenty eight times. For ten chromosomal DNA isolated from this patient three different PFGE profiles were obtained.     

Use of a methylene blue azide medium for isolation of enterococci

A methylene blue azide medium (MBA), developed by Schaedler, Dubos, and Costello to isolate enterococci from the gastrointestinal tract of animals, was evaluated. This was done by comparing the isolation of enterococci from feces and saliva on the medium. Fifty-two catalase-negative, gram-positive cocci from human feces isolated from MBA were classified as enterococci. All strains grew in S F, 6.5% NaCl, and streptomycin broths, and all fermented mannitol. The isolates were provisionally subdivided into Streptococcus faecalis and S. faecium groups. S. faecalis-like strains fermented glycerol and pyruvate aerobically and produced acid in Snyder's medium (initial pH, 4.8). The S. faecium group fermented raffinose. Among all strains, several tests were variable. These included growth at 45 C, in 0.1% tellurite and in methylene blue milk. Three methods were employed to isolate and identify enterococci from the oral cavity. Direct streaking of MBA with saliva failed to produce any growth on the medium. Two other methods, with the use of various selective broths to promote the recovery of oral enterococci, failed to produce any bacteria capable of growing on MBA. The MBA-isolated fecal strains and oral viridans streptococci were generally indistinguishable on Mitis-Salivarius and K F agars. In experiments with fecal material, no gram-negative bacilli were found among the isolates selected. The MBA medium was judged as a high selectivity-low yield medium, and may provide a means of separating fecal and nonfecal enterococci.     

Group Q Streptococci I. Ecology, Serology, Physiology, and Relationship to Established Enterococci

The group Q streptococci possess unique serological and physiological characteristics which differentiate them from established enterococci. The group Q antigen was not demonstrable in all strains; however, all possessed the group D antigen. All group Q strains were physiologically similar regardless of whether or not they possessed the group Q antigen. These strains differed from the established enterococcal species, as they neither hydrolyzed arginine nor initiated growth in 1.0% methylene blue-milk. They also differed radically in the fermentation of various carbohydrates, especially the polyhydric sugar alcohols. The results indicate that the group Q streptococci constitute a unique taxonomic entity; the species designation Streptococcus avium sp. n. is suggested, owing to their characteristic occurrence in chicken fecal specimens.     

Presumptive Identification of Group D Streptococci: the Bile-Esculin Test

Six tests commonly used for the presumptive identification of group D streptococci were evaluated. Strains tested included 282 group D streptococci and 366 non-group D. Ratios of percentages of group D to non-group D strains which gave positive reactions for each test are as follows: bile-esculin, 100:2; salt tolerance, 88:24; heat tolerance, 100:80; SF broth, 86:1; KF broth, 99:40; and methylene blue milk reduction, 90:17. These data indicate that the bile-esculin test provided a reliable means of identifying group D streptococci and differentiating them from non-group D streptococci. Methodology for reading and interpreting positive reactions and time of incubation of the bile-esculin medium was defined. Evidence of the need for standardization of salt and heat-tolerance tests was obtained.     

Microbiological Study of Streptococcal Bacteremia

During a 2.5-year study, streptococci were isolated from 280 patients (19% of those with bacteremia). Of this number, 54 had group D streptococci (45 were enterococci), 218 had α- or γ-hemolytic nongroup D streptococci, and 49 had β-hemolytic streptococci.