The enrichment and early detection of Streptococcus pyogenes in skin lesions, using colistin, oxolinic acid, Todd Hewitt broth and latex agglutination

An improved selective broth, colistin, oxolinic acid, Todd Hewitt broth (COTHB) for the isolation of Streptococcus pyogenes and other streptococci from swabs is described. Staphylococci, coryneforms and Gram negative organisms are inhibited in COTHB inoculated with swabs from skin lesions. Streptococcus pyogenes could be detected by a fluorescent antibody method and by the Streptex and Phadebact streptococcal grouping methods after 6 and 24 h incubation. Other streptococci of groups B, C and G, were also detected. Streptex was the most sensitive and specific method for the detection of streptococci of groups A, B, C, and G in this broth. The method detected 78% of these streptococci in COTHB after 6 h incubation and 93% after 24 h incubation. Of the Strep. pyogenes isolated, 82% were detected in the broth by the Streptex method.     

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.     

Evaluation of an improved rapid coagglutination method for the serological grouping of beta-hemolytic streptococci.

Grouping of beta-hemolytic streptococci was performed with the Phadebact Streptococcus Test, a coagglutination method, and the results compared with serological grouping by the standard Lancefield precipitin method. Of 171 clinical specimens examined, 169 (98.8%) were grouped correctly by the Phadebact Test after 24 h of continuous growth in Todd-Hewitt broth. In a parallel study, 96.9% of specimens that grew after only 4 h of incubation in broth were grouped correctly by the coagglutination method. In both studies, the accuracy of the coagglutination test was increased significantly by elimination of multiple-agglutination reactions through centrifugation of cultures and utilization of the supernatant fluid in the Phadebact Test.     

Nosopharyngeal microflora in ambulatory treated children and adults with upper respiratory tract infections

Upper respiratory tract consists resident and transient bacterial microflora, which in appropriate condition can cause infection. Bacteriological study was performed among 201 patients with upper respiratory tract infections treated in ambulatory. From nasal and pharyngeal swabs Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, and Streptococci group A, B, C, G were isolated. Antibiotic susceptibility testing of isolated strains was performed using CLSI criteria. All isolated strains of streptococci were susceptible to penicillin; some of them demonstrated resistance to macrolides and lincosamides. Few isolated strains of H. influenzae demonstrated resistance to penicillin and cotrimoxazole. Azitromycin resistant strains were not detected. All isolated strains of M. catarrhalis were beta-lactamase positive and demonstrated resistance to penicillin. Strains of methicillin sensitive S. aureus (MSSA) were isolated most frequently from pharyngeal swabs (35.4%) and S. pneumoniae (33.3)--from nasal swabs.     

Presence of chromosomal elements resembling the composite structure Tn3701 in streptococci.

Tn3701, carried by Streptococcus pyogenes A454, is the first chromosomal composite element to be described; it contains in its central region Tn3703, a transposon similar to Tn916. A comparison by DNA-DNA hybridization of Tn3701 with omega(cat-tet) and Tn3951, carried by Streptococcus pneumoniae BM6001 and by Streptococcus agalactiae B109, respectively, revealed that the two latter structures are also Tn3701-like composite elements. The DNAs of 27 other antibiotic-resistant group A, B, C, and G streptococci and of S. pneumoniae BM4200 showed sequence homologies to Tn3701 (14 strains, including BM4200), to the regions of Tn3701 outside of Tn3703 (5 strains), and to Tn916 alone (8 strains). The DNAs of five strains did not detectably hybridize with any probe. The tetM gene was identified in most chromosomal genetic elements coding for tetracycline-minocycline resistance. Since Tn3701-like elements are widely disseminated among antibiotic-resistant streptococci (47% of the 34 strains studied), we propose that Tn3701 be considered the prototype of chromosomal composite elements.     

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).     

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.     

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.     

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.     

A new simple method of curing plasmids in lactic streptococci (Streptococcus cremoris; Streptococcus lactis, plasmids)

A simple method for curing plasmid DNA from lactic streptococci is described. When strains of lactic streptococci are grown overnight at 32 degrees C in an unbuffered medium (M17-) and held at the same temperature for an extended period (96 h), the acid environment induces loss of plasmid DNA of different sizes. If the process of growth in M17- broth followed by extended incubation at 32 degrees C is repeated, most of the plasmids are lost, as revealed by gel electrophoretic profiles of DNA. The method is simple and efficient in curing plasmids of lactic streptococci without use of any mutagenic chemical.     

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.     

Development of high-level streptomycin resistance affected by a plasmid in lactic streptococci

Some lactose-negative (Lac-) mutants of Streptococcus lactis C2 and ML3 exhibited development of very high level streptomycin resistance after incubation with subinhibitory concentrations of the drug for 18 to 22 h. These drug-resistant mutants showed no loss of resistance even after 6 months of subculturing in broth without any drug. The parental Lac+ strains did not show mutation to high-level streptomycin resistance. The Lac+ characteristic of the parental strain was conjugally transferred to Lac- derivatives of C2 and ML3, showing the ability to mutate to high-level resistance. When transconjugants were analyzed for this characteristic, they showed both mutable and nonmutable Lac+ types. The results suggested that genetic information for mutation to high-level streptomycin resistance in lactic streptococci resides on the chromosome, and its expression is affected by a plasmid. The plasmid profiles of strains C2, ML3, C2 Lac-, ML3 Lac-, and two kinds of transconjugants confirmed the presence of a plasmid of approximately 5.5 megadaltons in strains showing no mutation to high-level streptomycin resistance, while strains missing such a plasmid exhibited high-level streptomycin resistance after incubation with subinhibitory concentrations of the drug.     

Development of high-level streptomycin resistance affected by a plasmid in lactic streptococci.

Some lactose-negative (Lac-) mutants of Streptococcus lactis C2 and ML3 exhibited development of very high level streptomycin resistance after incubation with subinhibitory concentrations of the drug for 18 to 22 h. These drug-resistant mutants showed no loss of resistance even after 6 months of subculturing in broth without any drug. The parental Lac+ strains did not show mutation to high-level streptomycin resistance. The Lac+ characteristic of the parental strain was conjugally transferred to Lac- derivatives of C2 and ML3, showing the ability to mutate to high-level resistance. When transconjugants were analyzed for this characteristic, they showed both mutable and nonmutable Lac+ types. The results suggested that genetic information for mutation to high-level streptomycin resistance in lactic streptococci resides on the chromosome, and its expression is affected by a plasmid. The plasmid profiles of strains C2, ML3, C2 Lac-, ML3 Lac-, and two kinds of transconjugants confirmed the presence of a plasmid of approximately 5.5 megadaltons in strains showing no mutation to high-level streptomycin resistance, while strains missing such a plasmid exhibited high-level streptomycin resistance after incubation with subinhibitory concentrations of the drug.     

Identification of a streptococcal penicillin-binding protein that reacts very slowly with penicillin.

Penicillin-binding protein (PBP) 5 of Streptococcus faecium ATCC 9790 has an unusually low affinity for penicillin (50% binding occurred at a penicillin level of 8 micrograms/ml after 60 min of incubation, and the protein only became labeled after 20 min of incubation with high concentrations of radioactive penicillin). PBPs with similar properties are carried by strains of Streptococcus durans, Streptococcus faecalis, and Streptococcus lactis but not by strains of groups A, B, C, and G streptococci or Streptococcus pneumoniae. The strains carrying the slow-reacting PBP demonstrated a sensitivity to penicillin that was several hundred times lower than that of strains not carrying it. Spontaneous mutants with minimal inhibitory concentrations of penicillin of 20, 40, and 80 micrograms/ml were isolated from S. faecium ATCC 9790. They all showed a dramatic increase in the amount of slow-reacting PBP produced. Mutants with increased penicillin resistance were also isolated from wild-type strains of S. durans, S. faecalis, and S. faecium. All of them carried a greater amount of the slow-reacting PBP than that carried by the parent. Finally, it was found that resistant S. faecium ATCC 9790 mutants grew normally in the presence of penicillin concentrations that were far above that saturating all PBPs except PBP 5. Cell growth was, on the contrary, inhibited by a penicillin concentration that saturated the slow-reacting PBP by 90%. This penicillin dose was equal to the minimal inhibitory concentration.     

Effect of Selected Lactic Acid Bacteria on Growth of Staphylococcus aureus and Production of Enterotoxin

Representative strains of 15 species of lactic acid bacteria were examined for their ability to influence growth of Staphylococcus aureus and production of enterotoxin in associative culture. Among the organisms used as effectors the streptococci were most inhibitory, followed by Pediococcus cerevisiae. The lactobacilli and Leuconostoc citrovorum were not inhibitory to growth and only slightly inhibitory to production of enterotoxin. Enterotoxin was detected in all cultures in which the population of S. aureus reached 8 x 10(7) per ml. At lower S. aureus populations no enterotoxin was detected after incubation for 48 h. Mechanisms of inhibition of growth and enterotoxin production by S. aureus strain 243 grown in association with Streptococcus lactis A64 or P. cerevisiae 10791 in APT broth were investigated. Competition for vital nutrients, especially niacin and biotin, and probably production of hydrogen peroxide contribute to inhibition. Production of lactic acid appears to inhibit growth of S. aureus in the early but not the late stages of incubation.     

Nucleic acid hybridization and cell wall composition studies of pyogenic streptococci

Abstract DNA-rRNA and DNA-DNA hybridization studies indicate that the classical pyogenic streptococci can be divided into five homology clusters. Based on these studies the term pyogenic streptococci should be confined to the first cluster consisting of serological groups A, A-variant, C, G ('large' colony, type II) and L.
Streptococci of serological groups B and M form the second cluster. The third cluster is composed of streptococci of serological groups R and S and serological groups U, V and P are found in the fourth cluster. The fifth cluster comprises strains of Streptococcus anginosus S. intermedius, Streptococcus MG and serological groups G ('minute' colony, type I) and F (type I). Most of the test strains contain the peptidoglycan type Lys-Ala_1–3. Only streptococci of serogroups R and S reveal a directly cross-linked peptidoglycan. Rhamnose was found as characteristic component of all cell wall polysaccharides. The impact of our results on the systematics of classical pyogenic streptococci will be discussed.     

Comparison of most probable number and pour plate procedures for isolation and enumeration of sulphite-reducing Clostridium spores and group D faecal streptococci from oysters

A comparative study of methods to enumerate sulphite-reducing Clostridium spores and Group D faecal streptococci in oysters demonstrated that pour plate solid agar techniques gave higher counts than liquid broth most probable number procedures. Reinforced clostridial broth with supplements to detect sulphite reduction was compared with pour plates of egg yolk-free tryptose sulphite cycloserine agar incubated at 37 degrees C for 24 h. Azide dextrose broth was compared with pour plates using Slanetz and Bartley (SB) agar or KF-streptococcus agar at 37 degrees C. Most probable number procedures used for both groups of organisms gave excessive numbers of improbable tube combinations. For enumeration of Group D faecal streptococci, a pour plate technique using SB agar incubated at 37 degrees C for 48 h is recommended.     

Inhibition ofCitrobacter freundii by lactic acid, ascorbic acid, citric acid,Thymus vulgaris extract and NaCl at 31 °C and 5 °C

Citrobacter freundii has been implicated in food spoilage and food poisoning outbreaks. This study examines the effects of some compounds (e.g. citric acid, ascorbic acid, lactic acid, sodium chloride, andThymus vulgaris extract) on growth of two strains of Citrobacter freundii at 31 °C and 5 °C. At 31 °C, lactic acid (0.2%) or ascorbic acid (0.2%) alone completely inhibited growth of the tested strains, as there was 100% reduction in growth of the strains after 24 h incubation in nutrient broth containing these compounds.Thymus vulgaris extract (0.3%) reduced the growth rate (p<0.05), the percentages of inhibition after 24 h incubation were about 60% for both strains. NaCl (5%) greatly reduced growth, the percentages of inhibition were about 84% for both strains. Combination ofT. vulgaris extract (0.3%) and NaCl (4%) together completely inhibited growth ofC. freundii species tested. Ascorbic acid (0.1%) or citric acid (0.03%) did not affect growth of the strains (p>0.05), but a lag occurred before increase in number could be observed. In chicken and fish homogenates, combination of NaCl (4%) and ascorbic acid (0.1%) reduced the growth (p < 0.05) (growth inhibition was 40%). At 5 °C, lactic acid (0.1%) alone greatly reduced the growth (p<0.05). The activity of NaCl, or ascorbic acid alone against the tested strains was greatly increased (p<0.05). ForC. freundii 4, the percentage of growth inhibition after 6 days incubation in broth containing 3% NaCl or 0.1% ascorbic acid were 88% and 72%, respectively. ForC. freundii 38, the percentage of growth inhibition after 6 days incubation in broth containing these compounds were 60% and 54%, respectively.     

Relationship of sporulation, enterotoxin formation, and spoilage during growth of Clostridium perfringens type A in cooked chicken

Sporulation and enterotoxin formation were determined for 17 strains of Clostridium perfringens type A in autoclaved chicken dark meat and in Duncan-Strong sporulation medium. The mean numbers of heat-resistant spores detected after 24 h at 37 degrees C were log10 1.13 to log10 7.64/ml in Duncan-Strong medium and log10 4.93 to log10 6.59/g in chicken. Of 17 strains, 7 formed enterotoxin in Duncan-Strong culture supernatant (1.0 to 60 microgram/ml) and 8 produced enterotoxin in chicken (0.21 to 24 microgram/g). Additional studies with chicken were conducted with C. perfringens NCTC 8239. With an inoculum of 10(6) cells per g, greater than log10 7.99 vegetative cells per g were detected by 4 h in chicken at 37 degrees C. Heat-resistant spores occurred by 4 and 6 h and enterotoxin occurred by 8 and 6 h in autoclaved chicken dark meat and barbecued chicken drumsticks, respectively. Enterotoxin was detected in autoclaved dark meat after incubation at 45 degrees C for 1.5 h followed by 37 degrees C for 4.5 h, but not after incubation at 45 degrees C for 1.5 to 8 h. With an inoculum of 10(2) cells per g in oven-cooked or autoclaved chicken, greater than log10 8.00 vegetative cells per g were detected by 6 to 8 h at 37 degrees C, heat-resistant spores were detected by 8 h, and enterotoxin was detected by 12 h. A statistical analysis of odor determinants of chicken after growth of C. perfringens indicated that, at the 95% confidence level, the product was considered spoiled (off or unwholesome odor) by the time spores or enterotoxin were formed.