Mutanolysin enhancement of serogrouping of single colonies of streptococci

Calandra, Gary B.; Henson, Charla

doi:10.1128/jcm.15.1.184-186.1982

J Clin Microbiol

1982

DOI: 10.1128/jcm.15.1.184-186.1982

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Mutanolysin enhancement of serogrouping of single colonies of streptococci

Gary B. Calandra¹,

Charla Henson²

Abstract: Single colonies of beta-hemolytic streptococci could be grouped by antibodycoated latex bead agglutination or coagglutination with staphylococci coated with antibody after incubation of the colonies with mutanolysin. This simple and quick procedure provided an enzymatic means of enhancing the sensitivity of tests such as Phadebact and SeroSTAT.

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1982

1987

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Cited by 4 publications

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“…Pronase is not effective in the extraction of group D streptococci (4,5,10). Group D streptococci may be extracted with mutanolysin only after a 4-h incubation (1). Achromopeptidase, although effective for the extraction of some strains of group D streptococci, would not be acceptable for the serogrouping of that serogroup.…”

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Serogrouping single colonies of beta-hemolytic streptococci with achromopeptidase extraction

Slifkin¹,

Cumbie²

1987

J Clin Microbiol

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Achromopeptidase (TBL-1; Wako Chemicals, USA, Inc., Dallas, Tex.) prepared as a 2,000-U/ml solution will extract the serogroup antigens from single colonies of groups A, B, C, F, and G streptococci in 1 min at room temperature. This enzyme extraction is not effective for the serogrouping of all group D streptococcus species. Achromopeptidase extracts can be used with latex or coagglutination reagents. Achromopeptidase is a bacteriolytic enzyme derived from Achromobacter lyticus (7, 8). This enzyme has been reported to lyse a relatively wide spectrum of bacteria including Micrococcus, Bacillus, Sarcina, Staphylococcus, Streptococcus, and Clostridium spp., as well as some gramnegative bacteria (Bacteriolytic Enzyme for Biochemical Use: TBL-1, Wako Chemicals, USA, Inc., Dallas, Tex.). Achromopeptidase has been used for the preparation of bacterial enzymes, DNA, and RNA (7, 8) and for the preparation of protoplasts and spheroplasts (8). The enzyme is now commercially available as a crude product (TBL-1) from Wako Chemicals, USA, Inc. This study demonstrates that achromopeptidase may be used to rapidly extract the specific antigens of groups A, B, C, F, and G streptococci from single colonies grown on sheep blood agar plates. The streptococci used in this investigation were obtained from stock cultures maintained in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) and 10% glycerol (vol/vol) at-70°C. The serogroups included groups A (15 strains), B (22 strains), C (15 strains), D (11 strains), F (8 strains), and G (10 strains). All of the bacteria used in this investigation were streaked on Columbia sheep blood agar (Scott Laboratories, Inc., Fiskeville, R.I.) and incubated at 35°C in ambient air for 12 to 18 h. Gram staining and the catalase test were done on all of the cultures to presumptively identify them as beta-hemolytic streptococci. The serogroups of the strains grown on the blood agar were confirmed by nitrous acid extraction of single colonies (9) and serogrouping with Streptex reagents (Wellcome Diagnostics, Research Triangle Park, N.C.). One colony of each strain of streptococcus was placed in 0.20 ml of 0.05 M Tris hydrochloride buffer solution (pH 8.0) containing 50 to 2,000 U of achromopeptidase per ml. The bacterial suspensions were incubated for 1 to 60 min at 37°C. In other experiments, incubations were done at room temperature. Serogrouping of the respective extracts of streptococci was done with Streptex latex reagents (Burroughs Wellcome Co., Research Triangle Park, N.C.) and the Phadebact coaggfutination reagents (Pharmacia Diagnostics, Piscataway, N.J.). A drop of each extract was placed on a glass microscope slide and mixed with a drop of the respective serodiagnostic reagent. The reactants were mixed by hand for 1 min to determine the agglutination or coagglutination response of each extract.

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mentioning

confidence: 99%

Serogrouping single colonies of beta-hemolytic streptococci with achromopeptidase extraction

Slifkin¹,

Cumbie²

1987

J Clin Microbiol

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Natural history of aortic valve endocarditis in rats

Héraïef¹,

Mp²,

Freedman³

1982

Infect Immun

123

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Sterile aortic vegetations were produced in rats by introducing a polyethylene catheter through the right carotid artery. The catheter was either left in place throughout the experiments or removed before bacterial challenge. Bacterial endocarditis was uniformly produced by intravenous injection of 10(7) colony-forming units of Staphylococcus aureus or Streptococcus intermedius, whether the catheter was left in place or removed. However, in rats with the catheter left in place, bacterial multiplication within the vegetations with both strains was accelerated, and mortality from Staphylococcus aureus infection was increased. Using 10(7) colony-forming units of serum-resistant Escherichia coli as a test microorganism, we found a marked difference in the production of endocarditis depending upon whether the catheter was left in place or removed before injection; only those animals infected with the catheter in place developed infection. From these experiments in rats, it was evident that the presence of a foreign body has a considerable influence on the ability of bacteria to grow within an intravascular vegetation. In addition, a striking difference in the virulence of the three strains studied was established; Staphylococcus aureus was the most, and E. coli the least, pathogenic.

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Enzyme immunoassay for the detection of group A streptococcal antigen

Knigge¹,

Babb²,

Firca³

et al. 1984

J Clin Microbiol

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A competitive inhibition enzyme immunoassay for the detection of Streptococcus pyogenes directly from throat specimens or from solid bacteriological medium is described. Group A-specific polysaccharide adsorbed onto treated polystyrene beads, in conjunction with rabbit antibody to S. pyogenes, was used to determine the presence of the polysaccharide antigen. Inhibition values in excess of 65% were observed with 104 or more CFU of S. pyogenes per test. An inhibition of 25% was demonstrated with as few as 103 CFU per test. Heterologous microorganisms tested at 106 CFU per test reacted at levels of inhibition less than 25%. Two types of bacterial transport medium and swabs of different fiber compositions did not alter the assay performance. Accurate identification of S. pyogenes was achieved by testing single colonies picked directly from blood agar plates which had been incubated for 18 to 24 h. In addition, the assay was performed on throat specimens from children and adults having pharyngitis. A single-swab, blind study was conducted in which enzyme immunoassay reactivity was compared with results of blood agar culture and bacitracin sensitivity. When there were discordant results, serological identification was used as the confirmatory test. At an optimal cutoff value of 40% inhibition, sensitivity and specificity by enzyme immunoassay were 97.0% and 97.9%, respectively, as compared with confirmed culture results. The assay has an incubation time of 3 h and is a sensitive and specific method for the detection of S. pyogenes antigen.

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Mutanolysin enhancement of serogrouping of single colonies of streptococci

Cited by 4 publications

References 9 publications

Serogrouping single colonies of beta-hemolytic streptococci with achromopeptidase extraction

Serogrouping single colonies of beta-hemolytic streptococci with achromopeptidase extraction

Natural history of aortic valve endocarditis in rats

Enzyme immunoassay for the detection of group A streptococcal antigen

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