Serogrouping single colonies of beta-hemolytic streptococci with achromopeptidase extraction

Abstract

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.

A comparison of different pre-lysis methods and extraction kits for recovery of Streptococcus agalacticae (Lancefield group B Streptococcus) DNA from whole blood

“…Previous studies used arbitrary and varying amounts of ACH: 1000U/ml (18), 1500U/ml (15), 2000U/ml (16) or 4000U/ml (19). Using 4000U/ml, Niwa et al (19) reported complete lysis of a range of Gram-positive bacteria in 10-15 minutes at 37ºC.…”

Pudding Proteomics: Cyclomaltodextrin Glucanotransferase and Microbial Proteases Can Liquefy Extended Shelf Life Dairy Products

“…Being able to break down casein into peptides and amino acids, this serine protease is alternatively named caseinase [ 28 ]. It is used to produce bacterial enzymes [ 29 ] and therefore most likely originates from production process of recombinant amylases.…”

Comparison of MIDI Sherlock system and pulsed-field gel electrophoresis in characterizing strains of methicillin-resistant Staphylococcus aureus from a recent hospital outbreak

“…We incubated the lysis mixture at 50ЊC on the basis of their recommendation. The manufacturer's application notes, however, cite procedures in which lysis occurs with ACP at 37ЊC (29,34). Our own experience has shown the plugs to be transparent when removed from the refrigerator, illustrating the enzyme's activity over a very broad temperature range.…”