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.     

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.     

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.

     

Comparison of Selective Media for Isolation of Presumptive Group D Streptococci from Human Feces

Pfizer Selective Enterococcus (PSE) agar, a medium containing bile, sodium azide, and esculin, was evaluated for its sensitivity and selectivity for detection and enumeration of presumptive group D streptococci in human feces. SF broth and SF broth plus agar (1.5%), representing selective media in common use, were studied simultaneously. Presumptive group D streptococci were recovered on PSE agar from the feces of all 25 subjects. No growth was observed in 8% of specimens in SF broth. No gram-negative organisms were recovered in any medium. PSE agar has the advantages of selecting out Streptococcus bovis, earlier appearance of distinctive reactions, and lack of requirement for special incubation temperature.     

Studies on Media for Enumerating Enterococci in Frozen Vegetables

A study was made of the relative sensitivity and specificity of presumptive and confirmatory media for the most probable number enumeration of enterococci in frozen vegetables. Azide dextrose broth yielded the highest numbers of confirmable enterococci and its sensitivity was shown to be comparable to nonselective media. The use of ethyl violet azide broth as a confirmatory medium resulted in a significant number of false positive tests. Growth in broth containing 6.5% sodium chloride incubated at 45 C for 2 days was found to be a more specific confirmatory test for enterococci.     

Evaluation of a new presumptive medium for group D streptococci.

A new medium designated as D streptococcus-enterococcus broth was formulated and evaluated for the enrichment and isolation of strains of serological group D streptococci. This medium was made by modifying Todd-Hewitt broth. Most-probable-number multiple-tube and membrane filter techniques were employed to estimate the numbers of enterococci in known cultures, wastewater, and other samples. Preliminary most-probable-number counts with this medium were as much as 3 logs higher than those counts obtained from four other media with which it was compared. The methodology for using this medium to estimate the numbers of group D streptococci in water is discussed.     

Evaluation of a new presumptive medium for group D streptococci.

A new medium designated as D streptococcus-enterococcus broth was formulated and evaluated for the enrichment and isolation of strains of serological group D streptococci. This medium was made by modifying Todd-Hewitt broth. Most-probable-number multiple-tube and membrane filter techniques were employed to estimate the numbers of enterococci in known cultures, wastewater, and other samples. Preliminary most-probable-number counts with this medium were as much as 3 logs higher than those counts obtained from four other media with which it was compared. The methodology for using this medium to estimate the numbers of group D streptococci in water is discussed.     

Differentiation of enterococci from other group D streptococci by means of a specific monoclonal antibody

We report on the specificity of a monoclonal antibody which reacts with autoclaved extracts of four species of enterococci but does not react to the same extent with similar extracts from two non-enterococcal group D streptococci. The monoclonal antibody also reacts specifically with purified lipoteichoic acid from Streptococcus faecalis but not significantly with purified lipoteichoic acid from the non-enterococcal species Streptococcus bovis and Streptococcus equinus. The specific antigen detected with this antibody could correlate with the definition of the enterococcus subgroup of the streptococci which would provide further evidence that this sub-group is taxonomically distinct from the other group D streptococci.     

Enrichment Procedure for Use with the Membrane Filter for the Isolation and Enumeration of Fecal Streptococci in Water

By the use of PYC enrichment medium, the recovery of fecal streptococci from river water has been increased more than twofold over that of M-Enterococcus Agar. Of the isolates tested, 94.6% could be classified as enterococci or enterococcus biotypes. This method seems to yield a larger number of strains which would not normally be revealed. Serological typing of atypical streptococcus strains isolated indicates that the majority of these “biotypes” can be placed in the enterococcus group.     

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.     

Evaluation of Thallium Acetate-Citrate Medium for Isolation of Enterococci

Thallous acetate-citrate (TAC) agar, a selective medium for isolating enterococci from frozen foods, was evaluated. Two types of bacteria, micrococci and group N streptococci, were responsible for erroneously high enterococcus counts on TAC agar. Selectivity of the medium was improved by the addition of 0.01% sodium azide.     

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.     

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.     

Evaluation of Pfizer selective enterococcus and KF media for recovery of fecal streptococci from water by membrane filtration.

Pfizer selective enterococcus (PSE) and KF agars were compared for their recovery of fecal streptococci from sewage effluent on membrane filters. The results showed that PSE agar is highly selective for the enterococci. The tan color resulting from esculin hydrolysis, which was not always visible on the surfaces of the colonies, is not considered a necessary differential characteristic on PSE agar since more than 90% of all colonies recovered on membrane filters were confirmed as fecal streptococci and 86% were confirmed as enterococci. The detection of esculin hydrolysis on membrane filters was not improved by using the new Millipore type HC filter. KF agar recovered significantly greater numbers of organisms but was not as selective, with 83% of the typical colonies being confirmed as fecal streptococci and 54% as enterococci. An attempt to improve the selectivity of KF agar while retaining its inclusiveness by incubation at 45 C was not successful.     

Evaluation of Selective Media for Enumeration of Group D Streptococci in Bovine Feces

Eight commercially available selective media were evaluated by the membrane filter technique. Enterococcosel broth (BBL) consistently gave the best recovery of group D streptococci, particularly of Streptococcus bovis.     

Hydrogen peroxide and superoxide radical formation in anaerobic broth media exposed to atmospheric oxygen.

Fourteen different broth media were autoclaved under anaerobic conditions and then exposed to atmospheric oxygen. The hydrogen peroxide and superoxide radical formation as well as the bactericidal effect of the media were studied. The rate of killing of Peptostreptococcus anaerobius VPI 4330-1 was high in media that rapidly autoxidized and accumulated hydrogen peroxide. In actinomyces broth (BBL), 50% of the cells were killed within 2 min, and in Brewer thioglycolate medium (Difco), 50% were killed within 11 min, whereas more than 50% of the cells survived for more than 2 h in Clausen medium (Oxoid), fluid thioglycolate medium (BBL), and thioglycolate medium without dextrose or indicator (Difco). Only media that contained phosphate and glucose had a tendency to accumulate hydrogen peroxide. A solution of phosphate and glucose autoxidized when it had been heated to 120 degrees C for at least 5 min and when the pH of the solution was higher than 6.5. Transitional metal ions catalyzed the autoxidation, but they were not necessary for the reaction to occur. Of the other substances heated in phosphate buffer, only alpha-hydroxycarbonyl compounds autoxidized with accumulation of hydrogen peroxide. Superoxide dismutase decreased the autoxidation rate of most of the broth media. This indicated that superoxide radicals were generated in these media.     

Serological identification of group D streptococci using commercial antisera

Three commercial group D streptococcal antisera were tested for the serological identification of 100 group D enterococci; 20 Streptococcus bovis; 5 isolated from each of the following streptococcal groups: A, B, C and G; and 3 isolates from serological group F. Antisera from Difco Laboratories, BBL, and Wellcome Reagents Limited were used in the classic capillary tube precipitin test on extracts prepared using the Rantz and Randall procedure. No false positive precipitin reactions were observed. Of the enterococcal isolates, all 100 reacted with the Wellcome, 99 reacted with the BBL, and 96 reacted with the Difco group D antisera. However, of the 20 S. bovis isolates, only 2 reacted with the BBL, and 1 reacted with both the Difco and the Wellcome antisera. Each antiserum was then used to prepare staphylococcal coagglutination (CoA) reagents and each isolate was subsequently tested. A simple extraction procedure was performed by suspending colonies of an isolate in a loopful of salin on a microscope slide and gently heating the slide directly in the flame of a Bunsen burner. All 100 enterococci and all 20 S. bovis gave positive results with the BBL and the Wellcome CoA reagents. Using the Difco reagent, 2 S. bovis isolates failed to produce postitive results. No false positive results were observed with the non-Group D isolates. Our results indicate that the CoA technique using commercial group D antisera may provide faster and sometimes more sensitive serological identification than the classic capillary tube precipitin test.     

Hydrogen peroxide and superoxide radical formation in anaerobic broth media exposed to atmospheric oxygen.

Fourteen different broth media were autoclaved under anaerobic conditions and then exposed to atmospheric oxygen. The hydrogen peroxide and superoxide radical formation as well as the bactericidal effect of the media were studied. The rate of killing of Peptostreptococcus anaerobius VPI 4330-1 was high in media that rapidly autoxidized and accumulated hydrogen peroxide. In actinomyces broth (BBL), 50% of the cells were killed within 2 min, and in Brewer thioglycolate medium (Difco), 50% were killed within 11 min, whereas more than 50% of the cells survived for more than 2 h in Clausen medium (Oxoid), fluid thioglycolate medium (BBL), and thioglycolate medium without dextrose or indicator (Difco). Only media that contained phosphate and glucose had a tendency to accumulate hydrogen peroxide. A solution of phosphate and glucose autoxidized when it had been heated to 120 degrees C for at least 5 min and when the pH of the solution was higher than 6.5. Transitional metal ions catalyzed the autoxidation, but they were not necessary for the reaction to occur. Of the other substances heated in phosphate buffer, only alpha-hydroxycarbonyl compounds autoxidized with accumulation of hydrogen peroxide. Superoxide dismutase decreased the autoxidation rate of most of the broth media. This indicated that superoxide radicals were generated in these media.     

Comparison of fluorescent gentamicin-thallous-carbonate and KF streptococcal agars to enumerate enterococci and fecal streptococci in meats.

Two selective and differential media were compared for their abilities to enumerate enterococci and fecal streptococci in pork, beef, and poultry products. Counts obtained on KF streptococcal (KF) agar were compared with counts obtained on fluorescent gentamicin-thallous-carbonate (fGTC) agar. Reactions of 13 known enterococcal species were also observed. All 13 species of enterococci as well as Streptococcus bovis and Streptococcus equinus grew equally well on fGTC agar. KF streptococcal medium allowed growth of most species of enterococci but not S. bovis and S. equinus. Quantitative comparisons between the two media inoculated with pure cultures of known species of enterococci revealed equivalent plate counts following incubation. However, when meat samples were plated, counts on fGTC agar were consistently and significantly higher than counts on KF agar for all sample sources.     

Evaluation of chromogenic media for the isolation of vancomycin-resistant enterococci from stool samples

Aims:  This study sought to evaluate the performance of two chromogenic media designed for the isolation of vancomycin-resistant enterococci (VRE) and compare them with a traditional bile-esculin medium for the isolation of VRE from stool samples.
Methods and Results:  A total of 285 stool samples were inoculated onto Chromogenic VRE Agar (AES VRE agar; AES Chemunex), chromID VRE (bioMérieux) and VRE Agar (Oxoid) both directly and also following broth enrichment. In total 18 strains of vancomycin-resistant Enterococcus faecium were recovered, including 17 harbouring the vanA gene and one with vanB . On direct culture, the sensitivity of the three media was 66·7%, 77·8% and 44·4% and after broth enrichment 66·7%, 83·3% and 77·8% using AES VRE Agar, chromID VRE and Oxoid VRE Agar respectively.
Conclusions:  All three media are useful tools for the isolation of VRE from stool samples. AES VRE Agar and bioMérieux chromID VRE are easier to use than Oxoid VRE Agar due to diffusion of black coloration from the latter.
Significance and Impact of the Study:  This is the first study to evaluate the performance of AES VRE Agar and the first to compare two media containing synthetic chromogens for the isolation of VRE.