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.     

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.     

Improved Procedure for Identification of Group D Enterococci with Two New Media

The aerosol survival in air was determined for Pasteurella tularensis live vaccine strain (LVS) as a function of relative humidity (RH). Three different preparations of bacteria were used: (i) liquid suspension of P. tularensis LVS in spent culture medium; (ii) powders of P. tularensis LVS freeze-dried in spent culture fluid; (iii) P. tularensis LVS freeze-dried in spent culture fluid and then reconstituted with distilled water and disseminated as a liquid suspension. Preparation (i) gave greatest survival at high RH and lowest survival at intermediate RH. Preparation (ii), in contrast, gave greatest survival at low RH and minimum survival at 81% RH. Preparation (iii) was the same as preparation (i), i.e., the process of freeze-drying and reconstituting with distilled water before aerosol formation had little or no effect upon aerosol survival as a function of RH. Hence, control of aerosol survival appears to be through the water content of P. tularensis LVS at the moment of aerosol generation rather than the water content of the bacteria in the aerosol phase.     

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.     

Fluorescent-Antibody Techniques for the Identification of Group D Streptococci: Direct Staining Method

Fluorescent-antibody (FA) techniques were employed in an attempt to develop a rapid test for the identification of group D streptococci. Fresh isolates were obtained from sewege and feces of sheep, cattle, horses, rabbits, chickens, geese, and rats. Identification to species were made by the conventional physiological, biochemical, and serological tests. Both whole and disrupted cells of representative strains of each species were used for the preparation of the group D streptococcus vaccine. Globulin fractions of individual and pooled antisera were labeled with fluorescein isothiocyanate, and the resulting conjugates were tested with homologous and heterologous antigens. The specificity of the conjugates and staining was assessed by adsorption and inhibition tests utilizing controls with homologous and heterologous antigens. Employing the direct staining method and individual and pooled conjugates, it was possible to obtain 84 and 85% positive FA reactions, respectively, with group D streptococcal strains. Trypsinization of the smears prior to staining eliminated all FA cross-reactions observed with non-group D streptococci and staphylococci. These findings suggest that the direct staining method will be of value in the rapid identification of group D streptococci.     

Species Differentiation of Group D Streptococci

Three hundred and fourteen strains of group D streptococci were studied by means of a number of tests. The majority of the strains were identified as Streptococcus faecalis (83 strains), Streptococcus faecium (131 strains), or Streptococcus bovis (32 strains). Several strains (47 or nearly 15%) either shared characteristics of two species or were completely atypical. S. faecalis and S. bovis were more easily identified than S. faecium, which is not sharply defined from the other species and could be subdivided into several fermentative types on the basis of fermentation of arabinose, mannitol, sorbitol, glycerol, and sucrose. The value of some characteristics in species identification is discussed. Growth in the presence of potassium tellurite 1:2,500 and in the presence of 6.5% NaCl and fermentation of arabinose, glycerol, and raffinose are very important tests for the identification of the three species. The reduction of tetrazolium salts, the reduction of litmus milk, and the fermentation of sorbitol may serve as complementary tests for the same purpose. For the differentiation of these three species the “pattern of reactions” is more important than single tests.     

Development of Chemically Defined Media Supporting High-Cell-Density Growth of Lactococci, Enterococci, and Streptococci

Lactococcus lactis IL1403 was used as an experimental strain to develop a chemically defined medium for study of the physiology and metabolic pathways of lactococci. An experimental leave-one-out technique was employed to determine the necessity of each of the 57 chemical components used in medium development. A statistical experimental design approach including three fractional factorial designs and a central composite design was used to optimize the fermentation process with 21 variables composed of 19 nutritional factors grouped from the 57 components and two environmental factors (initial pH and temperature). For L. lactis IL1403, the maximum biomass concentrations obtained with the two optimal chemically defined media developed in this study (ZMB1 and ZMB2) were generally 3.5- to 4-fold higher than the maximum biomass concentrations obtained with the previously described best synthetic media (SA) and 50% to 68% higher than the maximum biomass concentrations obtained with M17, a complex medium commonly used for lactococci. The new chemically defined media support high-cell-density growth of numerous strains of L. lactis, Enterococcus faecalis, and Streptococcus thermophilus.     

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.     

Immunofluorescent Identification of Type 12 Group A Streptococci

The fluorescent antibody (FA) conjugate prepared by labeling streptococcal M type 12 antibody with fluorescein isothiocyanate was found to exhibit considerable nonspecific FA staining with other group A M-serotypes. The cross-reactions could be reduced sufficiently or eliminated by the addition of adsorbed homologous blocking serum (AHB) but not by preimmune serum. The AHB was prepared by adsorbing type 12 antiserum with untreated homologous cells. Comparative staining with unblocked and AHB-blocked FA conjugates enabled type 12 streptococci from clinical specimens to be rapidly and accurately identified.     

Identification of Group A Streptococci by Direct Fluorometry

A simple direct fluorometric method for rapid identification of group A streptococci is described. The method permits the detection of the organism in mixed cultures without the aid of a microscope and is amenable to automated processing of specimens. Experience with the indirect fluorometric method revealed that nontrypsinized cells from a 10-fold dilution of overnight broth cultures could be stained with uniform brilliance with fluorescent antibody (1:15 dilution) and that fluorescent antibody dissociated from such cells at 55 C for 20 min gave serologically specific fluorometric values. With this information, it was possible to develop a simpler fluorometric test which gave results comparable to those obtained by conventional cultural-precipitin grouping techniques. In the direct test described, cultures from throat swabs were incubated overnight, and cells from a 10-fold dilution were stained with specific fluorescent antibody (1:50 dilution) and then rinsed. The stained specimens were transferred to a continuous-filter paper strip (Whatman 3 MM) and read serially in a Turner 110 fluorometer with Corning 5840 and Wratten 2A filters in place. The reagents used required careful standardization and testing to assure that fluorometric readings above a specified value would be indicative of the presence of group A streptococci.     

A Selective Medium for Isolation and Presumptive Identification of the Bacteroides fragilis Group

A new selective medium, Bacteroides fragilis ammonium-sulfate gentamicin (BFAG) agar, for isolation and presumptive identification of the Bacteroides fragilis group is presented in this paper. This semisynthetic medium includes 0.2 g of ammonium sulfate, 0.7 g of lactose, 10 mg of gentamicin, 0.1 mg of aminobenzylpenicillin, 60 units of bacitracin, 20 mg of sodium cholate and 1 mg of sodium azide per 100 ml of medium. Stock cultures of the B. fragilis group grew well on this medium. None of the other 126 gram-positive or negative strains belonging to 40 aerobic or 45 anaerobic species tested grew on this medium. Three of the seven specimens in the clinical trials yielded colonies of only the B. fragilis group on BFAG agar plates. Also BFAG agar plates inoculated with human feces and contents of the alimentary tract (stomach, small intestine, cecum and colon) of mice gave rise to colonies of only the B. fragilis group. The high selectivity and good plating efficiency of BFAG agar enabled us to isolate the B. fragilis group rapidly from various clinical specimens.     

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.     

Identification of Fluorescent-Antibody Labeled Group A Streptococci by Fluorometry

A quantitative system, amenable to automation, for determining the presence of group A streptococci in broth culture is described. After separation from the broth, the cells' protein coats are removed by digestion with 0.1% trypsin, and they are then stained with anti-A fluorescent antibody (FA). Excess FA is removed, and bound FA is put into solution by dissociation with demineralized distilled water. The amount of FA bound to the cells is quantitated by fluorometry of the solution. The level of nonspecific staining is measured by staining the cells with fluorescein-conjugated normal rabbit globulin absorbed with group A cells, dissociating, and quantifying, as above. The two quantities are subtracted to measure specific binding of FA to group A cells. A clinical trial showed 92% agreement with microscopists.