Identification of streptococci in a medical laboratory

A total of 965 cultures of streptococci received at a reference unit for identification were examined with API-20 Strep kits and also by established methods. The API method, although it needed to be supplemented with additional tests, largely overcame the difficulty that pyogenic streptococci are usually identified by their serological reactions and that biochemical tests are used for the identification of the other streptococci. Representatives of at least 24 established or possible species were identified.     

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.     

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.     

Taxonomic study of "tufted mitior" strains of streptococci (Streptococcus sanguinis biotype 11); recognition of a new genospecies

The taxonomic position of tufted strains of streptococci, phenotypically resembling Streptococcus mitis and previously referred to as 'tufted mitior' was investigated. By 16S rRNA sequence analysis, it was clear that the "tufted mitior" strains belonged to the mitis group of species within the genus Streptococcus. It was confirmed that these strains were taxonomically independent at the species level, sharing less than 43%, DNA-DNA similarity with all established species of the mitis group. However biochemical test data obtained, using three commercial identification kits (Rapid ID32 Strep, STREPTOGRAM, and Biolog GP-plate) together with in-house biochemical tests employing 4-MUF-linked fluorogenic substrates did not reveal sufficient differential tests with which to identify the "tufted mitior" strains unequivocally. From these data, we conclude that these "tufted mitior" strains represent a new taxon within the mitis group of the genus Streptococcus, and propose that they should be considered as a genospecies until differential phenotypic characteristics are found for their identification.     

Identification with the aid of various serological reactions of L forms of streptococci isolated from the live organism]

Growth inhibition, agglutination, precipitation, and passive hemagglutination tests were used for the identification of the L-forms of streptococci isolated from the organism of experimental rabbits both after the infection with the L-forms of streptococci and with the streptococci of group A. The tests were positive not only with the antiserum of homologous, but also of heterologous strains of the L-form of streptococcus, group A. The L-form cultures isolated from the experimental animals failed to differ from the laboratory strain of the L-forms of streptococcus, group A, by serological properties.     

Confirmatory identification of group D streptococci by slide co-agglutination

Group D streptococci were identified by slide co-agglutination and the results obtained were compared with conventional methods for the presumptive (bile esculin medium, salt tolerance) and confirmatory (Lancefield precipitin test, latex agglutination) identification of these bacteria. Of 137 clinical specimens examined, the co-agglutination procedure showed a 100% specificity and a 96.5–100% sensitivity (depending on the method of testing) in the identification of group D streptococci. The slide co-agglutination test was easy to perform and interpret and offers a valuable alternative to other less rapid techniques for the confirmatory identification of group D streptococci.     

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.     

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.     

Comparison of Several Laboratory Media for Presumptive Identification of Enterococci and Group D Streptococci

Bile-esculin (Difco), modified bile-esculin (Difco), selective enterococcus (Pfizer Co.), and eosin-methylene blue agar media were evaluated for accuracy in identifying group D streptococci. The regular and modified bile-esculin media performed equally well, but the selective enterococcus and eosin-methylene blue agars did not accurately differentiate the group D from non-group D streptococci. A modified 6.5% NaCl broth was compared with unmodified 6.5% NaCl broth and Streptococcus faecalis (SF; Difco) broth for accuracy in differentiating enterococci from non-enterococci. The modified and unmodified broths worked equally well in the salt tolerance test, but the lot-to-lot variability of SF broth made this medium unusable as an indicator for enterococci. With all seven media, the number of strains giving positive tests decreased when the tests were incubated at 45 C as compared with 35 C, and the number of strains giving negative tests increased. Thus, the number of false-positive identifications decreased, but the number of false-negative identifications increased. Variability in the susceptibility of group D non-enterococcal streptococci to oxacillin and methicillin sensitivity disks limited the usefulness of these tests for presumptive identification of either enterococci or group D streptococci.     

A note on a code for the identification of the 'viridans' streptococci

A simple numerical code for the identification of 'viridans' streptococci was devised with Facklam's system of classification. This is based on biochemical criteria and consists of nine tests, with an additional five for the identification of closely related species and the subspecies or biotype of Streptococcus mutans. The 'viridans' streptococci can be divided into 10 species with the aid of this scheme while five subspecies and an additional biotype of Streptococcus mutans can be recognized. Advantages of this scheme are that the numerical code differentiates between isolates of the same species, distinguishes between biochemically active and non-active species and indicates similarities between species.     

A note on a code for the identification of the 'viridans' streptococci

A simple numerical code for the identification of 'viridans' streptococci was devised with Facklam's system of classification. This is based on biochemical criteria and consists of nine tests, with an additional five for the identification of closely related species and the subspecies or biotype of Streptococcus mutans. The 'viridans' streptococci can be divided into 10 species with the aid of this scheme while five subspecies and an additional biotype of Streptococcus mutans can be recognized. Advantages of this scheme are that the numerical code differentiates between isolates of the same species, distinguishes between biochemically active and non-active species and indicates similarities between species.     

A short scheme for the identification of 'viridans' streptococci commonly isolated from the human mouth

A short scheme for the identification of those viridans streptococci commonly isolated from the human mouth was produced by selecting the most discriminating tests from the study made by Facklam (1977). The scheme comprised tests for the fermentation of mannitol, sorbitol, inulin, raffinose, melibiose and trehalose; the hydrolysis of arginine and aesculin and the production of dextran or levan from sucrose. The scheme was cost-effective in terms of preparation time and materials and was shown to reliably identify over 86% of the streptococci strains used in the study.     

Direct Identification of Bacteria in Positive Blood Culture Bottles by Matrix-Assisted Laser Desorption Ionisation Time-of-Flight Mass Spectrometry

Background

With long delays observed between sampling and availability of results, the usefulness of blood cultures in the context of emergency infectious diseases has recently been questioned. Among methods that allow quicker bacterial identification from growing colonies, matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry was demonstrated to accurately identify bacteria routinely isolated in a clinical biology laboratory. In order to speed up the identification process, in the present work we attempted bacterial identification directly from blood culture bottles detected positive by the automate.

Methodology/Principal Findings

We prospectively analysed routine MALDI-TOF identification of bacteria detected in blood culture by two different protocols involving successive centrifugations and then lysis by trifluoroacetic acid or formic acid. Of the 562 blood culture broths detected as positive by the automate and containing one bacterial species, 370 (66%) were correctly identified. Changing the protocol from trifluoroacetic acid to formic acid improved identification of Staphylococci, and overall correct identification increased from 59% to 76%. Lack of identification was observed mostly with viridans streptococci, and only one false positive was observed. In the 22 positive blood culture broths that contained two or more different species, only one of the species was identified in 18 samples, no species were identified in two samples and false species identifications were obtained in two cases. The positive predictive value of bacterial identification using this procedure was 99.2%.

Conclusions/Significance

MALDI-TOF MS is an efficient method for direct routine identification of bacterial isolates in blood culture, with the exception of polymicrobial samples and viridans streptococci. It may replace routine identification performed on colonies, provided improvement for the specificity of blood culture broths growing viridans streptococci is obtained in the near future.     

Amylase-binding as a discriminition among oral streptococci

The ability of 51 strains, belonging to Streptococcus sanguis, 'S. mitior', S. oralis and related groups, to bind salivary amylase was studied. Most strains were grouped according to their DNA-relatedness and then compared using 14 phenotypic tests. S. mitis, 'S. mitior' and three relatively new groups of strains ('CR', 'MGH' and 'Tufted mitior') bound salivary amylase, while strains of S. sanguis and S. oralis did not. The ability of strains to bind amylase or not was remarkably consistent within groups and the test proved to be reproducible, rapid and easy to perform. Combination of the amylase-binding test with 6 other conventional physiological tests allowed the construction of a dichotomous identification key which correctly identified 95% of strains for which genetic data was available. These findings suggest that the ability of organisms to bind salivary amylase could become a key test in identification schemes for certain oral streptococci.     

Phenotypic and molecular methods used for identification of oral streptococci and related microorganisms

The oral cavity contains the greatest biodiversity, over 70 species being isolated from mouth mucosa, saliva, denture surfaces and/or dental-plaque. The oral streptococci, representing over 80% of the mouth micro flora, are able to synthesize glucosyl-transferases, enzymes involved in glucans production. Glucans are involved in production of an extracellular slime layer promoting adhesion and formation of a dental plaque biofilm. The 43 isolates studied obtained from partially and/or totally edentulous, were identified by VITEK system using gram-positive identification cards. Species-specific regions within the genes coding for glucosyl-transferases (gtf genes) were targeted for PCR identification of isolates. Sequencing of 16S rRNA was used as gold standard for strain confirmation. VITEK system identified a number of 11 strains as S. mitis/oralis, 12 strains as S. anginosus/gordonii, 12 strains as S. sanguinis/parasanguinis, 3 strains as S. salivarius, 3 strains as S. plurianimalium, 1 strain as S. cristatus and 1 strain as S. alactolyticus, respectively. The PCR system targeting gtf genes was able to identify S. oralis, S. salivarius and S. gordonii strains. Sequence of 16S rRNA discriminated among streptococci species and revealed 16 strains of Leuconostoc mesenteroides. Many studies are needed in order to select the most reliable phenotypic and genotypic methods in order to improve the identification algorithm for oral streptococci used by clinical laboratories. Their accurate identification is mandatory for better understanding their role in human infections.     

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.     

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.     

Numerical classification and identification of Bacillus sphaericus including some strains pathogenic for mosquito larvae

Ninety-one strains of Bacillus sphaericus, including representatives of all the established DNA homology groups, related round-spored and oval-spored species, and six strains pathogenic for mosquito larvae, were examined for 155 characters. Numerical analyses (Jaccard coefficient/average linkage clustering) based on the 88 variable features revealed 14 clusters at the 79% similarity level that contained more than one strain and 17 single member clusters. All insect pathogenic strains were recovered in a single cluster and the classification was in accord with an established classification based on DNA sequence homology. Two frequency matrices for probabilistic identification were constructed and tested. A comprehensive matrix comprising 14 mesophilic, round-spored taxa and 27 tests gave good results for identification of hypothetical median organisms, cluster overlap and identifications of representative strains (based on data generated in the classification study). Reference strains for the 14 taxa and eight additional insect pathogenic strains were examined for the 27 tests and were correctly identified with high scores using this matrix. A second matrix comprising seven taxa and 13 tests also performed well in the theoretical evaluation and correctly identified the reference strains and insect pathogenic strains.     

The Use of Standardized Reference Antigens in the Serogrouping of Streptococci

Streptococci isolated from the dental plaque of five animal species were identified by physiological and serological methods. Twenty-nine strains of streptococci considered to resemble Streptococcus mitior or Strep. bovis on the basis of physiological data reacted with Lancefield group B or group K antisera respectively in tube precipitation tests. Further serological studies with standardized antigens from known serogroup B and K streptococci revealed that only three of these 29 isolates had been serogrouped accurately and carried the appropriate group antigen. Comparisons were made between the reactivity of the antisera produced by Difco and Wellcome Reagents with acid and autoclaved extracts of the strains. It was shown that the accuracy of serogrouping such isolates could be improved if the tests were made in a gel diffusion system that included a reference antigen.