Direct-Plate Serological Grouping of Beta-Hemolytic Streptococci from Primary Isolation Plates with the Phadebact Streptococcus Test

The grouping of beta-hemolytic streptococcal isolates by a new direct-plate procedure employing Phadebact Streptococcus Test reagents was compared with the results obtained with the 4- and 24-h Phadebact grouping procedure and with the Lancefield grouping obtained with a capillary precipitin test. The new procedure employed a modification of the Phadebact procedure that permitted the grouping of streptococci on glass slides with a minimum of five primary isolated colonies. When only five to eight colonies were available for direct testing with each Phadebact reagent, coagglutination was better manifested when the colonies were disaggregated on a glass slide in a loopful of Tween 80 solution. Further enhancement of the coagglutination reaction was effected when the respective Phadebact reagents were employed in relatively small volumes. The direct-plate procedure permitted the correct identification of 127 out of 129 betahemolytic isolates. The 4-h method correctly identified 192 of the 200 streptococci tested. All of the 200 isolates tested by the 24-h procedure and the Lancefield grouping were correctly identified. The direct-plate Phadebact procedure affords the clinical microbiologist a rapid and reliable means of identifying groups A, B, C, and G beta-hemolytic streptococci. When sufficient numbers of primary colonies are not available for the direct procedure, the 4- or 24-h procedures may be employed.     

Rapid slide coagglutination test for identifying and typing group B streptococci.

A Cowan I strain of Staphylococcus aureus was labeled with either group B streptococcal grouping or typing antiserum. These antibody-labeled reagent cells (ARC) were used in a slide coagglutination test to identify and type group B streptococci from blood agar plates. All streptococci were also identified by the standard Lancefield capillary precipitin test. In a blind study, all 141 group B streptococci were correctly identified by the coagglutination grouping test. None of the 148 non-group B streptococci caused agglutination of ARC. The coagglutination grouping test required an acid extract prepared from only four colonies and could be completed less than 30 min after colonies were removed from plates. The coagglutination typing test correctly identified 98.6% of the types of the 141 group B streptococcal strains tested. At least 88.6% of these streptococci could be typed directly from blood agar plates within 5 min by the coagglutination typing test. The remaining 11.4% of the group B streptococci were acid extracted (less than a 30-min procedure), and the extract was used for coagglutination typing. Coagglutination typing can be performed with only four colonies. The coagglutination grouping and typing tests are inexpensive, rapid, reliable, and easy to perform.     

Laboratory evaluation of a rapid four-hour serological grouping of groups A,B,C, and G beta-streptococci by the Phadebact streptococcus test.

Grouping of beta-hemolytic streptococcal isolates by staphylococcal coagglutination was performed with the Phadebact Streptococcus Test to determine whether such isolates could be accurately grouped serologically with 4 h after examination of the primary isolation plates. Of 132 clinical isolates, 131 were correctly grouped by the Phadebact method using the Lancefield precipitation method as the accepted standard. Of the correctly grouped streptococci, 119 were definitively grouped within 4 h after examination of the primary plates, and the remaining 12 isolates were grouped within 24 h. Since the Phadebact Streptococcus Test contains coagglutination reagents for groups A, B, C, and G, those isolates that failed to react were considered as positive for groups other than the four included in the test system. There were 23 such isolates in this study. Lancefield grouping of these isolates indicated that nine were group F, five were group D, and the remaining nine were not groupable with the Lancefield reagents employed in this study. The one Phadebact "failure" involved an isolate that produced a 4 + reaction with the Phadebact group A reagent and a 4 + reaction with Lancefield group F reagent.     

Rapid slide agglutination test for Lancefield grouping of streptococci.

A rapid slide agglutination test (the Phadebact [PB] Streptococcus test) was compared with the standard autoclave extraction method of Lancefield and presumptive clinical laboratory tests for grouping of streptococci (bacitracin disk sensitivity for group A and sodium hippurate hydrolysis for group B). Identification of group A streptococci by the PB kit was statistically as accurate as by the Lancefield method, whereas bacitracin grouping was significantly less accurate than the Lancefield method (P = less than .02). With regard to group B, there was no statistically significant difference between the PB test and the sodium hippurate test. The PB test correctly identified all group C and G streptococci. The PB kit provides a rapid and reliable method for Lancefield grouping of streptococci.     

The rapid recognition of Lancefield group B haemolytic streptococci

When grown overnight on Columbia agar in an atmosphere of hydrogen and carbon dioxide, haemolytic strains of Lancefield group B streptococci (Streptococcus agalactiae) produce orange or brown pigmented colonies. This production of pigmented colonies can be used for the rapid presumptive identification of these organisms as belonging to group B without the need for grouping by serological methods.     

Standardization and evaluation of the CAMP reaction for the prompt, presumptive identification of Streptococcus agalactiae (Lancefield group B) in clinical material.

Primary cultures of clinical material were screened for the presence of colonies suspected of being Streptococcus agalactiae (Lancefield group B). Sixty-three such cultures and 108 other isolates of beta-hemolytic streptococci (groups A, C, and G), encountered during the first 3 months of the investigation, were studied by Lancefield grouping, sodium hippurate hydrolysis, and a standardized CAMP test. All streptococci were inoculated perpendicularly to streaks of a beta-toxin-producing staphylococcus on sheep blood agar plates and incubated aerobically in a candle jar and anaerobically at 37 C. Plates were examined after 5 to 6 and 18 h of incubation. The production of a distinct "arrowhead" of hemolysis was indicative of a positive CAMP reaction. All group B streptococci produced a positive CAMP reaction in the candle jar or anaerobically, usually within 5 to 6 h, and aerobically after 18 h of incubation. All group A streptococci produced a positive reaction only under anaerobic conditions. Groups C and G streptococci were negative under all atmospheres. The CAMP reaction is a prompt and reliable procedure for the presumptive identification of group B streptococci when a candle jar atmosphere is used during incubation.     

Evaluation of spot CAMP test for identification of group B streptococci.

The CAMP (Christie-Atkins-Munch-Petersen) test is commonly used for the presumptive identification of Streptococcus agalactiae (Lancefield group B). Using 350 clinical isolates of beta-hemolytic streptococci, we compared a 30-min spot CAMP test with the standard overnight CAMP test and the Lancefield precipitin test. We found 99% agreement among all three tests for all streptococci tested. The spot CAMP test is a rapid, inexpensive, and accurate method for identifying group B streptococci.     

Évaluation du nouveau milieu chromogène StrepB Select™ pour le dépistage anténatal des streptocoques du groupe B chez la femme enceinte

Objectives

To compare and evaluate the new chromogenic StrepB Select™ (BioRad) medium to the Granada™ (bioMérieux) and Columbia horse blood agar plates (CH-BAP) for screening of Group B Streptococcus (GBS) vaginal colonization in pregnant women.

Methods

One hundred and ninety vaginal swabs were processed and the three media inoculated. All plates were examined after 18–24 h incubation at 37 °C and reincubated for an additional 24 h. All suspected colonies were identified as GBS by using a commercial Lancefield group-specific latex agglutination test.

Results

GBS were isolated in 32 samples (16.8%) by at least one medium. After 24 h of incubation, GBS were recovered on CH-BAP in 16 samples (50%) compared to 28 (87.5%) for StrepB Select™, and 27 (84.5%) for Granada™. After 48 h of incubation, 30 (93.7%) out of the 32 GBS vaginal carriers were positive with CH-BAP, StrepB Select™ and Granada™. Other streptococcal and enterococcal species gave GBS-like colonies on StrepB Select™ medium implying the use of other tests to confirm GBS identification. We demonstrated that direct agglutination of GBS isolated all media with Pastorex StrepB accurately identified GBS. The two StrepB Select™ false-negative results corresponded to a very low colonisation rate. Two Granada™ false-negative results corresponded to non-haemolytic and non-pigmented GBS strains which were correctly identified on StrepB Select™. Both selective media inhibit the growth of associated saprophytic flora.

Conclusion

The use of the new StrepB Select™ chromogenic medium in routine laboratories may therefore markedly facilitate the rapid and accurate detection of GBS in vaginal samples.     

Comparison of DNA dot blot hybridization and lancefield capillary precipitin methods for group B streptococcal capsular typing

Group B streptococci (GBS) (Streptococcus agalactiae) are a major cause of sepsis and meningitis in neonates and infants and of invasive disease in pregnant women, nonpregnant, presumably immunocompromised adults, and the elderly. Nine GBS serotypes based on capsular polysaccharide antigens have been described. The serotype distributions among invasive and colonizing isolates differ between pediatric and adult populations and have changed over time. Thus, periodic monitoring of GBS serotype distributions is necessary to ensure the proper formulation and application of an appropriate GBS vaccine for human use and to detect the emergence of novel serotypes. Since the mid-1990s, the proportion of GBS isolates that are nontypeable by standard serologic methods has increased, creating a need for more sensitive typing methods. We describe a typing method that uses DNA dot blot hybridization with probes generated by PCR from the GBS capsular genes for serotypes Ia, Ib, and II to VIII. PCR primers were designed to amplify type-specific GBS capsular gene sequences. Gene probes were constructed from the PCR products and used to classify isolates based on hybridization profiles. A total of 306 previously serotyped invasive and colonizing isolates were used to compare our dot blot capsular typing (DBCT) identification method with Lancefield serotyping (LS). A dot blot capsular type was assigned to 99% (303 of 306) of the isolates, whereas 273 of 306 isolates (89%) were assigned a Lancefield serotype. The overall agreement between the methods was 95% (256 of 270 isolates typeable by both methods). We conclude that the DBCT method is a specific and useful alternative to the commonly used LS method.     

Simplification of a Locus-Specific DNA Typing Method (Vir Typing) for Streptococcus pyogenes

We describe a simplification of a highly discriminatory molecular typing method, called Vir typing, for Streptococcus pyogenes (D. Gardiner, J. Hartas, B. Currie, J. D. Mathews, D. J. Kemp, and K. S. Sriprakash, PCR Methods Appl. 4:288–293, 1995). The procedure can be completed within a day, is reproducible, and can be applied directly to colonies growing on primary culture plates, allowing rapid establishment of strain identity in an outbreak.     

What happened to the streptococci: overview of taxonomic and nomenclature changes

Since the division of the Streptococcus genus into enterococci, lactococci, and streptococci in 1984, many changes in the nomenclature and taxonomy of the Streptococcus genus have taken place. The application of genetic comparisons has improved the proper classification of the different species. The Lancefield system of serogrouping the streptococci by the expression of beta-hemolysis on blood agar plates is still very useful for the identification of streptococci for patient management. The Lancefield grouping system cannot be used in itself for accurate identification of specific beta-hemolytic species, but it can be a useful part of the identification procedure. Except for identification of the "Streptococcus bovis group" of species and Streptococcus suis, Lancefield grouping is of little value in identification of the non-beta-hemolytic streptococci and related genera. In fact, identification of the non-beta-hemolytic species is problematic for conventional as well as commercially available identification procedures. A combination of conventional tests and specific chromogenic tests suggested by several investigators is presented and discussed. Tables are included that suggest tests and procedures to guide investigators attempting to identify all the species.     

Use of a serotype-specific DNA microarray for identification of group B Streptococcus (Streptococcus agalactiae)

Group B Streptococcus (GBS; Streptococcus agalactiae) is an important cause of sepsis and meningitis. Nine GBS serotypes, based on capsular polysaccharide (CPS) antigens, have been described. Their distribution varies worldwide and needs to be monitored to understand the epidemiology of GBS disease and inform the development of vaccines. In this study, we sequenced cpsH of GBS serotype II (cpsHII) and compared it with that of the other eight serotypes to identify serotype-specific regions. We then developed a DNA microarray based on the cpsH gene and used it to test 88 GBS isolates-9 serotype reference strains and 79 clinical isolates-and 7 other bacterial and fungal species which are commonly present in the vagina flora. The microarray was shown to be specific and reproducible. This is the first report of a microarray which can identify the nine GBS serotypes. The use of a microarray has advantages over traditional serotyping methods and will be of practical value in both reference and diagnostic laboratories.     

Reliable five-minute test strip method for identification ofStreptococcus pyogenes

A novel and rapid method (Strep Strip, Lab M) for identification ofStreptococcus pyogenes was evaluated. The method combines the established test for pyroglutamyl aminopeptidase (PYR) with a rapid chromogenic test for beta-glucosidase on a paper strip. The test was evaluated with 274 clinical isolates and 237 culture collection isolates of beta-haemolytic streptococci.Streptococcus pyogenes was identified with 100% specificity. Six isolates identified as Lancefield group A and which might therefore be assumed to beStreptococcus pyogenes were shown by the test strip method not to be this species. The beta-glucosidase test on the test strip allowed differentiation between enterococci andStreptococcus pyogenes (both PYR positive) in all cases. The test strip method represents an economical and accurate method for identification ofStreptococcus pyogenes in clinical material.     

Evaluation of a colour test for rapid detection of Salmonella

The performance of a colour test (Wellcolex) for rapid detection of the most frequently isolated O serogroups of salmonella (A, B, C, D, E/G) and the Vi antigen was evaluated using colonies grown on enteric differential agar media and in selenite broth. The test had excellent sensitivity (100 %) and specificity (100 %) with colonies taken directly from primary culture plates. Initial tests in selenite broth showed limited sensitivity (62.1 %) but by using high quality media and modifying the inoculation procedure, sensitivity was greatly improved (99 %).     

Modified coagglutination procedure for the serological grouping of streptococci.

Cowan I staphylococci coated with antisera to streptococcal groups A, B, C, D, F, and G were used as coagglutination reagents in a modified coagglutination procedure (MCAP). Streptococcal group antigens were extracted with a Streptomyces albus-lysozyme enzyme mixture for 30 min at 55 degrees C and centrifuged, and the supernatant was tested by slide coagglutination. Positive coagglutination reactions occurred within 30 s. The cell pellets from overnight broth cultures and colonies taken directly from sheep blood agar plates were tested and compared with the results of the Lancefield capillary precipitin method. Of the 102 strains of broth-grown cells tested, 100 were grouped by the MCAP and the Lancefield capillary precipitin method. The remaining two isolates were serologically identified only by the MCAP. Of the original 102 strains, 97 were tested by MCAP after extraction of five well-isolated colonies from a sheep blood agar plate. When this latter method was used, 95.9% of the strains were correctly identified. Nonspecific reactions were observed only while testing the MCAP with the direct plate assay. These cross-reactions were remedied promptly by either absorption or dilution of the antisera involved. The MCAP was found to be a rapid and reliable technique for the serological grouping of streptococci.     

Role of group C beta-hemolytic streptococci in pharyngitis: epidemiologic study of clinical features associated with isolation of group C streptococci.

All Lancefield group C beta-hemolytic streptococci isolated over 12 months from college students with clinical pharyngitis and age-matched healthy controls were identified. Clinical features of upper respiratory tract infection and pyogenic pharyngitis as well as colony counts were tabulated for each patient according to throat culture results. Of 1,480 patients, Lancefield group C Streptococcus equisimilis was isolated from 45 (3%) patients and Streptococcus anginosus ("Streptococcus milleri") was isolated from 164 (11.1%) patients. Patients from whom S. equisimilis was isolated had clinical features more suggestive of pyogenic infection than did patients from whom S. anginosus was isolated. Colony counts on primary throat culture plates from patients from whom S. equisimilis and Streptococcus pyogenes were isolated were higher than those from patients from whom S. anginosus was isolated. This study presents epidemiologic evidence supporting a role for S. equisimilis in causing pharyngeal infection and for S. anginosus as representing part of the normal oropharyngeal flora.     

Rapid, colorimetric test for the determination of hippurate hydrolysis by group B Streptococcus.

A colorimetric test for the determination of hippurate hydrolysis was developed. Brain heart infusion broth made with 1% sodium hippurate served as the test medium. Hydrolysis was determined by the addition of two chemical developers, M (rhodamine B) and A (uranium acetate). A dark pink color indicated hydrolysis; no color change indicated no hydrolysis. The method was efficacious in either rapid or overnight incubation. One hundred twenty-five strains of group B, 44 strains of group A, 15 strains of group C, and 10 strains of group G Streptococcus were tested. By using the Lancefield method as the standard, there was 100% agreement with both the colorimetric and ferric chloride tests for hippurate hydrolysis, and 96% agreement with the CAMP test.     

Mouse protection assay for group B streptococcus type III.

The mucin model for group B Streptococcus (GBS) type III was used to assay the protective effect of sera against a type III challenge in mice. Hyperimmune rabbit sera, prepared by the Lancefield method against the laboratory reference strain (SS620) and a clinical isolate (M732), protected against a lethal challenge with either strain of GBS type III. Absorption of the sera with either of these type III strains removed the protective effect. Neither normal rabbit sera nor heterologous antisera (anti-Ia, SS615) provided protection; however, protection was obtained with pooled human gamma globulin. Sera from adult volunteers were tested to assay protective levels in the mouse model. Human sera enhanced the mouse lethality of the clinical isolate, M732, but not the laboratory reference strain, SS620. Sera from adults vaccinated with type III polysaccharide of GBS were also tested. The murine-mucin-GBS model may be developed as a screening test to measure protective antibody levels in the pre- and postvaccine treatment period. The model may also be used to measure protective antibody in pooled human gamma globulin for use in the passive immunization of high-risk individuals.     

Rapid tube CAMP test for identification of Streptococcus agalactiae (Lancefield group B).

A rapid CAMP test for the presumptive identification of Streptococcus agalactiae (Lancefield group B) is described. Sheep erythrocytes, sensitized by staphylococcal beta-lysin and suspended in phosphate-buffered saline, were used to determine the lytic capacity of the neutralized supernatant fluids of 4-h broth cultures of streptococci being tested. A total of 96.2% of 130 group B streptococci gave positive CAMP tests, that is, lysis of the sheep erythrocytes after 10 min of exposure of streptococcal supernatants, whereas none of 381 non-group B streptococci tested produced any lysis. The test described provides presumptive identification of group B streptococci within 4 h and eliminates problems of intermediate reactions so that a positive test is indicative only of CAMP factor production.     

A new flow-cytometry-based opsonophagocytosis assay for the rapid measurement of functional antibody levels against Group B Streptococcus

Opsonophagocytosis is the primary mechanism for the clearance of Group B Streptococcus (GBS) by the host, and levels of opsonic antibodies may correlate with protection in preclinical models. A killing-based opsonophagocytosis assay (OPA), can be used to determine the functional activity of vaccine-induced GBS-specific antibodies. The assay, which measures the number of bacterial colonies surviving phagocytic killing in the presence of specific antibodies and complement, is rather expensive, time-consuming and poorly standardized. Here we describe a rapid, sensitive and reproducible fluorescent OPA assay (fOPA) based on flow cytometry analysis (FACS), which allows internalized bacteria to be distinguished from those associated to the plasma membrane of phagocytic cells. Fixed GBS were labeled with pHrodo?, a fluorescent dye which dramatically increases the emitted fluorescence at the acidic conditions present in the phagocytic endosomal compartment. Labeled bacteria were incubated with HL-60 cells differentiated to phagocytes, antibodies and complement, and then analyzed by FACS. A further improvement to our method, allowing to reduce assay variability, consisted on a step of selection of effector cells among the HL-60 population. Analysis of sera from mice immunized with different GBS vaccines revealed comparable sensitivity and specificity with the traditional killing OPA assay (kOPA), and a good correlation between the fluorescent signal of bacteria internalized by HL-60 phagocytes and killing. Remarkably, the pHrodo-based approach reduced the variability observed with other fOPA assays. The obtained data indicate the proposed fOPA as a reliable and useful tool for functional antibody assessment.