Serotyping Methods Research Articles

Coagglutination test for serotyping Pasteurella haemolytica.

A coagglutination test was described for simple, fast, and reliable detection of Pasteurella haemolytica type-specific antigens in lung lesions even in the absence of viable P. haemolytica. The coagglutinating reagents were prepared by coating protein A-producing Staphylococcus aureus cells with hyperimmune sera raised against P. haemolytica type strains. Bacterial suspensions, saline extracts, and boiled saline extracts of the bacteria were used as antigens. Homologous reactions with all types of antigens were precise. Some cross-reactions were similar to those obtained by the indirect hemagglutination test, and some additional one-way cross-reactions were identified. The coagglutination test was used for serotyping 65 P. haemolytica field strains and for the detection of P. haemolytica type-specific antigens in the lung specimens of 62 calves and 78 sheep. Ninety-four percent of the field strains could be serotyped by the coagglutination test. P. haemolytica type-specific antigens were detected in the lung specimens of 3 calves and 5 sheep that had succumbed to naturally occurring P. haemolytica pneumonia and in the lungs of 20 calves experimentally infected with P. haemolytica A1. The coagglutination test detected type-specific antigens in 36% of the lung specimens of slaughtered field sheep but not in the lungs of slaughtered field cattle with small chronic lung lesions. No reaction occurred in the case of nonpneumonic calves and sheep or when pneumonic lesions were caused by other bacteria. No P. haemolytica strains could be isolated from lung samples that were coagglutination test negative. This test is recommended as an additional method for fast and reliable serotyping of P. haemolytica.

Molecular and serologic characterization of a group A bovine rotavirus with a short genome pattern.

Rotaviruses are members of the family Reoviridae and are a widespread cause of diarrhea in the young of many mammalian species, including humans. The rotavirus particles are nonenveloped and possess an icosahedral symmetry. The genome consists of 11 discrete segments of linear doublestranded RNA (dsRNA) and is enclosed within a doublelayered capsid. The dsRNA fragments are numbered 1 to 11 on the basis of their order of migration during polyacrylamide gel electrophoresis (PAGE). Rotaviruses are classified into serotypes based on the specificity of the outer capsid proteins VP7 (G types) and VP4 (P types) The more common method of serotyping is on the basis of G types (13), and currently 14 G types have been identified among group A rotaviruses. Among group A bovine rotaviruses (BRVs), at least 4 G types (G1, G6, G8, G10) have been reported based on the results of enzymelinked immunosorbent assay (ELISA), virus neutralization assays, nucleic acid hybridization assays, or sequence analysis. Rotaviruses are also typed using similar procedures on the basis of VP4 specificity, and currently there are at least 4 P types of BRV (P1, P5, P11, and P12). Upon analysis by PAGE, the 11 dsRNA segments of rotaviruses produce characteristic patterns referred to as genome electropherotypes. 35 Such electrophoretic analysis of BRV isolates is useful for obtaining epidemiologic information regarding the origin of the isolates, because each isolate produces a unique pattern. There have been numerous reports of short genome electropherotypes among human rotaviruses. Most of these short electropherotypes are restricted to subgroup I and share serotype G2 specificity. All BRV isolates tested so far, regardless of genome electropherotype, belong to subgroup I, and most possess long electropherotypes.38 Recently, there have been reports of short electropherotypes among BRV isolates, all restricted to serotype G6 1,22,37 Because there have been no reports of non-G6 BRV with a short genome pattern, we wanted to investigate the possibility that BRV short pattern isolates are restricted to serotype G6, similar to the situation observed for human G2 rotaviruses. In addition, we wanted to correlate the hybridization data with serology and cross-protection studies. In this report, we describe the serologic and molecular characterization (G and P specificity) of a bovine rotavirus strain, 2292B, with a short genome pattern. The reference viruses were grown in rhesus monkey kidney (MA104) cells in roller bottles and titrated by a plaque assay as previously described. The sources and serotypes (P and

Characterization of monoclonal antibodies to O-antigen of lipopolysaccharide ofActinobacillus pleuropneumoniaeserotype 2 and their use in the classification of field isolates

Monoclonal antibodies (mAbs) against Actinobacillus pleuropneumoniae serotype 2 (reference strain Shope 4226 and field isolate F46) were produced. Twelve hybridoma clones were selected against both strains, and all the antibodies secreted were found to be reactive with whole-cell antigen of the homologous strain in ELISA, whereas only one mAb was reactive in slide agglutination test. The predominant antibody classes were IgG2b and IgG3, although IgG1 and IgM were also obtained. Immunoblot assay showed that mAbs could recognize a ladder band profile which is in accordance with the O-antigen of lipopolysaccharide. Most of the epitopes involved were resistant to proteinase K and also to boiling in the presence of sodium dodecyl sulfate and reducing conditions, but they were sensitive to periodic acid. The 12 mAbs recognized neither reference strains of the remaining A. pleuropneumoniae serotypes nor other taxonomically related Gram-negative organisms. The suitability of mAbs for serotyping of field isolates was also examined, and a high correlation (97.4%) was found between the results previously established by indirect hemagglutination with polyclonal rabbit sera and those obtained by ELISA with mAbs. The panel of mAbs described in this study was found to be extremely useful for identifying field isolates belonging to serotype 2 and could be used as a complementary serotyping method.

Characterization of monoclonal antibodies to O-antigen of lipopolysaccharide of Actinobacillus pleuropneumoniae serotype 2 and their use in the classification of field isolates

Monoclonal antibodies (mAbs) against Actinobacillus pleuropneumoniae serotype 2 (reference strain Shope 4226 and field isolate F46) were produced. Twelve hybridoma clones were selected against both strains, and all the antibodies secreted were found to be reactive with whole-cell antigen of the homologous strain in ELISA, whereas only one mAb was reactive in slide agglutination test. The predominant antibody classes were IgG2b and IgG3, although IgG1 and IgM were also obtained. Immunoblot assay showed that mAbs could recognize a ladder band profile which is in accordance with the O-antigen of lipopolysaccharide. Most of the epitopes involved were resistant to proteinase K and also to boiling in the presence of sodium dodecyl sulfate and reducing conditions, but they were sensitive to periodic acid. The 12 mAbs recognized neither reference strains of the remaining A. pleuropneumoniae serotypes nor other taxonomically related Gram-negative organisms. The suitability of mAbs for serotyping of field isolates was also examined, and a high correlation (97.4%) was found between the results previously established by indirect hemagglutination with polyclonal rabbit sera and those obtained by ELISA with mAbs. The panel of mAbs described in this study was found to be extremely useful for identifying field isolates belonging to serotype 2 and could be used as a complementary serotyping method.

Evaluation of microagglutination test for differentiation between Serpulina (Treponema) hyodysenteriae and S. innocens and serotyping of S. hyodysenteriae

Swine dysentery is a mucohemorrhagic diarrheal disease caused by the anaerobic spirochete Serpulina hyodysenteriae. At present, the serotyping is done by immunodiffusion testing with lipopolysaccharide (LPS) extract as antigen and rabbit hyperimmune sera produced against different serotypes of S. hyodysenteriae. Since the preparation of LPS is time-consuming and requires a large quantity of bacteria, it is desirable to use a serotyping method which does not require the extraction of LPS. In the present investigation, microagglutination was evaluated by using both formalinized whole- and boiled-cell suspensions as antigens and rabbit hyperimmune sera produced against formalinized whole-cell suspensions of reference strains of S. hyodysenteriae and S. innocens B256. Use of boiled cell suspension as antigen permitted the differentiation between isolates of S. hyodysenteriae and S. innocens as well as serotyping of S. hyodysenteriae strains accurately. A total of 18 isolates were identified as S. hyodysenteriae, and 3 isolates were identified as S. innocens. The microagglutination test was found specific, sensitive, and easy to perform; thus, it was judged suitable for routine identification and serotyping of S. hyodysenteriae isolates.

A southern analysis of Rh blood group genes: association between restriction fragment length polymorphism patterns and Rh serotypes

Polymorphisms within the Rh blood group system have been defined by serologic agglutination methods, but have not yet been defined at the DNA level. Two closely related genes associated with the Rh D antigen and with the Rh C/c and E/e antigens have been cloned. We used a Southern analysis incorporating probes to the 5′ and 3′ regions of the Rh C, E gene and D gene to identify polymorphisms associated with Rh C/c and E/e antigens, respectively. The D gene dosage could be determined by comparing the relative intensities of the D bands with bands from the 5′ and 3′ region of the Rh C, E gene. The concordance between restriction fragment length polymorphism (RFLP) patterns and serologic phenotypes for 102 randomly selected blood donors was 100% for C, e, and D, 94.8% for c, and 94.3% for E. The data are consistent with the sequences encoding the C/c epitopes residing on the 5′ side of those for the E/e epitopes. All samples discordant for the 3′ probe and E had the cE (r″) serotype. These data show that the gene coding for the cE serotype is different in Rh-positive and -negative individuals. The study demonstrates that Rh DNA typing, including D gene dosage measurements and Rh gene haplotyping, may supplement traditional serotyping methods in transfusion medicine.

A Southern Analysis of Rh Blood Group Genes: Association Between Restriction Fragment Length Polymorphism Patterns and Rh Serotypes

AbstractPolymorphisms within the Rh blood group system have been defined by serologic agglutination methods, but have not yet been defined at the DNA level. Two closely related genes associated with the Rh D antigen and with the Rh C/ c and E/e antigens have been cloned. We used a Southern analysis incorporating probes to the 5’ and 3’ regions of the Rh C,E gene and D gene to identify polymorphisms associated with Rh C/c and E/e antigens, respectively. The D gene dosage could be determined by comparing the relative intensities of the D bands with bands from the 5’ and 3’ region of the Rh C,E gene. The concordance between restriction fragment length polymorphism (RFLP) patterns and serologic phenotypes for 102 randomly selected blood donors was 100% for C, e, and O, 94.8% for c, and 94.3% for E. The data are consistent with the sequences encoding the C/c epitopes residing on the 5’ side of those for the E/e epitopes. All samples discordant for the 3’ probe and E had the cE (r“) serotype. These data show that the gene coding for the cE serotype is different in Rh-positive and -negative individuals. The study demonstrates that Rh DNA typing, including D gene dosage measurements and Rh gene haplotyping, may supplement traditional serotyping methods in transfusion medicine.

Rickettsia massiliae sp. nov., a new spotted fever group Rickettsia.

We propose the name Rickettsia massiliae sp. nov. (type strain, Mtu1 in the Collection of the World Health Organization Collaborative Center for Rickettsial Reference, Marseille, France) for a spotted fever group rickettsia determined to be distinct from previously recognized species by the serotyping method (L. Beati, J.-P. Finidori, B. Gilot, and D. Raoult, J. Clin. Microbiol. 30:1922-1930, 1992). This rickettsia has biological characteristics similar to those of the other spotted fever group rickettsiae. In addition, a sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein analysis, a polymerase chain reaction followed by a restriction fragment length polymorphism analysis of DNA fragments, and pulsed-field electrophoresis of the genome of R. massiliae revealed unique migration patterns distinct from those of all previously recognized spotted fever group rickettsiae. These additional characteristics (Beati et al., J. Clin. Microbiol. 30:1922-1930, 1992), together with the data usually considered sufficient for description of rickettsiae, are crucial to the proposal of this new species and should be helpful in species identification.

Common somatic O and heat-labile serotypes among Campylobacter strains from sporadic infections in the United States

Somatic O (formerly heat-stable) and heat-labile (HL) serotyping methods are commonly used to type Campylobacter jejuni and Campylobacter coli isolates. Although both systems are effective, the labor and time required for each have limited their application. These systems can be simplified by reducing the number of antisera used. To find an appropriate panel of antisera, we determined the distribution of common serotypes in the United States among a representative sample of 298 Campylobacter isolates. The strains, obtained between July 1989 and June 1990 from persons with sporadic cases of diarrhea, were collected from 19 randomly chosen counties in all geographic (census) regions of the United States. All strains were serotyped by the O and HL systems. By phenotypic methods, 288 C. jejuni, 9 hippurate-negative C. jejuni/C. coli, and 1 Campylobacter lari were identified. Of 57 O antisera, 24 typed 252 (84.6%) strains. Of the 55 HL antisera, 23 serotyped 253 (84.9%) strains. All strains were typeable in the unabsorbed O antisera. In the absorbed HL antisera, four strains were nontypeable and 14 were rough and untypeable. In each geographic region, 9 or more O and HL serotypes were found. Serotypes O:1, O:4, and O:13,16,43,50 and HL 1 were identified in all regions. The combination of both schemes gave greater discrimination than either system alone, but the maintenance of both requires a large resource investment. A serotyping scheme incorporating the 24 most prevalent O and 23 most prevalent HL serotypes could be useful for outbreak support and for surveillance. In the near future, we anticipate using a molecular subtyping method in combination with limited serotyping to distinguish Campylobacter strains.

Serotyping of Cryptococcus neoformans by using a monoclonal antibody specific for capsular polysaccharide

The importance of epidemiological studies of cryptococcosis has increased since the beginning of the AIDS epidemic. Cryptococcus neoformans exists in two varieties defining four serotypes, Cryptococcus neoformans var. neoformans (serotypes A and D) and Cryptococcus neoformans var. gattii (serotypes B and C). The varieties are easy to distinguish by their differences of growth on diagnostic media. We propose here an easy serotyping method combining diagnostic media and a direct immunofluorescence assay with one monoclonal antibody (E1) specific for cryptococcal polysaccharide. The method was validated by the blinded testing of four to five reference strains of each serotype. Immunofluorescence patterns were characteristic of a given serotype provided that the variety of the strain had been defined before. For C. neoformans var. neoformans, a bright, homogeneous staining with several cell aggregates was characteristics of serotype A, whereas only a few serotype D cells were positive. For C. neoformans var. gattii, a completely negative isolate was serotype C, whereas the population of serotype B included a majority of negative cells but also included positive cells with a speckled pattern. The method was then used to serotype 156 clinical isolates from France and isolates from areas where C. neoformans var. gattii was endemic before the AIDS epidemic (13 strains from Rwanda and Zaire and 5 strains from Australia). The specificity of E1 was defined by its reactivity with various Cryptococcus spp. and analyzed according to the described cryptococcal antigenic factors. We conclude from this study that E1 provides a rapid and reliable means to serotype multiple isolates of C. neoformans.

Serological characterization of Actinobacillus pleuropneumoniae strains isolated from pigs in Quebec

A total of 3306 isolates of A. pleuropneumoniae originating from lung tissues of pigs that died of acute pleuropneumonia and 140 isolates recovered from tonsils or nasal cavities of apparently healthy pigs from chronically infected herds were serotyped. Various serotyping methods, such as slide agglutination, tube agglutination, ring precipitation, coagglutination, immunodiffusion, indirect hemaglutination and counterimmunoelectrophoresis either alone or in combination were used. The techniques used for serotyping continued to evolve during the last 10 years depending on the problem encountered in serotyping. Antisera prepared in rabbits against formalinized whole cell suspensions of reference strains of A. pleuropneumoniae of serotypes 1 to 12 were employed for serotyping. Serotype 1 was predominant ranging from 55 to 87% from year to year during the last 10 years with an average prevalence of 68%. Serotype 5 was second in prevalence ranging from 9 to 30% with a mean of 23%. Both subtypes of serotype 5 (5a and 5b) were present in Quebec. Serotypes 3, 6, 7, 8, 10 and 12 were isolated in small numbers together accounting for about 9%. Serotypes 4, 9 and 11 were not present. Cross-reactions were observed among isolates of serotypes 3, 6 and 8, and 1, 9 and 11 were easily differentiated from each other by quantitation of type and group specific antigens by coagglutination and immunodiffusion tests. Serotypes 1, 5 and 7 were isolated most frequently from tonsils of pigs from chronically infected herds. Prevalence of different serotypes in different countries has also been reviewed.

Simplified microtiter cell culture method for rapid immunotyping of Chlamydia trachomatis

Serotyping of Chlamydia trachomatis strains usually requires three to six serial passages in shell vials to attain sufficient antigen for typing procedures. To circumvent this problem, we developed a rapid low-passage method for serotyping of C. trachomatis clinical isolates. Isolates with an inclusion count of greater than or equal to 500 per well in primary isolation were inoculated directly onto cell culture monolayers in microtiter plates for typing. Primary isolates with a lower initial inclusion count were passed one to two times in shell vials until there were greater than or equal to 20 inclusions per well and were then inoculated onto plates for typing. Inclusions were grown to maturity and reacted with a panel of 17 C. trachomatis-specific monoclonal antibodies in pools. Wells were then reacted with a fluorescein isothiocyanate conjugate and read by FA microscopy, and the reaction patterns were compared with prototype strain reaction patterns to determine the serotype. By the microtiter method, we successfully typed 1,711 consecutive C. trachomatis isolates; 1,215 isolates (71%) were typed with no or with one passage. The first 209 isolates typed by the microtiter method were also typed by the dot-enzyme-linked immunosorbent assay serotyping method; 100% agreement was demonstrated among strains that were typeable by both methods. We conclude that the microtiter method is extremely useful for accurate serotyping of large numbers of isolates and requires greatly reduced technician time.

Comparison between methods for serotyping of Candida albicans produces discrepancies in results

Serotyping of 101 clinical isolates of Candida albicans was done with two sets of Hasenclever original anti-Candida typing sera (HSN 1 and 2) and Iatron Candida Check factor 6 typing serum (IF6). The results of these two methods were compared with slide agglutination reactions of yeast with monoclonal antibody H9. Agglutination reactions with this antibody have been previously shown to correlate with serotype. Results indicate the following correlations: between HSN 1 and HSN 2 serotyping, 93% (kappa = 0.85; 95% confidence interval [CI], 0.70 to 0.99); between IF6 and HSN 1, 60% (kappa = 0.39, 95% CI, 0.19 to 0.58); and between IF6 and HSN 2, 74% (kappa = 0.77; 95% CI, 0.64 to 0.90). Results with HSN 1 and 2 antisera correlated with H9 reactivity at 85 and 89% (kappa = 0.88; 95% CI, 0.75 to 1.00; and kappa = 0.85; CI, 0.70 to 0.99, respectively), while agreement between IF6 and H9 reactivities was less than or equal to 64% (kappa less than or equal to 0.43; 95% CI, 0.14 to 0.60). Autoagglutination of yeast during IF6 serotyping occurred with 21 of the 101 (20.8%) yeast strains. In every case, these yeast strains were serotyped by the HSN methods without autoagglutination and were uniformly type B. This study implies that it may not be possible to make valid comparisons between studies which compare serotype prevalence unless the same methods are used to serotype the yeast. The practicality and utility of serotyping in epidemiological studies are discussed, as are some of the problems associated with the available methods.

Untersuchungen über die Möglichkeit zur serologischen Differenzierung von Clostridium species mittels Enzym‐Immun‐Verfahren

A method for serotyping and diagnosis of 30 selected Clostridia strains of 12 different species by an enzyme-immuno-assay using polystyrene balls as a solid phase is described and tested for its efficiency. The reference strains and the homologous test strains could be differentiated from the heterologous in principle. A reliable detection of the several serotypes of Cl. perfringens was not perfectly to achieve with the conjugates used here, cross-reaction with Cl. chauvoei could be removed by absorption. The conjugate of Cl. bifermentans reacted with Cl. sordellii and Cl. difficile too, and must be absorbed prior any diagnostic applications with vegetative cells of the corresponding strains. Concerning the antigenic relationships between the Clostridia strains used here, the results of Ellner and Green (7, 8) as well as of Poxton and Byrne (14) could be reconfirmed. Moreover the applied technique offers the possibility to come to a more detailed result concerning the degree of antigenic relationship. The developed method is a valuable tool for the differentiation of pathogenic Clostridia and within the group Cl. bifermentans/Cl. sordellii/Cl. difficile a rapid diagnosis at the species level could be achieved. Some limitations by cross-reactions are given, but results can be improved by absorption of the conjugates.

Restriction endonuclease analysis of chromosomal DNA from Listeria monocytogenes strains

Restriction endonuclease analysis of chromosomal DNA was applied to thirteen Listeria monocytogenes strains alongside the more conventional typing methods of serotyping and phage typing. The organisms were isolated from cases of sporadic listeriosis (nine strains); from an occasional nosocomial cluster (two strains); and from food samples (two strains). Purified DNAs were digested with EcoRI restriction endonuclease, and restriction fragments separated by electrophoresis. Restriction patterns correlated well with phage patterns, but also allowed typing of the phage-untypable strains. DNA fingerprinting appears to be a potentially helpful tool for epidemiological investigations of listeric infections, particularly when phage typing fails to determine the identity or diversity of the isolates.

DNA fingerprinting for differentiation of field isolates from reference vaccine strains of Pasteurella multocida in turkeys

SUMMARY The genomes from field isolates of Pasteurella multocida in turkeys and those of P multocida reference CU and M9 vaccine strains were analyzed and compared after cleavage with restriction endonucleases. The electrophoretic profiles obtained with DNA fragments from field isolates and vaccine strains of the same serotype were characteristic and reproducible. These features indicated the existence of differences among the isolates of the same serotype that cannot currently be detected, using available serotyping methods. However, several field isolates had electrophoretic profiles similar to those of either CU or M9 vaccine strain. It was concluded that restriction endonuclease analysis of DNA genomes from P multocida isolated from turkeys provides the information for differentiation of field isolates from vaccine strains of the same serotype.

Improved methods for detection and serotyping of coagulase from Staphylococcus aureus.

Improved methods for detection and serotyping of staphylocoagulase were concomitantly devised. We devised an improved method for detection of coagulase activity on agarose film in the same manner as single radial immunodiffusion. The amounts of reagents required for detection of coagulase on agarose film were successfully diminished by adding polyethylene glycol (PEG) to the original formula described by Boothby et al. Using microplates in another improved method for coagulase serotyping, the amount of reaction fluid required was considerably less compared with the conventional tube method. PEG was found to be also effective to increase the efficacy of coagulase serotyping. In the presence or absence of anti-coagulase antisera, culture supernatants of staphylococcal strain grown in brain heart infusion broth were incubated with the reaction fluid containing bovine fibrinogen, rabbit plasma, 6-amino caproic acid, polyethylene glycol 6,000. Coagulase activity was visualized as a turbid mass formed in the wells. Turbid mass formation due to coagulase activity was type-specifically inhibited in the presence of type-specific antisera. Detailed procedures of the methods are precisely described with some preliminary results obtained by the methods.

A Comparison between Methods of Identification and Serotyping of Encapsulated Strains of Haemophilus influenzae

Seven methods of serotyping of Haemophilus influenzae were evaluated. Comparing slide agglutination, staphylococcal coagglutination, latex agglutination, counterimmunoelectrophoresis, immunofluorescence, capsular swelling, and cultivation on antiserum agar the commercial coagglutination test was most reliable, most rapid, and easiest to perform. To identify all six serotypes this coagglutination test had to be combined with slide agglutination. With most methods best results were achieved by using cultures incubated at 37 degrees C for 6 h. As nonencapsulated strains often agglutinated unspecifically, selection of probably typeable strains was useful. Differentiation with help of colonial morphology and opalescent growth was facilitated by cultivation on Brain Heart Infusion (BHI) Chocolate Agar and testing of growth factor requirements on translucent BHI Agar with strips containing the growth factors V, X, and VX, respectively. In broth turbid growth was a hint for encapsulation. Nigrosin staining, a negative capsule staining, proved to be useful if specific antisera are not available. From 252 clinical isolates of H. influenzae 216 were not typeable. 36 strains could be serotyped. 27 (75%) belonged to serotype b, 6 (16.6%) were serotype e, 3 (8.3%) were serotype f. Serotype e and f were most difficult to identify. Spectrum of patients and diseases were corresponding to the findings of other authors. Less well-known infections like cellulitis (erysipelas of the cheeks) and arthritis were observed, too. Rapid identification of at least H. influenzae type b could render treatment in some cases more effective by early application of a suitable antibiotic.

The use of a monoclonal antibody serotyping system in the study of the epidemiology of Pseudomonas aeruginosa

A monoclonal antibody method was used as a primary O serotyping method for 1084 isolates of Pseudomonas aeruginosa, using commercially available antibody reagents in slide agglutination tests with whole-cell antigen. Differences in distribution of serogroups were found between various hospitals in the district. Serotyping provided useful information in the investigation of suspected incidents of cross-infection. Colonization at multiple sites, or over periods of time, in a single patient usually involved only a single serogroup.

New method of serotyping Escherichia coli: mathematical development

A mathematical analysis of serological data has provided the basis for a new serotyping routine. This paper describes the process whereby the results of agglutination reactions between the standard O antigens of Escherichia coli and rabbit antisera raised against them were used to produce a much simpler, faster, and cheaper method of serotyping. This new method for serotyping E. coli requires on average only one-fifth of the serum used in the previous method and requires less than half the laboratory time. By using this method, an efficient serotyping routine can be established for any set of comparable data regardless of the bacterial group.