The specificity of agglutination reactions of Pseudomonas aeruginosa with O antisera.

Polyagglutinable (PA) strains of Pseudomonas aeruginosa are agglutinated by more than one of the antisera prepared against antigenically unrelated O serogroups. They form c. 4% of strains--other than isolates from cystic-fibrosis units--submitted to us for typing. We were able to allocate 80% of PA strains to a single O serogroup by agglutination with typing sera that had been absorbed with the polyagglutinable strain SMC-247, or by co-agglutination tests with protein A-containing staphylococci coated with immunoglobulin from unabsorbed sera. Similar results were obtained by precipitation tests with crude bacterial extracts and unabsorbed sera, but these tests were less sensitive and less specific. Evidence is presented that PA antigen is a heat-stable cell constituent distinct from the O antigen. In rabbit antisera, anti-PA antibody is exclusively of the IgM class, but O antibody of both IgM and IgG classes is present.     

Pseudomonas aeruginosa isolates from patients with cystic fibrosis: a class of serum-sensitive, nontypable strains deficient in lipopolysaccharide O side chains

Twenty-six Pseudomonas aeruginosa strains from patients with cystic fibrosis were typed by the Fisher immunotyping scheme. Only 6 strains were agglutinated by a single typing serum, whereas 15 strains were agglutinated with more than one serum and 5 were not agglutinated by any serum. Neither the polyagglutinable nor the nonagglutinable strains were typable by hemagglutination inhibition or immunodiffusion, suggesting that these polyagglutinable strains did not express multiple serotype antigens, but were instead being agglutinated by antibody to nonserotype determinants. Four typable isolates were resistant to pooled normal human serum, whereas the 12 polyagglutinable and nonagglutinable isolates studied were very sensitive to normal human serum. The outer membranes of 16 strains were isolated and characterized. The data suggested, in general, strong conservation of outer membrane protein patterns. Lipopolysaccharides (LPS) were purified by a new technique which allowed isolation of both rough and smooth LPS in high yields. Three of four typable, serum-resistant strains examined had amounts of smooth, O-antigen-containing LPS equivalent to our laboratory wild type, P. aeruginosa PAO1 strain H103. In contrast, 10 of 12 polyagglutinable or nonagglutinable, serum-sensitive strains had very little or no smooth, O-antigen-containing LPS, and the other two contained less smooth LPS than our wild-type strain H103. In agreement with this data, five independent, rough, LPS O-antigen-deficient mutants of strain H103 were nontypable and serum sensitive. We suggest that the LPS defects described here represent a significant new property of many P. aeruginosa strains associated with cystic fibrosis.     

Epidemiological typing of Enterobacter aerogenes

The applicability of Enterobacter cloacae and Klebsiella typing reagents for classifying clinical strains of Enterobacter aerogenes was evaluated. Of 75 strains, none were agglutinated by E. cloacae O antisera or were sensitive to E. cloacae bacteriophages. In contrast, 70 strains reacted with Klebsiella capsular antisera. Two-thirds of the strains were lysed by Klebsiella typing phages. A set of five E. aerogenes bacteriocin producers classified 92% of strains into 15 sensitivity types. In conclusion, E. aerogenes may be typed with Klebsiella reagents, and the simple bacteriocin test provides further discrimination between strains. The limited number of capsular antigens in the species and their apparent similarity to Klebsiella capsular antigens warrant further investigation.     

Separate O-grouping schemes for serotyping clinical isolates of Proteus vulgaris and Proteus mirabilis.

Antisera were prepared against type strains of the original scheme of B. Perch (Acta Pathol. Microbiol. Scand. 25:703-714, 1948) and against newly defined types to produce separate schemes for O-grouping Proteus vulgaris and Proteus mirabilis. In assessing the schemes for their effectiveness it was found that 82% of 208 P. vulgaris isolates and 88% of 194 P. mirabilis isolates from two hospitals were typable. Only 3.4% of the P. vulgaris isolates agglutinated in P. mirabilis antisera, and 1.5% of the P. mirabilis agglutinated in P. vulgaris antisera, indicating that separation of the schemes would be more advantageous in routine typing. P. mirabilis of groups O3, O6, O10, O29, and O30 were most frequently isolated. Of the P. vulgaris isolates, 25% belonged to newly defined O-groups, and one of these was the largest with 14% of all isolates of this species. The application of serotyping using separate schemes for each species was advocated in epidemiological studies.     

Typing of polyagglutinable Pseudomonas aeruginosa isolates from cystic fibrosis patients

The study assesses the reproducibility, typability and discriminatory power of several typing methods for Pseudomonas aeruginosa isolated from cystic fibrosis patients. 178 polyagglutinable Pseudomonas aeruginosa isolates from cystic fibrosis patients were serotyped using polyclonal sera and monoclonal antibodies, phage typed, pyocin typed and reverse phage typed. 31 of these polyagglutinable isolates, six monoagglutinable isolates and three nontypable isolates were also typed by means of hybridization using a DNA probe. In a comparison of the methods used, on polyagglutinable isolates only, typability was 0% with polyclonal sera, 90% with monoclonal sera, 94% with phage typing, 85% with pyocin typing, 36% with reverse phage typing and 100% with DNA-prope typing. Using monoclonal antibodies, the reproducibility was 75%, while that of phage typing was 88%, pyocin typing 53% and reverse phage typing 62%. Typing with the DNA probe was not repeated. using polyclonal sera, repeated typing showed that 94% of the isolates were polyagglutinable. Using phage typing, 40% of the isolates belonged to phage type 31, while 60% were distributed amongst 32 phage types. Using monoclonal antibodies, 71% of the isolates belonged to 0-group 3, and these isolates showed 16 different phage types. Subdivision of the phage types was further achieved by both pyocin typing and reverse phage typing. The DNA probe typing made it possible in some cases to discriminate between isolates which were otherwise found identical with the conventional typing methods, while in other cases typing with the DNA probe recorded as identical isolates which conventional methods had typed as being different. These differences may be due to a high mutation rate caused by the selection pressure of antibiotics, and by the host immune response. According to our results, investigations of reproducibility and typability of old and new typing methods are essential when they are used in clinical situations. The low reproducibility of some of the typing methods in the present study affects the reliability of epidemiological investigations in cystic fibrosis patients. Usage of only one method may not be sufficient in cases of polyagglutinable strains from cystic fibrosis patients.     

Serogrouping of Clostridium difficile strains by slide agglutination.

Six different agglutinating antisera were obtained by immunizing rabbits with Formol-treated strains of Clostridium difficile. After appropriate absorption, these antisera were used to define six serogroups designated by the letters A, B, C, D, F, and G. Altogether, 315 strains of C. difficile from various origins were tested for slide agglutination by these antisera; 312 (99%) of them were agglutinated by one of these antisera. A and C were the most common serogroups. An excellent correlation, ranging from 85 to 100%, was found between the serogroup and the toxigenicity of the strains. The correlation between serogroup and sorbitol fermentation was higher, ranging from 89 to 100%. The results of this typing were compared with the clinical origin of the strains. Only strains of serogroups A, C, and D were isolated in 153 cases of antibiotic-associated diarrhea. This series included strains from three outbreaks; all the strains in two of the outbreaks belonged to serogroup C, and in the third, all the strains belonged to serogroup A. Strains of serogroups B, F, and G were only found in the stools of asymptomatic neonates or young children. In the latter samples, strains of serogroups A and D were found in the same ratio as in adults with antibiotic-associated diarrhea, but strains of serogroup C were seldom isolated. In patients treated with antineoplastic drugs and suffering from diarrhea, the distribution of the strains was the same as in cases of antibiotic-associated diarrhea.     

Rabbit antisera against three different bacteria which can induce reactive arthritis: analysis by ELISA, immunoprecipitation and Western Blot.

Three strains of bacteria which induce reactive arthritis were collected: a Shigella flexneri, designated 7060; another Sh. flexneri, designated 316; and a Yersinia enterocolitica of serotype 03. Rabbit antisera were generated against each of them to test for the extent and nature of cross-reactivity among these strains. When analysed by the ELISA technique, antisera against 7060 and 316 showed strong cross-reactivity with Y. enterocolitica. In contrast, the reaction of antisera prepared against putatively non-arthritis-causing bacteria reacted several-folds less. Using immunoprecipitation, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western Blot procedures, a 92,000 MW cross-reactive antigen on the Yersinia was identified. The antigen was present on the outer membranes of the Y. enterocolitica, and enzyme digestion experiments showed that this antigen was protein in nature.     

Serological characterisation of Ureaplasma urealyticum strains by enzyme-linked immunosorbent assay (ELISA).

A modification of enzyme-linked immunosorbent assay (ELISA) was developed for the serological characterisation and identification of strains of Ureaplasma urealyticum. The eight recognised human serotypes of U urealyticum and antisera produced against them were used as reference for the evaluation and standardisation of the method. The serological profile illustrating reactions of antigen with homologous and heterologous antisera was specific and reproducible for each serotype. The homologous reaction was always very prominent but some cross-reactivity was seen, most clearly between serotypes 2 and 5. The method was found to be suitable for serological typing of clinical isolates of U urealyticum because of rapid and simple technical procedure, good reproducibility of the results and economical consumption of antisera and other reagents.     

Expression of type-specific virion structural antigen(s) in L cells transformed by herpes simplex virus types 1 and 2.

Antisera prepared against thymidine kinaseless (tk^?) L cells (Ltk^?) transformed to the tk⁺ phenotype by herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) preferentially agglutinated purified homologous virions. Transformed cell antisera were much less effective in agglutinating nucleocapsids than they were in agglutinating envelope material from the homologous virus. Antiserum prepared against untransformed Ltk^? cells agglutinated about twice as much virus as did preimmune serum but only 13 to 19% as much as was agglutinated by transformed cell antisera. These results indicate that both HSV-1- and HSV-2-transformed L cells express an HSV type-specific structural antigen(s) and that this antigen is present in the envelope of the virion. Since the transformed cell antisera also agglutinated the heterologous virus but to a lesser extent than the homologous virus, these antisera were capable of detecting type-common antigenic determinants as well.     

Method for the serological typing of the capsular polysaccharides of Staphylococcus aureus.

A method is described for typing Staphylococcus aureus capsular polysaccharides that is based on direct bacterial cell agglutination and immunoprecipitation of cell extracts with monospecific antisera. Encapsulated strains were identified by their inagglutinability with teichoic acid antisera. The typing sera reacted specifically with extracts of eight prototype strains.     

Comparison of haplotypes of the major histocompatibility complex in the rat. II. Serological analysis of the haplotypes H-1a (Ag-B4), H-1d (Ag-B9) and H-1f (Ag-B10).

The strongly cross-reacting haplotypes of the inbred strains DA, ACI, ACP, MR, BD V and AS2, and of the congenic lines LEW.1A, LEW.1D and LEW.1F, were explored. All of these inbred strains and these congenic lines type with anti-Ag-B4 antisera raised against the DA strain, which behaved as operationally mono-specific in previously reported studies of the eight currently defined Ag-B haplotypes. Serological analyses showed that this cross-reactivity was due to antibodies against public antigenic specificities which were not previously recognized as being in the anti-Ag-B4 antisera, due to the fact that the LEW.1D and LEW.1F animals were not available for testing. With the use of appropriate antisera and of absorption studies, two haplotypes in the H-1 system were found not to have been previously identified in the Ag-B system. The animals carrying the H-1d haplotype (MR, BD V and LEW.1D) have been designated as Ag-B9, and those carrying the H-1f haplotype (AS2 and LEW.1F) have been designated as Ag-B10. With the proper absorptions, an anti-Ag-B4 antiserum can be prepared which reacts only with the DA, ACI and ACP strains, and it will henceforth be used as the operationally mono-specific Ag-B4 typing reagent. A variety of typing experiments showed that the results using antisera raised in Pittsburgh and in Prague and using the Ficoll and dextran haemagglutination methods were the same.     

Comparison of polyclonal rabbit antisera with monoclonal antibodies for serological typing of Pseudomonas aeruginosa.

A panel of 219 distinct strains of Pseudomonas aeruginosa were serotyped with a set of monoclonal antibodies prepared against the serotype strains of the Homma scheme (J. Y. Homma, Jpn. J. Exp. Med. 46:329-336, 1976). A total of 87.6% were typable, and there was very good agreement with the corresponding polyclonal serotype. A high proportion of strains that were polyagglutinating or nontypable with polyclonal antisera were agglutinated by antibody towards Homma group M.     

Autoagglutinin produced by organ fragment cultures of mouse spleen

Mouse spleen fragment microcultures produced a heat-labile substance which agglutinated syngeneic erythrocytes. The substance reacted with an antigen present on some strains of mouse erythrocytes and on rat erythrocytes, but absent from other strains of mouse erythrocytes and from guinea pig, rabbit or sheep erythrocytes. Agglutination was inhibited by antisera to mouse immunoglobulins except for antisera specific to mouse IgM which enhanced agglutination. The agglutinin sedimented with 19 S Ig on ultracentrifugation and was destroyed by pretreatment with mercaptoethanol. Using a rosette assay increased numbers of cells, specifically binding syngeneic erythrocytes by means of a surface Ig receptor were demonstrated in spleen fragment microcultures. It is considered that the agglutinin is a noncomplement-activating IgM autoantibody and that the precursors of the cells producing the autoantibody are detected by the rosette assay.     

COMPARISON OF HAPLOTYPES OF THE MAJOR HISTOCOMPATIBILITY COMPLEX IN THE RAT II. SEROLOGICAL ANALYSIS OF THE HAPLOTYPES H-1a (Ag-B4), H-1d (Ag-B9) and H-1f (Ag-B10)

The strongly cross-reacting haplotypes of the inbred strains DA, ACI, ACP, MR, BD V and AS2, and of the congenic lines LEW.1A, LEW.1D and LEW. 1F, were explored. All of these inbred strains and these congenic lines type with anti-Ag-B4 antisera raised against the DA strain, which behaved as operationally mono-specific in previously reported studies of the eight currently defined Ag-B haplotypes. Serological analyses showed that this cross-reactivity was due to antibodies against public antigenic specificities which were not previously recognized as being in the anti-Ag-B4 antisera, due to the fact that the LEW.1D and LEW. 1F animals were not available for testing. With the use of appropriate antisera and of absorption studies, two haplotypes in the H-1 system were found not to have been previously identified in the Ag-B system. The animals carrying the H-1^d haplotype (MR, BD V and LEW.1D) have been designated as Ag-B9, and those carrying the H-1^f haplotype (AS2 and LEW.1F) have been designated as Ag-B10. With the proper absorptions, an anti-Ag-B4 antiserum can be prepared which reacts only with the DA, ACI and ACP strains, and it will henceforth be used as the operationally mono-specific Ag-B4 typing reagent. A variety of typing experiments showed that the results using antisera raised in Pittsburgh and in Prague and using the Ficoll and dextran haemagglutination methods were the same.     

Serotypes of Pseudomonas aeruginosa in clinical specimens in relation to antibiotic susceptibility.

Ninety-eight hospital strains of Pseudomonas aeruginosa isolated from six different hospitals in Athens were serotyped by a slide agglutination test with unabsorbed commercial antisera. Serotypes O6, O11, O12, and "pool E" strains (strains that agglutinated only in pool E, which contained antisera against O2, O5, O15, and O16 antigens, but did not agglutinate in the individual antisera) predominated, accounting for more than 62% of all isolates tested. In respect to serotypes, (i) there was no apparent correlation with hospital of origin, (ii) most strains of serotypes O6 and O11 were sensitive to gentamicin and carbenicillin (iii) most strains of pool E were from urine and were resistant to these drugs, (iv) all 9 strains of serotype O12 tested were resistant to carbenicillin and all 5 strains tested hydrolyzed this drug, and (v) 24 of 25 strains of pool E were resistant to carbenicillin but only 2 of 17 strains hydrolyzed it.     

Virulence factors of avian Escherichia coli associated with swollen head syndrome.

Virulence characteristics of 50 strains of Escherichia coli isolated from chickens with swollen head syndrome were examined. The results were the following: in the absence of D-mannose, 74% of strains agglutinated guinea pig erythrocytes, but in the presence of D-mannose 32% agglutinated guinea-pig erythrocytes and 62% agglutinated human erythrocytes. When slide agglutination assays were carried out with antisera to adhesin of bovine and swine origin (K88, K99, F41, F42 987P and 2134P), only 14% of strains agglutinated with antiserum to F41. Colicin V was produced by 78% of the E. coli strains and 80% produced aerobactin. In the serum resistance test, 36 (72%) of strains showed resistance to normal chicken serum. Only seven (14%) strains expressed K1 capsular antigen, while motility was found in 62% of the strains.     

Purification and antigenic relatedness of proteins II of Neisseria gonorrhoeae.

Gonococcal proteins II from three strains were purified by chromatofocusing, and antisera was raised against them. These antisera were examined by immunoblotting to explore the antigenic relatedness of proteins II of seven different strains. The strongest reactions of the antisera were with the homologous proteins II. The antiserum against the proteins II of one strain also reacted with the proteins II present in all of the heterologous strains, whereas the antisera against the proteins II of two other strains showed little cross-reactivity with heterologous proteins II. Monoclonal antibodies produced against the three proteins II of strain F62 were specific for homologous proteins II and recognized epitopes unique to each individual protein II. These studies confirm the extensive intra- and interstrain variability in the antigenic structure of these proteins.     

The HLA-B12 and -B40 cross-reactive groups and their serological relationships

The serological analysis of 82 broad HLA-B antisera, produced by pregnancy alone, containing reactivity against HLA-B 12 and/or B40 positive cells and up to nine additional specificities was performed, using highly selected lymphocyte panels. The HLA typing of 76 of the antiserum donors and 75 of their husbands showed that the antisera were stimulated in response to one of 10 different HLA-B antigens. It also showed the influence of serum donor HLA-B antigens on the reaction range of the antiserum produced, as well as significant HLA-B antigen frequency disturbances within varous groups of the antiserum donors. Fifteen HLA-B specificities were found to comprise the B12 cross-reactive group and its related cross-reactions, with bidirectional cross-reactivity occurring between Bw44, Bw45, Bw49 and Bw50 (unidirectional between Bw44 and Bw50). Seventeen specificities were observed in the B40 cross-reactive group and its related crossreactions, with bidirectional cross-reactivity occurring between Bw41, Bw50, Bw60 and Bw61. Antisera were studied that showed: (1) cross-reactivity between Bw44, Bw45 and Bw60 stimulated antisera and both subdivisions of the B12 and B40 antigens; (2) cross-reactivity between B13, Bw44, Bw49, Bw60 and Bw61 stimulated antisera and Bw47; and (3) cross-reactivity between Bw44, Bw49, Bw60 and Bw61 stimulated antisera and B13, with bidirectional cross-reactivity occurring between B13, Bw60 and Bw61 and between B13 and Bw44. Bidirectional crossreactivity was also observed between B37 and Bw44 and between B7 and Bw60. HLA-Bw48 was shown to be included within the reaction range of both B7 and Bw60 stimulated antisera. Preferential cross-reactivity with one of two antigen subdivisions was extensively observed and occurred exclusively between antigens of the same Bw4/Bw6 association as the antiserum stimulating specificity. The results are discussed within the framework of the existence of multiple shared antigenic determinants.     

Serological Typing of Pseudomonas aeruginosa: Use of Commerical Antisera and Live Antigens

A practical slide agglutination test, with commercial antisera (Difco) and live antigens (antigens of live bacteria) taken directly from 24-h antimicrobial susceptibility plates, has been established for serotyping Pseudomonas aeruginosa. Until recently, the lack of both a standard antigenic scheme and a source of commercial antisera has made serological typing of this organism impractical. A simplified procedure with 17 unabsorbed antisera and live antigens prepared from materials readily available in most clinical microbiology laboratories makes epidemiological typing of this organism possible in hospital laboratories. The distribution of each serotype examined in this study was determined by using 425 consecutive patient isolates from six different hospitals. The distribution of O antigen groups (live antigen) was as follows: O1, 11.5%; O2, 1.6%; O3, 3.8%; O4, 7.8%; O5, 4.2% O6, 27.1%; O7,8, 5.9%; O9, 6.8%; O10, 2.4%; O11, 8.2%; O12 through O17, each less than 1%. Ten and six-tenths percent of the above agglutinated in two antisera, 3.3% agglutinated in more than two antisera, and 5.2% did not agglutinate in any antisera. A comparison of live and heated antigens shows that 93.2% were typable with the live antigen, and 94.5% were typable with the heated antigen. When both antigens were used, we typed 96.3% of 725 isolates. The reproductibility and specificity of the serological procedure were examined. We recommend using the live antigen for routine serological typing in clinical microbiology laboratories for "in house" epidemiology and reserving the heated antigen for reference and research typing (and for those few cases where results cannot be obtained using the live antigen). The application of serotyping in the study of outbreaks of P. aeruginosa is also presented.     

Serogrouping of Bacteroides vulgatus by the agglutination test

The agglutination technique was used to establish a serological classification scheme for Bacteroides vulgatus strains isolated from normal human feces and clinical specimens, especially from ulcerative colitis patients. Absorbed antisera to 10 strains of B. vulgatus were prepared. These 10 absorbed antisera were species specific. Of 90 B. vulgatus strains tested, 55 (61%) were agglutinated by one or more of these 10 absorbed antisera. A total of 27 serological patterns were grouped into 18 serogroups; 10 of these serogroups contained only one group component, whereas the other 8 serogroups were composed of more than one component. This serological classification could be used to study the epidemiology of this organism.