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Protein I serotype of serum-resistant versus serum-sensitive Neisseria gonorrhoeae strains.

In order to characterize serum-resistant and serum-sensitive strains of N. gonorrhoeae, the protein I serotype, auxotype, and penicillin susceptibility of 128 strains were tested. Sensitivity to the complement-dependent bactericidal activity of normal human serum was highly associated with protein I serotype (p less than 0.001). Thus 85% of serotype 1-3 strains were serum-resistant, whereas 86% of serotype 8 strains and all strains with serotypes 8 + 9 or 9 were serum-sensitive. Serum-resistance or sensitivity for a given serotype was independent of auxotype. The susceptibility to penicillin within the serotypes 1-3 was significantly associated with auxotype (p = 0.0016); all AHU- (requirement for arginine, hypoxanthine and uracil) strains had MICs of penicillin of 0.04 microgram/ml or less and were serotypes 1-3. Among the non-AHU-strains, serotype 9 was significantly more penicillin susceptible than the other serotypes (p less than 0.003).

Blood Bactericidal Activity↗

[Serotypes and electropherotypes of group A human rotaviruses in patients with acute gastroenteritis in Saitama area, 1988-1991].

Serotypes and RNA electropherotypes of group A human rotaviruses were identified in stool samples from patients with acute gastroenteritis in Kasukabe Kosei Hospital, Saitama, Japan, during three rotavirus seasons from 1988 to 1991. Of the 665 stool samples from patients with acute gastroenteritis, 169 (25.4%) stool samples were positive for group A human rotaviruses. Of these 169 samples, 98 (58.0%) were serotype 1, 7 (4.1%) serotype 2, 14 (8.3%) serotype 3, 3 (1.8%) serotype 4 and 47 (27.8%) were untypable. Serotype 1 was predominant over all three rotavirus seasons. Serotype 2 was most prevalent in the second rotavirus season, serotype 3 in the third season. Serotype 4 was detected only the second rotavirus season. The ratio of subtype I to subtype II was 4.5:95.5. Among the 162 strains examined, 139 specimens were available to be electropherotyped. Fifteen different RNA electropherotypes were detected. Three to four different electropherotypes were more prevalent in each rotavirus season and the detection rate in each year was different within the same electropherotypes. The rotavirus strains of the same electropherotypes were observed with high prevalence for every rotavirus seasons. The same electropherotypes was found in human rotavirus strains of different serotypes that were appeared at different epidemic seasons.

Acute Disease↗

Ingestion of Salmonella enterica serotype Poona by a free-living mematode, Caenorhabditis elegans, and protection against inactivation by produce sanitizers.

Free-living nematodes are known to ingest food-borne pathogens and may serve as vectors to contaminate preharvest fruits and vegetables. Caenorhabditis elegans was selected as a model to study the effectiveness of sanitizers in killing Salmonella enterica serotype Poona ingested by free-living nematodes. Aqueous suspensions of adult worms that had fed on S. enterica serotype Poona were treated with produce sanitizers. Treatment with 20 microg of free chlorine/ml significantly (alpha = 0.05) reduced the population of S. enterica serotype Poona compared to results for treating worms with water (control). However, there was no significant difference in the number of S. enterica serotype Poona cells surviving treatments with 20 to 500 microg of chlorine/ml, suggesting that reductions caused by treatment with 20 microg of chlorine/ml resulted from inactivation of S. enterica serotype Poona on the surface of C. elegans but not cells protected by the worm cuticle after ingestion. Treatment with Sanova (850 or 1,200 microg/ml), an acidified sodium chlorite sanitizer, caused reductions of 5.74 and 6.34 log(10) CFU/worm, respectively, compared to reductions from treating worms with water. Treatment with 20 or 40 microg of Tsunami 200/ml, a peroxyacetic acid-based sanitizer, resulted in reductions of 4.83 and 5.34 log(10) CFU/worm, respectively, compared to numbers detected on or in worms treated with water. Among the organic acids evaluated at a concentration of 2%, acetic acid was the least effective in killing S. enterica serotype Poona and lactic acid was the most effective. Treatment with up to 500 microg of chlorine/ml, 1% hydrogen peroxide, 2,550 microg of Sanova/ml, 40 microg of Tsunami 200/ml, or 2% acetic, citric, or lactic acid had no effect on the viability or reproductive behavior of C. elegans. Treatments were also applied to cantaloupe rind and lettuce inoculated with S. enterica serotype Poona or C. elegans that had ingested S. enterica serotype Poona. Protection of ingested S. enterica serotype Poona against sanitizers applied to cantaloupe was not evident; however, ingestion afforded protection of the pathogen on lettuce. These results indicate that S. enterica serotype Poona ingested by C. elegans may be protected against treatment with chlorine and other sanitizers, although the basis for this protection remains unclear.

Animals↗

Development of an enzyme-linked immunosorbent assay for serotyping ureaplasma urealyticum strains using monoclonal antibodies.

Ureaplasma urealyticum comprises 14 serotypes. The existing serotyping methods all use polyclonal antibodies. These methods are time-consuming and labor-intensive, and they cannot always be performed on primary isolates; in addition, the results are difficult to interpret. We developed a new enzyme-linked immunosorbent assay (ELISA) method using serotype-specific monoclonal antibodies (MAbs) to enable the serotyping of U. urealyticum isolates from primary broth cultures. Each of the 14 serotype reference strains was tested against 14 selected MAbs. Homologous reactions were very strong, while heterologous reactions were negligible. Three cross-reactions were observed: MAb 5 cross-reacted with serotype 2, MAb 14 cross-reacted with serotype 3, and MAb 8 cross-reacted with serotype 13. Despite the cross-reactions observed, all the serotype reference strains of U. urealyticum could be identified and differentiated using a combination of MAbs. Reproducibility was analyzed with a fractionated antigenic preparation and with several freshly prepared antigens of the same strain. No significant interrun variation was found with the fractionated antigen, but significant variations in optical density (OD) values were found when freshly prepared antigens were tested. However, the variation in OD values did not influence the overall interpretation of the ELISA: reactions with homologous MAbs were always prominent compared to those of the negative controls. This newly developed ELISA using MAbs seems promising for serotyping of U. urealyticum clinical isolates.

Antibodies, Bacterial↗

Course of infection and development of immunity in experimental infection of mice with Listeria serotypes.

NMRI mice were experimentally infected with Listeria monocytogenes serotypes 1/2b, 3a, 4b, and 4d and Listeria innocua serotype 6b by different means. The course of infection was monitored, using bacteriological and histological methods. The following typical features of experimental infection with the various L. monocytogenes and L. innocua serotypes were observed. (i) On the basis of the mean lethal dose, L. monocytogenes 4b, 4d, and 1/2b proved to be mouse pathogenic, although to different degrees, L. monocytogenes 3a and L. innocua can be regarded as nonpathogenic for NMRI mice. The virulence of L. monocytogenes serotype 4d was increased 1,000-fold after adaptation to mice. (ii) Primary infection with any serotype of L. monocytogenes or L. innocua resulted in protection against a lethal challenge with the most virulent serotype, 4b. This protective immunity could be transferred by spleen cells. Compared with the duration of immunity achieved by infection with L. monocytogenes serotype 4b, the protection induced by infection with L. innocua was short lived and dose dependent. The data obtained also suggest that immunity after experimental infection with any serotype of L. monocytogenes or L. innocua is produced only when the animal host is filled with bacteria. (iii) The distribution of the germs in the internal organs of the mouse shortly after infection was dependent on the route of infection rather than on the serotype used. (iv) The main difference among the Listeria serotypes tested was their ability to multiply within the host and to induce a granulomatous inflammation. The results indicate that mouse pathogenicity and virulence of Listeria spp. cannot be defined only by the capacity of the bacteria to infect or kill conventional mice. Such a definition should include an analysis of the immune system of the host, a kinetic study of experimental infection, and a histomorphological evaluation of the lesions induced.

Adaptation, Physiological↗

Production and characterization of monoclonal antibodies against specific serotypes of Mycobacterium avium and the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex.

Serotype-specific and Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex (MAIS complex)-specific monoclonal antibodies (MAbs) were prepared. A series of MAbs were obtained, five specific for serotype 2, three specific for serotype 4, eight against a strain of serotype 19, two specific for the MAIS complex, and two against a common glycolipid shared by all the mycobacteria tested so far. The serotype-specific and the MAIS complex-specific MAbs reacted in immunofluorescence with intact mycobacteria and in enzyme-linked immunosorbent assay and immuno-thin-layer chromatography with lipid extracts of mycobacteria. The two MAbs against a common mycobacterial glycolipid reacted only in lipid enzyme-linked immunosorbent assay and immuno-thin-layer chromatography. All MAbs were directed against glycopeptidolipids (GPLs), except for four MAbs against proteins of serotype 19. The serotype- and MAIS complex-specific epitopes on GPLs are exposed on the mycobacterial cell wall, in contrast with the common mycobacterial glycolipid, which is probably located inside the cell wall. The serotype-specific MAbs reacted with native as well as deacetylated GPLs, in contrast with the MAIS complex-specific MAbs, which reacted only with native GPLs. The MAbs will be useful for the identification of MAIS complex and M. avium serotypes 2 and 4 and a strain of serotype 19, GPL analyses with immuno-thin-layer chromatography, and the localization of GPL epitopes in mycobacteria.

Animals↗

Novel apoptosis-inducing activity in Bacteroides forsythus: a comparative study with three serotypes of Actinobacillus actinomycetemcomitans.

Bacteroides forsythus, which has been reported to be associated with periodontitis but has not been recognized as a key pathogen, was found to induce cytolytic activity against HL-60 and other human leukemic cells. This cytolytic activity was demonstrated according to three different criteria: (i) loss of both mitochondrial membrane potential and membrane integrity in cells treated with bacterial extracts and then with Rh123 and propidium iodide, respectively, as demonstrated by flow cytometry; (ii) damage to cytoplasmic membrane, as revealed by scanning electron microscopy (SEM); and (iii) DNA ladder formation and activation of caspase-3. These results indicate that B. forsythus produced an apoptosis-inducing factor(s) found to be composed of protein as judged by heat and trypsin sensitivity. In addition to extracts from B. forsythus, the culture supernatant of this bacterium has the ability to induce a cytolytic effect against peripheral white blood cells, especially lymphocytes. For comparison with B. forsythus, the same analyses were applied to two strains with different serotypes of Actinobacillus actinomycetemcomitans, serotypes a (ATCC 43717) and c (ATCC 43719), in addition to previously reported apoptosis-inducing serotype b (ATCC 43718), which was used as a positive control. The strains of A. actinomycetemcomitans serotypes a and b induced apoptosis in HL-60 cells as judged by the above three criteria but to a slightly lesser extent than did B. forsythus, while the serotype c strain produced apoptosis to a negligible extent. Detailed SEM images showed that the A. actinomycetemcomitans serotype a strain induced large-pore formation and the serotype b strain produced small pores with typical blebbing, while B. forsythus induced severe membrane ruffling. Further DNA ladder formation and caspase-3 activation were observed in the serotype a and b strains but not in the serotype c strain. The present paper is the first report of a protein factor(s) from B. forsythus and the A. actinomycetemcomitans serotype a strain which induces apoptotic cell death.

Aggregatibacter actinomycetemcomitans↗

Genetic basis for the structural difference between Streptococcus pneumoniae serotype 15B and 15C capsular polysaccharides.

In a search for the genetic basis for the structural difference between the related Streptococcus pneumoniae capsular serotypes 15B and 15C and for the reported reversible switching between these serotypes, the corresponding capsular polysaccharide synthesis (cps) loci were investigated by keeping in mind that at the structural level, the capsules differ only in O acetylation. The cps locus of a serotype 15B strain was identified, partially PCR amplified with primers based on the related serotype 14 sequence, and sequenced. Sequence analysis revealed, among other open reading frames, an intact open reading frame (designated cps15bM) whose product, at the protein level, exhibited characteristics of previously identified acetyltransferases. Genetic analysis of the corresponding region in a serotype15C strain indicated that the same gene was present but had a premature stop in translation. Closer analysis indicated that the serotype 15B gene contained a short tandem TA repeat consisting of eight TA units. In serotype 15C, this gene contained nine TA units that resulted in a frameshift and a truncated product. Genetic analysis of 17 serotype 15B and 15C clinical isolates revealed a perfect correlation between the serotype and the length of the short tandem repeat in the putative O-acetyltransferase gene. The number of TA repeating units varied between seven and nine in the various isolates. Together, the data strongly suggest that the structural difference between serotypes 15B and 15C is based on variation in the short tandem TA repeat in the O-acetyltransferase gene and that the transition between serotypes is due to slipped-strand mispairing with deletion or insertion of TA units in the cps15bM gene.

Acetyltransferases↗

Capsular serotype K1 or K2, rather than magA and rmpA, is a major virulence determinant for Klebsiella pneumoniae liver abscess in Singapore and Taiwan.

Capsular serotypes, magA, and rmpA have been documented in high prevalence for Klebsiella pneumoniae liver abscess. To investigate the regional difference and the correlation of capsular serotype, magA, and rmpA with virulence, 73 isolates were collected in Singapore and Taiwan. Capsular serotypes were determined by countercurrent immunoelectrophoresis, the presence of magA and rmpA was determined by PCR, and virulence was determined by phagocytosis and mouse inoculation. Isolates from Singapore were similar to those from Taiwan in genomic heterogeneity, prevalence of serotype, and the presence of magA and rmpA. The most common serotype was K1 (34/73; 46.6%), followed by K2 (15/73; 20.5%). magA was restricted to serotype K1. All K1 or K2 isolates and 66.7% (16/24) of isolates that were neither serotype K1 nor serotype K2 (non-K1/K2) carried rmpA. Serotype K1 or K2 isolates demonstrated significantly more phagocytic resistance and virulence than did rmpA-positive and -negative groups of non-K1/K2 isolates. In the non-K1/K2 group, the virulence profiles of rmpA-positive strains from Taiwan and Singapore were different by phagocytosis assay and in the mouse model, indicating that factors other than rmpA contributed to virulence. The characteristics of K. pneumoniae liver abscess in Singapore and Taiwan are similar. Capsular serotype K1 or K2 plays a more important role than magA and rmpA in determining virulence in K. pneumoniae liver abscess.

Animals↗

Identification and serotyping of Haemophilus pleuropneumoniae by coagglutination test.

A coagglutination test for the identification and serotyping of Haemophilus pleuropneumoniae is described. A total of 360 H. pleuropneumoniae strains were isolated from pulmonary tissues of feeder pigs which died of acute pleuropneumonia. All of the isolates were serotyped by coagglutination, and the results were confirmed by the ring precipitation test. The most common serotype isolated in Quebec was serotype 1, followed by serotypes 5, 2, and 7. None of the isolates belonged to serotypes 3, 4, or 6. Mixed infections due to H. pleuropneumoniae of more than one serotype in the same animal were encountered. Serotype 1 was the only common isolate among the mixed-serotype infections. The coagglutination test was more sensitive than was the ring precipitation test. Serotyping by the coagglutination test is inexpensive, rapid, reliable, and easy to perform.

Agglutination Tests↗

Genetic relatedness among human rotavirus genes coding for VP7, a major neutralization protein, and its application to serotype identification.

Antigenic characterization of human rotaviruses by plaque reduction neutralization assay has revealed four distinct serotypes. The outer capsid protein VP7, coded for by gene 8 or 9, is a major neutralization protein; however, studies of rotaviruses derived from genetic reassortment between two strains have confirmed that another outer capsid protein, VP3, is in some cases equally important in neutralization. In this study, the genetic relatedness of the genes coding for VP7 of human rotaviruses belonging to serotypes 1 through 4 was examined by hybridization of their denatured double-stranded genomic RNAs to labeled single-stranded mRNA probes derived from human-animal rotavirus reassortants containing only the VP7 gene of their human rotavirus parent. A high degree of homology was demonstrated between the VP7 genes of strain D and other serotype 1 human rotaviruses, strain DS-1 and other serotype 2 human rotaviruses, strain P and other serotype 3 human rotaviruses, and strain ST3 and other serotype 4 human rotaviruses. Hybrid bands could not be demonstrated between the VP7 gene of D, DS-1, P, or ST3 and the corresponding gene of human rotaviruses belonging to a different serotype. RNA specimens extracted from the stools of 15 Venezuelan children hospitalized with rotavirus diarrhea were hybridized to each of the reassortant probes representing the four human serotypes. All five viruses with short RNA patterns showed homology with the DS-1 strain VP7 gene; two of these were previously adapted to tissue culture and shown to be serotype 2 strains by tissue culture neutralization. Of the remaining 10 viruses with long RNA patterns, 2 hybridized only to the D strain VP7 gene, 6 hybridized only to the P strain VP7 gene, and 2 hybridized only to the ST3 strain VP7 gene. Hybridization using single human rotavirus gene substitution reassortants as probes may provide an alternative method for identifying the VP7 serotype of field isolates that would circumvent the need for tissue culture adaptation.

Capsid↗

Serotypic differentiation of group A rotaviruses with porcine rotavirus gene 9 probes.

The serotypic specificities of Gottfried and OSU porcine rotavirus gene 9 probes were investigated in a dot hybridization assay. The probes were reacted with homologous and heterologous serotypes of group A rotaviruses of human and animal origin. Hybridizations were conducted under relatively low-stringency (52 degrees C, no formamide, 5 x SSC) and high-stringency (52 degrees C, 50% formamide, formamide, 5 x SSC) conditions (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Under conditions of relatively low stringency, the Gottfried and OSU gene 9 probes demonstrated broad cross-reactivity and were useful in the detection of homologous and heterologous serotypes of group A rotaviruses. Under conditions of relatively high stringency, the Gottfried and OSU gene 9 probes were serotype specific. The Gottfried gene 9 probe (serotype 4) hybridized with homologous Gottfried porcine rotavirus as well as the serotype 4 human rotaviruses ST3 and VA70. The OSU gene 9 probe (serotype 5) hybridized with homologous OSU porcine rotavirus and the serotype 5 equine rotavirus H1. Hybridization was not observed with the antigenically distinct group B and C porcine rotaviruses or with other porcine enteric viruses, including calicivirus and a coronavirus, transmissible gastroenteritis virus, regardless of stringency conditions. Analysis of 14 group A rotavirus-positive field samples resulted in the serotypic differentiation, collectively, of six serotype 4 or 5 porcine rotaviruses. No field samples reacted with both the Gottfried and OSU gene 9 probes.

Animals↗

Characterization of Shigella dysenteriae serotypes 11, 12, and 13.

We conducted serologic and biochemical studies on strains of three provisional Shigella serotypes. Included were 19 strains of serotype 3873-50 from three countries (1 from Mexico, 1 from Netherlands Antilles, and 17 from the United States), 13 strains of serotype 3341-55 from three countries (1 from Canada, 1 from Bulgaria, and 11 from the United States), and 19 strains of serotype 19809-73 from two countries (16 from Israel and 3 from the United States). Reactions of these strains with homologous and heterologous Shigella antisera showed that serotypes 3873-50 and 19809-73 were unique. Strains of serotype 3341-55 shared an identical antigen with Shigella boydii 18; however, they were distinguished from S. boydii 18 biochemically and with absorbed sera. Strains of the three serotypes were biochemically most similar to Shigella dysenteriae. Like other shigellae, strains from each serotype were invasive in the Serény assay. Although biochemically similar to S. dysenteriae, none of the provisional serotypes produced Shiga toxin or the biologically related toxin, Shiga-like toxin II, produced by some strains of Escherichia coli. On the basis of these results and other published findings, we support earlier proposals to add the three provisional serotypes 3873-50, 3341-55, and 19809-73 to the Shigella schema and will include them as S. dysenteriae 11, 12, and 13, respectively.

Antibodies, Bacterial↗

Pyrazinamidase, CR-MOX agar, salicin fermentation-esculin hydrolysis, and D-xylose fermentation for identifying pathogenic serotypes of Yersinia enterocolitica.

We evaluated several simple laboratory tests that have been used to identify pathogenic serotypes of Yersinia enterocolitica or to indicate the pathogenic potential of individual strains. A total of 100 strains of Y. enterocolitica were studied, including 25 isolated during five outbreak investigations, 63 from sporadic cases, and 12 from stock cultures. The pyrazinamidase test, which does not depend on the Yersinia virulence plasmid, correctly identified 60 of 63 (95% sensitivity) strains of pathogenic serotypes and 34 of 37 (92% specificity) strains of nonpathogenic serotypes. Salicin fermentation-esculin hydrolysis (25 degrees C, 48 h) correctly identified all 63 (100% sensitivity) strains of the pathogenic serotypes and 34 of 37 (92% specificity) strains of the nonpathogenic serotypes. The results of the pyrazinamidase and salicin-esculin tests disagreed for only 7 of the 100 strains of Y. enterocolitica, and these would require additional testing. Congo red-magnesium oxalate (CR-MOX) agar determines Congo red dye uptake and calcium-dependent growth at 36 degrees C, and small red colonies are present only if the strain contains the Yersinia virulence plasmid. This test has proven to be extremely useful for freshly isolated cultures, but only 15 of 62 strains of pathogenic serotypes that had been stored for 1 to 10 years were CR-MOX positive. None of the 16 strains of Y. enterocolitica serotype O3 fermented D-xylose, so this test easily differentiated strains of this serotype, which now appears to be the most common in the United States. Although antisera that can actually be used to serotype strains of Y. enterocolitica are not readily available, the four simple tests described above can be used to screen for pathogenic serotypes.

Agar↗

Molecular subtyping of prevalent M serotypes of Streptococcus pyogenes causing invasive disease.

Reproducible methodologies and a scheme for high-resolution genotyping of Streptococcus pyogenes were defined with respect to a study of six predominant M serotypes causing invasive group A streptococcal disease in the United Kingdom. Serotype reference strains were compared with nine clinical isolates of each serotype from patients with diseases such as pneumonia, puerperal sepsis, toxic shock-like-syndrome, cellulitis, or necrotizing fasciitis. Four enzymes were evaluated for their discriminatory power in 16S rRNA gene-specific ribotyping. Discriminatory power was greatest with EcoRI, which generated serotype-specific ribotypes, and with SacI, which could subdivide strains of the same M serotype. Twenty-five combined ribotypes were found among the 60 strains, and the indices of discriminatory power (D values) of this method varied from 0.51 within serotype M1 to 0.98 within strains of serotype M5. Macrorestriction with the rarely cutting endonuclease SmaI and pulsed-field gel electrophoresis gave D values varying from 0.37 within serotype M1 to the maximal 1.0 within serotype M5. Comparison of macrorestriction profiles revealed various degrees of genetic heterogeneity within M serotypes. Strains of M1, M3, M6, and M11 exhibited clonally related macrorestriction profiles, while those of R28 and M5 strains were consistent with polyphyletic origin.

Antigens, Bacterial↗

Candida albicans serotype analysis by flow cytometry.

Candida albicans strains can be assigned to either of two major serogroups, A or B. Antigenic surface determinants present only in serotype A strains allow such a distinction, which has epidemiologic relevance. Reports have established that the relative distributions of the two serotypes can vary depending on the geographic origin of the isolates. A prevalence of susceptibility to an antifungal agent, flucytosine, was also observed with isolates of serotype A. More recently, it was suggested that the occurrence of serotype B isolates in various clinical forms of candidiasis is increasing. However, this latest finding remains controversial since serotyping results vary widely from one laboratory to another because of the lack of standardized methodologies. Difficulty in interpretation of results, which may lead to erroneous serotype identification, is the major setback associated with current methods. For this study, we thus devised a procedure that relies on flow cytometry and that may eliminate ambiguities in serotype determination. The validation of results was achieved with two types of serotype A-specific antisera, Iatron Factor 6 antiserum and an anti-C. albicans antiserum adsorbed on serotype B yeast cells. Agreement between results obtained with these two reagents was 100% with a wide array of Candida strains. These results confirmed the potential of the flow cytometric procedure as a reliable and reproducible method to establish the serotypes of C. albicans strains. Furthermore, some applications of this procedure to the epidemiological study of this human pathogen are presented.

Candida albicans↗

Genetic relationship between Cryptococcus neoformans var. neoformans strains of serotypes A and D.

Cryptococcus neoformans serotypes A and D are responsible for the overwhelming majority of infections in patients with AIDS. The genetic relationship between the serotypes is poorly understood, but there are significant differences in the epidemiology and clinical presentation of serotype A and D infections. We evaluated the genetic relationship between reference C. neoformans strains belonging to serotypes A and D by analyzing their URA5 sequences and restriction fragment length polymorphisms (RFLPs) with the C. neoformans repetitive element 1 (CNRE-1) probe. The results were compared to those previously obtained for isolates from Brazil and New York City by the same typing methods, and dendrograms were generated. Serotype A and D strains produced distinct RFLP patterns consistent with their separation into two major clusters in the dendrogram generated on the basis of RFLP data. Similarly, serotype A and D strains clustered independently of the basis of the nucleotide sequences of their URA5 genes. Pairwise comparisons revealed average numbers of nucleotide differences within serotypes A and D of 3.0 +/- 1.7 and 7.2 +/- 3.4, respectively (P < 0.0001), and between serotypes A and D of 41.9 +/- 2.7. In summary, our results indicate phylogenetic differences between the two serotypes of C. neoformans var. neoformans and suggest that these serotypes could probably be considered different varieties of C. neoformans.

AIDS-Related Opportunistic Infections↗

Clonal relationships among Shigella serotypes suggested by cryptic flagellin gene polymorphism.

The presence of cryptic fliC alleles in the genomes of 120 strains representative of the four Shigella species was investigated. One fragment was obtained by PCR amplification of fliC, with a size varying from 1.2 to 3.2 kbp, depending on the species or serotype. After digestion with endonuclease HhaI, the number of fragments in patterns varied from three to nine, with sizes of between 115 and 1,020 bp. Patterns sharing most of their bands were grouped to constitute an F type. A total of 17 different F types were obtained from all strains included in this study. A unique pattern was observed for each the following serotypes: Shigella dysenteriae 1, 2, 8, and 10 and S. boydii 7, 13, 15, 16, and 17. On the contrary, S. dysenteriae serotype 13 and S. sonnei biotype e were each subdivided into two different F types. S. flexneri serotypes 3a and X could be distinguished from the cluster containing S. flexneri serotypes 1 to 5 and Y. S. flexneri serotype 6 clustered with S. boydii serotypes 1, 2, 3, 4, 6, 8, 10, 11, 14, and 18 and S. dysenteriae serotypes 4, 5, 6, 7, 9, 11, and 12. Two other clusters were outlined: one comprising S. dysenteriae serotypes 3, 12, 13 (strain CDC598-77), 14, and 15 and the other one joining S. boydii serotypes 5 and 9. None of the 17 fliC patterns was found in the fliC HhaI pattern database previously described for Escherichia coli. Overall, this work supports the hypothesis that Shigella evolved from different ancestral strains of E. coli. Moreover, the method outlined here is a promising tool for the identification of some clinically important Shigella strains as well as for confirmation of atypical isolates as Shigella spp.

Deoxyribonucleases, Type II Site-Specific↗