Demonstration of Streptococcus mutans with a cell wall polysaccharide specific to a new serotype, k, in the human oral cavity

Streptococcus mutans organisms are occasionally isolated from the blood of patients with infective endocarditis, though the mechanisms of invasion and survival remain to be elucidated. Two of four blood isolates from patients with bacteremia or infective endocarditis (strains TW295 and TW871) were serologically untypeable by immunodiffusion testing, which was due to a lack of the glucose side chain of the serotype-specific polysaccharide antigen of S. mutans. Immunodiffusion analyses using antisera against these strains demonstrated that 2 of 100 isolates from 100 subjects showed a positive reaction, while further analysis of 2500 isolates from 50 subjects revealed that all 50 isolates from a single subject were not reactive with anti-c, -e, and -f antisera, though they were reactive with anti-TW295 and -TW871 antisera. The oral isolates showed biological properties similar to those of the reference S. mutans strain MT8148, including high levels of sucrose-dependent adhesion and cellular hydrophobicity, along with expression of glucosyltransferases and a protein antigen, PA. We designated these organisms serotype k. A glucose side chain-defective mutant strain was then constructed by insertional inactivation of the gluA gene of strain MT8148, which showed biological properties similar to those of serotype k of S. mutans. Serotype k oral isolates were less susceptible to phagocytosis, as were the gluA-inactivated mutant of strain MT8148 and blood isolates. These results indicate that S. mutans serotype k strains are present in the oral cavity in humans and may be able to survive longer in blood owing to their low susceptibility to phagocytosis.     

Serotype-specific amplification of Rickettsia tsutsugamushi DNA by nested polymerase chain reaction.

Polymerase chain reaction (PCR) with nested primer pairs was used to diagnose scrub typhus and identify the Rickettsia tsutsugamushi serotype. The primer pairs used for PCR were designed on the basis of the nucleotide sequence of the gene that encodes the 56-kDa antigen. Serotype-specific primers were used in the second PCR amplification. Five serovariants, the Gilliam, Karp, Kato, Kawasaki, and Kuroki strains of R. tsutsugamushi, were identified by nested PCR. In addition, the serotype identified by PCR with DNA from blood clots was the same as that of the strain isolated from five patients with scrub typhus. These findings indicate that this method is useful for diagnosis and identification of the rickettsial serotype in infected patients.     

PCR for detection and identification of Streptococcus sobrinus

Oligonucleotide primers were designed based upon a comparison of the dextranase gene (dex) sequences from Streptococcus sobrinus and S. mutans. The primers amplified a 1610-bp long DNA fragment on the dex gene by a PCR. The pair of primers was specific to S. sobrinus as the other members of the mutans streptococci - S. mutans, S. downei, S. cricetus, S. rattus, S. macacae and S. ferus - gave no PCR products. Other gram-positive oral bacteria (15 strains of 10 species of cocci and 18 strains of 12 species of rods) and gram-negative oral bacteria (3 strains of 3 species of cocci and 31 strains of 22 species of rods) also gave negative results in the PCR. The PCR procedure was able to detect as little as 100 fg of purified chromosomal DNA or as few as 9 cfu of S. sobrinus NIDR6715. Seven clinical isolates of S. sobrinus were also positive in the dex PCR. This laboratory developed the S. mutans-specific PCR (dexA PCR) method with the primers specific for a portion of the dextranase gene of S. mutans Ingbritt. Primers for the dex and dexA PCR methods detected two species exclusively from the mutans streptococci. Furthermore, these two species were effectively differentiated by the species-specific amplicons with different lengths. The application of the PCR method to human dental plaque showed that the prevalence of S. sobrinus (83%) in oral cavities was higher than currently supposed (0-50%). These results suggest that the described PCR method is suitable for the specific detection and identification of human cariogenic bacteria, S. sobrinus and S. mutans.     

Effect of peroral immunization of humans with Streptococcus mutans on induction of salivary and serum antibodies and inhibition of experimental infection

Naturally occurring antibodies reactive with Streptococcus mutans whole cells were assayed in whole saliva, parotid saliva, and blood samples collected from eight human volunteers. The levels and serotypes of indigenous S. mutans in plaque and whole saliva samples were also determined. After baseline sampling the teeth were cleaned and the subjects were inoculated with streptomycin-resistant S. mutans strains Ingbritt (serotype c) and OMZ65 (serotype g). The level of implantation and duration of colonization were determined in plaque and saliva, and antibodies reactive with these strains were monitored in saliva and serum. After the implanted bacteria were shed, the subjects wee immunized by the daily ingestion of an enteric-coated capsule containing 25 mg of Formalin-killed, freeze-dried OMZ65 cells for 3 days and inoculation was repeated. The levels of antibodies and of implantation and the duration of colonization were monitored as before. One month after the bacteria could no longer be detected, the immunization and inoculation cycle was repeated except that the subjects were immunized for 7 days. Five of the eight subjects were successfully colonized by strains Ingbritt and OMZ65. The remaining three did not become colonized with either strain. Strain OMZ65 implanted at a higher level than did strain Ingbritt. Oral immunization did not result in a detectable antibody response in saliva or serum to whole bacterial cells. However, after both the first and second immunizations there were marked reductions in the peak levels of infection and the duration of colonization of both OMZ65 and Ingbritt.     

Streptococcus mutans clonal variation revealed by multilocus sequence typing

Streptococcus mutans is the major pathogen of dental caries, a biofilm-dependent infectious disease, and occasionally causes infective endocarditis. S. mutans strains have been classified into four serotypes (c, e, f, and k). However, little is known about the S. mutans population, including the clonal relationships among strains of S. mutans, in relation to the particular clones that cause systemic diseases. To address this issue, we have developed a multilocus sequence typing (MLST) scheme for S. mutans. Eight housekeeping gene fragments were sequenced from each of 102 S. mutans isolates collected from the four serotypes in Japan and Finland. Between 14 and 23 alleles per locus were identified, allowing us theoretically to distinguish more than 1.2 x 10(10) sequence types. We identified 92 sequence types in these 102 isolates, indicating that S. mutans contains a diverse population. Whereas serotype c strains were widely distributed in the dendrogram, serotype e, f, and k strains were differentiated into clonal complexes. Therefore, we conclude that the ancestral strain of S. mutans was serotype c. No geographic specificity was identified. However, the distribution of the collagen-binding protein gene (cnm) and direct evidence of mother-to-child transmission were clearly evident. In conclusion, the superior discriminatory capacity of this MLST scheme for S. mutans may have important practical implications.     

Rapid detection of Helicobacter pylori in gastric biopsy material by polymerase chain reaction.

By using primers based on the sequence of a species-specific antigen of Helicobacter pylori (P. O'Toole, S.M. Logan, M. Kostrzynska. T. Wadstr?m, and T.J. Trust, J. Bacteriol. 173:505-513, 1991), a protocol was established for detection of this microorganism in gastric biopsy samples by the polymerase chain reaction (PCR). A single primer pair was used to specifically amplify a 298-bp sequence in a rapid two-step PCR. The primers exhibited the same specificity in PCR as that which we reported for the species-specific gene probe on which they were based. The sensitivity of the method was 20 copies of the target sequence, or 70 bacterial cells, under the lysis conditions used for patient-derived material. When amplification was performed for a saturating number of cycles, visual examination of ethidium bromide-stained gels successfully detected all samples subsequently judged to be positive by Southern hybridization of the gel with a probe specific for the PCR product. The bacterium could be detected in gastric biopsy samples from patients with various gastric diseases, including samples from which the bacterium could not be cultured. Only 9 of 19 patients who tested positive by PCR of gastric biopsy material were positive when a saliva sample was analyzed. Protocols for sample handling which minimized the risk of contamination while maximizing the sensitivity of the reaction were established. The results support a role for PCR in the rapid identification of H. pylori in clinical samples.     

Multiplex PCR assays for simultaneous detection of six major serotypes and two virulence-associated phenotypes of Streptococcus suis in tonsillar specimens from pigs

Multiplex PCR assays for the detection and identification of various Streptococcus suis strains in tonsillar specimens from pigs were developed and evaluated. In two separate reactions, five distinct DNA targets were amplified. Three targets, based on the S. suis capsular polysaccharide (cps) genes specific for serotypes 1 (and 14), 7, and 9, were amplified in multiplex PCR I. Two other targets, based on the serotype 2- (and 1/2-) specific cps gene and the epf gene, encoding the EF proteins of virulent serotype 2 and highly virulent serotype 1 strains, were amplified in multiplex PCR II. To identify false-negative results, firefly luciferase (luc) DNA and primers based on the luc gene were included in the assay. The multiplex PCR assays were evaluated with tonsillar specimens from pigs infected with S. suis strains. The results obtained with the PCR assays were compared with the results obtained with a bacteriological examination. Most (94%) of the results obtained with multiplex PCR assays were confirmed by the bacteriological examination. The PCR method seems to be more sensitive compared to the bacteriological method, since the remaining 6% of the samples were positive by PCR and negative by bacteriological examination. These results indicate that the PCR method is highly specific for the detection of S. suis strains most frequently involved in clinical disease in infected pig herds. The serotypes found by PCR in tonsillar specimens from diseased pigs were compared with the serotypes of the strains isolated from the affected tissues of the same pigs. The results showed that there is significant association between carriership and clinical illness for S. suis serotype 9 and EF-positive serotype 2 strains and not for serotype 7 and EF-negative serotype 2 (or 1/2) strains.     

Analysis of loci required for determination of serotype antigenicity in Streptococcus mutans and its clinical utilization

We recently identified the genes responsible for the serotype c-specific glucose side chain formation of rhamnose-glucose polysaccharide (RGP) in Streptococcus mutans. These genes were located downstream from the rgpA through rgpF locus that is involved in the synthesis of RGP. In the present study, the corresponding chromosomal regions were isolated from serotype e and f strains and characterized. The rgpA through rgpF homologs were well conserved among the three serotypes. By contrast, the regions downstream from the rgpF homolog differed considerably among the three serotypes. Replacement of these regions in the different serotype strains converted their serotypic phenotypes, suggesting that these regions participated in serotype-specific glucose side chain formation in each serotype strain. Based on the differences among the DNA sequences of these regions, a PCR method was developed to determine serotypes. S. mutans was isolated from 198 of 432 preschool children (3 to 4 years old). The serotypes of all but one S. mutans isolate were identified by serotyping PCR. Serotype c predominated (84.8%), serotype e was the next most common (13.3%), and serotype f occured rarely (1.9%) in Japanese preschool children. Caries experience in the group with a mixed infection by multiple serotypes of S. mutans was significantly higher than that in the group with a monoinfection by a single serotype.     

Isolation and detection of urease genes in Ureaplasma urealyticum.

Urease from ureaplasmas was purified by immunoaffinity chromatography, and the N-terminal amino acid sequence was determined for two of the three subunits. These sequences were used to design primers for a polymerase chain reaction (PCR) that amplified most of the gene coding for one of the subunits. By using a novel "PCR walking" technique, we synthesized almost the complete locus on two overlapping PCR products. We present here a partial nucleotide sequence of the urease locus from Ureaplasma urealyticum (serotype 8), which agrees with our N-terminal amino acid data but differs slightly from the sequence previously reported (A. Blanchard, Mol. Microbiol. 4:669-676, 1990). Also described are PCR primers, intended for diagnostic use, that amplify a sequence from all Ureaplasma strains tested but not from any other mycoplasmas or urease-positive bacteria.     

Serological and genetic examination of some nontypical Streptococcus mutans strains.

Thirty-four strains of Streptococcus mutans whose antigenic or genetic positions were unclear or unknown with respect to the serological scheme of Bratthall (1970) and Perch et al. (1974), or the genetic (deoxyribonucleic acid base sequence homology) scheme of Coykendall were analyzed to clarify their relationship to previously well-characterized strains. Strain OMZ175 of the "new" serotype f was genetically homologous with strains of S. mutans subsp. mutans. Strains of the "new" serotype g were homologous with serotype d strains (S. mutans subsp. sobrinus). Strains isolated from wild rats constituted a new genetic group but carried the c antigen. Thus, strains within a "genospecies" (subspecies) of S. mutans may not always carry a unique or characteristic antigen. We suggest that the existence of multiple serotypes within subspecies represents antigenic variation and adaptations to hosts.     

Salivary IgA antibody to glucosyltransferase in man.

Parotid salivas of 97 young adults were screened for IgA antibody to glucosyltransferase (GTF) from laboratory strains of Streptococcus mutans (serotypes c and g). Antibody levels to GTF from serotype c positively correlated with levels to serotype g GTF among these salivas. GTF's were prepared from S. mutans obtained from a subset of individuals in this population. All but one saliva showed IgA antibody activity to all of the GTF tested. In addition, the relative magnitude of each subject's antibody level was generally the highest to the GTF from their own S. mutans. Fractions, enriched for IgA by ammonium sulphate precipitation and gel filtration, showed patterns of functional inhibition of GTF activity which were consistent with patterns of IgA antibody activity in ELISA of unfractionated salivas. These data indicate that detectable levels of IgA antibody to S. mutans GTF exist in many young adult salivas, while this IgA antibody activity reacts with GTF from different biotypes, subjects generally show the highest secretory IgA antibody levels to their own GTF, and the relative amount of IgA antibody to GTF and the ability to inhibit GTF activity are roughly correlated.     

Listeria monocytogenes serotype identification by PCR

Serotyping is a universally accepted subtyping method for Listeria monocytogenes. Identification of the strain serotype permits differentiation between important food-borne strains (1/2a, 1/2b, and 4b) and provides a "gold standard" for comparing isolates analyzed in different labs and with different techniques. Although an efficient enzyme-linked immunosorbent assay serotyping protocol was described recently, identification of PCR serotyping primers would further increase the ease and accessibility of this classification system. Serotyping PCR primers were designed from variable regions of the L. monocytogenes genome. Three primer sets were used in conjunction with a previously described Division III primer set in order to classify 122 L. monocytogenes strains into five serotype groups [1/2a(3a), 1/2b, 1/2c(3c), 4b(d,e), and 4a/c]. Results of the PCR method agreed with those of the conventional slide agglutination method for 97, 100, 94, and 91% of strains belonging to serotypes 1/2a, 1/2b, 1/2c, and 4b, respectively.     

Longitudinal study of transmission, diversity, and stability of Streptococcus mutans and Streptococcus sobrinus genotypes in Brazilian nursery children

The aim of this study was to perform a follow-up evaluation of the Streptococcus mutans and Streptococcus sobrinus colonization profile of children's oral cavities, which included the pattern of vertical transmission from mother to child, genotypic diversity, and stability of the strains. The subjects were 16 mother-child pairs, who were monitored for 20 months. Samples of saliva, tongue dorsum, alveolar ridge mucosa, and dental plaque from the children were collected bimonthly. Saliva samples from the mothers were also collected. After isolation and identification, the arbitrarily primed PCR method was performed for the genotypic characterization of S. mutans (968 isolates) and S. sobrinus (111 isolates). At the time the strains were acquired, the children harbored one to four distinct genotypes of S. mutans and only one genotype of S. sobrinus. Although S. mutans prevalence and genotypic diversity were greater than those of S. sobrinus, the presence of matching genotypes of S. mutans and S. sobrinus was similar (in 81.25 and 83.33% of mother-child pairs, respectively), suggesting vertical transmission for both species. This longitudinal study showed an increase in genotypic diversity of S. mutans in the oral cavity during the follow-up period: most of the initially acquired genotypes persisted, normally those genotypes transmitted by the mother, and some were lost during follow-up; new strains were also acquired. In conclusion, S. mutans and S. sobrinus genotypes acquired from maternal or alternative sources may show effective persistence in the oral cavity and/or transitory detection in the children's mouths, reflecting the continuous development of oral microbiota in children.     

Clinical spectrum and molecular characteristics of Klebsiella pneumoniae causing community-acquired extrahepatic abscess

BACKGROUND AND PURPOSE: Klebsiella pneumoniae causes a wide spectrum of infections, including abscess and non-abscess formation. This study investigated the clinical spectrum and molecular characteristics of community-acquired Klebsiella infection with primary extrahepatic abscess. METHODS: From April 2004 through March 2007, a total of 18 strains of K. pneumoniae, 11 from blood and 7 from focal purulent specimens, were recovered from a medical center in southern Taiwan. The clinical data were collected from medical records. Hypermucoviscosity phenotype was defined as positive string test. The virulence genes, including rmpA (regulator of mucoid phenotype), magA (specific to K1 capsule serotype), k(2)A (specific to K2 capsule serotype), and kfu (an iron uptake system) were amplified by polymerase chain reaction using specific primers. RESULTS: Twelve men and 6 women with ages ranging from 37 to 74 years were enrolled. Fifteen patients had underlying diabetes mellitus. The duration of hospitalization ranged from 1 to 96 days. Three patients died by the end of treatment. All of the K. pneumoniae strains carried rmpA and 16 strains showed the hypermucoviscosity phenotype. Of the 18 strains, 7 strains were positive for k(2)A and 4 strains carried magA. kfu was identified in 4 magA-positive strains and 2 magA-negative/k(2)A-negative strains. CONCLUSION: Diabetes mellitus was the most frequent underlying disease among our patients. The rmpA and/or hypermucoviscosity phenotype were the most common virulence factors in K. pneumoniae isolates causing extrahepatic abscesses, among which K2 capsule serotype (k(2)A(+)) was more prevalent than K1 capsule serotype (magA(+)).     

Identification of antigenic epitopes in a surface protein antigen of Streptococcus mutans in humans.

The reactivities of antibodies in human serum and saliva to a cell surface protein antigen (PAc) of Streptococcus mutans and synthetic peptides covering the PAc molecule were examined. Both an enzyme-linked immunosorbent assay (ELISA) and Western blotting (immunoblotting) showed that all the serum samples from five adult subjects harboring serotype c S. mutans in their oral cavity reacted with recombinant PAc (rPAc). On the other hand, the serum from a 4-month-old infant did not react with rPAc in ELISA. The immunoglobulin A (IgA) antibodies in saliva samples from the five adult subjects reacted with rPAc. However, in saliva samples from these subjects, the titers of IgA antibody to rPAc did not correlate with the titers of serum antibody to the antigen. To map continuous antigenic epitopes in the PAc molecule, we synthesized 153 decapeptides covering the entire mature PAc molecule, 121 overlapping decapeptides covering the alanine-rich repeating region (A-region) of the PAc molecule, and 21 overlapping decapeptides covering the middle region (residues 824 to 853) according to multiple pin-coupled peptide synthesis technology. Of 153 decapeptides covering the mature PAc, 27 decapeptides showed a strong reaction with the antibodies in serum from the adult subjects. The epitope-scanning patterns in the serum samples from these subjects were also very similar to each other. The antigenic epitope patterns in the saliva resembled those in the serum. However, the ELISA titers of salivary IgA antibodies to these decapeptides differed from the titers of the serum antibody. Of the 121 overlapping decapeptides covering the A-region, 27 decapeptides showed a positive reaction with the antibodies in serum from the adult subjects. All of these 27 decapeptides had either one or two of the five common sequences YQAXL, NADAKA, VQKAN, NNAKNA, and IKKRNA. Six decapeptides of the 21 overlapping decapeptides covering the middle region reacted strongly with the serum antibodies from a high PAc responder, and each of the six decapeptides had one of the two common sequences KVTKEKP and VKPTAPTK. These epitopes might therefore be relevant to the humoral responses against the PAc protein during natural infection with S. mutans in humans.     

Prevalence, distribution of serotypes, and cariogenic potential in hamsters of mutans streptococci from elderly individuals.

The prevalence of mutans streptococci (Streptococcus mutans, Streptococcus cricetus, Streptococcus sobrinus, and Streptococcus rattus) was determined in the salivas of 169 elderly individuals ranging in age from 60 to 87 years. Approximately 40% of these individuals were edentulous and wore full upper and lower dentures. With the exception of a higher proportion of saliva counts below 1,000 CFU/ml in the full-denture wearers, the prevalence and the serotype and species distributions of the mutans streptococci were similar in the denture wearers and individuals with natural teeth only. The species and serotype distributions of mutans streptococci in this elderly population were also consistent with reported observations of other workers on younger, more caries-prone populations. A total of 87 representative isolates of the mutans streptococci were tested for cariogenic potential in a hamster model system. A considerable degree of variation in virulence between different strains was observed. However, these differences were not relatable to individual species or serotypes or to whether the organisms were isolated from denture wearers or naturally dentate subjects. The results of our studies indicate that elderly individuals with either natural or artificial dentitions may be a hitherto unrecognized reservoir of mutans streptococci having varying degrees of potential cariogenicity. Hence, in close family situations they could serve, along with parents and siblings, as vectors in the initial transmission of cariogenic microorganisms to young children.     

聚合酶链式反应快速检测E型肉毒神经毒素基因

目的Ｅ型肉毒中毒是人类肉毒中毒的主要型别之一，本试验旨在建立用于Ｅ型肉毒梭菌鉴定的ＰＣＲ方法。方法用人工合成的寡核苷酸引物扩增Ｅ型肉毒神经毒素基因的一段３６８ｂｐ的ＤＮＡ片段，快速检测Ｅ型肉毒神经毒素基因，对梭状芽胞杆菌属的４０株保藏菌株及３３份土壤标本进行了鉴定，用Ｅ型肉毒梭菌ＣＭＣＣ（Ｂ）６４５０１对其灵敏度进行了检查。结果从所有Ｅ型神经毒素原性菌株或标本均能扩增出目的片段，且能用特定的限制性内切酶切成相应的片段。其它菌株均未能扩增出目的片段。灵敏度试验可从３０个菌体扩增出目的片段。结论此方法用于Ｅ型神经毒素原性梭菌的鉴定具有较高的灵敏度及特异性。     

Evaluation of a multiplex PCR test for simultaneous identification and serotyping of Actinobacillus pleuropneumoniae serotypes 2, 5, and 6

Serotype-specific DNA regions involved in the biosynthesis of capsular polysaccharides (cps region) were used to develop a multiplex PCR test for the simultaneous species identification and serotyping of Actinobacillus pleuropneumoniae serotypes 2, 5, and 6. Primers specific for serotypes 2, 5, and 6 were combined with the already existing species-specific primers used in a PCR test based on the omlA gene. The PCR test was evaluated with serotype reference strains of A. pleuropneumoniae as well as 182 Danish field isolates previously serotyped by latex agglutination or immunodiffusion. For all serologically typeable strains, a complete correspondence was found between the results obtained by the multiplex PCR test and the results obtained by the traditional serotyping methods. Six of eight serologically nontypeable strains could be allocated to a serotype on the basis of the multiplex PCR results. The species specificity of the assay was evaluated with a collection of 93 strains representing 29 different species within the family Pasteurellaceae, as well as species normally found in the respiratory tracts of swine. All of these strains were negative by the multiplex PCR test, including 50 field isolates of the phylogenetically closely related species Actinobacillus lignieresii. When the multiplex PCR test was used to test Danish field strains, it was able to identify the serotypes of approximately 94% of all strains isolated from swine with clinical disease. More than 90% of the isolates that cross-reacted by the latex agglutination test were of serotype 2, 5, or 6. Determination of the serotype by PCR represents a convenient and specific method for the serotyping of A. pleuropneumoniae in diagnostic laboratories.     

In vitro colonization of Streptococcus mutans on enamel.

An in vitro model consisting of enamel from extracted human molars, suspended from wires in inoculated culture tubes, was used to study the adhesion of bacteria to enamel. Under conditions in which there was no macroscopically visible plaque formation, electron micrographs showed no bacterial deposits on the enamel surface. In samples where Streptococcus mutans attached to enamel, an extracellular, pellicle-like material was associated with the bacteria adjacent to the enamel. This material appeared to bind to the enamel surface and to mediate bacterial attachment. Membrane-filtered (Millipore Corp.) saliva deposited a thin surface layer on the enamel, but there were no observable alterations of S. mutans attachment to enamel pretreated with saliva. It was noted that Bratthall serotype c and e strains of S. mutans, when grown in glucose-containing medium, attached, although less tenaciously, to enamel and nichrome wires. Chemical and gas chromatographic analyses of cell-associated materials formed by serotype c and e strains cultured in glucose-containing medium revealed low amounts of glucose-positive material and no polymer linkages characteristic of glucan; yet the same strains cultured in sucrose-containing medium had relatively high amounts of glucose-positive material, with polymer linkages-characteristic of glucan. Serotype a, b, and d strains could attach only in sucrose-containing media.