Maternal Oral Health Influences Infant Salivary Microbiome.

Oral microbiomes vary in cariogenic potential; these differences may be established early in life. A major concern is whether mothers transmit cariogenic bacteria to their children. Here we characterize early salivary microbiome development and the potential associations of that development with route of delivery, breastfeeding, and mother's oral health, and we evaluate transmission of microbes between mother and child. We analyzed saliva and metadata from the Center for Oral Health Research in Appalachia. For this cohort study, we sequenced the V6 region of the 16S rRNA gene and used quantitative polymerase chain reaction to detect Streptococcus mitis, Streptococcus sobrinus, Streptococcus mutans, Streptococcus oralis, and Candida albicans in the saliva from mothers and their infants, collected at 2, 9, and 12 mo (Pennsylvania site) and 2, 12, and 24 mo (West Virginia site). Breastfed children had lower relative abundances of Prevotella and Veillonella. If mothers had decayed, missing, or filled teeth, children had greater abundances of Veillonella and Actinomyces. There was little evidence of maternal transmission of selected microbes. At 12 mo, children's microbiomes were more similar to other children's than to their mothers'. Infants' salivary microbiomes became more adult-like with age but still differed with mothers' microbiomes at 12 mo. There was little evidence supporting transmission of selected microbes from mothers to children, but risk of colonization was associated with tooth emergence. Children are likely to acquire cariogenic bacteria from a variety of sources, including foods and contact with other children and adults.

Caries resistance as a function of age in an initially caries-free population.

Using data from the Center for Oral Health Research in Appalachia Study, we examined variability in susceptibility to dental caries among children and adolescents in rural Appalachia. Among 210 participants who were caries-free at the initial visit, age at the baseline visit can be used as a proxy for the degree of caries resistance; probability of caries development at the tooth level decreased as age at the baseline visit increased. Participants who stayed caries-free for a longer period during childhood and adolescence experienced less extensive caries, as measured by the number of carious teeth. However, the probability of becoming caries-positive did not correlate with age at the baseline visit. For children between 1 and 18 years of age, there was not a "threshold age" after which a caries-free child's risk of caries onset is significantly reduced.

Comparative whole-genome analysis of Streptococcus mutans isolates within and among individuals of different caries status.

INTRODUCTION: Genotypic analyses of Streptococcus mutans using fingerprinting methods depend on a few genetic loci being different but do not reveal the underlying genome-wide differences between strains. METHODS: We used comparative genomic hybridization (CGH) with 70-mer oligonucleotide microarrays containing open reading frames (ORFs) from S. mutans strain UA159 to examine the genetic diversity of 44 isolates from nine children selected from a local study population in Eastern Iowa. RESULTS: Unique strains (clones) within each child initially identified by arbitrary-priming polymerase chain reaction were confirmed by CGH. There was a wide range of variation in the hybridization patterns of the 1948 ORFs among the test isolates examined. Between 87 and 237 ORFs failed to give a positive signal among individual isolates. A total of 323 of the UA159 ORFs were absent from one or more of the test strains. These 323 variable genes seemed to be distributed across the entire UA159 genome and across all the predicted functional categories. CONCLUSION: This set of very close geographically and temporally collected S. mutans isolates had a degree of gene content variation as high as a previously examined global set of strains. Comparing the frequency of these variable genes, the majority of which have unknown function, among strains of different origins (i.e. different caries status) could help to determine their relevance in S. mutans cariogenicity.

Streptococcus agalactiae pulsed-field gel electrophoresis patterns cross capsular types.

Streptococcus agalactiae is a genetically diverse organism; when typed by pulsed-field gel electrophoresis (PFGE), multiple types appear within a single serotype. We tested whether S. agalactiae PFGE types correspond to a specific serotype within individuals, and different individuals from the same geographic area. A total of 872 S. agalactiae isolates from 152 healthy individuals were classified by PFGE and capsular serotype. Serotype V was the most homogeneous (Simpson's diversity index 0.54); and types III, II and Ib were mostly heterogeneous (Simpson's diversity index 0.90). Within an individual, isolates with the same PFGE patterns had identical capsular types, but across individuals the same PFGE types sometimes occurred in different serotypes. Capsular type alone is insufficient to define epidemiological relatedness. Although PFGE types appear to be a valid surrogate for capsular typing of isolates from the same individual, it is not a valid surrogate for serotype in isolates from different individuals.

Risk factors for group B streptococcal colonization: potential for different transmission systems by capsular type.

PURPOSE: Group B Streptococcus (GBS) is a common inhabitant of the bowel and vaginal flora, with known transmission routes including sexual contact and vertical transmission from mother to infant. Food-borne transmission is also possible, as GBS is a known fish and bovine pathogen. We conducted a prospective cohort study in order to identify risk factors for acquisition. METHODS: We identified risk factors for GBS acquisition among college women (n = 129) and men (n = 128) followed at 3-week intervals for 3 months. RESULTS: A doubling in sex acts significantly increased incidence of GBS capsular type V by 80% (95% confidence interval [CI]: 1.19, 2.58), and other non-Ia or -Ib types combined by 40% (95% CI: 1.00, 2.06; incidence of capsular type Ia (odds ratio [OR] = 1.2; 95% CI: 0.71, 1.88; p = 0.57) and Ib (OR = 1.5, 95% CI: 0.75, 2.86; p = 0.27) were elevated, although not significantly. After adjustment for sexual activity and sexual history, gender, and eating venue, fish consumption increased risk of acquiring capsular types Ia and Ib combined 7.3 fold (95% CI: 2.34, 19.50), but not of acquiring other capsular types. Beef and milk were not associated with GBS incidence. CONCLUSIONS: Different GBS capsular types may have different transmission routes.

Distribution of novel and previously investigated virulence genes in colonizing and invasive isolates of Streptococcus agalactiae.

Although Streptococcus agalactiae has emerged as an important cause of invasive disease, relatively little is known regarding the genetic basis of virulence of this organism. Three novel genes with characteristics suggesting a role in virulence were identified via comparison of sequenced genomes of S. agalactiae. The presence of these genes and of the previously identified genes bac, bca, rib, and spb1 was determined, and isolates were assigned a binary genetic signature. It was found that isolates containing spb1, previously suggested to be limited to serotype III-3, were represented by 18 different genetic signatures and several serotypes, and that the presence of both sbp1 and rib was more predictive of invasive disease than spb1 alone. Additionally, bac-positive isolates, reported to be genetically homogeneous, were represented by 14 different genetic signatures. Finally, the majority of serotype V isolates examined contained zero or only one of the genes tested, suggesting that much remains undiscovered regarding important virulence factors in isolates of this serotype.

Probe hybridization array typing: a binary typing method for Escherichia coli.

The ability to distinguish between Escherichia coli strains is critical for outbreak investigations. Binary typing, based on the presence or absence of genetic material, provides a high-throughput alternative to gel- and PCR-based typing techniques that generate complex banding patterns and lack uniform interpretation criteria. We developed, validated, and determined the discriminatory power of an E. coli binary typing method, probe hybridization array typing (PHAT). In PHAT, the absence or presence of genetic material is identified by using DNA hybridization to produce a reproducible and portable fingerprint for each genome. PHAT probes were generated from genome subtractive hybridization experiments. We PHAT typed the ECOR collection of strains from a variety of geographical locations, and 33 rectal E. coli strains selected from college-aged women with urinary tract infection. In the set of 33 human rectal strains, the discriminatory power of PHAT (98%) equaled that of multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. However, for ECOR strains, which include nonhuman strains, the current set of PHAT probes was less discriminating than MLST, ribotyping, and enterobacterial repetitive intergenic consensus sequence PCR (80% versus 97, 92, and 97%, respectively). When we limited the analysis to ECOR strains of B2 and D lineage, which are associated with human infection, current PHAT probes were highly discriminatory (94%). PHAT can be applied in a high-throughput format (i.e., "library on a slide"), the discriminatory ability can be varied based on the probe set, and PHAT is readily adapted to other bacterial species with high variation in genetic content.

Association between Mycobacterium tuberculosis Beijing/W lineage strain infection and extrathoracic tuberculosis: Insights from epidemiologic and clinical characterization of the three principal genetic groups of M. tuberculosis clinical isolates.

Clinical strains of Mycobacterium tuberculosis can be divided into three principal genetic groups based on the single-nucleotide polymorphisms at the katG gene codon 463 and the gyrA gene codon 95. One subgroup of genetic group 1, the Beijing/W lineage, has been widely studied because of its worldwide distribution and association with outbreaks. In order to increase our understanding of the clinical and epidemiological relevance of the genetic grouping of M. tuberculosis clinical strains and the Beijing/W lineage, we investigated the genetic grouping of 679 clinical isolates of M. tuberculosis, representing 96.3% of culture-confirmed tuberculosis cases diagnosed in Arkansas between January 1996 and December 2000 using PCR and DNA sequencing. We assessed the associations of infections by different genetic groups of M. tuberculosis strains and infection by the Beijing/W lineage strains with the clinical and epidemiological characteristics of the patients using chi-square tests and multivariate logistic regression analysis. Of the 679 study isolates, 676 fell into one of the three principal genetic groups, with 63 (9.3%) in group 1, 438 (64.8%) in group 2, and 175 (25.9%) in group 3. After adjusting for potential confounding of age, gender, race/ethnicity, human immunodeficiency virus serostatus, and plcD genotype in a multivariate logistic regression model, patients infected by the Beijing/W lineage isolates were nearly three times as likely as patients infected with the non-Beijing/W lineage isolates to have an extrathoracic involvement (odds ratio [95% confidence interval], 2.85 [1.33, 6.12]). Thus, the Beijing/W lineage strains may have some special biological features that facilitate the development of extrathoracic tuberculosis.

Population-based study of deletions in five different genomic regions of Mycobacterium tuberculosis and possible clinical relevance of the deletions.

Regions of difference (RDs) have been described in clinical isolates of Mycobacterium tuberculosis, but the potential epidemiological and clinical relevance of the genotypes of these RDs remains to be investigated. We screened a population-based sample of 648 isolates for the deletion of five RDs, designated RD105, RD181, RD142, RD150, and RD239, using microarray-based hybridization, PCR, and DNA sequencing and assessed the associations between the RD deletions and the clinical characteristics of the patients using chi-square analysis and multivariate logistic regression model. Of the 648 isolates, 18 (2.8%) had the RD239 deletion and 39 (6.0%) had the RD105 deletion. The deletions of RD142, RD150, and RD181 subdivided the isolates with the RD105 deletion into four groups comprising a group with concurrent deletions of RD105, RD181, and RD142 (n = 13); a group with concurrent deletions of RD105, RD181, and RD150 (n = 5); a group with concurrent deletions of RD105 and RD181 (n = 13); and a group with a deletion of RD105 only (n = 8). Extrathoracic tuberculosis is statistically significantly associated with infection with the isolates with concurrent deletions of RD105, RD181, and RD142 (adjusted odds ratio [OR] = 3.05; 95% confidence interval [CI] = 1.58, 5.90) and the isolates with concurrent deletions of RD105, RD181, and RD150 (adjusted OR = 11.09; 95% CI = 4.27, 28.80), after controlling for the previously identified risk factors for extrathoracic tuberculosis (human immunodeficiency virus serostatus, race, gender, and the genotype of the plcD gene). These two combinations of RD deletions have the potential for predicting the clinical presentation of M. tuberculosis infection in the human host.

Distribution of insertion- and deletion-associated genetic polymorphisms among four Mycobacterium tuberculosis phospholipase C genes and associations with extrathoracic tuberculosis: a population-based study.

The Mycobacterium tuberculosis genome contains four phospholipase C (PLC)-encoding genes, designated plcA, plcB, plcC, and plcD, respectively. Each of the four genes contributes to the overall PLC activity of M. tuberculosis. PLC is hypothesized to contribute to M. tuberculosis virulence. Infection of M. tuberculosis strains carrying a truncated plcD gene is associated with the occurrence of extrathoracic tuberculosis. However, whether the other three plc genes are also associated with extrathoracic tuberculosis remains to be assessed. We investigated the insertion- and deletion-associated genetic diversity in all four plc genes among 682 epidemiologically and clinically well-characterized M. tuberculosis clinical isolates using PCR, DNA sequencing, and Southern hybridization. Two hundred sixty-six (39%) of the 682 isolates had an interruption in at least one of the four plc genes, most often associated with an IS6110 insertion. The plcD gene interruption was the most common: it was observed in 233 (34%) of the isolates, compared to 4.7%, 4.1%, and 5.9% for plcA, plcB, and plcC gene interruption, respectively. The association between the plc gene genotypes and disease presentation was adjusted for clustering using generalized estimating equations for both bivariate and multivariate analyses. After controlling for the genotypes of the plcABC genes and the host-related risk factors, interruption in the plcD gene remained significantly associated with extrathoracic tuberculosis (odds ratio, 3.27; 95% confidence interval, 1.32 to 8.14). The data suggest that the plcD gene might play a more important role in the pathogenesis of thoracic TB than it does in the pathogenesis of extrathoracic TB.

Identification of the lipooligosaccharide biosynthesis gene lic2B as a putative virulence factor in strains of nontypeable Haemophilus influenzae that cause otitis media.

Nontypeable (NT) strains of Haemophilus influenzae are an important cause of acute otitis media (OM). The pathogenic process by which NT H. influenzae strains cause OM is poorly understood. In order to identify specific virulence factors important for OM pathogenesis, genomic subtraction of the NT H. influenzae middle ear isolate G622 against H. influenzae strain Rd was conducted and the resulting subtraction products were used to screen a panel of H. influenzae isolates. Subtraction identified 36 PCR fragments unique to strain G622, which were used in a preliminary screen of 48 middle ear isolates and 46 nasopharyngeal and throat isolates to identify genes found more frequently among middle ear isolates. These experiments identified a PCR fragment with high homology to the lipooligosaccharide biosynthesis gene lic2B (originally identified in an H. influenzae type b strain) among 52% of the middle ear isolates and 9% of nasopharyngeal and throat isolates. The lic2B gene cloned from NT H. influenzae strain G622 was 99% identical at the amino acid level to that of the H. influenzae type b strain RM7004. The lic2B gene was used to screen a larger panel of H. influenzae isolates including the original 48 middle ear isolates, 40 invasive type b isolates, 90 NT H. influenzae throat isolates from children attending day care, and 32 NT H. influenzae nasopharyngeal clinical isolates. The lic2B gene was found 3.7 times more frequently among middle ear isolates than in throat isolates from children attending day care. These data suggest that a specific NT H. influenzae gene is associated with OM.

Use of pulsed-field gel electrophoresis, enterobacterial repetitive intergenic consensus typing, and automated ribotyping to assess genomic variability among strains of nontypeable Haemophilus influenzae.

We compared 75 nontypeable (NT) Haemophilus influenzae isolates by pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus (ERIC)-PCR, and automated ribotyping. PFGE was the most discriminatory of the techniques. ERIC-PCR provides a useful screen but should not replace other techniques as the sole method to group NT H. influenzae strains.

Analysis of pilus adhesins from Haemophilus influenzae biotype IV strains.

A subset of nontypeable Haemophilus influenzae (NTHI) biotype IV isolates from the human genital tract or from infected newborn infants forms a cryptic genospecies characterized by, among other features, the presence of peritrichous pili. The objective of this study was to determine the similarity of these pili to hemagglutinating, HifA- and HifE-containing pili expressed by respiratory H. influenzae isolates. For this analysis, the presence of hifA and hifE and their gene products in NTHI biotype IV strains was assessed, the binding of H. influenzae biotype IV strains to human epithelial cells was characterized, possible genital tissue tropism of these isolates was explored, and the role of HifA- and HifE-possessing pili in the adhesion of NTHI biotype IV strains to human epithelial cells was determined. None of the six biotype IV NTHI isolates tested agglutinated human red blood cells, nor could they be enriched for hemagglutinating variants. Although hifA, which encodes the major structural subunit of hemagglutinating pili, and hifE, which encodes the tip adhesin of hemagglutinating pili, were detected by PCR from six and five, respectively, of the six biotype IV strains tested, neither HifA nor HifE (the gene products of hifA and hifE) were detected in any of these strains by Western blot analysis using antisera that recognize HifA and HifE of respiratory strains. Transmission electron microscopy showed no surface pili on the two biotype IV H. influenzae isolates examined; strain 4162 containing an insertional mutation in hifA also showed no surface pili, whereas strain 1595 containing an insertional mutation in hifB showed pilus-like structures that were shorter and thicker than hemagglutinating pili of the respiratory strains AAr176 and M43. In enzyme-linked immunosorbent assays, biotype IV strains adhered to 16HBE14o(-) and HEp-2 cells of respiratory origin as well as to ME180 and HeLa cells of genital origin. This adherence was not pilus specific, however, as GM-1, a known pilus receptor analog, did not inhibit binding of biotype IV strains to ME180, HEp-2, or HeLa cells, and GM-1 inhibition of binding to 16HBE14o(-) cells did not correlate with the presence of hifE. While both nonpiliated variants and hifA and hifB (encoding the pilus chaperone) mutants of respiratory strain AAr176 showed reduced binding (64 to 87% of that of piliated AAr176) to 16HBE14o(-) and ME180 cells, hifA and hifB mutants of the biotype IV strains showed minimal reduction in binding to these cell lines (91 to 98% of that of wild-type strains). Thus, although biotype IV H. influenzae isolates of the cryptic genospecies possess the genes that code for HifA- and HifE-containing hemagglutinating pili, epithelial cell adherence exhibited by these strains is not mediated by expression of hemagglutinating pili.

Haemophilus influenzae - human specific bacteria.

Haemophilus influenzae is both a commensal and a pathogen specific to humans. Here we review this bacterium with special emphasis on characteristics that may be involved in virulence.

Prevalence of known P-fimbrial G alleles in Escherichia coli and identification of a new adhesin class.

Screening a large Escherichia coli collection for P-fimbrial adhesin classes identified 20 unclassifiable strains. Cloning and sequencing of papG from an unclassifiable strain identified another G allele. The novel adhesin gene has 65% identity to the class I adhesin gene, 44% identity to the class II adhesin gene, and 43% identity to the class III adhesin gene.

Optimization of a fluorescent-based phosphor imaging dot blot DNA hybridization assay to assess E. coli virulence gene profiles.

To increase the efficiency and consistency in screening Escherichia coli for virulence genes, a Phosphor Imager was adopted for signal detection in Dot Blot DNA hybridization replacing X-ray film read by eye. We assessed not only the reliability of the instrument-based procedure, but the impact of going from an outcome measured by visualization on a semi-quantitative scale to a digitized readout on an interval scale. We analyzed technical and biological variability of the assay and the factors contributing to the variability. In spite of high variability both within and between membranes in the Phosphor Imager readings, we were able to define classification rules for gene presence that were remarkably consistent. Using the X-ray film signal detection procedure with Southern confirmation as a gold standard, we obtained a sensitivity and specificity of 87-99% for a rule requiring no retesting for all but one gene probe.

Risk factors for otitis media and carriage of multiple strains of Haemophilus influenzae and Streptococcus pneumoniae.

We studied genetic diversity in Streptococcus pneumoniae and Haemophilus influenzae in throat culture isolates from 38 children attending two day-care centers in Michigan. Culture specimens were collected weekly; 184 S. pneumoniae and 418 H. influenzae were isolated from the cultures. Pulsed-field gel electrophoresis identified 29 patterns among the S. pneumoniae isolates and 87 among the H. influenzae isolates. Of the cultures, 5% contained multiple genetic types of S. pneumoniae, and 43% contained multiple types of H. influenzae. Carriage of multiple H. influenzae isolates, which was associated with exposure to smoking, history of allergies, and age 36 to 47 months, may increase risk for otitis media in children.

Risk factors for second urinary tract infection among college women.

To better understand the etiology of recurrent urinary tract infection (UTI), the authors followed a cohort of 285 female college students with first UTI for 6 months or until second UTI. A first UTI due to Escherichia coli was followed by a second UTI three times more often than was a non-E. coli first UTI (24 vs. 8%; p = 0.02). In a logistic regression analysis limited to the 224 women from the University of Michigan Health Service and the University of Texas at Austin Health Service from September 1992 to December 1994, with a first UTI due to E. coli, vaginal intercourse increased the risk of a second UTI with both a different (odds ratio (OR) = 1.60, 95% confidence interval (CI): 1.19, 2.15) and the same (OR = 1.37, 95% CI: 0.91, 2.07) uropathogen, as did using a diaphragm, cervical cap, and/or spermicide (same uropathogen: OR = 1.53, 95% CI: 0.95, 2.47; different uropathogen: OR = 1.77, 95% CI: 1.22, 2.58). Condom use decreased the risk of a second UTI caused by a different uropathogen (OR = 0.68, 95% CI: 0.48, 0.99) but had no effect on a second UTI caused by the same E. coli (OR = 0.99; 95% CI: 0.66, 1.50). Type or duration of treatment was not associated with a second UTI. Although the risk of second UTI is strongly influenced by sexual behavior, women with a first UTI caused by E. coli are more likely than are those with a non-E. coli first UTI to have a second UTI within 6 months.

Induction of proinflammatory cytokines from human respiratory epithelial cells after stimulation by nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) causes repeated respiratory infections in patients with chronic lung diseases. These infections are characterized by a brisk inflammatory response which results in the accumulation of polymorphonucleated cells in the lungs and is dependent on the expression and secretion of proinflammatory cytokines. We hypothesize that multiple NTHi molecules, including lipooligosaccharide (LOS), mediate cellular interactions with respiratory epithelial cells, leading to the production of proinflammatory cytokines. To address this hypothesis, we exposed 9HTEo- human tracheal epithelial cells to NTHi and compared the resulting profiles of cytokine gene expression and secretion using multiprobe RNase protection assays and enzyme-linked immunosorbent assays (ELISA), respectively. Dose-response experiments demonstrated a maximum stimulation of most cytokines tested, using a ratio of 100 NTHi bacterial cells to 1 9HTEo- tracheal epithelial cell. Compared with purified LOS, NTHi bacterial cells stimulated 3.6- and 4.5-fold increases in epithelial cell expression of interleukin-8 (IL-8) and IL-6 genes, respectively. Similar results were seen with epithelial cell macrophage chemotactic protein 1, IL-1alpha, IL-1beta, and tumor necrosis factor alpha expression. Polymyxin B completely inhibited LOS stimulation but only partially reduced NTHi whole cell stimulation. Taken together, these results suggest that multiple bacterial molecules including LOS contribute to the NTHi stimulation of respiratory epithelial cell cytokine production. Moreover, no correlation was seen between NTHi adherence to epithelial cells mediated by hemagglutinating pili, Hia, HMW1, HMW2, and Hap and epithelial cytokine secretion. These data suggest that bacterial molecules beyond previously described NTHi cell surface adhesins and LOS play a role in the induction of proinflammatory cytokines from respiratory epithelial cells.

Identification and genetic characterization of Haemophilus influenzae genetic island 1.

The type b capsule of pathogenic Haemophilus influenzae is a critical factor for H. influenzae survival in the blood and the establishment of invasive infections. Other pathogenic factors associated with type b strains may also play a role in invasion and sustained bacteremia, leading to the seeding of deep tissues. The gene encoding haemocin is the only noncapsular gene found to be specific to type b strains until now. Here we report the discovery of an approximately 16-kb genetic locus, HiGI1, that is present primarily in type b strains. Pulsed-field gel electrophoresis and Southern hybridization were used to map this new locus between secG (HI0445) and fruA (HI0446), which are contiguous in Rd, a nonpathogenic derivative of a serotype d strain. It is inserted at the 3' end of tRNA(4)(Leu) and has regions whose G+C content differs from the average genomic G+C content of H. influenzae. An integrase gene, which encodes a CP4-57 like integrase, is located downstream of tRNA(4)(Leu). Hybridization probes based on the sequences within the HiGI1 locus have been used to screen 61 H. influenzae strains (2 type a, 22 type b, 2 type c, 1 type d, 3 type e, 7 type f, and 21 nontypeable H. influenzae [NTHi]) from our collection. This HiGI1 locus exists in all 22 type b strains and two NTHi strains and is likely to have been acquired by an ancestral type b strain.