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Background : Dental caries results from a dysbiosis of tooth-associated biofilms and frequently extends through enamel into dentin which has a different structure and composition. Objective : To evaluate the metatranscriptome of caries to determine the metabolic potential of caries communities compared with health. Design : Samples from children, caries-free (CF: n  = 4) or with coronal (CC: n  = 5) or dentin (DC: n  = 5) caries were examined for gene expression potential. Functional profiling was performed using HUMAnN2 (HMP Unified Metabolic Analysis Network). Results : There was increased gene expression diversity in DC compared with CC and CF. Genes in CF included alcohol dehydrogenase from Neisseria sicca , methylenetetrahydrofolate reductase from Streptococcus sanguinis and choline kinase from streptococci. Genes in CC mapped mainly to Streptococcus mutans . Arginine deiminase in DC mapped to S. sanguinis and Actinomyces naeslundii . Glycerol kinase genes mapped to S. sanguinis in all groups whereas glycerol kinase in DC were from Rothia, Prevotella and streptococci. Uracil-DNA glycosylase in DC mapped to Prevotella denticola and Actinomyces . Repressor LexA in DC mapped to Scardovia wiggsiae, Dialister invisus and Veillonella parvula . Conclusions : Functional profiling revealed enzyme activities in both caries and caries-free communities and clarified marked differences between coronal and dentin caries in bacterial composition and potential gene expression.

Background and Objective: Bacterial invasion into pulps of primary teeth can lead to infection and premature tooth loss in children. This pilot study aimed to explore whether the microbiota of carious exposures of dental pulps resembles that of carious dentin or that of infected root canals., Design: Children with severe early childhood caries were studied. Children were consented and extent of caries, plaque, and gingivitis measured. Bacteria were sampled from carious lesion biofilms and vital carious exposures of pulps, and processed by anaerobic culture. Isolates were characterized from partial sequences of the 16S rRNA gene and identified by comparison with taxa in the Human Oral Microbiome Database (http://www.HOMD.org). The microbiotas of carious lesions and dental pulps were compared using univariate and multivariate approaches., Results: The microbiota of cariously exposed pulps was similar in composition to that of carious lesion biofilms except that fewer species/taxa were identified from pulps. The major taxa identified belonged to the phyla Firmicutes (mainly streptococci) and Actinobacteria (mainly Actinomyces species). Actinomyces and Selenomonas species were associated with carious lesions whereas Veillonella species, particularly Veillonella dispar was associated with pulps. Other bacteria detected in pulps included Streptococcus mutans, Parascardovia denticolens, Bifidobacterium longum, and several Lactobacillus and Actinomyces species. By principal, component analysis pulp microbiotas grouped together, whereas those in caries biofilms were widely dispersed., Conclusions: We conclude that the microbiota of cariously exposed vital primary pulps is composed of a subset of species associated with carious lesions. Vital primary pulps had a dominant Firmicutes and Actinobacteria microbiota which contrasts with reports of endodontic infections which can harbor a gram-negative microbiota. The microbiota of exposed primary pulps may provide insight into bacterial species at the forefront of caries invasion in dentinal lesions that can invade into the pulp and the nature of species that need suppressing for successful pulp therapy.

A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria, Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces species, and Veillonella parvula. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases.

Background: Lactobacillus species can contribute positively to general and oral health and are frequently acquired by breastfeeding in infancy. The present study aimed to identify oral lactobacilli in breast and formula-fed 4 month-old infants and to evaluate potential probiotic properties of the dominant Lactobacillus species detected. Saliva and oral swab samples were collected from 133 infants who were enrolled in a longitudinal study (n=240) examining the effect of a new infant formula on child growth and development. Saliva was cultured and Lactobacillus isolates were identified from 16S rRNA gene sequences. Five L. gasseri isolates that differed in 16S rRNA sequence were tested for their ability to inhibit growth of selected oral bacteria and for adhesion to oral tissues. Oral swab samples were analyzed by qPCR for Lactobacillus gasseri., Results: 43 (32.3%) infants were breastfed and 90 (67.7%) were formula-fed with either a standard formula (43 out of 90) or formula supplemented with a milk fat globule membrane (MFGM) fraction (47 out of 90). Lactobacilli were cultured from saliva of 34.1% breastfed infants, but only in 4.7% of the standard and 9.3% of the MFGM supplemented formula-fed infants. L. gasseri was the most prevalent (88% of Lactobacillus positive infants) of six Lactobacillus species detected. L. gasseri isolates inhibited Streptococcus mutans binding to saliva-coated hydroxyapatite, and inhibited growth of S. mutans, Streptococcus sobrinus, Actinomyces naeslundii, Actinomyces oris, Candida albicans and Fusobacterium nucleatum in a concentration dependent fashion. L. gasseri isolates bound to parotid and submandibular saliva, salivary gp340 and MUC7, and purified MFGM, and adhered to epithelial cells. L. gasseri was detected by qPCR in 29.7% of the oral swabs. Breastfed infants had significantly higher mean DNA levels of L. gasseri (2.14 pg/uL) than infants fed the standard (0.363 pg/uL) or MFGM (0.697 pg/uL) formula., Conclusions: Lactobacilli colonized the oral cavity of breastfed infants significantly more frequently than formula-fed infants. The dominant Lactobacillus was L. gasseri, which was detected at higher levels in breastfed than formula-fed infants and displayed probiotic traits in vitro.

Dengue is the most common vector-borne viral infection worldwide. In Brazil, the incidence has increased with successive epidemics, and an increasing proportion of patients present with severe forms of the disease. The prognosis for these patients is directly influenced by the quality of medical care. These guidelines present the management of the severe forms of dengue, including the recognition of warning signs, the treatment for prompt re-establishment of euvolemia and the evaluation and appropriate care of potential complications, thus reducing morbidity and mortality of infected children and adults.

Severe early childhood caries (ECC), while strongly associated with Streptococcus mutans using selective detection (culture, PCR), has also been associated with a widely diverse microbiota using molecular cloning approaches. The aim of this study was to evaluate the microbiota of severe ECC using anaerobic culture. The microbial composition of dental plaque from 42 severe ECC children was compared with that of 40 caries-free children. Bacterial samples were cultured anaerobically on blood and acid (pH 5) agars. Isolates were purified, and partial sequences for the 16S rRNA gene were obtained from 5,608 isolates. Sequence-based analysis of the 16S rRNA isolate libraries from blood and acid agars of severe ECC and caries-free children had >90% population coverage, with greater diversity occurring in the blood isolate library. Isolate sequences were compared with taxon sequences in the Human Oral Microbiome Database (HOMD), and 198 HOMD taxa were identified, including 45 previously uncultivated taxa, 29 extended HOMD taxa, and 45 potential novel groups. The major species associated with severe ECC included Streptococcus mutans, Scardovia wiggsiae, Veillonella parvula, Streptococcus cristatus, and Actinomyces gerensceriae. S. wiggsiae was significantly associated with severe ECC children in the presence and absence of S. mutans detection. We conclude that anaerobic culture detected as wide a diversity of species in ECC as that observed using cloning approaches. Culture coupled with 16S rRNA identification identified over 74 isolates for human oral taxa without previously cultivated representatives. The major caries-associated species were S. mutans and S. wiggsiae, the latter of which is a candidate as a newly recognized caries pathogen.

The human oral cavity contains a number of different habitats, including the teeth, gingival sulcus, tongue, cheeks, hard and soft palates, and tonsils, which are colonized by bacteria. The oral microbiome is comprised of over 600 prevalent taxa at the species level, with distinct subsets predominating at different habitats. The oral microbiome has been extensively characterized by cultivation and culture-independent molecular methods such as 16S rRNA cloning. Unfortunately, the vast majority of unnamed oral taxa are referenced by clone numbers or 16S rRNA GenBank accession numbers, often without taxonomic anchors. The first aim of this research was to collect 16S rRNA gene sequences into a curated phylogeny-based database, the Human Oral Microbiome Database (HOMD), and make it web accessible (www.homd.org). The HOMD includes 619 taxa in 13 phyla, as follows: Actinobacteria, Bacteroidetes, Chlamydiae, Chloroflexi, Euryarchaeota, Firmicutes, Fusobacteria, Proteobacteria, Spirochaetes, SR1, Synergistetes, Tenericutes, and TM7. The second aim was to analyze 36,043 16S rRNA gene clones isolated from studies of the oral microbiota to determine the relative abundance of taxa and identify novel candidate taxa. The analysis identified 1,179 taxa, of which 24% were named, 8% were cultivated but unnamed, and 68% were uncultivated phylotypes. Upon validation, 434 novel, nonsingleton taxa will be added to the HOMD. The number of taxa needed to account for 90%, 95%, or 99% of the clones examined is 259, 413, and 875, respectively. The HOMD is the first curated description of a human-associated microbiome and provides tools for use in understanding the role of the microbiome in health and disease.

For over 30 years, the African cichlid fish, Astatotilapia burtoni, has been an important model system for studying the mechanisms underlying socially mediated behavioral change, with the focus being the dominance behavior of males. A recently collected wild-stock (WS) of this species invigorates interest in parallel studies of females' behavior. Here, we describe a robust 'good-mother' phenotype, increased maternal affiliation in fry, and subtle differences in males' behavior that are exhibited by this new stock. While the females of both the laboratory-stock (LS) and the WS brood the developing fry in their buccal cavity, only the WS continues to provide maternal care after initial release of the fry while the LS engage in filial cannibalism. We show that weight loss during starvation, either during brooding or with restriction of food, is greater in the LS than in the WS; thus, the observed behavioral differences may be tied to metabolic differences. The WS also exhibits a robust androgen response to challenge during the maternal care phase. Given the increasing power of genomic tools available for this species, the comparison of these two stocks will offer the opportunity to investigate the genetic and genomic basis of behavioral differences.

Thirty-six cases of Salmonella enteritidis phage type (PT) 4 infection, mainly in children, were notified in one local authority in the two weeks beginning on 23 October 1995. Twenty-four cases and 42 controls nominated by cases were included in a case control study, which showed a significant association between illness and the consumption of marshmallow confectionery from a bakery. S. enteritidis PT4 was isolated from samples of the marshmallow, and all isolates had the same plasmid and pulsed field gel electrophoresis profiles. The marshmallow had been made using raw egg white, which shows that not all caterers follow the Department of Health's advice.

Experimental mycoplasmosis was induced in 1-day-old chicks by intrapulmonary inoculation of Mycoplasma gallisepticum (MG). This method of infection proved to be useful for evaluating the efficacy of antimicrobial medication, by measuring mortality, weight gain, pathological responses, frequency of reisolation of MG, and seroconversion. Using this model, the efficacies of danofloxacin (a novel fluoroquinolone) and tylosin were compared for two MG isolates, a reference isolate (the R-strain) and a field isolate from California. Danofloxacin administered in the water at 50 ppm for 3 days was equivalent to tylosin at 500 ppm for 3 days in the degree of control of mortality and maintenance of weight gain. Danofloxacin was superior to tylosin in preventing air-sac lesions, reducing the frequency of reisolation of MG, and preventing seroconversion in surviving birds.

The efficacy of danofloxacin was compared with that of tylosin in the control of induced mycoplasmosis. In three experiments, disease was induced in broiler chicks by intrapulmonary injection of field isolates of Mycoplasma gallisepticum (MG) originating from Brazil, a different isolate being used for each experiment. Starting the day after inoculation, groups of chicks were medicated for 3 days via the drinking water with danofloxacin (50 ppm) or tylosin (500 ppm) or were left as unmedicated controls. Chicks were observed for 21 days. A severe mycoplasmosis was induced in unmedicated birds, characterized by mortality, depression, and respiratory signs. Danofloxacin was highly efficacious in controlling this infection in all three experiments, whereas tylosin was efficacious in only one. This difference could be related directly to the reduced in vitro susceptibility to tylosin displayed by the two isolates of MG. In the two experiments where isolates were tylosin-resistant, danofloxacin was significantly (P < or = 0.05) superior to tylosin in reducing mortality, maintaining weight gain, and reducing the prevalence of air-sac lesions, isolation of MG, and seroconversion.

The in vitro susceptibility of Escherichia coli and Pasteurella multocida isolated from poultry was determined to danofloxacin, a novel fluoroquinolone, and five other commonly used antimicrobials. A total of 1737 E. coli field isolates and 107 P. multocida isolates were tested by veterinary diagnostic laboratories in Europe, Japan, South Africa, and North America during the period 1989-91. The antimicrobial susceptibility of these isolates was determined using the Sensititre broth microdilution technique. The minimum inhibitory concentrations (MIC) of danofloxacin, furaltadone, lincomycin, oxytetracycline, spectinomycin, and trimethoprim:sulfamethoxazole that prevented growth of 90% of the bacteria were 0.25 > 64, > 64, > 64, > 128, and > 16 micrograms/ml, respectively, against E. coli isolates and 0.25, 64, 64, 16, 128, and 8 micrograms/ml, respectively, against P. multocida isolates. Danofloxacin demonstrated considerable in vitro potency against these important poultry pathogens, many of which showed extensive resistance to the other antimicrobials tested.

A technique is described for determining the antimicrobial susceptibility of Mycoplasma gallisepticum, using the Sensititre broth microdilution system. Fourteen M. gallisepticum field isolates and one reference isolate (R-strain) were tested in duplicate against seven antimicrobials. Isolates were susceptible to oxytetracycline, furaltadone, and lincomycin/spectinomycin, but not to amoxycillin and apramycin. Susceptibility to erythromycin and tylosin varied. These data are in agreement with those reported by other workers using more traditional methods, but this adaptation of the broth microdilution technique eliminates any variation attributable to the time-consuming preparation of antimicrobial dilutions associated with these methods.

API ZYM and API An-Ident enzymatic substrate tests were done on six oral species which are difficult to characterize with conventional biochemical tests. "Bacteroides forsythus, the "fusiform" Bacteroides species (A. C. R. Tanner, M. A. Listgarten, M. N. Strzempko, and J. L. Ebersole, manuscript in preparation), is difficult to cultivate in broth media, yet it gave 15 positive tests in these series. The tests were able to separate this new species from species of Capnocytophaga and Fusobacterium. "B. forsythus" reactions were similar but not identical to those of reference Bacteroides species. Positive reactions for alpha-glucosidase, beta-glucosidase, alpha-fucosidase, and alpha-glucuronidase suggest that "B. forsythus" may be saccharolytic. It was the only species tested which was trypsin positive. Wolinella species, Campylobacter concisus, B. gracilis, and Eikenella corrodens are asaccharolytic, and characterization relies heavily on sensitivities to inhibitory agents. These species reacted weakly in the API ZYM and API An-Ident enzymatic substrate tests, and the reactions were not useful for separating these species. The enzyme reactions differentiated Wolinella recta and C. concisus from Selenomonas sputigena, another oral motile but saccharolytic organism.

Selected human oral and nonoral strains of the genera Actinobacillus and Haemophilus were examined by transmission and scanning electron microscopy. The strains examined were morphologically identical to recognized Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus. By transmission electron microscopy, the cells were typically gram negative in morphology, with several strains possessing some extracellular ruthenium red-staining polymeric material. Numerous vesicular structures, morphologically identical to lipopolysaccharide vesicles, were seen to originate from and be continuous with the surface of the outer membrane. Large numbers of these vesicles were also found in the external environment. Scanning electron microscopic observations revealed that both actinobacilli and haemophili possessed surface projections and an amorphous surface material which connected and covered adjacent cells.

A rapid method for identifying black-pigmented oral Bacteroides spp. is described. Species-specific rabbit antisera to Bacteroides gingivalis, B. intermedius, and B. melaninogenicus were used in an enzyme-linked immunosorbent assay to identify clinical isolates of black-pigmented Bacteroides spp. from humans. The results showed excellent agreement with biochemical identification of B. gingivalis and B. intermedius. Only 36% of the B. melaninogenicus isolates were identified with the enzyme-linked immunosorbent assay, suggesting that this group of black-pigmented Bacteroides spp. is made up of more than one serotype. The serological enzyme-linked immunosorbent assay should enable rapid identification of black-pigmented Bacteroides spp. isolated from sites of oral diseases and may also be used to identify the presence of these organisms in complex bacterial mixtures from oral sites.

Strains of Actinobacillus actinomycetemcomitans isolated from deep pockets of patients with juvenile periodontitis were analyzed for their content of cellular fatty acids. Oral Haemophilus strains, morphologically and biochemically similar to Haemophilus aphrophilus, were also examined for their content of cellular fatty acids. The extractable lipids of the actinobacilli represented approximately 10% of the cell dry weight, with the bound lipids representing 2 to 5%. The major fatty acids consisted of myristic (C14:0) and palmitic (C16:0) acids and a C16:1 acid, possibly palmitoleic acid, accounting for 21, 35, and 31% of the total extractable fatty acids, respectively. Haemophilus strains had a similar cellular fatty acid content.

The small asaccharolytic, nonpigmenting gram-negative rods of the human oral cavity are difficult to differentiate from each other. Protein profiles of sonicated cells of Wolinella species, Campylobacter concisus, Bacteroides gracilis, and Eikenella corrodens were obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized with a silver stain. The gels were scanned with a laser densitometer, and the similarity of strains was computed by determining correlation coefficients of normalized densities along the gels. The strains were grouped by cluster analysis of the correlation coefficients. All species were distinct from each other. Several groups were found within E. corrodens. A colored silver stain was found to highlight species differences and appears to be useful in the rapid identification of fresh isolates.