The Long and Short of Genome Sequencing: Using a Hybrid Sequencing Strategy to Sequence Oral Microbial Genomes.

Since our chapter on genome sequencing using the GS-FLX pyrosequencer in the First Edition of this book, significant advances have been made in next-generation DNA sequencing (NGS) technology. Not only has the GS-FLX become extinct, but the more recent introduction and establishment of the so-called third-generation DNA sequencers by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) has revolutionized genomics yet again by generating ultra-long (>100,000 basepair) sequence reads concomitant with an incredible reduction in cost per sequenced basepair. Unfortunately, the ultra-high sequence yields of third-generation sequencers are compromised by their inherent sequencing error rates, prompting an alternative sequencing strategy, i.e., a hybrid sequencing strategy, which combines PacBio/ONT primary datasets with complementary datasets generated by mainstream short-read NGS platforms, e.g., Illumina or Ion Torrent. Although the concept of a hybrid sequencing strategy is not new, existing yields and accuracy of ultra-long and short-read sequencing technologies makes such a strategy achievable, resulting in complete genome sequences in one hit. In this chapter, we describe our updated laboratory and bioinformatic protocols that will allow the average research group to obtain complete oral microbial genome sequences assembled from a combination of DNA sequence data generated by NGS and third-generation platforms.

Plaque and Dental Caries Risk in Midlife.

Dental caries is an endogenous microbial community-based disease resulting from an ecological shift from dynamic stability to metabolic imbalance in a consortium of acidogenic and aciduric bacteria comprising the dental plaque biofilm. Participants were members of the Dunedin Multidisciplinary Health and Development Study, a longitudinal investigation of health and behaviour in a cohort born in Dunedin, New Zealand. Oral biofilm samples (collected at age 32 years) from anterior labial supragingival, posterior lingual supragingival, posterior subgingival, and the dorsum of the tongue habitats for 841 participants were analysed using checkerboard DNA-DNA hybridisation (CKB), focussing on 30 ecologically significant bacteria. Associations of CKB data with dental caries at ages 32 and 45 years were assessed using regression modelling, adjusting for potential confounders including sex, xerostomia, and oral hygiene. The putative periodontitis pathobiont Tannerella forsythia (in the anterior supragingival biofilm) was associated with untreated caries at age 32 years. The percentage of total summed cell number counts for two putative periodontitis-associated species (T. forsythia and Micromonas micros) was associated with greater caries experience at age 32 years and the development of new caries between age 32 and 45 years. Additionally, severe caries (3 + cavities) was associated with putative caries pathobionts (Lactobacillus fermentum, Lactobacillus plantarum), periodontitis-associated species, and commensals (M. micros, Campylobacter rectus, Streptococcus mitis biovar I, Streptococcus mitis biovar II) in the subgingival biofilm. Participants with sustained poor oral hygiene through age 32 years not only had greater experience of caries by that age than those with good oral hygiene (fully adjusted incidence risk ratio = 5.10, 95% CI: 3.30, 7.89) but also experienced greater incidence of new caries from age 32 to 45 years (incidence risk ratio = 3.69, 95% CI: 2.62, 5.20). These findings provide evidence in support of the extended caries ecological plaque hypotheses, the polymicrobial aetiology of caries, and the integrated aetiology of dental caries and periodontal diseases. They also underscore the roles of poor oral self-care (particularly over the life course) and xerostomia in the occurrence and progression of caries.

Clinical and Microbiological Evaluation of a Chlorhexidine-Modified Glass Ionomer Cement (GIC-CHX) Restoration Placed Using the Atraumatic Restorative Treatment (ART) Technique.

The aims of this study were to investigate the clinical effectiveness and patient acceptability of a modified glass ionomer cement placed using the atraumatic restorative treatment (ART) technique to treat root caries, and to carry out microbiological analysis of the restored sites. Two clinically visible root surface carious lesions per participant were restored using ART. One was restored with commercial glass ionomer cement (GIC) (ChemFil® Superior, DENTSPLY, Konstonz, Germany) which acted as the control. The other carious root lesion was restored with the same GIC modified with 5% chlorhexidine digluconate (GIC-CHX; test). Patient acceptability and restoration survival rate were evaluated at baseline and after 6 months. Plaque and saliva samples around the test and control restorations were collected, and microbiological analysis for selected bacterial and fungal viability were completed at baseline, and after 1, 3, and 6 months. In total, 52 restorations were placed using GIC and GIC-CHX in 26 participants; 1 patient was lost to follow-up. After reviewing the restorations during their baseline appointments, participants indicated that they were satisfied with the appearance of the restorations (n = 25, 96%) and did not feel anxious during the procedure (n = 24, 92%). Forty-eight percent (n = 12) of the GIC-CHX restorations were continuous with the existing anatomic form as opposed to six for the GIC restorations (24%), a difference which was statistically significant (p = 0.036). There was no statistically significant reduction in the mean count of the tested microorganisms in plaque samples for either type of restorations after 1, 3, or 6 months. Restoration of carious root surfaces with GIC-CHX resulted in higher survival rates than the control GIC. ART using GIC-CHX may therefore be a viable approach for use in outreach dental services to restore root surface carious lesions where dental services are not readily available, and for older people and special needs groups.

Associations of sex, oral hygiene and smoking with oral species in distinct habitats at age 32 years.

The oral microbiome is ecologically diverse, complex, dynamic, and little understood. We describe the microbiota of four oral habitats in a birth cohort at age 32 and examine differences by sex, oral hygiene, and current smoking status, dental caries, and periodontal health. Oral biofilm samples collected from anterior labial supragingival, posterior lingual supragingival, subgingival, and tongue sites of 841 Dunedin Multidisciplinary Health and Development Study members were analysed using checkerboard DNA-DNA hybridization; focusing on 30 ecologically important bacterial species. The four habitats exhibited distinct microbial profiles that differed by sex. Streptococcus gordonii was more dominant in supragingival and tongue biofilms of males; Porphyromonas gingivalis exhibited higher relative abundance in subgingival biofilm of females. Males had higher scores than females for periodontal pathogens at supragingival sites. The relative abundance of several putative caries and periodontal pathogens differed in smokers and non-smokers. With poor oral hygiene significantly higher proportions of Gram-negative facultative anaerobes were present in subgingival biofilm and there were higher scores for the principal components characterised by putative cariogenic and periodontal pathogens at each site. Distinctive microenvironments shape oral biofilms and systematic differences exist by sex, oral hygiene, and smoking status.

Streptococcus salivarius Isolates of Varying Acid Tolerance Exhibit F1F0-ATPase Conservation.

Genes encoding the subunits of the membrane-bound F1F0-ATPase (responsible for exporting protons from the cytoplasm and contributing to acid tolerance) were sequenced for 24 non-mutans streptococci isolated from carious lesions. Isolates, mostly Streptococcus salivarius, displayed a continuum of acid tolerance thresholds ranging from pH 4.55 to 3.39, but amino acid alignments of F1F0-ATPase subunits revealed few non-synonymous substitutions and these were unrelated to acid tolerance. Thus, the F1F0-ATPase is highly-conserved among S. salivarius isolates despite varying acid tolerance thresholds, supporting the contention that acid tolerance is determined by the level of gene/protein expression rather than variation in molecular structure.

A TaqMan™-based quantitative PCR screening assay for the probiotic Streptococcus salivarius K12 based on the specific detection of its megaplasmid-associated salivaricin B locus.

In order to assess the colonization efficacy of the oral probiotic Streptococcus salivarius K12, a rapid method for specific detection and enumeration of the strain was developed. Here, we describe a two-step TaqMan™ quantitative PCR assay using primer-probe combinations targeting genes of the locus encoding the lantibiotic bacteriocin salivaricin B.

Oral bacteriome of HIV-1-infected children from Rio de Janeiro, Brazil: Next-generation DNA sequencing analysis.

BACKGROUND AND AIM: This study compared the oral bacteriome between HIV-1-infected and non-HIV-1-infected Brazilian children/teenagers. METHODS: Whole saliva, biofilm from the dorsal surface of the tongue and biofilm from supragingival and subgingival sites were collected from 27 HIV-1-infected and 30 non-HIV-1-infected individuals. Bacterial genomic DNA was extracted and 16S rRNA genes were sequenced using next-generation sequencing technology (Ion Torrent). RESULTS: In the supragingival biofilm, the phylum Firmicutes and genus Streptococcus sp. were more frequent in HIV-1-infected (95% and 78%, respectively) than in non-HIV-1-infected individuals (40% and 24%, respectively). In the subgingival biofilm of HIV-infected participants, the relative abundance of the Veillonella sp. and Prevotella sp. genera were higher than in non-HIV-1-infected participants. On the tongue, the genera with greater relative abundance in HIV-1-infected individuals were Neisseria sp. (21%). In saliva, the difference of the genus Prevotella sp. between non-HIV-1-infected and HIV-1-infected individuals was 15% and 7%, respectively. The Chao index revealed an increase in the richness of both sub- and supragingival biofilms in the HIV-1-infected samples compared with non-HIV-1-infected samples. CONCLUSION: HIV-1-infected children/teenagers have a higher frequency of the phyla Firmicutes and genus Streptococcus, and their oral microbiome shows more complexity than that of non-HIV-1-infected children/teenagers.

Three Bacteriocin Peptides from a Lactic Acid Bacterium Weissella confusa MBF8-1 with Spermicidal Activity.

BACKGROUND: The development of antibiotic resistance amongst bacterial pathogens and a population explosion, e.g. in countries such as Indonesia, are two issues the world is facing today. These issues have stimulated interest in the development of new antimicrobial therapeutic agents and contraceptive strategies, such as novel spermicides. Bacteriocins, which are bacterially-derived antimicrobial peptides, may fulfill some of the criteria for these new agents. METHODS: Weissella confusa MBF8-1, originally isolated from a homemade soy product, exhibits antibacterial activity that was subsequently found to be plasmid-encoded, presumably by three peptides Bac1, Bac2 and Bac3. In the present study, we tested cell-free MBF8-1 bacteriocin preparations and chemically-synthesized versions of Bac1, Bac2 and Bac3 peptides for (i) its antibacterial activity against the indicator bacterium Leuconostoc mesenteroides and (ii) its ability to affect the motility of spermatozoa. Nisin, a known lantibiotic bacteriocin, was used as the control. RESULTS: Here, we demonstrate that synthetic Bac1, in combination with synthetic Bac2, was sufficient to inhibit the growth of L. mesenteroides and affect sperm motility. However, the presence of all three synthetic peptides, s-Bac1, s-Bac2 and s-Bac3, was required for full potency. CONCLUSION: In summary, the bacteriocin-like peptides of W. confusa MBF8-1 have the potential to be developed as a narrow-spectrum antimicrobial agent and a novel spermicidal agent.

Bacteriocin-like inhibitory substance (BLIS) activity of Streptococcus macedonicus MBF10-2 and its synergistic action in combination with antibiotics.

OBJECTIVE: To characterize the bacteriocin-like inhibitory-substances (BLIS) activity of Streptococcus macedonicus MBF10-2 [named BLIS(MBF10-2)], a bacteriocinogenic strain isolated from an Indonesian tofu byproduct. METHODS: BLIS(MBF10-2) was obtained by culturing the bacterium, and standard deferred antagonism assays were used to demonstrate its activity. The antibacterial testing of fractions collected by filtration using 3-30 kDa cut-off membrane sizes were carried out by performing well diffusion method. RESULTS: The growth of Micrococcus luteus, Streptococcus pyogenes, Lactococcus lactis, Leuconostoc mesenteroides and Weissella confusa were inhibited by BLIS(MBF10-2). Interestingly, BLIS-containing fractions obtained from sequential application on ultrafiltration membranes indicated that this bacterium Streptococcus macedonicus MBF10-2 could produces at least two antimicrobial peptides activities, one of which is likely to be a lantibiotic peptide. Potential synergistic activity against certain Gram-positive (but not Gram-negative) species when partnered with antibiotics (ampicillin, tetracycline or kanamycin) were observed. CONCLUSION: Combination of some BLIS(MBF10-2) active fractions with antibiotics (ampicillin, tetracycline or kanamycin) could demonstrate synergistic activities against certain Gram-positive species.

Draft Genome Sequence of Weissella confusa MBF8-1, a Glucansucrase- and Bacteriocin-Producing Strain Isolated from a Homemade Soy Product.

We report here the draft genome sequence of Weissella confusa MBF8-1, an isolate from a homemade fermented soybean product that produces sucrases and exhibits antibacterial (bacteriocin) activity. The draft genome of W. confusa MBF8-1 comprises a 2.2-Mbp chromosome and a 17.8-kbp bacteriocin-encoding plasmid. Two putative glucansucrase genes were also identified.

Identification and sequence analysis of pWcMBF8-1, a bacteriocin-encoding plasmid from the lactic acid bacterium Weissella confusa.

Members of the Gram-positive lactic acid bacteria (LAB) are well-known for their beneficial properties as starter cultures and probiotics. Many LAB species produce ribosomally synthesized proteinaceous antibiotics (bacteriocins). Weissella confusa MBF8-1 is a strain isolated from a fermented soybean product that not only produces useful exopolysaccharides but also exhibits bacteriocin activity, which we call weissellicin MBF. Here, we show that bacteriocin production by W. confusa MBF8-1 is specified by a large plasmid, pWcMBF8-1. Plasmid pWcMBF8-1 (GenBank accession number KR350502), which was identified from the W. confusa MBF8-1 draft genome sequence, is 17 643 bp in length with a G + C content of 34.8% and contains 25 open reading frames (ORFs). Six ORFs constitute the weissellicin MBF locus, encoding three putative double-glycine-motif peptides (Bac1, Bac2, Bac3), an ABC transporter complex (BacTE) and a putative immunity protein (BacI). Two ORFs encode plasmid partitioning and mobilization proteins, suggesting that pWcMBF8-1 is transferable to other hosts. To the best of our knowledge, plasmid pWcMBF8-1 not only represents the first large Weissella plasmid to be sequenced but also the first to be associated with bacteriocin production in W. confusa.

Salivaricin E and abundant dextranase activity may contribute to the anti-cariogenic potential of the probiotic candidate Streptococcus salivarius JH.

Dental caries is an infectious disease that is continuing to increase in prevalence, reducing the quality of life for millions worldwide as well as causing considerable expense, with an estimated US$108 billion spent on dental care in the USA each year. Oral probiotics are now being investigated to determine whether they could play a role in the prevention and treatment of this disease. Streptococcus salivarius strain JH is a potential probiotic candidate that produces multiple proteinaceous antimicrobials (bacteriocins), the inhibitory spectrum of which includes Streptococcus mutans, one of the principal causative agents of dental caries. The genome of strain JH has previously been shown to contain the biosynthetic loci for the bacteriocins salivaricin A3, streptin and streptococcin SA-FF22. Here we show that strain JH also produces salivaricin E, a 32 aa lantibiotic with a mass of 3565.9 Da, which is responsible for the inhibition of S. mutans growth. In addition, strain JH was shown to produce dextranase, an enzyme that hydrolyses (1 → 6)-α-D-glucosidic linkages, at levels higher than any other S. salivarius tested. In vitro testing showed that partial hydrolysis of the exopolymeric substances of S. mutans, using strain JH dextranase, improved the anti-S. mutans inhibitory activity of the lytic bacteriocin, zoocin A. The multiple bacteriocin and dextranase activities of strain JH support its candidature for development as an oral probiotic.

Developing oral probiotics from Streptococcus salivarius.

Considerable human illness can be linked to the development of oral microbiota disequilibria. The predominant oral cavity commensal, Streptococcus salivarius has emerged as an important source of safe and efficacious probiotics, capable of fostering more balanced, health-associated oral microbiota. Strain K12, the prototype S. salivarius probiotic, originally introduced to counter Streptococcus pyogenes infections, now has an expanded repertoire of health-promoting applications. K12 and several more recently proposed S. salivarius probiotics are now being applied to control diverse bacterial consortia infections including otitis media, halitosis and dental caries. Other potential applications include upregulation of immunological defenses against respiratory viral infections and treatment of oral candidosis. An overview of the key steps required for probiotic development is also presented.

Periodontal pathogen load and increased antibody response to heat shock protein 60 in patients with cardiovascular disease.

AIM: To determine the relationship between periodontal pathogen load and anti-human heat shock protein 60 (hHSP60) antibodies in patients with established cardiovascular disease (CVD). MATERIALS AND METHODS: Participants were cardiovascular patients (n = 74) with a previous hospital admission for myocardial infarction. Concurrent periodontal pathogen load of Porphyromonas gingivalis, Fusobacterium nucleatum, Tannerella forsythia and Aggregatibacter actinomycetemcomitans was determined using quantitative real-time PCR. Serum antibodies to these pathogens, GroEL and hHSP60 were determined using an ELISA. RESULTS: There was a trend for increasing anti-hHSP60 antibody as the number of bacterial species increased. The strongest positive correlations were found between anti-hHSP60 levels and numbers of T. forsythia (r = 0.43; p < 0.001) and between anti-hHSP60 and anti-GroEL levels (r = 0.39; p = 0.001). Patients with extensive periodontal pocketing (≥4 mm) had higher numbers of P. gingivalis and T. forsythia (p < 0.05) and a higher subgingival pathogen load (p < 0.05) than patients with minimal pocketing (≤1 site ≥ 4 mm). They also had significantly elevated anti-hHSP60 levels (p < 0.05). Overall, the highest anti-hHSP60 levels were seen in patients with extensive periodontal pocketing and all four bacterial species. CONCLUSIONS: In cardiovascular patients, a greater burden of subgingival infection with increased levels of P. gingivalis and T. forsythia is associated with modestly higher anti-hHSP60 levels.

Genome sequence of the bacteriocin-producing oral probiotic Streptococcus salivarius strain M18.

Streptococcus salivarius is a Gram-positive bacterial commensal and pioneer colonizer of the human oral cavity. Many strains produce ribosomally synthesized proteinaceous antibiotics (bacteriocins), and some strains have been developed for use as oral probiotics. Here, we present the draft genome sequence of the bacteriocin-producing oral probiotic S. salivarius strain M18.

The evolution of host specialization in the vertebrate gut symbiont Lactobacillus reuteri.

Recent research has provided mechanistic insight into the important contributions of the gut microbiota to vertebrate biology, but questions remain about the evolutionary processes that have shaped this symbiosis. In the present study, we showed in experiments with gnotobiotic mice that the evolution of Lactobacillus reuteri with rodents resulted in the emergence of host specialization. To identify genomic events marking adaptations to the murine host, we compared the genome of the rodent isolate L. reuteri 100-23 with that of the human isolate L. reuteri F275, and we identified hundreds of genes that were specific to each strain. In order to differentiate true host-specific genome content from strain-level differences, comparative genome hybridizations were performed to query 57 L. reuteri strains originating from six different vertebrate hosts in combination with genome sequence comparisons of nine strains encompassing five phylogenetic lineages of the species. This approach revealed that rodent strains, although showing a high degree of genomic plasticity, possessed a specific genome inventory that was rare or absent in strains from other vertebrate hosts. The distinct genome content of L. reuteri lineages reflected the niche characteristics in the gastrointestinal tracts of their respective hosts, and inactivation of seven out of eight representative rodent-specific genes in L. reuteri 100-23 resulted in impaired ecological performance in the gut of mice. The comparative genomic analyses suggested fundamentally different trends of genome evolution in rodent and human L. reuteri populations, with the former possessing a large and adaptable pan-genome while the latter being subjected to a process of reductive evolution. In conclusion, this study provided experimental evidence and a molecular basis for the evolution of host specificity in a vertebrate gut symbiont, and it identified genomic events that have shaped this process.

Identification of DysI, the immunity factor of the streptococcal bacteriocin dysgalacticin.

DysI is identified as the protein that confers specific immunity to dysgalacticin, a plasmid-encoded streptococcal bacteriocin. dysI is transcribed as part of the copG-repB-dysI replication-associated operon. DysI appears to function at the membrane level to prevent the inhibitory effects of dysgalacticin on glucose transport, membrane integrity, and intracellular ATP content.

Oral bacterial genome sequencing using the high-throughput Roche Genome Sequencer FLX System.

For over 30 years, the chain termination method of DNA sequencing (commonly known as Sanger sequencing) has been the mainstay of any DNA sequencing project. In the past, whole-genome sequencing employing exclusively Sanger chemistry has been a labor-intensive and costly exercise and an option unfeasible for the average research group. However, within the last 4 years, the introduction of three high-throughput sequencing technologies (454, SOLiD, and Illumina) has revolutionized genomics by facilitating unprecedented levels (up to gigabasepairs) of reliable DNA sequence output in a relatively short time frame and at a much lower cost per sequenced basepair. Here, we provide laboratory and bioinformatic protocols that will allow the average research group to undertake high-throughput sequencing of oral bacterial genomes using the Roche Genome Sequencer FLX System which employs 454 pyrosequencing technology.

Something Old and Something New: An Update on the Amazing Repertoire of Bacteriocins Produced by Streptococcus salivarius.

Streptococcus salivarius has an exclusive and intimate association with humans. We are its sole natural host, and its contribution to the relationship appears overwhelmingly benevolent. Beautifully adapted to its preferred habitat, the human tongue, it only rarely ventures far from this location in the healthy host and indeed appears ill-equipped to become invasive due to a scarcity of virulence attributes. We consider that its strategically advantageous lingual location and numerical predominance allow S. salivarius to carry out a population surveillance and modulation role within the oral microbiota. Some strains are armed with complex arrays of targeted antibiotic weaponry, much of which belongs to the lantibiotic class of bacteriocins and a key to their ability to assemble and utilize this armament is their possession of transmissible multi-bacteriocin-encoding megaplasmid DNA. This review traces the origins of research into S. salivarius bacteriocins and bacteriocin-like inhibitory substances, showcases some of the inhibitory activities that we currently have knowledge of, and speculates about potential directions for ongoing investigation and probiotic application of this previously under-rated human commensal.

Streptococcal bacteriocins and the case for Streptococcus salivarius as model oral probiotics.

Members of the Gram-positive bacterial genus Streptococcus are a diverse collection of species inhabiting many body sites and range from benign, nonpathogenic species to those causing life-threatening infections. The streptococci are also prolific producers of bacteriocins, which are ribosomally synthesized proteinaceous antibiotics that kill or inhibit species closely related to the producer bacterium. With the emergence of bacterial resistance to conventional antibiotics, there is an impetus to discover, and implement, new and preferably 'natural' antibiotics to treat or prevent bacterial infections, a niche that bacterial interference therapy mediated by bacteriocins could easily fill. This review focuses on describing the diversity of bacteriocins produced by streptococci and also puts forth a case for Streptococcus salivarius, a nonpathogenic and numerically predominant oral species, as an ideal candidate for development as the model probiotic for the oral cavity. S. salivarius is a safe species that not only produces broad-spectrum bacteriocins but harbors bacteriocin-encoding (and bacteriocin-inducing) transmissible DNA entities (megaplasmids).