David A. Spratt

Statistical analyses of complex denaturing gradient gel electrophoresis profiles

ABSTRACT Studies using molecular techniques have demonstrated that a culture-based approach can severely underestimate the bacterial diversity in most environments. One of the molecular techniques that has been applied in microbial ecology is denaturing gradient gel electrophoresis (DGGE). The purpose of this study was to investigate differences in the microbiota of plaque, using a number of analysis techniques, from children without gingivitis (n = 30) and from those with gingivitis (n = 30). Extracted DNA from gingival margin plaque was subjected to PCR targeting the 16S rRNA gene using universal primers. DGGE profiles were analyzed in three ways. (i) Bacterial diversity was compared between cohorts by using the Shannon-Wiener index (also known as the Shannon-Weaver index). (ii) A hierarchical cluster analysis of the banding patterns was calculated and expressed as a dendrogram. (iii) Individual DGGE bands and their intensities for both cohorts were compared using a logistic regression analysis. The Shannon-Wiener indices demonstrated a greater bacterial diversity associated with no-gingivitis plaque (P = 0.009). Dendrograms demonstrated that seven clades associated with gingivitis and five clades associated with no gingivitis. The logistic regression demonstrated that one band was significantly associated with no gingivitis (P = 0.001), while two bands were significantly associated with gingivitis (P = 0.005 and P = 0.042). In conclusion, this study demonstrates that the development of gingivitis might be accompanied by a decrease in bacterial diversity. Furthermore, we have demonstrated that logistic regression is a good statistical method for analyzing and characterizing DGGE profiles.

Same Exposure but Two Radically Different Responses to Antibiotics: Resilience of the Salivary Microbiome versus Long-Term Microbial Shifts in Feces

ABSTRACT Due to the spread of resistance, antibiotic exposure receives increasing attention. Ecological consequences for the different niches of individual microbiomes are, however, largely ignored. Here, we report the effects of widely used antibiotics (clindamycin, ciprofloxacin, amoxicillin, and minocycline) with different modes of action on the ecology of both the gut and the oral microbiomes in 66 healthy adults from the United Kingdom and Sweden in a two-center randomized placebo-controlled clinical trial. Feces and saliva were collected at baseline, immediately after exposure, and 1, 2, 4, and 12 months after administration of antibiotics or placebo. Sequences of 16S rRNA gene amplicons from all samples and metagenomic shotgun sequences from selected baseline and post-antibiotic-treatment sample pairs were analyzed. Additionally, metagenomic predictions based on 16S rRNA gene amplicon data were performed using PICRUSt. The salivary microbiome was found to be significantly more robust, whereas the antibiotics negatively affected the fecal microbiome: in particular, health-associated butyrate-producing species became strongly underrepresented. Additionally, exposure to different antibiotics enriched genes associated with antibiotic resistance. In conclusion, healthy individuals, exposed to a single antibiotic treatment, undergo considerable microbial shifts and enrichment in antibiotic resistance in their feces, while their salivary microbiome composition remains unexpectedly stable. The health-related consequences for the gut microbiome should increase the awareness of the individual risks involved with antibiotic use, especially in a (diseased) population with an already dysregulated microbiome. On the other hand, understanding the mechanisms behind the resilience of the oral microbiome toward ecological collapse might prove useful in combating microbial dysbiosis elsewhere in the body. IMPORTANCE Many health care professionals use antibiotic prophylaxis strategies to prevent infection after surgery. This practice is under debate since it enhances the spread of antibiotic resistance. Another important reason to avoid nonessential use of antibiotics, the impact on our microbiome, has hardly received attention. In this study, we assessed the impact of antibiotics on the human microbial ecology at two niches. We followed the oral and gut microbiomes in 66 individuals from before, immediately after, and up to 12 months after exposure to different antibiotic classes. The salivary microbiome recovered quickly and was surprisingly robust toward antibiotic-induced disturbance. The fecal microbiome was severely affected by most antibiotics: for months, health-associated butyrate-producing species became strongly underrepresented. Additionally, there was an enrichment of genes associated with antibiotic resistance. Clearly, even a single antibiotic treatment in healthy individuals contributes to the risk of resistance development and leads to long-lasting detrimental shifts in the gut microbiome. Many health care professionals use antibiotic prophylaxis strategies to prevent infection after surgery. This practice is under debate since it enhances the spread of antibiotic resistance. Another important reason to avoid nonessential use of antibiotics, the impact on our microbiome, has hardly received attention. In this study, we assessed the impact of antibiotics on the human microbial ecology at two niches. We followed the oral and gut microbiomes in 66 individuals from before, immediately after, and up to 12 months after exposure to different antibiotic classes. The salivary microbiome recovered quickly and was surprisingly robust toward antibiotic-induced disturbance. The fecal microbiome was severely affected by most antibiotics: for months, health-associated butyrate-producing species became strongly underrepresented. Additionally, there was an enrichment of genes associated with antibiotic resistance. Clearly, even a single antibiotic treatment in healthy individuals contributes to the risk of resistance development and leads to long-lasting detrimental shifts in the gut microbiome.

Cultivable Oral Microbiota of Domestic Dogs

ABSTRACT Bacteria were isolated from the dental plaques of nine dogs and a sample of pooled saliva from five other dogs and were then identified by comparative 16S rRNA gene sequencing. Among 339 isolates, 84 different phylotypes belonging to 37 genera were identified. Approximately half of the phylotypes were identified to the species level, and 28% of these were considered members of the indigenous oral microbiota of humans. The 16S rRNA gene sequences of the remaining 44 phylotypes were not represented in GenBank, and most of these phylotypes were tentatively identified as candidate new species. The genera most frequently isolated from saliva were Actinomyces (26%), Streptococcus (18%), and Granulicatella (17%). The genera most frequently isolated from plaque were Porphyromonas (20%), Actinomyces (12%), and Neisseria (10%). A comparison of the DNA sequences from this study with sequences of the human microbiota available in GenBank showed that, on average, canine and human microbiotas differed by almost 7% in the 16S rRNA gene. In conclusion, this study has shown that the cultivable oral microbiotas of dogs and humans show significant differences.

Distribution of tetracycline and erythromycin resistance genes among human oral and fecal metagenomic DNA.

We have analyzed the total metagenomic DNA from both human oral and fecal samples derived from healthy volunteers from six European countries to determine the molecular basis for tetracycline and erythromycin resistance. We have determined that tet(M) and tet(W) are the most prevalent tetracycline resistance genes assayed for in the oral and fecal metagenomes, respectively. Additionally, tet(Q), tet(O), and tet(O/32/O) have been shown to be common. We have also shown that erm(B), erm(V), and erm(E) are common erythromycin resistance genes present in these environments. Further, we have demonstrated the ubiquitous presence of the Tn916 integrase in the oral metagenomes and the Tn4451 and Tn1549 integrase genes within the fecal metagenomes.

Duration, prevalence and intensity of bacteraemia after dental extractions in children

Objective: To investigate the duration, prevalence and intensity of bacteraemia after dental extractions in children by comparing within-patient bacteraemia before and after dental extraction. Methods: Children were randomly allocated to one of 10 postprocedure time groups from 10 s to 60 min. The differences between intensity and prevalence of the bacteraemia at each time after extractions were used to estimate the duration of the bacteraemia. After attainment of general anaesthesia, pre-extraction and postextraction blood samples were processed by broth culture and lysis filtration to isolate and quantify bacteria present in the patients’ blood. Results: 500 subjects between 3 and 16 years old were recruited. The estimated duration of bacteraemia was about 11 min. Conclusions: The duration of bacteraemia after dental extractions is less than previously thought. This has implications for the interpretation of odontogenic bacteraemia studies.

Prevalence of Periodontal Pathogens in Dental Plaque of Children

ABSTRACT Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Tannerella forsythensis have been implicated as the main etiological agents of periodontal disease. The purpose of this work was to estimate the prevalence of these organisms in plaque from children without gingivitis (group 1; n = 65) and from those with gingivitis (group 2; n = 53). Extracted DNA from plaque was subjected to two rounds of PCR targeting the 16S rRNA gene using both universal primers and species-specific primers. The results were as follows: group 1, P. gingivalis, 49%; A. actinomycetemcomitans, 55%; and T. forsythensis, 65%; group 2, P. gingivalis, 47%; A. actinomycetemcomitans, 59%; and T. forsythensis, 45%. T. forsythensis was detected more frequently in children with no gingivitis than in those with gingivitis (P = 0.03). There was no significant difference between the two groups with respect to the presence of P. gingivalis or A. actinomycetemcomitans in either group (P > 0.05). Logistic regression analysis revealed that the odds of a patient having gingivitis were 2.3 times greater in the absence of T. forsythensis. In conclusion, the results of this study have shown that the three pathogens can be detected in the dental plaque of healthy children and of those with gingivitis and that T. forsythensis is associated with dental plaque at sites with no gingivitis.

Viable Bacteria Present within Oral Squamous Cell Carcinoma Tissue

ABSTRACT Despite increasing interest in the possible relationships between bacteria and the different stages of cancer development, the association of bacteria with cancer of the oral cavity has yet to be adequately examined. With that in mind, the primary objective of this study was to identify any bacterial species within oral squamous cell carcinoma tissue using a standard microbiological culture approach. At the time of surgery, a 1-cm3 portion of tissue was harvested from deep within the tumor mass using a fresh blade for each cut. Whenever possible, “superficial” portions from the mucosa overlying the tumor and nontumorous control specimens from at least 5 cm away from the primary tumor site were also obtained. Surface contamination was eliminated by immersion in Betadine and washing with phosphate-buffered saline. Each specimen was aseptically macerated and cultured on nonselective media under both aerobic and anaerobic conditions. Isolates were identified by 16S rRNA gene sequencing. Twenty deep-tissue specimens, 19 with corresponding superficial tissues and 12 with control tissues, were successfully processed. A diversity of bacterial taxa were isolated and identified, including several putatively novel species. Most isolates were found to be saccharolytic and acid-tolerant species. Notably, some species were isolated only from either the tumorous or nontumorous tissue type, indicating a degree of restriction. Successful surface decontamination of the specimens indicates that the bacteria detected were from within the tissue. A diversity of bacterial groups have been isolated from within oral squamous cell carcinoma tissue. The significance of these bacteria within the tumor warrants further study.

Evaluation of Protocols for Field Decontamination Before Bacterial Sampling of Root Canals for Contemporary Microbiology Techniques

The effectiveness of sodium hypochlorite (NaOCl) (2.5%) or iodine (10%) for decontamination of the operation field (tooth, rubber dam, and gasket [Oraseal]) was compared by using bacterial cultivation. In addition, the final samples were also assessed for bacteria by using polymerase chain reaction. Teeth (n = 63) receiving root canal treatment were polished with pumice, isolated with rubber dam, and their margins sealed with Oraseal. The operation field was disinfected with hydrogen peroxide (30%), followed by iodine (n = 31) or NaOCl (n = 32), before and after access cavity preparation. The operation field was sampled before and after each decontamination, giving four samples per field. After the final decontamination, there was no significant difference (p = 0.602, 0.113, 0.204) in recovery of cultivable bacteria from various sites in either group. However, bacterial DNA could be detected significantly (p = 0.010) more frequently from the tooth surfaces after iodine (45%) compared with NaOCl (13%) decontamination, although on the rubber dam or Oraseal surfaces there was no difference. Root canal sampling for polymerase chain reaction might be better preceded by NaOCl decontamination than by iodine, based on the findings.

Characterisation of Eubacterium-like strains isolated from oral infections

The genus Eubacterium currently includes a heterogeneous group of gram-positive, non-spore-forming anaerobic bacilli, many of which are slow growing, fastidious and generally unreactive in biochemical tests. As a consequence, cultivation and identification of isolates are difficult and the taxonomy of the group remains indifferent. In this study, 105 isolates from odontogenic infections, infections associated with dental implants or saliva from healthy subjects and provisionally assigned to the genus Eubacterium were subjected to phenotypic and genotypic analysis. Ninety-one of the isolates were identified as belonging to one of 14 previously described species: Atopobium parvulum (5 isolates), A. rimae (29), Bulleidia extructa (2), Cryptobacterium curtum (1), Dialister pneumosintes (1), Eubacterium saburreum (2), E. sulci (8), E. yurii subsp. yurii (1), Filifactor alocis (3), Lactobacillus uli (1), Mogibacterium timidum (13), M. vescum (6), Pseudoramibacter alactolyticus (6) and Slackia exigua (13). The remaining 14 isolates did not correspond to existing species. This study confirms the diversity of organisms provisionally assigned to the genus Eubacterium by conventional identification methods. This group of organisms is frequently isolated from oral infections but their role in the aetiology of these conditions has yet to be determined.

Molecular detection of novel anaerobic species in dentoalveolar abscesses.

The ability to isolate in pure culture the bacteria that cause 1492R [2]. The amplified genes were singularized by cloning, reamplified with the same primers, and then together with amplidisease in humans has been a cornerstone of the advances made in medical microbiology over the past hundred years and an essential fied 16S rRNA genes from the cultivated bacteria were digested component of Koch’s postulates. The etiologic agents of the majorwith a range of restriction endonucleases. ity of the diseases suspected to be caused by bacteria have been The fragments were separated by electrophoresis to give restricdiscovered by this method. However, the advances made as a result tion-fragment-length polymorphism (RFLP) profiles, which were of the use of artificial culture media have perhaps been fortuitous used to group the cloned genes. Members of a group shared the because our knowledge of the microorganisms found on earth is same RFLP profiles for at least three restriction endonucleases. extremely limited; it has been estimated that õ1% of the bacteria The RFLP profiles from the cultivated isolates were then compared on earth have been cultured. It is also estimated that 50% of to those of the groups of cloned genes. Representative clones of the oral flora is unculturable, and the proportion of unculturable groups exhibiting profiles that did not match any of the cultivable organisms at other body sites remains unknown. organisms were sequenced. In recent years studies of the microbial composition of biomass Sequences were identified by means of Similarity-Rank software found at environmental sites have been facilitated by advances in [3]. The sequences were then aligned to related 16S rRNA molecular biology. More specifically, PCR has enabled the amplisequences, and phylogenetic analysis was performed with the fication of specific genes, and the development of automated DNA PHYLIP suite of programs [4]. sequencing methods has allowed the rapid characterization of both The bacteria isolated from two of the samples are shown in entire genomes and specific genes in large numbers of organisms. table 1. A similar range of organisms was recovered from both Genes encoding ribosomal RNA have been found to be particularly samples, and both the identity and numbers of the species isolated useful for the construction of phylogenetic trees reflecting the evowere typical. For each sample, there were three groups of cloned lution of bacteria [1]. This is because these genes have been con16S rRNA genes that did not match with the profiles of the cultured served throughout evolution because of the need to preserve funcorganisms. The results of the sequence analysis for these groups tion. are shown in table 2. Some regions are highly conserved, allowing the design of uniGroups 3.4 and 3.5 showed high similarity to Prevotella oris and versal PCR primers for the amplification of the gene, while other Porphyromonas endodontalis, respectively. P. oris was isolated in regions are more variable, with sufficient information present to the study, but the cultivated organism exhibited a different RFLP differentiate at the species level. The 16S rRNA gene has been profile to that of the cloned group. Group 3.6 showed only low found to be particularly useful because its size of Ç1,500 bases similarity to Prevotella oralis. Further phylogenetic analysis of is sufficient to generate meaningful information, while it is short this sequence indicated that this group may represent a new genus enough for easy sequencing. Thus, 16S rRNA genes have been related to Bacteroides and Prevotella. Group 9.2 was identified as amplified from a variety of sources, including the environment P. endodontalis, while 9.3 was found to show a similarity of 93.6% and human infections, and then sequenced to reveal the diversity to Prevotella buccae. This level of similarity is indicative of the of organisms present at the site. group representing a new species of Prevotella. Dentoalveolar abscesses are acute infections associated with the Group 9.4 showed 89.7% similarity to Eubacterium brachy. The teeth, arising from pulpal infection secondary to dental caries. oral asaccharolytic Eubacterium species are a heterogeneous group Cultural analyses have revealed they are associated with a reof organisms currently undergoing reclassification [5]; all of the stricted group of organisms, principally members of the genera currently recognized species are likely to be elevated to genus rank Fusobacterium, Peptostreptococcus, Prevotella, and Streptococcus. In the studies described here, we adapted methods for the detection of uncultivable bacteria in the environment to the analysis of the microflora associated with dentoalveolar abscesses. Table 1. Bacteria cultured from pus from dentoalveolar abscesses. Pus was aspirated from dentoalveolar abscesses by needle and