Haemophilus pittmaniae and Leptotrichia spp. Constitute a Multi-Marker Signature in a Cohort of Human Papillomavirus-Positive Head and Neck Cancer Patients.

OBJECTIVES: Human papillomavirus (HPV) is a known etiological factor of oropharyngeal head and neck cancer (HNC). HPV positivity and periodontal disease have been associated with higher HNC risk, suggesting a role for oral bacterial species. Our objective was to determine oral microbiome profiles in HNC patients (HPV-positive and HPV-negative) and in healthy controls (HC). METHODS: Saliva samples and swabs of buccal mucosa, supragingival plaque, and tongue were collected from HNC patients (N = 23 patients, n = 92 samples) before cancer therapy. Next-generation sequencing (16S-rRNA gene V3-V4 region) was used to determine bacterial taxa relative abundance (RA). ß-Diversities of HNC HPV+ (N = 16 patients, n = 64 samples) and HNC HPV- (N = 7 patients, n = 28 samples) groups were compared using PERMANOVA (pMonte Carlo < 0.05). LEfSe discriminant analysis was performed to identify differentiating taxa (Log LDA > 2.0). RA differences were analyzed by Mann-Whitney U-test (α = 0.05). CombiROC program was used to determine multi-marker bacterial signatures. The Microbial Interaction Network Database (MIND) and LitSuggest online tools were used for complementary analyses. RESULTS: HNC vs. HC and HNC HPV+ vs. HNC HPV- ß-diversities differed significantly (pMonte Carlo < 0.05). Streptococcus was the most abundant genus for HNC and HC groups, while Rothia mucilaginosa and Haemophilus parainfluenzae were the most abundant species in HNC and HC patients, respectively, regardless of antibiotics treatment. LEfSe analysis identified 43 and 44 distinctive species for HNC HPV+ and HNC HPV- groups, respectively. In HNC HPV+ group, 26 periodontal disease-associated species identified by LefSe had a higher average RA compared to HNC HPV- group. The significant species included Alloprevotella tannerae, Fusobacterium periodonticum, Haemophilus pittmaniae, Lachnoanaerobaulum orale, and Leptotrichia spp. (Mann-Whitney U-test, p < 0.05). Of 43 LEfSe-identified species in HPV+ group, 31 had a higher RA compared to HPV- group (Mann-Whitney U-test, p < 0.05). MIND analysis confirmed interactions between Haemophilus and Leptotrichia spp., representing a multi-marker signature per CombiROC analysis [area under the curve (AUC) > 0.9]. LitSuggest correctly classified 15 articles relevant to oral microbiome and HPV status. CONCLUSION: Oral microbiome profiles of HNC HPV+ and HNC HPV- patients differed significantly regarding periodontal-associated species. Our results suggest that oral bacterial species (e.g., Leptotrichia spp.), possessing unique niches and invasive properties, coexist with HPV within HPV-induced oral lesions in HNC patients. Further investigation into host-microbe interactions in HPV-positive HNC patients may shed light into cancer development.

Concordance of HOMIM and HOMINGS technologies in the microbiome analysis of clinical samples.

BACKGROUND: Over 700 bacterial species reside in human oral cavity, many of which are associated with local or distant site infections. Extensive characterization of the oral microbiome depends on the technologies used to determine the presence and proportions of specific bacterial species in various oral sites. OBJECTIVE: The objective of this study was to compare the microbial composition of dental plaque at baseline using Human Oral Microbe Identification Microarray (HOMIM) and Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS) technologies, which are based on 16S rRNA. METHODS: Dental plaque samples were collected from 96 patients at baseline prior to a dental procedure involving manipulation of gingival tissues. The samples were surveyed for 293 and 597 oral bacterial species via HOMIM and HOMINGS, respectively, based on 16S rRNA gene sequences. We determined the concordance between the two technologies for common species. Genus level analysis was performed using HOMINGS-specific genus identification capabilities. RESULTS: HOMINGS detected twice the number of species in the same dental plaque samples compared to HOMIM. For the species detected by both HOMIM and HOMINGS, there was no difference in relative proportions of overall bacterial composition at the species, genus or phylum levels. Additionally, there was no difference in relative proportion for total species per patient between the two technologies. CONCLUSION: HOMINGS significantly expanded oral bacterial species identification compared to HOMIM. The genus and species probes, combined in HOMINGS, provided a more comprehensive representation of oral bacterial community, critical for future characterization of oral microbes in distant site infections.

Microarray analyses of oral punch biopsies from acute myeloid leukemia (AML) patients treated with chemotherapy.

OBJECTIVE: Understanding the pathogenesis of chemotherapy-induced oral mucositis (CIOM) is vital to develop therapies for this common, dose-limiting side effect of cancer treatment. We investigated molecular events in CIOM from buccal mucosa tissue collected before and 2 days after chemotherapy from patients with acute myeloid leukemia (AML) and healthy controls by microarray analysis. METHODS: Microarray analysis was performed using Human Genome U133 Plus 2.0 Array on buccal mucosa punch biopsies from patients with AML before (n = 4) or after chemotherapy (n = 4), and from healthy controls (n = 3). Following Robust Multichip Average (RMA) normalization, we applied Linear Models for Microarray data (LIMMA) and Significance Analysis of Microarrays (SAM) for data analysis using the TM4/TMeV v4.5.1 program. RESULTS: LIMMA and SAM identified genes potentially affected by the presence of AML, including homeodomain-interacting protein kinase 1 (HIPK1), mex-3 homolog D (MEX3D), and genes potentially affected by chemotherapy, including argininosuccinate synthase 1 (ASS1), notch homolog 1 (NOTCH1), zinc transporter ZIP6 (SLC39A6), and TP53-regulated inhibitor of apoptosis 1 (TRIAP1). The expression of 2 genes with potential biological significance in oral mucositis, ASS1 and SLC39A6 (alias LIV-1), was confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). CONCLUSIONS: Our results suggest that AML-specific deregulated immune responses and inflammatory tissue damage to the oral mucosa caused by chemotherapy may not be overcome by the natural cellular repair processes and therefore contribute to CIOM.

Molecular methodology to assess the impact of cancer chemotherapy on the oral bacterial flora: a pilot study.

OBJECTIVE: This pilot study determined the profile of the oral bacterial flora in an outpatient cancer population before and after chemotherapy using molecular techniques. STUDY DESIGN: We recruited 9 newly diagnosed breast cancer patients scheduled for induction chemotherapy. All were seen immediately before chemotherapy, and 7 to 14 days later. At both visits, we performed oral evaluations and obtained mucositis grading (with the World Health Organization [WHO] scale), absolute neutrophil counts (ANC), and bacterial samples from the buccal mucosa. Bacterial DNA was isolated, and 16S ribosomal RNA gene clonal libraries were constructed. Sequences of genes in the library were used to determine species identity by comparison to known sequences. RESULTS: After chemotherapy, WHO scores of 0 and 1 were in 3 and 6 patients, respectively, and mean ANC (+/-SD) dropped from 3326 +/- 463 to 1091 +/- 1082 cells/mm(3). From pre- and post-chemotherapy samples, 41 species were detected, with a predominance of Gemella haemolysans and Streptococcus mitis. More than 85% of species have not been previously identified in chemotherapy patients. Seven species appeared exclusively before chemotherapy and 25 after chemotherapy. After chemotherapy, the number of species per patient increased by a mean of 2.6 (SD = 4.7, P = .052). CONCLUSION: We identified species not previously identified in chemotherapy patients. Our results suggest a shift to a more complex oral bacterial profile in patients undergoing cancer chemotherapy.

Poor oral hygiene as a risk factor for infective endocarditis-related bacteremia.

BACKGROUND: Infective endocarditis (IE) often is caused by bacteria that colonize teeth. The authors conducted a study to determine if poor oral hygiene or dental disease are risk factors for developing bacteremia after toothbrushing or single-tooth extraction. METHODS: One hundred ninety-four participants in a study were in either a toothbrushing group or a single-tooth extraction with placebo group. The authors assessed the participants' oral hygiene, gingivitis and periodontitis statuses. They assayed blood samples obtained before, during and after the toothbrushing or extraction interventions for IE-associated bacteria. RESULTS: The authors found that oral hygiene and gingival disease indexes were associated significantly with IE-related bacteremia after toothbrushing. Participants with mean plaque and calculus scores of 2 or greater were at a 3.78- and 4.43-fold increased risk of developing bacteremia, respectively. The presence of generalized bleeding after toothbrushing was associated with an almost eightfold increase in risk of developing bacteremia. There was no significant association between any of the measures of periodontal disease and the incidence of bacteremia after toothbrushing. The oral hygiene or disease status of a tooth was not significantly associated with bacteremia after its extraction. CONCLUSION: Bacteremia after toothbrushing is associated with poor oral hygiene and gingival bleeding after toothbrushing. CLINICAL IMPLICATIONS: Improvements in oral hygiene may reduce the risk of developing IE.

Systemic host immuno-inflammatory response to dental extractions and periodontitis.

OBJECTIVES: Little is known about the host immuno-inflammatory response to dental extractions. The purpose of this pilot clinical study was to explore the effect of both periodontitis and dental extractions on the host systemic immuno-inflammatory response. STUDY DESIGN: We recruited and collected baseline blood samples on 41 essentially healthy patients. A subset of 22 subjects underwent single (n = 11) or multiple (n = 11) tooth extractions with additional blood samples taken at 1 hour following single tooth extraction and at 8 and 24 hours following multiple tooth extractions. Samples were used for determination of an array of 12 cytokines known to play key roles in innate and adaptive immunity. RESULTS: There was no significant difference in cytokine levels between the pre- and post-tooth extraction samples for either extraction group for the time points measured. Nor was there a positive relationship between the level of any of the cytokines and periodontal disease status as measured by mean probing depths and other periodontal disease parameters. CONCLUSION: Our pilot data suggest that the body is well adjusted to deal with the inflammation and bacteremia associated with dental extractions and periodontal disease.

Bacteremia associated with toothbrushing and dental extraction.

BACKGROUND: Antibiotic prophylaxis recommendations for the prevention of infective endocarditis are based in part on studies of bacteremia from dental procedures, but toothbrushing may pose a greater threat. The purpose of this study was to compare the incidence, duration, nature, and magnitude of endocarditis-related bacteremia from single-tooth extraction and toothbrushing and to determine the impact of amoxicillin prophylaxis on single-tooth extraction. METHODS AND RESULTS: In this double-blind, placebo-controlled study, 290 subjects were randomized to (1) toothbrushing, (2) single-tooth extraction with amoxicillin prophylaxis, or (3) single-tooth extraction with identical placebo. Blood was drawn for bacterial culturing and identification at 6 time points before, during, and after these interventions. The focus of our analysis was on bacterial species reported to cause infective endocarditis. We identified 98 bacterial species, 32 of which are reported to cause endocarditis. Cumulative incidence of endocarditis-related bacteria from all 6 blood draws was 23%, 33%, and 60% for the toothbrushing, extraction-amoxicillin, and extraction-placebo groups, respectively (P<0.0001). Significant differences were identified among the 3 groups at draws 2, 3, 4, and 5 (all P<0.05). Amoxicillin resulted in a significant decrease in positive cultures (P<0.0001). CONCLUSIONS: Although amoxicillin has a significant impact on bacteremia resulting from a single-tooth extraction, given the greater frequency for oral hygiene, toothbrushing may be a greater threat for individuals at risk for infective endocarditis.

Identification of oral bacteria in blood cultures by conventional versus molecular methods.

OBJECTIVE: The purpose of this study was to compare two different methods of identifying oral bacteria from blood samples after dental extractions. STUDY DESIGN: Blood cultures were taken before, during, and after a single tooth extraction in 30 subjects and cultured using aerobic and anaerobic Bactec media. Positive cultures from 22 subjects were subcultured on selective and nonselective media, resulting in 58 isolates. Bacterial identification was performed by biochemical analysis and sequence analysis of 16S rRNA gene. RESULTS: Of the 58 isolates, only 10 (17%) were the same species by both methods. Thirty-two (55%) belonged to the same genus but different species, and 16 (28%) showed no correlation at all. There were 31 and 40 diverse species by the biochemical and the sequencing methods, respectively. Streptococci were the dominant species. CONCLUSIONS: DNA sequencing results in more accurate identification and a more diverse population of bacteria from bacteremia following dental extractions.

Diverse and novel oral bacterial species in blood following dental procedures.

We identified oral bacterial species in blood cultures following single-tooth extraction and tooth brushing. Sequence analysis of 16S rRNA genes identified 98 different bacterial species recovered from 151 bacteremic subjects. Of interest, 48 of the isolates represented 19 novel species of Prevotella, Fusobacterium, Streptococcus, Actinomyces, Capnocytophaga, Selenomonas, and Veillonella.

Molecular analysis of oral and respiratory bacterial species associated with ventilator-associated pneumonia.

Trauma intensive care unit (TICU) patients requiring mechanical respiratory support frequently develop ventilator-associated pneumonia (VAP). Oral and oropharyngeal bacteria are believed to be responsible for many cases of VAP, but definitive evidence of this relationship is lacking. Earlier studies used conventional culture-based methods for identification of bacterial pathogens, but these methods are insufficient, as some bacteria may be uncultivable or difficult to grow. The purpose of this study was to use a culture-independent molecular approach to analyze and compare the bacterial species colonizing the oral cavity and the lungs of TICU patients who developed VAP. Bacterial samples were acquired from the dorsal tongue and bronchoalveolar lavage fluid of 16 patients. Bacterial DNA was extracted, and the 16S rRNA genes were PCR amplified, cloned into Escherichia coli, and sequenced. The sequencing data revealed the following: (i) a wide diversity of bacterial species in both the oral and pulmonary sites, some of them novel; (ii) known and putative respiratory pathogens colonizing both the oral cavity and lungs of 14 patients; and (iii) a number of bacterial pathogens (e.g., Dialister pneumosintes, Haemophilus segnis, Gemella morbillorum, and Pseudomonas fluorescens) in lung samples that had not been reported previously at this site when culture-based methods were used. Our data indicate that the dorsal surface of the tongue serves as a potential reservoir for bacterial species involved in VAP. Furthermore, it is clear that the diversity of bacterial pathogens for VAP is far more complex than the current literature suggests.

Relationship between mucositis and changes in oral microflora during cancer chemotherapy.

It is thought that the incidence and severity of cancer chemotherapy-associated mucositis is caused in part by changes in the oral bacterial microflora. This systematic review examined the role of oral bacterial microflora changes in the development of oral mucositis during chemotherapy. Thirteen prospective clinical trials were identified, involving 300 patients with 13 different cancer diagnoses. There was great variability in patient populations, bacterial sample collection methodology, and oral sample sites. No clear pattern regarding qualitative and quantitative oral flora changes emerged among these studies. The most frequent Gram-negative species isolated during chemotherapy were from the Enterobacteriaceae family, Pseudomonas sp. and E. coli. The most common Gram-positive species isolated were Staphylococcus sp. and Streptococcus sp. Five studies assessed the role of oral flora changes in the genesis of oral mucosal changes, with no consensus among them. More detailed studies are required to understand the relationship between chemotherapy, alterations in the nature and magnitude of the oral microflora, and the presence of mucositis.

Identification and characterization of a novel uropathogenic Escherichia coli-associated fimbrial gene cluster.

Recently, we identified a fimbrial usher gene in uropathogenic Escherichia coli strain CFT073 that is absent from an E. coli laboratory strain. Analysis of the CFT073 genome indicates that this fimbrial usher gene is part of a novel fimbrial gene cluster, aufABCDEFG. Analysis of a collection of pathogenic and commensal strains of E. coli and related species revealed that the auf gene cluster was significantly associated with uropathogenic E. coli isolates. For in vitro expression analysis of the auf gene cluster, RNA was isolated from CFT073 bacteria grown to the exponential or stationary phase in Luria-Bertani broth and reverse transcriptase PCR (RT-PCR) with oligonucleotide primers specific to the major subunit, aufA, was performed. We found that aufA is expressed in CFT073 only during the exponential growth phase; however, no expression of AufA protein was observed by Western blotting, indicating that under these conditions, the expression of the auf gene cluster is low. To determine if the auf gene cluster is expressed in vivo, RT-PCR was performed on bacteria from urine samples of mice infected with CFT073. Out of three independent experiments, we were able to detect expression of aufA at least once at 4, 24, and 48 h of infection, indicating that the auf gene cluster is expressed in the murine urinary tract. Furthermore, antisera from mice infected with CFT073 reacted with recombinant AufA in an enzyme-linked immunosorbent assay. To identify the structure encoded by the auf gene cluster, a recombinant plasmid containing the auf gene cluster under the T7 promoter was introduced into the E. coli BL-21 (AI) strain. Immunogold labeling using AufA antiserum revealed the presence of amorphous material extending from the surface of BL-21 cells. No hemagglutination or cellular adherence properties were detected in association with expression of AufA. Deletion of the entire auf gene cluster had no effect on the ability of CFT073 to colonize the kidney, bladder, or urine of mice. In addition, no significant histological differences between the parent and aufC mutant strain were observed. Therefore, Auf is a uropathogenic E. coli-associated structure that plays an uncertain role in the pathogenesis of urinary tract infections.

Type 1 fimbriae and extracellular polysaccharides are preeminent uropathogenic Escherichia coli virulence determinants in the murine urinary tract.

Escherichia coli is the leading cause of urinary tract infections (UTIs). Despite the association of numerous bacterial factors with uropathogenic E. coli (UPEC), few such factors have been proved to be required for UTI in animal models. Previous investigations of urovirulence factors have relied on prior identification of phenotypic characteristics. We used signature-tagged mutagenesis (STM) in an unbiased effort to identify genes that are essential for UPEC survival within the murine urinary tract. A library of 2049 transposon mutants of the prototypic UPEC strain CFT073 was constructed using mini-Tn5km2 carrying 92 unique tags and screened in a murine model of ascending UTI. After initial screening followed by confirmation in co-infection experiments, 19 survival-defective mutants were identified. These mutants were recovered in numbers 101- to 106-fold less than the wild type in the bladder, kidneys or urine or at more than one site. The transposon junctions from each attenuated mutant were sequenced and analysed. Mutations were found in: (i) the type 1 fimbrial operon; (ii) genes involved in the biosyn-thesis of extracellular polysaccharides including group I capsule, group II capsule and enterobacterial common antigen; (iii) genes involved in metabolic pathways; and (iv) genes with unknown function. Five of the genes identified are absent from the genome of the E. coli K-12 strain. Mutations in type 1 fimbrial genes resulted in severely attenuated colonization, even in the case of a mutant with an insertion upstream of the fim operon that affected the rate of fimbrial switching from the 'off' to the 'on' phase. Three mutants had insertions in a new type II capsule biosynthesis locus on a pathogenicity island and were impaired in the production of capsule in vivo. An additional mutant with an insertion in wecE was unable to synthesize enterobacterial common antigen. These results confirm the pre-eminence of type 1 fimbriae, establish the importance of extracellular polysaccharides in the pathogenesis of UTI and identify new urovirulence determinants.