More Than Meets the Eye: Associations of Vaginal Bacteria with Gram Stain Morphotypes Using Molecular Phylogenetic Analysis

Bacterial vaginosis (BV) is a highly prevalent condition associated with adverse health outcomes. Gram stain analysis of vaginal fluid is the standard for confirming the diagnosis of BV, wherein abundances of key bacterial morphotypes are assessed. These Lactobacillus, Gardnerella, Bacteroides, and Mobiluncus morphotypes were originally linked to particular bacterial species through cultivation studies, but no studies have systematically investigated associations between uncultivated bacteria detected by molecular methods and Gram stain findings. In this study, 16S-rRNA PCR/pyrosequencing was used to examine associations between vaginal bacteria and bacterial morphotypes in 220 women with and without BV. Species-specific quantitative PCR (qPCR) and fluorescence in Situ hybridization (FISH) methods were used to document concentrations of two bacteria with curved rod morphologies: Mobiluncus and the fastidious BV-associated bacterium-1 (BVAB1). Rank abundance of vaginal bacteria in samples with evidence of curved gram-negative rods showed that BVAB1 was dominant (26.1%), while Mobiluncus was rare (0.2% of sequence reads). BVAB1 sequence reads were associated with Mobiluncus morphotypes (p<0.001). Among women with curved rods, mean concentration of BVAB1 DNA was 2 log units greater than Mobiluncus (p<0.001) using species-specific quantitative PCR. FISH analyses revealed that mean number of BVAB1 cells was 2 log units greater than Mobiluncus cells in women with highest Nugent score (p<0.001). Prevotella and Porphyromonas spp. were significantly associated with the “Bacteroides morphotype,” whereas Bacteroides species were rare. Gram-negative rods designated Mobiluncus morphotypes on Gram stain are more likely BVAB1. These findings provide a clearer picture of the bacteria associated with morphotypes on vaginal Gram stain.

[1]  R. Desmond,et al.  A randomized trial of the duration of therapy with metronidazole plus or minus azithromycin for treatment of symptomatic bacterial vaginosis. , 2007, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[2]  M. Collins,et al.  Proposal To Restrict the Genus Bacteroides (Castellani and Chalmers) to Bacteroides fragilis and Closely Related Species , 1989 .

[3]  P. Nyirjesy,et al.  The Effects of Intravaginal Clindamycin and Metronidazole Therapy on Vaginal Mobiluncus Morphotypes in Patients With Bacterial Vaginosis , 2007, Sexually transmitted diseases.

[4]  K. Holmes,et al.  Comparison of gram stain, DNA probe, and culture for the identification of species of Mobiluncus in female genital specimens. , 1985, The Journal of infectious diseases.

[5]  D R Hoover,et al.  Bacterial vaginosis and disturbances of vaginal flora: association with increased acquisition of HIV , 1998, AIDS.

[6]  A. Zeileis,et al.  Regression Models for Count Data in R , 2008 .

[7]  S. Garland,et al.  High recurrence rates of bacterial vaginosis over the course of 12 months after oral metronidazole therapy and factors associated with recurrence. , 2006, The Journal of infectious diseases.

[8]  C. Quince,et al.  Dirichlet Multinomial Mixtures: Generative Models for Microbial Metagenomics , 2012, PloS one.

[9]  P. Gajer,et al.  Vaginal microbiome of reproductive-age women , 2010, Proceedings of the National Academy of Sciences.

[10]  M. Vaneechoutte,et al.  Cloning of 16S rRNA genes amplified from normal and disturbed vaginal microflora suggests a strong association between Atopobium vaginae, Gardnerella vaginalis and bacterial vaginosis , 2004, BMC Microbiology.

[11]  Kevin W Eliceiri,et al.  NIH Image to ImageJ: 25 years of image analysis , 2012, Nature Methods.

[12]  J. Marrazzo,et al.  Temporal Variability of Human Vaginal Bacteria and Relationship with Bacterial Vaginosis , 2010, PloS one.

[13]  E. Virginia Armbrust,et al.  pplacer: linear time maximum-likelihood and Bayesian phylogenetic placement of sequences onto a fixed reference tree , 2010, BMC Bioinformatics.

[14]  R. Lillis,et al.  Quantitative PCR Assessments of Bacterial Species in Women with and without Bacterial Vaginosis , 2010, Journal of Clinical Microbiology.

[15]  K. Holmes,et al.  Diagnosis of bacterial vaginosis by direct gram stain of vaginal fluid , 1983, Journal of clinical microbiology.

[16]  James R. Cole,et al.  rrndb: the Ribosomal RNA Operon Copy Number Database , 2001, Nucleic Acids Res..

[17]  K. Holmes,et al.  Nonspecific vaginitis: Diagnostic criteria and microbial and epidemiologic associations , 1983 .

[18]  Mary Frances Cotch,et al.  Association between Bacterial Vaginosis and Preterm Delivery of a Low-Birth-Weight Infant , 1995 .

[19]  C. Ison,et al.  Validation of a simplified grading of Gram stained vaginal smears for use in genitourinary medicine clinics , 2002, Sexually transmitted infections.

[20]  J. Kendrick,et al.  The Prevalence of Bacterial Vaginosis in the United States, 2001–2004; Associations With Symptoms, Sexual Behaviors, and Reproductive Health , 2007, Sexually transmitted diseases.

[21]  Connie Celum,et al.  Bacterial Vaginosis Associated with Increased Risk of Female-to-Male HIV-1 Transmission: A Prospective Cohort Analysis among African Couples , 2012, PLoS medicine.

[22]  M A Krohn,et al.  Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation , 1991, Journal of clinical microbiology.

[23]  E. Bingen,et al.  Bacterial counts in cerebrospinal fluid of children with meningitis , 1990, European Journal of Clinical Microbiology and Infectious Diseases.

[24]  K. Holmes,et al.  Anaerobic bacteria in nonspecific vaginitis. , 1980, The New England journal of medicine.

[25]  H. Shah,et al.  Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides. , 1990, International journal of systematic bacteriology.

[26]  F. Dewhirst,et al.  Phylogeny of Bacteroides, Prevotella, and Porphyromonas spp. and related bacteria , 1994, Journal of bacteriology.

[27]  M. Vaneechoutte,et al.  Comparison between Gram stain and culture for the characterization of vaginal microflora: Definition of a distinct grade that resembles grade I microflora and revised categorization of grade I microflora , 2005, BMC Microbiology.

[28]  Mei-Cheng Wang,et al.  The Natural History of Bacterial Vaginosis Diagnosed by Gram Stain Among Women in Rakai, Uganda , 2011, Sexually transmitted diseases.

[29]  E. Holst,et al.  Anaerobic curved rods in genital samples of women. Performance of different selective media, comparison of detection by microscopy and culture studies, and recovery from different sampling sites. , 1984, Scandinavian journal of urology and nephrology. Supplementum.

[30]  Jeanne M. Marrazzo,et al.  Bacterial Communities in Women with Bacterial Vaginosis: High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria , 2012, PloS one.

[31]  M. Collins,et al.  Proposal for Reclassification of Bacteroides asaccharolyticus, Bacteroides gingivalis, and Bacteroides endodontalis in a New Genus, Porphyromonas , 1988 .

[32]  J. Marrazzo,et al.  Molecular identification of bacteria associated with bacterial vaginosis. , 2005, The New England journal of medicine.

[33]  J. Marrazzo,et al.  Changes in Vaginal Bacterial Concentrations with Intravaginal Metronidazole Therapy for Bacterial Vaginosis as Assessed by Quantitative PCR , 2009, Journal of Clinical Microbiology.

[34]  S. Acinas,et al.  Divergence and Redundancy of 16S rRNA Sequences in Genomes with Multiple rrn Operons , 2004, Journal of bacteriology.

[35]  E. Bukusi,et al.  In vitro inhibitory activity of human vaginal lactobacilli against pathogenic bacteria associated with bacterial vaginosis in Kenyan women. , 2010, Anaerobe.

[36]  J. Peipert,et al.  Efficacy of Clindamycin Vaginal Ovule (3-Day Treatment) vs. Clindamycin Vaginal Cream (7-Day Treatment) in Bacterial Vaginosis , 2001, Infectious diseases in obstetrics and gynecology.

[37]  Gregory B. Gloor,et al.  Deep Sequencing of the Vaginal Microbiota of Women with HIV , 2010, PloS one.