Objective
Conventional diagnosis of bacterial vaginosis contains some controversial points. To understand accurately the relationship between clinical stages and the microbiotas, the intravaginal microbial flora was analyzed by the clone library method.
Study Design
Vaginal fluid samples from 31 patients were examined. Lactobacillary grade, Nugent score, culture-based method, and clone library analysis using the 16SrRNA gene sequencing were performed and were compared with each other.
Results
Patients were categorized by Lactobacillary grade as I (normal) (n = 6), II (intermediate) (n = 11), and III (bacterial vaginosis) (n = 14). The clone library analysis detected 36 bacterial genera and 60 species from all 31 samples. A principal component analysis of the microbial proportions revealed a novel classification, which suggested the significance of the relative ratio of Lactobacillus iners , Atopobium vaginae and anaerobes in bacterial vaginosis.
Conclusion
Clone library analysis in combination with the conventional method provides substantial information for diagnosis of bacterial vaginosis.
During the last decade, there has been an increase of interest in bacterial vaginosis (BV) because of the reports of the adverse effects of this disorder such as preterm birth, pelvic inflammatory disease and postpartum endometritis. In addition, several publications reported that altered vaginal microflora are linked to an increased susceptibility to HIV and other sexually transmitted infectious agents such as Neisseria gonorrhoeae and Chlamydia trachomatis. BV is considered to have an abnormal intravaginal microflora that consists of diminished Lactobacilli and predominant BV-related bacteria, mainly, anaerobes. Gardnerella vaginalis, Mobilluncus, Prevotella, Porphyromonas, Bacteroides , and Mycoplasma species are thought to be the causative microorganisms of BV. The culture-based microbial detection method of the vaginal fluid is performed for the detection of the causative agents of intravaginal infections. However, the disadvantage of using a culture-based approach for bacterial identification is that it is dependent on the growth conditions being favorable to a select group of known microorganisms. Thus, “uncultivated” or “difficult to cultivate” bacteria will not be found if the culture conditions are not adequate.
BV is diagnosed by clinical symptoms (Amsel criteria ), Nugent score by Gram stain, and/or Lactobacillary grade (LAC grade) by wet mount smear. BV diagnosis contains some controversial points, for example: (1) Amsel criteria contains subjective elements and are poorly reproducible, (2) Nugent score is based on bacterial morphotypes and those ratios by Gram stain, which are not objective and fastidious because morphology and stainability are changeable depending on the environment. In addition, the meaning of “intermediate group” is ambiguous. Menard et al, reported that 57% of the intermediate group had a BV profile, (3) the culture-dependent microbial detection method is not good at detecting some bacterial species, especially anaerobic bacteria. Moreover, it takes at least 1 week to obtain the results, and some bacteria are often misevaluated.
Recently, some molecular methods using broad-range polymerase chain reactions (PCRs) of 16S rRNA gene have been applied to clinical practice to detect the causative agents of infectious diseases. Theoretically, this method is designed to detect most causative bacteria. This can help clinicians to detect uncultivable bacteria caused by cultural conditions and empirical antibiotic treatments. In addition, they will be able to identify novel pathogens that have never been recognized as a real etiologic agent. Some studies reported that a unique microbial flora existed in the vagina. However, no papers have ever compared the clone library method with conventional methods and each diagnostic tools requires that it be validated for evidence. In this article, we compare the results of clone library analysis with those of conventional methods and a relationship with BV is estimated by a principal component analysis.
Materials and Methods
Study population and specimen sampling
From October 2005 to August 2006, the samples of a 100 Japanese women from our university hospital were collected and a total of 31 patients were eligible with all data to be enrolled in this study. The ages of the cases were 20-53 years old and they did not receive any antibiotic treatment or other medication before sample collection that could have affected the study results. The sampling timing during menstrual cycle was not standardized in this study. Written informed consent was obtained from all participants in this prospective study, which was approved by the institutional review board at the University of Occupational and Environmental Health.
An unmoistened speculum was inserted before any other vaginal examination and vaginal pH was measured by use of color strips with a pH range of 3.6–6.1 (pH-Fix 3.6–6.1; Macherey-Nagel, Duren, Germany). The pH strip was placed on the lateral vaginal wall until wet, the color change of the strip was immediately compared with the colorimetric scale and the measurement was recorded. After making saline wet mount slides of vaginal fluid, specimens were collected by first inserting a sterile speculum into the vagina and wiping the vaginal discharge from the posterior fornix with sterile cotton swabs. The swabs were placed into an aerobic transport gel (Transwab, Medical Wire & Equipment Co. Ltd., Corsham, Wilts, England) and an anaerobic transport vial for up to 6 hours before assessment of microbial activity. The swabs were then rolled onto a glass slide for Gram staining to evaluate the Nugent score and to detect clue cells.
Lactobacillary grade (LAC grade)
All wet mount slides were evaluated for LAC grade and were classified according to Donders’ criteria. LAC grade I (normal flora) corresponds predominantly to Lactobacillary morphotypes, with very few coccoid bacteria present. LAC grade II (intermediate flora) corresponds to a diminished lactobacillary flora, which is mixed with other bacteria. LAC grade II was subdivided into 2 categories: one in which there was still a large amount of lactobacilli present (IIa), and a second group in which the coccoid flora outnumbered the lactobacillary flora (IIb). Finally, LAC grade III (BV) flora consists of numerous other bacteria, with no lactobacilli present. This grading system was chosen because of its simple and quick response, and its usefulness.
Grading of Nugent score and culture-based bacterial identification
Gram stained slides were examined for the presence of BV in accordance with standardized scoring criteria (Nugent score) by 1 trained technician who was blind to the origin of the slides and to the culture results, and reviewed by the author. BV was diagnosed if the score was 7-10; a score of 4-6 indicated intermediate vaginal flora; and a score of 0-3 indicated normal vaginal flora. Swabs of vaginal fluid were inoculated onto Drigalski agar medium, egg yolk salt agar medium, sheep blood agar medium (Nissui Pharmaceutical Co., Ltd., Tokyo), Desoxycholate agar (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan), CHROM agar Candida for aerobic culture (Kanto Chemical Co., Inc., Tokyo, Japan), Thayer Martin agar (Becton, Dickinson and Company, Franklin Lakes, NJ), Gardnerella agar for CO 2 culture (self-made), and Brucella HK agar for anaerobic culture (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan). Semiquantitation of 1+, 2+, and 3+ means the colonies occupied one-third, one-half, and the entire area of the agar plate, respectively. The 3+ growth of Lactobacilli indicates that it is a 10 7 colony forming unit.
Clone library method of broad-range 16S rRNA gene sequencing
For the clone library method, vaginal fluid was collected by the same method and was placed in a transport vial. Vaginal swabs were vigorously agitated in 2.0 mL Tris-HCl buffer (100 mM Tris-HCl, 50 mM EDTA-2Na, [pH 8.0]) to dislodge the cells. After treatment with a vortex mixer for 2 minutes, 900 μL aliquot of the cell suspension was transferred into a 2.5 mL polypropylene-tube. One hundred μL of 30% sodium dodecylsulfate (SDS, final concentration 3.0%) solution was added to the cell solution and approximately 0.3 g of a mixture of glass beads that consisted of equal weights of (a) 0.1 mm- and (b) 1 mm-diameter beads. The mixture was then vigorously shaken at 4500 rpm for 5 minutes on a MicroSmash MS-100 (Tomy Seiko Co., Ltd., Tokyo, Japan) and the supernatant was collected by centrifugation at 20,000 × g for 5 minutes at room temperature. This DNA extraction was carried out 3 times to improve a yield of DNA. The 3 supernatants were combined and treated with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1, vol/vol). The DNA in aqueous phase was concentrated and replaced by about 30 μL TE buffer using Montage PCR Centrifugal Filter Devices (Millipore, Bedford, MA). Using the extracted DNA as a template, the partial 16S rRNA gene fragments (approximately 550 base pair) were amplified by PCR method with a pair of universal primers (341F; 5′-CCTACGGGAGGCAGCAG-3′ and 907R; 5′-CCGTCAATTCMTTTRAGTTT-3′). Cycling conditions were as follows: 96C for 5 minutes, followed by 30 cycles of 96C for 30 seconds, 53C for 30 seconds and 72C for 1 minute, with final elongation at 72C for 7 minutes with a Gene Amp PCR system 9700 thermocycler (Applied Biosystems, Foster City, CA). The amplified products were cloned into Escherichia coli using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Nucleotide sequences of the randomly chosen 96 clones were determined using the Big Dye Terminator v3.1 cycle sequencing kit with ABI3130 sequencer (Applied Biosystems). Homology searches were performed with the basic local alignment search tool (BLAST) using an inhouse software system and database, which collected type strains from Ribosome Database Project II ( http://rdp.cme.msu.edu/ ). The species was identified with more than 95% similarity to the nucleotide sequence of the type strain.
Principal component analysis (PCA)
PCA analysis was performed by using the proportions of bacterial species level obtained by the clone library method to characterize intravaginal microbial flora. R software version 2.9.1. ( http://cran.r-project.org/ ) was used for the PCA software.
Results
A comparison between intravaginal bacterial flora analyzed by our clone library method and conventional methods
Table 1 shows age of the cases, the results of vaginal pH, Nugent score, and LAC grade of 31 cases. They were categorized by LAC grade: 6, 11, and 14 as normal (LAC grade I), intermediate (LAC grade IIa/IIb), and BV groups (bacterial vaginosis: LAC grade III). Table 1 also shows the results of our clone library analysis based on the nucleotide sequences of the 16S rRNA gene of the vaginal fluids. A total of 2759 clones (mean 89 clones per sample) were analyzed. The percentage ratios of each bacterial species of the total tested clones of each sample were determined and mapped. All cases of the normal group contained only Lactobacilli . In contrast, the intermediate, and the BV groups contained a wide variety of microbial flora. Focusing on Lactobacilli , L crispatus was identified in all cases of the normal group as the dominant species and also in 4 cases of the intermediate group, whereas none appeared in the BV group. L iners was not detected in the normal group. Thirty bacterial species in the intermediate group and 32 species in the BV group in addition to Lactobacillus spp were detected. Most of the anaerobes (ie, Atopobium vaginae, Dialister invisus, Gardnerella vaginalis, Veillonella dispar, Prevotella bivia, P buccalis, Clostridium akagii , and Gallicola barnesae) were detected in the intermediate and the BV group.
| Normal group | Intermediate group | Bacterial vaginosis group | |||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Case no. | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 |
| Age | 37 | 31 | 35 | 36 | 25 | 32 | 34 | 37 | 43 | 29 | 27 | 28 | 31 | 38 | 53 | 35 | 35 | 20 | 20 | 20 | 24 | 25 | 29 | 29 | 29 | 26 | 32 | 29 | 32 | 35 | 35 |
| Vaginal pH | 3.6 | 3.6 | 3.6 | 4.1 | 5 | 4.1 | 4.1 | 4.1 | 3.6 | 4.7 | 4.4 | 4.7 | 5 | 4.7 | 5 | 5 | 5 | 5 | 4.4 | 5 | 5 | 6.1 | 6.1 | 5 | 5 | 5 | 4.7 | 5 | 5 | 5 | 5 |
| Nugent score | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 3 | 0 | 0 | 1 | 4 | 4 | 5 | 4 | 6 | 8 | 4 | 8 | 8 | 8 | 8 | 7 | 8 | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
| LAC grade | I | I | I | I | I | I | IIa | IIa | IIa | IIa | IIa | IIa | IIa | IIa | IIa | IIb | IIb | III | III | III | III | III | III | III | III | III | III | III | III | III | III |
| Detected bacterial species and proportions by clone library method | |||||||||||||||||||||||||||||||
| Lactobacillus casei (L. cas) | 1 | ||||||||||||||||||||||||||||||
| Lactobacillus coleohominis (L col) | 9 | 1 | |||||||||||||||||||||||||||||
| Lactobacillus crispatus (L. cri) | 100 | 100 | 100 | 100 | 100 | 95 | 10 | 78 | 6 | 64 | |||||||||||||||||||||
| Lactobacillus delbrueckii (L. del) | 91 | ||||||||||||||||||||||||||||||
| Lactobacillus fornicalis (L. for) | 1 | ||||||||||||||||||||||||||||||
| Lactobacillus iners (L. ine) | 97 | 89 | 14 | 71 | 96 | 82 | 67 | 80 | 67 | 28 | 16 | 8 | 6 | ||||||||||||||||||
| Lactobacillus gasseri (L. gas) | 89 | 98 | 7 | ||||||||||||||||||||||||||||
| Lactobacillus jensenii (L. jen) | 7 | ||||||||||||||||||||||||||||||
| Lactobacillus mucosae (L. muc) | 3 | 2 | |||||||||||||||||||||||||||||
| Lactobacillus oris (L. ori) | 1 | ||||||||||||||||||||||||||||||
| Lactobacillus pontis (L. pon) | 5 | ||||||||||||||||||||||||||||||
| Lactobacillus rhamnosus (L. rha) | 1 | ||||||||||||||||||||||||||||||
| Achromobacter piechaudii (A. pie) | 2 | ||||||||||||||||||||||||||||||
| Aerococcus christensenii (A. chr) a | 1 | 6 | 2 | 1 | 1 | 1 | 1 | ||||||||||||||||||||||||
| Anaerococcus prevotii (A. pre) | 11 | 1 | 2 | 1 | 1 | ||||||||||||||||||||||||||
| Arcanobacterium hippocoleae (A. hip) | 2 | ||||||||||||||||||||||||||||||
| Atopobium vaginae (A. vag) | 6 | 4 | 10 | 7 | 6 | 5 | 8 | 24 | 25 | 48 | 74 | 21 | 72 | 18 | 26 | ||||||||||||||||
| Capnocytophaga ochracea (C. och) a | 4 | ||||||||||||||||||||||||||||||
| Clostridium akagii (C. aka) | 17 | 12 | 15 | 4 | 14 | 22 | 27 | ||||||||||||||||||||||||
| Clostridium hastiforme (C. has) | 4 | ||||||||||||||||||||||||||||||
| Clostridium novyi (C. nov) | 2 | 2 | |||||||||||||||||||||||||||||
| Clostridium polysaccharolyticum (C. pol) | 2 | 1 | |||||||||||||||||||||||||||||
| Corynebacterium accolens (C. acc) a | 1 | ||||||||||||||||||||||||||||||
| Desulfitobacterium chlororespirans (D. chl) | 1 | ||||||||||||||||||||||||||||||
| Dialister invisus (D. inv) | 2 | 3 | 1 | 1 | 2 | 1 | 1 | 1 | 2 | 2 | |||||||||||||||||||||
| Dialister pneumosintes (D. pne) | 1 | ||||||||||||||||||||||||||||||
| Eggerthella lenta (E. len) | 1 | ||||||||||||||||||||||||||||||
| Enterococcus faecalis (E. fae) | 9 | ||||||||||||||||||||||||||||||
| Eubacterium brachy (E. bra) | 1 | ||||||||||||||||||||||||||||||
| Fusobacterium nucleatum (F. nuc) | 90 | ||||||||||||||||||||||||||||||
| Gallicola barnesae (G. bar) | 1 | 2 | 15 | 2 | 5 | 1 | 2 | 2 | |||||||||||||||||||||||
| Gardnerella vaginalis (G. vag) | 7 | 5 | 10 | 1 | 1 | 1 | 4 | 1 | 1 | 3 | 9 | 11 | |||||||||||||||||||
| Gemella bergeri (G. ber) | 1 | ||||||||||||||||||||||||||||||
| Gemella palaticanis (G. pal) | 2 | 1 | 13 | 8 | |||||||||||||||||||||||||||
| Kingella denitrificans (K. den) a | 1 | ||||||||||||||||||||||||||||||
| Kingella kingae (K. kin) a | 1 | ||||||||||||||||||||||||||||||
| Leptotrichia buccalis (L. buc) | 1 | ||||||||||||||||||||||||||||||
| Megasphaera micronuciformis (M. mic) | 2 | 1 | |||||||||||||||||||||||||||||
| Mitsuokella jalaludinii (M. jal) | 2 | ||||||||||||||||||||||||||||||
| Mycoplasma hominis (M. hom) a | 1 | 3 | 1 | 2 | |||||||||||||||||||||||||||
| Neisseria flavescens (N. fla) a | 1 | ||||||||||||||||||||||||||||||
| Nitropropanol-degrading bacterium NPOH1 (NPH1) | 1 | 1 | 4 | 6 | 5 | 1 | 2 | 4 | |||||||||||||||||||||||
| Olsenellaprofusa (O. pro) | 1 | ||||||||||||||||||||||||||||||
| Peptoniphilus harei (P. har) | 1 | 1 | 3 | ||||||||||||||||||||||||||||
| Peptoniphilus lacrimalis (P. lac) | 5 | ||||||||||||||||||||||||||||||
| Peptostreptococcus anaerobius (P. ana) | 6 | 1 | |||||||||||||||||||||||||||||
| Prevotella bivia (P.biv) | 11 | 1 | 5 | 20 | 8 | 3 | 7 | 1 | |||||||||||||||||||||||
| Prevotella buccalis (P. buc) | 1 | 17 | 11 | 6 | 3 | 7 | 19 | ||||||||||||||||||||||||
| Prevotella corporis (P. cor) | 2 | ||||||||||||||||||||||||||||||
| Prevotella oralis (P. ora) | 1 | ||||||||||||||||||||||||||||||
| Serratia marcescens (S. mar) | 33 | ||||||||||||||||||||||||||||||
| Staphylococcus caprae (S. cap) | 1 | ||||||||||||||||||||||||||||||
| Stenotrophomonas maltophilia (S. mal) | 1 | ||||||||||||||||||||||||||||||
| Streptobacillus moniliformis (S. mon) | 21 | 1 | 1 | 36 | 1 | 15 | 15 | 39 | 24 | 11 | |||||||||||||||||||||
| Streptococcus agalactiae (S. aga) a | 2 | ||||||||||||||||||||||||||||||
| Streptococcus intermedius (S. int) a | 91 | ||||||||||||||||||||||||||||||
| Tannerella forsythensis (T. for) | 4 | ||||||||||||||||||||||||||||||
| Tissierella praeacuta (T. pra) | 1 | ||||||||||||||||||||||||||||||
| Veillonella dispar (V. dis) | 17 | 9 | 5 | 10 | 7 | 1 | 21 | 1 | 15 | 3 | |||||||||||||||||||||
| Unclassified | 1 | ||||||||||||||||||||||||||||||
| Number of tested clones | 91 | 93 | 92 | 88 | 88 | 93 | 93 | 90 | 96 | 93 | 82 | 93 | 96 | 85 | 83 | 84 | 89 | 88 | 84 | 79 | 94 | 79 | 86 | 94 | 88 | 85 | 90 | 82 | 93 | 95 | 93 |
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