Molecular evaluation of Brazilian vaginal lactobacilli microbiota and the role of hydrogen peroxide-producing isolates
Abstract number: P919
Martinez R.C.R., Franceschini S.A., Patta M.C., Quintana S.M., Nunes A.C., Moreira J.L., Anukam K.C., Reid G., De Martinis E.C.P.
Objectives: To study vaginal lactobacilli populations by culture-dependent [polymerase chain reaction and amplified ribosomal DNA restriction analysis (PCR-ARDRA)] and culture-independent [PCR and denaturing gradient gel electrophoresis (PCR-DGGE)] methods, and to evaluate the ability of isolates to produce H2O2.
Methods: This study, approved by local Ethics, comprised three groups of women recruited in the city of Ribeirão Preto, São Paulo State, Brazil: 64 healthy, 68 diagnosed with vulvovaginal candidiasis (VVC) and 64 with bacterial vaginosis (BV). Samples from lateral vaginal wall were obtained with swabs, transferred to tubes containing physiological saline and decimally diluted. Dilutions were surface plated on de Man Rogosa Sharpe (MRS) agar and incubated for 48 h at 37°C (aerobiosis and anaerobiosis). Isolates characterised as Gram-positive rods, catalase and oxidase negative were identified by PCR-ARDRA and evaluated semi-quantitatively for H2O2 production (Merckoquant Peroxide Test; Germany). Swabs obtained from patients were also evaluated by PCR-DGGE for detection of lactobacilli.
Results: Lactobacilli were isolated from 87.5% of controls, 88.2% of subjects with VVC and 32.8% of women with BV using PCR-ARDRA, compared to 98.4%, 94.1% and 57.8% assessed by PCR-DGGE, respectively. By PCR-ARDRA, L. crispatus was the most prevalent isolate in control group (37.0%), followed by L. johnsonii (16.4%). In VVC patients' group, L. crispatus was also the most common isolate (35.9%) followed by L. jensenii (21.8%), whereas in the group of BV, L. gasseri (36.4%) and L. crispatus (18.2%) were more frequently found. According to PCR-DGGE, L. iners was the most prevalent species in all groups. Only L. crispatus was detected accordingly by both methods in the three groups (Kappa coefficient = 0.531). From healthy and VVC groups, 1.4% and 2.6% respectively of the lactobacilli isolates did not produce H2O2, whereas in the BV group, this rate raised to 31.8%. In both healthy and VVC groups, L. crispatus and L. johnsonii produced, comparatively, the highest average amounts of H2O2.
Conclusions: The evaluation o vaginal microbiota was dependent on the methodology employed and the results also suggest the absence of H2O2-producing lactobacilli in the vaginal microbiota may be a contributing factor for the development of BV, whereas the presence of these isolates may not be protective against VVC.
Financial support: FAPESP # 04/145800 and # 06/065952; CAPES # 6159/062
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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