رسائل دكتوراة
Arabic
2017
Bacteriological and Molecular study of Gardnerella vaginalis association with preterm labor
ازل علاء عباس الربيعي, الهام عباس بنيان , لميس عبد الرزاق عبد اللطيف
كلية الطب-جامعة بابل · العراق
In this study, one hundred fifty samples were collected from patients with preterm labor (PTL), who had been attending Babylon Maternity and Pediatric Hospital and Al-Hilla Teaching Hospital, during the period from February to October 2016. Two swabs were collected: one for culturing and the other for direct extraction for isolation of Gardnerella vaginalis.
Out of the 150 samples, only 6 (4%) were positive by culture and 30 (20%) were positive at the molecular level, isolated from cases of preterm labor caused by bacterial vaginosis, urinary tract infection, and women with recurrent abortions. Laboratory diagnosis and biochemical tests confirmed that only 6 isolates belonged to G. vaginalis, which were further confirmed by the Vitek 2 system and molecular detection using specific primers.
Preterm birth is one of the most common causes of neonatal morbidity and mortality, and it is associated with subsequent preterm labor in up to 40% of cases, as shown in our results. The isolates varied in their ability to produce siderophores and extracellular protease, in addition to their hemolytic activity. Adherence activity was investigated and indicated that G. vaginalis has pili on its surface, which help it attach and initiate infection. Metronidazole was effective in preventing adherence at a low concentration (5%), achieving 100% inhibition.
All isolates were able to produce biofilm at different levels, which represents a key step in pathogenesis as it allows the bacteria to evade and stimulate the immune response and resist antibiotics. Alum, sodium chloride, and sodium bicarbonate were shown to be highly effective in eradicating G. vaginalis biofilm (0% survival), whereas thymol extract (carvacrol) was moderately effective, giving a 50% reduction in biofilm. Results also revealed that an increased pH provides an opportunity for the overgrowth of G. vaginalis.
The results indicated that lactic acid acts as a limiting factor preventing the growth of G. vaginalis. When grown with lactic acid, measured by optical density (OD), the highest production level was 1.901 at 2% concentration, whereas the lowest level was 0.32 at 10% concentration. The isolates also had the ability to produce a wide spectrum of bacteriocins, which inhibited the growth of both Gram-positive and Gram-negative bacteria found in the vaginal environment. Staphylococcus saprophyticus was the most inhibited bacterium.
Extracted vaginolysin exhibited complete hemolytic activity at different temperatures (37–38–40–42°C), and its activity increased with higher temperatures. Recent studies clarified that purified vaginolysin is active in the presence of cholesterol in the medium, giving a growth rate of 1.205 measured by optical density at 100% concentration. Vaginolysin is a cholesterol-dependent cytolysin (CDC) toxin. Antimicrobial activity of the extracted vaginolysin was evaluated based on the inhibition zone, showing that Candida species were the most affected among tested microorganisms (1.3 mm inhibition).
Some virulence factors were detected at the molecular level (Vly, SI, LuxS, LexA, recA, Pl, Fur, Omp, Gal, protease), which varied in production. This may be due to the absence of the gene in some bacteria, silent genes, or low bacterial quantity in the samples. Three antibiotics were examined at the molecular level (TetB), with varying resistance and sensitivity: 10 samples were resistant, RdxA was sensitive in all samples (100%), whereas the Erna gene was resistant in all samples (100%).
Results showed no sequence homology between Mycoplasma spp. and G. vaginalis at the adhesion level, which was detected using P30/P32 specific primers. The outcome of RFLP–PCR confirmed that G. vaginalis is virulent and plays a role in preterm labor by degrading membranes, associated with variable sialidase and β-galactosidase presence.
Mannose-binding lectin (MBL) levels have a direct role in pregnancy, playing an important role in defense against vaginal infection. MBL is essential for successful pregnancy regarding maternal defense against infection. Recent studies indicate that MBL deficiency is associated with adverse pregnancy outcomes. This study is considered the first study on MBL in Iraq.
DNA sequencing was performed for two main virulence factors: the phospholipase gene and quorum-sensing gene, which showed some variations and were recorded in NCBI Gene Sequencing as the first description in Iraq.