Indian Journal of Medical Research

ORIGINAL ARTICLE
Year
: 2019  |  Volume : 149  |  Issue : 1  |  Page : 57--61

Comparative analysis of virulence factors & biotypes of Gardnerella vaginalis isolated from the genital tract of women with & without bacterial vaginosis


Kumari Nisha1, Beena Antony1, Jeppu Udayalaxmi2,  
1 Department of Microbiology, Father Muller Medical College, Mangaluru, India
2 Department of Microbiology, Kasturba Medical College, Mangaluru, India

Correspondence Address:
Dr Beena Antony
Department of Microbiology, Father Muller Medical College, Kankanady P.O., Mangalore 575 002, Karnataka
India

Abstract

Background & objectives: Bacterial vaginosis (BV) involves the presence of a thick vaginal multispecies biofilm, where Gardnerella vaginalis is the predominant species. The reason for an increase in the number of G. vaginalis which are usually present as normal flora of the female genital tract in cases of BV, is not known. Hence, the objective of the present study was to compare the biotypes and virulence factors of G. vaginalis isolated from the genital tract of women with and without BV. Methods: High vaginal swabs collected from 811 women of reproductive age were cultured. G. vaginalis isolates were biotyped and tested for adherence to vaginal epithelial cells, biofilm formation, agglutination of human red blood cells (RBCs), protease production, phospholipase production and surface hydrophobicity. Results: Of the isolates from women with BV, 83.3 per cent (60/72) showed good adherence, 78.4 per cent (58/74) produced biofilm, 82.9 per cent (63/76) produced phospholipase, 67.1 per cent (51/76) produced protease, 77.3 per cent (58/75) were positive for surface hydrophobicity and 61.6 per cent (45/73) were positive for haemagglutination of human RBC. In case of G. vaginalis from non-BV women, 25 per cent (15/60) isolates showed good adherence, 18.4 per cent (9/49) biofilm production, 35 per cent (21/60) phospholipase, 36.6 per cent (22/60) protease, 41.7 per cent (25/60) surface hydrophobicity and 10.1 per cent (6/59) agglutination of human RBCs. Maximum number of isolates belonged to biotypes 6, 2 and 3. Biotype 3 was more associated with non-BV rather than BV; biotype 6, 2 and 1 were more associated with cases of BV. Maximum virulence factors were expressed by biotypes 6, 2 and 1. Interpretation & conclusions: Virulence factors were more expressed by G. vaginalis isolates obtained from women with BV rather than from non-BV. Biotypes 6, 2 and 1 were more associated with cases of BV and expressed maximum virulence factors.



How to cite this article:
Nisha K, Antony B, Udayalaxmi J. Comparative analysis of virulence factors & biotypes of Gardnerella vaginalis isolated from the genital tract of women with & without bacterial vaginosis.Indian J Med Res 2019;149:57-61


How to cite this URL:
Nisha K, Antony B, Udayalaxmi J. Comparative analysis of virulence factors & biotypes of Gardnerella vaginalis isolated from the genital tract of women with & without bacterial vaginosis. Indian J Med Res [serial online] 2019 [cited 2020 Apr 4 ];149:57-61
Available from: http://www.ijmr.org.in/text.asp?2019/149/1/57/256701


Full Text

Bacterial vaginosis (BV) is the most common genital tract infection in women of reproductive age group. BV is characterized by a marked reduction in counts of lactobacilli which is frequently present as normal flora of the healthy vagina and an increase in numbers of anaerobic bacteria, including Gardnerella vaginalis, Atopobium vaginae, Mobiluncus spp., Bacteroides spp., Prevotella spp. and various other anaerobes[1],[2],[3]. Being polymicrobial in nature, BV aetiology remains unclear. However, BV involves the presence of a thick vaginal multispecies biofilm, where G. vaginalis is the predominant species. Similar to what happens in many other biofilm-related infections, standard antibiotics like metronidazole are unable to fully eradicate the vaginal biofilm, which can explain the high recurrence rates of BV. BV is associated with a variety of obstetric and gynaecological complications such as preterm birth, low birth weight, postpartum endometritis and pelvic inflammatory disease. The factors which are associated with pathophysiology of BV are not completely known[1],[2],[3],[4],[5].

It is a known fact that the presence of virulence factors is one of the important determinants of the pathogenic potential of the organism. The various virulence factors of G. vaginalis are pili, microcapsule, surface hydrophobicity, adherence, vaginolysin, phospholipase C, protease and siderophores, sialidases and prolidases[6],[7]. The reason for an increase in the number of G. vaginalis which are usually present as normal flora of the female genital tract in cases of BV is not known. In the present study, the virulence factors and biotypes of G. vaginalis isolated from the genital tract of women with and without BV were compared.

 Material & Methods



This cross-sectional study was conducted in the department of Microbiology, Father Muller Medical College (FMMC), Mangaluru, India from January 2014 to December 2016. All consecutive women of 15-45 yr of age with complaints of vaginal discharge were included in the study. Women who were menstruating at the time of the specimen collection or were on medication for any bacterial, fungal, parasitic or viral infections for up to one month before the specimen collection were excluded. The study was approved by the Institutional Ethics Committee of FMMCH, Mangaluru, and written informed consents were obtained from the patients.

A detailed clinical history of each woman was taken, and their vaginal swabs were collected into sterile tube containing 0.5 ml normal saline and immediately transported to the microbiology laboratory. The vaginal swabs were subjected to wet mount preparation, Gram staining, p H determination and whiff test. BV cases were identified using Amsel's and Nugent's criteria[7],[8],[9]. Sample was inoculated onto human blood bilayer Tween 80 agar (Columbia blood agar base with 0.0075% Tween 80) with G. vaginalis selective supplement containing gentamicin sulphate 2 mg, nalidixic acid 15 mg, amphotericin B 1 mg (HiMedia Laboratories, Pvt. Ltd., Mumbai, India) and chocolate agar. The culture plates were incubated at 37°C for 48 h in candle jar which provided 5-10 per cent CO2. Colonies of G. vaginalis appeared small, smooth, round and β-haemolytic. G. vaginalis was identified as Gram-variable coccobacilli by Gram stain, catalase and oxidase negative, fermented glucose, maltose and starch and did not ferment mannitol, lactose, xylose and sucrose variable, α glucosidase positive and β glucosidase negative[10],[11]. The isolates were also tested for various virulence factors such as adherence to vaginal epithelial cells, biofilm formation, surface hydrophobicity, haemagglutination, phospholipase and protease production. All the tests were performed in triplicate. G. vaginalis ATCC 14018 was included as control with each test.

Evaluation of virulence factors: Vaginal swabs were collected from healthy asymptomatic women in the reproductive age group, and the vaginal discharge was eluted into 2 ml of 0.85 per cent sterile saline. The adherence of the isolates to these vaginal epithelial cells was determined[12]. An average of >10 adherent bacilli/cell was considered as good degree of adherence[7],[12]. Haemagglutination assay was performed according to Scott et al[13]. The suspension giving a carpet of erythrocytes over the bottom of the well was considered as positive. The same test was done using 2 per cent human, sheep and chick red blood cells (RBCs)[13]. Biofilm formation in microtiter plates was determined as done in previous studies[14],[15]. The biofilm formation was graded as, optical density (OD) <0.1 as weak or non-biofilm producers, OD 0.1-0.2 as moderate and an OD >0.2 as good biofilm producer. Pseudomonas aeruginosa ATCC 27853 was used as a positive control[14],[15]. For protease production G. vaginalis isolates were inoculated into skim milk agar with 5 per cent horse serum and incubated in the candle jar (5-10% CO2) at 37°C for 48 h. Clear zone around the growth indicated protease enzyme activity[16]. Brain heart infusion (BHI) agar supplemented with 1 per cent gelatin egg yolk was used for phospholipase production. G. vaginalis isolates were inoculated and incubated in the candle jar (5-10% CO2) at 37°C for 48 h. Pearly layer was seen around growth in lipase-positive organism. P. aeruginosa (ATCC 27853) and Staphylococcus aureus (25923) were used as negative reference strains[17]. Surface hydrophobicity of the isolates was determined as given in the previous studies[6],[18]. Surface hydrophobicity index ≤20 was graded as low and >20 as high. Biotyping was done using three tests - hippurate hydrolysis, phospholipase and O-Nitrophenyl β-D-galactopyranoside (ONPG) as described previously[7],[19].

Statistical analysis: Statistical analysis was done using SPSS version 16 (IBM, Chicago, USA). The various biotypes and virulence factors of G. vaginalis from women with or without BV were compared using Chi-square test.

 Results & Discussion



A total of 176 isolates of G. vaginalis were obtained from 811 women with abnormal vaginal discharge. According to Nugent's criteria, vaginal samples were classified into three groups. Group A consisted of 75 women with Nugent score 4-6 (not consistent with BV by Amsel's criteria). These were considered as non-BV cases. Group B included 89 women (Nugent score ≥7 and BV case by Amsel). These were considered as BV cases. Group C included 12 women with Nugent score 0-3 (non-BV case by Amsel) having other vaginal infections such as vulvovaginitis (n=6), cervicitis (n=4) and HIV reactive (n=2). These were also considered as non-BV cases. There were 23 women with Candida in group A, 49 in group B, and eight in group C. As group C has only 12 women, all comparisons were made between groups A (non-BV) and B (BV). [Table 1] shows the virulence factors expressed by the three groups.{Table 1}

A study proposed a conceptual model to explain the pathogenesis of BV[20]. In our study most isolates of G. vaginalis obtained from BV cases exhibited various virulence factors such as adherence to vaginal epithelial cells, biofilm formation, phospholipase C production, protease production, surface hydrophobicity and haemagglutination rather than the non-BV cases [Table 1].

According to a previous study, G. vaginalis was the first isolate to adhere to the vaginal epithelial cells causing the scaffolding to which other organism adhere[21]. However, another study reported that adherence of G. vaginalis was inhibited by the lactobacilli and adherent lactobacilli were displaced by G. vaginalis[22]. Other groups studied the influence of biofilm formation by G. vaginalis and other anaerobes from the time of their initial adhesion until biofilm formation in BV cases. It was reported that the synergistic interaction occurred between G. vaginalis and anaerobes from commensal status till biofilm formation[23],[24]. G. vaginalis was shown to have the highest virulence potential due to higher initial adhesion, cytotoxicity and the ability to form a biofilm[25].

Past studies have reported difference in genotype and in vitro expression of virulence factors between BV and non-BV isolates of G. vaginalis[26],[27]. The BV-associated isolate encoded a different variant of a biofilm-associated protein gene and demonstrated greater adherence, aggregation and biofilm formation than that isolated from healthy vagina[26]. In our study also BV-associated isolates showed better adherence, biofilm formation, haemagglutination, phospholipase and protease production in comparison to non-BV isolates. In our study, maximum number of isolates belonged to biotypes 6, 2 and 3. Biotype 3 was more associated with non-BV rather than BV [Table 2]; biotypes 6, 2 and 1 were more associated with cases of BV. Maximum virulence factors were expressed by biotypes 6, 2 and 1 [Table 3].{Table 2}{Table 3}

Our earlier study showed no relationship between biotypes and virulence factors or biotypes from BV and non-BV cases[7]. Another study showed that phospholipase-producing biotypes 1, 2, 3 and 4 were frequently associated with cases of BV rather than non-BV and also the patient acquired a different biotype after treatment[27]. In a longitudinal study on biotypes of G. vaginalis biotypes 2, 3 and 7 were more frequently isolated from cases of BV and biotype 7 was more isolated from non-BV cases[27]. In our study biotypes 6, 2 and 1 showed association with various virulence factors.

In conclusion, majority of G. vaginalis isolates from cases of BV exhibited more number of virulence factors than those from healthy women. Biotype 3 was more prevalent in non-BV cases while biotypes 6, 2 and 1 were associated with cases of BV and expressed maximum virulence factors.

Acknowledgment: Authors acknowledge the help provided by Dr Annie Rajaratnam and other staff of the department of Obstetrics and Gynaecology working in Father Muller Hospital in the collection of vaginal discharge samples.

Financial support & sponsorship: None.

Conflicts of Interest: None.

References

1Machado D, Castro J, Palmeira-de-Oliveira A, Martinez-de-Oliveira J, Cerca N. Bacterial vaginosis biofilms: Challenges to current therapies and emerging solutions. Front Microbiol 2015; 6 : 1528.
2Hardy L, Jespers V, Dahchour N, Mwambarangwe L, Musengamana V, Vaneechoutte M, et al. Unravelling the bacterial vaginosis-associated biofilm: A multiplex Gardnerella vaginalis and Atopobium vaginae fluorescence in situ hybridization assay using peptide nucleic acid probes. PLoS One 2015; 10 : e0136658.
3Huang B, Fettweis JM, Brooks JP, Jefferson KK, Buck GA. The changing landscape of the vaginal microbiome. Clin Lab Med 2014; 34 : 747-61.
4Menard JP, Mazouni C, Salem-Cherif I, Fenollar F, Raoult D, Boubli L, et al. High vaginal concentrations of Atopobium vaginae and Gardnerella vaginalis in women undergoing preterm labor. Obstet Gynecol 2010; 115 : 134-40.
5van Oostrum N, De Sutter P, Meys J, Verstraelen H. Risks associated with bacterial vaginosis in infertility patients: A systematic review and meta-analysis. Hum Reprod 2013; 28 : 1809-15.
6Cauci S, Culhane JF, Di Santolo M, McCollum K. Among pregnant women with bacterial vaginosis, the hydrolytic enzymes sialidase and prolidase are positively associated with interleukin-1beta. Am J Obstet Gynecol 2008; 198 : 132.e1-7.
7Udayalaxmi J, Bhat GK, Kotigadde S. Biotypes and virulence factors of Gardnerella vaginalis isolated from cases of bacterial vaginosis. Indian J Med Microbiol 2011; 29 : 165-8.
8Amsel R, Totten PA, Spiegal CA, Chen KC, Eschenbach D, Holmes KK. Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiological associations. Am J Med 1983; 74 : 14-22.
9Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a standardised method of gram stain interpretation. J Clin Microbiol 1991; 29 : 297-301.
10Winn WC, Allen SD, Janda WM, Koneman EW, Procop GW, Schreckenberger PC, et al., editors. Aerobic and facultative Gram-positive bacilli. In: Koneman's color atlas and textbook of diagnostic microbiology, 6th ed., Philadelphia: Lippincott Williams & Wilkins; 2006. p. 834-8.
11Piot P, Van Dyck E, Totten PA, Holmes KK. Identification of Gardnerella (Haemophilus) vaginalis. J Clin Microbiol 1982; 15 : 19-24.
12Peeters M, Piot P. Adhesion of Gardnerella vaginalis to vaginal epithelial cells: Variables affecting adhesion and inhibition by metronidazole. Genitourin Med 1985; 61 : 391-5.
13Scott TG, Smyth CJ, Keane CT. In vitro adhesiveness and biotype of Gardnerella vaginalis strains in relation to the occurrence of clue cells in vaginal discharges. Genitourin Med 1987; 63 : 47-53.
14Mathur T, Singhal S, Khan S, Upadhyay DJ, Fatma T, Rattan A, et al. Detection of biofilm formation among the clinical isolates of staphylococci: An evaluation of three different screening methods. Indian J Med Microbiol 2006; 24 : 25-9.
15Suman E, Singh S, Kotian S. Pseudomonas aeruginosa biofilms in hospital water systems and the effect of sub-inhibitory concentration of chlorine. J Hosp Infect 2008; 70 : 199-201.
16Folasada MO, Joshua OA. Production dynamics of extracellular protease from Bacillus species. Afr J Biotechnol 2005; 4 : 776-9.
17McGregor JA, Lawellin D, Franco-Buff A, Todd JK. Phospholipase C activity in microorganisms associated with reproductive tract infection. Am J Obstet Gynecol 1991; 164 : 682-6.
18Rosenberg M, Gubnick D, Rosenberg E. Adherence of bacteria to hydrocarbons: A simple method for measuring surface hydrophobicity. FEMS Microbiol Lett 1980; 9 : 29-33.
19Piot P, Van Dyck E, Peeters M, Hale J, Totten PA, Holmes KK, et al. Biotypes of Gardnerella vaginalis. J Clin Microbiol 1984; 20 : 677-9.
20Schwebke JR, Muzny CA, Josey WE. Role of Gardnerella vaginalis in the pathogenesis of bacterial vaginosis: A conceptual model. J Infect Dis 2014; 210 : 338-43.
21Verstraelen H, Swidsinski A. The biofilm in bacterial vaginosis: Implications for epidemiology, diagnosis and treatment. Curr Opin Infect Dis 2013; 26 : 86-9.
22Castro J, Henriques A, Machado A, Henriques M, Jefferson KK, Cerca N, et al. Reciprocal interference between Lactobacillus spp. and Gardnerella vaginalis on initial adherence to epithelial cells. Int J Med Sci 2013; 10 : 1193-8.
23Machado A, Cerca N. Influence of biofilm formation by Gardnerella vaginalis and other anaerobes on bacterial vaginosis. J Infect Dis 2015; 212 : 1856-61.
24Castro J, Cerca N. BV and non-BV associated Gardnerella vaginalis establish similar synergistic interactions with other BV-associated microorganisms in dual-species biofilms. Anaerobe 2015; 36 : 56-9.
25Pybus V, Onderdonk AB. Evidence for a commensal, symbiotic relationship between Gardnerella vaginalis and Prevotella bivia involving ammonia: Potential significance for bacterial vaginosis. J Infect Dis 1997; 175 : 406-13.
26Harwich MD Jr., Alves JM, Buck GA, Strauss JF 3rd, Patterson JL, Oki AT, et al. Drawing the line between commensal and pathogenic Gardnerella vaginalis through genome analysis and virulence studies. BMC Genomics 2010; 11 : 375.
27Briselden AM, Hillier SL. Longitudinal study of the biotypes of Gardnerella vaginalis. J Clin Microbiol 1990; 28 : 2761-4.