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BRIEF COMMUNICATION
Year : 2020  |  Volume : 38  |  Issue : 3  |  Page : 440-443
 

Characterisation of colonisation properties of vaginal lactobacilli from healthy Indian women: Implications for identification of potential probiotic candidates


ICMR-National AIDS Research Institute, 73 G Block, MIDC, Bhosari, Maharashtra, India

Date of Submission17-Mar-2020
Date of Decision24-May-2020
Date of Acceptance27-Jul-2020
Date of Web Publication4-Nov-2020

Correspondence Address:
Dr. Arati Mane
Division of Microbiology, ICMR-National AIDS Research Institute, 73 G Block, MIDC, Bhosari, Maharashtra - 411 026
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_20_108

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 ~ Abstract 


The ability of probiotic bacteria to colonise the target site is considered of utmost importance and used for preliminary screening of potential probiotic candidates. Thirty-eight vaginal Lactobacillus strains isolated from healthy women and characterised by 16S RNA sequencing were assessed for colonisation characteristics including adherence to human vaginal epithelial cells, auto-aggregation and hydrophobicity. No significant difference in adherence (P = 0.384), auto-aggregation (P = 0.282) and hydrophobicity (P = 0.084) abilities between Lactobacillus species was observed, however, significant difference in colonisation characteristics between strains of the same species was noted (P < 0.001). We identified seven Lactobacillus strains that may serve as best candidates for vaginal probiotic development.


Keywords: Adherence, auto-aggregation, cell surface hydrophobicity, probiotic, vaginal lactobacilli


How to cite this article:
Mane A, Khan I, Thakar M. Characterisation of colonisation properties of vaginal lactobacilli from healthy Indian women: Implications for identification of potential probiotic candidates. Indian J Med Microbiol 2020;38:440-3

How to cite this URL:
Mane A, Khan I, Thakar M. Characterisation of colonisation properties of vaginal lactobacilli from healthy Indian women: Implications for identification of potential probiotic candidates. Indian J Med Microbiol [serial online] 2020 [cited 2020 Nov 24];38:440-3. Available from: https://www.ijmm.org/text.asp?2020/38/3/440/299809





 ~ Introduction Top


The role of lactobacilli in maintenance of homeostasis within the vagina and in the prevention of sexually transmitted infections is well established.[1],[2] Studies have demonstrated that exogenously applied Lactobacillus-based probiotics reduce the risk of vaginal infections and hold promise to restore and maintain vaginal health.[3],[4] Research towards development of genetically engineered Lactobacillus-based probiotics is ongoing.

For successful administration of Lactobacillus-based probiotics to the vaginal niche, it is crucial to use indigenous Lactobacillus strains with optimal colonisation abilities to ensure desirable outcomes.[3] Adherence to the vaginal epithelial cells (VECs) and mucosal surfaces is a prerequisite for efficient colonisation.[5] Adherence, in turn, depends on the auto-aggregation ability of the strain and the physiochemical characteristics of cell surface such as hydrophobicity and electrical charge.[5],[6]

We had previously isolated Lactobacillus strains from the vaginal ecosystem of healthy Indian women, and in the present study, we characterised their colonisation properties, including adherence to VECs, auto-aggregation and cell surface hydrophobicity, in order to shortlist candidates for potential probiotic development.


 ~ Methods Top


The study was approved by the Institutional Ethics Committee of Indian Council of Medical Research-National AIDS Research Institute, Pune, India (NARI/CEM-CV/14-15/20).

Lactobacillusstrains

A total of 38 Lactobacillus strains belonging to nine different species isolated from vaginas of healthy women recruited in a previous study were used in the present study.[7] The strains were characterised by 16S RNA sequencing for species identification. The strains were Lactobacillus crispatus (n = 11; LC1 to LC11), Lactobacillus gasseri (n = 13; LG1 to LG13), Lactobacillus jensenii (n = 5; LJ1 to LJ5), Lactobacillus vaginalis (n = 4; LV1 to LV4), Lactobacillus mucosae (n = 1; LM), Lactobacillus reuteri (n = 1; LR), Lactobacillus fermentum (n = 1; LF), Lactobacillus oris (n = 1; LO) and Lactobacillus delbrueckii (n = 1; LD). Hydrogen peroxide (H2O2) production and pH of culture supernatants (as indirect measure of lactic acid production) was done. All Lactobacillus strains were lactic acid producers (pH ranging from 2.8 to 3.8), while all except LD1, LO1 and LV1 were strong H2O2 producers as determined previously.[7]

The isolates were stored at −70°C in deMan Rogosa Sharpe (MRS) broth (HiMedia, Mumbai, India) with 30% glycerol and were subcultured twice on MRS agar before experimental use in this study. For preparation of Lactobacillus suspension, strains were grown on MRS agar for 48 h. The cells washed twice with phosphate-buffered solution (PBS), harvested by centrifugation (1200 g, 10 min) and suspended in PBS to a final concentration of 1 × 107 cells per ml.

Detection of adherence to vaginal epithelial cells

Adherence to VEC was determined as per Boris et al.[8] Briefly, VECs were collected from healthy premenopausal women during proliferative phase of menstrual cycle at the same time of day each morning before performing the assay. VECs were washed twice and suspended in PBS to final concentration of 1 × 105 cells/ml. Equal volumes of Lactobacillus suspension and VEC were mixed and incubated at 37°C with orbital shaking (80 rpm/min) for 60 min. The suspension was passed through 8 μm millipore filter, and cells retained on the filter were Gram stained. VECs were scored for the presence and number of colonising lactobacilli microscopically. Adherence was expressed as percentage of colonised VEC. The average number of adherent lactobacilli per colonised VEC was determined, and the number of adherent lactobacilli per VEC (average number of adherent lactobacilli per colonised VEC × percentage of colonised VEC/100) was calculated.

Detection of cell surface hydrophobicity

Cell surface hydrophobicity was determined based on microbial adhesion to hydrocarbons method depending on the ability of lactobacilli to partition xylene from PBS as described by Rosenberg et al.[9] The initial optical density (OD) of Lactobacillus suspension was measured (540 nm). One millilitre of suspension was added to 1 ml of xylene and vortexed vigorously for 30 s. After phase separation for 30 min, OD of the aqueous phase was measured and compared with the initial value. Hydrophobicity percentage was calculated as (A540 initial − A540 after phase separation/A540 initial) × 100.

Detection of auto-aggregation

Auto-aggregation assay was performed according to Kos et al.[6] Two millilitres of Lactobacillus suspension was mixed by vortexing for 10 s and auto-aggregation was determined during 5 h of incubation at room temperature. At hourly intervals, 100 μl of upper part of suspension was transferred to another tube with 1.9 ml of PBS and the absorbance was measured (600 nm). Auto-aggregation percentage was expressed as (1 – At/A0) × 100, where At represents absorbance at time t = at 5 h and A0 is initial absorbance.

Statistical analysis

SPSS v. 15.0 software was used for statistical analysis SPSS Inc, USA. All assays were performed in triplicates. Data are expressed as mean ± standard deviation of results from three replicate experiments on the same strain. Lactobacillus strains scoring ≥80% for all three properties; adherence, auto-aggregation and hydrophobicity were considered as having optimal colonisation abilities. Analysis of variance and Games–Howell test were used to compare the colonisation characteristics within Lactobacillus species and between different isolates of the same Lactobacillus species. Mann–Whitney U-test was used to compare colonisation characteristics between groups. P < 0.05 was considered statistically significant.


 ~ Results Top


A total of 38 vaginal Lactobacillus strains isolated from healthy Indian women were evaluated for colonisation characteristics. Adherence to VEC amongst lactobacilli ranged from 25.2 ± 2.3 to 91.7 ± 1.9. The mean number of lactobacilli adhering per VEC ranged from 1.0 to 35.2. Adherence for different Lactobacillus species is presented in [Table 1]. L. reuteri showed the highest adherence, while L. delbrueckii the least. The adherence in different strains of the same Lactobacillus species is depicted in [Figure 1]. Adherence in strains of the same species ranged from 36.8 ± 3.6 to 91.7 ± 1.9 for L. crispatus, 38.7 ± 6.1–87.7 ± 2.3 for L. gasseri, 44 ± 1.0–91.1 ± 1.3 for L. jensenii and 25.2 ± 2.3–78.2 ± 2.1 for L. vaginalis.
Table 1: Adherence, auto.aggregation and hydrophobicity in different Lactobacillus species

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Figure 1: Adherence, auto-aggregation and hydrophobicity amongst different strains of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii and Lactobacillus vaginalis. The figure shows the colonisation characteristics expressed as percentages on Y-axis for different strains of a. Lactobacillus crispatus, b. Lactobacillus gasseri, c. Lactobacillus jensenii and d. Lactobacillus vaginalis (X-axis) with their standard deviations (error bars).

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Auto-aggregation amongst the lactobacilli ranged from 37.1 ± 4.0 to 89.3 ± 1.3. Auto-aggregation in different Lactobacillus species is presented in [Table 1]. L. reuteri showed the highest auto-aggregation, while L. mucosae the least. Auto-aggregation amongst different strains is depicted in [Figure 1]. Auto-aggregation ranged from 37.1 ± 4.0 to 88.8 ± 2.5 for L. crispatus, 54.2 ± 3.4–89.3 ± 2.1 for L. gasseri, 63.5 ± 1.7–87.5 ± 0.7 for L. jensenii and 51.5 ± 0.9–70 ± 1.6 for L. vaginalis.

Cell surface hydrophobicity amongst the lactobacilli ranged from 2.67 ± 1.9 to 92.5 ± 2.3. Hydrophobicity in different Lactobacillus species is presented in [Table 1]. L. reuteri showed the highest hydrophobicity, while L. fermentum the least. Hydrophobicity amongst different strains is depicted in [Figure 1]. Hydrophobicity ranged from 28 ± 3.9 to 91.1 ± 2.5 for L. crispatus, 16.4 ± 3.4–87.9 ± 1.3 for L. gasseri, 68.5 ± 4.0–91.3 ± 1.1 for L. jensenii and 2.7 ± 1.9–65.5 ± 1.2 for L. vaginalis.

No significant difference in adherence (P = 0.384), auto-aggregation (P = 0.282) and hydrophobicity (P = 0.084) abilities between different Lactobacillus species was noted, however, statistically significant difference in all three colonisation characteristics between different strains of the same species was noted (P < 0.001).

Seven strains LC4, LC6, LC10, LG7, LG10, LJ5 and LR exhibited ≥80% scores for all three colonisation characteristics tested. These strains were also strong H2O2 and lactic acid producers as determined previously.[7]


 ~ Discussion Top


Amongst the several properties, the ability of probiotic bacteria to colonise the target site is considered of utmost importance. Adherence to the VECs is an important prerequisite for colonisation of the probiotic strains in the vaginal tract. In the present study, we used fresh VECs collected from healthy women for the adherence assays. Although little is known about mechanisms by which lactobacilli adhere to the vaginal epithelium, multiple components of the bacterial cell surface seem to be involved.[5] Fresh VECs can be obtained with relative ease and may retain surface components that may be essential for adherence and hence serve as better candidates for adherence assays.

Auto-aggregation of probiotic strains is necessary for adherence to the vaginal epithelium. Self-aggregation may substantially increase colonisation potential of lactobacilli with short residence times and together with adherence favour colonisation of vaginal epithelium through formation of a bacterial biofilm.[10],[11] Cell surface hydrophobicity is reported to affect auto-aggregation and influence the strength of adherence as well. Our results confirm the finding that colonisation properties of lactobacilli are strain specific, rather than species specific.[12],[13]

To conclude, the study presents the first evaluation of colonisation characteristics of vaginal Lactobacillus strains isolated from healthy women from India for potential probiotic development. Seven Lactobacillus strains were identified that may serve as best candidates for vaginal probiotic development. These will be further tested for technological properties andin vivo animal studies for thorough understanding of their functional and immunomodulation capacities as potential vaginal probiotic candidates.

Financial support and sponsorship

This study was financially supported by the Department of Biotechnology, Government of India.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

1.
Amabebe E, Anumba DO. The vaginal microenvironment: The physiologic role of lactobacilli. Front Med (Lausanne) 2018;5:181.  Back to cited text no. 1
    
2.
Valenti P, Rosa L, Capobianco D, Lepanto MS, Schiavi E, Cutone A, et al. Role of lactobacilli and lactoferrin in the mucosal cervicovaginal defense. Front Immunol 2018;9:376.  Back to cited text no. 2
    
3.
Mastromarino P, Macchia S, Meggiorini L, Trinchieri V, Mosca L, Perluigi M, et al. Effectiveness of Lactobacillus-containing vaginal tablets in the treatment of symptomatic bacterial vaginosis. Clin Microbiol Infect 2009;15:67-74.  Back to cited text no. 3
    
4.
Pendharkar S, Brandsborg E, Hammarström L, Marcotte H, Larsson PG. Vaginal colonisation by probiotic lactobacilli and clinical outcome in women conventionally treated for bacterial vaginosis and yeast infection. BMC Infect Dis 2015;15:255.  Back to cited text no. 4
    
5.
Singh TP, Malik RK, Kaur G. Cell surface proteins play an important role in probiotic activities of Lactobacillus reuteri. Nutrire 2016;41:5.  Back to cited text no. 5
    
6.
Kos B, Susković J, Vuković S, Simpraga M, Frece J, Matosić S. Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. J Appl Microbiol 2003;94:981-7.  Back to cited text no. 6
    
7.
Mane A, Angadi M, Vidhate P, Bembalkar S, Khan I, Bichare S, et al. Characterization of vaginal lactobacilli from HIV-negative and HIV-positive Indian women and their association with genital HIV-1 shedding. J Med Microbiol 2017;66:1471-5.  Back to cited text no. 7
    
8.
Boris S, Suárez JE, Vázquez F, Barbés C. Adherence of human vaginal lactobacilli to vaginal epithelial cells and interaction with uropathogens. Infect Immun 1998;66:1985-9.  Back to cited text no. 8
    
9.
Rosenberg M, Gutnick D, Rosenberg E. Adherence of bacteria to hydrocarbons: A simple method for measuring cell-surface hydrophobicity. FEMS Microbiol Lett1980;9:29-33.  Back to cited text no. 9
    
10.
Borges S, Silva J, Teixeira P. The role of lactobacilli and probiotics in maintaining vaginal health. Arch Gynecol Obstet 2014;289:479-89.  Back to cited text no. 10
    
11.
Salas-Jara MJ, Ilabaca A, Vega M, García A. Biofilm forming Lactobacillus: New challenges for the development of probiotics. Microorganisms 2016;4:35.  Back to cited text no. 11
    
12.
Balakrishna A.In vitro evaluation of adhesion and aggregation abilities of four potential probiotic strains isolated from guppy (Poecilia reticulata). Braz Arch Biol Technol 2013;56:793-800.  Back to cited text no. 12
    
13.
Oh NS, Joung JY, Lee JY, Kim Y. Probiotic and anti-inflammatory potential of Lactobacillus rhamnosus 4B15 and Lactobacillus gasseri 4M13 isolated from infant feces. PLoS One 2018;13:e0192021.  Back to cited text no. 13
    


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