In vivo studies could not confirm in vitro prophylactic synergism between Moringa essential oil and Lactobacillus reuteri (MT180537)

Estudos in vivo não puderam confirmar o sinergismo profilático in vitro entre o óleo essencial de Moringa e Lactobacillus reuteri (MT180537)

K. Shazadi N. Arshad H. S. Ambreen A. Riaz A. Mehreen About the authors

Abstract

Aerobic vaginitis (AV) is a recently defined vaginal recurring infection, which is treated with antibiotics. However, excessive and prolonged use of antibiotics disrupts healthy vaginal microflora and leads to the emergence of antibiotic resistance among pathogens. This situation has directed researchers to explore alternative antimicrobials. The current study describes in vitro and in vivo antimicrobial efficacy and pharmaceutical interactions between plant essential oils (EOs) and five lactic acid bacteria (LABs), isolated from the healthy vagina, against E. faecalis, one of the major etiological agents of AV. In vitro experiments confirm good antimicrobial activity of both plant EOs and cell free supernatant (CFS) from LABs. Based on high antimicrobial efficacy, Moringa essential oil (MO) was selected to determine its nature of interaction with CFS of five LAB strains. Synergism was recorded between MO and CFS of L. reuteri (MT180537). To validate in vitro findings, prophylactic responses of individual and synergistic application of MO and L. reuteri (MT180537) were evaluated in an E. faecalis (MW051601) induced AV murine model. The prophylactic efficacy was evidenced by a reduction in intensity of clinical symptoms, E. faecalis (MW051601) count per vaginal tissue along with a reduction in AV associated changes in histological markers of infection in animals receiving Moringa essential oil and L. reuteri (MT180537) alone or in combination. However, significant synergism between Moringa essential oil and L. reuteri (MT180537) could not be observed. Our data confirms the importance of in vivo experiments in deducing pharmacological interactions.

Keywords:
aerobic vaginitis; essential oil; lactic acid bacteria; murine model; synergistic interaction

Resumo

Vaginite aeróbica (VA) é uma infecção vaginal recorrente definida recentemente, que é tratada com antibióticos. No entanto, o uso excessivo e prolongado de antibióticos perturba a microflora vaginal saudável e leva ao surgimento de resistência aos antibióticos entre os patógenos. Esta situação levou os pesquisadores a explorar antimicrobianos alternativos. O presente estudo descreve a eficácia antimicrobiana in vitro e in vivo e as interações farmacêuticas entre óleos essenciais vegetais (OE) e cinco bactérias lácticas (BAL), isoladas de vagina sã, contra E. faecalis, um dos principais agentes etiológicos da AV. Os experimentos in vitro confirmam a boa atividade antimicrobiana de ambos os EOs de plantas e sobrenadante livre de células (CFS) de LABs. Com base na alta eficácia antimicrobiana, o óleo essencial de Moringa (MO) foi selecionado para determinar sua natureza de interação com o sobrenadante livre de células (CFS) de cinco cepas de LAB. Sinergismo foi registrado entre MO e CFS de L. reuteri (MT180537). Para validar os resultados in vitro, as respostas profiláticas da aplicação individual e sinérgica de MO e L. reuteri (MT180537) foram avaliadas em um modelo murino AV induzido por E. faecalis (MW051601). A eficácia profilática foi evidenciada por uma redução na intensidade dos sintomas clínicos, contagem de E. faecalis (MW051601) por tecido vaginal, juntamente com uma redução nas alterações associadas a AV nos marcadores histológicos de infecção em animais que receberam óleo essencial de Moringa e L. reuteri (MT180537) sozinho ou em combinação. No entanto, não foi possível observar sinergismo significativo entre o óleo essencial de Moringa e L. reuteri (MT180537). Nossos dados confirmam a importância dos experimentos in vivo na dedução de interações farmacológicas.

Palavras-chave:
vaginite aeróbia; óleo essencial; bactéria láctica; modelo murino; interação sinérgica

1. Introduction

Vaginitis is an inflammation of the vagina that predominantly affects reproductive aged women (Jeng et al., 2020JENG, H.S., YAN, T.R. and CHEN, J.Y., 2020. Treating vaginitis with probiotics in non-pregnant females: A systematic review and meta-analysis. Experimental and Therapeutic Medicine, vol. 20, no. 4, pp. 3749-3765. http://dx.doi.org/10.3892/etm.2020.9090. PMid:32855726.
http://dx.doi.org/10.3892/etm.2020.9090...
). It is generally characterized by vaginal dysbiosis and is associated with an increased risk of preterm birth and pelvic inflammatory infections. The most common dysbiosis of the vaginal microbiome are vulvovaginal candidiasis (VVC), trichomoniasis, bacterial vaginosis (BV) and aerobic vaginitis (AV) (van de Wijgert and Jespers, 2017VAN DE WIJGERT, J.H. and JESPERS, V., 2017. The global health impact of vaginal dysbiosis. Research in Microbiology, vol. 168, no. 9-10, pp. 859-864. http://dx.doi.org/10.1016/j.resmic.2017.02.003. PMid:28257809.
http://dx.doi.org/10.1016/j.resmic.2017....
). Among these, AV is a newly defined vaginal infection, which is caused by aerobic pathogens such as Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and group B Streptococcus spp. (Tempera et al., 2004TEMPERA, G., BONFIGLIO, G., CAMMARATA, E., CORSELLO, S. and CIANCI, A., 2004. Microbiological/clinical characteristics and validation of topical therapy with kanamycin in aerobic vaginitis: a pilot study. International Journal of Antimicrobial Agents, vol. 24, no. 1, pp. 85-88. http://dx.doi.org/10.1016/j.ijantimicag.2003.12.013. PMid:15225868.
http://dx.doi.org/10.1016/j.ijantimicag....
). Enterococcus species were neglected as pathogens to humans for many years and believed to be insignificant medically (Moreno et al., 2006MORENO, M.F., SARANTINOPOULOS, P., TSAKALIDOU, E. and DE VUYST, L., 2006. The role and application of enterococci in food and health. International Journal of Food Microbiology, vol. 106, no. 1, pp. 1-24. http://dx.doi.org/10.1016/j.ijfoodmicro.2005.06.026. PMid:16216368.
http://dx.doi.org/10.1016/j.ijfoodmicro....
). Recently, enterococci have turned out to be one of the most imperative nosocomial pathogens, causing mortality up to 61% (Lopes et al., 2005LOPES, M.D.F.S., RIBEIRO, T., ABRANTES, M., MARQUES, J.J.F., TENREIRO, R. and CRESPO, M.T.B., 2005. Antimicrobial resistance profiles of dairy and clinical isolates and type strains of enterococci. International Journal of Food Microbiology, vol. 103, no. 2, pp. 191-198. http://dx.doi.org/10.1016/j.ijfoodmicro.2004.12.025. PMid:16083821.
http://dx.doi.org/10.1016/j.ijfoodmicro....
). E. faecalis is considered the etiological agent of almost 80 percent of human infections and is incriminated in the pathogenesis of AV at a rate of 32.26% followed by E. coli (25.8%) (Sangeetha et al., 2015SANGEETHA, K.T., GOLIA, S. and VASUDHA, C.L., 2015 [viewed 22 July 2021]. A study of aerobic bacterial pathogens associated with vaginitis in reproductive age group women (15-45 years) and their sensitivity pattern. International Journal of Research in Medical Science [online], vol. 3, no. 9, pp. 2268-2273. Available from: https://imsear.searo.who.int/handle/123456789/166467
https://imsear.searo.who.int/handle/1234...
; Daood et al., 2020DAOOD, I.I., SHAREEF, S.Y., AL JUBORY, I.H. and ALMUKHTAR, S.H., 2020. Evaluation and antimicrobial susceptibility testing of Enterococcus faecalis isolated from high vagina. EurAsian Journal of BioSciences, vol. 14, no. 1, pp. 1715-1720.). The symptoms of AV include thinned vaginal mucosa, increased vaginal inflammation, and invariably an abundant yellowish foul-odor vaginal discharge (Donders et al., 2009DONDERS, G.G., VAN CALSTEREN, K., BELLEN, G., REYBROUCK, R., VAN DEN BOSCH, T., RIPHAGEN, I. and VAN LIERDE, S., 2009. Predictive value for preterm birth of abnormal vaginal flora, bacterial vaginosis and aerobic vaginitis during the first trimester of pregnancy. An International Journal of Obstetrics & Gynaecology, vol. 116, no. 10, pp. 1315-1324. http://dx.doi.org/10.1111/j.1471-0528.2009.02237.x. PMid:19538417.
http://dx.doi.org/10.1111/j.1471-0528.20...
). Increased maternal age, multiple sex partners, previous spontaneous abortions and altered vaginal microbial flora including depressed vaginal Lactobacillus spp. are considered predisposing factors, which support pathogen colonization (Rampersaud et al., 2012RAMPERSAUD, R., RANDIS, T.M. and RATNER, A.J., 2012. Microbiota of the upper and lower genital tract. Seminars in Fetal & Neonatal Medicine, vol. 17, no. 1, pp. 51-57. http://dx.doi.org/10.1016/j.siny.2011.08.006. PMid:21920833.
http://dx.doi.org/10.1016/j.siny.2011.08...
).

Currently, there is no widely accepted standard approach for the treatment of AV except antibiotics. However, drug resistance among pathogens has resulted in treatment failures and frequent recurrent vaginal infections (Ben-Ami, 2018BEN-AMI, R., 2018. Treatment of invasive candidiasis: a narrative review. Journal of Fungi, vol. 4, no. 3, pp. 97. http://dx.doi.org/10.3390/jof4030097. PMid:30115843.
http://dx.doi.org/10.3390/jof4030097...
; Abd Ellah et al., 2019ABD ELLAH, N.H., ABDEL-ALEEM, J.A., ABDO, M.N., ABOU-GHADIR, O.F., ZAHRAN, K.M. and HETTA, H.F., 2019. Efficacy of ketoconazole gel-flakes in treatment of vaginal candidiasis: formulation, in vitro and clinical evaluation. International Journal of Pharmaceutics, vol. 567, pp. 118472. http://dx.doi.org/10.1016/j.ijpharm.2019.118472. PMid:31252146.
http://dx.doi.org/10.1016/j.ijpharm.2019...
). This situation has directed researchers to explore efficient alternative antimicrobials, preferably from natural sources.

Plant essential oils (EOs) have long been recognized for their antiviral, antifungal, antibacterial, antioxidant and insecticidal properties (Wang et al., 2018WANG, H., YANG, Z., YING, G., YANG, M., NIAN, Y., WEI, F. and KONG, W., 2018. Antifungal evaluation of plant essential oils and their major components against toxigenic fungi. Industrial Crops and Products, vol. 120, pp. 180-186. http://dx.doi.org/10.1016/j.indcrop.2018.04.053.
http://dx.doi.org/10.1016/j.indcrop.2018...
; Ma and Yao, 2020MA, L. and YAO, L., 2020. Antiviral effects of plant-derived essential oils and their components: an updated review. Molecules, vol. 25, no. 11, pp. 2627. http://dx.doi.org/10.3390/molecules25112627. PMid:32516954.
http://dx.doi.org/10.3390/molecules25112...
). Similarly, probiotics are recognized as live microorganisms that confer health benefits on the host when administered in adequate amounts (FAO, 2002FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS – FAO. World Health Organization – WHO, 2002 [viewed 22 July 2021]. Guidelines for the evaluation of probiotics in food [online]. London: FAO/WHO, pp. 1-11. Available from: https://www.who.int/foodsafety/fs_management/en/probiotic_guidelines.pdf
https://www.who.int/foodsafety/fs_manage...
). Probiotic lactobacilli are prompted to prevent the growth of pathogens by competitive exclusion and/or production of antimicrobial secondary metabolites (lactic acid, hydrogen peroxide and bacteriocins) (Tempera et al., 2004TEMPERA, G., BONFIGLIO, G., CAMMARATA, E., CORSELLO, S. and CIANCI, A., 2004. Microbiological/clinical characteristics and validation of topical therapy with kanamycin in aerobic vaginitis: a pilot study. International Journal of Antimicrobial Agents, vol. 24, no. 1, pp. 85-88. http://dx.doi.org/10.1016/j.ijantimicag.2003.12.013. PMid:15225868.
http://dx.doi.org/10.1016/j.ijantimicag....
; Bassolé and Juliani, 2012BASSOLÉ, I.H.N. and JULIANI, H.R., 2012. Essential oils in combination and their antimicrobial properties. Molecules, vol. 17, no. 4, pp. 3989-4006. http://dx.doi.org/10.3390/molecules17043989. PMid:22469594.
http://dx.doi.org/10.3390/molecules17043...
).

Previous studies have also suggested a potential synergy between plant EOs and other antimicrobial agents against multi-drug resistant pathogens (van Vuuren et al., 2009VAN VUUREN, S.F., SULIMAN, S. and VILJOEN, A.M., 2009. The antimicrobial activity of four commercial essential oils in combination with conventional antimicrobials. Letters in Applied Microbiology, vol. 48, no. 4, pp. 440-446. http://dx.doi.org/10.1111/j.1472-765X.2008.02548.x. PMid:19187494.
http://dx.doi.org/10.1111/j.1472-765X.20...
; Rakholiya and Chanda, 2012RAKHOLIYA, K. and CHANDA, S., 2012. In vitro interaction of certain antimicrobial agents in combination with plant extracts against some pathogenic bacterial strains. Asian Pacific Journal of Tropical Biomedicine, vol. 2, no. 3, pp. S1466-S1470. http://dx.doi.org/10.1016/S2221-1691(12)60439-0.
http://dx.doi.org/10.1016/S2221-1691(12)...
). These studies were based on the common assumption that essential oils can be used for their biological activities as well as synergistic enhancers in pharmaceutical formulations (Kamatou et al., 2006KAMATOU, G.P., VAN ZYL, R.L., VAN VUUREN, S.F., VILJOEN, A.M., FIGUEIREDO, A.C., BARROSO, J.G., PEDRO, L.G. and TILNEY, P.M., 2006. Chemical composition, leaf trichome types and biological activities of the essential oils of four related Salvia species indigenous to Southern Africa. The Journal of Essential Oil Research, vol. 18, no. 1, pp. 72-79. http://dx.doi.org/10.1080/10412905.2006.12067125.
http://dx.doi.org/10.1080/10412905.2006....
). The objective of the current work was to investigate the individual and synergistic antimicrobial efficacy of plant EOs and lactic acid bacteria against E. faecalis by employing both in vitro and in vivo approaches.

2. Materials and Methods

2.1. Essential oils

Naturally originated commercial essential oils of four medicinal plants Moringa oleifera Lam. (Drumstick), Colchicum luteum Baker. (Suranjan), Celastrus paniculatus Willd. (Black oil) and Sesamum indicum L. (Sesame) were purchased from the local market in Lahore, Pakistan. Stock solutions of each EO were prepared in DMSO (Dimethyl sulfoxide) and diluted to obtain a final concentration of 2% (v/v). Each EO was also buffered to pH 7.0 and to enhance solubility of EOs, Tween-80 (Sigma-Aldrich) was added at the final concentration of 0.001%. Moxifloxacin was used as a reference drug.

2.2. Lactic acid bacteria (source and characteristics)

Five strains of LABs were originally isolated from vaginal swabs of healthy women by professional clinicians. The strains were cultured on De Man, Rogosa and Sharpe (MRS) for 24-48 h at 37 °C under anaerobic conditions. The strains were screened morphologically and biochemically (Bergey et al., 1994BERGEY, D.H., HOLT, J.G. and KRIEG, P., 1994. Bergey’s manual of determinative bacteriology. Baltimore: Williams and Wilkins.). The strains were identified as Lactobacillus reuteri (MT180537), Pediococcus pentosaceus (MT176555), Lactobacillus pontis (MW362838), Lactobacillus brevis (MW051029) and Lactobacillus brevis (MW362790) based on 16S rRNA gene sequencing and accession numbers were obtained from NCBI (National Center for Biotechnology Information). L. acidophilus ATCC 4356 was included as a reference strain in in vitro antimicrobial assays. LABs were confirmed for their probiotic characteristics based on acid, bile salt, NaCl, lysozyme tolerance, self-aggregation, co-aggregation and good adherence ability (Data not shown).

2.3. Pathogen and growth conditions

E. faecalis (MW051601) was initially obtained from patients of AV. Pure culture was stored at -20 °C in Brain Heart Infusion broth (BHI) containing 25% (v/v) glycerol. Before each experiment, the strain was inoculated on a BHI agar plate to ensure purity and optimal growth.

2.4. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

The MIC and MBC values of Moringa essential oil and CFS from LABs were evaluated using microdilution assay against E. faecalis (MW051601) (CLSI, 2017CLINICAL LABORATORY STANDARDS INSTITUTE – CLSI, 2017 [viewed 22 July 2021]. Performance standards for antimicrobial susceptibility testing; 27th informational supplement [online]. Wayne: CLSI. Available from: https://webstore.ansi.org/preview-pages/CLSI/preview_CLSI+M100-S27.pdf
https://webstore.ansi.org/preview-pages/...
). Briefly, 50 µL of serial two-fold dilutions of both MO (25-0.05 mg mL-1) and CFS corresponding to 3.8-0.01 mg mL-1 were prepared by incorporating into Mueller-Hinton broth (MHB). Then, 50 µL of E. faecalis (MW051601) suspension containing 5x105 CFU mL-1 was added resulting in a final volume of 100 μL in each well. The last well, in each row, containing equal ratio of MHB and test strain without any antimicrobial was used as a positive control. For negative control, 50 μL of each antimicrobial at highest concentration was added to 50 μL of MHB. The plates were incubated at 37 °C for 24 h under aerobic conditions. The survival of E. faecalis (MW051601) was evidenced by appearance of red color following addition of 20 µL/well of 2,3,5-triphenyl tetrazolium chloride (TTC) and incubation at 37 °C for 30 min. The lower antimicrobials that restricted visible growth were termed the MIC while the lowest concentration of antimicrobial that kills 99.9% of the indicator strain was termed MBC. The experiments were performed in triplicate.

2.5. Determination of the Fractional Inhibitory Concentration (FIC) index

The interaction between Moringa essential oil and CFS from LABs against E. faecalis (MW051601) was determined using checkerboard assay following van Vuuren et al. (2009)VAN VUUREN, S.F., SULIMAN, S. and VILJOEN, A.M., 2009. The antimicrobial activity of four commercial essential oils in combination with conventional antimicrobials. Letters in Applied Microbiology, vol. 48, no. 4, pp. 440-446. http://dx.doi.org/10.1111/j.1472-765X.2008.02548.x. PMid:19187494.
http://dx.doi.org/10.1111/j.1472-765X.20...
. In brief, 50 µL of serially two-fold diluted antimicrobials were dispensed to the wells of 96-well microtiter plate in a horizontal (Moringa essential oil, 25-0.05 mg mL-1) and vertical direction (CFS, 3.8-0.01 mg mL-1) thus varying the concentrations of each antimicrobial along matrix. E. faecalis (MW051601) (5x105 CFU mL-1) was inoculated in each well except in row H. Wells (column 12) containing pathogen alone in absence of any antimicrobial were used as a positive control while wells (row H) with each antimicrobial alone were used as an additional media sterility control. After incubation at 37 °C overnight, results were interpreted as mentioned for MIC assay. The FIC index of interaction between Moringa essential oil (drug X) and CFS (drug Y) was calculated using the following Equation 1:

F I C i n d e x = F I C X + F I C Y = M I C X i n p r e s e n c e o f Y / M I C X a l o n e + M I C Y i n p r e s e n c e o f X / M I C Y a l o n e (1)

FICX is the MIC of Moringa essential oil alone, FICY is the MIC of CFS alone. Where X is the concentration of Moringa essential oil in a well that is MIC in its row, Y is the concentration of CFS in a well that is the MIC in its column. The nature of interaction was considered as synergistic (FIC ≤ 0.5), additive (0.5 < FIC ≤ 1), indifference if value is 1 < FIC ≤ 4 and antagonism at FIC > 4.0 (Hübsch et al., 2014HÜBSCH, Z., VAN ZYL, R.L., COCK, I.E. and VAN VUUREN, S.F., 2014. Interactive antimicrobial and toxicity profiles of conventional antimicrobials with Southern african medicinal plants. South African Journal of Botany, vol. 93, pp. 185-197. http://dx.doi.org/10.1016/j.sajb.2014.04.005.
http://dx.doi.org/10.1016/j.sajb.2014.04...
).

2.6. Experimental animals

Two-month old healthy female albino mice (n=50) weighing from 20 to 30 g were obtained from inbred stocks of Government College University (GCU) Lahore, Pakistan. Mice were housed in steel cages under standard conditions (12h light/dark period, 25-27 °C temperature and 45-60% humidity). Vital grower feed and fresh water were provided ad libitum and animals were acclimatized for one week prior to the commencement of the study.

2.7. Media for selective re-isolation of pathogen

Antibiotic susceptibility profile of mice flora and E. faecalis (MW051601) was performed initially to select the antibiotic/s to which mice flora was sensitive but E. faecalis (MW051601) was resistant. The MIC of selected antibiotic (Clindamycin) was performed using broth microdilution assay and antibiotic equivalent to 0.1 MIC was added in Eosin Methylene Blue (EMB) agar. The modified media (supplemented with Clindamycin) was confirmed for selective re-isolation of E. faecalis (MW051601), restricting the growth of indigenous enterococcus.

2.8. Inoculum preparation

Five serial 10-fold dilutions of suspensions (OD600nm=1.00 ± 0.02) of both L. reuteri (MT180537) and E. faecalis (MW051601) were prepared in sterile physiological saline. The 50 µL of different dilutions of L. reuteri (MT180537) and E. faecalis (MW051601) were spread on the surface of MRS and EMB agar plates respectively. The plates were incubated at 37 °C overnight and viable cells were counted by following Formula 2:

C F U / m L = n u m b e r o f c o l o n i e s / v o l u m e o f c u l t u r e s p r e a d o n p l a t e × D i l u t i o n f a c t o r (2)

The inoculum sizes of ∼ 1×108 and 5 × 105 of L. reuteri (MT180537) and E. faecalis (MW051601) respectively (each per 50 µL of physiological saline) were used for inoculation in mice (De Gregorio et al., 2014DE GREGORIO, P.R., TOMÁS, M.S.J., TERRAF, M.C.L. and NADER-MACÍAS, M.E.F., 2014. In vitro and in vivo effects of beneficial vaginal lactobacilli on pathogens responsible for urogenital tract infections. Journal of Medical Microbiology, vol. 63, no. 5, pp. 685-696. http://dx.doi.org/10.1099/jmm.0.069401-0. PMid:24523160.
http://dx.doi.org/10.1099/jmm.0.069401-0...
).

2.9. Experimental procedures

The schedule of treatments is given in Figure 1. Total fifty mice were randomized into five experimental groups (10 mice/group). Animals were immunocompromised with subcutaneous exposure to β-estradiol valerate at the concentration of 0.8 mg/kg/week. Mice in groups I-III were intravaginally (i.vag.) treated with 50 µL of Moringa essential oil (12.50 mg mL-1), L. reuteri (MT180537) (1×108 CFU) and mixture (1×108 CFU mixed in 12.50 mg mL-1 of Moringa essential oil) respectively twice a day for two consecutive days. Later on, mice of group I-IV were challenged with vaginal perfusion of 50 µL of E. faecalis (MW051601) (5×105 CFU) once daily for two days. Mice in Group V were treated similarly with physiological saline.

Figure 1
Experimental design, schedule of treatments and sampling days. H: β-Estradiol 17 valerate (0.8 mg/kg/week). The arrows illustrate i. vag. inoculations of: Saline (S), E. faecalis (MW051601) (EF=5×105 CFU), Moringa essential oil (MO=12.50 mg mL-1), L. reuteri (MT180537) (LR=1×108 CFU) and MO-LR mixture corresponding to 1×108 CFU of L. reuteri (MT180537) mixed in 12.50 mg mL-1 of Moringa essential oil) Sampling day (SD), Dissection (D).

2.10. Measurement of clinical signs

During the course of the study, animals were monitored for the severity of the AV using the clinical index, which consisted of five parameters viz., discharge turbidity, redness, inflammation, animal behavior and loss of fur around vagina. All symptoms were given equal weightage. Animals were scored (0-1) depending on presence or absence of symptom. After scoring, cumulative score of clinical symptoms was used in statistical analysis for comparison of clinical index among groups.

2.11. Sampling of vaginal tissue

On 12th dpi, animals were sacrificed under a high dose of ketamine (200 mg kg-1). Vaginal tissues were harvested, half part of vaginal tissue was weighed to determine bacterial load and the rest of the half part was fixed in neutral buffered-formalin (10%) for histopathological studies.

2.12. Bacterial load per vaginal tissue

The E. faecalis (MW051601) count in vaginal tissue was measured by homogenizing weighted tissue in 2 mL sterile saline. Different 10-fold dilutions of the homogenate (0.1 mL) were plated on clindamycin supplemented EMB and CHROM agar following incubation for 24 h at 37 °C. While indigenous enterococci are detected on CHROM agar, no mice were colonized with strains that resemble E. faecalis (MW051601), as no colonies appeared on EMB agar supplemented with clindamycin that selects for E. faecalis (MW051601).

2.13. Histopathology

The vaginal tissues fixed in 10% neutral buffered formalin, were processed for histopathological examination. Thin vaginal sections (5 µm) stained with hematoxylin-eosin were observed and images were captured using camera fitted light microscope (Labomed, USA). The transitional epithelial thickness and exfoliation were measured following Gilbert et al. (2013)GILBERT, N.M., LEWIS, W.G. and LEWIS, A.L., 2013. Clinical features of bacterial vaginosis in a murine model of vaginal infection with Gardnerella vaginalis. PLoS One, vol. 8, no. 3, e59539. http://dx.doi.org/10.1371/journal.pone.0059539. PMid:23527214.
http://dx.doi.org/10.1371/journal.pone.0...
with slight modifications.

2.14. Statistical analysis

Data were expressed as mean ± standard error and statistical analysis was performed in GraphPad Prism software (version 5.0.0). Non parametric data were analyzed by Kruskel Wallis H-test followed by Mann-Whitney U test for pairwise comparison while parametric data were analyzed with one-way Analysis of Variance (ANOVA) followed by Tukey’s test. Statistical significance was considered at p value < 0.05.

3. Results

3.1. Microdilution assay

Plant EOs and CFS from LABs produced variable MIC and MBC values as shown in Table 1. MO demonstrated MIC and MBC values of 12.5 mg mL-1 and 25 mg mL-1 respectively against E. faecalis (MW051601). Among LABs, MIC value ranged from 1.90 to 0.48 mg mL-1 and MBC from 3.80 to 0.95 mg mL-1 respectively. P. pentosaceus (MT176555) showed higher antimicrobial activity, with MIC and MBC values of 0.48 and 0.95 mg mL-1 respectively.

Table 1
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) in mg mL-1 of selected plant essential oils and cell free supernatant from lactic acid bacteria against E. faecalis (MW051601).

3.2. Determination of the Fractional Inhibitory Concentration (FIC) index

The data of the FIC index was expressed in terms of interaction between MO and CFS of LABs that is summarized in Table 2. Marked synergistic effect (FIC index = 0.25) was observed between MO with CFS of L. reuteri (MT180537). LABs including L. pontis (MW362838), L. brevis (MW051029), L. brevis (MW362790) and L. acidophilus ATCC 4356 (reference strain) showed indifferent outcomes. However, P. pentosaceus (MT176555) was antagonistic when combined with MO.

Table 2
Determination of Fractional Inhibitory Concentration (FIC) index (mg mL-1) between Moringa essential oils (MO) and Cell free Supernatant (CFS) from lactic acid bacteria against E. faecalis (MW051601).

3.3. Clinical symptoms

Mice exposed to E. faecalis (MW051601) (positive control) presented clinical signs (vaginal discharge turbidity, redness, inflammation, animal irritation and thinning of fur around vagina) when compared with the negative control. Animals treated with Moringa essential oil, L. reuteri (MT180537) or MO-LR (L. reuteri-MT180537 mixed in Moringa essential oil) showed a significantly lower clinical index as compared to positive control group (p < 0.05). Regarding the parameters of clinical index, few conditions (redness and discharge) initially appeared in all prophylactic groups that reduced with the passage of time. In addition, animals treated with mixture did not show any of the symptoms by 11 dpi (Table 3).

Table 3
Cumulative score of clinical symptoms in animals.

3.4. Tissue bacterial load

Bacterial load of E. faecalis (MW051601) was 5.58 ± 0.27 CFU g-1 in positive control (group IV) while significantly lower count (1.81 ± 0.68 CFU g-1) was observed in L. reuteri (MT180537) treated group (group II) with reference to positive control (group IV). Moreover, lowest E. faecalis (MW051601) count (1.17 ± 0.29) was detected in the group treated with the MO-LR mixture prior to E. faecalis-MW051601 exposure (group III) (Figure 2).

Figure 2
Bacterial load (CFU g-1 of vaginal tissue) at 11-day post infection. Data were analyzed using a one-way Analysis of Variance (ANOVA) followed by Tukey’s test. The results are expressed as Mean ± SEM. Different letters on bars show significant differences at p < 0.05. I= Moringa essential oil, II= L. reuteri (MT180537), III= MO-LR mixture, IV= E. faecalis (MW051601), V= Negative control.

3.5. Histopathological examination

E. faecalis (MW051601) treated mice (positive control) presented significant damage in mucosal tissue that was evident by increased thickness of epithelium and high epithelial cell exfoliation (Figure 3). Mice in Moringa essential oil, L. reuteri-MT180537 and MO-LR treated groups were found resistant to mucosal tissue damage that was evident by less degree of changes in epithelium similar to negative control (Figure 4).

Figure 3
Influence of prophylactic application of Moringa essential oil, L. reuteri (MT180537) and MO-LR mixture on vaginal histopathological changes; (a) epithelial thickness, (b) exfoliation in AV induced mice. Data were analyzed using a one-way Analysis of Variance (ANOVA) followed by Tukey’s test. The results are expressed as Mean ± SEM. Different letters on bars show significant differences at p < 0.05. I= Moringa essential oil, II= L. reuteri (MT180537), III= MO-LR mixture, IV= E. faecalis (MW051601), V= Negative control.
Figure 4
Influence of prophylactic application of MO, L. reuteri (MT180537) and MO-LR mixture on E. faecalis (MW051601) induced histopathological changes in vaginal transitional epithelium. Mice in groups I-III were (i.vag.) treated with 50 µL of Moringa essential oil (12.50 mg mL-1), L. reuteri (MT180537) (1×108 CFU) and mixture (1×108 CFU mixed in 12.50 mg mL-1 of Moringa essential oil) respectively twice a day for two consecutive days. Later on, mice of group I-IV were challenged with vaginal perfusion of 50 µL of E. faecalis (MW051601) (5×105 CFU) once daily for two days. Mice in Group V were treated similarly with physiological saline. All photomicrographs were captured at 100X magnification.

4. Discussion

For the last four decades, antibiotics have been used for the treatment of urogenital infections. However, antibiotic therapy, especially when prolonged, leads to side effects viz., neutropenia, peripheral neuropathy and pancreatitis (Ferreira et al., 2011FERREIRA, R.S., GUIDO, R.V., ANDRICOPULO, A.D. and OLIVA, G., 2011. In silico screening strategies for novel inhibitors of parasitic diseases. Expert Opinion on Drug Discovery, vol. 6, no. 5, pp. 481-489. http://dx.doi.org/10.1517/17460441.2011.563297. PMid:22646074.
http://dx.doi.org/10.1517/17460441.2011....
). Among natural products, plant EOs and probiotics have been investigated for their antiviral, antifungal and antibacterial properties by various authors (Kaur and Tiwari, 2016KAUR, R. and TIWARI, S.K., 2016. Isolation, identification and characterization of Pediococcus pentosaceus LB44 and Weissella confusa LM85 for the presence of bacteriocin-like inhibitory substances (BLIS). Microbiology, vol. 85, no. 5, pp. 540-547. http://dx.doi.org/10.1134/S0026261716050088.
http://dx.doi.org/10.1134/S0026261716050...
; Iseppi et al., 2020ISEPPI, R., TARDUGNO, R., BRIGHENTI, V., BENVENUTI, S., SABIA, C., PELLATI, F. and MESSI, P., 2020. Phytochemical composition and in vitro antimicrobial activity of essential oils from the lamiaceae family against Streptococcus agalactiae and Candida albicans biofilms. Antibiotics, vol. 9, no. 9, pp. 592. http://dx.doi.org/10.3390/antibiotics9090592. PMid:32927692.
http://dx.doi.org/10.3390/antibiotics909...
) but their synergistic antibacterial ability has not been reported so far. In the current study, in vitro and in vivo pharmaceutical interactions between four plant EOs and five strains of LABs were evaluated against E. faecalis, which is considered one of the most frequently isolated pathogens from AV cases.

Plant EOs are generally used as a promising alternative remedy for topical bacterial infections (Bogavac et al., 2017BOGAVAC, M.A., KARAMAN, M.A., SUĐI, J.J., RADOVANOVIĆ, B.B., JANJUŠEVIĆ, L.N., ĆETKOVIĆ, N.B. and TEŠANOVIĆ, K.D., 2017. Antimicrobial potential of Rosmarinus officinalis commercial essential oil in the treatment of vaginal infections in pregnant women. Natural Product Communications, vol. 12, no. 1, pp. 127-130. http://dx.doi.org/10.1177/1934578X1701200136. PMid:30549845.
http://dx.doi.org/10.1177/1934578X170120...
). The selection of essential oils was based both on ethnomedicinal use and on the proven antibacterial and/or antifungal activity of these oils. Several studies have reported an inverse relationship between the number of Lactobacillus spp. and pathogens in the female reproductive tract (Kamińska and Gajecka, 2017KAMIŃSKA, D. and GAJECKA, M., 2017. Is the role of human female reproductive tract microbiota underestimated? Beneficial Microbes, vol. 8, no. 3, pp. 327-343. http://dx.doi.org/10.3920/BM2015.0174. PMid:28504576.
http://dx.doi.org/10.3920/BM2015.0174...
). Lactobacillus spp. are known to provide protection against pathogens through their secondary metabolites (lactic acid, hydrogen peroxide and bacteriocins) (Tempera et al., 2004TEMPERA, G., BONFIGLIO, G., CAMMARATA, E., CORSELLO, S. and CIANCI, A., 2004. Microbiological/clinical characteristics and validation of topical therapy with kanamycin in aerobic vaginitis: a pilot study. International Journal of Antimicrobial Agents, vol. 24, no. 1, pp. 85-88. http://dx.doi.org/10.1016/j.ijantimicag.2003.12.013. PMid:15225868.
http://dx.doi.org/10.1016/j.ijantimicag....
; Bassolé and Juliani, 2012BASSOLÉ, I.H.N. and JULIANI, H.R., 2012. Essential oils in combination and their antimicrobial properties. Molecules, vol. 17, no. 4, pp. 3989-4006. http://dx.doi.org/10.3390/molecules17043989. PMid:22469594.
http://dx.doi.org/10.3390/molecules17043...
).

In the current study, the antimicrobial activity of EOs and CFS from LABs was determined by microdilution assays while their interaction was evaluated using a checkerboard assay. The results indicated that MO has superior antimicrobial activity among EOs. Virk et al. (2019)VIRK, A.K., KUMARI, C., TRIPATHI, A., KAKADE, A., LI, X. and KULSHRESTHA, S., 2019. Development and efficacy analysis of a Moringa oleifera based potable water purification kit. Journal of Water Process Engineering, vol. 27, pp. 37-46. http://dx.doi.org/10.1016/j.jwpe.2018.11.005.
http://dx.doi.org/10.1016/j.jwpe.2018.11...
has reported antagonistic activity of MO against S. aureus and E. coli. LABs showed MIC values ranging from 1.90 to 0.48 mg mL-1 against E. faecalis (MW051601) which is in accordance with Nazareth et al. (2019)NAZARETH, T.M., LUZ, C., TORRIJOS, R., QUILES, J.M., LUCIANO, F.B., MAÑES, J. and MECA, G., 2019. Potential application of lactic acid bacteria to reduce aflatoxin B1 and fumonisin B1 occurrence on corn kernels and corn ears. Toxins, vol. 12, no. 1, pp. 21. http://dx.doi.org/10.3390/toxins12010021. PMid:31906161.
http://dx.doi.org/10.3390/toxins12010021...
, who reported MIC values of LABs ranging from 125 to 4 g/L against fungi. Based on the higher antimicrobial efficiency of MO, further experiments were performed to evaluate the antimicrobial activity of MO, L. reuteri (MT180537) and MO-LR against E. faecalis (MW051601).

The synergistic interaction in combination therapies is believed to reduce the required concentration of the active molecules in each component and enhance their range of action, thereby decreasing the possible side effects, often related to monotherapy regimens (Walkenhorst, 2016WALKENHORST, W.F., 2016. Using adjuvants and environmental factors to modulate the activity of antimicrobial peptides. Biochimica et Biophysica Acta (BBA) - Biomembranes, vol. 1858, no. 5, pp. 926-935. http://dx.doi.org/10.1016/j.bbamem.2015.12.034. PMid:26751595.
http://dx.doi.org/10.1016/j.bbamem.2015....
). In the presence of MO, the antimicrobial potential of CFS from L. reuteri (MT180537) increased (FIC index = 0.25), suggesting marked synergism between them. In spite of the fact that the MIC of P. pentosaceus (MT176555) was lower than the MIC of the other Lactobacillus spp., it interacts negatively with MO resulting in an antagonistic outcome in checkerboard assay. The results indicated that the antibacterial potential does not depend only on the presence of secondary metabolites but also on the amount and type of interactions with their constituents. Dzotam et al. (2015)DZOTAM, J.K., TOUANI, F.K. and KUETE, V., 2015. Antibacterial and antibiotic-modifying activities of three food plants (Xanthosoma mafaffa Lam., Moringa oleifera (L.) Schott and Passiflora edulis Sims) against bacteria. BMC Complementary and Alternative Medicine, vol. 16, no. 1, pp. 1-8. http://dx.doi.org/10.1186/s12906-016-0990-7. PMid:26753836.
http://dx.doi.org/10.1186/s12906-016-099...
found synergistic effect among three plants (Xanthosoma mafaffa Lam., Moringa oleifera L.Schott and Passiflora edulis Sims.) against multi-drug resistant Gram negative bacteria (FIC index < 0.5). In another study, Aminnezhad et al. (2015)AMINNEZHAD, S., KERMANSHAHI, R.K. and RANJBAR, R., 2015. Evaluation of synergistic interactions between cell-free supernatant of Lactobacillus strains and amikacin and genetamicin against Pseudomonas aeruginosa. Jundishapur Journal of Microbiology, vol. 8, no. 4, e16592. http://dx.doi.org/10.5812/jjm.8(4)2015.16592. PMid:26034539.
http://dx.doi.org/10.5812/jjm.8(4)2015.1...
reported FIC indices (0.124 and 0.56) of CFS from L. rhamnosus and aminoglycoside antibiotics (Gentamicin and Amikacin) while FIC indices from L. casei with the same antibiotics were 0.124 and 0.312, respectively. Based on the FIC index, L. reuteri (MT180537) was selected as promising candidate to determine in vivo synergistic efficacy using an E. faecalis (MW051601) induced AV murine model. Additionally, L. reuteri (MT180537) rather than CFS was preferred due to the fact that L. reuteri (MT180537) will act as a continuous source of secondary metabolites in the murine model.

The murine model is an assessment tool for research outcomes of infections and treatments. AV murine model was based on the subcutaneous administration of β-estradiol valerate due to its role in colonization of pathogens in mice through induction of the pseudo estrus stage (Calderon et al., 2003CALDERON, L., WILLIAMS, R., MARTINEZ, M., CLEMONS, K.V. and STEVENS, D.A., 2003. Genetic susceptibility to vaginal candidiasis. Medical Mycology, vol. 41, no. 2, pp. 143-147. http://dx.doi.org/10.1080/mmy.41.2.143.147. PMid:12964847.
http://dx.doi.org/10.1080/mmy.41.2.143.1...
). Obvious, E. faecalis (MW051601) count emerged in the vaginal wash 11 days post infection at 0.8 mg/kg/week.

Previously, the link between clinical signs and pathogenesis of E. faecalis (MW051601) in the reproductive tract of females was not fully known. In this study, the first evidence of prophylactic effects of MO, L. reuteri (MT180537) and MO-LR was noticed by a decrease in the number of clinical symptoms (discharge turbidity, redness, inflammation, animal behavior and loss of fur around the vagina) in MO, L. reuteri (MT180537) and MO-LR treated mice as compared to untreated mice (negative control). The clinical symptoms improved in MO and L. reuteri (MT180537) treated groups but completely vanished in MO-LR treated animals. In a similar study, Zhou et al. (2019)ZHOU, Y., XU, W., HONG, K., LI, H., ZHANG, J., CHEN, X., ZHU, Y., ZHANG, Q., DING, F. and WANG, F., 2019. Therapeutic effects of probiotic Clostridium butyricum WZ001 on bacterial vaginosis in mice. Journal of Applied Microbiology, vol. 127, no. 2, pp. 565-575. http://dx.doi.org/10.1111/jam.14329. PMid:31102489.
http://dx.doi.org/10.1111/jam.14329...
recorded vaginal redness, swelling and turbidity of discharge as clinical parameters in the BV model and reported significant improvement in clinical symptoms following C. butyricum, another probiotic strain.

The growth inhibition of pathogens (lower bacterial load) is considered a gold standard for effective treatment outcome. In this study, colonization was determined in terms of E. faecalis (MW051601) count per vaginal tissue. The data further provided evidence in favor of prophylactic efficacy of three formulations and displayed significantly lower E. faecalis (MW051601) load as compared to positive control. The results indicated that prophylactic efficacy of lactobacilli may involve the blockage of receptor sites by steric hindrance, competition for receptors and production of secondary metabolites.

Consistent with other parameters, histopathological analysis also revealed the prophylactic efficacy of Moringa essential oil, L. reuteri (MT180537) and MO-LR that was evidenced by a lower degree of vaginal epithelial damage, decreased exfoliation and thickness on 12th dpi in prophylactic groups. Gilbert et al. (2013)GILBERT, N.M., LEWIS, W.G. and LEWIS, A.L., 2013. Clinical features of bacterial vaginosis in a murine model of vaginal infection with Gardnerella vaginalis. PLoS One, vol. 8, no. 3, e59539. http://dx.doi.org/10.1371/journal.pone.0059539. PMid:23527214.
http://dx.doi.org/10.1371/journal.pone.0...
reported that Gardnerella vaginalis (GV) induced BV caused abnormal pathological changes in the vaginal tissues of mice.

5. Conclusion

In culmination, plant essential oils and LABs show antagonistic potential against E. faecalis (MW051601). Moringa EO boosts antagonistic hallmarks of L. reuteri (MT180537) in in vitro findings. Moreover, prophylactic effects were observed in case of three formulations applied as a topical preventive strategy but no significant synergism was observed in the murine model employed in this study.

Acknowledgements

This study was supported by Higher Education Commission (HEC), Pakistan under grant (PIN # 315-9036-2BS3-105). We are extremely grateful to University of the Punjab for providing us laboratory facilities.

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Publication Dates

  • Publication in this collection
    18 Feb 2022
  • Date of issue
    2024

History

  • Received
    22 July 2021
  • Accepted
    20 Sept 2021
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