Antibacterial activity of Ocimum sanctum L. Essential Oil against multidrug resistance Bacteria Vaginosis

Full text

Antibacterial activity of Ocimum sanctum L. essential oil against multidrug
resistance bacteria vaginosis
Paul Akinniyi Akinduti
a,*
, Oluwashindara Lydia Osunlola
a
, Feyisikemi Adenike Adebekun
a
,
David Temiloluwa Viavonu
a
, Gift Nzubechi Elughi
a
, Oluwasegun Popoola
b
,
Somrat Adeola Abdulsalami
c
a
Department of Medical Laboratory Science, Babcock University, Ilishan, Ogun State, Nigeria
b
Golden Gate University School of Law, San Francisco, CA, USA
c
Department of Aquaculture and Fisheries, University of Ilorin, Ilorin Kwara State, Nigeria
ARTICLE INFO
Keywords:
Bacteria vaginosis
Multidrug resistance
Antibacterial activity
Phytochemicals
Metabolites
ABSTRACT
The antibiotic resistance of Bacteria Vaginosis (BV) isolates intensies vaginal morbidity and genital infections
facilitating poor treatment outcome and severe vaginal pathology. The phytochemicals in Ocimum sanctum
essential oil (OsEO) were investigated for their antibacterial activity against bacteria vaginosis and major me-
tabolites on multidrug resistance (MDR) strains. Bacteria pathogens isolated from vaginal samples (n =40)
obtained from patients with conrmed BV were analysed for hemolytic activity, biolm production and proled
for antibiotic resistance. Extracted OsEO was proled with GC-MS and analysed for antibacterial activity. Of the
recovered bacteria pathogens (n =241) associated with vaginosis including Streptococcus pyogenes (34 %),
Staphylococcus aureus (31 %) and Escherichia coli (10 %) and less than 10 % Klebsiella oxytoca, Enterobacter cloaca,
Pseudomonas aeruginosa and Citrobacter freundii were identied. Signicant rates of 21.6 %, 4.6 % and 2.3 % were
weak, mild and strong biolm producers respectively and overall 26.6 % were hemolytic strains (p <0.05). More
than 60 % resistance to ceftriaxone sulbactam, ampiclox, cefuroxime, cefotaxime, nalidixic acid and cefexime
was observed in BV with signicant proportion showing MARI>0.2 (p <0.05). Hierarchical clustering of MDR
BV strains provided related clustered bacteria pathogens having a very low susceptibility to iminepem, cefur-
oxime, and amoxycillin/clavulanate. More than 1.2 % saponin, alkaloids and avonoids levels in OsEO gave
signicant inhibitory activities at IC50 (25.0
μ
g/mL) and IC90 (50.0
μ
g/mL) and signicant inhibitory associ-
ation with phytochemical compounds (eta =0.457, p =0.015). OsEO cyclohexene and methanoazulene me-
tabolites showed signicant antibacterial association with BV strains (p <0.05). The OsEO phytochemical
metabolites showed antibacterial activity against multidrug resistance BV and identied cyclohexene and
methanoazulene are promising candidates for developing formulations as topical antimicrobial agents for BV
treatment.
1. Introduction
Over the years, less consideration was given to bacterial vaginosis
mostly among the women of reproductive ages. Bacterial vaginosis (BV)
is dened as an imbalance or shift in the vaginal microbiota leading to
reduction of predominant Lactobacilli and increase proliferation of
various aerobic, anaerobic and microaerophilic strains [1,2]. Several
reports made clear emphasizes on the characteristic increase and
abnormal level of aerobic and enteric bacteria including Staphylococcus
spp., Group B Streptococcus, Escherichia coli, Klebsiella spp., Acinetobacter
spp., and Enterococcus spp., causing vaginal inammation [3,4]. The
severity of BV inammations increases vaginal morbidity and was re-
ported to be associated with poor treatment outcome and increase
antibiotic resistance [2,4].
Although most women with BV are asymptomatic but the reported
prevalence rates among women ages 14–49 in USA [5], South and East
Africa including Mozambique, Lesotho, Kenya, Gambia [6–8], Europe
(Norway, Turkey, Poland) [9,10], Latin America and the Caribbean [11,
* Corresponding author.
E-mail addresses: akindutip@babcock.edu.ng (P.A. Akinduti), oluwashindara.osunlola@stu.cu.edu.ng (O.L. Osunlola), feyisikemi.adebekun@stu.cu.edu.ng
(F.A. Adebekun), david.viavonu@stu.cu.edu.ng (D.T. Viavonu), gift.elughipgs@stu.cu.edu.ng (G.N. Elughi), opopoola@my.ggu.edu (O. Popoola).
Contents lists available at ScienceDirect
Medicine in Microecology
journal homepage: www.journals.elsevier.com/medicine-in-microecology
https://doi.org/10.1016/j.medmic.2024.100115
Received 23 April 2024; Received in revised form 11 September 2024; Accepted 3 November 2024
Medicine in Microecology 22 (2024) 100115
Available online 8 November 2024
2590-0978/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (
http://creativecommons.org/licenses/by-
nc-nd/4.0/ ).

12] and 13.6 %–32.5 % prevalence rates in several locations in Nigerian
[13–15] provided global epidemiology of public health concern. The
reported epidemiological data indicate rising cases of BV that could give
rise to increase poor vaginal health, predisposing women to unfavorable
outcomes of early pregnancy, perinatal complications, pelvic inam-
matory diseases and sexually transmitted infections [16].
Due to high cost of diagnosis, limited diagnostic facilities and rising
cost of treatment in Low-income settings such as Nigeria, several women
had resolved to self-medication which has led to high antibiotic resis-
tance [13]. Similar resistance was reported in BV isolates in Addis Ababa
[17], France [18], USA [19], Gambia [20] and Nigeria [14,21]. The
increasing levels of antibiotic resistance of BV isolates further intensify
vaginal morbidity, secondary infertility, and genital infections [22].
Production of BV microbiota biolm allow resistance to various classes
of antibiotics, recurrence re-inoculation of BV strains through sexual
intercourse, tenacity to host while douching or use of IUD enhance va-
gina re-colonization [22]. As noted in recent reports, the increasing
prevalence rates of antibiotic-resistant strains of bacteria with high he-
molytic potential associated with BV, makes treatment more challenging
and increase severity [22]. In spite of the drug resistance BV, natural
antimicrobial source such as Ocimum sanctum L. Essential Oil (OsEO) has
proven to possess antimicrobial activity with no cytotoxic effect, cheap
and available for prevention and treatment of BV [23]. Not only has the
phytochemical and volatile compounds from OsEO give considerable
therapy for the management and treatment of soft tissue infections,
there are reports of the antimicrobial properties of OsEO against several
aerobic and anaerobic bacteria [24]. There are many advantages asso-
ciated with the use of Ocimum sanctum L. Essential Oil compared to other
metabolites with therapeutic activities apart from its cost-effectiveness
and global availability. Ocimum sanctum L. Essential Oil contains con-
tains camphor, eucalyptol, and eugenol.-caryophyllene which its com-
bination contributed to the antimicrobial activity of the oil [25,26]. Low
aqueous solubility and bioavailability are the main disadvantages for the
clinical applications of this compound but introduction of new micro
and nanoformulations would help to overcome these limitations [27]. In
recent past, there is paucity of OsEO antibacterial activity against BV
isolates and were largely unexplored. The main gap to be addressed is
the lack of comprehensive scientic evidence regarding the antibacterial
activity of OsEO against BV bacterial isolates. The present study in-
novates the detection of major metabolites in OsEO as antibacterial
molecules against BV strains using the combination of invitro assay and
statistical evaluation. Investigating the efcacy of metabolites from this
essential oil as inhibitory compounds against key bacterial species
commonly found in vaginosis, would provide valuable insights into its
potential as a natural and alternative therapeutic option for managing
BV. This study aims to determine the in vitro antimicrobial activity of
OsEO against bacteria isolates associated with vaginosis and investi-
gating major metabolites providing signicant antibacterial activity.
2. Methods
2.1. Ethical permission
Prior to sample collection, ethical approval was obtained from the
Covenant Health Research Ethics Committee (CU/HREC/EGN/206/23)
while the informed consent of each patients that were eligible for the
study was sought and kept condential.
Sampling: Vaginal samples (n =40) including high vaginal and
endocervical swab samples were aseptically collected from diagnosed
patients ages 20–45 years with conrmed bacteria vaginosis. The pa-
tients includes those attending the In- and Out-patients’ clinics of the
Federal Medical Center, Abeokuta, Nigeria; for clinical management.
Pregnant patients, post-partum women, immunosuppressive individuals
and women at menopausal stage were excluded from the study.
2.2. Bacteria strain and biolm assay
Collected vaginal swabs were inoculated in 5 mL glucose-nutrient
broth for 24 h at 37
0
C. After incubation, the swabs were further sub-
cultured for aerobic strains on heated Columbia blood and MacConkey
agars for further incubation at 37
0
C in 5 % CO
2
. For isolation of
anaerobic and Lactobacilli strains, the swabs were inoculated on 7 %
Columbia Blood and MRS (deMan, Rogosa, and Sharpe) agars, and
incubated in anaerobic jar and 5 % CO
2
respectively for 48 h. After
overnight incubation at 37
0
C, obtained isolates were examined for
colonial morphology and biotyped using the Analytical Prole Index
(API STAPH, API 20E, and API 50 CHL) identication kit (BioM´
erieux,
Inc, Durham, USA). Hemolytic activity of the obtained isolates was
determined as previously discussed [24]. Briey, each isolate was cul-
tures on 7 % Blood Agar, then incubated for 24 h at 37 ◦C and the
patterns of blood lysis were interpreted [24]. The ability of each isolates
to produce biolm was investigated using the micro-bioassay method as
described [28]. Aliquots of 200
μ
L of 0.5 McFarland turbid and freshly
prepared overnight broth culture was placed separately in 96-well
microtitre plate and incubated overnight at 37 ◦C. After incubation,
the microtiter plate was rinsed twice with water to remove non-adherent
cells, and crystal violet of 50
μ
L was added to each well and rinsed off
after 2 min. Following washing of the well, 100
μ
l of ethanol was added
to each well and the absorbance of the developed colour was estimated
using a UV spectrophotometer at 630 nm. The amount of biolm pro-
duced was determined according to Stepanovi´
c et al. [29]. Isolated
Lactobacilli commensals with no hemolytic and biolm production were
not further assayed for OsEO antibacterial.
2.3. Antibiotics susceptibility test
Susceptibility of the isolated bacterial strains to antibiotics was
evaluated using Kirby-Bauer disc diffusion method according to the
guideline of the European Committee on Antimicrobial Susceptibility
Testing [30,31]. A suspension of 0.5 McFarland adjusted broth of each
isolates was spread on Muller-Hinton agar (Oxoid, Basingstoke, UK)
using sterile swab sticks and incubated for 18 h at 37 ◦C. To each
inoculated MH plate, disc of each antibiotic including ceftriaxone sul-
bactam 45
μ
g (CRO), ampiclox 10
μ
g (ACX), cefuroxime 30
μ
g (CXM),
cefotaxime 25
μ
g (CTX), nalidixic acid 30
μ
g (NA), cefexime 5
μ
g (ZEM),
ooxacin 5
μ
g (OFX), imipenem 10/10
μ
g (IMP), nitrofurantoin 300
μ
g
(NF), levooxacin 5
μ
g (LBC), gentamycin 10
μ
g (GN), and amox-
icillin/clavulanate 30
μ
g (AUG) were place at 30 mm distance [32]. The
obtained zone of inhibition after incubation at 37 ◦C was estimated and
interpreted according to the CLSI, 2020 guidelines [33]. A minimum
inhibitory concentration (MICs) for each antibiotic agent against the
bacteria strains was determined using micro-broth dilution assay with
the previously used antibiotic panel [34]. The phenotypic resistance to
the antibiotic was interpreted based on the CLSI guidelines [33]. Bac-
teria strain showing resistance to more than two antibiotic classes was
classied as multi-drug resistant [35,36]. The multiple antibiotic resis-
tance index (MARI) was determined by dividing the total number of
drug resistance to antimicrobials for each isolate by the total number of
tested antimicrobials and interpreted within the range of 0–1 [37].
2.4. Plant selection and GC-MS proling
Based on the reported ethnopharmacological properties of Ocimum
sanctum for antibacterial activity as described in Table S1, fresh leaves of
Ocimum sanctum were selected from a single plant population. Prior to
the collection of the leaves from the premises of the Covenant University
in March 2023, approval was obtained from the oral management. As
previously reported, the plant was identied, authenticated and the
plant vouchers were deposited at the Herbarium of the Federal Uni-
versity of Agriculture, Abeokuta, Nigeria. After three days of air-drying
process, Ocimum sanctum leaves of 120 g was blended and the powdery
P.A. Akinduti et al.
Medicine in Microecology 22 (2024) 100115
2

form was dissolved in 1 L of sterile water. The ltrate of the aqueous
extract was hydro-distilled with distilled water of 400 mL in a
Clevenger-type apparatus for 6 h and further separated to obtain the
essential oil [38]. Quantitative analysis for the phytochemical sub-
stances was performed to estimate the constituted metabolites including
anthocyanin, alkaloid, glycosides, coumarins, avonoids, phenols, qui-
nons, saponins, tannins, and terpenoids as described by Chinnadurai
et al. [39]. The volatile metabolites in the essential oil were proled with
the GC-MS analytics using the Thermo Trace 1300 GC in conjunction
with the Thermo TSQ 800 Triple Quadrupole MS as previously described
[40]. To splitless mode, 6
μ
L of essential oil was introduced and allowed
to go through a warming process, and subsequently separated in a TG
5MS column. For the protocol, boiled deionized water was used as a
blank. The raw data from GC-MS were transformed into netCDF format
and further analysed to align for chromatographic peak areas based on
mass and time using XCalibur 2.2SP1 and Foundation 2.0SP1. The
characterization of the metabolites was performed with a comparative
analysis of retention time and mass spectra, with standards from the
NIST 2.0 Mass Spectral Library [41].
2.5. Antibacterial activity of OsEO
Susceptibility of the bacteria vaginosis to OsEO was determined
using the agar well diffusion method as previously described [42,43]. In
brief, overnight 0.5 McFarland turbid bacteria inoculum was spread
evenly on dried sterile Mueller–Hinton agar plates, and sterile paper disc
already impregnated with 20 mg/ml OsEO was rmly placed on the
agar. After 24 h incubation at 37 ◦C, the produced zone of inhibition was
measured and compared with the negative control disc containing
sterile physiological saline while 10ug/disc ciprooxacin serves as
positive control. Levels of antibacterial activity of the oil against the
strains were further conrmed using the standard microbroth dilution
assay. The inhibitory concentration (IC) of OsEO against each bacteria
pathogen was determined within the concentrations of 0.5–64 mg/ml.
In a sterile microtitre plate, 100
μ
l OsEO aliquot already diluted in
DMSO was serially diluted in equal volume of 100
μ
l each of nutrient
broth and 100
μ
l of 0.5 McFarland turbid bacteria inoculum was added
to each well. The growth medium without inoculum (serves as blank),
and combination of the growth medium, sterile normal saline with the
inoculum (control) with test plates were incubated aerobically at 37 ◦C
for 18–24 h. The minimum concentration (that is the highest dilution)
showing no turbidity (no visible bacterial growth) compared with the
blank and control tubes were recorded as the inhibitory concentrations.
2.6. Data analysis
The signicance levels of the bacteria vaginosis among the subjects,
signicance rates of biolm producers and hemolytic strains were
determined using Chi square taking the p-value <0.05. Comparative
evaluation of differences in MARI among the isolates was performed
with paired T-test at p <0.05 and within the MARI at p <0.05 (95 %
condence level) and p <0.01 (99 % condence level). To adjudged
differences between the signicance antibacterial activities at IC50 and
IC90, Wilcoxon Signed Rank Test was used for comparison at 95 %
probability level. The descriptive measure of the strength of association
between phytochemical compounds (independent variables) and anti-
microbial activities of plant oil extracts IC50 and IC90 (dependent var-
iables) was determined using the Eta-square calculations based on the
comparative level of inhibitory performance at 95 % probability. The
level of association of each volatile metabolites with antibacterial ac-
tivity according to obtained inhibitory concentrations was interpreted
by the Pearson’s coefcient taking the signicance p <0.05 at 95 %
condence interval.
3. Results
3.1. Prevalent bacterial pathogens associated with diagnosed vaginosis
Evaluation of recovered bacteria pathogens (n =241) associated
with vaginosis revealed high rates of Streptococcus pyogenes (34 %) and
Staphylococcus aureus (31 %), low rates of less than 10 % of Escherichia
coli, Klebsiella oxytoca, Enterobacter cloaca, Pseudomonas aeruginosa,
Citrobacter freundii and 1 % Lactobacillus species (including L. crispatus,
L. gasseri, L. iners, and L. jensenii) (Fig. 1A). Considering the level of the
strains biolm productions, signicant rates of 21.6 %, 4.6 % and 2.3 %
were weak, mild and strong biolm producers with overall lower rates of
26.6 % hemolytic strains (p <0.05; Fig. 1B).
3.2. Implications of the antibiotic resistance in bacteria vaginosis
Antibiotic resistance pattern of the bacteria isolates obtained from
the analysed BV cases revealed more than 60 % overall resistance to
ceftriaxone sulbactam, ampiclox, cefuroxime, cefotaxime, nalidixic acid
and cefexime. Less than 20 % susceptibility rates to ooxacin, imipenem
and levooxacin (Fig. 2A). A signicant proportion of the isolates
showed a MARI>0.2 compared to other strains with less than 0.2 MARI
(Fig. 2B). Obtained S. aureus, E. coli, K. oxytoca, S. pyogenes, P. aeruginosa
and C. freundii were resistant to more than two classes of antibiotic
suggesting multidrug resistance infection. Most resisted antibiotics be-
longs to different classes including gentamycin (aminoglycosides),
ampiclox (penicillin), cefotaxime, cefexime, ceftriaxone/sulbactam,
cefuroxime (cephalosporins) and low susceptibility to Levooxacin and
Ooxacin (uoroquinolones) (Fig. 2C). The hierarchical heatmap pro-
vided three clustered group of bacteria pathogens having a very low
susceptibility to iminepem, cefuroxime, ampiclox and amoxycillin/
clavulanate. It further reveals a related pattern of resistance among all
the isolates (Fig. 3).
3.3. Antibacterial activity of OsEO against the bacteria pathogens
More than 1.2 % level of saponin, alkaloids and avonoids were
estimated phytochemical compounds in the OsEO while other com-
pounds including phenol, glycoside, commanin, terpenoids and quinone
were less than 1.0 % (Fig. 4A). OsEO gave a signicant and higher
inhibitory activity against the bacteria pathogens at IC90 (50.0
μ
g/mL)
compared to its inhibition at IC50 (25.0
μ
g/mL). A signicant level of
association was observed in comparison of the inhibitory concentrations
(IC50 and IC90) with phytochemical compounds (eta =0.457, p =
0.015; Fig. 4B). The GC-MS proling of the OsEO metabolites is
graphically presented in a chromatogram as % concentration (Fig. S1)
indicated high percent methanoazulene (14.15 %) and benzodioxole-5-
carboxylic acid (10.61 %) (Table 1). The level of Carbonic acid, Cyclo-
dodecanemethanol, 2-Hydroxybenzeneacetic acid, Cholesta-6,22,24-
triene, Mercaptoethanol, Diethyl methylphosphonite, 2-hydroxybenzy-
lideneamino ranges between 5.22 and 8.30 % while other metabolites
were less than 5.00 %. Cyclohexene (r =0.770, 95%CI: 0.1111 to
0.9733; p =0.036) and methanoazulene (r =0.756, 95%CI: 0.1429 to
0.9716; p =0.040) showed a signicant level with antibacterial activity
against the strains (Table 1).
4. Discussion
Prevalent Bacterial Vaginosis (BV) observed among the recruited
subjects presents increasing gynaecological conditions characterized
with altered vaginal microbiota that often results in overgrowth of
pathogenic bacteria. The high rates of recovered aerobic bacteria
pathogens associated with vaginosis and very low Lactobacilli rates
indicate a typical shift in the vaginal ora. The shift in aerobic pathogens
observed in this study may be linked to severe vaginal dysbioses pre-
viously reported to be associated with unfavorable early pregnancy and
P.A. Akinduti et al.
Medicine in Microecology 22 (2024) 100115
3

adverse perinatal outcomes [44]. Similar bacteria pathogens in BV was
previously reported [4,9,10,15] but declining demographic prole,
multiple sexual partners, previous abortion, HIV infections, poor
reproductive health and vaginal hygiene were reported to exacerbates
the prevalence rates mostly among the vulnerable populations.
The rates of biolm producers implicated in the examined BV con-
ditions, indicate more risk for infection and re-infection with little or no
treatment outcome. The produced biolm enhance the bacteria adher-
ence to the vaginal epithelia and this extracellular matrix protects the
bacteria strains from antibiotics facilitating relapse of BV mostly among
the age bearing populations [16,45]. The interaction of bacterial species
producing polymicrobial BV biolms with vaginal epithelial is usually
aided through contaminated indwelling medical devices, inserted de-
vices (such as tampons, intra-uterine devices and vaginal rings), pro-
moting vaginal invasion [46]. The combine production of biolm and
hemolytic activity of these strains further intensify vaginal pathology
given rise to possible massive tissue damage, cellular necrosis and cell
death [47]. As host vaginal cell layers damage increases, more
cytotoxicity, epithelia bilayer structure deformities from immune
evasion and septic shock are evident mostly in severe conditions [23,
48]. Periodic vaginal examination for BV is required to prevent persis-
tent chronic infection with possible tissue degeneration.
Many of the characterized BV pathogens showed multiple resistances
to more than two classes of antibiotics, presenting multidrug resistance
infections. However, prevalence of BV with multiple drug resistance
have increase vaginal morbidity with high treatment failure and risk of
vaginal infection from mother to infant [49]. The implications of the
antibiotic resistance in BV portray signicant limitations to therapeutic
choice and selection of appropriate drugs for clinical management and
controlling the complication of BV among women of reproductive age.
Recording resistance to different classes of antibiotics including uo-
roquine (Levooxacin and Ooxacin); aminoglycosides (gentamycin);
penicillin (ampiclox); and cephalosporins (cefotaxime, cefexime, cef-
triaxone/sulbactam, cefuroxime) indicated different mechanism utilized
by these strains to evade cidal or static activities of the drugs. Possible
combined resistance to protein synthesis and cell wall inhibitors is
Fig. 1. (A). Distribution of recovered bacteria pathogens (B). Proportion of biolm and hemolytic strains.
Fig. 2. (A). Overall antibiotic resistance pattern; (B). Antibiotic resistance index of isolated bacteria pathogens; (C) Resistance rates of bacteria strain to different
antibiotics.
P.A. Akinduti et al.
Medicine in Microecology 22 (2024) 100115
4

strategy for development of multidrug and extensively drug-resistance
BV infections. Presence of BV biolm, resistance to narrow and
broad-spectrum antibiotics facilitates re-infection and increase severity.
Having a signicant proportion of the BV isolates with multi-antibiotic
resistance index (MARI) of more than 0.2 suggests possible environ-
mental or nosocomial source of infections with multiple antibiotic
resistance. There is need for hospital infection surveillance for resistance
bacteria strains with emphasis on the contamination of vaginal in-
struments mostly at local health facilities with inadequate
infrastructures.
To further investigate the antibiotic resistance relatedness of
different strains recovered from BV infections, the hierarchical clus-
tering provided clusters of bacteria pathogens having a very low sus-
ceptibility to iminepem, cefuroxime, ampiclox and amoxycilly/
clavulanic. These highlight the need to develop antibiotic monitoring
program for BV particularly for the pregnant women, in order to reduce
the BV resistant infections and spread from mother to child. Though BV
is reported to be associated with several health issues, colonization and
invasion of vaginal wall with production of biolm and resistance to
various classes of antibiotics is a major concern.
In spite of the growing vaginal morbidity with antibiotic resistance,
the use and application of plant extract as primary source of therapeutics
such as OsEO containing bioactive components and combinations of
many secondary metabolites has proved to be safe, non-toxic, cost-
effective, and globally available [23]. The observed level of saponin,
alkaloid, and avonoids in OsEO reveal a combined phytochemical with
potential antibacterial activity to inhibit or causes cidal effect on bac-
teria pathogens [23,50]. Saponin activity was reported to cause the
outer phospholipid membrane disruption rendering the integrity of the
structural lipopolysaccharide constituents of the cell wall inadequate
and give to permeability of lipophilic solutes with increase loss of cell
membrane osmotic activity leading to cell death [51]. Both alkaloids
and avonoids are very important group of antioxidants scavenging
reactive oxygen species including hydroxyl radicals and superoxide
anions causing oxidative stress, cytotoxic damage, by elimination of
Fig. 3. Heatmap of bacteria pathogen hierarchical clustering in relation to their
antibiotic resistance.
Fig. 4. (A). Level of phytochemical compounds in OsEO (B). Comparative inhibitory activity of OsEO with the phytochemical compounds.
Table 1
OsEO GC-MS metabolites proling and correlation analysis of its antibacterial activity.
Compound GC-MS prole Correlation analysis
Rt Area (%) r value 95%CI P value
Cyclohexane 28.951 4.33 0.142 −0.7566 to 0.8552 0.393
Carbonic acid 27.360 5.22 0.437 −0.5805 to 0.9217 0.193
Cholesta-6,22,24-triene 26.593 5.82 −0.488 −0.9310 to 0.5353 0.163
Palmitoleic acid 26.101 4.61 0.011 −0.8079 to 0.8152 0.492
Cyclododecanemethanol 25.266 5.63 0.135 −0.7599 to 0.8531 0.400
Propanamide 24.848 3.92 −0.567 −0.9441 to 0.4535 0.121
Mercaptoethanol 24.110 6.14 −0.221 −0.8755 to 0.7197 0.337
2-Hydroxybenzeneacetic acid 23.921 5.81 0.1888 −0.7355 to 0.8674 0.360
cyclohexene 23.234 3.29 0.770 −0.1111 to 0.9733 0.036
a
tetrahydro-thiazolo 20.310 3.72 0.085 −0.7805 to 0.8386 0.436
benzodioxole-5-carboxylic acid 15.029 10.61 −0.024 −0.8195 to 0.8033 0.482
Methanoazulene 14.176 14.15 0.756 −0.1429 to 0.9716 0.040
a
Diethyl methylphosphonite 12.586 7.33 −0.618 −0.9521 to 0.3884 0.096
Phenol 9.009 3.85 0.119 −0.7665 to 0.8487 0.411
2-hydroxybenzylideneamino 8.225 8.30 −0.402 −0.9150 to 0.6082 0.215
3(2H)-Benzofuranone 8.151 3.50 0.500 −0.5239 to 0.9330 0.156
9(2H)-acridone 3.173 3.76 −0.053 −0.8290 to 0.7926 0.460
a
p <0.05; signicant difference.
P.A. Akinduti et al.
Medicine in Microecology 22 (2024) 100115
5

cytosol free radicals (called metastable chemical species) leading to poor
cellular metabolism [52,53].
Moreover, the combined antibacterial activities of these phyto-
chemical substances possess ability to deplete the available oxygen
needed for the respiration or oxidation pathways, inhibition of protein
synthesis for bacteria cell replication, anti-oxidation of DNA, RNA,
proteins and lipids metabolic pathways [54]. Presence of several
phytochemical compounds (alkaloids, tannins, saponins, avonoids and
glycosides) in OsEO plays several key roles in synergistic antimicrobial
activity, having direct damaging interaction on bacteria cytosol and
DNA synthetic pathways [55]. Signicant and higher inhibitory activity
of OsEO against BV pathogens further supports the antibacterial activity
of the detected phytochemicals. In providing effective clinical man-
agement, OsEO would be effective in reducing the BV MDR infections.
Indicated high percent methanoazulene and benzodioxole-5-
carboxylic acid level with less amount of other metabolites including
reveal potent bioactive compounds previously identied to possess
antibacterial properties against Gram positive and resistant Gram-
negative bacteria [56,57]. These metabolites shared common proper-
ties against the bacteria hydrophilic outer membrane, via blockage of
hydrophobic compounds penetration required for cell membrane
metabolism [58]. Detected methanoazulene has potential to disrupt the
lipid structure of the bacteria cell, eliciting cytoplasmic leakages [56,
59]. And with the synergistic activity of benzodioxole-5-carboxylic acid,
more metabolic functional and cellular signal pathways could be
modulated [58]. Phenol has the ability to inhibit dihydrofolate reduc-
tase enzymes in bacterial cytosol, initiating the inactivation of the
supercoiling process of gyrase and also altering the ATP binding site of
bacteria gyrase B and DNA, thereby enhancing cleavage of topoisom-
erase IV enzyme-mediated DNA, leading to poor bacterial replication
[59]. In all, these metabolites cause structural changes and inadequate
functional activities of bacteria cytosol via disruption of several cellular
functions and cellular uidity [60].
Not only has these metabolites in OsEO are non-toxic [23], their
respective antibacterial properties enlisted it as suitable natural anti-
bacterial vaginosis compounds. Cyclohexene and methanoazulene pro-
vided a signicant association with antibacterial activity against the
strains. It is obvious that cyclohexene and methanoazulene have pros-
pect to be developed as lead antibacterial compounds and in formulation
of topical antibacterial agent for vaginal therapy against BV.
Study limitation: The heating effect of aqueous extract hydro-
distillation may reduce the potency of the oil metabolites thereby
reducing its antibacterial activities. The obtained small quantity of the
Essential oil having low aqueous solubility and bioavailability may limit
the clinical applications of this compound in a larger invitro study as
previously state [25,27].
5. Conclusion
Identication of MDR strains from the examined BV necessitates
empiric prescription of antibiotics therapy and substantive clinical
diagnosis. The biolm production and hemolytic activity of BV strains
would further intensify vaginal morbidity and tissue degeneration
causing more severe vaginal pathology. Since S. pyogenes, S. aureus, E.
coli, P. aeruginosa and C. freundii are major pathogens causing BV as
observed, Ocimum sanctum L. Essential Oil would be a valuable topical
antimicrobial agent for the management of vaginosis caused by these
organisms and could reduce the risk of development of resistance to
valuable antibiotics. Therefore, the choice of OsEO as antibacterial
therapy for BV and its signicant level of phytochemical metabolites
with associated antibacterial properties makes OsEO a promising anti-
bacterial agent for clinical management of BV. The presence of cyclo-
hexene and methanoazulene provide premise for developing
formulations as topical antimicrobial agents as prophylactics and addi-
tives in existing drug used for BV treatment.
CRediT authorship contribution statement
Paul Akinniyi Akinduti: Writing – review & editing, Writing –
original draft, Visualization, Validation, Supervision, Software, Re-
sources, Project administration, Methodology, Investigation, Funding
acquisition, Formal analysis, Data curation, Conceptualization. Olu-
washindara Lydia Osunlola: Software, Methodology, Investigation,
Data curation. Feyisikemi Adenike Adebekun: Methodology, Investi-
gation. David Temiloluwa Viavonu: Writing – review & editing, Su-
pervision, Investigation. Gift Nzubechi Elughi: Visualization,
Supervision, Methodology, Investigation, Conceptualization. Oluwase-
gun Popoola: Visualization, Software, Data curation. Somrat Adeola
Abdulsalami: Writing – review & editing, Visualization, Supervision,
Software, Methodology, Investigation, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Acknowledgements
The authors appreciate the management of Federal Medical Centre,
Abeokuta for assisting in sample storage and data collection.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.medmic.2024.100115.
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