Antimicrobial activities of some essential oils and antibiotics on multidrug-resistant microbial vaginitis

ABSTRACT


Introduction
Vaginitis is an inflammation of the vagina that can be caused by bacterial, fungal, parasites, viral infections, or chemical and physical irritation (Gupta et al., 2019). The vagina is usually acidic and contains normal flora of microorganisms. Certain conditions such as menstrual cycle, pregnancy, and cosmetic/hygienic agents (shampoos or shaving creams) can interfere with the acidic environment or normal flora. They can cause severe inflammation of the vagina and discharge. There are six types of vaginitis: bacterial (most common), candidal, parasitical, viral, atrophic and cytolytic vaginosis.

Bacterial vaginitis (BV) is an inflammation of the vagina caused by several bacterial species, including
Gardnerella vaginalis and Mobiluncus curtisii (Wilson and Wilson, 2021).
Hundreds of microorganisms were commonly isolated from vaginal swabs worldwide, which can be classified into normal microflora, opportunistic pathogens and true pathogens (Ravel et al., 2011). Bacterial vaginitis (BV) is an inflammation of the vagina caused by several bacterial species, including Gardnerella vaginalis, Mobiluncus curtisii and Neisseria gonorrhaeae. It is known to affect many women and is usually associated with other complications such as pelvic inflammatory disease (PID), preterm labor, and low birth weight, among others (Muzny and Schwebke, 2016).
The polymorphic opportunistic fungus Candida albicans is primarily responsible for vulvovaginal candidiasis (VVC), an incredibly prevalent mucosal infection of the lower female reproductive tract (FRT). C. albicans, a typical component of the human microbiota, frequently and asymptomatically colonies the vaginal lumen (Owolabi et al., 2018).
The goal of treating a candida infection is to lessen symptoms. Along with many topical azole formulations and regimens, oral fluconazole (Diflucan) in a single 150-mg dose is also readily accessible. When deciding between topical and oral treatment, there are various factors to consider (Roberts et al., 2015). Due to the calming nature of the topical treatment, topical medicines may offer more immediate relief. On the other hand, they may trigger localized hypersensitivity reactions that cause itching or burning. Available over the counter, many patients use topical antifungals to treat suspected vulvovaginal candidiasis (Paladine and Desai, 2018).
Antibiotic misuse has led to microorganism resistance, another issue harming public health. The appearance of multi-drug resistant bacteria and fungi leads to searching for new substances with antimicrobial activities. Plant extracts and essential oils are frequently used in popular medicine as remedies for many infectious diseases. Recently, medical plants that have fewer side effects and are more cost-effective have been considered due to the side effects, expense, and complexity of producing therapeutic-chemical materials. Salmonella spp., Staphylococcus aureus, Pseudomonas aeruginosa, and coagulase are part of pathogens acquired in the community and hospitals. Staphylococcus spp., Shigella spp, Enterococcus spp., Escherichia coli and Klebsiella pneumonia are some of the most common bacteria with multi-drug resistant organisms (MDROs). Consumers now have a high demand for novel antibiotics to combat diseases as a result of this (Sepahvand et al.,

2017).
Effective antiseptic, antibacterial, antiviral, antioxidant, antiparasitic, antifungal and insecticidal activity has been observed for essential oils. The ability to partition with the lipids found in bacteria and mitochondrial cell membranes, making them more effective by upsetting the cell structures, is a crucial property of essential oils and the substances that make them up. The significant loss of necessary chemicals and ions from the bacterial cell ultimately causes the bacterial cell's death. Some substances target the efflux pump systems in Gram-negative bacteria to regulate drug resistance (Evans et al., 2004). With the limitations of conventional drugs, using a new treatment for these diseases seems necessary. So, the purpose of the present work was to investigate the efficacy of some essential oils on bacterial and yeast vaginitis as a new promising alternative remedy.

Study the antibiotics sensitivity of the isolated microbial vaginitis isolates
Sixteen different commercial and broadspectrum antibiotic discs 6mm were selected for investigating their potency against the isolated bacterial isolates according to the standard Kirby-Bauer disk diffusion method (Bauer, 1966). Under sterilized conditions, four to five similar colonies from each bacterial isolate (overnight growth) were transferred separately into sterile distilled water and vigorously agitated to give turbidity that matches the 0.5 McFarland standard (approximately 10 6 CFU/ml) according to D'Amato and Hochstein, (1982). Within 15 min, a sterile cotton swab was dipped into the culture suspension for inoculating the surface of solidified Mueller-Hinton agar plates (Counts et al., 2007). Antibiotic discs were dispensed onto the inoculated plate's surfaces. After 15 minutes the plates were incubated at 37°C for 24h. The resulting diameters (mm) of inhibition zones around the antibiotic discs were measured. The results were interpreted according to protocols standardized for the assay of antibiotic compounds as guided by the National Committee for Clinical Laboratory Standards "NCCLS". The data were categorized as: R (resistant), I (intermediate sensitive), and S (sensitive) (Rijal et al., 2010). For sensitivity testing of yeast isolates, five broad-spectrum antifungal discs (Oxoid USA) from different families were used. The zone size chart 2018 was used for interpreting the data. The plates were inoculated by dipping a sterile swab into the inoculum suspension adjusted to the turbidity of a 0.5 McFarland standard (10 8 cells/ml) and streaking it across the surface of the SDA medium in all directions. Antibiotic discs were dispensed onto the inoculated plate surface. The plates were incubated at 28°C for 24h. The diameters (mm) of inhibition zones were measured (Khan et al., 2006).

Study the antimicrobial activities of the used essential oils against the most dominant MDR microbial vaginitis
In this experiment, seventeen essential oils were used for testing the antimicrobial activities against the pathogenic MDR bacterial and yeast vaginitis isolates according to the method described by Haller et al., (2015). A volume of 0.1 ml suspension of each of the selected pathogenic MDR bacterial and yeast isolates were inoculated separately on the surface of Mueller Hinton Agar plates and each was spread homogenously using a sterile glass rod and left to dry at 37 o C for 15 min. Three discs made from sterilized Whatman No.1 filter paper discs (3 replicates) with a diameter of 6 mm were applied to the prepared plates using sterile forceps and pressed gently against the agar surface. The sterile and refined essential oils were dissolved in sterilized 10% aqueous dimethyl sulfoxide (DMSO) with Tween 80 (0.5% v/v for easy diffusion) and impregnated with 10µl of each of the tested essential oils separately by using sterile automated pipette tips.
The plates were incubated at 37 o C for 24 h. The antimicrobial activity of each of the used essential oil against the selected MDR bacterial isolates was estimated by measuring the diameters (mm) of the inhibition zones by caliper and compared with negative control (Sterile filter paper saturated with sterile DMSO) and recorded as the average of three replicates.

Determination of the minimum inhibitory concentration (MIC) of the most effective essential oil against the most dominant MDR bacteria and Yeast isolates using ELISA reader
The (MIC) were determined by using the broth microdilution method as approved by the guidelines of Clinical and Laboratory Standards Institute (Mann and Markhon, 1998). Different concentrations of turmeric oil were prepared by suspending 1 gm of oil in 10 ml of 5% dimethyl sulfoxide solution (DMSO) to get stock solution of 100 mg/ml oil. Then half fold serial dilution was made for this stock with sterile distilled water to get oil concentrations of 50, 25, 12.5, 6.25, 3.125, 1.562, 0.781, 0.390, 0.195 and 0.097 mg/ml. The microtiter plates were prepared by adding 100μL of Mueller Hinton Broth (MHB) for bacteria and 100μL of Sabouraud Dextrose Broth (SDB) for yeast. Twenty microliters (20μL) of bacterial suspension (0.5 McFarland standards) and twenty microliters (20μL) of yeast suspension were added to each well except the control wells (control wells contained broth only and sterile distilled water only) (Thabaut and Meyran, 1984).

Effect of the combination between the most effective essential oils and the selected resisted antibiotics on the most dominant MDR bacterial and yeast isolates
The most effective essential oils Turmeric, Parsley, Garlic, and Black seed oils were tested in combination with the selected four resisted antibiotics Cefaclor, Nitrofurantion, Amoxicillin/Clavulinic acid and Levofloxacin, related to four different antibiotic families" patterns (Cephalosporins 2 nd generation, Nitrofurans, Penicillin combination and Quinolones/Floroquinolones families respectively) against the most dominant isolated MDR bacterial isolate. The selected bacterial isolate was cultured for study of the combination effects between the most effective essential oils and the four resisted antibiotics using disc diffusion method. A volume 0.1 ml of the bacterial suspension with final concentration 10 6 CFU/ml was dropped separately on the surface of the ( MHA) and was spread homogenously using sterile glass rod and left to let cells to settled down at room temperature for 15 min. Also the effect of the most effective essential oils in combination with the three selected resisted antibiotics; Metronidazole, Nystatin and Itraconazole which related to three different antibiotic families patterns (Nitroimidazole, Polyenes and Triazole families respectively) were tested against the most dominant MDR yeast (Van et al., 2009) using disc diffusion method as described above with the bacterial isolate but with inoculated the yeast isolate in sterile SDB and incubated for 48 h at 28 o C.

Molecular identification of the most inhibited pathogenic (MDR) bacteria by 16S rRNA
The extraction was done using Gene Jet genomic DNA purification Kit (Thermo) protocol (Tampieri et al., 2005). Amplification of the 16SRNA region was conducted in an automated thermal cycler (C1000 TM Thermal Cycler, Bio-RAD) using F (5`-AGA GTT TGA TCC TGG CTC AG -3`) and R (5`-GGT TAC CTT GTT ACG ACTT-3`) primers (Allen and Walter, 2016). Finally, sequencing was made to the PCR product on GATC Company using ABI 3730xl DNA sequencer using forward and reverse primers (Brugha et al., 2014).

Molecular identification of the most inhibited pathogenic (MDR) fungal by 16S rRNA
The surface of Sabrouaud's dextrose agar plate was scratched to remove the pseudomycelium of the most prevalent MDR yeast. In liquid nitrogen, 50 mg of fresh pseudomycelium was pulverized with a mortar and pestle. Using the genomic plant DNA extraction Mini Kit (iNtRON Biotechnology, Inc, Cat. Non 17371) and following the manufacturer's instructions, DNA was extracted from the powdered tissue. At -20 o C, the eluted DNA was kept. Internal transcribed spacer (ITS) region was conducted in an automated thermal cycler PCR machine. ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') primers were used in an automated thermal cycler (C1000TM Thermal Cycler, Bio-RAD) to amplify the ITS region (Hassan, et

Study the antivirulance activity of the most effective essential oils on formation of biofilm by the selected MDR isolates
The turmeric oil was tested for its potential to overcome and prevent biofilm formation of the most dominant MDR bacteria and yeast isolates. Different concentrations of turmeric oil (0. 097, 0.195, 0.390, 0.781, 1.562, 3.125, 6.25, 12.5, 25, 50 and 100 mg/ml) were tested against the most dominant MDR bacterial and yeast isolates. To acquire the initial concentrations in 100µl for microorganisms, an aliquot of two-fold serial dilutions was made on a 96-well microtiter plate with trypticase soy broth with 2% of resistant microbial vaginitis -Antimicrobial activities of some essential oils and antibiotics on multidrug glucose (TSBGlc). To get the initial concentrations in 100 µl for yeast, an aliquot of two-fold serial dilutions was made on a 96well microtiter plate using Sabouraud's dextrose broth. Then, bacterial suspensions (50 µl; final concentration: 5x10 5 CFU/ml) were poured into the plate. TSBGlc and Sabouraud's dextrose-containing distilled water were used as negative control. The positive control was inoculated TSBGlc and Sabouraud's dextrose without the essential oil

Detection the antimicrobial effect of turmeric oil on the structure of the most inhibited pathogenic MDR microbial vaginitis using Scanning Electron Microscope (SEM)
In this experiment, a scanning electron microscope (SEM) was applied to detect the effect of turmeric oil on the cellular structure of the pathogens compared with the control. The nutrient broth containing the growth of the most dominant MDR K. pneumoniae and C. albicans sensitive to Turmeric oil, control growth (without oil) and treated growth (with oil) were examined using SEM. It was analyzed using a scanning electron microscope (Model JEOL, JSM-5200 LV) and was applied in SEM Unit in the Faculty of Medicine, Tanta University. Treated cultures with Turmeric oil at (MIC) concentration of 0.781 mg/ml for the most dominant MDR bacterial and yeast isolates were incubated for 24h. At appropriate conditions for the type of bacteria and yeast. None treated bacterial, and yeast cultures from each bacterial type and yeast were included as a negative control, and then all of them were investigated by SEM (Altemimi et al., 2017).

Gas
Chromatography-Mass Spectrometry (GC-MS) analysis was used to determine the main compounds in turmeric essential oil, especially the antimicrobial agents. This analysis was carried out in the central lab of biochemistry, Tanta University (George et al., 2004). Turmeric essential oil was examined by gas chromatography, Mass spectroscopy in Claurs 580/560S. Work was done with column 30.0 m x 250 μm, Rtx-5MS (crossband 5% diphenyl 95% dimethyl polysiloxane). The GC conditions were employed using helium as carrier gas (0.8 ml/min), and the temperature program was 60°C for 1 min, followed by an increase of 10°C /min to 180°C for the remainder of the run. Detector and injection point heaters were 260 o C and 280°C, respectively and typically 0.1 or 1.0 µl was injected at a 20: 1 split (Peruzza et al., 2003).

Results
Fifty clinical vaginal swabs from ten different groups (each containing several cases) were investigated for their load of isolated bacteria, fungi, and parasites. These groups were arranged from 1 st to 10 th as the following: normal healthy, microbial vaginosis, pre-and post-vaginal area, pregnant, nosocomial infection, unexplained infertility, hyperglycemia with obesity, a child with poor hygiene, dysfunctions of the immune system and parasitic vaginal infection cases, respectively as shown in table (1). Table (2) showed that 47 microbial isolates were identified using an automated VITEK2 compact system, the identified microbial isolates belonged to ten genera, nine genera of bacteria and one genus of Candida, one species only related to each genus of bacteria and two species related to genus Candida. The results recorded 20 Klebsiella pneumoniae, 6 Escherichia coli, 4 Staphylococcus aureus, 1 Neisseria gonorrhoeae, 2 Pseudomonas aeruginosa, 1 Acinetobacter baumannii, 5 Lactobacillus plantarum, 1 Gardnerella vaginalis, 6 Candida albicans, 1 Candida lusitaniae and 1 Peptostreptococcus prevotii case no.33 specially identified by ID32A kit.

Antibiotics sensitivity testing for clinical vaginal bacterial and yeast isolates
The antibiotics sensitivity of the tested bacterial isolates showed different susceptibilities ranging from sensitive (S), intermediate (I) and resistant (R) reactions against 16 tested antibiotics as shown in table (2) , 9, 13, 14, 15, 25 28, 32, and 33 respectively and the numbers of antibiotics that were resisted by each MDR isolate were 2, 13,9,11,7,12,9,3 and 8 respectively where the inhibition zones ranged between 6 to 10 mm. in diameters. The remaining isolates exhibited various sensitive reactions as shown in table (3)

Effect of the combination between the most effective essential oils and the selected resisted antibiotics on the most dominant MDR bacterial and yeast isolates
The combination of the four selected resistant antibiotics with each of the most potent essential oils (turmeric, parsley, garlic, and black seed oils) against the pathogenic MDR K. pneumoniae was illustrated in Table  ( 5). The results showed the levofloxacin antibiotic with all essential oils (garlic, parsley, black seed, and turmeric oils) had the highest inhibition activity against the pathogenic MDR K. pneumoniae, according to the results, the combination of Cefaclor, Nitrofurantoin and Amoxicillin/Clavulanic acid antibiotics had no effect on the most dominant MDR bacterium. When compared to other garlic, black seed, and turmeric oils, the combination of levofloxacin antibiotic and parsley oil demonstrated the greatest impact against K. pneumoniae (the diameter of the inhibitory zone increases from 10 to 22 mm) ( Table 5). This result suggested that parsley oil and levofloxacin could be used to reduce K. pneumoniae"s resistance to the most efficient antimicrobial antibiotics. The results in table (6) demonstrated that the inhibitory effect of nystatin antibiotic appeared to increase when combined with all essential oils (garlic, parsley, resistant microbial vaginitis -Antimicrobial activities of some essential oils and antibiotics on multidrug black seed, and turmeric oils), while the combination with parsley oils and black seed showed the best effect against C. albicans (the diameter of the inhibition zone increases from 10 and 11 to 24 and 25 mm for parsley oils and black seed, respectively). Additionally, the combination of Metronidazole and Itraconazole antibiotics showed no effect on the most dominant MDR fungus (C. albicans). From the previous results the most effective combination between levofloxacin and nystatin antibiotics with parsley oils showed the highest effect against both MDR K. pneumoniae and C. albicans.  Molecular identification of the most inhibited MDR bacteria and yeast isolates.

Molecular identification of the most inhibited MDR bacterium (Klebsiella pneumoniae) by 16S rRNA
Klebsiella pneumoniae was the most dominant highly multidrug-resistant isolate from the nine MDR bacterial isolates and was the highly inhibited isolate by four of the tested essential oils. The identification of K. pneumoniae by VITEK 2 compact system was confirmed by molecular identification of 16S rRNA gene sequence using PCR. The results of identification were as shown in Fig. (3). By applying the biosystem 16S ribosomal RNA sequence, the tested Gram-negative bacterium isolate exhibited a similarity of 98% to the 16S ribosomal RNA sequence of Klebsiella pneumoniae subsp pneumoniae SUB11820947 Klebsiella-1 OP020451. The partial nucleotide sequence of 1021 bp 16s rDNA gene for K. pneumoniae isolate was done to determine the relationship with other recommended 16s DNA gene K. pneumoniae strains registered in GenBank. The sequencing was done from the forward direction at Mocrogen 3730X l6-1518-009 Korea Fig. (3).

Molecular identification of the most inhibited MDR yeast (Candida albicans) by 16S rRNA:
The pure band resulting from 18s rDNA required primer was partially sequenced. Sequences were then compared to the public database of the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool (BLAST).
The comparison gave 98% identity to Candida albicans SUB11826412 Candida -1 OP023836. The partial nucleotide sequence of 361 bp 18s rDNA gene for C. albicans isolate was done to determine the relationship with other recommended 16s DNA gene C. albicans strains registered in GenBank. The sequencing was done from the forward direction at Mocrogen 3730X l6-1518-009 Korea Fig. (4). The scanned images illustrated in (Fig.  5) showed severe damage to the tested microbe and lead to irregular cell shape with destroyed cell wall and shrinking of cells. Some of the cells were vacant, while others were flimsy. Additionally, most of them appeared to be melted and jammed together. Klebsiella that had been treated had enormous cells and appendages on their surface. Images of Candida (SEM) showed structures resembling pseudomycellium. The untreated (control) cells were whole and had a smooth surface. Generally, SEM images observations demonstrated physical damage and considerable morphological alteration to tested pathogenic microbial treated with turmeric oil extract. For confirmation of the antimicrobial activity of the turmeric oil against the tested vaginal pathogens GC-MS analysis was performed to detect the active ingredients and their concentrations in this extract, from table (7) and Fig. (6). The GC-MS determines the percentage and structure of major fragmentation ions component, Ar-turmerone as major ingredient 58.033%. The rest ingredients represented differential amounts as Curlone 14.000%, aromatic Ar-curcumene 7.025%, Phenol 4.515%, Zingiberene 3.480%, 1-Ethyl-4-isobutylbenzene 2.810%, α -Sesquiphellandrene 2.522%, 1,2,3,5tetramethyl-Benzene 1.761%, Benzene 1.381%, Benzaldehyde 1.330%, α -Bisabolene 1.207%, 4-Methyl-carbanilonitrile 1.094% and saline 0.842%. Ar-turmerone, Curlone, and Arcurcumene were represented with 58.033%, 14.000% and 7.025% respectively. These and other components in the analyzed oil were expected to be responsible for the previously recorded antimicrobial activity of turmeric oil against vaginal pathogens depending.

Discussion
The present study aimed to investigate the efficacy of some natural essential oils on the microbial vaginitis collected from different clinical labs. Fifty clinical vaginal microbes (each containing several cases) were investigated for their load of isolated bacteria, fungi, and parasites. The results showed that 47 microbial isolates were identified using an automated VITEK2 compact system, the identified microbial isolates as nine genera of bacteria and one genus of Candida, one species only related to each genus of bacteria and two species related to genus Candida. The results recorded 20 Klebsiella pneumoniae, 6 Escherichia coli, 4 Staphylococcus aureus, 1 Neisseria gonorrhoeae, 2 Pseudomonas aeruginosa, 1 Acinetobacter baumannii, 5 Lactobacillus plantarum, 1 Gardnerella vaginalis, 6 Candida albicans, 1 Candida lusitaniae and 1 Peptostreptococcus prevotii. The study by (Gokiladevi, 2019) bacterial vaginosis was diagnosed in 48%, candidiasis in 24%, and Trichomoniasis in 3.3% of the cases by clinical examination. Microbiological diagnosis indicated pathological organisms in 62% of cases, whereas in 38% of the cases, the discharge was physiological (Gokiladevi, 2019). Among the pathological organisms the common isolates were bacterial vaginosis 82%, candidiasis 14% and parasites 4% (Farhan et al., 2017).
The antibiotics sensitivity of the tested bacterial isolates showed different susceptibilities ranging from sensitive (S), intermediate (I) and resistant (R) reactions against 16 tested antibiotics. In this investigation, results showed that out of the thirty-seven pathogenic bacterial isolates, nine bacterial isolates exhibited multidrug resistance (MDR), theses bacterial isolates were L. plantarum, A. baumannii, N. gonorrhoeae, G. vaginalis, E. coli, P. aeruginosa, K. pneumoniae, S. aureus and P. prevotii. The remaining isolates exhibited various sensitive reactions. The antibiotic sensitivity of the most predominant vaginal isolates Klebsiella pneumonia isolated from the fifth group was designated as a multidrug resistance organism because it showed resistance to 9 different antibiotics and it possessed intermediate sensitivity to 4 antibiotics with sensitivity to only 3 ones (Barrett et al., 1999). Neisseria gonorrhoeae and Peptostreptococcus prevotii showed resistance same as K. pneumonia, however, they were not dominant isolates. This was coherent with studies in (Delgado et al., 2007), where they showed resistance of N. gonorrhoeae 37.5-42.5%, 50-94%, 25% and 50% resistance to ciprofloxacin, tetracycline, ampicillin, and erythromycin, respectively. In contrast, Staphylococcus aureus exhibited low levels of resistance to most of the tested antibiotics as well as Lactobacillus (Mwape et  al., 2021). The yeast isolates showed different susceptibilities ranging from sensitive (S) to resistant (R) reactions against the five tested antibiotics, Metronidazole (MTZ50), Fluconazole (FCA25), Itraconazole (IT30), Ketokenazole (KT10) and Nystatin (NS100). Two isolates out of the seven isolates of yeasts, C. albicans and C. lusitaniae showed MDR against Ketokenazole (KT10) and Itraconazole (IT30) of the used antibiotics respectively. The proportion of disease to be caused by Candida lusitaniae was about 2%. This proportion is like what was found in the most recent Centers for Disease Control and Prevention surveillance study (Barrett et al., 1999). Fluconazole still tends to be quite active against most isolates of Candida spp. (Tsega and mekonnen, 2019) showed a relatively stable C. albicans sensitivity to fluconazole this indicates that there is no ongoing decrease in the rate of fluconazole susceptibility.
Seventeen antibacterial plant essential oils were tested against nine MDR bacterial isolates. The results showed that turmeric, garlic, black seed and parsley oils exhibited the highest antibacterial and antifungal activities against the MDR bacteria and yeast isolates. However, the same bacterium and yeast isolates showed resistance to onion, green tea, peppermint, clove, lavender, marjoram, anise, fenugreek, basil, fennel, eucalypts, olive, and sage oils without any inhibition zones. (Enioutina et al., 2017). Azimi et al., (2011) studied the effect of anti-candida and antibacterial by different medical plants. The results showed that turmeric, parsley, garlic and black seed oils exhibited the highest antibacterial and antifungal activities. The antimicrobial activity of Nigella sativa oil (black seed oil) is attributed mainly to its phenolic constituents of the essential oil (Emeka et al., 2015). Other constituents, oleoresins, linoleic acid, and oleic acid may also have minor antimicrobial activity (Mohammed et al., 2019).
Klebsiella pneumoniae was the most dominant highly multidrug-resistant isolate from the nine MDR bacterial isolates and was the highly inhibited isolate by four of the tested essential oils. The identification of K. pneumoniae by VITEK 2 compact system was confirmed by molecular identification of 16S rRNA gene sequence using PCR. By applying the biosystem 16S ribosomal RNA sequence, the tested Gram-negative bacterium isolate exhibited a similarity of 98% to the 16S ribosomal RNA sequence of Klebsiella pneumoniae subsp. pneumoniae SUB11820947 Klebsiella-1 OP020451. In the case of fungal isolation by using 18s rDNA gene the comparison gave 98% identity to Candida albicans SUB11826412 Candida -1 OP023836.
The combination of the four selected resisted antibiotics with each of the most potent essential oils (turmeric, parsley, garlic, and black seed oils) against the pathogenic MDR K. pneumoniae. The results showed that Levofloxacin antibiotic with all essential oils (garlic, parsley, black seed, and turmeric oils) had the highest inhibition activity against the pathogenic MDR K. pneumoniae, the combination of levofloxacin antibiotic and parsley oil demonstrated the greatest impact against K. pneumoniae. This result suggested that parsley oil and levofloxacin could be used to reduce K. pneumoniae"s resistance to the most efficient antimicrobial antibiotics (Ribeiro et al., 2017). On the other hand, the combination with parsley oils and black seed showed the best effect against C. albicans. The previous results showed the most effective combination between levofloxacin and nystatin antibiotics with parsley oils showed the highest effect against both MDR K. pneumoniae and C. albicans. A value of combined essential oil situated between additive and antagonistic tendencies signifies an indifferent effect ( El  Atki et al., 2019; Sharma et al., 2020). The results of the current study show synergistic effects of situated essential oil and antibiotic mixtures against the tested bacterial and yeast isolates.
The antimicrobial effect of turmeric oil on the structure of the most inhibited pathogenic MDR microbial vaginitis using Scanning Electron Microscope (SEM) showed resistant microbial vaginitis -Antimicrobial activities of some essential oils and antibiotics on multidrug severe damage to the tested microbe and lead to irregular cell shape with destroyed cell wall and shrinking of cells. Some of the cells were vacant, while others were flimsy. Additionally, most of them appeared to be melted and jammed together. Klebsiella that had been treated had enormous cells and appendages on their surface ( Elhamy, et al., 2021). Images of Candida (SEM) showed structures resembling pseudomycellium. The untreated (control) cells were whole and had a smooth surface. Generally, SEM images observations demonstrated physical damage and considerable morphological alteration to tested pathogenic microbial treated with turmeric oil with turmeric oil at concentration 0.781 mg/ml.
For confirmation of the antimicrobial activity of the turmeric oil against the tested vaginal pathogens GC-MS analysis was performed to detect the active ingredients and their concentrations in this extract. It could be seen that curcumins and essential oils make up the majority of the extracts' ingredients. The essential oils contain Ar-turmerone (58.033%), Curlone (14%) and Ar-curcumene (7.025%) ( Crowley et al., 2012). Zingiberene (3.480%), phenol (4.51%), 1-Ethyl-4-isobutyl benzene (2.81%) and α -Sesquiphellandrene (2.52%) are the possibly active elements in essential oils that can cause apoptosis and have the potential to be used as new functional food ingredients for the treatment and prevention of non-small-cell lung cancer (Ma and Hovy, 2016). Both in vivo and in vitro, the ar-tumerone inhibits the proliferation of Hep-2 laryngeal carcinoma cells. (Candes, Romberg and Tao, 2006). 1,2,3,5-tetramethyl-Benzene (1.76%), Benzene (1.38%), Benzaldehyde (1.33%), α -Bisabolene (1.2%) and 4-Methylcarbanilonitrile (1.09%) in essential oil can protect against carbon tetrachloride (CCl 4 )induced liver fibrosis in rats by downregulating the expression levels of plasma endotoxin and serum tumor necrosis factor-α (TNF-α) (Qin et al., 2011). Thirteen compounds have been identified in essential oils, and the oils display remarkable antibacterial activity against S. aureus, L. monocytogenes, B. subtilis, P. aeruginosa, S. typhimurium and E. coli, (Rahman, Al-Reza and Kang, 2011). Essential oil's recognized constituents are frequently utilized as quality control indicators. Only a few of the purified single ingredients have reasonably high pharmacological effects, according to the pharmacological examination of those ingredients. Additionally, several studies show that the chemicals found in many Chinese medicine formulae and plants combine synergistically (Chen, et al., 2021). However, as secondary metabolites, essential oils vary greatly depending on the originating species, planting environment, and planting method. The quality and production of components are greatly influenced by the agroclimatic elements of growing conditions, such as rainfall, temperature, humidity, and soil nutrients