Abstract
Objective
To assess the probiotic nature of Lactobacillus in preventing cervical pathogens by studying the effectiveness of antimicrobial activity against vaginal pathogens.
Methods
Lactobacilli were isolated from healthy vaginal swabs on selective media and different pathogenic bacteria were isolated by using different selective media. The Lactobacillus strains were tested for the production of hydrogen peroxide and antimicrobial compounds along with probiotic properties.
Results
Of the 10 isolated Lactobacillus strains, strain 1, 3 and 6 are high hydrogen peroxide producers and the rest were low producers. Results of pH and amines tests indicated that pH increased with fishy odour in the vaginal fluids of cervicovaginal infection patients when compared with vaginal fluids of healthy persons. The isolates were found to be facultative anaerobic, Gram-positive, non-spore-forming, non-capsule forming and catalase-negative bacilli. The results of antimicrobial activity of compounds indicated that 280 and 140 µg/mL was the minimum concentration to inhibit the growth of both pathogens and test organisms respectively.
Conclusions
The results demonstrated that Lactobacillus producing antimicrobial compounds inhibits the growth of cervical pathogens, revealing that the hypothesis of preventing vaginal infection by administering probiotic organisms has a great appeal to patients, which colonize the vagina to help, restore and maintain healthy vagina.
Keywords: Antimicrobial compounds, Cervical infection, Hydrogen peroxide, Lactic acid bacteria, Minimal inhibitory concentrations
1. Introduction
The cervico-vaginal infections are one of the female morbid circumstances that account for the most frequent gynecological disorders and may cause serious abnormalities (tumors) associated with some viruses like human papilloma virus (HPV). HPV is one of the most common sexually transmitted pathogens and is strongly associated with pre-neoplastic and neoplastic lesions of the uterine cervix. It has been suggested that HPV infection alone may not be sufficient to promote cervical carcinogenesis and other co-factors could be involved, such as smoking, oral contraceptives, immuno suppression, vitamin deficiency, bacterial vaginosis and other sexually transmitted diseases[1]. A variety of microbial flora can be found in the vagina and cervix at different stages of health, growth or diseases[2]. An important group is the Lactobacillus bacteria, predominantly present in the urogenital microflora of healthy women and the obliteration of Lactobacilli in patients who develop urinary tract infections and also bacterial vaginosis and many other genital infections including cervical cancer, which has led to a focus on these bacteria[3],[4]. Lactobacilli, primarily facultative or strict anaerobes generally has a fastidious growth requirement. They prefer an acidic environment by producing lactic and other acids. In general, Lactobacilli have not been associated with disease and have been regarded as nonpathogenic members of the intestinal and urogenital flora[5]. Lactobacilli, through the antagonistic interaction with pathogenic bacteria, maintains the vaginal ecosystem in a healthy state. Regulatory processes are carried out by species of Lactobacillus that produces antibacterial compounds, such as lactic and other organic acids, hydrogen peroxide (H2O2) and bacteriocins. Bacteriocins are biologically active, low molecular-weight proteins or peptides that inhibit the growth of a variety of pathogenic bacteria which appear in the bacterial vaginosis or cervicovaginal infections.
Several investigators have isolated and partially purified bacteriocin from different species of Lactobacillus. Most of the investigations were conducted with nonhuman strains, predominantly isolated from food[6],[7]. Human isolates of Lactobacillus species were found to have more antagonistic activity against other pathogenic microorganisms. A strain isolated from human faeces produced a substance with potent inhibitory activity against a wide range of bacterial species. It inhibited anaerobic bacteria (Clostridium sp., Bacteroides sp., Bifidobacterium sp.) and members of the family Enterobacteriaceae, Pseudomonas sp., Staphylococcus sp. and Streptococcus sp.; however, it did not inhibit other Lactobacilli. The inhibitory activity occurred between pH 3 and pH 5 and was heat-stable[3]. Lactobacillus gasseri, the dominant species inhabiting human intestine[8], was found to produce bacteriocin that exhibited a wide spectrum of bactericidal activity against enteric pathogens[9]. Aroutcheva et al. isolated antibacterial proteins from Lactobacillus acidophilus obtained from urethral specimens that were active against Gardnerella vaginalis. A heat-resistant peptide was extracted from a vaginal isolate, Lactobacillus salivarius, which inhibited growth of Enterococcus faecalis, Enterococcus faecium, and Neisseria gonorrhoeae[10]. Therefore the present investigation is to study the role of Lactobacilli in preventing the cervical pathogens by producing antimicrobial compounds.
2. Materials and methods
2.1. Isolation of Lactobacillus
Cervicovaginal smear samples were collected from 20 patients who had abnormal smears or cervicograms from pathology laboratory, Department of Obstructions and Gynecology, Govt. Maternity Hospital, Tirupati, Andhra Pradesh, India. A total of four smears were also collected from healthy women who do not have any symptoms or vaginal diseases for the isolation of Lactobacilli species. Care was taken to avoid contamination of cervical smears and the swabs were immediately placed in thioglycolate transport medium.
2.2. Ethical approval
The present work was approved by the Institutional Ethical Committee (IEC), Sri Venkateswara Medical College, Tirupati along with the patient consent forms.
2.3. pH and amines test
Vaginal discharge and odour are frequent gynaecological complaints that result in women seeking medical care.
A portion of the undiluted vaginal material or one drop of the saline suspension was applied to the surface of a clean glass slide. One drop of 10% potassium hydroxide is added to the vaginal sample. The vapour layer above the surface of the slide was gently fanned to assess the presence of volatile amines which have a fishy odour[11].
2.4. Culturing
The collected healthy swabs were placed on the selective media for Lactobacilli, De Man, Rogosa and Sharpe (MRS) agar. The plates were incubated at 37 °C for 24-48 h under anaerobic conditions like candle light jar. The Lactobacillus was presumptively identified by their ability to grow well on MRS medium[5]. The pathogenic vaginal flora, isolated from the cervical swabs, were spread on different types of selective media like Salmonella-Shigella medium, Gardnerella selective agar with 5% Human Blood; Thayer-Martin agar medium, Trichomonas media and chocolate agar medium. The first three types of plates were incubated aerobically and the last one was incubated anaerobically in candle light jar for a period of 24-48 h at 37 °C[12].
2.5. Hydrogen peroxide production
All the isolated Lactobacillus species were tested for the production of H2O2 by exposure of organisms grown anaerobically on agar containing horseradish peroxidises and tetramethyl benzidine (TMB) agar[4]. Colonies of H2O2-producing organisms form a blue pigment as horseradish peroxidases oxidize TMB in the presence of bacterial derived H2O2.
2.6. Extraction of antimicrobial compound from broth
The isolated Lactobacillus grown on MRS agar for 24 h and the colonies were transferred into 15 mL of MRS broth and incubated in an anaerobic chamber at 37 °C for 18 h. This culture was used to inoculate one liter of MRS broth and incubated in an anaerobic chamber at 37 °C for 18 h. The bacteria were harvested in the early exponential growth phase. Bacterial cells were separated from the broth culture by centrifugation at 9 500 r/min for 25 min. The supernatant was filtered and the filtrate pH was adjusted to 5.5 with 12% ammonium hydroxide.
Protein from the supernatant was precipitated using the fractional precipitation method by adding increasing concentrations of ammonium sulfate (20%, 30%, 40%, 50%, 60% and 80%). After each precipitation, samples were centrifuged at 13 000 r/min for 15 min (4 °C). The sample was desalted by dialyzing at 5 °C using dialysis tubing against one liter deionized water. The entire dialysis required four changes of deionized water over 3 d. The dialyzed protein solution was frozen at -80 °C and lyophilized. Dried samples were stored at 4 °C. To separate protein agglomerated with Tween-80, the sample was defatted three times with chloroform/methanol in a ratio of 2:16. The aqueous and chloroform/methanol layers were air dried, dissolved in a small amount of phosphate buffer solution, and tested for antibacterial activity.
2.7. Estimation of total protein concentration
The total protein concentration was estimated in the supernatant by the modified Lowry's method in micro titer assay[13].
2.8. Demonstration of antimicrobial activity
2.8.1. Well diffusion method
The antimicrobial activity was determined by the well diffusion method. The isolated pathological strains were chosen as the target bacteria. The nutrient agar plates were inoculated with 100 µL of overnight cultures of pathogenic bacteria. Approximately 0.5 mm diameter wells were prepared by using borer and different concentrations (280, 140, 70, 35, 17.5, 71.25 and 8.6 µL) and standard streptomycin antibiotic for positive control of cell free supernatant was poured into the well and then incubated at 37 °C for 24 h.
2.8.2. Micro titer plate assay
Antimicrobial activity was assayed by determining the percentage of growth inhibition in pathogens by adding extracted compound[14]. A total of 50 µL of overnight culture of test [Escherichia coli (E. coli) and Bacillus] and the pathogens (Salmonella-1; Salmonella-2; Gardnerella; Chlamydia; Trichomonas; Neisseria) organisms were inoculated into 50 mL of nutrient broth and incubated at 37 °C for 1 to 3 h at 200 r/min until they grown to about 0.2 to 0.4 OD at 630 nm. Each well was filled with 100 µL of culture.
Different concentrations of cell free supernatants were prepared by serial dilution of 100 µL with 2 fold dilutions (280, 140, 70, 35, 17.5, 71.5 and 8.6 µg/mL of broth) and 100 µL of culture was added to each well which was made up to 200 µL with sterile broth. Microbial growth was measured in positive control (culture alone) and negative control (treated with 0.4% formaldehyde) after 12 h of incubation, read at 630 nm using ELISA reader (Bio-Rad, Germany). The percentage of reduction in growth of test bacteria due to the antimicrobial proteins was calculated as per the following formula mentioned below.
 |
2.9. Tests for probiotic characteristics
Screening of potential probiotic Lactobacillus was done by sequential probiotic characteristics like pH, temperature, and salt tolerance etc[15].
2.10. Morphological and biochemical tests for isolated strains
Isolates, grown on nutrient agar medium, were examined for Gram's staining, cell morphology and culture characteristics. Some biochemical characteristics of the cultures were studied by indole, methyl red, Voges Proskaur and citrate utilization tests, catalase, starch hydrolysis and lactose fermentation tests performed according to Bergey's manual of determinative bacteriology[16].
3. Results
3.1. Lactobacillus isolation
The vaginal samples, collected from 4 healthy women, were diluted and cultured on MRS agar plates. Colonies (196) were primarily picked up from MRS agar surface. Most of them showed round, small colonies without any pigment, and white to cream color. All of them were later tested for Gram's staining, catalase production and checked for spore formation. Only 24 of them showed Lactobacillius properties with Gram-positive, catalase-negative and, non-spore formation. Finally 10 potent antimicrobial isolates were selected for further analysis.
3.2. Isolation of pathogenic bacteria
A total of 6 bacterial pathogens were isolated from cervicovaginal infection patients by selective media and the pure cultures were maintained on nutrient agar medium for further testing. The cultures include Salmonella-1; Salmonella-2; Gardnerella; Chlamydia; Trichomonas; Neisseria.
3.3. Hydrogen peroxide production test
All the selected 10 isolates were tested for their ability to produce H2O2. Results are shown in Table 1. Isolates 1, 3 and 6 produced blue and 4, 7, 10 produced bluish brown color and the remaining isolates could not produce hydrogen peroxides, suggesting that the first 3 isolates were high H2O2 producers, and the next 3 isolates were medium producers and remaining were low producers.
Table 1. The colony colour of isolated Lactobacilli strains grown on agar plates containing TMB for the detection of H2O2 Production.
| Isolated strain | Colony colour | Level of H2O2 production |
| Isolate-1 | Blue | High |
| Isolate-2 | White | Low |
| Isolate-3 | Blue | High |
| Isolate-4 | Bluish brown | Medium |
| Isolate-5 | White | Low |
| Isolate-6 | Blue | High |
| Isolate-7 | Bluish brown | Medium |
| Isolate-8 | White | Low |
| Isolate-9 | White | Low |
| Isolate-10 | Bluish brown | Medium |
3.4. Extraction and estimation of total protein in cell free supernatant
Approximately 6 mL of cell free supernatant was obtained from the broth, and the supernatant was stored at -20 °C for further analysis. The total protein content of the supernatant was estimated 2.8 mg/mL.
3.5. Antimicrobial activity of extracted compound
Antimicrobial activity of the extracted compound was tested against the two laboratory organisms (Figure 1) and also tested against the isolated pathogenic bacteria (Figure 2). Results, presented in Table 2 and 3, demonstrated that 140 µg/mL was the minimum concentration to inhibit the growth of E. coli and Bacillus and 280 µg/mL was the minimum concentration to inhibit the growth of all pathogenic bacteria like Salmonella-1, Salmonella-2, Gardnerella, Chlamydia, Trichomonas and Neisseria, isolated from cervicovaginal infections. The same concentrations were also observed in micro-titer assay with a percentage of 96.2, 82.8, 91.5, 92.5, 79.2, 82.6, and 80.3, 97.1 for pathogens and laboratory organisms respectively.
Figure 1. Antimicrobial activity of lactic acid bacteria against laboratory test organisms (A: E. coli; B: Bacillus).
Figure 2. Antimicrobial activity of lactic acid bacteria against cervical pathogens (A: Salmonella; B: Gardnerella; C: Enterococci; D: Neisseria).
Table 2. Antimicrobial activity of bacteriocin by disk diffusion and micro-titer assay.
| Strains | Zone of Inhibition (cm) |
||||||||
| Laboratory organisms |
Pathogenic organisms |
||||||||
| E. coli | Bacillus | 1 | 2 | 3 | 4 | 5 | 6 | ||
| Isolate-1 | 0.4±0.2 | 0.2±0.0 | 0.2±0.1 | 0.3±0.2 | 0.4±0.2 | 0.6±0.0 | 0.2±0.0 | 0.2±0.2 | |
| Isolate-2 | 0.6±0.2 | 0.8±0.4 | 0.9±0.3 | 0.6±0.3 | 0.2±0.2 | 0.8±0.2 | 0.1±0.0 | 0.2±0.0 | |
| Isolate-3 | 0.4±0.2 | 0.4±0.2 | 0.8±0.4 | 0.4±0.2 | 0.1±0.0 | 0.3±0.2 | 0.2±0.2 | 0.1±0.0 | |
| Isolate-4 | 0.3±0.2 | 0.5±0.2 | 0.4±0.2 | 0.4±0.2 | 0.5±0.2 | 0.1±0.0 | 0.2±0.0 | - | |
| Isolate-5 | 0.5±0.2 | 0.2±0.2 | 0.4±0.2 | - | 0.2±0.2 | 0.2±0.2 | 0.4±0.2 | 0.1±0.0 | |
| Isolate-6 | 0.5±0.2 | 0.1±0.0 | - | 0.4±0.2 | 0.4±0.2 | 0.3±0.2 | 0.4±0.2 | - | |
| Isolate-7 | 0.5±0.2 | 0.1±0.2 | - | 0.3±0.2 | - | 0.2±0.2 | 0.5±0.3 | - | |
| Isolate-8 | 1.0±0.3 | 0.9±0.3 | 0.9±0.3 | 0.4±0.2 | - | 0.2±0.0 | 0.1±0.0 | - | |
| Isolate-9 | 1.5±0.2 | 1.0±0.0 | 0.9±0.4 | 0.5±0.2 | 0.8±0.3 | 0.4±0.2 | 0.2±0.0 | - | |
| Isolate-10 | 1.0±0.2 | 0.8±0.5 | 0.4±0.2 | 1.0±0.0 | 0.2±0.1 | 0.2±0.2 | 0.1±0.0 | - | |
| Control | 1.5±0.1 | 1.0±0.2 | 0.9±0.2 | 0.9±0.2 | 1.2±0.3 | 0.9±0.4 | 0.7±0.4 | 0.5±0.2 | |
1: Salmonella-1; 2: Salmonella-2; 3: Gardnerella; 4: Enterococci; 5: Neisseria-1; 6: Neisseria-2.
Table 3. Reduction of microbial growth by the antimicrobial compounds extracted from isolated laboratory strains against pathogenic and laboratory test organisms.
| Isolated laboratory strains | % of growth reduction in micro-titre assay |
||||||||
| Laboratory test organisms |
Pathogenic organisms |
||||||||
| E. coli | Bacillus | 1 | 2 | 3 | 4 | 5 | 6 | ||
| Isolate-1 | 51.6 | 97.1 | 96.2 | 80.0 | 84.0 | 85.6 | 66.9 | 71.1 | |
| Isolate-2 | 83.5 | 27.1 | 87.0 | 81.0 | 91.5 | 92.5 | 66.1 | 61.2 | |
| Isolate-3 | 30.5 | 80.6 | 21.0 | 82.8 | 79.5 | 91.5 | 77.7 | 41.4 | |
| Isolate-4 | 77.0 | 81.6 | 88.0 | 82.6 | 79.7 | 52.1 | 62.0 | 32.6 | |
| Isolate-5 | 30.5 | 82.8 | 81.0 | 12.4 | 87.9 | 79.4 | 53.4 | 80.2 | |
| Isolate-6 | 40.9 | 46.6 | 89.0 | 36.9 | 77.0 | 88.6 | 49.1 | 20.6 | |
| Isolate-7 | 80.3 | 82.6 | 21.0 | 66.0 | 7.0 | 36.1 | 60.1 | 22.4 | |
| Isolate-8 | 68.0 | 81.5 | 12.7 | 55.7 | 6.3 | 92.5 | 30.3 | 20.4 | |
| Isolate-9 | 21.6 | 84.9 | 49.0 | 65.3 | 63.5 | 81.6 | 26.5 | 17.9 | |
| Isolate-10 | 77.0 | 88.4 | 87.0 | 33.0 | 68.5 | 82.1 | 79.2 | 68.4 | |
1: Salmonella-1; 2: Salmonella-2; 3: Gardnerella; 4: Enterococci; 5: Neisseria-1; 6: Neisseria-2.
3.6. Tests for probiotic characteristics
All the 10 isolates were tested for their ability to test the probiotic property, and the results were tabulated in Table 4. The optimum pH for all the isolates was ranged from 2-5, beyond which they could not grow luxuriantly. Similarly the optimum temperature for all the isolates was found to be 30-40 °C except for the isolates 6 and 7, which grown even at 50 °C. Isolates 6, 7 and 10 showed high salt tolerance as evidenced by their conspicuous growth even at 6% NaCl where the remaining isolates grown at 4% NaCl concentration.
Table 4. Probiotic characters of the isolated strains grown at different NaCl concentrations.
| Strain | pH |
Temperature (°C) |
Concentration of NaCl (%) |
|||||||||||
| 2 | 5 | 7 | 9 | 30 | 40 | 50 | 60 | 3 | 4 | 6 | 7 | |||
| Isolate-1 | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | -Ve | -Ve | ||
| Isolate-2 | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | -Ve | -Ve | ||
| Isolate-3 | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | -Ve | -Ve | ||
| Isolate-4 | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | -Ve | -Ve | ||
| Isolate-5 | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | -Ve | -Ve | ||
| Isolate-6 | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | ||
| Isolate-7 | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | +Ve | +Ve | ||
| Isolate-8 | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | -Ve | -Ve | ||
| Isolate-9 | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | -Ve | -Ve | ||
| Isolate-10 | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | -Ve | -Ve | +Ve | +Ve | +Ve | +Ve | ||
+Ve: presence of growth; -Ve: no growth.
3.7. Morphological and biochemical tests
All the isolated Lactobacillus strains were tested according to Bergey's manual of determinative bacteriology, and the results were tabulated in Table 5, which shows that all of the Lactobacillus strains were rod shaped, Gram-positive bacteria with presumptive test of catalase-negative.
Table 5. Morphological and biochemical tests for isolated strains.
| Strain No. | Test | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 1 | Gram's staining | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve |
| 2 | Catalase | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve |
| 3 | Starch hydrolysis | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve |
| 4 | Indole test | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve |
| 5 | Methyl red | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve |
| 6 | Voges Proskaur test | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve |
| 7 | Citrate utilization test | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve |
| 8 | Lactose fermentation | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve | +Ve |
| 9 | Spore formation | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve |
| 10 | Capsule formation | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve | -Ve |
+Ve: positive; -Ve: negative.
4. Discussion
The presence of HPV in human cervical cells has been shown to be an initiating factor in the development of cervical dysplasia[17]. However, not all patients harboring HPV develop cervical dysplasia, indicating the presence of additional promoting factors. Multiple studies have shown that patients with bacterial vaginosis have a higher incidence of cervical dysplasia[18]–[20]. Bacterial infection or vaginosis is a polymicrobial syndrome involving the genital tract and cervix, characterized by the replacement of Lactobacilli- predominant flora with anaerobic pathogens[21]. Lactobacilli have been shown to be the predominant bacteria, found in the normal vaginal microbial flora of women in reproductive age. Lactobacilli, principally the strains that produce H2O2 and other antimicrobial compounds, may have a protective effect against vaginal pathogens including gonorrhea, Staphylococcus and Shigella[22]. In the present study, of the 196 isolated strains, 10 had inhibitory effects on laboratory test organisms and produced H2O2 at different concentrations. All the 10 strains were tested for probiotic characteristics; isolates 6 could grow even at 60 °C, and 7% NaCl concentrations, isolates 7 and 10 grown at 40 °C, and 7% NaCl respectively. Positive results of probiotic treatment were obtained from the studies of Anukam[23], showing 55% satisfaction of treatment of bacterial vaginosis with intravaginal capsules containing human derived H2O2-producing Lactobacillus. Lactobacilli prevent other bacteria by the production of H2O2, bacteriocin and some organic acids. The production of organic acids by Lactobacilli maintains the acidic pH and controls the other bacteria. In addition, the vaginal pH was identified as alkali ranging from 7-10, with fishy odour in patients suffering cervicovaginal infection. As stated above, Lactobacillus can produce lactic acid and some other organic acids which produces the antimicrobial activity of the micro-organisms[24]. Therefore the existence of antimicrobial substances present in the isolates studied would be partly due to organic acids, because the present investigation showed that the antimicrobial activity of the test isolates was more active at acidic pH than at alkali pH.
In the present investigations, the antimicrobial activity of probiotics was tested against the pathogenic bacteria isolated from vaginal swabs. The partially purified bacteriocin protein isolated from the culture medium inhibits the growth of all tested laboratory test organisms as well as pathogens. The present study correlates with many other studies which have revealed that Lactobacillus could produce organic acids, hydrogen peroxide and bacteriocins[25]. It is also noted that the extracted supernatant contains 2.8 mg/mL of total protein in a 100 mL of production medium. The extracted compound was tested for their antimicrobial activity, using a micro-titer assay method. The compound was serially diluted in a two-fold dilution to obtain minimal inhibitory concentration (MIC) for the inhibition of bacterial growth. Of the tested concentrations, 140 µg/mL was found to be the MIC for both the E. coli and Bacillus. However a higher concentration of the antibacterial compound was needed to inhibit the pathogenic bacteria like Gardnerella vaginalis with 280 µg/mL MIC and Chlamydia trachomatis with 140 µg/mL MIC.
In recent years, the use of probiotics has received greater attention as an alternative, inexpensive and natural remedy to restore and maintain health[26]. Two strains, Lactobacilli GG and Lactobacilli GR-1 appear to be effective in colonizing and protecting the intestine and urogenital tract respectively against microbial infection[27].
In conclusion, antimicrobial compounds present in the Lactobacillus isolated from healthy vaginal swabs shows in vitro antimicrobial activity against cervicovaginal pathogens and are more active under acidic conditions. Hence, it might be used as bioprotective agents to control the bacterial infections and maintain the normal healthy vagina that may lead to decreased risk assessment.
Acknowledgments
The first author extends his thanks to the Technopreneur Promotion Programme (TePP-DSIR), Ministry of Science and Technology, Govt. of India, the UGC-BSR-Non SAP, to DBT-BIF facility at Department of Zoology, Sri Venkateswara University, Tirupati, for their technical/scientific assistance.
Comments
Background
The health of the female genital tract depends significantly upon the composition of the vaginal micro flora. These organisms produce acid substances to avid other pathogenic microorganism including bacteria, fungi and other parasites from the vagina. Therefore only facts that decrease acidity of the vagina can cause a vaginal infections, and in most cases vaginal infections reach the cervix. the present paper clearly illustrates the role of Lactobacilli which are dominant in healthy vaginal micro flora on the pathogenic bacteria which causes cervicovaginal infections.
Research frontiers
The present study is being performed in order to determine the effect of healthy vaginal Lactobacilli on the bacteria which causes cervicovaginal infections. Nowadays use of probiotics has received great attention as an alternative inexpensive and natural remedy to restore and maintain health. In this paper the authors have experimentally proved that Lactobacilli isolated from healthy vaginal swabs showed an antimicrobial activity against cervicovaginal pathogen.
Related reports
In this study, heavy programme has been recorded in terms of treatment of cervicovaginal infections by increasing numerous pharmaceutically active substances as well as broadening the knowledge on the pathogenic and physiopathogenic mechanisms of genital infections.
Innovations & breakthroughs
In this study the probiotic bacterial strain Lactobacilli isolated from vaginal swabs shows in vitro antimicrobial activity against cervicovaginal pathogen. This study is very useful and innovative for modern medicine.
Applications
This study is useful for controlling the bacterial infections and maintaining the normal healthy vagina that may lead to decreased risk assessment.
Peer review
The results of the present study are very interesting and suggest that the Lactobacilli isolated from healthy vagina may serve as probiotics. This is a good study in which authors explained the role of Lactobacilli as probiotc to cure cervicovaginal infections.
Footnotes
Foundation Project: This work is supported by UGC-BSR-Non SAP (Grant No. 4-1/2008).
Conflict of interest statement: We declare that we have no conflict of interest.
References
- 1.Castel PE, Giuliano AR. Genital tract infections, cervical inflammation, and antioxidant nutrients-assessing their roles as human papillomavirus cofactors. J Natl Cancer Inst Monogr. 2003;2003(31):29–34. doi: 10.1093/oxfordjournals.jncimonographs.a003478. [DOI] [PubMed] [Google Scholar]
- 2.Stykova E, Valocky I, Novotny F, Guba P. Probiotic properties of vaginal lactic acid bacteria selected for harmonization of microenvironment of reproductive apparatus. J Microbiol Biotechnol Food Sci. 2012;2(1):359–405. [Google Scholar]
- 3.Ehrström S, Daroczy K, Rylander E, Samuelsson C, Johannesson U, Anzén B, et al. Lactic acid bacteria colonization and clinical outcome after probiotic supplementation in conventionally treated bacterial vaginosis and vulvovaginal candidiasis. Microbes Infect. 2010;12(10):691–699. doi: 10.1016/j.micinf.2010.04.010. [DOI] [PubMed] [Google Scholar]
- 4.Choi HS, Kim KM, Kim CH, Kim SM, Oh JS. Hydrogen peroxide producing Lactobacilli in women with cervical neoplasia. Cancer Res Treat. 2006;38(2):108–111. doi: 10.4143/crt.2006.38.2.108. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Ali OA. Inhibition of uropathogenic Citrobacter adhesion by biosurfactants extracted from vaginal Lactobacillus acidophilus. Anb Med J. 2012;10:59–67. [Google Scholar]
- 6.Sarika AR, Lipton AP, Aishwarya MS. Bacteriocin production by a new isolate of Lactobacillus rhamnosus GP1 under different culture conditions. Adv J Food Sci Technol. 2010;2(5):291–297. [Google Scholar]
- 7.Sankar NR, Priyanka VD, Reddy PS, Rajanikanth P, Kumar VK, Indira M, et al. Purification and characterization of bacteriocin produced by Lactobacillus plantarum isolated from cow milk. Int J Microbiol Res. 2012;3(2):133–137. [Google Scholar]
- 8.Morelli L. In vitro selection of probiotic Lactobacilli: a critical appraisal. Curr Issues Intest Microbiol. 2000;1(2):59–67. [PubMed] [Google Scholar]
- 9.Jena PK, Trivedi D, Chaudhary H, Sahoo TK, Seshadri S. Bacteriocin PJ4 active against enteric pathogen produced by Lactobacillus helveticus PJ4 isolated from gut microflora of wistar rat (Rattus norvegicus): partial purification and characterization of bacteriocin. Appl Biochem Biotechnol. 2013;169(7):2088–2100. doi: 10.1007/s12010-012-0044-7. [DOI] [PubMed] [Google Scholar]
- 10.Aroutcheva AA, Simoes JA, Faro S. Antimicrobial protein produced by vaginal Lactobacillus acidophilus that inhibits Gardnerella vaginalis. Infect Dis Obstet Gynecol. 2001;9(1):33–39. doi: 10.1155/S1064744901000060. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Thulkar J, Kriplani A, Agarwal N. Utility of pH test & whiff test in syndromic approach of abnormal vaginal discharge. Indian J Med Res. 2010;131:445–448. [PubMed] [Google Scholar]
- 12.Agbakoba NR, Adetosoye AI, Adewole IF, Chukwuma CM. Isolation of vaginal pathogens along with genital mycoplasmas from asymptomatic gynaecology and antenatal clinic attendees. Am Eurasian J Sci Res. 2008;3(2):195–198. [Google Scholar]
- 13.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193(1):265–275. [PubMed] [Google Scholar]
- 14.Hultmark D, Engstrom A, Anderson K, Steiner H, Bennich H, Boman HG. Insect immunity. Attacins, a family of antibacterial proteins from Hyalophora cecropia. EMBO J. 1983;2(4):571–576. doi: 10.1002/j.1460-2075.1983.tb01465.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Fernandez MF, Boris S, Barbes C. Probiotic properties of human Lactobacilli strains to be used in the gastrointestinal tract. J Appl Microbiol. 2003;94(3):449–455. doi: 10.1046/j.1365-2672.2003.01850.x. [DOI] [PubMed] [Google Scholar]
- 16.Bergey DH. Bergey's manual of sysytematic bacteriology. Baltimore: Williams and Wilkins; 1984. [Google Scholar]
- 17.Eileen MB. Human papillomavirus and cervical cancer. Clin Microbiol Rev. 2003;16:1–17. doi: 10.1128/CMR.16.1.1-17.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Cassisi JA, Davis J, Clark P, Duff P. The association between abnormal cervical cytology and bacterial vaginosis. Obstet Gynecol. 2000;95(Suppl 1):S53. [Google Scholar]
- 19.Janjić Z, Trajković ZP, Folić M, Popović J, Petrić A, Vukomanović P. Frequency of cervicalintraepithelial neoplasia and carcinomas in women with and without bacterial vaginosis. Acta Medica Medianae. 2011;50(1):5–10. [Google Scholar]
- 20.Gillet E, Meys JF, Verstraelen H, Verhelst R, De Sutter P, Temmerman M, et al. Association between bacterial vaginosis and cervical intraepithelial neoplasia: systematic review and meta-analysis. PLoS One. 2012;7(10):45201. doi: 10.1371/journal.pone.0045201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Luckic A, Canzio C, Patella A, Giovagnoli MR, Cipriani P, Frega A, et al. Determination of cervicivaginal microorganisms in women with abnormal cervical cytology: The role of Ureaplasma urealyticum. Anticancer Res. 2006;26:4843–4850. [PubMed] [Google Scholar]
- 22.Dembele T, Obdrazalek V, Votava M. Inhibition of bacterial pathogens by Lactobacilli. Zentralbl Bacterial. 1998;288(3):395–401. doi: 10.1016/s0934-8840(98)80013-3. [DOI] [PubMed] [Google Scholar]
- 23.Anukam KC, Osazuwa E, Osemene GI, Ehigiagbe F, Bruce AW, Reid G. Clinical study comparing probiotic Lactobacillus GR-1 and RC-14 with metronidazole vaginal gel to treat symptomatic bacterial vaginosis. Microbes Infect. 2006;8(12–13):2772–2776. doi: 10.1016/j.micinf.2006.08.008. [DOI] [PubMed] [Google Scholar]
- 24.Reid G. Probiotic agents to protect the urogenital tract against infection. Am J Clin Nutr. 2001;73(Suppl 2):S437–S443. doi: 10.1093/ajcn/73.2.437s. [DOI] [PubMed] [Google Scholar]
- 25.Soccol CR, Vandenberghe LP, Spier MR, Medeiros AB, Yamaguishi CT, Lindner JD, et al. The potential of probiotics: a review. Food Technol Biotechnol. 2010;48(4):413–434. [Google Scholar]
- 26.Gaggia F, Gioia DD, Baffoni L, Biavati B. The role of protective and probiotic cultures in food and feed and their impact in food safety. Trends Food Sci Technol. 2011;22(Suppl 1):S58–S66. [Google Scholar]
- 27.Marelli G, Papaleo E, Ferrari A. Lactobacilli for prevention of urogenital infections: a review. Eur Rev Med Pharmacol Sci. 2004;8(2):87–95. [PubMed] [Google Scholar]