Lactobacillus strains are thought to play a major role in protecting the vaginal environment from nonindigenous and potentially harmful microorganisms. This is accomplished through their production of lactic acid, resulting in a low and protective pH (3.5–4.5). Vaginal Lactobacillus species are also known to produce other antimicrobial compounds besides lactic acid, including target-specific bacteriocins,1 and broad-spectrum hydrogen peroxide.2Clostridium perfringens is an anaerobic Gram-positive bacterium known to be a common pathogen in humans, domestic animals, and wildlife, and is the primary cause of clostridial enteric disease in domestic animals. C. perfringens has 10 rRNA operons and 18 polymorphic sites among the 16S rRNA genes.3–5 A common feature of C. perfringens is the production of 17 exotoxins and enterotoxin (CPE). C. perfringens is subdivided into five toxinotypes (A–E). Isolates originating from humans with gastrointestinal diseases carrying both cpb2 and cpe have recently been described.6–8 Johansson et al9 reported that a wide genetic diversity of C. perfringens from isolates causing enteric diseases in humans, based on pulsed field gel electrophoresis analysis. C. perfringens is part of the normal vaginal flora of 1−27% of healthy women. Therefore, ascending infection from the vagina to the uterus may occur.6 Under appropriate conditions, the bacteria can cause endometritis that leads to sepsis.10 Meanwhile, C. perfringens infection may occur in the uterus, especially after abortions via the use of contaminated surgical instruments.10 However, the importance of Lactobacillus has been shown in preventing infections such as genital infections, urinary tract infections, and bacterial vaginitis, which are caused by reducing Lactobacillus and overgrowth of other microorganisms.11,12 Accordingly, this study was conducted with the aim of assessing the inhibitory capability of vaginal Lactobacillus metabolites against C. perfringens isolated from a woman's vagina.
2.1. Design of study
In this research, 100 reproductive-age women who were referred to the women's department of Imam Khomeini Hospital in Ahvaz, Iran, were considered for this study. The inclusion criteria called for healthy married women who were 25–50 years of age. The exclusion criteria included cervix injuries, previous abortion, and divorced women.
In a 35-year-old nonpregnant woman, the only bacterium isolated from a cervicovaginal smear sample was C. perfringens. On physical examination, she had high fever (39°C), a small amount of necrotic tissue, and mild inflammation of the uterus. The patient appeared anemic and icteric, although her blood pressure and pulse rate were normal. On examination, her abdomen was normal and no free air was seen on plain chest X-ray films. Blood tests showed evidence of mild inflammation, with a white blood cell count of 14,150 μL and a C-reactive protein level of 8.0 mg/dL. The cervicovaginal smear samples were collected from vaginal swabs of women, and the swabs were immediately placed in thioglycollate transport medium (Hi Media, Laboratories, Mumbai, India) and transported in ice bags to the university's laboratory. The samples were incubated for 48 hours at 37°C.
2.2. Isolation of bacteria
The collected swabs were placed on the selective medium for lactobacilli, De Man–Rogosa–Sharpe (MRS) agar (Conda, Madrid in Spain). The plates were incubated at 37°C for 24–48 hours under anaerobic conditions using a candle jar. The Lactobacillus were presumptively identified by their ability to grow well on MRS medium13 followed by differential biochemical tests including Gram staining, catalase test, fermentation of glucose, maltose, and sucrose in order to confirm the identification of Lactobacilli. Lactobacilli are rod-shaped, Gram-positive, fermentative, facultative anaerobic or microaerophilic organotrophs.
Isolation of C. perfringens from the vagina was accomplished by spreading the cervical swabs on a blood agar medium. Then plates were incubated anaerobically in a candle jar with gas packed for a period of 24–48 hours at 37°C.14 The colonies that showed double zone hemolysis on blood agar plates were considered C. perfringens. Thereafter, other diagnostic tests including stormy clot, Nagler test, gelatin hydrolysis test, and indole test were performed in order to confirm the identification of C. perfringens.
2.3. Antimicrobial compound extraction from Lactobacillus spp.
The isolated colonies of Lactobacillus grown on MRS agar were transferred into 100 mL of MRS broth and incubated in an anaerobic chamber at 37°C for 5 days. Then, the antimicrobial compound was extracted using the three following methods.
2.3.1. Method 1
After incubation, a part of the culture medium was directly mixed with ethyl acetate (50:50) and then stirred using a magnetic stirrer for 6 hours. The upper organic layer was separated using a separating funnel and centrifuged at 6000 rpm for 15 minutes. Then the ethyl acetate layer was removed and transferred into a clean flask. The extract was pooled and dried in a rotary evaporator (Heidolph in Schwabach, Germany) at 45°C. The yield from the extract was dissolved in methanol for antimicrobial susceptibility testing.
2.3.2. Method 2
The second part of the medium was shocked by immersion in boiling water for 1 minute, and then placed in cold water for 3 minutes. Then, the extraction was followed by adding ethyl acetate similar to the first method.
2.3.3. Method 3
The third part of the culture medium was stressed using an ultrasonic device for 3 minutes (160 W); then, similar to the first method, extraction of the antimicrobial compound was performed.
2.4. Determination of minimum inhibitory concentration of Lactobacillus spp. extracts
The minimum inhibitory concentrations (MICs) of Lactobacillus extracts against C perfringens isolates were determined using an improved E test method (AB Biodisk, Solna, Sweden) in order to show the antimicrobial activity of isolated Lactobacillus spp. In the improved E test, several AB Biodisks impregnated with different dilutions of the extracts were used instead of strips. In fact, it was a simulated version of the standard E test.
The C. perfringens suspensions of freshly grown cultures were prepared in sterile saline and adjusted to a density of 106 cells/mL, corresponding to 68–82% transmittance at 530 nm. The plate of Mueller−Hinton agar (Hi Media India in Mumbai, India) was inoculated by dipping a sterile cotton swab into the C. perfringens suspension and streaking it across the agar surface in three directions. The plates were dried at an ambient temperature for 15 minutes before applying the disks. Eight sterile disks (6 mm) were placed in a line on the agar surface. The Lactobacillus spp. extract was serially diluted in methanol, and 10 μL of each dilution was separately used to impregnate the disks. The plates were incubated for 24 hours at 37°C. The MIC values were read as the antimicrobial concentrations at the points where dense colonial growth intersected the disks. The test was performed in triplicate for each culture.15
A total of 59 Lactobacillus spp. strains and one strain of C. perfringens were isolated from 100 vaginal swabs by using different selective media and biochemical tests. The morphological and biochemical characteristics of all bacterial isolates were tested according to Bergey's Manual of Determinative Bacteriology.16
The antimicrobial activity of the extracted compounds from 10 Lactobacillus strains showed that the first method of extraction using an ultrasonic device has more activity compared with others. The obtained MICs from all tested Lactobacillus strains based on the modified E test ranged from 15.6 μg/mL to 31.2 μg/mL (Figs. 1 and 2).
It should be mentioned that the inhibitory capability of Lactobacillus metabolites against some vaginal/uterine opportunistic bacteria were also assessed as an ancillary study (Table 1). These pathogens included Candida albicans NCIM 7102, Staphylococcus aureus PTCC 1113, and Escherichia coli PTCC 1533.
C. perfringens, an anaerobic Gram-positive bacillus, is found in normal human intestinal and vaginal flora in approximately 25% of healthy women. The presence of C. perfringens in the vagina or uterus can be lethal because of the risk of developing sepsis and gas gangrene followed by genital sores or vaginal fissures that lead to additional gynecological examinations and even abortions. In cases involving gas gangrene, the mortality rate ranges from 50% to 85%.17 Although few cases of clostridial gas gangrene associated with uterine malignancy have been reported, further complication resulting in potentially fatal sepsis were possible outcomes in most of those cases.
The vaginal ecosystem is dynamic and contains microbiota that are protective against invading pathogens, including those causing urinary tract infections and sexually transmitted infections. Lactobacilli are the best-known bacteria of the normal vaginal microbiota as a probiotic.18 Antonio et al19 reported that women who were not colonized with H2O2-producing lactobacilli, such as Lactobacilli crispatus, Lactobacilli iners, Lactobacilli jensenii, Lactobacilli gasseri and Lactobacilli vaginalis, were 15 times more likely to have bacterial vaginosis than women who were colonized by these strains.19 In fact, owing to the rise of bacterial resistance against antibiotics, scientists are looking to benefit from probiotics in order to both strengthen the immune system and infection prevention and control.20,21 Previously, in many studies, the important role of vaginal lactobacilli as potential probiotics has been shown against such pathogens as Candida spp.,22 and the most common opportunistic bacterial (S. aureus, E. coli, S. agalactiae, and Klebsiella pneumoniae).23 In this study, the antimicrobial effect of the extracted compound of vaginal Lactobacilli was investigated on C. perfringens, in order to find the benefit of this probiotic to prevent the spread of clostridial infection in the uterus, reduce mortality, as well as reduce the use of antibiotics.
The results of this study showed that among the 100 vaginal samples, only one (1%) contained C. perfringens. Such frequency was reported to be 5% in the Jawetz book,24 1–27% of women in some published articles10,17 and also 1–27% of those patients who had optional abortions.25 Also, it was showed that 59 (59%) of the samples were Lactobacillus strains. These statistical results are consistent with other statistics that reported the prevalence of vaginal lactobacilli at a rate of 50–80%.26 Amin et. al.27 reported the prevalence of 49.5%. In the present study, all vaginal Lactobacillus strains showed significant antimicrobial activity (MIC ranged from 15.6 to 31.2 μg/ml) against C. perfringens like Amin's study (MIC against S. aureus: 4 μg/ml, MIC against Candida albicans: 1 μg/ml).27 Coolborn28 could isolate 8 Lactic acid bacteria from food sources and soil by using MRS medium and then evaluated their antimicrobial activity against some pathogenic bacteria with two methods of well-in agar and paper disc method. He defined that Lactic acid bacteria with inhibitory affinity over the bacterial indicators exhibited varying degrees of inhibitory zones.
In the present study, ethyl acetate was used to obtain the Lactobacillus extract from broth media similar to the antimicrobial extraction method of plants and also like Amin's study.27 Extracts obtained from all three methods were able to make zones of inhibition. However, inhibition zone diameter was much less in the second and third method than in first method. The weight of lactobacillus extract obtained from the second and third methods was reduced, as compared to the first method. In particular, it was reduced considerably more in the second method. In other studies, researchers used the filtered supernatant fluids from MRS liquid medium containing Lactobacillus cells as antimicrobial extract. In the ethyl acetate extraction method, we tried to partially purify the extract, and therefore the inhibition zones were much wider than in other studies. In our study, Sodium hydroxide (NaOH) was added to the extract in order to neutralize its PH. Therefore, the antimicrobial activity of extract was not related to its acidic nature. Moreover, the use of boiling method to obtain the extract indicated that the antimicrobial activity of extract was not related to bacteriocin (antibacterial proteins), which confirmed the results of Rokka's study.29
This study suggested that women without vaginal Lactobacillus strains might be susceptible to non-indigenous and potentially harmful microorganisms. Moreover, if the normal vaginal flora is not disturbed by antibiotics, vaginal lactobacilli can prevent pathogen growth and C. perfringens infections.
This work was financially supported by Grant Number: 95107 from Vice-Chancellor for Research Affairs of Ahvaz Jundishapur University of Medical Sciences.
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