Zainab A. Fadhil (1)
Background: Bacterial vaginosis (BV) is a common vaginal condition characterized by a shift from lactobacilli dominance to opportunistic bacteria. Specific Background: Recent observations indicate the increasing role of Staphylococcus spp., particularly multidrug-resistant strains, in persistent or recurrent BV. Knowledge Gap: Limited evidence exists regarding species-level identification and resistance profiles of Staphylococcus spp. in BV cases in Iraq. Aim: This study aimed to identify the bacterial species associated with BV and determine the antimicrobial susceptibility of the most resistant isolates. Results: Among 50 clinical samples, 20 yielded bacterial growth, dominated by Gram-positive isolates (65%), especially Staphylococcus haemolyticus (40%), S. aureus (15%), and S. epidermidis (10%). Gram-negative isolates included E. coli (20%) and Klebsiella pneumoniae (10%). Gardnerella vaginalis was detected in 5%. Antibiotic testing of 16 isolates showed high multidrug resistance, with S. haemolyticus exhibiting resistance to OFX, CRO, AMC, CTX, EM, CFM, and NA. Novelty: This study provides the first localized profiling of MDR Staphylococcus spp. in BV using VITEK-2 confirmation. Implications: Findings highlight the need for routine species identification, antimicrobial stewardship, and region-specific treatment guidelines to prevent rising resistance and recurrence.
Highlights• Dominance of MDR Staphylococcus spp. in BV• High resistance to β-lactams and macrolides• Importance of routine screening and antibiotic stewardship
Keywords: Bacterial Vaginosis, Staphylococcus Haemolyticus, Multidrug Resistance, VITEK-2, Vaginal Microbiota
One of the most prevalent illnesses among women of reproductive age is bacterial vaginosis (BV), which raises the risk of premature birth infections and pelvic inflammatory disease (PID). which are the second leading cause of neonatal death worldwide [1]. Numerous studies highlight the importance of normal vaginal flora. The presence of vaginal microorganisms, predominantly Lactobacillus species, plays a crucial role in regulating vaginal health and preventing diseases.[2] A decrease in Lactobacillus and an increase in the number or type of facultative and anaerobic bacteria alter the vaginal bacterial balance, leading to an increase in pathogenic vaginal bacteria and causing BV. This, in turn, increases vaginal pH, which has been linked to increased susceptibility to and transmission of sexually transmitted infections [3]. Lactobacillus regulates the acidic environment of the vagina by producing abundant lactic acid, which acidifies the vagina. A pH <4.5 effectively contributes to protecting the vagina from pathogenic bacterial and viral infections [4].Hormonal changes in the vagina play a significant role in altering the natural vaginal flora, leading to inflammation. Normal estrogen levels are essential for maintaining vaginal balance and resistance to bacterial infections, as this hormone stimulates and activates the growth and integrity of the vaginal epithelium [5].
Numerous studies have established that various types of bacteria cause bacterial vaginosis, a polymicrobial infection. There are numerous causes of female reproductive tract infections (particularly vaginal and cervical infections), such as bacteria. [6]. These constitute (40-50%) of cases, with yeasts accounting for (30-40%), in addition to parasitic and viral infections. Vaginitis occurs in 20-40% of cases as a result of mixed infections caused by several organisms simultaneously [7].
The primary line of therapy for bacterial vaginosis is antibiotics. Intravaginal clindamycin and oral or intravaginal metronidazole are suggested treatment plans. [8]. These treatments have similar short-term efficacy against the illness. Within six to twelve months after finishing antibiotic therapy, bacterial vaginosis recurs in 50% to 80% of women. Antimicrobial resistance, biofilm development, reinfection through sexual partners, and the inability to restore ideal vaginal flora are all hypothesised causes of this therapeutic failure. [9]. Inaccurate diagnosis of bacterial vaginosis in women and premature initiation of treatment without prior drug sensitivity testing and laboratory examinations can lead to the emergence of antibiotic-resistant bacterial strains. Furthermore, the indiscriminate and excessive use of antibiotics has contributed to the development of resistant bacterial strains, and this resistance often becomes widespread [10]. Antimicrobial resistance may be influenced by biofilm, which is more commonly seen in people with recurrent bacterial vaginosis than in healthy people or those who have only had one episode. Even if bacterial vaginosis is successfully treated with antibiotics, the bacterial biofilm decreases the penetration of antimicrobials. Bacterial vaginosis (BV) requires a variety of treatment and preventative approaches since the biofilm is clinically persistent.[11]. Controlling pH, disrupting biofilms, and adhering to dietary changes, hormonal contraceptives, and condom use are some of these tactics. [12].
In this study, (50) vaginal discharge samples were collected from pregnant women (35) and non-pregnant women (15), for the period from February 2023 to March 2023, from women arriving at Al-Batoul Teaching Hospital in Diyala Governorate and Al-Khansaa Teaching Hospital in Mosul. These samples included women aged 20-50 years, pregnant and non-pregnant, who had clinical symptoms associated with bacterial vaginosis (BV) and were transferred to the laboratory.
The bacterial isolates obtained were diagnosed according to the following criteria [13].
2.2.1 Morphological Identification
The morphological characteristics of the bacterial isolates were studied on blood agar and MacConkey agar, including colony size, colour, consistency, margins, and other properties [14].
2.2.2 Microscopic Diagnosis
Bacterial isolates were diagnosed using Gram staining. Several colonies growing on blood agar or MacConkey agar were placed on a clean glass slide containing a drop of normal saline. The slide was spread on the slide and allowed to dry. It was then heat-fixed by rapidly passing it over a flame two or three times. Finally, it was stained with Gram stain and examined under a microscope to observe the morphology, color, cell aggregation, and staining pattern [15].
Before biochemical diagnosis, bacteria were activated on blood agar. Diagnostic tests, as described in Forbes et al. (2007) [13]. were used to identify bacterial isolates at the species level, as follows:
2 .2.3.1 Catalase Test
By turning hydrogen peroxide into water and oxygen gas, this test was used to determine if bacterial isolates were capable of producing the catalase enzyme. A tiny amount of the 18–24-hour-old bacterial growth was transferred onto the surface of a dry, clean glass slide in order to conduct this test. Hydrogen peroxide (H2O2) was added in a few drops. A positive result, or the release of oxygen gas, is shown by the production of air bubbles on the glass slide's surface. This shows that the isolates are capable of producing the catalase enzyme. (14).
2.2.3.2 Oxidase Test
This test was used to detect the ability of bacterial isolates to produce the oxidase enzyme. Filter paper was saturated with several drops of oxidase reagent, and a portion of the colony under study was transferred to the filter paper using wooden sticks. A positive result was indicated by the appearance of a purple colour upon contact of the bacterial cells with the reagent on the paper (15)
The Vitek device is considered one of the best devices for identifying bacterial species quickly and accurately. Developed by the French company Biomeriex, it identifies the type of bacteria by performing 64 tests.
Kirby–Bauer disk diffusion on Mueller-Hinton agar using CLSI (2023) guidelines. Antibiotics tested included:
** MDR was defined as resistance to ≥3 antibiotic classes .
Between February and March of 2023, fifty samples were taken from pregnant and non-pregnant women at Al-Khansaa Hospital in Mosul City and Al-Batoul Teaching Hospital in Diyala Governorate. These samples comprised 15 (30%) non-pregnant women and 35 (70%) pregnant women who showed clinical signs of bacterial vaginosis (BV). The samples were examined morphologically, microscopically, biochemically, and molecularly.
The attending physician and laboratory technician collected clinical samples and categorized them according to the color, odour, and pH of vaginal discharge using a pH meter. According to the findings, the colour of the vaginal discharge varied from white to greenish-yellow (20%, 35%, and 45%, respectively). The vaginal discharge has a pH between 5.0 and 7.0. Based on their physical traits, the bacterial species responsible for bacterial vaginosis (BV) were first identified (Figure 4-1). Blood agar, chocolate agar, and MacConkey agar were used for direct culture of the samples, which were then incubated for 24 hours at 37°C. Twenty (40%) of the samples had positive bacterial growth, according to the results, and thirty (60%) had mixed growth (fungi and yeast), which led to their exclusion from the research.
The Vitek assay was used to authenticate the morphological and biochemical identities of the samples. While some isolates appeared white or grey on blood agar and chocolate agar, others exhibited a pink colour on MacConkey agar, suggest916ing their capacity or inability to ferment lactose. (Vaginal Infections Atlas [STDs], 2018) (16)
Figure 1. Figure ( 3- 1) Clinical samples of vaginal swabs: A) Positive for BV and B) Negative for BV
Figure 2. Figure ( 3- 1) Clinical samples of vaginal swabs: A) Positive for BV and B) Negative for BV
The bacteria appeared under the light microscope at a magnification of 100x after staining with Gram's stain. Gram-negative bacteria appeared red, and Gram-positive bacteria appeared purple (Figure 4-2). The percentage of Gram-positive bacteria (65%) was higher than the percentage of Gram-negative bacteria (35%), as shown in Table 4-1 and Figure 3-1.
Figure 3. Figure ( 3 -1): Distribution of bacterial isolates according to microscopic examination
Figure 4. A - Gram-negative isolates. Figure (4-2): Microscopic examination of bacterial isolates .
Figure 5. B - Gram-positive isolates Figure (4-2): Microscopic examination of bacterial isolates .
Every isolate underwent biochemical testing. K. pneumoniae, E. coli, and E. ae were among the bacteria that tested positive for catalase. These bacteria used the reagent to break down hydrogen peroxide (H2O2) into water and oxygen. These gas bubbles' emergence signifies a successful outcome (17). However, because the colonies did not become purple when the reagent was added, these bacteria tested negative for oxidase. This is due to the bacteria's lack of the hydrogen-accepting enzyme cytochrome oxidase. Every Gram-positive isolate, including Staphylococcus species, tested positive for catalase and negative for oxidase (16). Table (4-2) and Figure (4-3) illustrate this.
Figure 6. Figure (3-3): Oxidase A test: Catalase B test: for S. haemolyticus bacteria
The VITEK® 2 Compact system was used to diagnose bacterial isolates from vaginal fluids in order to guarantee the accuracy of the diagnosis and to determine their kinds. The VITEK technology produced results that were in line with conventional diagnostic techniques. (17) . Twenty pathogenic vaginal and intestinal bacterial isolates, distributed as Gram-positive, were among those causing vaginitis, according to our investigation employing VITEK diagnostics. Among them were S. haemolyticus, which grew at the fastest rate in vaginal secretions (8 (40%)), S. aureus (3 (15%), and S. epidermidis (2 (10)). According to Table (3-3), Gardnerella vaginalis bacteria had the lowest percentage (5%), whereas E. coli 4 (20%) and K. pneumoniae 2 (10%) were among the gram-negative bacteria. (Fig. 3-4)
Figure 7. Figure (3-4): Distribution of clinically isolated bacterial isolates from patients with vaginitis using the VITEK device.
( Diagnosis by Colour of Secretion )
According to the color of homogeneous vaginal discharge (white, yellow, or yellow-green) (17), the results of our study indicated that the highest percentage of bacterial isolation was found in yellow-green discharge (9%, 45%), followed by yellow discharge (7%, 35%), and white vaginal discharge (4%, 20%), as indicated in Table 4-4. These findings are in line with those of Al-Zubaidi (2012) (18), who discovered that the bacteria responsible for bacterial vaginosis were most prevalent in yellow-green discharge (6.7%), followed by yellow discharge (5.3%), and white discharge (4.3%) (19). The color, odor, and consistency of the vaginal discharge are used to identify bacterial vaginosis, confirming the existence of infection. Researchers' investigations verified this. (20). They said that although vaginal discharge color is a typical clinical indicator of bacterial vaginosis, vaginitis may be asymptomatic. Usually, the discharge is granular, white, or homogenous, and it may or may not smell bad. Additionally, they found that among the most significant indicators of vaginitis are changes in the consistency, viscosity, colour, and odour of vaginal discharge, as well as vaginal sensations including burning and itching (21). This is consistent with the research of Mengistie et al. (2014) (22), which showed that the diagnosis of bacterial vaginosis includes abnormal vaginal discharge. Table (3-4)
Figure 8. Table (3-4) : Distribution of Bacterial Isolates according to Colour of Secretion
To identify multidrug resistance in the bacterial isolates S. haemolyticus, S. aureus, E. coli, and K. pneumoniae, bacterial sensitivity testing was carried out using the Kirby-Bauer disk diffusion technique. Ofloxacin, Ceftriaxone, Clavulanic Acid-Amoxicillin, Cefotaxime, Vancomycin, Novobiocin, Imipenem, Cefixime, Erythromycin, and Nalidixic acid—ten of the most widely used antibiotics for treating specific illnesses brought on by various kinds of bacteria—were evaluated. This test is used to identify microorganisms that are very resistant to antibiotics. Our present study's findings demonstrated that S. haemolyticus bacterial isolates were the most resistant (Table 3-5), with 100% resistance to NA, CFM, EM, CTX, AMC, and CRO and 100% sensitivity to IPM, NV, and VA. As indicated in Table (3-5) and Figure (3-6), the majority of the bacterial isolates were resistant to the antibiotic OFX, with the exception of two isolates that were responsive.
Figure 9. Table (3-6): Multidrug resistance of S. haemolyticus bacterial isolates
This study highlights the emerging clinical significance of Staphylococcus spp. as contributors to bacterial vaginosis, especially among women with recurrent or persistent symptoms.
The findings of this study are in line with those of Hussein and Makhrmash (2023), who discovered that there were more Stain-positive bacteria (63% and 66%, respectively) and fewer Stain-negative bacteria (36% and 44%, respectively). These findings, however, contradict those of Al-Jamali and Al-Ghariri (2005) (24), who found that the proportion of stain-negative bacteria was greater than that of stain-positive bacteria.
. In addition to the indiscriminate use of antibiotics, birth control pills, and other substances that promote bacterial infection and the appearance of other symptoms like vaginal discharge, itching, changes in pH, and odor, the frequent appearance of these bacterial species that cause vaginal infections, particularly in pregnant women, is attributed to an imbalance in the microbial flora (the normal vaginal flora) (25).
Eight S. haemolyticus bacterial isolates were shown to be completely resistant to the medicines cefixime, cefotaxime, clavulanic acid/amoxicillin, ceftriaxime, nalidixic acid, and erythromycin. Regarding resistance to cefotaxime and amoxicillin (57.5% and 62%, respectively), their results were in line with those of Alwaily et al. (2022) and Shrestha et al. (2018). However, our study's sensitivity to Ceftriaxime (65%) and Cefotaxime (22%) was different from these two investigations. Additionally, our findings of 100% erythromycin resistance were comparable to those of Debnath et al. (2020), who discovered 60% resistance.
Our results are comparable with those of Debnath et al. (2020) (25) on multidrug resistance. However, our findings vary in that NV, NA, and IPM were effective against all S. haemolyticus isolates, in contrast to their investigation, which revealed no sensitivity and the inefficiency of these antibiotics against any isolate. Except for isolate 22, which was susceptible to the antibiotic, our findings are similarly consistent with those of Hussein and Makhrmash (2023) (26). Lastly, with the exception of isolates 21 and 10, OFX was ineffective against every bacterial strain. Table 3-5 illustrates how our findings align with those of Rashed et al. (2023) (27) and Shrestha et al. (2018) (28).
Multidrug resistance (MDR) is a characteristic of Staphylococcus aureus, which is frequently cultivated from hospitalised patients. Ten (10) antimicrobial drugs were evaluated for susceptibility in all eight (8) S. haemolyticus isolates. According to Table 4-6 above, three isolates (37.5%) were resistant to six agents or antibiotics, while five isolates (62.5%) were resistant to seven agents or antibiotics. The fact that S. haemolyticus has many mechanisms, such as enzymatic drug inactivation, target site alteration, efflux pump, and altered membrane permeability, is one of the primary causes of this multidrug resistance. (29)
The increasing frequency of MDR isolates is consistent with data from throughout the world showing that S. aureus and CoNS in the female genital tract are becoming more resistant. Our results are in line with several worldwide studies (30, 31) that reveal BV-associated Staphylococcus spp. exhibit strong resistance to β-lactams and macrolides.
With Gram-positive bacteria making up the majority (65%) of the isolates, the results of this investigation demonstrate a varied microbial profile linked to bacterial vaginosis. The most prevalent Gram-negative isolate was Escherichia coli, whereas the most common Gram-positive germs were Staphylococcus haemolyticus and Staphylococcus aureus. The 5% identification of Gardnerella vaginalis indicates that opportunistic and multidrug-resistant (MDR) bacteria are becoming more prevalent in the population under study, indicating a change in the etiological pattern of BV.
Testing for antibiotic susceptibility showed that the isolates had a worrying pattern of resistance. The S. Haemolyticus, S. aureus, E. coli, as well as K. Pneumoniae showed notable MDR behaviours by exhibiting resistance to several commonly prescribed antibiotics, such as ceftriaxone, amoxicillin/clavulanic acid, cefotaxime, erythromycin, and cefixime. Empirical treatment strategies for BV are further complicated by the high rate of resistance to macrolides and β-lactams. Moreover, E. Vancomycin-resistant coli isolates highlight the increasing risk of new resistant strains.
Overall, the study highlights the need for routine microbiological screening, species-level identification, and testing for antibiotic sensitivity in women who exhibit symptoms of BV. Rising antibiotic resistance and prolonged infection may be caused by empirical treatment without laboratory proof. To improve patient outcomes and stop the spread of MDR infections in the community, it is essential to update local treatment recommendations, promote early detection, and strengthen antimicrobial stewardship.
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