A study on microbiome diversity and antibiotic sensitivity pattern of vaginopathogens at gestation time in a tertiary care hospital

 Corresponding author: Lekshmi Gopal U. Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. A study on microbiome diversity and antibiotic sensitivity pattern of vaginopathogens at gestation time in a tertiary care hospital Lekshmi Gopal U 1, *, Devapriya Vijayan 1, Vandhana G 1, Akhila S Arjun 2, Chitra C Nair 3 and Beena P 4 1 Pharm D Intern, KVM College of Pharmacy, Cherthala, Kerala, India. 2 Assistant Professor, Department of Pharmacy Practice, KVM College of Pharmacy, Cherthala, Kerala, India. 3 Professor and HOD, Department of Pharmacy Practice, KVM College of Pharmacy, Cherthala, Kerala, India. 4 Principal, KVM College of Pharmacy, Cherthala, Kerala, India World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 276-287 Publication history: Received on 01 January 2025, revised on 08 February 2025; accepted on 11 February 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.21.2.0173 Abstract A six-month study was conducted in a tertiary care hospital in Kerala, India, to analyse the microbiome diversity and antibiotic sensitivity pattern of vaginopathogens in 75 pregnant women. Vaginal swabs were collected and sent for microbiological evaluation. Pregnancy causes significant changes in the vaginal microbiota, which plays a crucial role in protecting both the mother and foetus from infections. Case records were reviewed for age, presenting complaints, laboratory results, diagnostic reports, and therapeutic management. Out of 75 vaginal swab samples, 22 tested positives for bacterial growth, identifying 32 bacterial strains, which were classified into 9 distinct species. Klebsiella species were the most prevalent (21.8%), followed by coagulase-negative staphylococci (18.7%), beta-haemolytic streptococci (15.6%), and E. coli (12.5%). This study highlights the need for vigilant monitoring and targeted antibiotic therapy to optimize maternal and neonatal outcomes while addressing the growing challenge of antibiotic resistance. Keywords: Pregnancy; Vaginal Microbiota; Antibiotic Sensitivity; Vaginal Swab; Microbiome Diversity 1. Introduction The vagina represents a dynamic environment which is inhabited by diverse organisms that contribute nutrients. These one-of-a-kind surroundings undergo important changes in all levels of life, from start to the age of puberty until menopause. The equilibrium therein can be disturbed by physiological or non-physiological changes, mediated by hormonal status, sexual behavior, vaginal blood, foreign bodies and/or concurrent use of medications. The vaginal microbiota refers to the microorganisms that are living inside the vagina [1]. These microorganisms make contributions to a woman's reproductive and overall health. Lactobacillus species account for 95% of the vaginal flora. The 4 most common types of Lactobacilli dominant vaginal microbiome profiles are characterized by L. iners, L. crispatus, L. gasseri, or L. jensenii. Factors along with antibiotic use, sexual activity, and hormonal modifications can disrupt the stability of the vaginal microbiota, along with bacterial vaginosis. [2] During pregnancy, the vaginal microbiota’s composition undergoes significant changes and thereby acting as a protective shield against infection for both the mother and fetus. Hydrogen peroxide produced by Lactobacillus strains plays a critical role in preserving the microenvironment of the vagina and the inhibition of overgrowth of pathogenic microorganism. Lactobacillus and the predominance of anaerobic bacteria are the most common causes of vaginal infections in women of childbearing age. [3] World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 276-287 277 Intrapartum antibiotics play a crucial role in preventing Group B Streptococcus (GBS) transmission from pregnant women to their newborns during labor and delivery. GBS is a bacterium commonly found in the vaginal and rectal areas of healthy adults. While it typically does not cause harm in healthy adults, it can pose a significant risk to newborns if they are exposed to the bacteria during childbirth. In newborns, GBS infections can lead to serious complications such as pneumonia, sepsis, or meningitis, which can be life-threatening if not promptly treated. [4,5] Intrapartum antibiotics plays a crucial role in preventing Group B Streptococcus (GBS) transmission from pregnant women to their newborns during labor and delivery. GBS is a bacterium commonly found in the vaginal and rectal areas of healthy adults. While it typically does not cause harm in healthy adults, it can pose a significant risk to newborns if they are exposed to the bacteria during childbirth. In newborns, GBS infections can lead to serious complications such as pneumonia, sepsis, or meningitis, which can be life-threatening if not promptly treated. To mitigate the risk of GBS transmission to newborns, healthcare providers administer intrapartum antibiotics to pregnant women who are colonized with GBS or who have certain risk factors. [6,7] The administration of antibiotics during labor helps reduce the concentration of GBS bacteria in the birth canal, thereby lowering the likelihood of newborn exposure during delivery. The timing and duration of intrapartum antibiotics are critical to ensuring their effectiveness while minimizing potential risks associated with antibiotic use. Typically, intrapartum antibiotics are initiated at least four hours before delivery. This time frame allows sufficient time for the antibiotics to reach therapeutic levels in the mother's bloodstream, providing optimal protection against GBS transmission to the newborn. [8] The antibiotics are usually administered intravenously to ensure rapid absorption and distribution throughout the body. In cases where the mother's GBS status is unknown, or if she has certain risk factors such as preterm labor or prolonged rupture of membranes, healthcare providers may opt to initiate intrapartum antibiotics as a precautionary measure. Additionally, if a pregnant woman tests positive for GBS colonization earlier in her pregnancy but does not receive intrapartum antibiotics during labor due to premature delivery or other circumstances, antibiotics may still be administered at the time of delivery to provide protection to the newborn. While intrapartum antibiotics are highly effective in reducing the incidence of early-onset GBS infections in newborns, they are not without potential drawbacks. Antibiotic use during labor may contribute to the development of antibiotic resistance, both in the mother and in neonatal microbiota. Additionally, maternal antibiotic exposure can impact the newborn's microbiome, potentially affecting their long-term health outcomes. [9,10] 2. Materials and methods 75 cases were collected from the obstetrics and gynecology department. N= (𝑧1−𝛼 2)2𝜎2 𝐸2 σ is the standard deviation and E is the margin of error Data from a previous study found a standard deviation of 1.1 Here σ = 1.1 z= 1.96 (95% confidence level) E= 0.25 N= 𝑧2𝜎2 𝐸2 = 1.962×18.02 0.252 = 60 Anticipating loss to follow-up and missing of data, the minimum sample size is rounded to be 75. An observational prospective study, conducted among the pregnant women in the OBG department in a tertiary care hospital, SH Medical Centre, Kottayam, Kerala, India for 6-months period. After obtaining permission from the IEC and Informed consent from individual patients, the data collection was started. The study included all pregnant women with singleton gestation undergoing either vaginal delivery or cesarean section, while excluding those who were allergic to antibiotics, had incomplete medical records, or refused the vaginal swab. Case records are prospectively reviewed from World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 276-287 278 OPD and IP and the information includes age, presenting complaints, laboratory investigation data, other diagnostic reports, therapeutic management and any added complications. The vaginal swab was collected before the delivery and the swab was cultured at the microbiology lab and reports were collected and the isolated organisms will be noted along with their sensitivity and resistance pattern to the antibiotics. The data will be collected in a specially designed data collection form. 3. Results A total of 75 pregnant women meeting the inclusion criteria were included in the study. Most participants were aged 26–30 years. The primary reasons for admission were safe confinement (28%), complaints of pain (24%), induction of labour (22.7%), and leaking per vagina (13.3%). The majority of the study population were at a gestational age of 38 weeks, followed by 37 weeks, 39 weeks, and 36 weeks. Regarding medical history, gestational diabetes mellitus (29.3%) was the most common condition, followed by hypothyroidism (18.6%), pregnancy-induced hypertension (6.6%), and polycystic ovary syndrome (5.3%). Vaginal delivery was the predominant mode of delivery (62.7%), with 37.3% undergoing cesarean section. Vaginal swab cultures were obtained from all 75 participants, with 22 (29.3%) testing positive and 53 (70.6%) showing no significant flora (NSF). Among full-term pregnancies (63 cases), 18 (28.5%) had positive cultures, and 45 (71.5%) were NSF, while in preterm pregnancies (12 cases), 4 (33.3%) had positive cultures, and 8 (66.7%) were NSF. 3.1. Reason for admission Figure 1 Percentage distribution of reasons for admission 3.2. Gestation age in weeks Figure 2 Percentage distribution based on gestational age in weeks World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 276-287 279 3.3. Past medical history Figure 3 Percentage distribution of past medical history 3.4. Mode of delivery Figure 4 Percentage distribution based on mode of delivery 3.5. Vaginal swab culture Figure 5 Percentage distribution of vaginal swab culture World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 276-287 280 3.6. Vaginal swab culture in relation to gestational age Figure 6 Percentage distribution of vaginal swab culture based on gestational age 3.7. Microbiome diversity analysis Out of 75 vaginal swab samples, 22 tested positives for bacterial growth. A total of 32 bacterial strains were identified, with some microorganisms appearing in multiple samples. These 32 strains were ultimately classified into 9 distinct bacterial species. Klebsiella species were the most prevalent, found in 21.8% of samples, followed closely by Coagulase negative staphylococci at 18.7%. Beta haemolytic streptococci and E. coli were also notable constituents, occurring in 15.6% and 12.5% of samples, respectively. Table 1 Frequency distribution of microbiome diversity Organisms Frequency (n=32) Percentage (%) Klebsiella species 7 21.8 Coagulase negative Staphylococci 6 18.7 Beta-haemolytic Streptococci 5 15.6 E coli 4 12.5 Methicillin resistant coagulase negative Staphylococci 4 12.5 Non-haemolytic Streptococci 2 6.2 Staphylococcus aureus 2 6.2 Pseudomonas aeruginosa 1 3.1 Citrobacter diversus 1 3.1 3.8. Sensitivity and resistance pattern of isolated microorganisms from vaginal swab Klebsiella species demonstrated complete sensitivity to a range of antibiotics including Ampicillin/sulbactam, Meropenem, Imipenem, Cotrimoxazole, Chloramphenicol, and Nitillin, but were entirely resistant to Linezolid and Cefpodoxime. Coagulase negative Staphylococci were found to be highly sensitive to Ampicillin/sulbactam, Cefoperazone/sulbactam, Ceftriaxone, Meropenem, Cefepime, Imipenem, Vancomycin, Nitillin, Methicillin, Cephalexin, Linezolid, and Faropenem, with a notable resistance rate of 67% to Cefpodoxime. Beta-haemolytic Streptococci showed complete sensitivity to Ampicillin/sulbactam and Imipenem, while being completely resistant to Roxithromycin, Erythromycin, Colistin, and Lincomycin. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 276-287 281 E. coli strains exhibited full sensitivity to Meropenem, Gentamycin, Amikacin, Imipenem, Chloramphenicol, Nitillin, Cephalexin, and Linezolid, and complete resistance to Nalidixic acid and Cefpodoxime. Methicillin-resistant coagulase-negative staphylococci were completely sensitive to Imipenem, Cotrimoxazole, Vancomycin, Nitillin, Amoxicillin/clavulanic acid, and Tigecycline, but resistant to Roxithromycin, Erythromycin, Methicillin, Cephalexin, Lincomycin, Faropenem, and Cefpodoxime. Non-haemolytic Streptococci showed complete sensitivity to a broad spectrum of antibiotics, including Cefuroxime, Ampicillin/sulbactam, Cefoperazone/sulbactam, Levofloxacin, Cefixime, Ceftriaxone, Ciprofloxacin, Cefotaxime, Meropenem, Cefepime, Imipenem, Ofloxacin, Cotrimoxazole, Norfloxacin, Vancomycin, Nitillin, Cephalexin, Linezolid, Amoxicillin/sulbactam, Faropenem, Cefpodoxime, and Tigecycline, with complete resistance to Roxithromycin. Staphylococcus aureus was found to be completely sensitive to Cefuroxime, Ampicillin/sulbactam, Cefoperazone/sulbactam, Cefixime, Ceftriaxone, Clindamycin, Cefotaxime, Meropenem, Cefepime, Colistin, Amikacin, Imipenem, Cotrimoxazole, Cloxacillin, Piperacillin/Tazobactam, Nitillin, Methicillin, Cephalexin, Linezolid, Lincomycin, Amoxicillin/clavulanic acid, Faropenem, and Tigecycline. Pseudomonas aeruginosa and Citrobacter diversus were excluded from the study due to the presence of only a single isolate for each. cxmCefuroxime, CfmCefixime, CdClindamycin, AtRoxithromycin. Figure 7 Antibiotic Sensitivity and resistance Pattern of the various isolated microbes to Antimicrobial Agents World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 276-287 282 RC/CPCiprofloxacin, cpmCefepime, CLColistin, AkAmikacin. Figure 8 Antibiotic Sensitivity and resistance Pattern of the various isolated microbes to Antimicrobial Agents BA-Cotrimoxazole, Pr/CN-Cephalexin, Lz-Linezolid, amc-Amoxicillin/Clavulanic acid Figure 9 Antibiotic Sensitivity and resistance Pattern of the various isolated microbes to Antimicrobial Agents First-line antibiotics considered safe in pregnancy include Ampicillin/Sulbactam, effective against Klebsiella, CoNS, beta-haemolytic Streptococci, and Staphylococcus aureus, commonly used for respiratory, UTI, skin, and intraabdominal infections. Cephalexin (1st Gen) and Cefuroxime (2nd Gen) are also safe, targeting E. coli, Staphylococcus aureus, and Non-haemolytic Streptococci, suitable for UTIs, skin, and respiratory infections. Ceftriaxone (3rd Gen) is recommended for severe infections like pneumonia, meningitis, and sepsis, while Amoxicillin/Clavulanic Acid covers CoNS and Staphylococcus aureus, used for respiratory, UTI, and soft tissue infections. Vancomycin is reserved for serious Gram-positive infections, including MRSA. Antibiotics to avoid or use with caution include Meropenem and Imipenem, which are reserved for resistant cases, and Linezolid, which is used only when necessary. Aminoglycosides like Gentamycin and Amikacin should be used cautiously due to potential nephrotoxicity and ototoxicity. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 276-287 283 Table 2 Antibiotic sensitivity frequency of isolated organisms Organisms isolated n As Pr/CN cxm ctr amc VA mep Imp Lz Gm Ak Beta-haemolytic Streptococci 5 5 1 2 1 3 1 2 5 3 0 2 Citrobacter diversus 1 1 0 0 0 0 0 1 1 0 1 1 Coagulase negative Staphylococci 6 6 6 3 6 5 6 6 6 6 5 5 E coli 4 4 0 0 1 1 0 4 4 0 4 4 Klebsiella species 7 7 4 4 6 5 0 7 7 1 6 6 Methicillin resistant coagulase negative Staphylococci 4 3 0 1 1 4 4 2 4 3 3 3 Non-haemolytic Streptococci 2 2 2 2 2 2 2 2 2 2 1 1 Pseudomonas aeruginosa 1 1 0 0 0 0 0 1 1 0 1 1 Staphylococcus aureus 2 2 2 2 2 2 1 2 2 2 0 2 n= number of isolates of each organism; AsAmpicillin/Salbactam, Pr/CNCephalexin, cxmCefuroxime, ctrCeftriaxone, amcAmoxicillin/Clavulanic acid, VAVancomycin, mepMeropenem, ImpImipenem, LzLinezolid, GmGentamycin, AkAmikacin. Table 3 Antibiotic sensitivity patterns of isolated organisms in percentage (%) Organisms isolated n As Pr/CN cxm ctr amc VA mep Imp Lz Gm Ak Beta-haemolytic Streptococci 5 100 40.0 40.0 20.0 40.0 20.0 40.0 100 60.0 0.0 40.0 Citrobacter diversus 1 100 0.0 0.0 0.0 100 0.0 100 100 0.0 100 100 Coagulase negative Staphylococci 6 100 100 50.0 100 83.3 100 100 100 100 83.3 83.3 E coli 4 100 0.0 0.0 25.0 100 0.0 100 100 0.0 100 100 Klebsiella species 7 100 57.1 57.1 85.7 85.7 0.0 100 100 14.3 85.7 85.7 Methicillin resistant coagulase negative Staphylococci 4 75.0 0.0 25.0 25.0 75.0 100 50.0 100 75.0 75.0 75.0 Non-haemolytic Streptococci 2 100 100 100 100 50.0 100 100 100 100 50.0 50.0 Pseudomonas aeruginosa 1 100 0.0 0.0 0.0 100 0.0 100 100 0.0 100 100 Staphylococcus aureus 2 100 100 100 100 100 50.0 100 100 100 0.0 100 n= number of isolates of each organism; AsAmpicillin/Salbactam, Pr/CNCephalexin, cxmCefuroxime, ctrCeftriaxone, amcAmoxicillin/Clavulanic acid, VAVancomycin, mepMeropenem, ImpImipenem, LzLinezolid, GmGentamycin, AkAmikacin. Table 4 Antibiotic resistance frequency of isolated organisms Organisms isolated n As Pr/CN cxm ctr amc VA mep Imp Lz Gm Ak Beta-haemolytic Streptococci 5 0 4 3 3 1 2 0 0 1 4 3 Citrobacter diversus 1 0 1 1 1 0 0 0 0 1 0 0 Coagulase negative Staphylococci 6 0 0 1 0 1 0 0 0 0 1 0 E coli 4 0 4 4 2 3 0 0 0 4 0 0 Klebsiella species 7 0 3 3 1 2 0 0 0 6 0 0 Methicillin resistant coagulase negative Staphylococci 4 1 4 3 1 0 0 1 0 1 1 1 Non-haemolytic Streptococci 2 0 0 0 0 0 0 0 0 0 1 1 World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 276-287 284 Pseudomonas aeruginosa 1 0 1 1 0 1 0 0 0 1 0 0 Staphylococcus aureus 2 0 0 0 0 0 0 0 0 0 1 0 n= number of isolates of each organism; AsAmpicillin/Salbactam, Pr/CNCephalexin, cxmCefuroxime, ctrCeftriaxone, amcAmoxicillin/Clavulanic acid, VAVancomycin, mepMeropenem, ImpImipenem, LzLinezolid, GmGentamycin, AkAmikacin. Table 5 Antibiotic resistance patterns of isolated organisms in percentage (%) Organisms isolated n As Pr/CN cxm ctr amc VA mep Imp Lz Gm Ak Beta-haemolytic Streptococci 5 0.0 80.0 60 60.0 40.0 40.0 0.0 0.0 40.0 80.0 60.0 Citrobacter diversus 1 0.0 100 100 100 0.0 0.0 0.0 0.0 100 0.0 0.0 Coagulase negative Staphylococci 6 0.0 0.0 16.7 0.0 16.7 0.0 0.0 0.0 0.0 16.7 0.0 E coli 4 0.0 100 100 50.0 75.0 0.0 0.0 0.0 100 0.0 0.0 Klebsiella species 7 0.0 42.9 42.9 14.3 28.6 0.0 0.0 0.0 85.7 0.0 0.0 Methicillin resistant coagulase negative Staphylococci 4 25.0 100 75.0 25.0 0.0 0.0 25.0 0.0 25.0 25.0 25.0 Non-haemolytic Streptococci 2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 50.0 50.0 Pseudomonas aeruginosa 1 0.0 100 100 0.0 100 0.0 0.0 0.0 100 0.0 0.0 Staphylococcus aureus 2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 50.0 0.0 n= number of isolates of each organism.; AsAmpicillin/Salbactam, Pr/CNCephalexin, cxmCefuroxime, ctrCeftriaxone, amcAmoxicillin/Clavulanic acid, VAVancomycin, mepMeropenem, ImpImipenem, LzLinezolid, GmGentamycin, AkAmikacin. 3.9. Antibiogram Figure 10 Antibiogram of vaginal pathogens showing susceptibility patterns to commonly used antibiotic NUMBER OF ISOLATES Cefuroxime Ampicillin/sulbactam Cefaperasone/sulbactam Levofloxacin cefixime Ceftriaxone Clindamycin Roxithromycin Nalidixic acid Ciprofloxacin Cefotaxime Meropenem Cefpime Erythromycin Colistin Gentamycin Amikacin Imipenem Ofloxacin Tetracycline Cotrimoxazole Norfloxacin Cloxacillin Ceftizoxime Chloramphenicol Pipperacillin Piperacillin/ Tazobactam Vancomycin Netillin Methicillin cephalexin Linezolid Lincomycin Amoxicillin/ clavulanic acid Faropenem Cefpodoxime Ceftazidime Tigecycline S57 100 71 71 57 86 71 71 71 100 86 86 86 86 100 71 57 100 71 71 100 43 43 100 57 14 71 57 29 57 71 MS 14 29 29 14 14 14 14 29 43 29 14 43 29 29 29 R43 14 43 14 29 14 14 14 14 29 43 71 29 43 71 14 S100 75 75 25 75 100 75 50 100 100 100 50 25 75 75 25 100 50 50 100 25 75 75 MS 25 25 25 25 25 75 25 50 50 50 25 R100 75 50 100 25 25 50 50 75 25 25 100 100 75 25 100 75 25 S50 100 100 33 33 100 83 83 33 50 100 100 17 67 83 83 100 33 33 67 33 67 83 100 100 100 100 100 83 83 100 33 83 MS 33 33 33 33 33 50 17 33 33 33 16 17 R17 33 33 17 17 33 17 33 33 17 33 33 33 33 33 17 17 67 S25 75 50 25 25 25 25 50 25 75 75 75 100 25 50 100 25 50 50 100 100 75 100 100 MS 25 50 25 50 25 50 25 25 50 50 50 R75 25 25 25 75 25 75 100 25 50 25 25 100 25 25 25 25 50 25 50 50 100 100 25 100 100 100 S100 100 100 50 100 100 100 50 50 100 100 100 50 100 100 100 50 50 100 50 100 100 50 100 100 100 100 100 100 100 100 MS 50 50 50 50 50 50 50 50 R50 50 50 50 S40 80 20 40 40 40 20 100 20 80 20 60 20 80 20 60 60 20 20 20 MS 20 40 20 20 20 40 040 20 20 20 20 20 20 20 60 40 20 60 R60 20 80 60 80 60 80 100 60 60 20 40 100 100 80 60 60 80 20 60 20 40 40 20 80 40 100 20 80 80 20 S100 100 100 100 100 100 50 100 100 100 100 50 50 50 100 100 100 100 100 50 100 100 100 100 50 100 100 100 100 MS 50 50 50 R50 100 50 50 50 50 50 50 Staphylococcus aureus Beta-haemolytic Streptococci Non-haemolytic Streptococci Gram positive Coagulase negative Staphylococci ORGANISMS Klebsiella species E coli Gram negative Methicillin resistant coagulase negative Staphylococci 5 2 7 4 6 4 2