Int.J.Curr.Microbiol.App.Sci (2017) 6(1): 964-972

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 1 (2017) pp. 964-972 Journal homepage: http://www.ijcmas.com

Original Research Article http://dx.doi.org/10.20546/ijcmas.2017.601.114

Antibiogram of Bacterial Isolates from High Vaginal Swabs of Pregnant Women from Tertiary Care Hospital in Puducherry, India

N. Ravishankar1 and M. Prakash2*

1Research and Development Centre, Bharathiar University, Coimbatore-641 046, Tamil Nadu, India 2Department of Microbiology, Kanchi Shri Krishna College of Arts and Science, Kilambi, Kancheepuram-631 551, Tamil Nadu, India *Corresponding author:

ABSTRACT

Bacterial vaginosis is caused by an imbalance of the organisms that naturally exist in the

. The importance of with respect to pregnant women’s health is emphasized by the association between bacterial vaginosis and adverse outcome of pregnancy. The aim of present study was to evaluate the direct smear microscopy and culture for determination of bacteria from of pregnant women. Vaginal discharge in women is sometimes caused by candida and aerobic bacteria organisms like E.coli, staphylococcus aureus, and β- haemolytic streptococcus. Culture and sensitivity testing are done from high vaginal swab (HVS) specimen collected from women who come to the clinic complaining of vaginal discharge. Isolation and antibiotic sensitivity of K e yw or ds these organisms are key to the successful treatment of the cause of vaginal discharge. This Vaginal Discharge, study tends to evaluate the microbial and antibiotic sensitivity and resistance pattern of High Vaginal Swab, high vaginal swab culture results in the Rajeev Gandhi government women and children Bacterial Vaginosis, hospital, Puducherry India. A total of 500 High Vaginal Swabs (HVS) were collected from Vulvovaginal pregnant women and were screened for bacterial population. For each patient one swab Candidiasis . was used for smear preparation and Gram staining and the second swab was used for

Article Info cultivation. HVS Samples culture and sensitivity results were reviewed. Out of which

252(50.4%) are the pathogenic organisms and 248(49.6%) yielded growth of non Accepted: pathogenic organisms. Candida species were the predominant microbial organism, 27% 20 December 2016 (68/252). Bacterial Vaginosis causing bacteria identified were mostly the aerobic types Available Online: (184); with Escherichia coli being predominant, 38.8% (98/184) followed by Klebsiella 10 January 2017 sps, and Staph. aureus. Pregnant women of age group 20-30 are showing more pathogenic isolates growth. Amikacin (89.6%), Norfloxacin (75.6%), Ciprofloxacin (66.2%) and Gentamycin (68.4%) revealed high levels of susceptibility. Whereas high resistance rates were observed for Amoxicillin (83.3%) Tetracyclin (65.4%) and Cotrimoxizole (62.7%). The cephalosporins showed the best antibiotic sensitivity. Candida spp. and aerobic bacteria were the predominant microbial organisms identified from HVS results in the Puducherry. Proper laboratory diagnosis to identify causative organisms is vital for optimal therapeutic outcome.

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Introduction bacterial BV, a condition characterized by a raised vaginal pH and milky discharge, the The vaginal microflora constitutes a normal vaginal flora is replaced by a mixed complicated environment, composed of flora of aerobic, anaerobic and varying microbiological species in variable microaerophilic species (Schwebke, 2000). It quantities and proportions and their seems that BV is accompanied by a shift in concentrations are indicative of the vaginal the normal Lactobacillus flora to a mixed health of the individual (Donders et al., vaginal anaerobic flora including Gardnerella 2005). The microbial ecology subject to vaginalis, Bacteroides spp., and Mobiluncus remarkable changes over the course of spp. (Wilks et al., 2004). BV commonly lifetime a, induced by developmental and occurs in women of childbearing age and can hormonal changes (Pybus et al., 1999). In be especially substantial in pregnant women childhood, the vaginal flora contains skin (Witt et al., 2002). Prevalence of 10 to 31% commensals and bowel organisms. At has been reported in various populations menarche, the pH falls from neutral to (Wolrath et al., 2001; Chaudry et al., 2004). approximately 4, and the flora becomes Women with BV have fewer Lactobacillus dominated by lactobacilli. Many other organisms than normal and more of other organisms may be present in lower types of bacteria (Koumans et al., 2002). The concentrations, including anaerobic and importance of BV with respect to women’s facultative anaerobic bacteria and Candida health is emphasized by the association spp. (Donders, 1999). In women of between BV and pelvic inflammatory childbearing age this system is also diseases, adverse outcome of pregnancy, dominated by Lactobacillus spp., a defining postpartum endometritis, and cuff cellulites characteristic of which is the ability to grow (Donders et al., 2005; Berg, 2001). Women in acid media and tolerate acid conditions at with BV during pregnancy, have higher risks pH around 4.5; lactobacilli also ferment of miscarriage, early (preterm) delivery, and carbohydrates to produce lactic acid (Sobel, uterine infection after pregnancy (Leitich et 2000). The normal vaginal bacterial flora of al., 2003). Centers for disease control and healthy premenopausal women continues to prevention (CDC) advise that all pregnant consist predominantly of Lactobacillus spp. women with BV symptoms be screened and These are believed to play a protective role in treated with antibiotics (CDC, 2002). guarding the urogenital tract against infection by pathogens (Romanik and Martirosian, Materials and Methods 2004). Bacterial vaginosis (BV) is caused by an imbalance of the organisms (flora) that The study population was comprised of 500 naturally exist in the vagina. BV is among the pregnant women with gestation age ranging diseases that most frequently associated with between 18 to 40 years referred to Obstetrics vaginitis. The other diseases are vulvovaginal and Gynecology department of Rajiv Gandhi candidiasis, and trichomoniasis. Vaginitis is Government women and Child hospital, usually characterized by vaginal discharge, Pondicherry from January 2014 to June 2016. vulvar itching and irritation, or odor (Witt et al., 2002). Specimen Collection and Processing

Normally, about 95% of vaginal flora is Sampling was carried out by insertion of two lactobacillus bacteria. These lactobacilli help sterile cotton swabs for diagnosis of flora and keep the vaginal pH level low and prevent BV, one swab was obtained for smear overgrowth of other types of organisms. In preparation and Gram stain and the other 965

Int.J.Curr.Microbiol.App.Sci (2017) 6(1): 964-972 swab was obtained for culture. Samples were Results and Discussion immediately transferred to the microbiology laboratory. Gram stained smears were Total High Vaginal Sample (HVS): 500 examined under oil immersion (x 1,000) of light microscope for the following Total Positives: 252 (50.4%) morphotypes: large Gram positive rods (Lactobacillus morphotypes), small Gram Out of 500 samples enrolled in the study, 252 variable and Gram- negative rods (50.4%) had positive vaginal cultures. Fifteen (Gardnerella and Bacteroides morphotypes), microorganisms, including non pathogenic and curved Gram variable rods (Mobilincus bacteria like Micrococcai, Diphtheroid and morphotypes). Lactobacillus spp., and pathogenic gram- negative bacteria, gram positive coccai, Culture and Sensitivity candida spp., were isolated. The highest infection rate was observed among women High vaginal swabs specimen were received aged between 20 and 30 years (61.0%), from patients seen within the hospital either followed by those aged 31-40 years (27.0%) on admission or from gynecology outpatient and more than 40 years (6.5.0%), and the department. In either case, specimens were lowest frequency of infection was observed collected by trained nurses and Doctor’s. among those aged between 18 and 20 years Patients demographics were collected, and (5.5%), as shown in Figure.1. few drops of normal saline added to the collected specimen for microbial examination. According to Table 1. Incidence of E. coli is Specimens were then inoculated into the highest among all the cases i.e. 38.88%. MacConkey agar, blood agar and/or chocolate Then comes the Candidial species amounting agar, prepared from nutrient agar (Tulip 26.98% followed by Klebsiella sps 13.88%, Diagnostic Limited, India) for investigation of and Staphylococci 10%. At the lowest level BV and normal flora. Standard culture and come the Enterococcus 1.58%, Acinetobacter, sensitivity procedures were followed and the Streptococcus, Pseudomonas are 5% each, results interpreted. The culture plates were Coagulase Negative Staphylococcus (CoNS) incubated in 37°C in presence of 5% CO2 for and Proteus spps are 0.79% each, Providencia 24-48 h. The culture plates were then is 0.39%. Among the 252 isolates 146 examined and based on grown bacteria, the (57.93%) are the gram negative bacteria, 38 necessary biochemical tests were performed (15.07%) are the gram positive bacteria and and the organisms were identified as per 68 (26.98%) are the candida isolates. Gram standard criteria (Forbes et al., 2007). Negative bacteria are mostly resistant to Antibiotic susceptibility was tested using disc penicillins (amocyclav/amoxicillin), diffusion method (modified Kirby-Bauer’s Fluroquinoles (ciprofloxacin, levofloxacin, method). Drugs used for the susceptibility ofloxacin ). The antibiotic sensitive pattern of patterns: amikacin-AK, amoxyclav-AMC, E.coli showed a higher sensitivity towards azithromycin-AZM, chlorampinicol-C, cefta- piperacillin/tazobactam (85%), cefoperazone/ zidime-CAZ, ceforoxime-CXM, cephot- sulbactum (88) and amikacin (91%). The axime-CTX, ceftriaxone-CTR, ciprofloxacin- antibiotic sensitivity pattern of Klebsiella CIP, co-trimoxazole-COT, cepoperazone + showed a higher sensitivity towards sulbactam-CFS, doxycycline-DO, genta- cefoperazone/ sulbactum (89%), amikacin micin-G, norfloxacin-NA, ofloxacin-OF, (85%). E.coli strains were more resistant to levoflox-LE, oxacillin-OX, meropenem-MR, Amoxycillin 85%, cefuroxime 89%, pipercillin/tazobactam-PT, vancomycin-VA. ceftriaxone 69%, ciprofloxacin 63%, 966

Int.J.Curr.Microbiol.App.Sci (2017) 6(1): 964-972 cotrimaxole 54%. Klebsiella showed Lakshmi et al., which compared the comparatively less resistant to ciprofloxacin prevalence of vaginal infections between 51%, cotrimaxole 55%, cefuroxime 69%, and premenopausal and postmenopausal women. it was highly resistant to Amoxicillin 90%. Increased infections in post-menopausal Gram-positive organisms tested against women are due to the vagina being colonized vancomycin and oxacillin, resistance was by pathogenic organisms more than the found. In conclusion, E coli was found to be protective organisms. The highest frequency the common cause of UTI among the of infection was noted at an age of 20 to 30 pregnant women. Low to moderately high years with a fall in the frequency of infection level of resistance was found in first line as age advanced. Similar results with respect drugs while high level of resistance was found to age were seen in a Kenyan study, although in third generation cephalosporin. It is it was limited to the study of only one recommended to monitor the levels of organism. resistance for nitrofurantoin, fluoroquinolone (ciprofloaxacin, norfloxacin) and cefotaxime. Several microorganisms were isolated in Over the past years the susceptibility to present study, and those with the highest cephalosporins tends to decrease. frequencies were Escherichia coli, Klebsiella Amyloglycosides, piperacillin-tazobactam, sps, Staph aureus, Streptococcus, amikacin and cefoperazone/sulbactum show Pseudomonas aeruginosa, Proteus sp., high antibacterial activity as shown. Micrococci, Enterococci, Acinetobacter, and Candida albicans. Commensal growths were The majority of pregnant women were in age found in 25% of cases that need not be group of 21-30 years and gestational ages. treated, but necessary measures such as represent the relative frequencies of the most identification of risk factors (douching, common isolated normal flora and a few sprays, diabetes) and their prevention have to pathogenic bacteria in stained smears and be carried out. culture. The majority of pathogenic isolated bacteria in culture were Escherichia coli, The study also found Escherichia coli to be Klebsiella, yeast, and Staphylococci. Enteric the most common pathogenic bacteria Gram-negative rods and Streptococci were (38.8%) isolated from culture. In fact, isolated at lower rate. Based on the results of Escherichia coli was found to be the most Gram-stained smears, 78(65%) Gram positive prevalent pathogen isolated not only from rods and 54 (45%) Gram positive cocci were high vaginal swabs but also urine, pus, blood detected. Gram-stained smears were showed and wounds, as seen in a study conducted by to include polymorphonuclear leukocytes, Dutta et al., in Dhaka. Candida albicans is fungi and clue cells. tolerant to the acidic environment and is hence found in the vagina, but the Vaginal infections have wide implications for concentrations are too low to cause women’s health, being the most common symptoms. In conditions of decreased local gynaecological problem. It is believed that the immunity, the hyphae would multiply and lactobacilli play an important role in transform into infective patterns that result in maintaining normal vaginal ecosystem and symptomatic vaginitis.18 Colonization of preventing the growth of opportunistic Candida species also happens during bacteria. The present study demonstrates the pregnancy, resulting in symptomatic prevalence of potential vaginal pathogens in vaginitis.19. Infections with MRSA became a symptomatic pregnant women. The results of global health issue in 1960s, when the strains present study are comparable to the study by were first identified. 967

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Table.1 Distribution of organisms from HVS samples total with %

S.No. Isolated Organism No. of Positive Positive isolates isolates from HVS from HVS (%) 1 E.coli 98 38.88 2 Klebsiella species 35 13.88 3 Staphylococcus aureus 27 10.71 4 Enterococcus 4 1.58 5 Acinitobacter 5 1.98 6 CoNS 2 0.79 7 Streptococcus 5 1.98 8 Pseudomonas species 5 1.98 9 Proteus species 2 0.79 10 Providenciae 1 0.39 11 Candida 68 26.98 252 (50.4) 99.94

Table.2 Bacterial Isolates from HVS samples Resistance patterns to the antibiotics

Isolates β- Cepholosporin Tetracy- Carbape Amyloglycosides Quinolones Lactum cline -nems AMC CXM CTX CTR DO MR G AK LE OF E. coli 88 55 46 57 43 19 32 2 17 11 Klebsiella 29 25 25 28 15 12 11 4 16 17 Staphylococcus aureus 2 6 4 6 5 7 8 1 5 7 Enterococcus 2 2 1 2 1 1 0 0 1 2 Acinitobacter 1 4 2 4 2 2 1 0 3 2 CONS 1 1 1 2 0 0 0 1 1 0 Pseudomonas species 2 2 1 0 1 2 1 0 1 1 Proteus species 1 0 1 0 - - 1 0 2 1 Providenciae 2 1 1 1 2 3 - 0 1 - AMC-Amoxicillin, CXM-Ceforoxime, CTX-Cephotaxime, CTR-Ceftriaxone, DO-Doxycycline, G-Gentamicin, DO-Doxycyclin, MR-Meropenem, AK-Amikacin, NX-Norfloxacin, LE-Levofloxacin, OF-Oflxacin

Table.3 No. of MDR strains isolated from HVS samples of pregnant women

Isolates HVS samples E.coli 60 Klebsiella 21 S. aureus 2 Enterococcus 1 Acinetobacter 3 CONS 1 Pseudomonas 2 Proteus 2 Streptococcus 1

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Fig.1 Prevalence of Bacterial infection in pregnant women Age group

Fig.2 Distribution of Organisms isolated from HVS samples

They may be acquired nosocomially or from Coagulase-negative Staphylococci (CoNS), the community. What make them difficult to which are considered to be skin commensals, treat are their multiple antibiotic resistant were found in 0.79% of the cases. No cases of profiles and wide varying prevalence. One trichomoniasis, infection, and should keep in mind that higher antibiotics Neisseria gonorrhoeae infection were noted. may be required to treat these infections. The presence of co-morbidities like Maternal group B Streptococci (GBS) hospitalization, immunosuppression and co- colonization is a major risk factor for GBS existent reproductive tract infections have to disease in neonates. In pregnant women, GBS be evaluated accordingly. It is known that causes cystitis, amnionitis, endometritis and vaginal infections, due to a disruption of stillbirth; occasionally, it leads to endocarditis normal vaginal flora, increase the risk of or meningitis. sexually transmitted infection, especially

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Int.J.Curr.Microbiol.App.Sci (2017) 6(1): 964-972 human immunodeficiency virus (HIV). In conclusion, vaginal discharge in pregnancy However, our study did not identify any is common, but distinguishing abnormal association with HIV, and diabetic status vaginal discharge from normal leucorrhoea of records were not available. No patients had pregnancy is challenging. Bacterial vaginosis, taken hormone supplements or any other candidiasis are a common problem in women medications that could interfere with the reproductive age. Since findings have showed results. that the trio of vaginal candidiasis, trichomoniasis and bacterial vaginosis are Our study has several limitations. The common causes of abnormal vaginal practice of swab culture is done mostly in discharge in pregnancy; efforts must be made clinical microbiological laboratories, and to exclude these conditions in pregnant clinical diagnosis may be suboptimal. Only patients presenting with vaginal discharge so regularly used antibiotics were included in the that appropriate treatment can be instituted sensitivity testing, and socio-demographic timely. Moreover ciprofloxacin, norfloxacin, factors have not been considered. These and gentamycin are the recommended drugs limitations have to be overcome by future for empiric therapy and prophylaxis as studies, and proper practices have to be needed. implemented in order to preserve these lifesaving drugs for the future. References

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How to cite this article:

Ravishankar, N., and Prakash, M. 2017. Antibiogram of Bacterial Isolates from High Vaginal Swabs of Pregnant Women from Tertiary Care Hospital in Puducherry, India. Int.J.Curr.Microbiol.App.Sci. 6(1): 964-972. doi: http://dx.doi.org/10.20546/ijcmas.2017.601.114

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