Comparative Analysis of Microflora and Screening of Isolated Phages Against them in Pregnant and Non-Pregnant Females Suffering from Bacterial Vaginosis

Abstract

Bacterial Vaginosis (BV) is the infection of females’ genital system. It is a polymicrobial infection which shifts the paradigm of vaginal healthy microbiome to facultative anaerobe. Globally, BV prevalence in females is about 21.2 million, in Pakistan its prevalence is 35.3%. BV leads to serious complications in pregnancy, facilitates the onset of sexually transmitted diseases (STDs) and cervical cancer. Clindamycin and Metronidazole antibiotics are used for the treatment of BV worldwide. However, the relapse rate of BV infection in females indicate resistant against antibiotics treatment. Probiotic therapy alone or in combination with antibiotics is an alternative option for the treatment of BV infection. Nevertheless, large number of studies failed to reveal significant effects of probiotic therapy in BV patients. The pitfalls of currently available therapies against BV draw the attention of scientists to explore other treatment options. Phage therapy has shown a promising solution in this regard. The lytic activity of phages can be an effective strategy for combating antibiotic resistant bacteria. In addition to this advantage, phages are also host specific, safer, cost effective and easier to handle. Current study was designed to investigate the BV microflora of pregnant and non- pregnant females and their sensitivity towards antibiotics. The samples were collected from Obstetrics and Gynaecology Department of KRL Hospital, Islamabad, Pakistan. The consent of the patients was obtained before sampling. Total 60 samples were collected using high vaginal swabs (HVS), 30 from pregnant while 30 from non-pregnant females. The sample containing HVS were streaked on three different media i.e., LB agar, MRS agar and Blood agar for the isolation and comparison of BV microbiota. Total 150 colonies and 10 different bacterial strains were isolated from collected samples. Morphological characterization showed no difference in the microflora of BV infected pregnant and non- pregnant females, however, the number of isolates were different in both groups. Staphylococcus and Streptococcus species were prevalent in both pregnant and non- pregnant females. While Shigella flexneri was present only in non-pregnant females and was detected by 16S rRNA sequencing. Sensitivity and efficacy of antibiotics against isolated microbiota was also observed. Surprisingly, all of the bacterial strains showed

resistant against metronidazole and clindamycin, except 35 bacterial strains which showed sensitivity against clindamycin. Furthermore, isolation of phages was carried out from sewage water of slum areas of Islamabad, Pakistan. Lytic activity of isolated phages was examined against all isolates of BV samples. Three bacterial strains showed sensitivity to these isolated lytic phages. The 16S rRNA sequencing of these bacterial strains revealed that they belong to Enterococcus feacalis strain (QAUSD05), Enterococcus spp. (QAUEF03) and Shigella flexneri (HQ407262.1). Bioactivity analysis of isolated phages was observed within a pH range of 1-11. Phages against E. fecalis and Enterococcus QAUEF03 showed their maximum activity at neutral pH 7 while the phage against S. flexneri showed its maximum activity at pH 9. Similarly, thermal sensitivity of phages was checked against Enterococcus strains, showed their highest activity at 55⁰C and 70°C respectively. Phage of S. flexneri had an optimum activity at a temperature of 40⁰C. Morphological identification of phages was performed by Scanning Electron Microscopy (SEM). SEM results showed that all phages were spherical in shape. In order to analyze the genome nature of phages, organic extraction method was employed. It was observed that all phages had DNA genome with the size range in between 15-20kbp. This show they might share lineage with Podoviridae family as per reviewed from the literature. Overall, the current study provides the better understanding of BV microflora in pregnant and non-pregnant females. The isolation of phages provides a road map for the better treatment of BV infection microflora. Isolated phages can also be assessed to explore their potential to treat other bacterial diseases bacillary dysentery, shigellosis and urinary tract infections (UTIs) as well. Isolation of new phages against causative agents of BV, phages cocktails or combination with antibiotics may provide a pathway and a new hope to the world to combat the antibiotic resistance issue and highly effective treatment against BV infection.