Microbial Diversity in Complicated Urinary Tract Infection among Urolithiatic Patients

Abstract

Urolithiasis is a urological disorder associated with various complications including complicated urinary tract infection which can cause renal failure. Whole microbial diversity associated with complicated UTI (cUTI) in urolithiasis has not been deciphered completely. In present study, culture dependent and independent microbial diversity was investigated in urine specimens of urolithiatic patients with cUTI and compared with the urinary microbial diversity of urolithiatic patients without cUTI and healthy individuals. Standard microbiological procedures were used for culturable microbes and unculturable microbes were identified by targeting V4 region of 16S rRNA for bacterial and ITS gene for fungal diversity. Mid-stream urine samples were collected from 164 urolithiatic patients and 40 healthy controls. Overall, among urolithiatic patients 55.5% of the patients were males whereas 44.5% were females with mean age of 43yrs. cUTI was prevalent (64%) condition among these urolithiatic patients. Clinically, catheterized patients having stone disease for extended period were significantly positive for cUTI (P<0.001). By using culture dependent methods, different Gram positive and negative bacterial species were identified. These urolithiatic patients were predominantly (59%) colonized by bacterial species where K. pneumoniae (36%) was the most prevalent bacteria followed by E. coli (33%), S. aureus (9%), S. saprophyticus (5%) and S. epidermidis (2.4%). But in case of urolithiatic patients with cUTI, E. coli was the most prevalent. Female patients were more prone to be colonized with E. coli as compared to males (P=0.02). Upon analysis and comparison of virulence factors, 87% of K. pneumoniae showed strong biofilm formation ability and majority (80%) of these isolates were from urolithiatic patients with cUTI. Although E. coli isolates also had strong biofilm forming ability but their number was low. Furthermore, 22/69 K. pneumoniae isolates were hypervirulent strains. Majority of these cUTI patients’ isolates displayed hemolysis, K. pneumoniae isolates were alpha hemolysin producer and E. coli isolates had varying degree of hemolysin production. Most of these Gram negative bacterial isolates were xiv resistant against tested antibiotics. Among E. coli isolates, 44 were multi drug resistant (MDR) and 71% of these were isolated from cUTI patients. Thirty seven E. coli isolates were phenotypically ESBL positive, and 70% of these were from cUTI cases. In case of K. pneumoniae, 21/23 isolates from cUTI subjects were MDR. E. coli isolates from urolithiatic patients had 41%, 30% and 9% CTX-M, TEM and SHV genes prevalence respectively. In case of K. pneumoniae, 20%, 7% and 1.5% of isolates were positive for CTX-M, TEM and SHV genes respectively. Quinolone resistance genes were less prevalent among E. coli and K. pneumoniae isolates. Through culture dependent method, Candida was the only fungus which was isolated from the urine samples of urolithiatic patients as well as healthy controls. In these patients, 45% were colonized with three Candida species i.e. C. albicans, C. glabrata and C. krusei. The urolithiatic patients with cUTI had 1.8 fold increased risk for Candida colonization as compared to patients without cUTI. A higher isolation of C. albicans (66%) was seen than non-albicans (34%) in urolithiatic patients. Overall, in 64 Candida isolates, esterase production was in 44/51 C. albicans, 17/23 C. glabrata and 3/3 C.krusei. Of these esterase producers, 80% of Candida were from the cUTI patients (P<0.001). Phospholipase activity was seen in 38/51 C. albicans, 09/23 C. glabrata and 3/3 C. krusei. A significant esterase and phospholipase activity was detected in Candida isolates from urolithiatic patients with cUTI as compared to patients without cUTI. Among all Candida isolates, 43% showed strong biofilm forming activity, which was also found to be significantly high in isolates from urolithiatic patients with cUTI (P<0.05). Through culture independent diversity analysis, greater diversity richness and evenness in female urolithiatic patients with cUTI as compared to male urolithiatic patients and the healthy individuals was seen. Highest Bray-Curtis dissimilarity index was seen in male urolithiatic patients with cUTI as compared to healthy females which is an indication of microbial diversity change in number and type of microbes with gender and disease status. In female urolithiatic patients with cUTI, predominant bacterial genera found were Prevotella followed by Enterobacter, Achromobacter, Agrobacterium and Alcaligenes. However, Acinetobacter was the most prevalent (84%) among total genera detected in xv male urolithiatic patients with cUTI. Female urolithiatic patients with cUTI showed predominance of fungal genera Paraconiothyrium followed by Necteria, Cladosporium and Cryptococcus. While in male patients with cUTI Ustilago was predominant genera followed by Aspergillus and Necteria. Known prevalent uropathogens which are thought to be dominant were not predominating microbes in this condition. No E. coli and K. pneumoniae was observed in the microbiome through culture independent method. In present work, dual species biofilm assays for different combinations of isolated uropathogens Candida, E. coli and K. pneumoniae were studied as in vitro biofilm development model. A reduction in the biofilm biomass in dual species biofilm of C. albicans and E. coli isolates from cUTI was observed but, interestingly metabolic activity was enhanced. A possible antagonistic behaviour of Candida and K. pneumoniae in their dual biofilm was observed as both biofilm biomass formation and metabolic activity was reduced while in dual bacterial consortium of E. coli and K. pneumoniae a lowered biofilm biomass but enhanced metabolic activity was observed. Findings from the present work showed there was dysbiosis in urolithiasis where MDR isolates with enhanced virulence expression were present in culturable microbial diversity especially in cUTI cases. Various unculturable microbes were also detected through culture independent method. Data from the present work indicated for the first time, a snapshot of urinary microbial diversity in cUTI among urolithiatic patients. These findings suggest that there should be focus on evidence-based therapy using advanced techniques to determine the real picture of microbial diversity in cUTI patients for proper and targeted eradication of involved microbes in such infections to minimize the chances of antibiotic resistance in other commensal and disease burden.