Molecular Study of Some Virulence Factors and Antimicrobial Susceptibility Pattern of Citrobacter frundii Isolated from Human Diarrhea

Abstract

A total of (100) clinical specimens from Stool were collected under aseptic condition. These specimens were collected from patients attending to Al-Hilla Teaching Hospital in Babylon province from August (2019) and March (2020). Fecal samples were collected from (100) consecutive patients, with community-acquired gastro-enteritis. Quantitative biofilm formation experiments were performed in a microtiter (biofilm assay) using Trypticase Soy Broth supplemented with (1%) glucose. This assay was repeated as triplicate to increase the accuracy of assy. According to mean of value at (63) nm the results were interpreted as none, moderate and strong biofilm former when the mean of OD value were (0.240) respectively. The results revealed that seven isolates of Citrobacter freundii isolates were biofilm former (41.2%), the strong biofilm former were account for 5/7(71.4%) while isolates that express moderate biofilm formation were 1/7(14.3%), and 1/7(14.3%) isolates that express weak biofilm formation. The (17) C. freundii isolates were tested for susceptibility to (14) antibiotics belonging to (8) antibiotic classes using the disk diffusion method according to CLSI recommendations. Most of the (17) bacterial isolates were resistant to β-lactams, especially to Penicillins (35.29%), Cephalosporins (23.52%-70.58%). Resistance to the two quinolones (Ciprofloxacin and Levofloxacin) tested was (11.7%). Resistance to other antibiotics included Aminoglycosides (5.88%-11.7%), Tetracyclines (17.6%), Chloramphinicol (11.7%), Trimethoprim (23.52%) and Nitrofuran (23.52%). In this study, hemolysin production by all isolates were done, it was found that none of the isolates showed a typical hemolysis pattern on 5% sheep blood agar plates. Bacterial isolateswere screened for siderophores production the results showed 16(94.1%) isolates were able to produce siderophores. Ten isolates were positive for protease production (58.8%). The prevalence of viaB, hlyA, LT, and STp related genes in the bacterial isolates were low in this study. DNA was extracted from all (17) isolates; conventional PCR was carried out using these DNA samples for the amplification of specific viaB primer; after that gel electrophoresis showed that, out of the (17) samples, only 7(41.2%) produced the specific (516) bp DNA fragment when compared with allelic ladder. A PCR survey was performed using primers hlyA to determine whether a (597) bp hlyA gene fragment could be detected in the (17) Citrobacter freundii. After that gel electrophoresis showed that, out of the (17) samples, only 1(5.9%) produced the specific (597bp) DNA fragment when compared with allelic ladder. After that gel electrophoresis to detect TA gene showed that, out of the (17) samples, 8(47.1%) Produced the specific (273bp) DNA fragment when compared with allelic ladder. Moreover, 4(23.5%) isolates produced the specific (166 bp) DNA fragment as recorded heat stable toxin gene among isolates in recent study.