Phenotypic Detection of Adhesive Activity and Biofilm Formation among Prevotella spp. Isolated from Patients Suffering from Acute Inflammation in Root Canal Teeth

Abstract

In this study, 150 patients were visited to dental clinic in Al - Hilla city suffering from acute inflammation in root canal teeth, the samples were cultivated anaerobically culture at (37°C) for (48) hrs. Identification of Prevotella spp. was depended on the colonial morphology, microscopically, and biochemical tests as initial identification, after that, these isolates were identified by specific negative cards of Vitek 2 system, out of 150 specimens only 17(11.4%) isolates were belonged to Prevotella, while 133(88.6%) related to other types of microorganisms. Identification of Prevotella spp. by Vitek 2 system were classify these bacteria in to three species, the results showed that, out of 17 Prevotella isolate, 12(70.6%) were related to Prevotella intermedia, 3(17.7%) isolates were related to Prevotella nigresceens and 2(11.7%) isolates were related to Prevotella tannerae. In order to identify which Prevotella bacteria are present in the subject, the researchers extracted DNA from the numerous Prevotella strains that had previously been identified with the Vitek2 system. They then used the extracted DNA to conduct PCR with specific primers for the 16S RNA gene to amplify it. Gel electrophoresis revealed that all of the Prevotella intermedia samples (100%) generated a 660 bp DNA fragment, which was unique to the strain. Isolates that previously tested positive for Prevotella nigresceens using the Vitek2 method were identified with molecular testing for 16srRNA gene. The findings revealed that every one of the 3 test subjects (100%) was positive. Bands showing a higher yield as compared to the genomic sequence appear while looking for more than 800 bp of sequence. As well, 16srRNA gene detection was performed on 5 Prevotella tannerae isolates, and it was determined that the 2 (100%) isolates tested positive for this gene. The findings showed an increase in (1110 bp) band size compared to the normal allelic ladder. Epithelial cell adhesion is thought to be a vital component in bacterial infection. All the examined species of Prevotella (100% of the group) were positive for the findings. Additional studies to measure quantitatively the quantity of biofilm production were done in a microtiter with Trypticase Soy Broth plus (1 percent) glucose. To improve the accuracy of this test, it was performed three times. The study's findings were translated into three categories of biofilm formers: non- biopersistent, moderate, and chronic bacteria biofilm formers based on the level of OD value, which can be read as (OD<0.120, OD=0.120-0.240, and OD>0.240). The findings concluded that all Prevotella isolates produced biofilms (100 percent) and Prevotella intermedia biofilms were present in a majority of specimens (10 percent), the majority of Prevotella nigrescens also displayed biofilms (3 out of 3, 100 percent), and the minority of Prevotella tannerae expressed moderate biofilms (1 out of 2, 50 percent) while only the minority of Prevotella intermedia specimens showed any trace of biofilm (2 out of 12, percent) (50 percent). To test for in vitro antibiotic resistance in root tooth-related acute inflammation, Prevotella spp. isolates from all Prevotella isolates were put through the modified Kirby-Bauer disc diffusion technique. Clindamycin, Amoxillin, Ceftazidime, Tetracycline, Chloramphenicol, Aztreonam, and metronidazole are all antibiotics that have been shown to be effective against Prevotella spp. isolates. The results compare according to Clinical Laboratory Standard Institute guidelines (CLSI, 2019) as resistant. Highest rate of resistant is seen to almost antibiotics used in present study, 16(94.1%) isolates were resistant to Clindamycin, 15(88.2%) isolates were resistant to Amoxillin, 13(76.47%) isolates were resistant to Chloramphenicol, 11(64.7%) isolates were resistant to Tetracycline, 9(52.9%) isolates were resistant to Aztreonam and 3(17.6%) isolates were resistant to metronidazole.