Genomic diversity in biofilm forming pathogenic bacteria isolated from clinical samples

Abstract

Biofilms has become an emerging health concern as bacteria in biofilm are more resistant to antimicrobials and disinfectants than their planktonic counterparts. The multifactorial nature of biofilm development imposes great challenges for the use of conventional antimicrobials. It is therefore very important to understand the molecular basis of biofilm formation to find effective therapeutics to block the early steps of biofilm. In this study, a total of 45 MDR clinical isolates from different Bangladeshi hospitals were tested for their biofilm forming abilities. Of the 45 clinical isolates tested, 38 (85%) produced biofilm and 13 (29%) were characterized as strong biofilm formers (SBF). Among the SBFs, P. aeruginosa isolates were prevalent. Fluorescent image data analysis and viable cell count after 10 minute exposure with 0.1% SDS and NaOCl on P. aeruginosa biofilms did not show significant lethal effect on attached cells viability. On the other hand, 2 to 4 log cycle reduction on viable cell count observed after 70% ethanol and Savlon treatment, but the bacterial load remained as high as 103-104 cells/ mm2, indicating the failure of these agents to eradicate the biofilms completely. Despite of having variable biofilm forming ability, common biofilm related genes were detected in all of 10 the P. aeruginosa isolates tested by conventional PCR. Whole genome sequencing of a strong (27b), a moderate (20c) and a weak biofilm former (30b) isolate was performed and analysis of biofilm related genes in the sequenced genomes revealed that, 80 of the 88 biofilm related genes possess 98-100% sequence identity to the reference PA01 strain. Complete and partial sequence of LecB of 10 P. aeruginosa isolates revealed that, isolates that have PA14 like LecB sequence produced strong biofilms, while PAO1 like LecB containing isolates are either moderate or weak biofilm formers. All of the 7 Pel operon protein coding genes in weak biofilm former isolate 30b showed significant nucleotide and amino acid sequence variation with other tested isolates, and these proteins are 99% identical with the Pel operon proteins of PA7. Bioinformatics analyses identified distinct sequence and structural features that separates PA7 like Pel operon proteins from reference PAO1 like Pel operon. Congo red and Pellicle forming assays revealed that, the sequence and structure variations may have interfered with Pel production pathway and resulted in impaired Pel production in 30b. Expression analysis also showed that, both PelB and LecB proteins were about 5-6 folds upregulated in SBF 27b in comparison with WBF 30b. Our findings indicate significant genomic divergence in biofilm related genes of P. aeruginosa strains that affect their biofilm phenotypes.