Isolation and characterization of biocides resistant bacteria from dental unit water line biofilms

Abstract

Six biocides resistant isolates were isolated from dental unit water lines (DUWL) in Pakistan. All isolates could tolerate 150 microg ml(-1 )of biocides (5.25% sodium hypocholrite, 35% H2O2, 4% tween 20, 1% povidine iodine, 0.2% chlorohexidine gluconate, 1% ethylene di-amino tetra acetic acid and 1% phenol) on l-agar and 100 microg ml(-1 )in l-broth. The growth rate of all isolates was determined by generating growth curves at 37 degrees C for 48 h. The isolates were found to differ in growth rates with lag phase varying from (4-6 h) in biocides supplemented media compared to 2-4 h in biocides free medium. They have wide temperatures (24-42 degrees C) and pH (5-9) ranges. Traditional ways of identification of bacteria by phenotypic characteristics were accomplished by phenotypic and biochemical characterization. Heavy metals and antimicrobial susceptibility tests indicated that all isolates examined were resistant to trimethoprim, chloramphenicol while sensitive to HgCl2 and Pb (NO3)2. Almost all isolates were opportunistic pathogens. The 16S rRNA-encoding genes from these six isolates were sequenced to confirm the identity of these isolates. 5 different genera (Bacillus, Achromobacter, Pseudomonas and Klebsiella) of bacteria were identified by 16S rDNA genes amplified from genomic DNA of biocides resistant DUWL biofilm isolates. Analysis of 16S rDNA genes revealed a much more clear identification of microorganisms than culture methods. However, different species of the same genera can have the same 16S rRNA gene sequence but are different due to phenotypic differences or different clinical manifestations.