Plasposon mutagenesis in Klebsiella pneumoniae isolates reveals the function of a hypothetical protein in biofilm formation

Aims: Identify novel genes involved in biofilm formation in Klebsiella pneumoniae by plasposon mutagenesis technique using the suicide vector pTnMod-OCm.Methodology and results: Four hundred twenty-five samples were collected from pathogenic and environmental sources.Klebsiella was identified using selective media and the final diagnosis was proven using the VITEK 2 system and 16S rRNA gene sequencing.Accordingly, 12 isolates were identified as K. pneumoniae.Antibiotic susceptibility showed that the highest resistance rate after ampicillin was detected for cefotaxime (33.3%), while the lowest resistance rate was towards amikacin and ciprofloxacin (8.3%).Klebsiella pneumoniae isolates from environmental samples were more sensitive to antibiotics than clinical isolates.Biofilm production on microtiter plates was investigated and the result characterized 91% of strains as strong biofilm producers while 9% of the strains as moderate biofilm producers.The ability to produce slime was examined as an indirect method for biofilm production on Congo red agar.The results indicated that 9% of strains were strong biofilm producers and the other 11 strains (91%) were weak biofilm producers.Triparental mating was used to introduce pTnMod-OCm in recipient K. pneumoniae SE12 strain using E. coli HB101/ pRK2013 as a helper strain.Mutants were screened for biofilm defects by plating them on Congo red agar and microtiter plates.Analysis of mutant defective in producing a biofilm showed that the plasposon was inserted into an open reading frame for a hypothetical protein found in K. pneumoniae. Conclusion, significance and impact of study:The results identify, for the first time, the role of this open reading frame (ORF2) in biofilm formation in K. pneumoniae.