Prevalence of the fimbrial operon mrkABCD, mrkA expression, biofilm formation and effect of biocides on biofilm formation in carbapenemase-producing Klebsiella pneumoniae isolates belonging or not belonging to high-risk clones

Abstract

Background: The role of mrkA adhesin expression, biofilm production, biofilm viability and biocides in the biofilm of carbapenemase-producing Klebsiella pneumoniae isolates was investigated.

Methods: Seventeen isolates representing different sequence types and carbapenemases were investigated. mrkA expression was determined by real-time reverse transcription polymerase chain reaction. Biofilm production (25°C and 37°C, with and without humidity) was determined by the crystal violet assay. The effect of isopropanol, povidone-iodine, sodium hypochlorite, chlorhexidine digluconate, benzalkonium chloride, ethanol and triclosan on biofilm was determined. The effect of povidone-iodine on biofilm biomass and thickness was also determined by confocal laser scanning microscopy.

Results: mrkA expression ranged from 28.2 to 1.3 [high or intermediate level; 64% of high-risk (HR) clones] and from 21.5 to 1.3 (50% of non-HR clones). At 25°C, biofilm formation was observed in 41% of isolates (absence of humidity) and 35% of isolates (presence of humidity), whereas at 37°C, biofilm formation was observed in 76% of isolates with and without humidity. At 25°C, biofilm producers were more frequently observed in HR clones (45% with humidity and 55% without humidity) than non-HR clones (17% with and without humidity). Biofilm viability from day 21 was higher at 25°C than 37°C. The greatest decrease in biofilm formation was observed with povidone-iodine (29% decrease), which also decreased biofilm thickness.

Conclusions: Biofilm formation in carbapenemase-producing K. pneumoniae is related to mrkA expression. Biofilm formation is affected by temperature (37°C>25°C), whereas humidity has little effect. Biofilm viability is affected by temperature (25°C>37°C). At 25°C, HR clones are more frequently biofilm producers than non-HR clones. Povidone-iodine can decrease biofilm production and biofilm thickness.

Keywords: Biocide; Biofilm; Carbapenemase; Klebsiella pneumoniae; mrkA.