Induction of Biofilm Formation in Klebsiella pneumoniae ATCC 13884 by Several Drugs: The Possible Role of Quorum Sensing Modulation

Abstract

Bacterial resistance is caused by several biochemical factors, the formation of biofilm being one of the main causes. This process is triggered by Quorum Sensing (QS), through the production of endogenous molecules, although other substances such as natural products can also do this. In this work, we aimed to determine whether some drugs are involved in the induction of biofilm formation in Klebsiella pneumoniae ATCC 13884, and thus, increase bacterial resistance. For this, the effect of 22 drugs on K. pneumoniae ATCC 13884 growth was determined at sub-plasmatic concentrations; the production of autoinducer lactones was established by HPLC and with a biosensor. The induction of biofilm formation was determined through crystal violet assay at 585 nm in a microplate reader and using urethral catheters. According to the in vitro assays, some drugs were found to induce biofilm formation in K. pneumoniae ATCC 13884. The effect of acetaminophen, hydrochlorothiazide, and progesterone stood out. The first drug caused several changes in the biochemistry of K. pneumoniae ATCC 13884 related to QS: high synthesis of N-hexanoyl-homoserine lactone, increasing bacterial populations by 27% and biofilm formation by 49%, and a more gentamicin resistant biofilm. Furthermore, it increased the colonization area of urethral catheters. Hydrochlorothiazide showed the biggest increase in the induction of biofilm formation of 51%, and progesterone displayed the greatest ability to provoke bacterial mass adherence but had no effects on K. pneumoniae ATCC 13884 bacterial population growth.

Keywords: Klebsiella pneumoniae ATCC 13884; acetaminophen; biofilm; drugs; hydrochlorothiazide; quorum sensing; resistance; virulence.

Figure 1

Kinetics of Klebsiella pneumoniae ATCC 13884 biofilm formation. K. pneumoniae ATCC 13884 was incubated for 54 h and the biofilm was analyzed at 6, 12, 24, 30, 36, 48, and 54 h to determinate the best time for maximum biofilm formation. The highest value was 30 h and indicated the time required to obtain a mature biofilm. * Statistically significant differences were observed for 6 h, 12–30 h, and 36–54 h (* p < 0.05) (n = 15).

Figure 2

Kinetics of the production of C6-AHL in Klebsiella pneumoniae ATCC 13884 cultures. All samples were doped with 0.2 µg/mL C6-AHL; a control experiment was conducted but without K. pneumoniae ATCC 13884. * Statistically significative difference.

Figure 3

Mature biofilm elimination after application of antibiotics. Gentamicin eliminated 50% of the mature biofilm at between 0.12 and 1.0 µg/mL, while ciprofloxacin eliminated 50% of the mature biofilm at 0.03 µg/mL. Calculated values are in respect to the control group mean. ** Statistically significant difference between groups for gentamicin. * Statistically significant difference between groups for ciprofloxacin.

Figure 4

Effect of gentamicin on the stability of the biofilm formed under biofilm-inducing drugs at plasmatic concentrations. Mature biofilm formed after 30 h of drug applications was treated with gentamicin 0.1 μg/mL, and then, the mature biofilm remainder was quantified. In acetaminophen, a drug-dependent decrease was noticed at higher concentrations of 2.5 and 1.25 μg/mL. * Statistically significative difference.

Figure 5

K. pneumoniae ATCC 13884 biofilm formed on coverslip glass. The images were taken from three different zones on the coverslip glass. The brighter areas indicate greater accumulation of bacteria. (A) Biofilm assembled under acetaminophen has more bacterial aggrupation and a larger colonized area. (B) The control group has few bright areas and less bacteria adherence. Bar 50 μm.

Figure 6

Optical microscope image of the K. pneumoniae ATCC 13884 biofilm assembled on the urethral catheter: (A) biofilm formation during acetaminophen treatment; and (B) without treatment with acetaminophen; both stained with crystal violet at 0.05%. Quantification of the area for each treatment was carried out using the ImageJ program, processing the sample by the particle content. Thus, the colonized area in the control sample was 2.07%, while in the acetaminophen sample it was 12.17%.

Figure 7

Scanning electron microscope Image of K. pneumoniae ATCC 13884 biofilm assembled on urethral catheter. (A–C) Biofilm formed under the effects of acetaminophen displayed large colonization areas and compact masses bacteria in prominent islets. (D–F) Biofilm without treatment showed more empty zones with free bacterial masses. Bar 100 µm.