Effects of Extended Storage of Chlorhexidine Gluconate and Benzalkonium Chloride Solutions on the Viability of Burkholderia cenocepacia

Abstract

Chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) formulations are frequently used as antiseptics in healthcare and consumer products. Burkholderia cepacia complex (BCC) contamination of pharmaceutical products could be due to the use of contaminated water in the manufacturing process, over-diluted antiseptic solutions in the product, and the use of outdated products, which in turn reduces the antimicrobial activity of CHX and BZK. To establish a "safe use" period following opening containers of CHX and BZK, we measured the antimicrobial effects of CHX (2-10 μg/ml) and BZK (10-50 μg/ml) at sublethal concentrations on six strains of Burkholderia cenocepacia using chemical and microbiological assays. CHX (2, 4, and 10 μg/ml) and BZK (10, 20, and 50 μg/ml) stored for 42 days at 23°C showed almost the same concentration and toxicity compared with freshly prepared CHX and BZK on B. cenocepacia strains. When 5 μg/ml CHX and 20 μg/ml BZK were spiked to six B. cenocepacia strains with different inoculum sizes (10⁰ -10⁵ CFU/ml), their toxic effects were not changed for 28 days. B. cenocepacia strains in diluted CHX and BZK were detectable at concentrations up to 10² CFU/ml after incubation for 28 days at 23°C. Although abiotic and biotic changes in the toxicity of both antiseptics were not observed, our results indicate that B. cenocepacia strains could remain viable in CHX and BZK for 28 days, which in turn, indicates the importance of control measures to monitor BCC contamination in pharmaceutical products.

Keywords: Bactericidal effects; Burkholderia cenocepacia; antiseptic; benzalkonium chloride; chlorhexidine gluconate.

Fig. 1.

Change in the start of growth time (ΔSGT) by chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) against six Burkholderia cenocepacia strains after opening containers at initial (0) day and 42 days. (A) ΔSGT of 2 and 4 μg/ml CHX. (B) ΔSGT of 10 and 20 μg/ml BZK. Values represent the mean ± standard deviation of six B. cenocepacia strains in triplicate.

Fig. 2.

Monitoring of 15 μg/ml chlorhexidine gluconate (A) and 50 μg/ml benzalkonium chloride (B) concentrations with various cell numbers (10⁵, 10⁴, 10³, 10², and 10 CFU/ml) of Burkholderia cenocepacia for 28 days. Values represent the mean ± standard deviation of six B. cenocepacia strains in triplicate.

Fig 3.

Kinetics of the growth of Burkholderia cenocepacia J2315 in 5 μg/ml chlorhexidine gluconate (CHX) (A) and 20 μg/ml benzalkonium chloride (BZK) (B) preincubated with 10⁵ cells of strain HI2976 for 28days. Symbols represent averages of triplicate samples, and error bars represent the standard deviation. Closed circle: with B. cenocepacia HI2976 (approximately 10⁵ CFU/ml) for 28 days at 23°C. Open circle: without B. cenocepacia HI2976.

Fig. 4.

Recovery and detection with various cell numbers of six Burkholderia cenocepacia strains during incubation with 5 μg/ml chlorhexidine gluconate (A, C, E, and G) and 20 μg/ml benzalkonium chloride (B, D, F, and H) for 28 days. (A and B) 10⁵ CFU/ml inoculation, (C and D) 10⁴ CFU/ml inoculation, (E and F) 10³ CFU/ml inoculation, (G and H) 10² CFU/ml inoculation. Values represent the mean ± standard deviation of six B. cenocepacia strains in triplicate. Solid lines show the 95% upper and lower predicted interval. Dashed lines represent the mean. Six B. cenocepacia strains were not recovered at 2 days indicated by arrows.

Fig 5.

Comparison of the recovery of Burkholderia cenocepacia in 2 μg/ml chlorhexidine gluconate (CHX) and 10 μg/ml benzalkonium chloride (BZK) after 0, 14, and 28 days preincubation in distilled water. Values represent the mean ± standard deviation of six B. cenocepacia strains in triplicate. Solid lines show the 95% upper and lower predicted interval. The dashed line represents the mean.