Bacteria live primarily in microbial communities (biofilms), where they exhibit considerably higher biocide tolerance than their planktonic counterparts. Current standardized efficacy testing protocols of disinfectants, however, employ predominantly planktonic bacteria. In order to test the efficacy of biocides on biofilms in a standardized manner, a new assay was developed and optimized for easy-handling, quickness, low running costs, and above all—repeatability. In this assay, 5 mm glass- or polytetrafluoroethylene beads in 24 well microtiter plates served as substrate for Pseudomonas aeruginosa biofilms. After optimizing result-relevant steps, the actual performance of the assay was explored by treating P. aeruginosa biofilms with glutaraldehyde, isopropanol, or peracetic acid in predefined concentrations. The aspired 5 log10 reduction in CFU counts was achieved by glutaraldehyde at 5% (30 min), and by peracetic acid at 0.3% (10 min). In contrast, 80% isopropanol (30 min) failed to meet the reduction goal. However, the main accomplishment of this study was to unveil the potential of the array itself; most noteworthy here, a reliable repeatability of the results. The new bead assay for biofilms is a robust, quick and cost-effective method for assessing the efficacy of biocides against biofilms.
Background
One possible transmission route for nosocomial pathogens is contaminated medical devices. Formation of biofilms can exacerbate the problem. We report on a carbapenemase-producing Klebsiella pneumoniae that had caused an outbreak linked to contaminated duodenoscopes. To determine whether increased tolerance to disinfectants may have contributed to the outbreak, we investigated the susceptibility of the outbreak strain to disinfectants commonly used for duodenoscope reprocessing. Disinfection efficacy was tested on planktonic bacteria and on biofilm.
Methods
Disinfectant efficacy testing was performed for planktonic bacteria according to EN standards 13727 and 14561 and for biofilm using the Bead Assay for Biofilms. Disinfection was defined as ≥ 5log10 reduction in recoverable colony forming units (CFU).
Results
The outbreak strain was an OXA-48 carbapenemase-producing K. pneumoniae of sequence type 101. We found a slightly increased tolerance of the outbreak strain in planktonic form to peracetic acid (PAA), but not to other disinfectants tested. Since PAA was the disinfectant used for duodenoscope reprocessing, we investigated the effect of PAA on biofilm of the outbreak strain. Remarkably, disinfection of biofilm of the outbreak strain could not be achieved by the standard PAA concentration used for duodenoscope reprocessing at the time of outbreak. An increased tolerance to PAA was not observed in a K. pneumoniae type strain tested in parallel.
Conclusions
Biofilm of the K. pneumoniae outbreak strain was tolerant to standard disinfection during duodenoscope reprocessing. This study establishes for the first time a direct link between biofilm formation, increased tolerance to disinfectants, reprocessing failure of duodenoscopes and nosocomial transmission of carbapenem-resistant K. pneumoniae.
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