title: Biocide tolerance and antibiotic cross-resistance in human pathogenic bacteria
Creators: name: Rödel, Alice
Publisher: name: University of Bayreuth
Year: 2022
Subjects: subject: Biocide tolerance
subject: antibiotic resistance
subject: E. coli
subject: L. monocytogenes
subject: E. faecium
subject: 500
subject: 570

description: Biocides are applied to control harmful organisms. Especially the use of microbicides as disinfectants is a vital tool to prevent the spread of multidrug-resistant human pathogenic bacteria. Due to the increased use of disinfectants in recent years, concerns have been raised about biocide tolerance and the development of antibiotic cross-resistance. In vitro studies indicate biocide use as a risk factor for the emergence of antibiotic resistance. Still, a causal link between biocide usage and antibiotic resistance development in environmental settings needs to be verified. Thus, this thesis pursued three main objectives (I-III). The controversial current knowledge on the potential of bacteria to develop biocide tolerance and/or antibiotic resistance in response to biocide exposure was analyzed (objective I). Potential associations between biocide tolerance and antibiotic resistance, and underlying genetic determinants in field isolates were examined (objective II). The final aim of this thesis was to develop a ready-to-use test system enabling fast and accurate biocide susceptibility testing of bacteria (objective III). For the first aim, 78 in vitro biocide adaptation studies were evaluated. While literature demonstrated the general linkage between biocide exposure and antimicrobial cross-resistance, the potential of biocide–induced resistance development varied largely between biocides, bacterial target organisms, and experimental settings. Stable adaptation to biocides as well as altered antibiotic susceptibility profiles was frequently reported for biguanides, phenols, and quaternary ammonium compounds (QACs). For the second aim, comprehensive analyses were conducted of isolates from food (Listeria monocytogenes, n=93), livestock (Escherichia coli, n=93), and clinical environments (Enterococcus faecium, n=90) in Germany. Biocide and antibiotic susceptibility were determined for all isolates by broth microdilution methods according to ISO 20776-1. To differentiate susceptible isolates from those with reduced susceptibility to biocides, minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) representing 95% of the tested population (MIC95, MBC95 values) served as tentative epidemiological cut-offs. Underlying genetic determinants were detected by whole genome sequencing for L. monocytogenes and E. coli. The overall proportion of isolates with reduced biocide susceptibility was relatively low, and statistically significant phenotypic associations to antibiotic resistance were not found. However, genotypic associations were observed in E. coli. Biocide tolerance conferring genes co-located close to antibiotic resistance genes on plasmids were identified, highlighting the potential for co-selection. Biocide susceptibility of all tested isolates was well below in-use concentrations. That is why they are defined as non-resistant. Identified tolerances were associated with genetic determinants in several cases. Reduced susceptibility to QACs in L. monocytogenes could be mainly traced back to genes coding for small multidrug-resistance family efflux pumps. E. coli isolates with reduced formaldehyde susceptibility carried the gene coding for the degradative enzyme glutathione- and NAD-dependent formaldehyde dehydrogenase. In contrast to antibiotic susceptibility testing, no generally accepted, standardized biocide susceptibility test methods are available. Thus, the third aim was to establish a reliable, high-throughput screening system using vacuum dried biocide microtiter plates to identify bacteria resistant to cationic biocides frequently used in hospital settings. The developed test system was validated against the broth microdilution method using freshly prepared stocks of biocides. Biocide plates were evaluated for gram-positive Enterococcus spp. as well as gram-negative E. coli. This test system has been shown to provide highly reliable results. Subsequently, the test method served for the third susceptibility study of E. faecium from the clinical environment. The test system proved to be a fast and easy-to-handle surveillance tool to identify biocide tolerant isolates. Monitoring and early identification of clinical isolates tolerant to disinfectants applied in hospitals could help to adapt hygiene measures and control nosocomial infections while simultaneously reducing the frequency of antibiotic treatment.