The effect of biocidal residues on resistance phenotype in escherichia coli

Antimicrobial resistance (AMR) poses a threat to worldwide health, in particular in relation to multi-drug resistant organisms. Hygienic cleaning and disinfection can contribute in the prevention of AMR. There is ample evidence to support the use of disinfectants (biocides) in the decrease of healthcare acquired infections (HCAIs) (Weinstein and Hota, 2004, Maillard, 2018, Webber et al., 2015). However, there is also evidence of instances where disinfectant efficacy may be impeded resulting in microbial survival and emerging resistance (A Rutala and J Weber, 2007). Biocides are said to act as a selective pressure that encourages the acquisition of resistance traits in bacterial cells (Qiu et al., 2012). Furthermore, selective pressure may result from the overexposure of very low concentrations of biocides over long periods of time (Andersson et al., 2012, Gullberg et al., 2014, Gullberg et al., 2011, Thomas et al., 2000). Some biocidal products make claims of “residual biocidal activity” whereas efficacy is usually imparted to a much higher concentration. Some microbial populations may survive exposure to low biocide concentrations, and show decreased susceptibility or resistance to a biocide or consequentially other antimicrobials. This study aims to understand differences between bacterial selection and adaptation in Escherichia coli following exposure to realistic residual - during use - chlorhexidine (CHX) or benzalkonium chloride (BZC) concentrations. It was hypothesised that exposure to a high sub-biocide minimum inhibitory concentration (MIC) would exert a selective pressure enabling the least susceptible bacteria to survive resulting in a permanent change of susceptibility phenotype, whereas a low sub-MIC would be conducive to reactive metabolic shifts resulting in a transient change of susceptibility phenotype. iii Baseline biocide (CHX and BZC) and antibiotic susceptibility of E. coli isolates was obtained using a standard micro-dilution broth protocol, and EUCAST protocol. “Residual” CHX concentration left on surface over a 168 hours period was measured by HPLC. The impact of a range of biocide concentrations (including residual CHX ones) on growth kinetics was investigated. Any changes in susceptibility profile was assessed for stability. Efflux activity and metabolic regulation during exposure to low and high sub-CHX MIC were investigated aiming to identify a link with observed changes in susceptibility phenotype. Finally the propensity for different levels of CHX exposure to influence genetic transfer via conjugation was explored. It was demonstrated that a 0.006 ± 0.002 mg/mL is a realistic residual - during use exposure concentration of CHX. This concentration is 99% lower than the concentration initially applied (20 mg/mL). At this residual concentration, it was possible for CHX susceptible bacteria to survive the disinfection process. Five genotypically distinct strains (UCD-CFS ECP-1L3, UCD-CFS ECP-1L4, UCD-CFS ECP-1B2, UCD-CFS ECP-13P5, UCD-CFS ECP-13P4) demonstrated survival after a 5 min but not 24 hours CHX exposure. Surviving bacteria demonstrated elevated MIC and MBC values; the highest fold change was 32-fold (MIC) and 62- fold (MBC). The elevated MIC values obtained were higher than the average concentration of CHX found on surface. Decreases in MIC or MBC values were observed after residual BZC exposure. No stable changes in MIC and MBC were observed after exposure to residual CHX or BZC, but stable changes were observed for antibiotic resistance for amoxicillin/clavulanic acid, ampicillin, cefpodoxime and cephalothin. Efflux activity was observed during exposure to low (0.00005 mg/mL) but not for high (0.002mg/mL) sub-CHX and sub-BZC MIC. It was demonstrated that changes in susceptibility coincided with changes in the ability to metabolise certain substrates including salicin, L-alanine, betain, creatanine and iv phenylethlalamine. These substances were linked to cell wall and stress signalling regulatory processes. It was surmised that E. coli was able to adapt through metabolic alterations to produce transient changes in CHX susceptibility and stable changes in antibiotic susceptibility. Furthermore, our results show that a transiently adapted population may be selected amongst less tolerant sub-populations at the established CHX-during use concentration. Overall, this work suggests that the intended application concentration of a biocide may in fact be lower than the MIC of target organisms. It is concluded that residual concentrations of biocides do have the potential to drive resistance, particularly stable cross-resistance to antibiotics, through prolonged exposure to low level during use concentrations, driving metabolic modifications of the cell envelope. The potential risk of cross-resistance warrants further investigation

Use of a predictive protocol to measure the antimicrobial resistance risks associated with biocidal product usage

Background In this study we assessed the propensity of biocide exposure in the development of antimicrobial resistance in bacteria. Methods Our protocol is based on reporting changes in established antimicrobial susceptibility profiles in biocides and antibiotics after during use exposure to a product. The during use exposure reflects worse conditions of product use during application. It differs from the term low concentration, which usually reflects a concentration below the minimal inhibitory concentration, but not necessarily a concentration that occurs in practice. Results Our results showed that exposure to triclosan (0.0004%) was associated with a high risk of developing resistance and cross-resistance in Staphylococcus aureus and Escherichia coli. This was not observed with exposure to chlorhexidine (0.00005%) or a hydrogen peroxide–based biocidal product (in during use conditions). Interestingly, exposure to a low concentration of hydrogen peroxide (0.001%) carried a risk of emerging resistance to antibiotics if the presence of the oxidizing agent was maintained. We observed a number of unstable clinical resistances to antibiotics after exposure to the cationic biocide and oxidizing agent, notably to tobramycin and ticarcillin–clavulanic acid. Conclusions Using a decision tree based on the change in antimicrobial susceptibility test results, we were able to provide information on the effect of biocide exposure on the development of bacterial resistance to antimicrobials. Such information should address the call from the U.S. Food and Drug Administration and European Union Biocidal Products Regulation for manufacturers to provide information on antimicrobial resistance and cross-resistance in bacteria after the use of their product

Impact of standard test protocols on sporicidal efficacy

Background There has been an increase in the availability of commercial sporicidal formulations. Any comparison of sporicidal data from the literature is hampered by the number of different standard tests available and the use of diverse test conditions including bacterial strains and endospore preparation. Aim To evaluate the effect of sporicidal standard tests on the apparent activity of eight biocides against Clostridium difficile and Bacillus subtilis. Methods The activity of eight biocidal formulations including two oxidizing agents, two aldehydes, three didecyldimethylammonium chloride (DDAC) and amine formulations, and sodium hypochlorite were evaluated using four standard sporicidal tests (BS EN 14347, BS EN13704, ASTM E2197-11, and AOAC MB-15-03) against B. subtilis (ACTC 19659) and C. difficile (NCTC 11209) spores. Findings C. difficile spores were more susceptible to the sporicides than were B. subtilis spores, regardless of the method used. There were differences in sporicidal activity between methods at 5 min but not at 60 min exposure. DDAC and amine-based products were not sporicidal when neutralized appropriately. Neutralization validation was confirmed for these biocides using the reporting format described in the BS EN standard tests, although the raw data appear to indicate that neutralization failed. Conclusion The different methods, whether based on suspension or carrier tests, produced similar sporicidal inactivation data. This study suggests that detailed neutralization validation data should be reported to ensure that neutralization of active spores is effective. Failure to do so may lead to erroneous sporicidal claims

Impact of antimicrobial wipes compared with hypochlorite solution on environmental surface contamination in a health care setting: a double-crossover study

Objective Antimicrobial wipes are increasingly used in health care settings. This study evaluates, in a clinical setting, the efficacy of sporicidal wipes versus a cloth soaked in a 1,000 ppm chlorine solution. Intervention A double-crossover study was performed on 2 different surgical and cardiovascular wards in a 1,000-bed teaching hospital over 29 weeks. The intervention period that consisted of surface decontamination with the preimpregnated wipe or cloth soaked in chlorine followed a 5-week baseline assessment of microbial bioburden on surfaces. Environmental samples from 11 surfaces were analyzed weekly for their microbial content. Results A total of 1,566 environmental samples and 1,591 ATP swabs were analyzed during the trial. Overall, there were significant differences in the recovery of total aerobic bacteria (P < .001), total anaerobic bacteria (P < .001), and ATP measurement (P < .001) between wards and between the different parts of the crossover study. Generally, the use of wipes produced the largest reduction in the total aerobic and anaerobic counts when compared with the baseline data or the use of 1,000 ppm chlorine. Collectively, the introduction of training plus daily wipe disinfection significantly reduced multidrug-resistant organisms recovered from surfaces. Reversion to using 1,000 ppm chlorine resulted in the number of sites positive for multidrug-resistant organisms rising again. Conclusions This double-crossover study is the first controlled field trial comparison of using preimpregnated wipes versus cotton cloth dipped into a bucket of hypochlorite to decrease surface microbial bioburden. The results demonstrate the superiority of the preimpregnated wipes in significantly decreasing microbial bioburden from high-touch surfaces

Pathogen transfer and high variability in pathogen removal by detergent wipes

Background The rise in health care-associated infections has placed a greater emphasis on cleaning and disinfection practices. The majority of policies advocate using detergent-based products for routine cleaning, with detergent wipes increasingly being used; however, there is no information about their ability to remove and subsequently transfer pathogens in practice. Methods Seven detergent wipes were tested for their ability to remove and transfer Staphylococcus aureus, Acinetobacter baumannii, and Clostridium difficile spores using the 3-stage wipe protocol. Results The ability of the detergent wipes to remove S aureus, A baumannii, and C difficile spores from a stainless steel surface ranged from 1.50 log10 (range, 0.24-3.25), 3.51 log10 (range, 3.01-3.81), and 0.96 log10 (range, 0.26-1.44), respectively, following a 10-second wiping time. All wipes repeatedly transferred significant amounts of bacteria/spores over 3 consecutive surfaces, although the percentage of total microorganisms transferred from the wipes after wiping was low for a number of products. Conclusions Detergent-based wipe products have 2 major drawbacks: their variability in removing microbial bioburden from inanimate surfaces and a propensity to transfer pathogens between surfaces. The use of additional complementary measures such as combined detergent/disinfectant-based products and/or antimicrobial surfaces need to be considered for appropriate infection control and prevention

Hydroxyethoxy phenyl butanone, a new cosmetic preservative, does not cause bacterial cross-resistance to antimicrobials

Introduction. Biocide-induced cross-resistance to antimicrobials in bacteria has been described and is a concern for regulators. We have recently reported on a new protocol to predict the propensity of biocide to induce phenotypic resistance in bacteria. Aim. To measure bacterial propensity to develop antimicrobial resistance following exposure to a new cosmetic preservative developed by L’Oréal R and I. Methodology. Well-established antimicrobials including triclosan (TRI) and benzalkonium chloride (BZC) and a new molecule hydroxyethoxy phenyl butanone (HEPB) were investigated for their antimicrobial efficacy, effect on bacterial growth, and their potential to induce resistance to chemotherapeutic antibiotics using a new predictive protocol. Results. The use of this predictive protocol with Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa showed that TRI and BZC significantly affected bacterial growth, MICs and minimum bactericidal concentrations (MBCs). There was no change in antibiotic susceptibility profile following exposure to BZC, but E. coli became intermediate resistant to tobramycin following treatment with TRI (0.00002 % w/v). HEPB did not change the antimicrobial susceptibility profile in P. aeruginosa and S. aureus but E. coli became susceptible to gentamicin. TRI exposure resulted in bacterial susceptibility profile alteration consistent with the literature and confirmed the use of TRI as a positive control in such a test. Conclusion. Data produced on the propensity of a molecule to induce bacterial resistance is useful and appropriate when launching a new preservative

Ethylzingerone, a Novel Compound with Antifungal Activity

International audiencePreservatives increase the shelf life of cosmetic products by preventing growth of contaminating microbes, including bacteria and fungi. In recent years, the Scientific Committee on Consumer Safety (SCCS) has recommended the ban or restricted use of a number of preservatives due to safety concerns. Here, we characterize the antifungal activity of ethylzingerone (hydroxyethoxyphenyl butanone [HEPB]), an SCCS-approved new preservative for use in rinse-off, oral care, and leave-on cosmetic products. We show that HEPB significantly inhibits growth of Candida albicans, Candida giabrata, and Saccharomyces cerevisiae, acting fungicidally against C. albicans. Using transcript profiling experiments, we found that the C. albicans transcriptome responded to HEPB exposure by increasing the expression of genes involved in amino acid biosynthesis while activating pathways involved in chemical detoxification/oxidative stress response. Comparative analyses revealed that C. albicans phenotypic and transcriptomic responses to HEPB treatment were distinguishable from those of two widely used preservatives, triclosan and methylparaben. Chemogenomic analyses, using a barcoded S. cerevisiae nonessential mutant library, revealed that HEPB antifungal activity strongly interfered with the biosynthesis of aromatic amino acids. The trpl LI mutants in S. cerevisiae and C. albicans were particularly sensitive to HEPB treatment, a phenotype rescued by exogenous addition of tryptophan to the growth medium, providing a direct link between HEPB mode of action and tryptophan availability. Collectively, our study sheds light on the antifungal activity of HEPB, a new molecule with safe properties for use as a preservative in the cosmetic industry, and exemplifies the powerful use of functional genomics to illuminate the mode of action of antimicrobial agents