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

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

Increased Usage of Antiseptics Is Associated with Reduced Susceptibility in Clinical Isolates of Staphylococcus aureus

Hospital-acquired infection is a major cause of morbidity and mortality, and regimes to prevent infection are crucial in infection control. These include the decolonization of vulnerable patients with methicillin-resistant Staphylococcus aureus (MRSA) carriage using antiseptics, including chlorhexidine and octenidine. Concern has been raised, however, regarding the possible development of biocide resistance. In this study, we assembled a panel of S. aureus isolates, including isolates collected before the development of chlorhexidine and octenidine and isolates, from a major hospital trust in the United Kingdom during a period when the decolonization regimes were altered. We observed significant increases in the MIC and minimum bactericidal concentration (MBC) of chlorhexidine in isolates from periods of high usage of chlorhexidine. Isolates with increased MICs and MBCs of octenidine rapidly emerged after octenidine was introduced in the trust. There was no apparent cross-resistance between the two biocidal agents. A combination of variable-number tandem repeat (VNTR) analysis, PCR for qac genes, and whole-genome sequencing was used to type isolates and examine possible mechanisms of resistance. There was no expansion of a single strain associated with decreased biocide tolerance, and biocide susceptibility did not correlate with carriage of qac efflux pump genes. Mutations within the NorA or NorB efflux pumps, previously associated with chlorhexidine export, were identified, however, suggesting that this may be an important mechanism of biocide tolerance. We present evidence that isolates are evolving in the face of biocide challenge in patients and that changes in decolonization regimes are reflected in changes in susceptibility of isolates

Bacillus subtilis vegetative isolate surviving chlorine dioxide exposure: an elusive mechanism of resistance

Aims Oxidizing agents such as chlorine dioxide are widely used microbicides, including for disinfection of medical equipment. We isolated a Bacillus subtilis isolate from a washer-disinfector whose vegetative form demonstrated unique resistance to chlorine dioxide (0·03%) and hydrogen peroxide (7·5%). The aim of this study was to understand the mechanisms of resistance expressed by this isolate. Methods and Results A range of resistance mechanisms were investigated in the B. subtilis isolate and a reference B. subtilis strain (ATCC 6051) to include bacterial cell aggregation, the presence of profuse exopolysaccharide (EPS), and the expression of detoxification enzymes. The basis of resistance of the isolate to high concentrations of oxidizing agents was not linked to the presence of endospores. Although, the presence of EPS, aggregation and expression of detoxification enzymes may play a role in bacterial survival to low concentrations of chlorine dioxide, it is unlikely that the mechanisms helped tested to survive the bactericidal effect of higher oxidizer concentrations. Conclusions Overall, the mechanisms conferring resistance to chlorine dioxide and hydrogen peroxide remains elusive. Based on recent advances in the mode of action of oxidizing agents and notably hydrogen peroxide, we postulate that additional efficient intracellular mechanisms may be involved to explain significant resistance to in-use concentrations of commonly used high-level disinfectants

Understanding the risk of emerging bacterial resistance to over the counter antibiotics in topical sore throat medicines

Aims The aims of this study were to explore the development of bacterial resistance and cross‐resistance in four common human pathogens following realistic exposure to antibiotics found in over‐the‐counter (OTC) sore throat medicines: gramicidin, neomycin, bacitracin and tyrothricin. Methods and Results Bacterial exposure to in‐use (concentration in the product before use) and diluted concentration (i.e. during use ) of antibiotic where conducted in broth for 24 h or until growth was visible. The changes in bacterial susceptibility profile before and after exposure was determined using standardized ISO microdilution broth. Antibiotic testing was performed according to EUCAST guidelines. We demonstrated that test bacteria were able to survive exposure to the in‐use concentrations of some antibiotics used in OTC medicines. Exposure to during use concentrations of bacitracin resulted in stable increase in minimal inhibitory concentration (MIC) (>8‐fold) in Staphylococcus aureus and Acinetobacter baumannii . Exposure to tyrothricin resulted in a stable increase in MIC (2·4‐fold) in Klebsiella pneumoniae , and exposure to neomycin resulted in a stable increase MIC (5000‐fold higher than the baseline) in Streptococcus pyogenes . Clinical cross‐resistance to other antibiotics (ciprofloxacin, fusidic acid, gentamicin, cefpodoxime, amoxicillin/clavulanic acid and cefotaxime) was also demonstrated following exposure to bacitracin or tyrothricin. Bacitracin exposure lead to a stable bacterial resistance after 10 passages. Conclusions Our results indicate that OTC antibiotic medicines have the potential to drive resistance and cross‐resistance in vitro . Significance and Impact of the Study Tackling antibiotic resistance is a high worldwide priority. It is widely accepted that the overuse and misuse of antibiotics increase the risk of the development and spread of antibiotic resistance within communities. A number of OTC sore throat products, widely available across the world for topical use in respiratory indications, contain locally delivered antibiotics. Our findings showed that these antibiotics in OTC medicines present a risk for emerging cross‐resistance in a number of bacterial respiratory pathogens

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