Biocide Exposure Research Articles

Lung cancer mortality and exposure to synthetic metalworking fluid and biocides: controlling for the healthy worker survivor effect

ObjectivesSynthetic metalworking fluids (MWFs), widely used to cool and lubricate industrial machining and grinding operations, have been linked with increased risk of several cancers. Estimates of their relation with lung...

Resistance of Bacteria to Biocides.

Biocides and formulated biocides are used worldwide for an increasing number of applications despite tightening regulations in Europe and in the United States. One concern is that such intense usage of biocides could lead to increased bacterial resistance to a product and cross-resistance to unrelated antimicrobials including chemotherapeutic antibiotics. Evidence to justify such a concern comes mostly from the use of health care-relevant bacterial isolates, although the number of studies of the resistance characteristics of veterinary isolates to biocides have increased the past few years. One problem remains the definition of "resistance" and how to measure resistance to a biocide. This has yet to be addressed globally, although the measurement of resistance is becoming more pressing, with regulators both in Europe and in the United States demanding that manufacturers provide evidence that their biocidal products will not impact on bacterial resistance. Alongside in vitro evidence of potential antimicrobial cross-resistance following biocide exposure, our understanding of the mechanisms of bacterial resistance and, more recently, our understanding of the effect of biocides to induce a mechanism(s) of resistance in bacteria has improved. This article aims to provide an understanding of the development of antimicrobial resistance in bacteria following a biocide exposure. The sections provide evidence of the occurrence of bacterial resistance and its mechanisms of action and debate how to measure bacterial resistance to biocides. Examples pertinent to the veterinary field are used where appropriate.

Characterization of Heterogeneous MRSA and MSSA with Reduced Susceptibility to Chlorhexidine in Kuwaiti Hospitals.

The objective of this investigation was to identify the lineages of MRSA and MSSA with reduced susceptibility to chlorhexidine in Kuwaiti hospitals. 121 clinical MRSA and 56 MSSA isolates were included in this study. Antimicrobial susceptibility testing was performed for a selection of agents including chlorhexidine and resistance genes were amplified and sequenced. PFGE, spa typing, and MLST were completed for a selection of isolates. The results showed SCCmec II, III, IV, and V were present in 0.8, 21.5, 69.4, and 8.3% of the MRSA isolates. agr-1Sa was the most prevalent type in both MSSA (48%) and MRSA (54%). Forty-five percentage of MRSA contained pvl and 39% contained lukE-lukD, however, as many as 86% of MSSA contained pvl and 96.4% contained lukE-lukD. qac A-C genes were identified in 12.3% of MRSA, norA was present in 82.6% and blaZ in 94.2%. Among MSSA only 5.4% harbored qacA, 83% contained norA, and 91% blaZ. Multi-drug resistant ST239/t945 lineage containing a qac gene was the most identified S. aureus. However, other lineages, including ST772-MRSA-V/t4867/pvl(+)qacC/smr and non-qac harboring lineages of ST217-MRSAIV/t3244/pvl(–), ST34-MSSA/t161/pvl(+), ST5-MSSA/t688/pvl(+), ST5-MSSA/t4867/norA(+), and ST672-MSSA/t003/pvl(–), also showed reduced susceptibility to chlorhexidine. The observed reduced susceptibility of non-qac dependent MSSA isolates to chlorhexidine suggests the involvement of other elements in promoting higher MBC (≥30 mg/L). Our results confirm that monitoring MSSA is essential as they may have the potential to survive low level biocide exposure.

Effects of biocide exposure on P. Aeruginosa, E. coli and A. Baumannii complex isolates from hospital and household environments

Background: Bacterial responses to biocide exposure and its effects on survival and persistence remain to be studied in greater detail.Aim: To analyse the viability and survival of environmental isolates from household and hospital settings after biocide exposure.Methods: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of chlorhexidine (CHxG), benzalkonium chloride (BAC) and triclosan (TC) were determined in isolates of Pseudomonas aeruginosa, Acinetobacter baumannii complex and Escherichia coli collected from hospital and households environments. Viability was monitored after exposure and removal of biocides using agar cultures and flow cytometry.Findings: P. aeruginosa isolates showed greater tolerance for all biocides tested whereas A. baumannii complex and E. coli were less tolerant.When compared with reference strains, biocide tolerance was up to 8 to 13-fold higher for TC and BAC respectively. Flow cytometry showed that biocide exposure may induce viable but non-growing states in P. aeruginosa and E. coli isolates before becoming fully replicative. Changes in the susceptibility profile in one isolate of A. baumannii complex were observed after biocide exposure.Discussion: Bacteria isolates from hospital and households were able to recover after biocide exposure at bactericidal concentrations favouring persistence and spread of biocide-tolerant strains. This study reinforces that cleaning compliance should be monitored by non-culture based tests. Novel formulations in cleaning and disinfection protocols should be revisited in hospitals harbouring P. aeruginosa and A. baumannii multidrug resistant isolates.

Formulation of Biocides Increases Antimicrobial Potency and Mitigates the Enrichment of Nonsusceptible Bacteria in Multispecies Biofilms.

The current investigation aimed to generate data to inform the development of risk assessments of biocide usage. Stabilized domestic drain biofilm microcosms were exposed daily over 6 months to increasing concentrations (0.01% to 1%) of the biocide benzalkonium chloride (BAC) in a simple aqueous solution (BAC-s) or in a complex formulation (BAC-f) representative of a domestic cleaning agent. Biofilms were analyzed by culture, differentiating by bacterial functional group and by BAC or antibiotic susceptibility. Bacterial isolates were identified by 16S rRNA sequencing, and changes in biofilm composition were assessed by high-throughput sequencing. Exposure to BAC-f resulted in significantly larger reductions in levels of viable bacteria than exposure to BAC-s, while bacterial diversity greatly decreased during exposure to both BAC-s and BAC-f, as evidenced by sequencing and viable counts. Increases in the abundance of bacteria exhibiting reduced antibiotic or BAC susceptibility following exposure to BAC at 0.1% were significantly greater for BAC-s than BAC-f. Bacteria with reduced BAC and antibiotic susceptibility were generally suppressed by higher BAC concentrations, and formulation significantly enhanced this effect. Significant decreases in the antimicrobial susceptibility of bacteria isolated from the systems before and after long-term BAC exposure were not detected. In summary, dose-dependent suppression of bacterial viability by BAC was enhanced by formulation. Biocide exposure decreased bacterial diversity and transiently enriched populations of organisms with lower antimicrobial susceptibility, and the effects were subsequently suppressed by exposure to 1% BAC-f, the concentration most closely reflecting deployment in formulated products.IMPORTANCE Assessment of the risks of biocide use has been based mainly on the exposure of axenic cultures of bacteria to biocides in simple aqueous solutions. The current investigation aimed to assess the effects of formulation on the outcome of biocide exposure in multispecies biofilms. Formulation of the cationic biocide BAC significantly increased antimicrobial potency. Bacteria with lower antimicrobial susceptibility whose populations were enriched after low-level biocide exposure were more effectively suppressed by the biocide at in-use concentrations (1% [wt/vol]) in a formulation than in a simple aqueous solution. These observations underline the importance of simulating normal deployment conditions in considering the risks and benefits of biocide use.

Resistance to Biocides in Listeria monocytogenes Collected in Meat-Processing Environments.

The emergence of microorganisms exerting resistance to biocides is a challenge to meat-processing environments. Bacteria can be intrinsically resistant to biocides but resistance can also be acquired by adaptation to their sub-lethal concentrations. Moreover, the presence of biocide resistance determinants, which is closely linked to antibiotic resistance determinants, could lead to co-selection during disinfection practices along the food chain, and select cross-resistant foodborne pathogens. The purpose of this work was to test the resistance of wild strains of Listeria monocytogenes, isolated from pork meat processing plants, toward benzalkonium chloride (BC), used as proxy of quaternary ammonium compounds. Furthermore, the expression of two non-specific efflux pumps genes (lde and mdrL) under biocide exposure was evaluated. L. monocytogenes were isolated from five processing plants located in the Veneto region (northeast of Italy) before and after cleaning and disinfection (C&D) procedures. A total of 45 strains were collected: 36 strains before and nine after the C&D procedures. Collected strains were typed according to MLST and ERIC profiles. Strains sampled in the same site, isolated before, and after the C&D procedures and displaying the same MLST and ERIC profiles were tested for their sensitivity to different concentrations of BC, in a time course assay. The expression of non-specific efflux pumps was evaluated at each time point by qPCR using tufA gene as housekeeping. A differential expression of the two investigated genes was observed: lde was found to be more expressed by the strains isolated before C&D procedures while its expression was dose-dependent in the case of the post C&D procedures strain. On the contrary, the expression of mdrL was inhibited under low biocidal stress (10 ppm BC) and enhanced in the presence of high stress (100 ppm BC). These findings suggests a possible role for C&D procedures to select L. monocytogenes persisters, pointing out the importance of dealing with the identification of risk factors in food plants sanification procedures that might select more tolerant strains.

Role of Epidemiology in Boicide Deaths in a Korean Law Suit

In the spring of 2011 in Korea, a cluster of pregnant women and children were admitted to intensive care units, and a case-control study charged by Korean Center for Disease Control revealed the association between rapidly progressing pulmonary fibrosis and humidifier biocides. Animal experiments soon identified the same pulmonary lesions as in humans, and then toxicity of humidifier biocides were evaluated for the first time. In 2015, a criminal charge was brought against the manufacturing companies for the negligence in the product safety and all the evidences of the causality for deaths from biocide exposure were brought on the table of the court. The companies are denying the causality itself, and causality in the diagnosis of humidifier disinfectant lung injury becomes the central issue of the dispute. The following issues were noted in the midst of the controversies between the prosecutors and the companies. Case definition in the case control study was challenged that the patients studied in the case control study do not have the same diseases, which may make the conclusion invalid. (misclassification) Errors in the differential diagnosis of patients can result in confounding, especially by viral infections. (confounding) Double blinding was not followed in the case control study. (information bias) In bringing these issues, it was noted that distinctions between general causality determination among multiple individuals versus specific causality determination for an individual were lost in the argument of linking errors of differential diagnosis in clinical medicine to the defects of epidemiology study. Another issue to note is the influence of foreign consultants, such as Gradient, in raising doubt about the results of academic studies that were published in the peer-reviewed journals. Three lessons that I learned from this experience are 1) the need to clarify how the assessment of causal association in epidemiology can be different from the clinical diagnosis, 2) any need of differences in the rigorousness of evidence of causality between criminal versus civil law suits, 3) the pros and cons in the replacement of domestic authoritative opinions with foreign consultations.

Structural and Biochemical Characterization of Rm3, a Subclass B3 Metallo-β-Lactamase Identified from a Functional Metagenomic Study.

β-Lactamase production increasingly threatens the effectiveness of β-lactams, which remain a mainstay of antimicrobial chemotherapy. New activities emerge through both mutation of previously known β-lactamases and mobilization from environmental reservoirs. The spread of metallo-β-lactamases (MBLs) represents a particular challenge because of their typically broad-spectrum activities encompassing carbapenems, in addition to other β-lactam classes. Increasingly, genomic and metagenomic studies have revealed the distribution of putative MBLs in the environment, but in most cases their activity against clinically relevant β-lactams and, hence, the extent to which they can be considered a resistance reservoir remain uncharacterized. Here we characterize the product of one such gene, blaRm3, identified through functional metagenomic sampling of an environment with high levels of biocide exposure. blaRm3 encodes a subclass B3 MBL that, when expressed in a recombinant Escherichia coli strain, is exported to the bacterial periplasm and hydrolyzes clinically used penicillins, cephalosporins, and carbapenems with an efficiency limited by high Km values. An Rm3 crystal structure reveals the MBL superfamily αβ/βα fold, which more closely resembles that in mobilized B3 MBLs (AIM-1 and SMB-1) than other chromosomal enzymes (L1 or FEZ-1). A binuclear zinc site sits in a deep channel that is in part defined by a relatively extended N terminus. Structural comparisons suggest that the steric constraints imposed by the N terminus may limit its affinity for β-lactams. Sequence comparisons identify Rm3-like MBLs in numerous other environmental samples and species. Our data suggest that Rm3-like enzymes represent a distinct group of B3 MBLs with a wide distribution and can be considered an environmental reservoir of determinants of β-lactam resistance.

Multiple adaptive routes of Salmonella enterica Typhimurium to biocide and antibiotic exposure.

BackgroundBiocides and antibiotics are used to eradicate or prevent the growth of microbial species on surfaces (occasionally on catheters), or infected sites, either in combination or sequentially, raising concerns about the development of co-resistance to both antimicrobial types. The effect of such compounds on Salmonella enterica, a major food-borne and zoonotic pathogen, has been analysed in different studies, but only few works evaluated its biological cost, and the overall effects at the genomic and transcriptomic levels associated with diverse phenotypes resulting from biocide exposure, which was the aim of this work.ResultsExposure to triclosan, clorhexidine, benzalkonium, (but not to hypochlorite) resulted in mutants with different phenotypes to a wide range of antimicrobials even unrelated to the selective agent. Most biocide-resistant mutants showed increased susceptibility to compounds acting on the cell wall (β-lactams) or the cell membranes (poly-L-lysine, polymyxin B, colistin or toxic anions). Mutations (SNPs) were found in three intergenic regions and nine genes, which have a role in energy production, amino acids, carbohydrates or lipids metabolism, some of them involved in membrane transport and pathogenicity. Comparative transcriptomics of biocide-resistant mutants showed over-expression of genes encoding efflux pumps (sugE), ribosomal and transcription-related proteins, cold-shock response (cpeE) and enzymes of microaerobic metabolism including those of the phosphotransferase system. Mainly ribosomal, metabolic and pathogenicity-related genes had affected expression in both in vitro-selected biocide mutants and field Salmonella isolates with reduced biocide susceptibility.ConclusionsMultiple pathways can be involved in the adaptation of Salmonella to biocides, mainly related with global stress, or involving metabolic and membrane alterations, and eventually causing “collateral sensitivity” to other antimicrobials. These changes might impact the bacterial-environment interaction, imposing significant bacterial fitness costs which may reduce the chances of fixation and spread of biocide resistant mutants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2778-z) contains supplementary material, which is available to authorized users.

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

In this study we assessed the propensity of biocide exposure in the development of antimicrobial resistance in bacteria. 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. 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. 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.

Parallel evolutionary pathways to antibiotic resistance selected by biocide exposure

ObjectivesBiocides are widely used to prevent infection. We aimed to determine whether exposure of Salmonella to various biocides could act as a driver of antibiotic resistance.MethodsSalmonella enterica serovar Typhimurium was exposed to four biocides with differing modes of action. Antibiotic-resistant mutants were selected during exposure to all biocides and characterized phenotypically and genotypically to identify mechanisms of resistance.ResultsAll biocides tested selected MDR mutants with decreased antibiotic susceptibility; these occurred randomly throughout the experiments. Mutations that resulted in de-repression of the multidrug efflux pump AcrAB-TolC were seen in MDR mutants. A novel mutation in rpoA was also selected and contributed to the MDR phenotype. Other mutants were highly resistant to both quinolone antibiotics and the biocide triclosan.ConclusionsThis study shows that exposure of bacteria to biocides can select for antibiotic-resistant mutants and this is mediated by clinically relevant mechanisms of resistance prevalent in human pathogens.

Effect of sub-lethal concentrations of biocides on the susceptibility to antibiotics of multi-drug resistant Salmonella enterica strains

Ten multi-drug resistant Salmonella enterica isolates belonging to ten different serotypes were exposed to increasing sub-inhibitory concentrations of three biocides widely used in food industry facilities (trisodium phosphate, sodium nitrite, or sodium hypochlorite). Cultures were tested, before and after exposure to biocides, against 31 antibiotics of clinical significance by means of a standard disk-diffusion technique (CLSI). Exposed cultures displayed reduced susceptibility to a range of antibiotics, as compared with not exposed cultures. The impact of biocide exposure on reduced susceptibility to antibiotics was dependent on the Salmonella strain and the antibiotic family tested, susceptibility to aminoglycosides and cephalosporins being the most strongly affected. Results in the present study suggest that extensive use of biocides at sub-lethal concentrations could contribute to the emergence of antibiotic resistance in multi-drug resistant Salmonella enterica strains and therefore represent a public health risk. The intra-specific differences observed in antibiotic susceptibility underline the need to screen a wide range of strains.

Biocide resistance of Candida and Escherichia coli biofilms is associated with higher antioxidative capacities

Most clinical guidelines for the use of biocides have been developed for planktonic micro-organisms, but in nature, most micro-organisms live as surface-adherent communities or biofilms. To evaluate the effectiveness of commonly used biocides against Escherichia coli and Candida spp. in three distinct growth phases: planktonic, adhesion and biofilm. Ultrastructural, architectural and cellular viability changes following a 5 min exposure to biocide were monitored by scanning electron microscopy and confocal laser scanning microscopy using fluorescent dyes. Comparative transcript expression of the antioxidants SOD1 and CAT1 in the planktonic and biofilm phases was evaluated using quantitative real-time polymerase chain reaction. E. coli and Candida spp. in the planktonic phase were susceptible to all the tested biocides at the recommended concentrations. However, early adhesion and late biofilm phases of both were less susceptible to the biocides, and exceeded the recommended concentrations on several occasions. A short period of biocide exposure failed to fully eradicate the adherent microbial cells, and they recovered from the biocide challenge, forming biofilm on the biocide-treated surfaces. The biofilm phase showed higher expression of SOD1 and CAT1. The recommended concentrations of biocides for clinical disinfection in the hospital setting may not fully eradicate the adhesion or biofilm phases of E. coli and Candida spp. Higher antioxidative capacities in microbial biofilms may be responsible for the resistance of biofilms against clinical biocides.

Exposure of Salmonella enterica Serovar Typhimurium to High Level Biocide Challenge Can Select Multidrug Resistant Mutants in a Single Step

BackgroundBiocides are crucial to the prevention of infection by bacteria, particularly with the global emergence of multiply antibiotic resistant strains of many species. Concern has been raised regarding the potential for biocide exposure to select for antibiotic resistance due to common mechanisms of resistance, notably efflux.Methodology/Principal Findings Salmonella enterica serovar Typhimurium was challenged with 4 biocides of differing modes of action at both low and recommended-use concentration. Flow cytometry was used to investigate the physiological state of the cells after biocide challenge. After 5 hours exposure to biocide, live cells were sorted by FACS and recovered. Cells recovered after an exposure to low concentrations of biocide had antibiotic resistance profiles similar to wild-type cells. Live cells were recovered after exposure to two of the biocides at in-use concentration for 5 hours. These cells were multi-drug resistant and accumulation assays demonstrated an efflux phenotype of these mutants. Gene expression analysis showed that the AcrEF multidrug efflux pump was de-repressed in mutants isolated from high-levels of biocide.Conclusions/SignificanceThese data show that a single exposure to the working concentration of certain biocides can select for mutant Salmonella with efflux mediated multidrug resistance and that flow cytometry is a sensitive tool for identifying biocide tolerant mutants. The propensity for biocides to select for MDR mutants varies and this should be a consideration when designing new biocidal formulations.

Controlling the Invasive Diatom Didymosphenia geminata: An Ecotoxicity Assessment of Four Potential Biocides

In 2004, an invasive mat-forming freshwater diatom, Didymosphenia geminata (didymo), was found in New Zealand causing concern with regard to potential consequences for local freshwater ecosystems. A four-stage research program was initiated to identify methods to control D. geminata. This article reports the results of Stage 2, in which four potential control compounds [Gemex™ (a chelated copper formulation), EDTA, Hydrothol®191, and Organic Interceptor™ (a pine oil formulation)] selected in Stage 1 were evaluated for their biocidal efficacy on D. geminata and effects on non-target organisms using both artificial stream and laboratory trials. Artificial stream trials evaluated the mortality rates of D. geminata and fishes to three concentrations of the four biocides, whereas laboratory toxicity trials tested the response of green alga and cladocera to a range of biocide concentrations and exposure times. In artificial stream trials, Gemex and Organic Interceptor were the most effective biocides against D. geminata for a number of measured indices; however, exposure of fishes to Organic Interceptor resulted in high mortality rates. Laboratory toxicity testing indicated that Gemex might negatively affect sensitive stream invertebrates, based on the cladoceran sensitivity at the proposed river control dose. A decision support matrix evaluated the four biocides based on nine criteria stipulated by river stakeholders (effectiveness, non-target species impacts, stalk removal, degradation profile, risks to health and safety, ease of application, neutralization potential, cost, and local regulatory requirements) and Gemex was identified as the product warranting further refinement prior to an in-river trial.

Novel antifouling agent—Zinc pyrithione: Short- and long-term effects on survival and reproduction of the marine polychaete Dinophilus gyrociliatus

The recent ban on TBT in boat antifouling paints has resulted in a large employment of the biocide zinc pyrithione (ZnPT) as substitute. Despite concerns of its environmental toxicity at ultra-trace concentrations, ZnPT has received little attention, as it was assumed to photo-degrade easily. However, recent evidence has suggested that ZnPT degrades only partially, and persists in the marine environment, especially where the influence of light is limited, such as in harbours. Short-term acute toxicity tests and life table response experiments (LTREs) were performed to evaluate the effects of ZnPT on the polychaete Dinophilus gyrociliatus at both individual and population level. The 96h-LC 50 values for ZnPT on D. gyrociliatus were 7.8 and 11.5 nM under dark and 12 h light/12 h dark conditions, respectively. In LTREs, laboratory cultured cohorts of D. gyrociliatus were exposed to sublethal concentrations of ZnPT (0.5 and 1 nM) and compared to a control cohort. The survival among individuals exposed to the highest concentration decreased already during the 2nd week of life with respect to the control. The effects of the biocide on fecundity were even more evident: ZnPT caused a considerable reduction in both the exposed groups. The demographic approach applied here succeeded in identifying ZnPT effects both on the biological cycle and on the growth potential of polychaete D. gyrociliatus. The net growth rate ( R 0) appeared to be the demographic parameter most sensitive to ZnPT, as the biocide exposure was associated with a sharp decline of R 0 in both the 0.5 and the 1 nM groups (−41% and −63%, respectively) in comparison to the control cohort. The population growth rate λ (often used as an index of population fitness) and the life expectancy e 0 were also significantly reduced, while ZnPT exposure did not affect the generation time T. These results highlight the potential ecological threat posed by the biocide ZnPT, even at the very low tested concentrations, which are already detectable in some European marinas.

Use of biocides and insect repellents and risk of hypospadias

BackgroundThe relationship between the use of biocides and insect repellents and the risk of hypospadias was examined in a large case–control study in the South East of England.MethodsA case–control study...

Polyhexamethylene biguanide exposure leads to viral aggregation

This study reports the activity of two biguanides against MS2 bacteriophage used as a surrogate virus for nonenveloped mammalian viruses and provides an explanation as to their apparent limited efficacy. When tested in a standard suspension test, two polyhexamethylene biguanides (PHMB), VANTOCIL TG and COSMOCIL CQ, reduced the viability of MS2 by only 1-2 log(10) PFU ml(-1). Exposure time up to 30 min did not affect the activity of the biguanides, although both PHMB were shown to strongly interact with MS2 proteins. Inactivation kinetics and change in virus hydrophobicity suggested that PHMB induces the formation of viral aggregates. This hypothesis was supported using dynamic light scattering that showed an increase in viral aggregates sizes (up to 500 nm) in a concentration-dependent manner. It has been reported that viral aggregation is responsible for virus survival to the biocide exposure. Here, this might be the case, because the virucidal activity of the biguanides was modest and viral aggregation important. The formation of viral aggregates during virus exposure to PHMB was unlikely to overestimate the virucidal potential of the biguanides.

Effects of Biocides and Other Metal Removal Fluid Constituents on Mycobacterium immunogenum

Cells of Mycobacterium immunogenum are significantly more resistant to biocides and antimicrobial compounds used for disinfection of metal removal fluids (MRFs) than cells of the gram-negative bacterium Pseudomonas pseudoalcaligenes. To ensure accurate measurement of M. immunogenum susceptibility, a biocide inactivation step must be included to prevent an overestimation of killing for short-term exposures. Cell suspensions in 5% MRF for 30 min rather than direct plating following biocide exposure increased the killing of M. immunogenum cells, suggesting a heretofore undiscovered interaction between MRF components and biocides. Biocide killing was reduced at high M. immunogenum cell densities (>10(6)/ml). Among the chemicals tested, only Synergex Premier, Preventol CMK, and dicyclohexylamine killed greater than 90% of M. immunogenum cells in 6 h. M. immunogenum cells adhered to and grew rapidly on glass, copper, and galvanized surfaces, forming biofilms of 10(6) cells/cm(2) within 2 weeks and suggesting it is likely that the majority of M. immunogenum cells in MRF systems are on surfaces. M. immunogenum cells grown on surfaces in biofilms were three- to 100-fold more resistant to the biocides Synergex Premier and Preventol CMK than cells grown in suspension, compounding the problem of eradication of M. immunogenum cells from MRF systems.

Inactivation of Yersinia pseudotuberculosis, as a surrogate for Yersinia pestis, by liquid biocides in the presence of food residue.

The efficacy of liquid biocides is influenced by surface cleanliness, treatment time, and temperature. Experiments were completed to measure the impact of these variables on the ability of commercial biocides to inactivate Yersinia pseudotuberculosis ATCC 29910, as a surrogate for Yersinia pestis, in the presence of food residues. The test organism was mixed with water, milk, flour, or egg yolk and then dried onto stainless steel coupons. Coupons were then exposed to sodium hypochlorite, acidified sodium chlorite, a quaternary ammonium compound, an iodophor, hydrogen peroxide, peroxyacetic acid, or a peroxy-fatty acid mixture, for 10 or 30 min at 10, 20, or 30 degrees C. For all biocides except the iodophor, manufacturer-recommended disinfection levels applied for 10 min at 20 degrees C resulted in 5-log reductions of the test organism dried alone or with flour. However, in the presence of whole milk or egg yolk residue, markedly higher sodium hypochlorite, peroxyacetic acid, peroxy-fatty acid mixture, quaternary ammonium compound, and iodophor concentrations were needed to achieve the 5-log reductions. Further, the quaternary ammonium compound was incapable of achieving 5-log reductions in 10 min in the presence of milk and egg yolk residues. Hydrogen peroxide and acidified sodium chlorite disinfection levels (7.5% and 2500 ppm, respectively) achieved 5-log reductions under all test conditions. These results suggest that commercial disinfectants can adequately decontaminate clean surfaces contaminated with Y. pseudotuberculosis and Y. pestis. These results also provide guidance on the feasibility of overcoming the negative influence of food residues on disinfection by adjusting biocide exposure time, temperature, and concentration.