Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

Assessing decontamination and laundering processes for the removal of polycyclic aromatic hydrocarbons and flame retardants from firefighting uniforms.

Firefighter uniforms protect firefighters from exposure to potentially harmful chemicals including a range of semi-volatile organic compounds (SVOCs). Contaminated uniforms can become a secondary source of firefighters' exposure to these chemicals. There is inconsistency on the removal efficiency of SVOCs during the cleaning, laundering and field decontamination of firefighting uniforms. Therefore, this study aims to assess how effective decontamination and laundering processes are in reducing firefighter uniforms as a vector for transport and exposure to SVOCs. Firefighters who had attended a controlled house fire and simulated container burns had their uniforms sampled pre- and post-laundering. Clean station wear was laundered with contaminated uniforms and after a load of contaminated uniforms to assess inter and intra load contamination. Surface wipes were collected from uniforms across 12 fire stations, after they had returned from a laundering provider. Concentrations of 13 polycyclic aromatic hydrocarbons (PAHs), six organophosphate flame retardants (OPFRs) and seven polybrominated diphenyl ethers (PBDEs) were measured in the collected samples. The concentrations of ∑13 PAHs in firefighters uniforms ranged between 0.063 and 43 µg g-1, while concentration of ∑6 OPFRs were between 0.061 and 90 µg g-1 with ∑7 PBDEs concentrations being measured between 0.00054 and 0.97 µg g-1.The highest concentrations of ∑13 PAHs were measured on the outer layers of gloves at an average of 19 µg g-1, with the highest ∑6 OPFRs concentrations being measured in the middle layers of gloves at an average of 31 µg g-1. The highest ∑7 PBDEs concentrations were measured on the shell layers of turnout jackets at 0.42 µg g-1. The significant reduction in ∑13 PAHs after laundering or decontamination was only found in 3 of the 16 sampled areas from firefighting uniforms. No significant differences were found in the between pre- and post-laundering concentrations of ∑6 OPFRs or ∑7 PBDEs in firefighting uniforms. The current laundering techniques do not appear to effectively remove PAHs, OPFRs and PBDEs at the measured concentrations from firefighters' uniforms. Further research is required to assess if chemical exposure though firefighting uniforms poses a health risk to firefighters and to develop methods for the removal of SVOCs from firefighting uniforms.

Biomonitoring in firefighters for volatile organic compounds, semivolatile organic compounds, persistent organic pollutants, and metals: A systematic review.

Firefighters are exposed to a wide range of toxic chemicals due to combustion, with numerous biomonitoring studies completed that have assessed exposure. Many of these studies focus on individual classes of chemicals, with a few considering a broad range of systemic exposures. As yet, no review process has been undertaken to comprehensively examine these studies. The aims of this review are to: (1) ascertain whether biomonitoring studies pertaining to firefighters demonstrate occupational exposure to volatile organic compounds, semivolatile organic compounds, and metals; (2) determine and present results of biomonitoring studies; (3) provide any recommendations presented from the literature that may support exposure mitigation; and (4) suggest future study parameters that may assist in providing a greater understanding surrounding the occupational exposure of firefighters. A systematic review was undertaken with regards to firefighters and biomonitoring studies utilising the matrices of blood, urine, semen and breast milk. This yielded 5690 results. Following duplicate removal, inclusion and exclusion criteria screening and full text screening, 34 studies remained for review. Results of over 80% of studies analysed determined firefighters to experience occupational exposure. Results also show firefighters to be exposed to a wide range of toxic chemicals due to fire smoke; potentially exceeding the range of exposure of other occupations. As firefighters may face increased risk of health effects due to the additive, synergistic, and/or antagonistic effects of chemical exposure, all care must be taken to reduce exposure. This may be achieved by considering tactical decisions, increased personal hygiene, and thorough decontamination procedures. Future biomonitoring studies recognising and assessing the range of chemical exposure firefighters face would be beneficial.

Analysis of urinary metabolites of polycyclic aromatic hydrocarbons and cotinine in pooled urine samples to determine the exposure to PAHs in an Australian population.

Our previous biomonitoring study of hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) in a population in Australia found high levels of 1-naphthol, a metabolite of both naphthalene and carbaryl, in some adult samples. Here, we conducted a follow-up study to collect and analyse pooled urine samples, stratified by age and sex, from 2014 to 2017 using a GC-MS method. Geometric mean concentrations of 1-hydroxypyrene, the most common biomarker of PAH exposure, were 100 and 120 ng/L urine in 2014-2015 and 2016-2017, respectively. The concentrations of most OH-PAHs in this study except 1-naphthol are in line with those reported by biomonitoring programs in the US and Canada. In general, concentrations of OH-PAHs are lower in samples from small children (0-4 years) and school-aged children (5-14 years) compared with samples from the older age groups, except for some cases in the recent monitoring period. The concentrations of 1-naphthol in some adult samples of both sexes are very high, which is consistent with our previous findings. Such high concentrations of 1-naphthol together with the high 1-naphthol/2-naphthol ratio suggest potential exposure to the insecticide carbaryl in this population but other exposure sources and different rates of naphthalene metabolism should also be investigated.

The occurrence of PAHs and flame-retardants in air and dust from Australian fire stations.

Firefighters are exposed to a wide range of chemicals whilst on duty, including polycyclic aromatic hydrocarbons (PAHs), organophosphate flame-retardants (OPFRs), and polybrominated diphenyl ethers (PBDEs). These groups of chemicals are related to combustion emissions. PAHs are formed during combustion. OPFRs and PBDEs are flame-retardants and are inadvertently released during combustion. Exposure to these chemicals occurs when attending fire scenes, and firefighters can track these chemicals back into fire stations leading to further exposure. The objective of this study was to understand the concentrations of PAHs, OPFRs, and PBDEs in fire stations, to evaluate factors that affect chemical concentration, and to assess how air and dust could contribute to firefighters' relevant exposure risk. Concentrations of 13 PAHs, 9 OPFRs, and 8 PBDEs were quantified in fire station dust (n = 49) and air (n = 15) samples collected between November 2017 and February 2018. The median ∑13PAH concentration was 15 ng m-3 and 3.1 µg g-1 in air and dust, respectively, while the median ∑9 OPFR concentration was 56 ng m-3 in air and 84 µg g-1 in dust, and ∑8 PBDE had a median concentration of 0.78 ng m-3 in air and 26 µg g-1 in dust. The estimated daily intakes through dust and air for ∑13 PAHs, ∑9 OPFRs, and ∑8 PBDEs in firefighters were 3.6, 17, and 1.6 ng (kg body weight)-1 day-1, respectively. The worst-case estimated daily intakes were only 2% of the reference dose for individual chemicals. Pearson's correlations with chemical concentration for several PAHs, OPFRs, and PBDEs were found between the number of years since fire stations were last renovated, as well as the storage locations of firefighting ensembles. These results suggest chemicals are brought back to fire stations from fire scenes and that they are accumulating in fire stations. They also suggest soiled firefighting ensembles are a source of these chemicals in fire stations and that their proximity to the rest of the station determines the extent to which they contribute to chemical concentrations in fire stations.

Exposure to metals and semivolatile organic compounds in Australian fire stations.

Firefighting is an occupation with exposure to a wide range of chemicals by means of inhalation, ingestion or dermal contact. Although advancements in personal protective clothing and equipment have reduced the risks for acute exposure during fire suppression operations, chronic exposure may still be present at elevated levels in fire stations. The aim of this study was to assess chemicals in air and on surfaces in fire stations, compare this with other indoor environments, and use this data to estimate firefighter exposure within the fire station. Fifteen Australian fire stations were selected for chemical exposure assessment by means of 135 active air monitors, 60 passive air monitors, and 918 wipe samples. These samples were collected from the interior and exterior of fire stations, from personal protective clothing and equipment, and from within the cabins of vehicles. Chemicals analysed included polycyclic aromatic hydrocarbons, volatile organic compounds, metals, and diesel particulate matter. Specific chemicals were detected from within each class of chemicals, with metals being most frequently detected. Statistical analysis by means of Pearson's Correlations and threshold tests were used to consider the source of exposure, and a collective addition risk quotient calculation was used to determine firefighter exposure. The presence of metals in fire stations was compared with findings from global indoor dust measurements. Concentrations across firefighter ensemble, inside vehicle cabins, and within fire stations for chromium (39.5-493 µg/m2), lead (46.7-619 µg/m2), copper (594-3440 µg/m2), zinc (11100-20900 µg/m2), nickel (28.6-2469 µg/m2) and manganese (73.0-997 µg/m2) were in most instances orders of magnitude higher when compared with concentrations measured in homes and offices. Our study suggests that the elevated concentrations are associated with the transfer of chemicals from fire suppression operations. Due to this elevated concentration of chemicals, firefighters may face increased exposure, and in turn increased risk of adverse health effects. Data suggest that exposure may be mitigated by means of increased laundering frequency and increased decontamination at the scene of the fire.

σ1 receptor ligands control a switch between passive and active threat responses.

Humans and many animals show 'freezing' behavior in response to threatening stimuli. In humans, inappropriate threat responses are fundamental characteristics of several mental illnesses. To identify small molecules that modulate threat responses, we developed a high-throughput behavioral assay in zebrafish (Danio rerio) and evaluated 10,000 compounds for their effects on freezing behavior. We found three classes of compounds that switch the threat response from freezing to escape-like behavior. We then screened these for binding activity across 45 candidate targets. Using target profile clustering, we identified the sigma-1 (σ1) receptor as having a role in the mechanism of behavioral switching and confirmed that known σ1 ligands also disrupt freezing behavior. Furthermore, mutation of the gene encoding σ1 prevented the behavioral effect of escape-inducing compounds. One compound, which we call finazine, potently bound mammalian σ1 and altered threat-response behavior in mice. Thus, pharmacological and genetic interrogation of the freezing response revealed σ1 as a mediator of threat responses in vertebrates.

An Exploratory Analysis of Firefighter Reproduction through Survey Data and Biomonitoring.

Firefighters are occupationally exposed to chemicals that may affect fertility. To investigate this effect, firefighters were recruited to contribute blood, urine, breast milk or semen samples to (1) evaluate chemical concentrations and semen parameters against fertility standards and the general population; (2) assess correlations between chemical concentrations and demographics, fire exposure and reproductive history; and (3) consider how occupational exposures may affect reproduction. A total of 774 firefighters completed the online survey, and 97 firefighters produced 125 urine samples, 113 plasma samples, 46 breast milk samples and 23 semen samples. Blood, urine and breast milk samples were analysed for chemical concentrations (semivolatile organic compounds, volatile organic compounds, metals). Semen samples were analysed for quality (volume, count, motility, morphology). Firefighter semen parameters were below WHO reference values across multiple parameters. Self-reported rates of miscarriage were higher than the general population (22% vs. 12-15%) and in line with prior firefighter studies. Estimated daily intake for infants was above reference values for multiple chemicals in breast milk. More frequent fire incident exposure (more than once per fortnight), longer duration of employment (≥15 years) or not always using a breathing apparatus demonstrated significantly higher concentrations across a range of investigated chemicals. Findings of this study warrant further research surrounding the risk occupational exposure has on reproduction.

Effects of firefighting on semen parameters: an exploratory study.

Firefighters are occupationally exposed to heat intensities and chemical concentrations that may affect fertility. Twenty firefighters participated in an exploratory study assessing fertility of firefighters via an online survey and semen analysis. Data analysis included consideration of demographic characteristics, reproductive history and occupational exposures. Overall, firefighter semen parameters were below World Health Organisation reference values designating fertility in men. Firefighters younger than 45 years had a higher incidence of abnormal semen parameters (42%) than those aged 45 years or greater (9%). Increased rank and higher levels of occupational and/or personal hygiene were associated with improved semen quality. Increased frequency of fire exposure was associated with a reduction in normal forms, volume, sperm concentration and total sperm count. Sperm clumping was greater than 10% in 26% of samples, suggesting reduced semen quality. This exploratory study provides novel data that support the hypothesis of an association between semen quality and firefighter's occupational exposure to toxic environments.

Off-Gassing of Semi-Volatile Organic Compounds from Fire-Fighters' Uniforms in Private Vehicles-A Pilot Study.

Firefighters' uniforms become contaminated with a wide range of chemicals, including polycyclic aromatic hydrocarbons (PAHs), organophosphate flame retardants (OPFRs), and polybrominated diphenyl ethers (PBDEs). Laundering practices do not completely remove PAHs, OPFRs, and PBDEs from firefighting uniforms. This residual contamination of firefighting ensembles may be an ongoing source of exposure to firefighters. Firefighters are known to occasionally store firefighting ensembles in private vehicles. This study aimed to assess whether a firefighting uniform in a vehicle could act as a source for PAHs, OPFRs, and PBDEs to vehicle users. The shell layers of four laundered firefighting uniforms were sampled non-destructively. Three of these uniforms were heated in a laboratory oven (40, 60, and 80 °C) while the fourth was placed in a private vehicle on a summer day and off-gassing samples were collected from the uniforms. The off-gassing results for PAHs and OPFRs were relatively consistent between laboratory oven and the in-vehicle sample with ∑13 PAHs in off-gas ranging from 7800-23,000 ng uniform-1 day-1, while the ∑6 OPFRs off-gassed was an order of magnitude lower at 620-1600 ng uniform-1 day-1. The off-gassing results for PBDEs were much lower and less consistent between the experiments, which may reflect differences in uniform history. Currently, there is limited understanding of how PAHs, OPFRs, and PBDEs off-gassed from firefighting uniforms influence firefighter exposure to these chemicals. These findings suggest that firefighting ensembles off-gassing in private vehicles could be a relevant source of PAHs, OPFRs, and PBDEs that contributes to firefighters' exposure and that this warrants further investigation.

Characterising the exposure of Australian firefighters to polycyclic aromatic hydrocarbons generated in simulated compartment fires.

Firefighters are exposed to a wide variety of chemicals including polycyclic aromatic hydrocarbons (PAHs) while attending fire scenes. The objective of this study was to understand the exposure of firefighters to PAHs when attending simulated compartment fires that consisted of either a diesel pan or particleboard fire. Firefighters remained in the compartment fires for 15 min while using standard gear including self contained breathing apparatus (SCBA). Firefighters were able to remove firefighting clothing and shower within 10 min of leaving the burn. Air samples were collected from inside the compartment during the fire. Twenty-six (26) firefighters participated in the study providing urine and skin wipe samples collected from the wrist and neck before and after either one of the burn types. The concentrations of PAHs were measured in skin wipes and air samples, while concentrations of monohydroxy metabolites of PAHs (OH-PAHs) were measured in urine. The concentrations of all PAHs were significantly higher (p < 0.05) in the smoke layer of particleboard fires than in diesel pan fires. Correspondingly, the level of PAHs deposited on the wrists and necks of participants attending the particleboard fires was higher than those attending diesel pan fires. Urine samples from participants who attended diesel pan fires showed no significant difference (p > 0.05) in the concentration of all OH-PAHs between pre-burn and post-burn. Samples from participants who attended particleboard fires, showed no significant difference (p > 0.05) between 1-hydroxypyrene (1-OH-PYR) concentrations in urine pre- and post-burn. However, median concentrations of hydroxynaphthalenes (OH-NAPs), hydroxyfluorenes (OH-FLUs) and hydroxyphenanthrenes (OH-PHEs) increased significantly from 5.2, 0.44 and 0.88 µg g-1 creatinine pre-burn to 12, 1.4 and 1.2 µg g-1 creatinine post-burn, respectively. This suggests that in compartment burns with high concentrations of PAHs in the smoke layer, such as those created by the particleboard fires, exposure to PAHs can be observed though urinary OH-PAH metabolites. Overall, concentrations of urinary OH-PAHs were relatively low considering the potential exposure in these burns. This suggests protective equipment in combination with rapid removal of firefighting ensembles and showering are relatively effective in controlling exposure.

Comparison of lipid-normalised concentrations of persistent organic pollutants (POPs) between serum and adipose tissue.

Human biomonitoring of persistent organic pollutants (POPs) is typically based on serum analysis and for comparison and modelling purposes, data are often normalised to the lipid content of the serum. Such approach assumes a steady state of the compound between the serum lipids and for example lipid-rich adipose tissue. Few published data are available to assess the validity of this assumption. The aim of this study was to measure concentrations of POPs in both serum and adipose tissue samples from 32 volunteers and compare the lipid-normalised concentrations between serum and adipose tissue. For p,p'-DDE, PCB-138, PCB-153 and PCB-180, lipid-normalised adipose tissue concentrations were positively correlated to the respective serum concentrations but generally were more highly concentrated in adipose tissue. These results suggest that the investigated legacy POPs that were consistently found in paired samples may often not be in a steady state between the lipid compartments of the human body. Consequently, the analysis of serum lipids as a surrogate for adipose tissue exposure may more often than not underestimate total body burden of POPs. Further research is warranted to confirm the findings of this study.

Author Correction: Phase variation in pneumococcal populations during carriage in the human nasopharynx.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Phase variation in pneumococcal populations during carriage in the human nasopharynx.

Streptococcus pneumoniae is one of the world's leading bacterial pathogens, responsible for pneumonia, septicaemia and meningitis. Asymptomatic colonisation of the nasopharynx is considered to be a prerequisite for these severe infections, however little is understood about the biological changes that permit the pneumococcus to switch from asymptomatic coloniser to invasive pathogen. A phase variable type I restriction-modification (R-M) system (SpnIII) has been linked to a change in capsule expression and to the ability to successfully colonise the murine nasopharynx. Using our laboratory data, we have developed a Markov change model that allows prediction of the expected level of phase variation within a population, and as a result measures when populations deviate from those expected at random. Using this model, we have analysed samples from the Experimental Human Pneumococcal Carriage (EHPC) project. Here we show, through mathematical modelling, that the patterns of dominant SpnIII alleles expressed in the human nasopharynx are significantly different than those predicted by stochastic switching alone. Our inter-disciplinary work demonstrates that the expression of alternative methylation patterns should be an important consideration in studies of pneumococcal colonisation.

Polycyclic aromatic hydrocarbons, polychlorinated biphenyls and legacy and current pesticides in indoor environment in Australia - occurrence, sources and exposure risks.

Paired indoor air and floor dust samples were collected from residential houses and offices (n = 28) in two Australian cities in 2015. For the air samples, a modified passive air sampler (PAS) was used to collect semi-volatile organic compounds (SVOCs) in gaseous phase and airborne particles simultaneously. Sampling rates (R) of the PAS for gaseous SVOCs ranged from 0.69 to 3.4 m3 sampler-1 day-1. Out of the 33 analytes, 22, 14 and 17 compounds were detected (above the method detection limit) in over 50% of air, airborne particles and floor dust samples respectively. The highest median level in air, airborne particles and floor dust was observed for phenanthrene (2.0 ng m-3), permethrin (8800 ng g-1) and permethrin (5100 ng g-1) respectively. Among polychlorinated biphenyl (PCB) congeners, with few exceptions, the largest contribution was from 3,3'-dichlorobiphenyl (PCB11) for both indoor air and floor dust samples. In these houses and offices, the indoor level of polycyclic aromatic hydrocarbons (PAHs) was mainly influenced by ambient (outdoor) air. Primary sources of PCBs were from within indoor environments and generally older houses have higher concentrations in air. Among pesticides, hexachlorobenzene in indoor environments appeared to be due to transfer from outdoor sources whereas chlordanes and pyrethroids were associated with past and current household application respectively. Compared to data from other countries/regions, concentrations of chlordanes, chlorpyrifos and pyrethroids in indoor air and dust samples from Australia were among the highest whereas PCB and PAH levels were among the lowest. The sum of estimated daily intakes (EDIs) via inhalation and dust contact and ingestion were calculated. The highest median value of EDI was observed for permethrin at 2.8 (for adults) and 74 ng kg-1 day-1 (for toddlers), which are <0.15% of the U.S. EPA reference dose.

Evaluating the in-sewer stability of three potential population biomarkers for application in wastewater-based epidemiology.

Endogenous chemicals specific to human metabolism have been suggested to be good candidates for markers of population size in wastewater-based epidemiology (WBE). So far, creatinine is the only endogenous chemical to be assessed against the criteria of in-sewer stability. This study thus aimed to evaluate the fate of three other endogenous compounds, 5-hydroxy indole acetic acid (5-HIAA), cortisol and androstenedione, under different sewer conditions using laboratory-scale sewer reactors. The results showed that while all compounds were stable in wastewater only (i.e. without biofilm), cortisol and androstenedione degraded quickly in sewers with the presence of sewer biofilms. The degradation followed first-order kinetics similar to that of creatinine. In contrast, 5-HIAA was relatively stable in sewer reactors. This study also recognised the impact of wastewater pH on the detectability of 5-HIAA using a LC-MS/MS direct injection method. In samples acidified to pH 2, the method did not allow routine detection/quantification of 5-HIAA whereas in non-acidified samples the method was sufficiently sensitive for routine quantification of 5-HIAA. The stability of 5-HIAA in sewers and the possibility to measure it using a simple and rapid analytical method corroborate that 5-HIAA may be a suitable biomarker for estimation of population size in WBE.

The cytotoxic, inflammatory and oxidative potential of coconut oil-substituted diesel emissions on bronchial epithelial cells at an air-liquid interface.

Diesel emissions contain high levels of particulate matter (PM) which can have a severe effect on the airways. Diesel PM can be effectively reduced with the substitution of diesel fuel with a biofuel such as vegetable oil. Unfortunately, very little is known about the cellular effects of these alternative diesel emissions on the airways. The aim of this study was to test whether coconut oil substitution in diesel fuel reduces the adverse effect of diesel emission exposure on human bronchial epithelial cells. Human bronchial epithelial cells were cultured at air-liquid interface for 7 days and exposed to diesel engine emissions from conventional diesel fuel or diesel fuel blended with raw coconut oil at low (10%), moderate (15%) and high (20%) proportions. Cell viability, inflammation, antioxidant production and xenobiotic metabolism were measured. Compared to conventional diesel, low fractional coconut oil substitution (10% and 15%) reduced inflammation and increased antioxidant expression, whereas higher fractional coconut oil (20%) reduced cell viability and increased inflammation. Therefore, cellular responses after exposure to alternative diesel emission are dependent on fuel composition.

A National Wastewater Monitoring Program for a better understanding of public health: A case study using the Australian Census.

Wastewater contains a large range of biological and chemical markers of human activity and exposures. Through systematic collection and analysis of these markers within wastewater samples it is possible to measure the public health of whole populations. The analysis of effluent and biosolids can also be used to understand the release of chemicals from wastewater treatment plants into the environment. Wastewater analysis and comparison with catchment specific data (e.g. demographics) however remains largely unexplored. This manuscript describes a national wastewater monitoring study that combines influent, effluent and biosolids sampling with the Australian Census. An archiving program allows estimation of per capita exposure to and consumption of chemicals, public health information, as well as per capita release of chemicals into the environment. The paper discusses the study concept, critical steps in setting up a coordinated national approach and key logistical and other considerations with a focus on lessons learnt and future applications. The unique combination of archived samples, analytical data and associated census-derived population data will provide a baseline dataset that has wide and potentially increasing applications across many disciplines that include public health, epidemiology, criminology, toxicology and sociology.

Marked intra-strain variation in response of Listeria monocytogenes dairy isolates to acid or salt stress and the effect of acid or salt adaptation on adherence to abiotic surfaces.

During food processing, and particularly in cheese manufacturing processes, Listeria monocytogenes may be exposed routinely to environments of low pH or high salt concentration. It has been suggested that these environmental conditions may contribute to bacterial adherence to abiotic surfaces and increased resistance to disinfection. In this study strains isolated from the environment of artisanal cheese-making dairies were used to investigate the behaviour of L. monocytogenes in response to acid and salt stress and clear differences between strains was observed. In planktonic culture, strains varied in resistance to low pH or high NaCl concentration and in the occurrence of an adaptive response to moderate acid or NaCl. There was dislocation in responses to salt and acid. Strains resistant, or adaptive, to acid were not resistant or adaptive to NaCl. The reverse also was observed. Exposure to moderate acid did not promote adherence to polystyrene but survival, at low pH or high NaCl concentration, of cells adherent to stainless steel was increased, even for strains that had no adaptive response planktonically, but the detail of these observations varied between strains. In contrast to acid adaptation, with some strains salt adaptation enhanced adherence of L. monocytogenes to polystyrene but this was not true for all strains. For some strains salt- or acid adaptation may enhance the survival of sessile cells exposed to hypochlorite disinfection.