A long-amplicon quantitative PCR assay with propidium monoazide to enumerate viable Listeria monocytogenes after heat and desiccation treatments.

The objective of this study was to develop a qPCR method for specific enumeration of viable Listeria monocytogenes in food processing facilities and heat treated products. Primers specific for L. monocytogenes were designed to amplify a short (199 bp) or long (1561 bp) fragment of the listeriolysin (hly) gene. The short- and long-amplicon qPCR methods with and without propidium monoazide (PMA) treatment of the cells were tested for their ability to discriminate between viable (no heat) and heat-killed cells (90 °C, 10 min). The PMA-qPCR methods were subsequently used to assess the survival of L. monocytogenes during desiccation (33% RH, 15 °C) on stainless steel surfaces for ten days with and without prior biofilm formation. The long-amplicon qPCR method had a limit of quantification (LOQ) of 1.32 log CFU/reaction (efficiency 92%, R2 = 0.991), while the LOQ for the short-amplicon qPCR method was 1.44 log CFU/reaction (efficiency 102%, R2 = 0.991). PMA was essential for detection of viable cells, and the long-amplicon PMA-qPCR significantly (p < 0.05) reduced the signal from heat-killed cells compared to the short-amplicon method. L. monocytogenes survival during desiccation without biofilm formation was accurately enumerated with the long-amplicon PMA-qPCR method. However, when L. monocytogenes had formed biofilm prior to desiccation, the long-amplicon PMA-qPCR accurately measured the log fold inactivation but underestimated the number of viable cells even with use of an optimized DNA extraction method. This long-amplicon PMA-qPCR method can aid in the detection and enumeration of viable L. monocytogenes cells to further the understanding of its survival and persistence in food processing facilities. The developed method was demonstrated to work on both heat and desiccation treated cells and highlights the importance of amplicon size in viability-qPCR.

Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis.

Listeria monocytogenes is a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700 L. monocytogenes mutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm(2) relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (fliP, flhB, flgD, flgL) and motor control (motB, fliM, fliY), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (P < 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses.

Comparison of the Prevalences and Diversities of Listeria Species and Listeria monocytogenes in an Urban and a Rural Agricultural Watershed.

Foods and related processing environments are commonly contaminated with the pathogenic Listeria monocytogenes. To investigate potential environmental reservoirs of Listeria spp. and L. monocytogenes, surface water and point source pollution samples from an urban and a rural municipal water supply watershed in Nova Scotia, Canada, were examined over 18 months. Presumptive Listeria spp. were cultured from 72 and 35% of rural and urban water samples, respectively, with 24% of the positive samples containing two or three different Listeria spp. The L. innocua (56%) and L. welshimeri (43%) groups were predominant in the rural and urban watersheds, respectively. Analysis by the TaqMan assay showed a significantly (P < 0.05) higher prevalence of L. monocytogenes of 62% versus 17% by the culture-based method. Both methods revealed higher prevalences in the rural watershed and during the fall and winter seasons. Elevated Escherichia coli (≥ 100 CFU/100 ml) levels were not associated with the pathogen regardless of the detection method. Isolation of Listeria spp. were associated with 70 times higher odds of isolating L. monocytogenes (odds ratio = 70; P < 0.001). Serogroup IIa was predominant (67.7%) among the 285 L. monocytogenes isolates, followed by IVb (16.1%), IIb (15.8%), and IIc (0.4%). L. monocytogenes was detected in cow feces and raw sewage but not in septic tank samples. Pulsotyping of representative water (n = 54) and local human (n = 19) isolates suggested genetic similarities among some environmental and human L. monocytogenes isolates. In conclusion, temperate surface waters contain a diverse Listeria species population and could be a potential reservoir for L. monocytogenes, especially in rural agricultural watersheds.

Role of sigB and osmolytes in desiccation survival of Listeria monocytogenes in simulated food soils on the surface of food grade stainless steel.

This research aimed to determine whether the SigB (σ(B)) regulon and osmolytes impact the survival of the foodborne pathogen, Listeria monocytogenes, during desiccation in simulated food soils with varying salt and nutrient contents on food grade stainless steel (SS) surfaces. L. monocytogenes 568 (Lm568, serotype 1/2a), its isogenic sigB mutant (ΔsigB) and the back-complemented ΔsigB were desiccated in BHI, TSB with 1% glucose (TSB-glu), peptone physiological saline (PPS) and minimal media (MM) for 21 days at 43% relative humidity (RH) and 15 °C on SS. The effect of food related osmolytes (proline, betaine and carnitine) on desiccation survival was studied by (a) pre-culturing strains in MM with an osmolyte followed by desiccation in MM and (b) by desiccating strains in MM with an osmolyte. Desiccation survival of L. monocytogenes was positively correlated to the nutrient and osmolyte concentrations in the desiccation substrates. Initial Lm568 levels of 8 Log(CFU/cm(2)) decreased by 0.9 Log(CFU/cm(2)) in BHI and 1.1-2.9 Log(CFU/cm(2)) in TSB-glu, PPS and MM after 21 days. Comparatively, the initial survival of ΔsigB was reduced in PS and MM, while no differences were observed among the three strains in BHI and TSB-glu. Pre-culture in osmolyte containing MM enhanced (p < 0.05) desiccation survival of all strains. Desiccation in osmolyte-containing MM improved desiccation survival of all strains, albeit the protection was less than that observed after pre-culture with the osmolytes. Complementation of the ΔsigB mutant restored the wildtype phenotype. In conclusion, this work shows the protective effect of osmolytes in desiccation survival of L. monocytogenes, while the σ(B) regulon only improved the initial survival in nutrient and osmolyte poor environments.

Fecal Contamination in the Surface Waters of a Rural- and an Urban-Source Watershed.

Surface waters are commonly used as source water for drinking water and irrigation. Knowledge of sources of fecal pollution in source watersheds benefits the design of effective source water protection plans. This study analyzed the relationships between enteric pathogens ( O157:H7, spp., and spp. [, and ]), water quality (turbidity, temperature, and ), and human and ruminant-cow and mitochondrial DNA (mtDNA)-based fecal source tracking (FST) markers in two source watersheds. Water samples ( = 329) were collected at 10 sites (five in each watershed) over 18 mo. The human marker (HF183) occurred in 9 to 10% of the water samples at nine sampling sites; while a forested site in the urban watershed tested negative. Ruminant-cow markers (BacR and CowM2) only appeared in the rural watershed (6%). The mtDNA markers (HcytB and AcytB) showed the same pattern but were less sensitive due to lower fecal concentrations. Higher prevalences ( < 0.05) of spp. (41 vs. 16% for the rural and urban watershed, respectively) and O157:H7 (12 vs. 3%) were observed in the rural watershed, while spp. levels were comparable (23-28%). Densities of ≥100 colony-forming units (CFU) 100 mL increased the odds ( < 0.05) of detecting the enteric bacterial pathogens. The water turbidity levels (nephelometric turbidity units [NTU] ≥ 1.0) similarly predicted ( < 0.05) pathogen presence. Storm events increased ( < 0.01) pathogen and fecal marker concentrations in the waterways. The employment of multiple FST methods suggested failing onsite wastewater systems contribute to human fecal pollution in both watersheds.

Kinetics of biofilm formation and desiccation survival of Listeria monocytogenes in single and dual species biofilms with Pseudomonas fluorescens, Serratia proteamaculans or Shewanella baltica on food-grade stainless steel surfaces.

This study investigated the dynamics of static biofilm formation (100% RH, 15 °C, 48-72 h) and desiccation survival (43% RH, 15 °C, 21 days) of Listeria monocytogenes, in dual species biofilms with the common spoilage bacteria, Pseudomonas fluorescens, Serratia proteamaculans and Shewanella baltica, on the surface of food grade stainless steel. The Gram-negative bacteria reduced the maximum biofilm population of L. monocytogenes in dual species biofilms and increased its inactivation during desiccation. However, due to the higher desiccation resistance of Listeria relative to P. fluorescens and S. baltica, the pathogen survived in greater final numbers. In contrast, S. proteamaculans outcompeted the pathogen during the biofilm formation and exhibited similar desiccation survival, causing the N21 days of Serratia to be ca 3 Log10(CFU cm(-2)) greater than that of Listeria in the dual species biofilm. Microscopy revealed biofilm morphologies with variable amounts of exopolymeric substance and the presence of separate microcolonies. Under these simulated food plant conditions, the fate of L. monocytogenes during formation of mixed biofilms and desiccation depended on the implicit characteristics of the co-cultured bacterium.

Increased thermal and osmotic stress resistance in Listeria monocytogenes 568 grown in the presence of trehalose due to inactivation of the phosphotrehalase-encoding gene treA.

The food-borne pathogen Listeria monocytogenes is a problem for food processors and consumers alike, as the organism is resistant to harsh environmental conditions and inimical barriers implemented to prevent the survival and/or growth of harmful bacteria. One mechanism by which listeriae mediate survival is through the accumulation of compatible solutes, such as proline, betaine and carnitine. In other bacteria, including Escherichia coli, the synthesis and accumulation of another compatible solute, trehalose, are known to aid in the survival of stressed cells. The objective of this research was to investigate trehalose metabolism in L. monocytogenes, where the sugar is thought to be transferred across the cytoplasmic membrane via a specific phosphoenolpyruvate phosphotransferase system and phosphorylation to trehalose-6-phosphate (T6P). The latter is subsequently broken down into glucose and glucose-6-phosphate by α,α-(1,1) phosphotrehalase, the putative product of the treA gene. Here we report on an isogenic treA mutant of L. monocytogenes 568 (568:ΔTreA) which, relative to the wild-type strain, displays increased tolerances to multiple stressors, including heat, high osmolarity, and desiccation. This is the first study to examine the putative trehalose operon in L. monocytogenes, and we demonstrate that lmo1254 (treA) in L. monocytogenes 568 indeed encodes a phosphotrehalase required for the hydrolysis of T6P. Disruption of the treA gene results in the accumulation of T6P which is subsequently dephosphorylated to trehalose in the cytosol, thereby contributing to the stress hardiness observed in the treA mutant. This study highlights the importance of compatible solutes for microbial survival in adverse environments.

Growth of Listeria spp. in shredded cabbage is enhanced by a mild heat treatment.

Mild thermal processing can enhance the shelf life of cut fruits and vegetables by delaying the onset of spoilage and preserving the organoleptic properties of shredded cabbage. However, food safety issues related to this process have not been fully investigated. Therefore, the survival and growth of Listeria spp. on cabbage treated in this manner was examined. Experimentally, 24 strains of Listeria spp. (including L. monocytogenes) were inoculated onto cut and intact cabbage tissues and stored at 5 degrees C. All strains on intact tissues exhibited a moderate decline in numbers (up to 1.0 log CFU/cm(2)) over the 28-day storage period. Conversely, cut tissue supported growth of most strains during the first 7 to 14 days of incubation with maximum increases of 1.2 log CFU/cm(2). Subsequently, the survival or growth on heat-treated (50 degrees C for 3 min) and untreated shredded cabbage of four L. monocytogenes and four nonpathogenic Listeria spp. strains were compared during storage for 21 days at 5 degrees C. Growth on untreated shred for all strains was similar to the results observed on cut tissue with a maximum increase of approximately 1.0 log CFU/g. However, in the heat-treated cabbage shred all strains displayed a rapid increase in growth (up to 2.5 log CFU/g) during the first 7 days of incubation, which may be indicative of the destruction of an endogenous growth-inhibiting compound within the cabbage. In conclusion, this study shows that mild thermal treatments of cut cabbage may promote pathogen growth if other inimical barriers are not implemented downstream of the thermal treatment.

Cold-shock proteins affect desiccation tolerance, biofilm formation and motility in Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen whose biofilm formation and desiccation tolerance may contribute to its survival in the food industry. L. monocytogenes possesses three cold-shock domain family proteins (CspA, CspB and CspD) known to be essential for adaptation against various food-relevant stress conditions including cold growth. The role of Csps in desiccation tolerance and biofilm formation was investigated in csp mutants as well as twenty-one other wild-type (WT) strains. Mutants with a single (ΔcspA) or multiple (ΔcspAB, ΔcspAD and ΔcspABD) deletions of csp genes, in a desiccation sensitive WT background (L. monocytogenes EGD-e) were immotile and exhibited an elevated desiccation tolerance compared to the parent strain. However, deletion of cspA in the more desiccation resistant food and outbreak related L. monocytogenes strains 568 and 08-5578 had no impact on desiccation tolerance although compared to the parent strains the mutants were also immotile. A correlation between lower motility and higher desiccation tolerance was observed among the 20 WT strains (Spearman rank correlation, rs = -0.56, p = 0.01), although exceptions occurred indicating that multiple factors influence the diverse desiccation tolerance among L. monocytogenes strains. Expression of cspA was upregulated in WT EGD-e, 568 and 08-5578 strains after desiccation for seven days, while the 568 and 08-5578 ΔcspA mutants expressed elevated levels of cspD and cspB (>30 fold higher) compared to their WTs. This indicates that upregulation of the other csps compensates for the deleted cspA gene. Although biofilm formation was improved in all EGDe csp mutants relative to the WT strain, the opposite was observed for 568 and 08-5578 WT strains and their cspA deletion mutants. Only motile strains formed biofilm in the peg lid assay but a significant negative correlation (rs = -0.60, p = 0.01) was seen between higher motility and higher biofilm formation of WT strains. In conclusion, the survival of L. monocytogenes strains in the food processing environment may depend on the control of motility, which is a necessity for biofilm formation but disadvantageous for desiccation survival.

Initial Transcriptomic Response and Adaption of Listeria monocytogenes to Desiccation on Food Grade Stainless Steel.

The foodborne pathogen Listeria monocytogenes survives exposure to a variety of stresses including desiccation in the food industry. Strand-specific RNA sequencing was applied to analyze changes in the transcriptomes of two strains of L. monocytogenes (Lm 568 and Lm 08-5578) during desiccation [15°C, 43% relative humidity (RH)] on food grade stainless steel surfaces over 48 h to simulate a weekend with no food production. Both strains showed similar survival during desiccation with a 1.8-2 Log CFU/cm2 reduction after 48 h. Analysis of differentially expressed (DE) genes (>twofold, adjusted p-value <0.05) revealed that the initial response to desiccation was established after 6 h and remained constant with few new genes being DE after 12, 24, and 48 h. A core of 81 up- and 73 down-regulated DE genes were identified as a shared, strain independent response to desiccation. Among common upregulated genes were energy and oxidative stress related genes e.g., qoxABCD (cytochrome aa3) pdhABC (pyruvate dehydrogenase complex) and mntABCH (manganese transporter). Common downregulated genes related to anaerobic growth, proteolysis and the two component systems lmo1172/lmo1173 and cheA/cheY, which are involved in cold growth and flagellin production, respectively. Both strains upregulated additional genes involved in combatting oxidative stress and reactive oxygen species (ROS), including sod (superoxide dismutase), kat (catalase), tpx (thiol peroxidase) and several thioredoxins including trxAB, lmo2390 and lmo2830. Osmotic stress related genes were also upregulated in both strains, including gbuABC (glycine betaine transporter) and several chaperones clpC, cspA, and groE. Significant strain differences were also detected with the food outbreak strain Lm 08-5578 differentially expressing 1.9 × more genes (726) compared to Lm 568 (410). Unique to Lm 08-5578 was a significant upregulation of the expression of the alternative transcription factor σB and its regulon. A number of long antisense transcripts (lasRNA) were upregulated during desiccation including anti0605, anti0936, anti1846, and anti0777, with the latter controlling flagellum biosynthesis and possibly the downregulation of motility genes observed in both strains. This exploration of the transcriptomes of desiccated L. monocytogenes provides further understanding of how this bacterium encounters and survives the stress faced when exposed to dry conditions in the food industry.

Comparative Analysis of Listeria monocytogenes Plasmids and Expression Levels of Plasmid-Encoded Genes during Growth under Salt and Acid Stress Conditions.

Listeria monocytogenes strains are known to harbour plasmids that confer resistance to sanitizers, heavy metals, and antibiotics; however, very little research has been conducted into how plasmids may influence L. monocytogenes' ability to tolerate food-related stresses. To investigate this, a library (n = 93) of L. monocytogenes plasmid sequences were compared. Plasmid sequences were divided into two groups (G1 and G2) based on a repA phylogeny. Twenty-six unique plasmid types were observed, with 13 belonging to each of the two repA-based groups. G1 plasmids were significantly (p < 0.05) smaller than G2 plasmids but contained a larger diversity of genes. The most prevalent G1 plasmid (57,083 bp) was observed in 26 strains from both Switzerland and Canada and a variety of serotypes. Quantitative PCR (qPCR) revealed a >2-fold induction of plasmid-contained genes encoding an NADH peroxidase, cadmium ATPase, multicopper oxidase, and a ClpL chaperone protein during growth under salt (6% NaCl) and acid conditions (pH 5) and ProW, an osmolyte transporter, under salt stress conditions. No differences in salt and acid tolerance were observed between plasmid-cured and wildtype strains. This work highlights the abundance of specific plasmid types among food-related L. monocytogenes strains, the unique characteristics of G1 and G2 plasmids, and the possible contributions of plasmids to L. monocytogenes tolerance to food-related stresses.

Characterization of Listeria monocytogenes enhanced cold-tolerance variants isolated during prolonged cold storage.

In this study, we show that growth and prolonged storage of Listeria monocytogenes at 4 °C can promote the selection of variants with enhanced cold and heat tolerance. Enhanced cold-tolerance (ECT) variants (n = 12) were successfully isolated from a strain with impaired cold growth abilities following 84 days of storage at 4 °C in brain heart infusion broth (BHIB). Whole genome sequencing, membrane fatty acid analysis, and stress tolerance profiling were performed on the parent strain and two ECT variants: one displaying regular-sized colonies and the other displaying small colonies when grown at 37 °C on BHI agar. Under cold stress conditions, the parent strain exhibited an impaired ability to produce branched-chain fatty acids which are known to be important for cold adaptation in L.monocytogenes. The ECT variants were able to overcome this limitation, a finding which is hypothesized to be associated with the identification of two independent single-nucleotide polymorphisms in genes encoding subunits of acetyl-coA carboxylase, an enzyme critical for fatty acid biosynthesis. While the ECT phenotype was not found to be associated with improved salt (BHIB + 6% NaCl, 25 °C), acid (BHIB pH 5, 25 °C) or desiccation (33% RH, 20 °C) tolerance, the small-colony variant exhibited significantly (p < 0.05) enhanced heat tolerance at 52 °C in buffered peptone water compared to the parent strain and the other variant. The results from this study demonstrate that the continuous use of refrigeration along the food-supply chain has the potential to select for L.monocytogenes variants with enhanced cold and heat tolerance, highlighting the impact that microbial intervention strategies can have on the evolution of bacterial strains and likewise, food safety.

Screening-level microbial risk assessment of acute gastrointestinal illness attributable to wastewater treatment systems in Nunavut, Canada.

Most arctic communities use primary wastewater treatment systems that are capable of only low levels of pathogen removal. Effluent potentially containing fecally derived microorganisms is released into wetlands and marine waters that may simultaneously serve as recreation or food harvesting locations for local populations. The purpose of this study is to provide the first estimates of acute gastrointestinal illness (AGI) attributable to wastewater treatment systems in Arctic Canada. A screening-level, point estimate quantitative microbial risk assessment model was developed to evaluate worst-case scenarios across an array of exposure pathways in five case study locations. A high annual AGI incidence rate of 5.0 cases per person is estimated in Pangnirtung, where a mechanical treatment plant discharges directly to marine waters, with all cases occurring during low tide conditions. The probability of AGI per person per single exposure during this period ranges between 1.0 × 10-1 (shore recreation) and 6.0 × 10-1 (shellfish consumption). A moderate incidence rate of 1.2 episodes of AGI per person is estimated in Naujaat, where a treatment system consisting of a pond and tundra wetland is used, with the majority of cases occurring during spring. The pathway with the highest individual probability of AGI per single exposure event is wetland travel at 6.0 × 10-1. All other risk probabilities per single exposure are <1.0 × 10-1. The AGI incidence rates estimated for the other three case study locations are <0.1. These findings suggest that wastewater treatment sites may be contributing to elevated rates of AGI in some arctic Canadian communities. Absolute risk values, however, should be weighed with caution based on the exploratory nature of this study design. These results can be used to inform future risk assessment and epidemiological research as well as support public health and sanitation decisions in the region.

Wastewater treatment and public health in Nunavut: a microbial risk assessment framework for the Canadian Arctic.

Wastewater management in Canadian Arctic communities is influenced by several geographical factors including climate, remoteness, population size, and local food-harvesting practices. Most communities use trucked collection services and basic treatment systems, which are capable of only low-level pathogen removal. These systems are typically reliant solely on natural environmental processes for treatment and make use of existing lagoons, wetlands, and bays. They are operated in a manner such that partially treated wastewater still containing potentially hazardous microorganisms is released into the terrestrial and aquatic environment at random times. Northern communities rely heavily on their local surroundings as a source of food, drinking water, and recreation, thus creating the possibility of human exposure to wastewater effluent. Human exposure to microbial hazards present in municipal wastewater can lead to acute gastrointestinal illness or more severe disease. Although estimating the actual disease burdens associated with wastewater exposures in Arctic communities is challenging, waterborne- and sanitation-related illness is believed to be comparatively higher than in other parts of Canada. This review offers a conceptual framework and evaluation of current knowledge to enable the first microbial risk assessment of exposure scenarios associated with food-harvesting and recreational activities in Arctic communities, where simplified wastewater systems are being operated.

Disinfection and removal of human pathogenic bacteria in arctic waste stabilization ponds.

Wastewater stabilization ponds (WSPs) are commonly used to treat municipal wastewater in Arctic Canada. The biological treatment in the WSPs is strongly influenced by climatic conditions. Currently, there is limited information about the removal of fecal and pathogenic bacteria during the short cool summer treatment season. With relevance to public health, the objectives of this paper were to determine if treatment in arctic WSPs resulted in the disinfection (i.e., removal of fecal indicator bacteria, Escherichia coli) and removal of selected human bacterial pathogens from the treated effluent. The treatment performance, with focus on microbial removal, was assessed for the one-cell WSP in Pond Inlet (Nunavut [NU]) and two-cell WSP in Clyde River (NU) over three consecutive (2012-2014) summer treatment seasons (late June-early September). The WSPs provided a primary disinfection treatment of the wastewater with a 2-3 Log removal of generic indicator E. coli. The bacterial pathogens Salmonella spp., pathogenic E. coli, and Listeria monocytogenes, but not Campylobacter spp. and Helicobacter pylori, were detected in the untreated and treated wastewater, indicating that human pathogens were not reliably removed. Seasonal and annual variations in temperature significantly (p < 0.05) affected the disinfection efficiency. Improved disinfection and pathogen removal was observed for the two-cell system in Clyde River as compared to the one-cell system in Pond Inlet. A quantitative microbial risk assessment should be performed to determine if the release of low levels of human pathogens into the arctic environment poses a human health risk.

Fate of antibiotic resistance genes in two Arctic tundra wetlands impacted by municipal wastewater.

In the Canadian Arctic, it is common practice to discharge municipal wastewater into tundra wetlands. Antibiotic resistant bacteria and the antibiotic resistance genes (ARGs) they contain can be present in municipal wastewater and there is a scarcity of knowledge on ARGs in wastewater in Arctic environments. This study was initiated on the fate of ARGs in tundra wetland ecosystems impacted by anthropogenic wastewater sources in Arctic communities. In the summer season of 2016, two wetlands were studied in the Inuit communities of Sanikiluaq and Naujaat in Nunavut, Canada. Genomic DNA was extracted from both soil and water during the spring freshet and late summer in the wetlands, and a suite of nine clinically relevant ARGs (sul1, sul2, mecA, vanA, qnrS, ermB, tetO, blaTEM, blaCTX-M), and an integron gene (int1) were analyzed using quantitative polymerase chain reaction (qPCR). Hydrological and water quality measurements were conducted in conjunction with the microbiological sampling. Gene targets were consistently present in the wastewater, and throughout both wetlands, except for vanA and mecA. Concentrations of ARGs were greater during the spring freshet, due to short hydraulic retention times (<2 days), which coincided with decreased treatment performance. The environmental resistome in un-impacted wetlands had above detection limit concentrations of int1, sul1, sul2, blaCTX-M in water in Naujaat, and sul1, qnrS and tetO in soil in Sanikiluaq. First-order rate constants were widely variable and specific to the gene target. ARGs were present in concentrations elevated above baseline reference sites in tundra wetlands influenced by municipal wastewater, and hydrological conditions had a large impact on their spatial distribution and levels.

Removal of antibiotic resistance genes in two tertiary level municipal wastewater treatment plants.

Raw wastewater can contain high levels of antibiotic resistance genes (ARGs), making municipal wastewater treatment plants (WWTPs) critical for the control of the release of ARGs into the environment. The objective of this study was to investigate how individual treatment steps in two tertiary WWTPs affected the removal (copies/mL) and relative abundance of ARGs (copies/copies 16S rRNA genes). Nine ARG markers, representing resistance to commonly used antibiotics, as well as one integron gene (intl1) to assess ARG mobility potential, were quantified using quantitative real-time PCR (qPCR). Both WWTPs met provincial effluent regulations for removal of carbonaceous oxygen demand (CBOD5) and total suspended solids. Eight of the ten ARG markers (intl1, sul1, sul2, tet(O), ermB, blaCTX-M, blaTEM, qnrS) were detected in all samples. In contrast, mecA was detected intermittently and vanA remained below the detection limit in all samples. The total ARG marker abundances decreased by log 1.77 (p < 0.05) in the plant using an aerated lagoon (AL), and by 2.69 logs (p < 0.05) through treatment in the plant employing a biological nutrient removal (BNR) system. The BNR and secondary clarifier steps in both plants afforded the most removal of ARGs. The relative abundance of ARGs remained unchanged at the AL plant and showed a decreasing trend at the BNR plant. Levels of CBOD5, nitrate and the human Bacteroides fecal marker correlated with ARG concentrations, suggesting these variables may be useful in predicting ARG removal. In conclusion, the effluent coming from the WWTPs contained eight of the studied ARG markers in concentrations ranging from 0.01 to 3.6 log copies/mL, indicating their release into the environment, however, the relative abundance of ARGs was not enriched during treatment in the two WWTPs.

Chemical and microbial characteristics of municipal drinking water supply systems in the Canadian Arctic.

Drinking water in the vast Arctic Canadian territory of Nunavut is sourced from surface water lakes or rivers and transferred to man-made or natural reservoirs. The raw water is at a minimum treated by chlorination and distributed to customers either by trucks delivering to a water storage tank inside buildings or through a piped distribution system. The objective of this study was to characterize the chemical and microbial drinking water quality from source to tap in three hamlets (Coral Harbour, Pond Inlet and Pangnirtung-each has a population of <2000) on trucked service, and in Iqaluit (population ~6700), which uses a combination of trucked and piped water conveyance. Generally, the source and drinking water was of satisfactory microbial quality, containing Escherichia coli levels of <1 MPN/100 mL with a few exceptions, and selected pathogenic bacteria and parasites were below detection limits using quantitative polymerase chain reaction (qPCR) methods. Tap water in households receiving trucked water contained less than the recommended 0.2 mg/L of free chlorine, while piped drinking water in Iqaluit complied with Health Canada guidelines for residual chlorine (i.e. >0.2 mg/L free chlorine). Some buildings in the four communities contained manganese (Mn), copper (Cu), iron (Fe) and/or lead (Pb) concentrations above Health Canada guideline values for the aesthetic (Mn, Cu and Fe) and health (Pb) objectives. Corrosion of components of the drinking water distribution system (household storage tanks, premise plumbing) could be contributing to Pb, Cu and Fe levels, as the source water in three of the four communities had low alkalinity. The results point to the need for robust disinfection, which may include secondary disinfection or point-of-use disinfection, to prevent microbial risks in drinking water tanks in buildings and ultimately at the tap.

Anti-Bacterial Activity of Phenolic Compounds against Streptococcus pyogenes.

BACKGROUND: Worldwide, Streptococcus pyogenes is the leading cause of bacterial pharyngitis. To reduce the use of antibiotics, antimicrobial phytochemical-containing remedies, which have long been in use in traditional medicine, may provide new approaches for management of streptococcal pharyngitis. The objective of this study was to assess the inhibitory activities of 25 natural phenolic compounds against three strains of S. pyogenes. METHODS: After an initial screening, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the nine most effective phenolic compounds were determined. The effect of four compounds with the lowest MIC and MBC on streptococcal growth and biofilm formation was also studied. RESULTS: 1,2-Naphthoquinone and 5-hydroxy-1,4-naphthoquinone elicited the greatest anti-S. pyogenes activities with MICs ranging from 0.39 to 6.25 µg mL-1 and MBCs of 100 µg mL-1. Both naphthoquinones inhibited the biofilm formation at concentrations ranging from 12.5 to 50 µg mL-1. Biofilm reduction and altered bacterial cell structures were visible in scanning electron microscopy images of naphthoquinone-treated cells. CONCLUSION: In conclusion, 1,2-naphthoquinone and 5-hydroxy-1,4-naphthoquinone inhibit S. pyogenes and should be further investigated as candidates for the management of streptococcal pharyngitis.

Variations in biofilm formation, desiccation resistance and Benzalkonium chloride susceptibility among Listeria monocytogenes strains isolated in Canada.

Listeria monocytogenes is a pathogenic foodborne microorganism noted for its ability to survive in the environment and food processing facilities. Survival may be related to the phenotype of individual strains including the ability to form biofilms and resist desiccation and/or sanitizer exposure. The objectives of this research were to compare 14 L. monocytogenes strains isolated from blood (3), food (6) and water (5) with respect to their benzalkonium chloride (BAC) sensitivity, desiccation resistance, and ability to form biofilm. Correlations were tested between those responses, and the presence of the SSI-1 (Stress Survival Islet) and LGI1/CC8 (Listeria Genomic Island 1 in a clonal complex 8 background) genetic markers. Genetic sequences from four strains representing different phenotypes were also probed for predicted amino acid differences in biofilm, desiccation, and membrane related genes. The water isolates were among the most desiccation susceptible strains, while strains exhibiting desiccation resistance harboured SSI-1 or both the SSI-1 and LGI1/CC8 markers. BAC resistance was greatest in planktonic LGI1/CC8 cells (relative to non-LGI1/CC8 cells), and higher BAC concentrations were also needed to inhibit the formation of biofilm by LGI1/CC8 strains during incubation for 48h and 6days compared to other strains. Formation of biofilm on stainless steel was not significantly (p>0.05) different among the strains. Analysis of genetic sequence data from desiccation and BAC sensitive (CP4 5-1, CP5 2-3, both from water), intermediate (Lm568, food) and desiccation and BAC resistant (08 5578, blood, human outbreak) strains led to the finding of amino acid differences in predicted functional protein domains in several biofilm, desiccation and peptidoglycan related genes (e.g., lmo0263, lmo0433, lmo0434, lmo0771, lmo0973, lmo1080, lmo1224, lmo1370, lmo1744, and lmo2558). Notably, the LGI1/CC8 strain 08-5578 had a frameshift mutation in lmo1370, a gene previously associated with desiccation resistance. In conclusion, the more desiccation and BAC resistant LGI1/CC8 isolates may pose a challenge for sanitation efforts.