Evaluation of four human-associated fecal biomarkers in wastewater in Southern Ontario.

Human fecal biomarkers (HFBs) have a longstanding history in the field of microbial source tracking (MST) serving as indicators of human fecal contamination in drinking and recreational water. Further, HFBs have aided in recent efforts to monitor human pathogen transmission within communities. The dilution of wastewater from various sources throughout the sewershed cannot be controlled and human fecal biomarkers (HFBs) can be used to normalize target human pathogen concentrations so that fluctuations in fecal matter in wastewater can be accounted for. In the current study, we monitored the prevalence of four HFBs - including two viruses, Pepper mild mottle virus (PMMoV), cross-assembly phage (crAssphage), as well as two human-associated Bacteroides markers, HF183 and BacHuman - in wastewater samples from ten Southern Ontario wastewater treatment plants and evaluated their temporal and spatial variation in context of environmental factors that may impact the ability of HFB to normalize pathogen concentrations in wastewater. Environmental variables including precipitation, wastewater flow rate, temperature, and concentrated mass were also analyzed for their potential correlation with HFB variation in wastewater. The four HFBs were detected at high concentrations across all 10 sampling locations. The median concentrations across all sampling sites were: PMMoV 3.6 Log gene copies (GC)/mL; crAssphage 5.0 Log GC/mL; HF183 6.8 Log GC/mL and BacHuman 6.9 Log GC/mL. All HFBs were found to be similarly stratified across all 10 sites, and the bacterial markers were consistently found at higher concentration compared to the viral HFBs at all sites. The coefficient of variation (CV) for each HFB was used to characterize the variability of each biomarker at each sewershed. BacHuman and crAssphage were found to have lower CV than PMMoV and HF183, indicating that BacHuman and crAssphage may perform better in reflecting the variations in abundance of human feces in wastewater or MST applications.

Metagenomics of Wastewater Influent from Wastewater Treatment Facilities across Ontario in the Era of Emerging SARS-CoV-2 Variants of Concern.

We report metagenomic sequencing analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in composite wastewater influent from 10 regions in Ontario, Canada, during the transition between Delta and Omicron variants of concern. The Delta and Omicron BA.1/BA.1.1 and BA.2-defining mutations occurring in various frequencies were reported in the consensus and subconsensus sequences of the composite samples.

Passive sampling, a practical method for wastewater-based surveillance of SARS-CoV-2.

In search of practical and affordable tools for wastewater-based surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), three independent field experiments were conducted using three passive sampler sorbents (electronegative membrane, cotton bud, and gauze) in Guelph, Ontario, Canada. Total daily cases during this study ranged from 2 to 17/100,000 people and 43/54 traditionally collected wastewater samples were positive for SARS-CoV-2 with mean detectable concentrations ranging from 8.4 to 1780 copies/ml. Viral levels on the passive samplers were assessed after 4, 8, 24, 48, 72, and 96 hrs of deployment in the wastewater and 43/54 membrane, 42/54 gauze, and 27/54 cotton bud samples were positive. A linear accumulation rate of SARS-CoV-2 on the membranes was observed up to 48 hours, suggesting the passive sampler could adequately reflect wastewater levels for up to two days of deployment. Due the variability in accumulation observed for the cotton buds and gauzes, and the pre-processing steps required for the gauzes, we recommend membrane filters as a simple cost-effective option for wastewater-based surveillance of SARS-CoV-2.

Biogas production, waste stabilization efficiency, and hygienization potential of a mesophilic anaerobic plug flow reactor processing swine manure and corn stover.

Swine manure and corn stover are abundant agricultural wastes which contribute to greenhouse gas (GHG) emissions, nutrient runoff leading to eutrophication, and a biosafety risk with respect to improper swine manure handling. Anaerobic co-digestion (AcoD) of swine manure and corn stover can mitigate these negative impacts while producing biogas as a renewable energy source. Semi-continuous mesophilic plug flow reactor (PFR operation) was studied during a step-wise increase in organic loading rate (OLR) over the range of 0.25-4.7 kg volatile solids added (VS) m-3 d-1, which corresponded to total solids content (TS) of 1.5-9.0%. Process stability was observed at all OLR, with the highest total biogas yield and methane content of 0.674 ± 0.06 m-3 kg-1 and 62%, respectively, at 0.25 kg m-3 d-1. As OLR and TS increased, VS reduction decreased and volatile fatty acids (VFA) increased due to shorter hydraulic retention times (HRT). Hygienization potential was assayed using fecal indicator bacteria (FIB), with some groups being reduced (E. coli, fecal coliforms) and others not (Clostridia spp., fecal enterococci). Lignocellulolytic enzyme activity trended upward as OLR was increased, highlighting changes in microbial activity in response to feeding rate.

Terrestrial Isopods Porcellio scaber and Oniscus asellus (Crustacea: Isopoda) Increase Bacterial Abundance and Modify Microbial Community Structure in Leaf Litter Microcosms: a Short-Term Decomposition Study.

Invasive terrestrial isopods are likely to have altered leaf litter decomposition processes in North American forests, but the mechanisms underlying these alterations and the degree to which they differ among isopod species are poorly characterized. Using mixed-deciduous leaf litter microcosms, we quantified the effects of two common, invasive isopods (Oniscus asellus and Porcellio scaber) on short-term leaf litter decomposition and microbial community structure and function. Microcosms containing ground litter and a microbial inoculant were exposed to one of the two isopod species or no isopods for 21 days. Mass loss was then quantified as the change in litter dry mass after leaching, and microbial respiration was quantified as the mass of CO2 absorbed by soda lime. Litter leachates were plated on agar to quantify culturable bacterial and fungal abundance, and denaturing gradient gel electrophoresis of amplified leachate microbial DNA was used to characterize shifts in microbial community structure. Isopod presence increased litter mass loss by a modest ~ 6%, but did not affect litter microbial respiration. Bacterial abundance increased significantly in the presence of isopods, while fungal abundance was either unchanged or reduced. Overall litter microbial species richness was reduced by isopods, with O. asellus specifically reducing fungal abundance and diversity. Isopods modified the microbial community structure by suppressing four bacterial and one fungal species, while promoting growth of four other bacterial species (two unique to each isopod species) and two fungal species (one which was unique to O. asellus).

Hygienization and microbial metabolic adaptation during anaerobic co-digestion of swine manure and corn stover.

This study assessed the effect of different swine manure (SM)/corn stover (CS) mixtures based on total solids (TS) content with respect to hygienization, microbial community dynamics and methane yields on batch anaerobic co-digestion performance. Different ratios of SM and CS with TS content between 0.69 and 6% digested at 75 d revealed SM had the greatest methane yield at 403.9 mL g-1 volatile solids added (VS) and 86.31% VS reduction. BIOLOG AN microplates and lignocellulolytic enzyme assays proved to be rapid tools for characterizing microbial community metabolism as noted by the different carbon source utilization patterns between TS loadings. Hygienization of fecal indicator bacteria groups was achieved with some (E. coli) but not all groups (Clostridia spp.). The results showed that colorimetric biochemical assays and culture-based techniques can rapidly assess microbial community dynamics during co-digestion, and that TS- in the form of lignocellulosic biomass- influences microbial metabolic activities.

Potentiation of Tobramycin by Silver Nanoparticles against Pseudomonas aeruginosa Biofilms.

Increasing antibiotic resistance among pathogenic bacterial species is a serious public health problem and has prompted research examining the antibacterial effects of alternative compounds and novel treatment strategies. Compounding this problem is the ability of many pathogenic bacteria to form biofilms during chronic infections. Importantly, these communities are often recalcitrant to antibiotic treatments that show effectiveness against acute infection. The antimicrobial properties of silver have been known for decades, but recently silver and silver-containing compounds have seen renewed interest as antimicrobial agents for treating bacterial infections. The goal of this study was to assess the ability of citrate-capped silver nanoparticles (AgNPs) of various sizes, alone and in combination with the aminoglycoside antibiotic tobramycin, to inhibit established Pseudomonas aeruginosa biofilms. Our results demonstrate that smaller 10-nm and 20-nm AgNPs were more effective at synergistically potentiating the activity of tobramycin. Visualization of biofilms treated with combinations of 10-nm AgNPs and tobramycin reveals that the synergistic bactericidal effect may be caused by disrupting cellular membranes. Minimum biofilm eradication concentration (MBEC) assays using clinical P. aeruginosa isolates shows that small AgNPs are more effective than larger AgNPs at inhibiting biofilms, but that the synergy effect is likely a strain-dependent phenomenon. These data suggest that small AgNPs synergistically potentiate the activity of tobramycin against P. aeruginosain vitro and may reveal a potential role for AgNP/antibiotic combinations in treating patients with chronic infections in a strain-specific manner.

Draft Genome Sequence of Ureolytic Environmental Isolate Staphylococcus sp. NA309.

We report the 2.7 Mb draft genome sequence of Staphylococcus sp. NA309 isolated from poultry litter. The isolate was a dominant member of the cultivable aerobic bacteria identified to have ureolytic activity, responsible for ammonia generation in poultry litter residue.

Draft Genome Sequence of Ureolytic Environmental Isolate Bacillus galactosidilyticus PL133.

We report here the 5.19-Mb draft genome sequence of Bacillus galactosidilyticus PL133 isolated from poultry litter. The isolate was an important member of the cultivable aerobic bacteria identified to have ureolytic activity, which is responsible for ammonia generation in poultry litter residue.

Viability and infectivity of fresh and cryopreserved Nosema ceranae spores.

The microsporidium fungus Nosema ceranae is an intracellular parasite that infects the midgut of the honey bee, Apis mellifera. A major limitation of research on N. ceranae is that the fungus is non-culturable and thus studying it depends on the seasonal availability of Nosema spores. Also, spore viability and infectivity can vary considerably, and thus there is a need for reliable methods for determining those traits. This study examined different conditions for N. ceranae spore cryopreservation at -70°C, assessing spore viability and infectivity. Viability was determined by a staining procedure counting total spores numbers with bright field microscopy and un-viable spore numbers with the fluorescent dye, propidium iodide. Spore infectivity was determined with a dilution inoculation assay. Infectivity was dependent on the inoculum dose for the proportion of bees with detectable Nosema infections based on the number of spores per bee at 18days after inoculation; 4000 spores per bee or higher were needed to get approx. 100% of the inoculated bees infected. The median infective dose (ID50) was 149 spores per bee, and the minimum dose capable of causing a detectable infection was 1.28 spores. The proportion of N. ceranae infected bees correlated significantly with the number of spores per bee (r=0.98, P<0.0001). N. ceranae spores cryopreserved in water or 10% glycerol did not differ in viability compared to fresh spores, but lost infectivity when inoculated into bees. This study shows that while cryopreservation of N. ceranae spores can preserve viability, the spores can have reduced infectivity.

Evaluation of propidium monoazide and long-amplicon qPCR as an infectivity assay for coliphage.

Standardized and rapid assays for viable viral pathogens are needed to inform human health risk assessments. Conventional qPCR is designed to enumerate the gene copies of an organism in a sample, but does not identify those that originated from a viable pathogen. This study was undertaken to evaluate modified qPCR methods as infectivity assays for the enumeration of infectious MS2 coliphage. Propidium monoazide (PMA) treatment coupled with long-amplicon qPCR assays were assessed for their ability to quantify infectious MS2 in pure cultures and following inactivation by a range of UV light exposures and chlorine doses. The qPCR results were compared to the plaque assay, which was used as the standard to indicate the level of infectious MS2 in each sample. For pure cultures, PMA-qPCR results were not significantly different from the plaque assay (p>0.05). At >4 log inactivation, combined PMA and long-amplicon qPCR assays overestimated the level of infectious MS2 remaining (p<0.05). The most accurate long-amplicon qPCR infectivity assay targeted a 624-bp region at the 5' end of the genome. Modified qPCR approaches may be useful tools to monitor the loss of infectivity as a result of disinfection processes.

Metagenomic Comparison of Antibiotic Resistance Genes Associated with Liquid and Dewatered Biosolids.

Municipal biosolids (MBs) that are land-applied in North America are known to possess an active microbial population that can include human pathogens. Activated sludge is a hotspot for the accumulation of antibiotics and has been shown to be a selective environment for microorganisms that contain antibiotic resistance genes (ARGs); however, the prevalence of ARGs in MBs is not well characterized. In this study, we enriched the plasmid metagenome from raw sewage sludge and two CP2 MBs, a mesophilic anaerobic digestate and a dewatered digestate, to evaluate the presence of ARGs in mobile genetic elements. The CP2-class biosolids are similar to Class B biosolids in the United States. The CP2 biosolids must meet a microbiological cut off of 2 × 10 colony-forming units (CFU) per dry gram or 100 mL of biosolids. The enriched plasmid DNA was sequenced (Illumina MiSeq). Sequence matching against databases, including the Comprehensive Antibiotic Resistance Database (CARD), MG-RAST, and INTEGRALL, identified potential genes of interest related to ARGs and their ability to transfer. The presence and abundance of different ARGs varied between treatments with heterogeneity observed among the same sample types. The MBs plasmid-enriched metagenomes contained ARGs associated with resistance to a variety of antibiotics, including ß-lactams, rifampicin, quinolone, and tetracycline as well as the detection of extended spectrum ß-lactamase genes. Cultured bacteria from CP2 MBs possessed antibiotic resistances consistent with the MBs metagenome data including multiantibiotic-resistant isolates. The results from this study provide a better understanding of the ARG and MGE profile of the plasmid-enriched metagenome of CP2 MBs.

Determinants of tolerance to inhibitors in hardwood spent sulfite liquor in genome shuffled Pachysolen tannophilus strains.

Genome shuffling was used to obtain Pachysolen tannophilus mutants with improved tolerance to inhibitors in hardwood spent sulfite liquor (HW SSL). Genome shuffled strains (GHW301, GHW302 and GHW303) grew at higher concentrations of HW SSL (80 % v/v) compared to the HW SSL UV mutant (70 % v/v) and the wild-type (WT) strain (50 % v/v). In defined media containing acetic acid (0.70-0.90 % w/v), GHW301, GHW302 and GHW303 exhibited a shorter lag compared to the acetic acid UV mutant, while the WT did not grow. Genome shuffled strains produced more ethanol than the WT at higher concentrations of HW SSL and an aspen hydrolysate. To identify the genetic basis of inhibitor tolerance, whole genome sequencing was carried out on GHW301, GHW302 and GHW303 and compared to the WT strain. Sixty single nucleotide variations were identified that were common to all three genome shuffled strains. Of these, 40 were in gene sequences and 20 were within 5 bp-1 kb either up or downstream of protein encoding genes. Based on the mutated gene products, mutations were grouped into functional categories and affected a variety of cellular functions, demonstrating the complexity of inhibitor tolerance in yeast. Sequence analysis of UV mutants (UAA302 and UHW303) from which GHW301, GHW302 and GHW303 were derived, confirmed the success of our cross-mating based genome shuffling strategy. Whole-genome sequencing analysis allowed identification of potential gene targets for tolerance to inhibitors in lignocellulosic hydrolysates.

Genetic improvement of native xylose-fermenting yeasts for ethanol production.

Lignocellulosic substrates are the largest source of fermentable sugars for bioconversion to fuel ethanol and other valuable compounds. To improve the economics of biomass conversion, it is essential that all sugars in potential hydrolysates be converted efficiently into the desired product(s). While hexoses are fermented into ethanol and some high-value chemicals, the bioconversion of pentoses in hydrolysates remains inefficient. This remains one of the key challenges in lignocellulosic biomass conversion. Native pentose-fermenting yeasts can ferment both glucose and xylose in lignocellulosic biomass to ethanol. However, they perform poorly in the presence of hydrolysate inhibitors, exhibit low ethanol tolerance and glucose repression, and ferment pentoses less efficiently than the main hexoses glucose and mannose. This paper reviews classical and molecular strain improvement strategies applied to native pentose-fermenting yeasts for improved ethanol production from xylose and lignocellulosic substrates. We focus on Pachysolen tannophilus, Scheffersomyces (Candida) shehatae, Scheffersomyces (Pichia) stipitis, and Spathaspora passalidarum which are good ethanol producers among the native xylose-fermenting yeasts. Strains obtained thus far are not robust enough for efficient ethanol production from lignocellulosic hydrolysates and can benefit from further improvements.

Monitoring Bacteroides spp. markers, nutrients, metals and Escherichia coli in soil and leachate after land application of three types of municipal biosolids.

A lysimeter-based field study was done to monitor the transfer of culturable Escherichia coli, general (ALLBAC), human (Hf183) and swine (PIG-BAC-1) specific 16S rRNA Bacteroides spp. markers, nutrients and metals through soils and leachate over time following land application of a CP1/Class A as well as two CP2/Class B municipal biosolids (MBs). Hf183 markers were detected up to six days following application in soils receiving dewatered and liquid MBs, but not in leachate, suggesting their use in source tracking is better suited for recent pollution events. The CP2/Class B biosolids and swine manure contributed the highest microbial load with E. coli loads (between 2.5 and 3.7 log CFU (100 mL)(-1)) being greater than North American concentration recommendations for safe recreational water. ALLBAC persisted in soils and leachate receiving all treatments and was detected prior to amendment application demonstrating its unsuitability for identifying the presence of fecal pollution. A significant increase in NO3-N (for Lystek and dewatered MBs) and total-P (for dewatered and liquid MBs) in leachate was observed in plots receiving the CP1/Class A and CP2/Class B type MBs which exceeded North American guidelines, suggesting impact to surface water. Metal (As, Cd, Cr, Co, Cu, Pb, Mo, Ni, Se, Zn and Hg) transfer was negligible in soil and leachate samples receiving all treatments. This study is one of the first to examine the fate of E. coli and Bacteroides spp. markers in situ following the land application of MBs where surface runoff does not apply.

Genome Sequence of Pseudomonas mandelii PD30.

The genome sequence of Pseudomonas mandelii PD30 is reported in this announcement. The genes for the reduction of nitrate to dinitrogen were identified in the genome assembly and subsequently used in gene expression research.

Synergy of silver nanoparticles and aztreonam against Pseudomonas aeruginosa PAO1 biofilms.

Pathogenic bacterial biofilms, such as those found in the lungs of patients with cystic fibrosis (CF), exhibit increased antimicrobial resistance, due in part to the inherent architecture of the biofilm community. The protection provided by the biofilm limits antimicrobial dispersion and penetration and reduces the efficacy of antibiotics that normally inhibit planktonic cell growth. Thus, alternative antimicrobial strategies are required to combat persistent infections. The antimicrobial properties of silver have been known for decades, but silver and silver-containing compounds have recently seen renewed interest as antimicrobial agents for treating bacterial infections. The goal of this study was to assess the efficacy of citrate-capped silver nanoparticles (AgNPs) of various sizes, alone and in combination with the monobactam antibiotic aztreonam, to inhibit Pseudomonas aeruginosa PAO1 biofilms. Among the different sizes of AgNPs examined, 10-nm nanoparticles were most effective in inhibiting the recovery of P. aeruginosa biofilm cultures and showed synergy of inhibition when combined with sub-MIC levels of aztreonam. Visualization of biofilms treated with combinations of 10-nm AgNPs and aztreonam indicated that the synergistic bactericidal effects are likely caused by better penetration of the small AgNPs into the biofilm matrix, which enhances the deleterious effects of aztreonam against the cell envelope of P. aeruginosa within the biofilms. These data suggest that small AgNPs synergistically enhance the antimicrobial effects of aztreonam against P. aeruginosa in vitro, and they reveal a potential role for combinations of small AgNPs and antibiotics in treating patients with chronic infections.

A temporal examination of the planktonic and biofilm proteome of whole cell Pseudomonas aeruginosa PAO1 using quantitative mass spectrometry.

Chronic polymicrobial lung infections are the chief complication in patients with cystic fibrosis. The dominant pathogen in late-stage disease is Pseudomonas aeruginosa, which forms recalcitrant, structured communities known as biofilms. Many aspects of biofilm biology are poorly understood; consequently, effective treatment of these infections is limited, and cystic fibrosis remains fatal. Here we combined in-solution protein digestion of triplicate growth-matched samples with a high-performance mass spectrometry platform to provide the most comprehensive proteomic dataset known to date for whole cell P. aeruginosa PAO1 grown in biofilm cultures. Our analysis included protein-protein interaction networks and PseudoCAP functional information for unique and significantly modulated proteins at three different time points. Secondary analysis of a subgroup of proteins using extracted ion currents validated the spectral counting data of 1884 high-confidence proteins. In this paper we demonstrate a greater representation of proteins related to metabolism, DNA stability, and molecular activity in planktonically grown P. aeruginosa PAO1. In addition, several virulence-related proteins were increased during planktonic growth, including multiple proteins encoded by the pyoverdine locus, uncharacterized proteins with sequence similarity to mammalian cell entry protein, and a member of the hemagglutinin family of adhesins, HecA. Conversely, biofilm samples contained an uncharacterized protein with sequence similarity to an adhesion protein with self-association characteristics (AidA). Increased levels of several phenazine biosynthetic proteins, an uncharacterized protein with sequence similarity to a metallo-beta-lactamase, and lower levels of the drug target gyrA support the putative characteristics of in situ P. aeruginosa infections, including competitive fitness and antibiotic resistance. This quantitative whole cell approach advances the existing P. aeruginosa subproteomes and provides a framework for identifying and studying entire pathways critical to biofilm biology in this model pathogenic organism. The identification of novel protein targets could contribute to the development of much needed antimicrobial therapies to treat the chronic infections found in patients with cystic fibrosis.

Characterization of sources and loadings of fecal pollutants using microbial source tracking assays in urban and rural areas of the Grand River Watershed, Southwestern Ontario.

Sources of fecal water pollution were assessed in the Grand River and two of its tributaries (Ontario, Canada) using total and host-specific (human and bovine) Bacteroidales genetic markers in conjunction with reference information, such as land use and weather. In-stream levels of the markers and culturable Escherichia coli were also monitored during multiple rain events to gain information on fecal loadings to catchment from diffuse sources. Elevated human-specific marker levels were accurately identified in river water impacted by a municipal wastewater treatment plant (WWTP) effluent and at a downstream site in the Grand River. In contrast, the bovine-specific marker showed high levels of cattle fecal pollution in two tributaries, both of which are characterized as intensely farmed areas. The bovine-specific Bacteroidales marker increased with rainfall in the agricultural tributaries, indicating enhanced loading of cattle-derived fecal pollutants to river from non-point sources following rain events. However, rain-triggered fecal loading was not substantiated in urban settings, indicating continuous inputs of human-originated fecal pollutants from point sources, such as WWTP effluent. This study demonstrated that the Bacteroidales source tracking assays, in combination with land use information and hydrological data, may provide additional insight into the spatial and temporal distribution of source-specific fecal contamination in streams impacted by varying land uses. Using the approach described in this study may help to characterize impacted water sources and to design targeted land use management plans in other watersheds in the future.

Mutants of the pentose-fermenting yeast Pachysolen tannophilus tolerant to hardwood spent sulfite liquor and acetic acid.

A strain development program was initiated to improve the tolerance of the pentose-fermenting yeast Pachysolen tannophilus to inhibitors in lignocellulosic hydrolysates. Several rounds of UV mutagenesis followed by screening were used to select for mutants of P. tannophilus NRRL Y2460 with improved tolerance to hardwood spent sulfite liquor (HW SSL) and acetic acid in separate selection lines. The wild type (WT) strain grew in 50 % (v/v) HW SSL while third round HW SSL mutants (designated UHW301, UHW302 and UHW303) grew in 60 % (v/v) HW SSL, with two of these isolates (UHW302 and UHW303) being viable and growing, respectively, in 70 % (v/v) HW SSL. In defined liquid media containing acetic acid, the WT strain grew in 0.70 % (w/v) acetic acid, while third round acetic acid mutants (designated UAA301, UAA302 and UAA303) grew in 0.80 % (w/v) acetic acid, with one isolate (UAA302) growing in 0.90 % (w/v) acetic acid. Cross-tolerance of HW SSL-tolerant mutants to acetic acid and vice versa was observed with UHW303 able to grow in 0.90 % (w/v) acetic acid and UAA302 growing in 60 % (v/v) HW SSL. The UV-induced mutants retained the ability to ferment glucose and xylose to ethanol in defined media. These mutants of P. tannophilus are of considerable interest for bioconversion of the sugars in lignocellulosic hydrolysates to ethanol.