Composting reduces the risks of resistome in beef cattle manure at the transcriptional level.

Transcriptomic evidence is needed to determine whether composting is more effective than conventional stockpiling in mitigating the risk of resistome in livestock manure. The objective of this study is to compare composting and stockpiling for their effectiveness in reducing the risk of antibiotic resistance in beef cattle manure. Samples collected from the center and the surface of full-size manure stockpiling and composting piles were subject to metagenomic and metatranscriptomic analyses. While the distinctions in resistome between stockpiled and composted manure were not evident at the DNA level, the advantages of composting over stockpiling were evident at the transcriptomic level in terms of the abundance of antibiotic resistance genes (ARGs), the number of ARG subtypes, and the prevalence of high-risk ARGs (i.e., mobile ARGs associated with zoonotic pathogens). DNA and transcript contigs show that the pathogen hosts of high-risk ARGs included Escherichia coli O157:H7 and O25b:H4, Klebsiella pneumoniae, and Salmonella enterica. Although the average daily temperatures for the entire composting pile exceeded 55°C throughout the field study, more ARG and ARG transcripts were removed at the center of the composting pile than at the surface. This work demonstrates the advantage of composting over stockpiling in reducing ARG risk in active populations in beef cattle manure.IMPORTANCEProper treatment of manure before land application is essential to mitigate the spread of antibiotic resistance in the environment. Stockpiling and composting are two commonly used methods for manure treatment. However, the effectiveness of composting in reducing antibiotic resistance in manure has been debated. This work compared the ability of these two methods to reduce the risk of antibiotic resistance in beef cattle manure. Our results demonstrate that composting reduced more high-risk resistance genes at the transcriptomic level in cattle manure than conventional stockpiling. This finding not only underscores the effectiveness of composting in reducing antibiotic resistance in manure but also highlights the importance of employing RNA analyses alongside DNA analyses.

Effects of Nutrient Level and Growth Rate on the Conjugation Process That Transfers Mobile Antibiotic Resistance Genes in Continuous Cultures.

Bacteria in the effluent of wastewater treatment plants (WWTPs) can transfer antibiotic resistance genes (ARGs) to the bacteria in receiving water through conjugation; however, there is a lack of quantitative assessment of this phenomenon in continuous cultures. Our objective was to determine the effects of background nutrient levels in river water column and growth rates of bacteria on the conjugation frequency of ARGs from effluent bacteria to river bacteria, as well as on the resulting resistance level (i.e., MICs) of the river bacteria. Chemostats were employed to simulate the discharge points of WWTPs into rivers, where effluent bacteria (donor cells) meet river bacteria (recipient cells). Both donor and recipient cells were Escherichia coli cells, and the donor cells were constructed by filter mating with bacteria in the effluent of a local WWTP. Results showed that higher bacterial growth rate (0.45 h-1 versus 0.15 h-1) led to higher conjugation frequencies (10-4 versus 10-6 transconjugant per recipient). The nutrient level also significantly affected the conjugation frequency, albeit to a lesser extent than the growth rate. The MIC against tetracycline increased from 2 mg/L in the recipient to 64 to 128 mg/L in transconjugants. In comparison, the MIC only increased to as high as 8 mg/L in mutants. Whole-genome sequencing showed that the tet-containing plasmid in both the donor and the transconjugant cells also occur in other fecal bacterial genera. The quantitative information obtained from this study can inform hazard identification related to the proliferation of wastewater-associated ARGs in surface water. IMPORTANCE WWTPs have been regarded as an important hot spot of ARGs. The discharge point of WWTP effluent, where ARGs may be horizontally transferred from bacteria of treated wastewater to bacteria of receiving water, is an important interface between the human-dominated ecosystem and the natural environment. The use of batch cultures in previous studies cannot adequately simulate the nutrient conditions and growth rates in receiving water. In this study, chemostats were employed to simulate the continuous growth of bacteria in receiving water. Furthermore, the experimental setup allowed for separate investigations on the effects of nutrient levels (i.e., simulating background nutrients in river water) and bacterial growth rates on conjugation frequencies and resulting resistance levels. The study generates statistically sound ecological data that can be used to estimate the risk of wastewater-originated ARGs as part of the One Health framework.

Stockpiling versus Composting: Effectiveness in Reducing Antibiotic-Resistant Bacteria and Resistance Genes in Beef Cattle Manure.

Manure storage methods can affect the concentration and prevalence of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in cattle manure prior to land application. The objective of this study was to compare stockpiling and composting with respect to their effectiveness in reducing ARB and ARGs in beef cattle manure in a field-scale study. Field experiments were conducted in different seasons with different bulking agents for composting. For both the winter-spring cycle and the summer-fall cycle, ARB concentrations declined below the limit of quantification rapidly in both composting piles and stockpiles; however, ARB prevalence was significantly greater in the composting piles than in the stockpiles. This was likely due to the introduction of ARB from bulking agents. There was no significant change in ARG concentrations between initial and final concentrations for either manure storage treatment during the winter-spring cycle, but a significant reduction of the ARGs erm(B), tet(O), and tet(Q) over time was observed for both the composting pile and stockpile during the summer-fall cycle. Results from this study suggest that (i) bulking agent may be an important source of ARB and ARGs for composting; (ii) during cold months, the heterogeneity of the temperature profile in composting piles could result in poor ARG reduction; and (iii) during warm months, both stockpiling and composting can be effective in reducing ARG abundance. IMPORTANCE Proper treatment of manure is essential to reduce the spread of antibiotic resistance and protect human health. Stockpiling and composting are two manure storage methods which can reduce antibiotic-resistant bacteria and resistance genes, although few field-scale studies have examined the relative efficiency of each method. This study examined the ability of both methods in both winter-spring and summer-fall cycles, while also accounting for heterogeneity within field-scale manure piles. This study determined that bulking agents used in composting could contribute antibiotic-resistant bacteria and resistance genes. Additionally, seasonal variation could hinder the efficacy of composting in colder months due to heterogeneity in temperature within the pile; however, in warmer months, either method of manure storage could be effective in reducing the spread of antibiotic resistance.

Genomes of ubiquitous marine and hypersaline Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira spp. encode a diversity of mechanisms to sustain chemolithoautotrophy in heterogeneous environments.

Chemolithoautotrophic bacteria from the genera Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira are common, sometimes dominant, isolates from sulfidic habitats including hydrothermal vents, soda and salt lakes and marine sediments. Their genome sequences confirm their membership in a deeply branching clade of the Gammaproteobacteria. Several adaptations to heterogeneous habitats are apparent. Their genomes include large numbers of genes for sensing and responding to their environment (EAL- and GGDEF-domain proteins and methyl-accepting chemotaxis proteins) despite their small sizes (2.1-3.1 Mbp). An array of sulfur-oxidizing complexes are encoded, likely to facilitate these organisms' use of multiple forms of reduced sulfur as electron donors. Hydrogenase genes are present in some taxa, including group 1d and 2b hydrogenases in Hydrogenovibrio marinus and H. thermophilus MA2-6, acquired via horizontal gene transfer. In addition to high-affinity cbb3 cytochrome c oxidase, some also encode cytochrome bd-type quinol oxidase or ba3 -type cytochrome c oxidase, which could facilitate growth under different oxygen tensions, or maintain redox balance. Carboxysome operons are present in most, with genes downstream encoding transporters from four evolutionarily distinct families, which may act with the carboxysomes to form CO2 concentrating mechanisms. These adaptations to habitat variability likely contribute to the cosmopolitan distribution of these organisms.

Microbial Source Tracking Using Quantitative and Digital PCR To Identify Sources of Fecal Contamination in Stormwater, River Water, and Beach Water in a Great Lakes Area of Concern.

Areas of concern (AOCs) around the Great Lakes are characterized by historic and ongoing problems with microbial water quality, leading to beneficial use impairments (BUIs) such as beach postings and closures. In this study, we assessed river and beach sites within the Rouge River watershed, associated stormwater outfalls, and at Rouge Beach. The concentrations of Escherichia coli as well as human- and gull-specific qPCR microbial source tracking (MST) markers were assessed at all sites. A preliminary comparison of digital PCR (dPCR) methodologies for both MST markers was conducted regarding sensitivity and specificity. Within the watershed, the outfalls were found to be a prominent source of human fecal contamination, with two outfalls particularly affected by sewage cross-connections. However, the occurrence of human fecal contamination along Rouge Beach and in the lower portions of the watershed was largely dependent on rain events. Gull fecal contamination was the predominant source of contamination at the beach, particularly during dry weather. The multiplex human/gull dPCR methodology used in this study tended to be more sensitive than the individual quantitative PCR (qPCR) assays, with only a slight decrease in specificity. Both dPCR and qPCR methodologies identified the same predominance of human and gull markers in stormwater and beach locations, respectively; however, the dPCR multiplex assay was more sensitive and capable of detecting fecal contamination that was undetected by qPCR assays. These results demonstrate the dPCR assay used in this study could be a viable tool for MST studies to increase the ability to identify low levels of fecal contamination.IMPORTANCE Fecal contamination of recreational water poses a persistent and ongoing problem, particularly in areas of concern around the Great Lakes. The identification of the source(s) of fecal contamination is essential for safeguarding public health as well as guiding remediation efforts; however, fecal contamination may frequently be present at low levels and remain undetectable by certain methodologies. In this study, we utilized microbial source tracking techniques using both quantitative and digital PCR assays to identify sources of contamination. Our results indicated high levels of human fecal contamination within stormwater outfalls, while lower levels were observed throughout the watershed. Additionally, high levels of gull fecal contamination were detected at Rouge Beach, particularly during drier sampling events. Furthermore, our results indicated an increased sensitivity of the digital PCR assay to detect both human and gull contamination, suggesting it could be a viable tool for future microbial source tracking studies.

Comparison of Microbial and Chemical Source Tracking Markers To Identify Fecal Contamination Sources in the Humber River (Toronto, Ontario, Canada) and Associated Storm Water Outfalls.

Storm water runoff is a major source of pollution, and understanding the components of storm water discharge is essential to remediation efforts and proper assessment of risks to human and ecosystem health. In this study, culturable Escherichia coli and ampicillin-resistant E. coli levels were quantified and microbial source tracking (MST) markers (including markers for general Bacteroidales spp., human, ruminant/cow, gull, and dog) were detected in storm water outfalls and sites along the Humber River in Toronto, Ontario, Canada, and enumerated via endpoint PCR and quantitative PCR (qPCR). Additionally, chemical source tracking (CST) markers specific for human wastewater (caffeine, carbamazepine, codeine, cotinine, acetaminophen, and acesulfame) were quantified. Human and gull fecal sources were detected at all sites, although concentrations of the human fecal marker were higher, particularly in outfalls (mean outfall concentrations of 4.22 log10 copies, expressed as copy numbers [CN]/100 milliliters for human and 0.46 log10 CN/100 milliliters for gull). Higher concentrations of caffeine, acetaminophen, acesulfame, E. coli, and the human fecal marker were indicative of greater raw sewage contamination at several sites (maximum concentrations of 34,800 ng/liter, 5,120 ng/liter, 9,720 ng/liter, 5.26 log10 CFU/100 ml, and 7.65 log10 CN/100 ml, respectively). These results indicate pervasive sewage contamination at storm water outfalls and throughout the Humber River, with multiple lines of evidence identifying Black Creek and two storm water outfalls with prominent sewage cross-connection problems requiring remediation. Limited data are available on specific sources of pollution in storm water, though our results indicate the value of using both MST and CST methodologies to more reliably assess sewage contamination in impacted watersheds. IMPORTANCE: Storm water runoff is one of the most prominent non-point sources of biological and chemical contaminants which can potentially degrade water quality and pose risks to human and ecosystem health. Therefore, identifying fecal contamination in storm water runoff and outfalls is essential for remediation efforts to reduce risks to public health. This study employed multiple methods of identifying levels and sources of fecal contamination in both river and storm water outfall sites, evaluating the efficacy of using culture-based enumeration of E. coli, molecular methods of determining the source(s) of contamination, and CST markers as indicators of fecal contamination. The results identified pervasive human sewage contamination in storm water outfalls and throughout an urban watershed and highlight the utility of using both MST and CST to identify raw sewage contamination.

Comparative microbial source tracking methods for identification of fecal contamination sources at Sunnyside Beach in the Toronto region area of concern.

Several beaches within the Toronto region area of concern have persistent issues with fecal contamination, causing a beach beneficial use impairment (BUI). In this study, Escherichia coli, including ampicillin-resistant strains, were enumerated via culturable and quantitative polymerase chain reaction (qPCR) methods. Microbial source tracking (MST) markers (for general Bacteroidales, human, ruminant/cow, gull, and dog) were detected and enumerated via PCR and qPCR to identify sources of fecal contamination at Sunnyside Beach and in the Humber River. Human, cow, and dog markers had good host-specificity, while gull markers sometimes amplified a few other bird species. The ruminant endpoint PCR marker amplified a variety of other animal species rendering it less useful. Both human and gull fecal contamination were prevalent in the Humber River, while Sunnyside Beach was predominantly impacted by gull fecal contamination. Human sewage impacts were more prevalent in the lower Humber River, particularly in Black Creek. However, to reduce Sunnyside beach postings, reducing bird fecal contamination in the river and at the beach would be necessary. When there are high levels of E. coli throughout a beachshed, an MST toolbox approach can add value to discriminate source(s) of E. coli contamination and guide decisions relating to public health risk and remediation strategies.

A synthesis of the effects of pesticides on microbial persistence in aquatic ecosystems.

Pesticides have a pervasive presence in aquatic ecosystems throughout the world. While pesticides are intended to control fungi, insects, and other pests, their mechanisms of action are often not specific enough to prevent unintended effects, such as on non-target microbial populations. Microorganisms, including algae and cyanobacteria, protozoa, aquatic fungi, and bacteria, form the basis of many food webs and are responsible for crucial aspects of biogeochemical cycling; therefore, the potential for pesticides to alter microbial community structures must be understood to preserve ecosystem services. This review examines studies that focused on direct population-level effects and indirect community-level effects of pesticides on microorganisms. Generally, insecticides, herbicides, and fungicides were found to have adverse direct effects on algal and fungal species. Insecticides and fungicides also had deleterious direct effects in the majority of studies examining protozoa species, although herbicides were found to have inconsistent direct effects on protozoans. Our synthesis revealed mixed or no direct effects on bacterial species among all pesticide categories, with results highly dependent on the target species, chemical, and concentration used in the study. Examination of community-level, indirect effects revealed that all pesticide categories had a tendency to reduce higher trophic levels, thereby diminishing top-down pressures and favoring lower trophic levels. Often, indirect effects exerted greater influence than direct effects. However, few studies have been conducted to specifically address community-level effects of pesticides on microorganisms, and further research is necessary to better understand and predict the net effects of pesticides on ecosystem health.

Agrochemicals indirectly increase survival of E. coli O157:H7 and indicator bacteria by reducing ecosystem services.

Storm water and agricultural runoff frequently contain agrochemicals, fecal indicator bacteria (FIB), and zoonotic pathogens. Entry of such contaminants into aquatic ecosystems may affect ecology and human health. This study tested the hypothesis that the herbicide atrazine and the fungicide chlorothalonil indirectly affect the survival of FIB (Escherichia coli and Enterococcus faecalis) and a pathogen (E. coli O157:H7) by altering densities of protozoan predators or by altering competition from autochthonous bacteria. Streptomycin-resistant E. coli, En. faecalis, and E. coli O157:H7 were added to microcosms composed of Florida river water containing natural protozoan and bacterial populations. FIB, pathogen, and protozoan densities were monitored over six days. Known metabolic inhibitors, cycloheximide and streptomycin, were used to inhibit autochthonous protozoa or bacteria, respectively. The inhibitors made it possible to isolate the effects of predation or competition on survival of allochthonous bacteria, and each treatment increased the survival of FIB and pathogens. Chlorothalonil's effect was similar to that of cycloheximide, significantly reducing protozoan densities and elevating densities of FIB and pathogens relative to the control. Atrazine treatment did not affect protozoan densities, but, through an effect on competition, resulted in significantly greater densities of En. faecalis and E. coli O157:H7. Hence, by reducing predaceous protozoa and bacterial competitors that facilitate purifying water bodies of FIBs and human pathogens, chlorothalonil and atrazine indirectly diminished an ecosystem service of fresh water.

Lack of direct effects of agrochemicals on zoonotic pathogens and fecal indicator bacteria.

Agrochemicals, fecal indicator bacteria (FIB), and pathogens frequently contaminate water simultaneously. No significant direct effects of fertilizer, atrazine, malathion, and chlorothalonil on the survival of Escherichia coli, Enterococcus faecalis, Salmonella enterica, human polyomaviruses, and adenovirus were detected, supporting the assertion that previously observed effects of agrochemicals on FIB were indirect.

The Human Health Implications of Antibiotic Resistance in Environmental Isolates from Two Nebraska Watersheds.

One Health field-based approaches are needed to connect the occurrence of antibiotics present in the environment with the presence of antibiotic resistance genes (ARGs) in Gram-negative bacteria that confer resistance to antibiotics important in for both veterinary and human health. Water samples from two Nebraska watersheds influenced by wastewater effluent and agricultural runoff were tested for the presence of antibiotics used in veterinary and human medicine. The water samples were also cultured to identify the bacteria present. Of those bacteria isolated, the Gram-negative rods capable of causing human infections had antimicrobial susceptibility testing and whole-genome sequencing (WGS) performed to identify ARGs present. Of the 211 bacterial isolates identified, 37 belonged to pathogenic genera known to cause human infections. Genes conferring resistance to beta-lactams, aminoglycosides, fosfomycins, and quinolones were the most frequently detected ARGs associated with horizontal gene transfer (HGT) in the watersheds. WGS also suggest recent HGT events involving ARGs transferred between watershed isolates and bacteria of human and animal origins. The results of this study demonstrate the linkage of antibiotics and bacterial ARGs present in the environment with potential human and/or veterinary health impacts. IMPORTANCE One health is a transdisciplinary approach to achieve optimal health for humans, animals, plants and their shared environment, recognizing the interconnected nature of health in these domains. Field based research is needed to connect the occurrence of antibiotics used in veterinary medicine and human health with the presence of antibiotic resistance genes (ARGs). In this study, the presence of antibiotics, bacteria and ARGs was determined in two watersheds in Nebraska, one with agricultural inputs and the other with both agricultural and wastewater inputs. The results presented in this study provide evidence of transfer of highly mobile ARG between environment, clinical, and animal-associated bacteria.

Test of direct and indirect effects of agrochemicals on the survival of fecal indicator bacteria.

Water bodies often receive agrochemicals and animal waste carrying fecal indicator bacteria (FIB) and zoonotic pathogens, but we know little about the effects of agrochemicals on these microbes. We assessed the direct effects of the pesticides atrazine, malathion, and chlorothalonil and inorganic fertilizer on Escherichia coli and enterococcal survival in simplified microcosms held in the dark. E. coli strain composition in sediments and water column were positively correlated, but none of the agrochemicals had significant direct effects on E. coli strain composition or on densities of culturable FIBs. In a companion study, microcosms with nondisinfected pond water and sediments were exposed to or shielded from sunlight to examine the potential indirect effects of atrazine and inorganic fertilizer on E. coli. The herbicide atrazine had no effect on E. coli in dark-exposed microcosms containing natural microbial and algal communities. However, in light-exposed microcosms, atrazine significantly lowered E. coli densities in the water column and significantly increased densities in the sediment compared to controls. This effect appears to be mediated by the effects of atrazine on algae, given that atrazine significantly reduced phytoplankton, which was a positive and negative predictor of E. coli densities in the water column and sediment, respectively. These data suggest that atrazine does not directly affect the survival of FIB, rather that it indirectly alters the distribution and abundance of E. coli by altering phytoplankton and periphyton communities. These results improve our understanding of the influence of agricultural practices on FIB densities in water bodies impacted by agricultural runoff.

Using the heat generated from electrically conductive concrete slabs to reduce antibiotic resistance in beef cattle manure.

Proper treatment is necessary to reduce antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in livestock manure before land application. Conventional stockpiling suffers unreliable removal efficiency, while composting can be complicated and expensive. The objective of this study was to test the feasibility of a novel heat-based technology, i.e., stockpiling manure on conductive concrete slabs, to inactivate ARB and ARGs in beef cattle manure. In this study, two independent bench-scale trials were conducted. In both trials, samples were taken from manure piles on conductive concrete slabs and regular slabs (i.e., heated and unheated piles). In the heated pile of the first trial, 25.9% and 83.5% of the pile volume met the EPA Class A and Class B biosolids standards, respectively. For the heated pile of the second trial, the two values were 43.9% and 74.2%. In both trials, nearly all forms of the total and resistant Escherichia coli and enterococci were significantly lower in the heated piles than in the unheated piles. Besides, significant reduction of ARGs in heated piles was observed in the first trial. Through this proof-of-concept study, the new technology based on conductive concrete slabs offers an alternative manure storage method to conventional stockpiling and composting with respect to reduce ARB and ARGs in manure.

The effect of agrochemicals on indicator bacteria densities in outdoor mesocosms.

Water bodies, which are monitored for microbial water quality by quantification of faecal indicator organisms (IOs), can contain various zoonotic pathogens contributed by livestock waste and other sources. Sediments can serve as reservoirs of IOs and other enteric microorganisms, including pathogens. Agrochemicals may influence the survival of these microorganisms in water bodies impacted by livestock waste by enhancing or reducing their survival. Complex, 1100 l, freshwater mesocosms containing leaf litter, zooplankton, periphyton, phytoplankton, and invertebrate and vertebrate animals were used to investigate the response of Escherichia coli and enterococci to agrochemicals. Replicate tanks were treated with atrazine, malathion, chlorothalonil and inorganic fertilizer, either alone at 1× or 2× their expected environmental concentrations (EECs) or in pair-wise combinations at their EECs. IOs inoculated in sediment (∼104 cfu per 100 ml) were enumerated over 28 days. IOs generally declined over time, but manova revealed that addition of fertilizer and atrazine resulted in significantly greater IO densities. Malathion, chlorothalonil and agrochemical concentration (1× vs 2×) did not significantly affect IO densities and no significant interactions between agrochemicals were noted. The augmentation of IO densities in sediments by fertilizer and atrazine may impact their reliability as accurate predictors of water quality and human health risk, and indicates the need for a better understanding of the fate of IOs and enteric pathogens in sediments exposed to agrochemicals.

Antibiotic-Resistant Enterococcus Species in Marine Habitats: A Review.

Antibiotic-resistant Enterococcus (ARE) are among leading causes of nosocomial infections worldwide. Enterococcus spp. are ubiquitous in sewage, which can contaminate surface waters via many pathways, providing a route of exposure for humans. This review focuses on ARE in marine and estuarine habitats, including marine animals. Phylogenetic confirmation of the genus Enterococcus and intermediate or full resistance to clinically relevant antibiotics were inclusion criteria. The proportion of resistant isolates varied greatly among antibiotics, for example, 24.2% for ampicillin and 2.4% for vancomycin. The water column contained the highest proportion of ARE observations (18.8%), followed by animal feces and tissues (14.8%), sediment (9.4%), and sand (2.0%). The proportion of multidrug-resistant isolates was the greatest in animal tissue and fecal samples, followed by water and sediments. This review indicates that clinically relevant ARE are present in marine/estuarine habitats and that animals may be important reservoirs.

Corn stalk residue may add antibiotic-resistant bacteria to manure composting piles.

Manure is commonly used as a fertilizer or soil conditioner; however, land application of untreated manure may introduce pathogens and antibiotic-resistant bacteria (ARB) into the soil, with harmful implications for public health. Composting is a manure management practice wherein a carbon-rich bulking agent, such as corn (Zea mays L.) stalk residue, is added to manure to achieve desirable carbon/nitrogen ratios to facilitate microbial activities and generate enough heat to inactivate pathogens, including antibiotic-resistant pathogens. However, when comparing compost piles and stockpiles for ARB reduction, we noticed that bulking agents added ARB to composting piles and compromised the performance of composting in reducing ARB. We hypothesized that ARB could be prevalent in corn stalk residues, a commonly used bulking agent for composting. To test this hypothesis, corn stalk residue samples throughout Nebraska were surveyed for the presence of ARB. Of the samples tested, 54% were positive for antibiotic-resistant Escherichia coli or enterococci using direct plating or after enrichment. Although not statistically significant, there was a trend wherein the use of pesticides tended to result in a greater prevalence of some ARB. Results from this study suggest that bulking agents can be a source of ARB in manure composting piles and highlight the importance of screening bulking agents for effective ARB reduction in livestock manure during composting.

Impacts of a changing earth on microbial dynamics and human health risks in the continuum between beach water and sand.

Although infectious disease risk from recreational exposure to waterborne pathogens has been an active area of research for decades, beach sand is a relatively unexplored habitat for the persistence of pathogens and fecal indicator bacteria (FIB). Beach sand, biofilms, and water all present unique advantages and challenges to pathogen introduction, growth, and persistence. These dynamics are further complicated by continuous exchange between sand and water habitats. Models of FIB and pathogen fate and transport at beaches can help predict the risk of infectious disease from beach use, but knowledge gaps with respect to decay and growth rates of pathogens in beach habitats impede robust modeling. Climatic variability adds further complexity to predictive modeling because extreme weather events, warming water, and sea level change may increase human exposure to waterborne pathogens and alter relationships between FIB and pathogens. In addition, population growth and urbanization will exacerbate contamination events and increase the potential for human exposure. The cumulative effects of anthropogenic changes will alter microbial population dynamics in beach habitats and the assumptions and relationships used in quantitative microbial risk assessment (QMRA) and process-based models. Here, we review our current understanding of microbial populations and transport dynamics across the sand-water continuum at beaches, how these dynamics can be modeled, and how global change factors (e.g., climate and land use) should be integrated into more accurate beachscape-based models.

Fecal source tracking and eDNA profiling in an urban creek following an extreme rain event.

Fecal contamination of recreational waters (i.e. lakes, rivers, beaches) poses an on-going problem for environmental and public health. Heavy rainfall can exacerbate existing problems with fecal contamination. As there could be variable sources of fecal contamination, identifying the source is critical for remediation efforts. This study utilized microbial source tracking (MST), chemical source tracking (CST) markers and environmental DNA (eDNA) metabarcoding to profile sampling areas and identify sources of fecal contamination in creek, stormwater outfall and beach sites in the Etobicoke Creek watershed (Toronto, ON). Water samples were collected before and immediately following an extreme rain event. MST and CST identified stormwater outfalls as an important source of human fecal contamination during dry and wet conditions. eDNA metabarcoding allowed for potential identification of additional sources of fecal contamination and provided additional evidence of human fecal contamination. The extreme rainfall event altered the eDNA profiles, causing creek and beach sites to reflect a greater diversity of mammal and bird eDNA sequences. The profiles provided by eDNA metabarcoding provide a proof of concept suggesting that eDNA metabarcoding can be a useful tool to complement MST and CST methods for profiling sources of fecal contamination and studying impacts of extreme rain events.

Comparison of the occurrence and survival of fecal indicator bacteria in recreational sand between urban beach, playground and sandbox settings in Toronto, Ontario.

While beach sands are increasingly being studied as a reservoir of fecal indicator bacteria (FIB), less is known about the occurrence of FIB in other recreational sands (i.e., sandboxes and playgrounds). In this study, different culture-based FIB enumeration techniques were compared and microbial source tracking assays were conducted on recreational sand samples from beaches, playgrounds and sandboxes around Toronto, ON. FIB were detected in every sand sample (n=104) with concentrations not changing significantly over the five month sampling period. Concentrations of FIB and a gull-specific DNA marker were significantly higher in foreshore beach sands, and indicated these were a more significant reservoir of FIB contamination than sandbox or playground sands. Human- and dog-specific contamination markers were not detected. All culture-based FIB enumeration techniques were consistent in identifying the elevated FIB concentrations associated with foreshore beach sands. However, significant differences between differential agar media, IDEXX and Aquagenx Compartment Bag Test were observed, with DC media and Enterolert being the most sensitive methods to detect Escherichia coli and enterococci, respectively. To better understand the elevated occurrence of E. coli in foreshore sands, microcosm survival experiments were conducted at two different temperatures (15 °C and 28 °C) using non-sterile saturated foreshore beach sands collected from two urban freshwater beaches with different sand type (fine grain and sand-cobble). Microcosms were inoculated with a mixture of eight sand-derived E. coli strains and sampled over a 28-day period. E. coli levels were found to decline in all microcosms, although survival was significantly greater in the finer sand and at the cooler temperature (15 °C). These results indicate that FIB can be widespread in any type of recreational sand and, while E. coli can survive for many weeks, it is most likely to accumulate in cooler fine-grain sand as occurs below the foreshore sand surface.

Enterococci in the environment.

Enterococci are common, commensal members of gut communities in mammals and birds, yet they are also opportunistic pathogens that cause millions of human and animal infections annually. Because they are shed in human and animal feces, are readily culturable, and predict human health risks from exposure to polluted recreational waters, they are used as surrogates for waterborne pathogens and as fecal indicator bacteria (FIB) in research and in water quality testing throughout the world. Evidence from several decades of research demonstrates, however, that enterococci may be present in high densities in the absence of obvious fecal sources and that environmental reservoirs of these FIB are important sources and sinks, with the potential to impact water quality. This review focuses on the distribution and microbial ecology of enterococci in environmental (secondary) habitats, including the effect of environmental stressors; an outline of their known and apparent sources, sinks, and fluxes; and an overview of the use of enterococci as FIB. Finally, the significance of emerging methodologies, such as microbial source tracking (MST) and empirical predictive models, as tools in water quality monitoring is addressed. The mounting evidence for widespread extraenteric sources and reservoirs of enterococci demonstrates the versatility of the genus Enterococcus and argues for the necessity of a better understanding of their ecology in natural environments, as well as their roles as opportunistic pathogens and indicators of human pathogens.