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Antimicrobial resistance (AR) is a serious global problem due to the overuse of antimicrobials in human, animal, and agriculture sectors. There is intense research to control the dissemination of AR, but little is known regarding the environmental drivers influencing its spread. Although AR genes (ARGs) are detected in many different environments, the risk associated with the spread of these genes to microbial pathogens is unknown. Recreational microbial exposure risks are likely to be greater in water bodies receiving discharge from human and animal waste in comparison to less disturbed aquatic environments. Given this scenario, research practitioners are encouraged to consider an ecological context to assess the effect of environmental ARGs on public health. Here, we use a stratified, probabilistic survey of nearly 2000 sites to determine national patterns of the anthropogenic indicator class I integron Integrase gene (

Wastewater-based SARS-CoV-2 monitoring is an effective tool for rapid management of the pandemic. Evaluating methodologies during the height of this crisis strengthens the field to ensure reproducibility across states, nations, and the globe.

Correction for ‘Evaluation of intra- and inter-lab variability in quantifying SARS-CoV-2 in a state-wide wastewater monitoring network’ by Angela Davis et al., Environ. Sci.: Water Res. Technol., 2023, https://doi.org/10.1039/D2EW00737A.

Facing challenges in water demands and population size, particularly in the water-scarce regions in the United States, the reuse of treated municipal wastewater has become a viable potential to relieve the ever-increasing demands of providing water for (non-)potable use. The objectives of this study were to assess microbial quality of reclaimed water and to investigate treatability of microorganisms during different treatment processes. Raw and final treated effluent samples from three participating utilities were collected monthly for 16 months and analyzed for various microbial pathogens and fecal indicator organisms. Results revealed that the detectable levels of microbial pathogens tested were observed in the treated effluent samples from all participating utilities. Log10 reduction values (LRVs) of Cryptosporidium oocysts and Giardia cysts were at least two orders of magnitude lower than those of human adenovirus and all fecal indicator organisms except for aerobic endospores, which showed the lowest LRVs. The relatively higher LRV of the indicator organisms such as bacteriophages suggested that these microorganisms are not good candidates of viral indicators of human adenovirus during wastewater treatment processes. Overall, this study will assist municipalities considering the use of wastewater effluent as another source of drinking water by providing important data on the prevalence, occurrence, and reduction of waterborne pathogens in wastewater. More importantly, the results from this study will aid in building a richer microbial occurrence database that can be used towards evaluating reuse guidelines and disinfection practices for water reuse practices.

Wastewater monitoring of SARS-CoV-2 presents a means of tracking COVID-19 community infection dynamics on a broader geographic scale. However, accounting for environmental and sample-processing losses may be necessary for wastewater measurements to readily inform our understanding of infection prevalence. Here, we present measurements of the SARS-CoV-2 N1 and N2 gene targets from weekly wastewater samples at three sites in Hamilton County, Ohio, during an increase and subsequent decline of COVID-19 infections. The concentration of N1 or N2 RNA in wastewater, measured over the course of six months, ranged from below the detection limit to over 104 gene copies/L, and correlated with case data at two wastewater treatment plants, but not at a sub-sewershed-level sampling site. We also evaluated the utility of a broader range of variables than has been reported consistently in previous work, in improving correlations of SARS-CoV-2 concentrations with case data. These include a spiked matrix recovery control (OC43), flow-normalization, and assessment of fecal loading using endogenous fecal markers (HF183, PMMoV, CrAssphage). We found that adjusting for recovery, flow, and fecal indicators increased these correlations for samples from a larger sewershed (serving ~488,000 people) with greater industrial and stormwater inputs, but raw N1/N2 concentrations corresponded better with case data at a smaller, residential-oriented sewershed. Our results indicate that the optimal adjustment factors for correlating wastewater and clinical case data moving forward may not be generalizable to all sewersheds., Graphical abstract Unlabelled Image

Greywater, the wastewater from sinks, showers, and laundry, is an understudied environment for bacterial communities. Most greywater studies focus on quantifying pathogens, often via proxies used in other wastewater, like fecal indicator bacteria; there is a need to identify more greywater-appropriate surrogates, like Staphylococcus sp. Sequencing-based studies have revealed distinct communities in different types of greywater as well as in different parts of greywater infrastructure, including biofilms on pipes, holding tanks, and filtration systems. The use of metagenomic sequencing provides high resolution on both the taxa and genes present, which may be of interest in cases like identifying pathogens and surrogates relevant to different matrices, monitoring antibiotic resistance genes, and understanding metabolic processes occurring in the system. Here we review what is known about bacterial communities in different types of greywater and its infrastructure. We suggest that wider adoption of environmental sequencing in greywater research is important because it can describe the entire bacterial community along with its metabolic capabilities, including pathways for removal of nutrients and organic materials. We briefly describe a metagenomic dataset comparing different types of greywater samples in a college dormitory building to highlight the type of questions these methods can address. Metagenomic sequencing can help further the understanding of greywater treatment for reuse because it allows for identification of new pathogens or genes of concern.

Microbiota regulate important physiologic processes during early host development. They also biotransform xenobiotics and serve as key intermediaries for chemical exposure. Antimicrobial agents in the environment may disrupt these complex interactions and alter key metabolic functions provided by host-associated microbiota. To examine the role of microbiota in xenobiotic metabolism, we exposed zebrafish larvae to the antimicrobial agent triclosan. Conventionally colonized (CC), microbe-free axenic (AX), or axenic colonized on day 1 (AC1) zebrafish were exposed to 0.16–0.30 µM triclosan or vehicle on days 1, 6, 7, 8, and 9 days post fertilization (dpf). After 6 and 10 dpf, host-associated microbial community structure and putative function were assessed by 16S rRNA gene sequencing. At 10 dpf, triclosan exposure selected for bacterial taxa, including Rheinheimera. Triclosan-selected microbes were predicted to be enriched in pathways related to mechanisms of antibiotic resistance, sulfonation, oxidative stress, and drug metabolism. Furthermore, at 10 dpf, colonized zebrafish contained 2.5–3 times more triclosan relative to AX larvae. Nontargeted chemical analysis revealed that, relative to AX larvae, both cohorts of colonized larvae showed elevations in 23 chemical features, including parent triclosan and putative triclosan sulfate. Taken together, these data suggest that triclosan exposure selects for microbes that harbor the capacity to biotransform triclosan into chemical metabolites with unknown toxicity profiles. More broadly, these data support the concept that microbiota modify the toxicokinetics of xenobiotic exposure.

Estrogenic chemicals are widespread environmental contaminants associated with diverse health and ecological effects. During early vertebrate development, estrogen receptor signaling is critical for many different physiologic responses, including nervous system function. Recently, host-associated microbiota have been shown to influence neurodevelopment. Here, we hypothesized that microbiota may biotransform exogenous 17-βestradiol (E2) and modify E2 effects on swimming behavior. Colonized zebrafish were continuously exposed to non-teratogenic E2 concentrations from 1 to 10 days post-fertilization (dpf). Changes in microbial composition and predicted metagenomic function were evaluated. Locomotor activity was assessed in colonized and axenic (microbe-free) zebrafish exposed to E2 using a standard light/dark behavioral assay. Zebrafish tissue was collected for chemistry analyses. While E2 exposure did not alter microbial composition or putative function, colonized E2-exposed larvae showed reduced locomotor activity in the light, in contrast to axenic E2-exposed larvae, which exhibited normal behavior. Measured E2 concentrations were significantly higher in axenic relative to colonized zebrafish. Integrated peak area for putative sulfonated and glucuronidated E2 metabolites showed a similar trend. These data demonstrate that E2 locomotor effects in the light phase are dependent on the presence of microbiota and suggest that microbiota influence chemical E2 toxicokinetics. More broadly, this work supports the concept that microbial colonization status may influence chemical toxicity.

Background Across taxa, animals with depleted intestinal microbiomes show disrupted behavioral phenotypes. Axenic (i.e., microbe-free) mice, zebrafish, and fruit flies exhibit increased locomotor behavior, or hyperactivity. The mechanism through which bacteria interact with host cells to trigger normal neurobehavioral development in larval zebrafish is not well understood. Here, we monoassociated zebrafish with either one of six different zebrafish-associated bacteria, mixtures of these host-associates, or with an environmental bacterial isolate. Results As predicted, the axenic cohort was hyperactive. Monoassociation with three different host-associated bacterial species, as well as with the mixtures, resulted in control-like locomotor behavior. Monoassociation with one host-associate and the environmental isolate resulted in the hyperactive phenotype characteristic of axenic larvae, while monoassociation with two other host-associated bacteria partially blocked this phenotype. Furthermore, we found an inverse relationship between the total concentration of bacteria per larvae and locomotor behavior. Lastly, in the axenic and associated cohorts, but not in the larvae with complex communities, we detected unexpected bacteria, some of which may be present as facultative predators. Conclusions These data support a growing body of evidence that individual species of bacteria can have different effects on host behavior, potentially related to their success at intestinal colonization. Specific to the zebrafish model, our results suggest that differences in the composition of microbes in fish facilities could affect the results of behavioral assays within pharmacological and toxicological studies.

Antimicrobial resistance (AMR) in the environment is a growing global health concern, especially the dissemination of AMR into surface waters due to human and agricultural inputs. Within recent years, research has focused on trying to understand the impact of AMR in surface waters on human, agricultural and ecological health (One Health). While surface water quality assessments and surveillance of AMR have historically utilized culture-based methods, culturing bacteria has limitations due to difficulty in isolating environmental bacteria and the need for a priori information about the bacteria for selective isolation. The use of molecular techniques to analyze AMR at the genetic level has helped to overcome the difficulties with culture-based techniques since they do not require advance knowledge of the bacterial population and can analyze uncultivable environmental bacteria. The aim of this review is to provide an overview of common contemporary molecular methods available for analyzing AMR in surface waters, which include high throughput real-time polymerase chain reaction (HT-qPCR), metagenomics, and whole genome sequencing. This review will also feature how these methods may provide information on human and animal health risks. HT-qPCR works at the nanoliter scale, requires only a small amount of DNA, and can analyze numerous gene targets simultaneously, but may lack in analytical sensitivity and the ability to optimize individual assays compared to conventional qPCR. Metagenomics offers more detailed genomic information and taxonomic resolution than PCR by sequencing all the microbial genomes within a sample. Its open format allows for the discovery of new antibiotic resistance genes; however, the quantity of DNA necessary for this technique can be a limiting factor for surface water samples that typically have low numbers of bacteria per sample volume. Whole genome sequencing provides the complete genomic profile of a single environmental isolate and can identify all genetic elements that may confer AMR. However, a main disadvantage of this technique is that it only provides information about one bacterial isolate and is challenging to utilize for community analysis. While these contemporary techniques can quickly provide a vast array of information about AMR in surface waters, one technique does not fully characterize AMR nor its potential risks to human, animal, or ecological health. Rather, a combination of techniques (including both molecular- and culture-based) are necessary to fully understand AMR in surface waters from a One Health perspective.

Host-associated microbiota can biotransform xenobiotics, mediate health effects of chemical exposure, and play important roles in early development. Bisphenol A (BPA) is a widespread environmental chemical that has been associated with adverse endocrine and neurodevelopmental effects, some of which may be mediated by microbiota. Growing public concern over the safety of BPA has resulted in its replacement with structurally similar alternatives. In this study, we evaluated whether BPA and BPA alternatives alter microbiota and modulate secondary adverse behavioral effects in zebrafish. Zebrafish were developmentally exposed to BPA, Bisphenol AF (BPAF), Bisphenol B (BPB), Bisphenol F (BPF), or Bisphenol S (BPS). At 10 days post fertilization (dpf), toxicity assessments were completed and 16S rRNA gene sequencing was performed to evaluate potential chemical-dependent shifts in microbial community structure and predicted function. A standard light/dark behavioral assay was used to assess locomotor activity. Based on developmental toxicity assessments at 10 dpf, a range of potencies was observed: BPAF > BPB > BPF ∼ BPA > BPS. Analysis of 16S rRNA gene sequencing data showed significant concentration-dependent disruption of microbial community structure and enrichment of putative microbial functions with exposure to BPS, BPA, or BPF, but not BPB or BPAF. Interestingly, microbial disruption was inversely related to host developmental toxicity and estrogenicity. Exposure to BP analogs did not cause behavioral effects at 10 dpf. Our findings indicate that some BP analogs disrupt host microbiota early in life and demonstrate novel chemical-microbiota interactions that may add important context to current hazard identification strategies.

Metallic nanoparticles (NPs), the most abundant nanomaterials in consumer and industrial products, are the most probable class to enter the environment. In this study, wetland-derived microcosms were incubated with copper nanoparticles (Cu-NP) and ionic CuCl2 to investigate acute (10 days) and chronic (100 days) exposure towards nitrogen cycling microorganisms. The microbial ecology of wetlands play a crucial role in balancing nitrogen in pristine environments as well as in areas impacted by high nutrient loads (e.g., at wastewater effluent discharges). Gene abundance and expression changes were monitored using the GeoChip 5.0 high throughput functional gene microarray and metatranscriptomic shotgun sequencing (RNA-seq), respectively. After 10 days, the Cu-NP impacted microbial communities experienced structural shifts within microorganisms associated with dissimilatory nitrogen reduction accompanied by lower nitrate removal as compared to the unexposed controls. By day 100, these differences were largely resolved and nitrate removal was similar to the unexposed control. Furthermore, the Cu-NP exposed microcosms tolerated copper and were more resilient and adaptive than the unexposed controls based on the abundance and expression of other functions, including electron transfer, metal homeostasis, and stress response. These findings suggest sudden influxes of Cu-NPs into wetland systems may impair nitrogen removal initially, but long-term microbial shifts and functional redundancy would promote the net flux of total nitrogen out of the wetlands.

Drinking water treatment plants rely on purification of contaminated source waters to provide communities with potable water. One group of possible contaminants are enteric viruses. Measurement of viral quantities in environmental water systems are often performed using polymerase chain reaction(PCR) or quantitative PCR (qPCR). However, these results can often be underestimated due to challenges involved in a multi-step viral concentration process and PCR inhibition. In this study, water samples were concentrated from 25 drinking water treatment plants (DWTPs) across the US to study the occurrence of enteric viruses in source water and removal after treatment. The five different types of viruses studied were adenovirus, norovirus GI, norovirus GII, enterovirus, and polyomavirus. Quantitative PCR was performed on all samples to determine presence or absence of these viruses in each sample. Ten DWTPs showed presence of one or more viruses in source water, with four DWTPs having treated drinking water testing positive. Furthermore, PCR inhibition was assessed for each sample using an exogenous amplification control, which indicated that all of the DWTP samples, including source and treated water samples, had some level of inhibition, confirming that inhibition plays an important role in PCR based assessments of environmental samples. PCR inhibition measurements, viral recovery, and other assessments were incorporated into a Bayesian model to more accurately determine viral load in both source and treated water. Results of the Bayesian model documented that viruses are present in source water and treated water. By using a Bayesian framework that incorporates inhibition, as well as many other parameters that affect viral detection, this study offers an approach for more accurately estimating the occurrence of viral pathogens in environmental waters.

Antimicrobial resistance (AMR) is a global concern, pertaining not only to human health but also to the health of industry and the environment. AMR research has traditionally focused on genetic exchange mechanisms and abiotic environmental constraints, leaving important aspects of microbial ecology unresolved. The genetic and ecological aspects of AMR, however, not only contribute separately to the problem but also are interrelated. For example, mutualistic associations among microbes such as biofilms can both serve as a barrier to antibiotic penetration and a breeding ground for horizontal exchange of antimicrobial resistance genes (ARGs). In this review, we elucidate how species interactions promote and impede the establishment, maintenance, and spread of ARGs and indicate how management initiatives might benefit from leveraging the principles and tools of community ecology to better understand and manipulate the processes underlying AMR.

Somatic coliphages are alternative indicators of fecal pollution and attractive surrogates for viral pathogens. Here, we report the draft genome sequences of three replicate plaques from a novel Myoviridae bacteriophage isolated from raw wastewater. These genomes were similar to felix01virus phage and are predicted to contain up to 148 protein-coding genes.

Anaerobic treatment of domestic wastewater (DWW) produces dissolved methane that needs to be recovered for use as an energy product. Membrane-based recovery systems have been reported in the literature but are often limited by fouling. The objective of this study was to develop a methane producing biofilm on the shell side surface a membrane to allow for immediate recovery of methane as it was produced, negating mass transfer resistance caused by fouling. Between 89 and 96% of total methane produced was recovered via in-situ degassing without the need for fouling control or cleaning throughout 72 weeks of operation. High methane recovery efficiencies led to predictions of net positive energy yield in one reactor and a 32–61% reduction in energy demand in the others compared to the control. This research demonstrates the feasibility and usefulness of combining attached growth anaerobic wastewater treatment processes with hollow fiber membrane methane recovery systems for improved operation.

Risk-based treatment of onsite wastewaters for decentralized reuse requires information on the occurrence and density of pathogens in source waters, which differ from municipal wastewater due to scaling and dilution effects in addition to variable source contributions. In this first quantitative report of viral enteric pathogens in onsite-collected graywater and wastewater, untreated graywater (n = 50 samples) and combined wastewater (i.e., including blackwater; n = 28) from three decentralized collection systems were analyzed for two norovirus genogroups (GI/GII) and human adenoviruses using droplet digital polymerase chain reaction (ddPCR). Compared to traditional quantitative PCR (qPCR), which had insufficient sensitivity to quantify viruses in graywater, ddPCR allowed quantification of norovirus GII and adenovirus in 4% and 14% of graywater samples, respectively (none quantifiable for norovirus GI). Norovirus GII was routinely quantifiable in combined wastewater by either PCR method (96% of samples), with well-correlated results between the analyses (R2 = 0.96) indicating a density range of 5.2–7.9 log10 genome copies/L. These concentrations are greater than typically reported in centralized municipal wastewater, yet agree well with an epidemiology-based model previously used to develop pathogen log-reduction targets (LRTs) for decentralized non-potable water systems. Results emphasize the unique quality of onsite wastewaters, supporting the previous LRTs and further quantitative microbial risk assessment (QMRA) of decentralized water reuse.

Surveillance monitoring for microbial water quality typically involves collecting single discrete grab samples for analyzing only one contaminant. While informative, current approaches suffer from poor recoveries and only provide a limited snapshot of the microbial contaminants only at the time of collection. To overcome these limitations, bivalves have been proposed as effective biosentinels of water quality particularly for their ability to efficiently concentrate and retain microbial contaminants for long periods of time. In this study, we examined the use of indigenous blue mussels (Mytilus spp.) as biosentinels to monitor for the presence of Toxoplasma gondii and Cryptosporidium water. An efficient method to extract oocyst DNA from various mussel tissues followed by PCR-based detection of these pathogens was developed, which resulted in the detection down to 10 oocysts. This method was then used to conduct a small survey in Point Lobos and Morro Bay, California to determine prevalence T. gondii and Cryptosporidium. Results revealed that mussels from Morro Bay were contaminated with T. gondii (33 %), while mussels from Point Lobos were contaminated with T. gondii (54 %) and Cryptosporidium (26.9 %) oocysts. Phylogenetic analysis using the SSU rRNA gene identified two novel Cryptosporidium parvum-like genotypes. Overall, this study demonstrated the application of using native California Mytilus spp. as biosentinels for pathogen contamination along the central California shorelines. More importantly, T. gondii and Cryptosporidium were found at higher prevalence rates in Morro Bay and in Point Lobos, an area not previously reported to be contaminated with these pathogens.

Aims Development of efficacious grey water (GW) treatment systems would benefit from detailed knowledge of the bacterial composition of GW. Thus, the aim of this study was to characterize the bacterial composition from (i) various points throughout a GW recycling system that collects shower and sink handwash (SH) water into an equalization tank (ET) prior to treatment and (ii) laundry (LA) water effluent of a commercial-scale washer. Methods and Results Bacterial composition was analysed by high-throughput pyrosequencing of the 16S rRNA gene. LA was dominated by skin-associated bacteria, with Corynebacterium, Staphylococcus, Micrococcus, Propionibacterium and Lactobacillus collectively accounting for nearly 50% of the total sequences. SH contained a more evenly distributed community than LA, with some overlap (e.g. Propionibacterium), but also contained distinct genera common to wastewater infrastructure (e.g. Zoogloea). The ET contained many of these same wastewater infrastructure-associated bacteria, but was dominated by genera adapted for anaerobic conditions. Conclusions The data indicate that a relatively consistent set of skin-associated genera are the dominant human-associated bacteria in GW, but infrastructure-associated bacteria from the GW collection system and ET used for transient storage will be the most common bacteria entering GW treatment and reuse systems. Significance and Impact of the Study This study is the first to use high-throughput sequencing to identify the bacterial composition of various GW sources.

Changes in resident microbiota may have wide-ranging effects on human health. We investigated whether early life microbial disruption alters neurodevelopment and behavior in larval zebrafish. Conventionally colonized, axenic, and axenic larvae colonized at 1 day post fertilization (dpf) were evaluated using a standard locomotor assay. At 10 dpf, axenic zebrafish exhibited hyperactivity compared to conventionalized and conventionally colonized controls. Impairment of host colonization using antibiotics also caused hyperactivity in conventionally colonized larvae. To determine whether there is a developmental requirement for microbial colonization, axenic embryos were serially colonized on 1, 3, 6, or 9 dpf and evaluated on 10 dpf. Normal activity levels were observed in axenic larvae colonized on 1–6 dpf, but not on 9 dpf. Colonization of axenic embryos at 1 dpf with individual bacterial species Aeromonas veronii or Vibrio cholerae was sufficient to block locomotor hyperactivity at 10 dpf. Exposure to heat-killed bacteria or microbe-associated molecular patterns pam3CSK4 or Poly(I:C) was not sufficient to block hyperactivity in axenic larvae. These data show that microbial colonization during early life is required for normal neurobehavioral development and support the concept that antibiotics and other environmental chemicals may exert neurobehavioral effects via disruption of host-associated microbial communities.

Enterovirus infections are often not tracked or reported to health officials. This makes it hard to know how many people in a community are infected with these viruses at any given time. Here, we explored enterovirus in municipal wastewater to look at this issue. We show that enteroviruses are present year-round in municipal wastewater at levels of up to 800,000 genomic copies per liter. We estimate that, on average, 2.8% of the people contributing to the wastewater shed enterovirus daily. Sequence analysis of the viral capsid protein 4 gene shows that 8 enterovirus types are key drivers of seasonal trends. Populations of Enterovirus A members peak in the spring, while Enterovirus B types are most prevalent during the summer and fall months and Enterovirus C members influence the winter months. Enterovirus D was observed sporadically and did not influence seasonal trends., Enteroviruses are RNA viruses that are responsible for both mild gastroenteritis and mild respiratory illnesses as well as debilitating diseases such as meningitis and myocarditis. The disease burden of enteroviruses in the United States is difficult to assess because most infections are not recorded. Since infected individuals shed enterovirus in feces and urine, surveillance of municipal wastewater can reveal the diversity of enteroviruses circulating in human populations. Therefore, monthly municipal wastewater samples were collected for 1 year and enteroviruses were quantified by reverse transcriptase quantitative PCR and identified by next-generation, high-throughput sequencing. Enterovirus concentrations ranged from 3.8 to 5.9 log10 equivalent copies/liter in monthly samples. From the mean monthly concentration, it can be estimated that 2.8% of the contributing population was shedding enterovirus daily. Sequence analysis showed that Enterovirus A and Enterovirus B alternate in predominance, with Enterovirus B comprising over 80% of the reads during the summer and fall months and Enterovirus A accounting for >45% of the reads in spring. Enterovirus C was observed throughout the year, while Enterovirus D was present intermittently. Principal-component analysis further supported the date corresponding to enterovirus seasonal trends as CVA6 (Enterovirus A) was predominant in the spring months; CVB3, CVB5, and E9 (Enterovirus B) were predominant in the summer and fall months; and CVA1, CVA19, and CVA22 (Enterovirus C) and EV97 (Enterovirus B) were predominant in winter. Rhinoviruses were also observed. Wastewater monitoring of human enterovirus provided improved insight into the seasonal patterns of enteroviruses circulating in communities and can contribute to understanding of enterovirus disease burden. IMPORTANCE Enterovirus infections are often not tracked or reported to health officials. This makes it hard to know how many people in a community are infected with these viruses at any given time. Here, we explored enterovirus in municipal wastewater to look at this issue. We show that enteroviruses are present year-round in municipal wastewater at levels of up to 800,000 genomic copies per liter. We estimate that, on average, 2.8% of the people contributing to the wastewater shed enterovirus daily. Sequence analysis of the viral capsid protein 4 gene shows that 8 enterovirus types are key drivers of seasonal trends. Populations of Enterovirus A members peak in the spring, while Enterovirus B types are most prevalent during the summer and fall months and Enterovirus C members influence the winter months. Enterovirus D was observed sporadically and did not influence seasonal trends.

Aims This study developed and systematically evaluated performance and limit of detection of an off-the-slide genotyping procedure for both Cryptosporidium oocysts and Giardia cysts. Methods and Results Slide standards containing flow-sorted (oo)cysts were used to evaluate the off-the-slide genotyping procedure by microscopy and PCR. Results show approximately 20% of cysts and oocysts are lost during staining. Although transfer efficiency from the slide to the PCR tube could not be determined by microscopy, it was observed that the transfer process aided in the physical lysis of the (oo)cysts likely releasing DNA. PCR detection rates for a single event on a slide were 44% for Giardia and 27% for Cryptosporidium, and a minimum of five cysts and 20 oocysts are required to achieve a 90% PCR detection rate. A Poisson distribution analysis estimated the relative PCR target densities and limits of detection, it showed that 18 Cryptosporidium and five Giardia replicates are required for a 95% probability of detecting a single (oo)cyst on a slide. Conclusions This study successfully developed and evaluated recovery rates and limits of detection of an off-the-slide genotyping procedure for both Cryptosporidium and Giardia (oo)cysts from the same slide. Significance and Impact of the Study This off-the-slide genotyping technique is a simple and low cost tool that expands the applications of US EPA Method 1623 results by identifying the genotypes and assemblages of the enumerated Cryptosporidium and Giardia. This additional information will be useful for microbial risk assessment models and watershed management decisions.

Pneumocystis carinii is an unusual fungus that can cause pneumonitis in immunosuppressed laboratory rats. Reactions in sterol biosynthesis are attractive targets for development of antimycotic drugs. A key enzyme in sterol biosynthesis is sterol 14α-demethylase (14DM), which is coded by the erg11 gene. Here we describe detailed sterol analysis of wild-type Saccharomyces cerevisiae and in an erg11 knockout mutant expressing either P. carinii or S. cerevisiae 14DM from a plasmid-borne cDNA. Sterols of the three strains were qualitatively and quantitatively analyzed using thin-layer chromatography, high-performance liquid chromatography, and gas-liquid chromatography and mass spectrometry and nuclear magnetic resonance spectroscopy. Biochemical evidence for functional complementation was provided by detecting the same major sterols in all three strains with ergosterol being by far the most abundant. A total of 25 sterols was identified, 16 of which were identified in all three strains. The ratios of lanosterol:14-desmethyllanosterol in the three strains indicate that the mutant transformed with erg11 showed more 14DM activity than wild-type yeast. The sterol analyses also indicated that the P. carinii 14DM can utilize the sterol substrates used by the S. cerevisiae 14DM and suggested that the yeast 14DM in the yeast cell utilizes 4α-methyl sterols better than the P. carinii enzyme.

Metagenomics is a powerful tool for characterizing viral composition within environmental samples, but sample and molecular processing steps can bias the estimation of viral community structure. The objective of this study is to understand the inherent variability introduced when conducting viral metagenomic analyses of wastewater and provide a bioinformatic strategy to accurately analyze sequences for viral community analyses. A standard approach using a combination of ultrafiltration, membrane filtration, and DNase treatment, and multiple displacement amplification (MDA) produced DNA preparations without any bacterial derived genes. Results showed recoveries in wastewater matrix ranged between 60–100%. A bias towards small single stranded DNA (ssDNA; polyomavirus) virus types vs larger double stranded DNA (dsDNA; adenovirus) viruses was also observed with a total estimated recovery of small circular viruses to be as much as 173-fold higher. Notably, ssDNA abundance decreased with sample dilution while large dsDNA genomes (e.g., Caudovirales) initially increased in abundance with dilution before gradually decreasing with further dilution in wastewater samples. The present study revealed the inherent biases associated with different components of viral metagenomic methods applied to wastewater. Overall, these results provide a well-characterized approach for effectively conducting viral metagenomics analysis of wastewater and reveal that dilution can effectively mitigate MDA bias.

Humans are known to be the host to approximately 1500 infectious agents, out of which 66 are protozoa and 287 are helminths. Therefore, from a global perspective helminths and protozoan parasites account for approximately one fourth of the total infectious diseases of humans. A similar trend has been observed in waterborne infectious diseases, among which a significant part is caused by enteric parasites. Cryptosporidium and Giardia are the leading cause of waterborne outbreaks of gastroenteritis across the globe, and as such, will be discussed in length in this chapter. These parasites are particularly suited for waterborne transmission as the environmentally resistant cysts and oocysts, respectively are shed in large numbers in feces (108-109 oocysts/gram), have a low infectious dose, and are resistant to disinfection practices. Naegleria fowleri is a pathogenic free-living amoeba found in the environment in both water and soil. There have been over 40 species of Naegleria described to date, but only N. fowleri is pathogenic to humans. N. fowleri was first identified as a human pathogen in 1965 in Australia. The first case in the United States was reported in 1966 and was described as primary meningoencephalitis. Prior to this documented case, free-living amoebae were not considered to be pathogenic. Pathogenic Naegleria fowleri is not easily differentiated from other Naegleria species due to similarities including common morphology when observed microscopically and indistinguishable behavior in cell culture.

In the fungus Pneumocystis carinii, at least three gene families (PRT1, MSR, and MSG) have the potential to generate high-frequency antigenic variation, which is likely to be a strategy by which this parasitic fungus is able to prolong its survival in the rat lung. Members of these gene families are clustered at chromosome termini, a location that fosters recombination, which has been implicated in selective expression of MSG genes. To gain insight into the architecture, evolution, and regulation of these gene clusters, six telomeric segments of the genome were sequenced. Each of the segments began with one or more unique genes, after which were members of different gene families, arranged in a head-to-tail array. The three-gene repeat PRT1-MSR-MSG was common, suggesting that duplications of these repeats have contributed to expansion of all three families. However, members of a gene family in an array were no more similar to one another than to members in other arrays, indicating rapid divergence after duplication. The intergenic spacers were more conserved than the genes and contained sequence motifs also present in subtelomeres, which in other species have been implicated in gene expression and recombination. Long mononucleotide tracts were present in some MSR genes. These unstable sequences can be expected to suffer frequent frameshift mutations, providing P. carinii with another mechanism to generate antigen variation.

Organisms in the genus Pneumocystis are fungi that reside in the lungs of mammals that can cause a lethal pneumonia once the hosts lose immune function. The genus Pneumocystis contains many members, but only two species have been described formally to date, P. carinii, the type species found in rats, and P. jirovecii, resident in human beings. Rats have been shown to harbor another organism in addition to P. carinii, Pneumocystis wakefieldiae sp. nov., formerly known as Pneumocystis carinii f. sp. ratti, which is described here. Although often found together and morphologically similar, P. carinii and P. wakefieldiae are phenotypically and genetically divergent. We used the phylogenetic species recognition approach to distinguish these organisms as two distinct species and estimated the evolutionary time of their separation. Nucleotide sequence comparisons of seven homologous genes showed 4-7% divergence between the P. wakefieldiae and P. carinii sequences, which was in contrast to the 0-0.8% divergence observed within P. carinii species. Even greater divergence (30%) occurred in sequences located between genes. The MSG (major surface glycoprotein) gene families of P. carinii and P. wakefieldiae are 35% divergent from one another and differ with respect to sequence elements associated with regulation of their transcription. Differences in reactivity of monoclonal antibodies and polyclonal antisera reflected these genetically distinct surface antigens. Karyotypic analysis of P. wakefieldiae produced a single profile that was distinct from all 12 profiles known for P. carinii. Eight homologous genes were localized to chromosomes of different sizes in the two species. The cumulative genotypic and phenotypic data support a species distinction between these two organisms.

Pneumocystis carinii has a multigene family, PRT1, that encodes proteins with homology to KEX2-like proteases. PRT1 genes cluster with MSG genes near the telomeres and, like MSG, PRT1 proteins seem to be surface-expressed. The clustering of PRT1 and MSG genes suggested that expression of the two multigene families might be coordinated. Studying gene expression in P. carinii has been hampered by the lack of a culture system, and by lack of clonality in P. carinii populations in naturally infected rats, the host of this fungus. Heterogeneity can be reduced, however, by low-dose intratracheal inoculation, which can produce P. carinii populations dominated by organisms derived from a single progenitor. To study PRT1 expression, nude rats were inoculated with approximately 10 P. carinii each. The clonality of the P. carinii populations from inoculated rats was assessed by analysis of the UCS locus, a site in the genome that is known to be very heterogeneous in naturally infected rats, but nearly homogeneous in rats infected by low-dose intratracheal inoculation. Each of the populations had the same MSG gene at the UCS locus in at least 80 % of the organisms. To investigate PRT1 gene expression, RNA was amplified using primers that amplify numerous PRT1 genes. Seventy-four cloned cDNAs were sequenced, including at least 12 clones from each population of P. carinii. Many differently expressed PRT1 sequences were identified in each population, and a total of 45 different sequences were detected. However, the same PRT1 sequence was present in 15 of 74 plasmids and was found in 3 of the 5 P. carinii populations, suggesting that some PRT1 genes may be either more commonly expressed or expressed at a higher level. These data show that many members of the PRT1 gene family can be expressed in populations of P. carinii derived from few progenitors and suggest that the regulation of this family is different from that governing expression of the MSG gene family.

Pneumocystis is an opportunistic pathogen that can cause pneumonitis in immunodeficient people such as AIDS patients. Pneumocystis remains difficult to study in the absence of culture methods for luxuriant growth. Recombinant protein technology now makes it possible to avoid some major obstacles. The P. carinii expressed sequence tag (EST) database contains 11 entries of a sequence encoding a protein homologous to S-adenosyl-L-methionine (SAM):C-24 sterol methyl transferase (SMT), suggesting high activity of this enzyme in the organism. We sequenced the erg6 cDNA, identified the putative peptide motifs for the sterol and SAM binding sites in the deduced amino acid sequence and expressed the protein in Escherichia coli. Unlike SAM:SMT from other organisms, the P. carinii enzyme had higher affinities for lanosterol and 24-methylenelanosterol than for zymosterol, the preferred substrate in other fungi. Cycloartenol was not a productive substrate. With lanosterol and 24-methylenelanosterol as substrates, the major reaction products were 24-methylenelanosterol and pneumocysterol respectively. Thus, the P. carinii SAM:SMT catalysed the transfer of both the first and the second methyl groups to the sterol C-24 position, and the substrate preference was found to be a unique property of the P. carinii SAM:SMT. These observations, together with the absence of SAM:SMT among mammals, further support the identification of sterol C-24 alkylation reactions as excellent targets for the development of drugs specifically directed against this pathogen.

Previous graywater risk assessment studies have focused on fecal contamination, yet the low density of fecal indicators may not provide the most useful approach to assess pathogen removal during graywater treatment. In this study, we employed high throughput bacterial sequencing and qPCR to elucidate potential microbial surrogates in wastewater sourced from an industrial laundry. In addition, we explored human mitochondrial DNA (HmtDNA) as a new, potentially more reliable molecular marker, because it can be unambiguously sourced, has a high copy number per cell, and is persistent when released from cells with no self-replication in graywater. Pyrosequencing and qPCR revealed that laundry water microbiota was dominated by the skin-associated bacteria Staphylococcus, Corynebacterium, and Propionibacterium (6.5, 5.7, 5.4 log10 copies/100 mL, respectively). While HmtDNA was less abundant (2.8 log10 copies/100 mL), it showed a strong positive correlation with the opportunistic pathogen Staphylococcus aureus (r=0.54, P=3.2×10(-4)) and closely followed a first-order exponential decay model (R2=0.98), remaining detectable in stored laundry graywater for up to 6 days at 20 °C. Based on abundance and persistence, we propose HmtDNA and total Staphylococcus as future laundry graywater treatment surrogates to potentially assess a wide dynamic range of pathogen removal.

This article reviews the molecular genetic data pertaining to the major surface glycoprotein (MSG) gene family ofPneumocystis cariniiand its role in surface variation and compares this fungal system to antigenic variation systems in the protozoanTrypanosoma bruceiand the bacteriaBorreliaspp.

DNA was amplified from lung samples from three piglets infected with Pneumocystis carinii, using oligonucleotide primers designed to the P. carinii mitochondrial large subunit ribosomal RNA gene. The nucleotide sequence of the amplification product was determined and indicated lack of sequence variation among these pig-derived P. carinii samples at this locus. The data showed that porcine P. carinii was genetically distinct from P. carinii isolated from other mammalian host species.

Pneumocystis jirovecii is a fungus that causes Pneumocystis pneumonia in immunosuppressed patients and has been closely associated with AIDS since the beginning of the AIDS epidemic. Because in vitro cultivation of P. jirovecii is not possible, progress has been hindered in our understanding of its life cycle, mode of transmission, metabolic function, and genome. Limited amounts of P. jirovecii can be obtained from infected patients, but the occurrence of bacteria, other fungi, and human cells in clinical samples presents new challenges for whole-genome sequencing and downstream bioinformatic analysis. In a recent article, Cissé et al. used cell immunoprecipitation enrichment together with whole-genome amplification to generate sufficient quantities of DNA for Roche 454 and Illumina sequencing [O. H. Cissé, M. Pagni, and P. M. Hauser, mBio 4(1):e00428-12, 2012, doi:10.1128/mBio.00428-12]. In addition, a bioinformatic pipeline was devised to sort and assemble lung microbiome reads, thereby generating an 8.1-Mb P. jirovecii genome comprised of 356 contigs with an N 50 (median length of all contigs) of 41.6 kb. Knowledge of this genome will open new avenues of research, including the identification of nutritional requirements for in vitro cultivation as well as the identification of new and novel drug and vaccine targets.

Quantitative real-time polymerase chain reaction (qPCR) assays to detect Cryptosporidium oocysts in clinical samples are increasingly being used to diagnose human cryptosporidiosis, but a parallel approach for detecting and identifying Cryptosporidium oocyst contamination in surface water sources has yet to be established for current drinking water quality monitoring practices. It has been proposed that Cryptosporidium qPCR-based assays could be used as viable alternatives to current microscopic-based detection methods to quantify levels of oocysts in drinking water sources; however, data on specificity, analytical sensitivity, and the ability to accurately quantify low levels of oocysts are limited. The purpose of this study was to provide a comprehensive evaluation of TaqMan-based qPCR assays, which were developed for either clinical or environmental investigations, for detecting Cryptosporidium oocyst contamination in water. Ten different qPCR assays, six previously published and four developed in this study were analyzed for specificity and analytical sensitivity. Specificity varied between all ten assays, and in one particular assay, which targeted the Cryptosporidium 18S rRNA gene, successfully detected all Cryptosporidium spp. tested, but also cross-amplified T. gondii, fungi, algae, and dinoflagellates. When evaluating the analytical sensitivity of these qPCR assays, results showed that eight of the assays could reliably detect ten flow-sorted oocysts in reagent water or environmental matrix. This study revealed that while a qPCR-based detection assay can be useful for detecting and differentiating different Cryptosporidium species in environmental samples, it cannot accurately measure low levels of oocysts that are typically found in drinking water sources.

Objective : To investigate the hypothesis that P carinii special form hominis (Pc. hominis) reinfections occur in AIDS patients. Design : Polymerase chain reaction (PCR) was used to identify patients who had different Pc. hominis mitochondrial DNA (mtrRNA) genotypes in the two disease episodes (genotype switching). Pc. hominis from these patients were analysed with an allele-specific PCR (ASP) assay to determine whether the genotype found in a second disease episode were present in the first disease episode. To assess the possible contributions of other factors to genotype switching, data on the sampling method and drugs used to treat each patient were compiled. Methods : Bronchoalveolar lavage fluid (BALF) was subjected to PCR using primers that amplified a 346 base-pair region of the mtrRNA locus known to be polymorphic at site 85 of the amplicon. Samples from patients in whom the Pc. hominis mtrRNA sequence had changed at site 85 in the two disease episodes were studied by ASP in which primers designed to prime synthesis from the allele of the mtrRNA sequence found in second episodes of disease were used in PCR of Pc. hominis DNA from first episodes of P. carinii pneumonia. Results : In four of five patients who produced P.c. hominis with different mtrRNA genotypes during first and second episodes, ASP did not detect the second-episode genotype in first-episode BALF. There was no evidence that either sampling methods or drug-resistance contributed to genotype switching. Conclusions : P.c. hominis reinfections occur in AIDS patients.

Pneumocystis carinii is an unusual fungus that can cause pneumonitis in immunosuppressed laboratory rats. Reactions in sterol biosynthesis are attractive targets for development of antimycotic drugs. A key enzyme in sterol biosynthesis is sterol 14α-demethylase (14DM), which is coded by the erg11 gene. Here we describe detailed sterol analysis of wild-type Saccharomyces cerevisiae and in an erg11 knockout mutant expressing either P. carinii or S. cerevisiae 14DM from a plasmid-borne cDNA. Sterols of the three strains were qualitatively and quantitatively analyzed using thin-layer chromatography, high-performance liquid chromatography, and gas-liquid chromatography and mass spectrometry and nuclear magnetic resonance spectroscopy. Biochemical evidence for functional complementation was provided by detecting the same major sterols in all three strains with ergosterol being by far the most abundant. A total of 25 sterols was identified, 16 of which were identified in all three strains. The ratios of lanosterol:14-desmethyllanosterol in the three strains indicate that the mutant transformed with erg11 showed more 14DM activity than wild-type yeast. The sterol analyses also indicated that the P. carinii 14DM can utilize the sterol substrates used by the S. cerevisiae 14DM and suggested that the yeast 14DM in the yeast cell utilizes 4α-methyl sterols better than the P. carinii enzyme.

Publisher Summary This chapter studies the genus Pneumocystis. In the determination of the asexual reproduction, extracellular, obligate, host-specific, nonmycelial fungi that are restricted to mammalian lung tissues are seen. The fungi attach to Type I epithelial cells lining the aveoli and usually occur as clusters, which are composed of sexual and asexual developmental forms. A putative sexual cycle occurs after mating of trophic forms results in a diploid zygote that proceeds to form prototunicate asci, usually referred to as “cysts.” The eight-spored spherical ascus-like cysts, which range in size from 8 to 10 μm, are the most characteristic form of Pneumocystis species. Meiosis and an additional mitotic division produce eight ascospores within an ascus. The chapter also discusses physiology/biochemistry and phylogenetic placement of the genus. The type species taken is Pneumocystis carinii. In the systematic discussion of the species, synonyms, asexual reproduction, sexual reproduction, clinical description, growth in RPMI1640 or Dulbecco's minimum essential medium (DMEM), growth temperature, gene sequence accession numbers, cell carbohydrates, neotype, systematics, ecology, and clinical importance are determined.