SARS-CoV-2 monitoring at three sewersheds of different scales and complexity demonstrates distinctive relationships between wastewater measurements and COVID-19 case data.

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.

Human- and infrastructure-associated bacteria in greywater.

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 faecal 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.

Twenty-first century molecular methods for analyzing antimicrobial resistance in surface waters to support One Health assessments.

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.

Characterization of the relative importance of human- and infrastructure-associated bacteria in grey water: a case study.

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.

Development and evaluation of an off-the-slide genotyping technique for identifying Giardia cysts and Cryptosporidium oocysts directly from US EPA Method 1623 slides.

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.

Expression and complexity of the PRT1 multigene family of Pneumocystis carinii.

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.

Genetics of surface antigen expression in Pneumocystis carinii.

This article reviews the molecular genetic data pertaining to the major surface glycoprotein (MSG) gene family of Pneumocystis carinii and its role in surface variation and compares this fungal system to antigenic variation systems in the protozoan Trypanosoma brucei and the bacteria Borrelia spp.

Time between inoculations and karyotype forms of Pneumocystis carinii f. sp. Carinii influence outcome of experimental coinfections in rats.

The prevalence of Pneumocystis carinii pneumonia (PCP) in humans caused by more than a single genotype has been reported to range from 10 to 67%, depending on the method used for detection (3, 19). Most coinfections were associated with primary rather than recurrent disease. To better understand the factors influencing the development of coinfections, the time periods between inoculations and the genotype of the infecting organisms were evaluated in the chronically immunosuppressed-inoculated rat model of PCP. P. carinii f. sp. carinii infecting rats differentiated by karyotypic profiles exhibit the same low level of genetic divergence manifested by organisms infecting humans. P. carinii f. sp. carinii karyotype forms 1, 2, and 6 were inoculated into immunosuppressed rats, individually and in dual combinations, spaced 0, 10, and 20 days apart. Infections comprised of both organism forms resulted from admixtures inoculated at the same time. In contrast, coinfections did not develop in most rats, where a 10- or 20-day gap was inserted between inoculations; only the first organism form inoculated was detected by pulsed-field gel electrophoresis in the resultant infection. Organism burdens were reduced with combinations of forms 1 and 2 spaced 20 days apart but not in rats inoculated with forms 1 and 6. A role for the host response in the elimination of the second population and in reduction of the organism burden was suggested by the lack of direct killing of forms 1 and 2 in an in vitro ATP assay, by reduction of the burden by autoclaved organisms, and by the specific reactions of forms 1 and 2 but not forms 1 and 6. These studies showed that the time between inoculations was critical in establishing coinfections and P. carinii f. sp. carinii karyotype profiles were associated with differences in biological responses. This model provides a useful method for the study of P. carinii coinfections and their transmission in humans.

Genetic variation in Pneumocystis carinii isolates from different geographic regions: implications for transmission.

To study transmission patterns of Pneumocystis carinii pneumonia (PCP) in persons with AIDS, we evaluated P. carinii isolates from patients in five U.S. cities for variation at two independent genetic loci, the mitochondrial large subunit rRNA and dihydropteroate synthase. Fourteen unique multilocus genotypes were observed in 191 isolates that were examined at both loci. Mixed infections, accounting for 17.8% of cases, were associated with primary PCP. Genotype frequency distribution patterns varied by patients' place of diagnosis but not by place of birth. Genetic variation at the two loci suggests three probable characteristics of transmission: that most cases of PCP do not result from infections acquired early in life, that infections are actively acquired from a relatively common source (humans or the environment), and that humans, while not necessarily involved in direct infection of other humans, are nevertheless important in the transmission cycle of P. carinii f. sp. hominis.

Sequences of Pneumocystis carinii f. sp. hominis strains associated with recurrent pneumonia vary at multiple loci.

The sequences of the internal transcribed spacer (ITS) of Pneumocystis carinii f. sp. hominis strains from 7 of 15 AIDS patients were found to vary during discrete episodes of P. carinii pneumonia. Changes in the ITS sequence correlated with changes in the mitochondrial large-subunit rRNA sequence. The coincidence of changes in the sequences of the ITS, which is located in the nucleus, with changes in a mitochondrial gene excludes mutation as the cause of the genetic differences between P. carinii f. sp. hominis strains isolated during different episodes of P. carinii pneumonia and supports the hypothesis that recurrent P. carinii pneumonia is caused by reinfection rather than by reactivation of latent organisms. Thus, limiting the exposure of immunocompromised patients to P. carinii f. sp. hominis should help prevent P. carinii pneumonia.

Identification of porcine Pneumocystis carinii as a genetically distinct organism by DNA amplification.

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.

Source of Pneumocystis carinii in recurrent episodes of pneumonia in AIDS patients.

OBJECTIVE: To investigate the hypothesis that P.carinii special form hominis (P.c. hominis) reinfections occur in AIDS patients. DESIGN: Polymerase chain reaction (PCR) was used to identify patients who had different P.c. hominis mitochondrial DNA (mtrRNA) genotypes in the two disease episodes (genotype switching). P.c. 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 P.c. 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 P.c. 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.