Co-Carriage of Metal and Antibiotic Resistance Genes in Sewage Associated Staphylococci.

Controlling spread of resistance genes from wastewater to aquatic systems requires more knowledge on how resistance genes are acquired and transmitted. Whole genomic sequences from sewage-associated staphylococcus isolates (20 S. aureus, 2 Staphylococcus warneri, and 2 Staphylococcus delphini) were analyzed for the presence of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs). Plasmid sequences were identified in each isolate to investigate co-carriage of ARGs and MRGs within. BLASTN analysis showed that 67% of the isolates carried more than one ARG. The carriage of multiple plasmids was observed more in CC5 than CC8 S. aureus strains. Plasmid exchange was observed in all staphylococcus species except the two S. delphini isolates that carried multiple MRGs, no ARGs, and no plasmids. 85% of S. aureus isolates carried the blaZ gene, 76% co-carried blaZ with cadD and cadX, with 62% of these isolates carrying blaZ, cadD, and cadX on the same plasmid. The co-carriage of ARGs and MRGs in S. warneri isolates, and carriage of MRGs in S. delphini, without plasmids suggests non-conjugative transmission routes for gene acquisition. More studies are required that focus on the transduction and transformation routes of transmission to prevent interspecies exchange of ARGs and MRGs in sewage-associated systems.

Solar disinfection of turbid hygiene waters in Lexington, KY, USA.

Solar disinfection (SODIS) could be a key to providing a clean, hygiene water for birthing uses, but the recommended climate zone is limited, the microbial indicators are related to gastrointestinal illness and not wound infections. SODIS feasibility was investigated to remove Escherichia coli from turbid water at temperatures less than 50 °C in Lexington, KY. Increasing turbidity from 0 to 200 NTU decreased E. coli inactivation from 5 to 1 log. With the same experimental protocol, more than 4-log inactivation of Staphylococcus aureus and Staphylococcus epidermidis (common human-skin microorganisms related to serious post-partum infections of both mother and child) was achieved at different turbidity levels with a maximum, in-bottle temperature of 49.2 °C after 5.5 h. The thermal inactivation of the bacterial indicators was assessed without UV radiation and turbidity in water at 37 and 47 °C. Skin bacteria were inactivated completely after 9.5 h at 47 °C, but only 58% removal happened for thermo-tolerant E. coli. These results suggest that SODIS application may be expanded geographically to treat water for hygiene purposes. However, as E. coli is also capable of causing wound infections, UV with thermal inactivation may be required to produce safe hygiene water by SODIS outside of recommended latitudes.

Thiol-Affinity Immobilization of Casein-Coated Silver Nanoparticles on Polymeric Membranes for Biofouling Control.

Silver nanoparticles (AgNPs) have been widely studied for the control of biofouling on polymeric membranes due to their antimicrobial properties. However, nanoparticle leaching has posed a significant impediment against their widespread use. In this study, a one-step method of chemically embedding AgNPs on cellulose acetate (CA) membranes via their affinity to thiol group chemistry was investigated. The operational efficiency of the membranes was then determined via filtration and biofouling experiments. During filtration study, the average flux values of pure CA membranes was determined to be 11 ± 2 L/(m2·hr) (LMH), while membranes embedded with AgNPs showed significant increases in flux to 18 ± 2 LMH and 25 ± 9 LMH, with increasing amounts of AgNPs added, which is likely due to the NPs acting as pore formers. Leaching studies, performed both in dead-end and crossflow filtration, showed approximately 0.16 mg/L leaching of AgNPs after the first day of filtration, but afterwards the remaining chemically-attached AgNPs did not leach. Over 97% of AgNPs remained on the membranes after seven days of crossflow leaching filtration studies. Serratia marcescens were then used as target microorganisms in biofouling studies. It was observed that membranes embedded with AgNPs effectively suppressed the growth of Serratia marcescens, and specifically, membranes with AgNPs displayed a decrease in microbial growth by 59% and 99% as the amount of AgNP increased.

Use of acetaminophen and sucralose as co-analytes to differentiate sources of human excreta in surface waters.

Reducing pathogenic risks in surface waters impacted by leaking or overflowing sewage requires the ability to detect human excreta in raw sewage, discriminate human excreta from other types of animal excreta, and differentiate between treated wastewater and raw sewage. We used the relative concentrations of a degradable, human-specific pharmaceutical and a persistent artificial sweetener to indicate the presence of human excreta, its degree of environmental degradation, and the amount of dilution by freshwater sources. Samples were collected and analyzed for acetaminophen and sucralose between 2016 and 2018 from wastewater treatment plants (WWTPs) and streams in metropolitan Lexington, Kentucky (USA). Both co-analytes were consistently present in raw sewage, with acetaminophen in higher concentrations than sucralose. The presence of acetaminophen was related primarily to untreated human excreta, with concentrations rapidly decreasing upon treatment to nearly undetectable levels in WWTP effluents and streams. Sucralose in surface waters was related to inputs of both raw sewage and WWTP effluents. The ratio of acetaminophen to sucralose concentrations in raw sewage and spiked river waters exhibited linear decay kinetics with respect to time, with larger decay constants observed at higher temperatures. This co-analyte indicator approach was evaluated at a local site previously suspected of receiving raw sewage. The presence and ratios of the co-analytes indicated the presence of domestic sewage that was not fully treated.

Prevalence and characterization of Staphylococcus aureus in wastewater treatment plants by whole genomic sequencing.

Infections with Staphylococcus aureus are being spread through contact with the community environment, but the role of wastewater treatment plants in the transmission routes is not defined. This study investigated the prevalence, types, genetic elements, and potential for transmission of S. aureus by these engineered systems. Synchronized sampling events at two wastewater treatment plants were conducted with isolates of S. aureus obtained by a selective enrichment method using acriflavine that suppressed Staphylococcus epidermidis growth. DNA was extracted from a subset of the S. aureus isolates, checked by PCR to assure the absence of S. epidermidis, and sequenced to determine the multilocus sequence type, spa type, and carriage of the methicillin resistance and Panton-Valentine leukocidin genetic elements. Sequences were analyzed for single nucleotide polymorphism differences in pairwise comparison of isolates. There were two dominant S. aureus clonal complexes identified in the isolates, one commonly identified as hospital-related (CC5) and one community-related (CC8). Both types of isolates were found at both treatment facilities, even though only one facility had significant hospital sewage inputs. The presence of S. aureus persisted through treatment, with some isolates recovered from the final processes showing genetic diversity. The presence of the Panton-Valentine leukocidin genetic element was greater than the 1-5% expected from global reports. Our results suggest that treatment provides an opportunity for genetic shift, while the persistence and release of evolved strains of S. aureus may provide an environmentally relevant pathway to new hosts in the environment.

Use of Nitrogen-15-Enriched Escherichia coli as a Bacterial Tracer in Karst Aquifers.

Karst aquifers are susceptible to contamination by microorganisms, but relatively few studies have used bacteria as tracers. We demonstrate the utility of Escherichia coli enriched in the stable isotope nitrogen-15 (15 N) as a novel bacterial tracer. Nonpathogenic E. coli from two springs in central Kentucky were grown on 15 N-enriched media. Survival of E. coli and persistence of the isotopic signal were assessed in two sets of laboratory experiments conducted with sterilized spring water in dark microcosms at 14 °C. First, isotopically labeled bacteria survived for 130 d at concentrations within one log unit of the average initial value, and there was no significant difference in δ15 N values from Day 1 to Day 130. Second, water samples with E. coli were inoculated with either of two different species of protozoa (Tetrahymena pyriformis or Colpoda steinii). During 7 d, δ15 N values increased in T. pyriformis while bacterial populations decreased. In a field test, following a 2.1-cm rainfall, 15 N-labeled E. coli, solutes (rhodamine WT dye and bromide), and latex microspheres were injected into a sinkhole approximately 530 m upgradient of a spring. Breakthrough of all tracers coincided, but microspheres were remobilized by subsequent storms, unlike other tracers. Enriched E. coli exhibited more tailing than solute tracers during the initial storm-flow recession. These results indicate that 15 N-enriched E. coli is a viable tracer of bacterial transport in karst aquifers, although predation may attenuate the isotopic signal in systems that are not rapidly flushed.

Prevalence of and Relationship between Two Human-Associated DNA Biomarkers for Bacteroidales in an Urban Watershed.

Human-associated fecal biomarkers offer potent tools for the detection and control of human fecal pollution in watersheds. In some cases, the probability of false-negative findings may call for using a less specific biomarker that is present in higher quantities as long as it can be related to the more specific indicator. The objective of this study is to investigate the relationship between two previously published human-associated biomarkers for Bacteroidales bacteria in an urban watershed influenced by human fecal pollution and to determine if the less specific marker may be used to identify the locations of broken or leaking sewer lines. Samples were collected from 19 stream locations on 10 dates. Sample DNA was extracted and qPCR analysis was conducted for the HuBac and qHF183 biomarkers. The HuBac biomarker was detected more frequently than the qHF183 biomarker and in greater quantities when both were detected. A strong linear relationship ( = 0.91) between the two markers was observed in 219 samples taken from both the watershed and inlet sewage. The relationship between the two biomarkers showed less variance at higher concentrations. However, even when the inlet sewage samples were excluded from the dataset, a clear linear relationship remained ( = 0.74). The results indicate that use of a less specific, but more sensitive, biomarker may provide greater utility when the prevention of false negatives is necessary and the primary fecal source is known, as in spatial distribution studies of human fecal pollution in an urban watershed.

Peracetic acid as an alternative disinfection technology for wet weather flows.

Rain-induced wet weather flows (WWFs) consist of combined sewer overflows, sanitary sewer overflows, and stormwater, all of which introduce pathogens to surface waters when discharged. When people come into contact with the contaminated surface water, these pathogens can be transmitted resulting in severe health problems. As such, WWFs should be disinfected. Traditional disinfection technologies are typically cost-prohibitive, can yield toxic byproducts, and space for facilities is often limited, if available. More cost-effective alternative technologies, requiring less space and producing less harmful byproducts are currently being explored. Peracetic acid (PAA) was investigated as one such alternative and this research has confirmed the feasibility and applicability of using PAA as a disinfectant for WWFs. Peracetic acid doses ranging from 5 mg/L to 15 mg/L over contact times of 2 to 10 minutes were shown to be effective and directly applicable to WWF disinfection.

Occupational exposure to trichloroethylene and cancer risk for workers at the Paducah Gaseous Diffusion Plant.

OBJECTIVE: The Paducah Gaseous Diffusion Plant (PGDP) became operational in 1952; it is located in the western part of Kentucky. We conducted a mortality study for adverse health effects that workers may have suffered while working at the plant, including exposures to chemicals. MATERIALS AND METHODS: We studied a cohort of 6820 workers at the PGDP for the period 1953 to 2003; there were a total of 1672 deaths to cohort members. Trichloroethylene (TCE) is a specific concern for this workforce; exposure to TCE occurred primarily in departments that clean the process equipment. The Life Table Analysis System (LTAS) program developed by NIOSH was used to calculate the standardized mortality ratios for the worker cohort and standardized rate ratio relative to exposure to TCE (the U.S. population is the referent for ageadjustment). LTAS calculated a significantly low overall SMR for these workers of 0.76 (95% CI: 0.72-0.79). A further review of three major cancers of interest to Kentucky produced significantly low SMR for trachea, bronchus, lung cancer (0.75, 95% CI: 0.72-0.79) and high SMR for Non-Hodgkin's lymphoma (NHL) (1.49, 95% CI: 1.02-2.10). RESULTS: No significant SMR was observed for leukemia and no significant SRRs were observed for any disease. Both the leukemia and lung cancer results were examined and determined to reflect regional mortality patterns. However, the Non-Hodgkin's Lymphoma finding suggests a curious amplification when living cases are included with the mortality experience. CONCLUSIONS: Further examination is recommended of this recurrent finding from all three U.S. Gaseous Diffusion plants.

Time-period mortality patterns in a Gaseous Diffusion Plant workforce.

BACKGROUND: We sub-divided a cohort of 6820 workers at the Paducah (KY) Gaseous Diffusion Plant (PGDP) which was traced from 1953 to 2003. The subdivisions were made to assess the mortality risks in a sub-group of workers employed solely during the plant's refit period, a time of suspected higher exposure to metal dusts (nickel, arsenic, chromium and uranium) and trichloroethylene. METHODS: This article describes a comparison of exposures and causes of death for 754 workers employed exclusively during the period of 1975-1979, with 1554 workers who worked in this period as well as other years. This interval was when the gaseous diffusion cascade facilities were re-fit. The workers employed 'only' during this period have a variety of deterministic factors (age-at-hire, duration of employment) that distinguish this sub-group of employees from the 'long-term' workforce. RESULTS: The 'only 1975-1979' workers had a larger fraction of minorities and female workers. This 'only' sub-group was disproportionately employed in unskilled labor positions. The 'only' workers were younger than the referent group, and a 14-year earlier mean age at death. The all-cause mortality standardized rate ratio [SRR] was 1.58 [95% Cl: 0.97-2.42]. The 'only' group was significantly different from the 'ever' workers with respect to suicides, SRR = 3.74 [95% Cl: 1.86-6.69], and for homicides, SRR = 11.71 [95% Cl: 3.20-30.03]. CONCLUSIONS: These elevated mortality risks do not seem to be due to PGDP employment exposures to metal dusts or trichloroethylene. Socio-economic factors may be a determinant for the patterns of suicides and murders described for this sub-group of employees. These findings provide guidance for communities with a dominant local employer. Persons who experience short-term hiring may warrant public health services to mitigate their risk of tragic deaths. A case-control study of these deaths is recommended to clarify individual risk behaviors.

Mortality patterns among Paducah Gaseous Diffusion Plant workers.

OBJECTIVE: To determine whether Paducah Gaseous Diffusion Plant workers had mortality patterns that differed from the general US population and to investigate whether mortality patterns were associated with job title or workplace exposures. METHODS: A retrospective occupational cohort mortality study was conducted on 6759 workers. Standardized mortality ratio analyses compared the cohort with the referent US population. Internal comparisons producing standardized rate ratios were conducted by job title, metal exposure, and cumulative internal and external radiation exposures. RESULTS: Overall mortality and cancer rates were lower than the referent population, reflecting a strong healthy worker effect. Individual not significant standardized mortality ratios and standardized rate ratios were noted for cancers of the lymphatic and hematopoietic tissue. CONCLUSIONS: Although relatively low exposures to radiation and metals did not produce statistically significant health effects, no significant elevations for lymphatic and hematopoietic cancers were consistent with previous studies of nuclear workers.

Backfilling missing microbial concentrations in a riverine database using artificial neural networks.

Predicting peak pathogen loadings can provide a basis for watershed and water treatment plant management decisions that can minimize microbial risk to the public from contact or ingestion. Artificial neural network models (ANN) have been successfully applied to the complex problem of predicting peak pathogen loadings in surface waters. However, these data-driven models require substantial, multiparameter databases upon which to train, and missing input values for pathogen indicators must often be estimated. In this study, ANN models were evaluated for backfilling values for individual observations of indicator bacterial concentrations in a river from 44 other related physical, chemical, and bacteriological data contained in a multi-year database. The ANN modeling approach provided slightly superior predictions of actual microbial concentrations when compared to conventional imputation and multiple linear regression models. The ANN model provided excellent classification of 300 randomly selected, individual data observations into two defined ranges for fecal coliform concentrations with 97% overall accuracy. The application of the relative strength effect (RSE) concept for selection of input variables for ANN modeling and an approach for identifying anomalous data observations utilizing cross validation with ANN model are also presented.

Artificial neural network prediction of viruses in shellfish.

A database was probed with artificial neural network (ANN) and multivariate logistic regression (MLR) models to investigate the efficacy of predicting PCR-identified human adenovirus (ADV), Norwalk-like virus (NLV), and enterovirus (EV) presence or absence in shellfish harvested from diverse countries in Europe (Spain, Sweden, Greece, and the United Kingdom). The relative importance of numerical and heuristic input variables to the ANN model for each country and for the combined data was analyzed with a newly defined relative strength effect, which illuminated the importance of bacteriophages as potential viral indicators. The results of this analysis showed that ANN models predicted all types of viral presence and absence in shellfish with better precision than MLR models for a multicountry database. For overall presence/absence classification accuracy, ANN modeling had a performance rate of 95.9%, 98.9%, and 95.7% versus 60.5%, 75.0%, and 64.6% for the MLR for ADV, NLV, and EV, respectively. The selectivity (prediction of viral negatives) was greater than the sensitivity (prediction of viral positives) for both models and with all virus types, with the ANN model performing with greater sensitivity than the MLR. ANN models were able to illuminate site-specific relationships between microbial indicators chosen as model inputs and human virus presence. A validation study on ADV demonstrated that the MLR and ANN models differed in sensitivity and selectivity, with the ANN model correctly identifying ADV presence with greater precision.

Comparison of bacteriophages for use in iodine inactivation: batch and continuous flow studies.

Inactivation rates in batch studies for four commonly used surrogate bacteriophages were measured in stable aqueous iodine solutions for the purpose of determining which was the most suited to evaluate iodine disinfection efficacy in batch and continuous flow conditions. Two types of group Leviviridae bacteriophages were used, Type I (MS2) and Type II (GA), along with group Microviridae, Phi-X174, and group Tectiviridae, PRD1. Inactivation was compared at iodine doses of 1.0-1.5 mg l2/l. MS2 was the most susceptible to iodine inactivation of the four phages tested. Inactivation of naked, icosahedral bacteriophages, MS2 and Phi-X174 demonstrated removals to below detection limits (>99.99%) in less than 10 min. Lipid-containing PRD1 and F+ssRNA GA bacteriophages demonstrated the greatest iodine resistance in batch experiments with an average of 1.82 logs of inactivation (98.5%) after 60 min and 1.05 logs of inactivation (91.1%) after 30 min respectively. Similarly, in continuous flow studies through pentaiodide quaternary ammonium strong base resin, MS2, GA and Phi-X174 were more strongly inactivated than PRD1. The lipid component of PRD1 is thought to enhance resistance to iodine over non-lipid-containing bacteriophages by protecting easily oxidized groups on the protein capsid, but further research is needed before proving this hypothesis. The results from this research may provide a surrogate standard for more rigorous and developed research into the mode of iodine disinfection and its inactivation kinetics.

A neural-network-based classification scheme for sorting sources and ages of fecal contamination in water.

Artificial neural networks (ANNs) were successfully applied to data observations from a small watershed consisting of commonly measured indicator bacteria, weather conditions, and turbidity to distinguish between human sewage and animal-impacted runoff, fresh runoff from aged, and agricultural land-use-associated fresh runoff from that of suburban land-use-associated-fresh runoff. The ANNs were applied in a cascading, or hierarchical scheme. ANN performance was measured in two ways: (1) training and (2) testing. An ANN was able to sort sewage from runoff with < 1% error. Turbidity was found to be relatively unimportant for sorting sewage from runoff, while gross measurements of gram-negative and gram-positive bacteria were required. Predictions clustered tightly around the known values. ANN classification of aged suburban runoff from fresh, and agricultural runoff from suburban was accomplished with > 90% accuracy.