Persistence of fecal indicator bacteria and associated genetic markers from wastewater treatment plant effluents in freshwater microcosms

Limited information exists on the environmental persistence of genetic markers for fecal indicator bacteria (FIB) in treated wastewaters. Here, the decay rate constants of culturable cells and genetic markers for four diverse groups of FIBs, such as enterococci, Clostridium, Escherichia coli, and Bacteroides, were investigated in freshwater microcosms seeded with disinfected and non-disinfected secondary-treated wastewaters. Decay rate constants of genetic markers and culturable cells varied significantly among the different FIB groups. Water temperatures (winter vs. fall/spring/summer) significantly affected the decay of all genetic marker and cell types; however, genetic marker decay were not found to be significantly different in disinfected (chlorination/ultraviolet) and non-disinfected wastewater-seeded microcosms or, for example, lake- and river-receiving waters. No evidence was seen that decay rate constants of FIB genetic markers from treated wastewater were substantially different from those observed in similar, previously reported microcosm studies using raw sewage. Unexpected relationships between decay rate constants of genetic markers and culturable cells of Bacteroides were observed. Results suggest that decay rate constants of FIB genetic markers determined from other studies may be applicable to treated wastewaters. Results of this study should be informative for ongoing efforts to determine the persistence of FIB genetic markers relative to surviving pathogens after wastewater treatment.

Antibiotic Resistance Genes and Potentially Pathogenic Bacteria in the Central Adriatic Sea: Are They Connected to Urban Wastewater Inputs?

Despite last decades’ interventions within local and communitarian programs, the Mediterranean Sea still receives poorly treated urban wastewater (sewage). Wastewater treatment plants (WWTPs) performing primary sewage treatments have poor efficiency in removing microbial pollutants, including fecal indicator bacteria, pathogens, and mobile genetic elements conferring resistance to antimicrobials. Using a combination of molecular tools, we investigated four urban WWTPs (i.e., two performing only mechanical treatments and two performing a subsequent conventional secondary treatment by activated sludge) as continuous sources of microbial pollution for marine coastal waters. Sewage that underwent only primary treatments was characterized by a higher content of traditional and alternative fecal indicator bacteria, as well as potentially pathogenic bacteria (especially Acinetobacter, Coxiella, Prevotella, Streptococcus, Pseudomonas, Vibrio, Empedobacter, Paracoccus, and Leptotrichia), than those subjected to secondary treatment. However, seawater samples collected next to the discharging points of all the WWTPs investigated here revealed a marked fecal signature, despite significantly lower values in the presence of secondary treatment of the sewage. WWTPs in this study represented continuous sources of antibiotic resistance genes (ARGs) ermB, qnrS, sul2, tetA, and blaTEM (the latter only for three WWTPs out of four). Still, no clear effects of the two depuration strategies investigated here were detected. Some marine samples were identified as positive to the colistin-resistance gene mcr-1, an ARG that threatens colistin antibiotics’ clinical utility in treating infections with multidrug-resistant bacteria. This study provides evidence that the use of sole primary treatments in urban wastewater management results in pronounced inputs of microbial pollution into marine coastal waters. At the same time, the use of conventional treatments does not fully eliminate ARGs in treated wastewater. The complementary use of molecular techniques could successfully improve the evaluation of the depuration efficiency and help develop novel solutions for the treatment of urban wastewater.

Enhancement of sunlight irradiation for wastewater disinfection by mixing with seawater

There is a need for developing a simple and easy-to-maintain disinfection technique for sewage treatment for use in developing countries and disaster-affected areas. We propose a novel disinfection technology that inactivates bacteria in wastewater via sunlight irradiation under high salt concentration by mixing with seawater. The disinfection efficiency of the proposed method was quantitatively evaluated and examined using fecal indicator bacteria. When the salinity in wastewater was adjusted to 30 practical salinity units by mixing with seawater, the constant of inactivation irradiation energy Ks (m2/MJ) was 1.6–2.2-fold greater than that without seawater for total coliforms and Escherichia coli. By contrast, although enterococci were inactivated by sunlight irradiation, an increase in salinity did not enhance disinfection. On setting the irradiation energy of sunlight to 5.5 MJ/m2, >99% of the fecal indicator bacteria were inactivated. Finally, we examined the relationship between the attenuation of irradiance and water depth and accordingly proposed a design of a treatment system wherein wastewater and seawater were adequately mixed and passed via a disinfection tank under the natural flow with sunlight irradiation.

Systematic review of predictive models of microbial water quality at freshwater recreational beaches

Monitoring of fecal indicator bacteria at recreational waters is an important public health measure to minimize water-borne disease, however traditional culture methods for quantifying bacteria can take 18–24 hours to obtain a result. To support real-time notifications of water quality, models using environmental variables have been created to predict indicator bacteria levels on the day of sampling. We conducted a systematic review of predictive models of fecal indicator bacteria at freshwater recreational sites in temperate climates to identify and describe the existing approaches, trends, and their performance to inform beach water management policies. We conducted a comprehensive search strategy, including five databases and grey literature, screened abstracts for relevance, and extracted data using structured forms. Data were descriptively summarized. A total of 53 relevant studies were identified. Most studies (n = 44, 83%) were conducted in the United States and evaluated water quality using E. coli as fecal indicator bacteria (n = 46, 87%). Studies were primarily conducted in lakes (n = 40, 75%) compared to rivers (n = 13, 25%). The most commonly reported predictive model-building method was multiple linear regression (n = 37, 70%). Frequently used predictors in best-fitting models included rainfall (n = 39, 74%), turbidity (n = 31, 58%), wave height (n = 24, 45%), and wind speed and direction (n = 25, 47%, and n = 23, 43%, respectively). Of the 19 (36%) studies that measured accuracy, predictive models averaged an 81.0% accuracy, and all but one were more accurate than traditional methods. Limitations identifed by risk-of-bias assessment included not validating models (n = 21, 40%), limited reporting of whether modelling assumptions were met (n = 40, 75%), and lack of reporting on handling of missing data (n = 37, 70%). Additional research is warranted on the utility and accuracy of more advanced predictive modelling methods, such as Bayesian networks and artificial neural networks, which were investigated in comparatively fewer studies and creating risk of bias tools for non-medical predictive modelling.

Monitoring Urban Beach Quality on a Summer Day: Determination of the Origin of Fecal Indicator Bacteria and Antimicrobial Resistance at Prophète Beach, Marseille (France)

A highly frequented beach in Marseille, France, was monitored on an hourly basis during a summer day in July 2018, to determine possible water and sand fecal pollution, in parallel with influx of beach users from 8 a.m. to 8 p.m. Fecal indicator bacteria were enumerated, together with four host-associated fecal molecular markers selected to discriminate human, dog, horse, or gull/seagull origins of the contamination. The antimicrobial resistance of bacteria in water and sand was evaluated by quantifying (i) the class 1, 2, and 3 integron integrase genes intI, and (ii) blaTEM, blaCTX–M, and blaSHV genes encoding endemic beta-lactamase enzymes. The number of beach users entering and leaving per hour during the observation period was manually counted. Photographs of the beach and the bathing area were taken every hour and used to count the number of persons in the water and on the sand, using a photo-interpretation method. The number of beach users increased from early morning to a peak by mid-afternoon, totaling more than 1,800, a very large number of users for such a small beach (less than 1 ha). An increase in fecal contamination in the water corresponded to the increase in beach attendance and number of bathers, with maximum numbers observed in the mid-afternoon. The human-specific fecal molecular marker HF183 indicated the contamination was of human origin. In the water, the load of Intl2 and 3 genes was lower than Intl1 but these genes were detected only during peak attendance and highest fecal contamination. The dynamics of the genes encoding B-lactamases involved in B-lactams resistance notably was linked to beach attendance and human fecal contamination. Fecal indicator bacteria, integron integrase genes intI, and genes encoding B-lactamases were detected in the sand. This study shows that bathers and beach users can be significant contributors to contamination of seawater and beach sand with bacteria of fecal origin and with bacteria carrying integron-integrase genes and beta lactamase encoding genes. High influx of users to beaches is a significant factor to be considered in order to reduce contamination and manage public health risk.

Bacteriophages as Fecal Pollution Indicators

Bacteriophages are promising tools for the detection of fecal pollution in different environments, and particularly for viral pathogen risk assessment. Having similar morphological and biological characteristics, bacteriophages mimic the fate and transport of enteric viruses. Enteric bacteriophages, especially phages infecting Escherichia coli (coliphages), have been proposed as alternatives or complements to fecal indicator bacteria. Here, we provide a general overview of the potential use of enteric bacteriophages as fecal and viral indicators in different environments, as well as the available methods for their detection and enumeration, and the regulations for their application.