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.

Slovak Local Ewe’s Milk Lump Cheese, a Source of Beneficial Enterococcus durans Strain

Slovak ewe’s milk lump cheese is produced from unpasteurized ewe’s milk without any added culture. Because of the traditional processing and shaping by hand into a lump, this cheese was given the traditional specialty guaranteed (TSG) label. Up till now, there have existed only limited detailed studies of individual microbiota and their benefits in ewe’s milk lump cheese. Therefore, this study has been focused on the beneficial properties and safety of Enterococcus durans strains with the aim to contribute to basic dairy microbiology but also for further application potential and strategy. The total enterococcal count in cheeses reached 3.93 CFU/g (log 10) ± 1.98 on average. Based on a MALDI-TOF mass spectrometry evaluation, the strains were allotted to the species E. durans (score, 1.781–2.245). The strains were gelatinase and hemolysis-negative (γ-hemolysis) and were mostly susceptible to commercial antibiotics. Among the strains, E. durans ED26E/7 produced the highest value of lactase enzyme β-galactosidase (10 nmoL). ED26E/7 was absent of virulence factor genes such as Hyl (hyaluronidase), IS 16 element and gelatinase (GelE). To test safety, ED26E/7 did not cause mortality in Balb/c mice. Its partially purified bacteriocin substance showed the highest inhibition activity/bioactivity against Gram-positive indicator bacteria: the principal indicator Enterococcus avium EA5 (102,400 AU/mL), Staphylococcus aureus SA5 and listeriae (25,600 AU/mL). Moreover, 16 staphylococci (out of 22) were inhibited (100 AU/mL), and the growth of 36 (out of 51) enterococcal indicators was as well. After further technological tests, E. durans ED26E/7, with its bacteriocin substance, can be supposed as a promising additive to dairy products.

Bacterial Distribution and Community Structure in Beef Cattle Liver and Bile at Slaughter

In this study, the distribution of hygienic indicator bacteria in cattle livers and bile was examined at slaughterhouses. First, 127 cattle livers with gallbladders were carefully eviscerated from the carcasses at 10 slaughterhouses. Microbiological examination showed that 9 bile (7.1%) and 19 liver parenchyma (15.0%) samples were positive for the family Enterobacteriaceae (EB) with means ± SD of 3.68 ± 4.63 log CFU/mL and 1.59 ± 2.47 log CFU/g, respectively; thus, bacterial contamination was apparent even at the postevisceration stage. Subsequently, 70 cattle livers were obtained at the postprocessing/storage stage from 7 of the ten slaughterhouses; microbiological analysis revealed greater means of EB in the liver parenchyma (means ± SD of 3.00 ± 3.89 log CFU/g, P =0.011) than those at postevisceration stage, suggesting that bacterial dissemination and/or replication occurred in the liver parenchyma during processing and storage. According to 16S rRNA ion semiconductor sequencing analysis of representative samples from 12 cattle, Proteobacteria , Firmicutes , and Actinobacteria were dominant in both the parenchyma and bile, in which EB/ Escherichia coli were predominate among EB-rich livers. These results suggest that bile plays a role as a vehicle for bacterial transmission to the liver parenchyma. This is the first study to demonstrate bacterial distribution and community structure in the liver and biliary microecosystem of cattle at slaughter. Our data provide possible implication of EB testing in bile to screen cattle livers contaminated with high levels of fecal indicator bacteria.

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.

Sourcing Antibiotic-Resistant Escherichia coli in Aquatic Ecosystems: A Combined Laboratory and Field Module

The emergence of antibiotic-resistant bacteria represents a growing threat in aquatic ecosystems. In this combined field and laboratory activity, students will determine whether Escherichia coli , an indicator bacteria species commonly found in aquatic ecosystems, shows signs of resistance to common antibiotics.

The Influence of Environmental Conditions on the Antagonistic Activity of Lactic Acid Bacteria Isolated from Fermented Meat Products

The aim of this study was to determine the impact of environmental conditions on the antimicrobial properties of 21 lactic acid bacteria strains in the selected indicator bacteria. To assess the antimicrobial activity of the whole bacteria culture (WBC), the agar well diffusion method was used. The interference of LAB strains with the growth of the selected indicator bacteria was evaluated by incubating co-cultures in the food matrix. Based on the conducted research, it was found that environmental conditions have a significant impact on the antimicrobial activity of lactic acid bacteria strains. The highest antimicrobial activity was recorded under optimal conditions for the development of LAB, the incubation time being different depending on the indicator strain used. The tested LAB strains were characterized by a high ability to inhibit indicator strains, especially in the food matrix. These results led us to further characterize and purify the antimicrobial compound produced by lactic acid bacteria taking into account changing environmental conditions.

Systematic review of the relative concentrations of noroviruses and fecal indicator bacteria in wastewater: considerations for use in quantitative microbial risk assessment

Human noroviruses are a leading cause of food- and water-borne disease, which has led to an interest in quantifying norovirus health risks using quantitative microbial risk assessment (QMRA). Given the limited availability of quantitative norovirus data to input to QMRA models, some studies have applied a conversion factor to estimate norovirus exposure based on measured fecal indicator bacteria (FIB) concentrations. We conducted a review of peer-reviewed publications to identify the concentrations of noroviruses and FIB in raw, secondary-treated, and disinfected wastewater. A meta-analysis was performed to determine the ratios of norovirus-FIB pairs in each wastewater matrix and the variables that significantly impact these ratios. Norovirus-to-FIB ratios were found to be significantly impacted by the norovirus genotype, month of sample collection, geographic location, and the extent of wastewater treatment. Additionally, we evaluated the impact of using a FIB-to-virus conversion factor in QMRA and found that the choice of conversion ratio has a great impact on estimated health risks. For example, the use of a conversion ratio previously used in the World Health Organization Guidelines for the Safe Use of Wastewater, Excreta and Greywater predicted health risks that were significantly lower than those estimated with measured norovirus concentrations used as inputs. This work emphasizes the gold standard of using measured pathogen concentrations directly as inputs to exposure assessment in QMRA. While not encouraged, if one must use a FIB-to-virus conversion ratio to estimate norovirus dose, the ratio should be chosen carefully based on the target microorganisms (i.e., strain, genotype, or class), prevalence of disease, and extent of wastewater treatment.