Boot Swabs to Evaluate Cleaning and Disinfection Success in Poultry Barns

Due to the relevance of cleaning and disinfection in farm hygiene management, accurate evaluation of the success of such procedures remains a fundamental challenge for producers. This study aimed to use boot swab sampling to quantify the effects of such practices in poultry barns. For this purpose, the counts of both the total and fecal indicator bacteria were detected after the application of a cleaning and disinfection protocol in identical barns that were occupied by turkeys and broilers. Boot swab samples were compared to an established agar contact plating method to evaluate disinfection success. Statistical analyses showed no correlations between the bacterial counts that were obtained with either method. In contrast to the agar contact plating method, boot swab sampling permitted the assessment of the hygienic conditions of the barn floors before and after cleaning procedures. Furthermore, according to observations with the boot swab method, factors related to the species being farmed influenced the initial bacterial loads but did not affect the effectiveness of cleaning and disinfection. Species identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) also suggested that non-fecal bacteria grow on selective media. Further studies should validate the use of this sampling technique by comparing different cleaning and disinfection protocols.

Environmental and Fecal Indicator Organisms on Fruit Contact Surfaces and Fruit from Blueberry Mechanical Harvesters

Although previous studies have examined microbial loads on food contact surfaces in blueberry packing plants, there is currently no information regarding microbial risks associated with mechanical berry harvesters used in commercial blueberry production. In this study, we surveyed up to nine fruit contact surfaces on seven mechanical harvesters in each of 2015 and 2016 in the field. These surfaces included the shaking rods at the front of the harvester, the sidewalls of the harvesting tunnel behind the shaking mechanism, the catcher plates collecting the detached berries, horizontal and vertical fruit conveyor belts, and berry lugs collecting the fruit at the back of the harvester. Swab samples were collected from each surface three times a day (morning, noon, and evening) and assessed for environmental and fecal indicator organisms including total aerobes, total yeasts and molds, coliforms and fecal coliforms, and enterococci. At the same time points, fruit samples were assessed for microbial loads before the fruit entered each harvester and after they exited the harvester. Results showed statistically significant differences in microbial loads among harvester surfaces, whereas the effect of sampling time was generally not significant. High levels of total aerobes and total yeasts and molds were recorded, especially on horizontal surfaces and/or those located at the bottom of the harvester such as the lower sidewall, the catcher plates, and the horizontal conveyor belt. These surfaces therefore should be targeted by cleaning and sanitization practices. There was also statistical evidence that passage through the harvester may increase the levels of the environmental microorganisms on fruit in the field. In contrast, fecal indicator organisms such as fecal coliforms and enterococci were detected only sporadically and at very low densities on harvester surfaces and blueberry fruit, and there was no evidence that passage through the harvester increased their levels on the fruit. Berry lugs consistently harbored microbial loads, and given their movement back and forth between the field and the packing plant, deserve particular attention with regard to cleaning, sanitization, and storage protocols.

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.

A Model System for Sensitive Detection of Viable E. coli Bacteria Combining Direct Viability PCR and a Novel Microarray-Based Detection Approach

We established an innovative approach that included direct, viability, and nested PCR for rapid and reliable identification of the fecal indicator organism Escherichia coli (E. coli). Direct PCR enabled successful amplification of the target uidA gene, omitting a prior DNA isolation or purification step. Furthermore, we applied viability PCR (v-PCR) to ensure the detection of only relevant viable bacterial cells. The principle involves the binding of propidium monoazide (PMA), a selective nucleic acid intercalating dye, to accessible DNA of heat killed bacteria cells and, consequently, allows viable and heat killed E. coli cells to be discriminated. To ensure high sensitivity, direct v-PCR was followed by a nested PCR step. The resulting amplicons were analyzed by a rapid 30 min microarray-based DNA hybridization assay for species-specific DNA detection of E. coli. A positive signal was indicated by enzymatically generated silver nanoparticle deposits, which served as robust endpoint signals allowing an immediate visual readout. The presented novel protocol allows the detection of 1 × 101 viable E. coli cells per PCR run.

Evaluating Fecal Indicator and Pathogen Relationships in Sewage Impacted Surface Waters to Blend with Reclaimed Water for Potable Reuse in North Carolina

Surface waters used for drinking water supply often receive upstream wastewater effluent inputs, resulting in de facto wastewater reuse for drinking water and recreation. As populations grow, demands on water supplies increase. As this trend continues, it creates the need to understand the risks associated with such reuse. In North Carolina, potable reuse has been proposed as a combination of at least 80% surface water with up to 20% tertiary-treated, dual-disinfected, reclaimed wastewater, which is then stored for 5 days and further treated using conventional drinking water treatment methods. The state of North Carolina has set standards for both intake surface water and for the reclaimed water produced by wastewater utilities, using indicator microorganisms to measure compliance. The goal of this study was to quantify fecal indicator microorganisms, specifically E. coli, coliphages, and C. perfringens as well as key pathogens, specifically Salmonella spp. bacteria, adenoviruses, noroviruses, and the protozoan parasites Cryptosporidium and Giardia, in two types of water representing potential candidates for potable reuse in North Carolina, (1) run of river surface water and (2) sewage-impacted surface waters, with the purpose of determining if there are predictive relationships between these two microorganism groups that support microbial indicator reliability.

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.

Mapping global inputs and impacts from of human sewage in coastal ecosystems

Coastal marine ecosystems face a host of pressures from both offshore and land-based human activity. Research on terrestrial threats to coastal ecosystems has primarily focused on agricultural runoff, specifically showcasing how fertilizers and livestock waste create coastal eutrophication, harmful algae blooms, or hypoxic or anoxic zones. These impacts not only harm coastal species and ecosystems but also impact human health and economic activities. Few studies have assessed impacts of human wastewater on coastal ecosystems and community health. As such, we lack a comprehensive, fine-resolution, global assessment of human sewage inputs that captures both pathogens and nutrient flows to coastal waters and the potential impacts on coastal ecosystems. To address this gap, we use a new high-resolution geospatial model to measure and map nitrogen (N) and pathogen—fecal indicator organisms (FIO)—inputs from human sewage for ~135,000 watersheds globally. Because solutions depend on the source, we separate nitrogen and pathogen inputs from sewer, septic, and direct inputs. Our model indicates that wastewater adds 6.2Tg nitrogen into coastal waters, which is approximately 40% of total nitrogen from agriculture. Of total wastewater N, 63% (3.9Tg N) comes from sewered systems, 5% (0.3Tg N) from septic, and 32% (2.0Tg N) from direct input. We find that just 25 watersheds contribute nearly half of all wastewater N, but wastewater impacts most coastlines globally, with sewered, septic, and untreated wastewater inputs varying greatly across watersheds and by country. Importantly, model results find that 58% of coral and 88% of seagrass beds are exposed to wastewater N input. Across watersheds, N and FIO inputs are generally correlated. However, our model identifies important fine-grained spatial heterogeneity that highlight potential tradeoffs and synergies essential for management actions. Reducing impacts of nitrogen and pathogens on coastal ecosystems requires a greater focus on where wastewater inputs vary across the planet. Researchers and practitioners can also overlay these global, high resolution, wastewater input maps with maps describing the distribution of habitats and species, including humans, to determine the where the impacts of wastewater pressures are highest. This will help prioritize conservation efforts.Without such information, coastal ecosystems and the human communities that depend on them will remain imperiled.

Microbial Contamination Survey of Environmental Fresh and Saltwater Resources of Upolu Island, Samoa

Analysis of microbe diversity in freshwater resources and nearshore seawater samples of Upolu Island was performed to investigate the distribution of harmful bacteria. For this, 124 samples were collected from 23 river systems, two volcanic lakes, and 45 locations inside and outside the barrier reef of Upolu Island, Samoa. Physicochemical parameters for general water quality, detection of coliform bacteria and 16S rRNA amplicon sequencing were performed on all samples. Fecal indicator bacteria (FIB) testing indicated a wide distribution of coliform bacteria in all sampled freshwater sites with evidence of fecal coliform in most locations. Importantly, evidence of coliform bacteria was found in most seawater samples inside and along the reef, apart from those samples taken ~20 km offshore. Illumina 16S rRNA amplicon sequencing of the V4 hypervariable region confirmed the presence of various types of harmful bacterial species, namely from the Enterobacteriaceae, Enterococcae, Streptococcaceae, and Vibrionacea families. By combining the sensitivity of FIB testing and next-generation sequencing, we were able to show the extent of potential contaminations in fresh and seawater samples and simultaneously identify the potential pathogenic bacterial genera present. The wide distribution of potential harmful bacteria from river runoff or direct sewage dumping has an impact on human health, leading to many skin and intestinal diseases, and is potentially detrimental to coral reef community health.