The neutralizing effect of heparin on blood-derived antimicrobial compounds: impact on antibacterial activity and inflammatory response.

Acting through a combination of direct and indirect pathogen clearance mechanisms, blood-derived antimicrobial compounds (AMCs) play a pivotal role in innate immunity, safeguarding the host against invading microorganisms. Besides their antimicrobial activity, some AMCs can neutralize endotoxins, preventing their interaction with immune cells and avoiding an excessive inflammatory response. In this study, we aimed to investigate the influence of unfractionated heparin, a polyanionic drug clinically used as anticoagulant, on the endotoxin-neutralizing and antibacterial activity of blood-derived AMCs. Serum samples from healthy donors were pre-incubated with increasing concentrations of heparin for different time periods and tested against pathogenic bacteria (Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus) and endotoxins from E. coli, K. pneumoniae, and P. aeruginosa. Heparin dose-dependently decreased the activity of blood-derived AMCs. Consequently, pre-incubation with heparin led to increased activity of LPS and higher values of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6). Accordingly, higher concentrations of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa were observed as well. These findings underscore the neutralizing effect of unfractionated heparin on blood-derived AMCs in vitro and may lead to alternative affinity techniques for isolating and characterizing novel AMCs with the potential for clinical translation.

Performance of bacterial and mitochondrial qPCR source tracking methods: A European multi-center study.

With the advent of molecular biology diagnostics, different quantitative PCR assays have been developed for use in Source Tracking (ST), with none of them showing 100% specificity and sensitivity. Most studies have been conducted at a regional level and mainly in fecal slurry rather than in animal wastewater. The use of a single molecular assay has most often proven to fall short in discriminating with precision the sources of fecal contamination. This work is a multicenter European ST study to compare bacterial and mitochondrial molecular assays and was set to evaluate the efficiency of nine previously described qPCR assays targeting human-, cow/ruminant-, pig-, and poultry-associated fecal contamination. The study was conducted in five European countries with seven fecal indicators and nine ST assays being evaluated in a total of 77 samples. Animal fecal slurry samples and human and non-human wastewater samples were analyzed. Fecal indicators measured by culture and qPCR were generally ubiquitous in the samples. The ST qPCR markers performed at high levels in terms of quantitative sensitivity and specificity demonstrating large geographical application. Sensitivity varied between 73% (PLBif) and 100% for the majority of the tested markers. On the other hand, specificity ranged from 53% (CWMit) and 97% (BacR). Animal-associated ST qPCR markers were generally detected in concentrations greater than those found for the respective human-associated qPCR markers, with mean concentration for the Bacteroides qPCR markers varying between 8.74 and 7.22 log10 GC/10 mL for the pig and human markers, respectively. Bacteroides spp. and mitochondrial DNA qPCR markers generally presented higher Spearman's rank coefficient in the pooled fecal samples tested, particularly the human fecal markers with a coefficient of 0.79. The evaluation of the performance of Bacteroides spp., mitochondrial DNA and Bifidobacterium spp. ST qPCR markers support advanced pollution monitoring of impaired aquatic environments, aiming to elaborate strategies for target-oriented water quality management.

Genetic Microbial Source Tracking Support QMRA Modeling for a Riverine Wetland Drinking Water Resource.

Riverine wetlands are important natural habitats and contain valuable drinking water resources. The transport of human- and animal-associated fecal pathogens into the surface water bodies poses potential risks to water safety. The aim of this study was to develop a new integrative modeling approach supported by microbial source tracking (MST) markers for quantifying the transport pathways of two important reference pathogens, Cryptosporidium and Giardia, from external (allochthonous) and internal (autochthonous) fecal sources in riverine wetlands considering safe drinking water production. The probabilistic-deterministic model QMRAcatch (v 1.1 python backwater) was modified and extended to account for short-time variations in flow and microbial transport at hourly time steps. As input to the model, we determined the discharge rates, volumes and inundated areas of the backwater channel based on 2-D hydrodynamic flow simulations. To test if we considered all relevant fecal pollution sources and transport pathways, we validated QMRAcatch using measured concentrations of human, ruminant, pig and bird associated MST markers as well as E. coli in a Danube wetland area from 2010 to 2015. For the model validation, we obtained MST marker decay rates in water from the literature, adjusted them within confidence limits, and simulated the MST marker concentrations in the backwater channel, resulting in mean absolute errors of < 0.7 log10 particles/L (Kruskal-Wallis p > 0.05). In the scenarios, we investigated (i) the impact of river discharges into the backwater channel (allochthonous sources), (ii) the resuspension of pathogens from animal fecal deposits in inundated areas, and (iii) the pathogen release from animal fecal deposits after rainfall (autochthonous sources). Autochthonous and allochthonous human and animal sources resulted in mean loads and concentrations of Cryptosporidium and Giardia (oo)cysts in the backwater channel of 3-13 × 109 particles/hour and 0.4-1.2 particles/L during floods and rainfall events, and in required pathogen treatment reductions to achieve safe drinking water of 5.0-6.2 log10. The integrative modeling approach supports the sustainable and proactive drinking water safety management of alluvial backwater areas.

Bacteriophages Are Good Estimators of Human Viruses Present in Water.

The detection of fecal viral pathogens in water is hampered by their great variety and complex analysis. As traditional bacterial indicators are poor viral indicators, there is a need for alternative methods, such as the use of somatic coliphages, which have been included in water safety regulations in recent years. Some researchers have also recommended the use of reference viral pathogens such as noroviruses or other enteric viruses to improve the prediction of fecal viral pollution of human origin. In this work, phages previously tested in microbial source tracking studies were compared with norovirus and adenovirus for their suitability as indicators of human fecal viruses. The phages, namely those infecting human-associated Bacteroides thetaiotaomicron strain GA17 (GA17PH) and porcine-associated Bacteroides strain PG76 (PGPH), and the human-associated crAssphage marker (crAssPH), were evaluated in sewage samples and fecal mixtures obtained from different animals in five European countries, along with norovirus GI + GII (NoV) and human adenovirus (HAdV). GA17PH had an overall sensitivity of ≥83% and the highest specificity (>88%) for human pollution source detection. crAssPH showed the highest sensitivity (100%) and specificity (100%) in northern European countries but a much lower specificity in Spain and Portugal (10 and 30%, respectively), being detected in animal wastewater samples with a high concentration of fecal indicators. The correlations between GA17PH, crAssPH, or the sum of both (BACPH) and HAdV or NoV were higher than between the two human viruses, indicating that bacteriophages are feasible indicators of human viral pathogens of fecal origin and constitute a promising, easy to use and affordable alternative to human viruses for routine water safety monitoring.

Attachment and Detachment Behavior of Human Adenovirus and Surrogates in Fine Granular Limestone Aquifer Material.

The transport of human adenovirus, nanoparticles, and PRD1 and MS2 bacteriophages was tested in fine granular limestone aquifer material taken from a borehole at a managed aquifer recharge site in Adelaide, South Australia. Comparison of transport and removal of virus surrogates with the pathogenic virus is necessary to understand the differences between the virus and surrogate. Because experiments using pathogenic viruses cannot be done in the field, laboratory tests using flow-through soil columns were used. Results show that PRD1 is the most appropriate surrogate for adenovirus in an aquifer dominated by calcite material but not under high ionic strength or high pH conditions. It was also found that straining due to size and the charge of the colloid were not dominant removal mechanisms in this system. Implications of this study indicate that a certain surrogate may not represent a specific pathogen solely based on similar size, morphology, and/or surface charge. Moreover, if a particular surrogate is representative of a pathogen in one aquifer system, it may not be the most appropriate surrogate in another porous media system. This was apparent in the inferior performance of MS2 as a surrogate, which is commonly used in virus transport studies.

Enumerating Microorganism Surrogates for Groundwater Transport Studies Using Solid-Phase Cytometry.

Investigations on the pollution of groundwater with pathogenic microorganisms, e.g. tracer studies for groundwater transport, are constrained by their potential health risk. Thus, microspheres are often used in groundwater transport studies as non-hazardous surrogates for pathogenic microorganisms. Even though pathogenic microorganisms occur at low concentrations in groundwater, current detection methods of microspheres (spectrofluorimetry, flow cytometry and epifluorescence microscopy) have rather high detection limits and are unable to detect rare events. Solid-phase cytometry (SPC) offers the unique capability of reliably quantifying extremely low concentrations of fluorescently labelled microorganisms or microspheres in natural waters, including groundwater. Until now, microspheres have been used in combination with SPC only for instrument calibration purposes and not for environmental applications. In this study, we explored the limits of the SPC methodology for its applicability to groundwater transport studies. The SPC approach proved to be a highly sensitive and reliable enumeration system for microorganism surrogates down to a minimum size of 0.5 µm, in up to 500 ml of groundwater, and 0.75 µm, in up to 1 ml of turbid surface water. Hence, SPC is proposed to be a useful method for enumerating microspheres for groundwater transport studies in the laboratory, as well as in the field when non-toxic, natural products are used.