Coliphages as indicators of primary wastewater treatment efficiency by constructed wetlands.

Constructed wetlands are an efficient and cost-effective system for the treatment of wastewater that can be reused for diverse purposes, including irrigation; however, few studies have determined the efficiency of microbial removal by constructed wetlands in tropical regions. Therefore, the present study aimed to determine the microbial quality of the influent and effluent of a constructed wetland in Puerto Rico, using traditional bacterial indicators (i.e., thermotolerant coliforms and enterococci), as well as somatic and male-specific (F+) coliphages. Results showed that over 99.9 and 97.7% of thermotolerant coliforms and enterococci were removed after treatment by constructed wetlands, respectively. Notably, approximately 84.0% of male-specific (F+) coliphages were removed, while somatic and total coliphages exhibited differing removal percentages at different steps during treatment by constructed wetlands. The potential risk of the presence of enteric viruses in treated wastewater by constructed wetlands may increase when considering traditional bacterial indicators exclusively. The present study may aid in the efforts to determine public health concerns associated with the exposure of bioaerosols resulting from wastewater treatment by constructed wetlands.

MULTI-OMICS as Invaluable Tools for the Elucidation of Host-Microbe-Microbiota Interactions.

"Omics" is becoming an increasingly recognizable term, even to the general public, as it is used more and more often in everyday scientific research [...].

Multiomics and Health: A Holistic Approach to Better Understand the Role of the Microbiome.

The present Special Issue focuses on the latest approaches to health and public health microbiology using multiomics [...].

Microbiota dispersion in the Uyuni salt flat (Bolivia) as determined by community structure analyses.

Soil microbial communities are an important component of biological diversity and terrestrial ecosystems which is responsible for processes such as decomposition, mineralization of nutrients, and accumulation of organic matter. One of the factors that provide information on the mechanisms regulating biodiversity is spatial scaling. We characterized the microbial communities using 16S rRNA gene sequences from DNA isolated from halite at various locations and correlated these to geographic distance in the Uyuni salt flat (Bolivia). Sequences from each site were analyzed to determine any spatial patterns of diversity, as well as to describe the microbial communities. Results suggest that different taxa are able to disperse over Uyuni's surface crust regardless of distance. As expected, ubiquitous taxa included members of Halobacteriaceae such as Haloarcula, Halorubrum, Halorhabdus, Halolamina, and halophilic bacteria Salinibacter, Halorhodospira, and unclassified members of the Gammaproteobacteria. Archaeal communities were homogeneous across the salt flat. In contrast, bacterial communities present strong local variations which could be attributed to external factors. Likely sources for these variations are the Rio Grande river influent in the south shore and the Tunupa volcano influencing the northern area.

Assessing the microbial quality of a tropical watershed with an urbanization gradient using traditional and alternate fecal indicators.

Urbanization affects the microbial loading into tropical streams, but its impact on water quality varies across watersheds. Rainfall in tropical environments also complicates microbial dynamics due to high seasonal and annual variations. Understanding the dynamics of fecal contamination in tropical surface waters may be further hindered by limitations from the utilization of traditional microbial indicators. We measured traditional (Enterococcus spp. and Escherichia coli), as well as alternate (enterophages and coliphages) indicators of fecal contamination in a tropical watershed in Puerto Rico during a 1-year period, and examined their relationship with rainfall events across an urbanization gradient. Enterococcus spp. and E. coli concentrations were 4 to 5 logs higher in non-urbanized or pristine sites when compared to enterophages and coliphages, suggesting that traditional fecal indicator bacteria may be natural inhabitants of pristine tropical waters. All of the tested indicators were positively correlated with rainfall and urbanization, except in the most urbanized sites, where rainfall may have had a dilution effect. The present study indicates that utilizing novel indicators of microbial water quality may improve the assessment of fecal contamination and pathogen risk for tropical watersheds.

Prospective epidemiological pilot study on the morbidity of bathers exposed to tropical recreational waters and sand.

A prospective cohort epidemiological pilot study was performed at three tropical beaches with point- and non-point-sources of fecal pollution to characterize the risk of illness among swimmers and non-swimmers. There was an increased risk of illness in swimmers as compared to non-swimmers, even when waters met current microbial standards for recreational water quality. Illnesses included gastrointestinal (GI), skin and respiratory symptoms, earache and fever. Odds ratios (ORs) ranged from 0.32 to 42.35 (GI illness), 0.69 to 3.12 (skin infections), 0.71 to 3.21 (respiratory symptoms), 0.52 to 15.32 (earache) and 0.80 to 1.68 (fever), depending on the beach sampled. The indicators that better predicted the risks of symptoms (respiratory) in tropical recreational waters were total (somatic and male-specific) coliphages (OR = 1.56, p < 0.10, R(2) = 3.79%) and Escherichia coli (OR = 1.38, p < 0.10, R(2) = 1.97%). The present study supports the potential of coliphages as good predictors of risks of respiratory illness in tropical recreational waters. This is the first study that has determined risks of illness after exposure to tropical recreational waters with point- and non-point sources of fecal contamination. The results give an opportunity to perform epidemiological studies in tropical recreational waters in Puerto Rico which can include more participants and other indicators and detection techniques.

Tracking the primary sources of fecal pollution in a tropical watershed in a one-year study.

A study was conducted to determine the primary sources of fecal pollution in a subtropical watershed using host-specific assays developed in temperate regions. Water samples (n = 534) from 10 different sites along the Rio Grande de Arecibo (RGA) watershed were collected mostly on a weekly basis (54 sampling events) during 13 months. DNA extracts from water samples were used in PCR assays to determine the occurrence of fecal bacteria (Bacteroidales, Clostridium coccoides, and enterococci) and human-, cattle-, swine-, and chicken-specific fecal sources. Feces from 12 different animals (n = 340) and wastewater treatment samples (n = 16) were analyzed to determine the specificity and distribution of host-specific assays. The human-specific assay (HF183) was found to be highly specific, as it did not cross-react with nontarget samples. The cattle marker (CF128) cross-reacted to some extent with swine, chicken, and turkeys and was present in 64% of the cattle samples tested. The swine assays showed poor host specificity, while the three chicken assays showed poor host distribution. Differences in the detection of host-specific markers were noted per site. While human and cattle assays showed moderate average detection rates throughout the watershed, areas impacted by wastewater treatment plants and cattle exhibited the highest prevalence of these markers. When conditional probability for positive signals was determined for each of the markers, the results indicated higher confidence levels for the human assay and lower levels for all the other assays. Overall, the results from this study suggest that additional assays are needed, particularly to track cattle, chicken, and swine fecal pollution sources in the RGA watershed. The results also suggest that the geographic stability of genetic markers needs to be determined prior to conducting applied source tracking studies in tropical settings.

Antibiotic-resistance and virulence genes in Enterococcus isolated from tropical recreational waters.

The prevalence of enterococci harboring tetracycline- and vancomycin-resistance genes, as well as the enterococcal surface protein (esp) has mostly been determined in clinical settings, but their prevalence in tropical recreational waters remains largely unknown. The present study determined the prevalence of tetM (tetracycline-resistance), vanA and vanB (vancomycin-resistance) in the bacterial and viral fractions, enterococci and their induced phages isolated from tropical recreational marine and fresh waters, dry and wet sands. Since lysogenic phages can act as vectors for antibiotic-resistance and virulence factors, the prevalence of the mentioned genes, as well as that of an integrase-encoding gene (int) specific for Enterococcus faecalis phages was determined. Up to 60 and 54% of the bacterial fractions and enterococci, respectively, harbored at least one of the tested genes suggesting that bacteria in tropical environments may be reservoirs of antibiotic-resistance and virulence genes. int was detected in the viral fractions and in one Enterococcus isolate after induction. This study presents the opportunity to determine if the presence of bacteria harboring antibiotic-resistance and virulence genes in tropical recreational waters represents a threat to public health.

Evaluation of Enterococcus-infecting phages as indices of fecal pollution.

No microbial source tracking tool satisfies all the characteristics of an ideal indicator of human fecal pollution. For this reason, the potential of Enterococcus faecalis phages (enterophages) as markers of this type of contamination was tested by using eight Enterococcus type strains as the possible hosts. The prevalence of enterophages in animal feces and domestic sewage were determined, as were the inactivation rates in raw sewage at 4 °C and surface and tap waters at 22 °C. Enterophages were exclusively detected in raw sewage (up to 66.0 plaque forming units (PFU)/100 mL), suggesting a strictly human origin; and exhibited inactivation rates of approximately 0.002 to 0.05, 0.3 to 0.5 and 0.4 to 1.4 log day(-1) in raw sewage and surface and tap waters, respectively, similar to those of previous reports on human enteric viruses under similar conditions. Interestingly, phages infecting other Enterococcus type strains were detected in both animal feces and domestic sewage in concentrations of up to 335.8 PFU/g and 96.0 PFU/100 mL, and certain phage isolates infected several of the strains tested. This clearly indicates the possible promiscuous nature of some Enterococcus phages and thus opens up the opportunity to further characterize these as indices of specific fecal sources.

Deciphering Diets and Lifestyles of Prehistoric Humans through Paleoparasitology: A Review.

Parasites have affected and coevolved with humans and animals throughout history. Evidence of ancient parasitic infections, particularly, reside in archeological remains originating from different sources dating to various periods of times. The study of ancient parasites preserved in archaeological remains is known as paleoparasitology, and it initially intended to interpret migration, evolution, and dispersion patterns of ancient parasites, along with their hosts. Recently, paleoparasitology has been used to better understand dietary habits and lifestyles of ancient human societies. Paleoparasitology is increasingly being recognized as an interdisciplinary field within paleopathology that integrates areas such as palynology, archaeobotany, and zooarchaeology. Paleoparasitology also incorporates techniques such as microscopy, immunoassays, PCR, targeted sequencing, and more recently, high-throughput sequencing or shotgun metagenomics to understand ancient parasitic infections and thus interpret migration and evolution patterns, as well as dietary habits and lifestyles. The present review covers the original theories developed in the field of paleoparasitology, as well as the biology of some parasites identified in pre-Columbian cultures. Conclusions, as well as assumptions made during the discovery of the parasites in ancient samples, and how their identification may aid in better understanding part of human history, ancient diet, and lifestyles are discussed.

Edible flora in pre-Columbian Caribbean coprolites: Expected and unexpected data.

Coprolites, or mummified feces, are valuable sources of information on ancient cultures as they contain ancient DNA (aDNA). In this study, we analyzed ancient plant DNA isolated from coprolites belonging to two pre-Columbian cultures (Huecoid and Saladoid) from Vieques, Puerto Rico, using shotgun metagenomic sequencing to reconstruct diet and lifestyles. We also analyzed DNA sequences of putative phytopathogenic fungi, likely ingested during food consumption, to further support dietary habits. Our findings show that pre-Columbian Caribbean cultures had a diverse diet consisting of maize (Zea mays), sweet potato (Ipomoea batatas), chili peppers (Capsicum annuum), peanuts (Arachis spp.), papaya (Carica papaya), tomato (Solanum lycopersicum) and, very surprisingly cotton (Gossypium barbadense) and tobacco (Nicotiana sylvestris). Modelling of putative phytopathogenic fungi and plant interactions confirmed the potential consumption of these plants as well as edible fungi, particularly Ustilago spp., which suggest the consumption of maize and huitlacoche. These findings suggest that a variety of dietary, medicinal, and hallucinogenic plants likely played an important role in ancient human subsistence and societal customs. We compared our results with coprolites found in Mexico and the United States, as well as present-day faeces from Mexico, Peru, and the United States. The results suggest that the diet of pre-Columbian cultures resembled that of present-day hunter-gatherers, while agriculturalists exhibited a transitional state in dietary lifestyles between the pre-Columbian cultures and larger scale farmers and United States individuals. Our study highlights differences in dietary patterns related to human lifestyles and provides insight into the flora present in the pre-Columbian Caribbean area. Importantly, data from ancient fecal specimens demonstrate the importance of ancient DNA studies to better understand pre-Columbian populations.

Microbial quality of tropical inland waters and effects of rainfall events.

Novel markers of fecal pollution in tropical waters are needed since conventional methods recommended for other geographical regions may not apply. To address this, the prevalence of thermotolerant coliforms, enterococci, coliphages, and enterophages was determined by culture methods across a watershed. Additionally, human-, chicken-, and cattle-specific PCR assays were used to identify potential fecal pollution sources in this watershed. An enterococcus quantitative PCR (qPCR) assay was tested and correlated with culture methods at three sites since water quality guidelines could incorporate this technique as a rapid detection method. Various rainfall events reported before sample collection at three sites were considered in the data analyses. Thermotolerant coliforms, enterococci, coliphages, and enterophages were detected across the watershed. Human-specific Bacteroides bacteria, unlike the cattle- and chicken-specific bacteria, were detected mostly at sites with the corresponding fecal impact. Enterococci were detected by qPCR as well, but positive correlations with the culture method were noted at two sites, suggesting that either technique could be used. However, no positive correlations were noted for an inland lake tested, suggesting that qPCR may not be suitable for all water bodies. Concentrations of thermotolerant coliforms and bacteriophages were consistently lower after rainfall events, pointing to a possible dilution effect. Rainfall positively correlated with enterococci detected by culturing and qPCR, but this was not the case for the inland lake. The toolbox of methods and correlations presented here could be potentially applied to assess the microbial quality of various water types.

Mycobiome-Host Coevolution? The Mycobiome of Ancestral Human Populations Seems to Be Different and Less Diverse Than Those of Extant Native and Urban-Industrialized Populations.

Few data exist on the human gut mycobiome in relation to lifestyle, ethnicity, and dietary habits. To understand the effect of these factors on the structure of the human gut mycobiome, we analyzed sequences belonging to two extinct pre-Columbian cultures inhabiting Puerto Rico (the Huecoid and Saladoid) and compared them to coprolite samples found in Mexico and Ötzi, the Iceman's large intestine. Stool mycobiome samples from extant populations in Peru and urban cultures from the United States were also included. The ancient Puerto Rican cultures exhibited a lower fungal diversity in comparison to the extant populations. Dissimilarity distances showed that the Huecoid gut mycobiome resembled that from ancient Mexico. Fungal genera including Aspergillus spp., Penicillium spp., Rasamsonia spp., Byssochlamys spp., Talaromyces spp., Blastomyces spp., Monascus spp., and Penicilliopsis spp. were differentially abundant in the ancient and extant populations. Despite cultural differences, certain fungal taxa were present in all samples. These results suggest that culture and diet may impact the gut mycobiome and emphasize that modern lifestyles could be associated with the alteration of gut mycobiome diversity. The present study presents data on ancient and extant human gut mycobiomes in terms of lifestyle, ethnicity, and diet in the Americas.

Pre-Columbian zoonotic enteric parasites: An insight into Puerto Rican indigenous culture diets and life styles.

The pre-Columbian Huecoid and Saladoid cultures were agricultural ethnic groups that supplemented their diets by fishing, hunting and scavenging. Archaeological deposits associated to these cultures contained a variety of faunal osseous remains that hinted at the cultures' diets. The present study identified zoonotic parasites that may have infected these two cultures as a result of their diets. We used metagenomic sequencing and microscopy data from 540-1,400 year old coprolites as well as the zooarchaeological data to recreate the possible interactions between zoonotic parasites and their hosts. Microscopy revealed Diphyllobothrium spp. and Dipylidium caninum eggs along with unidentified cestode and trematode eggs. DNA sequencing together with functional prediction and phylogenetic inference identified reads of Cryptosporidium spp., Giardia intestinalis and Schistosoma spp. The complimentary nature of the molecular, microscopy and zooarchaeology data provided additional insight into the detected zoonotic parasites' potential host range. Network modeling revealed that rodents and canids living in close proximity to these cultures were most likely the main source of these zoonotic parasite infections.

Cryptic lineages of the genus Escherichia.

Extended multilocus sequence typing (MLST) analysis of atypical Escherichia isolates was used to identify five novel phylogenetic clades (CI to CV) among isolates from environmental, human, and animal sources. Analysis of individual housekeeping loci showed that E. coli and its sister clade, CI, remain largely indistinguishable and represent nascent evolutionary lineages. Conversely, clades of similar age (CIII and CIV) were found to be phylogenetically distinct. When all Escherichia lineages (named and unnamed) were evaluated, we found evidence that Escherichia fergusonii has evolved at an accelerated rate compared to E. coli, CI, CIII, CIV, and CV, suggesting that this species is younger than estimated by the molecular clock method. Although the five novel clades were phylogenetically distinct, we were unable to identify a discriminating biochemical marker for all but one of them (CIII) with traditional phenotypic profiling. CIII had a statistically different phenotype from E. coli that resulted from the loss of sucrose and sorbitol fermentation and lysine utilization. The lack of phenotypic distinction has likely hindered the ability to differentiate these clades from typical E. coli, and so their ecological significance and importance for applied and clinical microbiology are yet to be determined. However, our sampling suggests that CIII, CIV, and CV represent environmentally adapted Escherichia lineages that may be more abundant outside the host gastrointestinal tract.

Commensal and Pathogenic Members of the Dental Calculus Microbiome of Badia Pozzeveri Individuals from the 11th to 19th Centuries.

The concept of the human oral microbiome was applied to understand health and disease, lifestyles, and dietary habits throughout part of human history. In the present study, we augment the understanding of ancient oral microbiomes by characterizing human dental calculus samples recovered from the ancient Abbey of Badia Pozzeveri (central Italy), with differences in socioeconomic status, time period, burial type, and sex. Samples dating from the Middle Ages (11th century) to the Industrial Revolution era (19th century) were characterized using high-throughput sequencing of the 16S ribosomal RNA (rRNA) gene V4 region. Consistent with previous studies, individuals from Badia Pozzeveri possessed commensal oral bacteria that resembled modern oral microbiomes. These results suggest that members of the oral microbiome are ubiquitous despite differences in geographical regions, time period, sex, and socioeconomic status. The presence of fecal bacteria could be in agreement with poor hygiene practices, consistent with the time period. Respiratory tract, nosocomial, and other rare pathogens detected in the dental calculus samples are intriguing and could suggest subject-specific comorbidities that could be reflected in the oral microbiome.

On Controls in Ancient Microbiome Studies, and Microbial Resilience in Ancient Samples.

In the following comment, we reply to Eisenhofer and Weyrich's letter "Proper authentication of ancient DNA is still essential" responding to the article "Gut Microbiome and Putative Resistome of Inca and Italian Nobility Mummies" by Santiago-Rodriguez et al. One of the concerns raised was the possibility that the patterns noted in the gut microbiome of pre-Inca/Inca and Italian nobility mummies were due to contamination of the blank control. When examining the blank controls and filtering the operational taxonomic units (OTUs) present in the blank controls, and further performing in-silico contamination analyses, we noticed very similar patterns as those previously reported. We also discuss controls in ancient microbiome studies, and aspects of microbial resilience in ancient samples.

Definitions and Historical Perspectives in Environmental Forensics.

Environmental forensics is a tool that uses chemical, physical, and statistical techniques to investigate contaminants in the environment as a means to determine attribution for legal purposes. Environmental microbiology is a branch of science that has benefited from the use of metagenomics. The term microbial forensics, which includes nucleic acid sequencing methods, is now used to investigate the sources of microorganisms for attribution purposes as well. Environmental microbial forensics can fully address the questions that must be answered for attribution of causation and subsequent remedial actions within a reasonably short time frame. Although sensu stricto forensics refers to obtaining scientific evidence to be presented during legal proceedings, the term sensu lato is used as a description of the procedures used to reconstruct previous events, such as contamination. The term microbial forensics was first used to describe a forensic science approach for attribution purposes, specifically for bioterror as a purposeful release of pathogen microorganisms, but it also especially refers to investigations on the inadvertent or accidental release of pathogenic agents. However, microbial forensics can be used to determine the source of a microorganism or a group of microorganisms, regardless of whether they are pathogenic or not. Microbial forensics has limitations, but it should be used as part of a toolbox of methods to be relied upon when doing forensic studies. Environmental microbial forensics can only benefit from the development of new methods, and we already are experiencing a paradigm change in terms of approaches to the forensic sciences.

Future Technologies.

Microbiome analysis of environmental samples may represent the next frontier in environmental microbial forensics. Next-generation sequencing technologies significantly increased the available genetic data that could be used as evidentiary material. It is not clear, however, whether the microbiome can scale across institutions using forensic-based evidence due to the data resource requirements and the associated costs of maintaining these databases. A successful microbiome study is impacted by the quality of the information gathered and the steps in sample processing and data analysis. To ascertain the validity of methods and the results obtained, there needs to be a stringent procedure to validate the methods and ensure that the results are comparable and reproducible, not only within the laboratory but also between laboratories conducting similar research. Of primary importance for meaningful microbiome studies is an experimental design that leads to carefully executed, controlled, and reproducible studies. The microbiome literature contains a fair share of anecdotal descriptions of microbial community composition and "diagnostic" relative abundance of the taxa therein. These studies are now being supplemented by experimental designs that feature repeated measurements, error estimates, correlations of microbiota with covariates, and increasingly sophisticated statistical tests that enhance the robustness of data analysis and study conclusions. It is imperative to be careful, especially when carrying out attribution studies, to be fully aware of the possible biases included in a specific sample being analyzed.

Proteus vulgaris - Pt electrode system for urea to nitrogen conversion in synthetic urine.

One of the most challenging problems when trying to recycle urine for different purposes is the removal of urea. In this project we studied an ureolysis system using the bacterium Proteus vulgaris for the transformation of urea to ammonia and its subsequent oxidation to nitrogen at a Pt working electrode. Our system was tested under different pH, microbial reaction times, and urea and bacteria concentrations. Our results indicate that a pH8 is optimal for the combined Proteus vulgaris urease activity and the ammonia oxidation reaction at a Pt electrode. The reaction time and concentration dependence on the ammonia oxidation reaction current densities was also studied. Results showed limited ammonia oxidation under high urea concentrations in ~2.5×109cfu/mL Proteus vulgaris in synthetic urine.