COUPLING DNA LABELING AND NEXT-GENERATION SEQUENCING TECHNIQUES TO CHARACTERIZE METABOLICALLY-ACTIVE BACTERIA IN NONTRADITIONAL IRRIGATION WATER

Bacteria are ubiquitous in irrigation water resources and can include pathogens that may compromise food safety and public health. However, only a small fraction of total bacterial community members in water can be identified through standard culture-based laboratory methods. 16S rRNA and shotgun sequencing techniques have improved the identification of non-culturable bacteria in water resources. Nevertheless, because sequencing approaches are nucleic-acid based, they are unable to differentiate between the proportion of bacterial communities identified that are live and metabolically-active versus those that are represented by free, relic DNA, not present in viable cells. To bridge this knowledge gap, my dissertation research coupled DNA-labeling (using 5-bromo-2’- deoxyuridine (BrdU) and propidium monazide (PMA)) with next-generation sequencing approaches to identify and comprehensively characterize metabolically-active bacteria in multiple nontraditional irrigation water sources in the Mid-Atlantic region. My aims were as follows: 1) To characterize the metabolically-active fraction of bacterial communities, as well as antibiotic resistance genes and virulence gene profiles in nontraditional irrigation water sources; 2) To evaluate culture-dependent and -independent methods in the detection of metabolically-active pathogenic and non-pathogenic Vibrio species in four nontraditional irrigational water sources; and 3) To track metabolically-active bacterial communities from rooftop-harvested rainwater to irrigated produce in Maryland. Overall, we identified diverse metabolically-active bacterial communities in all nontraditional water sources. Notably, we observed the presence of viable bacteria of importance to both human and/or animal health (Actinobacterium spp., Flavobacterium spp., Aeromonas spp. Pseudomonas spp. and Vibrio spp.). Interestingly, diverse antimicrobial resistance and virulence genes were predominantly found in non-BrdU-treated samples, indicating that these genes can persist in relic DNA and could be transferred to other environmental bacteria through transformation events. We also source-tracked viable bacteria, including Sphingomonas spp., Enterobacter spp., Enterococcus spp, and Citrobacter spp. from rooftop-harvested irrigation water to produce. In summary, this work provides the first description of total, viable, and metabolically-active bacterial communities in different nontraditional irrigation water sources. These data can be used to improve risk characterization of these water sources, and ultimately inform the selection of appropriate cost-effective remediation methods to treat these waters prior to irrigation activities in order to prevent foodborne outbreaks

Coupled DNA-labeling and sequencing approach enables the detection of viable-but-non-culturable Vibrio spp. in irrigation water sources in the Chesapeake Bay watershed

Nontraditional irrigation water sources (e.g., recycled water, brackish water) may harbor human pathogens, including Vibrio spp., that could be present in a viable-but-nonculturable (VBNC) state, stymieing current culture-based detection methods. To overcome this challenge, we coupled 5-bromo-2′-deoxyuridine (BrdU) labeling, enrichment techniques, and 16S rRNA sequencing to identify metabolically-active Vibrio spp. in nontraditional irrigation water (recycled water, pond water, non-tidal freshwater, and tidal brackish water). Our coupled BrdU-labeling and sequencing approach revealed the presence of metabolically-active Vibrio spp. at all sampling sites. Whereas, the culture-based method only detected vibrios at three of the four sites. We observed the presence of V. cholerae, V. vulnificus, and V. parahaemolyticus using both methods, while V. aesturianus and V. shilonii were detected only through our labeling/sequencing approach. Multiple other pathogens of concern to human health were also identified through our labeling/sequencing approach including P. shigelloides, B. cereus and E. cloacae. Most importantly, 16S rRNA sequencing of BrdU-labeled samples resulted in Vibrio spp. detection even when our culture-based methods resulted in negative detection. This suggests that our novel approach can effectively detect metabolically-active Vibrio spp. that may have been present in a VBNC state, refining our understanding of the prevalence of vibrios in nontraditional irrigation waters.https://doi.org/10.1186/s40793-021-00382-

Primjena poluugniježđene lančane reakcije polimerazom za dokaz larvi oblića Spirocerca lupi u vrtnog guštera (Calotes versicolor).

The study reveals the usefulness of semi-nested PCR as a tool to identify larval stages of Spirocerca lupi in garden lizards (Calotes versicolor), the paratenic host for the parasite. Creamy white cysts present in the superficial muscles of seven out of thirteen garden lizards on the veterinary college campus, Pookode, were recovered and observed for the presence of live larvae. DNA isolated from these cysts and S. lupi worms recovered during postmortem examination of an infected dog were used for semi-nested PCR for amplifying an internal region of the S. lupi Cox-I gene, producing a specific 400 bp fragment. Thus the semi-nested PCR may be utilized for specific detection of larval stages of S. lupi in a paratenic host.Istraživanje je provedeno u svrhu provjere učinkovitosti primjene poluugniježđene lančane reakcije polimerazom za identifikaciju ličinaka oblića S. lupi u vrtnog guštera (Calotes versicolor). Žućkasto bijele ciste bile su izdvojene iz površinskih mišića sedam od 13 vrtnih guštera ulovljenih na području kampusa veterinarskog koledža Pookovode i pretražene na prisutnost živih ličinaka. DNA izdvojena iz tih cista i DNA iz oblića S. lupi izdvojenih tijekom razudbe invadiranoga psa rabljene su u poluugniježđenoj lančanoj reakciji polimerazom za umnožavanje unutarnjeg područja gena Cox-I i prepoznavanje specifičnog fragmenta od 400 bp. Zaključeno je da se poluugniježđena PCR može upotrijebiti za dokaz ličinaka oblića S. lupi u parateničnih domaćina

A roadmap from unknowns to knowns: advancing our understanding of the microbiomes of commercially available tobacco products.

Tobacco smoking is still the leading cause of preventable diseases and death in the USA and throughout the globe. Under Section 904(a)(3) of the US Federal Food, Drug, and Cosmetic Act, tobacco manufacturing companies need to report on quantities of harmful and potentially harmful constituents (HPHCs) in all tobacco products. While the extensive HPHC list of 2012 includes 93 chemicals, which are categorized as carcinogenic, respiratory, cardiovascular, or reproductive toxicants or addictive compounds, it fails to include microorganisms (bacteria and fungi) that have been shown to contribute to adverse health outcomes among tobacco users. Nevertheless, over the last 50 years, researchers have studied microorganisms in a variety of tobacco products using both culture-based and culture-independent techniques. In this mini-review, we provide an overview of this body of research, detailing the bacterial and fungal microbiomes residing in commercial tobacco products. Overall, studies have characterized over 89 unique bacterial genera and 19 fungal genera in cigarettes, cigars, cigarillos, hookah, and smokeless tobacco. The most predominant bacterial genera are Bacillus, Pseudomonas, and Staphylococcus. Fungal genera identified have included Aspergillus, Penicillium, Mucor, Alternaria, Cladosporium, Streptomyces, and Candida, to name a few. While some of the identified microorganisms are known human pathogens, others are potential opportunistic pathogens. Given the vast array of microorganisms that are present across diverse types of tobacco products, future research should be focused on the viability of these microorganisms, as well as their ability to transfer to the user's respiratory tract, potentially contributing to adverse health outcomes. KEY POINTS: • Commercial tobacco products harbor diverse bacterial and fungal communities. • Some of these microorganisms are known or opportunistic human pathogens. • Research on their viability and transmission to users' respiratory tracts is needed

Potential role of the skin and gut microbiota in premenarchal vulvar lichen sclerosus: A pilot case-control study.

The etiology of vulvar lichen sclerosus (LS) remains unclear; however, alterations in cutaneous and gut microbiota may be contributing to the pathogenesis of this inflammatory condition. To explore this hypothesis, we conducted a pilot case-control study, obtaining dermal swab and stool samples from prepubertal girls with vulvar LS (n = 5), girls with nonspecific vulvovaginitis (n = 5), and healthy controls (n = 3). Samples (n = 56) were subjected to total DNA extractions. Resulting DNA was purified, subjected to PCR (targeting the V3V4 region of the 16S rRNA gene), sequenced, and analyzed using QIIME, MetagenomeSeq, and DESeq2 software packages. Our findings showed that there were significant differences in the cutaneous and gut microbiotas of girls with LS compared to controls. On the skin, girls with LS had a statistically significantly higher relative abundance of Porphyromonas spp., Parvimonas spp., Peptoniphilus spp., Prevotella spp., Dialister spp., and Peptostreptococcus spp., but a lower relative abundance of Cornyebacterium compared to the control group. In the gut samples, girls with LS had a significantly higher relative abundance of Dialister spp., Clostridiales spp., Paraprevotella spp., Escherichia coli, Bifidobacterium adolescentis, and Akkermansia muciniphila, and a lower relative abundance of Roseburia faecis and Ruminococcus bromii compared to controls. These results suggest a potential association between cutaneous and gut dysbiosis and pediatric vulvar LS. Future studies involving larger samples sizes are warranted to further evaluate this association

Biochar and zero-valent iron sand filtration simultaneously removes contaminants of emerging concern and Escherichia coli from wastewater effluent

Abstract Advanced treated municipal wastewater is an important alternative water source for agricultural irrigation. However, the possible persistence of chemical and microbiological contaminants in these waters raise potential safety concerns with regard to reusing treated wastewater for food crop irrigation. Two low-cost and environmentally-friendly filter media, biochar (BC) and zero-valent iron (ZVI), have attracted great interest in terms of treating reused water. Here, we evaluated the efficacy of BC-, nanosilver-amended biochar- (Ag-BC) and ZVI-sand filters, in reducing contaminants of emerging concern (CECs), Escherichia coli (E. coli) and total bacterial diversity from wastewater effluent. Six experiments were conducted with control quartz sand and sand columns containing BC, Ag-BC, ZVI, BC with ZVI, or Ag-BC with ZVI. After filtration, Ag-BC, ZVI, BC with ZVI and Ag-BC with ZVI demonstrated more than 90% (> 1 log) removal of E. coli from wastewater samples, while BC, Ag-BC, BC with ZVI and Ag-BC with ZVI also demonstrated efficient removal of tested CECs. Lower bacterial diversity was also observed after filtration; however, differences were marginally significant. In addition, significantly (p < 0.05) higher bacterial diversity was observed in wastewater samples collected during warmer versus colder months. Leaching of silver ions occurred from Ag-BC columns; however, this was prevented through the addition of ZVI. In conclusion, our data suggest that the BC with ZVI and Ag-BC with ZVI sand filters, which demonstrated more than 99% removal of both CECs and E. coli without silver ion release, may be effective, low-cost options for decentralized treatment of reused wastewater. Graphical Abstrac

Canine Filarial Infections in a Human Brugia malayi Endemic Area of India

A very high prevalence of microfilaremia of 42.68 per cent out of 164 canine blood samples examined was observed in Cherthala (of Alappuzha district of Kerala state), a known human Brugia malayi endemic area of south India. The species of canine microfilariae were identified as Dirofilaria repens, Brugia malayi, and Acanthocheilonema reconditum. D. repens was the most commonly detected species followed by B. pahangi. D. immitis was not detected in any of the samples examined. Based on molecular techniques, microfilariae with histochemical staining pattern of “local staining at anal pore and diffuse staining at central body” was identified as D. repens in addition to those showing acid phosphatase activity only at the anal pore. Even though B. malayi like acid phosphatase activity was observed in few dogs examined, they were identified as genetically closer to B. pahangi. Hence, the possibility of dogs acting as reservoirs of human B. malayi in this area was ruled out

Variations in Bacterial Communities and Antibiotic Resistance Genes Across Diverse Recycled and Surface Water Irrigation Sources in the Mid-Atlantic and Southwest United States: A CONSERVE Two-Year Field Study

Reduced availability of agricultural water has spurred increased interest in using recycled irrigation water for U.S. food crop production. However, there are significant knowledge gaps concerning the microbiological quality of these water sources. To address these gaps, we used 16S rRNA gene and metagenomic sequencing to characterize taxonomic and functional variations (e.g., antimicrobial resistance) in bacterial communities across diverse recycled and surface water irrigation sources. We collected 1 L water samples (n = 410) between 2016 and 2018 from the Mid-Atlantic (12 sites) and Southwest (10 sites) U.S. Samples were filtered, and DNA was extracted. The V3-V4 regions of the 16S rRNA gene were then PCR amplified and sequenced. Metagenomic sequencing was also performed to characterize antibiotic, metal, and biocide resistance genes. Bacterial alpha and beta diversities were significantly different (p &lt; 0.001) across water types and seasons. Pathogenic bacteria, such as Salmonella enterica, Staphylococcus aureus, and Aeromonas hydrophilia were observed across sample types. The most common antibiotic resistance genes identified coded against macrolides/lincosamides/streptogramins, aminoglycosides, rifampin and elfamycins, and their read counts fluctuated across seasons. We also observed multi-metal and multi-biocide resistance across all water types. To our knowledge, this is the most comprehensive longitudinal study to date of U.S. recycled water and surface water used for irrigation. Our findings improve understanding of the potential differences in the risk of exposure to bacterial pathogens and antibiotic resistance genes originating from diverse irrigation water sources across seasons and U.S. regions