Gulshan Singh

All Publications

• The effect of gastric acid suppression on probiotic colonization in a double blinded randomized clinical trial. Clinical nutrition ESPEN Singh, G., Haileselassie, Y., Briscoe, L., Bai, L., Patel, A., Sanjines, E., Hendler, S., Singh, P. K., Garud, N. R., Limketkai, B. N., Habtezion, A. 1800; 47: 70-77

  Abstract

  BACKGROUND & AIMS: Probiotics contain living microorganisms consumed for their putative benefits on the intestinal microbiota and general health and a concept is emerging to use probiotic as a therapeutic intervention to reduce proton pump inhibitors (PPIs) negative effects, but data is lacking. The use of PPIs can result in disordered gut microbiota, leading to a risk of enteric infections. PPIs are frequently prescribed in the general practice setting for gastroesophageal reflux disease (GERD), peptic ulcer disease, and related conditions. Despite the availability and widespread use of probiotics and acid-suppressing medications, the effect of PPIs-induced gastric acid suppression on the survival and colonization of probiotics bacterial species is currently unclear. We hypothesized that gastric acid suppression may improve intestinal colonization of probiotics bacterial species and probiotic intervention may have a potential role in mitigating untoward effects of PPI.METHODS: In a randomized, double-blind, placebo-controlled study, healthy subjects were given either proton pump inhibitor (PPI, n=15) or placebo (n=15) over 6 weeks. All subjects then consumed multi-strain probiotics from weeks 2-6. Thirty participants (10 males, 20 females, age range: 18-56 years) were enrolled in the study. Shotgun metagenomic sequencing and untargeted metabolomics analyses were performed on stool samples collected at week 0, 2, and 6.RESULTS: Short term PPI treatment increased the microbial abundance of Streptococcaceae (p=0.004), Leuconostacaceae (p=0.001), and Pasteurellaceae (p=0.020) at family level and corresponding genus levels. The metabolomic analysis of the stools revealed a change in 10 metabolites where Gly Arg Val and phenylacetic acid were consistently increased compared to the baseline. Probiotic intervention inhibited PPI-induced microbial changes such as a decrease in Leuconostacaceae family (p=0.01) and led to an increase in metabolite 1H-Indole-4-carbaldehyde. Notably, PPI enhanced the colonization of certain probiotic bacterial species like Streptococcus thermophilus (p<0.05) along with other species present in the multi-strain probiotic.CONCLUSION: Acid suppression enhanced certain probiotic associated bacterial colonization and probiotics in turn suppressed PPI-mediated intestinal microbial alterations. Thus, probiotics in combination with PPI might be a beneficial strategy that allows probiotic colonization and suppress PPI-induced microbial perturbations. CLINICAL TRIALS.GOV, NUMBER: NCT03327051.

  View details for DOI 10.1016/j.clnesp.2021.11.005

  View details for PubMedID 35063245

• Novel circulating and tissue monocytes as well as macrophages in pancreatitis and recovery. Gastroenterology Manohar, M., Jones, E. K., Rubin, S. J., Subrahmanyam, P. B., Swaminathan, G., Mikhail, D., Bai, L., Singh, G., Wei, Y., Sharma, V., Siebert, J. C., Maecker, H. T., Husain, S. Z., Park, W. G., Pandol, S. J., Habtezion, A. 2021

  Abstract

  BACKGROUND AND AIMS: Acute pancreatitis (AP) is an inflammatory disease with mild to severe course that is associated with local and systemic complications and significant mortality. Uncovering inflammatory pathways that lead to progression and recovery will inform ways to monitor and/or develop effective therapies.METHODS: We performed single-cell mass cytometry (CyTOF) analysis to identify pancreatic and systemic inflammatory signals during mild (referred as AP), severe AP (SAP) and recovery using two independent experimental models and blood from AP and recurrent AP (RAP) patients. Flowcytometric validation of monocytes subsets identified by CyTOF analysis was performed independently.RESULTS: Ly6C+ inflammatory monocytes were most altered cells in the pancreas during experimental AP, recovery, and SAP. Deep profiling uncovered heterogeneity among pancreatic and blood monocytes and identified seven novel subsets during AP and recovery, and six monocyte subsets during SAP. Notably, a dynamic shift in pancreatic CD206+ macrophage population was observed during AP and recovery. Deeper profiling of the CD206+ macrophage identified seven novel subsets during AP, recovery and SAP. DE analysis of these novel monocyte and CD206+ macrophage subsets revealed significantly altered surface (CD44, CD54, CD115, CD140a, CD196, PDPN) and functional markers (IFN-gamma, IL-4, IL-22, LAP-TGF-beta, TNF-alpha, T-bet, RoRgammat) that were associated with recovery and SAP. Moreover, a targeted functional analysis further revealed distinct expression of pro- and anti-inflammatory cytokines by pancreatic CD206+ macrophage subsets as the disease either progressed or resolved. Similarly, we identified heterogeneity among circulating classical inflammatory monocytes (CD14+CD16-) and novel subsets in patients with AP and RAP.CONCLUSION: We identified several novel monocyte/macrophage subsets with unique phenotype and functional characteristics that are associated with AP, recovery, and SAP. Our findings highlight differential innate immune responses during AP progression and recovery that can be leveraged for future disease monitoring and targeting.

  View details for DOI 10.1053/j.gastro.2021.08.033

  View details for PubMedID 34450180

• Protective Effect of Saffron in Mouse Colitis Models Through Immune Modulation. Digestive diseases and sciences Singh, G., Haileselassie, Y., Ji, A. R., Maecker, H. T., Sinha, S. R., Brim, H., Habtezion, A., Ashktorab, H. 2021

  Abstract

  BACKGROUND: People with inflammatory bowel disease (IBD) including ulcerative colitis are at risk for colorectal cancer. Despite available effective drugs used to treat IBD, many patients fail or lose response over time with some displaying drug-induced adverse events. Saffron (Crocus sativus) has been reported to have anti-inflammatory properties. Its protective role in IBD has not been explored extensively.AIM: To establish whether saffron treatment alleviates inflammation in experimental colitis.METHODS: Colitis was induced in C57BL/6 mice with 3% DSS and treated with either saffron doses (7.5, 15, 20, 25mg/kg body weight) or vehicle through daily gavage. On day 11, mice were euthanized and analyzed for gross and microscopic inflammation. Distal colon segments were collected for mRNA and protein expression of HO-1 protein and GPX2, (the downstream targets of NRF-2). Nrf-2 translocation from cytosol to nucleus was confirmed by immunofluorescence, and further Nrf-2 protein expression in nuclear and cytosolic fraction of colon was analyzed by immunoblot. Immune cells were isolated from the lamina propria of mouse colon for flow cytometry-based immunophenotyping. Colitis was also induced in C57BL/6 Ahr knockout and wild type mice to explore the involvement of Ahr-dependent pathways in saffron's protective effect(s). The therapeutic effect of saffron was further validated in another TNBS model of colitis.RESULTS: Saffron 20mg/kg body weightshowed improved colon gross and histology features and led to better body weight, colon length, histology score, and reduced disease activity index (DAI). Saffron significantly decreased pro-inflammatory macrophages (M1), while increasing anti-inflammatory macrophages (M2) and IL10+dendritic cells. Saffron treatment also enhanced CD3+T and CD3+CD8+T cells followed by increase in different CD3+CD4+T cells subsets like CD25+T cells, FoxP3+CD25+regulatory T cells, and CD4+FOXP3+CD25-regulatory T cells. Immunoblot analysis showed a significant increase in HO-1/GPX2 protein expression. With saffron treatment, Nrf-2 translocation into nucleus from cytosol also supports the involvement of Nrf-2 and its downstream targets in the protective effect of saffron. Further, we demonstrated that saffron in part exert anti-inflammatory effect through activation of aryl hydrocarbon receptor (AhR)-nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent pathways.CONCLUSION: These data demonstrate saffron's therapeutic potential and its protective role in part via Ahr/Nrf-2 pathways and regulatory innate and adaptive immune cells.

  View details for DOI 10.1007/s10620-021-07163-3

  View details for PubMedID 34275090

• Dysbiosis-Induced Secondary Bile Acid Deficiency Promotes Intestinal Inflammation. Cell host & microbe Sinha, S. R., Haileselassie, Y., Nguyen, L. P., Tropini, C., Wang, M., Becker, L. S., Sim, D., Jarr, K., Spear, E. T., Singh, G., Namkoong, H., Bittinger, K., Fischbach, M. A., Sonnenburg, J. L., Habtezion, A. 2020

  Abstract

  Secondary bile acids (SBAs) are derived from primary bile acids (PBAs) in a process reliant on biosynthetic capabilities possessed by few microbes. To evaluate the role of BAs in intestinal inflammation, we performed metabolomic, microbiome, metagenomic, and transcriptomic profiling of stool from ileal pouches (surgically created resevoirs) in colectomy-treated patients with ulcerative colitis (UC) versus controls (familial adenomatous polyposis [FAP]). We show that relative to FAP, UC pouches have reduced levels of lithocholic acid and deoxycholic acid (normally the most abundant gut SBAs), genes required to convert PBAs to SBAs, and Ruminococcaceae (one of few taxa known to include SBA-producing bacteria). In three murine colitis models, SBA supplementation reduces intestinal inflammation. This anti-inflammatory effect is in part dependent on the TGR5 bile acid receptor. These data suggest that dysbiosis induces SBA deficiency in inflammatory-prone UC patients, which promotes a pro-inflammatory state within the intestine that may be treated by SBA restoration.

  View details for DOI 10.1016/j.chom.2020.01.021

  View details for PubMedID 32101703

• Saffron: The Golden Spice with Therapeutic Properties on Digestive Diseases NUTRIENTS Ashktorab, H., Soleimani, A., Singh, G., Amin, A., Tabtabaei, S., Latella, G., Stein, U., Akhondzadeh, S., Solanki, N., Gondre-Lewis, M. C., Habtezion, A., Brim, H. 2019; 11 (5)

  View details for DOI 10.3390/nu11050943

  View details for Web of Science ID 000471021600003

• Comparison of droplet digital PCR and quantitative PCR for the detection of Salmonella and its application for river sediments JOURNAL OF WATER AND HEALTH Singh, G., Sithebe, A., Enitan, A. M., Kumari, S., Bux, F., StenstrM, T. 2017; 15 (4): 505–8

  Abstract

  Despite advances in microbial detection that quantitative polymerase chain reaction (qPCR) has led to, complex environmental samples, such as sediments, remain a challenge due to presence of PCR inhibitors. Aquatic sediments accumulate particle-bound microbial contaminants and thereby reflect a cumulative microbial load over time. The relatively new droplet digital PCR (ddPCR) has emerged as a direct quantitative method, highly tolerant to PCR inhibitors and relinquishing the necessity for calibration/standard curves. Information is virtually absent where ddPCR has been applied to detect pathogenic organisms in aquatic sediments. This study compared the efficacy of ddPCR with qPCR, for quantification of Salmonella in sediments from the Palmiet River near an informal settlement in Durban, South Africa. ddPCR significantly improved both analytical sensitivity and detection of low concentrations of Salmonella as compared to qPCR. The expected copy numbers measured from both qPCR and ddPCR showed good R2 values (0.999 and 0.994, respectively). The site mostly affected by the informal settlements exhibited Salmonella in the range of 255 ± 37 and 818 ± 30 Salmonella/g (p ≤ 0.0001) in qPCR and ddPCR, respectively. The improved detection of Salmonella in sediments with ddPCR makes it a promising technical method for the quantification of Salmonella in multifarious environmental samples.

  View details for DOI 10.2166/wh.2017.259

  View details for Web of Science ID 000407037100003

  View details for PubMedID 28771147

• Exploring the potential reservoirs of non specific TEM beta lactamase (bla(TEM)) gene in the Indo-Gangetic region: A risk assessment approach to predict health hazards JOURNAL OF HAZARDOUS MATERIALS Singh, G., Vajpayee, P., Rani, N., Amoah, I., Stenstrom, T., Shanker, R. 2016; 314: 121–28

  Abstract

  The emergence of antimicrobial resistant bacteria is an important public health and environmental contamination issue. Antimicrobials of β-lactam group accounts for approximately two thirds, by weight, of all antimicrobials administered to humans due to high clinical efficacy and low toxicity. This study explores β-lactam resistance determinant gene (blaTEM) as emerging contaminant in Indo-Gangetic region using qPCR in molecular beacon format. Quantitative Microbial Risk Assessment (QMRA) approach was adopted to predict risk to human health associated with consumption/exposure of surface water, potable water and street foods contaminated with bacteria having blaTEM gene. It was observed that surface water and sediments of the river Ganga and Gomti showed high numbers of blaTEM gene copies and varied significantly (p<0.05) among the sampling locations. The potable water collected from drinking water facility and clinical settings exhibit significant number of blaTEM gene copies (13±0.44-10200±316 gene copies/100mL). It was observed that E.crassipes among aquatic flora encountered in both the rivers had high load of blaTEM gene copies. The information on prevalence of environmental reservoirs of blaTEM gene containing bacteria in Indo-Gangetic region and risk associated will be useful for formulating strategies to protect public from menace of clinical risks linked with antimicrobial resistant bacteria.

  View details for DOI 10.1016/j.jhazmat.2016.04.036

  View details for Web of Science ID 000377738300014

  View details for PubMedID 27111425

• Determination of viable Salmonellae from potable and source water through PMA assisted qPCR ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY Singh, G., Vajpayee, P., Bhatti, S., Ronnie, N., Shah, N., McClure, P., Shanker, R. 2013; 93: 121–27

  Abstract

  Resource constrained countries identified as endemic zones for pathogenicity of Salmonella bear an economic burden due to recurring expenditure on medical treatment. qPCR used for Salmonella detection could not discriminate between viable and nonviable cells. Propidium monoazide (PMA) that selectively penetrates nonviable cells to cross-link their DNA, was coupled with ttr gene specific qPCR for quantifying viable salmonellae in source/potable waters collected from a north Indian city. Source water (raw water for urban potable water supply) and urban potable water exhibited viable salmonellae in the range of 2.1×10(4)-2.6×10(6) and 2-7160CFU/100mL, respectively. Potable water at water works exhibited DNA from dead cells but no viable cells were detected. PMA assisted qPCR could specifically detect low numbers of live salmonellae in Source and potable waters. This strategy can be used in surveillance of urban potable water distribution networks to map contamination points for better microbial risk management.

  View details for DOI 10.1016/j.ecoenv.2013.02.017

  View details for Web of Science ID 000320080500017

  View details for PubMedID 23623706

• Bio-capture of S. Typhimurium from surface water by aptamer for culture-free quantification ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY Singh, G., Vajpayee, P., Rani, N., Jyoti, A., Gupta, K., Shanker, R. 2012; 78: 320–26

  Abstract

  In this study, a DNA aptamer was used to bio-capture Salmonella enterica serovar Typhimurium from surface water collected from highly endemic zone prior to culture-free detection through Molecular-Beacon based real-time PCR assay targeting invA gene. The assay could detect S. Typhimurium cells (1 CFU/PCR or 100 CFU/ml) selectively captured by serovar specific DNA aptamer. The observations indicate that all the water samples (n=40) collected from the river Gomti were contaminated by S. Typhimurium (31400-1 × 10(7) CFU/100 ml). The pre-analytical step in the form of serovar specific DNA aptamer based bio-capture of the bacterial cell was found to enhance the sensitivity of the florescent probe based real-time PCR assay during detection of S. Typhimurium in environmental samples exhibiting natural PCR inhibitors and high background bacterial flora. The assay could be used for the regular monitoring of surface waters for forecasting and management of non-typhoidal Salmonellosis in south Asia.

  View details for DOI 10.1016/j.ecoenv.2011.11.039

  View details for Web of Science ID 000301316500043

  View details for PubMedID 22226327

• Chromium Oxide Nano-Particles Induce Stress in Bacteria: Probing Cell Viability JOURNAL OF BIOMEDICAL NANOTECHNOLOGY Singh, G., Vajpayee, P., Khatoon, I., Jyoti, A., Dhawan, A., Gupta, K. C., Shanker, R. 2011; 7 (1): 166–67

  Abstract

  In this study, viability of an environmentally relevant bacterium, Escherichia coli exposed to 0-100 microg/mL chromium oxide nanoparticles (Cr2O3, Nps) for 120 min in Luria Bertani broth was evaluated by Propidium monoazide (PMA) assisted Q-PCR and standard plate count (SPC) method. Viable count for E. coli grown in Cr2O3, Nps amended medium was more by PMA assisted Q-PCR than SPC. Thus, the observations made in this study suggest that the inclusion of PMA assisted Q-PCR for viability assessment in Nps toxicity studies will provide the real count for the viable cells comprising of both viable and viable but not culturable (VBNC) cells.

  View details for DOI 10.1166/jbn.2011.1252

  View details for Web of Science ID 000291410800082

  View details for PubMedID 21485854

• Environmental Reservoirs for Enterotoxigenic Escherichia coli in South Asian Gangetic Riverine System ENVIRONMENTAL SCIENCE & TECHNOLOGY Singh, G., Vajpayee, P., Ram, S., Shanker, R. 2010; 44 (16): 6475–80

  Abstract

  Forecasting diarrheagenic E. coli contamination of aquatic resources to prevent outbreaks largely depends on rapid and accurate diagnostic testing in a few hours. Real-time PCR is widely used for quick culture-free quantitative enumeration of pathogenic bacteria in environmental samples. In this study, real-time PCR in molecular beacon format was used for detection and culture-free quantitative enumeration of enterotoxigenic Escherichia coli (ETEC) harboring LT1 gene in a sewage-impacted south Asian Gangetic riverine system. The quantitative budget for ETEC in surface water was observed to vary significantly (DMRT, p < 0.05) among the sites. Aquatic flora (Eichhornia crassipes, Potamogeton crispus, Potamogeton pectinatus, Ranunculus sceleratus, Polygonum glabrum, Pontederia cordata, Najas indica and strands of Spirogyra spp.) collected between sites 1 and 9 exhibited significant high levels of ETEC in comparison to their representatives collected from pristine area. The level of ETEC harboring LT1 gene observed in leafy vegetables cultivated along the banks was in the following order: mint leaves > coriander > spinach > methi leaves. The study suggests that the aquatic flora and cultivated leafy vegetables in the south Asian Gangetic riverine system are environmental reservoirs for enterotoxigenic Escherichia coli.

  View details for DOI 10.1021/es1004208

  View details for Web of Science ID 000280727400075

  View details for PubMedID 20704250

• Preventive effects of saffron in a colitis mouse model. Banskota, S., Khan, W., Singh, G., Habtezion, A., Brim, H., Ashktorab, H. AMER ASSOC CANCER RESEARCH. 2021

  View details for Web of Science ID 000680263503151

• Saffron Pre-Treatment Promotes Reduction in Tissue Inflammatory Profiles and Alters Microbiome Composition in Experimental Colitis Mice MOLECULES Banskota, S., Brim, H., Kwon, Y., Singh, G., Sinha, S. R., Wang, H., Khan, W. I., Ashktorab, H. 2021; 26 (11)

  View details for DOI 10.3390/molecules26113351

  View details for Web of Science ID 000660367900001

• Identification, antibiotic resistance, and virulence profiling of Aeromonas and Pseudomonas species from wastewater and surface water. Environmental monitoring and assessment Govender, R., Amoah, I. D., Adegoke, A. A., Singh, G., Kumari, S., Swalaha, F. M., Bux, F., Stenström, T. A. 2021; 193 (5): 294

  Abstract

  Aquatic environments are hotspots for the spread of antibiotic-resistant bacteria and genes due to pollution caused mainly by anthropogenic activities. The aim of this study was to evaluate the impact of wastewater effluents, informal settlements, hospital, and veterinary clinic discharges on the occurrence, antibiotic resistance profile and virulence signatures of Aeromonas spp. and Pseudomonas spp. isolated from surface water and wastewater. High counts of Aeromonas spp. (2.5 (± 0.8) - 3.3 (± 0.4) log10 CFU mL-1) and Pseudomonas spp. (0.6 (± 1.0) - 1.8 (± 1.0) log10 CFU mL-1) were obtained. Polymerase chain reaction (PCR) and MALDI-TOF characterization identified four species of Aeromonas and five of Pseudomonas. The isolates displayed resistance to 3 or more antibiotics (71% of Aeromonas and 94% of Pseudomonas). Aeromonas spp. showed significant association with the antibiotic meropenem (χ2 = 3.993, P < 0.05). The virulence gene aer in Aeromonas was found to be positively associated with the antibiotic resistance gene blaOXA (χ2 = 6.657, P < 0.05) and the antibiotic ceftazidime (χ2 = 7.537, P < 0.05). Aeromonas recovered from both wastewater and surface water displayed high resistance to ampicillin and had higher multiple antibiotic resistance (MAR) indices close to the hospital. Pseudomonas isolates on the other hand exhibited low resistance to carbapenems but very high resistance to the third-generation cephalosporins and cefixime. The results showed that some of the Pseudomonas spp. and Aeromonas spp. isolates were extended-spectrum β-lactamase producing bacteria. In conclusion, the strong association between virulence genes and antibiotic resistance in the isolates shows the potential health risk to communities through direct and indirect exposure to the water.

  View details for DOI 10.1007/s10661-021-09046-6

  View details for PubMedID 33893564

• Drinking water microbiology in a water-efficient building: stagnation, seasonality, and physicochemical effects on opportunistic pathogen and total bacteria proliferation ENVIRONMENTAL SCIENCE-WATER RESEARCH & TECHNOLOGY Ley, C. J., Proctor, C. R., Singh, G., Ra, K., Noh, Y., Odimayomi, T., Salehi, M., Julien, R., Mitchell, J., Nejadhashemi, A., Whelton, A. J., Aw, T. 2020; 6 (10): 2902–13

  View details for DOI 10.1039/d0ew00334d

  View details for Web of Science ID 000574031300021

• Eosinophilic pancreatitis: a rare or unexplored disease entity? Przeglad gastroenterologiczny Manohar, M. n., Verma, A. K., Singh, G. n., Mishra, A. n. 2020; 15 (1): 34–38

  Abstract

  Several case reports show accumulation of eosinophils in pancreatitis patients and term the disease as "eosinophilic pancreatitis (EP)". EP usually presents with a pancreatic tumour and abdominal pain in obstructive jaundice, which is generally not diagnosed until the patient undergoes pancreatic resection. Histologically, EP reveals distinct patterns like diffused, periductal, acinar, and septal inflammatory infiltrates with eosinophils, eosinophilic phlebitis, and localised extreme eosinophilic infiltrates related with pseudocyst formation. EP patients also have elevated serum IgE levels with high eosinophil counts in the pancreas as well as in other organs such as the gastrointestinal tract, which is termed as eosinophilic gastroenteritis. Due to the lack of knowledge based on just a few case reports, it is considered that eosinophilic infiltration is quite rare in the pancreas; therefore, the significance of eosinophils in pancreatitis is not yet established. This review assesses the current understanding of eosinophilic pancreatitis and the important role of eosinophils in promoting pancreatic fibrosis including malignancy.

  View details for DOI 10.5114/pg.2019.90631

  View details for PubMedID 32215125

  View details for PubMedCentralID PMC7089860

• Comparative assessment of DNA extraction procedures for Ascaris spp. eggs JOURNAL OF HELMINTHOLOGY Amoah, I. D., Singh, G., Troell, K., Reddy, P., Stenstrom, T. A., Bux, F. 2020; 94: e78

  Abstract

  A central and critical step in the molecular detection of soil-transmitted helminths from environmental sources is the extraction of DNA from the eggs. In this study, we investigated the yield of DNA extracted from known quantities (500, 100, 50, 20, 10 and 5) of Ascaris suum eggs, as well as directly from wastewater and sludge samples containing Ascaris spp. eggs, using six commercial DNA extraction kits. The amount of DNA extracted was quantified with NanoDrop, Qubit and Ct values from quantitative polymerase chain reaction (qPCR) assay using CFX96 Touch™ real-time PCR equipment. The PowerLyzer Ultraclean Microbial DNA isolation kit and PowerSoil DNA isolation kit gave the highest yield of DNA based on the NanoDrop, Qubit and Ct values. However, the qPCR results indicate that in some of the kits, PCR inhibitors may have been carried over to the PCR reaction. DNA extraction kits that incorporate a bead-beating step as well as other mechanical eggshell disruption steps were superior in extracting DNA from Ascaris spp. eggs. Additionally, for the accurate quantification of extracted DNA, the use of Ct values from qPCR and Qubit readings gives better results compared to the NanoDrop readings. For efficient downstream applications, the use of DNA extraction kits with superior inhibitor removal technology is essential, in addition to a high yield of DNA.

  View details for DOI 10.1017/S0022149X19000683

  View details for Web of Science ID 000538814800029

  View details for PubMedID 31455433

• Nanotechnology and detection of microbial pathogens Animal Biotechnology (Second Edition) Models in Discovery and Translation Shanker, R., Singh, G., Jyoti, A., Dwivedi, P. D., Singh, S. P. 2020: 593–611
• Efficiency of chlorine and UV in the inactivation of Cryptosporidium and Giardia in wastewater PLOS ONE Adeyemo, F., Singh, G., Reddy, P., Bux, F., Stenstrom, T. 2019; 14 (5): e0216040

  Abstract

  Wastewater from different sources is contaminated by protozoan parasites including Cryptosporidium and Giardia. Many protozoan parasites are becoming resistant to chemical treatment. The challenge of finding alternatives is presented to researchers by exploring other methods of eliminating protozoan parasites from wastewater. The aim of this study was to assess the speciation and the viability of Cryptosporidium and Giardia in environmental samples with the specific objective of evaluating if effluent chlorination and UV affect the viability. Different doses of chlorine with different exposure times were experimented with both distilled water and waste water spiked with (oo)cysts derived from environmental samples. UV irradiation at different doses was also experimented using the same spiked samples. Two methods of quantification and detection, namely, microscopy and flow cytometry, were used in the experiment. Two vital dyes, Syto-9+PI and DAPI+PI, were the used for staining the collected wastewater samples. It was found that the (oo)cysts responded to chlorination and UV treatments with Giardia responding better than Cryptosporidium. Giardia responded very well to UV irradiations with almost 0 percent remaining viable after a low dose of UV. Cryptosporidium was found to be resistant to chlorination even at high doses but responded well to high UV doses. DAPI+PI dye gave a lower mean percentage viability values than Syto-9+PI. Flow cytometry gave higher mean percentage than microscopy from the results. It is concluded that UV is a promising alternative to Chlorine in removing Cryptosporidium and Giardia from waste water. Appropriate treatment method for wastewater is necessary to minimize water resources pollution when wastewater is released into water systems.

  View details for DOI 10.1371/journal.pone.0216040

  View details for Web of Science ID 000467714000013

  View details for PubMedID 31083664

  View details for PubMedCentralID PMC6513095

• Detection of coliphages and human adenoviruses in a subtropical estuarine lake SCIENCE OF THE TOTAL ENVIRONMENT Cooksey, E. M., Singh, G., Scott, L. C., Aw, T. 2019; 649: 1514–21

  Abstract

  Fecal indicator bacteria (FIB) have been used to assess fecal contamination in recreational water. However, enteric viruses have been shown to be more persistent in the environment and resistant to wastewater treatment than bacteria. Recently, U.S Environmental Protection Agency has proposed the use of coliphages as viral indicators to better protect against viral waterborne outbreaks. This study aimed to detect and determine correlation between coliphages (F-specific and somatic), fecal indicator bacteria (enterococci and fecal coliforms), and human enteric viruses (human adenovirus) in a subtropical brackish estuarine lake. Water samples were collected from 9 estuarine recreation sites on Lake Pontchartrain in southeast Louisiana. Water samples (n = 222, collected weekly) were analyzed for coliphages and fecal indicator bacteria using culture-based methods and large volume water samples (n = 54, collected monthly) were analyzed for human adenovirus using quantitative PCR. Somatic coliphage and F-specific coliphage were found in 93.7 and 65.2% of samples with geometric mean concentrations of 30 and 3 plaque forming units (PFU) per 100 mL, respectively. Enterococci, fecal coliforms, and adenovirus were found in all samples with geometric mean concentrations of 27 most probable number (MPN), 77 MPN, and 3.0 × 104 gene copies per 100 mL, respectively. Watersheds in suburban areas exhibited significantly higher concentrations of coliphages and fecal indicator bacteria, indicating potential fecal contamination from septic systems. There was no significant correlation (p > 0.05) observed between the presence of adenoviruses and fecal indicator bacteria and coliphages. The presence of human adenovirus in Lake Pontchartrain poses a significant public health problem for both recreational use and seafood harvesting as it increases exposure risks. This study demonstrated the lack of relationship between fecal indicators and human viral pathogen in Lake Pontchartrain supporting an alternative microbial surveillance system such as direct pathogen detection.

  View details for DOI 10.1016/j.scitotenv.2018.08.322

  View details for Web of Science ID 000446076500142

  View details for PubMedID 30308919

• Methods for the detection of Cryptosporidium and Giardia: From microscopy to nucleic acid based tools in clinical and environmental regimes ACTA TROPICA Adeyemo, F., Singh, G., Reddy, P., Stenstrom, T. 2018; 184: 15–28

  Abstract

  The detection and characterization of genotypes and sub genotypes of Cryptosporidium and Giardia is essential for their enumeration, surveillance, prevention, and control. Different diagnostic methods are available for the analysis of Cryptosporidium and Giardia including conventional phenotypic tools that face major limitations in the specific diagnosis of these protozoan parasites. The substantial advancement in the development of genetic signature based molecular tools for the quantification, diagnosis and genetic variation analysis has increased the understanding of the epidemiology and preventive measures of related infections. The conventional methods such as microscopy, antibody and enzyme based approaches, offer better detection results when combined with advanced molecular methods. Gene based approaches increase the precision of identification, for example, many signatures detected in environmental matrices represent species/genotype that are not infectious to humans. This review summarizes the available methods and the advantages and limitations of advance detection techniques like nucleic acid-based approaches for the detection of viable oocysts and cysts of Cryptosporidium and Giardia along with the conventional and widely accepted detection techniques like microscopy, antibody and enzyme based ones. This technical article also encourages the wide application of molecular methods in genetic characterization of distinct species of Cryptosporidium and Giardia, to adopt necessary preventive measures with reliable identification and mapping the source of contamination.

  View details for DOI 10.1016/j.actatropica.2018.01.011

  View details for Web of Science ID 000441117000002

  View details for PubMedID 29395034

• Detection and quantification of soil-transmitted helminths in environmental samples: A review of current state-of-the-art and future perspectives ACTA TROPICA Amoah, I., Singh, G., Stenstrom, T., Reddy, P. 2017; 169: 187–201

  Abstract

  It is estimated that over a billion people are infected with soil-transmitted helminths (STHs) globally with majority occurring in tropical and subtropical regions of the world. The roundworm (Ascaris lumbricoides), whipworm (Trichuris trichiura), and hookworms (Ancylostoma duodenale and Necator americanus) are the main species infecting people. These infections are mostly gained through exposure to faecally contaminated water, soil or contaminated food and with an increase in the risk of infections due to wastewater and sludge reuse in agriculture. Different methods have been developed for the detection and quantification of STHs eggs in environmental samples. However, there is a lack of a universally accepted technique which creates a challenge for comparative assessments of helminths egg concentrations both in different samples matrices as well as between locations. This review presents a comparison of reported methodologies for the detection of STHs eggs, an assessment of the relative performance of available detection methods and a discussion of new emerging techniques that could be applied for detection and quantification. It is based on a literature search using PubMed and Science Direct considering all geographical locations. Original research articles were selected based on their methodology and results sections. Methods reported in these articles were grouped into conventional, molecular and emerging techniques, the main steps in each method were then compared and discussed. The inclusion of a dissociation step aimed at detaching helminth eggs from particulate matter was found to improve the recovery of eggs. Additionally the selection and application of flotation solutions that take into account the relative densities of the eggs of different species of STHs also results in higher egg recovery. Generally the use of conventional methods was shown to be laborious and time consuming and prone to human error. The alternate use of nucleic acid-based techniques has improved the sensitivity of detection and made species specific identification possible. However, these nucleic acid based methods are expensive and less suitable in regions with limited resources and skill. The loop mediated isothermal amplification method shows promise for application in these settings due to its simplicity and use of basic equipment. In addition, the development of imaging soft-ware for the detection and quantification of STHs shows promise to further reduce human error associated with the analysis of environmental samples. It may be concluded that there is a need to comparatively assess the performance of different methods to determine their applicability in different settings as well as for use with different sample matrices (wastewater, sludge, compost, soil, vegetables etc.).

  View details for DOI 10.1016/j.actatropica.2017.02.014

  View details for Web of Science ID 000396954000025

  View details for PubMedID 28214519

• Potential Biomedical Applications of Chitosan – and Chitosan‐Based Nanomaterials In Chitosan (eds S. Ahmed and S. Ikram) Singh, G., Manohar, M., Arya, S. ., Siddiqui, W. A., Stenstrom, T. A. Scrivener Publishing LLC. 2017

  View details for DOI 10.1002/9781119364849.ch14

• Antibiotic Resistant Superbugs: Assessment of the Interrelationship of Occurrence in Clinical Settings and Environmental Niches MOLECULES Adegoke, A., Faleye, A., Singh, G., Stenstroem, T. 2017; 22 (1)

  Abstract

  The increasing threat to global health posed by antibiotic resistance remains of serious concern. Human health remains at higher risk due to several reported therapeutic failures to many life threatening drug resistant microbial infections. The resultant effects have been prolonged hospital stay, higher cost of alternative therapy, increased mortality, etc. This opinionated review considers the two main concerns in integrated human health risk assessment (i.e., residual antibiotics and antibiotic resistant genes) in various compartments of human environment, as well as clinical dynamics associated with the development and transfer of antibiotic resistance (AR). Contributions of quorum sensing, biofilms, enzyme production, and small colony variants in bacteria, among other factors in soil, water, animal farm and clinical settings were also considered. Every potential factor in environmental and clinical settings that brings about AR needs to be identified for the summative effects in overall resistance. There is a need to embrace coordinated multi-locational approaches and interrelationships to track the emergence of resistance in different niches in soil and water versus the hospital environment. The further integration with advocacy, legislation, enforcement, technological innovations and further research input and recourse to WHO guidelines on antibiotic policy would be advantageous towards addressing the emergence of antibiotic resistant superbugs.

  View details for DOI 10.3390/molecules22010029

  View details for Web of Science ID 000395473500029

  View details for PubMedID 28035988

  View details for PubMedCentralID PMC6155606

• Biosynthesis of ZnO nanoparticles using Jacaranda mimosifolia flowers extract: Synergistic antibacterial activity and molecular simulated facet specific adsorption studies JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY Sharma, D., Sabela, M. I., Kanchi, S., Mdluli, P. S., Singh, G., Stenstrom, T. A., Bisetty, K. 2016; 162: 199–207

  Abstract

  The naturally occurring biomolecules present in the plant extracts have been identified to play an active role in the single step formation of nanoparticles with varied morphologies and sizes which is greener and environmentally benign. In the present work, spherical zinc oxide nanoparticles (ZnO NPs) of 2-4nm size were synthesized using aqueous extract of fallen Jacaranda mimosifolia flowers (JMFs), treated as waste. The microwave assisted synthesis was completed successfully within 5min. Thereafter, phase identification, morphology and optical band gap of the synthesized ZnO NPs were done using X-ray diffraction (XRD), high resolution transmission electron microscopy (HRTEM) and UV-Visible spectroscopy techniques. The composition of JMFs extract was analyzed by gas chromatography-mass spectrometry (GC-MS) and the ZnO NPs confirmation was further explored with fourier transform infrared spectroscopy (FTIR). The GC-MS results confirmed the presence of oleic acid which has high propensity of acting as a reducing and capping agent. The UV-Visible data suggested an optical band gap of 4.03eV for ZnO NPs indicating their small size due to quantum confinement. Further, facet specific adsorption of oleic acid on the surface of ZnO NPs was studied computationally to find out the impact of biomolecules in defining the shape and size of NPs. The viability of gram negative Escherichia coli and gram positive Enterococcus faecium bacteria was found to be 48% and 43%, respectively at high concentration of NPs.

  View details for DOI 10.1016/j.jphotobiol.2016.06.043

  View details for Web of Science ID 000383003800023

  View details for PubMedID 27380295

• Quantification of viable Enterococcus spp., and Psuedomonas aeruginosa through propidium monoazide dye assisted qPCR array in surface water and sediment Microbes in spotlight: recent progress in the understanding of beneficial and harmful microorganism.” Singh, G., Stenstrom, T. A., Shanker, R. Brown Walker Press. 2016: 121–125
• Nanotechnology and Detection of Microbial Pathogens First Edition: Animal Biotechnology: Models in Discovery and Translation, Shanker, R., Singh, G., Jyoti, A., Dwivedi, P. D., Singh, S. P. 2014: 525–539
• Identification of Environmental Reservoirs of Nontyphoidal Salmonellosis: Aptamer-Assisted Bioconcentration and Subsequent Detection of Salmonella Typhimurium by Quantitative Polymerase Chain Reaction ENVIRONMENTAL SCIENCE & TECHNOLOGY Jyoti, A., Vajpayee, P., Singh, G., Patel, C., Gupta, K., Shanker, R. 2011; 45 (20): 8996–9002

  Abstract

  In this study, identification of environmental reservoirs of Salmonella enterica subsp. enterica serovar Typhimurium (abbreviated as Salmonella Typhimurium) in sediments, water, and aquatic flora collected from the Ganges River (Ganges riverine material) was carried out by adopting a two-step strategy. Step 1 comprised a selective serovar-specific capture of Salmonella Typhimurium from potential reservoirs. Step 2 involved culture-free detection of selectively captured Salmonella Typhimurium by ttr gene-specific molecular beacon (MB) based quantitative polymerase chain reaction (q-PCR). The ttr gene-specific MB designed in this study could detect 1 colony-forming unit (cfu)/PCR captured by serovar-specific DNA aptamer. Sediments, water, and aquatic flora collected from the Ganges River were highly contaminated with Salmonella Typhimurium. The preanalytical step in the form of serovar-specific DNA aptamer-based biocapture of bacterial cells was found to enhance the sensitivity of the fluorescent probe in the presence of nonspecific DNA . Information about the presence of environmental reservoirs of Salmonella Typhimurium in the Ganges River region may pave the way for forecasting and management of nontyphoidal salmonellosis in south Asia.

  View details for DOI 10.1021/es2018994

  View details for Web of Science ID 000295704500054

  View details for PubMedID 21875107

• Determination of Internalization of Chromium Oxide Nano-Particles in Escherichia coli by Flow Cytometry JOURNAL OF BIOMEDICAL NANOTECHNOLOGY Khatoon, I., Vajpayee, P., Singh, G., Pandey, A. K., Dhawan, A., Gupta, K. C., Shanker, R. 2011; 7 (1): 168–69

  Abstract

  In this study, Escherichia coil DH5alpha (ATCC 35218) were exposed to 0-100 microg/mL chromium oxide nanoparticles (Cr2O3, Nps) for 15-120 min to study the internalization of Nps by flowcytometry. A concentration-duration dependent increased side scatter (SSC) confirmed the internalization of Cr2O3 NPs by the E. coli. This study suggests that the uptake of Nps by bacterial cells can be rapidly monitored with flow cytometry for toxicity and risk assessment.

  View details for DOI 10.1166/jbn.2011.1253

  View details for Web of Science ID 000291410800083

  View details for PubMedID 21485855

• Adverse Effects of Chromium Oxide Nano-Particles on Seed Germination and Growth in Triticum aestivum L. JOURNAL OF BIOMEDICAL NANOTECHNOLOGY Vajpayee, P., Khatoon, I., Patel, C., Singh, G., Gupta, K., Shanker, R. 2011; 7 (1): 205–6

  Abstract

  In this study, seeds of Triticum aestivum L. (Poaceae) were exposed to 0-100 microg/mL chromium oxide nanoparticles (Cr2O3, Nps) to study the phytotoxic effects on seed germination and seedling growth. It has been observed that 25-100 microg/mL Cr2O3, Nps inhibited the seed germination and seedling growth in concentration dependent manner. The present study suggests that release of Cr2O3, Nps in environment may adversely affect the wheat production.

  View details for DOI 10.1166/jbn.2011.1270

  View details for Web of Science ID 000291410800100

  View details for PubMedID 21485872

• Contamination of surface and potable water in South Asia by Salmonellae: Culture-independent quantification with molecular beacon real-time PCR SCIENCE OF THE TOTAL ENVIRONMENT Jyoti, A., Ram, S., Vajpayee, P., Singh, G., Dwivedi, P. D., Jain, S. K., Shanker, R. 2010; 408 (6): 1256–63

  Abstract

  Low numbers (15-100CFU) of Salmonella in food or water may pose a public health risk. The management of infections caused by Salmonella spp. during outbreaks or forecasting of contamination of aquatic resources largely depends on rapid, sensitive and accurate diagnostic in few hours. In this study, a real-time PCR assay in Molecular-Beacon format was developed and culture-independent quantitative enumeration of Salmonella spp. in surface and potable water is being reported for the first time from northern part of India. Molecular Beacon was designed in highly conserved region of invA gene (present in wide range of Salmonella serotypes including all subspecies) encoding an essential component of the invasion associated specialized type Ø protein secretion apparatus for detection of Salmonella spp. in water. The assay could detect directly 10 and 1 genomic equivalent of Salmonella typhimurium ATCC 14028 per PCR with detection probability of 100 and 20%, respectively. Further, the assay could detect 10CFU/PCR or more of reference strain (S. typhimurium ATCC 14028) without any enrichment in the presence of 10(8)CFUml(-1) of non-pathogenic E. coli (E. coli DH5alpha) with 100% detection probability. The assay could enumerate Salmonella spp. in surface (n=40) and potable waters (n=10) directly (without enrichment). Results indicate that northern India is at high risk of developing Salmonella borne infections. Further, real-time PCR assay in Molecular Beacon format can be used for identification of critical contamination points in natural water resources and potable water distribution systems, necessary to implement vaccination plan timely for prevention of waterborne outbreaks caused by Salmonella spp.

  View details for DOI 10.1016/j.scitotenv.2009.11.056

  View details for Web of Science ID 000274948000003

  View details for PubMedID 20035972