Exploring the potential of Meyerozyma caribbica and its combined application with bacteria for lindane bioremediation.

This study explored the degradation potential of a yeast strain, Meyerozyma caribbica, alone and in combination with Bacillus velezensis and Priestia megaterium, found novel for lindane biodegradation. Isolated from hexachlorocyclohexane (HCH)-contaminated sites, M. caribbica, B. velezensis, and P. megaterium demonstrated lindane reduction efficiencies of 86.5%, 78.6%, and 77.5%, respectively, at 750 mg L⁻1 within 10-day incubation period. Kinetic analysis revealed that M. caribbica followed the first-order degradation (r2 = 0.991; T1/2 = 4.3 days). Notably, M. caribbica exhibited the highest dechlorinase activity (9.27 U mL⁻1) in the cell supernatant. Co-cultivation as the mixed culture of M. caribbica and P. megaterium achieved maximum lindane reduction (90%) and dechlorinase activity (9.93 U mL⁻1). Whereas the mixed culture of M. caribbica and B. velezensis resulted in 80.9% reduction at 500 mg L⁻1 lindane with dechlorinase activity of 6.77 U mL⁻1. Growth kinetics, modelled using the Monod equation, showed a maximum specific growth rate of 0.416 h⁻1 for the mixed culture of M. caribbica and P. megaterium at 750 mg L⁻1 lindane. GC-MS analysis confirmed the presence of intermediate metabolites, viz., γ-pentachlorocyclohexane, 1,2,4-trichlorobenzene, 1,4-dichlorobenzene and maleyl acetate, validated successive dechlorination and oxidative-reduction processes during lindane biodegradation. The findings of the study highlighted the potential of these novel microbial strains and their mixed cultures for effective bioremediation of lindane-contamination.

Revelation of bioremediation approaches for hexachlorocyclohexane degradation in soil.

This review elucidates different bioremediation approaches used for degradation of HCH from contaminated sites. It highlights the significance of degradative pathways, microbial diversity and impact of different environmental factors for developing viable bioremediation strategies. The application of innovative biotechnological approaches and a thorough understanding of HCH biodegradation pathways show great promise for the creation of long-term solutions to HCH pollution and the restoration of polluted soil ecosystems. Bioremediation technologies viz. biostimulation, bioaugmentation, phytoremediation have been considered till date for treating HCH-contaminated sites. Different bacterial and fungal strains have been reported for degradation of HCH residues. However, these methods are limited to γ-HCH degradation, at laboratory scale and achieving lower success rate for large scale demonstration trials. This review presents a theoretical background for degradation of different HCH isomers in soil through plants, microbes and through their cooperative interactions. This work briefly overviews the substantial contamination of the environment by HCH residues, along with spontaneous evolution of degradation pathways through various HCH degrading microbes. Bioremediation mechanism and pathways of HCH degradation through plants and microbes have been discussed thoroughly. Through molecular and genetic investigations, the complex metabolic pathways used by these microbes, including reductive dechlorination, hydrolysis, and ring cleavage, has been clarified. This study seeks to give a thorough summary of recent discoveries and developments in bioremediation methods for soil HCH degradation. Numerous microbial consortia, including fungi, plants, and bacteria have been recognised as important participants in the transformation of HCH.

Role of ACC-deaminase synthesizing Trichoderma harzianum and plant growth-promoting bacteria in reducing salt-stress in Ocimum sanctum.

Salinity is a significant concern in crop production, causing severe losses in agricultural yields. Ocimum sanctum, also known as Holy Basil, is an important ancient medicinal plant used in the Indian traditional system of medicine. The present study explores the use of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing strains of plant-growth-promoting bacteria (PGPB) namely Str-8 (Halomonas desiderata), Sd-6 (Brevibacterium halotolerans), Fd-2 (Achromobacter xylosoxidans), Art-7 (Burkholderia cepacia), and Ldr-2 (Bacillus subtilis), and T. harzianum (Th), possessing multi-functional properties like growth promotion, stress alleviation, and for enhancing O. sanctum yield under salt stress. The results showed that co-inoculation of Th and PGPBs enhanced plant height and fresh herb weight by 3.78-17.65% and 7.86-58.76%, respectively; highest being in Th + Fd-2 and Th + Art-7 compared to positive control plants. The doubly inoculated plants showed increased pigments, phenol, flavonoids, protein, sugar, relative water content, and nutrient uptake (Nitrogen and Phosphorous) as compared to monocultures and untreated positive control plants. In addition, co-inoculation in plants resulted in lower Na+, MDA, H2O2, CAT, APX activities, and also lower ACC accumulation (49.75 to 72.38% compared to non-treated salt- stressed plant) in O. sanctum, which probably played a significant role in minimizing the deleterious effects of salinity. Finally, multifactorial analysis showed that co-inoculation of Th and PGPBs improved O. sanctum growth, its physiological activities, and alleviated salt stress compared to single inoculated and positive control plants. These microbial consortia were evaluated for the first time on O. sanctum under salt stress. Therefore, the microbial consortia application could be employed to boost crop productivity in poor, marginalized and stressed agricultural fields. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01328-2.

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.

The emergence of antimicrobial resistant bacteria is an important public health and environmental contamination issue. Antimicrobials of ß-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 ß-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.

Quantification of Salmonella Typhi in water and sediments by molecular-beacon based qPCR.

A molecular-beacon based qPCR assay targeting staG gene was designed for specific detection and quantification of S. Typhi and validated against water and sediment samples collected from the river Ganga, Yamuna and their confluence on two days during Mahakumbha mela 2012-2013 (a) 18 December, 2012: before six major religious holy dips (Makar Sankranti, Paush Poornima, Mauni Amavasya, Basant Panchami, Maghi Poornima and Mahashivratri) (b) 10 February, 2013: after the holy dip was taken by over 3,00,00,000 devotees led by ascetics of Hindu sects at Sangam on 'Mauni Amavasya' (the most auspicious day of ritualistic mass bathing). The assay could detect linearly lowest 1 genomic equivalent per qPCR and is highly sensitive and selective for S. Typhi detection in presence of non specific DNA from other bacterial strains including S. Paratyphi A and S. Typhimurium. It has been observed that water and sediment samples exhibit S. Typhi. The mass holy dip by devotees significantly affected the water and sediment quality by enhancing the number of S. Typhi in the study area. The qPCR developed in the study might be helpful in planning the intervention and prevention strategies for control of enteric fever outbreaks in endemic regions.

Determination of viable Salmonellae from potable and source water through PMA assisted qPCR.

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.

Bio-capture of S. Typhimurium from surface water by aptamer for culture-free quantification.

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.

Identification of environmental reservoirs of nontyphoidal salmonellosis: aptamer-assisted bioconcentration and subsequent detection of salmonella typhimurium by quantitative polymerase chain reaction.

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.

Determination of internalization of chromium oxide nano-particles in Escherichia coli by flow cytometry.

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.

Chromium oxide nano-particles induce stress in bacteria: probing cell viability.

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.

Adverse effects of chromium oxide nano-particles on seed germination and growth in Triticum aestivum L.

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.

Environmental reservoirs for enterotoxigenic Escherichia coli in south Asian Gangetic riverine system.

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.

Contamination of surface and potable water in South Asia by Salmonellae: culture-independent quantification with molecular beacon real-time PCR.

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.

Surface water of a perennial river exhibits multi-antimicrobial resistant shiga toxin and enterotoxin producing Escherichia coli.

The high incidences of waterborne diseases are frequently associated with shiga toxin (STEC) and enterotoxin producing Escherichia coli (ETEC). Therefore, in the present study, surface water samples collected from the river Saryu were analyzed for the presence of multi-antimicrobial resistant ETEC and STEC. Forty-two E. coli isolates were screened for virulence determinants of STEC and ETEC. Eighteen E. coli isolates exhibit both stx1 and stx2 genes (66.6%) or only stx1 (33.3%) gene. eaeA, hlyA, and chuA genes were present in 94.5%, 83.3%, and 55.6% of STEC, respectively. Further, it was observed that 12 isolates exhibit only ST1 gene (25%) or both LT1 and ST1 genes (75%). The resistance to multi-antimicrobials was observed in 100% and 27.7% of ETEC and STEC isolates, respectively. The presence of multi-antimicrobial resistant diarrheagenic E. coli in surface waters of south Asia is an important health concern due to risk of developing waterborne outbreaks.

Rapid culture-independent quantitative detection of enterotoxigenic Escherichia coli in surface waters by real-time PCR with molecular beacon.

Rapid and reliable detection of enterotoxigenic Escherichia coli (ETEC) is critical for the management of the waterborne diseases threatening human lives worldwide. In this study, a culture-independent real-time PCR assay, in molecular beacon format was designed and validated for detection and quantitative enumeration of ETEC harboring LT1 gene (encoding heat labile toxin) in surface waters contaminated by fecal pollutants of human and animal origin. It was observed that the assay was able to detect 2 CFU/mL of ETEC (r = 0.997; PCR efficiency = 99.8%) from water samples spiked by a reference organism (E. coli MTCC 723). In the presence of 10(6) CFU/mL of nonpathogenic E. coli(E. coli DH5alpha), the lowest detection limit from spiked water samples was 4 CFU/mL. The assay was 500 times more sensitive than conventional PCR using the same oligomers (Student's t test p < 0.05). The assay could specifically detect and quantify ETEC (1.2 x 10(3) to 1.4 x 10(6) CFU/100 mL) in polluted surface waters of river Gomti. The rapid culture-independent assay developed in this study for detection and quantitative enumeration of ETEC can be used for preliminary monitoring of surface waters to prevent waterborne outbreaks.

Contamination of potable water distribution systems by multiantimicrobial-resistant enterohemorrhagic Escherichia coli.

BACKGROUND: The contamination of processed or unprocessed drinking water by fecal coliform bacteria has been reported worldwide. Despite a high incidence of waterborne diseases, entero-hemorrhagic Escherichia coli (EHEC) is an underacknowledged pathogen of concern to public health in India. Although the presence of EHEC is recorded in surface water resources of India, drinking water sources are yet to be investigated. OBJECTIVES: The goal of this study was to analyze potable water samples for the presence of virulence determinants of EHEC and to determine the sensitivity of the virulence determinants to antimicrobials. METHODS: We enumerated coliform bacteria in potable water samples collected from six locations in Lucknow, a major city in northern India, using the most probable number method. E. coli (n = 81), randomly isolated by membrane-filtration technique from four sites, were identified by biochemical characterization. E. coli were not detected in samples from two other sites. We screened 15 randomly selected isolates from each site for virulence determinants of EHEC using polymerase chain reaction (PCR). The isolates positive for virulence determinants (n = 18) were screened for sensitivity to 15 antimicrobials by the disk diffusion method. RESULTS: Both stx1 and stx2 genes were present in 33.3% of isolates, whereas others possessed either stx1 (11.1%) or stx2 (55.6%). eaeA, hlyA, and chuA genes were present in 100, 23.3, and 16.7% of isolates, respectively. Resistance to multiple antimicrobials was observed in potential EHEC. CONCLUSIONS: The occurrence of multiantimicrobial-resistant EHEC in potable water is an important health concern because of the risk of waterborne outbreaks.

Prevalence of multi-antimicrobial-agent resistant, shiga toxin and enterotoxin producing Escherichia coli in surface waters of river Ganga.

The consumption of polluted surface water for domestic and recreational purposes by large populations in developing nations is a major cause of diarrheal disease related mortality. The river Ganga and its tributaries meet 40% of the water requirement for drinking and irrigation in India. In this study, Escherichia coli isolates (n=75) of the river Ganga water were investigated for resistance to antimicrobial agents (n=15) and virulence genes specific to shiga toxin (STEC) and enterotoxin producing E. coli (ETEC). E. coli isolates from the river Ganga water exhibit resistance to multiple antimicrobial agents. The distribution of antimicrobial agent resistance in E. colivaries significantly (chi2: 81.28 at df = 24, p < 0.001) between the sites. Both stx1 and stx2 genes were present in 82.3% of STEC (n=17) while remaining isolates possess either stxl (11.8%) or stx2 (5.9%). The presence of eaeA, hlyA, and chuA genes was observed in 70.6, 88.2, and 58.8% of STEC, respectively. Both LT1 and ST1 genes were positive in 66.7% of ETEC (n=15) while 33.3% of isolates harbor only LT1 gene. The prevalence of multi-antimicrobial-agent resistant E. coli in the river Ganga water poses increased risk of infections in the human population.

Evaluation of the alkaline Comet assay conducted with the wetlands plant Bacopa monnieri L. as a model for ecogenotoxicity assessment.

Wetlands play a key role in maintaining environmental quality, and wetlands plants could serve as model organisms for determining the genotoxic effects of pollutants contaminating these areas. In the present study, DNA damage was evaluated in a wetlands plant, Bacopa monnieri L., as a potential tool for the assessment of ecogenotoxicity. The Comet assay was used for detecting DNA damage in B. monnieri exposed to two model mutagens, ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS). Significant (P < 0.05) dose-dependent increases in DNA damage were observed following treatments conducted by exposing both isolated nuclei (acellular or in vitro exposure) and whole plants (in vivo exposure) to 0.01-5 mM EMS and 0.05-100 microM MMS for 2 hr at (26 +/- 2) degrees C. The assay was then used to evaluate the genotoxic potential of cadmium (Cd), a wetlands contaminant. In vitro exposure of nuclei from untreated leaves to 0.001-200 microM Cd for 2 hr resulted in significant (P < 0.05) levels of DNA damage. Cd concentrations >or=0.01 microM induced DNA damage as evidenced by increases in the Olive tail moment. In vivo exposure of plants to 0.01-500 microM Cd for 2, 4, and 18 hr resulted in dose- and time-dependent increases in DNA damage in the nuclei isolated from roots and leaves. Cd-induced DNA damage was greater in roots than leaves. To our knowledge, this is the first report describing the use of a wetlands plant for genotoxicity assessment, using the Comet assay.