BCIG-SMAC medium and PMA-qPCR for differential detection of viable Escherichia coli in potable water.

BACKGROUND AND OBJECTIVES: Public health protection requires timely evaluation of pathogens in potable water to minimize outbreaks caused by microbial contaminations. The present study was aimed at assessing the microbiological quality of water obtained from Shantinagar (a rural area in the South Goa region of Goa, India) using 5-Bromo-4-Chloro-3-Indoxyl ß-D-glucuronide-Sorbitol MacConkey agar (BCIG-SMAC) medium and, propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assay for differential detection and quantification of viable Escherichia coli cells in water samples. MATERIALS AND METHODS: Membrane filtration method was used for both BCIG-SMAC medium and PMA-qPCR methods. To determine the efficiency of detection of viable cells, we first evaluated the PMA treatment protocol and established the standard calibration curves using previously reported primers. RESULTS: PMA-qPCR detected as low as 7 femtograms of DNA of E. coli per qPCR reaction whereas the limit of detection (LOD) of BCIG-SMAC medium was 1.8 CFU/100mL. A total of 71 water samples spanning 2017-2018 have been analyzed using BCIG-SMAC medium and PMA-qPCR, of which 95.77% (68/71) and 7.04% (5/71) were found to be total E. coli and E. coli O157:H7, respectively. PMA-qPCR study showed the viable counts of total viable E. coli cells ranging from 3 CFU/100mL to 8.2×102 CFU/100mL. The total E. coli CFU/100mL quantified by PMA-qPCR significantly exceeded (paired t-test; P<0.05) the number on BCIG-SMAC medium. CONCLUSION: The present study indicates that the microbiological quality of environmental water samples analyzed do not comply with the regulatory standard. Therefore, special attention is warranted to improve the overall portable quality of water in the perspective of public health.

Ultrastructural changes in methicillin-resistant Staphylococcus aureus (MRSA) induced by a novel cyclic peptide ASP-1 from Bacillus subtilis: A scanning electron microscopy (SEM) study / Cambios ultraestructurales en Staphylococcusaureus resistente a meticilina (SARM) inducidos por un nuevo péptido cíclico ASP-1 de Bacillussubtilis: un estudio con microscopio electrónico de barrido (MEB)

ABSTRACT Increasing antimicrobial resistance amongStaphylococcus aureusnecessitates a new antimicrobial with a different site of action. We have isolated a novel cyclic peptide-1 (ASP-1) fromBacillussubtiliswith potent activity against methicillin-resistantS. aureus(MRSA) at a minimum inhibitory concentration (MIC) of 8-64μg/ml. Scanning electron micrographs demonstrated drastic changes in the cellular architecture of ASP-1 treated cells ofS. aureusATCC 29213 and an MRSA clinical isolate at MICs, with damages to the cell wall, membrane lysis and probable leakage of cytoplasmic contents at minimum bactericidal concentrations. The ultrastructure alterations induced by ASP-1 have also been compared with those of oxacillin-treated MRSA cells at its MIC using scanning electron microscopy.

RESUMEN El incremento de la resistencia antimicrobiana entre los tipos deS. aureusexige un nuevo agente antimicrobiano con un sitio de acción diferente. Aislamos un nuevo péptido cíclico (ASP-1) deBacillussubtiliscon potente actividad frente aS. aureusresistente a meticilina (SARM) en una concentración inhibitoria mínima (CIM) de 8-64μg/ml. Las micrografías obtenidas con microscopio electrónico de barrido mostraron cambios drásticos en la arquitectura celular de las células deS. aureusATCC 29213 tratadas con ASP-1, y un aislamiento clínico de SARM a la CIM, con daños a la pared celular, lisis de la membrana y probable fuga de contenido citoplasmático a concentraciones bactericidas mínimas. Comparamos también, las alteraciones de la ultraestructura inducidas por ASP-1 con las de células de SARM tratadas con oxacilina a su CIM, utilizando microscopio electrónico de barrido.

Attomolar analyte sensing techniques (AttoSens): a review on a decade of progress on chemical and biosensing nanoplatforms.

Detecting the ultra-low abundance of analytes in real-life samples, such as biological fluids, water, soil, and food, requires the design and development of high-performance biosensing modalities. The breakthrough efforts from the scientific community have led to the realization of sensing technologies that measure the analyte's ultra-trace level, with relevant sensitivity, selectivity, response time, and sampling efficiency, referred to as Attomolar Analyte Sensing Techniques (AttoSens) in this review. In an AttoSens platform, 1 aM detection corresponds to the quantification of 60 target analyte molecules in 100 µL of sample volume. Herein, we review the approaches listed for various sensor probe design, and their sensing strategies that paved the way for the detection of attomolar (aM: 10-18 M) concentration of analytes. A summary of the technological advances made by the diverse AttoSens trends from the past decade is presented.

Ultrastructural changes in methicillin-resistant Staphylococcus aureus (MRSA) induced by a novel cyclic peptide ASP-1 from Bacillus subtilis: A scanning electron microscopy (SEM) study.

Increasing antimicrobial resistance among Staphylococcus aureus necessitates a new antimicrobial with a different site of action. We have isolated a novel cyclic peptide-1 (ASP-1) from Bacillussubtilis with potent activity against methicillin-resistant S. aureus (MRSA) at a minimum inhibitory concentration (MIC) of 8-64µg/ml. Scanning electron micrographs demonstrated drastic changes in the cellular architecture of ASP-1 treated cells of S. aureus ATCC 29213 and an MRSA clinical isolate at MICs, with damages to the cell wall, membrane lysis and probable leakage of cytoplasmic contents at minimum bactericidal concentrations. The ultrastructure alterations induced by ASP-1 have also been compared with those of oxacillin-treated MRSA cells at its MIC using scanning electron microscopy.

A capacitive DNA sensor for sensitive detection of Escherichia coli O157:H7 in potable water based on the z3276 genetic marker: fabrication and analytical performance.

We report a label-free biosensor for the detection of Escherichia coli O157:H7 ATCC 43895 in potable water using a newly designed DNA sensing probe targeting the z3276 genetic marker. The surface of indium tin oxide (ITO) was functionalized with the novel sensing probe by covalent coupling using APTES as a crosslinker to fabricate the DNA sensor (dubbed ZEC [z[combining low line]3276 gene of E[combining low line]. c[combining low line]oli O157:H7 ATCC 43895]). The electrochemical characterization of the fabricated ZEC sensor was performed using cyclic voltammetry and electrochemical impedance spectroscopy. Atomic force microscopy and scanning electron microscopy revealed significant changes in the surface topographies of the fabricated ZEC sensor chip. Equivalent circuit analyses suggested the capacitive nature of the ZEC sensor chip, which demonstrated a declining trend of the capacitance value from 1.568 µF (Bare ITO) to 1.221 µF (after DNA hybridization). Non-faradaic sensing measurements revealed systematically declining capacitance values upon DNA hybridization, with a 10 min response time at 10 Hz frequency and 10 mV applied potential. The ZEC sensor chip exhibited linearity in the range of 0.5 to 25 pg per 10 mL for E. coli O157:H7, with ubiquitous cross-validation of each DNA concentration using quantitative PCR prior to the analyses of real water samples. The limit of detection (LOD) at 95% confidence estimated by logistic regression was 0.1 pg DNA per 10 mL of E. coli O157:H7 (equivalent to 13.67 CFU per 10 mL) with a p-value of 0.0237. Consequently, the obtained results demonstrate the possible application of the developed ZEC sensor chip for E. coli O157:H7 detection in real water samples.

A novel method for rapid and sensitive detection of viable Escherichia coli cells using UV-induced PMA-coupled quantitative PCR.

We report a specific and sensitive method to improve the coupling of propidium monoazide (PMA) with DNA derived from killed cells of Escherichia coli using UV light of 365 nm. UV light of three different intensities mainly 2.4 × 103, 4.8 × 103, and 7.2 × 103 µJ/cm2 was applied to E. coli cells each for 1, 3, and 5 min. PMA was found to be successfully cross-linked with the DNA from killed cells of E. coli at 4.8 × 103 µJ/cm2 in 3 min leading to the complete inhibition of PCR amplification of DNA derived from PMA-treated heat-killed cells. In spiked phosphate-buffered saline and potable water samples, the difference of the Cq values between PMA-treated viable cells and PMA-untreated viable cells ranged from -0.17 to 0.2, demonstrating that UV-induced PMA activation had a negligible effect on viable cells. In contrast, the difference of the Cq values between PMA-treated heat-killed cells and PMA-untreated heat-killed cells ranged from 8.9 to 9.99, indicating the ability of PMA to inhibit PCR amplification of DNA derived from killed cells to an equivalent as low as 100 CFU. In conclusion, this UV-coupled PMA-qPCR assay provided a rapid and sensitive methodology to selectively detect viable E. coli cells in spiked water samples within 4 h.

Xylooligosaccharides production by crude microbial enzymes from agricultural waste without prior treatment and their potential application as nutraceuticals.

Aspergillus fumigatus R1, on submerged fermentation using agricultural residues as carbon source produced extracellular xylanase (152IU/ml after 96h of incubation at 37°C with constant shaking at 100rpm). A maximum yield of 1gm% Xylooligosaccharides (XOS) mixture was obtained after 12h by enzymatic hydrolysis of xylan rich wheat husk without any prior pretreatment using the crude enzyme without any purification. HP-TLC data confirmed the presence of an array of XOS for its prebiotic properties by carrying out studies on ten standard probiotic cultures. Six of ten probiotic cultures were able to utilize XOS produced from agricultural wastes and showed remarkable growth in the media containing XOS as the sole source of carbon. XOS mixture also exhibited concentration dependent anti-oxidant activity. Thus, the results showed that XOS produced from agricultural residues have great prebiotic potential and good antioxidant activity; therefore, it can be used in food-related applications.

Recent developments in detection and enumeration of waterborne bacteria: a retrospective minireview.

Waterborne diseases have emerged as global health problems and their rapid and sensitive detection in environmental water samples is of great importance. Bacterial identification and enumeration in water samples is significant as it helps to maintain safe drinking water for public consumption. Culture-based methods are laborious, time-consuming, and yield false-positive results, whereas viable but nonculturable (VBNCs) microorganisms cannot be recovered. Hence, numerous methods have been developed for rapid detection and quantification of waterborne pathogenic bacteria in water. These rapid methods can be classified into nucleic acid-based, immunology-based, and biosensor-based detection methods. This review summarizes the principle and current state of rapid methods for the monitoring and detection of waterborne bacterial pathogens. Rapid methods outlined are polymerase chain reaction (PCR), digital droplet PCR, real-time PCR, multiplex PCR, DNA microarray, Next-generation sequencing (pyrosequencing, Illumina technology and genomics), and fluorescence in situ hybridization that are categorized as nucleic acid-based methods. Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence are classified into immunology-based methods. Optical, electrochemical, and mass-based biosensors are grouped into biosensor-based methods. Overall, these methods are sensitive, specific, time-effective, and important in prevention and diagnosis of waterborne bacterial diseases.

Purification, biochemical characterization and structural modelling of alkali-stable ß-1,4-xylan xylanohydrolase from Aspergillus fumigatus R1 isolated from soil.

BACKGROUND: Aspergillus fumigatus R1 produced xylanase under submerged fermentation which degrades the complex hemicelluloses contained in agricultural substrates. Xylanases have gained considerable attention because of their tremendous applications in industries. The purpose of our study was to purify xylanase and study its biochemical properties. We have predicted the secondary structure of purified xylanase and evaluated its active site residues and substrate binding sites based on the global and local structural similarity. RESULTS: Various microorganisms were isolated from Puducherry soil and screened by Congo-red test. The best isolate was identified to be Aspergillus fumigatus R1. The production kinetics showed the highest xylanase production (208 IU/ml) by this organism in 96 h using 1 % rice bran as the only carbon source. The purification of extracellular xylanase was carried out by fractional ammonium sulphate precipitation (30-55 %), followed by extensive dialysis and Bio-Gel P-60 Gel-filtration chromatography. The enzyme was purified 58.10 folds with a specific activity of 38196.22 IU/mg. The biochemical characterization of the pure enzyme was carried out for its optimum pH and temperature (5.0 and 50(0)C), pH and temperature stability, molecular mass (Mr) (24.5 kDa) and pI (6.29). The complete sequence of protein was obtained by mass spectrometry analysis. Apparent Km and Vmax values of the xylanase for birchwood xylan were 11.66 mg/ml and 87.6 µmol min(-1) mg(-1) respectively. CONCLUSION: Purified xylanase was analyzed by mass-spectrometry which revealed 2 unique peptides. Xylanase under current study showed significant production using agricultural residues and a broad range of pH stability in the alkaline region. Xylanase produced by Aspergillus fumigatus R1 could serve as the enzyme of choice in industries.