Assessment of bacterial viability: a comprehensive review on recent advances and challenges.

Assessing bacterial contamination in environmental samples is critical in determining threats to public health. The classical methods are time-consuming and only recognize species that grow easily on culture media. Viable but non-culturable (VBNC) bacteria are a possible threat that may resuscitate and cause infections. Recent dye-based screening techniques employ nucleic acid dyes such as ethidium monoazide (EMA) and propidium monoazide (PMA), along with many fluorescent dyes, which are an effective alternative for viability assessment. The measurement of cellular metabolism, heat flow and ATP production has also been widely applied in detection approaches. In addition, RNA-based detection methods, including nucleic acid sequence-based amplification (NASBA), have been applied for bacterial pathogen determination. Stable isotope probing using 13C, 15 N and 18O, which are mobilized by microbes, can also be used for effective viability assessment. Future detection tools, such as microarrays, BioNEMS and BioMEMS, which are currently being validated, might offer better microbial viability detection.

Prediction of B Cell Epitopes Among Hantavirus Strains Causing Hemorragic Fever With Renal Syndrome.

Hantavirus infections are now recognized to be a global problem. The hantaviruses include several genotypic variants of the virus with different distributions in varying geographical regions. The virus genotypes seem to segregate in association with certain manifestations specific for each syndrome. They primarily include HFRS, HCPS, febrile illness with or without mild involvement of renal diseases. In the course of our study on hantavirus etiology of febrile illnesses, we recovered a hantavirus strain identified by nPCR. This has been sequenced to be Hantaan-like virus (partial S segment). The current manuscript is focused on understanding the N protein coded by S segment in terms of variation of amino acid sequences of the virus genotypes associated with HFRS. The diagnosis of this infection is achieved by PCR testing of serum/plasma or demonstration of IgM/IgG in serum. The limitations of PCR are temporal often not positive after 7 days of onset of infection. IgM detection is possible around this period and up to 21 days. IgG detection is less definitive in acute infections. Here, we report characterization of the sequence diversity of HFRS strains, 3D structure of Hantaan N protein, and B-cell epitopes on this molecule. We predicted a 20 amino acid sequence length peptide by using BepiPred online server in IEDB analysis resource program. We suggest this peptide may be used for development of geographic region-specific immunoassays like EIAs for antibody detection, monoclonal antibody development, and immunoblots (line immunoassay). J. Cell. Biochem. 118: 1182-1188, 2017. © 2016 Wiley Periodicals, Inc.

Prediction of Pan-Specific B-Cell Epitopes From Nucleocapsid Protein of Hantaviruses Causing Hantavirus Cardiopulmonary Syndrome.

Hantaviruses are emerging viral pathogens that causes hantavirus cardiopulmonary syndrome (HCPS) in the Americas, a severe, sometimes fatal, respiratory disease in humans with a case fatality rate of ≥50%. IgM and IgG-based serological detection methods are the most common approaches used for laboratory diagnosis of hantaviruses. Such emerging viral pathogens emphasizes the need for improved rapid diagnostic devices and vaccines incorporating pan-specific epitopes of genotypes. We predicted linear B-cell epitopes for hantaviruses that are specific to genotypes causing HCPS in humans using in silico prediction servers. We modeled the Andes and Sin Nombre hantavirus nucleocapsid protein to locate the identified epitopes. Based on the mean percent prediction probability score, epitope IMASKSVGS/TAEEKLKKKSAF was identified as the best candidate B-cell epitope specific for hantaviruses causing HCPS. Promiscuous epitopes were identified in the C-terminal of the protein. Our study for the first time has reported pan-specific B-cell epitopes for developing immunoassays in the detection of antibodies to hantaviruses causing HCPS. Identification of epitopes with pan-specific recognition of all genotypes causing HCPS could be valuable for the development of immunodiagnositic tools toward pan-detection of hantavirus antibodies in ELISA. J. Cell. Biochem. 118: 2320-2324, 2017. © 2017 Wiley Periodicals, Inc.

Development & standardization of an in-house IgM indirect ELISA for the detection of parvovirus B19 infections.

BACKGROUND & OBJECTIVES: Parvovirus B19 infections occur worldwide; the infection is acquired early in childhood but could occur later. B19 is reported to cause infection in childhood febrile illnesses, and arthropathies in adults and children and in end-stage renal disease (ESRD) seen in adults. This study was designed to develop an in-house IgM indirect ELISA for serological screening among patients and controls, and to compare ELISA results with those of nested polymerase chain reaction (nPCR) assay. METHODS: An in-house IgM indirect ELISA was standardized using peptide sequence of VP1/VP2 region of parvovirus B19. A total of 201 children and adult with febrile illnesses, 216 individuals with non-traumatic arthropathies, 201 cases of chronic anaemia associated with ESRD and 100 healthy controls were tested. Serum was separated from the blood and subsequently used for DNA extraction. The nested polymerase chain reaction (nPCR) for the detection of B19V DNA was performed using primers targeting the overlapping region of VP1/VP2 capsid protein genes. RESULTS: A total of 618 samples were tested for parvovirus B19 by an in-house IgM indirect ELISA. Among these samples, six were positive by in-house ELISA. The inter-rater agreement between ELISA and PCR assays was calculated using kappa coefficient analysis. The value of κ was 0.77 and the strength of agreement was 'good' (P<0.001). INTERPRETATION & CONCLUSIONS: The in-house IgM indirect ELISA was found to be simple with high sensitivity and specificity when compared with nPCR and could be used as an alternative to expensive commercial kits in resource-poor settings.

Appraisal of the Possible Role of PPARγ Upregulation by CLA of Probiotic Pediococcus pentosaceus GS4 in Colon Cancer Mitigation.

The prevalence of colon cancer (CC) is increasing at the endemic scale, which is accompanied by subsequent morbidity and mortality. Although there have been noteworthy achievements in the therapeutic strategies in recent years, the treatment of patients with CC remains a formidable task. The current study focused on to study role of biohydrogenation-derived conjugated linoleic acid (CLA) of probiotic Pediococcus pentosaceus GS4 (CLAGS4) against CC, which induced peroxisome proliferator-activated receptor gamma (PPARγ) expression in human CC HCT-116 cells. Pre-treatment with PPARγ antagonist bisphenol A diglycidyl ether has significantly reduced the inhibitory efficacy of enhanced cell viability of HCT-116 cells, suggesting the PPARγ-dependent cell death. The cancer cells treated with CLA/CLAGS4 demonstrated the reduced level of Prostaglandin E2 PGE2 in association with reduced COX-2 and 5-LOX expressions. Moreover, these consequences were found to be associated with PPARγ-dependent. Furthermore, delineation of mitochondrial dependent apoptosis with the help of molecular docking LigPlot analysis showed that CLA can bind with hexokinase-II (hHK-II) (highly expressed in cancer cells) and that this association underlies voltage dependent anionic channel to open, thereby causing mitochondrial membrane depolarization, a condition that initiates intrinsic apoptotic events. Apoptosis was further confirmed by annexin V staining and elevation of caspase 1p10 expression. Taken all together, it is deduced that, mechanistically, the upregulation of PPARγ by CLAGS4 of P. pentosaceus GS4 can alter cancer cell metabolism in association with triggering apoptosis in CC.

Cell adherence efficacy of probiotic Pediococcus pentosaceus GS4 (MTCC 12683) and demonstrable role of its surface layer protein (Slp).

The current study examined the cell adherence property of probiotic Pediococcus pentosaceus GS4 (MTCC12683) with the characterization and functionality in adherence of its surface layer protein (GS4-Slp). The Slp of P. pentosaceus GS4 was extracted purified and detected using SDS-PAGE (98 kDa) and size exclusion chromatography. The cell adherence property of probiotic GS4 (Slp+/Slp-) was evaluated on buccal cells and HCT-116. Purified Slp was found neutralized with raised anti-Slp showing reduced adherence to HCT-116 as evident from SEM analysis. The structure of GS4-Slp was determined by MALDI-TOF analysis, CD analysis, atomic force microscopy (AFM), and FT-IR spectrometry. In Silico approach revealed its indirect similarity with cell membrane protein of Helicobacter pylori. Results thus reveal that GS4 has the potential of the production of 98 kDa Slp which facilitates the cell adherence property. This added probiotic attribute will enhance the probiotic potentials of P. pentosaceus GS4 to use it biotechnologically. SIGNIFICANCE: Probiotic Pediococcus pentosaceus GS4 facilitates demonstrable colonization by the elaboration of Slp. This property imparts a value to the strain and claims to be more useful biotechnologically.

Probiotic Enterococcus faecalis AG5 mitigated high fat diet induced obesity and produced propionic acid stimulated apoptosis in 3T3-L1 pre-adipocyte.

AIMS: Probiotics and their metabolites (SCFA) can regulate energy homeostasis. The present study thus evaluates the synergistic effect of probiotic Enterococcus faecalis AG5 on HFD induced obesity and the role of propionic acid (PA) in apoptosis induction of 3T3-L1 pre-adipocyte. METHODS: Male Wistar rats (n = 24) were used to develop an HFD induced obesity model for 24 weeks. The effect of the orally administered probiotic AG5 (18th-24th weeks, 1 × 109 CFU/ml) was evaluated using physiological, biochemical, anthropometry, histopathological and serological analyses. Apoptosis in 3T3-L1 pre-adipocyte was assessed using flow cytometry, protein expression of PPARγ, 5-LOX, NF-κB, p-AKT, caspase 10 and detection of caspase 3/7 by Immunofluorescence confirmed the apoptosis induced by PA. KEY FINDINGS: Probiotic AG5 significantly reduced body weight, BMI, serum cholesterol, triglycerides (p < 0.05) and improved HDL, insulin and leptin but lowered LDL and VLDL (p > 0.05). An inflammatory response was reduced as evident by TNF-α IHC. AG5 reduced adipocyte hypertrophy and fatty acid accumulation. Flow cytometry confirmed late apoptosis in PA-AG5 and standard PA treated 3T3-L1 cells. 5-LOX inhibition is associated with apoptosis induction, and increased caspase 1p 10 is related to cell death initiation. The study initially showed a low PPARγ activity inhibiting 5-LOX which may relate to adipose apoptosis. Finally caspase 3/7 detection using immunofluorescence proved the role of PA in adipocyte apoptosis. SIGNIFICANCE: The present study is a novel approach towards obesity mitigation involving adipocyte apoptosis. The role of SCFA in adipocyte apoptosis is very limited which can prove to be novel therapeutic approach in the future.

Purification and characterization of pediocin from probiotic Pediococcus pentosaceus GS4, MTCC 12683.

Pediococcus pentosaceus GS4 (MTCC 12683), a probiotic lactic acid bacterium (LAB), was found to produce bacteriocin in spent culture. Antibacterial and antagonistic potential of this bacteriocin against reference strains of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 25619), and Listeria monocytogenes (ATCC 15313) was proven by double-layer and well diffusion methods wherein nisin and ampicillin were used as positive controls. Bacteriocin in supernatant was purified and analyzed by SDS-PAGE, RP-HPLC, and circular dichroism (CD). The physico-chemical properties of purified bacteriocin were characterized being treated at different temperatures (30 to 110 °C), pH (3.0 to 12.0), with different enzymes (α-amylase, pepsin, and lysozyme), and organic solvents (hexane, ethanol, methanol, and acetone) respectively. The molar mass of bacteriocin (named pediocin GS4) was determined as 9.57 kDa. The single peak appears at the retention time of 2.403 with area amounting to 25.02% with nisin as positive control in RP-HPLC. CD analysis reveals that the compound appears to have the helix ratio of 40.2% with no beta sheet. The antibacterial activity of pediocin GS4 was optimum at 50 °C and at pH 5.0 and 7.0. The pediocin GS4 was not denatured by the treatment of amylase and lysozyme but was not active in the presence of organic solvents. This novel bacteriocin thus m ay be useful in food and health care industry.

Probiotic Enterococcus faecalis AG5 effectively assimilates cholesterol and produces fatty acids including propionate.

Probiotics are known for their health-promoting abilities through the production of various metabolites, and also benefits are membrane associated. Probiotic Enterococcus faecalis AG5 has been previously assessed for its probiotic properties and this study further strengthens that claim. Strain AG5 was inoculated with 0.3% sodium glychocholate (glycine conjugated) and bile salt mixture for different time periods. The percentage assimilation of cholesterol ranged from 14% to 33.1% in the bile salt mixture, but was slightly higher, ranging from 16.55% to 53.1%, in sodium thioglycocholate alone. Similarly, the bile salt hydrolysis activity was measured in terms of cholic acid release at different intervals. The overall range of cholic acid released ranged from 70.4 to 479 µg/mL. Fatty acid production is also an important criterion for a probiotic selection and it was found that the strain produced several long-chain fatty acids (LCFA) such as tetradecanoic acid, hexadecanoic acid, octadecanoic acid and bis(6-methylheptyl) phthalate. Besides LCFA, AG5 produced short-chain fatty acids (SCFA) such as propionic acid, which was estimated comparing liquid chromatography and mass spectrometry with standard SCFA. Thus the ability of deconjugation of bile salt and SCFA production is a value-added property of probiotic AG5, with the promise of being beneficial for human health.

Antimycobacterial potentials of quercetin and rutin against Mycobacterium tuberculosis H37Rv.

Quercetin and rutin, two flavonoids were examined for antimycobacterial activities against M. tuberculosis H37Rv (ATCC 27294). The quercetin exhibited (99.30 ± 0.268%) in (LRP) assay at 200 µg/ml and 56.21 ± 0.97% inhibition in (BMD) at 50 µg/ml, whereas rutin exhibited (90.40 ± 0.68%) in LRP assay at 200 µg/ml and 56.10 ± 0.67% inhibition in BMD at 50 µg/ml. The minimum inhibitory concentration (MIC) was found to be 6.25 µg ml-1 and 25 µg ml-1 respectively. The current investigation suggests that quercetin has better inhibitory activity than rutin.

Characterization of Functional, Safety, and Probiotic Properties of Enterococcus faecalis AG5 Isolated From Wistar Rat, Demonstrating Adherence to HCT 116 Cells and Gastrointestinal Survivability.

Gut microbiota remains a prominent source for a diverse range of potential probiotics. In this context, the current study explored the rectal region of experimental Wistar rat for the isolation of potent probiotic. Sixteen lactic acid bacteria (LAB), from rectal swab of Wistar rats, were subjected to evaluation of probiotic properties. Among all, AG5 was found unique with consistent probiotic properties and was further identified as Enterococcus faecalis AG5 (NCBI accession number KT248537) using 16S rDNA sequencing, followed by BLAST analysis. Since the Enterococci strains inhibit various food-borne pathogens efficiently while proving itself as a safe probiotic candidate, the study further evaluated the safety of the strain AG5 using primer specific PCR amplification which revealed the existence of gene encoding gelE, asa1, efaA, ace, vanA, and vanB and negative for cylA, hyl, and esp respectively. SEM analysis confirmed the adherence ability of AG5 to HCT 116 cells. Adherence was found to be non-colonial and scattered manner. Furthermore, the strain demonstrated a significant survivability during simulated gastrointestinal transit. Taken together, the E. faecalis AG5 was found potential probiotic candidate with future implication in both food and health industry.

Obesity: An overview of possible role(s) of gut hormones, lipid sensing and gut microbiota.

Obesity is one of the major challenges for public health in 21st century, with 1.9 billion people being considered as overweight and 600 million as obese. There are certain diseases such as type 2 diabetes, hypertension, cardiovascular disease, and several forms of cancer which were found to be associated with obesity. Therefore, understanding the key molecular mechanisms involved in the pathogenesis of obesity could be beneficial for the development of a therapeutic approach. Hormones such as ghrelin, glucagon like peptide 1 (GLP-1) peptide YY (PYY), pancreatic polypeptide (PP), cholecystokinin (CCK) secreted by an endocrine organ gut, have an intense impact on energy balance and maintenance of homeostasis by inducing satiety and meal termination. Glucose and energy homeostasis are also affected by lipid sensing in which different organs respond in different ways. However, there is one common mechanism i.e. formation of esterified lipids (long chain fatty acyl CoAs) and the activation of protein kinase C δ (PKC δ) involved in all these organs. The possible role of gut microbiota and obesity has been addressed by several researchers in recent years, indicating the possible therapeutic approach toward the management of obesity by the introduction of an external living system such as a probiotic. The proposed mechanism behind this activity is attributed by metabolites produced by gut microbial organisms. Thus, this review summarizes the role of various physiological factors such as gut hormone and lipid sensing involved in various tissues and organ and most important by the role of gut microbiota in weight management.

(E)-9-oxooctadec-10-en-12-ynoic acid from Ixora brachiata Roxb. increases glucose uptake in L6 myotubes by activating the PI3K pathway.

In an attempt to discover new scaffolds for anti-diabetic activity from plants, we screened extracts from Ixora brachiata Roxb. for their effect on glucose uptake in L6 myotubes. The petroleum (PE) extract of the plant showed a significant increase in insulin stimulated glucose uptake by L6 myotubes. The bioactivity guided fractionation of the crude extract yielded a compound (E)-9-oxooctadec-10-en-12-ynoic acid (OEA). The compound induced a dose dependent increase in insulin stimulated glucose uptake in L6 myotubes with an EC50 of 22.96µM. OEA also increased the phosphorylation of IRS-1, Akt and AS160 leading to increased GLUT4 translocation to the plasma membrane indicating that it promotes insulin stimulated glucose uptake in L6 myotubes by activating the PI3K pathway.

Analysis and modeling of heat-labile enterotoxins of Escherichia coli suggests a novel space with insights into receptor preference.

Features of heat-labile enterotoxins of Escherichia coli which make them fit to use as novel receptors for antidiarrheals are not completely explored. Data-set of 14 different serovars of enterotoxigenic Escherichia coli producing heat-labile toxins were taken from NCBI Genbank database and used in the study. Sequence analysis showed mutations in different subunits and also at their interface residues. As these toxins lack crystallography structures, homology modeling using Modeller 9.11 led to the structural approximation for the E. coli producing heat-labile toxins. Interaction of modeled toxin subunits with proanthocyanidin, an antidiarrheal showed several strong hydrogen bonding interactions at the cost of minimized energy. The hits were subsequently characterized by molecular dynamics simulation studies to monitor their binding stabilities. This study looks into novel space where the ligand can choose the receptor preference not as a whole but as an individual subunit. Mutation at interface residues and interaction among subunits along with the binding of ligand to individual subunits would help to design a non-toxic labile toxin and also to improve the therapeutics.

NFAT-133 increases glucose uptake in L6 myotubes by activating AMPK pathway.

NFAT-133 is an aromatic compound with cinammyl alcohol moiety, isolated from streptomycetes strain PM0324667. We have earlier reported that NFAT-133 increases insulin stimulated glucose uptake in L6 myotubes using a PPARγ independent mechanism and reduces plasma or blood glucose levels in diabetic mice. Here we investigated the effects of NFAT-133 on cellular signaling pathways leading to glucose uptake in L6 myotubes. Our studies demonstrate that NFAT-133 increases glucose uptake in a dose- and time-dependent manner independent of the effects of insulin. Treatment with Akti-1/2, wortmannin and increasing concentrations of insulin had no effect on NFAT-133 mediated glucose uptake. NFAT-133 induced glucose uptake is completely mitigated by Compound C, an AMPK inhibitor. Further, the kinases upstream of AMPK activation namely; LKB-1 and CAMKKß are not involved in NFAT-133 mediated AMPK activation nor does the compound NFAT-133 have any effect on AMPK enzyme activity. Further analysis confirmed that NFAT-133 indirectly activates AMPK by reducing the mitochondrial membrane potential and increasing the ratio of AMP:ATP.

Molecular insight of putative pathogenicity markers with ESBL genes and lipopolysaccharide in Laribacter hongkongensis.

Laribacter hongkongensis is an emerging bacterial pathogen causing gastroenteritis and traveller's diarrhoea. However, pathogenicity of L. hongkongensis has not yet been properly understood. We therefore, investigated putative pathogenicity markers like elt, est, stx1, stx2, eae, eaf, EAgg, bfpA, Int I, Int II, Int III and cnf respectively in strains received from Queen Mary Hospital, Hong Kong University. Above genes were commonly found among pathogenic member genera of Enterobacteriaceae-causing diarrhoea. Received strains were confirmed microbiologically and by 16S ribosomal RNA (rRNA) sequencing. Cultivability was examined using 23 different commercially available microbial growth media followed by antibiotic susceptibility test, lipopolysaccharide (LPS) extraction and raising antisera in rabbit against heat-killed L. hongkongensis. Moreover, Escherichia coli classification genes chuA, yjaA, TspE4 and extended spectrum beta lactamase (ESBL) genes like TEM, SHV, OXA, CTXM, CTXM1 and CTXM9 were also examined by PCR assay. Results showed the possession of eae, bfpA, Int and CTXM9 respectively for putative virulence. Furthermore, purity of extracted LPS was confirmed by HPLC, and raised serum was found useful in diagnosis of LPS; bacterium thus can be employed for immunodiagnostics.

Laribacter hongkongensis: an emerging pathogen of infectious diarrhea.

Laribacter hongkongensis is relatively a new name in the list of bacterial pathogens for gastroenteritis and travelers' diarrhea. Addition of another name increases burden on the enteric infections as a whole. L. hongkongensis belongs to Neisseriaceae family of ß subclass Proteobacteria. L. hongkongensis was initially isolated in Hong Kong from blood and empyema of an alcoholic cirrhotic patient in 2001, followed by reports from Korea and China, representing a total of 38 articles in PubMed until April 2013. As of now, there is no report from Indian subcontinent where infectious diarrhea is very much prevalent and a major burden. This review provides information about the microbiological characteristics, consideration of an emerging pathogen, relative pathogenicity, genome and proteome content, resistance toward multiple antibiotics, adaptability to different stress, and other features since its time of discovery. Investigation for this bacterium may avoid misidentification as other microbial flora. Further studies like the geographical distribution, type of infection, disease burden, pathogenicity, or genomic exploration of this bacterium will be useful in characterizing them properly. This bacterium may possibly be the emerging threat to public health.

An in silico model for rapid identification of multiple bacteria in resource limited laboratories.

We have developed an in silico model for simplified detection of five major food borne bacteria using common PCR primer based on conserved region flanking a variable region in the first rRNA operon sequences. The RFLP pattern exhibited on virtual SmaI and MboI digestion were unique to each genus. This design is based on the combination of comparative sequence analysis, conventional PCR and restriction digestion methods. We believe this approach as a better alternative to 16S rRNA sequencing based identification / detection of bacteria and suited for the resource limited academic research laboratories in which variety of bacteria are used in different students' projects.

Antimicrobial and antioxidant activities of Mimusops elengi seed extract mediated isotropic silver nanoparticles.

The present study reports the use of Mimusops elengi (M. elengi) fruit extract for the synthesis of silver nanoparticles (Ag NPs). The synthesized Ag NPs was initially noticed through visual color change from yellow to reddish brown and further confirmed by surface plasmonic resonance (SPR) band at 429 nm using UV-Visible spectroscopy. Morphology and size of Ag NPs was determined by Transmission Electron Microscopy (TEM) analysis. X-ray Diffraction (XRD) study revealed crystalline nature of Ag NPs. The prolonged stability of Ag NPs was due to capping of oxidized polyphenols which was established by Fourier Transform Infrared Spectroscopy (FTIR) study. The polyphenols present in M. elengi fruit extract was analyzed by High Pressure Liquid Chromatography (HPLC) and the results revealed the presence of ascorbic acid, gallic acid, pyrogallol and resorcinol. In order to study the role of these polyphenols in reducing Ag+ ions to Ag NPs, analyses of extracts before reduction and after reduction were carried out. In addition, the synthesized Ag NPs were tested for antibacterial and antioxidant activities against Staphylococcus aureus (S. Aureus) and Escherichia coli (E. coli). Ag NPs showed good antimicrobial activity against both gram positive (S. aureus) and gram negative (E. coli) bacteria. It also showed good antioxidant activity as compared to ascorbic acid as standard antioxidant.

Appraisal of microbial evolution to commensalism and pathogenicity in humans.

The human body is host to a number of microbes occurring in various forms of host-microbe associations, such as commensals, mutualists, pathogens and opportunistic symbionts. While this association with microbes in certain cases is beneficial to the host, in many other cases it seems to offer no evident benefit or motive. The emergence and re-emergence of newer varieties of infectious diseases with causative agents being strains that were once living in the human system makes it necessary to study the environment and the dynamics under which this host microbe relationship thrives. The present discussion examines this interaction while tracing the origins of this association, and attempts to hypothesize a possible framework of selective pressures that could have lead microbes to inhabit mammalian host systems.