Virulence and genotypic characterization of Listeria monocytogenes isolated from vegetable and soil samples.

BACKGROUND: Listeria monocytogenes, a foodborne pathogen is ubiquitous to different environments including the agroecosystem. The organism poses serious public health problem. Therefore, an attempt has been made to gain further insight to their antibiotic susceptibility, serotypes and the virulence genes. RESULTS: Out of the 10 vegetables selected, 6 (brinjal, cauliflower, dolichos-bean, tomato, chappan-kaddu and chilli), 20 isolates (10%) tested positive for L. monocytogenes. The prevalence of the pathogen in the respective rhizosphere soil samples was 5%. Noticeably, L. monocytogenes was absent from only cabbage, broccoli, palak and cowpea, and also the respective rhizospheric soils. The 30 isolates + ve for pathogenicity, belonged to serogroup 4b, 4d or 4e, and all were positive for inlA, inlC, inlJ, plcA, prfA, actA, hlyA and iap gene except one (VC3) among the vegetable isolates that lacked the plcA gene. ERIC- and REP-PCR collectively revealed that isolates from vegetables and their respective rhizospheric soils had distinct PCR fingerprints. CONCLUSIONS: The study demonstrates the prevalence of pathogenic L. monocytogenes in the selected agricultural farm samples. The increase in the number of strains resistant to ciprofloxacin and/or cefoxitin seems to pose serious public health consequences.

Attenuation of quorum sensing system and virulence in Vibrio cholerae by phytomolecules.

The Vibrio cholerae, a gram-negative bacterium, is the causative agent of cholera. Quorum sensing is a cell-to-cell communication that leads to gene expression, accumulation of signaling molecules, biofilm formation, and production of virulence factors. The quorum sensing pathway in V. cholerae is regulated by luxO, and biofilm formation and other virulence factors are positively controlled by aphA and negatively by hapR. Hence, targeting the global regulator luxO would be a promising approach to modulate the QS to curtail V. cholerae pathogenesis. The present study investigated the modulating activity of quercetin and naringenin on biofilm formation and quorum-sensing regulated phenotypes in V. cholerae. Then after we determined the anti-quorum sensing capability of phytomolecules against the model organism Chromobacterium violaceum. Also, we performed flow cytometry for live/dead bacteria, MTT assay, CLSM, and growth curve analysis to determine their role as QS modulators rather than anti-bacterial. V. cholerae strains VC287 and N16961 formed thick biofilm. We observed a two-fold reduction in the expression of biofilm-associated genes comprising gbpA, vpsA, rbmA, and mbaA in the presence of phytomolecules indicating that phytomolecules modulate quorum sensing pathway rather than killing the bacteria. These phytomolecules were non-toxic and non-hemolytic and had anti-adhesion and anti-invasion properties. In addition, quercetin and naringenin were found to be highly effective compared to known quorum-sensing inhibitors terrein and furanone C-30. Thus, this study provides evidence that phytomolecules: quercetin and naringenin modulate the quorum-sensing pathway rather than killing the bacteria and can be used as an anti-quorum-sensing molecule for therapy against the pathogen.

Escherichia coli, but Not Staphylococcus aureus, Functions as a Chelating Agent That Exhibits Antifungal Activity against the Pathogenic Yeast Candida albicans.

Humans are colonized by diverse populations of microbes. Infections by Candida albicans, an opportunistic fungal pathogen, are a result of imbalances in the gut microbial ecosystem and are due to the suppressed immunity of the host. Here, we explored the potential effects of the polymicrobial interactions of C. albicans with Staphylococcus aureus, a Gram-positive bacterium, and Escherichia coli, a Gram-negative bacterium, in dual and triple in vitro culture systems on their respective growth, morphology, and biofilms. We found that S. aureus promoted the fungal growth and hyphal transition of C. albicans through cell-to-cell contacts; contrarily, both the cell and cell-free culture filtrate of E. coli inhibited fungal growth. A yet to be identified secretory metabolite of E. coli functionally mimicked EDTA and EGTA to exhibit antifungal activity. These findings suggested that E. coli, but not S. aureus, functions as a chelating agent and that E. coli plays a dominant role in regulating excessive growth and, potentially, the commensalism of C. albicans. Using animal models of systemic candidiasis, we found that the E. coli cell-free filtrate suppressed the virulence of C. albicans. In general, this study unraveled a significant antimicrobial activity and a potential role in the nutritional immunity of E. coli, and further determining the underlying processes behind the E. coli-C. albicans interaction could provide critical information in understanding the pathogenicity of C. albicans.

Curcumin Encapsulated into Biocompatible Co-Polymer PLGA Nanoparticle Enhanced Anti-Gastric Cancer and Anti-Helicobacter Pylori Effect.

BACKGROUND: The current disadvantages (high cost, toxicity, resistance) of chemotherapy for gastric cancer opted people for alternative therapy from natural source. Curcumin (natural product) possess multiple biological activities but low bio-availability limits their uses as therapeutic. The Nano-formulation of curcumin increased the bioavailability and productivity of anti-cancer and anti-bacterial properties. The present study was initiated to determine the anti-cancer and anti-bacterial effect of Nano curcumin against gastric cancer and H. pylori. METHODS: Curcumin loaded PLGA nanoparticles (CUR-NPs) was prepared by single emulsion solvent evaporation method. The MIC were determined using agar dilution method to find the anti-H. Pylori activity of Nano curcumin. The cytotoxicity of Nano curcumin was evaluated by MTT assay and the apoptotic effect (cell cycle arrest and morphology change) was shown by PI staining and microscopy. RESULTS: The MIC of nanocurcumin and curcumin for all four H. pylori strains were 8 µg/ml and 16 µg/ml respectively. The inhibition rate of gastric cancer cells after treatment with curcumin was increased from 6% to 67% for 24h, from 8% to 75% for 48h, from 10% to 83% for 72h. In case of nanocurcumin, the inhibition rate increased from 7% to 69% for 24h, 11% to 87% for 48h and 16% to 97% for 72h. The IC50 of curcumin and Nano-curcumin were 24.20 µM and 18.78 µM respectively for 72 h. The population of cells in sub-G0 population increased from 4.1% in the control group to 24.5% and 57.8% when treated with curcumin and nanocurcumin respectively. After 72h of treatment with nanocurcumin, the apoptotic cells population increased as compared to native curcumin treated cells. CONCLUSION: The Nano curcumin might be used as a potential therapeutics against gastric cancer and H. Pylori. There is need of further in vivo study in order to validate CUR-NPs activity.

Virulence Pattern and Genomic Diversity of Vibrio cholerae O1 and O139 Strains Isolated From Clinical and Environmental Sources in India.

Vibrio cholerae is an autochthonous inhabitant of the aquatic environment. Several molecular methods have been used for typing V. cholerae strains, but there is no proper database for such scheme, including multilocus sequence typing (MLST) for V. cholerae O1 and O139 strains. We used 54 V. cholerae O1 and three O139 strains isolated from clinical and environmental sources and regions of India during the time period of 1975-2015 to determine the presence of virulence genes and production of biofilm. We devised a MLST scheme and developed a database for typing V. cholerae strains. Also, we performed pulsed-field gel electrophoresis to see the genomic diversity among them and compared it with MLST. We used the MEGA 7.0 software for the alignment and comparison of different nucleotide sequences. The advanced cluster analysis was performed to define complexes. All strains of V. cholerae, except five strains, showed variation in phenotypic characteristics but carried virulence-associated genes indicating they belonged to the El Tor/hybrid/O139 variants. MLST analysis showed 455 sequences types among V. cholerae strains, irrespective of sources and places of isolation. With these findings, we set up an MLST database on PubMLST.org using the BIGSdb software for V. cholerae O1 and O139 strains, which is available at https://pubmlst.org/vcholerae/ under the O1/O139 scheme. The pulsed-field gel electrophoresis (PFGE) fingerprint showed six fingerprint patterns namely E, F, G, H, I, and J clusters among 33 strains including strain N16961 carrying El Tor ctxB of which cluster J representing O139 strain was entirely different from other El Tor strains. Twenty strains carrying Haitian ctxB showed a fingerprint pattern classified as cluster A. Of the five strains, four carrying classical ctxB comprising two each of El Tor and O139 strains and one El Tor strain carrying Haitian ctxB clustered together under cluster B along with V. cholerae 569B showing pattern D. This study thus indicates that V. cholerae strains are undergoing continuous genetic changes leading to the emergence of new strains. The MLST scheme was found more appropriate compared to PFGE that can be used to determine the genomic diversity and population structure of V. cholerae.

Extracellular DNA builds and interacts with vibrio polysaccharide in the biofilm matrix formed by Vibrio cholerae.

Vibrio cholerae form biofilm, which is essential for their survival under harsh environmental conditions. The eDNA produced during biofilm formation and interaction with other components like vibrio polysaccharide is less studied in Vibrio cholerae despite its importance in biofilm structure and stability. In this study, we selected two strains of V. cholerae, which produced sufficient extracellular DNA in the biofilm, for characterization and studied its interaction with vibrio polysaccharide. Our data demonstrate that eDNA is present in the biofilm and interacts with VPS in V. cholerae. Our findings suggest that eDNA contributes to biofilm integrity by interacting with VPS and provides strength to the biofilm. Moreover, it might interact with other components of biofilm, which need further study.

Extracellular vesicles: An emerging platform in gram-positive bacteria.

Extracellular vesicles (EV), also known as membrane vesicles, are produced as an end product of secretion by both pathogenic and non-pathogenic bacteria. Several reports suggest that archaea, gram-negative bacteria, and eukaryotic cells secrete membrane vesicles as a means for cell-free intercellular communication. EVs influence intercellular communication by transferring a myriad of biomolecules including genetic information. Also, EVs have been implicated in many phenomena such as stress response, intercellular competition, lateral gene transfer, and pathogenicity. However, the cellular process of secreting EVs in gram-positive bacteria is less studied. A notion with the thick cell-walled microbes such as gram-positive bacteria is that the EV release is impossible among them. The role of gram-positive EVs in health and diseases is being studied gradually. Being nano-sized, the EVs from gram-positive bacteria carry a diversity of cargo compounds that have a role in bacterial competition, survival, invasion, host immune evasion, and infection. In this review, we summarise the current understanding of the EVs produced by gram-positive bacteria. Also, we discuss the functional aspects of these components while comparing them with gram-negative bacteria.

Antibiotic Resistance Profile, Outer Membrane Proteins, Virulence Factors and Genome Sequence Analysis Reveal Clinical Isolates of Enterobacter Are Potential Pathogens Compared to Environmental Isolates.

Outer membrane proteins (OMPs) of gram-negative bacteria play an important role in mediating antibacterial resistance, bacterial virulence and thus affect pathogenic ability of the bacteria. Over the years, prevalence of environmental antibiotic resistant organisms, their transmission to clinics and ability to transfer resistance genes, have been studied extensively. Nevertheless, how successful environmental bacteria can be in establishing as pathogenic bacteria under clinical setting, is less addressed. In the present study, we utilized an integrated approach of investigating the antibiotic resistance profile, presence of outer membrane proteins and virulence factors to understand extent of threat posed due to multidrug resistant environmental Enterobacter isolates. Also, we investigated clinical Enterobacter isolates and compared the results thereof. Results of the study showed that multidrug resistant environmental Enterobacter isolates lacked OmpC, lacked cell invasion abilities and exhibited low reactive oxygen species (ROS) production in neutrophils. In contrast, clinical isolates possessed OmpF, exhibited high invasive and adhesive property and produced higher amounts of ROS in neutrophils. These attributes indicated limited pathogenic potential of environmental Enterobacter isolates. Informations obtained from whole genome sequence of two representative bacterial isolates from environment (DL4.3) and clinical sources (EspIMS6) corroborated well with the observed results. Findings of the present study are significant as it highlights limited fitness of multidrug resistant environmental Enterobacter isolates.

Antibiotic Susceptibility, Virulence Pattern, and Typing of Staphylococcus aureus Strains Isolated From Variety of Infections in India.

Staphylococcus aureus is one of the major causes of nosocomial infections. This organism produces powerful toxins and cause superficial lesions, systemic infections, and several toxemic syndromes. A total of 109 S. aureus strains isolated from a variety of infections like ocular diseases, wound infection, and sputum were included in the study. Minimum inhibitory concentration (MIC) was determined against 8 antimicrobials. PCR determined the presence of 16S rRNA, nuc, mecA, czrC, qacA/B, pvl, and toxin genes in S. aureus isolates. Pulse-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), SCCmec, spa-, and agr-typing and serotyping determined the diversity among them. All isolates of S. aureus were resistant to two or more than two antibiotics and generated 32 resistance patterns. These isolates were positive for 16S rRNA and S. aureus-specific nuc gene, but showed variable results for mecA, czrC, and qacA/B and pvl genes. Of the 32 methicillin-resistant S. aureus (MRSA), 13 strains carried SCCmec type V, seven type IV, two type III, and nine carried unreported type UT6. Of the 109 strains, 98.2% were positive for hlg, 94.5% for hla, 86.2% for sei, 73.3% for efb, 70.6% for cna, 30.2% for sea, and 12.8% for sec genes. Serotypes VII and VI were prevalent among S. aureus strains. PFGE analysis grouped the 109 strains into 77 clusters. MLST classified the strains into 33 sequence types (ST) and eight clonal complexes (CCs) of which 12 were singletons, and two belong to new allelic profiles. Isolates showed 46 spa-types that included two new spa-types designated as t14911 and t14912. MRSA and methicillin-susceptible S. aureus (MSSA) isolates were diverse in terms of antibiotic resistance pattern, toxin genotypes, SCCmec types, serotypes and PFGE, MLST, and spa-types. However, few isolates from eye infection and wound infection belong to CC239, ST239, and spa-type t037/t657. The study thus suggests that S. aureus strains are multidrug resistant, virulent, and diverse irrespective of sources and place of isolation. These findings necessitate the continuous surveillance of multidrug-resistant and virulent S. aureus and monitoring of the transmission of infection.

Biofilm Formation by ica-Negative Ocular Isolates of Staphylococcus haemolyticus.

Staphylococcus haemolyticus is the second most frequently isolated CoNS from ocular infections and human blood cultures. In this study, we examined 18 ocular S. haemolyticus isolates for their capacity to form biofilm and conducted detachment assay to determine the composition of the biofilm matrix and involvement of various elements in cell lysis. PCR identified the presence of biofilm-associated genes, and ica operon and CLSM visualized the components of the biofilm matrix. We found that PIA-independent biofilm formation is the characteristic feature of S. haemolyticus isolates, irrespective of the sources of isolation, and protein or DNA or both are the major components of the biofilm matrix. Cell lysis enabling DNA release was an essential step for biofilm attachment during the initial stages of biofilm development. The srtA transcript expression study indicates its role in the early stages of biofilm development. We found the presence of antibiotic resistance genes in the eDNA and gDNA thus suggesting the possible role of biofilm in horizontal gene transfer of antibiotic resistance determinants. The overall study indicates that S. haemolyticus formed the biofilm comprising of protein or DNA or both and srtA play a role in the initial development of biofilm.

Analysis of 19 Highly Conserved Vibrio cholerae Bacteriophages Isolated from Environmental and Patient Sources Over a Twelve-Year Period.

The Vibrio cholerae biotype "El Tor" is responsible for all of the current epidemic and endemic cholera outbreaks worldwide. These outbreaks are clonal, and it is hypothesized that they originate from the coastal areas near the Bay of Bengal, where the lytic bacteriophage ICP1 (International Centre for Diarrhoeal Disease Research, Bangladesh cholera phage 1) specifically preys upon these pathogenic outbreak strains. ICP1 has also been the dominant bacteriophage found in cholera patient stools since 2001. However, little is known about the genomic differences between the ICP1 strains that have been collected over time. Here, we elucidate the pan-genome and the phylogeny of the ICP1 strains by aligning, annotating, and analyzing the genomes of 19 distinct isolates that were collected between 2001 and 2012. Our results reveal that the ICP1 isolates are highly conserved and possess a large core-genome as well as a smaller, somewhat flexible accessory-genome. Despite its overall conservation, ICP1 strains have managed to acquire a number of unknown genes, as well as a CRISPR-Cas system which is known to be critical for its ongoing struggle for co-evolutionary dominance over its host. This study describes a foundation on which to construct future molecular and bioinformatic studies of these V. cholerae-associated bacteriophages.

Phenotypic and genetic characteristics of Vibrio cholerae O1 carrying Haitian ctxB and attributes of classical and El Tor biotypes isolated from Silvassa, India.

Vibrio cholerae O1 biotype El Tor, the causative agent of the seventh pandemic, has recently been replaced by strains carrying classical and Haitian ctxB in India, Haiti and other parts of the world. We conducted phenotypic and genetic tests to characterize V. cholerae O1 isolated between 2012 and 2014 from Silvassa, India, to examine the presence of virulence and regulatory genes, seventh pandemic marker, ctxB type and biofilm formation and to study genomic diversity. Of the 59 V. cholerae O1, eight isolates belong to El Tor prototype, one to classical prototype and the remaining isolates have attributes of both classical and El Tor biotypes. PCR and ctxB gene sequencing revealed the presence of classical ctxB in four strains and Haitian ctxB in 55 isolates; indicating that isolates were either an El Tor or hybrid variant. All isolates carried virulence, regulatory, adherence, Vibrio seventh pandemic pathogenicity island I and seventh pandemic group-specific marker VC2346, in addition to tcpAET and rstRET, the features of seventh pandemic strains, and produced cholera toxin and biofilm. PFGE analysis showed that the majority of isolates are clonal and belong to fingerprint pattern A; however, pattern B is unrelated and patterns C and D are distinct, suggesting considerable diversity in the genomic content among them. These data thus show that isolates from Silvassa are genetically diverse and that Haitian ctxB and hybrid phenotypes are undergoing global dissemination.

Identification of Novel Sequence Types among Staphylococcus haemolyticus Isolated from Variety of Infections in India.

The aim of this study was to determine sequence types of 34 S. haemolyticus strains isolated from a variety of infections between 2013 and 2016 in India by MLST. The MEGA5.2 software was used to align and compare the nucleotide sequences. The advanced cluster analysis was performed to define the clonal complexes. MLST analysis showed 24 new sequence types (ST) among S. haemolyticus isolates, irrespective of sources and place of isolation. The finding of this study allowed to set up an MLST database on the PubMLST.org website using BIGSdb software and made available at http://pubmlst.org/shaemolyticus/. The data of this study thus suggest that MLST can be used to study population structure and diversity among S. haemolyticus isolates.

Pregnancy - associated human listeriosis: Virulence and genotypic analysis of Listeria monocytogenes from clinical samples.

Listeria monocytogenes, a life-threatening pathogen, poses severe risk during pregnancy, may cause abortion, fetal death or neonatal morbidity in terms of septicemia and meningitis. The present study aimed at characterizing L. monocytogenes isolated from pregnant women based on serotyping, antibiotic susceptibility, virulence genes, in vivo pathogenicity test and ERIC- and REP-PCR fingerprint analyses. The results revealed that out of 3700 human clinical samples, a total of 30 (0.81%) isolates [12 (0.80%) from placental bit (1500), 18 (0.81%) from vaginal swab (2200)] were positive for L. monocytogenes. All the isolates belonged to serogroup 4b, and were + ve for virulence genes tested i.e. inlA, inlC, inlJ, plcA, prfA, actA, hlyA, and iap. Based on the mice inoculation tests, 20 isolates showed 100% and 4 isolates 60% relative virulence while 6 isolates were non-pathogenic. Moreover, 2 and 10 isolates were resistant to ciprofloxacin and cefoxitin, respectively, while the rest susceptible to other antibiotics used in this study. ERIC- and REP-PCR collectively depicted that the isolates from placental bit and vaginal swab had distinct PCR fingerprints except a few isolates with identical patterns. This study demonstrates prevalence of pathogenic strains mostly resistant to cefoxitin and/or ciprofloxacin. The results indicate the importance of isolating and characterizing the pathogen from human clinical samples as the pre-requisite for accurate epidemiological investigations.

Purification and characterization of cytotoxin produced by a clinical isolate of Vibrio cholerae O54 TV113.

Vibrio cholerae O54 TV113 isolated from a diarrheal patient produces an extracellular cytotoxin that caused alteration in the morphology of Chinese hamster ovary cells manifested as cell shrinkage with intact cell boundaries and finally causing cell death. Syncase medium supplemented with lincomycin (50 µg/ml), pH 7.2, and 18 h incubation with shaking at 37 °C supported optimal cytotoxin production. We isolated and purified this cytotoxin to homogeneity by ultrafiltration, 40-80 % ammonium sulfate precipitation, gradient-anion exchange chromatography, stepwise-anion exchange chromatography, and size exclusion chromatography increasing the specific activity by 866-fold. The cytotoxin is heat-labile, sensitive to protease and papain, and has a molecular weight of 64 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and enterotoxic activity in rabbit ileal loop assay. Both cytotoxic and enterotoxic activity could be inhibited or neutralized by antiserum raised against purified cytotoxin but not by preimmune serum. Immunodiffusion test between purified cytotoxin and its antiserum gave a single well-defined precipitin band showing reaction of complete identity and a well-defined single band in an immunoblot assay. This study thus indicate that the cytotoxin expressed by strain TV113 has both cytotoxic and enterotoxic activity and appears to contribute in pathogenesis of non-O1, non-O139 strains.

Comparative structural analysis of two proteins belonging to quorum sensing system in Vibrio cholerae.

Vibrio cholerae uses quorum sensing communication system to interact with other bacteria and for gauzing environmental parameters. This organism dwells equally well in both human host and aquatic environments. Quorum sensing regulates multitude of activities and is one of the lucrative targets presently pursued for drug design in bacteria to encounter virulence. Histidine phosphotransfer protein LuxU and response regulator LuxO of V. cholerae are known to play important roles in biofilms and virulence machinery. In the present study, we used computational methods to model LuxU and LuxO and simulated the interactions of LuxO and LuxU. Since no structural details of the proteins were available, we employed homology modeling to construct the three-dimensional structures and then performed molecular dynamics simulations to study dynamic behavior of the LuxO and LuxU from V. cholerae. The modeled proteins were validated and subjected to molecular docking analyses. This allowed us to predict the binding modes of the proteins to elucidate probable sites of interference.

Binding efficiencies of carbohydrate ligands with different genotypes of cholera toxin B: molecular modeling, dynamics and docking simulation studies.

Vibrio cholerae produces cholera toxin (CT) that consists of two subunits, A and B, and is encoded by a filamentous phage CTXΦ. The A subunit carries enzymatic activity that ribosylates ADP, whereas the B subunit binds to monosialoganglioside (GM1) receptor in epithelial cells. Molecular analysis of toxigenic V. cholerae strains indicated the presence of multiple ctxB genotypes. In this study, we employed a comparative modeling approach to define the structural features of all known variants of ctxB found in O139 serogroup V. cholerae. Modeling, molecular dynamics and docking simulations studies suggested subtle variations in the binding ability of ctxB variants to carbohydrate ligands of GM1 (galactose, sialic acid and N-acetyl galactosamine). These findings throw light on the molecular efficiencies of pathogenic isolates of V. cholerae harboring natural variants of ctxB in causing the disease, thus suggesting the need to consider ctxB variations when designing vaccines against cholera.

Homology modelling of a sensor histidine kinase from Aeromonas hydrophila.

Aeromonas hydrophila has been implicated in extra-intestinal infection and diarrhoea in humans. Targetting unique effectors of bacterial pathogens is considered a powerful strategy for drug design against bacterial variations to drug resistance. The two-component bacterial system involving sensor histidine kinase (SHK) and its response regulators is considered a lucrative target for drug design. This is the first report describing a three-dimensional (3D) structure for SHK of A. hydrophila. The model was constructed by homology modelling using the X-ray structure of PleD--a response regulator--in conjunction with cdiGMP (PDB code 1W25) and HemAT sensor domain (PDB code 1OR4)--a globin coupled sensor. A combination of homology modelling methodology and molecular dynamics (MD) simulations was applied to obtain a reasonable structure to understand the dynamic behaviour of SHK. Homology modelling was performed using MODELLER9v2 software. The structure was relaxed to eliminate bad atomic contacts. The final model obtained by molecular mechanics and dynamics methods was assessed using PROCHECK and VERIFY 3D graph, which confirmed that the final refined model is reliable. Until complete biochemical and structural data of SHK are determined by experimental means, this model can serve as a valuable reference for characterising the protein and could be explored for drug targetting by design of suitable inhibitors.