Isolation and characterization of a moderately halophilic Marinobacter phage-host system from the Arabian Sea.

Marinobacter is an ecologically important genus of Gammaproteobacteria found in diverse marine habitats, many species of which are capable of degrading hydrocarbons. In this study, we isolated a Marinobacter phage-host system from the surface waters of the Arabian Sea using enrichment culture methods, studied their growth characteristics and investigated the effect of salinity and nitrate concentrations on phage-host interactions. The bacterial isolate had maximum identity to Marinobacter salsuginis based on 16S rRNA similarities and was termed as Marinobacter sp., strain D1S9. It could tolerate up to 14% of NaCl with maximum growth at 11% NaCl. The host grew optimally between 35 and 40 °C and at pH 8. It had a generation time of 3.7 h with a mean growth rate of 0.27 h-1. The phage infected the host forming clear, round plaques of 1-2 mm diameter. It had a narrow host range restricted to the strain Marinobacter D1S9. The latent period and burst size of the phage were estimated to be 30 min and 106 phages per infected cell, respectively. The phage had an adsorption rate of 3.4 × 10-8 ml min-1 and retained 40.4% of its adsorption efficiency at 16% NaCl with a maximum at 4% NaCl (76.1%). Inorganic nitrate was found to have a direct role in controlling host growth and phage burst size.

Diurnal variations in bacterial and viral production in Cochin estuary, India.

Microbes play a central role in the decomposition and remineralization of organic matter and recycling of nutrients in aquatic environments. In this study, we examined the influence of physical, chemical, and biological parameters on the rate of bacterial production (BP) and viral production (VP) with respect to primary production over a diurnal period in Cochin estuary. Time series measurements were made every 2 h for 12 h (6 a.m.-6 p.m.) during periods of low and high salinities. The light intensity as photosynthetically active radiation, temperature, salinity, nutrients like NO3-N, SiO4-Si, and PO4-P, and chlorophyll a (Chl a) were measured along with BP, VP, and net primary production (NPP). NPP showed a strong positive correlation with light and Chl a (r (2) = 0.56 and 0.47, respectively), while VP showed a strong positive correlation with light, salinity, and Chl a (r (2) = 0.37, 0.58, and 0.37, respectively) and a negative correlation with BP (r (2) = -0.39) at P ≤ 0.05. We observed a diurnal pattern in BP but did not have any significant correlation with light. Similar diurnal pattern was seen in VP, the peak of which was in succession with BP, suggesting that virus-mediated lysis plays an important role in loss processes of bacteria in Cochin estuary. The results of our study highlight the light-dependent and physicochemical-dependent diurnal variation in virioplankton production in a tropical estuarine ecosystem.

Comparative virulence genotyping and antimicrobial susceptibility profiling of environmental and clinical Salmonella enterica from Cochin, India.

Salmonella enterica serotype Newport is an important cause of non-typhoidal salmonellosis, a clinically less severe infection than typhoid fever caused by S. enterica serotype Typhi. In this investigation, the virulence genotypes of S. enterica Newport isolated from a backwater environment were compared with Salmonella Typhi from clinical cases in the same region where salmonellosis is endemic. Genotyping was done by PCR screening for virulence markers associated with Salmonella pathogenicity islands (SPIs) and plasmids. The virulence genes associated with SPIs I-VI were detected in 95-100% of all the isolates, while the viaB locus representing SPI-7 was detectable in 66 and 73% of the environmental and clinical isolates, respectively. A significant number of Salmonella Newport lacked virulence genes shdA and sopE compared to S. Typhi. All S. Typhi and S. Newport isolates lacked large plasmid-borne virulence genes spvR and pefA. Further investigations into the antimicrobial resistance of S. Newport revealed multiple drug resistance to ampicillin, amoxicillin/clavulanic acid, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline, cephalothin, and cephalexin. In comparison, S. Typhi were susceptible to all clinically relevant antimicrobials. The results of this study will help in understanding the spread of virulence genotypes and antibiotic resistance in S. Newport in the region of study.

Study of changes in bacterial and viral abundance in formaldehyde-fixed water samples by epifluorescence microscopy.

Accurate measurement of bacterial and viral abundance in coastal marine environments is important to understand the dynamics of microbial communities in these ecosystems. In this study, the effect of formaldehyde preservation on the abundance of bacteria and viruses in water samples from Cochin Backwater was determined by SYBR Green I staining and epifluorescence microscopy. The counts were determined for 45 days in samples fixed with 1-6% formaldehyde. The results suggest rapid decline in counts of bacteria and viruses in samples preserved in formaldehyde, and the decline increased with increase in the final concentration of formaldehyde in the sample. The initial bacterial and viral counts in the sampling site were 5.2 × 10(6)/ml and 3.9 × 10(7)/ml, respectively. The bacterial count dropped by 5.1%, 53.3%, 55.7%, and 85% after 1, 7, 15, and 45 days, respectively, in sample fixed with 1% formaldehyde. The decline in viral counts was higher, being 32.4%, 47.9%, 68.1%, and 93% after 1, 7, 15, and 45 days, respectively. Storage of fixed samples at - 20°C did not halt the decline in microbial counts, suggesting that, irrespective of storage temperature, formaldehyde-fixed samples lead to underestimation of microbial counts.

Influence of hydrological factors on bacterial community structure in a tropical monsoonal estuary in India.

In the present study, we analyzed variations in bacterial community structure along a salinity gradient in a tropical monsoonal estuary (Cochin estuary [CE]), on the southwest coast of India, using Illumina next-generation sequencing (NGS). Water samples were collected from eight different locations thrice a year to assess the variability in the bacterial community structure and to determine the physico-chemical factors influencing the bacterial diversity. Proteobacteria was the most dominant phyla in the estuary followed by Bacteroidetes, Cyanobacteria, Actinobacteria, and Firmicutes. Statistical analysis indicated significant variations in bacterial communities between freshwater and mesohaline and euryhaline regions, as well as between the monsoon (wet) and nonmonsoon (dry) periods. The abundance of Betaproteobacteria was higher in the freshwater regions, while Alphaproteobacteria and Epsilonproteobactera were more abundant in mesohaline and euryhaline regions of the estuary. Gammaproteobacteria was more abundant in regions with high nutrient concentrations. Various bacterial genera indicating the presence of fecal contamination and eutrophication were detected. Corrplot based on Pearson correlation analysis demonstrated the important physico-chemical variables (temperature, salinity, dissolved oxygen, and inorganic nutrients) that influence the distribution of dominant phyla, class, and genera. The observed spatio-temporal variations in bacterial community structure in the CE were governed by regional variations in anthropogenic inputs and seasonal variations in monsoonal rainfall and tidal influx.

Bacterial Resistance to Antimicrobial Agents.

Bacterial pathogens as causative agents of infection constitute an alarming concern in the public health sector. In particular, bacteria with resistance to multiple antimicrobial agents can confound chemotherapeutic efficacy towards infectious diseases. Multidrug-resistant bacteria harbor various molecular and cellular mechanisms for antimicrobial resistance. These antimicrobial resistance mechanisms include active antimicrobial efflux, reduced drug entry into cells of pathogens, enzymatic metabolism of antimicrobial agents to inactive products, biofilm formation, altered drug targets, and protection of antimicrobial targets. These microbial systems represent suitable focuses for investigation to establish the means for their circumvention and to reestablish therapeutic effectiveness. This review briefly summarizes the various antimicrobial resistance mechanisms that are harbored within infectious bacteria.

Functional and Structural Roles of the Major Facilitator Superfamily Bacterial Multidrug Efflux Pumps.

Pathogenic microorganisms that are multidrug-resistant can pose severe clinical and public health concerns. In particular, bacterial multidrug efflux transporters of the major facilitator superfamily constitute a notable group of drug resistance mechanisms primarily because multidrug-resistant pathogens can become refractory to antimicrobial agents, thus resulting in potentially untreatable bacterial infections. The major facilitator superfamily is composed of thousands of solute transporters that are related in terms of their phylogenetic relationships, primary amino acid sequences, two- and three-dimensional structures, modes of energization (passive and secondary active), and in their mechanisms of solute and ion translocation across the membrane. The major facilitator superfamily is also composed of numerous families and sub-families of homologous transporters that are conserved across all living taxa, from bacteria to humans. Members of this superfamily share several classes of highly conserved amino acid sequence motifs that play essential mechanistic roles during transport. The structural and functional importance of multidrug efflux pumps that belong to the major facilitator family and that are harbored by Gram-negative and -positive bacterial pathogens are considered here.

Identification of a novel UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Vibrio fischeri that confers high fosfomycin resistance in Escherichia coli.

MurA [UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase] is a key enzyme involved in bacterial cell wall peptidoglycan synthesis and a target for the antimicrobial agent fosfomycin, a structural analog of the MurA substrate phosphoenol pyruvate. In this study, we identified, cloned and sequenced a novel murA gene from an environmental isolate of Vibrio fischeri that is naturally resistant to fosfomycin. The fosfomycin resistance gene was isolated from a genomic DNA library of V. fischeri. An antimicrobial agent hypersensitive strain of Escherichia coli harboring murA from V. fischeri exhibited a high fosfomycin resistance phenotype, with minimum inhibitory concentration of 3,000 microg/ml. The cloned murA gene was 1,269 bp long encoding a 422 amino acid polypeptide with an estimated pI of 5.0. The deduced amino acid sequence of the putative protein was identified as UDP-NAG enolpyruvyl transferase by homology comparison. The MurA protein with an estimated molecular weight of 44.7 kDa was expressed in E. coli and purified by affinity chromatography. MurA of V. fischeri will be a useful target to identify potential inhibitors of fosfomycin resistance in pharmacological studies.

Prevalence and heterogeneity of hemolysin gene vhh among hatchery isolates of Vibrio harveyi in India.

Vibrio harveyi, pathogenic to fish, harbor a hemolysin gene vhh, the homologues of which are found in many species of the Genus Vibrio. In this study, we investigated the prevalence of vhh gene among V. harveyi isolated from Penaeus monodon hatcheries in India by polymerase chain reaction (PCR). The vhh was detected in 67 of the 70 V. harveyi isolates tested in this study using different combinations of PCR primers. A variant vhh gene detected in a minority of strains was cloned, sequenced, and the recombinant protein was expressed in Escherichia coli. The deduced amino acid sequence of the cloned gene was 86% similar to the previously reported amino acid sequences of VHH. The results of this study suggest that though V. harveyi strains invariably harbor vhh, the sequence variants of the hemolysin gene exist that may impede their detection by PCR.

Biochemical and molecular characterization of Bacillus pumilus isolated from coastal environment in Cochin, India.

Bacillus species constitute a diverse group of bacteria widely distributed in soil and the aquatic environment. In this study, Bacillus strains isolated from the coastal environment of Cochin, India were identified by detailed conventional biochemical methods, fatty acid methyl ester (FAME) analysis and partial 16S rDNA sequencing. Analysis of the data revealed that Bacillus pumilus was the most predominant species in the region under study followed by B. cereus and B. sphaericus. The B. pumilus isolates were further characterized by arbitrarily primed PCR (AP-PCR), antibiotic sensitivity profiling and PCR screening for known toxin genes associated with Bacillus spp. All B. pumilus isolates were biochemically identical, exhibited high protease and lipase activity and uniformly sensitive to antibiotics tested in this study. One strain of B. pumilus harboured cereulide synthetase gene cesB of B. cereus which was indistinguishable from rest of the isolates biochemically and by AP-PCR. This study reports, for the first time, the presence of the emetic toxin gene cesB in B. pumilus.

Development and evaluation of a polymerase chain reaction (PCR) assay for the detection of Opisthorchis viverrini in fish.

Human opisthorchiais caused by the liver fluke Opisthorchis viverrini is a major fish-borne trematode infection endemic in the Southeast Asian countries. The infection is acquired through consumption of raw fish harboring metacercariae of O. viverrini. Owing to potential risk of transmission of opisthorchiasis through fish trade, rapid and reliable detection methods have gained importance to ensure food safety. In the study described here, we report successful development and evaluation of a polymerase chain reaction (PCR) assay for the detection of O. viverrini, based on the nucleotide sequence derived in this study. The assay is specific with no cross-reaction with other trematodes commonly found in fish including the closely related species, Clonorchis sinensis. The sensitivity of the assay was determined to be 10(-12)ng of O. viverrini DNA while in artificially spiked fish meat, 3 metacercariae could be detected. The results suggest that the PCR method described here is specific to O. viverrini with potential application in fish quarantining.

Opisthorchis viverrini: detection by polymerase chain reaction (PCR) in human stool samples.

A polymerase chain reaction (PCR) assay was evaluated for detection of Opisthorchis viverrini eggs in the stool specimens of light and heavily infected individuals in Khon Kaen province of Thailand. A total of 75 fecal specimens were analyzed by PCR following DNA extraction. All the microscopically positive samples were positive by PCR, while 23 of 30 (76.6%) microscopically negative samples were also PCR positive. The sensitivity of the assay was 5 eggs/g of stool. This method is potentially useful in the diagnosis of human opisthorchiasis in endemic areas for treatment and in epidemiological investigations.

Genetic and functional diversity of double-stranded DNA viruses in a tropical monsoonal estuary, India.

The present study illustrates the genetic diversity of four uncultured viral communities from the surface waters of Cochin Estuary (CE), India. Viral diversity inferred using Illumina HiSeq paired-end sequencing using a linker-amplified shotgun library (LASL) revealed different double-stranded DNA (dsDNA) viral communities. The water samples were collected from four stations PR1, PR2, PR3, and PR4, during the pre-monsoon (PRM) season. Analysis of virus families indicated that the Myoviridae was the most common viral community in the CE followed by Siphoviridae and Podoviridae. There were significant (p < 0.05) spatial variations in the relative abundance of dominant families in response to the salinity regimes. The relative abundance of Myoviridae and Podoviridae were high in the euryhaline region and Siphoviridae in the mesohaline region of the estuary. The predominant phage type in CE was phages that infected Synechococcus. The viral proteins were found to be involved in major functional activities such as ATP binding, DNA binding, and DNA replication. The study highlights the genetic diversity of dsDNA viral communities and their functional protein predictions from a highly productive estuarine system. Further, the metavirome data generated in this study will enhance the repertoire of publicly available dataset and advance our understanding of estuarine viral ecology.

Differential impact of lytic viruses on prokaryotic morphopopulations in a tropical estuarine system (Cochin estuary, India).

Our understanding on the importance of viral lysis in the functioning of tropical estuarine ecosystem is limited. This study examines viral infection of prokaryotes and subsequent lysis of cells belonging to different morphotypes across a salinity gradient in monsoon driven estuarine ecosystem (Cochin estuary, India). High standing stock of viruses and prokaryotes accompanied by lytic infection rates in the euryhaline/mesohaline region of the estuary suggests salinity to have an influential role in driving interactions between prokaryotes and viruses. High prokaryotic mortality rates, up to 42% of prokaryote population in the pre-monsoon season is further substantiated by a high virus to prokaryote ratio (VPR), suggesting that maintenance of a high number of viruses is dependent on the most active fraction of bacterioplankton. Although myoviruses were the dominant viral morphotype (mean = 43%) throughout the study period, there was significant variation among prokaryotic morphotypes susceptible to viral infection. Among them, the viral infected short rod prokaryote morphotype with lower burst estimates (mean = 18 viruses prokaryote-1) was dominant (35%) in the dry seasons whereas a substantial increase in cocci forms (30%) infected by viruses with high burst size (mean = 31 viruses prokaryote-1) was evident during the monsoon season. Such preferential infections of prokaryotic morphopopulations with respect to seasons can have a strong and variable impact on the carbon and energy flow in this tropical ecosystem.

High Incidence of Lysogeny in the Oxygen Minimum Zones of the Arabian Sea (Southwest Coast of India).

Though microbial processes in the oxygen minimum zones (OMZs) of the Arabian Sea (AS) are well documented, prokaryote-virus interactions are less known. The present study was carried out to determine the potential physico-chemical factors influencing viral abundances and their life strategies (lytic and lysogenic) along the vertical gradient in the OMZ of the AS (southwest coast of India). Water samples were collected during the southwest monsoon (SWM) season in two consecutive years (2015 and 2016) from different depths, namely, the surface layer, secondary chlorophyll a maxima (~30⁻40 m), oxycline (~70⁻80 m), and hypoxic/suboxic layers (~200⁻350 m). The high viral abundances observed in oxygenated surface waters (mean ± SD = 6.1 ± 3.4 × 106 viral-like particles (VLPs) mL-1), drastically decreased with depth in the oxycline region (1.2 ± 0.5 × 106 VLPs mL-1) and hypoxic/suboxic waters (0.3 ± 0.3 × 106 VLPs mL-1). Virus to prokaryote ratio fluctuated in the mixed layer (~10) and declined significantly (p < 0.001) to 1 in the hypoxic layer. Viral production (VP) and frequency of virus infected cells (FIC) were maximum in the surface and minimum in the oxycline layer, whereas the viral lysis was undetectable in the suboxic/hypoxic layer. The detection of a high percentage of lysogeny in suboxic (48%) and oxycline zones (9⁻24%), accompanied by undetectable rates of lytic viral infection support the hypothesis that lysogeny may represent the major survival strategy for viruses in unproductive or harsh nutrient/host conditions in deoxygenated waters.

Viral-Induced Mortality of Prokaryotes in a Tropical Monsoonal Estuary.

Viruses are recognized as the most abundant and dynamic biological entities in the marine and estuarine environment. However, studies on the dynamics and activity of viruses in transient estuarine systems are limited. This study examines temporal and spatial variations in viral abundance (VA) and viral activity across the salinity gradient in a monsoon-driven tropical estuarine system (Cochin estuary, CE) along the southwest coast of India. Water samples were collected from five stations (with different hydrological settings) every 3 h for 24 h period during two distinct seasons, namely pre-monsoon (PRM, dry season) and monsoon (MON, wet season). Time series measurements were made for a spring and neap tidal cycle for each season at all the stations. The results showed marked spatial and seasonal variability with relatively low diel and tidal variations in VA and lytic activity. Viral activity was found to be distinct in five stations studied with the maximum activity in the mesohaline regions (salinity <20) of the estuary. This region was characterized by high VA, lytic infection and viral production, accompanied by low (BGE) and high bacterial respiration. Based on viral lytic production, lytic viruses were found to be responsible for the release of ca. 72.9 ± 58.5 µg C L-1d-1 of bacterial carbon. The contribution of the viral shunt to the dissolved organic carbon (DOC) pool was higher during the dry season (PRM) than MON. Statistical analysis confirmed a significant association of viruses with the host availability and salinity. This work demonstrates the spatiotemporal distribution of viruses in a tropical estuarine ecosystem and highlights their role in microbial mortality across different salinity gradients. This study forms the first report on viral processes from a monsoon-driven tropical estuarine ecosystem.

A gyrB-based PCR for the detection of Vibrio vulnificus and its application for direct detection of this pathogen in oyster enrichment broths.

A polymerase chain reaction (PCR) method based on the gyrB (encoding gyrase B or topoisomerase II) gene sequence was developed for the detection of Vibrio vulnificus in seafood. The gyrB primers detected all laboratory isolates of V. vulnificus and did not cross react with other Vibiro and non-Vibrio species examined in this study. The sensitivity of detection of V. vulnificus by gyrB PCR was 300 CFU/g in artificially seeded oyster homogenate without enrichment while, 30 CFU/g could be detected following 18 h enrichment in alkaline peptone water (APW). The gyrB-specific PCR was employed for the direct detection of V. vulnificus in oyster enrichment broths. The assay detected V. vulnificus in 75% of natural oyster samples after 18 h enrichment in APW. The gyrB-based PCR described here offers a simple and specific one step PCR method for the detection of V. vulnificus in seafood enrichment broths.

Distribution of Putative Virulence Genes and Antimicrobial Drug Resistance in Vibrio harveyi.

The marine-estuarine bacterium Vibrio harveyi is an important pathogen of invertebrates, most significantly, the larvae of commercially important shrimp Penaeus monodon. In this study, we analyzed V. harveyi isolated from shrimp hatchery environments for understanding the distribution of putative virulence genes and antimicrobial drug resistance. The putative genes targeted for PCR detection included four reversible toxin (Rtx)/hemolysin genes, a gene encoding homologue of Vibriocholerae zonula occludens toxin (Zot) and a hemolysin-coregulated protein gene (hcp) by polymerase chain reaction (PCR). Of the four putative reversible toxin genes, vhh-1 was detected in 31% of the isolates, vhh-2 in 46%, vhh-3 in 23% and vhh-4 was detected in 27% of the isolates. A zot-like sequence of bacteriophage f237 was present in 15%, while hcp sequence was detected in 48% of the isolates. The antimicrobial susceptibility test revealed resistance to several groups of antibiotics including ß-lactams, cephalosporins, macrolides, quinolones, nitrofurantoin and tetracycline.

Clonorchis sinensis: development and evaluation of a nested polymerase chain reaction (PCR) assay.

Clonorchis sinensis is a fish-borne trematode endemic to East Asia, which infects over 35 million people globally. In the study described here, we developed a nested polymerase chain reaction (PCR) method for the specific and reliable detection of C. sinensis. The primers designed from the nucleotide sequence data derived in this study were evaluated for their specificity and sensitivity for the detection of C. sinensis. The specific amplification products were obtained only with C. sinensis and no amplifications occurred with the DNA of closely related trematodes including Opisthorchis viverrini demonstrating the specificity of the assay. The novel PCR method described here will be useful for the quarantine of fishery products and evaluation of transmission status of clonorchiasis in the endemic areas.

Molecular characterization of thermostable direct haemolysin-related haemolysin (TRH)-positive Vibrio parahaemolyticus from oysters in Mangalore, India.

Pathogenic Vibrio parahaemolyticus strains producing either or both of a thermostable direct haemolysin (TDH) and a TDH-related haemolysin (TRH) encoded by tdh and trh genes, respectively, are isolated at a low rate from the environment. However, recently we observed that a considerable percentage of APW (alkaline peptone water) enrichment broths of oysters collected off Mangalore India, were trh(+), rather than tdh(+) by PCR. In order to further investigate the prevalence and genetic diversity of trh bearing V. parahaemolyticus in our coast, we attempted to isolate and characterize trh(+)V. parahaemolyticus from oysters. A total of 27 trh(+) strains were isolated during the period between March 2002 and February 2004, of which nine were also tdh(+). All the trh(+) isolates were positive for urease phenotype. The isolates belonged to diverse phenotypes. In order to explore the possible presence of heterogeneity in the trh gene region among trh(+)V. parahaemolyticus, a 1.5 kb region around trh gene was PCR amplified and restriction digested using selected restriction enzymes. The whole genome comparison of strains was performed by randomly amplified polymorphic DNA PCR (RAPD PCR). The PCR-RFLP results revealed fairly well conserved nature of the trh gene region studied in different serotypes. Though 11 strains were positive by PCR for a genomic fragment that has been reported to be amplified in pandemic strains, all strains were negative by group-specific PCR (GS-PCR), orf8 PCR and showed a different RAPD pattern compared with pandemic strains. The results suggest that genetically diverse V. parahaemolyticus carrying virulence genes are associated with the aquatic environment in this region.