Characterization of a hemolytic and antibiotic-resistant Pseudomonas aeruginosa strain S3 pathogenic to fish isolated from Mahananda River in India.

Virulent strain Pseudomonas aeruginosa isolated from Mahananda River exhibited the highest hemolytic activity and virulence factors and was pathogenic to fish as clinical signs of hemorrhagic spots, loss of scales, and fin erosions were found. S3 was cytotoxic to the human liver cell line (WRL-68) in the trypan blue dye exclusion assay. Genotype characterization using whole genome analysis showed that S3 was similar to P. aeruginosa PAO1. The draft genome sequence had an estimated length of 62,69,783 bp, a GC content of 66.3%, and contained 5916 coding sequences. Eight genes across the genome were predicted to be related to hemolysin action. Antibiotic resistance genes such as class C and class D beta-lactamases, fosA, APH, and catB were detected, along with the strong presence of multiple efflux system genes. This study shows that river water is contaminated by pathogenic P. aeruginosa harboring an array of virulence and antibiotic resistance genes which warrants periodic monitoring to prevent disease outbreaks.

Siderophore-producing Bacillus amyloliquefaciens BM3 mitigate arsenic contamination and suppress Fusarium wilt in brinjal plants.

AIM: Arsenic contamination in agricultural soils poses a serious health risk for humans. Bacteria that produce siderophores, primarily for iron acquisition, can be relevant in combating arsenic toxicity in agricultural soils and simultaneously act as biocontrol agents against plant diseases. We evaluated the arsenic bioremediation and biocontrol potential of the rhizosphere isolate Bacillus amyloliquefaciens BM3 and studied the interaction between the purified siderophore bacillibactin and arsenic. METHODS AND RESULTS: BM3 showed high arsenic resistance [MIC value 475 and 24 mM against As(V) and As(III), respectively] and broad spectrum in-vitro antagonism against several phytopathogenic fungi. BM3 was identified by biochemical characterization and 16S rRNA gene sequencing. Scanning electron microscopy (SEM) analysis revealed increased cell size of BM3 when grown in presence of sub-lethal arsenic concentrations. Bioremediation assays showed a 74% and 88.1% reduction in As(V) and As(III) concentrations, respectively. Genetic determinants for arsenic resistance (arsC and aoxB) and antifungal traits (bacAB and chiA) were detected by PCR. Arsenic chelating ability of bacillibactin, the siderophore purified from culture filtrate of BM3 and identified through spectroscopic data analysis, was observed in CAS assay and fluorescence spectrometry. In-vivo application of talc-based formulation of BM3 in brinjal seedlings showed significant reduction in Fusarium wilt disease. CONCLUSION: Strain B. amyloliquefaciens BM3 may be useful in arsenic bioremediation and may be considered for large field trials as an alternative to chemical fungicides by inhibiting soil borne pathogens.

Synthesis of Cu (II) and Zn (II) Complexes of a Quinoline Based Flexible Amide Receptor as Fluorescent Probe for Dihydrogen Phosphate and Hydrogen Sulphate and Their Antibacterial Activity.

A novel 8-hydroxy quinoline-derived amide receptor, in conjunction with its Cu (II) and Zn (II) complexes, has been strategically developed to function as remarkably efficient fluorescent receptors with a distinct capability for anion sensing. The comprehensive characterization of the synthesized compounds were achieved through UV-Vis, IR, NMR, and HRMS spectroscopic techniques. Among the Cu (II) and Zn (II) complexes, the latter exhibits superior selectivity for anions, specifically dihydrogen phosphate and hydrogen sulfate, as their tetrabutylammonium salts in a 9:1 acetonitrile-water (v/v) mixture. The Cu (II) complex demonstrates enhanced anion binding compared to the amide ligand, albeit with reduced selectivity. Furthermore, the affinity was evaluated using the Benesi-Hildebrand plot. The binding constants and Limit of Detection (LOD) for both complexes were precisely quantified. The Job plot illustrates a clear 1:1 binding interaction between the metal complexes and the guest anions. Significantly, both metal-complex receptors display a broad spectrum of antibacterial activity, against both gram-positive and gram-negative bacteria. It is worth highlighting that the Zn (II) complexed receptor outperforms the Cu (II) complexed receptor, as evidenced by its considerably lower Minimum Inhibitory Concentration (MIC) value against both bacterial strains.

Detection of Virulence-Associated Genes and in vitro Gene Transfer From Aeromonas sp. Isolated From Aquatic Environments of Sub-himalayan West Bengal.

Aeromonas is omnipresent in aquatic environments and cause disease within a wide host range. A total of thirty-four isolates from water samples of small fish farms were identified as Aeromonas based on biochemical characteristics and 16S rRNA gene sequence. A total of six virulent factors were analyzed which indicated 100% of isolates as beta-haemolytic and proteolytic, whereas 44.1, 38.2, and 70.6% of isolates produced DNAse, siderophore, and amylase, respectively. Studies on the occurrence of four genetic determinants of virulence factors revealed that aer/haem (haemolytic toxin) and flaA (polar flagella) genes were present in 44.1% of strains whereas ascV (type 3 secretion system) and aspA (serine protease) genes were detected in 21.5 and 8.82% of strains, respectively. Fish (Anabas testudineus) challenge studies showed that the isolate GP3 (Aeromonas veronii) bearing five virulent factors with the combination of aer/haem + /ascV + /fla + genes induced severe lesions leading to 100% of mortality. In contrast, RB7 possessing four virulence factors and three genes (aer/haem + /ascV + /aspA +) could not produce severe lesions and any mortality indicating the absence of correlation between the virulence factors, its genes, and the pathogenicity in fishes. GP3 was cytotoxic to human liver cell line (WRL-68) in trypan blue dye exclusion assay. The 431 bp aer/haem gene of GP3 was transferable to E. coli Dh5α with a conjugational efficiency of 0.394 × 10 -4 transconjugants per recipient cell. The transfer was confirmed by PCR and by the presence of 23-kb plasmids in both donor and transconjugants. Therefore, the occurrence of mobile genetic elements bearing virulence-associated genes in Aeromonas indicates the need for periodic monitoring of the aquatic habitat to prevent disease outbreaks.

Oxidative Stress, Mitochondrial Dysfunction, and Premature Ageing in Severe Acute Malnutrition in Under-Five Children.

OBJECTIVES: To assess oxidative stress, mitochondrial dysfunction, and premature ageing in children with severe acute malnutrition (SAM). METHODS: This cross-sectional study was conducted in children (1 mo-5 y) with SAM (defined as per WHO criteria) presenting to Pediatrics inpatient department. Oxidative stress, mitochondrial dysfunction, and premature ageing were assessed by measuring and comparing total antioxidant status (TAOS), mitochondrial DNA (mtDNA) content, and telomere length (TL), respectively in 40 under-five children with SAM and 40 age- and sex-matched non-malnourished controls. RESULTS: Oxidative stress was significantly increased in children with SAM, reflected by lower median (IQR) TAOS in cases as compared to controls [10.78 (9.08, 12.3) vs. 16.63 (15.20, 18.03) mM Trolox, p < 0.001]. Median (IQR) mtDNA content was significantly increased in children with SAM [188.7 (105.2, 398.9) vs. 116.2 (67.2, 154.6), p < 0.001]. There was no significant difference in telomere length between cases and controls [1184.5 (894, 1408) vs.1082.6 (823.3, 1479), p = 0.747]. CONCLUSION: Children with SAM had significantly increased oxidative stress that possibly caused mitochondrial dysfunction but no premature ageing.

Evaluation of the biocontrol efficacy of a Serratia marcescens strain indigenous to tea rhizosphere for the management of root rot disease in tea.

The aim of the present study is to evaluate plant growth promoting and biocontrol efficacy of a Serratia marcescens strain ETR17 isolated from tea rhizosphere for the effective management of root rot disease in tea. Isolated bacterial culture ETR17 showed significant level of in vitro antagonism against nine different foliar and root pathogens of tea. The phenotypic and molecular characterization of ETR17 revealed the identity of the bacterium as Serratia marcescens. The bacterium was found to produce several hydrolytic enzymes like chitinase, protease, lipase, cellulase and plant growth promoting metabolites like IAA and siderophore. Scanning electron microscopic studies on the interaction zone between pathogen and antagonistic bacterial isolate revealed severe deformities in the fungal mycelia. Spectral analyses (LC-ESI-MS, UV-VIS spectrophotometry and HPLC) and TLC indicated the presence of the antibiotics pyrrolnitrin and prodigiosin in the extracellular bacterial culture extracts. Biofilm formation by ETR17 on polystyrene surface was also observed. In vivo application of talc-based formulations prepared with the isolate ETR17 in tea plantlets under green house conditions revealed effective reduction of root-rot disease as well as plant growth promotion to a considerable extent. Viability studies with the ETR17 talc formulation showed the survivability of the isolate up to six months at room temperature. The sustenance of ETR17 (concentration of 8-9x108 cfu g-1) in the soil after the application of talc formulation was recorded by ELISA. Safety studies revealed that ETR17 did not produce hemolysin as observed in pathogenic Serratia strains. The biocontrol strain reported in this study can be used for field application in order to minimize the use of chemical fungicides for disease control in tea gardens.

Urine Culture Follow-up and Antimicrobial Stewardship in a Pediatric Urgent Care Network.

BACKGROUND AND OBJECTIVES: Empiric antibiotic therapy for presumed urinary tract infection (UTI) leads to unnecessary antibiotic exposure in many children whose urine culture results fail to confirm the diagnosis. The objective of this quality improvement study was to improve follow-up management of negative urine culture results in the off-campus urgent care network of Nationwide Children's Hospital to reduce inappropriate antibiotic exposure in children. METHODS: A multidisciplinary task force developed and implemented a protocol for routine nurse and clinician follow-up of urine culture results, discontinuation of unnecessary antibiotics, and documentation in the electronic medical record. Monthly antibiotic discontinuation rates were tracked in empirically treated patients with negative urine culture results from July 2013 through December 2015. Statistical process control methods were used to track improvement over time. Fourteen-day return visits for UTIs were monitored as a balancing measure. RESULTS: During the study period, 910 patients received empiric antibiotic therapy for UTIs but had a negative urine culture result. The antibiotic discontinuation rate increased from 4% to 84%, avoiding 3429 (40%) of 8648 antibiotic days prescribed. Among patients with discontinued antibiotics, none was diagnosed with a UTI within 14 days of the initial urgent care encounter. CONCLUSIONS: Implementation of a standard protocol for urine culture follow-up and discontinuation of unnecessary antibiotics was both effective and safe in a high-volume pediatric urgent care network. Urine culture follow-up management is an essential opportunity for improved antimicrobial stewardship in the outpatient setting that will affect many patients by avoiding a substantial number of antibiotic days.

Influence of drought stress on cellular ultrastructure and antioxidant system in tea cultivars with different drought sensitivities.

Drought is the major yield-limiting abiotic factor of tea cultivation. In the present study, influence of drought stress on cellular ultrastructure and antioxidants was studied drought-tolerant (TV-23) and -sensitive (S.3/A3) tea cultivars by imposing drought stress for 21 days. Drought stress led to considerable structural alterations in mitochondria, chloroplast and vacuole. Lesser membrane integrity and higher structural damage was observed in S.3/A3. Chlorophyll a, chl-b and carotenoids content in leaves decreased in each cultivar; however, the decrement was more brisk in S.3/A3. Proline, total soluble sugar, ascorbic acid and abscisic acid were elevated in TV-23 whereas hydrogen peroxide, superoxide anion, lipid peroxidation and electrolyte leakage increased rapidly in S.3/A3. Starch content decreased both in leaves and roots of each cultivar and was more pronounced in roots of TV-23. Under drought, enhanced activities of ascorbate peroxidase, catalase, peroxidase and superoxide dismutase were recorded in both roots and leaves of each cultivar, but the rate of enhancement was more in TV-23. This indicated that tolerant cultivar exhibited higher antioxidant capacity and a stronger protective mechanism such that their ultrastructural integrity was better maintained during exposure to drought stress.

Phylogeny and synonymous codon usage pattern of Papaya ringspot virus coat protein gene in the sub-Himalayan region of north-east India.

Sub-Himalayan West Bengal is favorable for the production of several fruits and vegetables. Papaya is one of the common plants cultivated in the area. Most of the papaya plants of the area are susceptible to Papaya ringspot virus (PRSV). Coat protein genes of 6 PRSV isolates of the area were sequenced following RT-PCR. Phylogenetic study of the PRSV isolates showed about 80%-90% similarity with Cuban isolates. The codon usage pattern of our isolates was also analyzed, along with several other isolates. PRSV isolates of our study showed a preference for 8 putative optimal codons. Correspondence analysis of the genes of different isolates along the first 2 major axes were done, as the first 2 axes contributed more in shaping codon usage pattern. In the phylogenetic tree constructed by the neighbour-joining method, our isolates clustered together with the east Indian, north Indian, and Bangladeshi isolates. The diversity and codon usage pattern of the PRSV isolates of different regions were studied, and it was observed that the codon usage pattern of PRSV isolates is probably influenced by translational selection along with mutational bias.

Molecular detection and partial characterization of a begomovirus causing leaf curl disease of potato in sub-Himalayan West Bengal, India.

The characteristic disease symptoms of apical leaf curl, crinkled leaves and conspicuous mosaic were observed in potato plants grown in areas of Coochbehar, one of the distinct agroclimatic zones of sub-Himalayan West Bengal. Whitefly (Bemisia tabaci) population was also observed in and around the infected plants. The characteristic disease symptoms and presence of whitefly (Bemisia tabaci) population indicated the possibility of begomovirus infection. Total DNA was extracted from infected samples and PCR was carried out using begomovirus specific primers. PCR product of 1539 nucleotide long containing pre-coat protein, coat protein, AC5, AC3, AC2 and partial AC1 gene of DNA-Asegment and 1001 nucleotide long DNA-B containing BV1 and BC1 genes were submitted to the GenBank (accession numbers HQ597033 for DNA-A and JN390432 for DNA-B). The amplified DNA-A and DNA-B segment showed highest 98% and 99% nucleotide sequence similarity respectively with Tomato leaf curl New Delhi virus (ToLCNDV). In phylogenetic analysis also the virus sequence clustered with ToLCNDV isolates. The disease was successfully transmitted to healthy tomato plants using both whitefly vector, B. tabaci and mechanical sap inoculation using sap of infected potato leaves. It is the first record of begomovirus infection of cultivated potato in sub-Himalayan West Bengal of Eastern India.

A new antifungal coumarin from Clausena excavata.

A new γ-lactone coumarin, named as excavarin-A, showing antifungal activity was isolated from the leaves of Clausena excavata by bioassay guided fractionation method. The structure was elucidated by spectroscopic data analysis and identified as 7((2E)-4(4,5-dihydro-3-methylene-2-oxo-5-furanyl)-3-methylbut-2-enyloxy) coumarin. Minimum inhibitory concentration (MIC) was determined against fifteen fungal strains pathogenic against plants and human. The least MIC was recorded against the human pathogen, Candida tropicalis and the plant pathogens Rhizoctonia solani and Sclerotinia sclerotiorum. Antifungal activities against the human pathogens, Aspergillus fumigatus and Mucor circinelloides and plant pathogens, Colletotrichum gloeosporioides, Lasiodiplodia theobromae, Fusarium oxysporum and Rhizopus stolonifer were stronger than that of the standard antimicrobials.

Resistance to the Pseudomonas syringae effector HopA1 is governed by the TIR-NBS-LRR protein RPS6 and is enhanced by mutations in SRFR1.

The Pseudomonas syringae-Arabidopsis (Arabidopsis thaliana) interaction is an extensively studied plant-pathogen system. Arabidopsis possesses approximately 150 putative resistance genes encoding nucleotide binding site (NBS) and leucine-rich repeat (LRR) domain-containing proteins. The majority of these belong to the Toll/Interleukin-1 receptor (TIR)-NBS-LRR (TNL) class. Comparative studies with the coiled-coil-NBS-LRR genes RPS2, RPM1, and RPS5 and isogenic P. syringae strains expressing single corresponding avirulence genes have been particularly fruitful in dissecting specific and common resistance signaling components. However, the major TNL class is represented by a single known P. syringae resistance gene, RPS4. We previously identified hopA1 from P. syringae pv syringae strain 61 as an avirulence gene that signals through ENHANCED DISEASE SUSCEPTIBILITY1, indicating that the corresponding resistance gene RPS6 belongs to the TNL class. Here we report the identification of RPS6 based on a forward-genetic screen and map-based cloning. Among resistance proteins of known function, the deduced amino acid sequence of RPS6 shows highest similarity to the TNL resistance protein RAC1 that determines resistance to the oomycete pathogen Albugo candida. Similar to RPS4 and other TNL genes, RPS6 generates alternatively spliced transcripts, although the alternative transcript structures are RPS6 specific. We previously characterized SRFR1 as a negative regulator of avrRps4-triggered immunity. Interestingly, mutations in SRFR1 also enhanced HopA1-triggered immunity in rps6 mutants. In conclusion, the cloning of RPS6 and comparisons with RPS4 will contribute to a closer dissection of the TNL resistance pathway in Arabidopsis.