A clinical pilot study for the detection of sphingomyelinase in leptospirosis patient's urine at tertiary care hospital.

Purpose: Leptospirosis is a perplexing mystification for many clinicians. Clinically often underdiagnosed due to lack of a rapid, sensitive, and specific diagnostic test. Currently available diagnostic tests have their own limitations; therefore, monitoring biomarkers that contribute an essential role in pathogenesis is crucial. Herein, a pilot study was conducted to detect the presence of sphingomyelinase in urine of leptospirosis patients. Methods: Blood and urine samples were collected from 140 patients having febrile illness. Samples were analyzed through culturing, dark-field microscopy, detecting anti-leptospiral antibodies by MAT, IgM ELISA, Leptocheck-WB and screening for sphingomyelinase using a sphingomyelinase assay kit. Results: Out of 140 febrile illness patients, 22.14 % were tested leptospirosis, 33.57 % were dengue, 25 % scrub typhus, 18.57 % malaria and 0.71 % co-infection (dengue-leptospirosis). MAT seropositivity of 19.28 % (27/140) was confirmed with the highest agglutinant determined against serovar Icterohaemorrhagiae RGA followed by Autumnalis, Australis, and Pyrogens. IgM ELISA and Leptocheck-WB positivity was 16.42 % and 13.57 % respectively. Whereas culture and dark-field microscopy showed a sensitivity of 4.28 % and 2.1 %, respectively. Out of 31 confirmed cases of leptospirosis, sphingomyelinase was detected in the urine of 25 (80.64 %) patients, MAT positivity was seen in 87.09 % and culture positivity was seen in 12.90 % of cases. Conclusion: Detection of sphingomyelinase in the urine of a leptospirosis patient and its absence in other febrile illnesses like dengue, malaria and scrub typhus establish evidence of secretion of sphingomyelinase in urine during leptospiral infection. Hence, sphingomyelinase could be used as a potential diagnostic biomarker to detect leptospirosis in a non-invasive way.

Leptospiral sphingomyelinase Sph2 as a potential biomarker for diagnosis of leptospirosis.

Leptospirosis is an underestimated infectious tropical disease caused by the spirochetes belonging to the genus Leptospira. Leptospirosis is grossly underdiagnosed due to its myriad symptoms, varying from mild febrile illness to severe haemorrhage. Laboratory tests for leptospirosis is an extremely important and potent way for disease diagnosis, as the clinical manifestations are very similar to other febrile diseases. Currently available diagnostic techniques are time-consuming, require expertise and sophisticated instruments, and cannot identify the disease at an early phase of infection. Early diagnosis of leptospirosis is the need of the hour while considering the severe complications after the infection and the rate of mortality after misdiagnosis. Secretion of Leptospira-specific sphingomyelinases in leptospirosis patient's urine within a few days of the onset of infection is quite common and is a virulence factor present only in pathogenic Leptospira species. Herein, the structural and functional importance of leptospiral sphingomyelinase Sph2 in leptospirosis pathogenesis, as well as the potential of screening urinary Sph2 for diagnosis and the scope for developing a rapid and easily affordable point-of-care test for urinary leptospiral sphingomyelinase Sph2 as an alternative to current diagnostic methods are discussed.

Evaluation of anti-LipL32 carbon nanotube immunofluorescence probe (carbo-lip) and comparison with MAT, IgM ELISA, IgM spot test and culture for early detection of leptospirosis at local hospital.

Leptospirosis is an emerging public health problem affecting people mainly from tropical and subtropical regions. Therefore, there is a need for rapid and sensitive tests for proper and prompt treatment. Recently we have demonstrated Carbo-Lip probe, which was fabricated through immuno recognition method with fluorescent dye functionalized LipL32 monoclonal antibodies, secondary antibody and Leptospira for rapid and accurate diagnosis. In an effort to validate Carbo-Lip, we collected clinical samples from a cohort of 104, consisting of 26 positive, 40 negative and 38 unconfirmed cases of Leptospirosis. Subsequently, the test was also compared and validated with the gold standard method microscopic agglutination test (MAT), IgM ELISA, IgM spot test, and culture. Carbo-Lip exhibited a sensitivity of 75% with specificity of 92.3% for Leptospirosis in comparison with MAT. The fabricated Carbo-Lip sensor could be used as a potential diagnostic tool for early detection of Leptospirosis in patients from endemic areas.

Genome analysis of a halophilic bacterium Halomonas malpeensis YU-PRIM-29T reveals its exopolysaccharide and pigment producing capabilities.

Halomonas malpeensis strain YU-PRIM-29T is a yellow pigmented, exopolysaccharide (EPS) producing halophilic bacterium isolated from the coastal region. To understand the biosynthesis pathways involved in the EPS and pigment production, whole genome analysis was performed. The complete genome sequencing and the de novo assembly were carried out using Illumina sequencing and SPAdes genome assembler (ver 3.11.1) respectively followed by detailed genome annotation. The genome consists of 3,607,821 bp distributed in 18 contigs with 3337 protein coding genes and 53% of the annotated CDS are having putative functions. Gene annotation disclosed the presence of genes involved in ABC transporter-dependent pathway of EPS biosynthesis. As the ABC transporter-dependent pathway is also implicated in the capsular polysaccharide (CPS) biosynthesis, we employed extraction protocols for both EPS (from the culture supernatants) and CPS (from the cells) and found that the secreted polysaccharide i.e., EPS was predominant. The EPS showed good emulsifying activities against the petroleum hydrocarbons and its production was dependent on the carbon source supplied. The genome analysis also revealed genes involved in industrially important metabolites such as zeaxanthin pigment, ectoine and polyhydroxyalkanoate (PHA) biosynthesis. To confirm the genome data, we extracted these metabolites from the cultures and successfully identified them. The pigment extracted from the cells showed the distinct UV-Vis spectra having characteristic absorption peak of zeaxanthin (λmax 448 nm) with potent antioxidant activities. The ability of H. malpeensis strain YU-PRIM-29T to produce important biomolecules makes it an industrially important bacterium.

First Report of Pathogenic Bacterium Kalamiella piersonii Isolated from Urine of a Kidney Stone Patient: Draft Genome and Evidence for Role in Struvite Crystallization.

Uropathogenic bacteria are widely distributed in the environment and urinary tract infection is implicated in kidney stone disease. Here, we report on a urease negative bacterium Kalamiella piersonii (strain YU22) isolated from the urine of a struvite stone (MgNH4PO4·6H2O) patient. The closest species, K. piersonii IIIF1SW-P2T was reported from International Space Station samples. However, there are no earlier reports on its human association. Using whole genome and experimental analysis, its involvement in urinary tract colonization and struvite crystallization was explored. The strain YU22 showed many virulence factors that are needed for host cell invasion and colonization including cell adhesion factors, swimming and swarming motilities, biofilm and siderophore among others. In vitro infection studies in HEK-293T cells demonstrated the host cell attachment and killing. It was able to utilize amino acids as sole carbon source and showed growth in synthetic and healthy urine establishing metabolic adaptation to urinary tract. Increased pH and availability of ammonium ions from amino acid breakdown promoted struvite crystallization. The results from this study support the involvement of urease negative uropathogen in the struvite lithogenesis. Further studies on other isolates of K. peirsonii are warranted to assess its health risks.

Oral microbial profile in oral cancer patients before and after radiation therapy in a cancer care center - A prospective study.

BACKGROUND: Head and neck cancer is the sixth most common cancer reported worldwide. In many cases, the level of aggressiveness of therapy adopted in cancer patients may cause the alteration in oral microbiota; the emergence of potential pathogens may cause opportunistic infections in already immune-compromised individuals leading to increases in morbidity and mortality. Hence, this study was conducted to assess the oral microbial profile in oral cancer patients before and after radiotherapy. MATERIALS AND METHODS: A total of 145 oral swabs were collected before radiotherapy (n = 96), 3 months postradiotherapy (n = 25), 6 months postradiotherapy (n = 12) and controls (n = 12). The samples were inoculated into brain-heart infusion broth and later in different media for bacterial isolation. The isolates were subjected to phenotypic characterization by automatic identification system. RESULTS: Among the 96 samples studied from the preradiotherapy patient samples, Streptococcus species (n = 28) were the predominant isolate, followed by Staphylococcus species (n = 16), Enterobacter species (n = 6) and Enterococcus species (n = 6). Of the 25 samples studied 3 months after radiotherapy, Klebsiella pneumoniae (n = 4) was isolated and 12 samples studied after 6 months of radiotherapy Candida species (n = 4) and Pediococcus species (n = 3) were isolated. Among the control group (n = 12) screened, Streptococcus acidominimus (n = 3) is the predominant bacteria isolated. CONCLUSION: High prevalence of Streptococcus sp. was found in patients of oral cancer before radiotherapy, while Candida albicans and Klebsiella species and Pediococcus species are the significant pathogens isolated in postradiotherapy cancer patients.

Anti-biofilm and cytoprotective activities of quercetin against Pseudomonas aeruginosa isolates.

Increase in infection with multidrug resistant Pseudomonas aeruginosa is a serious global challenge in healthcare. Pseudomonas aeruginosa is capable of causing human infection in various sites and complicates the infection due to its virulence factors. This study was aimed to investigate the effect of quercetin, a dietary flavonoid against the virulence factors of P. aeruginosa and its cell protective effects on epithelial cells. Bactericidal activity, anti-biofilm activity and effect on different virulence factors were carried out using standard methods by using five P. aeruginosa isolates. Cytotoxicity and cell protective effect of quercetin was evaluated by trypan blue dye exclusion assay. All the tested isolates were completely inhibited (100%) by quercetin at a concentration of 500 µg ml-1 . It showed significant (P < 0·05) inhibitory effect on virulence factors including biofilm formation and showed significant protective effect on HEK 293T cells infected with P. aeruginosa strains. This study supports the role of quercetin against P. aeruginosa, by inhibiting virulence factors as well as its cytoprotective activity during bacterial infection either by attenuating the virulence or providing direct protective effect to the host cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The increase in infections caused by opportunistic pathogen Pseudomonas aeruginosa is a serious concern in the health care system. This study describes the beneficial effects of a dietary flavonoid, quercetin against pathogenic P. aeruginosa strains and its protective effect against the P. aeruginosa infection in HEK 293T cells in vitro.

Adaptation and diversification in virulence factors among urinary catheter-associated Pseudomonas aeruginosa isolates.

AIM: The aim of this study was to investigate the natural variation in the antibiotic sensitivity, biofilm formation and virulence among Pseudomonas aeruginosa isolated from the catheter-associated urinary tract infection (CAUTI) from a single centre. METHODS AND RESULTS: Pseudomonas aeruginosa strains were isolated from the patients with CAUTI after obtaining informed consent. These isolates were identified by routine biochemical methods and 16S rRNA gene sequencing. Antibiotic sensitivity and virulence factors were measured using standard protocols. Crystal violet staining, scanning electron microscopy and confocal laser scanning microscopy were used for the biofilm studies. The extent of infectivity of the strains to induce cell lysis was studied in vitro using the Human Embryonic Kidney cells (HEK 293T). Association between virulence factors, biofilm formation and antibiotic resistance among the strains was analysed statistically. Among the 1266 patients admitted during the 2016-2017 period, 98 cases of CAUTI were reported and 18·36% (n = 18) was due to P. aeruginosa infection. Antibiogram showed that 94·4% of isolates were resistant to multiple antibiotics and 73·7% were carbapenem-resistant. All the isolates formed biofilm on different material surfaces with varying intensity (OD580 ≥0·20-1·11). The biofilm intensity on silicone-latex material was significantly higher compared to the polystyrene surface (P > 0·05). All the strains were highly virulent and able to cause cell killing of HEK 293T cells with a rate ranging from 69·35 to 100% and showed very low sensitivity to healthy human serum. CONCLUSIONS: Antibiotic sensitivity and association between the virulence factors and biofilm formation in the P. aeruginosa clinical strains showed complex natural diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the natural diversity and adaptation in virulence factors, biofilm formation and host-pathogen interaction among catheter-associated P. aeruginosa strains. The findings from the study urge for developing individualized drug strategy for targeting these multidrug-resistant pathogens.

Halomonas malpeensis sp. nov., isolated from rhizosphere sand of a coastal sand dune plant.

A Gram-stain-negative, aerobic, non-endospore-forming organism, isolated from the rhizosphere sand of a coastal sand dune plant was studied for its taxonomic position. On the basis of 16S rRNA gene sequence similarity comparisons, strain YU-PRIM-29T was grouped within the genus Halomonas and was most closely related to Halomonas johnsoniae (97.5 %). The 16S rRNA gene sequence similarity to other Halomonas species was <97.5 %. Strain YU-PRIM-29T grew optimally at 28 °C (growth range, 10-36 °C), at a pH of 7-9 (growth range, pH 5.5-12.0) and in the presence of 0.5 to 5 % (w/v) NaCl (growth up to 20 % NaCl). The fatty acid profile from whole-cell hydrolysates supported the allocation of the strain to the genus Halomonas. The fatty acids C18 : 1ω7c and C16 : 0 were found as major compounds, followed by the hydroxylated fatty acid C12 : 0 3-OH. The quinone system consisted predominantly of ubiquinone Q-9. The polar lipid profile was composed of the major lipids diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. In the polyamine pattern, spermidine was the predominant compound. The DNA G+C content was 64.8 mol%. In addition, the results of physiological and biochemical tests also allowed phenotypic differentiation of strain YU-PRIM-29T from its closest-related species. Hence, YU-PRIM-29T represents a new species of the genus Halomonas, for which we propose the name Halomonas malpeensis sp. nov., with YU-PRIM-29T (=LMG 28855T=CCM 8737T) as the type strain.

Characterization and Antibiotic Sensitivity Profile of Bacteria in Orofacial Abscesses of Odontogenic Origin.

BACKGROUND: Odontogenic infections range from peripheral abscess to superficial and deep infections leading to severe infections in head and neck region. This study was aimed to assess bacterial isolates responsible for orofacial infection of odontogenic origin and their drug susceptibility patterns so as to provide better perceptive for the management of odontogenic infections. METHODS: The study was made in a selected cohort of patients, irrespective of age and gender having moderate and severe orofacial infections of odontogenic origin admitted to Yenepoya University Hospital. Pus samples were collected and identification of bacteria was performed by 16S rRNA gene sequencing. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion method. RESULT: A total of 37 study subjects were included, with bacterial isolation rate of 31 (83.7 %). The mean age presented of all patients was 40.62. Of all, 24 (64.9 %) were males. Staphylococcus aureus, Enterobacter claocae subsp. dissolvens, Klebsiella quasipneumoniae subsp. similipneumoniae, Staphylococcus aureus subsp. anaerobius and Klebsiella pneumoniae subsp. ozaenae were the most prevalent isolates. Result showed that 58.6 % of the isolates were resistant to gentamicin, 52.5 % for ampicillin, 51.3 % for piperacillin; least resistant being 18.9 % for azithromycin. CONCLUSION: High prevalence of bacterial isolates was found, Staphylococcus aureus being the dominant. Most of the bacteria were resistant to different classes of antibiotics. Appropriate antibiotics should be given based on the bacterial isolates, culture sensitivity and clinical course of the disease.

A medicinal herb Cassia alata attenuates quorum sensing in Chromobacterium violaceum and Pseudomonas aeruginosa.

Quorum sensing (QS) has been shown to play a crucial role in the pathogenesis in many bacteria, and attenuation of QS is one of the targets of antimicrobial therapy with particular interest in combating drug resistance. This study reports the QS inhibitory activity of metabolites from Cassia alata L. (Ca. alata), an important medicinal herb widely used in the treatment of microbial infections. For investigating the QS inhibition (QSI), the potential of Ca. alata L., initially, metabolites of the leaves extracted using ethanol was tested against biosensor strain Chromobacterium violaceum CV026 and C. violaceum wild-type strains. Furthermore, a purified fraction rich in flavonoids (F-AF) was used for establishing QSI activity by studying the inhibition of violacein production in C. violaceum, and QS controlled virulence and biofilm formation in Pseudomonas aeruginosa PAO1. The study results showed 50% inhibition of violacein production in C. violaceum at 0·05 mg ml-1 concentration of F-AF. In P. aeruginosa PAO1, it inhibited the tested virulence factors and biofilm formation significantly. The F-AF contained major flavonoids namely, quercetin, quercetrin and kaempferol displaying QSI activity individually against the test organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: Present study demonstrates the quorum sensing inhibitory activity of metabolites from Cassia alata, an important medicinal herb which is commonly used worldwide in the treatment of infections caused by microorganisms. An extract prepared from the leaves of the plant showed activity against quorum sensing in Chromobacterium violaceum and was also effective against attenuating the quorum sensing controlled virulence factors in Pseudomonas aeruginosa. Activity is attributed to the rich flavonoid composition of the plant. Results of the present investigation throw an insight into the possibility of developing drug formulations using the isolated compounds against infections caused by quorum sensing-mediated pathogenicity of bacteria.

Biochemical and histopathological responses of the Swiss albino mice treated with uranyl nitrate and its recovery.

Uranium is a radioactive heavy metal ubiquitous in the natural environment. In its chemical form, it is known to induce nephrotoxicity both in human and in animals. Its toxicity is dose and time dependent, also varies with form of uranium. In the present study, we assessed the nephrotoxicity induced by a single dose of uranyl nitrate (UN) in mice at different time intervals and recovery from its toxicity. Two doses of 2 and 4 mg/kg body weight of uranyl nitrate was injected intraperitoneally and animals were sacrificed after 1, 3, 5, 14, and 28 d of administration. Histopathological and biochemical alterations of post-UN dosing in comparison to control were evaluated. Tubular damage to about 75% was observed after 3 d (4 mg/kg) and the biochemical parameters such as serum creatinine, urea, and blood urea nitrogen levels were also significantly increased. Progression of tubular damage was not found after 5 d. Dose-dependent recovery of uranyl nitrate-treated animals was observed after 14 and 28 d of dosing. The concentration of uranium retained in kidney correlates with biochemical and histopathological analysis.

Functional and cell proliferative properties of an exopolysaccharide produced by Nitratireductor sp. PRIM-31.

Exopolysaccharides (EPS) produced by bacteria are important source of natural biomolecules with therapeutic applications. In this study EPS produced by a strain PRIM-31 isolated from seawater from south west coast of India identified by 16S rRNA gene sequencing as Nitratireductor sp. was investigated for its biotechnological applications. The isolate produced 650 mg L(-1) EPS under optimum growth conditions. The purified EPS contained 58.3% carbohydrates with 7.08% uronic acid containing sugars, functional groups such as sulfate (2.68%) and trace amounts of proteins (0.65%). Molecular weight of the EPS was 90 kDa and monosaccharide composition was arabinose, glucose, xylose, glucuronic acid and galactose (6.6: 3.5: 2.1: 1.3: 1). The EPS displayed antioxidant activity in terms of total antioxidant capacity, ferric reducing power, scavenging of DPPH (IC50 390 µg mL(-1)) and superoxide radicals (IC50 340 µg mL(-1)). Cell proliferative activity of the EPS was evidenced by significant improvement in human dermal fibroblast (HDF) proliferation compared to control. Further, significant improvement (41%) in HDF cell migration was observed in in vitro scratch assay with EPS treatment. The EPS also showed antiproliferative activity against glioblastoma cells with an IC50 of 234.04 µg mL(-1). These functional properties of the EPS prospect its biotechnological applications.

Anti-quorum sensing activity of flavonoid-rich fraction from Centella asiatica L. against Pseudomonas aeruginosa PAO1.

BACKGROUND/PURPOSE: Inhibition of quorum sensing (QS), a cell-density dependent regulation of gene expression in bacteria by autoinducers is an attractive strategy for the development of antipathogenic agents. METHODS: In this study, the anti-QS activity of the ethanolic extract of the traditional herb Centella asiatica was investigated by the biosensor bioassay using Chromobacterium violaceum CV026. The effect of ethyl acetate fraction (CEA) from the bioassay-guided fractionation of ethanol extract on QS-regulated violacein production in C. violaceum ATCC12472 and pyocyanin production, proteolytic and elastolytic activities, swarming motility, and biofilm formation in Pseudomonas aeruginosa PAO1 were evaluated. Possible mechanism of QS-inhibitory action on autoinducer activity was determined by measuring the acyl homoserine lactone using C. violaceum ATCC31532. Anti-QS compounds in the CEA fraction were identified using thin layer chromatography biosensor overlay assay. RESULTS: Ethanol extract of C. asiatica showed QS inhibition in C. violaceum CV026. Bioassay-guided fractionation of ethanol extract revealed that CEA was four times more active than the ethanol extract. CEA, at 400 µg/mL, completely inhibited violacein production in C. violaceum ATCC12472 without significantly affecting growth. CEA also showed inhibition of QS-regulated phenotypes, namely, pyocyanin production, elastolytic and proteolytic activities, swarming motility, and biofilm formation in P. aeruginosa PAO1 in a concentration-dependent manner. Thin layer chromatography of CEA with biosensor overlay showed anti-QS spot with an Rf value that corresponded with that of standard kaempferol. CONCLUSION: The anti-QS nature of C. asiatica herb can be further exploited for the formulation of drugs targeting bacterial infections where pathogenicity is mediated through QS.

Versatile properties of an exopolysaccharide R-PS18 produced by Rhizobium sp. PRIM-18.

Exopolysaccharides (EPS) produced by bacteria have attracted scientific and industrial attention due to their multifunctional properties and relatively easier production. In this study, an EPS viz., R-PS18 produced by Rhizobium sp. PRIM-18 was characterized and its functional properties were assessed. Cell proliferative and in vitro wound healing activities of the EPS were established using human dermal fibroblast (HDF) cells. The isolate produced 2.1 g L(-1) purified EPS (molecular weight 9.33×10(6) Da) comprising of glucose, galactose, and mannose (6.1:1.8:1). Viscosity of 0.25% solution was 23.4 mPa s (shear rate 75 s(-1)) and it showed pseudoplastic and thixotropic behavior. High emulsification, iron chelation, and superoxide scavenging abilities were also observed. Significant increase in HDF cell proliferation and wound healing in vitro was achieved by R-PS18 treatment. Sulfation of R-PS18 significantly enhanced the cell proliferative and wound healing activities. In conclusion, these findings indicate potential applications of R-PS18.

Chelativorans intermedius sp. nov. and proposal to reclassify Thermovum composti as Chelativorans composti comb. nov.

Two Gram-stain-negative, non-endospore-forming, rod-shaped bacteria, strains CC-MHSW-5(T) and A1392, were isolated from water of coastal hot springs located in Taiwan and China, respectively, and investigated for their taxonomic position. The two strains shared identical 16S rRNA gene sequences, a DNA-DNA hybridization value >80% and similar genomic DNA G+C contents (64.3 and 64.6 mol%), but showed different genomic fingerprint patterns generated by BOX-PCR and three random amplification polymorphic DNA PCRs. The strains shared highest 16S rRNA gene sequence similarities with the type strains of Chelativorans multitrophicus (96.7 and 96.1%), Thermovum composti (96.2 and 96.1%) and Chelativorans oligotrophicus (96.1 and 95.8%). Phylogenetic trees (based on 16S rRNA and recA gene sequence comparisons) showed a distinct clustering of both strains with the type strains of species of the genus Chelativorans and T. composti Nis3(T). The quinone systems of strains CC-MHSW-5(T) and Nis3(T) contained ubiquinone Q-10 as the major component. The major polyamine in both strains was sym-homospermidine. Putrescine, spermidine and, for strain CC-MHSW-5(T), spermine were found in minor concentrations. Their polar lipid profiles consisted of phosphatidylmonomethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and diphosphatidylglycerol. The fatty acid profile contained major amounts of C18 : 1ω7c and C19 : 0 cyclo ω8c. On the basis of these results, the two strains are considered to represent a novel species of the genus Chelativorans , for which the name Chelativorans intermedius sp. nov. is proposed. The type strain is CC-MHSW-5(T) ( =CCM 8543(T) =LMG 28482(T) =DSM 29391(T) =CIP 110825(T)). Based on both genotypic and phenotypic characters, it is proposed that T. composti be reclassified within the genus Chelativorans as Chelativorans composti comb. nov.

Rhizobium capsici sp. nov., isolated from root tumor of a green bell pepper (Capsicum annuum var. grossum) plant.

A novel, Gram-staining-negative, rod-shaped, aerobic and motile bacterium, designated strain CC-SKC2(T), was isolated from the root tumor of a green bell pepper (Capsicum annuum var. grossum) plant in Taiwan. Cells were positive for oxidase and catalase activities and exhibited growth at 25-37 °C, pH 4.0-9.0 and tolerated NaCl concentrations up to 4.0 % (w/v). Strain CC-SKC2(T) is able to trigger nodulation in soybean (Glycine max Merr.), but not in Capsicum annuum var. grossum, red bean (Vigna angularis), sesbania (Sesbania roxburghii Merr.) or alfalfa (Medicago varia Martin.). The novel strain shared highest 16S rRNA gene sequence similarity to Rhizobium rhizoryzae KCTC 23652(T) and Rhizobium straminoryzae CC-LY845(T) (both 97.5 %) followed by Rhizobium lemnae L6-16(T) (97.3 %), Rhizobium pseudoryzae KCTC 23294(T) (97.1 %), and Rhizobium paknamense NBRC 109338(T) (97.0 %), whereas other Rhizobium species shared <96.7 % similarity. The DNA-DNA relatedness values of strain CC-SKC2(T) with R. rhizoryzae KCTC 23652(T), R. pseudoryzae KCTC 23294(T) and R. paknamense NBRC 109338(T) were 11.4, 17.2 and 17.0 %, respectively (reciprocal values were 11.1, 28.3 and 24.0 %, respectively). Phylogenetic analysis based on 16S rRNA, atpD and recA genes revealed a distinct taxonomic position attained by strain CC-SKC2(T) with respect to other Rhizobium species. The major fatty acids in strain CC-SKC2(T) were C16:0, C19:0 cyclo ω8c, C14:0 3-OH and/or C16:1 iso I and C18:1 ω7c and/or C18:1 ω6c. The polyamine pattern showed predominance of spermidine and moderate amounts of sym-homospermidine. The predominant quinone system was ubiquinone (Q-10) and the DNA G+C content was 60.5 mol%. On the basis of polyphasic taxonomic evidence presented here, strain CC-SKC2(T) is proposed to represent a novel species within the genus Rhizobium, for which the name Rhizobium capsici sp. nov. is proposed. The type strain is CC-SKC2(T) (=BCRC 80699(T) = JCM 19535(T)).

Carbon nanotube-reinforced hydroxyapatite composite and their interaction with human osteoblast in vitro.

As a bone mineral component, hydroxyapatite (HA) has been an attractive bioceramic for the reconstruction of hard tissues. However, its poor mechanical properties, including low fracture toughness and tensile strength, have been a substantial challenge to the application of HA for the replacement of load-bearing and/or large bone defects. In this study, HA is reinforced with high-purity and well-functionalized multiwalled carbon nanotubes (MWCNTs; >99 wt%) having an average diameter of 15 nm and length from 10 to 20 µm. The cellular response of these functionalized CNTs and its composites were examined in human osteoblast sarcoma cell lines. Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) and diammonium hydrogen phosphate ((NH4)2HPO4) were used to synthesize HA in situ. MWCNTs were functionalized by heating at 100°C in 3:1 ratio of sulfuric acid and nitric acid for 60 min with stirring and dispersed in sodium dodecyl benzene sulfonate by sonication. HA particles were produced in MWCNTs solution by adding Ca(NO3)2·4H2O and (NH4)2HPO4 under vigorously stirring conditions. The composite was dried and washed in distilled water followed by heat treatment at 250°C to obtain CNT-HA powder. Physiochemical characterization of the composite material was carried out using Fourier transform infrared spectroscopy, field-emission scanning electron microscopy, energy-dispersive X-ray spectrometer, and X-ray diffractometer. Furthermore, this study investigates the cytotoxic effects of functionalized-MWCNTs (f-MWCNTs) and its composites with HA in human osteoblast sarcoma cell lines. Human osteoblast cells were exposed with different concentrations of f-MWCNTs and its composite with HA. The interactions of f-MWCNT and MWCNT-HA composites were analyzed by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The results indicate no detrimental effect on survival or mitochondrial activity of the osteoblast cells. Cell viability decreased with an increase in CNT concentration indicating that MWCNTs and its composite can be cytotoxic at higher dosages. This result provides further evidence that the bionano interface can be developed for CNT-reinforced HA composites for load-bearing bone implants, drug delivery, and tissue engineering.

Zeaxanthin biosynthesis by members of the genus Muricauda.

Zeaxanthin, a C40xanthophyll carotenoid, has potential biological applications in nutrition and human health. In this study we characterized carotenoid composition in 5 taxonomically related marine bacterial isolates from the genus Muricauda. The pigment was characterized using high performance liquid chromatography (HPLC) and mass spectrometry, which confirmed the presence of all-trans-zeaxanthin. Muricauda strains produced zeaxanthin as a predominant carotenoid. M. flavescens JCM 11812(T) produced highest yield (4.4 +/- 0.2 mg L(-1)) when cultured on marine broth at 32 degrees C for 72 h. This is the first report on the presence of zeaxanthin among the majority of species from the genus Muricauda.

Zeaxanthin production by novel marine isolates from coastal sand of India and its antioxidant properties.

Zeaxanthin carotenoids are class of commercially important natural products and diverse biomolecules produced by plants and many microorganisms. Bacteria often produce a cocktail of polar and nonpolar carotenoids limiting their industrial applications. Marine members of the family Flavobacteriaceae are known to produce potential carotenoids such as astaxanthin and zeaxanthin. A few bacterial species have been reported for the predominant production zeaxanthin. Here, we report the molecular identification of the zeaxanthin as a major carotenoid produced by two novel bacteria (YUAB-SO-11 and YUAB-SO-45) isolated from sandy beaches of South West Coast of India and the effect of carbon sources on the production of zeaxanthin. The strains were identified based on the 16S rRNA gene sequencing as a member of genus Muricauda. The closest relatives of YUAB-SO-11 and YUAB-SO-45 were Muricauda aquimarina (JCM 11811(T)) (98.9 %) and Muricauda olearia (JCM 15563(T)) (99.2 %), respectively, indicating that both of these strains might represent a novel species. The highest level of zeaxanthin production was achieved (YUAB-SO-11, 1.20 ± 0.11 mg g(-1)) and (YUAB-SO-45, 1.02 ± 0.13 mg g(-1)) when cultivated in marine broth supplemented with 2 % NaCl (pH 7) and incubated at 30 °C. Addition of 0.1 M glutamic acid, an intermediate of citric acid cycle, enhanced the zeaxanthin production as 18 and 14 % by the strains YUAB-SO-11 and YUAB-SO-45 respectively. The zeaxanthin showed in vitro nitric oxide scavenging, inhibition of lipid peroxidation, and 2,2-diphenyl-1-picryl hydrazyl scavenging activities higher than the commercial zeaxanthin. The results of this study suggest that two novel strains YUAB-SO-11 and YUAB-SO-45 belonging to genus Muricauda produce zeaxanthin as a predominant carotenoid, and higher production of zeaxanthin was achieved on glutamic acid supplementation. The pigment showed good in vitro antioxidant activity, which can be exploited further for commercial applications.