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.

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.

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.

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.

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)).

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.

Bhargavaea ullalensis sp. nov., isolated from coastal sand.

A Gram-positive-staining, aerobic, non-endospore-forming bacterium, isolated from Ullal coastal sand, Mangalore, Karnataka, India, on marine agar 2216, was studied in detail for its taxonomic position. Based on 16S rRNA gene sequence similarity comparisons, strain ZMA 19(T) was grouped into the genus Bhargavaea with high 16S rRNA gene sequence similarities to all currently described species of the genus Bhargavaea, Bhargavaea cecembensis (99.3 %), Bhargavaea beijingensis (98.8 %) and Bhargavaea ginsengi (98.6 %). GyrB amino acid sequence-based analysis supported the phylogenetic position and also distinguished strain ZMA 19(T) from the three other species of the genus Bhargavaea. Amino acid sequence similarities were only 85.6 to 89.5 % between strain ZMA 19(T) and the type strains of members of the genus Bhargavaea, which shared higher similarities among each other (93.0 to 96.2 %). The chemotaxonomic characterization supported the allocation of the novel strain to the genus Bhargavaea. The major menaquinone was MK-8. The polar lipid profile contained predominantly diphosphatidylglycerol and moderate amounts of phosphatidylglycerol. The diagnostic peptidoglycan diamino acid was lysine and the polyamine pattern contained spermidine and spermine. The major fatty acids were iso- and anteiso-branched fatty acids. DNA-DNA hybridization with the types strains Bhargavaea cecembensis LMG 24411(T), Bhargavaea beijingensis DSM 19037(T) and Bhargavaea ginsengi DSM 19038(T) resulted in values (reciprocal values in parentheses) of 26 % (29 %), 18 % (15 %) and 21 % (12 %), respectively. The results of physiological and biochemical tests allowed phenotypic differentiation of strain ZMA 19(T) from all other species of the genus Bhargavaea. Thus, ZMA 19(T) represents a novel species of this genus, for which the name Bhargavaea ullalensis sp. nov. is proposed, with ZMA 19(T) ( = LMG 27071(T) = CCM 8429(T)) as the type strain.

Stappia taiwanensis sp. nov., isolated from a coastal thermal spring.

A beige-coloured, Gram-stain-negative, aerobic, non-motile moderately thermotolerant, rod-shaped organism, strain CC-SPIO-10-1(T), was isolated from a coastal hot spring of Green Island (Lutao), located off Taituang, Taiwan, on Marine Agar 2216. Based on 16S rRNA gene sequence analysis, this organism was grouped into the genus Stappia, showing 98.3 % sequence similarity to Stappia indica B106(T) and 98.2 % gene sequence similarity to Stappia stellulata IAM 12621(T). Ubiquinone Q-10 was the major respiratory quinone and C18 : 1ω7c and C18 : 1ω7c 11-methyl were detected as the major fatty acids. The hydroxylated fatty acid C18 : 0 3-OH was detected as well. Predominant polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, unidentified aminolipid AL1 and unidentified phospholipid PL1. Minor amounts of several unidentified lipids (PL2 and L1-L7) were present as well. The polyamine pattern contained the major compounds spermidine and spermine. Strain CC-SPIO-10-1(T) could be differentiated from the type strains of S. stellulata and S. indica by a set of biochemical tests. On the basis of the 16S rRNA gene sequence analysis and the chemotaxonomic and physiological data, it is concluded that strain CC-SPIO-10(T) represents a novel species of the genus Stappia for which the name Stappia taiwanensis sp. nov. is proposed. The type strain is CC-SPIO-10 (T) ( = CCUG 59208(T) = LMG 25538 (T) = CCM 7757(T)).

Ruegeria intermedia sp. nov., a moderately thermophilic bacterium isolated from a coastal hot spring.

A cream-coloured, Gram-negative, aerobic, non-motile moderately thermophilic, rod-to-irregular-shaped bacterium, CC-GIMAT-2(T), was isolated from a coastal hot spring of Green Island (Lutao), located off Taituang, Taiwan, on marine agar 2216. The 16S rRNA gene sequence analysis and subsequent comparisons showed that it was placed into the genus Ruegeria with 97.4 % similarity to Ruegeria lacuscaerulensis ITI-1157(T), and a lower sequence similarity to all other species of the genus Ruegeria. Reconstruction of phylogenetic trees indicated that strain CC-GIMAT-2(T) clustered within the genus Ruegeria. Robust tree topology for the genus Ruegeria including the new strain was only obtained by including all Rhodobacteraceae type strains but not if the analysis was limited to few selected taxa. The quinone system contained exclusively ubiquinone Q-10 and the fatty acid profile consisted mainly of C18 : 1ω7c, 11-methyl C18 : 1ω7c and C12 : 0 3-OH. The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol and an unidentified aminolipid. Other lipids were detected in moderate to minor amounts. The characteristic feature of the polyamine pattern was the predominant triamine spermidine. On the basis of the 16S rRNA gene sequence analysis and of the chemotaxonomic and physiological data, strain CC-GIMAT-2(T) represents a novel species of the genus Ruegeria, for which the name Ruegeria intermedia sp. nov. is proposed. The type strain is CC-GIMAT-2(T) ( = CCUG 59209(T) = LMG 25539(T) = CCM 7758(T)).

Description of Noviherbaspirillum malthae gen. nov., sp. nov., isolated from an oil-contaminated soil, and proposal to reclassify Herbaspirillum soli, Herbaspirillum aurantiacum, Herbaspirillum canariense and Herbaspirillum psychrotolerans as Noviherbaspirillum soli comb. nov., Noviherbaspirillum aurantiacum comb. nov., Noviherbaspirillum canariense comb. nov. and Noviherbaspirillum psychrotolerans comb. nov. based on polyphasic analysis.

An aerobic, Gram-negative, rod-shaped bacterium with polar flagella, strain CC-AFH3(T), was isolated from an oil-contaminated site located in Kaohsiung county, Taiwan. Strain CC-AFH3(T) grew at 20-40 °C, pH 5.0-10.0 and <2 % (w/v) NaCl. 16S rRNA gene sequence analysis indicated that strain CC-AFH3(T) showed the greatest degree of similarity to Herbaspirillum soli SUEMI10(T) (96.5 %), H. aurantiacum SUEMI08(T) (96.3 %), H. canariense SUEMI03(T) (96.0 %), H. psychrotolerans PB1(T) (95.4 %) and members of other Herbaspirillum species (94.1-95.2 %), and lower similarity to members of other genera (<94 %). Phylogenetic analyses also positioned the novel strain in the genus Herbaspirillum as an independent lineage. The major fatty acids in strain CC-AFH3(T) were C10 : 0 3-OH, C12 : 0, C14 : 0 2-OH, C16 : 0, iso-C15 : 0 3-OH, C17 : 0 cyclo, C16 : 1ω7c/C16 : 1ω6c and C18 : 1ω7c/C18 : 1ω6c. The major polar lipids of strain CC-AFH3(T) were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. The predominant quinone was ubiquinone 8 (Q-8) and the DNA G+C content was 63.4 mol%. On the basis of 16S rRNA gene sequence analysis in combination with physiological and chemotaxonomic data, strain CC-AFH3(T) represents a novel species in a new genus, for which we propose the name Noviherbaspirillum malthae gen. nov., sp. nov.; the type strain of Noviherbaspirillum malthae is CC-AFH3(T) ( = BCRC 80516(T) = JCM 18414(T)). We also propose the reclassification of Herbaspirillum soli, Herbaspirillum aurantiacum, Herbaspirillum canariense and 'Herbaspirillum psychrotolerans' as Noviherbaspirillum soli comb. nov. (type strain SUEMI10(T) = LMG 26149(T) = CECT 7840(T)), Noviherbaspirillum aurantiacum comb. nov. (type strain SUEMI08(T) = LMG 26150(T) = CECT 7839(T)), Noviherbaspirillum canariense comb. nov. (type strain SUEMI03(T) = LMG 26151(T) = CECT 7838(T)) and Noviherbaspirillum psychrotolerans comb. nov. (type strain PB1(T) = DSM 26001(T) = LMG 27282(T)), respectively. An emended description of Herbaspirillum seropedicae is also presented.

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.

Aquabacterium limnoticum sp. nov., isolated from a freshwater spring.

A Gram-negative, facultatively anaerobic, short-rod-shaped, non-motile and non-spore-forming bacterial strain, designated ABP-4(T), was isolated from a freshwater spring in Taiwan and was characterized using the polyphasic taxonomy approach. Growth occurred at 20-40 °C (optimum, 30-37 °C), at pH 7.0-10.0 (optimum, pH 7.0-9.0) and with 0-3% NaCl (optimum, 0%). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain ABP-4(T), together with Aquabacterium fontiphilum CS-6(T) (96.4% sequence similarity), Aquabacterium commune B8(T) (96.1%), Aquabacterium citratiphilum B4(T) (95.5%) and Aquabacterium parvum B6(T) (94.7%), formed a deep line within the order Burkholderiales. Strain ABP-4(T) contained summed feature 3 (comprising C(16:1) ω7c and/or C(16:1) ω6c), C(18:1) ω7c and C(16:0) as predominant fatty acids. The major cellular hydroxy fatty acid was C(10:0) 3-OH. The major isoprenoid quinone was Q-8 and the DNA G+C content was 68.6 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, diphosphatidylglycerol and several uncharacterized phospholipids. The DNA-DNA relatedness of strain ABP-4(T) with respect to recognized species of the genus Aquabacterium was less than 70%. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain ABP-4(T) represents a novel species in the genus Aquabacterium, for which the name Aquabacterium limnoticum sp. nov. is proposed. The type strain is ABP-4(T) (=BCRC 80167(T)=KCTC 23306(T)).

Paracoccus rhizosphaerae sp. nov., isolated from the rhizosphere of the plant Crossostephium chinense (L.) Makino (Seremban).

A Gram-negative, coccoid-shaped bacterium, strain CC-CCM15-8(T), was isolated from a rhizosphere soil sample of the plant Crossostephium chinense (L.) Makino (Seremban) from Budai Township, Chiayi County, Taiwan. 16S rRNA gene sequence analysis clearly allocated strain CC-CCM15-8(T) to the Paracoccus cluster, showing highest similarities to the type strains of 'Paracoccus beibuensis' (98.8%), Paracoccus homiensis (97.6%), Paracoccus aestuarii (97.7%) and Paracoccus zeaxanthinifaciens (97.7%). The fatty acid profile, comprising C(18:1)ω7c as the major component and C(10:0) 3-OH as the characteristic hydroxylated fatty acid, supported the placement of strain CC-CCM15-8(T) within the genus Paracoccus. The polyamine pattern consisted of putrescine and spermidine as major components. Ubiqinone Q-10 was the major quinone type (95%); ubiquinone Q-9 was also detected (5%). The complex polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, and unidentified phospholipids, lipids and glycolipids. Levels of DNA-DNA relatedness between strain CC-CCM15-8(T) and 'P. beibuensis' LMG 25871(T), P. aestuarii DSM 19484(T), P. zeaxanthinifaciens LMG 21993(T) and P. homiensis KACC 11518(T) were 24.9% (34.8%, reciprocal analysis), 15.7% (17.5%), 17.7% (23.4%) and 16.0% (25.4%), respectively. Physiological and biochemical test results allowed the phenotypic differentiation of strain CC-CCM15-8(T) from its closest relatives in the genus Paracoccus. Based on the data presented, it is concluded that strain CC-CCM15-8(T) represents a novel species of the genus Paracoccus, for which the name Paracoccus rhizosphaerae sp. nov. is proposed. The type strain is CC-CCM15-8(T) (=LMG 26205(T)=CCM 7904(T)).

Microbulbifer taiwanensis sp. nov., isolated from coastal soil.

A Gram-negative, non-spore-forming rod (CC-LN1-12(T)) was isolated from coastal soil samples of Lutao Island (Green Island), Taiwan, and its taxonomic position was studied. 16S rRNA gene sequence analysis showed that isolate CC-LN1-12(T) was grouped into the Microbulbifer cluster, with the highest similarities to Microbulbifer okinawensis ABABA23(T) (97.9 %), Microbulbifer maritimus TF-17(T) (97.7 %) and Microbulbifer donghaiensis CN85(T) (97.7 %), similarities to all other species of the genus Microbulbifer were lower than 96.8 %. The polyamine pattern contained the major compounds spermidine and cadaverine. The fatty acid profile, comprising the major fatty acids iso-C(15 : 0), iso-C(17 : 1)ω9c, C(18 : 1)ω7c and iso-C(11 : 0) 3-OH as the major hydroxylated fatty acid, supported the affiliation of strain CC-LN1-12(T) to the genus Microbulbifer. DNA-DNA hybridizations between strain CC-LN1-12(T) and Microbulbifer okinawensis ABABA23(T), M. donghaiensis CN85(T) and M. maritimus JCM 12187(T) resulted in relatedness values of 21.5 % (14.3 %, reciprocal analysis), 35.9 % (48.5 %, reciprocal analysis) and 48.1 % (52.1 %, reciprocal analysis), respectively. From these data, as well as from physiological and biochemical tests, strain CC-LN1-12(T) could be clearly differentiated from the most closely related species of the genus Microbulbifer. It is concluded that strain CC-LN1-12(T) represents a novel species, for which the name Microbulbifer taiwanensis sp. nov. is proposed. The type strain is CC-LN1-12(T) ( = LMG 26125(T) = CCM 7856(T)).

Sphingomicrobium lutaoense gen. nov., sp. nov., isolated from a coastal hot spring.

A yellowish pigmented, Gram-negative, rod-shaped, non-spore-forming bacterium (strain CC-TBT-3(T)), was isolated on marine agar 2216 from a coastal hot spring of Green Island (Lutao), located off Taituang, Taiwan. 16S rRNA gene sequence analysis of strain CC-TBT-3(T) showed a relatively low similarity (<95.5 %) to representatives of the genera Novosphingobium, Sphingosinicella and Sphingomonas of the Sphingomonadaceae, with the most related strain being the type strain of Novosphingobium soli. In addition to the relatively low 16S rRNA gene sequence similarity to members of established species, the isolate also showed some unique chemotaxonomic features, including the presence of some glycolipids with unusual chromatographic behaviour. The major components of the polar lipid profile were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid and three unidentified glycolipids. The major respiratory quinone was ubiquinone Q-10. The polyamine pattern was characterized by the triamine sym-homospermidine as a major component. Although the predominant fatty acids were C(18:1)ω7c and summed feature 3 (C(16:1)ω7c and/or iso-C(15:0) 2-OH), the isolate did not show the typical hydroxyl fatty acids, such as C(14:0) 2-OH, C(15:0) 2-OH and C(16:0) 2-OH, found in members of the genera Novosphingobium, Sphingomonas and Sphingosinicella, but showed instead high amounts of C(18:1) 2-OH (12.0 %). The DNA G+C content of strain CC-TBT-3(T) was 63.4 mol%. 16S rRNA gene sequence, chemotaxonomic and physiological analyses revealed that strain CC-TBT-3(T) represents a novel species in a new genus in the family Sphingomonadaceae for which the name Sphingomicrobium lutaoense gen. nov., sp. nov. is proposed; the type strain is of the type species S. lutoaense, CC-TBT-3(T) ( = DSM 24194(T) = CCM 7794(T)).

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.

Proposal of Solimonas aquatica sp. nov., reclassification of Sinobacter flavus Zhou et al. 2008 as Solimonas flava comb. nov. and Singularimonas variicoloris Friedrich and Lipski 2008 as Solimonas variicoloris comb. nov. and emended descriptions of the genus Solimonas and its type species Solimonas soli.

A bacterial strain designated NAA16(T) was isolated from a freshwater spring in Taiwan and was characterized using a polyphasic taxonomic approach. Strain NAA16(T) was aerobic, Gram-staining-negative, rod-shaped, non-spore-forming and motile by means of a single polar flagellum. Growth occurred at 20-40 °C (optimum, 25 °C), at pH 7.0-8.0 (optimum, pH 7.5) and with up to 1 % NaCl (optimum, 0.5 %). Phylogenetic analyses based on 16S rRNA gene sequences showed that the closest relatives of strain NAA16(T) were Singularimonas variicoloris MN28(T), Sinobacter flavus CW-KD 4(T) and Solimonas soli DCY12(T), with respective sequence similarities of 96.7, 96.6 and 96.2 %. Phylogenetic trees reconstructed from 16S rRNA gene or rpoB sequences (encoding the ß-subunit of the RNA polymerase) revealed that the novel strain NAA16(T) and these three closest relatives formed an independent phylogenetic clade within the Gammaproteobacteria. Strain NAA16(T) contained C16:0, C18:1ω7c and summed feature 3 (C16:1ω7c and/or C16:1ω6c) as predominant fatty acids and possessed phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an uncharacterized aminophospholipid as dominant polar lipids. The major isoprenoid quinone was Q-8. The DNA G+C content of strain NAA16(T) was 66.2 mol%. The taxonomic relationship of strain NAA16(T), Singularimonas variicoloris DSM 15731(T), Sinobacter flavus DSM 18980(T) and Solimonas soli LMG 24014(T) was clarified by means of a direct experimental comparison. Based on phenotypic, chemotaxonomic and phylogenetic data, the descriptions of the genus Solimonas and its type species Solimonas soli are emended. Members of the genus are Gram-negative, oxidase- and catalase-positive, aerobic or facultatively anaerobic and chemo-organotrophic. Chemotaxonomically, members of the genus Solimonas possess Q-8 as the major respiratory quinone, C16:0 and C18:1ω7c as predominant fatty acids and phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an uncharacterized aminophospholipid as dominant polar lipids; the DNA G+C content is 64.9-68.4 mol%. Phylogenetic evidence, supported by chemotaxonomic and phenotypic data, allowed us to assign strain NAA16(T) to the genus Solimonas within the novel species Solimonas aquatica sp. nov. (type strain NAA16(T)  = BCRC 17835(T)  = LMG 24500(T)). The reclassification of Sinobacter flavus as Solimonas flava comb. nov. (type strain CW-KD 4(T)  = DSM 18980(T)  = KCTC 12881(T)  = CCTCC AB 206145(T)) and Singularimonas variicoloris as Solimonas variicoloris comb. nov. (type strain MN28(T)  = DSM 15731(T)  = LMG 22844(T)) is also proposed.