Subcellular localization and function study of a secreted phospholipase C from Nocardia seriolae.

Fish nocardiosis is a chronic systemic granulomatous disease, and Nocardia seriolae is the main pathogen that causes it. The pathogenesis and virulence factors of N. seriolae are not fully understood. A phospholipase C (PLC), which is likely to be a secreted protein targeting host cell mitochondria, was found by a bioinformatics analysis of the whole genome sequence of N. seriolae. In order to determine the subcellular localization and study the preliminary function of PLC from N. seriolae (NsPLC), in this study gene cloning, secreted protein identification, subcellular localization in host cells and apoptosis detection of NsPLC were carried out. Mass spectrometry analysis of extracellular products from N. seriolae showed that NsPLC was a secreted protein. Subcellular localization of NsPLC-GFP fusion protein in fathead minnow (FHM) cells revealed that the green fluorescence exhibited a punctate distribution near the nucleus and did not co-localize with mitochondria. In addition, an apoptosis assay suggested that apoptosis was induced in FHM cells by the overexpression of NsPLC. This study may lay the foundations for further studies on the function of NsPLC and promote the understanding of the virulence factors and pathogenic mechanism of N. seriolae.

Dual color fluorescence in situ hybridization (FISH) assays for detecting Mycobacterium tuberculosis and Mycobacterium avium complexes and related pathogens in cultures.

Two rapid dual color fluorescence in situ hybridization (FISH) assays were evaluated for detecting M. tuberculosis and related pathogens in cultures. The MN Genus-MTBC FISH assay uses an orange fluorescent probe specific for the Mycobacterium tuberculosis complex (MTBC) and a green fluorescent probe specific for the Mycobacterium and Nocardia genera (MN Genus) to detect and distinguish MTBC from other Mycobacteria and Nocardia. A complementary MTBC-MAC FISH assay uses green and orange fluorescent probes specific for the MTBC and M. avium complex (MAC) respectively to identify and differentiate the two species complexes. The assays are performed on acid-fast staining bacteria from liquid or solid cultures in less than two hours. Forty-three of 44 reference mycobacterial isolates were correctly identified by the MN Genus-specific probe as Mycobacterium species, with six of these correctly identified as MTBC with the MTBC-specific probe and 14 correctly as MAC by the MAC-specific probe. Of the 25 reference isolates of clinically relevant pathogens of other genera tested, only four isolates representing two species of Corynebacterium gave a positive signal with the MN Genus probe. None of these 25 isolates were detected by the MTBC and MAC specific probes. A total of 248 cultures of clinical mycobacterial isolates originating in India, Peru and the USA were also tested by FISH assays. DNA sequence of a part of the 23S ribosomal RNA gene amplified by PCR was obtained from 243 of the 248 clinical isolates. All 243 were confirmed by DNA sequencing as Mycobacterium species, with 157 and 50 of these identified as belonging to the MTBC and the MAC, respectively. The accuracy of the MN Genus-, MTBC-and MAC -specific probes in identifying these 243 cultures in relation to their DNA sequence-based identification was 100%. All ten isolates of Nocardia, (three reference strains and seven clinical isolates) tested were detected by the MN Genus-specific probe but not the MTBC- or MAC-specific probes. The limit of detection for M. tuberculosis was determined to be 5.1x104 cfu per ml and for M. avium 1.5x104 cfu per ml in liquid cultures with the respective MTBC- and MAC-specific probes in both the MN Genus-MTBC and MTBC-MAC FISH assays. The only specialized equipment needed for the FISH assays is a standard light microscope fitted with a LED light source and appropriate filters. The two FISH assays meet an important diagnostic need in peripheral laboratories of resource-limited tuberculosis-endemic countries.

Nocardia zapadnayensis sp. nov., isolated from soil.

A novel Gram-stain positive, rod-shaped, non-motile and mycolic acid containing strain, FMN18(T), isolated from soil, was characterised using a polyphasic approach. The organism showed a combination of morphological, biochemical, physiological and chemotaxonomic properties that were consistent with its classification in the genus Nocardia and it formed a phyletic line in the Nocardia 16S rRNA gene tree. The cell wall contained meso-diaminopimelic acid (type IV) and whole cell sugars were galactose, glucose, arabinose and ribose. The predominant menaquinone was MK-8(H4ω-cyclo). The major phospholipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. Major fatty acids are C16:0, 10-methyl C18:0 (TBSA), C18:1 cis9 and C16:1 trans9. These chemotaxonomic traits are in good agreement with those known for representatives of the genus Nocardia. The phylogenetic analysis based on the 16S rRNA gene sequence of strain FMN18(T) showed it to be closely related to Nocardia grenadensis GW5-5797(T) (99.2 %), Nocardia speluncae N2-11(T) (99.1 %), Nocardia jinanensis 04-5195(T) (99.0 %) and Nocardia rhamnosiphila 202GMO(T) (98.3 %). The phylogenetic analysis based on the gyrB gene sequence of strain FMN18(T) showed it to be closely related to N. rhamnosiphila 202GMO(T) (99.0 %), N. grenadensis DSM 45869(T) (96.6 %), N. jinanensis DSM 45048(T) (93.1 %), N. carnea IFM 0237(T) (89.7 %) and N. speluncae DSM 45078(T) (89.1 %). A combination of DNA-DNA hybridization results and phenotypic properties demonstrated that strain FMN18(T) was clearly distinguished from all closely related Nocardia species. It is proposed that the organism be classified as representing a novel species of the genus Nocardia, for which the name Nocardia zapadnayensis (type strain FMN18(T) = DSM 45872(T) = KCTC 29234(T)) is proposed.

Atomic force microscopy in biofilm study.

Biofilms have been classically visualized by Scanning Electron Microscopy (SEM). The complex operating procedure of SEM restricts its use in routine practice. There is a need of newer visualizing techniques for examining surfaces of biofilms, in particular under ambient conditions. We have presented the unique advantages of atomic force microscopy (AFM) in studying surfaces of biofilms through analyses of the height images obtained on biofilms of two gram positive and one gram negative bacteria, namely Staphylococcus aureus, Nocardia brasiliensis and Pseudomonas aeruginosa, respectively. Biofilm quality of the three different bacteria, ageing effects on Nocardia spp. biofilm surface and effects of the antibiotic ciprofloxacin at different doses on Staphylococcus and Pseudomonas biofilm surfaces have been investigated under ambient conditions and distinctive features have been observed.

Improvement of the production efficiency of L-(+)-tartaric acid by heterogeneous whole-cell bioconversion.

An Escherichia coli-engineered bacterium with cis-epoxysuccinate hydrolase (ESH) activity was used to catalyze the stereospecific hydrolysis of cis-epoxysuccinic acid to L-(+)-tartaric acid. The effect of the substrate composition on the production efficiency of L-(+)-tartaric acid was investigated. Based on the sodium-type homogeneous substrate system, a heterogeneous substrate system, composed of 1.2 M sodium-type substrate and 1.8 M calcium-type substrate, was designed to improve ESH catalytic efficiency. After process optimization, a catalytic efficiency of 9.37 × 10(-3) g U(-1) h(-1) was obtained with fed-batch mode in the heterogeneous substrate system, about a twofold increase compared to the traditional bioconversion process with Nocardia tartaricans cells. The scale-up tests were carried out in a 15-m(3) stirred tank reactor, which indicated that the heterogeneous substrate system had great application prospect for the L-(+)-tartaric acid industrial production.

Characterization of Nocardia farcinica, a filamentous bacterium isolated from foaming activated sludge samples.

The identification and characterization of filamentous bacteria and their association with specific plant operating conditions and influent characteristics has been hampered because of morphological variations and differences between process configurations. A study was conducted to isolate and characterize the predominant filamentous bacteria observed in a foaming activated sludge treatment plant. The predominant foam-forming filament was isolated and characterized using microscopic, biochemical and molecular techniques. The phylogenetic analysis of the 16S rRNA gene confirmed that it was Nocardia farcinica, a typical filamentous foam-foaming pathogenic bacterium which is not widely reported outside of South Africa. The bacterium used a variety of substrates for its growth and showed greater affinity to larger and slowly biodegradable compounds. The N. farcinica grew well at temperatures ranging from 12 to 30 degrees C in R2A medium and with a pH ranging from 5.5 to 8.0 and an NaCl concentration of 1 to 5%. This range of conditions shows that N. farcinica can withstand extreme conditions, which results in its proliferation in foaming samples.

Study on mechanical properties of a new type micro/nano metal material based on bacteria shape.

The functional groups and mechanical properties of Nocadia, a kind of bacteria with submicrometer in diameter and 3-10 microm in length, before and after metallization are determined by fourier transform infrared spectroscopy (FT-IR) and nanoindentation technology. The group -COOH exists on surface of Nocadia and the function groups of Nocadia decreases due to metallization. The elastic modulus of metallized Nocadia, Nocadia and resin is 42.583 GPa, 9.501 GPa and 5.723 GPa, respectively, and the hardness is 1.940 GPa, 0.265 GPa and 0.301 GPa, respectively. There is a great improvement of 5 times in elastic modulus and 9 times in hardness compared with bare Nocadia.

Actinomycetoma in arm disseminated to lung with grains of Nocardia brasiliensis with peripheral filaments.

Actinomycetomas represent 97.8% of mycetomas in Mexico, where 86.6% are produced by Nocardia brasiliensis. We report a case of actinomycetoma in the arm by Nocardia brasiliensis disseminated to lung. Uncommon grains were observed which present outside peripheral filaments and also numerous filaments loosing the grains. These characteristics of the grains are due probably because for the long treatment with antibiotics of the patient. In situ antibiotic action against the microcolonies is discussed.

Cytological diagnosis of pulmonary nocardiosis in an immunocompromised patient.

We report a case of pulmonary nocardiosis in an immunosuppressed patient having vasculitis who presented with fever, cough and chest pain. A suspicion of nocardiosis was made on auramine O staining of material procured by CT guided fine needle aspiration cytology right lung. Modified Ziehl-Neelsen staining was useful in confirming the diagnosis. The patient showed remarkable recovery after treatment with co-trimoxazole. Quick identification of this uncommon pathogen in the cytological material using special stains helped in timely diagnosis and successful treatment of the patient.

Nocardia ninae sp. nov., isolated from a bronchial aspirate.

A Gram-positive and acid-fast filamentous bacterium (OFN 02.72(T)) was isolated from a bronchial aspirate from a 53-year-old patient. Chemotaxonomic data supported the affiliation of this organism to the genus Nocardia, and the phenotypic characteristics demonstrated that the strain differed from all previously described Nocardia species. Restriction enzyme analysis of the hsp65 gene (encoding the 65 kDa heat-shock protein) and sequencing of the 16S rRNA and hsp65 genes confirmed that this isolate is unique. The most closely related type strains were Nocardia alba YIM 30243(T) (=DSM 44684(T)) and Nocardia jejuensis N3-2(T) (=JCM 13281(T)) (with 16S rRNA gene sequence similarities of 98.3 and 97.2 %, respectively). On the basis of this polyphasic study, strain OFN 02.72(T) represents a novel species within the genus Nocardia, for which the name Nocardia ninae sp. nov. is proposed. The type strain is OFN 02.72(T) (=CIP 108955(T)=DSM 44978(T)).

Disseminated nocardiosis diagnosed by fine needle aspiration biopsy: quick and accurate diagnostic approach.

Nocardia is an uncommon pathogen in immunocompetent patients; however, it has been increasingly recognized as a significant opportunistic pathogen in organ transplant patients. Diagnosis of Nocardiosis is usually made by microbiologic culture or cytologic examination of pulmonary specimens including, sputum, and brushing/washings or by histologic evaluation of tissue biopsy material. We report a case of subcutaneous Nocardiosis diagnosed by Fine-needle aspiration biopsy (FNA). The patient is a 66-year-old man with a history of lung transplantation and posttransplant lymphoproliferative disorder who presented with subcutaneous masses in the right upper arm and the left shoulder. FNA was performed in an outpatient clinic setting, with immediate morphologic assessment revealing filamentous branching organisms suspicious for Nocardiosis. Subsequent examination with special stains and microbiologic culture confirmed the diagnosis. The quick and accurate diagnosis by FNA led to emergent and appropriate treatment.

Cytologic diagnosis of pulmonary nocardiosis: a report of 3 cases.

BACKGROUND: Nocardiosis is an uncommon infection and presents as an opportunistic infection in an immunocompromised host. Pulmonary infection by Nocardia may be difficult to diagnose based on clinical and radiologic features, as these are not specific. Sputum examination, bronchoalveolar lavage and transthoracic ultrasound/computed tomography-guided fine needle aspiration cytology offer a simple means of procuring material for diagnostic evaluation. Very few articles have described the morphologic appearance of this uncommon pathogen in cytologic material. CASES: Three cases occurred in patients with an underlying immunocompromised state. Patient 1 was on steroid therapy for nephrotic syndrome, patient 2 was on immunosuppressant therapy after renal transplantation, and patient 3 was HIV positive. A diagnosis of pulmonary nocardiosis was suspected on Papanicolaou stain. Modified Ziehl-Neelsen stain and silver methanamine stains were useful in confirming the diagnosis. CONCLUSION: A high index of suspicion for nocardiosis must be maintained while assessing cytologic material in immunosuppressed individuals as it may be masked by the intense inflammatory exudate associated with this infection. A meticulous search may reveal the presence of delicate, thin, faintly stained, branching filaments of Nocardia on routine Papanicolaou stain. Special stains and culture studies are useful in confirming the diagnosis.

Reclassification of Nocardia corynebacterioides Serrano et al. 1972 (Approved Lists 1980) as Rhodococcus corynebacterioides comb. nov.

The type strain of Nocardia corynebacterioides was the subject of a polyphasic taxonomic study. The 16S rRNA gene sequence was aligned with the sequences of representatives of the genera Corynebacterium, Dietzia, Gordonia, Mycobacterium, Nocardia, Rhodococcus, Skermania, Tsukamurella and Williamsia, and phylogenetic trees were constructed by using maximum-parsimony, maximum-likelihood and neighbour-joining methods. It was evident from the phylogenetic analysis that N. corynebacterioides represents a distinct phyletic line within the genus Rhodococcus. Menaquinone analysis showed that the organism contained dihydrogenated menaquinone with eight isoprene units, MK-8(H(2)), as the major isoprenologue. The genealogical evidence, together with chemotaxonomic and phenotypic data from this and previous studies, indicates that N. corynebacterioides DSM 20151(T) (= CIP 104510(T)) should be reclassified in the genus Rhodococcus as Rhodococcus corynebacterioides comb. nov.

Nocardia araoensis sp. nov. and Nocardia pneumoniae sp. nov., isolated from patients in Japan.

Two actinomycete strains isolated from two patients with lung nocardiosis between 1995 and 1997 in Japan were assigned to novel species of the genus Nocardia based on morphological and chemical criteria. Comparative 16S rRNA gene sequence analysis of the two strains revealed that they belong to the genus Nocardia and are most closely related to the species Nocardia beijingensis. Determination of DNA-DNA relatedness indicated that these strains could be assigned to two novel species. Based on their phenotypic and phylogenetic characters, two novel species of the genus Nocardia are proposed: Nocardia araoensis sp. nov. for IFM 0575(T) (=NBRC 100135(T)=JCM 12118(T)=DSM 44729(T)) and Nocardia pneumoniae sp. nov. for IFM 0784(T) (=NBRC 100136(T)=JCM 12119(T)=DSM 44730(T)).

Nocardia pigrifrangens sp. nov., a novel actinomycete isolated from a contaminated agar plate.

A polyphasic study was undertaken to establish the taxonomic position of an actinomycete strain isolated from a contaminated agar plate. The strain, designated 7031T, had morphological and chemotaxonomic properties typical of the genus Nocardia. An almost-complete 16S rRNA gene sequence determined for the strain was aligned with available sequences for nocardiae, and phylogenetic trees were inferred using three tree-generating algorithms. Strain 7031T clustered with the type strains of Nocardia carnea and Nocardia flavorosea, showing low 16S rRNA gene sequence similarities to these species (97.2 and 97.5 %, respectively). The strain was also distinguished from the closest species by a range of phenotypic properties. It is proposed that the strain be recognized as a novel species of Nocardia, Nocardia pigrifrangens sp. nov., the type strain of which is 7031T (= AS 4.1808T = JCM 11884T).

Nocardia alba sp.nov., a novel actinomycete strain isolated from soil in China.

A novel actinomycete strain, designated YIM 30243T, was isolated from a soil sample in Yunnan Province, China. Based on the results of phenotypic and genotypic characteristics, strain YIM 30243T should be assigned to a new species of the genus Nocardia, for which the name Nocardia alba sp. nov. is proposed. The type strain is YIM 30243T (= CCTCC AA001030T = DSM 44684T).

Nocardia brasiliensis: from microbe to human and experimental infections.

Nocardia brasiliensis is a Gram-positive bacterium that lives as a saprophyte in soil. In this article the physical properties, chemical composition and taxonomic position of this species is reviewed. Human infections and an experimental model of actinomycetoma in BALB/c mice as well as the host-immune response is described.

Nocardia uniformis nom. rev.

A soil isolate representing the putatively novel species 'Nocardia uniformis' was found to have morphological, staining and chemotaxonomic properties consistent with its classification in the genus Nocardia. An almost complete sequence of the 16S rDNA of the strain was determined following cloning and sequencing of the amplified gene. The sequence was aligned with those available for nocardiae and phylogenetic trees were inferred using four tree-making algorithms. The organism was consistently associated with the type strain of Nocardia otitidiscaviarum albeit with a relatively low bootstrap value recorded for neighbour-joining analysis. The strain was also readily separated from representatives of all validly described Nocardia species using a set of phenotypic properties. The genotypic and phenotypic data indicate that the strain should be assigned to the genus Nocardia as a new species. The name proposed for the new species is Nocardia uniformis. The type strain is JCM 3224T.

Actinomycosis and other bronchopulmonary infections with bacterial granules.

Pulmonary infections with formation of bacterial granules are rare. We reviewed the clinical and pathologic data from 18 cases diagnosed using surgical specimens in our department during the last 10 years. Three clinicopathologic forms were observed: endobronchial infections complicating tuberculous sequelae or bronchiectases (n = 7), tumor-like lesions (n = 8), and diffuse pneumonia (n = 3). The two latter forms contrasted with the former by a male predominance, association with general debilitating conditions and inflammatory syndrome, and pathologically by smaller granules often located in parenchymal abscesses or excavations. The pathologic examination of the bacteria forming the granules permitted the diagnoses of actinomycosis (n = 10), botryomycosis (n = 7), or nocardiosis (n = 1). The latter case corresponded to an endobronchial infection. Both actinomycosis and botryomycosis were encountered in every clinicopathologic form. At present, pulmonary actinomycosis and related infections rarely seems to present with chest wall invasion. On the contrary, purely endobronchial forms represented a large proportion of our cases. Cultures are often difficult and the clinical appearance is not specific. However, pathologic examination with special stains must indicate the type of involved microorganism.

Local expression of cytokines in rat bladder carcinoma tissue after intravesical treatment with Nocardia rubra cell wall skeleton and bacille-Calmette-Guerin.

This study aimed to investigate local immunotherapy with Nocardia rubra cell wall skeleton (N-CWS) and bacille-Calmette-Guerin (BCG) in chemically induced rat bladder carcinoma. After being fed with 0.025% N-butyl-N-(4-hydroxybutyl) nitrosamine for 26 weeks, female Wistar rats were treated once a week by intravesical instillation of 100 microg N-CWS or 5 x 10(6) Colony-Forming Units (CFU) BCG. Tissue specimens were obtained 4 h after the ninth instillation and analyzed by reverse transcription polymerase chain reaction (RT-PCR) for mRNA expression of rat cytokines. The analysis showed high expression of interleukin (IL)-1 alpha, tumor necrosis factor (TNF)-alpha, IL-2, and interferon (IFN)-tau in BCG (7/7, 7/7, 7/7, 5/7) and N-CWS (9/9, 9/9, 8/9, 8/9) treated tumors in comparison to low expression in controls (3/9, 0/9, 3/9, 3/9). Interestingly, intravesical instillation of N-CWS tended to be less effective at preventing local invasion of the tumors. It is speculated that this difference may result from a more strongly induced expression of T-helper cell-derived lymphokines (IL-2, IFN-tau) by BCG.