Nocardia veterana isolated from ascitic fluid of a patient with human immunodeficiency virus infection.
ABSTRACT: Nocardia veterana is a recently characterized species within the genus Nocardia, and only three human clinical isolates have been reported for this species. We describe a case of ascitic fluid infection in an immunocompromised patient due to N. veterana. To our knowledge, this is the first report of a Nocardia sp. strain from ascitic fluid and the fourth report of N. veterana isolated from human samples. Chemotaxonomic methods showed the strain to belong to the genus Nocardia, and identification to the species level was done by 16S ribosomal DNA gene sequencing. The antibiotic susceptibility profile of N. veterana is reported here for the second time. The strain was deposited in the Collection of the Pasteur Institute and in the Culture Collection of the University of Göteborg (CIP 107497 and CCUG 46576). The corresponding 16S ribosomal DNA gene sequence is available from the GenBank database under accession number AY149599. A phylogenetic analysis was conducted and showed that N. veterana was most closely related to the recently characterized species Nocardia africana rather than to Nocardia vaccinii, as previously reported.
Project description:Molecular methodologies have become useful techniques for the identification of pathogenic Nocardia species and for the recognition of novel species that are capable of causing human disease. Two isolates recovered from immunocompromised patients were characterized as Nocardia nova by biochemical and susceptibility testing results. The restriction fragment length polymorphism (RFLP) patterns obtained by restriction endonuclease analysis (REA) of an amplified portion of the heat shock protein gene were identical to those obtained with the type strain of N. nova. REA of an amplified portion of the 16S rRNA gene showed RFLP patterns that were unlike those obtained for the type strain of N. nova but that were similar to those obtained for the type strains of N. africana and N. veterana. Subsequent sequencing of a portion of the 16S rRNA gene produced identical results for the two patient isolates. Sequence analysis of 1,352-bp portions of the 16S rRNA gene indicated that these isolates were 99.8% similar to the recently described species N. veterana but were only 99.3, 98.1, and 98.1% similar to the type strains of N. africana, N. nova, and N. vaccinii, respectively. DNA-DNA hybridization studies confirmed that the two patient isolates belonged to the same species but were not closely related to N. africana, N. nova, N. vaccinii, or N. veterana. The patient isolates have been designated N. kruczakiae sp. nov. Because N. africana, N. veterana, and the new species are not readily differentiated from N. nova by phenotypic methods alone, the designation "N. nova complex" can be used to designate isolates such as these that phenotypically resemble N. nova but that have not been definitively characterized by 16S rRNA gene sequencing or DNA-DNA hybridization.
Project description:The molecular methodologies used in our laboratories have allowed us to define a group of Nocardia isolates from clinical samples which resemble the type strain of Nocardia veterana. Three patient isolates and the type strain of N. veterana gave identical and distinctive restriction fragment length polymorphisms (RFLPs) for an amplified portion of the 16S rRNA gene. These three isolates and the N. veterana type strain also gave identical RFLPs for an amplified portion of the 65-kDa heat shock protein gene, but this pattern was identical to that obtained for the Nocardia nova type strain. Sequence analysis of both a 1,359-bp region of the 16S rRNA gene and a 441-bp region of the heat shock protein gene of the patient isolates showed 100% identities with the same regions of the N. veterana type strain. DNA-DNA hybridization of the DNA of one of the patient isolates with the DNA of the N. veterana type strain showed a relative binding ratio of 82%, with 0% divergence, confirming that the isolate was N. veterana. Biochemical and susceptibility testing showed no significant differences among the patient isolates and the N. veterana type strain. Significantly, the results of antimicrobial susceptibility testing obtained for our isolates were similar to those obtained for N. nova, indicating that susceptibility testing alone cannot discriminate between these species. We present two case studies which show that N. veterana is a causative agent of pulmonary disease in immunocompromised patients residing in North America. We also describe difficulties encountered in using 16S rRNA gene sequences alone for discrimination of N. veterana from the related species Nocardia africana and N. nova because of the very high degree of 16S rRNA gene similarity among them.
Project description:Nocardia veterana is a newly described species named after the veteran's hospital where it was first isolated. This initial type strain was not thought to be clinically significant. We describe three cases of pulmonary disease attributable to N. veterana: two cases in patients presenting with multiple pulmonary nodules in a setting of immunocompromise and one case of exacerbation of chronic pulmonary disease. The isolates were susceptible to ampicillin, imipenem, gentamicin, amikacin, and trimethoprim-sulfamethoxazole and had reduced susceptibilities to ceftriaxone, cefotaxime, minocycline, and ciprofloxacin. The MICs of amoxicillin-clavulanate were higher than that of ampicillin alone, and the bacteria produced a beta-lactamase detectable only after induction with clavulanic acid. Phenotypically, the isolates could not be characterized beyond the Nocardia genus level. All three isolates were definitively identified as N. veterana by PCR and sequencing of the 16S rRNA gene. On the basis of their susceptibility and restriction enzyme analysis profiles, our findings indicate that they could potentially be misidentified as N. nova. These cases illustrate the pathogenic potential of this newly described species and emphasize the importance of accurate identification of Nocardia isolates to the species level by integrated use of phenotypic and genotypic methods.
Project description:Sequence analysis of the Nocardia essential secretory protein SecA1 gene (secA1) for species identification of 120 American Type Culture Collection (ATCC) and clinical isolates of Nocardia (16 species) was studied in comparison with 5'-end 606-bp 16S rRNA gene sequencing. Species determination by both methods was concordant for all 10 ATCC strains. secA1 gene sequencing provided the same species identification as 16S rRNA gene analysis for 94/110 (85.5%) clinical isolates. However, 40 (42.6%) isolates had sequences with <99.0% similarity to archived secA1 sequences for the species, including 29 Nocardia cyriacigeorgica (96.6 to 98.9% similarity) and 4 Nocardia veterana (91.5 to 98.9% similarity) strains. Discrepant species identification was obtained for 16 (14.5%) clinical isolates, including 13/23 Nocardia nova strains (identified as various Nocardia species by secA1 sequencing) and 1 isolate each of Nocardia abscessus (identified as Nocardia asiatica), Nocardia elegans (Nocardia africana), and Nocardia transvalensis (Nocardia blacklockiae); both secA1 gene sequence analysis and deduced amino acid sequence analysis determined the species to be different from those assigned by 16S rRNA gene sequencing. The secA1 locus showed high sequence diversity (66 sequence or genetic types versus 40 16S rRNA gene sequence types), which was highest for N. nova (14 secA1 sequence types), followed by Nocardia farcinica and N. veterana (n = 7 each); there was only a single sequence type among eight Nocardia paucivorans strains. The secA1 locus has potential for species identification as an adjunct to 16S rRNA gene sequencing but requires additional deduced amino acid sequence analysis. It may be a suitable marker for phylogenetic/subtyping studies.
Project description:16S rDNA sequence analysis is the most accurate method for definitive species identification of nocardiae. However, conflicting results can be found due to sequence errors in gene databases. This study tested the feasibility of species identification of Nocardia by partial (5'-end 606-bp) 16S rDNA sequencing, based on sequence comparison with "reference" sequences of well-annotated strains. This new approach was evaluated using 96 American Type Culture Collection (n=6), and clinical (n=90) Nocardia isolates. Nucleotide sequence-based polymorphisms within species were indicative of "sequence types" for that species. Sequences were compared with those in the GenBank, Bioinformatics Bacteria Identification and Ribosomal Database Project databases. Compared with the reference sequence set, all 96 isolates were correctly identified using the criterion of ?99% sequence similarity. Seventy-eight (81.3%) were speciated by database comparison; alignment with reference sequences resolved the identity of 14 (15%) isolates whose sequences yielded 100% similarity to sequences in GenBank under >1 species designation. Of 90 clinical isolates, the commonest species was Nocardia nova (33.3%) followed by Nocardia cyriacigeorgica (26.7%). Recently-described or uncommon species included Nocardia veterana (4.4%), Nocarida bejingensis (2.2%) and, Nocardia abscessus and Nocardia arthriditis (each n=1). Nocardia asteroides sensu stricto was rare (n=1). There were nine sequence types of N. nova, three of Nocardia brasiliensis with two each of N. cyriacigeorgica and Nocardia farcinica. Thirteen novel sequences were identified. Alignment of sequences with reference sequences facilitated species identification of Nocardia and allowed delineation of sequence types within species, suggesting that such a barcoding approach can be clinically useful for identification of bacteria.
Project description:gyrB is used to improve the identification of the Nocardia species N. brasiliensis, N. higoensis, N. ignorata, N. otitidiscaviarum, N. paucivorans, N. pneumoniae, N. puris, N. takedensis, N. veterana, and N. vinacea, but it does not improve the identification of another 12 Nocardia studied species. gyrB provides typing and phylogenetic markers for N. carnea, N. transvalensis, N. brasiliensis, and N. otitidiscaviarum.
Project description:Identification of pathogenic bacteria in ascites correlates with poor clinical outcomes. Ascites samples are commonly reported culture-negative, even where frank infection is indicated. Culture-independent methods have previously reported bacterial DNA in ascites, however, whether this represents viable bacterial populations has not been determined. We report the first application of 16S rRNA gene pyrosequencing and quantitative PCR in conjunction with propidium monoazide sample treatment to characterise the viable bacterial composition of ascites. Twenty five cirrhotic patients undergoing paracentesis provided ascites. Samples were treated with propidium monoazide to exclude non-viable bacterial DNA. Total bacterial load was quantified by 16S rRNA Q-PCR with species identity and relative abundance determined by 16S rRNA gene pyrosequencing. Correlation of molecular microbiology data with clinical measures and diagnostic microbiology was performed. Viable bacterial signal was obtained in 84% of ascites samples, both by Q-PCR and pyrosequencing. Approximately 190,000 ribosomal pyrosequences were obtained, representing 236 species, including both gut and non gut-associated species. Substantial variation in the species detected was observed between patients. Statistically significant relationships were identified between the bacterial community similarity and clinical measures, including ascitic polymorphonuclear leukocyte count and Child-Pugh class. Viable bacteria are present in the ascites of a majority of patients with cirrhosis including those with no clinical signs of infection. Microbiota composition significantly correlates with clinical measures. Entry of bacteria into ascites is unlikely to be limited to translocation from the gut, raising fundamental questions about the processes that underlie the development of spontaneous bacterial peritonitis.
Project description:BACKGROUND:Nocardia species are rare, opportunistic organisms that cause disease in both immunocompetent and immunocompromised individuals. OBJECTIVE:To investigate the clinical presentations of various Nocardia infections based on the 16S ribosomal RNA gene of the isolate, as well as related risk factors and susceptibility patterns to antimicrobial agents. METHODS:Thirteen patients with a diagnosis of nocardiosis were included in the present study. Seven Nocardia species were identified by 16S ribosomal RNA. Susceptibility testing was performed using six antimicrobial agents. RESULTS:Five patients were immunocompromised, and eight were immunocompetent with predisposing factors including cystic fibrosis, tuberculosis and ophthalmic infections. Nocardia caused pulmonary infections in eight patients (61.5%), invasive systemic infections in three patients (23%) and local (ophthalmic) infections in two patients (15.4%). In the patients with pulmonary disease, nocardiosis was caused by six species (Nocardia cyriacigeorgica, Nocardia otitidiscaviarum, Nocardia farcinica, Nocardia carnea, Nocardia testacea and Nocardia asiatica). The seventh species identified in the present study was Nocardia crassostreae. DISCUSSION:N crassostreae is a multidrug-resistant organism that was reported to be an emerging human pathogen causing invasive nocardiosis in a patient with non-Hodgkin's lymphoma. N farcinica was isolated from blood in a patient with breast cancer. None of the Nocardia isolates were resistant to linezolid. One N otitidiscaviarum isolate was a multidrug-resistant organism. All patients in the present study were treated with the appropriate antibiotics and their condition resolved without further sequelae. CONCLUSIONS:The present study is the first report on N crassostreae as a human pathogen. The detection of multidrug-resistant species necessitate molecular identification and susceptibility testing, and should be performed for all Nocardia infections. Nocardiosis manifests various clinical features depending on the Nocardia species and underlying conditions.
Project description:Nocardia strain NRRL 5646, isolated from a garden soil sample in Osceola, Iowa, USA, was initially of interest as an antibiotic producer. It contained biocatalytically important enzymes and represented the first described nitric oxide synthase enzyme system in bacteria. The present polyphasic taxonomic study was undertaken to differentiate strain NRRL 5646(T) from related species of the genus Nocardia. Chemotaxonomic analyses included determinations of the fatty acid methyl ester profile (C(16 : 1)omega6c/C(16 : 1)omega7c, C(16 : 0), C(18 : 1)omega9c and C(18 : 0) 10-methyl as major components), quinone [cyclo MK-8(H(4)) as the major component], polar lipid (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside as major components) and mycolic acid. These results supported its placement within the genus Nocardia. Biochemical testing and 16S rRNA, 65-kDa heat-shock protein (hsp65) and preprotein translocase (secA1) gene sequence analyses differentiated strain NRRL 5646(T) from recognized Nocardia species. Previous studies have demonstrated that other genetic sequences (carboxylic acid reductase, Nocardia phosphopantetheinyl transferase and GTP cyclohydrolase I) from strain NRRL 5646(T) can also be used to substantiate its uniqueness. The level of 16S rRNA gene sequence similarity between strain NRRL 5646(T) and the type strains of Nocardia tenerifensis and Nocardia brasiliensis was 98.8 %. However, strain NRRL 5646(T) could be clearly distinguished from these Nocardia species based on DNA-DNA hybridization data. Consequently, strain NRRL 5646(T) is considered to represent a novel species of the genus Nocardia, for which the name Nocardia iowensis sp. nov. is proposed. The type strain is NRRL 5646(T) (=UI 122540(T)=NRRL B-24671(T)=DSM 45197(T)).
Project description:Five clinical isolates of Nocardia that showed ambiguous bases within the variable region of the 16S rRNA gene sequence were evaluated for the presence of multiple copies of this gene. The type strains of three Nocardia species, Nocardia concava, Nocardia ignorata, and Nocardia yamanashiensis, which also showed ambiguous bases in the variable region, were also examined. Cloning experiments using an amplified region of the 16S rRNA that contains the variable region showed that each isolate possessed 16S rRNA genes with at least two different sequences. In addition, hybridization studies using a 16S rRNA gene-specific probe and extracted genomic DNA of the patient isolates and of the type strain of N. ignorata showed that each isolate possessed at least three copies of the gene. These multiple differing copies of the 16S rRNA gene and the results of DNA-DNA hybridization studies indicate problems of species definition and identification for such isolates. A broader species concept than that currently in vogue may be required to accommodate such organisms.