The Complexities of Nocardia Taxonomy and Identification.

Nocardia species are a complex group of organisms considered to belong to the aerobic actinomycetes. Of the validly described species, many have been implicated as the cause of serious human infections, especially in immunocompromised patients. The genus has a complicated taxonomic history; this is especially true for Nocardia asteroides, the type species of the genus and previously the most frequently reported nocardial taxon from human specimens. We provide background on the current taxonomy of Nocardia, with a focus on clinically relevant species, and discuss the currently available methods used to accurately identify isolates to the species, complex, or group level.

FDA-CDC Antimicrobial Resistance Isolate Bank: a Publicly Available Resource To Support Research, Development, and Regulatory Requirements.

The FDA-CDC Antimicrobial Resistance Isolate Bank was created in July 2015 as a publicly available resource to combat antimicrobial resistance. It is a curated repository of bacterial isolates with an assortment of clinically important resistance mechanisms that have been phenotypically and genotypically characterized. In the first 2 years of operation, the bank offered 14 panels comprising 496 unique isolates and had filled 486 orders from 394 institutions throughout the United States. New panels are being added.

Multicenter Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Study for Identification of Clinically Relevant Nocardia spp.

This multicenter study analyzed Nocardia spp., including extraction, spectral acquisition, Bruker matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification, and score interpretation, using three Nocardia libraries, the Bruker, National Institutes of Health (NIH), and The Ohio State University (OSU) libraries, and compared the results obtained by each center. A standardized study protocol, 150 Nocardia isolates, and NIH and OSU Nocardia MALDI-TOF MS libraries were distributed to three centers. Following standardized culture, extraction, and MALDI-TOF MS analysis, isolates were identified using score cutoffs of ≥2.0 for species/species complex-level identification and ≥1.8 for genus-level identification. Isolates yielding a score of <2.0 underwent a single repeat extraction and analysis. The overall score range for all centers was 1.3 to 2.7 (average, 2.2 ± 0.3), with common species generally producing higher average scores than less common ones. Score categorization and isolate identification demonstrated 86% agreement between centers; 118 of 150 isolates were correctly identified to the species/species complex level by all centers. Nine strains (6.0%) were not identified by any center, and six (4.0%) of these were uncommon species with limited library representation. A categorical score discrepancy among centers occurred for 21 isolates (14.0%). There was an overall benefit of 21.2% from repeat extraction of low-scoring isolates and a center-dependent benefit for duplicate spotting (range, 2 to 8.7%). Finally, supplementation of the Bruker Nocardia MALDI-TOF MS library with both the OSU and NIH libraries increased the genus-level and species-level identification by 18.2% and 36.9%, respectively. Overall, this study demonstrates the ability of diverse clinical microbiology laboratories to utilize MALDI-TOF MS for the rapid identification of clinically relevant Nocardia spp. and to implement MALDI-TOF MS libraries developed by single laboratories across institutions.

New rapid scheme for distinguishing the subspecies of the Mycobacterium abscessus group and identifying Mycobacterium massiliense isolates with inducible clarithromycin resistance.

Mycobacterium abscessus (M. abscessus sensu lato, or the M. abscessus group) comprises three closely related taxa whose taxonomic statuses are under revision, i.e., M. abscessus sensu stricto, Mycobacterium bolletii, and Mycobacterium massiliense. We describe here a simple, robust, and cost-effective PCR-based method for distinguishing among M. abscessus, M. massiliense, and M. bolletii. Based on the M. abscessus ATCC 19977(T) genome, regions that discriminated between M. abscessus and M. massiliense were identified through array-based comparative genomic hybridization. A typing scheme using PCR primers designed for four of these locations was applied to 46 well-characterized clinical isolates comprising 29 M. abscessus, 15 M. massiliense, and 2 M. bolletii isolates previously identified by multitarget sequencing. Interestingly, 2 isolates unequivocally identified as M. massiliense were shown to have a full-length erm(41) gene instead of the expected gene deletion and showed inducible clarithromycin resistance after 14 days. We propose using this PCR-based typing scheme combined with erm(41) PCR for straightforward identification of M. abscessus, M. massiliense, and M. bolletii and the assessment of inducible clarithromycin resistance. This method can be easily integrated into a routine workflow to provide subspecies-level identification within 24 h after isolation of the M. abscessus group.

Adaptability and persistence of the emerging pathogen Bordetella petrii.

The first described, environmentally isolated, Bordetella petrii was shown to undergo massive genomic rearrangements in vitro. More recently, B. petrii was isolated from clinical samples associated with jaw, ear bone, cystic fibrosis and chronic pulmonary disease. However, the in vivo consequences of B. petrii genome plasticity and its pathogenicity remain obscure. B. petrii was identified from four sequential respiratory samples and a post-mortem spleen sample of a woman presenting with bronchiectasis and cavitary lung disease associated with nontuberculous mycobacterial infection. Strains were compared genetically, phenotypically and by antibody recognition from the patient and from inoculated mice. The successive B. petrii strains exhibited differences in growth, antibiotic susceptibility and recognition by the patient's antibodies. Antibodies from mice inoculated with these strains recapitulated the specificity and strain dependent response that was seen with the patient's serum. Finally, we characterize one strain that was poorly recognized by the patient's antibodies, due to a defect in the lipopolysaccharide O-antigen, and identify a mutation associated with this phenotype. We propose that B. petrii is remarkably adaptable in vivo, providing a possible connection between immune response and bacterial evasion and supporting infection persistence.

Genomic insights into the emerging human pathogen Mycobacterium massiliense.

Mycobacterium massiliense (Mycobacterium abscessus group) is an emerging pathogen causing pulmonary disease and skin and soft tissue infections. We report the genome sequence of the type strain CCUG 48898.

Multisite reproducibility of the broth microdilution method for susceptibility testing of Nocardia species.

Antimicrobial susceptibility testing (AST) of clinical isolates of Nocardia is recommended to detect resistance to commonly used antimicrobial agents; such testing is complicated by difficulties in inoculum preparation and test interpretation. In this study, six laboratories performed repetitive broth microdilution testing on single strains of Nocardia brasiliensis, Nocardia cyriacigeorgica, Nocardia farcinica, Nocardia nova, and Nocardia wallacei. For each isolate, a total of 30 microdilution panels from three different lots were tested at most sites. The goal of the study was to determine the inter- and intralaboratory reproducibility of susceptibility testing of this group of isolates. Acceptable agreement (>90% agreement at ±1 dilution of the MIC mode) was found for amikacin, ciprofloxacin, clarithromycin, and moxifloxacin. After eliminating MIC values from single laboratories whose results showed the greatest deviation from those of the remaining laboratories, acceptable agreement was also found for amoxicillin-clavulanic acid, linezolid, minocycline, and tobramycin. Results showed unsatisfactory reproducibility of broth microdilution testing of ceftriaxone with N. cyriacigeorgica and N. wallacei, tigecycline with N. brasiliensis and N. cyriacigeorgica, and sulfonamides with N. farcinica and N. wallacei. N. nova ATCC BAA-2227 is proposed as a quality control organism for AST of Nocardia sp., and the use of a disk diffusion test for sulfisoxazole is proposed as a check of the adequacy of the inoculum and to confirm sulfonamide MIC results.

Sulfonamide resistance in isolates of Nocardia spp. from a US multicenter survey.

Recent reports of increasing in vitro sulfonamide resistance in Nocardia prompted us to investigate the findings. Despite the reports, there is a paucity of clinical reports of sulfonamide failure in treatment of nocardia disease. We reviewed 552 recent susceptibilities of clinical isolates of Nocardia from six major laboratories in the United States, and only 2% of the isolates were found to have resistant MICs of trimethoprim-sulfamethoxazole and/or sulfamethoxazole. We hypothesize that the discrepancies in the apparent sulfonamide resistance between our study and the previous findings may be associated with difficulty in the laboratory interpretation of in vitro MICs for trimethoprim-sulfamethoxazole and sulfamethoxazole and the lack of quality controls for Nocardia for these agents.

Nucleic acid amplification tests for diagnosis of smear-negative TB in a high HIV-prevalence setting: a prospective cohort study.

BACKGROUND: Nucleic acid amplification tests are sensitive for identifying Mycobacterium tuberculosis in populations with positive sputum smears for acid-fast bacilli, but less sensitive in sputum-smear-negative populations. Few studies have evaluated the clinical impact of these tests in low-income countries with high burdens of TB and HIV. METHODS: We prospectively enrolled 211 consecutive adults with cough ≥2 weeks and negative sputum smears at Mulago Hospital in Kampala, Uganda. We tested a single early-morning sputum specimen for Mycobacterium tuberculosis DNA using two nucleic acid amplification tests: a novel in-house polymerase chain reaction targeting the mycobacterial secA1 gene, and the commercial Amplified® Mycobacterium tuberculosis Direct (MTD) test (Gen-Probe Inc, San Diego, CA). We calculated the diagnostic accuracy of these index tests in reference to a primary microbiologic gold standard (positive mycobacterial culture of sputum or bronchoalveolar lavage fluid), and measured their likely clinical impact on additional tuberculosis cases detected among those not prescribed initial TB treatment. RESULTS: Of 211 patients enrolled, 170 (81%) were HIV-seropositive, with median CD4+ T-cell count 78 cells/µL (interquartile range 29-203). Among HIV-seropositive patients, 94 (55%) reported taking co-trimoxazole prophylaxis and 29 (17%) reported taking antiretroviral therapy. Seventy-five patients (36%) had culture-confirmed TB. Sensitivity of MTD was 39% (95% CI 28-51) and that of secA1 was 24% (95% CI 15-35). Both tests had specificities of 95% (95% CI 90-98). The MTD test correctly identified 18 (24%) TB patients not treated at discharge and led to a 72% relative increase in the smear-negative case detection rate. CONCLUSIONS: The secA1 and MTD nucleic acid amplification tests had moderate sensitivity and high specificity for TB in a predominantly HIV-seropositive population with negative sputum smears. Although newer, more sensitive nucleic acid assays may enhance detection of Mycobacterium tuberculosis in sputum, even currently available tests can provide substantial clinical impact in smear-negative populations.

Evaluation of the integrated database network system (IDNS) SmartGene software for analysis of 16S rRNA gene sequences for identification of Nocardia species.

16S rRNA gene sequences of 102 Nocardia isolates were analyzed using the Integrated Database Network System (IDNS) SmartGene centroid database. A total of 76% of the isolates were correctly identified. Discordant identifications were due to inadequate centroid length (3 species), inaccurate or insufficient entries in the public databases (5 species), and heterogeneous sequences among members of a species (1 species).

Restriction fragment length polymorphism typing demonstrates substantial diversity among Pneumocystis jirovecii isolates.

Better understanding of the epidemiology and transmission patterns of human Pneumocystis should lead to improved strategies for preventing Pneumocystis pneumonia (PCP). We have developed a typing method for Pneumocystis jirovecii that is based on restriction fragment length polymorphism (RFLP) analysis after polymerase chain reaction amplification of an approximately 1300 base-pair region of the msg gene family, which comprises an estimated 50-100 genes/genome. The RFLP pattern was reproducible in samples containing >1000 msg copies/reaction and was stable over time, based on analysis of serial samples from the same patient. In our initial analysis of 48 samples, we found that samples obtained from different individuals showed distinct banding patterns; only samples obtained from the same patient showed an identical RFLP pattern. Despite this substantial diversity, samples tended to cluster on the basis of country of origin. In an evaluation of samples obtained from an outbreak of PCP in kidney transplant recipients in Germany, RFLP analysis demonstrated identical patterns in samples that were from 12 patients previously linked to this outbreak, as well as from 2 additional patients. Our results highlight the presence of a remarkable diversity in human Pneumocystis strains. RFLP may be very useful for studying clusters of PCP in immunosuppressed patients, to determine whether there is a common source of infection.

Nocardia iowensis sp. nov., an organism rich in biocatalytically important enzymes and nitric oxide synthase.

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)).

"Mycobacterium tilburgii," a newly described, uncultivated opportunistic pathogen.

Molecular methods are increasingly used to identify pathogens that are difficult to cultivate. We report a case of disseminated infection with "Mycobacterium tilburgii," a proposed species that has never been cultivated. The case illustrates the diagnostic utility of sequence analysis of the 16S rRNA gene directly from clinical specimens.

Nocardia wallacei sp. nov. and Nocardia blacklockiae sp. nov., human pathogens and members of the "Nocardia transvalensis Complex".

Nocardia isolates that share the property of in vitro amikacin resistance are grouped together by some authors in the Nocardia transvalensis complex. Our examination of 13 isolates that are amikacin resistant has revealed the existence of three distinct species. Sequence analysis of the 16S rRNA, 65-kDa heat shock protein, and secA1 genes, coupled with DNA-DNA hybridization, indicated that "N. asteroides drug pattern IV," "N. transvalensis new taxon 1," and N. transvalensis sensu stricto should each be considered a distinct species. The phenotypic and molecular characteristics of the proposed new species Nocardia wallacei (N. asteroides drug pattern IV) and N. blacklockiae (N. transvalensis new taxon 1) are presented and compared with those of N. transvalensis sensu stricto. The relative genetic diversity of isolates best placed with the species N. blacklockiae is also discussed. Case studies demonstrating the pathogenicity of N. wallacei and N. blacklockiae are presented. The type strain of N. wallacei is ATCC 49873 (DSM 45136), and that of N. blacklockiae is ATCC 700035 (DSM 45135).

Organisms designated as Nocardia asteroides drug pattern type VI are members of the species Nocardia cyriacigeorgica.

Nocardia cyriacigeorgica has recently been described as an "emerging" pathogen. However, DNA-DNA hybridization results confirm that Nocardia asteroides drug pattern type VI, which has long been recognized as a common and significant pathogen in the United States, belongs to the species N. cyriacigeorgica.

Analysis of multiple differing copies of the 16S rRNA gene in five clinical isolates and three type strains of Nocardia species and implications for species assignment.

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.

Granulibacter bethesdensis gen. nov., sp. nov., a distinctive pathogenic acetic acid bacterium in the family Acetobacteraceae.

A Gram-negative, aerobic, coccobacillus to rod-shaped bacterium was isolated from three patients with chronic granulomatous disease. The organism was subjected to a polyphasic taxonomic study. A multilocus phylogenetic analysis based on the 16S rRNA gene, the internal transcribed spacer (ITS) region and the RecA protein demonstrated that the organism belongs to a new sublineage within the acetic acid bacteria in the family Acetobacteraceae. Phenotypic features are summarized as follows: the organism grew at an optimum temperature of 35-37 degrees C and optimum pH of 5.0-6.5. It produced a yellow pigment, oxidized lactate and acetate, the latter weakly, produced little acetic acid from ethanol and could use methanol as a sole carbon source. The two major fatty acids were a straight-chain unsaturated acid (C18:1omega7c) and C16:0. The DNA base composition was 59.1 mol% G+C. The very weak production of acetic acid from ethanol, the ability to use methanol, the yellow pigmentation and high optimum temperature for growth distinguished this organism from other acetic acid bacteria. The unique phylogenetic and phenotypic characteristics suggest that the bacterium should be classified within a separate genus, for which the name Granulibacter bethesdensis gen. nov., sp. nov. is proposed. The type strain is CGDNIH1T (=ATCC BAA-1260T=DSM 17861T).

Analysis of secA1 gene sequences for identification of Nocardia species.

Molecular methodologies, especially 16S rRNA gene sequence analysis, have allowed the recognition of many new species of Nocardia and to date have been the most precise methods for identifying isolates reliably to the species level. We describe here a novel method for identifying Nocardia isolates by using sequence analysis of a portion of the secA1 gene. A region of the secA1 gene of 30 type or reference strains of Nocardia species was amplified using secA1-specific primers. Sequence analysis of 468 bp allowed clear differentiation of all species, with a range of interspecies similarity of 85.0% to 98.7%. Corresponding 16S rRNA gene sequences of a 1,285-bp region for the same isolates showed a range of interspecies similarity of 94.4 to 99.8%. In addition to the type and reference strains, a 468-bp fragment of the secA1 gene was sequenced from 40 clinical isolates of 12 Nocardia species previously identified by 16S rRNA gene sequence analysis. The secA1 gene sequences of most isolates showed >99.0% similarity to the secA1 sequences of the type or reference strain to which their identification corresponded, with a range of 95.3 to 100%. Comparison of the deduced 156 amino acid sequences of the SecA1 proteins of the clinical isolates showed between zero and two amino acid residue differences compared to that of the corresponding type or reference strain. Sequencing of the secA1 gene, and using deduced amino acid sequences of the SecA1 protein, may provide a more discriminative and precise method for the identification of Nocardia isolates than 16S rRNA gene sequencing.

Clinical and laboratory features of the Nocardia spp. based on current molecular taxonomy.

The recent explosion of newly described species of Nocardia results from the impact in the last decade of newer molecular technology, including PCR restriction enzyme analysis and 16S rRNA sequencing. These molecular techniques have revolutionized the identification of the nocardiae by providing rapid and accurate identification of recognized nocardiae and, at the same time, revealing new species and a number of yet-to-be-described species. There are currently more than 30 species of nocardiae of human clinical significance, with the majority of isolates being N. nova complex, N. abscessus, N. transvalensis complex, N. farcinica, N. asteroides type VI (N. cyriacigeorgica), and N. brasiliensis. These species cause a wide variety of diseases and have variable drug susceptibilities. Accurate identification often requires referral to a reference laboratory with molecular capabilities, as many newer species are genetically distinct from established species yet have few or no distinguishing phenotypic characteristics. Correct identification is important in deciding the clinical relevance of a species and in the clinical management and treatment of patients with nocardial disease. This review characterizes the currently known pathogenic species of Nocardia, including clinical disease, drug susceptibility, and methods of identification.