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.     

First Spanish Case of Nocardiosis Caused by Nocardia takedensis

Nocardia takedensis is a recently described species isolated from soil. The first clinical isolate in Japan has recently been reported. This report describes the first clinical isolate of N. takedensis in Spain from a respiratory specimen.Nocardia species are aerobic gram-positive bacteria of the Actinomycetales order, soil saprophytes involved in the decomposition of plant material (2, 7, 10). Isolation from respiratory samples may be indicative of colonization or invasive infection. The criteria used to determine the clinical significance of a culture positive for a Nocardia sp. include signs and symptoms in the patient and the clinical setting (e.g., corticosteroid therapy, transplantation). The respiratory tract is the most common site of infection or colonization. Most Nocardia infections are believed to be acquired by inhalation of airborne spores or mycelial fragments from environmental sources (3). The diagnosis of nocardiosis is usually based on direct examination since conventional cultures are complex, as well as long and time-consuming (4). However, the classical identification process is complicated and incomplete and therefore the current identification of Nocardia is being mainly based on molecular phylogenetic information (3). With the introduction of genetic technologies, the reports of new species of Nocardia have increased. Nocardia takedensis has been recently described as a new species of the genus (14). The type strain of this species was isolated from soil, and only a clinical case of a patient with T-cell lymphoma has been reported in Japan so far (13). This report describes the first clinical isolate of N. takedensis from a respiratory specimen in Spain.The strain of N. takedensis was isolated from a 41-year-old female patient with type 2 diabetes mellitus, eosinophilic granuloma, and lung damage. The patient was diagnosed after a lung biopsy with typical histological findings. She was successfully treated with steroids for about 6 months.In the laboratory, sputum was processed for mycobacterial study so the strain was isolated from mycobacterial culture medium. Gram-stained smears revealed gram-positive short filaments, coccoid forms, and branching rods. In the modified Ziehl-Neelsen stain, the strain was partially acid fast. The identification to the genus level as Nocardia was based on macroscopic, microscopic, and biochemical characteristics. The methods described by Boiron et al. (1) were used to determine the decomposition of adenine, casein, hypoxanthine, tyrosine, and xanthine. The isolated strain did not degrade any of them. Regarding the culture conditions, this strain grew well at 30°C but did not grow at 45°C.DNA was extracted for PCR amplification and sequencing on both strands of the 16S rRNA gene as previously described by Rodriguez-Nava et al. (10). A 606-nucleotide amplified fragment was obtained with primers described by Rodriguez-Nava et al. (10) (Noc1, 5′-GCTTAACACATGCAAGTCG-3′; Noc2, 5′-GAATTCCAGTCTCCCCTG-3′), confirming the diagnosis of nocardiosis. The clinical isolate sequence was then compared with those of representatives species classified in the genus Nocardia in the GenBank and BIBI databases (5). Our clinical isolate was identified as N. takedensis with 99.5% sequence similarity (3 nucleotide differences out of 606 nucleotides) to the type strain of N. takedensis. It has been demonstrated by 16S rRNA gene sequencing that N. takedensis is most closely related to two pathogenic Nocardia species, Nocardia beijingensis (98.1 to 98.3% similarity) and Nocardia brasiliensis (97.9 to 98.0% similarity) (14), which have been reported as responsible for pulmonary abscesses and actinomycetoma, respectively.The susceptibility of the isolate to different antimicrobials was determined by using a commercial broth microdilution method. Appropriate dilutions for MIC determinations were obtained from EMIZA 9EF Sensititre plates. Reference strains Escherichia coli ATCC 35218 and Staphylococcus aureus ATCC 29213 were used as controls (8). We used the recommended primary antimicrobials (amikacin, amoxicillin-clavulanic acid, ciprofloxacin, imipenem, tetracycline, and trimethoprim-sulfamethoxazole) and one secondary antimicrobial (cefotaxime) for susceptibility testing. The plate was incubated at 37°C for 72 h and read manually with a mirrored box.Nocardia species can vary in their antimicrobial susceptibility patterns (11). In vitro, this isolate was susceptible to amikacin (≤4 μg/ml), cefotaxime (8 μg/ml), trimethoprim-sulfamethoxazole (2 and 38 μg/ml, respectively), imipenem (4 μg/ml), and tetracycline (≤4 μg/ml) and was resistant to amoxicillin-clavulanic acid (≥16/8 μg/ml) and ciprofloxacin (≥4 μg/ml). We used Clinical and Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Standards) susceptibility criteria for the interpretation of susceptible, intermediate, and resistant isolates (8).This is the first report of N. takedensis isolation in Spain. The patient did not show any symptoms of pulmonary nocardiosis or any radiographic manifestations, so this isolation represented transient colonization. An association such as the one seen in our patient between nocardiosis and steroid therapy has already been reported in previous studies (12). Pulmonary nocardiosis should be suspected in patients with a history of prior steroid use since this risk factor has been highly correlated with the development of nocardiosis (6, 9). The patient reported here was an immunocompromised patient who received steroid treatment, so it is possible that aerosolization of grass and soil facilitated the lung colonization seen.In conclusion, the colony morphology or morphological characteristics of Nocardia do not allow differentiation of the numerous species. The new molecular methods based on the 16S rRNA gene for Nocardia identification are crucial. Besides, the different species of Nocardia show species-specific drug susceptibility patterns, and patients are most frequently immunosuppressed and generally require antibiotic treatment. Finally, reports of isolates from clinical specimens of new species such as N. takedensis underline the need to provide clinical data to establish their significance in every patient but especially in patients with risk factors.     

Candida inconspicua and Candida norvegensis: New Insights into Identification in Relation to Sexual Reproduction and Genome Organization

Candida inconspicua and Candida (Pichia) norvegensis are two emerging pathogenic species that exhibit reduced susceptibility to azole derivatives. Conventional (biochemical) approaches do not readily differentiate between the two species. The first aim of this work was to analyze the performance of biochemical, proteomic (matrix-assisted laser desorption ionization–time of flight [MALDI-TOF]), and molecular approaches in the precise identification of these species. These results then led us to sequence 3 genomic loci, i.e., the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA), the D1/D2 domain of the 28S rDNA, and the elongation factor 1α (EF-1α) gene, either directly or following cloning, of 13 clinical isolates and 9 reference strains belonging to the 5 species included in the Pichia cactophila clade, namely, Pichia cactophila, Pichia insulana, C. inconspicua, C. norvegensis, and P. pseudocactophila. Finally, isolates of C. inconspicua were challenged for sexual reproduction on the appropriate medium. Our results show that EF-1α sequencing and proteic profiling by MALDI-TOF are the two most efficient approaches to distinguish between C. norvegensis and C. inconspicua. As a characteristic of the P. cactophila clade, we found multiple alleles of the rDNA regions in certain strains belonging to the tested species, making ITS or D1/D2 sequencing not appropriate for identification. Whatever the method of identification, including MALDI-TOF and EF-1α sequencing, none could differentiate C. inconspicua from P. cactophila. The results of phylogenetic analysis and the generation of asci from pure cultures of all C. inconspicua strains both support the identification of P. cactophila as the teleomorph of C. inconspicua.     

Clinical Spectrum of Exophiala Infections and a Novel Exophiala Species,Exophiala hongkongensis

We characterized 12 Exophiala strains isolated from patients over a 15-year period to the species level using phenotypic tests and internal transcribed spacer (ITS) and Rpb1 sequencing and described the clinical spectrum of the 12 patients. Eight patients had nail or skin infections, two had invasive infections, and two had colonization of the gastrointestinal tract. ITS and Rpb1 sequencing showed that 11 of the 12 strains were known Exophiala species (E. oligosperma [n = 3], E. jeanselmei [n = 2], E. lecanii-corni [n = 2], E. bergeri [n = 1], E. cancerae [n = 1], E. dermatitidis [n = 1], and E. xenobiotica [n = 1]), which included the first reported cases of onychomycosis caused by E. bergeri and E. oligosperma. The 12th strain (HKU32^T), isolated from the nail clipping of the right big toe of a 68-year-old female patient with onychomycosis, possessed unique morphological characteristics distinct from other Exophiala species. It grew very slowly and had a velvety colony texture after 28 days, short conidiophores of the same olivaceous color as the supporting hyphae, numerous spores, and no chlamydospore-like cells. ITS, Rpb1, β-tubulin, and β-actin gene sequencing unambiguously showed that HKU32^T was clustered with but formed branches distinct from other Exophiala species in phylogenetic trees. We propose the new species Exophiala hongkongensis to describe this novel fungus.     

Inducible β-Lactam Resistance in Aeromonas hydrophila: Therapeutic Challenge for Antimicrobial Therapy

Despite the abundant amount of knowledge about inducible chromosomally mediated β-lactamases among Aeromonas species, extended-spectrum β-lactam-resistant A. hydrophila strains selected in clinical practice were rarely reported. In the present study, two strains of A. hydrophila, A136 and A139, with markedly different susceptibilities to extended-spectrum cephalosporins were isolated from blood and the tip segment of an arterial catheter of a burn patient. Another strain (A136m) was selected in vitro by culturing A136 in a subinhibitory concentration of cefotaxime, the β-lactam agent administered for the treatment of Aeromonas bacteremia in this patient. Typing studies by arbitrarily primed PCR and pulsed-field gel electrophoresis indicated a clonal relationship among strains A136, A136m, and A139. These strains were identified to be of DNA hybridization group 1. Wild-type strain A136 was resistant only to ampicillin and cephamycins, but A136m and A139 were highly resistant to the expanded- and broad-spectrum cephalosporins. The presence of increased β-lactamase activity in A139 suggests that A139 is a derepressed mutant which overexpresses β-lactamases. These results call attention to the use of β-lactam agents for the treatment of invasive Aeromonas infections.     

Integrins αvβ3 and α5β1 Mediate Attachment of Lyme Disease Spirochetes to Human Cells

Borrelia burgdorferi (sensu lato), the agent of Lyme disease, is able to cause chronic, multisystemic infections in human and animal hosts. Attachment of the spirochete to host cells is likely to be important for the colonization of diverse tissues. The platelet-specific integrin α_IIbβ₃ was previously identified as a receptor for all three species of Lyme disease spirochetes (B. burgdorferi sensu stricto, B. garinii, and B. afzelii). Here we show that B. burgdorferi also recognizes the widely expressed integrins α_vβ₃ and α₅β₁, known as the vitronectin and fibronectin receptors, respectively. Three representatives of each species of Lyme disease spirochete were tested for the ability to bind to purified α_vβ₃ and α₅β₁. All of the strains tested bound to at least one integrin. Binding to one integrin was not always predictive of binding to other integrins, and several different integrin preference profiles were identified. Attachment of the infectious B. burgdorferi strain N40 to purified α_vβ₃ and α₅β₁ was inhibited by RGD peptides and the appropriate receptor-specific antibodies. Binding to α_vβ₃ was also shown by using a transfected cell line that expresses this receptor but not α_IIbβ₃. Attachment of B. burgdorferi N40 to human erythroleukemia cells and to human saphenous vein endothelial cells was mediated by both α₅β₁ and α_vβ₃. Our results show that multiple integrins mediate attachment of Lyme disease spirochetes to host cells.     

secA1 Gene Sequence Polymorphisms for Species Identification of Nocardia Species and Recognition of Intraspecies Genetic Diversity

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.Nocardia spp. are Gram-positive saprophytic bacteria capable of causing suppurative infections, including pulmonary, cutaneous, central nervous system, and disseminated diseases. To date, approximately 90 species have been described (NCBI taxonomy for Nocardia [http://www.ncbi.nlm.nih.gov/Taxonomy/]; http://www.bacterio.cict.fr/n/nocardia.html), at least 33 of which have been implicated in human disease (2). Identification of clinical isolates to the species level is important to characterize associated disease manifestations and to predict antimicrobial susceptibility and for epidemiological and ecological purposes (2, 17).Because of the difficulty of identifying Nocardia isolates by standard phenotypically based methods and the inability of such methods to identify novel species (2, 17), various nucleic acid amplification methods targeting conserved Nocardia gene regions have been proposed to provide accurate species determination. Of these, sequence analysis of the 16S rRNA gene has become the gold standard for definitive species identification (2, 5, 6, 8, 19). Certain closely related species, however, may not be distinguished by this method due to insufficient interspecies polymorphisms within the 16S rRNA gene sequences (2, 5, 14). Other practical limitations include potential misidentifications as a result of multiple but different copies of the 16S rRNA gene in species such as Nocardia nova (7, 9) and/or the presence of intraspecies 16S rRNA gene sequence polymorphisms (or “sequence types” [STs]) in N. nova, Nocardia cyriacigeorgica, and other species (14, 21).As such, the continuing evaluation of alternate gene targets to facilitate species identification is important. Sequence polymorphisms within the Nocardia 65-kDa heat shock protein (hsp65), essential secretory protein SecA1 (secA1), gyrase B (gyrB), and 16S-23S rRNA intergenic spacer (ITS) region genes have been reported to enable species level identification (10, 18, 22-24). In particular, sequence variability within a portion (470 bp) of the secA1 gene locus (in conjunction with analysis of deduced amino acid sequences of the SecA1 protein) has shown promise in recognizing and discriminating between the major Nocardia spp. (10). However, data on the application of secA1 gene sequencing in the clinical microbiology laboratory for the identification of Nocardia isolates are few. In one report, reference (n = 30 species), and clinical Nocardia isolates were correctly identified by secA1 gene sequencing (10); in the only other published study, this approach assisted with identification of a novel Nocardia species from soil (16). Evaluation of larger numbers of clinical isolates is essential for establishing a robust repository of secA1 gene sequences.Our laboratory, which provides regional microbiology services to a large number of health care institutions, has undertaken routine species identification by partial (5′-end 606-bp) 16S rRNA gene sequencing of Nocardia isolates since 2005. In the course of evaluating this approach to providing species identification, we identified significant intraspecies sequence heterogeneity within certain species, such as N. nova and Nocardia brasiliensis (14), highlighting the need to recognize species-specific sequence-based genetic types, or sequence types. Here, to explore the potential of sequence analysis of the secA1 gene as an adjunct to, or a possible substitute for, 16S rRNA gene sequencing, we performed species identification of 120 Nocardia reference and clinical isolates representing the 16 most clinically relevant species by secA1 gene sequence analysis and compared the results with 5′-end 606-bp 16S rRNA gene sequencing. We also report on the genetic diversity of the Nocardia secA1 gene.     

The GraS Sensor in Staphylococcus aureus Mediates Resistance to Host Defense Peptides Differing in Mechanisms of Action

Staphylococcus aureus uses the two-component regulatory system GraRS to sense and respond to host defense peptides (HDPs). However, the mechanistic impact of GraS or its extracellular sensing loop (EL) on HDP resistance is essentially unexplored. Strains with null mutations in the GraS holoprotein (ΔgraS) or its EL (ΔEL) were compared for mechanisms of resistance to HDPs of relevant immune sources: neutrophil α-defensin (human neutrophil peptide 1 [hNP-1]), cutaneous β-defensin (human β-defensin 2 [hBD-2]), or the platelet kinocidin congener RP-1. Actions studied by flow cytometry included energetics (ENR); membrane permeabilization (PRM); annexin V binding (ANX), and cell death protease activation (CDP). Assay conditions simulated bloodstream (pH 7.5) or phagolysosomal (pH 5.5) pH contexts. S. aureus strains were more susceptible to HDPs at pH 7.5 than at pH 5.5, and each HDP exerted a distinct effect signature. The impacts of ΔgraS and ΔΕL on HDP resistance were peptide and pH dependent. Both mutants exhibited defects in ANX response to hNP-1 or hBD-2 at pH 7.5, but only hNP-1 did so at pH 5.5. Both mutants exhibited hyper-PRM, -ANX, and -CDP responses to RP-1 at both pHs and hypo-ENR at pH 5.5. The actions correlated with ΔgraS or ΔΕL hypersusceptibility to hNP-1 or RP-1 (but not hBD-2) at pH 7.5 and to all study HDPs at pH 5.5. An exogenous EL mimic protected mutant strains from hNP-1 and hBD-2 but not RP-1, indicating that GraS and its EL play nonredundant roles in S. aureus survival responses to specific HDPs. These findings suggest that GraS mediates specific resistance countermeasures to HDPs in immune contexts that are highly relevant to S. aureus pathogenesis in humans.     

Helcococcus ovis, an emerging pathogen in bovine valvular endocarditis

The initial isolation of Helcococcus ovis from a valvular thrombus prompted us to investigate the prevalence of this bacterium in bovine valvular endocarditis. Specimens from 55 affected hearts were examined by culture using Columbia blood agar and cross streaking the inoculated plate with a Staphylococcus aureus strain. As confirmed by 16S rRNA gene sequencing, H. ovis was isolated with an unexpectedly high frequency of 33%, predominantly as heavy growth and pure culture. The majority of H. ovis isolates showed distinct satellitism around S. aureus and pyridoxal dependency, resembling “nutritionally variant streptococci” (now assigned to the genera Abiotrophia and Granulicatella). Using the API rapid ID 32 Strep, API ZYM, and Rosco Diatabs systems, incongruent results were obtained for alkaline phosphatase, β-galactosidase, β-glucuronidase, and leucine aminopeptidase activities. Based on the satellitism/pyridoxal dependency; hemolysis on blood agar; the API rapid ID 32 Strep results for arginine dihydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, and pyroglutamic acid arylamidase activities; hippurate hydrolysis; and acidification of sucrose, a scheme for the identification of H. ovis and its differentiation from other members of the Helcococcus genus and the pyridoxal-dependent species Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans is proposed. By establishing specific fluorescence in situ hybridization, large H. ovis aggregates were specifically detected within the fibrinous exudate of the valvular thrombi. Our results demonstrate for the first time that H. ovis represents an emerging pathogen in bovine valvular endocarditis that is frequently isolated if appropriate culture conditions are used.     

Can Results Obtained with Commercially Available MicroScan Microdilution Panels Serve as an Indicator of β-Lactamase Production among Escherichia coli and Klebsiella Isolates with Hidden Resistance to ExpandedSpectrum Cephalosporins and Aztreonam?

Among clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca, there is an ever-increasing prevalence of β-lactamases that may confer resistance to newer β-lactam antibiotics that is not detectable by conventional procedures. Therefore, 75 isolates of these species producing well-characterized β-lactamases were studied using two MicroScan conventional microdilution panels, Gram Negative Urine MIC 7 (NU7) and Gram Negative MIC Plus 2 (N+2), to determine if results could be utilized to provide an accurate indication of β-lactamase production in the absence of frank resistance to expanded-spectrum cephalosporins and aztreonam. The enzymes studied included Bush groups 1 (AmpC), 2b (TEM-1, TEM-2, and SHV-1), 2be (extended spectrum β-lactamases [ESBLs] and K1), and 2br, alone and in various combinations. In tests with E. coli and K. pneumoniae and the NU7 panel, cefpodoxime MICs of ≥2 μg/ml were obtained only for isolates producing ESBLs or AmpC β-lactamases. Cefoxitin MICs of >16 μg/ml were obtained for all strains producing AmpC β-lactamase and only 1 of 33 strains producing ESBLs. For the N+2 panel, ceftazidime MICs of ≥4 μg/ml correctly identified 90% of ESBL producers and 100% of AmpC producers among isolates of E. coli and K. pneumoniae. Cefotetan MICs of ≥ 8 μg/ml were obtained for seven of eight producers of AmpC β-lactamase and no ESBL producers. For tests performed with either panel and isolates of K. oxytoca, MICs of ceftazidime, cefotaxime, and ceftizoxime were elevated for strains producing ESBLs, while ceftriaxone and aztreonam MICs separated low-level K1 from high-level K1 producers within this species. These results suggest that microdilution panels can be used by clinical laboratories as an indicator of certain β-lactamases that may produce hidden but clinically significant resistance among isolates of E. coli, K. pneumoniae, and K. oxytoca. Although it may not always be possible to differentiate between strains that produce ESBLs and those that produce AmpC, this differentiation is not critical since therapeutic options for patients infected with such organisms are similarly limited.     

A PCR-Based Intergenic Spacer Region-Capillary Gel Electrophoresis Typing Method for Identification and Subtyping of Nocardia Species

While 16S rRNA sequence-based identification of Nocardia species has become the gold standard, it is not without its limitations. We evaluated a novel approach encompassing the amplification of the Nocardia 16S-23S rRNA intergenic spacer (IGS) region followed by fragment analysis by capillary gel electrophoresis (CGE) of the amplified product for species identification of Nocardia. One hundred forty-five Nocardia isolates (19 species) and four non-Nocardia aerobic actinomycetes were studied. Reproducibility testing was performed in a subset (21%) of isolates. Ninety-five different electropherograms were identified, with heterogeneity within species being a general observation. Among common Nocardia species (e.g., Nocardia cyriacigeorgica, N. nova, N. farcinica), 2 or 3 dominant electropherogram subgroups were typical. While only a minority (8/19; 42%) of the different Nocardia species contained isolates displaying unique fragment sizes that were predictive of a particular species, virtually all isolates (142/145; 98%) could be assigned to the correct species using IGS-CGE typing based on the number and size of amplified fragments. The median number of fragments for each isolate was 2 (range, 1 to 5) with only a minority (17%) having a single fragment detected. The majority (93%) of amplified fragments were between 408 and 461 bp. The technique was also non-operator dependent, highly reproducible, and quicker and less expensive than 16S sequencing. In summary, PCR-based IGS-CGE typing is relatively simple, accurate, reproducible, and cost-effective and offers a potential alternative to 16S rRNA sequencing for identifying and subtyping Nocardia isolates.     

Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Nocardia Species

The identification of Nocardia species, usually based on biochemical tests together with phenotypic in vitro susceptibility and resistance patterns, is a difficult and lengthy process owing to the slow growth and limited reactivity of these bacteria. In this study, a panel of 153 clinical and reference strains of Nocardia spp., altogether representing 19 different species, were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). As reference methods for species identification, full-length 16S rRNA gene sequencing and phenotypical biochemical and enzymatic tests were used. In a first step, a complementary homemade reference database was established by the analysis of 110 Nocardia isolates (pretreated with 30 min of boiling and extraction) in the MALDI BioTyper software according to the manufacturer''s recommendations for microflex measurement (Bruker Daltonik GmbH, Leipzig, Germany), generating a dendrogram with species-specific cluster patterns. In a second step, the MALDI BioTyper database and the generated database were challenged with 43 blind-coded clinical isolates of Nocardia spp. Following addition of the homemade database in the BioTyper software, MALDI-TOF MS provided reliable identification to the species level for five species of which more than a single isolate was analyzed. Correct identification was achieved for 38 of the 43 isolates (88%), including 34 strains identified to the species level and 4 strains identified to the genus level according to the manufacturer''s log score specifications. These data suggest that MALDI-TOF MS has potential for use as a rapid (<1 h) and reliable method for the identification of Nocardia species without any substantial costs for consumables.Nocardia spp. are ubiquitous bacteria dispersed in vegetation, dust, soil, freshwater, and salt water that are isolated with increasing frequency from clinical specimens, especially those from immunocompromised patients (1).The taxonomy of the genus Nocardia has undergone major changes during the last decades, and currently more than 50 species have been characterized by phenotypic and molecular methods, besides a number of unnamed genomospecies (2). Not all of them have been found in humans, and Nocardia asteroides, previously considered the species most frequently isolated from clinical specimens, has been shown to be heterogeneous and has been divided into several species (2). Moreover, several additional species of human origin have been recently described and reported (5, 6).The routine identification of Nocardia strains to the species level by conventional phenotypical methods is a fastidious and time-consuming process owing to the limited biochemical reactivity of these organisms, often requiring 1 or more days to complete identification. Moreover, the available tests may be difficult to interpret and inconclusive and require dedicated trained staff. In order to overcome these drawbacks, molecular methods such as 16S rRNA gene sequencing and PCR-restriction fragment length polymorphism analysis of both the 65-kDa heat shock protein-encoding gene (hsp65) and the 16S rRNA gene have been recently advocated for Nocardia species identification (3). However, these methods remain accessible to reference laboratories only and are difficult to implement for routine bacterial identification in a clinical laboratory.Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can analyze the protein composition of a bacterial cell and has emerged as a new technology for species identification. By measuring the exact sizes of peptides and small proteins, which are assumed to be characteristic for each bacterial species, it allows determination to the species level within a few minutes when the analysis is performed on whole cells, cell lysates, or crude bacterial extracts (8). Through the improvement of the technique, MALDI-TOF MS has proved over the recent years to be a rapid, accurate, easy-to-use, and inexpensive method for the universal identification of microorganisms (11). Until now, MALDI-TOF MS has been challenged for the identification of various groups of microorganisms, including Gram-positive bacteria, Enterobacteriaceae, Gram-negative nonfermenters, mycobacteria, anaerobes, and yeasts (8, 9, 11-13). In this respect, the use of MALDI-TOF MS as a tool for the identification of fastidious, slow-growing organisms such as Nocardia species, which are notoriously difficult to identify by conventional tests, in the routine laboratory appeared to us of major interest. One factor limiting the use of MALDI-TOF MS remains the limited availability of reference data sets for microorganisms that are infrequently isolated from clinical specimens, and it has been shown previously that the absence or the availability of only a small number of isolates of a given species in the reference database may account for most of the cases in which no identification can be obtained by the MALDI-TOF MS method (11).In this study, we therefore aimed to establish a large reference database for the MALDI-TOF MS-based identification of Nocardia species isolates. In a first step, we developed a simple modified extraction procedure based on boiling for 30 min, followed by ethanol-formic acid extraction, and we generated our own spectrum database issued from a large collection of clinical and reference Nocardia sp. isolates. Following the establishment of our reference database, we subsequently evaluated the methodology against 43 blind-coded clinical isolates of Nocardia species that were analyzed by phenotypical, biochemical, and enzymatic tests and by full-length 16S rRNA gene sequencing, which was used as the reference identification method.     

Evaluation of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Rapid Detection of β-Lactam Resistance in Enterobacteriaceae Derived from Blood Cultures

The identification of pathogens directly from blood cultures by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) can be a valuable tool for improving the treatment of patients with sepsis and bacteremia. However, the increasing incidence of multidrug-resistant Gram-negative bacteria makes it difficult to predict resistance patterns based only on pathogen identification. Most therapy regimens for sepsis caused by Gram-negative rods consist of at least one β-lactam antibiotic. Thus, it would be of great benefit to have an early marker of resistance against these drugs. In the current study, we tested 100 consecutive blood cultures containing Enterobacteriaceae for resistance against 3rd-generation cephalosporins in a MALDI-TOF MS β-lactamase assay. Escherichia coli was also tested for resistance against aminopenicillins. The results of the β-lactamase assay were compared with those of conventional methods. The assay permitted discrimination between E. coli strains that were resistant or susceptible to aminopenicillins with a sensitivity and a specificity of 100%. The same was true for resistance to 3rd-generation cephalosporins in Enterobacteriaceae that constitutively produced class C β-lactamases. Discrimination was more difficult in species expressing class A β-lactamases, as these enzymes can generate false-positive results. Thus, the sensitivity and specificity for this group were 100% and 91.5%, respectively. The test permitted the prediction of resistance within 2.5 h after the blood culture was flagged as positive.     

High Frequency of Acinetobacter soli among Acinetobacter Isolates Causing Bacteremia at a Tertiary Hospital in Japan

Acinetobacter baumannii is generally the most frequently isolated Acinetobacter species. Sequence analysis techniques allow reliable identification of Acinetobacter isolates at the species level. Forty-eight clinical isolates of Acinetobacter spp. were obtained from blood cultures at Tohoku University Hospital. These isolates were identified at the species level by partial sequencing of the RNA polymerase β-subunit (rpoB), 16S rRNA, and gyrB genes. Then further characterization was done by using the PCR for detection of OXA-type β-lactamase gene clusters, metallo-β-lactamases, and carO genes. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing were also performed. The most frequent isolate was Acinetobacter soli (27.1%). Six of the 13 A. soli isolates were carbapenem nonsusceptible, and all of these isolates produced IMP-1. PFGE revealed that the 13 A. soli isolates were divided into 8 clusters. This study demonstrated that A. soli accounted for a high proportion of Acinetobacter isolates causing bacteremia at a Japanese tertiary hospital. Non-A. baumannii species were identified more frequently than A. baumannii and carbapenem-nonsusceptible isolates were found among the non-A. baumannii strains. These results emphasize the importance of performing epidemiological investigations of Acinetobacter species.     

Rapid Identification of Clinically Relevant Nocardia Species to Genus Level by 16S rRNA Gene PCR

Two regions of the gene coding for 16S rRNA in Nocardia species were selected as genus-specific primer sequences for a PCR assay. The PCR protocol was tested with 60 strains of clinically relevant Nocardia isolates and type strains. It gave positive results for all strains tested. Conversely, the PCR assay was negative for all tested species belonging to the most closely related genera, including Dietzia, Gordona, Mycobacterium, Rhodococcus, Streptomyces, and Tsukamurella. Besides, unlike the latter group of isolates, all Nocardia strains exhibited one MlnI recognition site but no SacI restriction site. This assay offers a specific and rapid alternative to chemotaxonomic methods for the identification of Nocardia spp. isolated from pathogenic samples.     

Phylogeny and Identification of Nocardia Species on the Basis of Multilocus Sequence Analysis

Nocardia species identification is difficult due to a complex and rapidly changing taxonomy, the failure of 16S rRNA and cellular fatty acid analysis to discriminate many species, and the unreliability of biochemical testing. Here, Nocardia species identification was achieved through multilocus sequence analysis (MLSA) of gyrase B of the β subunit of DNA topoisomerase (gyrB), 16S rRNA (16S), subunit A of SecA preprotein translocase (secA1), the 65-kDa heat shock protein (hsp65), and RNA polymerase (rpoB) applied to 190 clinical, 36 type, and 11 reference strains. Phylogenetic analysis resolved 30 sequence clusters with high (>85%) bootstrap support. Since most clusters contained a single type strain and the analysis corroborated current knowledge of Nocardia taxonomy, the sequence clusters were equated with species clusters and MLSA was deemed appropriate for species identification. By comparison, single-locus analysis was inadequate because it failed to resolve species clusters, partly due to the presence of foreign alleles in 22.1% of isolates. While MLSA identified the species of the majority (71.3%) of strains, it also identified clusters that may correspond to new species. The correlation of the identities by MLSA with those determined on the basis of microscopic examination, biochemical testing, and fatty acid analysis was 95%; however, MLSA was more discriminatory. Nocardia cyriacigeorgica (21.58%) and N. farcinica (14.74%) were the most frequently encountered species among clinical isolates. In summary, five-locus MLSA is a reliable method of elucidating taxonomic data to inform Nocardia species identification; however, three-locus (gyrB-16S-secA1) or four-locus (gyrB-16S-secA1-hsp65) MLSA was nearly as reliable, correctly identifying 98.5% and 99.5% of isolates, respectively, and would be more feasible for routine use in a clinical reference microbiology laboratory.As part of the aerobic actinomycetes, Nocardia is a group of filamentous branching bacilli that are characteristically Gram positive and modified acid fast. Although Nocardia species normally exist as soil saprophytes, they have increasingly been isolated as infectious agents in immunosuppressed patients and, in some cases, even healthy individuals. Infections range from pulmonary nocardiosis, characterized by necrotizing pneumonia, to cutaneous nocardiosis and even brain abscess (25).For nearly a century, since its inception in 1888 by Edmund Nocard, the genus Nocardia comprised only about a dozen species (26), largely because the somewhat biochemically inert nature of the group inhibited characterization (6). However, in 1988, Wallace et al. (38) uncovered latent diversity when they described six antimicrobial susceptibility pattern types among clinical isolates. DNA (e.g., 16S rRNA [16S] gene) sequencing confirmed and further expanded knowledge of the genetic diversity within the genus (6, 22). To date, the National Center for Biotechnology Information (NCBI) lists 86 recognized species (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi). However, the species differ in their abilities to cause human infection and their responses to antimicrobials (2, 6, 21, 25, 27, 33, 38). For this reason, species identification of Nocardia isolates from clinical specimens is relevant to patient treatment and provides important epidemiological information.Beyond Gram and modified-acid-fast staining, species identification of Nocardia relies heavily on biochemical tests and cellular fatty acid analysis, which are cumbersome, time-consuming, and not definitive. Various molecular identification schemes investigated to date represent promising alternatives (7, 10, 29, 32, 36). However, 16S rRNA gene sequencing, considered to be the “gold standard” for bacterial identification, fails to discriminate many species (7), and the reliability of identification methods on the basis of the DNA sequence of a single housekeeping gene suffers from stochastic genetic variation and horizontal gene transfer and recombination (12).Recently, multilocus sequence analysis (MLSA) has been suggested as a method to examine prokaryotic taxonomy. From phylogenetic analysis of a concatenated sequence typically consisting of 5 to 7 housekeeping genes, MLSA assigns a species designation on the basis of the assumption that sequence clusters represent species clusters (12). MLSA has been employed to identify the species of a number of genera with very promising results (1, 4, 5, 11, 14, 15, 16, 18, 20, 24, 28, 40). Furthermore, because of its ease of use, accuracy, and discriminatory power, MLSA may soon surpass DNA-DNA hybridization (DDH) as the gold standard for the investigation of prokaryotic taxonomy, species identification, and determination of genetic diversity (34).The purpose of this study was to develop an MLSA scheme for the species identification of Nocardia clinical isolates. Through phylogenetic analysis of concatenated sequences consisting of partial fragments of gyrase B, the β subunit of a type II DNA topoisomerase (gyrB), 16S, subunit A of the SecA preprotein translocase (secA1), the 65-kDa heat shock protein (hsp65), and RNA polymerase (rpoB) genes, we delineated 30 species clusters. Since most clusters contained a single type strain, the results correlated well with existing knowledge of Nocardia taxonomy and provided a means of species assignment for the clinical isolates on the basis of strain placement within the phylogenetic analysis. Furthermore, the MLSA identifications were consistent with, although more discriminatory than, species assignments based on traditional microscopic evaluation, biochemical testing, and cellular fatty acid analysis. We present MLSA as a practical tool for routine Nocardia species identification in a clinical reference microbiology laboratory.     

Beta-galactosidase and lactose fermentation in the identification of enterobacteria including salmonellae

One hundred and fourteen strains of non-lactose fermenters and 127 lactose fermenters on MacConkey's agar have been compared in the 5% and 1% lactose tests and in β-galactosidase production, using ortho-nitro-phenyl-β-D-galactopyranoside (O.N.P.G.) as a test substance. The superiority of the O.N.P.G. test in the number of positive results and its rapidity is shown. In general, late or non-lactose fermenting strains of genera, usually lactose-positive, yield a rapidly positive O.N.P.G. reaction. Forty-one wild strains of Salmonella, Proteus, Providencia, and Pseudomonas aeruginosa were found negative in all three tests.     

Identification of Nocardia species from clinical isolates using MALDI-TOF mass spectrometry

The identification of Nocardia isolates still represents a challenge for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) despite its acceptance for most bacterial and fungal isolates. In this study we evaluate the identification of Nocardia isolates using direct spotting and an updated database.Overall, 82 Nocardia isolates belonging to 13 species were identified by DNA sequence analysis of the 16S rRNA and secA1 genes. Nine of these well-characterized isolates from 6 Nocardia species were used to create an in-house library. The remaining 73 isolates were directly spotted on the target plate and on-plate protein extraction was performed. The protein spectra obtained were analyzed by MALDI-TOF MS using the BDAL database (Bruker Daltonics) updated with 6,903 MSPs or the combination of this commercial database and our in-house library.As a result, the use of the commercial database alone and in combination with the in-house library yielded 94.5% and 95.9% of correct species-level identifications, respectively, No isolate was misidentified at the genus level with either database. Besides, the use of the in-house library allowed the species-level identification of a N. otitidiscaviarum isolate that could only be identified at the genus-level with a score value <1.6 using the commercial database.In conclusion, the implementation of the direct spotting method and the in-house database provided a high rate of correct species assignment of Nocardia isolates despite the low number of isolates added. Further addition of well-characterized Nocardia isolates may ensure the rapid, accurate and inexpensive identification of most isolates encountered in the routine of the microbiology laboratory.     

Molecular Characterization of Nonhemolytic and Nonpigmented Group B Streptococci Responsible for Human Invasive Infections

Group B Streptococcus (GBS) is a common commensal bacterium in adults, but is also the leading cause of invasive bacterial infections in neonates in developed countries. The β-hemolysin/cytolysin (β-h/c), which is always associated with the production of an orange-to-red pigment, is a major virulence factor that is also used for GBS diagnosis. A collection of 1,776 independent clinical GBS strains isolated in France between 2006 and 2013 was evaluated on specific medium for β-h/c activity and pigment production. The genomic sequences of nonhemolytic and nonpigmented (NH/NP) strains were analyzed to identify the molecular basis of this phenotype. Gene deletions or complementations were carried out to confirm the genotype-phenotype association. Sixty-three GBS strains (3.5%) were NH/NP, and 47 of these (74.6%) originated from invasive infections, including bacteremia and meningitis, in neonates or adults. The mutations are localized predominantly in the cyl operon, encoding the β-h/c pigment biosynthetic pathway and, in the abx1 gene, encoding a CovSR regulator partner. In conclusion, although usually associated with GBS virulence, β-h/c pigment production is not absolutely required to cause human invasive infections. Caution should therefore be taken in the use of hemolysis and pigmentation as criteria for GBS diagnosis in routine clinical laboratory settings.     

Effects of Activated Macrophages on Nocardia asteroides

The mechanism(s) of host resistance against Nocardia asteroides has not been well defined. Since disease due to N. asteroides frequently occurs in patients with impaired cell-mediated immunity, we studied the interaction of N. asteroides with activated and control mouse peritoneal macrophages. Activated macrophages were from mice infected with Toxoplasma gondii or injected with Corynebacterium parvum. N. asteroides in the early stationary phase (>99% in the coccobacillary form) was used for challenge of macrophage monolayers. Growth of two strains of N. asteroides was markedly inhibited in activated macrophages, whereas N. asteroides grew well in control macrophages. Quantitation of macrophage-associated N. asteroides indicated that activated macrophages killed 40 to 50% of N. asteroides within 6 h (P < 0.002). In control macrophage preparations, it appeared as if Nocardia filaments extended from within macrophages to the outside, and many of these filaments appeared to have extended to and then grown through neighboring macrophages. In activated macrophage preparations, Nocardia remained in the coccobacillary form in most macrophages. Control macrophage monolayers were almost completely overgrown with and destroyed by Nocardia 20 h after challenge, whereas activated macrophage monolayers remained intact. Nocardia that grew in control macrophages were not acid-alcohol fast or only weakly so, whereas the few Nocardia that grew in activated macrophages were strongly acid-alcohol fast. Our results indicate that activated macrophages may be important in host defense against N. asteroides.