www.nature.com/scientificreports OPEN Identifcation and antimicrobial susceptibility profles of Nocardia species clinically isolated in Japan Masahiro Toyokawa 1,2,4*, Noboru Ohana2, Akiko Ueda3, Minako Imai4, Daiki Tanno2,4, Mutsuko Honda4, Yukiko Takano4, Kazutaka Ohashi4, Kyoichi Saito2 & Hiroki Shimura2,4 The aims of the present study were to profle the antimicrobial susceptibility patterns of a diverse range of Nocardia species isolated in Japan, and to determine the ability of matrix-assisted laser desorption ionization-time of fight mass spectrometry (MALDI-TOF MS) for species/complex identifcation. Identifcation of 153 clinical isolates was performed by full-length 16S rRNA gene sequencing as a reference method to evaluate the usefulness of MALDI-TOF MS identifcation. Antimicrobial susceptibility testing (AST) for 14 antibiotics was performed using the broth microdilution method against 146 of the isolates. Among the total 153 clinical isolates, Nocardia farcinica complex (25%) was the most common species, followed by Nocardia cyriacigeorgica (18%), Nocardia brasiliensis (9%), Nocardia nova (8%), and Nocardia otitidiscaviarum (7%). Among 150 isolates identifed to the species/complex level by 16S rRNA gene sequencing, MALDI-TOF MS with the use of a supplemental Nocardia library (JMLD library ver.ML01) correctly identifed 97.3% (n = 146) to the species/complex level and 1.3% (n = 2) to the genus level. Among the 146 Nocardia isolates that underwent AST, the susceptibilities were 100% to linezolid, 96% to amikacin, 94% to trimethoprim- sulfamethoxazole, and 76% to imipenem. None of the trimethoprim-sulfamethoxazole-resistant isolates carried either plasmid-mediated sulfonamide-resistant genes (sul1, sul2) or trimethoprim- resistant genes (dfrA). Nocardia species are ubiquitous environmental organisms that can cause local or disseminated infection in humans. Te lung is the most common primary site of infection, and central nervous system infection is ofen encountered through hematogenous dissemination from a pulmonary focus, particularly in immunocompro- mised hosts1. Prompt diagnosis and appropriate treatment of nocardiosis are required, because it is a fatal infection2. Trimethoprim-sulfamethoxazole (TMP-SMX) is the frst-line agent for initial therapy for nocardiosis3, thus accurate determination of the susceptibility to TMP-SMX in clinical isolates is crucial. Conville et al. reported that the standard broth microdilution method for TMP-SMX may cause false resistance because of difculties in end-point interpretation, and they recommend performing a confrmation test via sulfsoxazole disk difusion testing 4. Unfortunately we are unable to purchase sulfsoxazole disks in Japan; therefore there is no reliable data concerning Nocardia isolates resistant to TMP-SMX in Japan. On the other hands, identifcation of clinical isolates to the species/complex level is important, because Nocardia species difer in clinical spectrum and susceptibility patterns5–7. Gene sequencing such as 16S rRNA gene is currently used for the identifcation of Nocardia species; however, recently, matrix-assisted laser desorption ionization-time of fight mass spectrometry (MALDI-TOF MS)-based identifcation has been identifed as a rapid, easy and reliable method 8,9. Te aims of the present study were to profle the antimicrobial susceptibility patterns of a diverse range of Nocardia species isolated from clinical specimens in Japan, and to determine the utility of MALDI-TOF MS for the routine identifcation of Nocardia species. Materials and methods Bacterial isolates and identifcation. A total of 153 clinical isolates of Nocardia species recovered from patients in 25 microbiology laboratories in Japan were studied. Te isolates were cultured from respiratory tract specimens (n = 90), skin and sof tissue (n = 14), blood (n = 5), deep abscess (n = 6), pleural efusion (n = 1), ascitic 1Preparing section for New Faculty of Medical Science, Fukushima Medical University, 1 Hikariga-oka, Fukushima City 960-1295, Japan. 2Department of Laboratory Medicine, Fukushima Medical University, Fukushima, Japan. 3Laboratory for Clinical Investigation, Osaka University Hospital, Osaka, Japan. 4Department of Clinical Laboratory Medicine, Fukushima Medical University Hospital, Fukushima, Japan. *email: [email protected] Scientifc Reports | (2021) 11:16742 | https://doi.org/10.1038/s41598-021-95870-2 1 Vol.:(0123456789) www.nature.com/scientificreports/ fuid (n = 1), synovial fuid (n = 1), others (n = 5) and unknown (n = 30). Identifcation of Nocardia species was based on Gram stain, colonial morphology and molecular technique. All isolates were identifed by full-length 16S rRNA gene sequencing, for which the universal primers 8UA (5′-AGA GTT TGATCMTGG CTC AG-3′) and 1485B (5′-ACG GGC GGT GTG TRC-3′) were used, as described previously10. We performed sequencing analysis using a GenBank BLAST search and EzBioCloud (https:// www. ezbio cloud. net/ ident ify/ result? id= 5ef99 cb3c3 9ad46 1094e 9aa3). Previously established criteria for identifcation of Nocardia isolates to the species or complex level were followed11,12. MALDI-TOF MS identifcation. All Nocardia species isolates were analyzed using a Microfex LT bench top mass spectrometer (Bruker Daltonics, Germany). MALDI Biotyper 3.1 sofware (Bruker Daltonics, MALDI Biotyper reference library version 8.0.0.0) was applied with the use of a supplemental Nocardia library (JMLD library ver.ML01, containing 114 Main Spectras for 46 Nocardia species) provided by BCKK MALDINOMICS (Beckman Coulter Japan, Tokyo, Japan). Te isolates were cultivated on 5% sheep blood agar plates at 35℃, and tested at 18 and 48 h, an early stage of growth8. Samples were prepared as previously described (on-plate extrac- tion)8. Protein extraction was also performed using the formic acid/ethanol method according to the Bruker Daltonics’ protocol for any isolate failed to be identifed by on-plate extraction. A spectral score of ≥ 2.00 was considered identifcation to the species level, a score of 1.700–1.999 indicated identifcation at the genus level, and a score of < 1.70 was considered unreliable identifcation. Complex level identifcation was performed on some Nocardia species according to Conville’s criteria11. Nocardia asteroides ATCC 23206, Nocardia brasiliensis ATCC 23238, Nocardia farcinica ATCC 23157, and Nocardia otitidiscaviarum ATCC 23240 were used as the quality control strains. Antimicrobial susceptibility testing (AST). AST was performed using the broth microdilution method with frozen panels (Eiken Chemical, Tokyo, Japan), according to the Clinical and Laboratory Standards Institute (CLSI) M24-A2 guidelines13 against 146 clinical isolates. In brief, a heavy organism suspension was prepared in a small volume of sterile saline with 7–10 3-mm glass beads and was vortexed vigorously. Clumps were allowed to settle for 15 min, and the supernatant was adjusted to a 0.5 McFarland standard using a calibrated neph- elometer. For frozen panel inoculation, the adjusted 0.5 McFarland suspension was diluted 30-fold with sterile saline and 10 µl of the diluted solution was dispensed into each well of the panel. Te panels were incubated at 35℃ for 72 h until moderate growth was observed in the growth control wells. For TMP-SMX, the MICs were determined as the wells corresponding to 80% inhibition of growth compared to the controls. Te MICs were determined for TMP-SMX, amikacin, tobramycin, cefriaxone, imipenem, minocycline, linezolid, ciprofoxa- cin, moxifoxacin, clarithromycin, cefotaxime (100 isolates only), meropenem (100 isolates only), tigecycline (100 isolates only), and arbekacin (100 isolates only), and interpreted as recommended by CLSI. Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853 and Enterococcus faecalis ATCC 29212 were used as the quality control strains. For confrmation of TMP-SMX resistance, disk difusion testing with a 250-μg sulfsoxazole disk (Hardy Diagnostics, CA, USA)4 was performed in all 21 TMP-SMX-resistant isolates determined by AST (MIC ≥ 4/76 μg/ ml). Moreovere, re-analysis of the broth microdilution method using panels with diferent lots and inoculum colony count were also performed. For this analysis, Nocardia nova ATCC BAA-2227 and Escherichia coli ATCC 25922 were used as the quality control strains. Detection of plasmid-mediated TMP-SMX-resistant genes. Te plasmid-mediated sulfonamide- resistant genes (sul1, sul2)14 and trimethoprim-resistant gene (dfrA)15 were detected by PCR in 21 TMP-SMX- resistant isolates, determined by AST (MIC ≥ 4/76 μg/ml) (see Table S1 for the primer sequence and Figure S1 for a gel electrophoresis image of positive controls in the supplemental material). Te present study was conducted in accordance with the ethical guidelines of the Ministry of Health, Labor and Welfare of Japan. No ethical committee approvals or informed consent were needed for this study. Conference presentation. A part of this study was presented at the 27th European Congress of Clinical Microbiology and Infectious Diseases, 22 to 25 April 2017, Vienna, Austria [abstr. #316]. Results Identifcation of clinical isolates by full-length 16S rRNA gene sequencing. Among the 153 clin- ical isolates, 150 were identifed to the species/complex level, including 24 diferent species/complexes, and the remaining three isolates were identifed to the genus level (Table 1). Nocardia farcinica complex (n = 39; 25%) was the most common species, followed by Nocardia cyriacigeorgica (n = 27; 18%), Nocardia brasiliensis (n =