Identification and antimicrobial susceptibility profiles of Nocardia species clinically isolated in Japan

The aims of the present study were to profile 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 flight mass spectrometry (MALDI-TOF MS) for species/complex identification. Identification 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 identification. 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 identified 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 identified 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).

www.nature.com/scientificreports/ fluid (n = 1), synovial fluid (n = 1), others (n = 5) and unknown (n = 30). Identification of Nocardia species was based on Gram stain, colonial morphology and molecular technique. All isolates were identified 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 previously 10 . 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 identification of Nocardia isolates to the species or complex level were followed 11,12 . MALDI-TOF MS identification. All Nocardia species isolates were analyzed using a Microflex LT bench top mass spectrometer (Bruker Daltonics, Germany). MALDI Biotyper 3.1 software (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). The isolates were cultivated on 5% sheep blood agar plates at 35℃, and tested at 18 and 48 h, an early stage of growth 8 . Samples were prepared as previously described (on-plate extraction) 8 . Protein extraction was also performed using the formic acid/ethanol method according to the Bruker Daltonics' protocol for any isolate failed to be identified by on-plate extraction. A spectral score of ≥ 2.00 was considered identification to the species level, a score of 1.700-1.999 indicated identification at the genus level, and a score of < 1.70 was considered unreliable identification. Complex level identification was performed on some Nocardia species according to Conville's criteria 11 . 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 guidelines 13 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 nephelometer. 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. The 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. The MICs were determined for TMP-SMX, amikacin, tobramycin, ceftriaxone, imipenem, minocycline, linezolid, ciprofloxacin, moxifloxacin, 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 confirmation of TMP-SMX resistance, disk diffusion testing with a 250-μg sulfisoxazole 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 different 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. The plasmid-mediated sulfonamideresistant genes (sul1, sul2) 14 and trimethoprim-resistant gene (dfrA) 15 were detected by PCR in 21 TMP-SMXresistant 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).
The 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.
N. cyriacigeorgica was the most frequently isolated Nocardia species from the respiratory tract (28%; 25/90), followed by N. farcinica complex (21%; 19/90). N. brasiliensis was isolated in one-half (50%; 7/14) of the skin and soft tissue samples. Figure 1 shows the alignment of 1405 bases of the 16SrRNA gene of all clinical isolates of Nocardia with those of closely related species obtained using the neighbor-joining method 16,17 with MEGA X software 18 .
Antimicrobial susceptibility testing. The MIC range, MIC 50 , MIC 90 and susceptibility for the seven most frequently isolated Nocardia species/complexes are shown in Table 2. The antibiograms of the uncommon Nocardia species are listed in Table 3.
Overall, linezolid was the most active drug across all species, with no in vitro resistance. Among the 146 Nocardia isolates that underwent AST, 96% were susceptible to amikacin; 86% were susceptible to TMP-SMX; and 76% were susceptible to imipenem. In contrast, about 80% of the Nocardia isolates were not susceptible to clarithromycin, minocycline or ciprofloxacin. Six amikacin-resistant isolates were N. transvalensis complex. The results of disk diffusion testing with a 250-μg sulfisoxazole disk and re-analysis of the broth microdilution method against 21 TMP-SMX-resistant isolates are shown in Table 4. Of these 21 isolates, five were interpreted as being TMP-SMX-resistant, and 12 were susceptible, while four were not interpretable. Finally, 94% (137/146) of the Nocardia isolates were determined to be susceptible to TMP-SMX. The isolates that were not susceptible to TMP-SMX (including isolates not interpretable) were found among N. otitidiscaviarum (27%; 3/11), N. farcinica complex (8%; 3/37), N. cyriacigeorgica (4%; 1/27), N. thailandica (1/2) and N. mexicana (1/1).
Susceptibility varied according to Nocardia species. Among the frequently isolated species, nonsusceptibility to imipenem was high in N. otitidiscaviarum (100%) and N. brasiliensis (86%), while that was low in N. nova complex (0%), N. cyriacigeorgica (4%) and N. farcinica complex (5%). More than 80% susceptibility to ceftriaxone was shown in N. abscessus complex (89%), N. cyriacigeorgica (85%) and N. transvalensis complex (83%). N. nova complex showed a good susceptibility to clarithromycin, although its resistance rate in other frequently isolated species was high. Susceptibility to fluoroquinolones including moxifloxacin was low among all the Nocardia species except N. transvalensis complex and N. brasiliensis. Table 1. Comparison of full-length 16SrRNA gene sequencing and MALDI-TOF MS identification for 153 Nocardia spp. isolates using the manufactuer's library combined with a custom library. a 2/3 isolates were identified as N. testacea/N. sienata, and 1/3 isolates was identified as N. testacea/N. flavorosea/N. sienata/N. rhamnosiphila. b Two isolates were identified as N. araoensis with scores of 1.91 and 1.99, respectively. c misidentified as N. brasilliensis with a score of 2.033. N. abscessus complex  The evolutionary history was inferred using the Neighbor-Joining method 16 . The optimal tree is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method 17 and are in the units of the number of base substitutions per site. The proportion of sites where at least 1 unambiguous base is present in at least 1 sequence for each descendent clade is shown next to each internal node in the tree. This analysis involved 59 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1405 positions in the final dataset. Evolutionary analyses were conducted in MEGA X 18 . a The read length of N75 strain was 1023 bp with a good quality sequence.     N. thailandica (2) N. asteroides (1) N. takedensis (1) N. yamanashiensis (1) N. mexicana (1) N. vinacea (1   www.nature.com/scientificreports/ The cumulative percentages of the 100 Nocardia isolates inhibited by each concentration of arbekacin, amikacin and tobramycin are shown in Fig. 2. The MIC 50 and MIC 90 values of arbekacin, amikacin and tobramycin were 0.25 and 1, 1 and 4, and 4 and 128 μg/ml, respectively. Arbekacin showed low MIC values (4-16 μg/ml) even against N. transvalensis complex, which included high-level amikacin-resistant isolates (> 256 μg/ml).

Discussion
There is limited information about the distribution and antimicrobial susceptibility of various Nocardia species in Japan. In the present study, N. farcinica complex (25%) was the most common species, followed by N. cyriacigeorgica (18%), N. brasiliensis (9%), N. nova (8%), and N. otitidiscaviarum (7%), according to full-length 16S rRNA gene sequence identification. When using the complex criteria for MALDI-TOF MS identification 11 , N. farcinica complex (25%) remained the most predominant, but the next most dominant species were N. cyriacigeorgica and N. nova complex (18.3% each), followed by the N. abscessus complex (12%), and N. brasiliensis (9%). These epidemiological data, taken together with the antimicrobial susceptibility profiles of different species/complexes, may contribute to accurate empirical treatment decisions.
The current study demonstrates that MALDI-TOF MS is useful for rapidly and accurately providing species/complex identification of Nocardia species. The direct spotting and standard bacterial extraction methods developed for MALDI-TOF MS are suboptimal for Nocardia species, due to the hardness and composition of the cell wall 7 . Previous studies have stressed the need for enhanced sample preparation methods to sufficiently identify Nocardia species 9,19-21 . Khot et al. reported that the age of Nocardia cultures plays an important role in the success of MALDI-TOF MS identification, and recommended to use a colony at an early stage of growth 8 . Their method correctly identified 82.8% (72/87) to the species/complex level and 11.5% (10/87) to the genus level if the cut-off for species-level identification was lowered from a score of ≥ 2.00 to ≥ 1.90. The results of the current study indicate that our method is more reliable than Khot's method, despite the strict threshold value for species-level identification being used. The point of the method used in the present study was to use a colony at an early stage of growth (18-48 h cultivation), and to use a considerably augmented reference spectrum database created with well-characterized strains cultured in the same condition (18-48 h cultivation).
Our results indicate that TMP-SMX still has a good activity against Nocardia species isolated in Japan. TMP-SMX-resistant isolates were found among N. otitidiscaviarum, N. cyriacigeorgica and N. mexicana. The mechanism of resistance to TMP-SMX is being studied mainly in clinically important bacteria such as Escherichia coli and Salmonella species. It has been reported that acquisition of plasmid-mediated resistance genes (sul and dfr) and chromosomal gene mutations in the dhps and dhfr genes coding for the target enzymes dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR), respectively, is the major resistance mechanisms [22][23][24] in such bacteria. To the best of our knowledge, the mechanism of TMP-SMX resistance in Nocardia species has not yet been clarified, although a recent study reported that the acquisition of plasmid-mediated resistance genes is   26 . While their study did not include sequence data of clinically TMP-SMX-resistant Nocardia strains, they suggested that chromosomal gene mutations may be implicated in low-level TMP-SMX resistance identical to that of other bacterial species, such as E. coli 27 .
On the other hand, it is known that TMP-SMX therapy is strongly associated with the emergence of thymidine-dependent small colony variants (SCVs) in Staphylococcus species 28 . Underlying mutations have been identified for thymidine-dependent SCVs in S. aureus, and mutations of the thyA gene have been shown to be responsible for the SCV phenotype 29 . The SCVs have also been found in some clinically important bacteria such as Stenotrophomonas maltophilia, P. aeruginosa, E. coli, Salmonella species, and Enterococcus species 30,31 . The SCV phenotype is characterized by small colony size, slow growth on agar media compared to wild-type isolates, and the inability to generate in vitro susceptibility results under standard conditions, as defined by CLSI 30 . Unfortunately, to date there have been no reports on SCVs in Nocardia species. Mehta et al. reported that a point mutation was observed at 16 bp upstream of thyA, which is an operon with the DHFR gene (folA), in experimental de novo TMP-SMX-resistant Nocardia strains, although they did not investigate the relationship between the mutation and SCV phenotype 26 . Nocardia infections are not uncommon in immunosuppressed patients receiving TMP-SMX for prophylaxis 1,32 ; therefore, the existence of SCVs in Nocardia species cannot be denied. Further, TMP-SMX is frequently used not only for prophylaxis, but also for long-term treatment over 6 months 1 , so it is necessary to elucidate the resistance mechanisms, including chromosomal gene mutations and SCVs, and to develop an accurate detection method for TMP-SMX-resistant strains.
Tigecycline is the first in a new class of antimicrobials, a member of the glycylcyclines, and is an analogue of minocycline with additional properties that negate most mechanisms, mediating resistance to tetracyclines 33 . In vitro testing has revealed that tigecycline is active against Gram-positive cocci, including Enterococcus species, S. aureus and Streptococcus pneumoniae, and many species of multi-drug-resistant Gram-negative bacteria 33 . Lai et al. investigated 151 clinical isolates of Nocardia species, and reported that tigecycline had a low MIC 90 (1 μg/ ml), and that MIC values were ≤ 8 μg/ml against all of the tested isolates, suggesting the potential clinical application of tigecycline for the treatment of nocardiosis 34 . In the present study, tigecycline had a low MIC distribution only for N. brasiliensis, N. otitidiscaviarum and some clinically unusual Nocardia species. Some researchers have reported that N. farcinica complex, N. nova complex and N. transvalensis complex isolates were less susceptible to tigecycline than N. abscessus, N. brasiliensis, or N. otitidiscaviarum 35,36 . Further studies are needed to demonstrate the clinical role of tigecycline in the management of nocardiosis.
To our knowledge, the present study is the first to have evaluated the activity of arbekacin against a diverse range of Nocardia species. Arbekacin is a broad-spectrum aminoglycoside licensed for systemic use in Japan and Korea, where it is usually used to treat methicillin-resistant S. aureus infections 37-39 . Matsumoto et al. reported that arbekacin is stable against aminoglycoside-inactivating enzymes such as (3′) aminoglycoside-phosphotransferase, (4′) aminoglycoside-adenyltransferase (AAD), or AAD (2″) and has a weak affinity for (6′-IV) aminoglycoside-acetyltransferase 40,41 . Therefore, arbekacin has antimicrobial activity against Gram-positive and -negative pathogens, including strains resistant to gentamicin, tobramycin, and amikacin 40,42 . In this study, arbekacin was four-fold more active than amikacin, and showed low MIC values even against N. transvalensis complex, which is reported to be resistant to all aminoglycosides 43 . These results indicate that arbekacin has a good potential to be a concomitant antibiotic for empirical therapy or therapy for serious nocardiosis infections.
In conclusion, the current study demonstrated that MALDI-TOF MS is a quick, easy and reliable method for the species/complex identification of Nocardia species. Accurate identification by MALDI-TOF MS and antimicrobial susceptibility profiles together can help earlier implementation of appropriate antimicrobial treatment and improvement of patient prognosis.