Due to the large number of Nocardia species, biochemical methods are insufficient to identify the clinically relevant species . MALDI-TOF MS can rapidly identify the frequently encountered Nocardia species, but it is limited by the database and distinguishing closely related species . The 16S rRNA gene sequencing is robust for accurate identification of Nocardia species, but it is also limited by discriminating closely related species due to high conservation . The current study accurately identified the species by MLSA and determined the antimicrobial susceptibility by BMD method of clinically isolated Nocardia from three tertiary hospitals in China. The MLSA which is promising as the primary method in identification of prokaryotic species has powerful interspecies and intraspecies discrimination [14, 15, 27]. In our previous work, a fact has been proved by digital DNA-DNA hybridization analysis that three-locus MLSA is superior to five-locus MLSA for Nocardia species identification . Therefore, three-locus MLSA was carried out in this study.
The geographical distribution of the Nocardia species has unique characteristics around the world. Figure 1 shows that N. cyriacigeorgica (40.2%) was the most encountered species in this study that is similar to the reports from Iran, Spain, and the USA [9, 28, 29]. The prevalent species are slightly different in different regions [28, 30, 31]. Even within China, the prevalent species also have regional characteristics. N. otitidiscaviarum appears to be more prevalent in the eastern and southern coastal areas of China, and N. abscessus prefers to be distributed in the neighboring northern provinces of China, including Shanxi, Hebei, Beijing, and Shandong [20, 32]. N. aobensis and N. nova which are close species on 16S rRNA and/or secA1 genes and even classified into N. nova complex by Conville et al. , are distributed in Jiangsu in this study, while N. nova is distributed in Shandong in the study by Huang et al.  Actually, Jiangsu and Shandong are adjacent to each other. N. asiatica is mainly distributed in Hunan in this study, while it is distributed in Chongqing and Guangxi, which are adjacent to Hunan in the study by Huang et al. 
Based on the current study and the related references mentioned above [20, 32], an interesting phenomenon was found: Some species prefer to be distributed according to the climate type, while others prefer to be distributed along the coast in China. Shanxi, Hebei, Beijing, and Shandong all belong to the monsoon climate of medium latitudes, and N. abscessus is prevalent. Hunan, Chongqing, and Guangxi all belong to the subtropical monsoon climate, and N. asiatica is prevalent. N. otitidiscaviarum and N. nova complex tend to be distributed in coastal provinces (Beijing, Hebei, Shandong, Jiangsu, Zhejiang, and Guangxi). This phenomenon suggests that the distribution of Nocardia is affected by the climate type and the sea.
With the increase of sample size, the incidence of nocardiosis in men increased significantly, which is different from our previous conclusion that there is no difference between men and women , but the new finding is similar to the report by Martínez-Barricarte, who summarized the gender distribution of patients with isolated nocardiosis worldwide . According to Hernandez Hernandez et al., the female hormone estradiol shows inhibitory effect on Nocardia brasiliensis , which suggests the gender difference may be caused by estradiol. The proportion of nocardiosis for age ranging from 50 to 69 years was more than half in this study (58.5%), which is similar to the report by Huang et al. (54.7%) , but it is different from the data reported by Martínez-Barricarte that age ranging from 31 to 40 and from 51 to 60 years are the maximum proportion . It suggests that nocardiosis in China has unique characteristics in terms of age. Nocardiosis most often shows up as a pulmonary infection [1, 12], confirmed by this study (90.2%).
There are few large-scale studies [20, 21] about the antimicrobial susceptibility pattern of various Nocardia species based on BMD method that is recommended by the CLSI  in China. This study provides a relatively large collection of clinical Nocardia isolates to explore the correlation between antibiotics and species and reach a guideline for the nocardiosis treatment in China. SXT is the drug of the first choice for the treatment of nocardiosis, but some studies report a high level of resistance to the drug . It is urgent to survey the SXT susceptibility in China. Overall, 98.8% of isolates were susceptible to SXT, and only one isolate of N. wallacei was resistant to the drug (Table 1). Similar results are reported by Lu et al. and other researchers [20, 36,37,38,39]. Therefore, the present study indicates that SXT remains high-level of activity against Nocardia in vivo and can still be considered as the first-line therapeutic drug of choice for nocardiosis in China. The discrepancy may be caused by geographic differences, and/or SXT exposure before testing . According to the reports [28, 31], the isolation rates of N. nova complex and N. farcinica which have high resistance rates to SXT were more than 10% in Spain and the USA. However, the former is rarely isolated in China [20, 32], and the latter may be divided into different types due to the different geographic areas.
Linezolid, that shows 100% activity against clinical Nocardia isolates in several large-scale studies [24, 25, 35, 38, 40], has become popular in treating nocardiosis recently, and the present study showed the same results. Amikacin is also an effective drug for all Nocardia isolates except for N. transvalensis complex, which is intrinsic resistant to the drug [37, 38, 40,41,42]. However, N. transvalensis complex is not prevalent in China [20, 32]. In the study, N. wallacei, which belongs to N. transvalensis complex, is the only resistant species to amikacin. The treatment of nocardiosis commonly requires a combination of antibiotics. Imipenem is usually used in combination with SXT for adequate therapy of invasive Nocardia infections . Imipenem showed good activity (susceptibility rate: 73.2%) in the current study which supports it to participate in the combination regimen in empirical therapy. Amoxicillin/clavulanic acid, minocycline, or doxycycline can be also selected in a combination therapy, because the frequently encountered Nocardia species exhibit low-level resistance to the three antibiotics in China.
However, the remaining antimicrobials showed low activity against Nocardia isolates, and the susceptibility had species-specific. Compared with the studies by Wallace et al.  and Schlaberg et al. , a similar correlation between the antimicrobials and Nocardia species is observed, but there are some differences as well (Table S1). In particular, Wallace et al. reported that N. abscessus was resistant to imipenem, but the resistance rates were 40% in this study and 69% in the study by Schlaberg et al. Unlike Wallace et al. and Schlaberg et al., who indicated that N. wallacei was susceptible to ciprofloxacin, the current study indicated 50% susceptibility rate to the drug. For N. farcinica, Wallace et al. and this study reported that it was susceptible to imipenem, while Schlaberg et al. reported that the susceptibility rate was only 33%. Besides, ciprofloxacin was less active to N. farcinica in the study by Schlaberg et al. and the current study, but was active in the study by Wallace et al. However, moxifloxacin, which is a higher generation quinolone antibiotic, was much more active than ciprofloxacin against N. farcinica in the study by Schlaberg et al. (susceptibility rate: 79%) and the current study (75%). It may be caused by the longer exposure time of ciprofloxacin compared to moxifloxacin . For N. cyriacigeorgica, this study indicated that it was susceptible to imipenem (87.9%), which is similar to the study by Wallace et al., but Schlaberg et al. reported the susceptibility rate was only 43%. For N. otitidiscaviarum, ciprofloxacin was much active in the study by Wallace et al., but it was almost inactive in the study by Schlaberg et al. and the present study. The small differences mentioned above need to be further confirmed because the reproducibility of the BMD method for susceptibility testing of Nocardia species is not always very stable reported by Conville et al. 
For empirical treatment of nocardiosis, clarithromycin should be avoided due to the high resistance rate in this study. Ciprofloxacin is much less active to N. cyriacigeorgica, and ceftriaxone, cefepime, and tobramycin are much less active to N. farcinica. Therefore, these antibiotics also should avoid being used unless the species has been identified and/or the susceptibility test has been done, as N. cyriacigeorgica and N. farcinica are the most prevalent strains in China.
In addition to the major epidemic strains in China, the isolation rate of N. otitidiscaviarum in eastern and southern coastal areas is relatively high. The use of β-lactam antibiotics should be paid attention to because of its high resistance rate to this kind of antibiotics [20, 32].