MLSA phylogeny and antimicrobial susceptibility of clinical Nocardia isolates: a multicenter retrospective study in China

With the increase of detection rate and long treatment period, nocardiosis has become a noticeable problem in China. However, there are limited large-scale studies on the epidemiology and antimicrobial susceptibility profiles of clinical Nocardia spp. in China. The present study aimed to explore the species distribution and drug susceptibility pattern of 82 clinical Nocardia isolates from three tertiary hospitals in China by multilocus sequence analysis (MLSA) and broth microdilution (BMD) method. Pulmonary nocardiosis (90.2%) was the most common clinical presentation of infection. N. cyriacigeorgica (n = 33; 40.2%) and N. farcinica (n = 20; 24.4%) were the most frequently encountered Nocardia species, followed by N. otitidiscaviarum (n = 7; 8.5%), N. abscessus (n = 5; 6.1%), N. asiatica (n = 4; 4.9%), and N. wallacei (n = 4; 4.9%). Trimethoprim/sulfamethoxazole (SXT) remained high activity against all Nocardia isolates (susceptibility rate: 98.8%). Linezolid and amikacin were also highly active; 100 and 95.1% of all isolates demonstrated susceptibility, respectively. Except for N. otitidiscaviarum, all the Nocardia isolates exhibited high susceptibility rates to imipenem. The resistance rates of all isolates to clarithromycin and ciprofloxacin were 92.7 and 73.2%, respectively, but the resistance rate of N. farcinica to ciprofloxacin was only 25%. The clinically isolated Nocardia spp. had diverse antimicrobial susceptibility patterns, which were similar to the reports by other groups elsewhere, but some differences were also observed, mainly including imipenem and ciprofloxacin. According to this study, SXT still can be the first choice for empirical therapy due to the low resistance rate. Linezolid can be chosen when a patient is allergic to SXT, and amikacin and imipenem can be the choice in a combination regimen.

specific drug susceptibility patterns [8,9], but it is always a complex problem in clinical practice. With the development of matrix-assisted laser desorption ionizationtime of flight mass spectrometry (MALDI-TOF MS), the common clinical Nocardia species (e.g., N. cyriacigeorgica and N. farcinica) can be identified rapidly. However, the available database of MALDI-TOF MS is limited for Nocardia strains leading to some uncommon isolates with no or false identification [10,11]. The 16S rRNA gene sequencing is generally considered as the primary means for accurate identification of the clinically encountered Nocardia isolates [12], but it cannot discriminate closely related species due to high conservation, unless it combines with a housekeeping gene, such as gyrB or rpoB [13]. In recent years, multilocus sequence analysis (MLSA) has become more and more critical in bacterial taxonomy and species identification [14][15][16], because it has the advantages of a comparable database, robust discrimination, and relatively low cost. Our recent work shows that three-locus (gyrB-16S rRNA-secA1) MLSA for identification of clinical Nocardia species is superior to five-locus (gyrB-16S rRNA-secA1-hsp65-rpoB) MLSA which leads to misidentification for N. abscessus confirmed by digital DNA-DNA hybridization [17]. Therefore, the three-locus MLSA was carried out in this study to accurately identify clinical isolates from three tertiary care centers.
Although trimethoprim/sulfamethoxazole (SXT) has long been considered as the primary choice for therapy, it combines with other antibiotics that are always used for severe or systemic infections [12]. It is essential to carry out accurately antimicrobial susceptibility tests for clinical Nocardia isolates in China due to not only treatment for the individual patients but also providing guidance on empirical therapy in China.
The broth microdilution (BMD) method was recommended by the Clinical and Laboratory Standards Institute (CLSI) to determine antimicrobial susceptibility of Nocardia isolates [18], but few research groups carried out the susceptibility test with a relatively large number of isolates using this method in China (< 30 strains in the related references) [19][20][21]. Therefore, this study aimed to provide the susceptibility profile of 82 clinical Nocardia isolates using the BMD method.

Species distribution and geographic characteristics
In the phylogenetic tree formed by three-locus MLSA (Fig. 1), these sequence clusters were considered to represent species clusters [14]. A phylogenetic tree was constructed from the 1902-bp concatenated gyrB-16S rRNA-secA1 sequences of 23 Nocardia type strains and 82 clinical Nocardia isolates by the neighbor-joining method [22] and Kimura two-parameter distances [23].
The geographic distribution of the clinical isolates is shown in Fig. 2. N. cyriacigeorgica and N. farcinica were the most widely distributed species in this study, being distributed in 64.3% (9/14) and 57.1% (8/14) of provinces, respectively. N. otitidiscaviarum isolates were distributed in eastern coastal regions (Beijing, Hebei, Jiangsu, and Zhejiang). N. abscessus isolates were distributed in three adjacent provinces of north China (Beijing, Hebei, and Shanxi).

Demographic characteristics and infection types.
The demographic characteristics and infection types of 82 patients with nocardiosis are shown in Table 2. More than half of the patients age ranged from 50 to 69 years old. Of the 82 patients, pulmonary infection was the primary type of infection.

Antimicrobial susceptibility testing
There are no CLSI clinical breakpoints for cefoxitin and tigecycline, but the studied 82 Nocardia clinical strains showed an overall susceptibility rate of the other 13 antimicrobial drugs according to species that is summarized in Table  were high, but for tigecycline (2 and 4 μg/ml) were low, which is similar to the report by Tan et al. [24] As previously described [12,25], we noted a strong correlation between the drug susceptibility pattern types and Nocardia species. The type I, III, IV, V, and VI were displayed by the N. abscessus, N. nova, N. wallacei, N. farcinica, and N. cyriacigeorgica, respectively. In addition, the species that were not defined drug susceptibility pattern types also had unique drug susceptibility profiles. The susceptibility profiles were varied by species of Nocardia which are shown in Table S1. The susceptibility rates to amoxicillin/clavulanic acid and moxifloxacin were high for N. farcinica (90 and 75%), but they were low for N. cyriacigeorgica (6.1 and 6.1%), N. otitidiscaviarum (0 and 14.3%), and N. asiatica (0 and 0), respectively. N. wallacei was the only species that was resistant         to amikacin. Imipenem was effective against most Nocardia spp., but it was ineffective against N. otitidiscaviarum isolates.
Table S1 also shows that the drug susceptibility patterns reported by Wallace et al. [26] and Schlaberg et al. [25] The similarities and slight differences are all displayed.

Discussion
Due to the large number of Nocardia species, biochemical methods are insufficient to identify the clinically relevant species [4]. MALDI-TOF MS can rapidly identify the frequently encountered Nocardia species, but it is limited by the database and distinguishing closely related species [10]. 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 [12]. 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 [17]. 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. [4], are distributed in Jiangsu in this study, while N. nova is distributed in Shandong in the study by Huang et al. [32] 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. [32] 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 [19], but the new finding is similar to the report by Martínez-Barricarte, who summarized the gender distribution of patients with isolated nocardiosis worldwide [33]. According to Hernandez Hernandez et al., the female hormone estradiol shows inhibitory effect on Nocardia brasiliensis [34], 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%) [32], 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 [33]. 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 [18] 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 [35]. 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 [36]. 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 [43]. 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. [26] and Schlaberg et al. [25], 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 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. [45] 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].

Conclusion
In summary, N. cyriacigeorgica and N. farcinica, which are widely distributed in China, were the most frequently isolated species, as well as they were the most common species causing pulmonary infection in this study. The clinically isolated Nocardia spp. had diverse antimicrobial susceptibility patterns, which were similar to the reports by other groups elsewhere, but some differences were also observed. It indicates the specific characteristics and provides the basis of empirical therapy in China. According to the current study, SXT can still be the first choice due to the low resistance rate. Linezolid can be chosen when a patient is allergic to SXT, and amikacin and imipenem can be the choice in a combination regimen.

Strains
A total of 82 non-repetitive clinical isolates of Nocardia from three tertiary hospitals in Beijing were studied between 2010 and 2020; seventy were from Beijing Chao-Yang Hospital, of which 26 isolates have been tested for antimicrobial susceptibility with self-made drug susceptibility plates and published in our previous work [19], seven from Beijing Ditan Hospital, and five from Aerospace Center Hospital. The three hospitals have 1880, 1158, and 1050 beds, respectively. Observation of grampositive beaded branching filaments on a direct gramstained smear and positive Kinyoun acid-fast stain under the microscope, as well as white to orange colonies on culture plates, indicated that the isolates were presumptive Nocardia [46]. The type strains in this study were selected as previously described [17]. In short, 23 type strains were used to construct phylogenetic relationships based on three-locus MLSA (gyrB-16S rRNA-secA1), and their GenBank accession numbers were shown in Table  S2. Most of the type strains were selected based on their similarity to 16S rRNA gene sequences of the clinical isolates.

DNA extraction, PCR, and sequencing
The DNA was extracted by boiling method, which was the same as our previous work [17]. The primers of 16S rRNA gene, and the primers of gyrB gene and secA1 gene referred to the work published by Carrasco et al. [13] and McTaggart et al. [16], respectively. The forward and reverse primers were listed in Table S3. The PCR experiments were carried out as previously described [13,16]. An ABI 3730XL DNA sequencer (Applied Biosystems) was used to sequence the PCR products, and the SeqMan program in Lasergene 7.1 (DNASTAR, Inc., Madison, WI) was used to assemble the sequences.

Construction of phylogenetic tree
The gene sequences of gyrB, 16S rRNA, and secA1 was aligned and trimmed by Mega (version 6.0) software [47] to generate the fragments of 482 bp, 1026 bp, and 394 bp, respectively. The concatenation of gyrB-16S rRNA-secA1 (1902 bp sequence) was used to constructed phylogenetic