Clinical Nocardia species: Identification, clinical characteristics, and antimicrobial susceptibility in Shandong, China

Nocardia is a pathogen responsible for a variety of clinical infections. Here, we aimed to investigate the species distribution, clinical manifestations, and antimicrobial susceptibility of Nocardia species over 3 years in two tertiary general hospitals in China. In this retrospective study, a total of 27 Nocardia species were isolated from 27 individuals between January 2017 and December 2019. Nocardia isolates were identified to species level by mass spectrometry and 16S rRNA PCR sequencing. Clinical data were collected from medical records. Antimicrobial susceptibility was determined by the standard Broth microdilution method. The 27 patients with Nocardia infection included 12 males and 15 females with a mean age of 60.11 years. Among 27 Nocardia isolates, 7 species were identified, with the most common species being Nocardia otitidiscaviarum (40.7%). The antimicrobial susceptibility profiles varied between different Nocardia species. Notably, all Nocardia isolates were linezolid susceptible. The majority of Nocardia isolates were collected from a department of respiratory medicine (55.56%) and sputum specimen (44.44%). Pulmonary region was the most involved body site (70.37%) followed by skin (7.4%) and pleural cavity (7.4%). Most patients with Nocardia infection needed combination antibiotic therapy. Two deaths were reported during the treatment period and 24 patients achieved improvement after antibiotic therapy. The clinical manifestations of Nocardia infection and antimicrobial susceptibility profiles varied with diverse Nocardia species. Thus, the accurate identification of these species is crucial for the diagnosis and the selection of antibiotic treatment.


INTRODUCTION
Nocardia species, an aerobic actinomycete, exist in a wide range of environments around the world [1]. Currently, only a small proportion of described Nocardia species are known to be human pathogens that affect patients [2]. Nocardia species are facultative intracellular pathogens, which are capable of causing either a localized or disseminated infection both in immunocompetent and immunocompromised hosts [3]. In recent decades, an increased number of Nocardia infection cases have been reported worldwide [4]. Nocardia infection is characterized by a variety of clinical manifestations with the involvement of the lungs, central nervous system, skin, and other organs. More seriously, Nocardia can cause severe, life-threatening disseminated infections, such as osteomyelitis and nocardial sepsis [5,6]. Early recognition and effective therapy are imperative to achieve successful outcomes. Thus, elaboration of the molecular characteristics, infection, and clinical manifestations of Nocardia species is necessary for timely detection and diagnosis.
Despite numerous Nocardia species have been characterized both phenotypically and genotypically within the genus, the genotype remains heterogeneous and continues to evolve [7]. In addition, the genus of Nocardia is rapidly expanding and the species distribution varies with different geographical locations [8]. Reports about Nocardia species in China are limited to a few case reports, case series, and research studies [9][10][11][12]. Particularly, there is only limited information about the species distribution and drug susceptibility of Nocardia [12]. Thus, the present study was designed to identify Nocardia species using 16SrRNA and mass spectrometry (MS) in two tertiary hospitals in China and to investigate the species distribution, clinical manifestations, microbiological characteristics, and antimicrobial susceptibility of the Nocardia species. In addition, we retrospectively analyzed the therapeutic effects and prognosis of patients with Nocardia infection. 532 www.bjbms.org

Bacterial isolates
This retrospective study was conducted at two tertiary hospitals in China, namely, the Affiliated Hospital of Jining Medical University and the Shengli Oilfield Central Hospital. A total of 27 patients with culture-proven Nocardia infection from January 2017 through December 2019 were included in this analysis. Nocardia infection was defined as a positive culture in the presence of clinical and/or radiological features of infection. Ethics approvals were obtained from the participating centers. Clinical specimens, including sputum, bronchoalveolar lavage fluid (BALF), alveolar pus, and puncture fluid were incubated on the Columbia blood agar plates at 35°C for 3-7 days. The bacterial isolates were then stored at -80°C until used. The demographic characteristics, including age, years and diseases history, as well as clinical features, laboratory tests, therapeutic regimen and outcome of included patients, were retrospectively reviewed from the medical records.

Antimicrobial susceptibility tests
Antimicrobial susceptibility was determined using the standard Broth microdilution method following the recommendations of the Clinical and Laboratory Standards Institute (CLSI, 24 th edition) [13]. Antibiotics were selected according to the first-line and second-line drugs used for Nocardia rec- Identification of Nocardia species Initial presumptive identification was performed according to the colony morphology on solid medium, Gram stain appearance, acid-fast staining and modified acid-fast staining, following the standard protocol. White colonies on culture plates, branching Gram-positive bacilli, positive acid-fast staining, and positive partial acid-fast staining were identified as Nocardia species.
Nocardia species were further verified by MS identification using an automated VITEK-MS system (BioMerieux, Marcy l'Etoile, France). Briefly, the bacteria isolates were mixed with 0.5 mm glass beads containing 70% ethanol (500 μL) in a pre-sterilized centrifuge tube and incubated at room temperature for 10 min. Then, the suspension was transferred into a 2.0 mL tube and centrifuged at 14,000 × g for 2 min. After ethanol was removed, the bacteria isolates were resuspended with 70% formic acid (10 μL) followed by acetonitrile (10 μL) for protein extraction. Next, the suspension was centrifuged at 14,000 × g for 2 min again and 1 μL supernatant was transferred onto target slide. After drying, 1 μL α-cyano-4-hydroxycinnamic acid (CHCA) matrix was added to complete the process. Results were analyzed using the VITEK-MS system with knowledgebase 3.0.
Bacterial genotypic identification of Nocardia species was further performed by 16S rRNA gene polymerase chain reaction (PCR) analysis. DNA was prepared using Bacterial Genomic DNA Extraction Kit according to the manufacturer' s instructions (TIANGEN, Beijing, China). The primer sequences of 16S rRNA used were as follows: forward primer (5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ) and reverse primer (5ʹ-GGTTACCTTGTTACGACTT-3ʹ). PCR reactions were conducted with a total reaction volume of 25 μL per well containing Phanta Max Master Mix (12.5 μL, Vazyme, Nanjing, China), forward and reverse primer (each 0.5 μL), DNA template (2.5 μL), and RNase-free water (9 μL). Amplification was performed using Veriti Thermal Cycler (Applied Biosystems, USA) under the following conditions: 95°C for 2 min, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. PCR product identity was confirmed by BLAST searches of the GenBank database (https://blast.ncbi.nlm.nih.gov/Blast. cgi). Species identification results of 16S rRNA with at least 99.0% similarity were regarded as positive.

Statistical analysis
All statistical analyses were performed by IBM SPSS Statistics for Windows, Version 22.0. (IBM Corp., Armonk, NY, USA). Quantitative data were presented by mean ± standard deviation (SD). Qualitative data were described by number or percentage.

Phenotype identification of Nocardia species
Phenotype identification of Nocardia species was performed by colony morphology, Gram staining, acid-fast staining, and modified acid-fast staining. Bacterial cultures showed that Nocardia species were aerobic, but with a slow reproduction rate. It generally takes 3-5 days to form visible colonies and present as white, yellow, or orange colonies on culture plates ( Figure 1A). Clinical isolates with branching Gram-positive bacilli ( Figure 1B), negative acid-fast staining ( Figure 1C), and positive partial acid-fast staining ( Figure 1D) were identified as Nocardia species.

Molecular identification and distribution of Nocardia species
The 27 Nocardia isolates were firstly identified using the automated VITEK-MS system. Among the 27 Nocardia isolates, 7 species and 1 genus were confirmed. The representative MS identification results are shown in Figure 2. N. otitidiscaviarum was the most common species, accounting for 40.7% (11/27)

Antimicrobial susceptibility
The susceptibility to 13 kinds of commonly-used antibiotics for Nocardia species is shown in Figure 3.

Treatment and outcome
Treatment information was available for all included patients. Among them, 2 patients were administrated with TMP-SMX monotherapy and 24 were administrated with the combination of TMP-SMX and other antibacterial agents such as MIN, AMK, etimicin sulfate, CIP, LNZ, and MOX. One patient was administrated with combination therapy of LNZ and cephalosporins because of allergy to sulfonamides. The treatment duration in surviving patients ranged from 7 days to 6 months. The duration of treatment in the majority of patients (81.48%) was 3 months. During treatment, no patient had abnormal blood test results nor the dysfunction of liver and kidney. Two patients experienced gastrointestinal discomfort and were relieved after omeprazole administration. Another patient had leukocytopenia and received treatment with Diyushengbai tablet. After treatment, a total of 24 patients achieved improvement and were discharged. Two deaths were reported during the treatment period. Another patient repeatedly experienced Nocardia infection and had been hospitalized 3 times for Nocardia infection of the joint until December 31, 2019.

DISCUSSION
Previous reports indicated an increasing trend of Nocardia infection over the past decade [4]. In addition, Nocardia species are distributed differently across various geographic regions. Thus, it is necessary to investigate the epidemiology, clinical characteristics, and antimicrobial susceptibility of Nocardia species in different geographic regions, especially in  [12]. Based on this evidence, we speculated that Nocardia infection occurred more frequently in our two centers over the past 3 years, in accordance with the overall epidemiological trends of Nocardia infection.
To date, more than 80 species of Nocardia have been described and 50 species have been identified to be human pathogens [8,12]. Nevertheless, only a few species can be reliably identified by the traditional biochemical methods, including N. farcinica, N. brasiliensis, N. asteroides and N. pseudobrasiliensis [7]. In general, the traditional biochemical methods are very time-consuming, resulting in a delayed diagnosis of infection. In recent years, 16S rRNA gene sequencing technique has become a reliable method for Nocardia identification [15]. However, the high cost and lack of standardization have been the major obstacle for the application of 16S rRNA gene sequencing in China. Notably, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS has emerged as an alternative method in the routine laboratory identification of Nocardia [16] and also has been widely applied in a large number of tertiary general hospitals in China [17]. In our study, we performed both the sequencing of 16S rRNA gene and MALDI-TOF MS to identify Nocardia species and obtained consistent identification www.bjbms.org results. Only one strain of N. wallacei was not identified to the specific species level by MS and whether this was due to technical operation or the limitations of MS remains to be further confirmed. Overall, MS has the potential to be a reliable technique for the identification of Nocardia species, according to the experience at our centers.
In the present study, a total of 7 Nocardia species were identified, of which N. otitidiscaviarum was the most frequently isolated, followed by N. cyriacigeorgica. Inconsistently, in previous studies, the predominant Nocardia species varied in different regions; namely, N. nova, N. abscessus and N. cyriacigeorgica were, respectively, the most predominant species in the United States [18], Italy [19], and Spain [20]. Even in China, the distributional features of Nocardia species varied in different regions [12,[21][22][23], indicating that the distribution of Nocardia species might be geographically-related.
Treatment for Nocardia infection according to the susceptibility of the isolated species is essential whenever possible, thereby, understanding the species-specific antimicrobial susceptibility patterns is very important. We reported the antimicrobial susceptibility patterns of the first-line and second-line drugs used for Nocardia infection. Here, the antimicrobial susceptibility profile was largely consistent with the results of the antibiograms available in the literature [19,22]. We found that the Nocardia species had good susceptibility to LNZ, TMP-SMX, and AMK in the present study. Furthermore, our isolates showed varying susceptibilities to different antibiotics. However, each species had a limited number of strains, which may not be enough to accurately represent the antimicrobial susceptibility. Thus, further studies with a larger sample size are still needed to confirm the antibiograms from our region.
In general, the majority of patients with Nocardia infection have a certain degree of immune deficiency, particularly patients with underlying diseases or immunosuppressive treatment. In the present study, approximately 93% of patients had at least one known underlying disease responsible for immune deficiency, which was consistent with the previous data [4,23]. The most common underlying diseases were type 2 diabetes mellitus, hypertension, and COPD. It is reported that impaired local pulmonary deficiency caused by COPD predispose to pulmonary nocardiosis, particularly in patients requiring long-term corticosteroid treatment [24]. Among the 27 included cases, 2 (7.41%) cases showed no evidence of underlying diseases. This data ranges from 10% to 25% in other reports [4]. Thus, Nocardia infection can occur both in immunocompetent and immunocompromised hosts.
With regard to the site of involvement, the pulmonary region was the most commonly involved body site of the patients in our two centers, which is consistent with the previous findings [14,19]. In addition, a literature review that included more than 1000 patients also reported that the pulmonary apparatus was the most commonly involved site [25]. In fact, it is well known that, because of its transmission route (direct inhalation or inoculation), Nocardia infections are mainly localized in the lower respiratory tract and the soft tissue. Nevertheless, nocardiosis is mostly an opportunistic infection leading to severe, life-threatening disseminated infections. Occasionally, some patients with disseminated infection may show the brain abscess, brain involvement, or neurological deficits [26,27]. No brain involvement was reported in our retrospective analysis, however, this may be due to the small sample size and the possibility should not be discounted. These findings suggest that either lung or brain computed tomography should be performed routinely in patients with Nocardia infections.
The laboratory test results were available in all 27 patients and showed that most patients had positive hs-CRP and approximately 20% of patients had positive PCT. Similarly, Guo et al. reported that most patients with nocardiosis had an elevated CRP level, whereas PCT levels were normal or slightly elevated [28]. These results suggest some typical indicators of Nocardia infection and increasing PCT levels may indicate a severe infection.
At present, antibiotic treatment is a major therapeutic strategy for Nocardia infection and, among them, TMP-SMX has been preferred and used as the first-line therapy [29]. For some patients with a serious infection and long duration, TMP-SMX-based combination therapy is recommended in clinical setting [30]. Unfortunately, an optimal therapeutic strategy has not been well defined in clinical practice. In the present study, TMP-SMX was the most commonly administered treatment and is still considered as a primary treatment option at our centers. The majority of our patients received TMP-SMX-based combination therapy. Recently, LNZ showed favorable clinical effectiveness in Nocardia infections, and it has the potential to be a useful alternative therapeutic agent due to its oral availability and activity against most Nocardia species [31]. In our study, since one patient was allergic to SMX, the combination therapy of LNZ and cephalosporins was administrated. After the corresponding therapy, nearly 90% of our patients achieved improvement and were discharged. The mortality rates associated with Nocardia infection reported in the literature range from 26% to 63% [32]. The mortality rate was 7.41% in our study, which is at a relatively low level. It is demonstrated that the mortality rate depends on the underlying disease, degree of immunosuppression, and disseminated form of Nocardia infection [33]. The clinical course of one patient in the present study, who experienced recurrent infection during treatment, was very complicated. This case suggested that follow-up treatment is necessary and may protect against relapses and dissemination. 537 www.bjbms.org

CONCLUSION
The present study retrospectively analyzed 27 non-repetitive clinical Nocardia isolates from two centers in Shandong, China. We found that the clinical manifestations of Nocardia infection and antimicrobial susceptibility profiles varied with different Nocardia species. Besides, MALDI-TOF MS was proved as a rapid and accurate identification for the Nocardia species. Our findings suggest the necessity of accurate species identification and confirmation of antimicrobial susceptibility patterns in the diagnosis and the choice of antibiotic treatment.