Clinical characteristics and drug resistance of Nocardia in Henan, China, 2017–2023

Background The aim of this study was to investigate the clinical features of Nocardia infections, antibiotic resistance profile, choice of antibiotics and treatment outcome, among others. In addition, the study compared the clinical and microbiological characteristics of nocardiosis in bronchiectasis patients and non-bronchiectasis patients. Methods Detailed clinical data were collected from the medical records of 71 non-duplicate nocardiosis patients from 2017 to 2023 at a tertiary hospital in Zhengzhou, China. Nocardia isolates were identified to the species level using MALDI-TOF MS and 16S rRNA PCR sequencing. Clinical data were collected from medical records, and drug susceptibility was determined using the broth microdilution method. Results Of the 71 cases of nocardiosis, 70 (98.6%) were diagnosed as pulmonary infections with common underlying diseases including bronchiectasis, tuberculosis, diabetes mellitus and chronic obstructive pulmonary disease (COPD). Thirteen different strains were found in 71 isolates, the most common of which were N. farcinica (26.8%) and N. cyriacigeorgica (18.3%). All Nocardia strains were 100% susceptible to both TMP-SMX and linezolid, and different Nocardia species showed different patterns of drug susceptibility in vitro. Pulmonary nocardiosis is prone to comorbidities such as bronchiectasis, diabetes mellitus, COPD, etc., and Nocardia is also frequently accompanied by co-infection of the body with pathogens such as Mycobacterium and Aspergillus spp. Sixty-one patients underwent a detailed treatment regimen, of whom 32 (52.5%) received single or multi-drug therapy based on TMP-SMX. Bronchiectasis was associated with a higher frequency of Nocardia infections, and there were significant differences between the bronchiectasis and non-bronchiectasis groups in terms of age distribution, clinical characteristics, identification of Nocardia species, and antibiotic susceptibility (P < 0.05). Conclusions Our study contributes to the understanding of the species diversity of Nocardia isolates in Henan, China, and the clinical characteristics of patients with pulmonary nocardiosis infections. Clinical and microbiologic differences between patients with and without bronchiectasis. These findings will contribute to the early diagnosis and treatment of patients. Supplementary Information The online version contains supplementary material available at 10.1186/s12941-024-00677-4.


Introduction
Nocardia, a Gram-positive filamentous bacterium, is commonly found in soil, water, and air [1,2].It can enter the body through inhalation of hyphal fragments, broken skin, or the digestive tract, leading to pneumonia, brain abscesses, and skin and soft tissue infections [1].Hosts with suppressed immune function, such as those on long-term glucocorticoid therapy, patients undergoing radiotherapy and chemotherapy for malignant tumors, organ transplantation and hematopoietic stem cell transplantation recipients, and individuals infected with human immunodeficiency virus (HIV), often develop primary pulmonary infections [3].Non-immunosuppressed hosts primarily suffer from structural lung diseases like cystic fibrosis and bronchiectasis [4,5].The distribution of Nocardia species varies geographically [3].There is limited research on the clinical characteristics of nocardiosis in mainland China, particularly regarding patient treatment, prognosis, species identification of Nocardia, and drug susceptibility testing [6,7].To address this knowledge gap, we conducted a retrospective study, collecting detailed clinical data on nocardiosis patients in a tertiary hospital in Henan over a 6-year period.Additionally, we performed species identification of all included Nocardia isolates using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and genetic sequencing, as well as tested their antibiotic susceptibility spectrum.In recent years, the prevalence of pulmonary nocardiosis has significantly increased among patients with bronchiectasis.Although the exact reasons for this increase are not fully understood, they may be attributed to environmental exposure and microbial surveillance, among other factors [5].Therefore, our study aimed to compare the clinical and microbiological characteristics between nocardiosis patients with and without bronchiectasis.To our knowledge, no studies comparing the characteristics of these two patient groups have been conducted in mainland China.

Collection of bacterial strains
We conducted a retrospective analysis of 71 unique cases of Nocardia infection that occurred at Henan Chest Hospital from January 2017 to April 2023.These cases involved patients from 14 different cities in Henan Province.Patient medical records were collected, including demographic information, underlying diseases, coinfections, imaging data, laboratory data, antimicrobial treatment, and prognosis.The inclusion criteria for this study were as follows: Nocardia isolation from qualified sputum specimens with at least two positive cultures, or from lung tissue or bronchoalveolar lavage fluid (BALF) obtained under sterile operating conditions, along with the presence of clinical signs of infection and/or radiographic evidence (pulmonary, skin, and soft tissue) of organ involvement.The exclusion criteria included patients with only one positive sputum culture and cases where all Nocardia isolates originated from blood agar plate (BAP) culture and BACTEC-MGIT 960 culture system (MGIT960).

Identification of strains
Suspected Nocardia specimens found in smear microscopy of clinical samples were inoculated onto BAP and incubated in a (35 ± 2) °C incubator for 1-7 days, with daily observations of growth.Nocardia identification was carried out based on colony morphology on the culture medium, positive Gram stain (Gram-positive branching, moniliform, and filamentous bacilli), acid-fast staining, and modified acid-fast staining for preliminary presumptive identification.Further species identification was performed using MALDI-TOF MS.In cases where MALDI-TOF MS failed to provide species-level identification, final identification was achieved through polymerase chain reaction (PCR) amplification and sequencing of the full length of the 16S rRNA gene.The 16S rRNA primers used were: forward primer (5'-AGA GTT TGA TCC TGG CTC AG-3'), reverse primer (5'-CGG TTA CCT TGT TAC GAC TT-3'), with an amplification length of approximately 1500 bp.The amplified products were purified by Sangon Biotech (Shanghai, China) and sequenced using an ABI 3730XL gene sequencer.At the species level, the identity of the PCR products was confirmed by searching the 16S rRNA gene sequence in the NCBI GenBank using BLAST software (http:// www.ncbi.nlm.nih.gov).Nocardia isolates were considered identified at the species level when similarity values ≥ 99.0% were obtained.

Antimicrobial susceptibility testing
Antibiotic susceptibility testing (AST) was performed using the microbroth dilution method provided by Thermo Fisher (USA).In brief, colonies growing on BAP were collected using a swab and suspended in deionized water.The turbidity was adjusted to a 0.5 McFarland standard by visual inspection or with the help of a Sensititre ® Turbidimeter.Next, 50μL of the bacterial suspension was transferred into a test tube containing cation-adjusted Mueller-Hinton broth medium with TES buffer added, and mixed thoroughly.Subsequently, 100 μL of the bacterial suspension was transferred onto a drug susceptibility plate and incubated at 35℃ for 72 h.The results were then observed and the minimum inhibitory concentration (MIC) was determined based on the instructions provided on the drug susceptibility plate.Interpretations of the results were made according to clinical and laboratory standards institute (CLSI) recommendations for Nocardia.Quality control strains, Staphylococcus aureus ATCC29213 and Escherichia coli ATCC35218, were included in the testing.The MIC was defined as the lowest concentration of a drug that inhibited visible growth.

Comparison between drug sensitivity data
Accurate identification of Nocardia species provides the potential to partially predict antimicrobial susceptibility and helps in the selection of appropriate therapeutic approaches, for this reason we we reviewed the literature with drug susceptibility data between 2014-2023, from which we chose four representative papers (large size, large number, reliable drug susceptibility methods, etc.) to do a cross-sectional comparative study with our findings and with CLSI M62 in the The results were compared with our study in a cross-sectional study and with the CLSI M62 drug susceptibility patterns of different Nocardia species to find out the similarities and differences between them.

Statistical analysis
The MIC data for each antibiotic were recorded and analyzed using WHONET 5.6 software.The MIC50 and MIC90 were calculated as well.Additionally, data analysis was performed using SPSS 25.0 statistical software.Normally distributed measurement data were presented as mean ± standard deviation (x ± s), while non-normally distributed measurement data were presented as the median.A comparison was made between patients with bronchiectasis and those non-bronchiectasis in terms of the distribution, drug susceptibility, and clinical characteristics of Nocardia strains.Categorical variables were compared using the x 2 test or Fisher exact test.

Demographic characteristics and geographical distribution
A total of 71 cases of nocardiosis were collected, with ages ranging from 18 to 85 years and an average age of 56 years.Among these cases, 31 (43.6%) were aged 60 years or older, including 42 males and 29 females.The majority of the patients were farmers (67.6%, 48), and the main department involved was respiratory medicine (60.6%, 43).The most common specimen sources were sputum (52.1%, 37) and alveolar lavage fluid (46.5%, 33).Please refer to Table 1 for more details.The geographical distribution of Nocardia is depicted in Fig. 1.The city with the highest number of sources was Zhengzhou (21), followed by Zhoukou (12), Zhumadian (9), Shangqiu and Xuchang (6 strains each), and the classification of Nocardia species in various municipalities shows different.

Clinical characteristics
Among patients infected with Nocardia, 23.9% had a history of smoking, and 12.7% had a history of alcohol consumption.Out of 71 patients, 90.1% had at least one underlying disease.These included bronchiectasis (39), tuberculosis (16), type 2 diabetes (12), chronic obstructive

Molecular identification and distribution of Nocardia species
We initially identified the Nocardia species using MALDI-TOF MS and confirmed it further through 16S rRNA sequencing.However, the results of MALDI-TOF MS identification for the rare Nocardia species (N.africana, N.pseudobrasiliensis, N.flavorosea, N.amamiensis and N.rhamnosiphila) were inadequate and inconsistent with the sequencing results.Conversely, the identification results for N.cyriacigeorgica,N.farcinica,N.abscessus, N.beijingensis,N.otitidiscaviarum,N.asiatica,N.puris and N.wallacei were generally consistent between the two methods.

Antibiotic sensitivity
Table 2 provides a summary of Nocardia's sensitivity to 15 antibiotics, including the MIC inhibiting 50% (MIC50) and 90% (MIC90) of strains, along with the MIC range for all Nocardia isolates.It displays the sensitivity rate, intermediate rate, and resistance rate for each antibiotic.All Nocardia strains were 100% sensitive to TMP-SMX and linezolid.It is worth noting that different species of Nocardia demonstrate different antibiotic resistances, as shown in Table 2, N. farcinica has higher resistance rates to imipenem (47.4%), ceftriaxone (78.9%), tigecycline (78.9%) and clarithromycin (89.4%), while N.cyriacigeorgica has lower rates of resistance to imipenem(15.4%),ceftriaxone (15.4%), tigecycline (0%),and clarithromycin (23.1%)In addition, N. farcinica and N. abscessus exhibited different sensitivities to doxycycline and minocycline.One isolate of N. pseudobrasiliensis exhibited resistance to ceftriaxone, ciprofloxacin, doxycycline and minocycline, and intermediate susceptibility  to imipenem, moxifloxacin, and amoxicillin-clavulanic acid.
The drug sensitivity results for each Nocardia species in this study were compared with the predicted antimicrobial drug sensitivity patterns provided by the CLSI standard M62 and larger-scale data studies.Table 3 showed a strong correlation between the drug pattern types and the identification of Nocardia species.Nonetheless, some differences were observed.For example, although only 52.6% of the cutaneous Nocardia isolates were sensitive to amoxicillin-clavulanate, the drug pattern indicated sensitivity according to the CLSI M62.The sensitivity rate of amoxicillin-clavulanate in N.abscessus complex isolates was only 43.8%, despite the drug pattern indicating sensitivity.The sensitivity rate of ciprofloxacin in N.otitidiscaviarum isolates was 20%, with the drug sensitivity pattern also indicating sensitivity.Furthermore, despite the sensitivity rate of 100% for amikacin in N.wallacei isolates, the drug sensitivity pattern indicated resistance.

Comparison between pulmonary nocardiosis complicated with bronchiectasis group and non-bronchiectasis group
Bronchiectasis was found to be the predominant underlying disease among the patients based on the data presented in Table 1.The collected cases of nocardiosis were classified into two groups: the bronchiectasis group (39) and the non-bronchiectasis group (29), based on the patients' radiological examination results.We conducted a comprehensive comparison of the clinical characteristics, distribution of bacterial species, and drug sensitivity results between these two groups.Further detailed comparisons are provided in Tables 4 and 5.
It can be concluded from Table 4 that there was no statistically significant difference in the distribution of age groups between the two groups (p > 0.05), but in terms of gender composition, the bronchiectasis group was predominantly female, whereas the non-bronchiectasis group was predominantly male.There was a statistical difference between the two groups in terms of smoking history, with patients with a history of smoking in the non-bronchodilated group being more susceptible to nocardiosis than those with a history of smoking in the bronchodilated group.In terms of comorbidities between the two groups, we learnt that patients with diabetes mellitus in the non-bronchiectasis group were more likely to develop nocardiosis.Other differences are detailed in Table 4.
In Table 5, our study found that there was a statistical difference between the two groups in terms of species distribution of Nocardia isolates, in both N.abscessus (p < 0.05) and N.farcinica (p < 0.05) detection rates, with patients with bronchodilatation being more likely to be detected with N. abscessus than those with non-bronchodilatation.In antibiotic susceptibility testing, there was a statistical difference between the two groups in ceftriaxone and tobramycin (p < 0.05) antimicrobial drugs.

Treatment and outcome
Out of the 71 cases of nocardiosis that were collected, treatment details were available for 61 patients, while the treatment plans for 10 patients were missing.Based on the data presented in Table 1, out of the 61 patients with treatment plans, 32 patients (52.5%) received combination therapy comprising of TMP-SMX.Among these patients, 10 received TMP-SMX combined with a single drug, while 22 patients received TMP-SMX combined with two or more antibiotics as part of a multidrug treatment plan.Moreover, 20 patients (32.8%) received a multidrug regimen containing amikacin and other antibiotics, while 20 patients (32.8%) received a regimen consisting of linezolid combined with other antibiotics.Furthermore, 24 patients (39.3%) were treated with a combination of quinolone antibiotics and other antibiotics, and 17 patients were prescribed alternative antibiotic regimens during the treatment.Out of the 71 patients' treatment outcomes, 3 patients were lost to follow-up with unknown treatment results, 58 patients (85.3%) achieved a cure or improvement in clinical symptoms, and 10 patients (14.7%) were discharged without being cured.

Discussion
Nocardia, an important group of actinomycetes in the environment, can lead to human infections through traumatic inoculation or inhalation [1].The latest taxonomic study of Nocardia reveals the existence of 119 species, with 54 of them being associated with human infections [12,13].Some medically relevant bacterial species include N. asterosa, N.brasiliensis, N.farcinica and N.abscessus.Over the past decade, China has witnessed an increasing trend in Nocardia infections [7,10,14,15].However, there is a scarcity of reports on Nocardia in Henan.Given the variation in Nocardia distribution across different geographical regions, it is essential to investigate the epidemiology, clinical characteristics, and antibiotic resistance of Nocardia in various areas.
The incidence of nocardiosis is influenced by age.Our data showed that the majority of patients were older with a slight male predominance, which is consistent with previous literature [7,10].Occupational classification was dominated by farmers, who may be more susceptible to environmental Nocardia infections due to their greater outdoor exposure to contaminated soil.In our study, the diagnostic methods for Nocardia infection include BAP culture and MGIT960 culture.As shown in Fig. 2, N.farcinica was most frequently detected through the MGIT 960 culture method (n = 9, 60%), which is consistent with the literature [16,17].A study by Hu et al. [16] found that the high recovery rate of N.farcinica in MGIT 960 and the growth of other Nocardia species are due to their resistance to trimethoprim-sulfamethoxazole.The main species of Nocardia varies from region to region, in Australia [18] and USA [9], the most common is N. nova complex. in Iran [19], France [20] and Japan [21], the most prevalent species are N.asteroides, N.farcinica and N.cyriacigeorgica.Even in China, the distribution characteristics of Nocardia vary from region to region.In Taiwan, China [22] and Hebei province, China [10], the most frequently occurring species are N.brasiliensis and N.cyriacigeorgica.Nocardia is mainly transmitted by inhalation and pulmonary nocardiosis usually affects frail patients, especially immunocompromised patients due to organ transplantation and/or treatment with corticosteroids and COPD patients, and forms infected lesions in the lungs [23,24].Bronchiectasis was the most common (54.9%) among the patients with pulmonary nocardiosis combined with the underlying disease in our study.In a study by Huang et al. [25].bronchiectasis was most common (30.4%) among the underlying diseases.In a study by Yang et al. bronchiectasis was comorbid in 6 out of 12 (50%) patients diagnosed with nocardiosis [26].A study in Taiwan reported that the most common comorbidities were diabetes mellitus (30%) and COPD (26.7%) [27], which is similar to our report.Data show that an increasing number of patients with bronchiectasis are being diagnosed with pulmonary nocardiosis, but the reasons for this are not fully understood and may be due to environmental exposures, microbiological surveillance, and other factors [5].Woodworth et al. concluded that N.nova complex was more likely to be detected in patients with bronchiectasis than in other patients [5], but in our study data (Table 5) N.abscessus was more likely to be detected in patients with bronchiectasis, which is clearly inconsistent.
In this study, the clinical symptoms and CT manifestations of patients with pulmonary nocardiosis lacked specificity, making it difficult to distinguish them from other diseases such as filamentous fungi (e.g., Aspergillus and trichothecenes) or mycobacterial infections [28].Pulmonary nocardiosis may be mistaken for tuberculosis, and tuberculosis and HIV are common co-infections [29].Of 10 patients diagnosed with nocardiosis after death, 40% were misdiagnosed with TB before death [30].A study evaluating patients with suspected tuberculosis in Ghana found that 16.7% were co-infected with HIV and Nocardia spp.[31] The antibiotic regimen for nocardiosis and other diseases varies considerably, making accurate diagnosis critical to treatment.
To this day, that TMP-SMX is the main drug used for the treatment of nocardiosis, however, some studies have raised concerns about the increased resistance of Nocardia isolates in particular to TMP-SMX.In the study of Lebeaux et al. [20], 5.4% of isolates were insusceptible to TMP-SMX; in the study of Uhde et al., the rate of resistance to TMP-SMX was 42% [32]; these discrepancies may be caused by inter-labortory differences or differences in species distribution in different geographical regions.A multicentre study of 441 Nocardia strains in China showed a resistance rate to TMP-SMX of only 0.9% [7], which is more consistent with the results of this study.In Table 3 the main results of studies reported from antibiotic sensitivity testing of large-scale clinical Nocardia isolates published between 2014 and 2023 show a strong correlation between the type of drug pattern and the identification of Nocardia spp.species, with some differences between different strains and the corresponding drug susceptibility patterns; Therefore, for the optimal treatment of nocardiosis, the Nocardia spp.species should be identified as accurately as possible and antimicrobial drug susceptibility testing should be performed.
TMP-SMX is typically used as the drug of choice for the treatment of nocardiosis, either alone or in combination with other drugs such as amikacin, imipenem, or third-generation cephalosporins.Amikacin can be used in combination with TMP-SMX or other drugs for the treatment of critical nocardiosis [33].Imipenem is more active than meropenem or ertapenem against most Nocardia [34], and the combination of amikacin and imipenem is more effective in treating cerebral and pulmonary nocardiosis than TMP-SMX alone in a mouse model [35,36].The combination of imipenem and cefotaxime, amikacin and TMP-SMX, imipenem and TMP-SMX, amikacin and cefotaxime, or amikacin and imipenem provided enhanced activity [37].For most forms of nocardiosis, initial combination drug therapy is recommended [28].Among our 61 patients with a treatment plan for pulmonary nocardiosis, TMP-SMX in combination with amikacin and linezolid was the more common regimen.Although TMP-SMX is a common treatment option for nocardiosis, some patients in our study cases opted for other effective antimicrobials due to allergy to oral sulfonamides.Linezolid has shown good clinical efficacy in Nocardia infections and can be recommended as an alternative therapy to TMP-SMX due to its oral availability and activity against most Nocardia species [38].In our patients with nocardiosis, after aggressive clinical treatment, 14.7% of them still failed, and the failure may be due to the severity and complexity of the patient's own underlying disease.
The study has several limitations.Firstly, being a retrospective study, it carries inherent limitations of this study design,especially in terms of data loss, such as missed patient cases and loss to follow-up.Secondly, the study's small scale may introduce biases in epidemiology and prognosis.The data may not be representative, indicating the need for further research with a larger sample size.
In summary,This is the first study on the epidemiological and clinical characteristics of nocardiosis on a larger scale in Henan, China, and describes the distribution, clinical characteristics and antibiotic drug sensitivity of the identified Nocardia species.Drug susceptibility varies among different Nocardia species, and accurate species identification and confirmation of antimicrobial susceptibility patterns are necessary in diagnosis and selection of antibiotic therapy.Pulmonary nocardiosis is prone to comorbidities with other underlying diseases such as bronchiectasis, tuberculosis, diabetes mellitus and COPD.nocardia infections are susceptible to concurrent comorbidities with a variety of pathogens such as Mycobacterium and Aspergillus.Our study also showed that bronchiectasis occurs more frequently with Nocardia infections, and the data from the bronchiectasis and non-bronchiectasis groups showed statistical differences in clinical characteristics and drug sensitivity.Our study adds new value to the characterisation of nocardiosis in China, and a better understanding of the characteristics of Nocardia infections will help physicians make better decisions in the diagnosis and treatment of nocardiosis.

Fig. 1
Fig. 1 Geographical and spatial distribution of Nocardia isolates in Henan Province,China

5 Table 3 1
Main results of antibiotic susceptibility testing of clinical Nocardia isolates, derived from large-scale studies published beteen 2014 and 2023 % of susceptible isolates First author, year AST method Break-points N of isolates Amikacin AUG Ceftriaxone Ciprofloxacin Imipenem Linezolid Minocycline Moxifloxacin Han et al.Ann Clin Microbiol Antimicrob (2024) 23:23 AST antimicrobial susceptibility testing, BMD broth microdilution, CLSI Clinical and Laboratory Standards Institute, N number, N/A not available a Expected antimicrobial susceptibility patterns of the most com monly isolated Nocardia species or species complexes provided by CLSI standard M62 [11]; the expected pattern "R/S/V" represents resistant/susceptible/ variable b Except for moxifloxacinTable 3 (continued) % of susceptible isolates First author, year AST method Break-points N of isolates Amikacin AUG Ceftriaxone Ciprofloxacin Imipenem Linezolid Minocycline Moxifloxacin

Table 1
Basline characteristics of included patients 69% (29/42) showed elevated CRP levels.Additionally, among the 31 patients who underwent ESR examination, 24 had elevated ESR levels.Concurrent infections with other pathogens were present in 37 patients.These included 16 cases of Mycobacterium tuberculosis(MTB) infection, 5 cases of Aspergillus.sppinfection, and 2 cases of non-tuberculous mycobacterium infection.Please refer to Table 1 for further details.

Table 2
Antimicrobial susceptibility and MICs of 71 Nocardia isolates in clinical infections in Henan China from 2017 to 2023

Table 4
Characteristics of patients with nocardiosis*Due to total number < 40 and some of the expected number < 5, Fisher exact test's result was used a Three patients were lost to follow-up (including two pulmonary and one cutaneous), whose presence or absence of bronchiectasis was unknown due to incomplete case data

Table 5
Nocardia species identification and antibiotic risistance *Due to total number < 40 and some of the expected number < 5, Fisher exact test's result was used a Three patients were lost to follow-up (including two pulmonary and one cutaneous), whose presence or absence of bronchiectasis was unknown due to incomplete case data