Molecular identification and antibiotic resistance pattern of actinomycetes isolates among immunocompromised patients in Iran, emerging of new infections

Recent advancements in DNA-based approaches have led to the identification of uncommon and rare bacterial pathogens. In this study, by utilizing a DNA-based approach, a total of 1043 clinical specimens were processed for the identification of actinobacteria targeting the 16S rRNA and gyrB genes. Drug susceptibility testing was also conducted using micro-broth dilution and PCR. Two isolates of Nocardia flavorosea and Rhodococcus erythropolis were reported for the first time in Iran. Also, Nocardiopsis dassonvillei, Streptomyces olivaceus, and Streptomyces griseus were reported for the first time in Asia. Infections caused by Nocardia caishijiensis and Prauserella muralis have also been reported in this study. The first Asian case of pulmonary infection caused by Nocardia ignorata and the first global case of brain abscess caused by Nocardia ninae and Nocardia neocaledoniensis have been reported in this study. Overall 30 isolates belonging to 6 genera (Nocardia, Streptomyces, Rodoccoccus, Nocardiopsis, Rothia, and Prauserella) were detected in 30 patients. All 30 isolates were susceptible to amikacin and linezolid. Three isolates including Nocardia otitidiscaviarum (n = 2) and Nocardia flavorosea (n = 1) were resistant to trimethoprim-sulfamethoxazole which were the first trimethoprim-sulfamethoxazole resistant clinical actinomycetes in Iran. Isolation of rare species of actinomycetes particularly Nocardia spp. requires urgent action before they spread clinically particularly among immunocompromised patients.


Patients and specimens.
A total of 1043 consecutive non-duplicate clinical samples including sputum, bronchoalveolar lavage (BAL), blood, drainage of the brain, or cutaneous abscesses and corneal scraping suspected to be actinomycetes infections were collected from patients in this study. The specimens were then transferred to the microbiology laboratory while observing standard safety protocols.
Recruited patients had the following inclusion criteria: (1) at least one underlying condition supporting infection by actinomycetes such as HIV infection, cancer, diabetes, autoimmune disorders, transplantation, immunodeficiency and chronic respiratory complications, (2) clinical or radiological findings consistent with a diagnosis of infection by actinomycetes such as lung consolidation, nodule, and cavitation in radiography, cutaneous lesions, solid and soft organ abscess. Patients with previous antibiotic therapy and those with evidence of tuberculosis or non-tuberculosis mycobacterial infections as well as fungal infections were excluded from the experiment.
Isolation of actinomycetes. The direct microscopical examination was performed using gram and partial acid-fast staining. Specimens were cultured on blood and chocolate agar plates containing cycloheximide, vancomycin, and polymyxin B as well as antibiotic-free media 7,11,12 . After 3 weeks of incubation at 37 °C, suspicious colonies were selected for further investigation. Conventional biochemical tests including hydrolysis of hypoxanthine, tyrosine and xanthine, resistance to lysozyme, growth at 45 °C were performed and the colony morphology were assessed. Using these phenotypic tests, identification of Nocardia spp. at genus level was done 11,12 . Because phenotypic identification of all Actinomycetales is not feasible, suspicious colonies other than Nocardia were directly subjected to molecular identification. Molecular identification. Bacterial DNA was extracted using the boiling method 12 . In brief, PCR test was performed using primers 27F: 5′-AGA GTT TGA TCC TGG CTC AG-3′ and 1525R: 5′-AAG GAG GTG WTC CAR CC-3′ 13 targeting 16S rRNA gene as the primary target for identification. For rare Nocardia species, a fragment of gyrB gene, as a confirmatory target, was amplified and sequenced using primers F: 5'-CTT CGC CAA CAC CAT CAA CAC-3' and R: 5'-TGA TGA TCG ACT GGA CCT CG-3′ 13 . PCR reactions were performed in a mixture of 25 μL containing 12.5 μL of the master mix, (Thermo Fisher Scientific, USA), 1 μL (0.2 µM) of each forward and reverse primers and 1 μL template DNA under following conditions: 5 min of preheating at 94 °C, 32 cycles of 30 s denaturation at 95 °C, 30 s of primer annealing at 58.5 °C, 1 min extension step at 72 °C and post cycling extension of 5 min at 72 °C. The process of amplification was carried out in a thermal cycler (Biorad Thermal Cycler, USA) 25 .
Electrophoresis of PCR products was performed in 1% agarose gel prepared in 1 × TAE buffer with 100 mV for 30 min. Initial quality assessment of PCR products (single and sharp bands) was performed using a UV transilluminator. The amplicons were subsequently sent for sequencing using both primers (Bioneer, South Korea). Results were trimmed and aligned with verified sequences in GenBank using the Basic Local Alignment Search Tool (BLAST). Standard databases for Nucleotide collection (nr/nt) and Highly similar sequences (megablast) program were used for alignment (https:// blast. ncbi. nlm. nih. gov/ Blast).
Streptomyces olivaceus and Streptomyces griseus were non-susceptible to 3 and 4 out of 10 tested antimicrobial drugs, respectively. Regarding the rare species Prauserella muralis, Rhodococcus erythropolis, and Rothia dentocariosa, a non-susceptible phenotype was observed to 2 out of 10, 5 out of 11 and one out of 8 tested antimicrobial drugs, respectively. Susceptibility pattern of all isolates is presented in Table 2.
Due to the common prescription of trimethoprim/sulfamethoxazole in actinomycetes infections, especially nocardiosis, resistant isolates to this antibiotic were further studied. A total of 3 isolates including 2 Nocardia otitidiscaviarum and one Nocardia flavorosea isolates were resistant to trimethoprim/sulfamethoxazole. Genes int1, sulf1 were detected in all three isolates (Fig. 1).

Discussion
Actinomycetes can be isolated from different environmental sources including soil, water, decaying plants, and animals 11,12 . Unlike many bacterial communities including Bacillus and Clostridium which produce endospore as a dormant structure, in actinomycetes, mycolic acid and a peptidoglycan layer form a non-productive and tough structure to survive under harsh conditions for a long period of time [29][30][31][32] .
Immunodeficiency, transplantation procedure, and old age predispose individuals to bacterial infection particularly caused by opportunistic pathogens. Meanwhile, with the exception of a few studies, a comprehensive study on the evaluation of actinomycetes is not available. On the other hand, traditional and culture-based methods have inadequacies in the detection of unexpected and rare pathogens such as actinomycetes in clinical specimens. Therefore, further investigation using sequencing-based approaches is required to identify actinomycetes at species level accurately and evaluate antibiotic resistance patterns in order to prevent the progression of infection and extend the current knowledge regarding the epidemiology of infection [13][14][15][16][17]33 .
The prevalence of actinomycetes infections varies geographically. In this study, the prevalence of actinomycetes infections was 2.88% (30 out of 1043). The prevalence of Nocardia infections was 2.21% (23 out of 1043) which was slightly higher than the estimated prevalence of nocardiosis in Iran (1.88%) 34 . From 1970 to 2005, the prevalence of nocardiosis in Asia has been estimated to range between 0.001% in the United Arab Emirates to 1.9% in China, while in Africa, it was estimated to be 1.8% in Congo to 4.1% in Nigeria 34 . However, due to the paucity of information in this regard, almost all the recent studies were focused on previously isolated actinomycetes or retrospective reviews of medical records and were unable to provide a comprehensive prevalence of the infection. Similar prospective studies are needed to improve our understanding of the exact prevalence of actinomycetes in different countries.
In this study, pulmonary infection (n = 24, 80%) was the most common complication among patients and was higher than previous reports in Iran (50%) 13 , the United States (57.4%) and China (65.22%). Extrapulmonary infection was found in 6 patients, of them 3 were diagnosed with brain abscess. The brain abscesses made 10% of all cases in this study and is close to the previous reports in Iran (9%) 13 . Based on these results, nocardiosis should be considered in all patients with pulmonary infections as the lungs are the most common site of infection. However, extrapulmonary infections, especially brain abscesses should not be overlooked. Although the mortality associated with brain abscesses is high (40-50%), it can also be misdiagnosed with neoplasms, fungal or mycobacterial abscesses 19 .
Due to the immune deficiency and low CD4+ count, HIV patients are prone to actinomycetes infections. In this study, HIV/AIDS was the most common underlying condition which was found in 8 patients (26.67%) and was consistent with the previous study in Iran 13 . Delayed diagnosis and treatment of actinomycetes infections among HIV patients can lead to a high mortality rate from 18 to 66% 35 13,36 . Therefore, in the presence of relevant symptoms, actinomycetes infections should be considered in the differential diagnosis, not only in immunocompromised patients but also in immunocompetent individuals.
Nocardia cyriacigeorgica (n = 7, 23.33%) followed by Nocardia otitidiscaviarum (n = 4, 13.33%) were the most common identified species in this study which was not in agreement with the previous study in which  www.nature.com/scientificreports/ Nocardia asteroides (n = 31, 24.41%) and Nocardia cyriacigeorgica (n = 25, 19.69%) were the most common isolated species 13 . Our findings are not in agreement with other countries. For instance, a study conducted on hematopoietic stem cell transplant recipients in Japan 37 revealed the Nocardia farcinica (n = 3, 42.86%) as the leading species, similar to studies conducted in China with 42.1% 40 and 34.78% 38 of cases caused by this pathogen. However, in the United States 39 and Spain 40 , Nocardia nova has been reported as the most common species contributing to 28% and 29.57% of cases, respectively. Therefore, molecular-based studies providing more precise results are needed in all countries for the identification of common and uncommon species in actinomycetes infections. Using molecular-based approaches, more unexpected and rare bacterial species are expected to be detected in similar cases.
In the present study, infections due to a set of rare actinomycetes were identified. To the best of our knowledge, in this study, Nocardia flavorosea and Rhodococcus erythropolis were reported for the first time in Iran, and Nocardiopsis dassonvillei, Streptomyces olivaceus, and Streptomyces griseus for the first time in Asia, and infections caused by Nocardia caishijiensis and Prauserella muralis were reported for the first time in the world. We also reported the first Asian case of pulmonary infection caused by Nocardia ignorata and the first global cases of brain abscess cause by Nocardia ninae and Nocardia neocaledoniensis in this study.
From the treatment point of view, trimethoprim/sulfamethoxazole has been the most common antibiotic prescribed in actinomycetes infections, particularly nocardiosis. In the present study, 3 isolates (10.71%) (2 Nocardia otitidiscaviarum and 1 Nocardia flavorosea) were resistant to trimethoprim/sulfamethoxazole. By excluding non-Nocardia isolates, this rate was 13.04% among Nocardia species which was higher than the previous report in Iran (1.57%) 13 . The rate of trimethoprim/sulfamethoxazole resistance varies in different studies. While all isolates were susceptible to this antibiotic in one study 36  Because of a high rate of the susceptibility of Nocardia species to linezolid and amikacin, both antibiotics have been prescribed to treat the infections caused by these organisms 13 . High susceptibility of isolates to linezolid in this study may imply the effectivity of this antibiotic in similar cases. Also, all Nocardia isolates were susceptible to amikacin and only Rhodococcus erythropolis isolates (3.45%) were resistant to this antibiotic. Three trimethoprim/ sulfamethoxazole-resistant isolates were susceptible to linezolid and amikacin in this study demonstrating the application of linezolid and amikacin in infections caused by trimethoprim/sulfamethoxazole-resistant isolates. However, it is worth pointing out that there are also few studies reporting amikacin and linezolid resistance 40,42,43 .  www.nature.com/scientificreports/ Also, the int1 and sulf1 were detected in all trimethoprim/sulfamethoxazole-resistant isolates which were consistent with previous findings 26 . Regarding the non-Nocardia species, the susceptibility patterns were speciesspecific. In general, there was no infection with imipenem, trimethoprim/sulfamethoxazole, and linezolid resistance isolates. However, resistance to amikacin was observed in the Rhodococcus erythropolis isolate and with the exception of cefazolin, the Rothia dentocariosa isolate was susceptible to the all tested antibiotics.
Based on our findings, there is an urgent need for proper identification and antibiotic susceptibility testing of actinomycetes isolates in all regions particularly in vulnerable patients to actinomycetes infections.

Conclusion
Based on the results, a wide range of actinomycetes species can lead to severe infections in vulnerable patients. Although Nocardia was reported as the leading genus in this study, emerging new infections due to Nocardia ignorata, Nocardiopsis dassonvillei, Nocardia ninae, Rhodococcus erythropolis, Nocardia kruczakiae, Nocardia flavorosea, Streptomyces griseus, Nocardia cerradoensis, Nocardia caishijiensis, Prauserella muralis were also identified. Inter-species differences were observed in the antimicrobial resistance pattern in isolates harboring trimethoprim/sulfamethoxazole-resistance genes. Precise identification of actinomycetes isolates using molecular-based approaches is required for a better understanding of the epidemiology of infection. Although accurate identification of this group of bacteria is challenging in many laboratories, evaluation of this pathogen particularly in patients with underlying medical conditions should not be overlooked. Furthermore, the evaluation of the antibiotic resistance pattern of actinomycetes isolates should not be limited to a period of time and continuous monitoring is required for the prevention of infection and the improvement of therapeutic approaches.