The case report of Mycobacterium arupense wound infection in diabetes mellitus patients; the first report and literature review

Mycobacterium arupense is among the opportunist pathogens of atypical mycobacteria emergence (atypical mycobacteria) that is one of the isolated and reported environmental and clinical specimens. Numerous cases of osteo-articular infections of this bacterium are reported nowadays, while the pulmonary infection is rare. We identified Mycobacterium arupense in non-healing wound infection of an elderly woman with history of diabetes mellitus. She has negative tests for HIV, HBV and HCV, but was positive for HTLV-1. The patient was referred according to mild-fever, non-healing, destructive, and swelled lesion on her left foot. The mycobacterial wounds infection was suspected due to her non-conclusive previous treatment. The pathology, acid-fast staining, conventional and 16S rRNA sequencing confirmed the micro-organism to be M. arupense . Finally, the patient recovered following two-week consumption of clarithromycin, ethambutol and rifabutin. The results of this study provide evidence on the potential pathogenicity, clinical outcomes and treatment of infections caused by this bacterium.


InTROduCTIOn
Non-tuberculosis mycobacteria (NTM) are a group of 'Mycobacteria' that live in environmental resources such as saprophytes and that enter their body through inhalation and traumatic inclusion, causing the mycobacterosis infection [1,2]. The incidence rate of NTM infections is increasing nowadays [3,4]. The improved diagnostic methods, especially the molecular diagnostic methods, and the increased number of immune-disorders have increased the rate of NTM infections [5].
Mycobacterium arupense was first isolated from a tendon sample in 2006 and identified by Cloud et al. [6]. M. arupense is part of the M. terrae complex and is very similar to M. nonchromogenicum [6,7]. Identification of this type of clinical sample is quite difficult due to the similarity of phenotypic tests with the members of M. terrae complex. However, the 16S rRNA gene in M. arupense is as a signature sequence and the sequencing of housekeeping genes, especially 16S rRNA, is able to correctly identify this species [6,8]. According to the American Thoracic Society (ATS) guidelines, it is recommended that the NTM isolates isolated from clinical specimens should be identified to the species level for the final diagnosis, accurate identification, patient management, appropriate treatment and epidemiological goals [9].

CASE PRESEnTATIOn
A 51-year-old woman referred to Al-Zahra Hospital in Isfahan (Isfahan, Iran) in June 2018 due to non-healing foot ulcers in her left foot. She was a housewife living in a rural area near Faridan, Iran, working on farms and having a previous experience of foot ulcers. However, she stated that her recent foot ulcer had not healed in the last 1.5 months. The patient had a history of diabetes mellitus (since 2011). On initial examination, the patient had a mild fever (37.8 °C), and a swollen, necrotic ulcer was evident on the toes, and according to the patient, the lesions were not very painful. Sampling was done from the ulcer. Based on the microbiology lab reports, Staphylococcus aureus and Klebsiella pneumonia were isolated and penicillin, doxycycline, imipenem and bandage with Betadine were prescribed for the patient.
The patient returned again about a month later due to failure to respond to treatment, although she reported painful scarring and pale discharge; lesion depth was 1.5 cm and also extended to her sole (Fig. 1). The patient had a temperature of 38.2 °C and according to MRI abdominal cavity and chest X-ray, she had no signs of inflammation in her lungs and internal organs.
The pathology evaluation revealed the presence of granuloma. Blood culture of the patient was negative, and wound exudate samples were examined using Gram-staining and Ziehl-Neelsen staining. Acid-fast bacilli were confirmed in the wound exudate, and subsequently, wound exudate samples were cultured on blood agar and Lowenstein Jensen slant. Two weeks later small colonies appeared on LJ enriched in Sauton's broth (Fig. 2).
The considered isolate was identified as rapidly growing mycobacteria (RGM) due to the growth rate (<7 days), lack of pigment production, negative results for niacin and nitrate reductase as well as urease and heat stable catalase production (68 °C). Molecular analysis was performed to identify to the species level. Simple boiling method was used to extract the DNA, the amplification of nearly full-length of 16S rRNA was performed by primers pA: 5′ AG-AGA GTTTGATCCTGGCTCAG-3′ and pI: 5′-TGCACACAGGCCACAAGGGA-3′ according to Rogall et al., and the sequence of PCR product was analysed [13]. NTM spp. can be differentiated by high-precision via nucleotide sequence of the hypervariable regions A (125-270) and B (408-503) of the 16S rRNA. In addition, the nucleotide sequence of rRNA gene of a short helix region is between the 451-482 positions that is a signature for RGM [14]. Based on the results by Blast, it was found that the partial sequence 16S rRNA of the considered isolation was 100 % similar to Mycobacterium arupense (DQ157760). A phylognic-relationship analysis based on closely related mycobacterial species also identified the isolate as M. arupense as accession number: MN865166 (Fig. 3).   Drug susceptibility test (DST) was performed according to CLSI M24-A2 recommendations by the broth micro-dilution method. Based on the DST results, the considered isolate was sensitive to clarithromycin, ethambutol, and rifabutin antibiotics and resistant to isoniazid, rifampicin, amikacin, moxifloxacin, ciprofloxacin and linezolid. Finally, the treatment was done with purulent drainage, initiation with clarithromycin, ethambutol and rifabutin together with once applying of interferon alpha to reduce the proviral load HTLV-1. After two weeks of antibiotictherapy the foot wound infection of the patient recovered and the patient was discharged with personal consent.

dISCuSSIOn
There are various evidences of isolation of M. arupense from environmental samples nowadays, such as surface water, soil, fish tanks, animal urine, and duck houses [15,16]. Despite the widespread presence of this bacterium, there have been limited reports of human infections with M. arupense [16,17]. The present study was the first case report of a diabetic person foot ulcer infection by M. arupense. Due to the limitations of M. arupense's clinical reports, there is no standard guideline for the treatment of infections of this bacterium [17]. According  [21] to the review of the literature, M. arupense infections are more common in people with immune-disorders (Table 1).
Currently, human infections caused by M. arupense are divided into two categories: pulmonary and extra-pulmonary infections. Based on the existing reports, most of these people have trauma, HIV, or corticosteroids use [17,18]. Regarding the limited available information, it is not possible to fully understand the clinical significance, clinical outcome and duration of treatment of this bacterium [17].
However, surgical and antimicrobial therapy methods are commonly used for tenosynovitis and osteo-articular infections, whereas disseminated infections initiated with rifabutin, clatrithomycin and ethambutol have had satisfactory results. Furthermore, treatment regarding the pulmonary infection is based on ethambutol, clatrithomycin, rifabutin and drug susceptibility test; TMP-SXT results were also varied ( Table 1).
According to the review of the literatures, the duration of treatment for M. arupense infections varies between 6 and 24 months, depending on the type of infection and the involved tissue, and includes a combination of surgery and antibiotic therapy. No signs of relapse or re-infection were reported after the treatment (Table 1). Also, most reports have shown that M. arupense clinical isolates are sensitive to clarithromycin, rifabutin, ethambutol and rarely to quinolones (Table 1).
In a study on M. arupense infections in cancer patients, Hamal et al. observed that the clinical outcome showed no significant difference between the treated M. arupense infected cancer patients treated and the untreated group; there were no reports of relapse or death from M. arupense [18]. Vasireddy et al. reported in their studies 10 strains of M. arupense tissue specimens that most of these patients had experience of trauma or using corticosteroids [19]. Currently, M. arupense is considered an emergent pathogen for osteoarticular infection. However, the role of this bacterium as a respiratory system pathogen is still unknown [20][21][22]. Pulmonary infections caused by M. arupense have been so far observed only in immune-deficiency patients (Table 1).
In this study, we present the first report of an unusual cutaneous infection caused by M. arupense in Iran. Patient's immune system of the present study was weakened by infection with HTLV-1 and diabetes mellitus, and according to the evidence, this bacterium is more likely to cause opportunistic infections in the individuals with immune system deficiency.
Identification of M. arupense is very important in TB-indemic regions, especially in Iran. Due to the slow growth of mycobacterium tuberculosis in the developing countries such as Iran, the considered patient affected by TB is reported only by observing acid-fast bacilli in smears of clinical specimens and considering a TB-endemic area [23]. This report demonstrates the importance of culture and identification to the species level of mycobacteria for appropriate diagnosis and treatment [6]. Based on the available evidence, two reports of infection with M. arupense in Iran have been reported indicating circulation of this bacterium in this geographical area [8]. The study was also the first report of cutaneous infection by this bacterium, indicating the potential pathogenicity of this microorganism.
Finally, the importance of molecular methods in identifying NTM spp. should be mentioned. Conventional and culture methods are expensive due to the slow growing nature of mycobacteria, and are not quite appropriate due to their inconclusive state, whereas molecular methods, especially 16S rRNA sequencing, are able to identify NTM species in high accuracy, in addition to being non-expensive and fast [24].

Funding information
The authors received no specific grant from any funding agency.