TB or Not TB: Emerging Mycobacteriaceae Detected in a Human Patient, Tree Shrews, and Soil

TB or Not TB: Emerging Mycobacteriaceae Detected in a Human Patient, Tree Shrews, and Soil | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report TB or Not TB: Emerging Mycobacteriaceae Detected in a Human Patient, Tree Shrews, and Soil Zhen Yun Siew, Chia Ting Khoo, Ghee Khang Ong, Siti Norazlin Muhamad Nor, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4999589/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract This study investigated the extrapulmonary pathogenesis of a novel Mycobacteroides abscessus subsp. massiliense strain in a young adult, manifesting as a chronic cutaneous infection. Epidemiological investigation proposed pathogen transmission via contaminated Java apple fruit, likely facilitated by tree shrews. The strain, identified through next-generation sequencing, demonstrated resistance to 76.7% of tested antibiotics, and potential heteroresistance among the M. abscessus subsp. massiliense subpopulations were suggested. Mycobacteroides abscessus complex (MAC) is a group of non-tuberculous mycobacteria, typically associated with soil and water, and poses significant clinical challenges due to its resistance to conventional treatments and its similarity to Mycobacterium tuberculosis . The findings underscore the necessity for further genetic studies to confirm heteroresistant subpopulations and address the complexities of MAC infections. Mycobacterium Mycobacteroides abscessus Emerging infectious disease Epidemiology Antibiotic Tree shrew Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Mycobacteroides abscessus complex (MAC) is a group of non-tuberculous Mycobacteroides abscessus subspecies sharing a similar genomic relationship, including Mycobacteroides abscessus subspecies abscessus (MA), Mycobacteroides abscessus subspecies massiliense , and Mycobacteroides abscessus subspecies bolletii [ 1 , 2 ]. Intriguingly, MAC is not only predominantly detected in natural mineral-rich soils but is also found in biofilms within stream water and domestic shower fixtures [ 3 , 4 ]. However, Johansen et al. [ 5 ] recently argued that human-to-human transmission might be a more significant source of MA infection than contaminated soil and water. In addition to natural sources, MAC biofilms are also discovered in hospital equipment, reused acupuncture needles, and tattoo instruments or ink [ 6 – 8 ]. Exposure to such contaminated sources frequently results in acquired or nosocomial chronic pulmonary disease followed by extrapulmonary infections, including the integumentary, circulatory, central nervous, ocular, and musculoskeletal systems, which resemble the pathogenicity of Mycobacterium tuberculosis complex [ 1 , 9 , 10 ]. It is intuitive that, aside from direct contact, MAC pulmonary infections are transmitted through aerosols, as demonstrated in vivo [ 11 ]. MAC produces membrane glycopeptidolipids, proteins, enzymes, and surface antigens that facilitate infection in pulmonary alveolar tissues, immune evasion, and severe inflammation [ 12 ]. Analogous to tuberculous mycobacteria, MAC commonly induces caseating granulomatous inflammation in pulmonary and extrapulmonary sites, particularly the skin [ 8 , 9 ]. This histological finding is a typical characteristic of both tuberculous and non-tuberculous mycobacteria (NTM). Ultimately, MAC infection leads to complications of disseminated disease and even fatality [ 1 , 13 ]. In the previous century, MAC infection was mistaken for tuberculous mycobacteria when it was first isolated from chronic abscesses of knee synovium and gluteal tissues [ 14 ]. Until recently, the Centers for Disease Control and Prevention (CDC) reported that extrapulmonary NTM infections are relatively less common than pulmonary ones [ 15 ]. In Malaysia, MAC has been one of the most significant causative pathogens of NTM infections from 2018 to 2022 [ 16 ]. Patients who suffer from primary or secondary lung conditions (cystic fibrosis, bronchiectasis, emphysema) and immunodeficiency diseases (glucocorticoid use, human immunodeficiency virus infection) are at increased risk of acquiring MAC infections and their complications [ 9 , 17 , 18 ]. Remarkably, cutaneous lesions caused by MAC are reported to be associated with acupuncture and tattooing [ 7 , 8 ]. It is widely acknowledged that MAC are superbug bacteria due to their resistance to various antibiotics typically used to treat NTM infections, including amikacin and tigecycline, as well as their tolerability to commonly used surface disinfectants [ 6 , 19 ]. Therefore, it is evident that MAC diseases are difficult to manage and easily neglected owing to their clinical resemblance to tuberculous diseases. Despite tuberculous mycobacterial infection receiving more attention due to being a notifiable disease in Malaysia, it is wise to recognize MAC as a clinically relevant pathogen in medical practice. In this article, the extrapulmonary pathogenesis of a mycobacterium species with a case presentation of cutaneous mycobacterium infection in a young immunocompetent adult is discussed. The same mycobacterium species was isolated from the patient and animal samples and identified through prokaryote DNA barcoding. The genome of the mycobacterium isolate was then sequenced through next-generation sequencing (NGS). Methodology Case Study A healthy young adult male Asian (Chinese ethnic) visited the hospital due to a chronic skin infection that lasted for at least 8 months. The patient was examined and a series of pathogen isolation and identification procedures were performed. He was then treated with antibiotics similar to the case described by Tiong et al. [ 20 ] and Huth et al. [ 13 ]. Epidemiological Investigation and Sample Collection A few locations in the Selangor state near the patient’s house were inspected. An area where the patient visited before the skin inflammation presented was observed with some animal activities. The tree in that area was identified using PlantNet [ 21 , 22 ] and also morphological identification. The small mammal was identified and faecal samples were collected. Briefly, a plastic sheet was fixed on the ground of locations with frequent tree shrew activities observed. Bananas were placed in the middle of the plastic sheet. Wet faeces and urine were collected immediately with a swab (as described in a companion article submitted) [ 23 ]. This non-invasive method did not involve trapping and hurting the animal. The soil sample from the vicinity was collected as well. Pathogen Isolation The virus isolation protocol was performed as described previously [ 24 ]. Briefly, all the collected samples from the patient and animal were pooled and co-cultured with Vero (ATCC CCL81) and C6/36 (ATCC CRL1660) cells and observed for cytopathic effects (CPE). Furthermore, bacterial and fungal isolations were performed. The samples were streaked onto Nutrient agar (NA), Sabouraud dextrose agar (SDA) and Sheep blood agar (SBA). Then, colonies formed on the agar were further cultured on different selective agar which include Columbia nalidixic acid agar (CNA) and Löwenstein–Jensen agar (LJ). All agar were purchased from Thermo Fisher Scientific, USA. The discovery of the mycobacteria from the tree shrew sample was accidental as described in our companion paper [ 23 ]. Prokaryote DNA Barcoding and Sanger Sequencing DNA barcoding of isolated colonies was conducted using the Bacterial DNA Barcoding Kit (1st BASE, Malaysia) adhering to the manufacturer’s protocol. The PCR amplification protocol of dos Santos et al. [ 25 ] was slightly modified. The universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3') were employed, incorporating a C-to-M degenerate nucleotide substitution in the 27F primer. The thermal conditions were as follows: 2 min of hot-start at 95°C; 25–35 cycles of denaturation (96°C for 30 sec), annealing (53°C − 58°C for 40 sec), and extension (72°C for 2 minutes); final extension step at 72°C for 3 min. Then, the DNA band obtained from gel electrophoresis was extracted using the PrimeWay Gel Extraction/PCR Purification Kit (1st BASE, Malaysia). The purified PCR products were subjected to bidirectional sequencing with universal primers 785F and 907R using BigDye® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, USA). Whole Genome Sequencing, Genome Analysis and Data Repository DNA samples were analyzed for purity and processed using the Illumina DNA Library Prep according to the manufacturer’s instructions. Samples were sequenced on the Illumina Novaseq 6000 system (Illumina, USA). Libraries were normalized to 1.75nM and pooled before loading into the NovaSeq sequencing platform. The output of the demultiplexing was raw sequencing data, which is also known as FASTQ files. Subsequently, the paired-end reads were aligned to the reference genome of Mycobacterium abscessus downloaded from NCBI, using the Illumina DRAGEN (Dynamic Read Analysis for GENomics) Bio-IT Platform. Variant calling was executed using the same platform, producing a Variant Call File (VCF). The generated variants were subsequently annotated using SnpEff [ 26 ]. De novo assembly was performed using the unmapped reads to generate contigs in FASTA format. The contigs were subsequently processed with the Whole Genome Alignment pipeline built in CLC Genomics Workbench (Qiagen, Germany), which includes analysis of Average Nucleotide Identity (ANI), Phylogenetic Tree and Heatmap Construction, and Multiple Sequence Alignment (MSA). The obtained sequence was also counter-checked using BLAST-XYPlot Viewer [ 27 ] using the following parameters: Blast Type: Genome; Cut Off E-value: 1e-08; Results Limit: 50000. The newly isolated strain was designated as Mycobacteroides abscessus subsp. massiliense strain Siew/H.sapiens/Malaysia/2023 and Mycobacteroides abscessus subsp. massiliense strain SIEW/TupaiaGlis/UNM/2023. Their 16S rRNA (GenBank accession: PP939676) and genome (GenBank accession: CP163267) sequences were submitted to the National Center for Biotechnology Information (NCBI) GenBank ( https://www.ncbi.nlm.nih.gov/genbank/ ). The genome sequence was redeposited under BioProject PRJNA1119049 and BioSample SAMN41636034. Antibiotic Screening Antibiotic screening was performed using the disk diffusion (Kirby-Bauer) method according to the Clinical and Laboratory Standards Institute (CLSI) [ 28 , 29 ]. Briefly, a bacterial suspension containing mycobacteria at 0.5 McFarland (approximately OD600 0.08–0.1) was evenly distributed on Mueller Hinton II Agar (Cation-Adjusted Mueller Hinton Agar, CAMHB) (Thermo Fisher Scientific, USA). The antibiotic disks used in this study were Hexa G-Plus 20, Dodeca Staphylococci-2, and Icosa G-I Plus, which were obtained from HiMedia, India. Escherichia coli (Migula) Castellani and Chalmers (ATCC 8739) (denoted as EC) and Staphylococcus aureus subsp. aureus Rosenbach (ATCC 11632) (denoted as SA) were used as positive controls. In total, five independent experiments were performed. Results and Discussion Case Presentation A healthy young Chinese man has been experiencing relapsing-remitting itchy rashes on his hands, particularly on the left little, ring, and middle fingers, since late 2022 or early 2023. For the first few months, the patient applied common topical antibiotics, but saw no improvement. The rashes were characterized by small, superficial vesicles or blisters, measuring 1–2 mm in diameter. Lesions were present, with those on the left ring and little fingers being more severe. The lesions tended to recur and occasionally formed small vesicles that would rupture, leaving multiple small punctate erosions. Through successful isolation and next-generation sequencing (NGS), the pathogen was identified as Mycobacteroides abscessus subsp. massiliense (hereafter referred to as "MAM"). The patient’s condition eventually improved after treatment with clarithromycin, tigecycline, and amikacin (Fig. 1 ). Fortunately, the patient’s condition did not deteriorate following treatment. Notably, infections caused by mycobacteria can be fatal, particularly in cases of endocarditis, meningitis, and disseminated infection [ 30 – 33 ]. The significant improvement observed in the patient’s condition may be attributed to his overall health and immunocompetence. Furthermore, it is possible that the MAM infection was confined to a localized cutaneous site, without dissemination to other organs. Epidemiological Investigation Tree shrews ( Tupaia glis ), squirrels, fruit bats ( Cynopterus brachyotis ), civets, birds, and other animals were observed near the trees the patient visited before the onset of symptoms. These trees were identified as Java apple ( Syzygium samarangense or Syzygium aqueum ) trees (Fig. 2 ). Small mammals and humans often visit these trees for the Java apple, which might serve as a potential pathway for pathogen transmission to humans (Fig. 2 d). As illustrated in Fig. 2 d, a tree shrew may have picked up MAM from the soil, as M. abscessus is widely distributed in the environment [ 1 ]. The Java apple could have become contaminated with MAM when the tree shrew moved around the tree. Ultimately, MAM was transmitted to humans when the contaminated Java apple was collected. Pathogen Isolation After 3 to 5 days of co-culturing samples with Vero and C6/36 cells, cytopathic effects (CPE) can be observed. The CPE in the cell culture appeared very similar to a viral infection. However, it was caused by MAM, not a viral infection. MAM lyses all the cells, even at very low densities, demonstrating significant cytotoxicity compared to some other bacteria. Additionally, the isolation culture was initially mistaken for a virus because the medium never turned cloudy, and debris and small fragments were observed (Fig. 3 ). Furthermore, 5 days after the patient and tree shrew samples were streaked onto different agar plates, tiny white colonies appeared on all agar plates (Table 1 ). These colonies were collected, and DNA barcoding was performed. For pooled soil samples, bacterial colonies with colour and morphology similar to those of the tree shrew and patient samples were selected, and their 16S rRNA was sequenced as well. Table 1 Results of microbe isolation using different agar Types of Agar Colony Formation Controls Nutrient agar Positive N/A Sabouraud dextrose agar Positive Sheep blood agar Positive Columbia nalidixic acid agar Positive SA: Positive; EC: Negative Löwenstein–Jensen agar Positive SA: Negative; EC: Negative N/A, not applicable; SA, Staphylococcus aureus subsp. aureus Rosenbach (ATCC 11632); EC, Escherichia coli (Migula) Castellani and Chalmers (ATCC 8739). Prokaryote DNA Barcoding and Sanger Sequencing Prokaryote DNA barcoding of the patient, tree shrew, and soil samples revealed that Mycobacteroides abscessus subsp. massiliense strain Siew/H.sapiens/Malaysia/2023 (GenBank accession: PP939676) was the only bacterium detected in all three sample sources. However, the approximately 1500 base pairs (bp) of the 16S rRNA sequence are insufficient to determine the subspecies of MAC, as it has 100% nucleotide sequence identity among the three subspecies of MAC. Therefore, if genome sequencing is not available, subspecies identification of MAC often requires sequencing of specific genes (genotyping) or MALDI-TOF mass spectrometry (proteotyping) [ 2 , 34 – 36 ]. In this study, the genome of the isolated MAM strain was sequenced. Whole Genome Sequencing and Genome Analysis The genome of MAM was sequenced and deposited in GenBank. This newly isolated strain was designated Mycobacteroides abscessus subsp. massiliense strain SIEW/TupaiaGlis/UNM/2023 (GenBank accession: CP163267) under BioProject PRJNA1119049 and BioSample SAMN41636034. The pairwise comparison table and phylogenetic analysis (Fig. 4 ) indicated that MAM aligns best with Mycobacteroides abscessus M154 (GenBank accession: NZ_AJMA00000000), with an alignment percentage (AP) of 74.31%. Additionally, MAM has the highest average nucleotide identity (ANI) with Mycobacteroides abscessus M159 (GenBank accession: NZ_AJSD00000000) at 99.53%. Both reference strains are from Malaysia. The BLAST results using BLAST-XYPlot Viewer were consistent with the genome pairwise comparison table and phylogenetic analysis. Antibiotic Screening In total, 30 antibiotics (some in combination) were tested, and only 7 (23.3%) were consistently effective against MAM across all independent experiments (Table 2 ). These effective antibiotics included erythromycin, gentamicin, azithromycin, amikacin, clarithromycin, tigecycline, and pristinomycin. Additionally, MAM was susceptible to linezolid at 5 days post-treatment (DPT), but resistance developed by 7 DPT. A similar pattern was observed with teicoplanin (TEI) and moxifloxacin (MO). However, some replicates exhibited resistance to TEI and MO even at 5 DPT. This suggests that heteroresistance may have occurred within the subpopulations of this isolated MAM strain. Theoretically, there could be at least two subpopulations of MAM, one resistant to TEI and/or MO and the other susceptible (Fig. 5 ) [ 37 ]. Conclusion In summary, an epidemiological investigation was conducted following a patient’s report of a chronic skin infection. The investigation indicated that the tree shrew might serve as a potential carrier, transferring the pathogen—commonly found in soil—to Java apple fruit. The patient likely contracted the pathogen through contact with the contaminated fruit. Fortunately, the patient’s condition improved significantly after a series of treatments. Next-generation sequencing identified the pathogen as a novel strain of Mycobacteroides abscessus subsp. massiliense , which exhibited resistance to 76.7% of the antibiotics tested. The possibility of heteroresistance among subpopulations of the isolated strain could be due to the development of antibiotic resistance and variability in the strain’s susceptibility to certain antibiotics. However, this study has limitations: the minimum inhibitory concentration (MIC) test was not performed, and further isolation and genetic studies are necessary to confirm the presence of heteroresistant subpopulations. Statements and Declarations Ethics approval and consent to participate Animal ethics is not required in this study as no trapping, catching or invasive methods were used to collect the animal samples. Informed consent was obtained from the participants to participate in the current study. The patient agrees and supports this submission for publication. All procedures performed involving a human patient in this study were following the ethical standards of the FOSE Faculty Research Integrity and Research Ethics Committee (FOSE FRIREC) from the University of Nottingham Malaysia, adhering to the tenets of the Declaration of Helsinki. This study was approved (application ID: KV230924) by the FOSE FRIREC from the University of Nottingham Malaysia. Acknowledgements We extend our sincere gratitude to the lab technicians and staff from the hospital and the University of Nottingham Malaysia for their invaluable contributions and support. Author Contributions Siew Z.Y. contributed to the whole study. Khoo C. T. & Muhamad Nor S. N. contributed to the microbiology works and antibiotic tests. Ong G. K., Leong P.P., Wong S.T., Low D. E. and Voon K. contributed to the epidemiological study and case presentation. Tan B. S. and Leong C. O. contributed to the molecular works and next-generation sequencing. All authors were involved in writing and reviewing the manuscript. Funding This study was supported in part by the UNM pump priming grant (F0013.54.04). Competing Interests The authors have no competing interests to declare that are relevant to the content of this article. 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Pathogens and disease 75(3):10.1093/femspd/ftx020. https://doi.org/10.1093/femspd/ftx020 Table Table 2 is available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Table.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 16 Oct, 2024 Reviews received at journal 15 Oct, 2024 Reviews received at journal 11 Oct, 2024 Reviewers agreed at journal 08 Oct, 2024 Reviewers agreed at journal 08 Oct, 2024 Reviewers invited by journal 01 Oct, 2024 Editor assigned by journal 30 Sep, 2024 Submission checks completed at journal 30 Sep, 2024 First submitted to journal 29 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4999589","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":366591963,"identity":"737411b0-e47d-4fb3-b7f1-511d39b400dc","order_by":0,"name":"Zhen Yun Siew","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYDACZjApIQciDzwgQYuNMVhLAgl2pSU2gCiitMi3Mx+T/Nl2OH1+2OGHQFvs5HQbCGgxOMyWJiHZdjh34+00A6CWZGOzA4S0MPOY3TAEaZmdANJyIHEbIS3yzfzfbiQCHWY4O/0DcVoYDvOw3TjYlpYgL51DpC1Av5j/bDhnY7hBOqfgQIIBEX6R7z/82PBHmYS8/Oz0zR8+VNjJEdQCBoxsQOvAKg2IUQ4Gf4DWNRCtehSMglEwCkYaAACmfUZjhZezmgAAAABJRU5ErkJggg==","orcid":"","institution":"University of Nottingham Malaysia","correspondingAuthor":true,"prefix":"","firstName":"Zhen","middleName":"Yun","lastName":"Siew","suffix":""},{"id":366591966,"identity":"3b4a0f82-63a9-4eb8-9338-97e8bb321666","order_by":1,"name":"Chia Ting Khoo","email":"","orcid":"","institution":"University of Nottingham Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Chia","middleName":"Ting","lastName":"Khoo","suffix":""},{"id":366591969,"identity":"ca76e017-62eb-4e23-9f88-52747397a9d6","order_by":2,"name":"Ghee Khang Ong","email":"","orcid":"","institution":"IMU University","correspondingAuthor":false,"prefix":"","firstName":"Ghee","middleName":"Khang","lastName":"Ong","suffix":""},{"id":366591970,"identity":"79ce1bab-7205-449b-a140-000aaacf9a3b","order_by":3,"name":"Siti Norazlin Muhamad Nor","email":"","orcid":"","institution":"University of Nottingham Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Siti","middleName":"Norazlin Muhamad","lastName":"Nor","suffix":""},{"id":366591971,"identity":"e83866e0-9f06-440c-b001-e3375c652bd1","order_by":4,"name":"Pooi Pooi Leong","email":"","orcid":"","institution":"University Tunku Abdul Rahman","correspondingAuthor":false,"prefix":"","firstName":"Pooi","middleName":"Pooi","lastName":"Leong","suffix":""},{"id":366591972,"identity":"c1481c4a-2dfe-471f-94fa-744099f40d26","order_by":5,"name":"Siew Tung Wong","email":"","orcid":"","institution":"IMU University","correspondingAuthor":false,"prefix":"","firstName":"Siew","middleName":"Tung","lastName":"Wong","suffix":""},{"id":366591973,"identity":"746844c6-063b-4f6d-b525-34a44ba28de5","order_by":6,"name":"Boon Shing Tan","email":"","orcid":"","institution":"AGTC Genomics","correspondingAuthor":false,"prefix":"","firstName":"Boon","middleName":"Shing","lastName":"Tan","suffix":""},{"id":366591974,"identity":"b69a7fa3-1d5a-44bc-a179-507a51507340","order_by":7,"name":"Chee-Onn Leong","email":"","orcid":"","institution":"AGTC Genomics","correspondingAuthor":false,"prefix":"","firstName":"Chee-Onn","middleName":"","lastName":"Leong","suffix":""},{"id":366591976,"identity":"47383172-70f1-43a0-9634-831263f308fd","order_by":8,"name":"Dyoi-E Low","email":"","orcid":"","institution":"Pantai Hospital Cheras, Taman Cheras Makmur","correspondingAuthor":false,"prefix":"","firstName":"Dyoi-E","middleName":"","lastName":"Low","suffix":""},{"id":366591979,"identity":"c457c69c-d8d8-455d-8925-fe6d1dfe2b56","order_by":9,"name":"Kenny Voon","email":"","orcid":"","institution":"University of Nottingham Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Kenny","middleName":"","lastName":"Voon","suffix":""}],"badges":[],"createdAt":"2024-08-29 18:38:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4999589/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4999589/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69236352,"identity":"d1c43d41-655a-4476-8748-146821013017","added_by":"auto","created_at":"2024-11-18 09:44:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":244689,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Ruptured lesions (red arrow) were observed. Sometimes, multiple lesions may rupture and bleed at the same location. Occasionally, a few lesions may develop concurrently, presenting with bleeding as the most severe manifestation. The RF and LF were more reddish and swelled compared to IF and MF. (b) The condition of RF and LF improved slightly but lesions were still present. The relapsing-remitting conditions (from a to b, then b to a) occurred from time to time for at least 8 months. (c) The condition of the RF and LF improved greatly after antibiotic treatments. IF, index finger; MF, middle finger; RF, ring finger; LF, little finger.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4999589/v1/47db23c5ca637aa61c265484.png"},{"id":69237036,"identity":"99c9f8b2-0aa8-46b0-963e-c5a43b3dba81","added_by":"auto","created_at":"2024-11-18 09:52:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":516832,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Java apple (yellow arrow) and white flower (red arrow) can be observed from the tree. (b) A tree shrew (white arrow) was observed with a Java apple in its mouth. (c) Civet (blue arrow) was also observed at night. (d) Possible mechanism of pathogen cross-species transmission from carrier/vector (animal) to humans. Created with BioRender.com.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4999589/v1/f1c6bcf2ba9760877d24a483.png"},{"id":69236350,"identity":"6ac00a42-c412-4fcc-ba6d-1906b1445e80","added_by":"auto","created_at":"2024-11-18 09:44:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":799005,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The culture media turned yellow and cloudy when co-cultured with SA, turned cloudy without changing colour when co-cultured with EC, and showed no obvious changes when co-cultured with MAM. A very high density of bacteria can be observed in the co-culture of Vero cells with SA (b \u0026amp; c) and EC (d \u0026amp; e). The density of MAM (f \u0026amp; g) is relatively lower than that of SA and EC, and cell debris can be observed. The micrographs were captured using the Zeiss Inverted Microscope at 100X (b, d \u0026amp; f) and 400X (c, e \u0026amp; g) magnifications. SA, \u003cem\u003eStaphylococcus aureus subsp. aureus\u003c/em\u003eRosenbach (ATCC 11632); EC, \u003cem\u003eEscherichia coli\u003c/em\u003e (Migula) Castellani and Chalmers (ATCC 8739); MAM, \u003cem\u003eMycobacteroides abscessus subspecies massiliense\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4999589/v1/dc30c349a49980e797696fce.png"},{"id":69237035,"identity":"bda51ee0-5043-4342-bb2d-a3a419b8c568","added_by":"auto","created_at":"2024-11-18 09:52:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":295821,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Pairwise comparison table of \u003cem\u003eMycobacteroides abscessus subsp. massiliense \u003c/em\u003estrain SIEW/TupaiaGlis/UNM/2023 (highlighted in yellow) and reference strains. The upper-right values represent the Average Nucleotide Identity (ANI), and the lower-left values represent the Alignment Percentage (AP). These two different values are separated by a blank diagonal line. (b) Phylogenetic tree constructed based on ANI value.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4999589/v1/e621b625f79782d4a775b714.png"},{"id":69236351,"identity":"a68f871b-7759-4088-a375-9a5eb0f1fc60","added_by":"auto","created_at":"2024-11-18 09:44:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":53344,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical illustration of theoretical heteroresistance among subpopulations of \u003cem\u003eMycobacteroides abscessus\u003c/em\u003e subsp. \u003cem\u003emassiliense\u003c/em\u003estrain SIEW/TupaiaGlis/UNM/2023 (MAM).The illustration depicts at least two subpopulations of MAM: one resistant to antibiotics A and/or B, and another that is susceptible. Additionally, the resistant subpopulation may exhibit resistance to multiple antibiotics. Created with BioRender.com.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4999589/v1/ea3de462deb7ad7a118b2338.png"},{"id":69237043,"identity":"167017da-e08e-4dfd-a4ca-d18cbcf6e684","added_by":"auto","created_at":"2024-11-18 09:52:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2543119,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4999589/v1/1e411c14-7626-4c65-932a-2ead335d29b9.pdf"},{"id":69236354,"identity":"d60ff639-a52a-44c8-9c87-77038687db76","added_by":"auto","created_at":"2024-11-18 09:44:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17479,"visible":true,"origin":"","legend":"","description":"","filename":"Table.docx","url":"https://assets-eu.researchsquare.com/files/rs-4999589/v1/6395efd6de80630e5ba54d7b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"TB or Not TB: Emerging Mycobacteriaceae Detected in a Human Patient, Tree Shrews, and Soil","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eMycobacteroides abscessus\u003c/em\u003e complex (MAC) is a group of non-tuberculous \u003cem\u003eMycobacteroides abscessus\u003c/em\u003e subspecies sharing a similar genomic relationship, including \u003cem\u003eMycobacteroides abscessus subspecies abscessus\u003c/em\u003e (MA), \u003cem\u003eMycobacteroides abscessus subspecies massiliense\u003c/em\u003e, and \u003cem\u003eMycobacteroides abscessus subspecies bolletii\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Intriguingly, MAC is not only predominantly detected in natural mineral-rich soils but is also found in biofilms within stream water and domestic shower fixtures [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, Johansen et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] recently argued that human-to-human transmission might be a more significant source of MA infection than contaminated soil and water.\u003c/p\u003e \u003cp\u003eIn addition to natural sources, MAC biofilms are also discovered in hospital equipment, reused acupuncture needles, and tattoo instruments or ink [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Exposure to such contaminated sources frequently results in acquired or nosocomial chronic pulmonary disease followed by extrapulmonary infections, including the integumentary, circulatory, central nervous, ocular, and musculoskeletal systems, which resemble the pathogenicity of Mycobacterium tuberculosis complex [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It is intuitive that, aside from direct contact, MAC pulmonary infections are transmitted through aerosols, as demonstrated in vivo [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. MAC produces membrane glycopeptidolipids, proteins, enzymes, and surface antigens that facilitate infection in pulmonary alveolar tissues, immune evasion, and severe inflammation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnalogous to tuberculous mycobacteria, MAC commonly induces caseating granulomatous inflammation in pulmonary and extrapulmonary sites, particularly the skin [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This histological finding is a typical characteristic of both tuberculous and non-tuberculous mycobacteria (NTM). Ultimately, MAC infection leads to complications of disseminated disease and even fatality [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the previous century, MAC infection was mistaken for tuberculous mycobacteria when it was first isolated from chronic abscesses of knee synovium and gluteal tissues [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Until recently, the Centers for Disease Control and Prevention (CDC) reported that extrapulmonary NTM infections are relatively less common than pulmonary ones [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In Malaysia, MAC has been one of the most significant causative pathogens of NTM infections from 2018 to 2022 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Patients who suffer from primary or secondary lung conditions (cystic fibrosis, bronchiectasis, emphysema) and immunodeficiency diseases (glucocorticoid use, human immunodeficiency virus infection) are at increased risk of acquiring MAC infections and their complications [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Remarkably, cutaneous lesions caused by MAC are reported to be associated with acupuncture and tattooing [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is widely acknowledged that MAC are superbug bacteria due to their resistance to various antibiotics typically used to treat NTM infections, including amikacin and tigecycline, as well as their tolerability to commonly used surface disinfectants [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Therefore, it is evident that MAC diseases are difficult to manage and easily neglected owing to their clinical resemblance to tuberculous diseases. Despite tuberculous mycobacterial infection receiving more attention due to being a notifiable disease in Malaysia, it is wise to recognize MAC as a clinically relevant pathogen in medical practice.\u003c/p\u003e \u003cp\u003eIn this article, the extrapulmonary pathogenesis of a mycobacterium species with a case presentation of cutaneous mycobacterium infection in a young immunocompetent adult is discussed. The same mycobacterium species was isolated from the patient and animal samples and identified through prokaryote DNA barcoding. The genome of the mycobacterium isolate was then sequenced through next-generation sequencing (NGS).\u003c/p\u003e"},{"header":"Methodology","content":"\u003cdiv id=\"Sec3\" type=\"CaseStudy\" class=\"Section2\"\u003e \u003ch2\u003eCase Study\u003c/h2\u003e \u003cp\u003eA healthy young adult male Asian (Chinese ethnic) visited the hospital due to a chronic skin infection that lasted for at least 8 months. The patient was examined and a series of pathogen isolation and identification procedures were performed. He was then treated with antibiotics similar to the case described by Tiong et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and Huth et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEpidemiological Investigation and Sample Collection\u003c/h3\u003e\n\u003cp\u003eA few locations in the Selangor state near the patient\u0026rsquo;s house were inspected. An area where the patient visited before the skin inflammation presented was observed with some animal activities. The tree in that area was identified using PlantNet [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and also morphological identification. The small mammal was identified and faecal samples were collected. Briefly, a plastic sheet was fixed on the ground of locations with frequent tree shrew activities observed. Bananas were placed in the middle of the plastic sheet. Wet faeces and urine were collected immediately with a swab (as described in a companion article submitted) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This non-invasive method did not involve trapping and hurting the animal. The soil sample from the vicinity was collected as well.\u003c/p\u003e\n\u003ch3\u003ePathogen Isolation\u003c/h3\u003e\n\u003cp\u003eThe virus isolation protocol was performed as described previously [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, all the collected samples from the patient and animal were pooled and co-cultured with Vero (ATCC CCL81) and C6/36 (ATCC CRL1660) cells and observed for cytopathic effects (CPE). Furthermore, bacterial and fungal isolations were performed. The samples were streaked onto Nutrient agar (NA), Sabouraud dextrose agar (SDA) and Sheep blood agar (SBA). Then, colonies formed on the agar were further cultured on different selective agar which include Columbia nalidixic acid agar (CNA) and L\u0026ouml;wenstein\u0026ndash;Jensen agar (LJ). All agar were purchased from Thermo Fisher Scientific, USA. The discovery of the mycobacteria from the tree shrew sample was accidental as described in our companion paper [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eProkaryote DNA Barcoding and Sanger Sequencing\u003c/h3\u003e\n\u003cp\u003eDNA barcoding of isolated colonies was conducted using the Bacterial DNA Barcoding Kit (1st BASE, Malaysia) adhering to the manufacturer\u0026rsquo;s protocol. The PCR amplification protocol of dos Santos et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] was slightly modified. The universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3') were employed, incorporating a C-to-M degenerate nucleotide substitution in the 27F primer. The thermal conditions were as follows: 2 min of hot-start at 95\u0026deg;C; 25\u0026ndash;35 cycles of denaturation (96\u0026deg;C for 30 sec), annealing (53\u0026deg;C \u0026minus;\u0026thinsp;58\u0026deg;C for 40 sec), and extension (72\u0026deg;C for 2 minutes); final extension step at 72\u0026deg;C for 3 min. Then, the DNA band obtained from gel electrophoresis was extracted using the PrimeWay Gel Extraction/PCR Purification Kit (1st BASE, Malaysia). The purified PCR products were subjected to bidirectional sequencing with universal primers 785F and 907R using BigDye\u0026reg; Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, USA).\u003c/p\u003e\n\u003ch3\u003eWhole Genome Sequencing, Genome Analysis and Data Repository\u003c/h3\u003e\n\u003cp\u003eDNA samples were analyzed for purity and processed using the Illumina DNA Library Prep according to the manufacturer\u0026rsquo;s instructions. Samples were sequenced on the Illumina Novaseq 6000 system (Illumina, USA). Libraries were normalized to 1.75nM and pooled before loading into the NovaSeq sequencing platform. The output of the demultiplexing was raw sequencing data, which is also known as FASTQ files. Subsequently, the paired-end reads were aligned to the reference genome of \u003cem\u003eMycobacterium abscessus\u003c/em\u003e downloaded from NCBI, using the Illumina DRAGEN (Dynamic Read Analysis for GENomics) Bio-IT Platform. Variant calling was executed using the same platform, producing a Variant Call File (VCF). The generated variants were subsequently annotated using SnpEff [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDe novo assembly was performed using the unmapped reads to generate contigs in FASTA format. The contigs were subsequently processed with the Whole Genome Alignment pipeline built in CLC Genomics Workbench (Qiagen, Germany), which includes analysis of Average Nucleotide Identity (ANI), Phylogenetic Tree and Heatmap Construction, and Multiple Sequence Alignment (MSA).\u003c/p\u003e \u003cp\u003eThe obtained sequence was also counter-checked using BLAST-XYPlot Viewer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] using the following parameters: Blast Type: Genome; Cut Off E-value: 1e-08; Results Limit: 50000.\u003c/p\u003e \u003cp\u003eThe newly isolated strain was designated as \u003cem\u003eMycobacteroides abscessus\u003c/em\u003e subsp. \u003cem\u003emassiliense\u003c/em\u003e strain Siew/H.sapiens/Malaysia/2023 and \u003cem\u003eMycobacteroides abscessus\u003c/em\u003e subsp. \u003cem\u003emassiliense\u003c/em\u003e strain SIEW/TupaiaGlis/UNM/2023. Their 16S rRNA (GenBank accession: PP939676) and genome (GenBank accession: CP163267) sequences were submitted to the National Center for Biotechnology Information (NCBI) GenBank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/genbank/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/genbank/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The genome sequence was redeposited under BioProject PRJNA1119049 and BioSample SAMN41636034.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAntibiotic Screening\u003c/h2\u003e \u003cp\u003eAntibiotic screening was performed using the disk diffusion (Kirby-Bauer) method according to the Clinical and Laboratory Standards Institute (CLSI) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Briefly, a bacterial suspension containing mycobacteria at 0.5 McFarland (approximately OD600 0.08\u0026ndash;0.1) was evenly distributed on Mueller Hinton II Agar (Cation-Adjusted Mueller Hinton Agar, CAMHB) (Thermo Fisher Scientific, USA). The antibiotic disks used in this study were Hexa G-Plus 20, Dodeca Staphylococci-2, and Icosa G-I Plus, which were obtained from HiMedia, India. \u003cem\u003eEscherichia coli\u003c/em\u003e (Migula) Castellani and Chalmers (ATCC 8739) (denoted as EC) and \u003cem\u003eStaphylococcus aureus subsp. aureus\u003c/em\u003e Rosenbach (ATCC 11632) (denoted as SA) were used as positive controls. In total, five independent experiments were performed.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eCase Presentation\u003c/h2\u003e\n \u003cp\u003eA healthy young Chinese man has been experiencing relapsing-remitting itchy rashes on his hands, particularly on the left little, ring, and middle fingers, since late 2022 or early 2023. For the first few months, the patient applied common topical antibiotics, but saw no improvement. The rashes were characterized by small, superficial vesicles or blisters, measuring 1\u0026ndash;2 mm in diameter. Lesions were present, with those on the left ring and little fingers being more severe. The lesions tended to recur and occasionally formed small vesicles that would rupture, leaving multiple small punctate erosions. Through successful isolation and next-generation sequencing (NGS), the pathogen was identified as \u003cem\u003eMycobacteroides abscessus\u003c/em\u003e subsp. \u003cem\u003emassiliense\u003c/em\u003e (hereafter referred to as \u0026quot;MAM\u0026quot;). The patient\u0026rsquo;s condition eventually improved after treatment with clarithromycin, tigecycline, and amikacin (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eFortunately, the patient\u0026rsquo;s condition did not deteriorate following treatment. Notably, infections caused by mycobacteria can be fatal, particularly in cases of endocarditis, meningitis, and disseminated infection [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. The significant improvement observed in the patient\u0026rsquo;s condition may be attributed to his overall health and immunocompetence. Furthermore, it is possible that the MAM infection was confined to a localized cutaneous site, without dissemination to other organs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eEpidemiological Investigation\u003c/h2\u003e\n \u003cp\u003eTree shrews (\u003cem\u003eTupaia glis\u003c/em\u003e), squirrels, fruit bats (\u003cem\u003eCynopterus brachyotis\u003c/em\u003e), civets, birds, and other animals were observed near the trees the patient visited before the onset of symptoms. These trees were identified as Java apple (\u003cem\u003eSyzygium samarangense\u003c/em\u003e or \u003cem\u003eSyzygium aqueum\u003c/em\u003e) trees (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Small mammals and humans often visit these trees for the Java apple, which might serve as a potential pathway for pathogen transmission to humans (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). As illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed, a tree shrew may have picked up MAM from the soil, as \u003cem\u003eM. abscessus\u003c/em\u003e is widely distributed in the environment [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. The Java apple could have become contaminated with MAM when the tree shrew moved around the tree. Ultimately, MAM was transmitted to humans when the contaminated Java apple was collected.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003ePathogen Isolation\u003c/h2\u003e\n \u003cp\u003eAfter 3 to 5 days of co-culturing samples with Vero and C6/36 cells, cytopathic effects (CPE) can be observed. The CPE in the cell culture appeared very similar to a viral infection. However, it was caused by MAM, not a viral infection. MAM lyses all the cells, even at very low densities, demonstrating significant cytotoxicity compared to some other bacteria. Additionally, the isolation culture was initially mistaken for a virus because the medium never turned cloudy, and debris and small fragments were observed (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eFurthermore, 5 days after the patient and tree shrew samples were streaked onto different agar plates, tiny white colonies appeared on all agar plates (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). These colonies were collected, and DNA barcoding was performed. For pooled soil samples, bacterial colonies with colour and morphology similar to those of the tree shrew and patient samples were selected, and their 16S rRNA was sequenced as well.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eResults of microbe isolation using different agar\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTypes of Agar\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eColony Formation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControls\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNutrient agar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSabouraud dextrose agar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSheep blood agar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eColumbia nalidixic acid agar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSA: Positive; EC: Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL\u0026ouml;wenstein\u0026ndash;Jensen agar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSA: Negative; EC: Negative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eN/A, not applicable; SA, \u003cem\u003eStaphylococcus aureus subsp. aureus\u003c/em\u003e Rosenbach (ATCC 11632); EC, \u003cem\u003eEscherichia coli\u003c/em\u003e (Migula) Castellani and Chalmers (ATCC 8739).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eProkaryote DNA Barcoding and Sanger Sequencing\u003c/h2\u003e\n \u003cp\u003eProkaryote DNA barcoding of the patient, tree shrew, and soil samples revealed that \u003cem\u003eMycobacteroides abscessus\u003c/em\u003e subsp. \u003cem\u003emassiliense\u003c/em\u003e strain Siew/H.sapiens/Malaysia/2023 (GenBank accession: PP939676) was the only bacterium detected in all three sample sources. However, the approximately 1500 base pairs (bp) of the 16S rRNA sequence are insufficient to determine the subspecies of MAC, as it has 100% nucleotide sequence identity among the three subspecies of MAC. Therefore, if genome sequencing is not available, subspecies identification of MAC often requires sequencing of specific genes (genotyping) or MALDI-TOF mass spectrometry (proteotyping) [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this study, the genome of the isolated MAM strain was sequenced.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eWhole Genome Sequencing and Genome Analysis\u003c/h2\u003e\n \u003cp\u003eThe genome of MAM was sequenced and deposited in GenBank. This newly isolated strain was designated \u003cem\u003eMycobacteroides abscessus\u003c/em\u003e subsp. \u003cem\u003emassiliense\u003c/em\u003e strain SIEW/TupaiaGlis/UNM/2023 (GenBank accession: CP163267) under BioProject PRJNA1119049 and BioSample SAMN41636034. The pairwise comparison table and phylogenetic analysis (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) indicated that MAM aligns best with \u003cem\u003eMycobacteroides abscessus\u003c/em\u003e M154 (GenBank accession: NZ_AJMA00000000), with an alignment percentage (AP) of 74.31%. Additionally, MAM has the highest average nucleotide identity (ANI) with \u003cem\u003eMycobacteroides abscessus\u003c/em\u003e M159 (GenBank accession: NZ_AJSD00000000) at 99.53%. Both reference strains are from Malaysia.\u003c/p\u003e\n \u003cp\u003eThe BLAST results using BLAST-XYPlot Viewer were consistent with the genome pairwise comparison table and phylogenetic analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eAntibiotic Screening\u003c/h2\u003e\n \u003cp\u003eIn total, 30 antibiotics (some in combination) were tested, and only 7 (23.3%) were consistently effective against MAM across all independent experiments (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). These effective antibiotics included erythromycin, gentamicin, azithromycin, amikacin, clarithromycin, tigecycline, and pristinomycin. Additionally, MAM was susceptible to linezolid at 5 days post-treatment (DPT), but resistance developed by 7 DPT. A similar pattern was observed with teicoplanin (TEI) and moxifloxacin (MO). However, some replicates exhibited resistance to TEI and MO even at 5 DPT. This suggests that heteroresistance may have occurred within the subpopulations of this isolated MAM strain. Theoretically, there could be at least two subpopulations of MAM, one resistant to TEI and/or MO and the other susceptible (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e) [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, an epidemiological investigation was conducted following a patient\u0026rsquo;s report of a chronic skin infection. The investigation indicated that the tree shrew might serve as a potential carrier, transferring the pathogen\u0026mdash;commonly found in soil\u0026mdash;to Java apple fruit. The patient likely contracted the pathogen through contact with the contaminated fruit. Fortunately, the patient\u0026rsquo;s condition improved significantly after a series of treatments. Next-generation sequencing identified the pathogen as a novel strain of \u003cem\u003eMycobacteroides abscessus\u003c/em\u003e subsp. \u003cem\u003emassiliense\u003c/em\u003e, which exhibited resistance to 76.7% of the antibiotics tested. The possibility of heteroresistance among subpopulations of the isolated strain could be due to the development of antibiotic resistance and variability in the strain\u0026rsquo;s susceptibility to certain antibiotics. However, this study has limitations: the minimum inhibitory concentration (MIC) test was not performed, and further isolation and genetic studies are necessary to confirm the presence of heteroresistant subpopulations.\u003c/p\u003e"},{"header":"Statements and Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003eAnimal ethics is not required in this study as no trapping, catching or invasive methods were used to collect the animal samples. Informed consent was obtained from the participants to participate in the current study. The patient agrees and supports this submission for publication. All procedures performed involving a human patient in this study were following the ethical standards of the FOSE Faculty Research Integrity and Research Ethics Committee (FOSE FRIREC) from the University of Nottingham Malaysia, adhering to the tenets of the Declaration of Helsinki. This study was approved (application ID: KV230924) by the FOSE FRIREC from the University of Nottingham Malaysia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eWe extend our sincere gratitude to the lab technicians and staff from the hospital and the University of Nottingham Malaysia for their invaluable contributions and support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003eSiew Z.Y. contributed to the whole study. Khoo C. T. \u0026amp;\u0026nbsp;Muhamad Nor S. N. contributed to the microbiology works and antibiotic tests. Ong G. K., Leong P.P., Wong S.T., Low D. E. and Voon K. contributed to the epidemiological study and case presentation. Tan B. S. and Leong C. O. contributed to the molecular works and next-generation sequencing. All authors were involved in writing and reviewing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis study was supported in part by the UNM pump priming grant (F0013.54.04).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u0026nbsp;In addition, funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the article, or in the decision to publish the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement (DAS)\u0026nbsp;\u003c/strong\u003eAll\u0026nbsp;data\u0026nbsp;generated or analysed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLee MR, Sheng WH, Hung CC, Yu CJ, Lee LN, Hsueh PR (2015) Mycobacterium abscessus Complex Infections in Humans. \u003cem\u003eEmerging infectious diseases\u003c/em\u003e 21(9):1638\u0026ndash;1646. https://doi.org/10.3201/2109.141634\u003c/li\u003e\n \u003cli\u003eNg HF, Ngeow YF (2020) A single-gene approach for the subspecies classification of \u003cem\u003eMycobacteroides abscessus\u003c/em\u003e. \u003cem\u003ePathog Dis\u003c/em\u003e 78(8):ftaa055. https://doi.org/10.1093/femspd/ftaa055\u003c/li\u003e\n \u003cli\u003eGlickman CM, Virdi R, Hasan NA et al. 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(2023) Epidemiology of Pulmonary and Extrapulmonary Nontuberculous Mycobacteria Infections at 4 US Emerging Infections Program Sites: A 6-Month Pilot. \u003cem\u003eClin Infect Dis\u003c/em\u003e 77:629-637. https://doi.org/10.1093/cid/ciad214\u003c/li\u003e\n \u003cli\u003eOschmann-Kadenbach AM, Schaudinn C, Borst L et al. (2024) Impact of \u003cem\u003eMycobacteroides abscessus\u003c/em\u003e colony morphology on biofilm formation and antimicrobial resistance. \u003cem\u003eInt J Med Microbiol\u003c/em\u003e 314:151603. https://doi.org/10.1016/j.ijmm.2024.151603\u003c/li\u003e\n \u003cli\u003eTiong CW, Nack T, Tai AYC, Friedman ND (2019) Medical management of atraumatic \u003cem\u003eMycobacterium abscessus\u003c/em\u003e cutaneous infection: A case report. \u003cem\u003eJournal of clinical tuberculosis and other mycobacterial diseases\u003c/em\u003e 17:100132. https://doi.org/10.1016/j.jctube.2019.100132\u003c/li\u003e\n \u003cli\u003eLi D, Shi G, Li J, Chen Y, Zhang S, Xiang S, Jin S (2022) PlantNet: a dual-function point cloud segmentation network for multiple plant species. \u003cem\u003eISPRS J Photogramm Remote Sens\u0026nbsp;\u003c/em\u003e184:243\u0026ndash;263. https://doi.org/10.1016/j.isprsjprs.2022.01.007.\u003c/li\u003e\n \u003cli\u003eYang Z, He W, Fan X, Tjahjadi T (2022) PlantNet: transfer learning-based fine-grained network for high-throughput plants recognition. \u003cem\u003eSoft Comput\u003c/em\u003e 26:10581\u0026ndash;10590. https://doi.org/10.1007/s00500-021-06689-y.\u003c/li\u003e\n \u003cli\u003eAnother submission to Discover medicine by Tang et al. 2024\u003c/li\u003e\n \u003cli\u003eSiew ZY, Lai ZJ, Ho QY, Ter HC, Ho SH, Wong ST, Gani M, Leong PP, Voon K (2023) Bat coronavirus was detected positive from insectivorous bats in Krau Wildlife Reserve Forest. \u003cem\u003eTropical biomedicine\u003c/em\u003e 40(4):462\u0026ndash;470. https://doi.org/10.47665/tb.40.4.012\u003c/li\u003e\n \u003cli\u003edos Santos HRM, Argolo CS, Arg\u0026ocirc;lo-Filho RC, Loguercio LL (2019) A 16S rDNA PCR-based theoretical to actual delta approach on culturable mock communities revealed severe losses of diversity information. \u003cem\u003eBMC Microbiol\u003c/em\u003e 19:74. https://doi.org/10.1186/s12866-019-1446-2\u003c/li\u003e\n \u003cli\u003eCingolani P, Platts A, Wang LL, Coon M, Nguyen T, Wang L, Land SJ, Lu X, Ruden DM (2012) A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. \u003cem\u003eFly\u003c/em\u003e 6(2):80\u0026ndash;92. https://doi.org/10.4161/fly.19695\u003c/li\u003e\n \u003cli\u003ePedraza-P\u0026eacute;rez Y, Cuevas-Vede RA, Canto-G\u0026oacute;mez \u0026Aacute;B, L\u0026oacute;pez-Pliego L, Guti\u0026eacute;rrez-R\u0026iacute;os RM, Hern\u0026aacute;ndez-Lucas I, Rub\u0026iacute;n-Linares G, Mart\u0026iacute;nez-Laguna Y, L\u0026oacute;pez-Olgu\u0026iacute;n JF, Fuentes-Ram\u0026iacute;rez LE (2018) BLAST-XYPlot Viewer: A Tool for Performing BLAST in Whole-Genome Sequenced Bacteria/Archaea and Visualize Whole Results Simultaneously. \u003cem\u003eG3 (Bethesda, Md.)\u003c/em\u003e 8(7):2167\u0026ndash;2172. https://doi.org/10.1534/g3.118.200220\u003c/li\u003e\n \u003cli\u003eWoods GL, Brown-Elliott BA, Conville PS, Desmond EP, Hall GS, Lin G, Pfyffer GE, Ridderhof JC, Siddiqi SH, Wallace RJ Jr, Warren NG, Witebsky FG (2011) \u003cem\u003eSusceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes\u003c/em\u003e. (2nd ed.). Clinical and Laboratory Standards Institute.\u003c/li\u003e\n \u003cli\u003eBrown-Elliott BA, Nash KA, Wallace RJ Jr (2012) Antimicrobial susceptibility testing, drug resistance mechanisms, and therapy of infections with nontuberculous mycobacteria. \u003cem\u003eClinical microbiology reviews\u003c/em\u003e 25(3):545\u0026ndash;582. https://doi.org/10.1128/CMR.05030-11\u003c/li\u003e\n \u003cli\u003eOka K, Morioka H, Eguchi M, Sato Y, Tetsuka N, Iguchi M, Kanematsu T, Fukano H, Hoshino Y, Kiyoi H, Yagi T (2021) Bursitis, Bacteremia, and Disseminated Infection of Mycobacteroides (Mycobacterium) abscessus subsp. massiliense. \u003cem\u003eInternal medicine (Tokyo, Japan)\u003c/em\u003e 60(18):3041\u0026ndash;3045. https://doi.org/10.2169/internalmedicine.6189-20\u003c/li\u003e\n \u003cli\u003eTsai WC, Hsieh HC, Su HM, Lu PL, Lin TH, Sheu SH, Lai WT (2008) Mycobacterium abscessus endocarditis: a case report and literature review. \u003cem\u003eThe Kaohsiung journal of medical sciences\u003c/em\u003e 24(9):481\u0026ndash;486. https://doi.org/10.1016/S1607-551X(09)70005-1\u003c/li\u003e\n \u003cli\u003eMorimoto K, Nakagawa T, Asami T, Morino E, Fujiwara H, Hase I, Tsujimoto Y, Izumi K, Hayashi Y, Matsuda S, Murase Y, Yano R, Takasaki J, Betsuyaku T, Aono A, Goto H, Nishimura T, Sasaki Y, Hoshino Y, Kurashima A et al.\u0026nbsp;(2018) Clinico-microbiological analysis of 121 patients with pulmonary Mycobacteroides abscessus complex disease in Japan - An NTM-JRC study with RIT. \u003cem\u003eRespiratory medicine\u003c/em\u003e 145:14\u0026ndash;20. https://doi.org/10.1016/j.rmed.2018.10.012\u003c/li\u003e\n \u003cli\u003eOmori K, Kitagawa H, Tadera K, Naka Y, Sakamoto S, Kamei N, Nomura T, Shigemoto N, Hattori N, Ohge H (2022) Vertebral osteomyelitis caused by Mycobacteroides abscessus subsp. abscessus resulting in spinal cord injury due to vertebral body fractures. \u003cem\u003eJournal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy\u003c/em\u003e 28(2):290\u0026ndash;294. https://doi.org/10.1016/j.jiac.2021.09.013\u003c/li\u003e\n \u003cli\u003eMinias A, Żukowska L, Lach J, Jagielski T, Strapagiel D, Kim SY, Koh WJ, Adam H, Bittner R, Truden S, Žolnir-Dovč M, Dziadek J (2020)\u0026nbsp;Subspecies-specific sequence detection for differentiation of Mycobacterium abscessus complex. \u003cem\u003eScientific reports\u003c/em\u003e 10(1):16415. https://doi.org/10.1038/s41598-020-73607-x\u003c/li\u003e\n \u003cli\u003eRodr\u0026iacute;guez-Temporal D, Herrera L, Alcaide F, Domingo D, H\u0026eacute;ry-Arnaud G, van Ingen J, Van den Bossche A, Ingebretsen A, Beauruelle C, Terschl\u0026uuml;sen E, Boarbi S, Vila N, Arroyo MJ, M\u0026eacute;ndez G, Mu\u0026ntilde;oz P, Mancera L, Ruiz-Serrano MJ, Rodr\u0026iacute;guez-S\u0026aacute;nchez B (2023) Identification of Mycobacterium abscessus Subspecies by MALDI-TOF Mass Spectrometry and Machine Learning. \u003cem\u003eJournal of clinical microbiology\u003c/em\u003e 61(1):e0111022. https://doi.org/10.1128/jcm.01110-22\u003c/li\u003e\n \u003cli\u003eTakei S, Teramoto K, Sekiguchi Y et al.\u0026nbsp;(2024) Identification of \u003cem\u003eMycobacterium abscessus\u003c/em\u003e using the peaks of ribosomal protein L29, L30 and hemophore-related protein by MALDI-MS proteotyping. \u003cem\u003eSci Rep\u003c/em\u003e 14:11187. https://doi.org/10.1038/s41598-024-61549-7\u003c/li\u003e\n \u003cli\u003eMcIvor A, Koornhof H, Kana BD (2017) Relapse, re-infection and mixed infections in tuberculosis disease. \u003cem\u003ePathogens and disease\u003c/em\u003e 75(3):10.1093/femspd/ftx020. https://doi.org/10.1093/femspd/ftx020\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table ","content":"\u003cp\u003eTable 2 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Medicine](https://link.springer.com/journal/44337)","snPcode":"44337","submissionUrl":"https://submission.springernature.com/new-submission/44337/3","title":"Discover Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mycobacterium, Mycobacteroides abscessus, Emerging infectious disease, Epidemiology, Antibiotic, Tree shrew","lastPublishedDoi":"10.21203/rs.3.rs-4999589/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4999589/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated the extrapulmonary pathogenesis of a novel \u003cem\u003eMycobacteroides abscessus subsp. massiliense\u003c/em\u003e strain in a young adult, manifesting as a chronic cutaneous infection. Epidemiological investigation proposed pathogen transmission via contaminated Java apple fruit, likely facilitated by tree shrews. The strain, identified through next-generation sequencing, demonstrated resistance to 76.7% of tested antibiotics, and potential heteroresistance among the \u003cem\u003eM. abscessus subsp. massiliense\u003c/em\u003e subpopulations were suggested. \u003cem\u003eMycobacteroides abscessus complex\u003c/em\u003e (MAC) is a group of non-tuberculous mycobacteria, typically associated with soil and water, and poses significant clinical challenges due to its resistance to conventional treatments and its similarity to \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e. 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