Application of Genomic Epidemiology of Pathogens to Farmed Yellowtail Fish Mycobacteriosis in Kyushu, Japan.

To investigate mycobacterial cases of farmed yellowtail fish in coastal areas of western Japan (Kagoshima, Kyushu), where aquaculture fisheries are active, Mycobacterium pseudoshottsii, the causative agent, was isolated from six neighboring fishing ports in 2012 and 2013. A phylogenetic ana-lysis revealed that the strains isolated from one fishing port were closely related to those isolated from other regions of Japan, suggesting the nationwide spread of a single strain. However, strains from Japan were phylogenetically distinct from those from the Mediterranean and the United States; therefore, worldwide transmission was not observed based on the limited data obtained on the strains exami-ned in this study. The present results demonstrate that a bacterial genomic ana-lysis of infected cases, a mole-cular epidemiology strategy for public health, provides useful data for estimating the prevalence and transmission pathways of M. pseudoshottsii in farmed fish. A bacterial genome ana-lysis of strains, such as that performed herein, may play an important role in monitoring the prevalence of this pathogen in fish farms and possible epidemics in the future as a result of international traffic, logistics, and trade in fisheries.

Changes in skin discoloration according to clofazimine dosage in nontuberculous mycobacterial pulmonary disease.

Although clofazimine is currently one of the standard regimens for Mycobacterium abscessus, it frequently causes skin discoloration, posing esthetic concerns for patients. We studied thirteen Asian patients with pulmonary nontuberculous mycobacterial disease treated with clofazimine at the NHO Kinki Chuo Chest Medical Center. In three patients (two women and one man) whose dosing regimens were altered owing to skin discoloration, we continuously measured luminance (L*), red-green (a*), and yellow-blue (b*) values (using a colorimeter) in both sun-exposed and sun-unexposed skin areas at each visit. Compared to baseline L* and a* values, the ΔL* values were negative (decreased brightness) and Δa* values were positive (increased redness) while patients received daily clofazimine. After switching to intermittent or reduced dosing, these changes gradually diminished. If such a dose reduction does not affect the therapeutic outcome, an even lower clofazimine dose may be attempted to minimize skin adverse effects.

Efficacy of an oscillating positive expiratory pressure device in patients with Mycobacterium avium complex pulmonary disease.

Patients with Mycobacterium avium complex pulmonary disease (MAC-PD) often suffer from chronic symptoms such as sputum production, which reduces quality of life. Oscillatory positive expiratory pressure (OPEP) devices are used in physiotherapy to promote the clearance of respiratory secretions. We report two cases of improved lung function and improved scores on the Leicester Cough Questionnaire (LCQ) and the Breathlessness, Cough and Sputum Scale (BCSS) after the use of OPEP in patients with MAC-PD where treatment with guideline-based therapy, including amikacin liposome inhalation suspension, had proved ineffective for symptoms. Use of OPEP might maximize the efficacy of therapy and thereby improves outcomes in patients with MAC-PD. It is important to use both guideline-based therapy and OPEP, especially in patients whose health-related quality of life is affected by sputum symptoms. Further prospective studies are warranted to assess the benefit of adding OPEP to guidelines concerning therapy for patients with MAC-PD and sputum symptoms.

A Case of Lymphangioleiomyomatosis Showing the Development of Mycobacterium abscessus subsp. massiliense Infection During Sirolimus Therapy.

Among nontuberculous mycobacterial pulmonary diseases (NTM-PDs), Mycobacterium abscessus species pulmonary disease (MABS-PD) is one of the most severe and intractable infections. We herein report a 45-year-old woman with advanced lymphangioleiomyomatosis (LAM) who developed MABS-PD while undergoing sirolimus therapy. MABS-PD was immediately controlled using antibiotic therapy, although the patient's lung transplant registration was significantly delayed. To our knowledge, this is the first case report on the development of NTM-PD in a patient with LAM before lung transplantation. This case suggests that the early diagnosis and optimal treatment of NTM-PD are crucial in patients with advanced LAM.

Retrospective identification of pathogenic mycobacterial species in fish: Mycobacterium pseudoshottsii YM-3, isolated from a yellowtail fish in 1986 in Kochi, Japan.

The complete genome sequence of mycobacterial strain YM-3, isolated from cultured yellowtail in 1986, was determined. The strain was Mycobacterium pseudoshottsii, a closely related subspecies of Mycobacterium marinum, so the strain was isolated earlier than the first report of the subspecies in 2005.

Unique genomic sequences in a novel Mycobacterium avium subsp. hominissuis lineage enable fine scale transmission route tracing during pig movement.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most prevalent mycobacteria causing non-tuberculous mycobacterial disease in humans and animals. Of note, MAH is a major cause of mycobacterial granulomatous mesenteric lymphadenitis outbreaks in pig populations. To determine the precise source of infection of MAH in a pig farm and to clarify the epidemiological relationship among pig, human and environmental MAH lineages, we collected 50 MAH isolates from pigs reared in Japan and determined draft genome sequences of 30 isolates. A variable number of tandem repeat analysis revealed that most pig MAH isolates in Japan were closely related to North American, European and Russian human isolates but not to those from East Asian human and their residential environments. Historical recombination analysis revealed that most pig isolates could be classified into SC2/4 and SC3, which contain MAH isolated from pig, European human and environmental isolates. Half of the isolates in SC2/4 had many recombination events with MAH lineages isolated from humans in East Asia. To our surprise, four isolates belonged to a new lineage (SC5) in the global MAH population. Members of SC5 had few footprints of inter-lineage recombination in the genome, and carried 80 unique genes, most of which were located on lineage specific-genomic islands. Using unique genetic features, we were able to trace the putative transmission route via their host pigs. Together, we clarify the possibility of species-specificity of MAH in addition to local adaptation. Our results highlight two transmission routes of MAH, one exposure on pig farms from the environment and the other via pig movement. Moreover, our study also warns that the evolution of MAH in pigs is influenced by MAH from patients and their residential environments, even if the MAH are genetically distinct.

Cases of severe Mycobacterium avium lung disease occurred in a married couple caused by identical strain.

Bacteria of the Mycobacterium avium complex, which are environmental organisms found in soil and water, have been found to cause human lung diseases. Although infection is reported to occur in cohabiting patients, the incidence of infection from the single clone remains rarely documented. Herein, we report a case of M. avium lung disease caused by specimens with the same clone strains in a married couple. The wife, a 67-year-old female, had severe M. avium lung disease despite receiving multidrug chemotherapy for eleven years. The husband, a 68-year-old male, died of acute lung injury complicated by M. avium pleurisy. The result of the variable-number tandem-repeat analysis of isolates from serial sputum specimens of both patients indicated that the severe M. avium lung disease in a married couple was caused by the isolates with identical pattern. This case were considered to have acquired clarithromycin resistance during each clinical course, revealing the possibility of infection with a strain that may induce severe pulmonary condition.

Mycobacterium avium Pleurisy with Bronchopleural Fistula Resulting in Rapidly Progressive Respiratory Failure Diagnosed by Transbronchial Autopsy.

Nontuberculous mycobacterial lung disease usually manifests as a chronic pulmonary infection. We herein report a fatal case of Mycobacterium avium pleurisy in a man with a refractory bronchopleural fistula that led to rapidly progressive pneumonia. A post-mortem transbronchial biopsy was performed. Histopathology revealed an acute lung injury pattern and epithelioid granulomas. Variable number tandem repeat analyses and drug susceptibility testing revealed Mycobacterium avium had acquired macrolide resistance during chemotherapy with rifampicin, ethambutol, and clarithromycin. Clinicians should be aware that Mycobacterium avium pleurisy with bronchopleural fistula can lead to fatal pneumonia, especially in patients with persistently positive cultures despite multidrug treatment.

Effect of sputum quality on Mycobacterium avium-intracellulare complex lung disease diagnosis and treatment initiation according to disease type.

We evaluated sputum quality to establish a diagnostic and treatment strategy for Mycobacterium avium-intracellulare complex lung disease (MAC-LD). Of all 905 MAC-LD patients, 564 (62.3%) had nonpurulent sputum. Furthermore, 88 treated patients were associated with purulent sputum. Therefore, sputum quality may be useful for diagnosing MAC-LD and deciding treatment initiation.

Monitoring IgG against Mycobacterium tuberculosis proteins in an Asian elephant cured of tuberculosis that developed from long-term latency.

Tuberculosis (TB) is fatal in elephants, hence protecting elephants from TB is key not only in the conservation of this endangered animal, but also to prevent TB transmission from elephants to humans. Most human TB cases arise from long-term asymptomatic infections. Significant diagnostic challenges remain in the detection of both infection and disease development from latency in elephants due to their huge bodies. In this study, we assessed cryopreserved sera collected for over 16 years, from the first Japanese treatment case of elephant TB. Semi-quantification of IgG levels to 11 proteins showed high detection levels of 3 proteins, namely ESAT6/CFP10, MPB83 and Ag85B. The level of IgG specific to these 3 antigens was measured longitudinally, revealing high and stable ESAT6/CFP10 IgG levels regardless of onset or treatment. Ag85B-specifc IgG levels were largely responsive to onset or treatment, while those of MPB83 showed intermediate responses. These results suggest that ESAT6/CFP10 is immunodominant in both asymptomatic and symptomatic phases, making it useful in the detection of infection. On the other hand, Ag85B has the potential to be a marker for the prediction of disease onset and in the evaluation of treatment effectiveness in elephants.

Comparison of drug-susceptibility patterns and gene sequences associated with clarithromycin and azithromycin resistance in Mycobacterium abscessus complex isolates and evaluation of the accumulation of intrinsic macrolide resistance.

Introduction. Mycobacterium abscessus complex (MABC) is an infectious agent associated with macrolide resistance and treatment failure.Hypothesis/Gap Statement. Despite drug-susceptibility testing for MABC isolates including clarithromycin (CAM), long-term treatment with azithromycin (AZM) for MABC disease is recommended.Aim. We compared phenotypic and genotypic resistance to AZM and CAM in clinical isolates and evaluated the accumulation of intrinsic macrolide resistance (AIM) and morphological changes by macrolides exposure.Methodology. Forty-nine isolates were characterized regarding erm(41) sequevars. Sequencing data were compared to the nucleotide sequence of rrl and whiB7. The AIM MIC was performed in three reference strains and 15 isolates were randomized [each set of five isolates with M. abscessus subsp. abscessus (MAA) T28, MAA C28 and subsp. massiliense (MAM)].Results. The 49 isolates were distributed as 24 MAA T28, 5 MAA C28 and 20 MAM. The MIC50 values to CAM at day 3 in MAA T28, C28 and MAM were 1, 0.12 and 0.12 µg ml-1, while those at day 14 were 32, 0.5 and 0.12 µg ml-1, respectively. The AZM-MIC50 values at day 3 of the above isolates were 4, 0.25 and 0.5 µg ml-1, while those at day 14 were >64, 0.5 and 0.5 µg ml-1, respectively. Neither mutations in rrl of MAA T28 with acquired resistance nor deletions in whiB7 of MAA T28 without inducible resistance were observed . For AIM MIC, MAA T28 showed that the time-to-detection of AZM resistance was significantly faster over that of CAM (P<0.05). Morphological changes were not determined in all isolates.Conclusion. Our findings did not support the suggestion for the preferential use of AZM for, at least, MAA T28 disease due to the high-level MIC value and the increased AIM. The long duration of AZM-based treatment eventually may favour the emergence of isolates with a high-level of intrinsic resistance.

Resumption/efficacy and safety of an azithromycin-containing regimen against Mycobacterium avium complex lung disease in patients who experienced adverse effects with a clarithromycin-containing regimen.

BACKGROUND: Antibiotic therapy, including clarithromycin (CLR), has been widely used for the management of Mycobacterium avium complex (MAC) lung disease in clinical settings. When patients develop adverse events (AEs) during CLR-based treatment, the treatment regimen is modified or chemotherapy itself is discontinued. The need for alternative macrolide treatment strategies is emphasized due to the high rate of AEs possibly caused by CLR. Thus, the current study aimed to examine the efficacy and safety of azithromycin (AZM) in patients with MAC lung disease whose treatment was switched from CLR to AZM. METHODS: We performed a retrospective study of patients with MAC lung disease. The inclusion criteria were as follows: (1) patients who experienced AEs during treatment with antibiotics, including CLR, between December 2012 and November 2017, and (2) those who had antimicrobial therapy that was switched from CLR to AZM. The efficacy and safety of AZM during the clinical course of the disease after switching the regimen from CLR to AZM were investigated. RESULTS: Antibiotic therapy was switched in 31 patients who presented with AEs including drug-induced fever, rash, dysgeusia, liver dysfunction, and neutropenia during treatment with CLR-containing regimens. After switching to AZM, the median duration of treatment was 1286 (364-4615) days. During follow-up, 13 patients had a negative conversion of sputum culture. CONCLUSIONS: AZM may be safe and effective for patients with MAC lung disease who have difficulty tolerating CLR. In patients who experienced AEs possibly caused by CLR, switching from CLR to AZM might be an appropriate strategy.

INTENSIVE TREATMENT OF A CAPTIVE BORNEAN ELEPHANT (ELEPHAS MAXIMUS BORNEENSIS) INFECTED WITH MYCOBACTERIUM CAPRAE IN JAPAN.

In 2015, an estimated 17-year-old female Bornean elephant (Elephas maximus borneensis) at Fukuyama Zoo in Japan exhibited anorexia and significant weight loss. Pan-susceptible Mycobacterium tuberculosis complex (MTBC) was isolated from vaginal discharge, oral mucus, urine, and fecal samples by culture. The isolate was identified as Mycobacterium caprae by genetic analysis. Isoniazid, pyrazinamide, and levofloxacin were administered rectally. Body weight increased to normal, but subsequently decreased again. Elevation of liver enzymes occurred, likely related to the increase in isoniazid dosage. After recovery from side effects, the elephant's weight increased further. However, isoniazid-resistant M. caprae was isolated from oral mucus after anti-tuberculosis drug treatment for 9 mo. The regimen was changed to rifampicin, pyrazinamide, ethambutol, and levofloxacin, administered orally or rectally. The 18-mo treatment was completed in October 2018. This elephant has shown no clinical sign since. No MTBC-positive sample had been obtained as of March 2020.

Genomic features of Mycobacterium avium subsp. hominissuis isolated from pigs in Japan.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most important agents causing non-tuberculosis mycobacterial infection in humans and pigs. There have been advances in genome analysis of MAH from human isolates, but studies of isolates from pigs are limited despite its potential source of infection to human. Here, we obtained 30 draft genome sequences of MAH from pigs reared in Japan. The 30 draft genomes were 4,848,678-5,620,788 bp in length, comprising 4652-5388 coding genes and 46-75 (median: 47) tRNAs. All isolates had restriction modification-associated genes and 185-222 predicted virulence genes. Two isolates had tRNA arrays and one isolate had a clustered regularly interspaced short palindromic repeat (CRISPR) region. Our results will be useful for evaluation of the ecology of MAH by providing a foundation for genome-based epidemiological studies.

Two New Cases of Pulmonary Infection by Mycobacterium shigaense, Japan.

We report 2 case-patients in Japan with Mycobacterium shigaense pulmonary infections. One patient was given aggressive treatment and the other conservative treatment, according to distinctive radiologic evidence. A close phylogenetic relationship based on whole-genome sequencing was found between strain from the conservatively treated patient and a reference strain of cutaneous origin.

Serum immunoglobulin a antibodies to glycopeptidolipid core antigen for Mycobacteroides abscessus complex lung disease.

Background: Mycobacteroides abscessus complex (MABC) exhibits smooth morphotypes, expressing glycopeptidolipid (GPL), and rough morphotypes, expressing diminished GPL, on the MABC cell wall. Few reports have focused on the relationship between anti-GPL-core immunoglobulin A (IgA) antibody and colony morphology in MABC lung disease. Methods: This study aimed to test GPL core antigen in patients with MABC lung disease to investigate the relationship between coinfection/contamination in other nontuberculous mycobacteria species and colony morphology variant in MABC isolates. Patients with MABC lung disease and contamination diagnosed between 2012 and 2017 at our hospital were enrolled retrospectively. Results: Of the assessed patients, 43 patients with MABC lung disease and 13 with MABC contamination were included. There was a significant difference in anti-GPL-core IgA antibody levels between them (P = 0.02). Forty-three patients with MABC lung disease were divided into two groups as positive and negative antibodies groups. A significant increase in the positive anti-GPL-core IgA antibody was observed in coexistence with both Mycobacterium avium complex (MAC) (P = 0.02) and the isolate of the smooth variant (P = 0.03) in MABC. Conclusions: Anti-GPL-core IgA antibodies in patients with MABC are greatly influenced by MAC coexistence, and colony morphology variant of the MABC isolate.

ISOLATION AND ANTIMICROBIAL SUSCEPTIBILITIES OF NONTUBERCULOUS MYCOBACTERIA FROM WILDLIFE IN JAPAN.

Nontuberculous mycobacteria (NTM) are opportunistic pathogens of humans and animals and are transmitted among the environment, wildlife, livestock, and humans. The aim of this study was to investigate the rate of isolation and antimicrobial susceptibility of NTM in wildlife. In total, 178 samples of feces (n=131) and tissues (n=47) were collected from 11 wildlife species in Gifu Prefecture and Mie Prefecture, Japan, between June 2016 and October 2018. We isolated NTM from 15.3% (20/ 131) of fecal samples using Ogawa medium, and isolates were identified by sequencing the rpoB and hsp65 genes. The rpoB sequences were compared with those from other strains of human and environmental origin. The NTM isolates were obtained from sika deer (Cervus nippon), wild boar (Sus scrofa), Japanese monkey (Macaca fuscata), raccoon dog (Nyctereutes procyonoides), masked palm civet (Paguma larvata), and Japanese weasel (Mustela itatsi) and were classified as rapidly growing mycobacteria (RGM) and slowly growing mycobacteria (SGM). The 12 RGM identified were Mycolicibacterium peregrinum (n=5), Mycolicibacterium fortuitum (n=3), Mycolicibacterium septicum (n=3), and Mycolicibacterium thermoresistibile (n=1), and the eight SGM were Mycobacterium paraense (n=4), Mycolicibacter arupensis (n=2), Mycolicibacter virginiensis (n=1), and Mycobacterium nebraskense (n=1). The NTM from wildlife showed ≥99% similarity with strains from different sources including humans. The RGM were susceptible to the antimicrobial agents tested except for M. fortuitum, which was resistant to azithromycin and clarithromycin. The SGM showed multiple drug resistance qualities but were susceptible to amikacin, clarithromycin, and rifabutin. These results indicate that wildlife may be reservoir hosts of NTM in Japan. The presence of antimicrobial-resistant NTM in wildlife suggests that the trends of NTM antimicrobial susceptibility in wildlife should be monitored.

Bacterial population kinetics in heteroresistant Mycobacterium tuberculosis harbouring rare resistance-conferring mutations in gyrA and rpoB imply an epistatic interaction of mutations in a pre-XDR-TB patient.

OBJECTIVES: Bacterial population kinetics of strains harbouring drug resistance-conferring mutations within a patient often show cryptic resistance in clinical practice. We report a case that showed emergence and dominance of Mycobacterium tuberculosis with uncommon rpoB and gyrA mutations, followed by an rpoC compensatory mutation, during treatment. METHODS: A pre-XDR-TB patient showed heteroresistance to rifampicin and levofloxacin during treatment as a result of intermittent self-cessation. WGS was applied to investigate intra-host strain composition using five pairs of isolates from sputum samples. RESULTS: The subclone in this study possessed rare mutations conferring resistance to rifampicin (rpoB V170F) and levofloxacin (gyrA S91P) and it rapidly outcompeted other subclones during treatment that included levofloxacin but not rifampicin (<7 days). The high-probability compensatory mutation rpoC V483A also emerged and became dominant subsequent to the rpoB V170F mutation. CONCLUSIONS: To the best of our knowledge, this is the first case showing the emergence of such a rare variant that dominated the population within a patient during treatment of TB.

Effective treatment for clarithromycin-resistant Mycobacterium avium complex lung disease.

Clinical management of macrolide-resistant Mycobacterium avium complex (MR-MAC) lung disease is difficult. To date, there only exist a limited number of reports on the treatment of clarithromycin-resistant MAC (CR-MAC) lung disease. This study aimed to evaluate prognostic factors and identify effective treatments in CR-MAC lung disease. We retrospectively collected clinical data of patients newly diagnosed with CR-MAC lung disease at the Kinki-Chuo Chest Medical Center between August 2010 and June 2018. Altogether, 37 patients with CR-MAC lung disease were enrolled. The median age was 69 years; 30, 22, and 21 patients received clarithromycin, ethambutol, and rifampicin, respectively, on their own or in drug combination. The observed sputum culture conversion rate was 29.7% (11/37 patients). In univariate analysis, ethambutol significantly increased the rate of sputum culture conversion (p = 0.027, odds ratio (OR) 10; 95% confidence interval (CI) 1.11-89.77). Multivariate analysis confirmed that ethambutol increased sputum culture conversion rate (p = 0.026; OR 21.8; 95% CI 1.45-329) while the existence of lung cavities decreased it (p = 0.04; OR 0.088; 95% CI 0.009-0.887). The combined use of ethambutol with other drugs may improve sputum culture conversion rate in CR-MAC lung disease.

Soft tissue infection caused by Mycolicibacter kumamotonensis.

Mycolicibacter kumamotonensis (M. kumamotonensis), formerly Mycobacterium kumamotonense, is a nontuberculous mycobacteria species, which was first separated from Mycobacterium terrae complex in 2006. Reports about infections caused by M. kumamotonensis are extremely rare, with most of them being lung infection. Here, we report the case of a 68-year-old man with a hobby of gardening who developed swelling in his right middle finger. He underwent surgical debridement at a previous hospital and was diagnosed with nontuberculous mycobacteria infection based on positive findings of acid-fast staining of pus obtained from the surgical specimen. He was treated with rifampicin, ethambutol, and clarithromycin, but the swelling worsened. Therefore, he was referred to our hospital for further examination and treatment. We performed a second debridement and added isoniazid to the treatment regimen, but the swelling continued to worsen. We then administered levofloxacin, but his condition did not change. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and DNA sequencing analysis confirmed M. kumamotonensis as the causative bacterium. Since the finger swelling did not improve, the patient underwent a third debridement and amikacin was added to the treatment regimen. Finally, the infection was controlled. He completed amikacin therapy and will continue treatment with the other five antibiotics for a total of 24 months. To the best of our knowledge, this is the first report of a patient with M. kumamotonensis soft tissue infection. We consider this case might provide important insights into the diagnosis and treatment of soft tissue infections caused by M. kumamotonensis.