Fortuitum Group Research Articles

NONTUBERCULOUS MYCOBACTERIA DIVERSITY IN KARST WATERS AND BIOFILMS IN BULGARIAN CAVES

Background: Nontuberculous Mycobacteria (NTM) are emerging pathogens causing opportunistic infections in humans and animals. Their distribution in the waters and caves of Bulgaria is poorly studied. Climatic changes are associated with changes in the amplitudes of ambient and water temperature, as well as changes in the amount of precipitation which play an essential role in the creation of reservoirs of some types of NTM in the environment. Material and Methods: We optimized the methods for successful isolation of environmental NTM and then used molecular genetic methods for identification. Results: A total of 235 samples (karst water, sediments, soil, bat guano) were collected in some caves of the following karst regions: 203 in Vratsa Karst area, 204 in Ponor Karst area, 205 in Bezdenski area and 303 in Karst and caves of Bosnek region. Primary isolation of mycobacteria by Löwenstein–Jensen at room temperature was more successful than on liquid media at 37°C. We identified NTM in 10% (n=24) from these materials. Diverse NTM included: M. chelonae (n=3), M. gordonae (n=2), M. intermedium (n=3), M. scrofulaceum (n=1), M. szulgai (n=4), M. fortuitum group (n=4), NTM mix culture (n=5), M. terrae complex (n=1), Mycobacterium sp. (n=1). Rapidly growing NTM (M. chelonae, M. fortuitum group) were the most common. The isolates belonged to group of environmental saprophytes (Risk group 1) and potential pathogens (Risk group 2). Conclusions: We successfully implemented a procedure for decontamination and isolation of NTM from the environment. For the first time in the country, NTM species were identified in biofilms, karst waters, soil and bat guano within caves. The presence of NTM in cave ecosystems represents a potential source for human infection.

An Unusual Case of Renal Infection with Mycobacterium senegalense

Mycobacterium senegalense is a Non Tuberculous Mycobacteria (NTM) belonging to rapid growers Mycobacterium fortuitum group. Authors describe a unique case of Mycobacterium senegalense infection in a 62-year-old diabetic male with bilateral pyelonephritis and acute kidney injury, as patient had complaints of haematuria on and off for a week. Mycobacterium senegalense is difficult to diagnose as the symptoms are expansive and routine diagnostic tests have lesser sensitivity. It remains resistant to the commonly used antibiotics. The patient was under multiple antibiotic regimens for a long period of time and the isolate grown from the urine sample was identified by Matrix Assisted Laser Desorption Ionisation- Time of Flight (MALDI-TOF) as Mycobacterium senegalense. Later, the patient was started on antitubercular therapy and finally responded.

Reply to Pavlik et al. Clinical Relevance and Environmental Prevalence of Mycobacterium fortuitum Group Members. Comment on "Mugetti et al. Gene Sequencing and Phylogenetic Analysis: Powerful Tools for an Improved Diagnosis of Fish Mycobacteriosis Caused by Mycobacterium fortuitum Group Members. Microorganisms 2021, 9, 797".

We appreciate the valuable comment of Pavlik et al. [...].

1392. Nontuberculous Mycobacteria Isolated from Wisconsin Residents, 2010-2018

BackgroundWisconsin is one of a handful of states in which laboratory identification of nontuberculous mycobacteria (NTM) from clinical samples is reportable to public health. The aims of this study were to characterize the demographic features of Wisconsin adults with NTM, assess the relative abundance of NTM species recovered, and describe trends in NTM isolation over the study period.MethodsWe conducted a retrospective cohort study of Wisconsin residents 18 years of age and older from whom NTM isolates were recovered and reported to the Wisconsin Electronic Disease Surveillance System (WEDSS) between 2010 and 2018. Isolates of M. gordonae were excluded. For the analysis of NTM frequency, multiple reports from the same individual were enumerated as separate isolates when non-identical or collected from different sites. Because NTM were usually reported into WEDSS without clinical data, this study couldn’t discern the clinical significance of the isolates.ResultsA total of 9,032 NTM isolates from 7,722 adults were analyzed. The average annual number of reported NTM cases was 950 (21.7/100,000 adults) during 2011-2018. Table 1 shows the demographic characteristics of individuals with NTM isolates, stratified by specimen collection site and NTM species. M. avium complex (MAC) accounted for 75.7% of respiratory isolates. An important pathogenic NTM, M. xenopi, accounted for 8.9% of non-MAC respiratory isolates. As shown in Table 2, M. chelonae, a rapidly growing mycobacteria (RGM), was the most common species isolated from skin and soft tissue, head, ears, nose and throat, and eye specimens. MAC was the most common isolate from other tissue sites. Table 1. Demographic characteristics of individuals with NTM isolates. Categorization was based upon the initially recovered sample when multiple samples were obtained from a given individual. “Respiratory” samples included sputum, bronchoalveolar lavage, and tracheal aspirate specimens. IQR, interquartile range. RGM, rapidly growing mycobacteria (M. chelonae and the M. abscessus, M. chelonae-abscessus, and M. fortuitum groups). SST, skin and soft tissue.Table 2. Most common NTM species isolated from non-respiratory sites. *’Respiratory specimens’ was inclusive of sputum, bronchoalveolar lavage, and tracheal aspirate specimens. CNS, central nervous system. HENT, head, ears, nose, or throat. SST, skin and soft tissue.ConclusionConsistent with prior studies, MAC is the predominant NTM isolated from respiratory specimens in Wisconsin. RGM are important minority respiratory pathogens, and predominate as skin and soft tissue NTMs. We highlight M. xenopi as an important pathogen in Wisconsin compared to other parts of the United States. In contrast to recent reports of increasing incidence of NTM disease, we found a stable annual incidence of NTM isolation between 2010 and 2018. Disclosures All Authors: No reported disclosures

Bloodstream infections with rapidly growing nontuberculous mycobacteria

BackgroundBloodstream infections (BSI) with rapidly growing mycobacteria (RGM) resulted in recent nosocomial outbreaks predominantly in immunocompromised patients. A little is known about the clinical implications of RGM BSI with different species. MethodsWe conducted a multicenter retrospective cohort study of patients with RGM BSI from November 2011 to December 2020. Demographic data, clinical presentation, laboratory and radiographic findings and microbiological characteristics were used to tabulate descriptive statistics. We performed a comparative analysis of patients with BSI due to Mycobacterium abscessus complex (MABC) vs. other RGM. ResultsWe identified 32 patients with positive blood cultures for RGM, 4/32 (12.5%) were considered to have unclear significance. The most common source for RGM BSI was intravascular catheters (14/28, 50%). Compared to other sources, patients with catheter-related bloodstream infection (CRBSI) received a shorter course of antimicrobial therapy (median [IQR]: one month [0.37–2.25] vs. six months [2–12]), (P = 0.01). The most common species isolated were MABC (12/28, 42.9%), followed by Mycobacterium fortuitum group (6/28, 21.4%) and Mycobacterium chelonae (6/28, 21.4%). Compared to other RGM, MABC BSI was more likely to be secondary to skin and soft tissue infection, associated with longer hospital stay (P = 0.04) and higher death rates despite a higher number of antimicrobial agents used for empirical and directed therapy per patient. ConclusionMABC BSI is associated with an overall more resistant profile, longer hospital stay, and higher death rate despite a more aggressive therapy approach.

Clinical Relevance and Environmental Prevalence of Mycobacterium fortuitum Group Members. Comment on Mugetti et al. Gene Sequencing and Phylogenetic Analysis: Powerful Tools for an Improved Diagnosis of Fish Mycobacteriosis Caused by Mycobacterium fortuitum Group Members. Microorganisms 2021, 9, 797.

Mycobacterium fortuitum group (MFG) members are able to cause clinical mycobacteriosis in fish and other animals including humans. M. alvei, M. arceuilense, M. brisbanense, M. conceptionense, M. fortuitum, M. peregrinum, M. porcinum, M. senegalense, M. septicum, and M. setense were isolated from fish with mycobacteriosis. In other animals only three MFG species have been isolated: M. arceuilense from camels’ milk, M. farcinogenes from cutaneous infections often described as “farcy”, and M. fortuitum from different domestic and wild mammals’ species. Out of 17, only 3 MFG species (M. arceuilense, M. lutetiense and M. montmartrense) have never been reported in humans. A total of eight MFG members (M. alvei, M. brisbanense, M. conceptionense, M. fortuitum subsp. acetamidolyticum, M. houstonense, M. peregrinum, M. porcinum, and M. septicum) have been isolated from both pulmonary and extrathoracic locations. In extrathoracic tissues five MFG species (M. boenickei, M. farcinogenes, M. neworleansense, M. senegalense, and M. setense) have been diagnosed and only one MFG member (M. fortuitum subsp. acetamidolyticum) has been isolated from pulmonary infection.

Mycobacterium porcinum Skin and Soft Tissue Infections After Vaccinations - Indiana, Kentucky, and Ohio, September 2018-February 2019.

During December 2018-February 2019, a multistate investigation identified 101 patients with vaccination-associated adverse events among an estimated 940 persons in Kentucky, Indiana, and Ohio who had received influenza; hepatitis A; pneumococcal; or tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis (Tdap) vaccines at the workplace during September 11-November 28, 2018. These vaccines had been administered by staff members of a third-party health care company contracted by 24 businesses. Company A provided multiple vaccine types during workplace vaccination events across 54 locations in these adjoining states. Injection-site wound isolates from patients yielded Mycobacterium porcinum, a nontuberculous mycobacteria (NTM) species in the Mycobacterium fortuitum group; subtyping using pulsed-field gel electrophoresis of all 28 available isolates identified two closely related clusters. Site visits to company A and interviews with staff members identified inadequate hand hygiene, improper vaccine storage and handling, lack of appropriate medical record documentation, and lack of reporting to the Vaccine Adverse Event Reporting System (VAERS). Vaccination-associated adverse events can be prevented by training health care workers responsible for handling or administering vaccines in safe vaccine handling, administration, and storage practices, timely reporting of any suspected vaccination-associated adverse events to VAERS, and notifying public health authorities of any adverse event clusters.

A Case of Laryngeal Infection Secondary to Mycobacterium mageritense in an Immunocompetent Patient

Abstract Mycobacterium mageritense is a rare rapidly growing nontuberculous mycobacterium classified as a member of the Mycobacterium fortuitum group. It has been described in the literature in case reports as causing varied types of infections, primarily in patients with an identifiable predisposition such as direct inoculation or immunosuppression. Here we describe the first known case of this bacteria causing a laryngeal infection. The patient in this case did not have any identifiable exposures or immunosuppression leading to the infection.

Mycobacterium setense isolated from a cat with atypical mycobacterial panniculitis.

Atypical mycobacterial panniculitis was diagnosed in a cat. Mycobacterium setense was identified as causative agent by 16 S rRNA gene sequence analysis. This a gram-positive rod-shaped acid-fast bacterium belonging to Mycobacterium fortuitum group was never reported before in diseased animals. Resistance to doxycycline and clarithromycin was detected. During treatment with pradofloxacin, additional resistance to fluoroquinolones developed which was due to a mutation in the gyrase gene gyrA (S90W exchange). Despite of antimicrobial treatment for 33 months the patient did not fully recover. Species identification and susceptibility testing for choosing adequate antimicrobial treatment is recommended in cases of feline mycobacterial panniculitis.

General characteristics, features of cultivation
 and antibiotic resistance representatives of mycobacterium fortuitum group representatives (review of literature)

Recently, more and more scientific works have been devoted to non-tuberculous mycobacteria, both by domestic and foreign researchers. One of the main reasons for this is the increase in patients with immunosuppression of various origins, improvement of the quality of laboratory and instrumental diagnostics of mycobacteriosis. This article focuses on the representatives of the M. fortuitum group, as the main pathogens among the group of fast-growing mycobacteria. The data on the modern classification based on the use of molecular genetic studies are indicated. The M. fortuitum group includes: Mycobacterium fortuitum, M. peregrinum, M. senegalense, M. porcinum, M. houstonense, M. neworleansense, M. boenickei, M. conceptionense, M. septicum, M. alvei. According to the new data, mycobacteria were divided into 5 clades (Abscessus-Chelonae, Fortuitum-Vaccae, Terrae, Triviale, Tuberculosis-Simiae), and based on molecular genetic studies, new genera in the Mycobacteriaceae family were isolated: Mycolicibacter spp., Mycolicibacillus spp., Mycolicibacillus spp., Mycobacteroides spp., Mycolicibacterium spp. In accordance with the new classification, representatives of the Mycobacterium fortuitum group belong to the genus Mycolicibacterium. The main epidemiological features of the main sources of the spread of mycobacteria, factors and ways of their transmission are indicated. Due to their wide distribution in the environment, representatives of the M. fortuitum group are capable of causing diseases of the pulmonary and extrapulmonary localization. The distinctive features of pathogenicity factors, due to which the course of the disease is determined, are noted. The article also indicates the main difficulties and features of determining the sensitivity to antimicrobial chemotherapy drugs, provides data on the main features of antibiotic resistance of M.fortuitum group. In preparing the review, literature sources obtained from international and domestic databases were used: Scopus, Web of Science, Springer, RSCI.

Gene Sequencing and Phylogenetic Analysis: Powerful Tools for an Improved Diagnosis of Fish Mycobacteriosis Caused by Mycobacterium fortuitum Group Members.

The Mycobacterium fortuitum group (MFG) consists of about 15 species of fast-growing nontuberculous mycobacteria (NTM). These globally distributed microorganisms can cause diseases in humans and animals, especially fish. The increase in the number of species belonging to MFG and the diagnostic techniques panel do not allow to clarify their real clinical significance. In this study, biomolecular techniques were adopted for species determination of 130 isolates derived from fish initially identified through biochemical tests as NTM belonging to MFG. Specifically, gene sequencing and phylogenetic analysis were used based on a fragment of the gene encoding the 65 KDa heat shock protein (hsp65). The analyzes made it possible to confirm that all the isolates belong to MFG, allowing to identify the strains at species level. Phylogenetic analysis substantially confirmed what was obtained by gene sequencing, except for six strains; this is probably due to the sequences present in NCBI database. Although the methodology used cannot represent a univocal identification system, this study has allowed us to evaluate its effectiveness as regards the species of MFG. Future studies will be necessary to apply these methods with other gene fragments and to clarify the real pathogenic significance of the individual species of this group of microorganisms.

1274. Evaluating the Activity of SPR719, a Novel Aminobenzimidazole, against Nontuberculous Mycobacteria

BackgroundPulmonary infections caused by Nontuberculous Mycobacteria (NTM) are increasing in prevalence and are associated with high mortality and morbidity. Members of the Mycobacterium avium complex (MAC; primarily M. avium and M. intracellulare) and M. abscessus are most commonly associated with NTM pulmonary disease. Treatment options are limited and new agents with potent activity are needed. In this study, the activity of SPR719, a novel aminobenzimidazole, against NTM is reported.MIC range and MIC50/90 summary table MethodsThe susceptibility of 58 non-consecutive, non-duplicate clinical NTM isolates was determined in accordance with the Clinical and Laboratory Standards Institute (CLSI) standard M24. Isolates included 20 rapidly-growing mycobacteria (10 M. abscessus/chelonae Group, 6 M. fortuitum Group, and 4 M. mucogenicum Group) and 38 slow-growing mycobacteria (28 MAC and 10 M. kansasii). SPR719 and comparators clarithromycin (CLA), amikacin (AMK), moxifloxacin (MXF), rifabutin (RFB), minocycline (MIN), and imipenem (IPM) were evaluated. Minimum bactericidal concentrations (MBC) for SPR719, CLA, and AMK were determined in accordance with CLSI M26.ResultsThe activity of SPR719 and comparators by MIC range and MIC50/90 (µg/mL) is summarized in the accompanying table. SPR719 activity was not affected by resistance to CLA, AMK, or MXF. MBC:MIC ratios for SPR719 and CLA were typically >8 which indicates a bacteriostatic mode of action; AMK MBC:MIC ratios were typically ≤ 4 indicative of bactericidal activity.ConclusionSPR719 had potent activity by both MIC50/90 and MIC range across the evaluated NTM species. The SPR719 activity against clinically relevant MAC and M. abcessus/chelonae Group isolates was comparable or superior to the evaluated comparators, and SPR719 was active against isolates resistant to currently utilized agents. These results highlight the potential of SPR719 in the treatment of NTM pulmonary disease.DisclosuresNicole S. Cotroneo, BS, Spero Therapeutics (Employee, Shareholder) Ian Critchley, PhD, Spero Therapeutics (Employee, Shareholder) Michael Pucci, PhD, Spero Therapeutics (Employee, Shareholder) Suzanne Stokes, PhD, Spero Therapeutics (Employee, Shareholder)

Endophthalmitis caused by Mycobacterium houstonense: case report

BackgroundMycobacterium houstonense is rapidly growing mycobacteria (RGM) that belongs to M. fortuitum group. So far, there have been few associated reports of human diseases induced by M. houstonense worldwide.Case presentationWe present a delayed-onset postoperative endophthalmitis caused by M. houstonense after glaucoma drainage implant (GDI) surgery. The ocular infection lasted for 2 months without appropriate treatment that developed into endophthalmitis and the patient underwent an emergency enucleation.ConclusionImplant erosion and a delay in diagnosis of ocular infection could lead to irreversible damage as observed in our case. Ophthalmologists should be alert for ocular RGM infection, and prompt laboratory diagnosis with initiation of effective multidrug therapy might prevent loss of vision.

A Current Microbiological Picture of Mycobacterium Isolates from Istanbul, Turkey.

Despite advances in diagnosis and treatment, tuberculosis (TB) continues to be one of the essential health problems throughout the world. Turkey is considered to be endemic for TB. In this study, we analyzed the distribution of Mycobacterium species, compare the diagnostic methods, and susceptibilities to anti-tuberculosis drugs of TB isolates. The aim was to document the current status and to provide a frame of reference for future studies. In this study, 278 Mycobacterium species isolated from 7,480 patients between September 2015 and June 2019 were included. Löwenstein-Jensen medium (LJ) and MGIT 960 were used for the isolation of strains. Susceptibility to 1st-line anti-tuberculosis drugs was determined. Positivity rates in clinical samples were as follows: 1.4% for direct microscopic acid-fast bacilli (AFB) detection, 3.4% for growth on the LJ, and 3.7% for growth on MGIT-960. Two hundred thirty-three isolates were identified as Mycobacterium tuberculosis complex (MTBC) and 45 were non-tuberculous mycobacteria (NTMs). Eleven of the NTMs (24.4%) were Mycobacterium fortuitum group isolates, and eight NTMs (17.7%) were Mycobacterium abscessus complex isolates. A number of patients diagnosed with tuberculosis peaked twice between the ages of 20–31 and 60–71. A hundred and eighty-two MTBC isolates (78.1%) were susceptible to all 1st-line anti-tuberculosis drugs, while 51 isolates (21.9%) were resistant to at least one drug tested. The multidrug-resistant tuberculosis rate was 13.7% among resistant strains and 3% in all strains. The liquid cultures were better for detection of both MTBC and NTMs isolates. The data demonstrate that MTBC continues to be challenge for this country and indicates the need for continued surveillance and full-spectrum services of mycobacteriology laboratory and infectious diseases.

Nontuberculous mycobacteria: A report of eighteen cases from a tertiary care center in India.

Context:Nontuberculous mycobacteria (NTM) are ubiquitous mycobacteria present in environment and generally affect patients with either structural lung disease or immunosuppression and commonly involve lungs, lymph node, or skin.Materials and Methods:Between July 2016 and February 2019, 18 cases of NTM were diagnosed and their relevant clinical, diagnostic, and treatment details were recorded after taking informed consent.Results:We report 18 cases of NTM involving lungs (n = 11), skin and soft tissue (n = 3), joint (n = 2), genitourinary (n = 1), and central nervous system (n = 1). History of immunosuppression was present in two patients, whereas history of some form of intervention was seen in six patients. Mycobacterium fortuitum group (n = 5) was the most commonly isolated organism, followed by Mycobacterium avium complex (n = 4), Mycobacterium abscessus (n = 3), Mycobacterium kansasii (n = 2), and Mycobacterium chelonae (n = 1). In two patients, M. chelonae and M. abscessus were isolated in succession. Of these 18 patients, clinical response was present in 15 of the patients. Diagnosis and treatment of NTM in resource limited settings is extremely challenging.Conclusion:Most of the patients with NTM are misdiagnosed and are treated as tuberculosis in India, sometimes with a multidrug resistance regimen, which results in significant morbidity and mortality. We present these cases to shed some light on the epidemiology of NTM in this part of India.

Drug susceptibility patterns of rapidly growing mycobacteria isolated from skin and soft tissue infections in Venezuela.

To our knowledge, this is the first work on drug susceptibility patterns of rapid growing mycobacteria from Latin America. The susceptibility patterns for 14 antimicrobial agents of the three most important species that cause skin infections in Venezuela were determined as follows: 63 strains belonging to Mycobacterium abscessus group, 66 strains of the Mycobacterium fortuitum group, and 13 Mycobacterium chelonae strains. The M. abscessus group strains were resistant to most antibiotics tested while M. fortuitum strains were relatively susceptible to a large number of antibiotics. We demonstrate the presence of an inducible and truncated erm(41) gene in M. abscessus group, namely M. abscessus subsp. massiliense. We show the variations in susceptibility to antimicrobial agents within and between the mycobacterial species and compare our susceptibility patterns with those reported from other countries. We conclude that the identification of mycobacteria to the species level can guide the antibiotic treatment, but that it is always important to consider drug susceptibility testing when rapidly mycobacteria are isolated.

Surgical wound infection following open humeral fracture caused by Mycobacterium houstonense: a case report

BackgroundHistorically Mycobacterium houstonense belongs to the unnamed third biovariant complex of the Mycobacterium fortuitum group, which are sorbitol positive. To date, there have been few reports of human infection induced by M. houstonense worldwide.Case presentationWe describe the case of a 68-year-old man with surgical wound infection, following an open humeral fracture, caused by M. houstonense and Escherichia coli. An implant bone plate had been embedded for internal fixation during surgery on the humeral fracture previously. A week later E. coli was isolated from the skin wound secretions. Cefoperazone-sulbactam was used for treatment for two weeks but the infection was not controlled, with a subsequent risk of deep wound infection. External fixation of the fracture was then performed instead of internal fixation. Ten days later, M. houstonense was isolated from new wound secretions. M. houstonense was identified by the molecular sequencing method. The TREK Diagnostic System was used to test the susceptibility to antibiotics by the microbroth dilution method. Levofloxacin and amikacin were used for treatment according to the results of the susceptibility test and the patient’s condition obviously improved.ConclusionTo the best of our knowledge, this is the first case in China of human surgical wound infection caused by M. houstonense following open humeral fracture. The combination of levofloxacin and amikacin was effective in the treatment of M. houstonense infection.

Antimicrobial susceptibility testing of rapidly growing mycobacteria isolated in Japan

BackgroundDifficult-to-treat infections caused by rapidly growing mycobacteria (RGM) are increasingly observed in clinical settings. However, studies on antimicrobial susceptibilities and effective treatments against RGM in Japan are limited.MethodsWe conducted susceptibility testing of potential antimicrobial agents, including tigecycline and tebipenem, against RGM. Clinical RGM isolates were collected from a university hospital in Japan between December 2010 and August 2013. They were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the sequencing of 16S rRNA, rpoB, and hsp65 genes. The samples were utilized for susceptibility testing using 16 antimicrobials, with frozen broth microdilution panels.ResultsForty-two isolates were obtained: 13, Mycobacterium abscessus complex; 12, Mycobacterium chelonae; 9, Mycobacterium fortuitum; and 8, M. fortuitum group species other than M. fortuitum. Different antimicrobial susceptibility patterns were observed between RGM species. Clarithromycin-susceptible strain rates were determined to be 0, 62, and 100% for M. fortuitum, M. abscessus complex, and M. chelonae, respectively. M. abscessus complex (100%) and >80% M. chelonae isolates were non-susceptible, while 100% M. fortuitum group isolates were susceptible to moxifloxacin. Linezolid showed good activity against 77% M. abscessus complex, 89% M. fortuitum, and 100% M. chelonae isolates. Regardless of species, all tested isolates were inhibited by tigecycline at very low minimal inhibitory concentrations (MICs) of ≤0.5 μg/mL. MICs of tebipenem, an oral carbapenem, were ≤4 μg/mL against all M. fortuitum group isolates.ConclusionsOur study demonstrates the importance of correct identification and antimicrobial susceptibility testing, including the testing of potential new agents, in the management of RGM infections.

Real time PCR for the rapid identification and drug susceptibility of Mycobacteria present in Bronchial washings.

BackgroundMycobacteria have a spectrum of virulence and different susceptibilities to antibiotics. Distinguishing mycobacterial species is vital as patients with non-tuberculous mycobacterial (NTM) infections present clinical features that are similar to those of patients with tuberculosis. Thus, rapid differentiation of Mycobacterium tuberculosis complex from NTM is critical to administer appropriate treatment. Hence the aim of the study was to rapid identification of mycobacterial species present in bronchial washings using multiplex real time Polymerase Chain Reaction (PCR) and to determine the drug susceptibility in identified mycobacterial species.MethodsSputum smear negative bronchoscopy specimens (n = 150) were collected for a period of one year, from patients attending the General Hospital Kandy, Sri Lanka. The specimens were processed with modified Petroff’s method and were cultured on Löwenstein– Jensen medium. DNA, extracted from the mycobacterial isolates were subjected to a SYBR green mediated real time multiplex, PCR assay with primers specific for the M. tuberculosis complex, M. avium complex, M. chelonae-M.abscessus group and M. fortuitum group. DNA sequencing was performed for the species confirmation, by targeting the 16S rRNA gene and the drug susceptibility testing was performed for the molecularly identified isolates of M. tuberculosis and NTM.ResultsThe optimized SYBR Green mediated multiplex real-time PCR assay was able to identify the presence of genus Mycobacterium in 25 out of 26 AFB positive isolates, two M. tuberculosis complex, three M. avium complex and two isolates belonging to M. chelonae-M. abscessus group. DNA sequencing confirmed the presence of M. tuberculosis, M. chelonae-M. abscessus, M. intracellulare, M. avium, Rhodococcus sp. and M. celatum. Remaining isolates were identified as Mycobacterium sp. All the NTM isolates were sensitive to amikacin and seven were resistant to ciproflaxacin. Twenty two of the NTM isolates and the isolate Rhodococcus was resistant to clarithromycin. The two isolates of M. tuberculosis were sensitive to all first line anti tuberculosis drugs.ConclusionThe optimized SYBR Green mediated multiplex real time PCR assay could be an effective tool for the rapid differentiation of pathogenic M. tuberculosis complex from the opportunistic nontuberculous mycobacteria and also it confirmed the presence of NTM in 15.3 % of the study population.

Mycobacteria in aquarium fish: results of a 3-year survey indicate caution required in handling pet-shop fish.

Fish are commonly infected with non-tuberculous mycobacteria (NTM), which should be regarded as potential pathogens when handling aquarium fish and equipment. This study examined 107 aquarium fish from pet shops. Cultivation of the fish samples using different selective media was conducted for identification of NTM. Isolates were identified using the GenoType Mycobacterium common mycobacteria and additional species assays, sequencing of the 16S rRNA and rpoB genes, and real-time PCR assay for identification of Mycobacterium (M.) marinum. Among the investigated fish, 79.4% (85/107) were positive for mycobacteria, with 8.2% (7 of 85) having two mycobacterial species present. Among the positive fish, the common pathogens M.marinum, Mycobacterium fortuitum (M.fortuitum group) and Mycobacterium chelonae were identified in approx. 90% of fish and other NTM species in 10%, including Mycobacterium peregrinum/septicum, Mycobacterium gordonae, Mycobacterium arupense, Mycobacterium kansasii, Mycobacterium ulcerans and Mycobacterium setense. The well-known human pathogen M.marinum was present in 10.6% of the positive fish (9 of 85). The species of mycobacteria identified in the study are not only recognized as aquarium fish pathogens, but can also cause pathology in humans. Microbiological and clinical communities should therefore be sensitized to the role of NTM in infections associated with exposure to aquarium fish.