New RAPMYCOI SensititreTM Antimicrobial Susceptibility Test for Atypical Rapidly Growing Mycobacteria (RGM)
Department of Microbiology National Tuberculosis and Lung Diseases Research Institute, 01-138 Warsaw, Poland
*
Author to whom correspondence should be addressed.
Diagnostics 2022, 12(8), 1976; https://doi.org/10.3390/diagnostics12081976
Submission received: 3 June 2022
/
Revised: 12 August 2022
/
Accepted: 12 August 2022
/
Published: 15 August 2022
(This article belongs to the Special Issue Challenges and Advances in Tuberculosis and Mycobacterial Lung Diseases)
Abstract
:Rapidly growing mycobacteria (RGM) cause an increasing international concern, mainly due to their natural resistance to many antibiotics. The aim of this study was to conduct species identification and determine the antimicrobial susceptibility profiles of RGM isolated in Poland. Antimicrobial susceptibility was tested using broth microdilution and the RAPMYCOI panel. A total of 60 strains were analysed, including the following species: M. fortuitum complex (30), M. abscessus subsp. abscessus (16), M. abscessus subsp. massiliense (7), M. chelonae (5), and M. mucogenicum (2). For 12 M. abscessus subsp. abscessus strains, the presence of the erm 41T28 genotype associated with inducible macrolide resistance and a functional erm gene was confirmed. A MUT2 mutation in the rrl gene (constitutive resistance) was identified for two strains from the subtype M. abscessus subsp. massiliense. Among the 15 tested antibiotics, amikacin and linezolid had the strongest antimycobacterial activity. Most of the tested strains were resistant to doxycycline and trimethoprim/sulfamethoxazole. Tigecycline MICs were low for all tested strains. Findings from our study highlight the importance of correct identification of clinical isolates and antimicrobial susceptibility testing.
1. Introduction
Nontuberculous mycobacteria (NTM), also known as mycobacteria other than tuberculosis (MOTT), are ubiquitous environmental microorganisms [1]. Currently, more than 150 species are known worldwide and many of them are increasingly recognized as important human pathogens [2,3]. Based on their growth rate, NTM species are divided into slowly growing mycobacteria (SGM) and rapidly growing mycobacteria (RGM) [4]. To date, more than 75 RGM species have been identified, which represents approximately 50% of all known mycobacterial species [5]. Due to advances in molecular research, the number of newly discovered species continues to increase.
RGM species are classified into six main taxonomic groups, distinguished based on genetic relatedness and the presence of pigment. These are:
- (1)
- M. fortuitum (M. fortuitum, M. peregrinum, M. sengalense, M. porcinum, M. neworleansense, M. boenickei, M. houstonense, M. brisbanense, M. septicum, and M. setense),
- (2)
- M. chelonae/M. abscessus complex (M. chelonae, M. immunogenum, M. franklinii, M. salmoniphilum, M. abscessus subsp. abscessus, M. abscessus subsp. Massiliense, and M. abscessus subsp. bolletii),
- (3)
- M. smegmatis (M. smegmatis and M. goodii),
- (4)
- M. mucogenicum (M. mucogenicum, M. phocaicum, and M. aubagnense),
- (5)
- M. mageritense/M. wolinskyi,
- (6)
Tuberculosis caused by Mycobacterium tuberculosis complex remains a serious global health problem in developing countries [13]. However, in recent decades, the number of reported cases of mycobacteriosis, a disease caused by atypical mycobacteria, has increased significantly [14]. Factors contributing to the increased incidence of NTM infections include: demographic changes, ageing of the population, increased incidence of comorbidities, and immunosuppression [15]. However, the epidemiology of NTM infections remains unknown as the reporting of mycobacterial cases to public health authorities is not mandatory in most countries [16]. Undoubtedly, the diagnosis of the disease is facilitated by recently improved testing methods [17].
Epidemiological statistics indicate that people living in Asia are particularly susceptible to NTM infections. In 2014, the incidence of NTM in the Japanese population was estimated at 14.7/100,000 [18,19,20]. In Great Britain, the incidence of NTM infection increased from 0.9 to 2.9/100,000 between 1995 and 2006 [21]. Studies from North America and Australia revealed that the annual incidence of NTM in these regions in 1997–2010 was 3.2–9.8/100,000 [14]. In Denmark, the incidence of NTM increased between 2003 and 2008 from 0.6 to 1.5/100,000 [22]. In Poland, statistics published by the National Institute of Public Health (PZH) and the Chief Sanitary Inspectorate show that the incidence rate of mycobacteriosis was 0.69 in 2017, 0.63 in 2018, and 0.61/100,000 in 2019 [23].
Atypical mycobacteria are opportunistic pathogens, ubiquitous in the environment, and are found in fresh and marine water, soil, and on biofilms [24]. Infections mainly concern the population of high-risk patients, which includes patients with cystic fibrosis (CF), bronchiectasis, emphysema, chronic obstructive pulmonary disease (COPD), and immunoincompetence (human immunodeficiency virus (HIV) infection, organ transplant, diabetes mellitus, renal failure). Among rapidly growing mycobacteria, the highly pathogenic non-pigmented species include M. fortuitum, M. abscessus, and M. chelonae, which are responsible for more than 80% of all clinical cases [25].
The clinical manifestations of RGM infections are very diverse. They most often concern the respiratory tract, skin, soft tissues, bones and joints, lymphadenitis, or disseminated infections [26]. Chronic lung infections are usually caused by M. abscessus subsp. abscessus and M. abscessus subsp. massiliense [25,27]. In patients with cystic fibrosis, these pulmonary infections are associated with a very high mortality. M. fortuitum complex is most frequently isolated from infected skin after accidental injuries, cosmetic procedures, and laser surgery. Reportedly, M. abscessus is responsible for 90% of respiratory diseases caused by RGM, and M. fortuitum is responsible for 60–80% of postsurgical and catheter-related infections [26]. The most common symptoms of infection caused by M. chelonae are diseases of the skin, bones, and soft tissues as well as ophthalmic infections, including keratitis. Rapidly growing mycobacteria are also isolated from patients with catheter-related bloodstream infection. In this case, the causative pathogens are M. mucogenicum and M. fortuitum, but also M. neoaurum and M. bacteremicum [28,29,30,31].
To determine the etiological factors of mycobacteriosis, it is necessary to correctly identify atypical mycobacteria to the species level. This is due to the different antimicrobial susceptibilities of mycobacteria. The management of a wide spectrum of NTM infections is a serious challenge worldwide. The selection of the appropriate antibiotic therapy for the patient should be based on the results of in vitro antimicrobial susceptibility testing.
However, the suitability of antimicrobial susceptibility testing in the treatment of patients with mycobacteriosis remains controversial due to the discrepancy between test results and clinical response [27,32]. Good correlations demonstrated in the studies carried out to date have been shown for two groups of antibiotics: macrolides and aminoglycosides.
Macrolides (clarithromycin and azithromycin) are among the basic antibiotics used in the treatment of mycobacteriosis. All macrolides bind to the V domain in 23S rRNA on the 50S ribosome subunit [33]. Two mechanisms of resistance to this class of drugs have been identified so far among atypical mycobacteria. The first mechanism is the constitutive resistance associated with a point mutation at either the A2058 or A2059 position of the 23S rRNA (rrl gene). The second mechanism, defined as inducible macrolide resistance, is associated with functional erm genes encoding ribosomal methyltransferase. The erm genes have been identified in the following species: erm (41) in M. abscessus subsp. abscessus (serovars I, VI, VII (80% of isolates)) and M. abscessus subsp. bolletii; erm (39) in M. fortuitum, M. houstonense, M. porcinum, and M. neworleansense; erm (38) in M. smegmatis and M. goodie; erm (40) in M. mageritense and M. wolinskyi. Clarithromycin-sensitive strains lack or have damaged erm genes. This group includes the following species: M. abscessus subsp. abscessus serovar II (Mab30), M. abscessus subsp. massiliense, M. chelonae, M. immunogenum, M. mucogenicum group, M. peregrinum, and M. senegalense [13,34,35].
The aminoglycosides (amikacin and tobramycin) act by binding stably to the 30S ribosomal subunit in bacterial cells, leading to misreading of the genetic code and inhibition of protein synthesis and consequently to cell death. Resistance to aminoglycosides in atypical mycobacteria is associated with single-point mutations in the 16S rRNA (rrs gene) [36].
According to the Clinical and Laboratory Standards Institute (CLSI), the broth microdilution method is considered the gold standard for testing the drug sensitivity of atypical RGM strains. Antimicrobial susceptibility testing should include the following antibiotics: clarithromycin, amikacin, moxifloxacin, linezolid, imipenem, cefoxitin, ciprofloxacin, doxycycline, trimethoprim/sulfamethoxazole, and tobramycin (only for M. chelonae). It is also recommended to determine the minimal inhibitory concentration (MIC) value for tigecycline, but to date there are no consensus breakpoints or guidelines for the interpretation of results [34,37].
There is a commercially available RAPMYCOI test for RGM from Thermo Fisher Scientific (Waltham, MA, USA) that includes all the antibiotics recommended for the treatment of RGM infections. M. fortuitum complex, M. abscessus subsp. abscessus, M. abscessus subsp. massiliense and M. chelonae are the most common rapidly growing mycobacteria (RGM) isolated in Poland.
In the presented study, the susceptibility of 60 RGM strains to 15 antibiotics was determined using the RAPMYCOI panels. The obtained results were compared with data published worldwide, which made it possible to obtain a complete picture of the drug resistance in this group of mycobacteria.
2. Materials and Methods
2.1. Bacterial Strains and Growth Conditions
The study was conducted on 60 strains of atypical mycobacteria (RGM) originally isolated from respiratory specimens (sputum, bronchial washings), in the period from 2019 to 2020 in mycobacterial laboratories in Poland.
The respiratory specimens were decontaminated with the sodium hydroxide and N-acetyl-L-cysteine (NaOH/NALC) (Chempur, Poland) method. The strains were cultured on solid media: egg-based Lowenstein-Jensen medium, Stonebrink medium, and in automated system MGIT (Becton Dickinson, Franklin Lakes, NJ, USA).
2.2. Strain Identification
For DNA extraction, the GenoLyse (Hain Lifescience, Nehren, Germany) kit was used according to protocol.
The strains were identified using the GenoType Mycobacterium CM assay ver. 2.0 (Hain Lifescience, Nehren, Germany) in accordance with the manufacturer’s instructions.
Mycobacteria from the Mycobacterium abscessus complex (MABC) were identified using the GenoType NTM-DR assay (Hain Lifescience, Nehren, Germany). M. mucogenicum was identified using the GenoType Mycobacterium AS assay (Hain Lifescience, Nehren, Germany).
The collection of RGM strains from patients with suspected tuberculosis included the following species: M. abscessus subsp. abscessus (16), M. abscessus subsp. massiliense (7), M. fortuitum complex (30), M. chelonae (5), and M. mucogenicum (2).
2.3. Molecular Determination of Antimicrobial Susceptibility to Macrolides and Aminoglycosides
GenoType NTM-DR assay enabled the detection of resistance to macrolides (erm (41) and rrl genes) and aminoglycosides (rrs genes).
Erm (41) gene was only detected in members of the M. abscessus complex.
The above test detected mutations at position 28 of the erm (41) gene:
- If the strain had a genotype in which C was at position 28 it meant that the tested strain was sensitive to macrolides.
- If the strain had a genotype in which T was at position 28 it meant that the tested strain was resistant to macrolides.
2.4. Phenotypic Determination of Antimicrobial Susceptibility Profile
Antimicrobial susceptibility was tested using broth microdilution. For this purpose, 96-well RAPMYCOI Sensititre ™ titration plates (Thermo Fisher Scientific, Waltham, MA, USA) were used, which allow for the simultaneous determination of susceptibility to 15 antibiotics.
RAPMYCOI plates contain freeze-dried antibiotics in a range of concentrations (μg/mL). The plate design and the tested antibiotic concentrations are presented in Figure 1.
At the first stage of the test, an inoculum of a mycobacterial suspension at the optical density of 0.5 McFarland scale was prepared. A total of 50 µL of inoculum was transferred to 10 mL of CAMHB medium (cation-supplemented Mueller-Hinton broth and TES buffer) (Thermo Fisher Scientific, Waltham, MA, USA). The 100 μL suspension prepared according to this protocol was pipetted onto a 96-well titration plate and incubated at 30 °C ± 2 °C. Plates with RGM were incubated for 3 to 5 days. Only for clarithromycin, the incubation period was prolonged to 14 days in order to detect inducible resistance associated with the presence of the erm genes. If microbial growth in the positive control sample was sufficient, MICs were measured. In cases of difficulties with visual reading, 10 µL of Alamar Blue (BIO-RAD, Hercules, CA, USA) reagent and 25 µL of 5% Tween 80 (Fisher Scientific, Hampton, NH, USA) were added. A colour change from blue to pink indicated the growth of a strain. Measured MICs were interpreted and each strain was classified into one of three groups (sensitive (S), intermediate (I), and resistant (R)) in accordance with the CLSI guidelines (document M62, 1st edition) (37) (Table 3).
3. Results
Table 4 below presents the percentage of strains that are sensitive, intermediate, and resistant to particular antibiotics. The classification was made on the basis of the obtained MIC values.
The data obtained in the performed antimycobacterial susceptibility test showed that amikacin and linezolid had the strongest antituberculotic activity against RGM. Most of the analysed strains were resistant to doxycycline and trimethoprim/sulfamethoxazole.
The following tables (Table 5, Table 6, Table 7, Table 8 and Table 9) present the obtained results separately for each RGM species.
3.1. Mycobacterium abscessus subsp. abscessus
All strains representing Mycobacterium abscessus subsp. abscessus were sensitive only to amikacin. Of the 16 strains, 12 (75%) were also sensitive to linezolid. However, they were all resistant to minocycline, trimethoprim/sulfamethoxazole, and doxycycline. Of the 16 strains, 13 (81%) were resistant to ciprofloxacin and moxifloxacin. Among the 16 strains from this subtype, 12 (75%) were clarithromycin-resistant (MIC > 16 µg/mL) (Table 5). The GenoType NTM-DR assay confirmed the presence of the functional erm (41) gene in these strains, associated with inducible macrolide resistance (erm41T28 genotype). Another four strains were sensitive to clarithromycin (erm41C28 genotype).
3.2. Mycobacterium abscessus subsp. massiliense
Strains representing Mycobacterium abscessus subsp. massiliense were sensitive to linezolid (100%) and amikacin (86%). They were all resistant to trimethoprim/sulfamethoxazole, doxycycline, and ciprofloxacin. Of the 7 strains, 2 (29%) representing the above subtype were resistant to clarithromycin (Table 6). The GenoType NTM-DR assay revealed the presence of the MUT2 mutation in the rrl gene (constitutive resistance) (Table 1). One strain representing Mycobacterium abscessus subsp. massiliense and sensitive to clarithromycin, with the MUT1 mutation in the rrs gene, was resistant to amikacin (MIC > 64 µg/mL) (Table 2).
3.3. Mycobacterium chelonae
All Mycobacterium chelonae strains (5) were sensitive to amikacin, clarithromycin, linezolid, and tobramycin, but resistant to trimethoprim/sulfamethoxazole, ciprofloxacin, and doxycycline (Table 7).
3.4. Mycobacterium mucogenicum
Two tested Mycobacterium mucogenicum strains (100%) were sensitive to clarithromycin, amikacin, cefoxitin, ciprofloxacin, moxifloxacin, and trimethoprim/sulfamethoxazole. One strain was resistant to doxycycline (MIC >16 µg/mL) (Table 8).
3.5. Mycobacterium fortuitum Complex
All strains representing Mycobacterium fortuitum complex (30) were sensitive to amikacin and moxifloxacin, 29 out of 30 strains were also sensitive to ciprofloxacin, 23 (77%) were resistant to clarithromycin, 17 (57%) were resistant to doxycycline, and 21 (70%) were resistant to trimethoprim/sulfamethoxazole (Table 9).
In the analysed collection of RGM, all 60 (100%) strains had low MIC values (from 0.06 to 1 µg/mL) for tigecycline, an antibiotic considered as a potential therapeutic agent and a drug of last resort in the treatment of severe cases of mycobacteriosis.
4. Discussion
With advances in molecular techniques and genetic tools, including whole genome sequencing (WGS), knowledge about the genetic diversity of NTM species and genes determining resistance to antibiotics continues to grow. Long treatment (18 to 24 months on average) and the need to use a combination of antibiotics with multiple side effects increase the importance of drug resistance testing, especially in RGM strains naturally resistant to first-line antituberculotic drugs.
Guidelines on antimicrobial susceptibility testing (AST) of atypical mycobacteria were developed by the CLSI and last updated in December 2018. Currently, CLSI M24 (3rd edition) provides recommendations on AST for slowly growing non-tuberculous mycobacteria, including M. avium complex (MAC), M. kansasii, and M. marinum, as well as rapidly growing mycobacteria (RGM) [34]. Since atypical mycobacteria may colonize the respiratory tract, their isolation from clinical specimens does not always correlate with the identification of an etiological factor responsible for the observed changes. This primarily refers to single sputum cultures. A negative sputum smear indicates a small number of microorganisms that are unlikely to be clinically significant, i.e., insufficient to establish a diagnosis of NTM. Therefore, detailed criteria for the diagnosis of mycobacteriosis have been developed for clinically significant isolates from the respiratory tract [27,38]. These criteria include the following:
- at least two NTM culture-positive sputa or one bronchial wash or lavage sample,
- a transbronchial or lung biopsy specimen with supporting mycobacterial histopathology and a positive NTM culture.
According to the current CLSI recommendations, AST includes antimicrobial agents for RGM such as amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline (or minocycline), imipenem, linezolid, moxifloxacin, trimethoprim-sulfamethoxazole, and tobramycin (for M. chelonae only) (Table 3). Worth noting is the fact that there are insufficient data to establish MIC breakpoints for tigecycline and clofazimine, and therefore for these agents a MIC without interpretation should be given [34].
The results of AST with selected drugs may concern specific species of atypical mycobacteria. For this reason, CLSI and most experts in RGM recommend identifying RGM strains at the species or even subspecies level (Table 10), especially for the M. abscessus complex, before performing a new AST RAPMYCOI and initiating treatment [5,34,39,40].
Because the incubation period for most RGM species ranges from 2 to 5 days, the final MIC reading in the RAPMYCOI test should be performed <5 days. This is mainly due to the instability of some drugs, including carbapenems and tetracyclines. There are only two exceptions where this incubation time should be extended when performing the RAPMYCOI test. The first case concerns strains representing M. abscessus complex isolated from patients who had a history of long-term treatment, including patients with cystic fibrosis. Mycobacterial strains isolated from this population of patients need a longer incubation period; therefore, in some cases it may be helpful to change the incubation temperature or to establish a culture in a shaking incubator. However, if the culture incubation period is longer than 5 days, results are only reliable for AST related to two drugs: clarithromycin and amikacin. The CLSI recommends a comment on the AST report such as: this NTM strain required extended incubation and results for only clarithromycin and amikacin are reliable after incubation for >5 days (Table 10) [34].
The second exception in the RAPMYCOI test to the incubation period longer than 5 days is clarithromycin. Phenotypical detection of inducible resistance to macrolides is achieved by extending the incubation of clarithromycin to 14 days unless the MIC is ≥16 μg/mL at an earlier time point. If the clarithromycin MIC is 4 or 8 μg/mL after 14 days of incubation, the test should be repeated. If the MIC is 4 or 8 μg/mL in the retest, sequencing of the erm gene for the given strain is recommended.
Worth noting is the fact that several RGM species have a non-functional or absent erm gene and are naturally sensitive to clarithromycin [41]. Therefore, sensitivity to clarithromycin can be reported at the initial MIC reading as no prolonged incubation is required for these specific species [42].
In the presented study, we identified 60 rapidly growing mycobacterial strains and determined their antimicrobial susceptibility in accordance with CLSI guidelines.
The most frequently isolated species was Mycobacterium fortuitum complex, which accounted for 50% (30/60) of all identified strains. The tests demonstrated that among all RGM species this group is characterized by high sensitivity to antibiotics. Our study confirmed this thesis and showed that 100% (30/30) of the strains from this group were sensitive to amikacin and moxifloxacin, 97% (29/30) were also sensitive to ciprofloxacin, and 93% (28/30) were sensitive to linezolid. In contrast, tests with clarithromycin showed a high level of resistance for 77% (23/30) of the strains. This resistance is higher compared to that reported by Sriram et al. (100% of sensitive strains among 30 tested) and Bhalla et al. (94.1% of sensitive strains among 17 tested) [43,44].
A low rate of drug resistance according to CLSI was also found for Mycobacterium chelonae. In our study, 100% of the strains (5/5) were sensitive to amikacin, clarithromycin, linezolid, and tobramycin. Our findings are consistent with those reported by Bhalla et al. In the cited study, no resistance to the four above-mentioned antibiotics was found for the three tested isolates [43].
Mycobacterium chelonae and Mycobacterium mucogenicum are classified into the group of species lacking functional erm genes. In our study, all strains of M. chelonae (5/5) and M. mucogenicum (2/2) were sensitive to clarithromycin and no erm genes were detected. However, Esteban et al. detected resistance to clarithromycin associated with the presence of erm genes in two strains of M. chelonae [45]. In a study by Davalos et al., 100% (2/2) of M. chelonae strains were sensitive to clarithromycin. However, one strain (25%) of M. mucogenicum resistant to this antibiotic was detected [46]. In our study, two strains representing M. mucogenicum were sensitive to most of the tested antibiotics. Only one strain was resistant to doxycycline and minocycline. A different antimicrobial susceptibility profile for this species was found by Faridah et al., who reported resistance to ciprofloxacin, doxycycline, clarithromycin, and tobramycin in a strain isolated from blood [47].
Isolates representing the Mycobacterium abscessus complex accounted for 38% (23/60) of all identified strains and it was the second largest group. Most strains (16) represented M. abscessus subsp. abscessus subtype, while M. abscessus subsp. massiliense subtype was less frequently identified (seven strains). We did not identify M. abscessus subsp. bolletii. In our study, most strains of M. abscessus subsp. abscessus (75%) were resistant to clarithromycin (MIC >16 µg/mL). This resistance was associated with the presence of a functional erm gene. The situation was different for M. abscessus subsp. massiliense. Only 28% of strains representing this subtype were clarithromycin-resistant, and the MUT2 mutation in the rrl gene was responsible for the resistance mechanism. Our findings confirm the worldwide reports on the more frequent resistance of M. abscessus subsp. abscessus to clarithromycin compared to M. abscessus subsp. massiliense [48,49,50]. Considering amikacin, the vast majority of strains were sensitive to this antibiotic. Only one strain (4%) was resistant to amikacin (MIC > 64 µg/mL) and had the MUT1 mutation in the rrs gene. Similar findings were reported by Bhalla et al., who found 92.3% of sensitive strains [43].
Among the three tested tetracyclines (doxycycline, minocycline, and tigecycline), the lowest MICs (from 0.06 to 1 µg/mL) were found for tigecycline. If we assume the criteria for interpretation proposed by Wallace et al. (resistant strain when MIC ≥ 8 µg/mL), all tested strains (60/60) were sensitive to tigecycline [51]. Similar relationships were observed by Pang et al.: sensitivity to tigecycline was found for 96% (53/55) of strains from the M. abscessus complex, 91% (10/11) of M. fortuitum strains, and 100% (3/3) of M. chelonae strains [52]. Similarly, in a study by Comba et al., the MIC value was <0.25 µg/mL for 45.7% of the strains (16/35), and from 0.25 µg/mL to 0.5 µg for 54.3% of the strains (19/35) [53]. According to worldwide reports, tigecycline is used in the treatment of the most severe infections with RGM mycobacteria, but to date there are no CLSI guidelines for the interpretation of MIC values in the AST.
5. Conclusions
The new RAPMYCOI test is a rapid tool for the determination of drug resistance profile in RGM. The obtained results are reliable and reproducible, and the test setup is not time-consuming. The broth microdilution method on which the test is based and the selection of antibiotics are consistent with the CLSI guidelines.
Taken together, the findings from the presented study highlight the importance of a correct identification of clinical isolates to the species and subtype level and the role of antimicrobial susceptibility testing, especially for highly resistant rapidly growing mycobacteria (RGM). The obtained results confirm previous assumptions published worldwide according to which there are predictable drug resistance profiles depending on the identified mycobacterial species. However, there are some exceptions to this rule, and therefore the drug resistance of individual strains should be tested as standard practice. The correlation between data obtained from AST with clinical findings proving the effectiveness of treatment will enable the development of new therapeutic regimens. As a result, effective drugs can be selected and the patient’s treatment optimized at an early stage.
Author Contributions
Conceptualization, A.B. and E.A.-K.; methodology, A.B., A.G. and D.F.; formal analysis, A.B.; writing—original draft preparation, A.B.; writing—review and editing, A.G., A.Z. and E.A.-K.; supervision, A.Z. and E.A.-K. All authors have read and agreed to the published version of the manuscript.
Funding
This research was funded by the statutory activity of National Tuberculosis and Lung Diseases Research Institute, Task No 1.8/2019.
Institutional Review Board Statement
The study was approved by the Ethics Committee of the National Tuberculosis and Lung Diseases Research Institute (KB-64/2018, 14 December 2018) as a part of research on mycobacterial diseases in human.
Informed Consent Statement
Not applicable.
Data Availability Statement
Data supporting reported results can be found in source data collected in National Tuberculosis and Lung Diseases Research Institute.
Conflicts of Interest
The authors declare no conflict of interest.
References
- Daley, C.L.; Iaccarino, J.M.; Lange, C.; Cambau, E.; Wallace, R.J., Jr.; Andrejak, C.; Böttger, E.C.; Brozek, J.; Griffith, D.E.; Guglielmetti, L.; et al. Treatment of nontuberculous mycobacterial pulmonary disease: An official ATS/ERS/ESCMID/IDSA clinical practice guideline. Eur. Respir. J. 2020, 56, 2000535. [Google Scholar] [CrossRef] [PubMed]
- Heifets, L. Mycobacterial infections caused by nontuberculous mycobacteria. Semin. Respir. Crit. Care Med. 2004, 25, 283–295. [Google Scholar] [CrossRef] [PubMed]
- Van Ingen, J. Diagnosis of nontuberculous mycobacterial infections. Semin. Respir. Crit. Care Med. 2013, 34, 103–109. [Google Scholar] [CrossRef] [PubMed]
- Wen, S.; Gao, X.; Zhao, W.; Huo, F.; Jiang, G.; Dong, L.; Zhao, L.; Wang, F.; Yu, X.; Huang, H. Comparison of the in vitro activity of linezolid, tedizolid, sutezolid, and delpazolid against rapidly growing mycobacteria isolated in Beijing, China. Int. J. Infect. Dis. 2021, 109, 253–260. [Google Scholar] [CrossRef] [PubMed]
- Brown-Elliott, B.A.; Philley, J.V. Rapidly Growing Mycobacteria. Microbiol. Spectr. 2017, 5, 703–723. [Google Scholar] [CrossRef] [PubMed]
- Schinsky, M.F.; Morey, R.E.; Steigerwalt, A.G.; Douglas, M.P.; Wilson, R.W.; Floyd, M.M.; Butler, W.R.; Daneshvar, M.I.; Brown-Elliott, B.A.; Wallace, R.J.; et al. Taxonomic variation in the Mycobacterium fortuitum third biovariant complex: Description of Mycobacterium boenickei sp. nov., Mycobacterium houstonense sp. nov., Mycobacterium neworleansense sp. nov. and Mycobacterium brisbanense sp. nov. and recognition of Mycobacterium porcinum from human clinical isolates. Int. J. Syst. Evol. Microbiol. 2004, 54, 1653–1667. [Google Scholar] [CrossRef] [PubMed]
- Adékambi, T.; Berger, P.; Raoult, D.; Drancourt, M. rpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacterium bolletii sp. nov., Mycobacterium phocaicum sp. nov. and Mycobacterium aubagnense sp. nov. Int. J. Syst. Evol. Microbiol. 2006, 56, 133–143. [Google Scholar] [CrossRef] [PubMed]
- Brown-Elliott, B.A.; Wallace, R.J., Jr. Mycobacterium: Clinical and Laboratory Characteristics of Rapidly Growing Mycobacteria. In Manual of Clinical Microbiology, 11th ed.; Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S., Warnock, D.W., Eds.; ASM Press: Washington, DC, USA, 2015; pp. 595–612. [Google Scholar]
- Brown, B.A.; Springer, B.; Steingrube, V.A.; Wilson, R.W.; Pfyffer, G.E.; Garcia, M.J.; Menendez, M.C.; Rodriguez-Salgado, B.; Jost, K.C., Jr.; Chiu, S.H.; et al. Mycobacterium wolinskyi sp. nov. and Mycobacterium goodii sp. nov., two new rapidly growing species related to Mycobacterium smegmatis and associated with human wound infections: A cooperative study from the International Working Group on Mycobacterial Taxonomy. Int. J. Syst. Bacteriol. 1999, 49, 1493–1511. [Google Scholar] [CrossRef]
- Wallace, R.J., Jr.; Brown-Elliott, B.A.; Wilson, R.W.; Mann, L.; Hall, L.; Zhang, Y.; Jost, K.C., Jr.; Brown, J.M.; Kabani, A.; Schinsky, M.F.; et al. Clinical and laboratory features of Mycobacterium porcinum. J. Clin. Microbiol. 2004, 42, 5689–5697. [Google Scholar] [CrossRef]
- Jiménez, M.S.; Campos-Herrero, M.I.; García, D.; Luquin, M.; Herrera, L.; García, M.J. Mycobacterium canariasense sp. nov. Int. J. Syst. Evol. Microbiol. 2004, 54, 1729–1734. [Google Scholar] [CrossRef]
- Whipps, C.M.; Butler, W.R.; Pourahmad, F.; Watral, V.G.; Kent, M.L. Molecular systematics support the revival of Mycobacterium salmoniphilum (ex Ross 1960) sp. nov., nom. rev., a species closely related to Mycobacterium chelonae. Int. J. Syst. Evol. Microbiol. 2007, 57, 2525–2531. [Google Scholar] [CrossRef]
- Huh, H.J.; Kim, S.Y.; Jhun, B.W.; Shin, S.J.; Koh, W.J. Recent advances in molecular diagnostics and understanding mechanisms of drug resistance in nontuberculous mycobacterial diseases. Infect. Genet. Evol. 2019, 72, 169–182. [Google Scholar] [CrossRef]
- Prevots, D.R.; Marras, T.K. Epidemiology of human pulmonary infection with nontuberculous mycobacteria: A review. Clin. Chest Med. 2015, 36, 13–34. [Google Scholar] [CrossRef]
- Larsson, L.O.; Polverino, E.; Hoefsloot, W.; Codecasa, L.R.; Diel, R.; Jenkins, S.G.; Loebinger, M.R. Pulmonary disease by non-tuberculous mycobacteria-clinical management, unmet needs and future perspectives. Expert Rev. Respir. Med. 2017, 11, 977–989. [Google Scholar] [CrossRef]
- Stout, J.E.; Koh, W.J.; Yew, W.W. Update on pulmonary disease due to non-tuberculous mycobacteria. Int. J. Infect. Dis. 2016, 45, 123–134. [Google Scholar] [CrossRef]
- Park, S.C.; Kang, M.J.; Han, C.H.; Lee, S.M.; Kim, C.J.; Lee, J.M.; Kang, Y.A. Prevalence, incidence, and mortality of nontuberculous mycobacterial infection in Korea: A nationwide population-based study. BMC Pulm. Med. 2019, 19, 140. [Google Scholar] [CrossRef]
- Namkoong, H.; Kurashima, A.; Morimoto, K.; Hoshino, Y.; Hasegawa, N.; Ato, M.; Mitarai, S. Epidemiology of Pulmonary Nontuberculous Mycobacterial Disease, Japan. Emerg. Infect. Dis. 2016, 22, 1116–1117. [Google Scholar] [CrossRef]
- Simons, S.; van Ingen, J.; Hsueh, P.R.; Van Hung, N.; Dekhuijzen, P.N.; Boeree, M.J.; van Soolingen, D. Nontuberculous mycobacteria in respiratory tract infections, eastern Asia. Emerg. Infect. Dis. 2011, 17, 343–349. [Google Scholar] [CrossRef]
- Yang, S.C.; Hsueh, P.R.; Lai, H.C.; Teng, L.J.; Huang, L.M.; Chen, J.M.; Wang, S.K.; Shie, D.C.; Ho, S.W.; Luh, K.T. High prevalence of antimicrobial resistance in rapidly growing mycobacteria in Taiwan. Antimicrob. Agents Chemother. 2003, 47, 1958–1962. [Google Scholar] [CrossRef]
- Moore, J.E.; Kruijshaar, M.E.; Ormerod, L.P.; Drobniewski, F.; Abubakar, I. Increasing reports of non-tuberculous mycobacteria in England, Wales and Northern Ireland, 1995–2006. BMC Public Health 2010, 10, 612. [Google Scholar] [CrossRef]
- Benfield, T.L.; Duhaut, P.; Sørensen, H.T.; Lescure, F.X.; Thomsen, R.W. Nontuberculous pulmonary mycobacteriosis in Denmark: Incidence and prognostic factors. Am. J. Respir. Crit. Care Med. 2010, 181, 514–521. [Google Scholar] [CrossRef]
- Czarkowski, M.P.; Cieleba, E.; Staszewska-Jakubik, E.K.B. Infectious Diseases and Poisoning in Poland 2017–2019; Bulletin of the National Institute of Public Health-National Institute of Hygiene: Warsaw, Poland, 2020. [Google Scholar]
- Jang, M.A.; Koh, W.J.; Huh, H.J.; Kim, S.Y.; Jeon, K.; Ki, C.S.; Lee, N.Y. Distribution of nontuberculous mycobacteria by multigene sequence-based typing and clinical significance of isolated strains. J. Clin. Microbiol. 2014, 52, 1207–1212. [Google Scholar] [CrossRef]
- Brown-Elliott, B.A.; Wallace, R.J., Jr. Clinical and taxonomic status of pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria. Clin. Microbiol. Rev. 2002, 15, 716–746. [Google Scholar] [CrossRef]
- Brown-Elliott, B.A.; Nash, K.A.; Wallace, R.J., Jr. Antimicrobial susceptibility testing, drug resistance mechanisms, and therapy of infections with nontuberculous mycobacteria. Clin. Microbiol. Rev. 2012, 25, 545–582. [Google Scholar] [CrossRef]
- Griffith, D.E.; Aksamit, T.; Brown-Elliott, B.A.; Catanzaro, A.; Daley, C.; Gordin, F.; Holland, S.M.; Horsburgh, R.; Huitt, G.; Iademarco, M.F.; et al. ATS Mycobacterial Diseases Subcommittee; American Thoracic Society; Infectious Disease Society of America. An official ATS/IDSA statement: Diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am. J. Respir. Crit. Care Med. 2007, 175, 367–416. [Google Scholar] [CrossRef]
- Brown-Elliott, B.A.; Wallace, R.J., Jr.; Petti, C.A.; Mann, L.B.; McGlasson, M.; Chihara, S.; Smith, G.L.; Painter, P.; Hail, D.; Wilson, R.; et al. Mycobacterium neoaurum and Mycobacterium bacteremicum sp. nov. as causes of mycobacteremia. J. Clin. Microbiol. 2010, 48, 4377–4385. [Google Scholar] [CrossRef]
- Raad, I.I.; Vartivarian, S.; Khan, A.; Bodey, G.P. Catheter-related infections caused by the Mycobacterium fortuitum complex: 15 cases and review. Rev. Infect. Dis. 1991, 13, 1120–1125. [Google Scholar] [CrossRef]
- Washer, L.L.; Riddell, J., IV; Rider, J.; Chenoweth, C.E. Mycobacterium neoaurum bloodstream infection: Report of 4 cases and review of the literature. Clin. Infect. Dis. 2007, 45, e10–e13. [Google Scholar] [CrossRef]
- Martínez López, A.B.; Álvarez Blanco, O.; Ruíz Serrano, M.J.; Morales San-José, M.D.; Luque de Pablos, A. Mycobacterium fortuitum as a cause of peritoneal dialysis catheter port infection. A clinical case and a review of the literature. Nefrologia 2015, 35, 584–586. [Google Scholar] [CrossRef]
- Haworth, C.S.; Banks, J.; Capstick, T.; Fisher, A.J.; Gorsuch, T.; Laurenson, I.F.; Leitch, A.; Loebinger, M.R.; Milburn, H.J.; Nightingale, M.; et al. British Thoracic Society Guideline for the management of non-tuberculous mycobacterial pulmonary disease (NTM-PD). BMJ Open Respir. Res. 2017, 4, e000242. [Google Scholar] [CrossRef]
- Markiewicz, Z.; Korsak, D.; Popowska, M. Antibiotics in the Era of Increasing Drug Resistance, 1st ed.; PWN: Warsaw, Poland, 2021; p. 43. [Google Scholar]
- Clinical and Laboratory Standards Institute. Susceptibility Testing of Mycobacteria, Nocardia spp., and Other Aerobic Actinomycetes, 3rd ed.; CLSI Standard Document M24; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2018. [Google Scholar]
- Huang, W.C.; Yu, M.C.; Huang, Y.W. Identification and drug susceptibility testing for nontuberculous mycobacteria. J. Formos. Med. Assoc. 2020, 119 (Suppl. S1), S32–S41. [Google Scholar] [CrossRef] [PubMed]
- Kotra, L.P.; Haddad, J.; Mobashery, S. Aminoglycosides: Perspectives on mechanisms of action and resistance and strategies to counter resistance. Antimicrob. Agents Chemother. 2000, 44, 3249–3256. [Google Scholar] [CrossRef] [PubMed]
- Clinical and Laboratory Standards Institute. Performance Standards for Susceptibility Testing of Mycobacteria, Nocardia spp., and Other Aerobic Actinomycetes, 1st ed.; CLSI Document M62; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2018. [Google Scholar]
- Clinical and Laboratory Standards Institute. Laboratory Detection and Identification of Mycobacteria; CLSI Guideline M48; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2018. [Google Scholar]
- Brown-Elliott, B.A.; Vasireddy, S.; Vasireddy, R.; Iakhiaeva, E.; Howard, S.T.; Nash, K.A.; Parodi, N.; Strong, A.; Gee, M.; Smith, T.; et al. Utility of sequencing the erm(41) gene in isolates of Mycobacterium abscessus subsp. abscessus with low and intermediate clarithromycin MICs. J. Clin. Microbiol. 2016, 53, 1211–1215, Erratum in J. Clin. Microbiol. 2016, 54, 1172. [Google Scholar] [CrossRef] [PubMed]
- Brown-Elliott, B.A. Laboratory diagnosis and antimicrobial susceptibility testing of nontuberculous mycobacteria. In Nontuberculous Mycobacterial Disease; Griffith, D.E., Ed.; Respiratory Medicine Humana Press: Cham, Switzerland, 2018; pp. 15–59. [Google Scholar]
- Nash, K.A.; Brown-Elliott, B.A.; Wallace, R.J., Jr. A novel gene, erm(41), confers inducible macrolide resistance to clinical isolates of Mycobacterium abscessus but is absent from Mycobacterium chelonae. Antimicrob. Agents Chemother. 2009, 53, 1367–1376. [Google Scholar] [CrossRef]
- Brown-Elliott, B.A.; Woods, G.L. Antimycobacterial Susceptibility Testing of Nontuberculous Mycobacteria. J. Clin. Microbiol. 2019, 57, e00834-19. [Google Scholar] [CrossRef]
- Bhalla, G.S.; Grover, N.; Singh, L.; Sarao, M.S.; Kalra, D.; Pandey, C. RAPMYCO: Mitigating conventional broth microdilution woes. J. Health Res. Rev. 2018, 5, 93–97. [Google Scholar]
- Sriram, R.; Sarangan, P. Antimicrobial susceptibility testing of rapidly growing mycobacteria isolated from cases of surgical site infections by microbroth dilution method at a Tertiary Care Center. J. Mar. Med. Soc. 2017, 19, 6–10. [Google Scholar] [CrossRef]
- Esteban, J.; Martín-de-Hijas, N.Z.; García-Almeida, D.; Bodas-Sánchez, A.; Gadea, I.; Fernández-Roblas, R. Prevalence of erm methylase genes in clinical isolates of non-pigmented, rapidly growing mycobacteria. Clin. Microbiol. Infect. 2009, 15, 919–923. [Google Scholar] [CrossRef]
- Dávalos, A.F.; Garcia, P.K.; Montoya-Pachongo, C.; Rengifo, A.; Guerrero, D.; Díaz-Ordoñez, L.; Díaz, G.; Ferro, B.E. Identification of Nontuberculous Mycobacteria in Drinking Water in Cali, Colombia. Int. J. Environ. Res. Public Health 2021, 18, 8451. [Google Scholar] [CrossRef]
- Faridah, S.; Siti Asma’, H.; Zeti, N.S.; Tuan Noorkorina, T.K.; Intan Baiduri, B.; Azura, H. Fatal outcome of catheter-related bloodstream infection caused by Multidrug-Resistant Mycobacterium mucogenicum. Med. J. Malays. 2021, 76, 248–250. [Google Scholar]
- Aono, A.; Morimoto, K.; Chikamatsu, K.; Yamada, H.; Igarashi, Y.; Murase, Y.; Takaki, A.; Mitarai, S. Antimicrobial susceptibility testing of Mycobacteroides (Mycobacterium) abscessus complex, Mycolicibacterium (Mycobacterium) fortuitum, and Mycobacteroides (Mycobacterium) chelonae. J. Infect. Chemother. 2019, 25, 117–123. [Google Scholar] [CrossRef]
- Lee, S.H.; Yoo, H.K.; Kim, S.H.; Koh, W.J.; Kim, C.K.; Park, Y.K.; Kim, H.J. The drug resistance profile of Mycobacterium abscessus group strains from Korea. Ann. Lab. Med. 2014, 34, 31–37. [Google Scholar] [CrossRef]
- Koh, W.J.; Jeon, K.; Lee, N.Y.; Kim, B.J.; Kook, Y.H.; Lee, S.H.; Park, Y.K.; Kim, C.K.; Shin, S.J.; Huitt, G.A.; et al. Clinical significance of differentiation of Mycobacterium massiliense from Mycobacterium abscessus. Am. J. Respir. Crit. Care Med. 2011, 183, 405–410. [Google Scholar] [CrossRef]
- Wallace, R.J., Jr.; Brown-Elliott, B.A.; Crist, C.J.; Mann, L.; Wilson, R.W. Comparison of the in vitro activity of the glycylcycline tigecycline (formerly GAR-936) with those of tetracycline, minocycline, and doxycycline against isolates of nontuberculous mycobacteria. Antimicrob. Agents Chemother. 2002, 46, 3164–3167. [Google Scholar] [CrossRef]
- Pang, H.; Li, G.; Zhao, X.; Liu, H.; Wan, K.; Yu, P. Drug Susceptibility Testing of 31 Antimicrobial Agents on Rapidly Growing Mycobacteria Isolates from China. Biomed. Res. Int. 2015, 2015, 419392. [Google Scholar] [CrossRef]
- Comba, I.Y.; Tabaja, H.; Almeida, N.E.C.; Fida, M.; Saleh, O.A. Bloodstream infections with rapidly growing nontuberculous mycobacteria. J. Clin. Tuberc. Other Mycobact. Dis. 2021, 25, 100288. [Google Scholar] [CrossRef]
Figure 1.
RAPMYCOI plate design: positive control (POS), amikacin (AMI), amoxicillin/clavulanic acid (AUG2), cefepime (FEP), cefoxitin (FOX), ceftriaxone (AXO), ciprofloxacin (CIP), clarithromycin (CLA), doxycycline (DOX), imipenem (IMI), linezolid (LZD), minocycline (MIN), moxifloxacin (MXF), trimethoprim/sulfamethoxazole (SXT), tigecycline (TGC), and tobramycin (TOB). The number under the antibiotic abbreviation shows its concentration in μg/mL.
Figure 1.
RAPMYCOI plate design: positive control (POS), amikacin (AMI), amoxicillin/clavulanic acid (AUG2), cefepime (FEP), cefoxitin (FOX), ceftriaxone (AXO), ciprofloxacin (CIP), clarithromycin (CLA), doxycycline (DOX), imipenem (IMI), linezolid (LZD), minocycline (MIN), moxifloxacin (MXF), trimethoprim/sulfamethoxazole (SXT), tigecycline (TGC), and tobramycin (TOB). The number under the antibiotic abbreviation shows its concentration in μg/mL.
Table 1.
Mutations determining resistance to macrolides detected using the GenoType NTM-DR assay within the rrl gene.
Table 1.
Mutations determining resistance to macrolides detected using the GenoType NTM-DR assay within the rrl gene.
| Absence of Wild-Type Band | Analysed Nucleic Acid Positions | Mutation Bands Present | Mutation | Phenotypic Resistance |
|---|---|---|---|---|
| rrl WT | 2058–2059 | rrl MUT1 | A2058C | macrolides |
| rrl MUT2 | A2058G | |||
| A2058T | ||||
| rrl MUT3 | A2059C | |||
| rrl MUT4 | A2059G | |||
| A2059T |
Table 2.
Mutations determining resistance to aminoglycosides detected using the GenoType NTM-DR assay within the rrs gene.
Table 2.
Mutations determining resistance to aminoglycosides detected using the GenoType NTM-DR assay within the rrs gene.
| Absence of Wild-Type Band | Analysed Nucleic Acid Positions | Mutation Bands Present | Mutation | Phenotypic Resistance |
|---|---|---|---|---|
| rrs WT | 1406–1409 | rrs MUT1 | A1408G | aminoglycosides |
| T1406A | ||||
| C1409T |
Table 3.
Antimicrobial agents and susceptibility breakpoints (MICs) for testing rapidly growing mycobacteria.
Table 3.
Antimicrobial agents and susceptibility breakpoints (MICs) for testing rapidly growing mycobacteria.
| Antimicrobial Agent | MIC (μg/mL) | Comment | ||
|---|---|---|---|---|
| S | I | R | ||
| AMI | ≤16 | 32 | ≥64 | M. abscessus complex isolates with MIC of ≥64 μg/mL should be retested and/or the 16S rRNA gene sequenced to check for mutation |
| FOX | ≤16 | 32–64 | ≥128 | |
| CIP | ≤1 | 2 | ≥4 | Ciprofloxacin and levofloxacin are interchangeable, but both are less active than the newer B-methoxy-fluoroquinolones |
| CLA | ≤2 | 4 | ≥8 | See text for information on the erm gene; clarithromycin and azithromycin are interchangeable clinically |
| DOX | ≤1 | 2–4 | ≥8 | |
| MIN | ≤1 | 2–4 | ≥8 | |
| IMI | ≤4 | 8–16 | ≥32 | All isolates of M. fortuitum, M. smegmatis, and the M. mucogenicum group are presumed imipenem susceptible; imipenem MICs do not predict meropenem or ertapenem susceptibility |
| LZD | ≤8 | 16 | ≥32 | |
| MXF | ≤1 | 2 | ≥4 | |
| TMP-SMX | ≤2/38 | ≥4/76 | MIC is 80% inhibition | |
| TOB | ≤2 | 4 | ≥8 | Predominantly for M. chelonae; if MIC >4 μg/mL, the test should be repeated and/or the identification confirmed by rpoβ gene sequencing |
| TGC | Insufficient data to establish breakpoints; only MIC should be reported | |||
Table 4.
Classification of analysed RGM species into groups: (S)-sensitive, (I)-intermediate, and (R)-resistant, based on the measured MIC values.
Table 4.
Classification of analysed RGM species into groups: (S)-sensitive, (I)-intermediate, and (R)-resistant, based on the measured MIC values.
| M. abscessus subsp. abscessus n = 16 | M. abscessus subsp. massiliense n = 7 | M. chelonae n = 5 | M. mucogenicum n = 2 | M. fortuitum complex n = 30 | |
|---|---|---|---|---|---|
| ANTIBIOTIC AGENT | values in (%) | ||||
| AMI | 100 (S) | 86 (S) 14 (R) | 100 (S) | 100 (S) | 100 (S) |
| FOX | 100 (I) | 86 (I) 14 (S) | 80 (S) 20 (R) | 100 (S) | 67 (I) 33 (S) |
| CIP | 81 (R) 19 (I) | 100 (R) | 100 (R) | 100 (S) | 97 (S) 3 (I) |
| CLA | 75 (R) 25 (S) | 71 (S) 29 (R) | 100 (S) | 100 (S) | 77 (R) 23 (S) |
| IMI | 100 (I) | 100 (I) | 80 (R) 20 (I) | 50 (S) 50 (I) | 63 (I) 27 (S) 10 (R) |
| LZD | 75 (S) 25 (I) | 100 (S) | 100 (S) | 100 (S) | 93 (S) 7 (I) |
| DOX | 100 (R) | 100 (R) | 100 (R) | 50 (S) 50 (R) | 56,6 (R) 43,3 (S) |
| MIN | 100 (R) | 57 (S) 43 (R) | 100 (R) | 50 (S) 50 (I) | 56,6 (R) 43,3 (S) |
| MXF | 81 (R) 19 (I) | 86 (R) 14 (I) | 80 (R) 20 (S) | 100 (S) | 100 (S) |
| SXT | 100 (R) | 100 (R) | 100 (R) | 100 (S) | 70 (R) 30 (S) |
| TOB | 100 (S) | ||||
Table 5.
Results of in vitro susceptibility testing for M. abscessus subsp. abscessus strains.
| M. abscessus subsp. abscessus (n = 16) | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| AMI | FOX | CIP | CLA | DOX | IMI | LZD | MIN | MXF | TGC | SXT | |
| 1 | 4 (S) | 32 (I) | 2 (I) | 0.5 (S) | >16 (R) | 16 (I) | 8 (S) | >8 (R) | 4 (R) | 0.5 | >8/152 (R) |
| 2 | 8 (S) | 32 (I) | 4 (R) | >16 (R) | 16 (R) | 16 (I) | ≤1 (S) | >8 (R) | 2 (I) | 0.5 | 8/152 (R) |
| 3 | 4 (S) | 32 (I) | 4 (R) | >16 (R) | >16 (R) | 16 (I) | 16 (I) | >8 (R) | 4 (R) | 0.5 | >8/152 (R) |
| 4 | 2 (S) | 32 (I) | 4 (R) | >16 (R) | >16 (R) | 16 (I) | 8 (S) | >8 (R) | 4 (R) | 0.5 | >8/152 (R) |
| 5 | 4 (S) | 32 (I) | 4 (R) | >16 (R) | >16 (R) | 16 (I) | 8 (S) | >8 (R) | 4 (R) | 0.12 | >8/152 (R) |
| 6 | 2 (S) | 32 (I) | >4 (R) | >16 (R) | >16 (R) | 8 (I) | 8 (S) | >8 (R) | 8(R) | 0.5 | >8/152 (R) |
| 7 | 4 (S) | 32 (I) | 4 (R) | >16 (R) | >16 (R) | 16 (I) | 2 (S) | >8 (R) | 4 (R) | 0.06 | 8/152 (R) |
| 8 | 4 (S) | 32 (I) | 2 (I) | 2 (S) | >16 (R) | 8 (I) | 8 (S) | >8 (R) | 4 (R) | 1 | 8/152 (R) |
| 9 | 8 (S) | 32 (I) | 4 (R) | 1 (S) | >16 (R) | 8 (I) | 4 (S) | >8 (R) | 4 (R) | 0.25 | >8/152 (R) |
| 10 | 4 (S) | 32 (I) | 4 (R) | >16 (R) | >16 (R) | 8 (I) | 8 (S) | >8 (R) | 4 (R) | 0.25 | >8/152 (R) |
| 11 | 4 (S) | 32 (I) | >4 (R) | >16 (R) | >16 (R) | 16 (I) | 16 (I) | >8 (R) | >8(R) | 1 | >8/152 (R) |
| 12 | 8 (S) | 32 (I) | >4 (R) | >16 (R) | >16 (R) | 16 (I) | 16 (I) | >8 (R) | >8 (R) | 1 | >8/152 (R) |
| 13 | 4 (S) | 32 (I) | 2 (I) | >16 (R) | >16 (R) | 16 (I) | 2 (S) | >8 (R) | 2 (I) | 0.25 | 4/76 (R) |
| 14 | 4 (S) | 32 (I) | 4 (R) | >16 (R) | >16 (R) | 16 (I) | 4 (S) | >8 (R) | 2 (I) | 0.25 | >8/152 (R) |
| 15 | 4 (S) | 64 (I) | 4 (R) | >16 (R) | >16 (R) | 16 (I) | 16 (I) | >8 (R) | 8 (R) | 0.5 | >8/152 (R) |
| 16 | 4 (S) | 32 (I) | >4 (R) | 0.12 (S) | >16 (R) | 16 (I) | 4 (S) | >8 (R) | 4 (R) | 0.25 | >8/152 (R) |
Table 6.
Results of in vitro susceptibility testing for M. abscessus subsp. massiliense strains.
| M. abscessus subsp. massiliense (n = 7) | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| AMI | FOX | CIP | CLA | DOX | IMI | LZD | MIN | MXF | TGC | SXT | |
| 1 | 4 (S) | 32 (I) | 4 (R) | 0.25 (S) | >16 (R) | 16 (I) | 8 (S) | >8 (R) | 8 (R) | 1 | >8/152 (R) |
| 2 | >64 (R) | 16 (S) | 4 (R) | ≤0.06 (S) | >16 (R) | 16 (I) | 2 (S) | >8 (R) | 2 (I) | 0.12 | 8/152 (R) |
| 3 | 4 (S) | 32 (I) | 4(R) | 0.12 (S) | 8 (R) | 16 (I) | 8 (S) | 2 (S) | 4 (R) | 0.25 | >8/152 (R) |
| 4 | 8 (S) | 32 (I) | >4 (R) | 0.25 (S) | >16 (R) | 16 (I) | 8 (S) | >8 (R) | >8 (R) | 0.5 | >8/152 (R) |
| 5 | 4 (S) | 32 (I) | 4 (R) | >16 (R) | 16 (R) | 16 (I) | 8 (S) | 2 (S) | 8 (R) | 0.5 | 8/152 (R) |
| 6 | 4 (S) | 32 (I) | 4 (R) | >16 (R) | 16 (R) | 16 (I) | 8 (S) | 2 (S) | 8 (R) | 0.5 | 8/152 (R) |
| 7 | 8 (S) | 32 (I) | >4 (R) | 0.25 (S) | >16 (R) | 16 (I) | 8 (S) | 2 (S) | >8 (R) | 0.5 | >8/152 (R) |
Table 7.
Results of in vitro susceptibility testing for M. chelonae strains.
| M. chelone (n = 5) | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| AMI | FOX | CIP | CLA | DOX | IMI | LZD | MIN | MXF | TGC | TOB | SXT | |
| 1 | 16 (S) | >128 (R) | 4(R) | 0.5 (S) | >16 (R) | 16 (I) | 4 (S) | >8 (R) | 1 (S) | 0.5 | 2 (S) | 8/152 (R) |
| 2 | 8 (S) | 64 (I) | 4(R) | ≤0.06 (S) | >16 (R) | 32 (R) | 4 (S) | >8 (R) | 4(R) | 0.25 | ≤1 (S) | 8/152 (R) |
| 3 | 4 (S) | 64 (I) | 4(R) | 0.25 (S) | >16 (R) | 32 (R) | 4 (S) | >8 (R) | 4(R) | 0.5 | ≤1 (S) | >8/152 (R) |
| 4 | 4 (S) | 64 (I) | 4(R) | 0.25 (S) | >16 (R) | 32 (R) | 4 (S) | >8 (R) | 4(R) | 0.5 | ≤1 (S) | 8/152 (R) |
| 5 | 8 (S) | 64 (I) | 4(R) | 0.25 (S) | >16 (R) | 64(R) | 4 (S) | >8 (R) | 4(R) | 0.25 | ≤1 (S) | >8/152 (R) |
Table 8.
Results of in vitro susceptibility testing for M. mucogenicum strains.
| M. mucogenicum (n = 2) | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| AMI | FOX | CIP | CLA | DOX | IMI | LZD | MIN | MXF | TGC | SXT | |
| 1 | 2 (S) | 16 (S) | 0.25 (S) | 0.25 (S) | >16 (R) | 8 (I) | 2 (S) | >8 (R) | ≤0.25 (S) | 0.25 | 1/19 (S) |
| 2 | ≤1 (S) | 8 (S) | 0.5 (S) | 0.12 (S) | ≤0.12 (S) | 4 (S) | 2 (S) | ≤1 (S) | 0.5 (S) | 0.12 | 0.5/9.5 (S) |
Table 9.
Results of in vitro susceptibility testing for M. fortuitum complex strains.
| M. fortuitum Complex (n = 30) | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| AMI | FOX | CIP | CLA | DOX | IMI | LZD | MIN | MXF | TGC | SXT | |
| 1 | ≤1 (S) | 16 (S) | 0.5 (S) | 0.5 (S) | 0.5 (S) | ≤2 (S) | 4 (S) | ≤1 (S) | ≤0.25 (S) | 0.12 | 2/38 (S) |
| 2 | ≤1 (S) | 16 (S) | ≤0.12 (S) | 0.12 (S) | >16 (R) | ≤2 (S) | 2 (S) | >8 (R) | ≤0.25 (S) | 0.5 | 4/76 (R) |
| 3 | ≤1 (S) | 32 (I) | ≤0.12 (S) | >16 (R) | >16 (R) | 4 (S) | 2 (S) | >8 (R) | ≤0.25 (S) | 0.25 | 0.5/9.5 (S) |
| 4 | ≤1 (S) | 16 (S) | ≤0.12 (S) | 0.12 (S) | >16(R) | 4 (S) | ≤1 (S) | >8 (R) | ≤0.25 (S) | 0.25 | 1/19 (S) |
| 5 | ≤1 (S) | 16 (S) | ≤0.12 (S) | >16 (R) | ≤0.12 (S) | 8 (I) | ≤1 (S) | ≤1 (S) | ≤0.25 (S) | 0.25 | 2/38 (S) |
| 6 | ≤1 (S) | 32 (I) | ≤0.12 (S) | >16 (R) | >16 (R) | 8 (I) | 16 (I) | >8 (R) | ≤0.25 (S) | 0.5 | 8/152 (R) |
| 7 | ≤1 (S) | 16 (S) | 0.25 (S) | >16 (R) | 0.5 (S) | 4 (S) | ≤1 (S) | ≤1 (S) | ≤0.25 (S) | 0.25 | 1/19 (S) |
| 8 | ≤1 (S) | 16 (S) | ≤0.12 (S) | 0.25 (S) | >16(R) | 4 (S) | ≤1 (S) | >8 (R) | ≤0.25 (S) | 0.25 | 1/19 (S) |
| 9 | ≤1 (S) | 64 (I) | 2(I) | >16 (R) | 0.5 (S) | 64(R) | 8 (S) | >8 (R) | 1 (S) | 1 | >8/152 (R) |
| 10 | ≤1 (S) | 64 (I) | 0.25 (S) | >16 (R) | >16 (R) | 16 (I) | 8 (S) | >8 (R) | ≤0.25 (S) | 0.25 | >8/152 (R) |
| 11 | 2 (S) | 64 (I) | 0.25 (S) | >16 (R) | >16 (R) | 16 (I) | 8 (S) | >8 (R) | 0.5 (S) | 0.25 | 4/76 (R) |
| 12 | 4 (S) | 64 (I) | 0.25 (S) | >16 (R) | 0.25 (S) | 64 (R) | 4 (S) | ≤1 (S) | ≤0.25 (S) | 0.06 | 2/38 (S) |
| 13 | ≤1 (S) | 32 (I) | ≤0.12 (S) | >16 (R) | ≤0.12 (S) | 4 (S) | 4 (S) | ≤1 (S) | ≤0.25 (S) | 0.03 | 0.5/9.5 (S) |
| 14 | ≤1 (S) | 32 (I) | 0.25 (S) | >16 (R) | >16 (R) | 8 (I) | 8 (S) | >8 (R) | ≤0.25 (S) | 0.25 | 8/152 (R) |
| 15 | ≤1 (S) | 32 (I) | ≤0.12 (S) | >16 (R) | >16 (R) | 8 (I) | 8 (S) | >8 (R) | ≤0.25 (S) | 0.03 | 2/38 (S) |
| 16 | ≤1 (S) | 8 (S) | ≤0.12 (S) | >16 (R) | 8 (R) | 4 (S) | 2 (S) | ≤1 (S) | ≤0.25 (S) | 0.12 | 4/76 (R) |
| 17 | ≤1 (S) | 32 (I) | 0.25 (S) | >16 (R) | >16 (R) | 8 (I) | 8 (S) | >8 (R) | ≤0.25 (S) | 0.25 | >8/152(R) |
| 18 | ≤1 (S) | 32 (I) | 0.25 (S) | 16 (R) | >16 (R) | 8 (I) | 8 (S) | >8 (R) | ≤0.25 (S) | 0.03 | >8/152(R) |
| 19 | ≤1 (S) | 32 (I) | 0.25 (S) | 16 (R) | >16 (R) | 8 (I) | 8 (S) | >8 (R) | ≤0.25 (S) | 0.06 | >8/152(R) |
| 20 | ≤1 (S) | 32 (I) | ≤0.12 (S) | >16 (R) | 0.25 (S) | 16 (I) | 4 (S) | ≤1 (S) | ≤0.25 (S) | 0.06 | 4/76 (R) |
| 21 | ≤1 (S) | 32 (I) | ≤0.12 (S) | >16 (R) | >16(R) | 8 (I) | 2 (S) | >8(R) | ≤0.25 (S) | 0.12 | 4/76 (R) |
| 22 | ≤1 (S) | 32 (I) | 0.25 (S) | >16 (R) | 0.5 (S) | 8 (I) | 8 (S) | ≤1 (S) | ≤0.25 (S) | 0.5 | >8/152(R) |
| 23 | ≤1 (S) | 32 (I) | ≤0.12 (S) | >16 (R) | 0.12 (S) | 8 (I) | 4 (S) | ≤1 (S) | ≤0.25 (S) | 0.25 | 4/76 (R) |
| 24 | ≤1 (S) | 32 (I) | ≤0.12 (S) | >16 (R) | 0.25 (S) | 8 (I) | 4 (S) | ≤1 (S) | ≤0.25 (S) | 0.25 | 8/152 (R) |
| 25 | ≤1 (S) | 16 (S) | 0.5 (S) | 1 (S) | 0.25 (S) | 8 (I) | 8 (S) | ≤1 (S) | ≤0.25 (S) | 0.25 | 4/76 (R) |
| 26 | ≤1 (S) | 32 (I) | ≤0.12 (S) | >16 (R) | >16 (R) | 16 (I) | 16 (I) | >8 (R) | ≤0.25 (S) | 0.25 | 4/76 (R) |
| 27 | ≤1 (S) | 32 (I) | 0.5 (S) | 0.25 (S) | 0.25 (S) | 32R | 8 (S) | ≤1 (S) | ≤0.25 (S) | 0.25 | 4/76 (R) |
| 28 | ≤1 (S) | 16 (S) | ≤0.12 (S) | >16 (R) | 0.25 (S) | 16 (I) | 4 (S) | ≤1 (S) | ≤0.25 (S) | 0.25 | 8/152 (R) |
| 29 | ≤1 (S) | 16 (S) | ≤0.12 (S) | 0.12 (S) | 16 (R) | 8 (I) | 8 (S) | 8 (R) | ≤0.25 (S) | 0.12 | 4/76 (R) |
| 30 | ≤1 (S) | 32 (I) | ≤0.12 (S) | >16(R) | >16(R) | 16 (I) | 8 (S) | >8(R) | ≤0.25 (S) | 0.06 | >8/152 (R) |
Table 10.
Interpretation of AST results for M. abscessus complex and clarithromycin.
| Sensitivity to Clarithromycin on Days 3–5 of Incubation | Sensitivity to Clarithromycin on Day 14 of Incubation | Genetic Mechanisms | Subspecies of M. abscessus | Phenotypic Sensitivity to Macrolides |
|---|---|---|---|---|
| sensitive | sensitive | non-functional erm gene (41) | M. a. massiliense | sensitive to macrolides |
| sensitive | resistant | functional erm gene (41) | M. a. abscessus M. a. bolletii | inducible resistance to macrolides |
| resistant | resistant | 23S point mutation in rRNA | any of the above listed | high constitutive resistance to macrolides |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
MDPI and ACS Style
Borek, A.; Zabost, A.; Głogowska, A.; Filipczak, D.; Augustynowicz-Kopeć, E. New RAPMYCOI SensititreTM Antimicrobial Susceptibility Test for Atypical Rapidly Growing Mycobacteria (RGM). Diagnostics 2022, 12, 1976. https://doi.org/10.3390/diagnostics12081976
AMA Style
Borek A, Zabost A, Głogowska A, Filipczak D, Augustynowicz-Kopeć E. New RAPMYCOI SensititreTM Antimicrobial Susceptibility Test for Atypical Rapidly Growing Mycobacteria (RGM). Diagnostics. 2022; 12(8):1976. https://doi.org/10.3390/diagnostics12081976
Chicago/Turabian StyleBorek, Anna, Anna Zabost, Agnieszka Głogowska, Dorota Filipczak, and Ewa Augustynowicz-Kopeć. 2022. "New RAPMYCOI SensititreTM Antimicrobial Susceptibility Test for Atypical Rapidly Growing Mycobacteria (RGM)" Diagnostics 12, no. 8: 1976. https://doi.org/10.3390/diagnostics12081976
Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.
