Over the past three decades, the incidence of infections caused by non-tuberculous mycobacteria (NTM) has increased. The prevalence of lung infections caused by NTM, representing approximately 200 species and subspecies, has increased dramatically over the last three decades, particularly among patients with cystic fibrosis (CF) (Floto et al. 2016; Abdelaal et al. 2022). NTM are facultative pathogenic microorganisms primarily responsible for opportunistic infections. They are widely distributed in the environment and can be found in diverse ecological niches, including water sources such as freshwater lakes, rivers, hot tubes, and soil. Moreover, NTM can form biofilms, facilitating their spread and colonization within the environment and among patients. Infections caused by NTM are primarily acquired through environmental exposure, although person-to-person transmission has been reported in some instances. NTM infections can affect lungs, skin and soft tissues, lymph nodes, joints, and bones, and disseminated infections can occur. Symptoms of NTM infections can range from mild to severe, and the clinical presentation often depends on a host’s immune status. Patient populations at a higher risk of NTM infections are individuals with cystic fibrosis, bronchiectasis, emphysema, chronic obstructive pulmonary disease, and immunocompromised conditions (Jang et al. 2014; Qvist et al. 2014; Kalaiarasan et al. 2020).
One of the most clinically relevant among rapidly growing mycobacteria (RGM) and very common among patients with CF are bacteria belonging to
MABC is responsible for about 90% of respiratory tract disorders caused by RGM. Moreover, the mortality in patients with CF is very high. Other infections concern the skin, soft tissue, bone, and central nervous system (Medjahed et al. 2010; Cho et al. 2013; Abdalla et al. 2015). Furthermore,
MABC showed resistance to the majority of routinely used antituberculosis drugs listed by the World Health Organization (WHO), and macrolides and amikacin remain the main therapeutic options. Most MABC infections are challenging to treat due to the numerous resistance mechanisms (Boeck et al. 2022; Li et al. 2022). Resistance to macrolides (clarithromycin, azithromycin) and aminoglycosides (kanamycin, amikacin, gentamicin) are associated with point mutations. While acquired macrolide resistance is associated with point mutations in a region of the
Resistance to aminoglycosides (kanamycin, amikacin, gentamicin) is the result of point mutations in the
Currently, the
The aim of the study was the evaluation of a rapid and effective assay to detect and discriminate species among NTM as well as among MABC and their mechanisms of resistance. Local monitoring of the prevalence of non-tuberculosis mycobacteria and their resistance profile enables proper antibiotic administration and ensures successful eradication therapy. Rapid detection of an etiological factor and molecular resistance profiles shortens the waiting time for results of microbiological designation. Thus, there is an urgent need to evaluate the performance of a new molecular assay for the rapid detection of clarithromycin and aminoglycoside resistance in the main clinically encountered NTM, including
The study covered 223 NTM clinical strains from patients diagnosed in the Malopolska Central Laboratory of Tuberculosis Diagnostics, The Saint John Paul II Specialist Hospital, in Cracow, Poland. Isolates were collected between June 2018 and December 2021. Strains were isolated from the materials collected from the lower respiratory tract, such as samples from bronchial lavage (137/223) and sputum (62/223), extrapulmonary materials were minor: (13/223) lung tumor section (8/223), fragment of lymph nodes (2/223), peritoneal fluid (1/223), one from gastric lavage and one from a wound swab. According to the Infectious Disease Society of America recommendations, all strains were isolated from patients with confirmed mycobacteriosis (Daley et al. 2020).
The respiratory samples were decontaminated with 4% sodium hydroxide and 2,9% N-acetyl-L-cysteine (NaOH-NALC) (Merck KGaA, Germany). The samples were cultured on solid Löwenstein-Jensen medium (LJ) (Becton, Dickinson and Company, USA) and BBL MGIT tube using the automatic mycobacterial detection system BACTEC™ MGIT™ supplemented with OADC (Becton, Dickinson and Company, USA). All specimens were processed for acid-fast-bacilli (AFB) smear microscopy and culture on the collection day. Smears were screened by Auramine-O fluorescence microscopy under 400 × magnification (Fig. 1). Positive smears were re-examined with Ziehl-Neelsen staining to identify AFB under oil immersion (1,000 × magnification) (Fig. 2).
All strains of Group I, photochromogenic mycobacteria – producing pigment after exposition to light, Group II, scotochromogenic mycobacteria – producing tint regardless of access to or a lack of light, Group III, nonchromogenic organisms – do not produce color under any conditions, Group IV, organisms are rapidly growing mycobacteria (5–7 days) that do not produce pigment.
International standard rapid growth mycobacteria species were used as the corresponding quality control strains for the clinical isolates tested:
At first, a bacterial colony was suspended in 150 ml of Vircell sample solution and incubated at 95°C for 60 min, according to the manufacturer’s recommendation. The supernatant, including extracted DNA, was used for further PCR analyses. Identification of 223 NTM strains was performed, using a Speed-Oligo® MYCOBACTERIA kit (Vircell Microbiologists, Spain) based on the amplification of a fragment of mycobacterial genomes and enabling for identification of
Total DNA was extracted using GenoLyse® VER. 2.0 (Hain Lifescience GmbH, Germany). Genomic DNA was extracted from a pure mycobacterial culture grown on Löwenstein-Jensen’s solid medium (Becton, Dickinson and Company, USA). The strains identified as
Interpretation of susceptibility pattern of rapidly growing mycobacteria according to the CLSI recommendations.
Antimycobacterial agents | MIC breakpoints (μg/ml) | ||
---|---|---|---|
S | I | R | |
Amikacin | ≤ 16 | 32 | ≥ 64 |
Cefoxitin | ≤ 16 | 32-64 | ≥ 128 |
Doxycycline | ≤ 1 | 2-4 | ≥ 32 |
Ciprofloxacin | ≤ 1 | 2 | ≥ 4 |
Clarithromycin | ≤ 2 | 4 | ≥ 8 |
Linezolid | ≤ 8 | 16 | ≥ 32 |
Moxifloxacin | ≤ 1 | 2 | ≥ 4 |
Trimthoprim/sulfamethoazole | ≤ 2/38 | – | ≥ 4/76 |
Imipenem | ≤ 4 | 8-16 | ≥ 32 |
Tigecycline* | – | – | – |
S – susceptible, I – intermediate, R – resistant
* – insufficient data on tigecycline’s correlation between in vitro results and clinical response hinders the establishment of breakpoints to differentiate susceptible from resistant strain, thus only MIC should be reported
MALDI-TOF MS (Matrix-assisted laser desorption ionization-time of flight mass spectrometry) identification of the selected strains of NTM was performed using the MALDI Biotyper® sirius IVD System (Bruker Daltonics, France). The protein extraction procedure for analysis was carried out under the manufacturer’s recommendations. Mycobacteria extraction MycoEX method was conducted according to standard operating procedure. All samples were prepared from solidmedium. Firstly, three 10 μl loops of mycobacterial biomass were transferred into 300 μl of deionized water in Eppendorf tubes and inactivated at 95°C by boiling them for 30 min. 900 μl EtOH was pipetted into an Eppendorf tube and centrifuged for 2 minutes at maximum speed. Then, the supernatant was discarded. The pellet was left at room temperature to dry. Silica beads were added to the Eppendorf tubes to thoroughly break up the mycobacterial colonies, followed by adding 20 μl of pure acetonitrile and vortexed at full speed for 1 minute. A 20 μl volume of 70% formic acid was added, vortexed, and then centrifuged at maximum speed for 2 min. Finally, 1 μl of the supernatant was placed on the MALDI-TOF target plate and allowed to dry at room temperature. Immediately after the sample spot had dried, the spot was overlaid with 1 μl of HCCA matrix. The species identification was conducted using Bruker software to compare the protein profile of the bacteria obtained from a database of protein profiles. The MBT Mycobacteria RUO Library 7.0 covers 182 of 201 mycobacteria species. A range 1.8–3.0 indicates a high-confidence identification of genus and species. A score > 1.7 indicates the identification of genus but not species, and a score lower than 1.7 indicates no identification of bacteria.
Minimum inhibitory concentrations (MICs) of clarithromycin (PHR1038; Merck KGaA, Germany) and amikacin (PHR1654; Merck KGaA, Germany) were determined by the broth microdilution method according to CLSI recommendation (CLSI 2018a) and ISO norm (ISO 20776-1:2019 2019).
The analysis of 11 strains
Simultaneously, a commercially available Sensititre™ RAPMYCO2 Susceptibility Testing Plate (Thermo Fisher Scientific, Inc., USA) was used to evaluate MIC by the broth microdilution method. The broth microdilution method provided by Sensititre™ RAPMYCO2 was a validated method for the susceptibility testing of RGM including
Molecular evaluation of susceptibility to aminoglycosides and macrolides was determined by GenoType NTM-DR VER. 1.0 that enables the detection of point mutations referring to resistance to these classes of drugs. The analysis was conducted as described above. Based on hybridization and alkaline phosphatase reaction on a membrane strip, specific strips allow for the determination of the resistance mechanisms (Table II). Wild strains without macrolides resistance mechanisms reveal
Interpretation of the mutation type conferring to the macrolides and aminoglicosides resistance according to the GenoType NTM-DR VER 1.0 test instructions.
Discernible phenotypic pattern resistance | Target nucleic acid sequence | Nascent mutation band | Mutation |
---|---|---|---|
Macrolides | 2058–2059 | A2058C | |
A2058G | |||
– | A2058T | ||
A2059C | |||
A2059T | |||
Aminoglycosides | 1406–1409 | A1408G | |
– | T1406A | ||
– | C1409T |
Based on phenotypic and molecular assays, as well as the MALDI-TOF analysis, the species identification was conducted. In total, 223 NTM strains were isolated from patients between 2018–2021. Among them, 35% (77/223) were
The analysis of the frequency of NTM occurrence over the years revealed 66 strains in 2018, 48 in 2019, 39 in 2020, and 70 in 2021. Within isolated strains, the most frequent was
The initial phenotypic identification showed that among all 223 NTM strains, 19 belonged to the RGM group. Molecular discrimination (CM Direct) confirmed 12 isolates as
Strip 1 | – a clinical strain of |
Strips 2-6, 10, 12 - | –clinical strains of |
Strips 7, 9 | – clinical strains of |
Strips 8 | – clinical strains of |
Strip 11 | – clinical strains of |
Antimicrobial susceptibility testing was performed for 11
Results of susceptibility testing to antimycobacterial agents obtained by Sensititre™ RAPMYCO2 plates. Interpretation criteria following CLSI.
MIC values (μg/ml) (Interpretation S/I/R) | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
No. | Species | AN | FOX | CIP | DOX | LZD | MXF | SXT | IMI | TGC | CLA after 5d | CLA after 14 d |
1 | Mabs | 16 (S) | 64 (I) | > 4 (R) | > 16 (R) | ≤ 1 (S) | 4 (R) | 4/76 (R) | 16 (I) | MIC = 1 | 0,06 (S) | 0,125 (S) |
2 | Mabs | 16 (S) | >128 (R) | > 4 (R) | > 16 (R) | 4 (S) | 2 (I) | > 8/152 (R) | 16 (I) | MIC = 0,5 | 0,06 (S) | > 16 (R) |
3 | Mabs | 16 (S) | 32 (I) | > 4 (R) | > 16 (R) | 4 (S) | > 8 (R) | 4/76 (R) | 16 (I) | MIC = 0,5 | 0,5 (S) | > 16 (R) |
4 | Mabs | 16 (S) | >128 (R) | > 4 (R) | > 16 (R) | 16 (I) | > 8 (R) | > 8/152 (R) | 16 (I) | MIC = 2 | 0,06 (S) | > 16 (R) |
5 | Mabs | 16 (S) | 64 (I) | > 4 (R) | > 16 (R) | 16 (I) | > 8 (R) | > 8/152 (R) | 16 (I) | MIC = 1 | 0,06 (S) | > 16 (R) |
6 | Mabs | 16 (S) | 64 (I) | > 4 (R) | > 16 (R) | 4 (S) | > 8 (R) | > 8/152 (R) | 16 (I) | MIC = 2 | 4 (I) | > 16 (R) |
7 | Mmas | - | - | - | - | - | - | - | - | - | - | - |
8 | Mmas | 16 (S) | >128 (R) | > 4 (R) | > 16 (R) | 16 (I) | 4 (R) | 4/76 (R) | 16 (I) | MIC = 0,25 | 0,06 (S) | 0,125 (S) |
9 | Mabs | 16 (S) | >128 (R) | > 4 (R) | > 16 (R) | 16 (I) | 4 (R) | 4/76 (R) | 16 (I) | MIC = 0,5 | 0,06 (S) | 0,125 (S) |
10 | Mmas | 16 (S) | 32 (I) | > 4 (R) | 1 (S) | 1 (S) | 2 (I) | 2/38 (S) | 16 (I) | MIC = 0,5 | 0,06 (S) | 0,06 (S) |
11 | Mbol | 4(S) | 32 (I) | > 4 (R) | > 16 (R) | 4 (S) | > 8 (R) | > 8/152 (R) | 16 (I) | MIC = 0.25 | 0.06 (S) | > 16 (R) |
12 | Mabs | 4 (S) | 33 (I) | > 4 (R) | > 16 (R) | 8 (S) | > 8 (R) | > 8/152 (R) | 16 (I) | MIC = 0.12 | 4 (I) | > 16 (R) |
Mabs –
Results of susceptibility testing to amikacin and clarithromycin obtained by manually conducted microdilution method. Interpretation criteria following CLSI.
No. | Species | AN | Interpretation* | CLA after 5 days of incubation | Interpretation* | CLA after 14 days of incubation | Interpretation* |
---|---|---|---|---|---|---|---|
1 | Mabs | 16 | (S) | 0.06 | (S) | 0,5 | (S) |
2 | Mabs | 8 | (S) | 0.06 | (S) | > 64 | (R) |
3 | Mabs | 16 | (S) | 0.06 | (S) | = 32 | (R) |
4 | Mabs | 16 | (S) | 0.06 | (S) | = 32 | (R) |
5 | Mabs | 16 | (S) | 0.06 | (S) | = 32 | (R) |
6 | Mabs | 16 | (S) | 0.06 | (S) | = 32 | (R) |
7 | Mmas | – | – | – | – | – | – |
8 | Mmas | 16 | (S) | 0.06 | (S) | = 0.125 | (S) |
9 | Mabs | 16 | (S) | 0.06 | (S) | = 0.125 | (S) |
10 | Mmas | 16 | (S) | 0.06 | (S) | = 0.06 | (S) |
11 | Mbol | 0.5 | (S) | 0.06 | (S) | > 64 | (R) |
12 | Mabs | 0.250 | (S) | 4 | (R) | = 16 | (R) |
Mabs –
S – susceptible, I – intermediate, R – resistant
AN – amikacin, CLA – clarithromycin
* – interpretation of the result was done according CLSI
Results of molecular identification of mechanisms of resistance reveal that only one strain exhibits point mutation in
Results of molecular identification of resistance mechanisms to amikacin obtained by GenoType NTM in relation to phenotypic susceptibility testing results.
No. of strain | Species | GenoType NTM-DR results | Phenotypic results of susceptibility to amikacin | |
---|---|---|---|---|
Type of bands (WT or mutation) | Molecular mechanisms of resistance (Yes/No) | |||
1 | Mabs | No | (S) | |
2 | Mabs | No | (S) | |
3 | Mabs | No | (S) | |
4 | Mabs | No | (S) | |
5 | Mabs | No | (S) | |
6 | Mabs | No | (S) | |
7 | Mmas | No | nt | |
8 | Mmas | No | (S) | |
9 | Mabs | Yes | (S) | |
10 | Mmas | No | (S) | |
11 | Mb ol | No | (S) | |
12 | Mabs | No | (S) |
Mabs –
Mmas –
Mbol –
S – susceptible, nt – not tested
A molecular assay to determine the resistance of
Results of molecular identification of resistance mechanisms to clarithromycin by GenoType NTM in relation to phenotypic susceptibility testing results.
No. of strain | Species | GenoType NTM-DR results | Phenotypic results of susceptibility to clarithromycin | |
---|---|---|---|---|
Type of bands (WT or mutation) | Molecular mechanisms of resistance (Yes/No) | |||
1 | Mabs | No | (S) | |
2 | Mabs | Yes | (R) | |
3 | Mabs | Yes | (R) | |
4 | Mabs | Yes | (R) | |
5 | Mabs | Yes | (R) | |
6 | Mmas | Yes | (R) | |
7 | Mmas | No | nt | |
8 | Mmas | No | (S) | |
9 | Mabs | Yes | (S) | |
10 | Mb ol | No | (S) | |
11 | Mabs | Yes | (R) | |
12 | Mabs | Yes | (R) |
Mabs –
Mmas –
Mbol –
S – susceptible, R – resistant, nt – not tested
* – due to the deletion in the
The prevalence of MABC and other mycobacteria species was investigated in our research conducted between 2018 and 2021. Notably, only strains isolated for the first time from clinically confirmed mycobacteriosis patients were included in the analysis. Consistent with the literature on the epidemiology of NTM in Europe (Wassilew et al. 2016), as well as the previous investigation in Poland (Hoefsloot et al. 2013), our study results demonstrated a predominant prevalence of the
In our study, the average prevalence of slow-growing mycobacteria between 2018–2021 amounted to 91.93%, while bacteria belonging to rapid-growing mycobacteria were on average 8.07%. These data closely resembled those obtained within the NTM-NET collaborative study (Hoefsloot et al. 2013), 80% SGM and 20% RGM, respectively. The prevalence of NTM can vary across different regions and may be influenced by factors such as population demographics, geographic location, and diagnostic practices. In total, 5% of pulmonary infections are caused by rapidly growing mycobacteria, with approximately 65–80% of RGM infections specifically attributed to
A notable finding is the complete absence of the MAB complex isolates in 2020. The decline in the number of isolates was undoubtedly attributed to the ongoing COVID-19 pandemic, which resulted in a reduced frequency of isolation and access to mycobacterial diagnostics. During this period, diagnostic efforts were primarily directed toward detecting COVID-19 cases, which may have led to a decline in identifying mycobacterial infections, including MABC, from pulmonary samples. Our results confirmed these assumptions. In 2018, the number of all cultures performed in the Malopolska Central Laboratory of Tuberculosis Diagnostics, amounted to 9,882, including positive results for NTM in 69 samples, which accounted for 0,7% of all tests performed. During the announcement of the pandemic in 2020, the number of tests ordered was 7,120, and a positive result was obtained from 39 samples, which was 0,54 %. The number of ordered tests in the first year of the pandemic decreased by nearly 30%. In the case of molecular tests, the number of tests performed in 2020 decreased by 14% compared to 2018 (3,326 and 2,869 tests, respectively). The drastic decrease in research was also visible in relation to specific months in which successive ‘waves’ of infections and peaks of COVID-19 cases occurred. Research was often reduced by as much as 60% during these periods. The COVID-19 pandemic has significantly affected the number of mycobacterial diagnoses. The decrease in ordered tests was related to changes in the organization of medical facilities, the transformation of departments or entire hospitals into single-name infectious disease hospitals, and the abandonment of diagnostics for other diseases, including mycobacteriosis.
The proper diagnosis and detection of infections caused by NTM, as well as accurate species identification and drug susceptibility profiling, hold immense significance in guiding appropriate treatment strategies. This aspect becomes particularly crucial in the case of managing infections caused by MAB complex, because of their high level of resistance. A high level of resistance limits the treatment of MABC infections to most available anti-mycobacterial drugs. Therefore, precise and timely identification of the causative species and their drug resistance profile is paramount for optimizing treatment outcomes in patients affected by MABC infections. Compared to other non-tuberculous mycobacterial NTM infections, MAB complex infections tend to have relatively poorer treatment outcomes, with an in-hospital mortality rate of up to 16% (Choi et al. 2017). In particular, MABC is associated with intrinsic and acquired resistance to most anti-mycobacterial agents, including macrolides. Macrolide antibiotics, specifically clarithromycin and azithromycin, are the cornerstone of treatment for mycobacteriosis. These antibiotics exert their therapeutic effect by binding to the V domain of the 23S rRNA in the 50S ribosomal subunit. Macrolide resistance is induced upon exposure to these drugs by the expression of the ribosome methylase-encoding
Another important group of drugs is aminoglycosides. However, MABC has also developed a range of resistance mechanisms against this class of antibiotics. Hence treatment of
In our research, we assessed the resistance mechanisms of MABC to aminoglycosides and macrolides. Additionally, we compared the results of molecular analysis with their correlations to phenotypic resistance determination using microdilution methods. We obtained mostly concordant results, except for one strain, which accounted for approximately 8.33% (1 out of 12) of the discordant cases. The molecular analyze of
Among MAB complex subspecies there are significant differences in susceptibility patterns.
According to the IDSA (Infectious Diseases Society of America) guidelines, the recommended approach for treating lung infections is to customize the treatment based on drug susceptibility testing of macrolides and aminoglycosides, instead of using empirical therapy. To identify macrolide resistance, a 14-day incubation and/ or sequencing of the
It is important to highlight that not all major anti-mycobacterial drugs presented well-established links between laboratory and clinical outcomes. Thus it is recommended to conduct baseline susceptibility testing for specific drugs for each patient with a confirmed NTM disease, following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Although testing additional drugs may offer potential benefits, there is currently limited data to provide specific recommendations in this context. These guidelines align with those set forth by the Infectious Diseases Society of America (Daley et al. 2020; CLSI 2018a; 2018b).
Limited therapeutic options for MABC infections associated with natural and acquired resistance to most anti-mycobacterial drugs pose a high risk of treatment failure. Differentiation of subspecies within MABC, the simultaneous determination of genes for resistance to macrolides and aminoglycosides is crucial to undertaking the correct eradication therapy.
To conclude, high clarithromycin resistance of
Our report is a retrospective