Mycobacterium chimaera is a slow-growing nontuberculous Mycobacterium species belonging to the Mycobacterium avium complex (MAC). It has been identified globally as the cause of a large outbreak of cardiovascular infections following open heart surgery, but it can also cause respiratory infections in individuals with underlying structural pulmonary disease. Invasive M. chimaera infections are associated with poor clinical responses, and the optimal antibiotic treatment regimen for these infections is not known.
KEYWORDS: antimicrobial susceptibility testing, mycobacteria, Mycobacterium avium complex, Mycobacterium chimaera
ABSTRACT
Mycobacterium chimaera is a slow-growing nontuberculous Mycobacterium species belonging to the Mycobacterium avium complex (MAC). It has been identified globally as the cause of a large outbreak of cardiovascular infections following open heart surgery, but it can also cause respiratory infections in individuals with underlying structural pulmonary disease. Invasive M. chimaera infections are associated with poor clinical responses, and the optimal antibiotic treatment regimen for these infections is not known. In this study, the drug susceptibility profiles of clinical and environmental M. chimaera isolates for antimicrobial agents that are commonly considered for treatment of MAC infections were determined. All M. chimaera isolates were susceptible to clarithromycin, with a median MIC of 2 μg/ml, while 98% (85/87 isolates) were susceptible to amikacin. Twenty-five percent of isolates (22/87 isolates) had intermediate susceptibility and 52% (46/87 isolates) were resistant to moxifloxacin. Similarly, 39% of isolates (34/87 isolates) had intermediate susceptibility and 39% (34/87 isolates) were resistant to linezolid. MIC breakpoints derived from the literature were used to determine resistance to rifampin (16/87 isolates [18%]), ethambutol (10/87 isolates [11%]), rifabutin (2/87 isolates [2%]), and streptomycin (1/87 isolates [1%]). In conclusion, our results showed that clarithromycin, amikacin, rifabutin, and streptomycin had the best activity against M. chimaera isolates, while susceptibility rates were lower for rifampin and ethambutol. In contrast, there was a high prevalence of isolates that were not susceptible to moxifloxacin or linezolid. While factors in addition to antibiotic susceptibility may determine the outcomes of treatment of M. chimaera infections, our results should inform the selection of antimicrobials as part of the overall therapeutic strategy.
INTRODUCTION
Mycobacterium chimaera is a slow-growing nontuberculous Mycobacterium (NTM) species that belongs to the Mycobacterium avium complex (MAC) (1). In recent years, M. chimaera has been reported as a cause of respiratory infections in patients with preexisting lung diseases such as chronic obstructive pulmonary disease, bronchiectasis, and cystic fibrosis (1–3). The growing importance of this emerging pathogen is emphasized by the recent global outbreak of invasive cardiovascular M. chimaera infections associated with exposure to aerosols generated by heater-cooler units (HCUs) during open heart surgery (OHS) (4).
Studies regarding the in vitro antimicrobial susceptibility of MAC strains are sparse, and those available in the literature have mainly evaluated the drug susceptibility patterns of members of the MAC, including M. avium and Mycobacterium intracellulare, rather than M. chimaera specifically (5–7). The Clinical and Laboratory Standards Institute (CLSI) recommends that broth microdilution MIC assays should be used to test for the antimicrobial susceptibility of MAC strains (8–10). However, routine susceptibility testing is recommended only for clarithromycin, as it is the primary drug used for treatment and results correlate well with clinical responses (8). The CLSI guidelines also recommend reporting the susceptibility of MAC isolates to moxifloxacin and linezolid in cases of clarithromycin-resistant MAC infections (8). Rifampin, rifabutin, ethambutol, amikacin, and streptomycin are also considered clinically useful for treatment of MAC infections; however, the role of susceptibility testing for these agents is controversial, as there are often poor correlations between MIC results and treatment outcomes (11).
Nevertheless, the guidelines of both the American Thoracic Society and the British Thoracic Society for treatment of NTM infections recommend that a combination of a macrolide (clarithromycin or azithromycin), a rifamycin (rifampin or rifabutin), and ethambutol should be used for the treatment of MAC pulmonary infections, for a period of 12 months (12, 13). Treatment with an injectable aminoglycoside such as amikacin or streptomycin is recommended as an addition for individuals with severe MAC pulmonary disease (13). Treatment of invasive cardiovascular M. chimaera infections subsequent to OHS is particularly challenging, as there is a high rate of therapeutic failure despite targeted antimicrobial therapy (14–16). Therefore, the optimal treatment regimen for invasive M. chimaera infections is unknown; to date, therapy has been based largely on treatment recommendations for pulmonary MAC infections (1, 3, 17). The aim of this study was to determine the susceptibility profiles of M. chimaera clinical isolates of both respiratory and cardiovascular origin, as well as environmental M. chimaera isolates recovered from the water reservoirs of HCUs, for the antimicrobial agents that are commonly considered for use in the treatment of MAC infections.
RESULTS
The MIC ranges, MIC50 and MIC90 values, and numbers of resistant isolates are presented in Table 1. Using the CLSI breakpoints, 100% of M. chimaera isolates (87/87 isolates) were susceptible to clarithromycin (MICs of ≤8 μg/ml), with the distribution of MICs for susceptible isolates ranging from 0.25 μg/ml to 4 μg/ml. Based on CLSI breakpoints for moxifloxacin, 25% of isolates (36/87 isolates) had intermediate susceptibility (MICs of 2 μg/ml) and 52% of isolates (46/87 isolates) were resistant (MICs of ≥4 μg/ml). Similarly, using breakpoints for linezolid, 39% of isolates (34/87 isolates) had intermediate susceptibility (MICs of 16 μg/ml), while 39% of isolates (34/87 isolates) were resistant (MICs of ≥32 μg/ml). When the suggested pharmacokinetics (PK)/pharmacodynamics (PD)-derived breakpoints for rifampin and ethambutol were applied, 18% of isolates (16/87 isolates) were resistant to rifampin (MICs of ≥8 μg/ml) and 11% of isolates (10/87 isolates) were resistant to ethambutol (MICs of ≥8 μg/ml). When proposed CLSI breakpoints for amikacin (18) were applied, all isolates were susceptible except for 2 isolates that had intermediate susceptibility (MICs of 32 μg/ml). Epidemiological cutoff (ECOFF) values showed that only 2/87 M. chimaera isolates had non-wild-type profiles for rifabutin and 1/87 had a non-wild-type profile for streptomycin.
TABLE 1.
Antimicrobial susceptibility profiles of Mycobacterium chimaera isolates (n = 87)
| Antimicrobial agent | MIC range (μg/ml) | MIC50 (μg/ml) | MIC90 (μg/ml) | No. of resistant isolates/total no. of isolates (%) according to: |
||
|---|---|---|---|---|---|---|
| CLSI breakpointa | Tentative ECOFFb | PK/PD breakpointc | ||||
| Clarithromycin | 0.25 to 4 | 2 | 4 | 0 | ||
| Moxifloxacin | 1 to >8 | 4 | 4 | 46/87 (52) | ||
| Linezolid | 4 to 64 | 16 | 32 | 34/87 (39) | ||
| Rifampin | 0.5 to >8 | 4 | 8 | 16/87 (18) | ||
| Ethambutol | ≤0.5 to >16 | 4 | 8 | 10/87 (11) | ||
| Rifabutin | ≤0.25 to >8 | ≤0.25 | 1 | 2/87 (2) | ||
| Amikacin | 2 to 32 | 8 | 16 | 0 | ||
| Streptomycin | 4 to >64 | 16 | 32 | 1/87 (1) | ||
CLSI breakpoints were used to interpret susceptible, intermediate, and resistant MICs for the following antimicrobial agents: clarithromycin: susceptible, ≤8 μg/ml; intermediate, 16 μg/ml; resistant, ≥32 μg/ml; moxifloxacin: susceptible, ≤1 μg/ml; intermediate, 2 μg/ml; resistant, ≥4 μg/ml; linezolid: susceptible, ≤8 μg/ml; intermediate, 16 μg/ml; resistant, ≥32 μg/ml. CLSI breakpoints proposed by Brown-Elliott et al. (18) were used to interpret results for amikacin (susceptible, ≤16 μg/ml; intermediate, 32 μg/ml; resistant, ≥64 μg/ml).
Tentative ECOFF values published by Maurer et al. (23) were used to determine wild-type (susceptible) and non-wild-type (resistant) isolates for rifabutin and streptomycin. ECOFF values were as follows: rifabutin, 2 μg/ml; streptomycin, 64 μg/ml.
Resistance breakpoints were derived from a PK/PD study by van Ingen et al. (27). M. chimaera isolates were classified as resistant if the MIC for rifampin was ≥8 μg/ml, that for ethambutol was ≥8 μg/ml, and that for amikacin was ≥64 μg/ml, as used previously by Kim et al. (26) and Schön and Chryssanthou (7).
Table 2 presents the MIC distributions for all clinical and environmental isolates of M. chimaera. All respiratory, OHS, and environmental isolates were susceptible to clarithromycin. Of the moxifloxacin-resistant isolates, 38/61 (62%) were of respiratory origin, 2/15 (13%) were from OHS-related sources, and 6/11 (55%) were from environmental HCU water sources. Of the linezolid-resistant isolates, 26/61 (43%) were from respiratory sources, 2/15 (13%) were from OHS sources, and 6/11 (55%) were from environmental HCU water sources. Using the published breakpoints and ECOFF values, 8/61 isolates (13%) from respiratory sources, 1/15 isolates (6.6%) from OHS sources, and 7/11 isolates (63%) from HCU water were classified as resistant to rifampin. Of the ethambutol-resistant isolates, 9/61 (14.7%) were from respiratory sources and 1/15 (6.6%) was from an OHS-related source. Eighty-five of 87 M. chimaera isolates were classified as susceptible to amikacin, with 1/15 (6.6%) from an OHS source and 1/11 (9%) from a HCU water source being classified as intermediate. Of the rifabutin-resistant isolates (non-wild-type profiles), 1/15 (6.6%) was from an OHS source and 1/11 (9%) was from a HCU water source. Similarly, of the streptomycin-resistant M. chimaera isolates (non-wild-type profiles), 1 was a respiratory isolate and none was from an OHS or environmental source.
TABLE 2.
In vitro susceptibility testing of Mycobacterium chimaera isolates collected and tested between 2007 and 2017
| Isolate source and antimicrobial agenta | No. of isolatesb
with MICs of: |
No. of resistant isolatesc | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.25 μg/ml | 0.5 μg/ml | 1 μg/ml | 2 μg/ml | 4 μg/ml | 8 μg/ml | 16 μg/ml | 32 μg/ml | 64 μg/ml | >64 μg/ml | ||
| Respiratory (n = 61) | |||||||||||
| CLR | 1 | 4 | 21 | 28 | 7 | 0 | |||||
| MXF | 1 | 22 | 34 | 4 | 38 | ||||||
| LZD | 10 | 25 | 24 | 2 | 26 | ||||||
| RIF | 5 | 21 | 27 | 8 | 8 | ||||||
| EMB | 11 | 41 | 6 | 3 | 9 | ||||||
| RFB | 27 | 23 | 9 | 1 | 1 | 1 | |||||
| AMK | 1 | 4 | 29 | 11 | 0 | ||||||
| STR | 1 | 12 | 20 | 24 | 4 | 1 | 1 | ||||
| OHS related (n = 15) | |||||||||||
| CLR | 1 | 3 | 6 | 4 | 1 | 0 | |||||
| MXF | 2 | 11 | 2 | 2 | |||||||
| LZD | 2 | 5 | 6 | 2 | 2 | ||||||
| RIF | 3 | 5 | 6 | 1 | 1 | ||||||
| EMB | 2 | 4 | 8 | 1 | 1 | ||||||
| RFB | 11 | 3 | 1 | 0 | |||||||
| AMK | 1 | 6 | 6 | 1 | 1 | 0 | |||||
| STR | 1 | 9 | 5 | 0 | |||||||
| Environmental HCU water (n = 11) | |||||||||||
| CLR | 3 | 1 | 5 | 2 | 0 | ||||||
| MXF | 2 | 3 | 4 | 2 | 6 | ||||||
| LZD | 1 | 1 | 3 | 6 | 6 | ||||||
| RIF | 1 | 1 | 2 | 7 | 7 | ||||||
| EMB | 0 | 3 | 8 | 0 | |||||||
| RFB | 7 | 2 | 1 | 1 | 1 | ||||||
| AMK | 3 | 5 | 2 | 1 | 0 | ||||||
| STR | 2 | 6 | 3 | 0 | |||||||
Antimicrobial agents included clarithromycin (CLR), moxifloxacin (MXF), linezolid (LZD), rifampin (RIF), ethambutol (EMB), rifabutin (RFB), amikacin (AMK), and streptomycin (STR).
The numbers of resistant isolates are highlighted in bold.
CLSI breakpoints were used to interpret susceptible, intermediate, and resistant MICs for the following antimicrobial agents: clarithromycin: susceptible, ≤8 μg/ml; intermediate, 16 μg/ml; resistant, ≥32 μg/ml; moxifloxacin: susceptible, ≤1 μg/ml; intermediate, 2 μg/ml; resistant, ≥4 μg/ml; linezolid: susceptible, ≤8 μg/ml; intermediate, 16 μg/ml; resistant, ≥32 μg/ml. M. chimaera isolates were classified as resistant if the MIC for rifampin was ≥8 μg/ml, that for ethambutol was ≥8 μg/ml, and that for amikacin was ≥64 μg/ml, as used previously by Kim et al. (26), Brown-Elliott et al. (18), and Schön and Chryssanthou (7). ECOFF values used to determine non-wild-type isolates were as follows: rifabutin, 2 μg/ml; streptomycin, 64 μg/ml.
DISCUSSION
Publications on the in vitro susceptibility of M. chimaera to antimicrobial agents that are commonly considered for therapy of MAC infections are limited, and different methods of susceptibility testing have been used (19–21). Treatment of M. chimaera infection is based on treatment guidelines for the MAC as a group, as there are limited data from clinical experience (12, 13). Clarithromycin is the antibiotic of first choice for treatment of MAC infections, and this study showed that clarithromycin had very good in vitro activity against M. chimaera. Generally, wild-type M. chimaera isolates not previously exposed to antibiotic treatment are susceptible to clarithromycin; in some cases, however, isolates from patients who were previously treated have become resistant (22). CLSI breakpoints for moxifloxacin and linezolid are also available for reporting in cases of clarithromycin-resistant MAC strains, and moxifloxacin is commonly used as part of a multidrug regimen for treatment of invasive M. chimaera infections associated with OHS (14). The clinical efficacy of moxifloxacin and linezolid in MAC infections is unproven, although the CLSI recommends breakpoints (10). In our study, we observed that a large proportion of isolates were not fully susceptible to moxifloxacin or linezolid, thereby demonstrating poor activity against M. chimaera. In vitro susceptibility data may have an influence on whether these are efficacious therapeutic agents.
Rifampin and rifabutin, together with ethambutol, are often included in treatment regimens for MAC infections to prevent the emergence of clarithromycin resistance, and an injectable aminoglycoside such as amikacin may also be included for patients with severe MAC pulmonary disease or invasive M. chimaera infections associated with OHS (12–14). Similar to the findings of Maurer et al. (23), we found that M. chimaera isolates were susceptible to amikacin, rifabutin, and streptomycin, with up to 2% not being fully susceptible.
In vitro susceptibility testing results for amikacin have recently been found to correlate with positive treatment outcomes, and breakpoints for amikacin proposed by Brown-Elliott et al. were recently accepted by the CLSI (9, 18). CLSI guidelines for susceptibility testing of MAC strains have also been recently published, along with defined breakpoints for intravenous and liposomal amikacin (9, 10). It has also been suggested that streptomycin can be used in place of amikacin; however, no studies have been performed to correlate streptomycin MIC results and clinical responses (10). In contrast, 18% and 11% of M. chimaera isolates were not susceptible to rifampin and ethambutol, respectively. However, in vitro results for rifampin, rifabutin, and ethambutol for MAC strains have not reliably correlated with clinical responses and treatment outcomes, and the clinical usefulness of testing these agents for M. chimaera is subject to debate (24). More studies are needed to adequately determine the correlation between MIC data for rifampin, rifabutin, and ethambutol and treatment outcomes for M. chimaera infections.
A limitation of in vitro susceptibility testing of M. chimaera and other MAC species is that breakpoints for agents that are considered clinically useful for the treatment of MAC infections have not been approved by the CLSI. PK/PD breakpoints are often used to guide breakpoint development and may aid interpretation in situations in which there are no species-specific breakpoints (25). Previously, breakpoints based on PK/PD studies were used to determine rifampin and ethambutol resistance (7, 26, 27). These breakpoints were based on Bactec 460 broth macrodilution determinations, and the application of these breakpoints for antimicrobial susceptibility testing (AST) by broth microdilution may vary, which is a limitation of this study (27). Recent studies on the drug susceptibility of M. chimaera and other MAC species showed that ECOFF values could not be determined for rifampin and ethambutol, due to truncated MIC distributions (23). Similarly, recent tentative ECOFF values for moxifloxacin and linezolid proposed by Maurer et al. suggested that the CLSI breakpoints for these antibiotics should be reevaluated for MAC susceptibility testing (23).
A limitation of this work was the relatively small number of M. chimaera isolates tested, particularly from OHS and environmental HCU sources. The MIC distributions showed that respiratory M. chimaera isolates appeared to have higher rates of resistance to moxifloxacin (38/61 isolates), linezolid (26/61 isolates), rifampin (8/61 isolates), and ethambutol (9/61 isolates), compared to isolates relating to OHS. Similarly, M. chimaera isolates from environmental HCU water appeared to have high rates of resistance to moxifloxacin (6/11 isolates), linezolid (6/11 isolates), and rifampin (7/11 isolates). AST of more M. chimaera isolates is required to determine whether the rates of resistance among M. chimaera strains reported here represent valid observations and are reproducible in other centers.
Environmental M. chimaera isolates were recovered from water samples taken from HCUs as part of the national incident investigation in Ireland (28). AST of environmental M. chimaera isolates recovered from HCUs is important because whole-genome sequencing studies have linked the majority of invasive M. chimaera infections to strains recovered from contaminated HCU devices globally (4). Treatment of invasive M. chimaera infections associated with OHS has been challenging (14, 16, 17, 29). Like other NTM species, M. chimaera appears to be an effective biofilm producer (30). Further research is required to investigate the antimicrobial susceptibility of M. chimaera grown in biofilms, as this may lead to better choices of antimicrobial agents with improved clinical outcomes.
Compared with Mycobacterium tuberculosis, antibiotic resistance mechanisms in NTM species are not as well studied (31). Treatment failure for MAC infections has been associated with clarithromycin monotherapy in individual patients whose isolates developed mutations in the 23S rRNA gene, also known as rrl (32–34). Amikacin-resistant MAC isolates have also been reported, and resistance is linked to mutations in the 16S rRNA gene, also known as rrs (18). A commercial molecular assay for determining drug susceptibility to macrolides and aminoglycosides has recently been developed for MAC strains, and results appear to show good correlation with in vitro susceptibility testing results (35). However, the molecular mechanisms for resistance to other antimicrobial agents have not been well defined for MAC strains, and very few reports of resistance mechanisms associated with rifampin and ethambutol resistance have been published (36, 37).
Further in vitro susceptibility testing of M. chimaera with bedaquiline and clofazimine should also be investigated. Although there are no specific CLSI guidelines for testing with these agents, limited reports have shown that bedaquiline appears to be useful in treating MAC infections with low MICs (38, 39).
Clofazimine and amikacin have shown significant synergistic activities against MAC strains in vitro and may be useful for treatment of M. chimaera infections (40). More recently, in vitro data showed that a combination of bedaquiline and clofazimine may add activity for the treatment of MAC infections (41).
In conclusion, treatment of M. chimaera infections is challenging, and optimal treatment regimens are not established. However, in vitro AST should help inform the choice of antimicrobial agents as part of the overall therapeutic strategy. Further research is needed to determine correlations between antimicrobial therapy and treatment outcomes for patients with M. chimaera respiratory or cardiovascular infections. Whole-genome sequencing may facilitate exploration of the diversity of M. chimaera strains associated with antimicrobial resistance and their drug-resistance-associated gene targets.
MATERIALS AND METHODS
Background.
This study was undertaken, in part, in response to a national Health Services Executive investigation of M. chimaera cardiovascular infections associated with OHS (28). The study was conducted between 2007 and 2017, and no personal patient information was used. Isolates were provided by the Irish Mycobacteria Reference Laboratory and were handled thereafter using alphanumeric codes, with no patient identifiers.
Collection of M. chimaera isolates.
M. chimaera isolates (n = 87) included an archival collection collected between 2007 and 2017 and more recent clinical and environmental isolates referred from Irish hospitals to the Irish Mycobacteria Reference Laboratory in St. James Hospital, Dublin, Ireland. All isolates were identified by sequencing the 16S rRNA gene and internal transcribed spacer (ITS) region, and a subset of isolates were also identified using the GenoType NTM-DR v1.0 assay (Hain Lifescience), as described previously (42). M. chimaera isolates included for AST were selected on the basis of 1 isolate per patient per year. Duplicate isolates were excluded unless an isolate had been recovered from a different specimen type. Of these isolates, 61 were previously recovered from respiratory specimens, 15 isolates were from diagnostic samples that had been taken during or following OHS, and 11 environmental M. chimaera isolates were recovered from water samples taken from HCUs used in Irish cardiothoracic surgery centers, as part of the HCU surveillance program in Ireland from 2015 (18). M. chimaera strain DSM 44623, which was first described by Tortoli et al., was also tested (1).
Antimicrobial susceptibility testing.
AST was performed on all M. chimaera isolates (n = 88), using a broth microdilution method recommended by the CLSI for susceptibility testing of MAC strains (8). The SlowMyco Sensititre panel (TREK Diagnostic Systems, Cleveland, OH) was used, as described previously (19). MICs were determined for clarithromycin, moxifloxacin, linezolid, rifabutin, rifampin, ethambutol, amikacin, and streptomycin. M. chimaera isolates were cultured on Lowenstein-Jensen medium supplemented with pyruvate (E & O Laboratories, United Kingdom) or on Middlebrook 7H11 agar (E & O Laboratories) for approximately 3 weeks or until confluent growth was present. A confluent portion of growth was used to adjust to a 0.5 McFarland standard with sterile glass beads, and 50 μl of the suspension was transferred to 11 ml of Sensititre Mueller-Hinton broth with 5% (vol/vol) oleic acid-albumin-dextrose-catalase (OADC) growth supplement. All broth microdilution plates were incubated at 35°C and were read after 7 days or up to 14 days if growth was insufficient in the positive-control well, as recommended (8). A quality control strain of Mycobacterium avium ATCC 700898 was also used and tested with each batch of isolates (8).
Interpretation of MICs and breakpoints.
The positive-growth well was read first, and MIC results were interpreted using the Sensititre Manual Viewbox (Thermo Fisher Scientific). The MIC was recorded as the lowest concentration of antimicrobial agent that inhibited visible growth (8). Breakpoints available from the CLSI were used to interpret isolates as being susceptible, intermediate, or resistant to clarithromycin (susceptible, ≤8 μg/ml; intermediate, 16 μg/ml; resistant, ≥32 μg/ml), and breakpoints from the CLSI were used for both moxifloxacin (susceptible, ≤1 μg/ml; intermediate, 2 μg/ml; resistant, ≥4 μg/ml) and linezolid (susceptible, ≤8 μg/ml; intermediate, 16 μg/ml; resistant, ≥32 μg/ml) (8). Because CLSI breakpoints for amikacin, rifampin, ethambutol, rifabutin, and streptomycin were not defined at the time of the study, MICs for amikacin were interpreted according to breakpoints proposed by Brown-Elliott et al. (susceptible, ≤16 μg/ml; intermediate, 32 μg/ml; resistant, ≥64 μg/ml) (18). M. chimaera isolates were classified as resistant if the MICs met the breakpoints for rifampin (≥8 μg/ml) and ethambutol (≥8 μg/ml) used previously by Kim et al. and Schön and Chryssanthou (7, 26), derived from PK/PD studies by van Ingen et al. (27). Tentative ECOFF values reported by Maurer et al. were also used to determine wild-type versus non-wild-type isolates for rifabutin (2 μg/ml) and streptomycin (64 μg/ml) (23). The MIC50 was determined as the minimum concentration required to inhibit 50% of the isolates, and the MIC90 was determined as the minimum concentration required to inhibit 90% of the isolates.
ACKNOWLEDGMENTS
We acknowledge support and funding received in part from the Health Services Executive of Ireland, the Department of Clinical Microbiology of Trinity College Dublin, the Irish Mycobacteria Reference Laboratory, and the Microbiology Department, LabMed Directorate, St. James Hospital, Dublin, Ireland.
We acknowledge the many Irish hospitals, clinicians, and laboratory staff members, in addition to our own, for referring isolates and/or samples that generated the collection of M. chimaera isolates used in this study.
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