Resistance mechanisms and drug susceptibility testing of nontuberculous mycobacteria

Drug Resistance Updates 15 (2012) 149–161

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Resistance mechanisms and drug susceptibility testing of nontuberculous mycobacteria Jakko van Ingen a,∗ , Martin J. Boeree b , Dick van Soolingen a,b , Johan W. Mouton a a b

Department of Clinical Microbiology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands Department of Respiratory Diseases, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands

a r t i c l e

i n f o

Keywords: Mycobacteria Nontuberculous mycobacteria Drug susceptibility Macrolides Resistance

a b s t r a c t Nontuberculous mycobacteria (NTM) are increasingly recognized as causative agents of opportunistic infections in humans. For most NTM infections the therapy of choice is drug treatment, but treatment regimens differ by species, in particular between slow (e.g. Mycobacterium avium complex, Mycobacterium kansasii) and rapid growers (e.g. Mycobacterium abscessus, Mycobacterium fortuitum). In general, drug treatment is long, costly, and often associated with drug-related toxicities; outcome of drug treatment is poor and is likely related to the high levels of natural antibiotic resistance in NTM. The role of drug susceptibility testing (DST) in the choice of agents for antimicrobial treatment of NTM disease, mainly that by slow growers, remains subject of debate. There are important discrepancies between drug susceptibility measured in vitro and the activity of the drug observed in vivo. In part, these discrepancies derive from laboratory technical issues. There is still no consensus on a standardized method. With the increasing clinical importance of NTM disease, DST of NTM is again in the spotlight. This review provides a comprehensive overview of the mechanisms of drug resistance in NTM, phenotypic methods for testing susceptibility in past and current use for DST of NTM, as well as molecular approaches to assess drug resistance. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Nontuberculous mycobacteria (NTM) are increasingly recognized as causative agents of opportunistic infections in humans. Pulmonary infections are most frequent and tend to affect patients with pre-existing pulmonary diseases. Cervicofacial lymphadenitis is the second most common and relatively benign manifestation and affects immunocompetent children. In severely immunocompromised patients, NTM can cause localized extrapulmonary or disseminated disease. NTM can be acquired by inhalation, ingestion or direct inoculation in the skin (Griffith et al., 2007). Treatment of infections caused by NTM is either by surgery, drug therapy, or both. Drug therapy of NTM disease is long, costly, and often associated with drug-related toxicities. The treatment regimens differ by species with the most important distinction being that between slow and rapid growing NTM (see Table 1). For most slow growers, the recommended regimen consists of rifampicin (or rifabutin), ethambutol and a macrolide antibiotic and is given

∗ Corresponding author at: Radboud University Nijmegen Medical Center, Department of Clinical Microbiology (574), PO Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: +31 24 3614356; fax: +31 24 3540216. E-mail addresses: [email protected], [email protected] (J. van Ingen). 1368-7646/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.drup.2012.04.001

for 18–24 months; amikacin or streptomycin can be added in the initial 3–6 months in cases of severe disease. For the rapid growers, regimens are primarily based on in vitro drug susceptibility test (DST) results. For Mycobacterium abscessus, the most notorious causative agent of disease among rapid growers, these regimens often include a macrolide antibiotic, amikacin and either cefoxitin, imipenem or tigecycline (Jarand et al., 2011). Treatment for NTM disease, in particular pulmonary NTM disease, can be disappointing and clinical improvement and prolonged culture conversion is not achievable for all patients. Cure rates of pulmonary NTM disease differ by species, ranging from 30–50% in M. abscessus disease, to 50–70% in Mycobacterium avium complex and 80–90% in Mycobacterium malmoense and Mycobacterium kansasii disease (Hoefsloot et al., 2009; Jarand et al., 2011; van Ingen et al., 2009). The exact role of DST remains subject of debate, in particular for slow growers. One of the reason is that there are important discrepancies between drug susceptibility measured in vitro and the activity of the drug observed in vivo (Griffith et al., 2007; Society, 2001). In part, these discrepancies derive from laboratory technical difficulties of DST, standardization of methods and a lack of clinical validation. Yet, with the increasing clinical importance of NTM disease, DST of NTM is again in the spotlight. In this review we provide a comprehensive overview of the mechanisms of drug resistance in NTM, phenotypic methods in past and current use for DST of NTM, as well as molecular approaches to assess drug resistance.

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Table 1 Common NTM species/complexes, grouped by growth rate. Slowa growers

Intermediate

Rapid growers

Mycobacterium avium complex M. kansasii M. xenopi M. malmoense M. simiae

M. marinum

M. abscessus group M. chelonae M. fortuitum M. smegmatis

a

Slow is determined as >7 days required for visible growth from subculture.

We also discuss the usefulness of these tests in clinical practice. This review is limited to susceptibility and susceptibility testing to antimicrobial compounds in past or current clinical use; susceptibility to disinfectants or non-medical substances falls outside the scope of this review. 2. Literature search strategy Relevant publications for review were identified using the PubMed database. The following Medical Subject Heading (MeSH) terms were used alone and in combination: “Mycobacteria, Atypical” or “Mycobacterium” and “Drug Resistance, Bacterial”. We restricted the search to English-, German-, French- and Dutchlanguage papers only. The search was last performed on March 31st, 2011. The search based on “Mycobacteria, Atypical” and “Drug Resistance, Bacterial” yielded 221 results. The use of “Mycobacterium” and “Drug Resistance, Bacterial” yielded 1141 results. We excluded case reports and implementation studies of established techniques. For remaining studies, we screened title and abstracts for their relevance. 3. Drug resistance mechanisms in nontuberculous mycobacteria DST measures the result of a highly complex interplay between natural resistance, inducible resistance and mutational resistance acquired during suboptimal drug exposure and selection. The role of these three determinants of drug susceptibility differs for the various drugs used to treat NTM disease. Knowledge of their relative importance is essential for the selection and optimization of drug treatment regimens. A simplified overview of the various determinants of resistance is presented in Fig. 1. 3.1. Natural resistance 3.1.1. The role of the cell wall Natural resistance to antimicrobial drugs is conferred by a variety of mechanisms that interfere with uptake of the drug, enable its biotransformation in the cell or decrease the affinity with the drug target. The first physical barrier is the mycobacterial cell wall. Natural drug resistance in mycobacteria is related, in large part, to mechanisms that affect the content, hydrophobicity and thereby permeability of that cell wall. The lipid-rich cell wall of mycobacteria forms an important barrier to the penetration of antimicrobial compounds (Lambert, 2002). Several genes and systems involved in cell wall maintenance are important to maintain the multidrug resistant phenotype; these include protein kinase G, fbpA (encoding the so-called ‘antigen 85 complex’) and asnB in Mycobacterium smegmatis, the species that serves as a model organism for the genus Mycobacterium, the mtrAB two-component system in M. smegmatis and M. avium, kasB in Mycobacterium marinum and Maa2520 and pks12 of M. avium. Disruption of these genes generally reduces to hydrophobicity of the mycobacterial cell wall and lead to increased

susceptibility to lipophilic antibiotics including the rifamycins, macrolides, ciprofloxacin, vancomycin, imipenem and penicillins (Cangelosi et al., 2006; Gao et al., 2003; Nguyen et al., 2005, 2010; Philalay et al., 2004; Ren and Liu, 2006; Wolff et al., 2009). Transport of molecules across the membrane into the mycobacterial cell is partly controlled by porins, channel proteins that cross the outer membrane. In mycobacteria, porins are important in nutrient acquisition (Niederweis, 2008) but their number is significantly lower than in gram negative bacteria (Lambert, 2002); only the porin mspA in M. smegmatis has been extensively studied and its activity determines susceptibility to small hydrophilic antibiotic molecules including norfloxacin, chloramphenicol and ␤lactam antibiotics (Danilchanka et al., 2008; Stephan et al., 2004), but also the hydrophobic vancomycin, erythromycin and rifampicin (Stephan et al., 2004). For the clinically important NTM species M. avium and M. abscessus distinct colony variants are known that result from differences in the cell wall content. Cell wall glycopeptidolipid content is low in the rough, invasive M. abscessus phenotype, but high in the non-invasive, colonizing smooth phenotype associated with biofilm formation (Howard et al., 2006). For M. avium, smooth transparent and smooth opaque colony types, as well as rare rough types are discerned, though the link between particular phenotypes and virulence is less strong than in M. abscessus (Schorey and Sweet, 2008). The smooth opaque colony type is more susceptible to ciprofloxacin, clarithromycin and penicillin (Cangelosi et al., 1999); this increased susceptibility is regulated by the mtrAB two-component system (Cangelosi et al., 2006). The differences in drug susceptibility between these variants may in part result from a switch to a stationary metabolic phase of the bacteria during biofilm formation (Falkinham, 2007; Greendyke and Byrd, 2008; Howard et al., 2006); the metabolic state of mycobacteria is an important determinant of their drug susceptibility (Nguyen and Thompson, 2006). The cell wall-related mechanisms that confer resistance to particular antimycobacterial drugs are presented in Table 2. 3.1.2. Biotransformation in the intracellular environment Biotransformation of the antimicrobial compounds by mycobacteria has been described for penicillins, quinolones, aminoglycosides and rifampicin. Penicillins are not used in the treatment of mycobacterial infections, owing to ␤-lactamase activity in all mycobacteria (Flores et al., 2005; Nash et al., 1986). In M. smegmatis, the major ␤-lactamase is blaS and blaE is a minor ␤-lactamase, actually a cephalosporinase (Flores et al., 2005). Acetylation and nitrosation of both norfloxacin and ciprofloxacin has been noted in various rapidly growing Mycobacterium species; the acetylation and nitrosation create molecules that have 2–1000 times less antimycobacterial activity (Adjei et al., 2006, 2007). Aminoglycoside susceptibility is influenced by three distinct classes of aminoglycoside modifying enzymes: aminoglycoside O-nucleotidyltransferases, aminoglycoside Ophosphotransferases and aminoglycoside N-acetyltransferases. The latter two have been identified in the genomes of Mycobacterium species: a phosphotransferase conveys streptomycin resistance in Mycobacterium fortuitum and twelve homologues have been identified in the genome of M. abscessus (Ramirez and Tolmasky, 2010; Ripoll et al., 2009). Distinct N-acetyltransferases have been identified in the genomes of the Mycobacterium tuberculosis complex (M. tuberculosis, Mycobacterium bovis), M. kansasii (Ho et al., 2000), the rapid growers M. fortuitum, M. smegmatis (Ramirez and Tolmasky, 2010) and, again, M. abscessus (Ripoll et al., 2009). Whether these aminoglycoside converting enzymes actually confer resistance to the clinically used amikacin remains controversial; the affinity of these enzymes for aminoglycosides was low and the presence of these enzymes was not associated with increased MICs in either M. kansasii or M. fortuitum (Ho et al.,

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Fig. 1. Overview of the mycobacterial cell wall and resistance determinants. Natural drug resistance in mycobacteria is conferred by their highly lipophilic cell wall and the various mechanisms that control the cell wall content, a low number of porins, broad range of efflux pumps, active biotransformation by cytosolic enzymes and inducible resistance mechanisms (see text for details). Note: the position of the cell wall maintenance mechanisms need not reflect their true position in the cell wall.

2000; Hull et al., 1984). Their homology with similar enzymes in other genera may imply that they have been acquired by lateral gene transfer (Ramirez and Tolmasky, 2010; Ripoll et al., 2009). Last, chromosomally encoded rifampicin ADP-ribosyltransferase (Arr) proteins modify and thereby inactivate rifampicin in select Mycobacterium spp.; this mechanism has been studied in M. smegmatis, but is present in many genera and likely in multiple Mycobacterium species (Baysarowich et al., 2008). The biotransformation mechanisms implied in resistance to common antimycobacterial drugs are presented in Table 2. 3.1.3. Inducible resistance: the role and control of target binding disruption and active efflux The best known inducible resistance mechanism in mycobacteria is the group of erythromycin resistance methylase (erm) genes that confer macrolide resistance through methylation of the 23S ribosomal RNA which impairs binding of the macrolides to the ribosomes (Nash et al., 2005, 2006, 2009). These methylases have been characterized in the clinically important rapidly growing nontuberculous mycobacteria (RGM) M. abscessus and M. fortuitum (and,

in fact, M. tuberculosis) as well as in several clinically less relevant NTM species (Nash et al., 2005, 2006, 2009), but are absent in Mycobacterium chelonae (Nash et al., 2009). The exact influence of this inducible macrolide resistance mechanism on treatment outcome of macrolide-based regimens in disease caused by M. abscessus remains to be investigated. An inducible mechanism by which mycobacteria can increase their tolerance to rifampicin is the RNA polymerase binding protein A (RbpA), which has been characterized in M. tuberculosis and M. smegmatis; this protein binds to the RNA polymerase, where it hampers binding of rifampicin (Dey et al., 2010). Whether this protein is also present in slowly growing nontuberculous mycobacteria (SGM) that are treated with rifampicin based regimens and the possible impact on the efficacy of these regimens remain unknown. Whereas porins can restrict entry of molecules into the cell, efflux pumps are utilized to evacuate potentially harmful substances out of the mycobacterial cell. In recent years, the role of efflux pumps as well as methods to inhibit their activity, have gained significant attention. The P55 is an efflux pump that is likely present in all Mycobacterium species that permits efflux of at least

Table 2 Antimycobacterial drugs and mechanisms of resistance in nontuberculous mycobacteria. Drug

Cell wall

Biotransformation

Resistance inducers

Efflux pumps

Acquired mutations in target gene

Rifampicin

PknG, kasB, Maa2520, pks12, asnB, fbpA, mtrAB, mspA porin PknG, kasB, Maa2520, pks12, mtrAB PknG, asnB, kasB, Maa2520, pks12, fbpA, mtrAB, mspA porin

ADP ribosyltransferase

RbpA

efpA

rpoB

lfrA, efpA, pstB gene

gyrA, gyrB 23S rRNA

Aminoglycoside modifying enzymesa

tetV, tap, P55

16S rRNA 23S rRNA

blaS, blaE

lfrA

Ethambutol Quinolones Macrolides Aminoglycosides Linezolid Penicillins Carbapenems Tetracyclines

Maa2520, pks12, mtrAB, mspA porin PknG, fbpA, mtrAB,

Note: See text for relevant references. a Clinical significance controversial.

Acetylation, nitrosation erm

tetV, tap, P55

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tetracycline and aminoglycosides (Silva et al., 2001). The best characterized efflux pumps in nontuberculous mycobacteria are the tap (Ainsa et al., 1998; Ramon-Garcia et al., 2006), tetV (De Rossi et al., 1998), lfrA (Sander et al., 2000) and efpA (Li et al., 2004) efflux pumps that confer tetracycline, aminoglycoside, hydrophilic quinolone and ␤-lactam antibiotic and fluoroquinolones, rifamycins and isoniazid resistance, respectively, to M. fortuitum and M. smegmatis. One member of the ABC transporter superfamily has been found in nontuberculous mycobacteria, the phosphate transporter encoded by the pstB gene of M. smegmatis. This pump is important for fluoroquinolones efflux and its overexpression confers resistance (Bhatt et al., 2000). An overview of the roles of inducible resistance mechanisms and efflux pumps in resistance to particular antibiotics is presented in Table 2. Part of this extensive armament of mycobacteria is controlled by a single putative transcriptional activator, whiB7, also dubbed the ‘resistome’. WhiB7 is induced by antibiotics and controls the expression of at least erm and the tap efflux pump. Given the presence of the whiB7 system in all Streptomyces and Mycobacterium species sequenced to date, it is likely an ancestral trait of a presumed soil dwelling ancestor (Morris et al., 2005).

4. Phenotypic drug susceptibility testing of mycobacteria Assessments of drug susceptibility of NTM at first were not a clinical tool. In the early 1950s, DST of NTM started as a laboratory diagnostic tool to discern the more resistant nontuberculous mycobacteria from the more susceptible M. tuberculosis. An assessment of susceptibility to the newly developed antituberculosis drugs isoniazid, streptomycin and para-aminosalicylic acid (PAS) was combined with phenotypic features including pigmentation, biochemical properties and pace of growth to identify mycobacteria as either M. tuberculosis complex or NTM (Manten, 1957; Runyon, 1959). The drug susceptibility test results of isolated NTM were of limited clinical significance at the time, as the pathogenic potential of NTM was not apparent at the time. Their clinical relevance only became apparent after Buhler and Pollack described two cases of pulmonary disease caused by “the yellow bacillus” (now known as M. kansasii), in 1953 (Buhler and Pollak, 1953), and Runyon reviewed the literature on these infections in 1959 (Runyon, 1959).

4.1. Overview of applied methods 3.2. Acquired genomic mutations The emergence of multidrug- and later extensively drugresistant tuberculosis resulted in an enormous research effort to study acquired genomic mutations underlying this drug resistance. In NTM, protein sequences of key drug targets are similar or identical to those in M. tuberculosis, despite in vitro resistance in the NTM, which implies that in NTM the impermeability of the cell wall is a more important driver of drug resistance than polymorphism in the drug target (van Ingen et al., 2011). Perhaps as a result, the topic of acquired resistance related to genomic mutations has not been studied extensively. Since the macrolide antibiotics play a key role in treatment of NTM disease, mutational resistance to this group has received most attention. Mutational resistance to macrolides in M. avium complex (MAC) disease can be prevented by the use of multidrug regimens that also include rifampicin and ethambutol (Griffith et al., 2006). Mutations in codon 2058 or 2059 of the 23S ribosomal RNA gene (rrl) have been associated with high level macrolide resistance in both the M. avium complex species and the rapid growers of the M. abscessus group (Bastian et al., 2011; Meier et al., 1994); in a case series of patients with macrolide resistant MAC disease, 96% of patients had isolates with mutations in these two codons. Prior macrolide monotherapy or regimens including only quinolones and macrolides were risk factors for the development of macrolide resistance (Griffith et al., 2006). The rrl is also the target of linezolid. In experimental settings, mutations in codons inside as well as outside the peptidyl transferase center, the target of linezolid, have decreased susceptibility to linezolid in M. smegmatis (Long et al., 2010). Mutational resistance to aminoglycosides has been observed in M. abscessus, particularly in cystic fibrosis patients and patients with otomastoiditis, who receive long-term amikacin therapy. A mutation in codon 1408 of the 16S ribosomal RNA (rrs) gene is responsible for high level resistance in both M. abscessus and M. chelonae after therapy as well as in vitro selection (Prammananan et al., 1998). Rifampicin is the key component of treatment regimens for disease causes by M. kansasii. Acquired rifampicin resistance with mutations in codons 513, 526 and 531 of the rpoB gene has been observed in M. kansasii. These mutations are identical to those observed in rifampicin-resistant M. tuberculosis complex isolates (Klein et al., 2001). Table 2 provides an overview of the target genes for which acquired mutations have been associated with drug resistance.

Susceptibility testing of mycobacteria became more streamlined after the publication of the consensus statement by Canetti et al. (1963). Three procedures based on dilution of the antimycobacterial drug in the culture medium were described, with proposals for standardization of the procedures and the interpretation of their results: the absolute concentration method, the resistance ratio method and the proportion method. All these three early methods applied Löwenstein-Jensen medium and were standardized for tests of isoniazid, streptomycin and PAS only, although advice on testing other compounds (kanamycin, cycloserine, viomycin, thioacetazone, ethionamide) was provided. Moreover, the statement only refers to testing M. tuberculosis complex bacteria, not NTM. For all three techniques, cut-off points for resistance were provided; these were derived from MIC distributions in series of wild type M. tuberculosis strains of treatment naïve patients, who had not had contact with other patients who had received drug treatment for tuberculosis (Canetti et al., 1963). In the following decade similar strategies were applied using more sensitive media that produce faster growth of mycobacteria, particularly the 7H10 medium developed by Middlebrook and Cohn (1958) and Molavi and Weinstein (1971). All methods in past or current clinical use are presented underneath, ordered by year of first description.

4.1.1. Absolute concentration method In the absolute concentration method, standardized inoculums of mycobacteria are incubated on media that contain the test drug in various concentrations including the critical concentration to determine minimum inhibitory concentrations (MIC); growth at and above the critical concentration is interpreted as resistance and the amount of growth, compared to growth of controls on drug-free medium, determines whether there is total or partial resistance.

4.1.2. Resistance ratio method The resistance ratio method uses similar methodology as the absolute concentration method, but the MIC is determined and divided by that of the M. tuberculosis H37Rv reference strain, to come to a ratio. A ratio of 2 or lower is interpreted as susceptible, ratios of 8 or higher as resistant; owing to the comparison with M. tuberculosis H37Rv, even for NTM, this method is more relevant for M. tuberculosis than for NTM.

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4.1.3. Proportion method The proportion method, which is still propagated for second line drug testing of M. tuberculosis by the World Health Organization, also uses drug containing media, but applies multiple standardized dilutions of the initial inoculum; two dilutions are incubated on drug-containing and drug free media. The number of colonies formed on drug containing medium from the most diluted inoculum (the number of resistant bacillary units) is counted and compared to the number of colonies formed from the inoculums of equal dilution on drug free medium (the viable bacillary units). If the number of resistant bacillary units is >1% of the viable bacillary units (i.e. <99% inhibition of the isolate), the isolate is considered resistant to the drug at the tested concentration. 4.1.4. Disk diffusion/disk elution In the late 1970s, a series of studies examined the utility of novel susceptibility testing methods, with varying degrees of success. Many of these techniques were adopted from general bacteriology and were first applied to RGM. Disk diffusion was the first method to be tested. In disk diffusion a disc with an established quantity of the antimicrobial drug is placed on solid medium inoculated with the test strain. The size of the zone of growth inhibition is measured and compared to established breakpoints (if available). In early studies, disk diffusion on solid Mueller-Hinton medium yielded inhibition zones which diameters showed a near linear correlation with MIC determined by agar dilution with Mueller-Hinton medium. Its use of commercially available products in routine use in bacteriology laboratories was a benefit, but its downsides were that the content of the cefoxitin disk (30 ␮g) was too low, the zone of inhibition around the sulfonamide disk (300 or 250 ␮g) could be hard to read, and that the technique was difficult to adapt to slow growing organisms: even isolates of M. marinum, which is characterized by a pace of growth intermediate between rapid and slow growers (Table 1), grow too slowly to be tested according to unpublished observations by Richard Wallace Jr. (Stone et al., 1983). Moreover, the solid Mueller-Hinton medium used in the first published study did not support growth of all strains (Wallace et al., 1979); this issue was later overcome by the addition of oleic acid, albumin, dextrose and catalase (OADC) (Stone et al., 1983). Much later, a single study re-examined disk diffusion to test clarithromycin susceptibility in MAC using a Middlebrook 7H11 medium and showed 98% agreement with broth microdilution; the cut-off point for susceptibility (zone >10 mm) was set based on the bimodal inhibition zone diameter distribution observed within the study (Jarboe et al., 1998). In disk elution, antibiotic-containing disks are added to liquid OADC supplement first, to which melted medium is then added; contents are mixed by stirring prior to solidification of the medium, so disks are in rather than on the medium and the drug is mixed in rather than diffused through the medium. Disk elution is used to add fixed quantities of the antibiotic to the medium and the results is read as growth or no growth at that (breakpoint) concentration, not by reading inhibition zone diameter as in disk diffusion. To overcome the problems observed in disk diffusion, disk elution was applied to M. fortuitum and M. marinum, where its results were comparable to that of broth microdilution in liquid Mueller-Hinton medium (Stone et al., 1983). 4.1.5. Broth macrodilution In the same period in the late 1970s, broth macrodilution methods to determine drug susceptibility of M. tuberculosis were first developed. MIC determination by broth macrodilution is performed by inoculating a set of vials with media containing the various concentrations of the antibiotics to be tested, as well as a 1:100 diluted inoculum in a drug-free control vial, with the organism to be tested; this methodology is similar to the proportion method and

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is sometimes referred to as proportion method in liquid media. To represent the lowest drug concentration inhibiting more than 99% of the bacterial population, the MIC is then defined as the lowest drug concentration that yields growth index readings lower than those in the drug-free vial. These relatively fast methods were based on either measurement of 14 CO2 produced by the oxidation of formate labeled with 14 C or incorporation of 3H-uracil into ribonucleic acid (Snider et al., 1981). The latter format proved unreliable and was abandoned; the former ultimately became commercially available as the BacTec460 system. Soon thereafter, this method was adapted to testing SGM (Heifets et al., 1987; Hoffner et al., 1987). Results of broth macrodilution of rapid growers proved difficult to interpret clinically (Hansen et al., 1994; Yew et al., 1994) and the technique is no longer considered appropriate for RGM. The radiometric BacTec460 system has been largely abandoned as a tool for primary culture of mycobacteria. Production of its supplies has been terminated in the year 2011. Its successor, the Mycobacterial Growth Indicator Tube (MGIT) system, has been set up for primary culture as well as DST of M. tuberculosis, for which it is likely to become the gold standard (CLSI, 2011; Drobniewski et al., 2007). It has not yet been adapted to DST of NTM at the same rate. More extensive studies are still needed to establish the potential of MGIT-based DST for NTM. 4.1.6. Broth microdilution SGM and RGM drug susceptibility testing developed along mostly separate lines. For RGM, test methods used in general bacteriology were adapted. In the 1970s, broth microdilution became the gold standard in general bacteriology, after the report by Ericsson and Sherris (1971). In 1982, the first report on the use of broth (Mueller-Hinton) microdilution for drug susceptibility testing of RGM was published (Swenson et al., 1982), which was followed up by a larger study with more isolates tested by broth microdilution in liquid Mueller-Hinton medium (Swenson et al., 1985). Broth microdilution is performed similar to macrodilution, except for the fact that a 96 wells microtitre plate format with broth volumes of 100 ␮l per well and an inoculum of 5 × 105 CFU is used and growth density is measured optically and compared to growth in drug-free control vials, to determine MICs. After its successful introduction for the RGM, The microdilution method was also adapted to testing of slowly growing nontuberculous mycobacteria (SGM). For SGM, Middlebrook 7H9 medium was used as a compromise between the Middlebrook 7H10 medium that was used in the classic agar proportion method and supports growth of all important species and the faster, more versatile and better standardized microdilution method (Wallace et al., 1986). Microdilution proved suitable to determine MICs for SGM, although the MICs for rifampicin, ethambutol and streptomycin proved lower in broth than in agar. For streptomycin, this lead to discrepant interpretations (Wallace et al., 1986). In a multisite reproducibility study, DST for MAC by broth microdilution in both 7H9 and Mueller-Hinton medium were compared (Woods et al., 2003). Results in 7H9 medium were more reproducible than those in Mueller-Hinton medium and end point readings were easier in 7H9 than in Mueller-Hinton (Woods et al., 2003). 4.1.7. Epsilon tests In the early 1990s, the newly developed Epsilon tests (E-tests) were first applied to mycobacteria. These tests allow MIC determinations for antimicrobial agents based on a predefined antibiotic gradient on a plastic strip calibrated with a continuous logarithmic MIC scale that covers 15 twofold dilutions. Early studies noted that for slowly growing M. kansasii and M. avium complex (MAC) bacteria as well as for the rapidly growing M. chelonae and M. fortuitum, MICs determined by E-tests were similar to those measured by the proportion method or absolute concentration method

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on Löwenstein-Jensen medium for most drugs (Fabry et al., 1995, 1996; Hoffner et al., 1994b). The enthusiasm for these E-tests dampened when multisite reproducibility studies revealed that for RGM, reproducibility of the E-tests was inferior to that of broth microdilution for most drugs (Woods et al., 2000). One of the reasons could be that E-tests were never calibrated for slow growing microorganisms. 4.1.8. Rapid analysis of mycolic acids The latest addition to this field is the use of the rapid analysis of mycolic acids method. This method is based on broth macrodilution using the MGIT960 automated system. However, rather than reading the growth index over time and at the moment of growth detection in the drug-free vial, the tubes are spun down after 72 h of incubation and cell wall mycolic acids of actively growing NTM are visualized by high-performance liquid chromatography; their intensity is compared to that in the drug-free vial to determine the MIC. This method has the advantage that it can detect very early growth of M. avium complex bacteria, significantly reducing the turnaround time (Flauta et al., 2010). 4.2. Pitfalls in drug susceptibility testing of nontuberculous mycobacteria 4.2.1. Medium The choice of medium is critical to the test results and their interpretation. Trimethoprim/sulfamethoxazole susceptibility tests should not be performed on media that contain thymidine, which allows mycobacteria to bypass the folic acid biosynthesis pathway, the target of trimethoprim/sulfamethoxazole. The use of agar-based media largely circumvents this problem, although potential presence of trace elements dictates the use of 80% growth reduction as the cut-off for MIC determination (Wallace et al., 1982, 1985). In turn, the use of the agar-based Middlebrook 7H10 medium is known to yield higher MICs for amikacin and doxycycline in RGM than those measured in Mueller-Hinton medium. This would require separate breakpoints and helped Mueller-Hinton medium to become the medium of choice in RGM (Stone et al., 1983). Last, testing susceptibility to cycloserine is only possible in media devoid of pyruvate, as pyruvate inactivates cycloserine (Tison et al., 1963). 4.2.2. Degradation of antimicrobials Degradation of antibiotics in the test medium adds another layer of difficulty. Doxycycline is known to degrade rapidly, i.e. in 14 days, in Mueller-Hinton medium. Test materials thus need to be prepared fresh (Wallace and Wiss, 1981). Imipenem degrades very rapidly in the media used for broth macrodilution DST and daily addition of the antibiotic is necessary to obtain useful test results (Watt et al., 1992). Broth microdilution plates must be read no later than after 3 days of incubation; high imipenem MICs (>8 ␮g/ml) merit retesting (Woods et al., 1999). In Middlebrook 7H10 medium, reduction to <50% activity was noted within 1 week for trimethoprim and minocycline and within two weeks for kanamycin, amikacin, trimethoprim/sulfamethoxazole and rifampicin (Davis et al., 1987; Rynearson et al., 1971). 4.2.3. Effects of pH The pH of the medium is important when testing macrolide susceptibility (Heifets, 1996). In vitro, the activity of clarithromycin decreases at an acidic pH lower (Truffot-Pernot et al., 1991). The radiometric broth macrodilution method employs Middlebrook 7H12B medium, which is 7H9 medium supplemented with bovine serum albumin, casein hydrolysate, catalase and 14 C-labeled palmitic acid (Middlebrook et al., 1977). This medium has a slightly acidic pH of 6.8, thought to reflect the conditions encountered within macrophages (CLSI, 2011). The broth microdilution method

commonly applies cation-adjusted Mueller-Hinton broth, which has a pH of 7.3–7.4. At this pH clarithromycin MICs are twofold lower than at pH 6.8 and separate interpretation criteria must thus be used (CLSI, 2011). Similar decreases of activity at lower pH have been recorded for ciprofloxacin, clofazimine and ethambutol (Heginbothom et al., 1998). The more acidophilic NTM species M. malmoense and M. genavense grow best, in vitro, at pH 5.5–6.0 and DST at this pH has been performed by macrodilution using acidic media intended for pyrazinamide testing of M. tuberculosis and Middlebrook 7H12B medium at its standard pH, 6.8 (Realini et al., 1997, 1999; Carlson et al., 1998; Heginbothom et al., 1998). This issue shows parallels with DST for fungi, where activity of key drugs including amphotericin B and itraconazole is strongly influenced by the pH of the medium (Te Dorsthorst et al., 2004). 4.2.4. Establishment of breakpoint concentrations The clinical utility of DST relies on consistency between in vitro susceptibility to a drug, a clinically (i.e. at the site of infection) achievable drug exposure and good outcome of treatment with the respective drug, in vivo. Hence, breakpoint concentrations must reflect MIC distributions of wild-types and resistant mutants, pharmacokinetics and pharmacodynamics of the drug in human infections and must aid to predict outcome of treatment. For MAC, only macrolide susceptibility testing is advised and clarithromycin is the preferred class representative (CLSI, 2011). The cut-off for defining acquired resistance has in part been inferred from wild-type MIC distributions and studies of macrolideresistant isolates with proven mutations in the 23S rRNA gene that encodes the drug target (Meier et al., 1994; Nash and Inderlied, 1995). The breakpoint has been validated clinically in a trial of clarithromycin monotherapy in HIV-related disseminated MAC disease (Chaisson et al., 1994) and in pulmonary MAC disease (Wallace et al., 1996a; Tanaka et al., 1999). This susceptibility breakpoint does not take into account that MAC disease is mostly treated with a combination of a rifamycin (rifampicin or rifabutin), ethambutol and a macrolide (Griffith et al., 2007). Simultaneous use of rifamycins significantly decreases the serum concentrations of clarithromycin (Wallace et al., 1995), hence the exposure of the MAC bacteria to the macrolide is likely decreased. As a result, the breakpoint concentration based on efficacy of clarithromycin monotherapy may be too high and may not correctly predict the outcome if rifamycins and clarithromycin are used in combination. The latest CLSI document also features breakpoint concentrations for linezolid and moxifloxacin (CLSI, 2011), although the clinical role of these compounds has not been established. The role of quinolones may be limited, as existing pharmacodynamic indices are hardly met in patient with MAC lung disease (van Ingen, personal communication) and existing studies with a regimen of rifampicin, ethambutol and ciprofloxacin have yielded outcomes no better than those of just rifampicin and ethambutol (Jenkins et al., 2008). For M. kansasii, most breakpoint concentrations have been extrapolated from M. tuberculosis complex (where clinical validation has also been limited, especially for so-called second-line drugs) or from MIC distributions in laboratory-driven studies; only the rifampicin breakpoint has been validated clinically and hence only rifampicin testing is currently recommended (CLSI, 2011). Treatment failure has been associated with high MICs for rifampicin (Wallace et al., 1994). In cases of rifampicin resistance, CLSI recommends, with caveats, testing of clarithromycin, ciprofloxacin, moxifloxacin, amikacin, ethambutol, linezolid, rifabutin, streptomycin and trimethoprim-sulfamethoxazole, with most breakpoints extrapolated from MAC (clarithromycin) or M. tuberculosis (CLSI, 2011). The clinical implications of susceptibility or resistance to these drugs have not been established, although

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clarithromycin-based regimens (including rifampicin) have led to good outcomes in a case series from Israel (Shitrit et al., 2006). For the RGM, CLSI recommends susceptibility testing of macrolides, aminoglycosides, fluoroquinolones, imipenem, doxycycline, tigecycline, cefoxitin, co-trimoxazole and linezolid (CLSI, 2011). Their breakpoint concentrations have been established in laboratory driven studies, though many most (except for the newer tigecycline and linezolid) have been validated clinically, mostly in patients with extrapulmonary disease in the studies by Wallace et al. (1985); some breakpoints, notably that of cefoxitin, have later been altered, based on increased clinical experience (Woods et al., 1999). Some of the laboratory-driven studies that underlie CLSIrecommended breakpoints, both in RGM, MAC and M. kansasii, have only been presented as posters in conferences (CLSI, 2011), rather than in peer-reviewed journals; this limits interpretation of the underlying data. The clinical and pharmacokinetic studies are described in more detail in Section 6.1. 4.3. Comparative performance of currently recommended methods Forty years of experiments and experience ultimately culminated in the National Committee on Clinical Laboratory Standards (NCCLS, now Clinical Laboratory Standards Institute – CLSI) document M24-A, which for the first time provided standards for drug susceptibility testing of mycobacteria, Nocardia, and aerobic actinomycetes; the second edition was published in 2011 (CLSI, 2011). Whereas prior NCCLS documents had only covered M. tuberculosis, this document specifically covered NTM, Nocardia species and aerobic actinomycetes. The current recommendations for NTM are summarized in Table 3. The Clinical Laboratory Standards Institute is at present the only organization that has published guidelines for susceptibility testing of NTM. Its European counterpart, the European Committee on Antimicrobial Susceptibility Testing (EUCAST), has not yet done so. These CLSI approved methods can thus be regarded as the current gold standard. Comparative studies of various methods have been performed and these have shaped the current CLSI recommendations. Broth microdilution using cation-adjusted Mueller-Hinton medium is set as the standard for testing drug susceptibility of RGM, M. kansasii and non-fastidious SGM. For MAC, broth macrodilution (using the radiometric method with Middlebrook 7H12B medium) and broth microdilution (using either Middlebrook 7H9 or 10% OADC [oleic acid-albumin-dextrose-catalase]-enriched MuellerHinton supplemented with OADC) are the standards for drug susceptibility testing. The recommendations were shaped by a series of comparative studies. 4.3.1. The M. avium complex Broth macrodilution became the standard for M. avium complex (MAC) drug susceptibility testing after a comparative study with classic agar dilution. This studied revealed largely identical results, except for lower MICs by broth macrodilution, particularly for amikacin and ethambutol (Inderlied et al., 1987). The decreased turnaround time and superior interlaboratory reproducibility (Woods et al., 2003) compared to agar dilution (Woods and Witebsky, 1996) ultimately rendered broth macrodilution by the BacTec 460 method the gold standard. Later, broth macrodilution and microdilution, which had become standardized for RGM, were compared. Two broth microdilution assays, using either cation-adjusted Mueller-Hinton or Middlebrook 7H9 liquid medium were compared to broth macrodilution using the commercial BacTec460 system at standard condition (Middlebrook 7H12B medium at pH 6.8) and using a 7H12B medium at pH 7.3–7.4 (Woods et al., 2003). Both methods with both media yielded similar

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results in terms of interpretative category (susceptible, intermediate, resistant), provided correct reference ranges for the various pH were used. Nonetheless, end point reading and interpretation was more difficult in the microdilution assays, particularly in cationadjusted Mueller-Hinton medium. As a result, reproducibility of macrodilution was superior to that of microdilution (Woods et al., 2003). Given the fact that production of the BacTec460 system and its supplies have been halted, its non-radiometric successor, the MGIT960 automated system, may become a new standard for DST of MAC isolates and possibly other SGM, alongside broth microdilution. Comparative studies have already noted largely identical results in BacTec460 and MGIT DST for MAC (Piersimoni et al., 1998) including the veterinary pathogen M. avium subsp. paratuberculosis (Krishnan et al., 2009). A comparative study of MGIT and agar dilution DST for MAC revealed mostly concordant results, except for amikacin and ciprofloxacin (Marone et al., 1997). 4.3.2. M. kansasii and other SGM For the SGM M. kansasii, several platforms for DST have been used successfully, including both micro- and macrodilution, agar dilution and agar proportion methods (van Ingen et al., 2010; Wallace et al., 1986, 1994). Results of the various methods are largely identical, except for streptomycin and the aminoglycosides, to which resistance is recorded by the agar proportion method whereas susceptibility is measured in broth (Middlebrook 7H9) microdilution; in absence of established breakpoints for microdilution (and, in fact, NTM) agar proportion breakpoints of M. tuberculosis were used for both techniques (Wallace et al., 1986). This explains part of the supposed discrepancies. The susceptibility in broth correlated well with clinical response in a small series of patients with rifampicin-resistant M. kansasii disease. This clinical correlation was important to settle broth microdilution as the method of choice for M. kansasii (CLSI, 2011; Wallace et al., 1986). For SGM other than MAC or M. kansasii, the optimal method has not been set and broth macro- and microdilution, absolute concentration methods on Middlebrook 7H10 medium as well as agar proportion methods have all been used (CLSI, 2011; van Ingen et al., 2010); results should be interpreted with caution as breakpoints have not been validated for these SGM. 4.3.3. RGM Susceptibility testing of RGM has always developed along separate lines. Methods from general bacteriology have been applied, because classic agar-based methods intended for M. tuberculosis testing did not support growth of all RGM, in particular of M. chelonae. Broth microdilution in Mueller-Hinton medium, as used for other aerobic rapidly growing bacteria was adopted early. After modification to the medium, i.e. the adjustment of cation concentrations, and the use of the OADC supplement, this medium supports growth of virtually all RGM (Swenson et al., 1982, 1985). Broth microdilution proved superior over macrodilution as well as agar proportion and agar dilution, in particular for testing susceptibility to amikacin (Hansen et al., 1994; Swenson et al., 1982, 1985). By the late 1990s, both E-tests and broth microdilution were frequently used methods (Hoffner et al., 1994b). Since the reproducibility of broth microdilution proved superior to that of E-tests (Woods et al., 2000), broth microdilution has remained the CLSIrecommended method for RGM. 4.3.4. M. marinum M. marinum forms an intermediate category between the slow and rapid growers. When relevant, CLSI recommends microdilution with cation-adjusted Mueller-Hinton broth supplemented with 5% AODC, although agar dilution methods on Middlebrook 7H10 medium and E-tests have also been used successfully (Aubry et al.,

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Table 3 Clinical and Laboratory Standards Institute recommendations for drug susceptibility testing. Grouping/species

First choice

Alternative(s)

M. avium complex M. kansasii other slow growers Fastidious species M. marinum Rapid growers

Broth macrodilution in 12B medium Broth microdilution in CAMH Broth microdilution in CAMHa no recommendation Broth microdilution in CAMH Broth microdilution in CAMH

Broth microdilution in CAMH Macrodilution, agar proportion not established no recommendation Macrodilution, agar dilution not established

CAMH: cation-adjusted Mueller-Hinton broth with OADC supplement. a M. xenopi grows poorly in this medium.

2000; CLSI, 2011; van Ingen et al., 2010). Agar dilution is preferred over E-tests because of limited reproducibility of the latter (Aubry et al., 2000). Similar to M. kansasii, the various methods yield largely identical results, except for streptomycin and the aminoglycosides. Agar dilution methods tend to measure higher MICs than broth microdilution, which yield differences in interpretation (resistant versus susceptible) if identical breakpoints, extrapolated from M. tuberculosis DST, are used for both assays (Wallace et al., 1986). Because of the growth characteristics of M. marinum, microdilution trays should be incubated at 28–30 ◦ C for 7 days. 5. Molecular methods for assessment of drug susceptibility Thus far, no commercial assays exist for the detection of resistance-conferring mutations in NTM. Nonetheless, speciesspecific methods have been developed. Most of these are based on sequencing of the target gene and comparisons with wild type strains of the same species and include rpoB gene mutation analysis to assess rifamycin susceptibility in M. kansasii, 23S rRNA and erm gene mutation analysis to assess macrolide susceptibility in MAC and rapid growers and 16S rRNA gene mutation analysis to assess aminoglycoside resistance in MAC and M. abscessus (Klein et al., 2001; Meier et al., 1996; Nash and Inderlied, 1996; Wallace et al., 1996b). Owing to the central role of the macrolides in treatment of NTM disease and the fact that MAC and M. abscessus group organisms are the most frequent causative agents, molecular analysis of macrolide susceptibility in MAC and M. abscessus have received most attention (Bastian et al., 2011; Meier et al., 1996; Wallace et al., 1996b). 6. Ongoing controversy – in vitro drug susceptibility versus in vivo outcome of treatment In this review, we have summarized the various methods that have been used to determine drug susceptibility of NTM, for mostly clinical purposes. Yet, clinical validation of these methods, i.e. relating MICs and breakpoint concentrations for resistance to pharmacokinetic data and treatment outcome, has hardly been done. 6.1. The role of drug susceptibility testing in currently established treatment regimens The discrepancies between in vitro drug susceptibility and in vivo outcome of treatment became obvious as soon as NTM were acknowledged to be causative agents of human lung disease (Goldman, 1968). At the time, the standard regimen for tuberculosis treatment, PAS, isoniazid and streptomycin was also administered to patients with NTM despite MICs several orders of magnitude higher than those of M. tuberculosis. Nonetheless, a sizeable number of patients, especially those with M. kansasii disease, responded to treatment. Outcome was much worse in patients infected with M. avium complex bacteria or RGM (dubbed Runyon group III and IV organisms at the time) (Goldman, 1968; Manten, 1957).

6.1.1. Phenotypic susceptibility versus treatment outcome – MAC Trials of monotherapy with rifampicin, ethambutol, clofazimine or clarithromycin for HIV-associated disseminated M. avium disease established that only drug susceptibility testing results for clarithromycin predicted outcome of treatment with this drug. In this trial, 99% of all pretreatment isolates were susceptible to 4 ␮g/ml of clarithromycin in broth macrodilution; 46% developed MICs >32 during treatment and follow-up and these increased MICs were associated with recrudescence of symptoms and increases in bacterial load in blood cultures (Table 4) (Chaisson et al., 1994). In vitro drug susceptibility tests for rifampicin, ethambutol and clofazimine could not predict outcome of monotherapy with the respective drugs (Chaisson et al., 1994; Sison et al., 1996). In lung disease caused by MAC, the macrolide antibiotic clarithromycin was also the first drug for which a clear relationship between in vitro activity and in vivo efficacy could be demonstrated: case series from the United States (Wallace et al., 1996a) and Japan (Tanaka et al., 1999) have assessed the efficacy of triple drug regimens of rifampicin, ethambutol and a macrolide in MAC lung disease. In these series, treatment outcome was significantly worse in patients with macrolide resistant MAC bacteria, either at baseline or acquired during therapy (Table 4); Tanaka and colleagues revealed that culture conversion rates of the triple drug regimens of rifampicin, ethambutol and a macrolide with adjunctive aminoglycosides during the first months of therapy were 71.8% overall, though dropped to 25% in patients whose initial isolates were resistant to macrolides (Tanaka et al., 1999). More recently, the British Thoracic Society has performed 2 consecutive trials of regimens based on rifampicin and ethambutol for MAC, M. xenopi and M. malmoense lung disease. Within the first trial, single drug DST for isoniazid, rifampicin and ethambutol was performed by the resistance ratio method, only to note that in vitro resistance, using breakpoints extrapolated from M. tuberculosis DST, was commonplace and could not be linked to the outcome of treatment in vivo (Table 4) (Society, 2001). In the second trial, which compared a regimen of rifampicin, ethambutol and clarithromycin to rifampicin, ethambutol and ciprofloxacin, drug susceptibility testing was not performed at all (Jenkins et al., 2008), presumably based on the results in study 1. In MAC disease, the clinical utility of aminoglycoside susceptibility testing is limited. On basis of in vitro data, amikacin and streptomycin are the most active aminoglycosides against MAC (Heifets and Lindholm-Levy, 1989). Clinically, there is no clear superiority of streptomycin over amikacin or vice versa (Griffith et al., 2007); yet, the few studies that have examined the effect of adjunctive aminoglycoside treatment in MAC disease have primarily used streptomycin; although adjunctive streptomycin was associated with more rapid culture conversion, it had no effect on clinical outcomes (Kobashi et al., 2007). The in vitro susceptibility to streptomycin does not predict the outcome of treatment regimens that include this drug, as shown in Table 4. For the older antituberculosis drugs sporadically used in disease caused by MAC (e.g. clofazimine, ethionamide, cycloserine) a relation between in vitro susceptibility and in vivo treatment outcome

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Table 4 Cure or conversion rates in relation to phenotypic drug susceptibility in clinical trials. Study

Species/disease type

Regimen

RIF R

EMB R

STR R

CLA R

Method

Cure rate, relation

Chaisson et al. (1994) Sison et al. Wallace (1996) et al. (1996a) Tanaka et al. (1999) Kobashi et al. (2007) Kobashi et al. (2006) Society (2001)

M. avium/Diss M. avium/Diss MAC/LD MAC/LD MAC/LD MAC/LD MAC, M. xenopi, M. malmoense/LD

Cla R E R/Rb, E, Cla, S R, E, Cla, K R, E, Cla, S R, E, Cla, S H, R, E

n.a. 50%, 86%, 57% n.a. ND ND ND 75% 67%

n.a. n.a. 50%, 92%, 50% ND ND ND 100% 42%

n.a. n.a. n.a. ND ND 48% 90% n.a.

46% n.a. n.a. 0% 21% ND 25% n.a.

MaD MiD, AD, MaD MiD, AD, MaD MiD MiD MiD MiD RRM

n.a., Cla only 43%, no relation 75%, no relation 82%, n.a. 71.8%, Cla only 71.2%, no relation 59.6%, Cla only 33%, no relation

MAC, Mycobacterium avium complex; LD, lung disease; Diss, disseminated; R, rifampicin; Rb, rifabutin; E, ethambutol; Cla, clarithromycin; S, streptomycin; K, kanamycin; H, isoniazid, MiD, broth microdilution; AD, agar dilution; MaD, broth macrodilution; RRM, resistance ratio method; ND, not done; n.a., not applicable. Note: Both intermediate and full resistance are interpreted as “resistance” for this Table.

has never been established (Table 4) (Kuze, 1984; Kuze et al., 1981). Routine single-drug testing of these drugs is not recommended (CLSI, 2011). 6.1.2. Phenotypic susceptibility versus treatment outcome – M. kansasii and other SGM For M. kansasii, the currently recommended treatment regimen consists of isoniazid, rifampicin and ethambutol for an 18 month duration, or 12 months after culture conversion (Griffith et al., 2007). Rifampicin plays a key role in treatment regimens and rifampicin resistance leads to failure of rifampicin-based treatment regimens (Ahn et al., 1987; Griffith et al., 2007; Wallace et al., 1994). Resistance to isoniazid or ethambutol also occurs, but is generally associated with rifampicin resistance (Ahn et al., 1987). Hence, rifampicin is the key drug to be tested (CLSI, 2011). In rifampicin resistant M. kansasii, additional tests of isoniazid, amikacin, streptomycin, ciprofloxacin, moxifloxacin, clarithromycin, rifabutin and co-trimoxazole are advised. For clinically significant slow growers other than MAC or M. kansasii, it has been recommended to test rifampicin and the set of drugs tested for rifampicin-resistant M. kansasii (CLSI, 2011; Griffith et al., 2007). The interpretation of their results should be cautious as in vitro activity has not been correlated with in vitro efficacy for any of these drugs. 6.1.3. Phenotypic susceptibility versus treatment outcome – RGM For rapid growers, treatment regimens are designed using DST results and often feature a macrolide, an aminoglycoside, ciprofloxacin or moxifloxacin, imipenem, doxycycline or tigecycline, cefoxitin, co-trimoxazole, or linezolid (Griffith et al., 2007). Hence, these drugs are candidates for in vitro DST. For the rapid growers, relationships between in vitro drug susceptibility and treatment outcome, particularly in extrapulmonary disease, are more straightforward. Richard Wallace Jr. and colleagues published outcome data for patients with extrapulmonary RGM disease who were treated with regimens selected on basis of DST (Wallace et al., 1985); cure rates of 90% after mostly monotherapy with trimethoprim-sulfamethoxazole (M. fortuitum) and 72% after mostly amikacin with cefoxitin treatment (M. chelonei; now separated in M. chelonae and the M. abscessus group) were observed. Nonetheless, in M. abscessus lung disease, the outcome of treatment using drugs to which susceptibility is noted in vitro is very limited. In a recent study of 69 patients, the culture conversion rate was just 48% (Jarand et al., 2011). The relationship between inducible macrolide resistance, especially in M. abscessus, and outcome of treatment with macrolide-based regimens remains uncertain (Jarand et al., 2011; Nash et al., 2009); this may alter the interpretation of macrolide susceptibility test results in the future. Recently, the activity of clofazimine, in vitro, against RGM has been demonstrated (Shen et al., 2010). Yet, this drug is rarely used in treatment regimens and interpretative criteria are non-existent, limiting the utility of susceptibility testing to this drug.

6.1.4. Phenotypic susceptibility versus treatment outcome – M. marinum For M. marinum, routine drug susceptibility is not recommended because of the generally excellent response to monotherapy with clarithromycin, doxycycline or minocycline, rifampicin, cotrimoxazole or clarithromycin, or combinations thereof. In vitro susceptibility to these compounds is commonplace (Aubry et al., 2002). DST is only warranted in treatment failure and should cover all above-mentioned drugs (CLSI, 2011). 6.1.5. The association between pharmacokinetic data and treatment outcome There is very little supporting pharmacokinetic data to support the MIC breakpoint concentrations or to guide therapeutic decisions based on the exact MICs. The regimens that have been established for treatment of disease by slow growing NTM use drugs that have important interactions. Rifampicin is the best studied and lowers clarithromycin (Wallace et al., 1995) and moxifloxacin (Nijland et al., 2007) serum levels. Co-administration of rifampicin likely increases metabolization of clarithromycin to 14OH-clarithromycin (Alffenaar et al., 2010), which is 8–32 times less active in vitro (based on a median MIC 8.0 versus 0.5 ␮g/ml for clarithromycin) (Cohen et al., 1992; Heifets, 1991). This is an area of research that deserves more attention. 7. The role of synergy in current drug regimens The current CLSI recommendations all involve testing of single drugs. There is evidence of synergy, in vitro, between the various drugs used to treat NTM disease. Three drugs are particularly known for their synergistic effects, in vitro: ethambutol, clarithromycin and clofazimine. For ethambutol, the best known synergistic activity is with rifampicin. The combination of rifampicin and ethambutol has synergistic activity against SGM, particularly MAC, M. malmoense and M. xenopi. The synergistic activity is mostly based on increased cell wall permeability caused by one drug leading to increased uptake of the second. Ethambutol is known to increase mycobacterial cell wall permeability (Hoffner et al., 1990) and for its synergistic activity with a wide array of antimycobacterial drugs with intracellular targets, including rifampicin, streptomycin, quinolones and macrolides against MAC (Banks and Jenkins, 1987; Heifets, 1982; Hoffner et al., 1987, 1989, 1990; Piersimoni et al., 1995; Yajko et al., 1996). In vitro synergy between rifampicin and ethambutol was observed in most, but not all MAC strains (Banks and Jenkins, 1987; Heifets, 1982; Hoffner et al., 1987). All M. xenopi and M. malmoense strains proved highly susceptible to this combination (Banks and Jenkins, 1987; Heifets, 1982; Hoffner et al., 1993). There is no synergistic activity of this combination against M. simiae. The macrolide clarithromycin, too, exerts synergistic activity in vitro with other commonly used antimycobacterial drugs. These

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include rifabutin, ethambutol and clofazimine, at least against MAC organisms (Piersimoni et al., 1995). Finally, clofazimine, too, can act as a “Trojan horse” to increase exposure to other drugs (most importantly macrolides) is. This synergistic activity of clofazimine and drugs with intracellular targets was evident in broth (Piersimoni et al., 1995; Yajko et al., 1996), though even more so in infected macrophages (Yajko et al., 1996). Recently, the synergistic activity of clofazimine and amikacin against the RGM M. abscessus has been demonstrated in vitro, using broth microdilution (Shen et al., 2010). Follow-up experiments have revealed that this synergy is also observed against MAC and M. simiae (van Ingen, submitted for publication). The clinical implications of testing synergistic activities in individual strains remain to be elucidated. Although rifampicinethambutol synergy is considered clinically important (Jenkins et al., 2008; Society, 2001), the effect of the absence of synergy in some MAC strains on clinical outcome has not been thoroughly studied. One study has performed rifampicin-ethambutol and rifampicin-streptomycin synergy tests in five patients with MAC disease who failed treatment, to reveal that resistance to previously synergistic combinations arose during therapy (Hoffner et al., 1994a). It is well known that the results of the rifampicin, ethambutol regimen with or without macrolides in M. simiae disease are disappointing (Griffith et al., 2007; van Ingen et al., 2008), in line with the absence of in vitro synergy (van Ingen et al., 2012).

8. Perspective Despite the fact that DST of NTM is now an established tool in the laboratory, its clinical value has been not been sufficiently studied. Natural drug resistance determines a large part of the multidrug-resistance that is commonplace in NTM. This multidrugresistance, in turn, is a likely explanation of the limited efficacy of current treatment regimens for NTM disease. Testing the susceptibility of individual clinical isolates is of limited value for drugs to which natural resistance is to be expected. Moreover, for most drugs there is no clear correlation between in vitro activity and the outcome of treatment, in vivo. This should still be the prominent subject of research to clinicians and microbiologists involved in mycobacterial disease treatment. The influence of pharmacokinetic interactions between the various drugs and the formulation of pharmacodynamic parameters that predict outcomes should be accounted for in such correlation studies. For the moment, laboratories with isolation frequencies that warrant the local initiation of drug susceptibility testing are best advised to use the CLSI-recommended methods, to at least ensure some level of (international) standardization. Yet, it is important to realize the shortcomings of the currently recommended methods and it is time that an international consensus is reached, in line with the international standard method for fast growing bacteria (International Organization for Standardization, 2006). Currently established breakpoint concentrations for many drugs have a very limited clinical evidence base, and extrapolation of these breakpoints to other DST methods may not be possible. Low MICs of the tetracyclines, aminoglycosides, cefoxitin, sulfonamides and macrolides have been related to favorable treatment outcomes of mostly monotherapy in extrapulmonary RGM disease (Wallace et al., 1985), but not of multi-drug treatment in the (more frequent) pulmonary infections (Jarand et al., 2011; Jeon et al., 2009). For MAC, too, the clarithromycin breakpoint comes from monotherapy in disseminated infections. For the newly proposed breakpoints for linezolid and moxifloxacin no clinical validation has been performed. Pharmacokinetic studies have revealed significant interactions between key drugs in NTM treatment, including between rifamycins and both macrolides (Wallace et al., 1995) and

moxifloxacin (Nijland et al., 2007); these may further complicate interpretation of MICs and a reevaluation of breakpoints seems warranted. Further international standardization of methodology offers the advantage that clinical validation studies with sizeable patient cohorts can be performed. Incorporation of NTM DST into a general bacteriology laboratory remains costly as the recommended methods mostly require equipment specific to mycobacteriology; this pertains particularly to the broth macrodilution systems, although broth microdilution in cation-adjusted Mueller-Hinton medium is also not a commonly applied approach in general bacteriology laboratories, where automated systems such as VitekTM (BioMerieux, Durham, NC, USA) and PhoenixTM (Becton & Dickinson, Sparks, MD, USA) are popular for their broad coverage. It is also important to note that the radiometric BacTec460 method is slowly phased out and, for laboratories set to apply broth macrodilution, it may be preferable to start using the MGIT960 method for all SGM, even though there is limited evidence that it yields results comparable to the reference BacTec460 method. Broth microdilution offers the advantage that it can be applied to RGM, M. kansasii and MAC, which, together, encompass the large majority of clinical NTM isolates (Griffith et al., 2007). This technique and the interpretation of its results demand reasonable experience (Woods et al., 1999, 2003), which reconfirms that DST should only be performed in centers that isolate sufficiently high numbers of NTM. The EUCAST methodology for breakpoint concentration establishment, which uses a synthesis of wild-type MIC distributions, human pharmacokinetics/pharmacodynamics and treatment outcome studies as well as data from standardized animal and in vitro (e.g. hollow fiber model) experimentation (Mouton et al., 2011), may be of interest to NTM as well. The wild-type MIC distributions, pharmacokinetics, as well as relevant and pharmacodynamic indices for the key drugs used in treatment regimens still need to be determined, prior to adaptation of such an approach. Here, too, the correlation with clinical outcomes should have the highest priority. For selected drugs, combination tests rather than single drug testing could be useful, although methodology is not standardized as yet. Synergistic activity between compounds to which natural resistance exists may help to overcome this natural resistance. Among SGM, the synergy between rifampicin and ethambutol is the best example, in which ethambutol likely facilitates the entry of rifampicin into the mycobacteria; it may do the same for the aminoglycosides (Hoffner et al., 1990). Clofazimine exerts a similar function, at least for the aminoglycosides, in both RGM (Shen et al., 2010) and SGM (van Ingen, personal communication). All these examples suggest that testing drug combinations may be more worthwhile than single drug tests for selected drugs including rifampicin and ethambutol. Nonetheless, there is a significant lack of good quality trials that clearly show the efficacy of these combinations in clinical practice. The only existing study has examined just five patients who failed therapy and in whose isolates new resistance to drug combinations (rifampicin and ethambutol and/or rifampicin and streptomycin) was measured (Hoffner et al., 1994a). Most of these in vitro observations await a proof of concept study, in vivo. Given the importance of natural drug resistance in NTM and the treatment of NTM infections, novel therapies could use compounds that destabilize the mycobacterial cell wall and increase its permeability. The various genes and systems described in this review all provide potential drug targets. In addition, efflux pump inhibitors may help to improve the efficacy of antimycobacterial drugs (Rodrigues et al., 2011). Various efflux pump inhibitors are known, of which the phenothiazines (notably thioridazine) and verapamil have been best studied. The phenothiazines have the additional advantage of intrinsic antimycobacterial activity, both in vitro (van Ingen, 2011) and in vivo (van Soolingen et al., 2010).

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The introduction of new drugs for (adjunctive) treatment of NTM disease should be accompanied by extensive studies of the in vitro activity and its correlation to in vivo outcome of treatment. References Adjei, M.D., Heinze, T.M., Deck, J., Freeman, J.P., Williams, A.J., Sutherland, J.B., 2006. Transformation of the antibacterial agent norfloxacin by environmental mycobacteria. Applied and Environment Microbiology 72, 5790–5793. Adjei, M.D., Heinze, T.M., Deck, J., Freeman, J.P., Williams, A.J., Sutherland, J.B., 2007. Acetylation and nitrosation of ciprofloxacin by environmental strains of mycobacteria. Canadian Journal of Microbiology 53, 144–147. Ahn, C.H., Wallace Jr., R.J., Steele, L.C., Murphy, D.T., 1987. Sulfonamide-containing regimens for disease caused by rifampin-resistant Mycobacterium kansasii. American Review of Respiratory Disease 135, 10–16. Ainsa, J.A., Blokpoel, M.C., Otal, I., Young, D.B., De Smet, K.A., Martin, C., 1998. Molecular cloning and characterization of Tap, a putative multidrug efflux pump present in Mycobacterium fortuitum and Mycobacterium tuberculosis. Journal of Bacteriology 180, 5836–5843. Alffenaar, J.W., Nienhuis, W.A., de Velde, F., Zuur, A.T., Wessels, A.M., Almeida, D., Grosset, J., Adjei, O., Uges, D.R., van der Werf, T.S., 2010. Pharmacokinetics of rifampin and clarithromycin in patients treated for Mycobacterium ulcerans infection. Antimicrobial Agents and Chemotherapy 54, 3878–3883. Aubry, A., Jarlier, V., Escolano, S., Truffot-Pernot, C., Cambau, E., 2000. Antibiotic susceptibility pattern of Mycobacterium marinum. Antimicrobial Agents and Chemotherapy 44, 3133–3136. Aubry, A., Chosidow, O., Caumes, E., Robert, J., Cambau, E., 2002. Sixty-three cases of Mycobacterium marinum infection: clinical features, treatment, and antibiotic susceptibility of causative isolates. Archives of Internal Medicine 162, 1746–1752. Banks, J., Jenkins, P.A., 1987. Combined versus single antituberculosis drugs on the in vitro sensitivity patterns of non-tuberculous mycobacteria. Thorax 42, 838–842. Bastian, S., Veziris, N., Roux, A.L., Brossier, F., Gaillard, J.L., Jarlier, V., Cambau, E., 2011. Assessment of clarithromycin susceptibility in strains belonging to the Mycobacterium abscessus group by erm(41) and rrl sequencing. Antimicrobial Agents and Chemotherapy 55, 775–781. Baysarowich, J., Koteva, K., Hughes, D.W., Ejim, L., Griffiths, E., Zhang, K., Junop, M., Wright, G.D., 2008. Rifamycin antibiotic resistance by ADP-ribosylation: structure and diversity of Arr. Proceedings of the National Academy of Sciences of the United States of America 105, 4886–4891. Bhatt, K., Banerjee, S.K., Chakraborti, P.K., 2000. Evidence that phosphate specific transporter is amplified in a fluoroquinolone resistant Mycobacterium smegmatis. European Journal of Biochemistry 267, 4028–4032. Buhler, V.B., Pollak, A., 1953. Human infection with atypical acid-fast organisms: report of two cases with pathologic findings. American Journal of Clinical Pathology 23, 363–374. Canetti, G., Froman, S., Grosset, J., Hauduroy, P., Langerova, M., Mahler, H.T., Meissner, G., Mitchison, D.A., Sula, L., 1963. Mycobacteria: laboratory methods for testing drug sensitivity and resistance. Bulletin of the World Health Organization 29, 565–578. Cangelosi, G.A., Palermo, C.O., Laurent, J.P., Hamlin, A.M., Brabant, W.H., 1999. Colony morphotypes on Congo red agar segregate along species and drug susceptibility lines in the Mycobacterium avium–intracellulare complex. Microbiology 145 (Pt 6), 1317–1324. Cangelosi, G.A., Do, J.S., Freeman, R., Bennett, J.G., Semret, M., Behr, M.A., 2006. The two-component regulatory system mtrAB is required for morphotypic multidrug resistance in Mycobacterium avium. Antimicrobial Agents and Chemotherapy 50, 461–468. Carlson, L.D., Wallis, C.K., Coyle, M.B., 1998. Standardized BACTEC method to measure clarithromycin susceptibility of Mycobacterium genavense. Journal of Clinical Microbiology 36, 748–751. Chaisson, R.E., Benson, C.A., Dube, M.P., Heifets, L.B., Korvick, J.A., Elkin, S., Smith, T., Craft, J.C., Sattler, F.R., 1994. Clarithromycin therapy for bacteremic Mycobacterium avium complex disease. A randomized, double-blind, dose-ranging study in patients with AIDS. AIDS Clinical Trials Group Protocol 157 Study Team. Annals of Internal Medicine 121, 905–911. CLSI, 2011. Susceptibility testing of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes; Approved Standard – Second Edition. CLSI document M24-A2. Clinical and Laboratory Standards Institute, Wayne, PA. Cohen, Y., Perronne, C., Truffot-Pernot, C., Grosset, J., Vilde, J.L., Pocidalo, J.J., 1992. Activities of WIN-57273, minocycline, clarithromycin, and 14-hydroxyclarithromycin against Mycobacterium avium complex in human macrophages. Antimicrobial Agents and Chemotherapy 36, 2104–2107. Danilchanka, O., Pavlenok, M., Niederweis, M., 2008. Role of porins for uptake of antibiotics by Mycobacterium smegmatis. Antimicrobial Agents and Chemotherapy 52, 3127–3134. Davis Jr., C.E., Carpenter, J.L., Trevino, S., Koch, J., Ognibene, A.J., 1987. In vitro susceptibility of Mycobacterium avium complex to antibacterial agents. Diagnostic Microbiology and Infectious Disease 8, 149–155. De Rossi, E., Blokpoel, M.C., Cantoni, R., Branzoni, M., Riccardi, G., Young, D.B., De Smet, K.A., Ciferri, O., 1998. Molecular cloning and functional analysis of a novel tetracycline resistance determinant, tet(V), from Mycobacterium smegmatis. Antimicrobial Agents and Chemotherapy 42, 1931–1937.

159

Dey, A., Verma, A.K., Chatterji, D., 2010. Role of an RNA polymerase interacting protein, MsRbpA, from Mycobacterium smegmatis in phenotypic tolerance to rifampicin. Microbiology 156, 873–883. Drobniewski, F., Rusch-Gerdes, S., Hoffner, S., 2007. Antimicrobial susceptibility testing of Mycobacterium tuberculosis (EUCAST document E.DEF 8.1)—report of the Subcommittee on Antimicrobial Susceptibility Testing of Mycobacterium tuberculosis of the European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID). Clinical Microbiology and Infection 13, 1144–1156. Ericsson, H.M., Sherris, J.C., 1971. Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathologica et Microbiologica Scandinavica Section B: Microbiology and Immunology 217 (Suppl. 217), 211. Fabry, W., Schmid, E.N., Ansorg, R., 1995. Comparison of the E test and a proportion dilution method for susceptibility testing of Mycobacterium kansasii. Chemotherapy 41, 247–252. Fabry, W., Schmid, E.N., Ansorg, R., 1996. Comparison of the E test and a proportion dilution method for susceptibility testing of Mycobacterium avium complex. Journal of Medical Microbiology 44, 227–230. Falkinham 3rd, J.O., 2007. Growth in catheter biofilms and antibiotic resistance of Mycobacterium avium. Journal of Medical Microbiology 56, 250–254. Flauta, V., Osterhout, G., Ellis, B., Grayson, M., Sasser, M., Cohen, S., Sander, M., Dionne, K., Carroll, K., Parrish, N., 2010. Use of the RAM susceptibility testing method for rapid detection of clarithromycin resistance in the Mycobacterium avium complex. Diagnostic Microbiology and Infectious Disease 67, 47–51. Flores, A.R., Parsons, L.M., Pavelka Jr., M.S., 2005. Genetic analysis of the betalactamases of Mycobacterium tuberculosis and Mycobacterium smegmatis and susceptibility to beta-lactam antibiotics. Microbiology 151, 521–532. Gao, L.Y., Laval, F., Lawson, E.H., Groger, R.K., Woodruff, A., Morisaki, J.H., Cox, J.S., Daffe, M., Brown, E.J., 2003. Requirement for kasB in Mycobacterium mycolic acid biosynthesis, cell wall impermeability and intracellular survival: implications for therapy. Molecular Microbiology 49, 1547–1563. Goldman, K.P., 1968. Treatment of unclassified mycobacterial infection of the lungs. Thorax 23, 94–99. Greendyke, R., Byrd, T.F., 2008. Differential antibiotic susceptibility of Mycobacterium abscessus variants in biofilms and macrophages compared to that of planktonic bacteria. Antimicrobial Agents and Chemotherapy 52, 2019–2026. Griffith, D.E., Brown-Elliott, B.A., Langsjoen, B., Zhang, Y., Pan, X., Girard, W., Nelson, K., Caccitolo, J., Alvarez, J., Shepherd, S., Wilson, R., Graviss, E.A., Wallace Jr., R.J., 2006. Clinical and molecular analysis of macrolide resistance in Mycobacterium avium complex lung disease. American Journal of Respiratory and Critical Care Medicine 174, 928–934. 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., Iseman, M., Olivier, K., Ruoss, S., von Reyn, C.F., Wallace Jr., R.J., Winthrop, K., 2007. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. American Journal of Respiratory and Critical Care Medicine 175, 367–416. Hansen, K.T., Clark, R.B., Sanders, W.E., 1994. Effects of different test conditions on the susceptibility of Mycobacterium fortuitum and Mycobacterium chelonae to amikacin. Journal of Antimicrobial Chemotherapy 33, 483–494. Heginbothom, M.L., Lindholm-Levy, P.J., Heifets, L.B., 1998. Susceptibilities of Mycobacterium malmoense determined at the growth optimum pH (pH 6.0). International Journal of Tuberculosis and Lung Disease 2, 430–434. Heifets, L.B., 1982. Synergistic effect of rifampin, streptomycin, ethionamide, and ethambutol on Mycobacterium intracellulare. American Review of Respiratory Disease 125, 43–48. Heifets, L.B., 1991. In: Heifets, L.B. (Ed.), Drug susceptibility in the chemotherapy of mycobacterial infections. CRC Press, Boca Raton, FL. Heifets, L., 1996. Susceptibility testing of Mycobacterium avium complex isolates. Antimicrobial Agents and Chemotherapy 40, 1759–1767. Heifets, L., Lindholm-Levy, P., 1989. Comparison of bactericidal activities of streptomycin, amikacin, kanamycin, and capreomycin against Mycobacterium avium and M. tuberculosis. Antimicrobial Agents and Chemotherapy 33, 1298–1301. Heifets, L.B., Iseman, M.D., Lindholm-Levy, P.J., 1987. Determination of MICs of conventional and experimental drugs in liquid medium by the radiometric method against Mycobacterium avium complex. Drugs Under Experimental and Clinical Research 13, 529–538. Ho, I.I., Chan, C.Y., Cheng, A.F., 2000. Aminoglycoside resistance in Mycobacterium kansasii, Mycobacterium avium–M. intracellulare, and Mycobacterium fortuitum: are aminoglycoside-modifying enzymes responsible? Antimicrobial Agents and Chemotherapy 44, 39–42. Hoefsloot, W., van Ingen, J., de Lange, W.C., Dekhuijzen, P.N., Boeree, M.J., van Soolingen, D., 2009. Clinical relevance of Mycobacterium malmoense isolation in The Netherlands. European Respiratory Journal 34, 926–931. Hoffner, S.E., Svenson, S.B., Kallenius, G., 1987. Synergistic effects of antimycobacterial drug combinations on Mycobacterium avium complex determined radiometrically in liquid medium. European Journal of Clinical Microbiology 6, 530–535. Hoffner, S.E., Kratz, M., Olsson-Liljequist, B., Svenson, S.B., Kallenius, G., 1989. In vitro synergistic activity between ethambutol and fluorinated quinolones against Mycobacterium avium complex. Journal of Antimicrobial Chemotherapy 24, 317–324. Hoffner, S.E., Svenson, S.B., Beezer, A.E., 1990. Microcalorimetric studies of the initial interaction between antimycobacterial drugs and Mycobacterium avium. Journal of Antimicrobial Chemotherapy 25, 353–359.

160

J. van Ingen et al. / Drug Resistance Updates 15 (2012) 149–161

Hoffner, S.E., Hjelm, U., Kallenius, G., 1993. Susceptibility of Mycobacterium malmoense to antibacterial drugs and drug combinations. Antimicrobial Agents and Chemotherapy 37, 1285–1288. Hoffner, S.E., Heurlin, N., Petrini, B., Svenson, S.B., Kallenius, G., 1994a. Mycobacterium avium complex develop resistance to synergistically active drug combinations during infection. European Respiratory Journal 7, 247–250. Hoffner, S.E., Klintz, L., Olsson-Liljequist, B., Bolmstrom, A., 1994b. Evaluation of E test for rapid susceptibility testing of Mycobacterium chelonae and M. fortuitum. Journal of Clinical Microbiology 32, 1846–1849. Howard, S.T., Rhoades, E., Recht, J., Pang, X., Alsup, A., Kolter, R., Lyons, C.R., Byrd, T.F., 2006. Spontaneous reversion of Mycobacterium abscessus from a smooth to a rough morphotype is associated with reduced expression of glycopeptidolipid and reacquisition of an invasive phenotype. Microbiology 152, 1581–1590. Hull, S.I., Wallace Jr., R.J., Bobey, D.G., Price, K.E., Goodhines, R.A., Swenson, J.M., Silcox, V.A., 1984. Presence of aminoglycoside acetyltransferase and plasmids in Mycobacterium fortuitum. Lack of correlation with intrinsic aminoglycoside resistance. American Review of Respiratory Disease 129, 614–618. Inderlied, C.B., Young, L.S., Yamada, J.K., 1987. Determination of in vitro susceptibility of Mycobacterium avium complex isolates to antimycobacterial agents by various methods. Antimicrobial Agents and Chemotherapy 31, 1697–1702. International Organization for Standardization, 2006. ISO 20776-1. Clinical laboratory testing and in vitro diagnostic test systems – susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices – part 1: reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases. Geneva, Switzerland. Jarand, J., Levin, A., Zhang, L., Huitt, G., Mitchell, J.D., Daley, C.L., 2011. Clinical and microbiologic outcomes in patients receiving treatment for Mycobacterium abscessus pulmonary disease. Clinical Infectious Diseases 52, 565–571. Jarboe, E., Stone, B.L., Burman, W.J., Wallace Jr., R.J., Brown, B.A., Reves, R.R., Wilson, M.L., 1998. Evaluation of a disk diffusion method for determining susceptibility of Mycobacterium avium complex to clarithromycin. Diagnostic Microbiology and Infectious Disease 30, 197–203. Jenkins, P.A., Campbell, I.A., Banks, J., Gelder, C.M., Prescott, R.J., Smith, A.P., 2008. Clarithromycin vs. ciprofloxacin as adjuncts to rifampicin and ethambutol in treating opportunist mycobacterial lung diseases and an assessment of Mycobacterium vaccae immunotherapy. Thorax 63, 627–634. Jeon, K., Kwon, O.J., Lee, N.Y., Kim, B.J., Kook, Y.H., Lee, S.H., Park, Y.K., Kim, C.K., Koh, W.J., 2009. Antibiotic treatment of Mycobacterium abscessus lung disease: a retrospective analysis of 65 patients. American Journal of Respiratory and Critical Care Medicine 180, 896–902. Klein, J.L., Brown, T.J., French, G.L., 2001. Rifampin resistance in Mycobacterium kansasii is associated with rpoB mutations. Antimicrobial Agents and Chemotherapy 45, 3056–3058. Kobashi, Y., Yoshida, K., Miyashita, N., Niki, Y., Oka, M., 2006. Relationship between clinical efficacy of treatment of pulmonary Mycobacterium avium complex disease and drug-sensitivity testing of Mycobacterium avium complex isolates. Journal of Infection and Chemotherapy 12, 195–202. Kobashi, Y., Matsushima, T., Oka, M., 2007. A double-blind randomized study of aminoglycoside infusion with combined therapy for pulmonary Mycobacterium avium complex disease. Respiratory Medicine 101, 130–138. Krishnan, M.Y., Manning, E.J., Collins, M.T., 2009. Comparison of three methods for susceptibility testing of Mycobacterium avium subsp. paratuberculosis to 11 antimicrobial drugs. Journal of Antimicrobial Chemotherapy 64, 310–316. Kuze, F., 1984. Experimental chemotherapy in chronic Mycobacterium avium–intracellulare infection of mice. American Review of Respiratory Disease 129, 453–459. Kuze, F., Kurasawa, T., Bando, K., Lee, Y., Maekawa, N., 1981. In vitro and in vivo susceptibility of atypical mycobacteria to various drugs. Reviews of Infectious Diseases 3, 885–897. Lambert, P.A., 2002. Cellular impermeability and uptake of biocides and antibiotics in Gram-positive bacteria and mycobacteria. Journal of Applied Microbiology 92 (Suppl.), 46S–54S. Li, X.Z., Zhang, L., Nikaido, H., 2004. Efflux pump-mediated intrinsic drug resistance in Mycobacterium smegmatis. Antimicrobial Agents and Chemotherapy 48, 2415–2423. Long, K.S., Munck, C., Andersen, T.M., Schaub, M.A., Hobbie, S.N., Bottger, E.C., Vester, B., 2010. Mutations in 23S rRNA at the peptidyl transferase center and their relationship to linezolid binding and cross-resistance. Antimicrobial Agents and Chemotherapy 54, 4705–4713. Manten, A., 1957. Antimicrobial susceptibility and some other properties of photochromogenic Mycobacteria associated with pulmonary disease. Antonie Van Leeuwenhoek 23, 357–363. Marone, P., Bono, L., Carretto, E., Barbarini, D., Telecco, S., 1997. Rapid drug susceptibility of Mycobacterium avium complex using a fluorescence quenching method. Journal of Chemotherapy 9, 247–250. Meier, A., Kirschner, P., Springer, B., Steingrube, V.A., Brown, B.A., Wallace Jr., R.J., Bottger, E.C., 1994. Identification of mutations in 23S rRNA gene of clarithromycin-resistant Mycobacterium intracellulare. Antimicrobial Agents and Chemotherapy 38, 381–384. Meier, A., Heifets, L., Wallace Jr., R.J., Zhang, Y., Brown, B.A., Sander, P., Bottger, E.C., 1996. Molecular mechanisms of clarithromycin resistance in Mycobacterium avium: observation of multiple 23S rDNA mutations in a clonal population. Journal of Infectious Diseases 174, 354–360. Middlebrook, G., Cohn, M.L., 1958. Bacteriology of tuberculosis: laboratory methods. American Journal of Public Health and the Nations Health 48, 844–853.

Middlebrook, G., Reggiardo, Z., Tigertt, W.D., 1977. Automatable radiometric detection of growth of Mycobacterium tuberculosis in selective media. American Review of Respiratory Disease 115, 1066–1069. Molavi, A., Weinstein, L., 1971. In vitro susceptibility of atypical mycobacteria to rifampin. Applied Microbiology 22, 23–25. Morris, R.P., Nguyen, L., Gatfield, J., Visconti, K., Nguyen, K., Schnappinger, D., Ehrt, S., Liu, Y., Heifets, L., Pieters, J., Schoolnik, G., Thompson, C.J., 2005. Ancestral antibiotic resistance in Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences of the United States of America 102, 12200–12205. Mouton, J.W., Brown, D.F., Apfalter, P., Canton, R., Giske, C.G., Ivanova, M., Macgowan, A.P., Rodloff, A., Soussy, C.J., Steinbakk, M., Kahlmeter, G., 2011. The role of pharmacokinetics/pharmacodynamics in setting clinical MIC breakpoints: the EUCAST approach. Clinical Microbiology and Infection 18, E37–E45. Nash, K.A., Inderlied, C.B., 1995. Genetic basis of macrolide resistance in Mycobacterium avium isolated from patients with disseminated disease. Antimicrobial Agents and Chemotherapy 39, 2625–2630. Nash, K.A., Inderlied, C.B., 1996. Rapid detection of mutations associated with macrolide resistance in Mycobacterium avium complex. Antimicrobial Agents and Chemotherapy 40, 1748–1750. Nash, D.R., Wallace Jr., R.J., Steingrube, V.A., Udou, T., Steele, L.C., Forrester, G.D., 1986. Characterization of beta-lactamases in Mycobacterium fortuitum including a role in beta-lactam resistance and evidence of partial inducibility. American Review of Respiratory Disease 134, 1276–1282. Nash, K.A., Zhang, Y., Brown-Elliott, B.A., Wallace Jr., R.J., 2005. Molecular basis of intrinsic macrolide resistance in clinical isolates of Mycobacterium fortuitum. Journal of Antimicrobial Chemotherapy 55, 170–177. Nash, K.A., Andini, N., Zhang, Y., Brown-Elliott, B.A., Wallace Jr., R.J., 2006. Intrinsic macrolide resistance in rapidly growing mycobacteria. Antimicrobial Agents and Chemotherapy 50, 3476–3478. Nash, K.A., Brown-Elliott, B.A., Wallace Jr., R.J., 2009. A novel gene, erm(41), confers inducible macrolide resistance to clinical isolates of Mycobacterium abscessus but is absent from Mycobacterium chelonae. Antimicrobial Agents and Chemotherapy 53, 1367–1376. Nguyen, L., Thompson, C.J., 2006. Foundations of antibiotic resistance in bacterial physiology: the mycobacterial paradigm. Trends in Microbiology 14, 304–312. Nguyen, L., Chinnapapagari, S., Thompson, C.J., 2005. FbpA-dependent biosynthesis of trehalose dimycolate is required for the intrinsic multidrug resistance, cell wall structure, and colonial morphology of Mycobacterium smegmatis. Journal of Bacteriology 187, 6603–6611. Nguyen, H.T., Wolff, K.A., Cartabuke, R.H., Ogwang, S., Nguyen, L., 2010. A lipoprotein modulates activity of the MtrAB two-component system to provide intrinsic multidrug resistance, cytokinetic control and cell wall homeostasis in Mycobacterium. Molecular Microbiology 76, 348–364. Niederweis, M., 2008. Nutrient acquisition by mycobacteria. Microbiology 154, 679–692. Nijland, H.M., Ruslami, R., Suroto, A.J., Burger, D.M., Alisjahbana, B., van Crevel, R., Aarnoutse, R.E., 2007. Rifampicin reduces plasma concentrations of moxifloxacin in patients with tuberculosis. Clinical Infectious Diseases 45, 1001–1007. Philalay, J.S., Palermo, C.O., Hauge, K.A., Rustad, T.R., Cangelosi, G.A., 2004. Genes required for intrinsic multidrug resistance in Mycobacterium avium. Antimicrobial Agents and Chemotherapy 48, 3412–3418. Piersimoni, C., Tortoli, E., Mascellino, M.T., Passerini Tosi, C., Sbaraglia, G., Mandler, F., Bistoni, F., Bornigia, S., De Sio, G., Goglio, A., et al., 1995. Activity of seven antimicrobial agents, alone and in combination, against AIDS-associated isolates of Mycobacterium avium complex. Journal of Antimicrobial Chemotherapy 36, 497–502. Piersimoni, C., Nista, D., Bornigia, S., De Sio, G., 1998. Evaluation of a new method for rapid drug susceptibility testing of Mycobacterium avium complex isolates by using the mycobacteria growth indicator tube. Journal of Clinical Microbiology 36, 64–67. Prammananan, T., Sander, P., Brown, B.A., Frischkorn, K., Onyi, G.O., Zhang, Y., Bottger, E.C., Wallace Jr., R.J., 1998. A single 16S ribosomal RNA substitution is responsible for resistance to amikacin and other 2-deoxystreptamine aminoglycosides in Mycobacterium abscessus and Mycobacterium chelonae. Journal of Infectious Diseases 177, 1573–1581. Ramirez, M.S., Tolmasky, M.E., 2010. Aminoglycoside modifying enzymes. Drug Resistance Updates 13, 151–171. Ramon-Garcia, S., Martin, C., Ainsa, J.A., De Rossi, E., 2006. Characterization of tetracycline resistance mediated by the efflux pump Tap from Mycobacterium fortuitum. Journal of Antimicrobial Chemotherapy 57, 252–259. Realini, L., Van Der Stuyft, P., De Ridder, K., Hirschel, B., Portaels, F., 1997. Inhibitory effects of polyoxyethylene stearate, PANTA, and neutral pH on growth of Mycobacterium genavense in BACTEC primary cultures. Journal of Clinical Microbiology 35, 2791–2794. Realini, L., De Ridder, K., Hirschel, B., Portaels, F., 1999. Blood and charcoal added to acidified agar media promote the growth of Mycobacterium genavense. Diagnostic Microbiology and Infectious Disease 34, 45–50. Ren, H., Liu, J., 2006. AsnB is involved in natural resistance of Mycobacterium smegmatis to multiple drugs. Antimicrobial Agents and Chemotherapy 50, 250–255. Ripoll, F., Pasek, S., Schenowitz, C., Dossat, C., Barbe, V., Rottman, M., Macheras, E., Heym, B., Herrmann, J.L., Daffe, M., Brosch, R., Risler, J.L., Gaillard, J.L., 2009. Non mycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus. PLoS One 4, e5660.

J. van Ingen et al. / Drug Resistance Updates 15 (2012) 149–161 Rodrigues, L., Ainsa, J.A., Amaral, L., Viveiros, M., 2011. Inhibition of drug efflux in mycobacteria with phenothiazines and other putative efflux inhibitors. Recent Patents on Antiinfective Drug Discovery 6, 118–127. Runyon, E.H., 1959. Anonymous mycobacteria in pulmonary disease. Medical Clinics of North America 43, 273–290. Rynearson, T.K., Shronts, J.S., Wolinsky, E., 1971. Rifampin: in vitro effect on atypical mycobacteria. American Review of Respiratory Disease 104, 272–274. Sander, P., De Rossi, E., Boddinghaus, B., Cantoni, R., Branzoni, M., Bottger, E.C., Takiff, H., Rodriquez, R., Lopez, G., Riccardi, G., 2000. Contribution of the multidrug efflux pump LfrA to innate mycobacterial drug resistance. FEMS Microbiology Letters 193, 19–23. Schorey, J.S., Sweet, L., 2008. The mycobacterial glycopeptidolipids: structure, function, and their role in pathogenesis. Glycobiology 18, 832–841. Shen, G.H., Wu, B.D., Hu, S.T., Lin, C.F., Wu, K.M., Chen, J.H., 2010. High efficacy of clofazimine and its synergistic effect with amikacin against rapidly growing mycobacteria. International Journal of Antimicrobial Agents 35, 400–404. Shitrit, D., Baum, G.L., Priess, R., Lavy, A., Shitrit, A.B., Raz, M., Shlomi, D., Daniele, B., Kramer, M.R., 2006. Pulmonary Mycobacterium kansasii infection in Israel, 1999–2004: clinical features, drug susceptibility, and outcome. Chest 129, 771–776. Silva, P.E., Bigi, F., Santangelo, M.P., Romano, M.I., Martin, C., Cataldi, A., Ainsa, J.A., 2001. Characterization of P55, a multidrug efflux pump in Mycobacterium bovis and Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 45, 800–804. Sison, J.P., Yao, Y., Kemper, C.A., Hamilton, J.R., Brummer, E., Stevens, D.A., Deresinski, S.C., 1996. Treatment of Mycobacterium avium complex infection: do the results of in vitro susceptibility tests predict therapeutic outcome in humans. Journal of Infectious Diseases 173, 677–683. Snider Jr., D.E., Good, R.C., Kilburn, J.O., Laskowski Jr., L.F., Lusk, R.H., Marr, J.J., Reggiardo, Z., Middlebrook, G., 1981. Rapid drug-susceptibility testing of Mycobacterium tuberculosis. American Review of Respiratory Disease 123, 402–406. Society, R.C.o.t.B.T., 2001. First randomised trial of treatments for pulmonary disease caused by M. avium intracellulare, M. malmoense, and M. xenopi in HIV negative patients: rifampicin, ethambutol and isoniazid versus rifampicin and ethambutol. Thorax 56, 167–172. Stephan, J., Mailaender, C., Etienne, G., Daffe, M., Niederweis, M., 2004. Multidrug resistance of a porin deletion mutant of Mycobacterium smegmatis. Antimicrobial Agents and Chemotherapy 48, 4163–4170. Stone, M.S., Wallace Jr., R.J., Swenson, J.M., Thornsberry, C., Christensen, L.A., 1983. Agar disk elution method for susceptibility testing of Mycobacterium marinum and Mycobacterium fortuitum complex to sulfonamides and antibiotics. Antimicrobial Agents and Chemotherapy 24, 486–493. Swenson, J.M., Thornsberry, C., Silcox, V.A., 1982. Rapidly growing mycobacteria: testing of susceptibility to 34 antimicrobial agents by broth microdilution. Antimicrobial Agents and Chemotherapy 22, 186–192. Swenson, J.M., Wallace Jr., R.J., Silcox, V.A., Thornsberry, C., 1985. Antimicrobial susceptibility of five subgroups of Mycobacterium fortuitum and Mycobacterium chelonae. Antimicrobial Agents and Chemotherapy 28, 807–811. Tanaka, E., Kimoto, T., Tsuyuguchi, K., Watanabe, I., Matsumoto, H., Niimi, A., Suzuki, K., Murayama, T., Amitani, R., Kuze, F., 1999. Effect of clarithromycin regimen for Mycobacterium avium complex pulmonary disease. American Journal of Respiratory and Critical Care Medicine 160, 866–872. Te Dorsthorst, D.T., Mouton, J.W., van den Beukel, C.J., van der Lee, H.A., Meis, J.F., Verweij, P.E., 2004. Effect of pH on the in vitro activities of amphotericin B, itraconazole, and flucytosine against Aspergillus isolates. Antimicrobial Agents and Chemotherapy 48, 3147–3150. Tison, F., Tacquet, A., Guillaume, J., Devulder, B., 1963. Unsuitability of the Basic Coletsos Medium for the Measurement of Sensitivity to Cycloserine. Inactivation of Cycloserine by Sodium Pyruvate. Annales de l Institut Pasteur de Lille 14, 117–124. Truffot-Pernot, C., Ji, B., Grosset, J., 1991. Effect of pH on the in vitro potency of clarithromycin against Mycobacterium avium complex. Antimicrobial Agents and Chemotherapy 35, 1677–1678. van Ingen, J., 2011. The broad-spectrum antimycobacterial activities of phenothiazines, invitro: somewhere in all of this there may be patentable potentials. Recent Patents of Antiinfective Drug Discovery 6, 104–109. van Ingen, J., Boeree, M.J., Dekhuijzen, P.N., van Soolingen, D., 2008. Clinical relevance of Mycobacterium simiae in pulmonary samples. European Respiratory Journal 31, 106–109. van Ingen, J., Bendien, S.A., de Lange, W.C., Hoefsloot, W., Dekhuijzen, P.N., Boeree, M.J., van Soolingen, D., 2009. Clinical relevance of non-tuberculous mycobacteria isolated in the Nijmegen-Arnhem region, The Netherlands. Thorax 64, 502–506. van Ingen, J., van der Laan, T., Dekhuijzen, R., Boeree, M., van Soolingen, D., 2010. In vitro drug susceptibility of 2275 clinical non-tuberculous Mycobacterium

161

isolates of 49 species in The Netherlands. International Journal of Antimicrobial Agents 35, 169–173. van Ingen, J., Boeree, M.J., van Soolingen, D., Iseman, M.D., Heifets, L.B., Daley, C.L., 2011. Are phylogenetic position, virulence, drug susceptibility and in vivo response to treatment in mycobacteria interrelated? Infection, Genetics and Evolution (Epub ahead of print). van Ingen, J., Totten, S.E., Heifets, L.B., Boeree, M.J., Daley, C.L., 2012. Drug susceptibility testing and pharmacokinetics question current treatment regimens in Mycobacterium simiae complex disease. International Journal of Antimicrobial Agents 39, 173–176. van Soolingen, D., Hernandez-Pando, R., Orozco, H., Aguilar, D., Magis-Escurra, C., Amaral, L., van Ingen, J., Boeree, M.J., 2010. The antipsychotic thioridazine shows promising therapeutic activity in a mouse model of multidrug-resistant tuberculosis. PLoS One 5, e12640. Wallace Jr., R.J., Wiss, K., 1981. Susceptibility of Mycobacterium marinum to tetracyclines and aminoglycosides. Antimicrobial Agents and Chemotherapy 20, 610–612. Wallace Jr., R.J., Dalovisio, J.R., Pankey, G.A., 1979. Disk diffusion testing of susceptibility of Mycobacterium fortuitum and Mycobacterium chelonei to antibacterial agents. Antimicrobial Agents and Chemotherapy 16, 611–614. Wallace Jr., R.J., Wiss, K., Bushby, M.B., Hollowell, D.C., 1982. In vitro activity of trimethoprim and sulfamethoxazole against the nontuberculous mycobacteria. Reviews of Infectious Diseases 4, 326–331. Wallace Jr., R.J., Swenson, J.M., Silcox, V.A., Bullen, M.G., 1985. Treatment of nonpulmonary infections due to Mycobacterium fortuitum and Mycobacterium chelonei on the basis of in vitro susceptibilities. Journal of Infectious Diseases 152, 500–514. Wallace Jr., R.J., Nash, D.R., Steele, L.C., Steingrube, V., 1986. Susceptibility testing of slowly growing mycobacteria by a microdilution MIC method with 7H9 broth. Journal of Clinical Microbiology 24, 976–981. Wallace Jr., R.J., Dunbar, D., Brown, B.A., Onyi, G., Dunlap, R., Ahn, C.H., Murphy, D.T., 1994. Rifampin-resistant Mycobacterium kansasii. Clinical Infectious Diseases 18, 736–743. Wallace Jr., R.J., Brown, B.A., Griffith, D.E., Girard, W., Tanaka, K., 1995. Reduced serum levels of clarithromycin in patients treated with multidrug regimens including rifampin or rifabutin for Mycobacterium avium–M. intracellulare infection. Journal of Infectious Disease 171, 747–750. Wallace Jr., R.J., Brown, B.A., Griffith, D.E., Girard, W.M., Murphy, D.T., 1996a. Clarithromycin regimens for pulmonary Mycobacterium avium complex. The first 50 patients. American Journal of Respiratory and Critical Care Medicine 153, 1766–1772. Wallace Jr., R.J., Meier, A., Brown, B.A., Zhang, Y., Sander, P., Onyi, G.O., Bottger, E.C., 1996b. Genetic basis for clarithromycin resistance among isolates of Mycobacterium chelonae and Mycobacterium abscessus. Antimicrobial Agents and Chemotherapy 40, 1676–1681. Watt, B., Edwards, J.R., Rayner, A., Grindey, A.J., Harris, G., 1992. In vitro activity of meropenem and imipenem against mycobacteria: development of a daily antibiotic dosing schedule. Tubercle and Lung Disease 73, 134–136. Wolff, K.A., Nguyen, H.T., Cartabuke, R.H., Singh, A., Ogwang, S., Nguyen, L., 2009. Protein kinase G is required for intrinsic antibiotic resistance in mycobacteria. Antimicrobial Agents and Chemotherapy 53, 3515–3519. Woods, G.L., Witebsky, F.G., 1996. Susceptibility testing of Mycobacterium avium complex in clinical laboratories. Results of a questionnaire and proficiency test performance by participants in the College of American Pathologists Mycobacteriology E Survey. Archives of Pathology and Laboratory Medicine 120, 436–439. Woods, G.L., Bergmann, J.S., Witebsky, F.G., Fahle, G.A., Wanger, A., Boulet, B., Plaunt, M., Brown, B.A., Wallace Jr., R.J., 1999. Multisite reproducibility of results obtained by the broth microdilution method for susceptibility testing of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium fortuitum. Journal of Clinical Microbiology 37, 1676–1682. Woods, G.L., Bergmann, J.S., Witebsky, F.G., Fahle, G.A., Boulet, B., Plaunt, M., Brown, B.A., Wallace Jr., R.J., Wanger, A., 2000. Multisite reproducibility of Etest for susceptibility testing of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium fortuitum. Journal of Clinical Microbiology 38, 656–661. Woods, G.L., Williams-Bouyer, N., Wallace Jr., R.J., Brown-Elliott, B.A., Witebsky, F.G., Conville, P.S., Plaunt, M., Hall, G., Aralar, P., Inderlied, C., 2003. Multisite reproducibility of results obtained by two broth dilution methods for susceptibility testing of Mycobacterium avium complex. Journal of Clinical Microbiology 41, 627–631. Yajko, D.M., Sanders, C.A., Madej, J.J., Cawthon, V.L., Hadley, W.K., 1996. In vitro activities of rifabutin, azithromycin, ciprofloxacin, clarithromycin, clofazimine, ethambutol, and amikacin in combinations of two, three, and four drugs against Mycobacterium avium. Antimicrobial Agents and Chemotherapy 40, 743–749. Yew, W.W., Piddock, L.J., Li, M.S., Lyon, D., Chan, C.Y., Cheng, A.F., 1994. In vitro activity of quinolones and macrolides against mycobacteria. Journal of Antimicrobial Chemotherapy 34, 343–351.