Species Identification and Clarithromycin Susceptibility Testing of 278 Clinical Nontuberculosis Mycobacteria Isolates

Purpose of this paper is to analyze different species' proportion of nontuberculosis mycobacteria (NTM) and susceptibility to clarithromycin of different species. 278 clinical NTM isolates were identified into species by using 16S rRNA, rpoB and hsp65. Then clarithromycin susceptibility testing against different species was done separately, using microplate Alamar Blue assay. Finally, resistance isolates' erm(41) of M. abscessus were sequenced in order to analyze mechanisms for clarithromycin resistant. In this test, 131 isolates (47%) belonged to M. avium complex (MAC), and 70 isolates (25%) belonged to M. abscessus. Nearly all the M. abscessus subsp. abscessus resistant to clarithromycin had T28 in erm(41). However, all the M. abscessus subsp. abscessus susceptible to clarithromycin had C28 in erm(41). In this study, we find that MAC was the most common pathogens of NTM, and the second one was M. abscessus. However, M. chelonei, M. fuerth, and M. gordon were rare. Clarithromycin had a good inhibition activity against all the NTM species except M. abscessus subsp. abscessus. The erm(41) genotype is of high relevance to clarithromycin resistance.


Introduction
Nontuberculosis mycobacteria (NTM) are globally recognized as pathogens especially in immunocompromised population including HIV/AIDS (Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome) patients. In addition to causing infections in immune-compromised patients, this group of organisms has been reported to be increasingly recognized as pathogens from immunocompetent individuals as well [1]. Different species have different susceptibility to antibiotics, so it is important to identify NTM into different species in order to make the respective therapy plan. The American Thoracic Society (ATS) and the Infectious Diseases Society of America (IDSA) recommended treatment for NTM pulmonary and disseminated disease is a macrolide-containing, multidrug regimen. And clarithromycin, a macrolide drug, is described as the key therapy for NTM diseases [2]. Clarithromycin forms the cornerstone of NTM treatment and is the only drug recommended by the Clinical and Laboratory Standards Institute (CLSI) for drug susceptibility testing (DST) [3]. However, antibiotic treatment of pulmonary M. abscessus infections remains problematic. Infections due to the M. abscessus group are difficult to treat because these mycobacteria are intrinsically resistant not only to the classical antituberculous drugs but also to most of the antibiotics that are currently available. Clarithromycin had been the drug of choice for M. abscessus infections for the past 20 years before a resistance gene, erm(41), was described in M. abscessus. The discovery of a novel inducible erm gene, erm(41), provides an explanation for the lack of efficacy for macrolides against M. abscessus infections [4]. A polymorphism in erm(41) (T or C) was described at position 28 and its association with the susceptibility was examined. Our objective was to identify NTM isolates into different species and to do the DST separately and then to seek the presence of erm(41) in a large collection of clinical strains precisely identified as M. abscessus and finally to correlate these results with clarithromycin susceptibility.

Species Identification.
All of the 278 clinical strains in Reference Lab of Beijing Chest Hospital were isolated from sputum samples of patients, which were previously identified as NTM strain using growth experiments of pnitrobenzoic acid (PNB) by Reference Lab of Beijing Chest Hospital. All the strains were subcultured on the Lowenstein-Jensen medium at 37 ∘ C to observe colony morphology. The incubation time ranged from 4 to 28 days. Then colonies were used for species identification based on 16S rRNA sequence analysis. Preparation of the PCR reaction mixture and amplification were done as described previously [5,6]. PCR products were purified and sequenced by the BGI corporation, using forward and reverse primers. Both strands were sequenced as a crosscheck. Species identification of these strains was accomplished by the sequencing of 16S rRNA, using BLAST search to measure the similarities.

Drug Susceptibility Testing.
We performed DST of NTM isolates by broth microdilution using 96-well plates, which is a reference method as recommended by CLSI [3]. DST of these NTM isolates was done as described previously [6]. To M. abscessus, plates were submitted to an extended incubation of 14 days' incubation at 30 ∘ C. However, to other NTM isolates, the incubation time ranged from 3 to 10 days. A color change from blue to pink indicates bacterial growth. MIC was defined as the lowest concentration of the drug that showed no color change, which was the lowest concentration of drug capable of inhibiting the visible growth of tested isolates. MIC 50 and MIC 90 values were defined as drug concentrations, which inhibit 50% and 90% of isolates, respectively. Susceptibility was evaluated according to CLSI breakpoint recommendations [3]. The type strains of American Type Culture Collection (ATCC) were used for comparison.

Clarithromycin Resistant Mechanism.
Crude DNA preparations suitable for PCR were obtained by heating suspensions of mycobacteria in Tris-EDTA buffer at 100 ∘ C for 15 to 20 min. Debris was removed from the preparations by centrifugation at 16,000 rpm for 3 min. The basic 40 L PCR mixture comprised 2 L DNA, 2 L DNA polymerase, and 1 L each primer and finally was complete to 40 L by double distilled water. Reactions were run for 3 min at 94 ∘ C, followed by 30 cycles at 94 ∘ C for 1 min, 61 ∘ C for 1 min, and 72 ∘ C for 1 min. For cloning of erm(41), gene segments were amplified with primers 5-ACG TTG GAT CCG AGC GCC GTC ACA AGA TGC ACA-3 and 5-GCG AGA AGC TTG ACT TCC CCG CAC CGA TTC CAC-3. The resulting amplification products were purified and sequenced by the BGI corporation, using forward and reverse primers. Both strands were sequenced as a crosscheck. The sequence results were compared with M. abscessus standard strains.

Species Identification.
Of all the 278 NTM clinical strains (Table 1), 100 (36%) isolates belonged to rapidly growing Mycobacterium (RGM), and 178 (64%) isolates belonged to  Table 3, respectively. As RGM, M. fortuitum should be incubated for 3 days (CLSI recommends), and then the MIC value is read. In Table 3, it shows that most of M. fortuitum (92%) strains were susceptible to clarithromycin, and the resistant proportion was low (4%     [7]. This is the first study to focus primarily on the clinical relevance of the species massiliense, and M. abscessus subsp. bolletii is challenging for clinical microbiology laboratories. Recent work proved the inaccuracy of single-target sequencing for separating these three taxa within the M. abscessus group. In some strains, additional housekeeping genes were analyzed because of the discordant results between rpoB and hsp65 genes analysis [5,12]. Sequencing of more other targets (such as secA1, soda, and ITS), combined with phylogenomic analysis, has been shown to increase identification accuracy among these different taxa [8]. More recently, a multilocus sequence analysis targeting 8 housekeeping genes and a multispacer sequence analysis were reported [13,14]. However, all of these methods require genomic sequencing, which is relatively costly and time consuming and may not be available in all clinical microbiology laboratories.

Drug Susceptibility
Testing. NTM infection is a refractory disease that, unlike tuberculosis, does not respond well to conventional antituberculosis drug. With the progress of mycobacterial taxonomy and speciation, it appeared that antibiotic susceptibility of NTM is tightly related to mycobacterial species and, conversely, that a homogenous intrinsic susceptibility pattern may reflect the homogeneity of the species [2,7]. Since many new species have been described recently with the assistance of molecular biology and genomics, we need to assess their intrinsic susceptibility patterns species by species. The macrolide clarithromycin is considered a cornerstone in antimicrobial chemotherapy of NTM infections. Clarithromycin is a 14-membered ring macrolide that binds to the large ribosomal subunit in the vicinity of the peptidyl transferase center and inhibits protein synthesis, which results in the arrest of bacterial growth. Clarithromycin is given orally and is highly active against many species of NTM. It is the only drug of demonstrated efficacy that can be administered orally. In all of the studies so far, most variations were reported in the clarithromycin susceptibility results within different NTM species even with strains precisely identified and even with reference strains [5,15,16]. For example, M. abscessus subsp. bolletii was shown to be clarithromycin resistant by Adékambi and Drancourt [15] and susceptible by Leao et al. [16]. NTM species show a characteristically different response to clarithromycin from others, and precise identification of these species would be important for the treatment of infected patients. This is why we would like to propose that clarithromycin susceptibility be assessed on a molecular basis for all new cases of M. abscessus group infections and that the outcomes of clarithromycin therapy be observed as follows. In this study, we evaluated clarithromycin's activity against different NTM species, since this is the single drug CLSI recommended in in vitro testing. The CLSI recommends the use of the radiometric BACTEC 460, the broth-based macrodilution, or the broth microdilution method for the testing of NTM susceptibility to clarithromycin, and it requires weeks to complete from the time of sample collection because of the growing rate of organism.
As the most common NTM species, the treatment outcome of MAC pulmonary disease was disappointing before the use of newer macrolide antibiotics such as clarithromycin and azithromycin. Since 1990 introduction of newer macrolide antibiotics, treatment of pulmonary MAC disease has improved patient outcome. The ATS and IDSA recommend that a three-drug regimen be utilized for the treatment of MAC infection, including a macrolide (clarithromycin or azithromycin). The macrolides are the only class of drugs for which a correlation has been demonstrated between in vitro susceptibility results and in vivo clinical response in MAC disease (Griffith, 2007). For this reason, the ATS only recommends routine susceptibility testing for clarithromycin, the macrolide most commonly used for treating MAC disease (Griffith, 2007). In this test clarithromycin showed an excellent inhibition activity against MAC. 95% MAC were susceptible to it. And the resistance proportion was low. Considering the big sample size of 131 isolates, this result again proves clarithromycin's good inhibition activity against MAC. However, ATS guidelines also reported that, in contrast to Mycobacterium tuberculosis, DST maybe of little use for the treatment of MAC infection, although clarithromycin is the only drug for which the results of susceptibility testing should be taken into consideration in the treatment of MAC infection [2]. We need to conduct a prospective clinical study in order to compare efficacy in vivo and susceptibility data in vitro.
As the second-largest NTM species and one of the most notorious causative agents of disease, drug therapy of M. abscessus disease is long, costly, resistant to many antibiotics, and often associated with drug-related toxicities, leading to unsatisfactory treatment results. In the 1990s, clarithromycin became the drug of choice for M. abscessus infections, and therapeutic successes were reported. Combination therapy of intravenous amikacin with cefoxitin or imipenem and an oral macrolide has been recommended by the ATS/IDSA and many other experts [2]. However, treatment response rates are not satisfactory and optimal therapeutic regimens and treatment durations are not well established.  [12,17]. For instance, an experiment in Japan shows that with M. abscessus subsp. abscessus bronchiectasia may occur more frequently than with M. abscessus subsp. bolletii in vitro, and the resistant proportion of imipenem against M. abscessus subsp. abscessus is lower than M. abscessus subsp. bolletii [17]. An experiment in Korea shows that M. abscessus subsp. abscessus may have a worse radiographic response on HRCT and microbiologic response than M. abscessus subsp. bolletii in vitro, and similarly, the resistant proportion of imipenem against M. abscessus subsp. abscessus is lower than M. abscessus subsp. bolletii [12]. massiliense's MIC to clarithromycin is small, and nearly all of the isolates were susceptible to it. From a clinical standpoint, our study has some important therapeutic implications. The inducible resistance to clarithromycin of M. abscessus subsp. abscessus isolates means that it can be much more difficult to treat M. abscessus subsp. abscessus lung disease. A much longer duration of intravenous antibiotic therapy and more effective oral antibiotics may be needed to improve treatment outcomes in M. abscessus subsp. abscessus lung disease. The low MIC for clarithromycin and the absence of inducible resistance to clarithromycin suggest that M. abscessus subsp. massiliense lung disease may be more effectively treated with a clarithromycin-based antibiotic regimen [12].
M. kansasii is the second most common cause of NTM disease in the United States. In our test it is the thirdlargest species, but the sample size is small. In this test, M. kansasii showed an excellent susceptibility to clarithromycin. All the M. kansasii isolates were susceptible to it. However, according to ATS and IDSA, routine susceptibility testing of M. kansasii isolates is recommended for rifampin only, and the recommended treatment of M. kansasii pulmonary disease does not include clarithromycin. But the excellent in vitro activity of clarithromycin against M. kansasii suggests that these agents may be useful in retreatment regimens. M. fortuitum infections are rare, and they are usually susceptible to multiple oral antimicrobial agents. In this test, M. fortuitum showed a good susceptibility to clarithromycin. All of the M. fortuitum isolates were susceptible to clarithromycin, except 1 isolate resistant to it. In vitro susceptibility data are limited because of the extreme fastidiousness of the organism [2]. Optimal therapy is not determined, but ATS and IDSA also recommend that multidrug therapies including clarithromycin appear to be more effective than those without clarithromycin. M. chelonei and M. fuerth are extremely rare species. In our test, isolates of these two species are very few, so Table 4 only shows every isolate's MIC value. In Table 4, it shows that the MIC value is very low and all the M. chelone and M. fuerth strains were susceptible to clarithromycin. Although the sample size is small, we may still suppose that clarithromycin may have a good inhibition activity against these two species.

Clarithromycin Resistant Mechanism.
For M. abscessus, one of the most notorious causative agents of disease, the macrolide clarithromycin is considered as cornerstone in antimicrobial chemotherapy of pulmonary M. abscessus infection. Paradoxically, however, M. abscessus infections of the lung are often intractable to clarithromycin chemotherapy, although high doses of clarithromycin may lead to clinical improvement in some patients [2,4]. The lack of efficacy of clarithromycin is puzzling, as pretreatment isolates are usually reported as susceptible to clarithromycin when CLSI procedures are used [3,4]. Intriguingly, Brown et al. reported that the clarithromycin MIC increased for some M. abscessus isolates if the incubations for the susceptibility assays were extended, although these isolates would still be deemed clarithromycin susceptible if an incubation period based on the previous CLSI guidelines was used [4,18]. Similarly, Nash and colleagues showed that although a strain might appear susceptible after 3 days of in vitro incubation [3], due to induction of the synthesis of a methyltransferase [4], the strain was clarithromycin resistant if incubation was extended to 14 days or if the strain was preincubated with clarithromycin. So in 2012, CLSI guideline extends M. abscessus DST incubation time from 3 days to 14 days. Although M. abscessus subsp. massiliense remained a high susceptible proportion after this adjustment, the resistant proportion of M. abscessus subsp. abscessus increased to a high level. An explanation for the instability of clarithromycin MIC is that M. abscessus might have inducible resistance to clarithromycin. Inducible resistance to clarithromycin has been suggested as an explanation for the lack of efficacy of clarithromycin-based treatments against M. abscessus infection [12]. An erythromycin ribosomal methylase gene, erm(41), has been identified in several isolates of M. abscessus and the presence of the gene was associated with inducible resistance to clarithromycin. The induction of resistance was shown to result from increased expression of erm(41) as demonstrated by an increase in erm(41) RNA levels after exposure to clarithromycin. The functionality of the methylase is dependent on the nucleotide at position 28 of the erm(41) gene. Wild type T28 sequevars show inducible clarithromycin resistance, while C28 sequevars do not [4]. In our test, of all the 40 M. abscessus subsp. abscessus isolates which were sequenced successfully, 35 isolates were resistant to clarithromycin, and 34 isolates in them had T28 in erm(41). However, all the 5 M. abscessus subsp. abscessus isolates which were susceptible to clarithromycin had C28 in erm(41). This result also highlights the resistant mechanism above, which means that erm(41) may be of relevance to clarithromycin resistance. There is also 1 isolate which has C28 in erm(41) and still resistant to clarithromycin. Maybe there are some other resistant mechanisms needed to be analyzed. Although the therapeutic implications of erm(41) require further study, it is anticipated that expression of this gene does confer clinically significant resistance to clarithromycin. And still, the clinical impact of inducible resistance remains to be determined.

Conclusions
In this study, we find that MAC was the most common pathogen of NTM, and the second one was M. abscessus. However, M. chelonei, M. fuerth, and M. gordon were rare. Clarithromycin had a good inhibition activity against all the NTM species except M. abscessus subsp. abscessus. erm(41) genotype which is likely to be a major determinant of clarithromycin resistance of M. abscessus.