Absence of a Functional erm Gene in Isolates of Mycobacterium immunogenum and the Mycobacterium mucogenicum Group, Based on In Vitro Clarithromycin Susceptibility

Abstract

Macrolide resistance has been linked to the presence of a functional erythromycin ribosomal methylase (erm) gene in most species of pathogenic rapidly growing mycobacteria (RGM). For these Mycobacterium isolates, extended incubation in clarithromycin is necessary to determine macrolide susceptibility. In contrast, the absence of a detectable erm gene in isolates of M. chelonae, M. senegalense, and M. peregrinum and a nonfunctional erm gene in M. abscessus subsp. massiliense and 15% to 20% of M. abscessus subsp. abscessus isolates renders these species intrinsically macrolide susceptible. Not all RGM species have been screened for the presence of an erm gene, including the Mycobacterium mucogenicum group (M. mucogenicum, M. phocaicum, and M. aubagnense) and Mycobacterium immunogenum. A total of 356 isolates of these two pathogenic RGM taxa from two reference laboratories (A.R.U.P. Reference Laboratories and the Mycobacteria/Nocardia Laboratory at the University of Texas Health Science Center at Tyler) underwent clarithromycin susceptibility testing with readings at 3 to 5 days and 14 days. Only 13 of the 356 isolates had resistant clarithromycin MICs at initial extended MIC readings, and repeat values on all available isolates were ≤2 μg/ml. These studies suggest that these two additional RGM groups do not harbor functional erm genes and, like M. chelonae, do not require extended clarithromycin susceptibility testing. We propose to the Clinical Laboratory and Standards Institute that isolates belonging to these above-mentioned six rapidly growing mycobacterial groups based on molecular identification with no known functional erm genes undergo only 3 to 5 days of susceptibility testing (to exclude mutational resistance).

INTRODUCTION

The discovery of a family of erythromycin ribosomal methylase (erm) genes [erm(38), erm(39), erm(40), and erm(41)] that confer inducible macrolide resistance to rapidly growing mycobacteria (RGM) changed the Clinical and Laboratory Standards Institute (CLSI) recommendations for susceptibility testing of macrolides for RGM (1–4). Previously, susceptibility to clarithromycin (the class drug for macrolides and azalides) was reported only after 3 to 5 days of incubation. However, molecular studies have shown that inducible macrolide resistance in several of these genes is detected only following extended incubation of the isolates in the presence of clarithromycin (3). Because of this relatively slow inducible resistance, the interpretation of clarithromycin MICs with rapidly growing species that contain functional erm genes requires up to 14 days to reach a final MIC result.

Previous studies of the erm gene in RGM have shown the importance of detecting inducible macrolide resistance following extended incubation of Mycobacterium isolates in clarithromycin (3). Isolates of M. chelonae, M. peregrinum, and M. senegalense do not appear to contain an erm gene, while isolates of M. abscessus subsp. massiliense have a large deletion in their erm(41) gene that renders them nonfunctional (2–6). A recent study showed that extended incubation for macrolide susceptibility testing is not necessary if the isolate is confirmed by molecular testing to be M. chelonae (7). In contrast, the majority of the isolates confirmed as M. abscessus subsp. abscessus harbor a functional erm(41) gene (3, 8) and require extended incubation to ensure the detection of inducible macrolide resistance. Only about 20% of the isolates of M. abscessus subsp. abscessus in the United States have a single base pair substitution that results in a nonfunctional erm(41) gene, and untreated isolates remain macrolide susceptible with extended incubation (8).

Not all RGM groups or species have been evaluated for the presence of an erm gene. Two such taxa are the nonpigmented pathogens, M. immunogenum, and members of the M. mucogenicum group (M. mucogenicum, M. phocaicum, and M. aubagnense).

In this study, we present extensive macrolide susceptibility data for M. immunogenum and the M. mucogenicum group from two large reference laboratories. We also propose that the CLSI consider modification of the current recommendations for 14-day extended incubation for reporting clarithromycin susceptibility to a reading of 3 to 5 days in the current six RGM taxa not known to harbor a functional erm gene.

MATERIALS AND METHODS

Isolates.

This retrospective study was performed in two reference laboratories with experience in susceptibility testing of RGM. Between 2009 and 2014, 199 isolates of the M. mucogenicum group (including M. mucogenicum, M. phocaicum, and/or M. aubagnense) and 157 isolates of M. immunogenum were submitted to A.R.U.P. Reference Laboratories and the Mycobacteria/Nocardia Laboratory at the University of Texas Health Science Center at Tyler (UTHSCT). There were 79 isolates of M. immunogenum and 96 isolates of the M. mucogenicum group tested at the UTHSCT and 78 isolates of M. immunogenum and 103 isolates of the M. mucogenicum group tested at the A.R.U.P. Laboratories. The same isolates were not tested in both laboratories; each laboratory had separate personnel assigned to read the MICs. All 356 isolates were identified by molecular methods using region 5 rpoB gene sequencing (9) and/or partial 16S rRNA gene sequencing (10–12) or PCR restriction fragment length analysis (PRA) using the CLSI-recommended guidelines for the interpretation of sequence identity as previously described (10, 13–15). Institutional review board approvals for the use of data from human subjects were obtained by the UTHSCT and the A.R.U.P. Laboratory.

CST.

Clarithromycin susceptibility testing (CST) was performed using the CLSI-recommended method of broth microdilution using Rapmyco Sensititre MIC plates (Thermo Fisher, Cleveland, OH) (1). Clarithromycin MICs were read initially at 3 to 5 days of incubation in the UTHSCT Laboratory and again at 14 days or until the time resistance was first detected. The A.R.U.P. Laboratory protocol records only 3- to 5-day readings for isolates that are resistant at that time; thus, 3-day MICs were not available for comparison from that laboratory. Current MIC breakpoint recommendations for clarithromycin with RGM are ≤2 μg/ml (susceptible), 4 μg/ml (intermediate), and ≥8 μg/ml (resistant).

RESULTS

A total of 157 isolates of M. immunogenum were tested for extended (14-day) clarithromycin susceptibilities. Of these isolates, 142 (90%) had a 14-day clarithromycin MIC of ≤2 μg/ml. Only 7% of the isolates had an initial extended clarithromycin MIC of ≥8 μg/ml. Initially, four isolates had an extended clarithromycin MIC of 4 μg/ml, six isolates had an MIC of 8 μg/ml, and five isolates had an MIC of >16 μg/ml. Four isolates with a 14-day clarithromycin MIC of 8 μg/ml and four isolates with an MIC of >16 μg/ml had a repeat extended clarithromycin MIC of ≤2 μg/ml. Four isolates with a clarithromycin MIC of 4 μg/ml were retested, and 3 had an MIC of ≤2 μg/ml on repeat testing. The MIC of one isolate remained at 4 μg/ml, the current CLSI-recommended clarithromycin intermediate MIC breakpoint.

A total of 199 isolates of the M. mucogenicum group had extended clarithromycin MICs. Of these isolates, 96% had a clarithromycin MIC of ≤2 μg/ml. Less than 2% had an extended clarithromycin MIC of ≥8 μg/ml. Initially, four isolates had an extended clarithromycin MIC of 4 μg/ml; three of these isolates were available for repeat testing and showed a 14-day clarithromycin MIC of ≤2 μg/ml upon repeat testing. Three isolates had an extended clarithromycin MIC of ≥8 μg/ml. Only one isolate was available for retesting, and it showed an extended clarithromycin MIC of 2 μg/ml. Retest clarithromycin MICs are shown in Table 1.

TABLE 1.

Clarithromycin susceptibilities of 356 isolates of Mycobacterium immunogenum and the Mycobacterium mucogenicum group^a after initial (3- to 5-day) and 14-day incubations

Species	No. tested	No. (cumulative %) of isolates inhibited at each clarithromycin MIC according to indicated incubation time
		≤0.25 μg/ml		0.5 μg/ml		1 μg/ml		2 μg/ml		4 μg/ml	8 μg/mlb	≥16 μg/mlb
		3 daysc	14 days	3 days	14 days	3 days	14 days	3 days	14 days	14 days	14 days	14 days
M. immunogenumd	157	55 (70)	16 (10)	20 (90)	39 (35)	4 (100)	60 (73)		38 (97)	1 (98)	2 (99)	1 (100)
M. mucogenicum groupe	199	66 (69)	60 (30)	25 (95)	58 (59)	4 (99)	50 (84)	1 (100)	28 (98)	1 (99)	1 (99)	1 (100)

^aIncludes M. mucogenicum, M. phocaicum, and M. aubagnense.

^bThe 14-day MICs represent repeat testing values. Some isolates with an MIC of ≥8 μg/ml were not available for repeat testing. All other isolates with an MIC in this range had an MIC of ≤2 μg/ml on repeat testing.

^cThree- to 5-day readings were available from only one laboratory (UTHSCT).

^dSeventy-nine isolates of M. immunogenum were from UTHSCT; 78 isolates were from A.R.U.P.

^eNinety-six isolates of M. mucogenicum group were from UTHSCT; 103 isolates were from A.R.U.P.

For one laboratory, there was a 2.5 mean dilution difference (±1.5 standard deviation [SD]) and a 1.8 mean dilution difference (±1.0 SD) between the 3- to 5-day initial reading and the 14-day reading for isolates of M. immunogenum and the M. mucogenicum group, respectively (data not shown).

The range of initial (3-day) clarithromycin MICs for M. immunogenum was ≤0.06 to 1 μg/ml, and the range at 14 days was ≤0.12 to >16 μg/ml (MICs of ≥4 μg/ml were retested as previously noted). In comparison, the range of initial (3- to 5-day) MICs for the M. mucogenicum group was ≤0.06 to 1 μg/ml, and the 14-day range was ≤0.06 to >16 μg/ml.

DISCUSSION

For more than 20 years, macrolides have been the cornerstone of therapy for most infections caused by nontuberculous mycobacteria (NTM), including RGM (16, 17). Recent studies illustrating the significance of the presence of a functional erm gene in determining the macrolide susceptibility of isolates of the RGM have shed new light on the importance of in vitro susceptibility testing results among these species in correlation to the therapeutic response (3, 5, 6, 18). In vitro studies have confirmed that the presence of a functional erm gene confers inducible macrolide resistance, which is evidenced by increases in in vitro clarithromycin MIC values with extended incubation times (3).

M. abscessus subsp. massiliense has an erm(41) gene, but because of a large deletion, it is not functional; thus, isolates remain susceptible to clarithromycin throughout the extended 14-day incubation. Recently, two strains of M. abscessus subsp. massiliense with full-length functional erm genes were described. However, these genes were unusual in that they showed >1% mismatches compared to the erm(41) genes of the type strain of M. abscessus subsp. abscessus and/or M. abscessus subsp. bolletii. To our knowledge, these are the only examples to date of M. abscessus subsp. massiliense with a functional erm gene (19).

A recent study of 427 isolates of M. chelonae by Hanson and colleagues suggested that the elimination of 14-day clarithromycin susceptibility testing for M. chelonae may streamline workflow and perhaps also be more cost-effective for the clinical laboratory (7). Likewise, the current study suggests that 14-day clarithromycin susceptibility testing can also be eliminated for isolates molecularly identified as M. immunogenum and for those in the M. mucogenicum group.

Isolates of the M. mucogenicum group are sometimes resistant to often-used oral antimicrobials, including doxycycline, minocycline, and ciprofloxacin. Thus, clarithromycin is an attractive choice for oral medication for infections with this group.

Isolates of M. immunogenum often show intermediate-to-resistant MICs to quinolones (ciprofloxacin and moxifloxacin), doxycycline, minocycline, trimethoprim-sulfamethoxazole (TMP-SMX), and linezolid (15). Thus, the susceptibility of these species to clarithromycin often shows that clarithromycin is the only available oral option for therapy against these organisms.

The purpose of this study was to confirm that clinical isolates of M. mucogenicum and M. immunogenum consistently exhibit extended clarithromycin MICs of ≤4 μg/ml and that, as with M. chelonae, extending the clarithromycin MIC incubation of these two species to exclude inducible macrolide resistance is unnecessary.

Based on the results of the current study of 356 isolates (199 of the M. mucogenicum group and 157 isolates of M. immunogenum), we propose to the CLSI Antimicrobial Susceptibility Testing Subcommittee that isolates previously genotypically identified as belonging to the M. mucogenicum group and M. immunogenum do not require extended clarithromycin susceptibility testing. We also propose that the other four RGM species/taxa without a functional erm gene do not require extended clarithromycin susceptibility testing. Only those isolates of these species with a point mutation in the 23S rRNA gene (exhibiting resistance in the standard 3- to 5-day incubation period, as noted with other RGM species with the same mutation) (2, 4, 20) should be deemed macrolide resistant.

Less than 1% of isolates of M. immunogenum and the M. mucogenicum group exhibited an MIC in the current intermediate range (4 μg/ml) at 14 days. Most isolates of these taxa exhibited a rise in their MICs with prolonged incubation, presumably related to the instability of the drug in 30°C incubation. It seems likely that the few isolates with an MIC of 4 μg/ml after 14 days of incubation were not truly intermediate, as upon retesting all but one isolate had a lower MIC.

The elimination of extended clarithromycin susceptibility testing for isolates of the M. mucogenicum group and M. immunogenum along with the previously studied isolates of M. chelonae enables the clinician to select a treatment plan more rapidly and potentially helps to decrease the laboratory work load.