Standardized BACTEC Method To Measure Clarithromycin Susceptibility of Mycobacterium genavense

Abstract

A standardized clarithromycin susceptibility test for Mycobacterium genavense is reported. The BACTEC radiometric broth dilution test method recommended for Mycobacterium avium complex was modified to develop a reliable and reproducible procedure. Test development involved optimization of medium pH and inoculum densities for antibiotic vials as well as growth control vials. MIC control organisms included Mycobacterium simiae, Mycobacterium avium, and Mycobacterium xenopi. Growth control vials required two to three inoculum dilutions, which varied for each species. Clarithromycin MICs and MBCs for 12 isolates and 1 colonial variant of M. genavense ranged from ≤0.06 to 0.25 μg/ml.

With the emergence of fastidious Mycobacterium genavense as an opportunistic pathogen in patients with AIDS, there is a need for susceptibility testing of this organism against drugs such as clarithromycin which seem to have good clinical potency (1, 15). M. genavense is associated with fatal disseminated infections in patients with AIDS (2, 8, 21, 29). It has been cultured from a cervical lymph node of an otherwise healthy child (14) and detected by DNA amplification in colon biopsies of human immunodeficiency virus-negative patients with colonic cancer (4). This organism is believed to cause fatal infections in birds (9, 22) and has been isolated from cervical lymph nodes of a dog (13).

A number of AIDS patients infected with M. genavense have improved when treated with clarithromycin alone (1) or in multiple-drug regimens (1, 15, 18). Use of clarithromycin in multiple-drug therapy is now recommended for infections with M. genavense (1, 15, 27), but there is no guideline for susceptibility testing of this unusually fastidious mycobacterium, which grows best in acidic broth media. Multicenter studies have shown that radiometric broth methods can be standardized to yield reproducible MIC results for Mycobacterium avium complex strains tested against a variety of drugs (25), including clarithromycin (23). However, susceptibility tests of M. genavense against clarithromycin are confounded by the incompatibility of the organism’s marked preference for acidic media (26, 28) and the reduced activity of clarithromycin at a pH below 7.0 (24). Results from two reports of M. genavense clarithromycin susceptibility tests suggest that there are technical difficulties. In Middlebrook 7H9 broth medium (pH 6.6), two M. genavense isolates were inhibited by 2.5 to 5.0 μg of clarithromycin per ml (15), whereas a study with a BACTEC broth at pH 6.0 reported that the MIC for their two isolates was relatively low, 0.5 μg/ml (28). The present study was designed to provide a standardized and reproducible procedure for testing the clarithromycin susceptibility of M. genavense.

MATERIALS AND METHODS

Strains.

Clarithromycin MICs were determined for 13 M. genavense cultures from 12 patients. Strains included isolates from 10 patients in three Seattle hospitals, an isolate from Minnesota, and another from Ohio. The remaining Seattle isolate was a creamy colonial variant from a repeat blood culture of a patient with an initial dry colony type. Seven of these isolates have been described previously (3, 29). The Seattle isolates included strains ATCC 51233, with predominantly smooth, creamy colonies, and ATCC 51234, with typically flat, dry colonies. Mycobacterium simiae (ATCC 25275) was the resistant control. Initially M. avium (ATCC 25291) and later Mycobacterium xenopi (ATCC 19970) served as susceptible controls. This laboratory’s procedures for routine growth of M. genavense in liquid and on solid media have been described previously (3).

MIC tests.

Routine BACTEC 12B broth at pH 6.8 was converted to pH 7.2 by addition of 0.1 ml of 200 mM NaOH to each BACTEC vial. Fresh stock solutions of clarithromycin were prepared for each run by suspending the powder in sterile water and adding 1 N HCl dropwise until it dissolved. Drug dilutions were introduced to the vials by injection of 0.1 ml from tuberculin syringes. Every experiment included blank vials with and without pH adjustment that were read in parallel with test vials and had final pHs determined after 14 days.

Organisms were grown for 2 weeks in 7H9 broth before BACTEC vials were inoculated with 0.1 ml of standardized suspensions. Table 1 summarizes the inoculum cell densities for antibiotic vials and growth control vials of M. genavense, M. avium, M. xenopi, and M. simiae.

TABLE 1.

Inocula for testing M. genavense and control species

Species	McFarland standard for drug vial inoculuma	Dilution of standard inoculum for growth control vialsb
		Set 1	Set 2	Set 3
M. genavense	2.0	1:5	1:10	1:20
M. avium	0.5	1:50	1:100	1:200
M. xenopi	0.5	1:20	1:50	1:100
M. simiae	0.05	1:1,000	1:2,000	NDc

^aDrug vials were inoculated with 0.1 ml of standard inoculum. 

^bGrowth control vials were inoculated with 0.1 ml of the respective dilution. 

^cND, not done. 

MICs of clarithromycin in broth were measured with twofold dilutions that usually ranged from 0.06 to 8.0 μg/ml. Growth was recorded daily with the BACTEC 460TB system. MICs were tested with parallel sets of vials at pH 6.8 and 7.2 incubated at 35°C.

Interpretation of MICs.

Growth results were interpreted by a modification of the BACTEC-recommended method for testing Mycobacterium tuberculosis. MICs were interpreted when one or more growth control vials achieved a growth index (GI) of ≥50 between days 4 and 10. If more than one vial met this criterion, the MIC was determined relative to the most dilute growth control vial. The MIC was defined as the concentration at which the delta GI in the antibiotic vial was less than that of the control vial. Results were considered uninterpretable when the growth control vials grew too slowly (GI of ≥50 after day 10) or too rapidly (GI of ≥50 before day 4).

MBC tests.

Tests of MBCs were attempted for four of the M. genavense strains by subculturing 10-fold dilutions of broth from antibiotic-inhibited vials to Middlebrook 7H11 agar plates supplemented with 2 μg of mycobactin J (Remel, Lenexa, Kans.) per ml. Plates were incubated in zipper closure polyethylene bags at 35°C in 5% CO₂ for 16 weeks. MBC₉₉s were calculated as the minimal concentrations of drug which reduced the colony counts by 99% compared with those of untreated controls.

RESULTS

Clarithromycin MICs are shown in Table 2. On initial testing, the MIC for 9 of 13 M. genavense isolates was ≤0.06 μg/ml at both pHs 6.8 and 7.2. The results from the remaining four strains were as follows: MIC = 0.25 μg/ml at both pHs, MIC = 0.12 μg/ml at pH 6.8 and ≤0.06 at pH 7.2, and MIC = 0.12 μg/ml at pH 6.8 and there was insufficient growth at pH 7.2. One strain had uninterpretable results because of insufficient growth at both pHs.

TABLE 2.

Distribution of clarithromycin MICs

Species	MIC (μg/ml)	No. of tests with MIC at pH:
		6.8	7.2
M. genavense (n = 13 [+ 9 repeats])	≤0.06	17a	16a
	0.12	2
	0.25	1	1
	UIb	2	5
M. avium (ATCC 25291)	≤0.06	1
	0.12
	0.25
	>0.5	1	1
	UI	1	2
M. xenopi (ATCC 19970)	≤0.06	1	3
	0.12	2
M. simiae (ATCC 25275)	4.0	1	3
	8.0	4	3
	>8.0	1

^aIncludes results from strains ATCC 51233 and ATCC 51234. 

^bUI, uninterpretable, because no control reached a GI of 50 by day 10 or all control vials exceeded a GI of 50 before day 4. 

Tests of MIC reproducibility were done with seven strains that had adequate growth at pH 6.8 in their initial MIC tests. Six of seven repeat MICs at pH 6.8 were ≤0.06 μg/ml. The MICs for three strains were identical to their original values, while two MICs were 1 dilution lower, and one MIC was 2 dilutions lower. One repeat MIC test at pH 6.8 was uninterpretable. At pH 7.2, four of seven repeat MICs were ≤0.06 μg/ml. Two of the four repeat MICs confirmed the original MICs, whereas the original MIC results of the remaining two strains had been 0.25 μg/ml and an uninterpretable test. Three of the repeat MIC tests at pH 7.2 were not interpretable. In addition to these seven strains, the strain that initially had uninterpretable results at both pHs was retested twice: one result was uninterpretable at pH 7.2, and three MICs were ≤0.06 μg/ml. The MICs for the two colonial variants from a single patient were not consistently different.

The 1:20 growth control vial of M. genavense at pH 6.8 was used to interpret 17 of 22 MICs, the 1:10 vial was used once, and the 1:5 vial was used twice (data not shown). In two tests, the strains grew too poorly to interpret the MIC. At pH 7.2, the 1:20 growth control vial was acceptable for 7 of 22 MIC tests, the 1:10 vial was used for 8 tests, the 1:5 vial was used for 2 tests, and the remaining 5 tests were uninterpretable. The MICs for the two colonial variants represented by the ATCC strains were ≤0.06 μg/ml at both pHs, with the exception of one MIC, which was 0.12 μg/ml at pH 6.8.

Table 2 also summarizes the MIC results for the control strains. In three experiments, M. avium clarithromycin MICs at pH 6.8 were ≤0.06 μg/ml, >0.5 μg/ml, and uninterpretable. At pH 7.2, one MIC was >0.5 μg/ml and two were uninterpretable. In three tests of M. xenopi, the MICs at pH 6.8 were ≤0.06 or 0.12 μg/ml, while at pH 7.2, all MICs were ≤0.06 μg/ml. The MICs of the resistant control organism, M. simiae, ranged from 4.0 to >8.0 μg/ml at pH 6.8 and were 4.0 or 8.0 μg/ml at pH 7.2.

The MBC₉₉s for three strains of M. genavense at pH 6.8 were ≤0.06 to 0.25 μg/ml (Table 3). At pH 7.2, the MBC₉₉s were ≤0.06 and 0.12 μg/ml. MBC₉₉s could not be determined for one strain that failed to grow on viability plates.

TABLE 3.

Summary of clarithromycin MBC₉₉s^a

Strain	MIC or MBC99 (μg/ml) at:
	pH 6.8		pH 7.2
	MIC	MBC99	MIC	MBC99
M. genavense
1924	UIb	≤0.06	UI	≤0.06
2635	≤0.06	0.25	≤0.06	0.12
2682	≤0.06	UCc	≤0.06	UC
2709	≤0.06	0.12	UI	≤0.06
M. xenopi ATCC 19970	≤0.06	>1.0	≤0.06	>1.0
M. simiae ATCC 25275	4.0	>8.0	8.0	8.0

^aBased on 99% reduction in viability. 

^bUI, data uninterpretable, because control vials reached a GI of 50 after day 10 or before day 4. 

^cUC, unable to calculate, because the inoculum viability cultures failed to grow. 

After 14 days of daily reading in the BACTEC 460, the average pH values of blank vials initially at pH 6.8 and 7.2 were 6.8 (range, 6.46 to 6.97) and 7.1 (range, 7.02 to 7.25), respectively.

DISCUSSION

Although routine susceptibility testing of slow-growing mycobacteria other than M. tuberculosis is not recommended (10, 20), clinicians caring for our AIDS patients with M. genavense sepsis requested susceptibility data on their patients’ isolates. Since most patients infected with M. genavense experience clinical improvement (1, 15, 27) and clearance of bacteremia with clarithromycin therapy (1), it could be argued that susceptibility testing of this organism with clarithromycin is not necessary. Furthermore, the levels of clarithromycin in macrophages (17), neutrophils (11), and lung tissue (5) far exceed the extracellular levels or the peak levels in serum of 4 μg/ml. However, clarithromycin resistance due to spontaneous mutations can be expected to occur in M. genavense as it does in M. avium complex and in the Mycobacterium chelonae group when patients receive monotherapy with clarithromycin over an extended period. Treatment failures of AIDS patients receiving clarithromycin for M. avium sepsis have been shown to be due to point mutations in the 23S rRNA gene (16, 19). The mutation rate to clarithromycin resistance in M. avium is 1 × 10⁻⁸ to 1 × 10⁻⁹ (12), which suggests that patients with heavy loads of M. genavense may also harbor clarithromycin-resistant mutants.

The results from the 13 isolates of M. genavense in this study are likely to be representative of the diversity within clinical isolates of this species, since the literature to date describes no more than 110 isolates from humans. To the best of our knowledge, there is no published information on the genetic diversity within the species M. genavense. The challenge with this dysgonic species is getting sufficient cell mass for adequate DNA yields.

Interpretation of clarithromycin MICs for mycobacteria cannot be based on the fact that the usual peak level in serum is 4.0 μg/ml, because clarithromycin concentrations in macrophages are 16-fold higher than those in the extracellular fluid (17). In the absence of resistant isolates or clinical trials with clarithromycin treatment for M. genavense infections, tentative interpretations of clarithromycin MICs for this species probably can be extrapolated from the recommendations for M. avium complex by Heifets, who suggested a pH 7.4 broth-determined MIC of 2.0 μg/ml as the susceptible breakpoint and a MIC of >32.0 μg/ml as the resistant breakpoint (7). For convenience, Heifets recommended a MIC of >32.0 μg/ml as the resistant breakpoint rather than >256 μg/ml, which was the MIC for all M. avium isolates obtained from patients who relapsed during treatment. Since Heifets found clarithromycin MICs at pH 6.8 were two- to eightfold higher than those at pH 7.4, a conservative estimate of the susceptible breakpoint at pH 6.8 would be 4 μg/ml. We did not attempt to determine the exact clarithromycin MICs of the M. genavense isolates for which the MICs were ≤0.06 μg/ml, because the additional expense would not be clinically justified.

When there is clinical cause to do susceptibility testing of slow-growing mycobacteria, most authorities recommend that tests be done with the BACTEC radiometric system, which includes 12B broth at pH 6.8 (6, 10, 26). When clarithromycin became available as an antimycobacterial drug, Rastagi and colleagues studied the effect of pH on many mycobacterial species by measuring MICs in BACTEC vials at pHs 6.0, 6.8, and 7.4. Their studies confirmed the fact that clarithromycin behaves like all other macrolides, which are much less active in acid than alkaline solutions (24).

We have found that the optimal growth of M. genavense tested in the BACTEC system occurs in 12B broth adjusted to pH 5.4 (2a). For the present study, we measured clarithromycin MICs at pH 6.8, the standard for mycobacterial broth, and at pH 7.2, which allowed good clarithromycin activity as well as some growth of M. genavense. Although pH 6.8 is suboptimal for clarithromycin activity, the satisfactory growth of M. genavense and the relatively reproducible MICs at this pH indicate that testing at pH 6.8 is preferable to that at pH 7.2. The results from pH 6.8 instead of 7.2 are conservative estimates of this species’ susceptibility to clarithromycin. At pH 7.2, we could not distinguish enhanced activity of the drug from failure of M. genavense to thrive. In addition, the enhanced drug activity at pH 7.2 would not reflect the situation in the more acidic intracellular milieu.

In preliminary studies, we evaluated both sodium hydroxide and sodium bicarbonate for adjusting the pH of BACTEC 12B medium to 7.0 and 7.2. Siddiqi suggested bicarbonate for adjusting the pH of BACTEC vials to pH 7.2, because it provided a more stable pH after multiple readings on the BACTEC 460 instrument, which injects CO₂ into the vial headspace (24a). In our experience, both sodium bicarbonate and sodium hydroxide allowed a 0.1-pH-unit shift after 14 days of testing in the BACTEC 460. We used sodium hydroxide in the present experiments, because the slight acidic shift was less inhibitory for the growth of M. genavense and M. avium than the slight alkaline shift with sodium bicarbonate. The medium at pH 7.0 was discontinued when preliminary experiments showed M. genavense growth to be essentially identical to that at pH 7.2.

M. avium ATCC 25291 was not a suitable control strain for our studies, because it exhibited surprisingly erratic growth, which prompted a search for a better clarithromycin-susceptible control organism. The poor reproducibility of the M. avium MICs may have reflected its moderate susceptibility to this drug, its marked preference for more acidic growth media, and the loss of clarithromycin activity at pHs below 7.4. In the search for an alternative susceptible control, Mycobacterium szulgai, M. kansasii, and M. xenopi were compared, and M. xenopi was found to have the most uniform growth rates in control vials and more reproducible MICs. The MICs for the M. xenopi control and M. simiae, the resistant control, varied by no more than 1 dilution from their respective modal values.

In one experiment, false resistance in four isolates (data not shown) was attributed to a clarithromycin stock solution that had been refrigerated for 1 week and perhaps had been degraded by the HCl initially used to dissolve the drug. We recommend using freshly prepared clarithromycin solutions.

BACTEC MICs in broth can be determined for M. genavense in a timely fashion (4 to 10 days), but the poor reproducibility of the growth control vials requires routine use of growth control vials with three different inocula. In our experience, the same inoculum could reach a GI of 50 before day 4 or after day 10. With multiple growth control vials, the number of uninterpretable tests decreased dramatically. Graphing the growth indices of the antibiotic vials in comparison to those of the control vials was attempted as an alternate method of MIC interpretation, but technologists accustomed to the BACTEC susceptibility procedure for M. tuberculosis found the calculation of MICs based on delta GIs to be less time-consuming and more straightforward.

Agar MIC determinations were not attempted with M. genavense, because this species frequently fails to grow on appropriately supplemented agar media even after prolonged incubation. Growth on agar media is particularly unpredictable when M. genavense is subcultured from broth media. Only two of our four attempted MBC tests were interpretable at 10 weeks and remained unchanged through week 16. However, one strain produced increasing colony counts between weeks 10 and 15, and the fourth strain yielded no colonies.

The present study indicates that the initial isolates of M. genavense are likely to be susceptible to clarithromycin. However, based on experience with other mycobacteria, we believe that any patient infected with M. genavense who does not respond or who relapses while on clarithromycin therapy should have the benefit of a MIC test of their pre- and posttherapy isolates. With the modifications developed in this study, a standardized BACTEC broth susceptibility method appears to be acceptable for testing M. genavense.