A new selective medium for rapidly growing mycobacteria (RGM medium) was evaluated on respiratory specimens from non-cystic fibrosis patients and compared to the mycobacterial growth indicator tube (MGIT) system and Middlebrook 7H11 agar for the isolation of all nontuberculous mycobacteria (NTM). A total of 203 mucolyzed respiratory specimens collected from 163 patients were inoculated on RGM medium and incubated at both 30°C (RGM30) and 35°C (RGM35) over a 28-day period.
KEYWORDS: MGIT, Middlebrook, Mycobacterium, RGM, nontuberculous mycobacteria, respiratory culture, selective medium
ABSTRACT
A new selective medium for rapidly growing mycobacteria (RGM medium) was evaluated on respiratory specimens from non-cystic fibrosis patients and compared to the mycobacterial growth indicator tube (MGIT) system and Middlebrook 7H11 agar for the isolation of all nontuberculous mycobacteria (NTM). A total of 203 mucolyzed respiratory specimens collected from 163 patients were inoculated on RGM medium and incubated at both 30°C (RGM30) and 35°C (RGM35) over a 28-day period. N-Acetyl-l-cysteine–sodium hydroxide (NALC-NaOH)-decontaminated specimens were inoculated into MGIT and Middlebrook 7H11 agar and incubated at 35°C for 42 days. NTM were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) or gene sequencing. A total of 133 NTM isolates were recovered overall from 101 (49.8%) specimens collected from 85 (52.1%) patients by a combination of all culture methods. The sensitivity of RGM30 for the recovery of NTM was significantly higher than that of either the MGIT system (76.7% versus 59.4%; P = 0.01) or Middlebrook 7H11 agar (76.7% versus 47.4%; P = 0.0001) alone, but it was not significantly different from that of an acid-fast bacillus culture (AFC) which includes both MGIT and Middlebrook 7H11 agar (76.7% versus 63.9%; P = 0.0647). RGM35 had significantly lower sensitivity than the MGIT system (49.6% versus 59.4%; P = 0.0367) and AFC (49.6% versus 63.9%; P = 0.0023). RGM medium was highly effective at inhibiting the growth of nonmycobacterial organisms in the respiratory specimens, with breakthrough contamination rates of 5.4% and 4.4% for RGM30 and RGM35, respectively.
INTRODUCTION
Nontuberculous mycobacteria (NTM) are significant pulmonary pathogens in patients with cystic fibrosis (CF), as well as those with structural lung conditions such as bronchiectasis and chronic obstructive pulmonary disease (COPD) (1–4). Non-CF bronchiectasis may be caused by severe respiratory infections from bacterial pneumonia or tuberculosis, impaired ciliary clearance, immunodeficiency, and allergic bronchopulmonary aspergillosis (5). Rapidly and slowly growing NTM are commonly isolated from patients with non-CF bronchiectasis, with Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABSC) as the most frequently isolated organisms (6–8).
The isolation of NTM from respiratory specimens from CF and non-CF bronchiectasis patients is laborious due to potential overgrowth of coexisting non-acid-fast bacillus (non-AFB) bacteria such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus (2, 3) and fungi such as Aspergillus fumigatus (9). Therefore, a decontamination step should be performed for respiratory specimens by using, for example, sodium hydroxide (NaOH) prior to plating on culture media to reduce overgrowth of nonmycobacterial organisms. However, NTM, especially rapidly growing mycobacteria (RGM), are particularly susceptible to such decontamination procedures, leading to suboptimal recovery on culture (10). Standard procedures for NTM culture from clinical specimens include both solid and liquid media for optimal recovery and enhancement of mycobacterial growth (11). The liquid medium provides a higher yield of mycobacterial isolation and shorter time to detection, but the solid medium allows for direct observation of mycobacterial colony morphology, growth rates, recognition of mixed mycobacterial colonies, and quantitation of organisms (10). Our current protocol of mycobacterial culture of respiratory specimens (acid-fast bacillus culture [AFC]) at the Microbiology service, Clinical Center, National Institutes of Health, uses both Middlebrook 7H11 agar and mycobacterial growth indicator tube (MGIT) following N-acetyl-l-cysteine–sodium hydroxide (NALC-NaOH) digestion/decontamination procedures. However, we have experienced relatively high contamination rates of nonmycobacterial organisms in both culture methods that reduce and/or delay the recovery of NTM from clinical specimens. In addition, NTM viability may be affected by the decontamination procedures.
Recently, a new selective medium (RGM medium), which is composed of Middlebrook 7H9-based agar supplemented with oleic acid-albumin-dextrose-catalase (OADC) and four antimicrobial agents (12, 13), has been shown to have high performance for the recovery of rapidly growing mycobacteria from respiratory specimens from CF patients (12–15). RGM medium was superior to Burkholderia cepacia complex selective medium and showed performance equivalent to or higher than that of the MGIT system. An added advantage was the ability of RGM medium to inhibit growth of other nonmycobacterial organisms in CF patients’ respiratory specimens without requirement for a decontamination step (12–15). The use of an extended incubation period of 28 days allows RGM medium to be used for the isolation of all NTM, rather than simply rapidly growing species, but there are only limited data on the recovery of slowly growing species (15). Furthermore, while several studies have evaluated the performance of RGM medium in the setting of CF, RGM medium has yet to be studied in the non-CF patient population with underlying lung conditions.
This study assessed the performance of RGM medium for the isolation of rapidly and slowly growing NTM from respiratory specimens from non-CF patients with bronchiectasis, COPD and other lung conditions seen at the Clinical Center, National Institutes of Health. The performance of RGM medium was evaluated by a comparison to the conventional AFC method using the MGIT system and Middlebrook 7H11 agar.
MATERIALS AND METHODS
A total of 203 respiratory specimens were obtained between October 2017 and March 2018 from 163 non-CF patients with underlying lung conditions such as bronchiectasis. The patients, ranging in age from 10 to 91 years, were enrolled in Institutional Review Board-approved protocols at the National Institutes of Health. The respiratory specimens comprised 160 sputa/tracheal aspirates and 43 bronchoalveolar lavage (BAL) fluids which were submitted for routine AFC without additional specimens requested for the purposes of this study. The RGM medium was prepared and kindly provided by the Microbiology Department, Freeman Hospital, Newcastle upon Tyne, UK, as previously described (12, 13). RGM plates may be obtained from this source until they are commercially available.
For RGM medium, a 1-ml aliquot of sputum and 5 ml of mucoid BAL specimens were digested with 1 mg/ml mucolytic agent (Mucolyse sputum digestant; Prolab Diagnostics, Round Rock, TX) at a ratio of 1:1. The specimens were vortex mixed until homogeneous and then incubated for 15 min at room temperature. A 5-ml aliquot of nonmucoid BAL fluid or 10 ml of mucolyzed BAL fluid was centrifuged at 2,800 × g for 10 min, and then pellets were resuspended in 2 ml of supernatant and vortex mixed. A volume of 100 μl of mucolyzed specimen or resuspended pellet was plated on RGM medium and spread to obtain isolated colonies. Inoculated RGM plates were incubated for 28 days at 30°C air (RGM30) or at 35°C with 8% CO2 (RGM35) and examined for growth after 4, 7, 10, 14, 21, and 28 days of incubation.
For the AFC method, a 3- to 5-ml aliquot of respiratory specimen was processed by digestion/decontamination procedures using 0.5% NALC–4% NaOH (NAC-PAC; AlphaTec, Vancouver, WA), followed by a neutralization step with phosphate buffer (pH 6.8) according to the manufacturer’s instructions. The neutralized specimens were then centrifuged for 15 min at 3,000 × g in a refrigerated centrifuge (4°C). After discarding the supernatant, a smear was prepared from the concentrated pellet to evaluate for the presence of AFB with the auramine rhodamine (AR) stain. The pellets were resuspended in 0.8 ml of phosphate buffer, and culture media were inoculated as follows: 0.3 ml on Middlebrook 7H11 agar (Remel/Thermo Fisher Scientific, Lenexa, KS) and 0.5 ml into a MGIT (Becton Dickinson, Franklin Lakes, NJ). The MGIT was supplemented with 0.8 ml of BBL MGIT PANTA according to the manufacturer’s procedures. The inoculated plates were placed at 35°C with 8% CO2 and observed 21 and 42 days after incubation or at the time the culture was determined positive in liquid medium. MGITs were loaded onto the Bactec MGIT 960 instrument and incubated for 42 days or until determined to be positive by the instrument.
All colonies that grew on RGM medium and Middlebrook 7H11 agar were confirmed as AFB by Kinyoun stain and subcultured onto Middlebrook 7H11 agar incubated at 35°C with 8% CO2 or at 30°C air (depending on the temperature of the primary isolation). Colonies with negative Kinyoun staining were subjected to Gram staining. MGITs that flagged positive were examined for initial detection of contaminating organisms and AFB using Gram stain and AR stain, respectively. AFB-positive MGITs were subcultured onto Middlebrook 7H11 agar and incubated at 35°C until colonies were visible for isolation and further identification.
Identification of mycobacteria and other microorganisms.
NTM were primarily identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS; Microflex; Bruker Daltonics, Inc., Billerica, MA). Proteins were extracted from mycobacterial colonies using a rapid extraction method developed at the Microbiology Service, Clinical Center, National Institutes of Health, based on a previous protocol (16). Briefly, mycobacterial proteins were extracted by 200 μl of 70% formic acid and 100% acetonitrile (1:1), along with 50 μl of silica beads in 1.5-ml screw-cap tubes using a PowerLyzer 24 high-power homogenizer (Mo Bio Laboratories, Inc., Qiagen, Germantown, MD) for two cycles of 45 s, with a 30-s rest interval between the cycles. A 1-μl volume of extract was spotted onto a target plate for MALDI-TOF MS analysis according to the manufacturer’s instructions using the MALDI-TOF MS parameters described previously (16). Nonmycobacterial organisms recovered on RGM medium were subcultured on Trypticase soy agar with 5% sheep blood (Remel/Thermo Fisher Scientific) and identified by MALDI-TOF MS as described previously (17).
Further identification by gene sequencing targeting the secA gene and/or 16S rRNA was performed for mycobacterial isolates with scores below 1.8 log after twice testing by MALDI-TOF MS. Briefly, primers complementary to secA gene (18) and/or 16S rRNA (MicroSEQ 1500 Applied Biosystems, Foster, CA) were used to amplify DNA with a BigDye Terminator X kit (Life Technologies, Tarrytown, NY). Sequencing was performed on an ABI Prism 3130 genetic analyzer (Applied Biosystems). The resulting sequences were analyzed for quality and compared against the GenBank database through nucleotide BLAST queries.
Statistical analysis.
The statistical significance of the difference in two culture methods was compared by using McNemar’s test with the continuity correction applied. Statistical significance was assigned when the P value was ≤0.05.
RESULTS
Recovery of NTM from respiratory specimens by different culture methods.
A total of 203 respiratory specimens were collected from 163 non-CF patients seen at the Clinical Center, National Institutes of Health. The samples included 43 (21.2%) BAL fluids and 160 (78.8%) sputa/tracheal aspirates. NTM were isolated from 101 of 203 (49.8%) specimens from 85 patients by using a combination of all methods: RGM30 (day 28), RGM35 (day 28), the MGIT system, and Middlebrook 7H11 agar. The prevalence of NTM among this patient population was 52.1% (85/163). The 101 specimens that yielded NTM included 95 (94.1%) sputa/tracheal aspirates and 6 BAL fluids (5.9%). A total of 53 (26.1%) specimens were AFB smear positive using the AR stain (Table 1). RGM30 and RGM35 recovered NTM from 35 (23.3%) and 15 (10%) AR-negative specimens, respectively (P = 0.0002). RGM30 significantly differed from Middlebrook 7H11 agar (35 versus 11, P = 0.0001) but was not significantly different from the MGIT system (35 versus 25, P = 0.1003) for the NTM recovery from AR-negative specimens. The percent recoveries of NTM from AR-positive specimens were 94.3% (50/53), 90.6% (48/53), 98.1% (52/53), and 94.3% (50/53) for the RGM30, RGM35, MGIT, and Middlebrook 7H11 methods, respectively, showing no significant difference (P > 0.05) (Table 1).
TABLE 1.
AFB smear and culture results for 203 clinical samplesa
| AR results | NTM isolation | No. of specimens |
|||||
|---|---|---|---|---|---|---|---|
| Totalb | RGM30 | RGM35 | MGIT | 7H11 | AFC | ||
| Negative | NTM isolated | 48 | 35 | 15 | 25 | 11 | 27 |
| Negative | 102 | 115 | 135 | 125 | 139 | 123 | |
| Total | 150 | 150 | 150 | 150 | 150 | 150 | |
| Positive | NTM isolated | 53 | 50 | 48 | 52 | 50 | 52 |
| Negative | 0 | 3 | 5 | 1 | 3 | 1 | |
| Total | 53 | 53 | 53 | 53 | 53 | 53 | |
| Grand total | 203 | 203 | 203 | 203 | 203 | 203 | |
AR, auramine rhodamine stain; MGIT, mycobacterial growth indicator tube; 7H11, Middlebrook 7H11 agar. RGM30 and RGM35 are RGM media incubated at 30°C and 35°C, respectively. AFC is a culture method using the MGIT system and Middlebrook 7H11 agar.
The total number of specimens refers to all specimens from which NTM were isolated by a combination of all culture test methods (RGM30, RGM35, the MGIT system, and Middlebrook 7H11 agar).
From 101 respiratory specimens yielding NTM, a total of 133 NTM were isolated by using a combination of all methods. The overall sensitivities of RGM30 and RGM35 for the recovery of all NTM were 76.7% (102/133) and 49.6% (66/133), respectively (P = 0.0001) (Table 2; see also Fig. S1 in the supplemental material). The sensitivity of RGM30 was significantly higher than that of the MGIT method (76.7% versus 59.4%; P = 0.01) and Middlebrook 7H11 agar (76.7% versus 47.4%; P = 0.0001) alone but it was not significantly different from that of the AFC method (76.7% versus 63.9%; P = 0.0647). The sensitivity of RGM35 was significantly lower than that of the MGIT system (49.6% versus 59.4%; P = 0.0367) and AFC method (49.6% versus 63.9%; P = 0.0023) but was not significantly different from that of Middlebrook 7H11 agar (49.6% versus 47.4%; P = 0.6892) (Table 2). RGM medium could recover and differentiate rough and smooth colony variants of NTM such as MABSC (Fig. S2).
TABLE 2.
Numbers of nontuberculous mycobacteria isolated and percent sensitivities of different culture test methods for recovery of nontuberculous mycobacteria
| Organism | Total no. of mycobacterial isolatesb | No. of mycobacterial isolates (% sensitivity)a |
|||||
|---|---|---|---|---|---|---|---|
| RGM30 | RGM35 | MGIT | 7H11 | AFC | MGIT+RGM30 | ||
| Rapidly growing mycobacteria | |||||||
| M. abscessus complex | 28 | 27 (96.4) | 27 (96.4) | 25 (89.3) | 24 (85.7) | 25 (89.3) | 28 (100) |
| M. chelonae | 5 | 5 (100) | 0 | 0 | 0 | 0 | 5 (100) |
| M. fortuitum group | 6 | 4 (66.7) | 3 (50) | 2 (33.3) | 2 (33.3) | 3 (50) | 4 (66.7) |
| M. immunogenum | 1 | 1 (100) | 0 | 0 | 0 | 0 | 1 (100) |
| M. llatzerense | 1 | 1 (100) | 0 | 0 | 0 | 0 | 1 (100) |
| M. mucogenicum group | 16 | 16 (100) | 1 (6.3) | 0 | 1 (6.3) | 1 (6.3) | 16 (100) |
| Total | 57 | 54 (94.7) | 31 (54.4) | 27 (47.4) | 27 (47.4) | 29 (50.9) | 55 (96.5) |
| Slowly growing mycobacteria | |||||||
| M. avium complex | 48 | 29 (60.4) | 32 (66.7) | 42 (87.5) | 34 (70.8) | 45 (93.8) | 45 (93.8) |
| M. avium | 24 | 13 (54.2) | 17 (70.8) | 21 (87.5) | 16 (66.7) | 23 (95.8) | 22 (91.7) |
| M. colombiense | 1 | 1 (100) | 1 (100) | 1 (100) | 1 (100) | 1 (100) | 1 (100) |
| M. intracellulare/M. chimaera | 22 | 15 (68.2) | 14 (63.6) | 19 (86.4) | 16 (72.7) | 20 (90.9) | 21 (95.5) |
| M. yongonense | 1 | 0 | 0 | 1 (100) | 1 (100) | 1 (100) | 1 (100) |
| M. gordonae | 14 | 10 (71.4) | 0 | 5 (35.7) | 0 | 5 (35.7) | 14 (100) |
| M. lentiflavum | 1 | 0 | 1 (100) | 1 (100) | 0 | 1 (100) | 1 (100) |
| M. paragordonae | 7 | 4 (57.1) | 0 | 3 (42.9) | 0 | 3 (42.9) | 7 (100) |
| M. paraffinicum | 1 | 0 | 0 | 0 | 1 (100) | 1 (100) | 0 |
| M. simiae | 1 | 1 (100) | 1 (100) | 1 (100) | 1 (100) | 1 (100) | 1 (100) |
| Mycobacterium sp. | 4 | 4 (100) | 1 (25) | 0 | 0 | 0 | 4 (100) |
| Total | 76 | 48 (63.2) | 35 (46.1) | 52 (68.4) | 36 (47.4) | 56 (73.7) | 72 (94.7) |
| Grand total | 133 | 102 (76.7) | 66 (49.6) | 79 (59.4) | 63 (47.4) | 85 (63.9) | 127 (95.5) |
MGIT, mycobacterial growth indicator tube; 7H11, Middlebrook 7H11 agar. RGM30 and RGM35 are RGM media incubated at 30°C and 35°C, respectively. AFC is a culture method using the MGIT system and Middlebrook 7H11 agar.
That is, the total number of nontuberculous mycobacteria isolated from a combination of all culture test methods (RGM30, RGM35, the MGIT system, and Middlebrook 7H11 agar).
For rapidly growing mycobacteria, the sensitivity of RGM30 (94.7%) was significantly higher than for RGM35 (54.4%), the MGIT method (47.4%), Middlebrook 7H11 agar (47.4%), and AFC (50.9%) (P = 0.0001) (Table 2). MABSC predominated among the rapidly growing mycobacteria isolated by a combination of all culture methods (49.1% [28/57]). RGM30 or RGM35 yielded 27 isolates of MABSC, which was higher than, although not significantly different from, results obtained by the MGIT method (96.4% versus 89.3%; P = 0.6171), Middlebrook 7H11 agar (96.4% versus 85.7%; P = 0.3711), and the AFC method (96.4% versus 89.3%; P = 0.6171). RGM30 recovered rapidly growing mycobacteria which were not recovered by other methods such as M. chelonae, M. immunogenum, and M. llatzerense. Moreover, RGM30 recovered more M. mucogenicum group and M. fortuitum group than other methods (Table 2 and Fig. S1).
For the recovery of slowly growing mycobacteria, the sensitivity of RGM30 was not significantly different from that of the MGIT method (63.2% versus 68.4%; P = 0.6511), Middlebrook 7H11 agar (63.2% versus 47.4%; P = 0.0592), or the AFC method (63.2% versus 73.7%; P = 0.3020) but was significantly different from that of RGM35 (63.2% versus 46.1%; P = 0.0209). MAC, including M. avium, M. colombiense, M. intracellulare/M. chimaera, and M. yongonense, constituted the majority of the slowly growing mycobacteria (63.2% [48/76]) isolated by a combination of all culture methods. RGM30 and RGM35 recovered 29 (60.4%) and 32 (66.7%) MAC isolates, respectively (P = 0.5050). However, the sensitivity of RGM30 was significantly lower than that of the MGIT method (60.4% versus 87.5%; P = 0.0059) and AFC method (60.4% versus 93.8%; P = 0.0008) but not significantly lower than for the Middlebrook 7H11 agar (60.4% versus 70.8%; P = 0.3017) for the recovery of MAC. Similarly, the sensitivity of RGM35 for the recovery of MAC was significantly lower than that of the MGIT method (66.7% versus 87.5%; P = 0.0244) and the AFC method (66.7% versus 93.8%; P = 0.0036) but not significantly lower than for the Middlebrook 7H11 agar (66.7% versus 70.8%; P = 0.7893). In addition, M. gordonae, M. paragordonae, M. simiae, and other Mycobacterium spp. whose species could not be identified by either MALDI-TOF MS or secA and/or 16S rRNA sequencing were isolated from RGM30. RGM35 yielded M. lentiflavum, M. simiae, and Mycobacterium sp. (Table 2 and Fig. S1).
An improved recovery of NTM was observed from a combination of RGM30 and MGIT methods (MGIT+RGM30). The overall sensitivity increased from 76.7% by using RGM30 alone to 95.5% by a combination with the MGIT method (P = 0.0001). In addition, the sensitivity of a combination of RGM30 and the MGIT method was significantly higher than that of our current AFC method using a combination of the MGIT system and Middlebrook 7H11 agar (95.5% versus 63.9%; P = 0.0001) for the isolation of NTM. An increase of sensitivity to 100% was observed for the recovery of MABSC, M. gordonae, M. lentiflavum, and M. paragordonae. However, the sensitivity for recovery of MAC by this combination method could not reach 100% (93.8%) because two isolates of M. avium were only isolated by RGM35 and Middlebrook 7H11 agar and one isolate of M. intracellulare/M. chimaera grew only on Middlebrook 7H11 agar (Table 2 and Fig. S1).
Cumulative recovery of NTM on RGM medium.
RGM plates were incubated at a temperature of 30°C in order to enhance the recovery of mycobacteria preferring low temperature to grow. In addition, the incubation period was extended to 28 days for the recovery of slowly growing mycobacteria. The extension of incubation period from 10 to 28 days significantly increased the NTM recovery rate from 57.1% (day 10) to 76.7% (day 28) by RGM30 (P = 0.0001) and from 42.9% (day 10) to 49.6% (day 28) by RGM35 (P = 0.0077) (Table 3 and Fig. S3). However, extended incubation of RGM medium beyond 28 days (to 42 days) did not yield additional NTM (data not shown). For rapidly growing mycobacteria, the recovery rate was increased from 84.2% (day 10) to 94.7% (day 28) by RGM30 (P = 0.0412) and from 50.9% (day 10) to 54.4% (day 28) by RGM35 (P = 0.4795). The large majority of MABSC (92.9% [26/28]) was recovered on both RGM30 and RGM35 by day 4 of incubation; only one strain was detected by day 7 (Table 3 and Fig. S3).
TABLE 3.
Cumulative number and percent recovery of nontuberculous mycobacteria isolated from RGM media incubated at 30°C (RGM30) and 35°C (RGM35)
| Organism | Total no. of mycobacterial isolatesa | No. of mycobacterial isolates (% recovery) |
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| RGM30 |
RGM35 |
||||||||||||
| D4 | D7 | D10 | D14 | D21 | D28 | D4 | D7 | D10 | D14 | D21 | D28 | ||
| Rapidly growing mycobacteria | |||||||||||||
| M. abscessus complex | 28 | 26 (92.9) | 27 (96.4) | 27 (96.4) | 27 (96.4) | 27 (96.4) | 27 (96.4) | 26 (92.9) | 27 (96.4) | 27 (96.4) | 27 (96.4) | 27 (96.4) | 27 (96.4) |
| M. chelonae | 5 | 3 (60) | 3 (60) | 4 (80) | 5 (100) | 5 (100) | 5 (100) | 0 | 0 | 0 | 0 | 0 | 0 |
| M. fortuitum group | 6 | 1 (16.7) | 3 (50) | 3 (50) | 3 (50) | 4 (66.7) | 4 (66.7) | 0 | 1 (16.7) | 1 (16.7) | 1 (16.7) | 3 (50) | 3 (50) |
| M. immunogenum | 1 | 0 | 0 | 1 (100) | 1 (100) | 1 (100) | 1 (100) | 0 | 0 | 0 | 0 | 0 | 0 |
| M. llatzerense | 1 | 0 | 1 (100) | 1 (100) | 1 (100) | 1 (100) | 1 (100) | 0 | 0 | 0 | 0 | 0 | 0 |
| M. mucogenicum group | 16 | 4 (25) | 9 (56.3) | 12 (75) | 15 (93.8) | 15 (93.8) | 16 (100) | 0 | 0 | 1 (6.3) | 1 (6.3) | 1 (6.3) | 1 (6.3) |
| Total | 57 | 34 (59.6) | 43 (75.4) | 48 (84.2) | 52 (91.2) | 53 (93) | 54 (94.7) | 26 (45.6) | 28 (49.1) | 29 (50.9) | 29 (50.9) | 31 (54.4) | 31 (54.4) |
| Slowly growing mycobacteria | |||||||||||||
| M. avium complex | 48 | 4 (8.3) | 17 (35.4) | 24 (50) | 26 (54.2) | 28 (58.3) | 29 (60.4) | 6 (12.5) | 17 (35.4) | 27 (56.3) | 30 (62.5) | 30 (62.5) | 32 (66.7) |
| M. avium | 24 | 1 (4.2) | 5 (20.8) | 10 (41.7) | 10 (41.7) | 12 (50) | 13 (54.2) | 2 (8.3) | 6 (25) | 12 (50) | 15 (62.5) | 15 (62.5) | 17 (70.8) |
| M. colombiense | 1 | 0 | 1 (100) | 1 (100) | 1 (100) | 1 (100) | 1 (100) | 0 | 1 (100) | 1 (100) | 1 (100) | 1 (100) | 1 (100) |
| M. intracellulare/M. chimaera | 22 | 3 (13.6) | 11 (50) | 13 (59.1) | 15 (68.2) | 15 (68.2) | 15 (68.2) | 4 (18.2) | 10 (45.5) | 14 (63.6) | 14 (63.6) | 14 (63.6) | 14 (63.6) |
| M. yongonense | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| M. gordonae | 14 | 0 | 0 | 1 (7.1) | 4 (28.6) | 8 (57.1) | 10 (71.4) | 0 | 0 | 0 | 0 | 0 | 0 |
| M. lentiflavum | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 (100) | 1 (100) |
| M. paragordonae | 7 | 0 | 0 | 1 (14.3) | 2 (28.6) | 4 (57.1) | 4 (57.1) | 0 | 0 | 0 | 0 | 0 | 0 |
| M. paraffinicum | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| M. simiae | 1 | 0 | 1 (100) | 1 (100) | 1 (100) | 1 (100) | 1 (100) | 0 | 1 (100) | 1 (100) | 1 (100) | 1 (100) | 1 (100) |
| Mycobacterium sp. | 4 | 0 | 0 | 1 (25) | 2 (50) | 3 (75) | 4 (100) | 0 | 0 | 0 | 0 | 0 | 1 (25) |
| Total | 76 | 4 (5.3) | 18 (23.7) | 28 (36.8) | 35 (46.1) | 44 (57.9) | 48 (63.2) | 6 (7.9) | 18 (23.7) | 28 (36.8) | 31 (40.8) | 32 (42.1) | 35 (46.1) |
| Grand total | 133 | 38 (28.6) | 61 (45.9) | 76 (57.1) | 87 (65.4) | 97 (72.9) | 102 (76.7) | 32 (24.1) | 46 (34.6) | 57 (42.9) | 60 (45.1) | 63 (47.4) | 66 (49.6) |
That is, the total number of nontuberculous mycobacteria isolated from a combination of all culture test methods (RGM30, RGM35, the MGIT system, and Middlebrook 7H11 agar).
The extension of incubation period from 10 to 28 days was useful for the recovery of slowly growing mycobacteria such as MAC, M. gordonae, M. lentiflavum, and M. paragordonae (Table 3 and Fig. S3). The recovery rate of slowly growing mycobacteria increased from 36.8% (day 10) to 63.2% (day 28) by RGM30 (P = 0.0001) and from 36.8% (day 10) to 46.1% (day 28) by RGM35 (P = 0.0233). In conclusion, the incubation time of 28 days increased recovery of both rapidly and slowly growing mycobacteria.
Breakthrough contamination of nonmycobacterial organisms on RGM medium.
The contamination of nonmycobacterial organisms was observed on RGM medium after incubation either at 30°C air or at 35°C with 8% CO2 for 28 days. From 203 respiratory specimens, only 11 (5.4%) and 9 (4.4%) specimens plated on RGM30 and RGM35, respectively, showed contamination of nonmycobacterial organisms (P = 0.6831), listed in Table 4. In contrast, higher contamination rates were observed for the MGIT method (12.3% [25/203]) and Middlebrook 7H11 agar (34.5% [70/203]) than for RGM medium (P < 0.05). In conclusion, RGM medium could inhibit other nonmycobacterial growth and had a lower rate of breakthrough contamination than the MGIT system and Middlebrook 7H11 agar.
TABLE 4.
Breakthrough contaminants and contamination rates of RGM media incubated at 30°C (RGM30) and 35°C (RGM35)
| Organism | No. of contaminants (contamination rate [%]) |
|
|---|---|---|
| RGM30 | RGM35 | |
| Achromobacter sp. | 1 (0.49) | 1 (0.49) |
| Burkholderia cepacia complex | 1 (0.49) | 1 (0.49) |
| Herbaspirillum sp. | 1 (0.49) | 1 (0.49) |
| Klebsiella pneumoniae | 1 (0.49) | 0 |
| Candida glabrata | 3 (1.48) | 2 (0.99) |
| Candida tropicalis | 1 (0.49) | 0 |
| Saccharomyces cerevisiae | 2 (0.99) | 2 (0.99) |
| Aspergillus fumigatus | 1 (0.49) | 2 (0.99) |
| Total | 11 (5.42) | 9 (4.43) |
DISCUSSION
This study evaluated the performance of RGM medium for the recovery of NTM from respiratory specimens from non-CF patients with underlying lung conditions. Respiratory specimens require decontamination procedures such as NALC-NaOH prior to plating on culture media to reduce overgrowth of nonmycobacterial organisms. NTM, especially rapidly growing mycobacteria, are particularly susceptible to decontamination procedures, leading to suboptimal recovery on culture. RGM medium was designed to enhance the recovery of rapidly growing mycobacteria from CF patients by inhibiting growth of nonmycobacterial organisms such as P. aeruginosa (12) and does not require decontamination of clinical specimens.
We studied the recovery of NTM at two temperature settings, i.e., 30°C air and 35°C with 8% CO2, to cover the optimal temperatures that promote growth of most mycobacteria. The lower temperature supported the growth of several mycobacteria such as M. chelonae, M. immunogenum, M. llatzerense, M. mucogenicum group, M. paragordonae, M. gordonae, and an unidentified slowly growing Mycobacterium sp. However, the incubation at 35°C (with 8% CO2) supported growth of certain slowly growing mycobacteria, including MAC, M. lentiflavum, and M. simiae. In this study, we extended the incubation time of RGM plates to 28 days as described in a previous study (15) with the goal of increasing recovery of slowly growing mycobacteria. Our results showed increased recovery of NTM from RGM medium, including both rapidly and slowly growing mycobacteria upon extended incubation from 10 to 28 days. Although the sensitivity of RGM30 with a 28-day incubation period was comparable to that of the MGIT system (P = 0.6171) and Middlebrook 7H11 agar (P = 0.3711) for the recovery of MABSC, RGM30 performed better for the recovery of all rapidly growing mycobacteria isolated in this study (P = 0.0001). This finding was consistent with previous studies (14, 15), demonstrating that RGM medium provided performance equivalent to that of the MGIT system or even better for the recovery of MABSC and other rapidly growing mycobacteria. Notably, the time to recovery of MABSC on RGM medium was comparable to the average time to positivity of MABSC on the MGIT system (∼4 days). RGM35 showed a higher (but not significantly different) sensitivity than the MGIT system and Middlebrook 7H11 agar for the recovery of MABSC and other rapidly growing mycobacteria (P > 0.05). For slowly growing mycobacteria, RGM30 and RGM35 failed to recover 13 and 10 isolates of MAC, respectively, compared to the MGIT system (P < 0.05). The difference between RGM30 and the MGIT system for the recovery of MAC from clinical specimens had not been shown in previous studies (14, 15).
RGM medium showed potential as an alternative medium for recovery of mycobacteria, especially rapidly growing mycobacteria. The incubation at 30°C showed a better performance for recovery of NTM from respiratory specimens than 35°C. Based on our results, the MGIT system is still required for optimal recovery of MAC and possibly other slowly growing mycobacteria. However, positive MGIT cultures required additional time until mycobacterial identification. The subculture from positive MGIT culture to solid medium such as Middlebrook 7H11 agar was needed to obtain pure culture and to observe colony morphology, thus prolonging the time until diagnosis. In contrast, colony morphology and occasional contamination in culture could be observed directly on RGM medium.
Based on this study, we propose using a combination of RGM30 and the MGIT system for mycobacterial culture of respiratory specimens. This combination produces improved recovery of NTM, notably MABSC (100% versus 96.4%) and MAC (93.8% versus 60.4%), as well as M. gordonae (100% versus 71.4%), M. lentiflavum (100% versus 0), and M. paragordonae (100% versus 57.1%) (Table 2), compared to the use of RGM30 alone. Replacing Middlebrook 7H11 agar with RGM medium incubated at 30°C can improve the recovery of NTM from respiratory specimens from non-CF patients. However, it is important to emphasize that the presence of certain NTM isolated from pulmonary specimens in our patient population does not necessarily indicate pulmonary infection or disease because NTM may be present in the respiratory system as colonizers without fulfilling microbiological criteria for NTM disease (7).
In addition, RGM medium offers selectivity by inhibiting growth of nonmycobacterial organisms. From our study, RGM medium has a low contamination rate (RGM30, 5.4%; RGM35, 4.4%) from non-CF patients’ specimens, which was lower than that observed with the MGIT method (12.3%) and Middlebrook 7H11 agar (34.5%). RGM medium showed the capability to inhibit other nonmycobacterial organisms which grew on routine bacterial and fungal cultures such as Escherichia coli, Klebsiella pneumoniae, and Penicillium sp. However, some Gram-negative bacteria, such as B. cepacia complex and Achromobacter sp., could break through and grow on RGM medium. This observation was consistent with the results from previous studies (12, 13, 15). In our routine mycobacterial culture, we also use additional medium (Mitchison 7H11 selective agar) to enhance growth of mycobacteria by inhibiting other nonmycobacterial organisms. However, we have observed that some mycobacterial growth is suppressed on Mitchison 7H11 selective agar (data not shown), as described in a previous study (19). While our study demonstrated the usefulness of RGM medium at 30°C, one limitation is that we did not evaluate Middlebrook 7H11 agar at 30°C, which was shown as an optimal temperature for growth of several rapidly growing mycobacteria. We chose to compare RGM medium to our current routine AFC, which does not include an incubation at 30°C for respiratory specimens. Although the incubation of Middlebrook 7H11 agar at 30°C might enhance the recovery of several mycobacteria, RGM medium is still superior to Middlebrook 7H11 agar for the inhibition of nonmycobacterial organisms.
In conclusion, RGM medium provides an alternative method for the recovery of NTM from respiratory specimens from non-CF patients by offering a simple and rapid method for specimen processing. However, the performance for recovery of MAC was inferior to that of the MGIT system. The combination of the MGIT system and RGM medium with incubations at 30°C could increase the recovery rate of NTM, including both rapidly and slowly growing mycobacteria.
Supplementary Material
ACKNOWLEDGMENTS
We thank the staff members in the Microbiology Service, in particular the Specimen Processing and Mycology and Mycobacteriology sections, Department of Laboratory Medicine, Clinical Center, National Institutes of Health, for their work on this study.
This research was supported by the Intramural Research Program of the NIH Clinical Center, Department of Laboratory Medicine.
The Freeman Hospital Microbiology Department (represented by Dominic Stephenson and John Perry) has received funding from bioMérieux for the development and evaluation of culture media.
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/JCM.01519-18.
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