Validation of the MYChrOme™ Culture Plate for Detection and Differentiation of Rapid-Growing Nontuberculous Mycobacteria in Potable and Non-Potable Water: AOAC Performance Tested MethodSM 062101.

BACKGROUND: The MYChrOme™ Culture Plate is a chromogenic media for the detection and differentiation of rapid-growing nontuberculous mycobacteria (NTM) in water, aided by MYCOn™ decontamination to reduce background microbiota.
OBJECTIVE: Evaluate the MYChrOme Culture Plates for the detection of rapid-growing NTM in potable and non-potable water as part of the AOAC Performance Tested Method(s)SM program.
METHODS: Inclusivity and exclusivity of MYChrOme were evaluated with 50 target and 30 non-target organisms. Method robustness and lot stability of MYChrOme were analyzed. The candidate method was compared to a modified US Food and Drug Administration (FDA) Method: U.S. FDA-Isolation and Identification of Nontuberculous Mycobacteria in Tattoo Inks using an equivalency test. The matrix study consisted of artificially contaminated potable water and naturally contaminated non-potable water. Independent laboratory testing was conducted to verify method performance in non-potable water.
RESULTS: The inclusivity of MYChrOme was 94% within one week, and 98% within two weeks. The exclusivity was 96% for untreated samples and 100% for treated samples. The candidate method remained statistically equivalent for robustness and a three-month shelf-life was confirmed. For both matrixes, the candidate and reference methods were not equivalent, with more colonies enumerated on the candidate method except for one contamination level of the potable matrix.
CONCLUSION: The MYChrOme culture method can successfully detect and differentiate rapid-growing NTM in the matrixes tested, with sensitivity equivalent or higher than the reference method. HIGHLIGHTS: The MYChrOme culture plate offers differentiation of rapid-growing NTM colonies, improved detection in non-potable samples with MYCOn decontamination, and results within 7 days. © AOAC INTERNATIONAL 2021.

Principle of the Method

The MYChrOme™ Culture Plate includes a unique formula for the detection and differentiation of rapid-growing nontuberculous mycobacteria (NTM) in environmental water samples. On this media, rapid-growing NTM colonies remain white/non-colorized and other bacteria are colorized by crystal violet dye (usually purple). An example of the differentiation is shown in Figure 1 where Mycobacterium porcinum (Phigenics Culture Collection) forms white colonies and Klebsiella pneumoniae (NCTC 13340) forms purple colonies on the MYChrOme Culture Plate. This is due to the Mycobacterium genus’s ability to metabolize and decolorize the crystal violet. The high level of crystal violet in the media also inhibits the growth of most gram-positive bacteria. In order to further decontaminate the sample, a novel decontamination step is used. The MYCOn™ decontamination reagent relies on low pH and sodium dodecyl sulfate (SDS) to kill the non-mycobacteria. The strong cell wall and hardy nature of mycobacterial cells allow them to survive the decontamination step. Non-potable water samples are plated with the decontamination step and potable water samples with <1000 colony-forming units (CFU)/mL of background microbiota are plated without the decontamination step. After 7 days of incubation at 30 ± 1°C, the white colonies are counted and confirmed to be NTM using serological or molecular methods, such as acid-fast staining or real-time PCR.

Figure 1.

Example of the MYChrOme Culture Plate chromogenic differentiation of NTM and non-mycobacteria. (A) Klebsiella pneumoniae (NCTC 13340) plated on MYChrOme forms purple colonies. (B) Mycobacterium porcinum (Phigenics Culture Collection) plated on MYChrOme forms white colonies.

General Information

NTM are a category of the genus Mycobacterium that excludes the species that cause tuberculosis (Mycobacterium tuberculosis complex) and leprosy (Mycobacterium leprae complex). NTM are acid-fast, non-motile, and have a unique and complex cell wall structure made up of mycolic acids. This waxy, hydrophobic, and thick cell wall make NTM extremely hardy and thus difficult to disinfect (1). There are two different types of NTM, rapid-growing NTM, which take less than two weeks to form colonies, and slow-growing NTM, which take 2–8 weeks to form colonies.

NTM can cause serious infections, such as skin/soft tissue infections, lymphadenitis, disseminated disease, and most commonly, lung disease presenting similarly to tuberculosis. Exposure to NTM is most common through water systems, especially in monochloraminated systems. NTM can grow and proliferate in biofilms and tend to aggregate in water. This is especially a problem in facilities such as hospitals with large populations of immunocompromised individuals that can potentially be exposed to NTM through sinks and showers (1).

Current methods for detecting NTM are adapted from methods for the isolation of M. tuberculosis and involve spread-plating a sample onto a limited nutrient agar such as Middlebrook 7H10 or Middlebrook 7H11 Selective (7H11S). An additional decontamination step with cetylpyridinium chloride (CPC) is often utilized before plating but has been noted to be overly harsh to several species of NTM (2). Differentiation of NTM from other bacteria is not possible on these agars.

Analyte.—Rapid-growing nontuberculous Mycobacterium spp.

Matrixes.—Potable and non-potable water (200 mL).

Summary of validated performance claims.—The MYChrOme Culture Plate method was found to be as equivalent or better than the US Food and Drug Administration (FDA).—Isolation and Identification of Nontuberculous Mycobacteria in Tattoo Inks (3), modified for water samples.

Repeatability (s—Standard deviation of replicates for each analyte at each concentration of each matrix for each method.

Mean difference between candidate and reference methods.—Mean difference between candidate and reference method transformed results with 90% confidence interval for each analyte at each concentration of each matrix.

Selectivity.—Ability of the method to detect analyte without interference from matrix or other components of similar behavior.

Bias.—Bias is the difference between the candidate method mean result and the true value or reference method value, meancand−known spike or meancand−meanref.

Standard deviation of repeatability.—sr =

Relative standard deviation of repeatability.—RSDr = [sr/meancand] × 100.

Kit name.—MYChrOme Culture Plate.

Product number.—MB-2650.

Ordering information.—Contact Phigenics, LLC 3S701 West Ave. Suite 100 Warrenville, IL 60555; Tel: 1.844.850.4087. Website: info.phigenics.com, for more information.

MYChrOme Culture Plate.

MYCOn decontamination reagent.

Water collection bottles.—Sterile with 100 mg/L sodium thiosulfate.

Reusable filter unit.—Thermo Scientific Cat. No. 09-740-23A, or equivalent.

0.22 µm filters.—47 mm, track-etched polycarbonate.

15 mL conical tubes.

Sterile forceps.

1:40 Ringer's Solution.—Diluted from 1:4 Ringer’s (Sigma-Aldrich, Cat. No. 96724-100TAB).

Disposable plastic cell spreaders.—Sterile.

Disposable plastic loops.—Sterile.

Micropipet and tips.—2–20 µL.

Micropipet and tips.—100–1000 µL.

Microcentrifuge tubes.—1.5 mL.

Mycobacterium genus-specific real-time colony PCR.—For colony confirmation.

Incubator.—Set to 30 ± 1°C.

Real-time PCR Thermocycler.—Bio-Rad, CFX96 Deep Well Touch, or equivalent.

Vortex.

Materials and Methods

Reference Materials

Bacterial strains used in this study were sourced from the following institutions:

American Type Culture Collection (ATCC).—Manassas, VA.

National Collection of Type Cultures (NCTC).—Porton Down, UK.

Phigenics Culture Collection (Phigenics).—Reno, NV.

Culture Collection University of Gothenburg (CCUG).—Gothenburg, Sweden.

US FDA.—Irvine, CA.

Public Health England (PHE).—London, UK.

National Collection of Industrial, Food, and Marine Bacteria (NCIMB).—Aberdeen, UK.

Safety Precautions

Follow biosafety level 2 precautions and wear appropriate personal protective equipment. All work should be conducted in properly equipped facilities utilizing the appropriate safety equipment (for example, physical containment devices). Biological samples have the potential to transmit infectious diseases.

Decontaminate all surfaces and equipment prior to and after use.

It is the responsibility of each laboratory to handle waste and effluents processed according to their nature and degree of hazardness and to treat and dispose of them in accordance with applicable local, state, and federal regulations.

Potable and non-potable water.—Collect water samples in sterile bottles with sodium thiosulfate (100 mg/L) to neutralize the residual oxidant. Filter concentrates 200 mL of the water sample on a 0.22 µm track-etched 47 mm polycarbonate membrane. Testing must be performed within 36 h of sample collection.

Resuspend the filter in 10 mL of one of the following: Sterile 1:40 Ringer's solution, filtrate, sterile deionized (DI) water, or sterile phosphate buffered saline (PBS).

Vortex the sample for 30 s.

For potable samples, 100 µL of the concentrated sample is spread plated onto a MYChrOme Culture Plate Note.—Some potable water samples with background microbiota >1000 CFU/mL will need to follow the non-potable water method.

For non-potable water, transfer 981 µL of concentrated sample to 19 µL of MYCOn decontamination reagent, pipet up and down to mix, and incubate at room temperature for 5 min. After the incubation period, 100 µL of the decontaminated sample is spread plated onto a MYChrOme Culture Plate.

Incubate the MYChrOme plates at 30 ± 1°C for 7 days.

After 7 days of incubation, enumerate all white colonies, very light purple, grey colonies, or bright yellow colonies (some NTM form yellow colonies). These are the suspect rapid-growing NTM colonies that require confirmation.

All purple, brown, or pink colonies are not NTM and do not need to be analyzed further.

Confirm that the suspect colonies identified are NTM using serological (acid-fast) or molecular methods (Mycobacterium genus-specific real-time PCR).

Recommended confirmation method.—Real-time PCR for NTM that targets the atpE gene using the primers and probe from Radomski et al. (4). The following thermocycling conditions were used: The activation step (1 cycle) is 95°C for 3 min, followed by 40 cycles of 95°C for 10 s, and 60°C for 30 s. The suspect colony is picked and directly inoculated into 40 µL of PCR master mix.

Validation Study

This validation study was conducted under the AOAC Research Institute Performance Tested Method(s)SM program and the AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces (5). Method developer studies were conducted in the laboratory of Phigenics, LLC (Reno, NV), and included the inclusivity/exclusivity study, matrix studies for potable and non-potable, product consistency and stability studies, and robustness testing. The independent laboratory study was conducted by Silliker Food Science Center: Mérieux NutriSciences (Crete, IL), and included a matrix study for non-potable water.

Method Developer Studies

Inclusivity testing

Methodology.—A total of 50 rapid-growing NTM strains were used for inclusivity testing (Table 1). Each strain was cultured in 1395 Middlebrook 7H9 broth (Hardy Diagnostics, Santa Maria, CA) for 5–7 days at 30 ± 1°C. Each culture was diluted in sterile PBS and inoculated into 200 mL of sterile DI water to achieve a concentration of approximately 1–100 CFU/mL. This 200 mL sample was filter-concentrated through a 0.22 µm track-etched 47 mm polycarbonate membrane. The filter was placed into a 15 mL conical tube, resuspended in 10 mL of sterile DI water, and vortexed for 30 s. Each sample was inoculated onto MYChrOme media by spread plating 100 µL in a randomized, blind-coded fashion. A separate portion of each sample was decontaminated by adding 981 µL of the concentrated sample to 19 µL of MYCOn in a microcentrifuge tube. The tube was mixed twice by pipetting up and down and incubated at room temperature for 5 min. Then, 100 µL of the decontaminated sample was spread plated onto MYChrOme. The inclusivity plates were incubated at 30 ± 1°C for 7 days. The plates were counted, and the colony color was recorded.

Table 1.

Inclusivity testing results for the MyChrOme culture plate

Genus	Species	Source	Origin	Result
				Untreated, Log10 CFU/plate	Treated, Log10 CFU/plate
Mycobacterium	abscessus	FDA 858508-1	Not available	2.48	2.48
Mycobacterium	abscessus	FDA 923093-1075	Not available	2.10	2.10
Mycobacterium	abscessus subsp abscessus	CCUG 71636	Human blood	0.48	0.04
Mycobacterium	abscessus subsp. bolletii	CCUG 50184	Human bronchial lavage	1.00	0.48
Mycobacterium	abscessus subsp. massiliense	CCUG 48898	Human sputum	0.48	0.30
Mycobacterium	agri	CCUG 37673	Soil	2.48	2.48
Mycobacterium	aubagnense	CCUG 50186	Human bronchial aspirate	0.04	0.04
Mycobacterium	aurum	CCUG 70546	Soil	2.48	2.48
Mycobacterium	barrassiae	CCUG 50398	Human bronchial lavage	2.48	2.48
Mycobacterium	boenickei	CCUG 47580	Human wound	1.08	0.70
Mycobacterium	brisbanense	CCUG 47584	Antral sinus	0.30	0.60
Mycobacterium	canariasense	CCUG 47953	Human blood	0.85	0.04
Mycobacterium	chelonae	PHE	Not available	1.18	0.90
Mycobacterium	chelonae	Phigenics	Env. isolate	2.00	1.90
Mycobacterium	chelonae	CCUG 72969	Human eye	0.48	0.48
Mycobacterium	chelonae	CCUG 37827	Human wound	0.60	0.04
Mycobacterium	chelonae	FDA 858509-1-1-1	Not available	2.48	2.48
Mycobacterium	chelonae	FDA 858509-2-3-2	Not available	2.54	2.48
Mycobacterium	chitae	CCUG 39181	Soil	1.15	0.04
Mycobacterium	cosmeticum	CCUG 55442	Human feces	0.04	0.04
Mycobacterium	fortuitum	PHE	Not available	0.95	0.90
Mycobacterium	fortuitum	ATCC 6841	Cold abscess	0.30	0.04
Mycobacterium	fortuitum	FDA 858508-10	Not available	2.48	2.48
Mycobacterium	fortuitum	FDA 923093-1278	Not available	2.48	2.48
Mycobacterium	fortuitum subsp. fortuitum	CCUG 46694	Human blood	0.48	0.48
Mycobacterium	franklinii	Phigenics	Env. isolate	1.23	0.04
Mycobacterium	gadium	CCUG 37515	Human sputum	2.30	1.93
Mycobacterium	goodii	CCUG 5204	Human blood	0.60	0.30
Mycobacterium	hodleri	CCUG 38151	Chemical contaminate	1.34	1.20
Mycobacterium	immunogenum	Phigenics	Env. isolate	0.78	0.78
Mycobacterium	immunogenum	CCUG 52935	Water for injection	0.60	0.04
Mycobacterium	iranicum	CCUG 52297	Human sputum	1.89	1.04
Mycobacterium	mageritense	CCUG 51275	Human calf	0.95	1.11
Mycobacterium	moriokaense	CCUG 37671	Soil	1.74	0.48
Mycobacterium	mucogenicum	Phigenics	Env. isolate	2.00	0.48
Mycobacterium	mucogenicum	FDA 858510-2	Not available	0.30	0.04
Mycobacterium	mucogenicum	FDA 858510-4	Not available	1.81	1.60
Mycobacterium	mucogenicum	FDA 858510-9	Not available	2.46	2.12
Mycobacterium	murale	CCUG 57579	Wall material	ND	ND
Mycobacterium	neoaurum	Phigenics	Env. isolate	2.16	1.23
Mycobacterium	peregrinum	CCUG 41354	Human bronchial aspiration	2.00	1.38
Mycobacterium	phocaicum	Phigenics	Env. Isolate	1.23	0.48
Mycobacterium	phocaicum	CCUG 50185	Human bronchial aspirate	0.30	0.04
Mycobacterium	phocaicum	FDA 858510-1	Not available	2.20	2.10
Mycobacterium	porcinum	Phigenics	Env. Isolate	1.91	1.28
Mycobacterium	porcinum	CCUG 37674	Swine lymph node	0.70	0.30
Mycobacterium	senegalense	CCUG 59339	Human sputum	1.70	0.04
Mycobacterium	septicum	CCUG 47583	Not available	0.78	0.04
Mycobacterium	smegmatis	ATCC 14468	Not available	1.23	0.48
Mycobacterium	wolinskyi	CCUG 47168	Human abscess	1.57	1.15

Results.—Results are shown in Table 1. Out of 50 rapid-growing NTM strains tested, 47 (94.0%) grew on MYChrOme (treated and untreated) in 7 days. Mycobacterium chitae was detected on MYChrOme within 12 days, and Mycobacterium moriokaense grew within 14 days. Mycobacterium murale was not detected on MYChrOme, but did grow on M7H10. All detected strains had white colonies, except for Mycobacterium aurum and Mycobacterium neoaurum, which retained their yellow pigment. In addition, Mycobacterium abscessus subsp. massiliense colonies were white with a light purple hue. Due to trial and error in spiking the water samples and the aggregative nature of NTM, some samples had a colony count in excess of 100 CFU/mL.

Exclusivity testing

Methodology.—A total of 30 non-mycobacterial strains were used for exclusivity testing (Table 2). These strains were chosen because they often inhabit the same water systems as Mycobacterium spp. Each non-target organism was inoculated into 10 mL of buffered yeast extract broth from a glycerol stock. Each culture was incubated at optimal conditions (35 or 30°C) for 24 to 48 h. The entire 10 mL of culture was then poured into 190 mL of sterile DI water to achieve a cell concentration of >106 CFU/mL. The resulting 200 mL sample was filter concentrated through a 0.22 µm track-etched 47 mm polycarbonate membrane. The filter was placed into a 15 mL conical tube and resuspended in 10 mL of sterile DI water. The resuspended sample was vortexed for 30 s. In a randomized, blind-coded fashion each sample was inoculated onto MYChrOme media by spread plating 100 µL. A separate portion of each sample was decontaminated by adding 981 µL of the concentrated sample to 19 µL of MYCOn in a microcentrifuge tube. The tube was mixed twice by pipetting up and down and incubated at room temperature for 5 min. Then, 100 µL of the decontaminated sample was spread plated onto MYChrOme. The exclusivity plates were incubated at 30 ± 1°C for 7 days. Then the plates were counted, and the colony color was recorded.

Table 2.

Exclusivity testing results for the MyChrOme culture plate method

Genus	Species	Source	Origin	Result
				Untreated, CFU/mL-color	Treated, CFU/mL-color
Acinetobacter	baumannii	NCIMB 12457	Urine	Lawn-purple	NDa
Aeromonas	hydrophila	ATCC 35654	Not available	Lawn-purple	ND
Alcaligenes	faecalis	ATCC 35655	Not available	Lawn-purple	ND
Bacillus	subtilis	ATCC 14990	Nose	ND	ND
Burkholderia	cepacia	ATCC 25608	Incision wound	Lawn-purple	ND
Chryseobacterium	shigense	ATCC 51823	Milk	Lawn-purple	23-dark purple
Elizabethkingia	meningoseptica	ATCC 13253	Spinal fluid	Lawn-purple	ND
Escherichia	coli	ATCC 10536	Not available	Lawn-purple	ND
Klebsiella	aerogenes	ATCC 13048	Sputum	Lawn-purple	ND
Klebsiella	pneumonia	NCTC 13340	Not available	Lawn-purple	ND
Legionella	Anisa	Phigenics	Env. isolate	ND	ND
Legionella	birminghamensis	CCUG 31233	Human lung biopsy	0.5-purple	ND
Legionella	bozemanii	CCUG 16416	Lung aspirate	ND	ND
Legionella	Feelei	CCUG 29668	Human lung tissue	3-slate gray	ND
Legionella	Jordansis	CCUG 16413	Jordan river	ND	ND
Legionella	longbeachae	ATCC 33462	Human lung	ND	ND
Legionella	pneumophila sg 1	CCUG 9568T	Human lung	ND	ND
Legionella	pneumophila sg 7	ATCC 33823	Human lung	ND	ND
Legionella	sainthelensi	CCUG 29672T	Stream water	ND	ND
Legionella	wadsworthii	CCUG 16415T	Human sputum	ND	ND
Methylobacterium	spp.	Phigenics	Env. isolate	ND	ND
Microbacterium	oxydans/maritypicum	Phigenics	Env. isolate	ND	ND
Nocardia	brasiliensis	ATCC 19296	Not available	15.5-off white	ND
Pseudomonas	aeruginosa	ATCC 27853	Blood	Lawn-purple	1.5-purple
Pseudomonas	Fragi	ATCC 51821	Milk	Lawn-purple	ND
Pseudomonas	Mosseli	ATCC 49838	Not available	Lawn-purple	ND
Pseudomonas	Stutzeri	ATCC 17588	Spinal fluid	Lawn-purple	ND
Sphingomonas	paucimobilis	ATCC 29837	Hospital respirator	Lawn-purple	ND
Staphylococcus	Aureus	ATCC 25923	Clinical	200-purple	ND
Stenotrophomonas	maltophilia	ATCC 17666	Tissue culture	Lawn-purple	ND

ND = No detection.

Results.—Results are shown in Table 2. All 30 non-mycobacterial strains (100%) were not detected (n = 28) or had purple colonies (n = 2) for the treated samples. The two strains with purple colony growth after the treatment were Chryseobacterium shigense and Psudomonas aeruginosa, with 46 and 3 colonies, respectively. For the untreated samples, 29 (96.6%) strains were either not detected (n = 11) or had purple colonies (n = 19) on MYChrOme. The one strain with an off-white colony color was Nocardia brasiliensis which had 31 colonies on the untreated plate and zero colonies on the treated plate (see discussion).

Robustness study

Methodology.—The MYChrOme protocol, including sample concentration, decontamination, and plating, was analyzed for its ability to remain unaffected by small variations in method parameters. The parameters analyzed in a factorial design were sample volume, filtrate volume, and decontamination incubation time (Table 3). Sterile DI water artificially contaminated with 1 × 106 CFU/mL of Acinetobacter baumannii (NCIMB 12457) was used for the non-target organism samples and spiked with two levels of Mycobacterium chelonae (CCUG 37827) for the target organism samples. The low-level target organism samples were spiked with 25–50 CFU/mL and the high-level target organism samples were spiked with approximately 5000 CFU/mL. Five replicates per sample type were analyzed in a randomized, blind-coded fashion by each of the nine factorial methods by plating 100 µL of treated sample onto MYChrOme. The plates were incubated at 30 ± 1°C for 7 days and colonies were counted. Each modified method was compared to the standard MYChrOme method (number 9) using an unpaired equivalency test assuming equal variances with a 90% confidence interval.

Table 3.

Robustness of the MYChrOme culture plate, bias, and repeatability

Parameter test combinationa	Parameters
	Sample volume, mL	Filtrate volume, mL	Decontamination incubation time, min	Nb	Meanc	srd	Nominal condition	N	Mean	sr	Mean differencee	LCLf	UCLg
Non-potable water with Mycobacterium—low level
1	175	9	4	5	1.598	0.231	9	5	1.763	0.05	–0.165	–0.362	0.032
2	175	9	6	5	1.525	0.234	9	5	1.763	0.05	–0.238	–0.437	–0.04
3	175	11	4	5	1.745	0.204	9	5	1.763	0.05	–0.018	–0.193	0.156
4	175	11	6	5	1.726	0.19	9	5	1.763	0.05	–0.037	–0.2	0.126
5	225	9	4	5	1.662	0.282	9	5	1.763	0.05	–0.101	–0.339	0.136
6	225	9	6	5	1.652	0.147	9	5	1.763	0.05	–0.111	–0.24	0.018
7	225	11	4	5	1.956	0.218	9	5	1.763	0.05	0.193	0.007	0.379
8	225	11	6	5	1.747	0.189	9	5	1.763	0.05	–0.016	–0.179	0.146

Each parameter test combination is being compared to the nominal test condition—9 = 200 mL sample volume, 10 mL filtrate volume, 5 min decontamination incubation.

N = Number of test portions.

Mean of five replicate portions after logarithmic transformation: Log10[CFU/g + (0.1)f].

Repeatability standard deviation.

Mean difference between the candidate and reference methods.

90% Lower confidence limit for difference of means.

90% Upper confidence limit for difference of means.

Results.—Robustness results are shown in Table 3. For the low-level target organism, the colony counts for the eight modified methods were statistically equivalent to the nominal method. For the high-level target organism, all nine methods resulted in too numerous to count MYChrOme plates with all white colonies. For the non-target organism, all nine methods resulted in zero colonies on the MYChrOme plates.

Product consistency (lot-to-lot) and stability study

Methodology.—Three lots of MYChrOme plates were analyzed for consistency, stability, bias, and repeatability over three months at 4°C. Lot 1 (end of shelf-life) was manufactured on September 1st, 2020; lot 2 (middle of shelf-life) was manufactured on October 10th, 2020; and lot 3 (beginning of shelf-life) was manufactured on November 15th, 2020. All three lots were manufactured by the Phigenics Analytical Services Laboratory in Fayetteville, AR. This study was performed on December 2nd, 2020. To evaluate the product consistency five replicates of high and low-level target organism (Mycobacterium fortuitum, ATCC 6841) were analyzed in addition to five replicates of the non-target organism (A. baumannii, NCIMB 12457). The M. fortuitum stock was cultured on MYChrOme for 5 days at 30 ± 1°C and used to inoculate sterile PBS and diluted in order to plate approximately 5000 CFU/mL for the high-level and approximately 50 CFU/mL for the low-level. The cell suspension for the high-level target organism was diluted to 10−2 and plated in quintuplicate in order to recover countable plates. For the non-target organism, A. baumannii was grown in non-selective broth for 24 h at 30 ± 1°C and was not diluted. 100 µL of each cell suspension was plated onto five replicates of each MYChrOme lot in a randomized, blind-coded fashion. After 7 days of incubation at 30 ± 1°C, the plates were counted and colony color was recorded. Each lot was compared to the other with an unpaired equivalency test assuming equal variances and using a 90% confidence interval.

Results.—The stability study results are summarized in Table 4. All MYChrOme lots were statistically equivalent for the high- and low-level target organism samples. All of the high-level target organism plates had too numerous to count white colonies, so the dilution plates were used for accurate colony counts. One dilution plate of the high-level target organism was contaminated on lot 1 and could not be counted. All lots of the non-target organism were too numerous to count with purple colonies, as expected.

Table 4.

Product consistency (lot-to-lot) and stability of MYChrOme culture plate, bias, and repeatability

Level	Na	Meanb	src	Lotg	Mean	sr	Mean differenced	LCLe	UCLf
1—High	5	4.105	0.015	2	4.076	0.034	0.792	–0.063	0.006
2—High	5	4.076	0.034	3	4.024	0.015	–0.052	–0.083	–0.022
3—High	5	4.024	0.015	1	4.105	0.015	0.740	–0.1	–0.062
1—Low	5	2.493	0.051	2	2.411	0.063	–0.082	–0.15	–0.015
2—Low	5	2.411	0.063	3	2.351	0.052	–0.06	–0.128	0.008
3—Low	5	2.351	0.052	1	2.493	0.051	–0.142	–0.203	–0.082

N = Number of test portions.

Mean of five replicate portions after logarithmic transformation: Log10[CFU/g + (0.1)f].

Repeatability standard deviation.

Mean difference between the candidate and reference methods.

90% Lower confidence limit for difference of means.

90% Upper confidence limit for difference of means.

Lot 1 was beginning of expiration period. Lot 2 was middle of expiration period. Lot 3 was end of expiration period.

Matrix study

Methodology.—Two matrixes, potable and non-potable water, were evaluated for NTM detection by the MYChrOme Culture Plate and a modified FDA method (larger test portion size validated to accommodate water sample). The study included five replicate test portions at each contamination level. There were low, medium, and high levels of contamination for each matrix, with each level being approximately 1 log10 higher than the previous so that the range of samples covered at least two log10 units. Potable sink water (e.g., tap water) was inoculated at three contamination levels (10–100, 100–1000, and 1000–10 000 CFU/mL) with an unstressed liquid culture of M. fortuitum (ATCC 6841). An uncontaminated control was also analyzed. Samples were equilibrated at 4°C for 48 h prior to testing. Non-potable outdoor fountain water was evaluated containing natural contamination of NTM and was tested without an equilibration phase. The candidate method and reference method were performed on the same day for each contamination level on split 400 mL samples. The candidate and reference methods were evaluated using unpaired test portions.

Modified US FDA—Isolation and Identification of Nontuberculous Mycobacteria in Tattoo Inks.—Methodology.—To each 200 mL test portion, 8 mL of 0.04% (w/v) CPC was added and left at room temperature for 30 min. A 200 mL test portion was vacuum filtered using a 0.45 µm black grid HABG 47 mm filter. The filter was rinsed with sterile phosphate buffer, placed in separate 15 mL conical tubes, and resuspended with 10 mL of sterile PBS. The concentrated sample was vortexed for 30 s. Each sample was spread plated using 1.0 mL (0.33, 0.33, and 0.34 mL) onto three separate M7H10 and M7H11S agar plates. Each replicate was plated in duplicate. A 0.1 mL sample was plated in duplicate onto separate M7H10 and M7H11S agar plates. Two 10-fold serial dilutions were prepared by transferring 1.0 mL into 9.0 mL of PBS of the previous dilution. From each dilution, 0.1 mL was spread plated in duplicate onto separate M7H10 and M7H11S agar plates. Plates were allowed to dry and sealed in a gas permeable container. The plates were incubated at 37 ± 1°C. The plates were examined every 7 days for up to 2 weeks. One to two isolated colonies were selected from each test portion and resuspended in 100 µL of sterile water in a 1.5 mL microcentrifuge tube. A 50 µL aliquot was transferred into a 1.5 mL microcentrifuge tube containing 100 µL of InstaGene Matrix (Bio-Rad, Hercules, CA) for DNA extraction. The microcentrifuge tubes were vortexed for 10 s and incubated at 56 ± 1°C for 15 min. The microcentrifuge tubes were vortexed for 10 s, then heated at 100 ± 1°C for 8 min. The tubes were centrifuged at 12 000 rpm for 2 min, then analyzed by PCR.

The instructions in the FDA method were followed for setting up the PCR thermocycler—7500 Fast—parameters. The activation step was set (1 cycle) to 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s with “collect data on hold”. Following the last cycle of the PCR reaction, the temperature ramped from 60°C for 1 min to 95°C for 15 s at a 1% ramp rate. The master mix was prepared with n + 4 reactions, with each reaction containing 1.25 µL of 10 µM (acid-fast bacilli) AFB primer mix, 12.5 µL 2x FastStart Universal SYBR green master mix, and 9.25 µL molecular-grade water. Aliquots of 23 µL of the master mixes were dispensed into each PCR tube. Two microliters of extracted DNA or positive/negative control were added. The caps of the PCR tubes were closed, mixed, centrifuged briefly, then placed into the thermocycler and analyzed.

MYChrOme Culture Plate.—Methodology.—A 200 mL test portion was filter-concentrated using a 0.22 µm pore size track-etched 47 mm polycarbonate membrane. The filter was placed in a separate 15 mL conical tube, resuspended with 10 mL of 1:40 sterile Ringer's solution, and vortexed for 30 s. For the non-potable matrix, a 981 µL aliquot of the concentrated sample was added to 19 µL of MYCOn in a microcentrifuge tube. This sample was mixed twice by pipetting up and down, then incubated at room temperature (20–25°C) for 5 min. A 100 µL aliquot of treated or untreated sample was plated onto a MYChrOme culture plate. Serial dilutions were prepared in sterile PBS and plated in the same manner. Plates were incubated at 30 ± 1°C for 7 days after which typical colonies were enumerated. White colonies were confirmed as NTM with a Mycobacterium genus-specific real-time PCR (see Confirmation section).

Results.—Matrix study results are shown in Table 5. Each contamination level of the candidate method was compared to the modified FDA method using an unpaired equivalency test assuming equal variances with a 90% confidence interval. For the potable water matrix study, in comparison to both 7H10 and 7H11S plates used in the reference method, MYChrOme had consistently higher mean log10 results; therefore, the candidate method is not equivalent to the reference method but demonstrated higher recovery of the target organisms. The one exception being the medium contamination level in comparison to the 7H10 plate, where the reference method had a slightly higher average enumeration of NTM colonies.

Table 5.

Matrix study results for potable and non-potable water matrixes

Matrix	Cont. levela	Nb	MYChrOme Culture Plate			FDA Method			Mean diff.f	90% CIg
			Mean Log10 CFU/mLc	srd	RSDr, %e	Mean Log10 CFU/mL	sr	RSDr, %		LCLh	UCLi
	Untreated			M7H10
Potable water	Low	5	0.86	0.38	43.46	0.34	0.59	172.07	0.52	–0.05	1.10
	Medium	5	1.97	0.47	23.80	1.99	0.30	15.10	–0.08	–0.54	0.39
	High	5	3.50	0.44	12.44	3.48	0.44	12.66	0.02	–0.49	0.54
			Untreated			M7H11S
	Low	5	0.86	0.38	43.46	0.28	0.50	180.53	0.59	0.06	1.11
	Medium	5	1.92	0.47	24.51	1.90	0.41	21.72	0.02	–0.51	0.54
	High	5	3.50	0.44	12.44	3.44	0.40	11.55	0.06	–0.43	0.55
Non-potable water (method developer)			Treated			M7H10
	Low	5	1.41	0.28	19.67	0.80	0.22	27.39	0.61	0.32	0.91
	Medium	5	1.88	0.27	14.12	1.18	0.08	6.83	0.69	0.46	0.92
	High	5	2.86	0.06	2.16	1.31	0.14	10.82	1.55	1.42	1.67
			Treated			M7H11S
	Low	5	1.41	0.28	19.67	–0.35	0.39	–109.67	1.77	1.37	2.16
	Medium	5	1.88	0.27	14.12	0.38	0.10	26.03	1.49	1.26	1.73
	High	5	2.86	0.06	2.16	0.27	0.08	30.69	2.59	2.50	2.67
Non–potable water (independent lab)			Treated			M7H10
	Low	5	2.24	0.25	11.2	2.28	0.64	28.1	–0.042	–0.62	0.53
	Medium	5	2.99	0.24	8.00	2.24	0.22	9.96	0.75	0.48	1.02
	High	5	3.91	0.58	14.7	2.83	0.61	21.7	1.08	0.38	1.78
			Treated			M7H11S
	Low	5	2.24	0.25	11.25	1.48	0.79	53.3	0.77	0.08	1.45
	Medium	5	2.99	0.24	8.00	2.00	0.00	0.00	0.99	0.79	1.19
	High	5	3.91	0.58	14.7	2.00	0.00	0.00	1.91	1.43	2.39

All potable matrixes are artificially contaminated, all non-potable matrixes are naturally contaminated.

Number of test portions.

Mean of five replicate portions, plated in duplicate, after logarithmic transformation: Log10[CFU/g + (0.1)f].

Repeatability standard deviation.

Relative standard deviation for repeatability.

Mean difference between the candidate and reference methods.

Confidence interval based on paired statistical analysis.

90% Lower confidence limit for difference of means.

90% Upper confidence limit for difference of means.

For the non-potable matrix study, the candidate method was not equivalent to the reference method, with significantly more NTM colonies enumerated on MYChrOme. The relative standard deviation for both reference and candidate method were low (<0.4 and <0.3, respectively).

Independent Laboratory Studies

Methodology.—Non-potable water was evaluated for rapid-growing NTM by the MYChrOme Culture Plate and modified US FDA method. The same matrix study design was followed as above. Non-potable water (residential laundry rinse water) was inoculated with M. chelonae (CCUG 37827) in liquid form. The samples were allowed to equilibrate at 4°C for 72 h after contamination. The candidate method and reference method were performed on the same day for each contamination level. The candidate and reference methods were evaluated using unpaired test portions.

The same methods used in the method developer matrix study for the reference method and candidate method were followed with the following modifications. Suspect colonies from both methods were confirmed using the US FDA AFB PCR method. After concentration of the MYChrOme protocol test portion, the filter was resuspended in 10 mL of filtrate.

Results.—The results are summarized in Table 5. The MYChrOme Culture Plate method and modified US FDA method were not equivalent with results favoring the candidate method for each contamination level in comparison to 7H11S plates. The same was observed in comparison to the 7H10 plates with the exception of the low contamination level that had a difference of means of –0.042. The MYCOn decontamination reagent appeared to decrease background microflora on the plates for some samples but not all samples. Naturally occurring Mycobacterium was detected in the uninoculated non-potable water which is indicative of real-world samples.

Discussion

The results of this validation study indicate that the MYChrOme method is equivalent, and in some areas, an improvement upon the reference method for the detection of NTM. These results also validate that the MYChrOme method is successful in differentiating NTM from other bacteria.

Fifty rapid-growing NTM strains were tested in the inclusivity portion of this study. Ninety-four percent of the strains grew on MYChrOme within 7 days and 98% of the strains grew within 14 days. The only NTM strain that did not grow on MYChrOme was M. murale, a rare species of NTM that has only been isolated from the walls of a daycare in Finland (6). This species was included to be able to evaluate 50 diverse strains and would not be expected to be found in product testing. All inclusivity strains that grew on MYChrOme were not colorized by the crystal violet dye and thus were easily distinguishable on the media except for M. abscessus subsp. massiliense, which was white with a slight purple pigment.

There were 30 non-mycobacteria species that were tested for exclusivity and 29 were either not detected or grew purple colonies on MYChrOme (treated and untreated). Nocardia brasiliensis grew only 31 off-white colonies after being plated at >106 CFU/mL. This high concentration of the bacteria indicates a worst-case scenario and typically would not be found in real-world samples. N. brasiliensis is an acid-fast bacteria, so it was expected to produce white colonies on the MYChrOme culture plate if any cells could survive on the crystal violet. There was approximately a 4 log10 reduction in N. brasiliensis colonies on the untreated MYChrOme Culture Plate, and all colonies were eliminated on the treated plate. Colony confirmation with colony PCR eliminated this false-positive result.

The robustness study showed that the MYChrOme Culture Plate method can withstand modest variation in three critical parameters simultaneously. The product consistency study supports a 3-month shelf-life at 4°C from the time of manufacturing.

For both method developer and independent laboratory non-potable matrix studies, the candidate method achieved higher plate counts than the reference method. One exception being the low contamination level for the independent laboratory study in comparison to the 7H10 plates, where slight differences were observed. All non-potable samples analyzed by the method developer with the MYChrOme method had white colonies with all non-mycobacteria eliminated by the decontamination step. On 7H10 and 7H11S plates, some background microbiota were present that increased analysis time. The MYChrOme protocol was innovated specifically to address the issue of high background microbiota in non-potable water samples. Similar results were observed in the potable water matrix study, with a difference of means in favor of the candidate method, in all but one sample set. A slightly higher difference of means in favor of the 7H10 data set for the medium contamination level was noted. The disparities in colony counts between the two methods could be due to the fact that the FDA method is meant for tattoo ink analysis; however, minor modifications were included in the approved study outline to make the reference method more applicable to water samples. Overall, the same number of matrix study samples had positive NTM detections by the candidate method and the 7H10 reference method. Additionally, the MYChrOme method achieved results in 7 days versus 14 days. These results indicate that the MYChrOme Culture Plate is a comparable method to current standards for the detection of NTM in water samples.

Conclusions

Based on the results of this validation study, it is recommended that the MYChrOme Culture Plate be granted Performance Tested Method status for detection of rapid-growing NTM in potable and non-potable water sources.

Credit Author Statement

Katherine Fisher: Conceptualization, Investigation, Formal Analysis, Writing—original draft. Avneet Chhabra: Conceptualization, Investigation, Methodology, Writing—original draft. Leah Wickenberg: Resources, Project Administration, Writing—review & editing. William McCoy: Supervision, Funding acquisition.

Acknowledgments

Submitting Company

Phigenics, LLC

3S701 West Ave. Suite 100

Warrenville, IL 60555

Independent Laboratory

Silliker, Inc. dba Mérieux NutriSciences

3600 Eagle Nest Drive

Crete, IL 60417

Reviewers

James Agin

Ohio Department of Agriculture (Retired)

Columbus, OH

Wayne Ziemer

USDA FERN (Retired)

Loganville, GA

Thomas Hammack

Office of Regulatory Science

Center for Food Safety and Applied Nutrition

U.S. Food and Drug Administration

5100 Paint Branch Parkway

College Park, MD 20740

Conflict of Interest

During this validation study, all authors were employed by Phigenics, LLC.