Recovery of Nontuberculous Mycobacteria and Nocardiae Causing Skin/Soft Tissue Infections by Use of the Copan ESwab Collection and Transport System

LETTER

Recent reports have shown that infections with nontuberculous mycobacteria (NTM) are increasing worldwide and often are difficult to diagnose and to treat (1–3). Consequently, improved diagnostics are needed. Clinical and Laboratory Standards Institute (CLSI) guidelines (4) describe methods for assessing the ability of transport devices to maintain microorganisms in a viable condition for up to 48 h during transport at room temperature (RT) (20 to 25°C) and refrigerator temperature (FT) (2 to 8°C). There are no formal published data on the viability of NTM using swab-based systems; however, a study conducted only at RT was presented at the 110th General Meeting of the American Society for Microbiology (5). The present investigation was designed, using CLSI methods, for evaluation of the Copan ESwab transport system (Copan Diagnostics Inc., Murrieta, CA) for NTM species commonly causing skin and soft tissue infections.

Nine NTM clinical isolates (Mycobacterium porcinum, Mycobacterium abscessus subsp. abscessus, Mycobacterium senegalense, Mycobacterium mucogenicum, Mycobacterium fortuitum, Mycobacterium avium, Mycobacterium chelonae, Mycobacterium marinum, and Mycobacterium haemophilum), which had been previously identified using the Bruker MALDI Biotyper CA system (Bruker Daltonics Inc., Billerica, MA, USA), and Nocardia asteroides ATCC 19247 were evaluated in this study. After 5 to 7 days of growth on blood agar plates, a heavy stock suspension of each organism was prepared in 4.0 ml of sterile saline and vigorously vortex mixed. From this, suspensions were prepared in triplicate and adjusted to match a 0.5 MacFarland turbidity standard (1.5 × 10⁸ CFU/ml), using a Vitek nephelometer (6). Each suspension was serially diluted 1:10 in normal saline to obtain working suspensions of ∼1.5 × 10⁷ CFU/ml to ∼1.5 × 10³ CFU/ml. One hundred microliters of each working suspension was placed in 18 wells of a microtiter plate. Flocked swabs were placed in the wells, allowed to absorb the inoculum for ∼10 s, and returned to their respective labeled ESwab transport vials. Each microorganism/device combination was assayed in triplicate at three time points (0, 24, and 48 h). The 0-h swabs were removed from the transport vials, containing 1.0 ml of Amies broth, after ∼15 min of incubation and discarded; 100 μl of the inoculum was plated onto a Middlebrook 7H10 agar plate, streaked for isolation, and incubated at 30°C in O₂. The remaining ESwabs were incubated at RT or FT for 24 or 48 h. Results were calculated by taking an average of the colony counts from triplicate tests. Counts at 24 and 48 h were compared to 0-h reference counts.

Results are summarized in Table 1. The CLSI criterion for acceptable recovery using the roll plate method (i.e., ≥5 colonies recovered from the dilution yielding a baseline count closest to 250 colonies [1]) was met for 6 of the 10 organisms tested at 24 h in RT incubation. Of these 6, 5 also met the CLSI criterion at 48 h at RT. Another 4 NTM organisms met the criteria within 10% at 48 h at RT. Nine organisms produced CFU within ∼50% of the baseline CFU at 24 h and 48 h at FT. The exception was M. marinum, which showed overgrowth after 48 h of storage. At the 10⁶ dilution, 7 organisms yielded colonies too numerous to count (TNTC). At 10⁵ and/or 10⁴ dilutions, the same 7 yielded countable colonies within ∼250 CFU/ml at both 24 and 48 h, compared to 0-h counts, except for M. marinum, which at 10⁵ yielded colonies TNTC at 48 h at RT. M. mucogenicum, M. fortuitum, and N. asteroides had lower counts and produced countable colonies at 10⁶ dilution. Mycobacterial cells are a bit larger than routine bacteria; therefore, initial serial dilutions made from a 0.5 McFarland standard contained the desired concentrations of bacteria at higher dilutions.

TABLE 1.

Recovery of NTM and N. asteroides held for 24 or 48 h at RT or FT, using the Copan ESwab transport system

Organism and starting concn (CFU/ml)a	Recovery (CFU/ml)b after:
	0 h	24 h at RT	48 h at RT	24 h at FT	48 h at FT
M. porcinum
106	TNTC	TNTC	TNTC	TNTC	TNTC
105	82	75	80	61	48
104	6	4	5	7	5
M. abscessus
106	TNTC	TNTC	TNTC	TNTC	TNTC
105	335	370	276	293	284
104	36	43	27	35	30
M. senegalense
106	TNTC	TNTC	TNTC	TNTC	TNTC
105	246	241	237	238	239
104	30	30	32	25	25
M. mucogenicum
106	107	108	108	112	106
105	4	6	5	5	6
104	0	0	0	0	0
M. fortuitum
106	205	290	240	184	155
105	12	17	14	12	12
104	1	3	2	1	1
M. avium
106	TNTC	TNTC	TNTC	TNTC	TNTC
105	143	150	152	129	123
104	17	19	21	16	17
M. chelonae
106	TNTC	TNTC	TNTC	TNTC	TNTC
105	270	263	243	242	230
104	25	22	16	17	20
M. marinum
106	TNTC	TNTC	TNTC	TNTC	TNTC
105	166	235	TNTC	148	138
104	9	22	433	7	6
M. haemophilum
106	TNTC	TNTC	TNTC	TNTC	TNTC
105	257	282	298	258	257
104	30	33	34	35	28
N. asteroides
106	125	114	92	91	60
105	11	8	7	11	6
104	0	0	0	0	0

^aTenfold (10⁶, 10⁵, and 10⁴) serial dilutions starting from a 0.5 McFarland standard were made.

^bData are expressed as the average CFU (performed in triplicate).

Our results indicate that the Copan ESwab collection and transport device appears to be an appropriate system for the maintenance, transport, and recovery of select NTM and Nocardia species, by maintaining the viability of NTM species known to cause skin and soft tissue infections and an ATCC strain of N. asteroides with fair efficiency for up to 48 h at RT. However, until further studies are carried out in clinical settings to confirm the utility of swab specimens, tissue/fluid samples remain the preferred specimen type for detection of NTM. Studies directly comparing flocked swabs to paired tissue/fluid specimens are needed. Further study is also required to determine the utility of the ESwab in recovering NTM and Nocardia species from swab specimens collected from patients with suspected infections.