Comparison of the Copan ESwab System with an Agar Swab Transport System for Maintenance of Fastidious Anaerobic Bacteria Viability

Abstract

We compared the eSwab system to a swab with an anaerobic transport semi-solid agar system for their capacities to maintain viability of 20 species of fastidious anaerobes inoculated on the bench and held at ambient and refrigerator temperature for 24 and 48h. On average, both systems maintained similar viability among analogous groups of organisms at both temperatures although there were quantitative differences among some species.

Introduction

Suitable specimen transport from collection to the laboratory is essential for accurate laboratory diagnosis. Given increasing laboratory centralization, transport times have increased as well, requiring systems to be robust enough to ensure sufficient organism collection, viability and release. Specimens with anaerobic organisms have the added requirement of anaerobiasis for at least 48 hours. The eSwab (Copan Diagnostics, Inc., Murrieta, CA) is a relatively new system compared to conventional gel-tube systems and lends itself to automation. The eSwab consists of a nylon-flocked swab, which provides better capillary action and strong hydraulic uptake of liquids compared to spun-fiber nylon or rayon swabs (1) and a screw-top tube containing liquid modified Amies medium. After specimen collection, the swab is inserted into the tube and the scored shaft of the swab is easily broken to the length of the tube. A swab capture system in the cap locks the broken shaft into the lid of the tube after it is fully closed. Favorable release studies comparing the flocked swab to convention rayon or Dacron swabs have been performed (2), as well as other studies comparing viability of aerobic and a small number of anaerobic organisms (1, 3-7). The recommended CLSI standard control strains have been shown in a previous study (1) to meet the requirements of the M40-A recommendations for transport systems (8). This is the first study comparing numerous fastidious anaerobic bacteria. We compare the eSwab with Anaerobic Transport Medium (ATM) (Anaerobe Systems, Morgan Hill, CA), both of which use modified Amies medium in liquid and gel form respectively, for the release and recovery of fastidious anaerobic bacteria from the swabs after 24 and 48h at 4°C and room temperature (RT).

Materials and Methods

Twenty fastidious anaerobes, nine Gram-positive and 11 Gram-negative, from various sources were selected for study (Table 1). The organisms were identified by standard (9, 10) or molecular methods. This feasibility study of the recovery of various fastidious anaerobic bacteria was based on the CLSI (NCCLS) document M40-A (8) the approved standard for quality control of transport media. A 24–48h subculture of each organism was suspended in saline in the anaerobe chamber to a turbidity of 0.5 McFarland (~1.5×108 CFU/ml). To mimic clinical settings, the inoculation suspension was transferred to room air and 0.1 ml aliquots were pipetted into microcentrifuge tubes to inoculate eSwabs and rayon swabs for the ATM system. Each system was set up for recovery testing at room temperature and 4°C; each temperature had separate tubes set up for subculture at t=0, 24 and 48h. At each sampling time a suspension was made from each tube. The eSwab tube was vortexed for 5 sec, whereas the rayon swabs were removed from the ATM, the tip placed in 0.9 ml saline and vortexed for 5 sec. Each suspension was serially diluted, plated onto Brucella agar, incubated in an anaerobic chamber for 24–72h at 37°C and colony counts determined. The inoculum suspension was also serially diluted and colony counts were performed. Although the CLSI M40A quality control standard recommends dilutions in triplicate and platings in duplicate, because this was a performance study of each transport system and not a quantitative quality control analysis, each organism was studied once and each dilution was plated once. If, however, the colony counts from the serial dilutions were inconsistent, the organism was repeated. In addition, C. difficile the dilutions were also plated onto CCFA-HT in order to better recover spores, which germinate better in the presence of taurocholate (11).

Release of Sample From Swabs

The eSwabs released more organisms than did the rayon swabs although, on average, the difference was minor (Table 2). There were some exceptions (Figure 1a, 1b).

In the Gram-negative group the eSwabs and rayon swabs retained 1.5 and 1.9 log10 CFU/ml on average, respectively. All Fusobacterium spp. were retained ~1 log10 CFU/ml more than the Gram-negative group average by both swab systems. In the Gram-positive group, the eSwabs and rayon swabs retained 1.5 and 1.6 log10 CFU/ml on average, respectively. Finegoldia magna was retained by both swab systems ~1.5 log10 CFU/ml more than the Gram-positive group average. In the Clostridium spp. group, the eSwabs and rayon swabs were retained 1.4 and 2.1 log10 CFU/ml on average, respectively. C. ramosum was retained by 0.7 log10 CFU/ml more with the eSwab and 1.6 log10 CFU/ml more than the rayon swab compared to the Clostridium spp. group average.

Recovery of Sample

All organisms were recovered at room temperature and 4°C at t = 0, 24 and 48h (Figures 2–4). Overall, both Gram-positive and Gram-negative organisms maintained similar average viability in both systems at room temperature (RT) and 4°C (Table 2); however, there were some exceptions.

In the Gram-negative group (Figures 2a, 2b), the best recovery of all organisms over t0–24 and t24–48 at 4°C and RT were Bacteroides spp. and Bilophila wadsworthia, with an average loss of only 0.1 log10 CFU/ml over 48h.

At 24h, F. necrophorum lost 0.9 log10 CFU/ml in ATM at 4°C and RT but had almost no loss in the eSwab. At 48 h, there was 0.8 log10 CFU/ml loss in ATM at 4°C and RT, however, in the eSwab there was a loss of 1.4 log10 CFU/ml at RT but only 0.3 log10 6 CFU/ml at 4°C. Best performance for F. necrophorum was the eSwab at 4°C. The two F. nucleatum species had mixed results. One strain lost >1 log10 CFU/ml at 24h in both systems and temperatures; the loss was less at 48h for the eSwab at RT and the ATM at 4°C and RT, but the eSwab lost >1 log10 CFU/ml at 4°C. The other F. nucleatum strain lost an average of 0.5 log10 CFU/ml in the eSwab at RT and ATM at 4°C and RT but lost 2.2 log10 CFU/ml in the eSwab at 4°C. Fusobacteria had the most loss in the Gram negative group in both systems.

P. asaccharolytica and P. gingivalis had <1 log10 CFU/ml loss in both systems and temperatures over 48 h despite their very fastidious nature.

On average, the Prevotella species lost most during the first 24h, 0.9 log10 CFU/ml t0–24 and 0.5 log10 CFU/ml t24–48. After 48h, P. buccae decreased only 0.5 log10 on average at 4°C and RT in the eSwab, but in the ATM lost 2.5 log10 CFU/ml at RT and 1.1 log10 CFU/ml at 4°C. P. melaninogenica lost 2.2 and 1.0 log10 CFU/ml in the ATM at RT and 4°C; the eSwab loss was 1.3 and 0.7 log10 CFU/ml at RT and 4°C. P. intermedia performed similarly to P. melaninogenica except the eSwab loss at RT was 3.3 log10 CFU/ml. The best performance for all Prevotella species was the eSwab at 4°C.

In the Gram-positive group (Figure 3), the average loss was 0.3 and 0.2 log10 CFU/ml at t0–24h and t24–48h respectively. Both systems performed similarly on average at RT and 4°C with the exception of P. anaerobius, which lost 2.2 and 1.9 log10 CFU/ml at RT and 4°C over 48h.

Clostridium spp. varied considerably (Figure 4), those strains known for producing more spores (e.g. C. difficile, ribotype 027) lost less in the first 24h than in the second. The average loss of sample of C. clostridioforme and C. ramosum was 1.1 log10 CFU/ml at t0–24h and no average loss at t24–48h. The average loss of sample of C. difficile ribotype 027 was greater at t24–48h than at t0–24h. C. clostridioforme did not perform as well in the eSwab system at t0–24h and t24–48h.

All counts were higher on HT than Brucella with the exception of the 027 ribotype of C. difficile, indicating more organism recovery from HT than suggested by Brucella (results not shown).

The eSwab is an all-in-one collection device that was shown to provide equal or superior release, viability and recovery performance for 48h at both room temperature and 4°C with most fastidious anaerobic bacteria compared to the conventional anaerobic transport system consisting of a rayon swab and an anaerobic transport tube. In addition, the eSwab provides the added ability to be used in automated specimen plating devices.

Acknowledgements

This study was funded by a research grant from Copan Diagnostics, Inc.

References

1. Van Horn KG, Audette CD, Sebeck D, Tucker KA. 2008. Comparison of the Copan eSwab system with two Amies agar swab transport systems for maintenance of microorganism viability. J Clin Microbiol 46:1655-1658.
2. Van Horn KG, Audette CD, Tucker KA, Sebeck D. 2008. Comparison of 3 swab transport systems for direct release and recovery of aerobic and anaerobic bacteria. Diagn Microbiol Infect Dis 62:471-473.
3. Fontana C, Favaro M, Limongi D, Pivonkova J, Favalli C. 2009. Comparison of the eSwab collection and transportation system to an amies gel transystem for Gram stain of clinical specimens. BMC Res Notes 2:244.
4. Nys S, Vijgen S, Magerman K, Cartuyvels R. 2010. Comparison of Copan eSwab with the Copan Venturi Transystem for the quantitative survival of Escherichia coli, Streptococcus agalactiae and Candida albicans. Eur J Clin Microbiol Infect Dis 29:453-456.
5. Silbert S, Kubasek C, Uy D, Widen R. 2014. Comparison of eSwab with traditional swabs for detection of methicillin-resistant Staphylococcus aureus using two different walk-away commercial real-time PCR methods. J Clin Microbiol 52:2641-2643.
6. Smismans A, Verhaegen J, Schuermans A, Frans J. 2009. Evaluation of the Copan eSwab transport system for the detection of methicillin-resistant Staphylococcus aureus: a laboratory and clinical study. Diagn Microbiol Infect Dis 65:108-111.
7. Stoner KA, Rabe LK, Austin MN, Meyn LA, Hillier SL. 2008. Quantitative survival of aerobic and anaerobic microorganisms in Port-A-Cul and Copan transport systems. J Clin Microbiol 46:2739-2744.
8. NCCLS. 2003. Quality control of microbiological transport systems; Approved Standard. NCCLS document M40-A. National Committee for Clinical Laboratory Standards, Wayne, Pa.
9. Jousimies-Somer HR, Summanen P, Citron DM, Baron EJ, Wexler HM, Finegold SM. 2002. Wadsworth-KTL Anaerobic Bacteriology Manual, vol 6th ed. Star Publishing, Belmont, CA.
10. Versalovic J, Carrol KC, Funke G, Jorgensen JH, Landry ML, Warnock DW. 2011. Manual of clinical microbiology, vol 10th. ASM Press, Washington, DC.
11. Tyrrell KL, Citron DM, Leoncio ES, Merriam CV, Goldstein EJ. 2013. Evaluation of cycloserine-cefoxitin fructose agar (CCFA), CCFA with horse blood and taurocholate, and cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme for recovery of Clostridium difficile isolates from fecal samples. J Clin Microbiol 51:3094-3096.