Comparison of Copan ESwab™ with the Copan Venturi Transystem for the quantitative survival of Escherichia coli, Streptococcus agalactiae and Candida albicans

Abstract

Swab transport systems should preserve the viability and stability of micro-organisms in clinical specimens throughout transport and storage. ESwab™, a nylontipped swab in liquid medium, designed for better specimen collection and less micro-organism entrapment, was evaluated for the maintenance of viability and quantitative survival of Escherichia coli, Streptococcus agalactiae and Candida albicans. The quantitative elution method was used to evaluate ESwab™ in vitro. In vitro, the recovery of the three micro-organisms was higher in ESwab™ (97–100%) as compared to the Copan Venturi Transystem (CVT) (86– 96%) at room temperature (RT) for time point 0 h and remained similar after 6 h. E. coli and C. albicans proliferated in both transport systems when preserved beyond 6 h. At 4°C, the recovery of ESwab™ was higher (>94%) compared to CVT (77–94%) for the microorganisms tested. ESwab™ did not only meet the Clinical Laboratory and Standards Institute (CLSI) criteria for microbiological transport devices, but as its recovery rate in vitro was higher than that of CVT, it might also enhance the sensitivity of bacterial culture in the future.

Introduction

Swab transport systems should preserve the viability and stability of micro-organisms in clinical specimens throughout the transport and storage process. Most swab collection devices currently on the market are prepared with rayon, Dacron or cotton fibres wound onto the tip of the swab shaft. The flocked swab (COPAN) is a nylon-tipped swab prepared by a spray-on flocked fibre technique, developed for the transport of bacteria, yeast and viruses [1, 2]. This technique provides stronger capillary action and strong hydraulic uptake of liquids, which should result in better specimen collection. This design should also provide more efficient uptake and release of specimen material and, therefore, less entrapment of specimens. The new transport device, ESwab™, is the combination of the new nylon-flocked swab with the modified liquid Amies transport medium. Up until now, only two studies describe the comparison of this new flocked transport system with the current systems available for the recovery and maintenance of aerobic and anaerobic bacteria [1, 2]. Both studies describe the performance of ESwab™ for the strains described in the CLSI document M40-A [3], i.e. fastidious organisms and strains difficult to culture. These data are also indicated on the package insert provided by Copan. On easy-growing aerobic bacteria or yeasts however, no information is available yet. Therefore, in this study, ESwab™ was compared with an Amies agar swab system for the maintenance of viability and the quantitative survival of Escherichia coli, Streptococcus agalactiae and Candida albicans in vitro.

Materials and Methods

Strains The three strains tested were E. coli ATCC 25922, S. agalactiae ATCC 13813 and C. albicans ATCC 90028.

Quantitative elution The quantitative elution method, as described in the CLSI document M40-A [3], was used to evaluate the performance of ESwab™ (480CE, COPAN) and the Copan Venturi Transystem (CVT, 108C.USE, COPAN). For each microorganism, inoculated swabs were preserved at room temperature (RT) and 4°C for 0 (15 min after inoculation), 6, 24 and 48 h. Also, three mixtures of E. coli and S. agalactiae (900/100 μL, 500/500 μL and 100/900 μL) were tested under the same conditions.

In each experiment, strains were freshly sub-cultured twice onto Columbia agar plates supplemented with 5% sheep blood (COL5%, 221165, BD Diagnostics, US) and incubated in 5% CO2 at 35°C. In the single-strain experiments, an initial organism suspension of 0.5 McFarland standard (approximately 108cfu/mL) in sterile saline (0.85% NaCl w/v) was prepared and 10-fold-diluted to obtain a suspension of 107cfu/mL (also used as the positive control suspension). For each time point (n=4) and each temperature (n=2), one swab was inoculated with 100 μL of the 107-cfu/mL suspension for the three organisms tested and allowed to absorb the fluid completely (∼30 s). Subsequently, all swabs (24 CVT and 24 ESwab™) were closed in their respective tube, i.e. ESwab™ or CVT, and stored at the proper conditions. This experiment was performed in triplicate. The swabs for time point zero (T=0) were processed 15 min after inoculation.

The CVTswabs were mixed well into 1 mL of sterile saline in order to obtain ∼106cfu/mL, as already present in the ESwab™ liquid medium. Subsequently, the CVT suspension and ESwab™ medium were serially 10-fold-diluted. A 100-μL aliquot of each dilution was inoculated to duplicate COL5% and spread over the entire surface using a sterile L-spatula (174CS01, COPAN). Plates were incubated for 18–24 h in 5% CO2 at 35°C. Colony counts were obtained by reading plates with 30 to 300 countable colonies. A series of 10-fold dilutions of the positive control suspension was used as growth controls.

In the mixed experiments of both E. coli and S. agalactiae, an initial organism suspension of 0.5 McFarland (approximately 108cfu/mL) in sterile saline was prepared and 10-fold-diluted to obtain a suspension of 107 cfu/mL. Of both 107-cfu/mL suspensions, a mixture was made with ratios of 900/100 μL, 500/500 μL and 100/ 900 μL E. coli/S. agalactiae, respectively. These mixed suspensions were also used as positive growth controls. Swabs were further inoculated and processed with the mixtures as described above for the single-organism suspensions. This experiment was performed in triplicate. After preservation for 0, 6, 24 or 48 h at RT or 4°C, a 100- μL aliquot of the diluted sample was inoculated onto duplicate MacConkey (221270, BD Diagnostics, US) and CNA agar (221353, BD Diagnostics, US) for the enumeration of E. coli and S. agalactiae, respectively. MacConkey plates were incubated for 18–24 h at 35°C in ambient air, whereas CNA plates where incubated in 5% CO2 at 35°C. Colony counts were obtained by reading plates with 30 to 300 countable colonies. A series of 10-fold dilutions of the positive control suspension was used as growth controls.

The log10 of the average cfu recovered was determined for the growth controls (100%) and for each swab type, temperature and time combination, and based upon a cfu comparison with the growth controls expressed in % recovery. According to the CLSI guideline M40-A [3], for samples stored at RT, no more than a 3 log10 decline in cfu is accepted. At RT, for overgrowth, no limit for acceptance is defined. For samples stored at 4°C, no more than a 1 log10 increase in cfu and 3 log10 decline in cfu is acceptable.