Comparison of FecalSwab and ESwab™ Devices for Storage and Transportation of Diarrheagenic Bacteria
Using a collection (n12) of ATCC and known stock isolates, as well as 328 clinical stool specimens, we evaluated the ESwab™ and the new FecalSwab liquid-based microbiology (LBM) devices for storing and transporting diarrheagenic bacteria. The stock isolates were stored in these swab devices up to 48 h at refrigeration (4°C) or room (25°C) temperature and up to 3 months at 20°C or70°C. With the clinical stool specimens, the performances of the ESwab™ and FecalSwab were compared to those of routinely used transport systems (Amies gel swabs and dry containers). At a refrigeration temperature, all isolates survived in FecalSwab up to 48 h, while in ESwab™, only 10 isolates (83.3%) out of 12 survived. At70°C, all isolates in FecalSwab were recovered after 3 months of storage, whereas in ESwab™, none of the isolates were recovered. At20°C, neither of the swab devices preserved the viability of stock isolates after 2 weeks of storage, and at room temperature, 7 (58.3%) of the stock isolates were recovered in both transport devices after 48 h. Of the 328 fecal specimens, 44 (13.4%) were positive for one of the common diarrheagenic bacterial species with all transport systems used. Thus, the suitability of the ESwab™ and FecalSwab devices for culturing fresh stools was at least equal to those of the Amies gel swabs and dry containers. Although the ESwab™ was shown to be an option for collecting and transporting fecal specimens, the FecalSwab device had clearly better preserving properties under different storage conditions.
Appropriate specimen collection and transport are essential for accurate laboratory diagnosis of bacterial infections. Swab collection has been the most frequently used method in health care settings because swabs are inexpensive and specimens are easy to collect, although it may not be the best approach for detecting, e.g., anaerobic and fastidious organisms (1, 2). Recent improvements in the swab tip material and the transport medium have, however, greatly enhanced the recovery and viability of various microorganisms present in specimens (3–6). The flocked nylon swabs (FLOQSwabs) with liquid transport media have shown to yield greater organism release than cotton swabs in dry containers (6) and rayon- or Dacron-based swabs in Amies gel (4).
In this study, we evaluated the performances of the ESwab™ (COPAN) and the recently launched FecalSwab (COPAN) liquid-based microbiology (LBM) devices for maintaining the viability of diarrheagenic bacteria at different temperatures. In addition, the suitability of these swab systems for the recovering enteric pathogens in stool specimens was assessed in comparison with the suitability of the routinely used dry containers and Amies gel swabs (COPAN).
(These results were presented in part at the 113th General Meeting of the American Society for Microbiology, 18 to 21 May 2013, Denver, CO .)
MATERIALS AND METHODS
The survival of gastrointestinal bacterial pathogens in the ESwab™ and FecalSwab devices was investigated using four ATCC bacterial strains and eight known clinical bacterial stock isolates. The ATCC strains were Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028, Shigella sonnei ATCC 9290, Yersinia enterocolitica ATCC 23715, and Campylobacter jejuni ATCC 33291. The clinical isolates were enterohemorrhagic Escherichia coli serotype O157:H7, S. enterica subsp. enterica serovar Enteritidis,Shigella flexneri, Campylobacter coli, Vibrio cholerae, Aeromonas hydrophila, Plesiomonas shigelloides, and the tcdB gene-positive Clostridium difficile. All strains except Campylobacter species and C. difficile were cultured from70°C stocks on 5% sheep blood agar (Becton, Dickinson, Sparks, MD, USA) at 35°C for 16 to 24 h. C. coli and C. jejuni were cultured on Campylobacter blood-free selective medium (Oxoid Ltd., Thermo Fisher Scientific, Inc., Basingstoke, Hampshire, United Kingdom) and incubated at 42°C in a microaerophilic atmosphere for 48 h. C. difficile was cultured on fastidious anaerobe agar (Lab M Ltd., Lancashire, United Kingdom) at 35°C in an anaerobic atmosphere for 48 h.
The inocula of the stock isolates were prepared in 0.9% NaCl to equal a 0.5 McFarland standard (approximately 1.5 108 CFU/ml) using a nephelometer (DensiCHEK Plus; bioMérieux, Inc., Durham, NC, USA). Next, each preparation was serially diluted (10-fold dilutions) in order to get 1.5 103 to 1.5 104 CFU/ml to be inoculated in duplicate into the ESwab™ and FecalSwab devices. The amount of inoculum that was added into each device was 100 l. Each inoculated swab device was vortexed for 15 s and stored for 48 h at room temperature (RT) (25°C) or a refrigeration temperature (4°C), and for 3 months at 20°C or 70°C. The colony counts in each swab device was determined at storage times of 0, 6, 24, and 48 h (from devices stored at RT and 4°C) or at storage times of 0 h, 2 weeks, 1 month, and 3 months (from devices stored at 20°C or 70°C) by triplicate plating of 10 l ESwab™ and FecalSwab medium on either 5% sheep blood agar, Campylobacter blood-free selective medium, or fastidious anaerobe agar. The plates for each organism were incubated as mentioned above. In addition, mixtures of E. coli strain ATCC 25922, Enterococcus faecalis strain ATCC 29212, and S. enterica serovar Typhimurium strain ATCC 14028 or S. sonnei strain ATCC 9290 were prepared in duplicate and stored at RT and 4°C for 48 h. The simulated mixed specimens were plated on cystine lactose electrolyte-deficient (CLED) agar (Oxoid Ltd., Thermo Fisher Scientific, Inc., Basingstoke, Hampshire, United Kingdom) at 0, 6, 24, and 48 h of storage and incubated at 35°C for 16 to 24 h. Fisher’s exact test was used to determine the statistical significance of the differences in performance between the ESwab™ and FecalSwab devices.
Clinical stool specimens were collected from 328 patients presenting with gastroenteritis (n 228) or antibiotic-associated diarrhea (n 100) at Vaasa Central Hospital from December 2012 to February 2013. The specimens from the patients with gastroenteritis were aliquoted into one dry container, one Amies gel swab, two FecalSwab devices, and two ESwab™ devices and were cultured immediately after receiving them at the hospital laboratory on xylose-lysine-deoxycholate (XLD) agar (Oxoid Ltd., Thermo Fisher Scientific, Inc., Basingstoke, Hampshire, United Kingdom), cefsulodin-irgasan-novobiocin (CIN) agar (Oxoid Ltd., Thermo Fisher Scientific, Inc.), Campylobacter blood-free selective agar plates, and in selenite broth (Oxoid Ltd., Thermo Fisher Scientific, Inc.). The specimens from the patients with antibiotic-associated diarrhea were aliquoted into one dry container, two FecalSwab devices, and two ESwab™ devices and cultured immediately on cycloserine-cefoxitin-egg-yolk (CCEY) agar (Oxoid Ltd., Thermo Fisher Scientific, Inc.). In addition, these specimens were tested directly for C. difficile toxins with an immunoassay (IA) targeting toxins A and B (Alere Tox A/B Quik Chek; Tech Lab, Waltham, MA, USA) and with the GenomEra C. difficile assay (Abacus Diagnostica, Turku, Finland) targeting the tcdB gene. The IA and GenomEra C. difficile assay were performed according to each manufacturer’s instructions. In addition to the immediate culturing and testing, all clinical specimens in the FecalSwab and ESwab™ devices were recultured and retested, as mentioned above, after being stored at 4°C and RT for 20 h.
The inoculated plates were incubated either aerobically (CIN and XLD) at 35°C or in a microaerophilic atmosphere at 42°C (Campylobacter blood-free selective agar) for 48 h. The selenite broth was incubated at 4°C for 24 h and then subcultured onto an additional XLD plate. The plates for C. difficile were incubated anaerobically (in CCEY) at 35°C for 48 h. Biochemical analyses were performed on all suspected colonies, and isolates preliminarily identified as being pathogenic were sent to the bacteriology unit of the National Institute for Health and Welfare (THL) for confirmation and strain typing, excluding C. difficile. The presumptive growth of C. difficile was confirmed by Gram staining, UV light, and IA (Alere C. diff Quik Chek Complete) targeting C. difficile-specific glutamate dehydrogenase (GDH). The toxigenic nature of the suspected isolate growing on the culture medium was confirmed by the same above-mentioned IA.
Recovery of stock isolates. In both the ESwab™ and FecalSwab devices, the number of viable organisms remained stable for up to 6 h at RT storage (Table 1). At 24 and 48 h of storage, however, a clear increase in growth was observed for all other isolates, except for C. jejuni, C. coli, and C. difficile. Campylobacter spp. yielded no growth after 24 h of storage at RT. Also, the vegetative growth of C. difficile ceased after 24 h of storage. However, in these cases, high numbers of thin curved Gram-negative bacilli (presumably Campylobacter spp.) or large poorly Gram staining bacilli with spore-like structures (presumably C. difficile) were seen when the preservation media of the devices were Gram stained.
At a refrigeration temperature, the recovery of stock isolates in ESwab™ and FecalSwab was more stable over time (Table 2) than that at RT. Heavy proliferation was not seen, except with Salmonella spp., which began to grow in both swab devices after 24 h of storage. C. difficile survived for48 h in FecalSwab at 4°C, while in ESwab™, the amount of viable cells began to decline rapidly after inoculation, and no colonies were recovered at 48 h of storage. In contrast to the other species, the viability of Campylobacter spp. started to reduce in both swab devices after 6 h of storage, and by 24 h, no growth of C. coli in FecalSwab and C. jejuni in either of the swab devices were observed. However, at 48 h of storage in FecalSwab, both Campylobacter spp. were again recovered, although in concentrations 6% of the initial values. From ESwab™, only C. jejuni was recovered at 48 h of incubation.
At 70°C, all stock isolates (n 12) survived 3 months of storage in FecalSwab, while with ESwab™, the viability of the isolates decreased rapidly after 2 weeks, and only Salmonella spp. (n 2) survived up to 1 month. The difference in the survival of the stock isolates at70°C was significant (P0.0001). However, for most isolates, the cell concentrations in FecalSwab were clearly lower 3 months after inoculation than at 0 h. While the viability of E. coli and Yersinia spp. remained stable, with no significant reduction at 3 months of storage, a 1 log reduction in the number of viable Shigella and Salmonella cells, a 1.5 log reduction in the number of viable Aeromonas and Plesiomonas cells, and a 2 log reduction in the number of viable Vibrio, Campylobacter, and Clostridium cells were observed in FecalSwab. At 20°C, a notable reduction in viable cells was seen in both swab devices 2 weeks after inoculation, after which time we could not recover the growth of any isolates. Thus, for longer preservation, storage at 70°C maintained the viability of stock isolates significantly better than at 20°C (P 0.0001).
For the mixed specimens of E. coli, E. faecalis, and S. enterica serovar Typhimurium, or E. coli, E. faecalis, and S. sonnei, we observed results similar to those in the survival of the separate enteric pathogens (Tables 3 and 4). At RT, a notable proliferation of all isolates was seen after 24 h of storage in both transport devices. Mixing different microorganisms together did not seem to influence the growth processes of individual organisms. At 4°C, the concentration of mixed isolates remained stable at least up to 48 h.
Performance with clinical stool specimens. Of the 228 gastroenteritis stool specimens, 24 (10.5%) were positive for one of the common enteric bacterial pathogens, Yersinia (n2), Salmonella (n17), or Campylobacter (n5) species, from all four specimen collection systems (ESwab™, FecalSwab, Amies gel swab, and dry container) used in this study. However, cell recovery was slightly higher using the semiquantitative culture method from ESwab™ and FecalSwab devices than from routinely used transport systems. For example, when scanty growth of suspected pathogen was observed from the routinely used transport systems, moderate growth was seen from ESwab™ and FecalSwab. Moreover, the additional 20 h of storage at RT improved the yields (e.g., from moderate to substantial growth) of Yersinia and Salmonella spp. from ESwab™ and FecalSwab, while at 4°C, the cell concentrations remained stable. Any Campylobacter spp., on the other hand, were not recovered at 20 h of storage at RT or at 4°C from either of the swab devices.
Of the 100 antibiotic-associated diarrheal specimens, 20 (20%) were positive by toxigenic C. difficile culture and by PCR, from both swab devices and routinely used transport systems. A direct IA of C. difficile toxins, however, revealed only 6 positive specimens. The results from the FecalSwab and ESwab™ devices were identical even after 20 h of storage at RT or at 4°C.
Although no significant differences were seen between the ESwab™ and FecalSwab devices for the storage of various microorganisms at room temperature or 4°C, at70°C, FecalSwab maintained the viability of stock isolates significantly better than ESwab™. In ESwab™, the recovery of 10 stock isolates out of 12 ceased during 2 weeks of storage, and only two Salmonella spp. survived up to 1 month, while in FecalSwab, all isolates survived at least up to 3 months. Moreover, for longer preservation, storage at 70°C proved to be more reliable, as at 70°C, the reduction of viable cells after 2 weeks was significantly less than that at storage at 20°C.
Compared to the other isolates, C. difficile survived better in FecalSwab than in ESwab™ at lower temperatures, while at room temperature, the recoveries were equal in both swab devices. At 4°C, C. difficile remained viable up to 48 h in FecalSwab, even though it was stored aerobically. Similar results have also been demonstrated with other transport systems, such as Amies gel swabs (8). Furthermore, as C. difficile cytotoxins have been demonstrated to remain highly stable for up to several months at 4°C (9), we performed a preliminary investigation of the preservation of C. difficile toxins and toxin genes in FecalSwab and ESwab™ for an extended period of time as well (data not shown). After 48 h of storage at 4°C, all initially C. difficile toxin A/B- and tdcB gene-positive clinical stool specimens were positive from both the FecalSwab and ESwab™ devices with the IA and PCR assay used in our study. At 7 days of storage, all PCR-positive and approximately 67% of the IA-positive specimens from both devices were still positive. PCR positivity lasted for up to 1 month, after which time the follow-up ended. Of the IA-positive specimens, 50% remained positive until 1 month.
Most interestingly, the extension of storage time from 20 to 72 h seemed to improve gradually the cell viability of Campylobacter spp. at 4°C. Higher recovery rates of Campylobacter stock isolates were seen at 72 h of storage than at 48 h, and the storage of clinical stool samples containing Campylobacter spp. for 20 h (72 h) at 4°C enabled the recovery of growth on selective culture medium, although no growth was seen at 20 h of storage (data not shown). The frequency of and reason for this odd growth behavior of Campylobacter isolates at 4°C in FecalSwab and ESwab™ devices are not known. We did not find any previous reports with similar findings. It is known, though, that Campylobacter organisms may undergo a temporal physiological and morphological transition into a viable but nonculturable stage, whereby they retain basal metabolic activity yet fail to grow or multiply in cultures when translocated from their intestinal niche into an aquatic environment (10). However, at 4°C and, e.g., in Cary-Blair medium, which is the medium base in FecalSwab, Campylobacter spp. have been shown to remain culturable for days (11), albeit with reducedCFU counts (10, 11). We observed a nonculturable stage of Campylobacter spp. rapidly after 6 h of storage at 4°C, mainly in FecalSwab, which then returned to a culturable stage after 48 h of inoculation. However, aerated specimens, e.g., those prepared with shaking, may demonstrate a more rapid decrease in the recoverability of Campylobacter organisms than specimens held in a stationary state (10). In our study, all specimens in the FecalSwab or ESwab devices were vigorously mixed by vortexing at each time point prior to plating onto the culture medium. Accordingly, the detection of Campylobacter spp. in fecal specimens with culture may vary depending on the storage time and growth stage of the bacteria.
In conclusion, the ESwab proved to be a well-suited swab device for short-term storage and transportation of enteric pathogens. The FecalSwab, on the other hand, proved to be suitable even for extended storage and transportation of enteric pathogens, enabling successful and reliable microbiological analysis when specimens are sent to either a local laboratory or a more distant reference laboratory. Furthermore, due to a more homogenized form, specimens in both the ESwab and FecalSwab devices are easier to handle and use for various laboratory tests, e.g., IA or PCR assay, than specimens in dry containers. In addition, flocked swabs in liquid media are more suitable for culture automation than the gel swabs (12) or even stools in dry containers.