APMIS 100: 1048-1052, 1992

Experimental studies of survival of anaerobic bacteria at 4OC and 22OC in two different transport systems Brief report MICHAEL TVEDE'" and NIELS HOIBY' 'Dept. of Clinical Microbiology, Rigshospitalet, University of Copenhagen, Copenhagen, and '.*Department of Clinical Microbiology, Viborg Sygehus, Viborg, Denmark

Tvede, M. & Heriby, N. Experimental studies of survival of anaerobic bacteria at 4°C and 22°C in two different transport systems. APMIS 100: 1048-1052, 1992. The survival of anaerobic bacteria on swabs in two different transport systems at 4°C and 22°C was evaluated. The transport systems were a charcoal-impregnated cotton swab in modified Stuart transport medium (MST), and a viscose swab in modified SIFF transport medium (BTM) (BionorTM). The following eight clinical strains of anaerobic bacteria were tested for quantitative recovery at 24 h, 48 h, 12 h, and 96 h; Fusobacterium necrophorum, Bacteroides melaninogenicus, Bacteroides intermedius, Peptostreptococcus anaerobius, Peptococcus magnus, Clostridium perfringens, Clostridium tetani, and Actinomyces israelii. Additionally, a mixture of Staphylococcus aureus and Escherichia coli was tested together with Bacteroides fragilis, Fusobacterium necrophorum and Clostridium perfringens. Both transport systems preserved the bacteria, but the BTM medium gave a better quantitative recovery of the bacteria than the MST in 29/64 (45%) of the experiments, whereas the opposite was the case in 15/64 (23%) of the experiments (p < 0.05). There was no significant strain-related difference between the recovery of 10 different B. fragilis strains. There was no major difference in the recovery of the anaerobes in the two systems at 4°C compared to 2 2 T , except for Fusobacterium necrophorum, which survived best at 4°C in the BionorTMtransport system. Key words: Anaerobes; transport media. M. Tvede, Department of Clinical Microbiology, Viborg Sygehus, LI. Sct. Mikkelsgade 29, Postbox 130, DK-8800 Viborg, Denmark.

Transport of samples containing fastidious bacteria to the laboratory is generally done using non-nutrient transport media (1). Several transport media exist, most being modifications of the Stuart medium (5, 6, 7 ) . Such media should be suitable for the transport of fastidious aerobes like N . gonorrhoeae as well as oxygen-sensitive Gram-negative and Gram-positive anaerobes. Modified Stuart Transport medium has previously been shown to fulfill these requirements (2, 4, 5 ) , and this transport medium is generally used in Denmark. It has, however,

Received March 16, 1992. Accepted August 21, 1992. 1048

some drawbacks: as it is provided in glass tubes, it is rather heavy, and the shelf life is only two months or six months if vacuum packed. Recently, a modified Stuart transport medium (SIFF medium (6)) with improved ability t o preserve N. gonorrhoeae has been developed. This medium has also been found t o be a t least as good as the Stuart medium for preservation of

Bordetella pertussis, Haemophilus injluenzae, Neisseria meningitidis, Escherichia coli, Proteus mirabilis, Klebsiella species, Pseudomonas aeruginosa, Bacteroides fragilis, Staphylococcus aureus, Enterococcus species, Streptococcus pneumoniae, Streptococcus pyogenes gr. A , Listeria monocytogenes, and Candida albicans (P Sandvek, National Institute of Public Health (SIFF),

TRANSPORT MEDIA FOR ANAEROBES

Oslo, Norway, personal communication). This medium is provided in low-weight impact-resistant plastic tubes with sealed screw caps and has a shelf life of one year. We therefore decided to compare the two media with respect to recovery of Actinomyces israelii and a number of strictly anaerobic microorganisms alone and in mixed cultures with E. coli and S. aureus.

MATERIALS AND METHODS Bacteria One strain of each of the following species was used; Bacteroides melaninogenicus, Bacteroides inrermedius, Fusobacterium necrophorum, Peptostreptococcus anaerobius. Peptococcus magnus, Clostridium perfringens, Clostridium tetani, and A . israelii. These anaerobes represent the range of sensitivity to oxygen relevant to clinical microbiology laboratories. To examine if individual differences occur within the most commonly isolated anaerobic bacteria, 10 clinical strains of B. fragilis were examined in one experiment. For the recovery of anaerobes in mixed cultures, E. coli and S. aureus were employed. All strains were clinical isolates that had been identified on the basis of Gram stain, morphology, volatile fatty acid production, and biochemical tests according to Holdeman et al. (3). Preparation of bacterial suspensions Each strain to be tested was grown in supplemented Brain Heart Infusion Broth (Difco) for 24 h in an anaerobic glove-box chamber (Forma Scientific anaerobe system, Marietta, Ohio, USA) with an atmosphere of 80% N2, 10% H,, and 10% COz. The bacteria were then suspended in inactivated human serum, pretreated at 56°C for 30 min, at a standard concentration equal to McFarland 4 (approximately lo9 colony-forming units (CFU)/ml). The two different swabs were allowed to absorb 0.1 ml of this suspension for 10 min before they were inserted into the appropriate transport media. Four different tubeswab combinations were used: 1) MST with a wooden charcoal swab, 2) MST with a viscose polystyrene swab, 3) BTM with a viscose polystyrene swab, and 4) BTM with a wooden charcoal swab. To evaluate whether a mixture of S. aureus and E. coli could interfere with the recovery of anaerobes from the transport systems, these two bacterial strains were mixed with B. fragilis, F: necrophorum and CI. pecfringens before the swabs were inoculated with 0.1 ml of this mixture. To evaluate whether the swabs and media differed in absorption and release of the bacteria, and estimate the variations in CFU from swab to swab, 10 different swabs and tubes of each transport system were

tested for recovery of S. aureus after absorption of bacteria in inactivated human serum, as described above, followed by culture immediately after the swabs had been placed in the media.

Transport systems Two transport systems were compared. A) the MST ( 5 ) routinely used at our hospital was obtained from Statens Seruminstitut, Copenhagen, Denmark (formula: 1.67% agar, 10/0 sodium glycerophosphate, 0.01% CaCI,, 0.76% thioglycollate, 0.0002% methylene blue, pH 7.4). The medium is distributed in sterile glass tubes (15 by 155 mm) and sealed with cotton wool plugs. The height of the agar column is approximately 10 cm. Shelf life is two months (six months if vacuum packed; this option was not tested in the present work since it is not routinely used at our hospital). The MST is provided with separately packed wooden swabs with heads covered with cotton wool impregnated with charcoal, sterilized by autoclaving. By microscopy the cotton wool appears loosely woven and contains loose and adherent charcoal particles (size of the particles: 10-100 pm). Weight of the total system, including tube container, envelope and the laboratory’s paper form, is 73 g. B) the BTM (slightly modified from Sandven et al. (6)) was obtained from Bionor, Skien, Norway (formula: 0.50% agar, 0.10% sodium glycerophosphate, 0.40%) soluble starch, NaCl/KCl 0.35%/0.30% Na,HPO,/ KH2P04 0.30%/0.04%, MgCI,ICaCl, O.O02O/u/ 0.0015%, cysteine 0.0025%, thioglycolic acid 0.08%, and finally 0.0003% methylene blue, pH 7.4). The medium is distributed in a transparent plastic tube 15 by 120 nun, and provided with a screw cap with an inside socket to fit the swab. The height of the medium is approximately 7 cm. Shelf life is one year. This system is provided with separately packed polystyrene swabs with heads covered with viscose. By microscopy the viscose appears tightly woven and without particles. Weight of the total system (no tube container is necessary in Norway according to the manufacturer), including envelope and the laboratory’s paper form, is 49 g. Storage The transport systems containing the inoculated swabs were stored either in the refrigerator at 4’C or at room temperature (approximately 22°C). All strains were thus tested under four different conditions on five separate occasions after placing the swab with the inoculum in the transport medium (0 h, 24 h, 48 h, 72 h, and 96 h). Recovery of bacteria After the above-mentioned time intervals the appropriate swabs were transferred to glass tubes containing 4 ml of BHI broth. After 10 min vigorous shaking in a whirl-mixer the swabs were discharged and 100 p1 of the mixture was spread on a Brain Heart

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Infusion (BHI) agar plate. After 48 h of anaerobic incubation at 35'C (except for the A. israelii which was incubated for 96 h) the number of colonies was counted. The results were given as CFU. Recovery of B. jragilzs from the mixed solution was examined on kanamycin-containing BHI, S. uureus was examined on agar plates containing 7.5% NaCl, and E. coli on modified Conradi-Drigalski agar, which also allowed counting of CFU. Recovery of F necrophorum and CI. prrfringens from the mixed solution was scored as and 0, as the concurrent growth of the facultative microorganisms did not allow counting of the exact number of CFU for these two set-ups.

+ + +, + +, +

Stability of' rnediu Five tubes of each of the transport media were placed protected from daylight at room temperature and at 4 ' C for one month, and were observed for changes in color and evaporation of media.

RESULTS AND DISCUSSION The reproducibility of the ability of the two swabs to absorb and release bacteria was tested with a S. aureus strain diluted in heat-inactivated human serum followed by transfer to media. Total count of the suspension before inoculation into media was 8.5 x 10' CFU. The results showed that the mean CFU released by the cotton wool charcoal swab combined with the MST was 7.8 x 10' (coefficient of variation: 17%) equal to 92%) recovery. For the viscose swab combined with the BTM, the mean CFU released was 7.2 x 10' (coefficient of variation: 28%) equal to 85% recovery. No significant difference was found between the two swabs (p= 0.4, Mann-Whitney rank sum test, two-tailed). More evaporation occurred from the MST medium (1 5% decrease in height of medium in the tubes) than the BTM (3%) when kept in the dark at room temperature for one month, and the oxygenation of MST medium during that period as judged by blue coloring was highest (30%), while only 6%0 of the BMT medium turned blue. Except for A . israelii, both media preserved the bacteria for the 96 h period and growth of the tested microorganisms occurred from all different series of tubes. The BMT medium was generally found to yield higher CFU compared to the MST medium when the swabs were cultured after 24-96 h. This was especially the case 1050

when the two strict anaerobes l? necrophorum and B. melaninogenicus were examined. These findings may be due to the screw cap of the BTM system, which excludes oxygen from the media better than the MST system, which is sealed by a cotton wool plug. When all 64 different variations of the tests were compared between the two systems, the BTM yielded the highest recovery of CFU. In 29/64 (45'Yn) series of tubes the CFU were significantly higher in BMT (> + 2 x standard deviation above the CFU of the MST), while the MST was significantly better in 15/64 (23%) different series of tubes (pCin modqied SIFF transport medium ( B T M . BionorflMI and in modified Stuart transuort medium (MST. Statens Serurninstitut J Bacterial species Initial Bacterial recovery (%I) after suspension (CFU) 24 h 48 h 72 h 96 h BMT MST BMT MST BMT MST BMT MST BMT MST E necrophorum (4 C) 1.3 1.4 41* 33 50* 3 13* 1.1 8* 0.4 122-C) x106 x106 23 54* 30 29 8* 2.4 1.6* 0.2 B. intermedius (4OC) 4.3 4.2 686* 214 216 335* 282* 251 64 141* ( 2 2 ~ ) 105 x105 203 212 317* 235 259 256 272* 192 B. rnelaninogenicus (4’C) 1.2 1.2 57* 4.3 25* 2.1 6.7* 0.1 1.6* 0.4 (22’C) x loh 35* 0.2 32* 13 x lo6 7.4* 1.9 4.1* 3.0 5.2 Peptococcus mugnus (4’C) 9.7 104* 88 104 103 43 58* 24 50* 102* 88 30 36 46 105 i50* 44 49 105 (22 c) 8.2 176* 137 Peptostreptococcus (4°C) 1.1 103 144 89 112 46 142* x 105 54 anaerobius (22-c) x 106 58 106 99 38 75* 38* 30 Clostridium (4 C) 3.4 2.3 555 941* 347 616* 188 444* 193 504* 177 342* 647* 538 248 400* 267 390 x lo’ perjringens (22 C) x 10’ 4.5 98 105 Clostriilium (4’C) 4.5 200* 100 45 80* 26 60* x 104 i40* 99 200 200 70 70 tetani (22 c) x 104 50 50 Actinomycrs i.sraelii** (4 C) n d . n.d. 450 450 1755 1530 0 0 0 0 (CFU/ml) (22 C) 450* 0 1935* 0 450* 0 0 0 * Difference in CFU 2 2 x standard deviation (Poisson distribution) compared to CFU of the other medium. ** The exact inoculum could not be determined due to clumping (microcolony formation),and the ‘%I recovery has therefore not been calculated. ~~~

A mixed culture of S. aureus and E. coli with E necrophorum of CLperfringens did not inhibit detectable growth of the anaerobes and no difference was observed between the results obtained with the two media. Likewise, mixed cultures of S. uureus, E. coli and B. fragilis did not inhibit the recovery of any of the three species after storage in the two media with the two different swabs at 4°C and 22°C for 24-96 h. This experiment was repeated three times. It is often regarded as a disadvantage for transport systems when growth occurs in the media, but in this respect both media were capable of producing growth for some of the anaerobes, as shown previously by Justesen et al. (4).This is not surprising as both starch from the BMT medium and glycerophosphate in both media may serve as nutrient for some strains, in this case B. in(ermedius and Cl.perfringens. B. melaninogenicus did not increase in CFU although both glycerophosphate and starch act as nutritients for this bacteria. This can be explained by the lack of other growth factors, i.e. vitamin K. The optimal temperature for storage has been a matter of discussion. Holdeman et al. (3) and Balows et al. (1)

advise against refrigeration, wheras Hoffmann et al. (2) and Justesen et al. (4) recommend refrigeration for transport of anaerobes. In the present study the total CFU were higher for some strains kept at 4”C, while other strains survived better when kept at 22°C and gave diverging results with the two transport media used. These findings may be caused by species differences, and this underlines that no general guidelines can be given. The BMT medium has previously proved superior to other transport media using clinical specimens containing N . gonorrhoeae (6). Our study did not include clinical specimens which may contain components, for example antibiotics, mucus and leukocytes, inhibitory for anaerobes and other bacteria. A study including clinical specimens containing anaerobes would be valuable, although our present results and the composition of the BMT medium do not indicate any difference in this respect between the BMT and the MST. In conclusion, BMT proved as reliable a transport medium for anaerobes as it has previously been shown to be for N . gonorrhoeae and other aerobic and facultative bacterial spe1051

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cies, and BMT proved t o be marginally but significantly better than MST. Technician Annette Gregersen is thanked for skilful technical assistance.

REFERENCES 1. Balows, A . , Hausler, W J. Jr., Herrmann, K. L., Isenberg, H. D.& Shadomy, H. J. (Eds.): Manual of Clinical Microbiology. 5th ed. American Society for Microbiology, Washington 1991. 2. Hoffmann, S., Jensen, A. M . & Justesen, T.: The recovery of anaerobic bacteria from swabs in three transport systems. Acta path. microbiol. immunol. scand. Sect. B, 91: 23-26, 1983.

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3. Holdeman, L. V , Cato, E. I? & Moore, W E. C. (Eds.): Anaerobic laboratory manual. 4th ed. Virginia Polytechnic Institute and State University, Blacksburg, Virginia 1977. 4. Justesen, T , Jensen, A . M . & Hoffmann, S.: The survival of anaerobic bacteria at 4°C and 22°C on swabs in three transport systems. Acta path. microbiol. immunol. scand. Sect. B, 91: 17-22, 1983. 5. Reyn, A . .I Laboratory diagnosis of gonococcal infections. Bull. Wld. Hlth. Org. 32: 449-469, 1965. 6 . Sandven, P, Solberg, O., 0degaard, K. C? Myhre, G.: Improved medium for the transportation of gonococcal specimens. Acta path. microbiol. immunol. scand. Sect. B, 90: 73-77, 1982. 7. Stuart, R. D.: The diagnosis and control of gonorrhoea by bacteriological cultures. Glasg. Med. J. 27: 131-142, 1946.

Experimental studies of survival of anaerobic bacteria at 4 degrees C and 22 degrees C in two different transport systems.

The survival of anaerobic bacteria on swabs in two different transport systems at 4 degrees C and 22 degrees C was evaluated. The transport systems we...
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