Journal of Microbiological Methods 109 (2015) 20–24
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Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Non-selective and selective enrichment media for the recovery of Clostridium difficile from chopped beef Changhoon Chai, Kyung-Soo Lee, Dayoung Lee, Soyeon Lee, Se-Wook Oh ⁎ Department of Food and Nutrition, Kookmin University, Seoul 136-702, Korea
a r t i c l e
i n f o
Article history: Received 13 October 2014 Received in revised form 1 December 2014 Accepted 1 December 2014 Available online 9 December 2014 Keywords: Antibiotics Beef Clostridium difficile Cooked meat medium Enrichment medium
a b s t r a c t Clostridium difficile exists within the intestines of animals and in meat products. Enrichment of C. difficile in an appropriate medium is necessary for the detection of C. difficile in meat products. Non-selective media (brain heart infusion medium [TBHI] and cooked meat medium containing sodium taurocholate [TCM]) and selective media (cycloserine-cefoxitin-fructose medium [TCCFB] and C. difficile moxalactam-norfloxacin medium containing antibiotics and sodium taurocholate [TCDMN]) can be used to enrich C. difficile. This study aimed to evaluate non-selective and selective enrichment media for the recovery of C. difficile from beef specimens. The efficiency of the enrichment media was investigated on the basis of the recovery frequency of C. difficile from beef specimens inoculated with C. difficile. The beef specimens were inherently contaminated with bacteria (around 104 CFU g−1), and further inoculated with C. difficile (around 100 CFU g−1). The antibiotics in TCCFB and TCDMN adversely affected C. difficile growth. The bacteria inherent to these specimens exhibited resistance to antibiotics and grew during the enrichment of C. difficile-inoculated chopped beef in TCCFB and TCDMN, which hindered the recovery of C. difficile. The frequency of recovery of C. difficile from beef specimens in TCM was higher than that from any other enrichment medium. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Clostridium difficile, a Gram-positive spore-forming anaerobic bacterium, is a causative pathogen of mild diarrhea or life-threatening pseudomembranous colitis (Poutanen and Simor, 2004). The incidence of C. difficile-associated diseases has increased over the past decade (Lessa et al., 2012). C. difficile is found in the intestines of humans and animals (Poutanen and Simor, 2004; Songer and Anderson, 2006) and in meat products (Jöbstl et al., 2010). It may be transmitted to humans via contact with infected humans or animals or by consumption of contaminated food (Jöbstl, et al., 2010; Rupnik et al., 2009). Clinical and environmental samples contain small amounts of C. difficile, and enrichment in an appropriate medium is frequently required to diagnose C. difficile infection and contamination (Arroyo et al., 2005; Levett and Margaritis-Bassoulis, 1985). C. difficile can be enriched in common media used for bacteria, and can be selectively enriched using C. difficile-specific carbohydrates or C. difficile-resistant antibiotics (Arroyo, et al., 2005; Tyrrell et al., 2013). The recovery of C. difficile spores from stool and clinical samples can be improved by incubation in cycloserine-cefoxitin-fructose medium (TCCFB) or C. difficile moxalactam-norfloxacin medium containing sodium taurocholate (TCDMN) (Jöbstl, et al., 2010; Tyrrell, et al., 2013). However, studies using enrichment media for the recovery of C. difficile from meat ⁎ Corresponding author. Tel.: +82 2 910 5778; fax: +82 2 910 5249. E-mail address: [email protected] (S.-W. Oh).
http://dx.doi.org/10.1016/j.mimet.2014.12.001 0167-7012/© 2014 Elsevier B.V. All rights reserved.
products have not yet been reported, despite the public safety concerns regarding C. difficile-associated diseases and the urgent need for research in this area. This study aimed to evaluate non-selective and selective enrichment media for the efficient recovery of C. difficile from beef specimens. In this study, brain heart infusion (TBHI) and cooked meat medium containing sodium taurocholate (TCM) were used as the non-selective enrichment media for C. difficile (Sorg and Dineen, 2009), and TCCFB and TCDMN were used as the selective media (Jöbstl, et al., 2010; Tyrrell, et al., 2013). The growth of C. difficile in TBHI, TCM, TCCFB, and TCDMN was measured, and the growth characteristics in each enrichment medium were analyzed using a modified Gompertz growth model (Zwietering et al., 1990). The efficiency of the enrichment media was evaluated on the basis of the frequency of recovery of C. difficile after incubation of C. difficile-inoculated chopped beef specimens in TBHI, TCM, TCCFB, and TCDMN. 2. Materials and methods 2.1. C. difficile strains and inoculum preparation C. difficile ATCC 9689, ATCC 43593, and ATCC 43601 were obtained from Korean Collection for Type Cultures (KCTC). Prior to inoculation in enrichment medium and chopped beef, each C. difficile strain was separately cultivated in BHI broth (OXOID, UK) at 37 °C for 14 h under anaerobic conditions (80% N2, 10% H2, and 10% CO2) in a Whitley A35
C. Chai et al. / Journal of Microbiological Methods 109 (2015) 20–24
Anaerobic Workstation (Don Whitley Scientific, UK). A three-strain C. difficile cocktail was prepared by mixing an equal volume of each C. difficile culture. Freshly cultivated C. difficile ATCC 9689 suspension and freshly prepared three-strain C. difficile cocktail were plated on BHI agar plates for enumeration and immediately used to inoculate the enrichment media and chopped beef. 2.2. Preparation of enrichment media TBHI and TCM were prepared by adding 10% (w/v) sodium taurocholate solution (Sigma-Aldrich, USA) to sterilized BHI (OXOID, UK) and CM broths (OXOID, UK) to a final concentration of 0.1% (Songer et al., 2009). TCCFB and TCDMN were prepared in accordance with a previously described method (Aspinall and Hutchinson, 1992; Tyrrell, et al., 2013). Proteose peptone (40 g; OXOID., UK), disodium hydrogen phosphate (5 g), potassium dihydrogen phosphate (1 g), magnesium sulfate (0.1 g), sodium chloride (2 g), and fructose (6 g) were dissolved in 910 mL of distilled water. After sterilization, 70 mL of horse blood (Hanil Komed, Korea) and 10 mL of 10% (w/v) sodium taurocholate solution were added. Subsequently, 10 mL of either C. difficile-selective supplement solution containing 25 mg L−1 d-cycloserine and 0.8 mg L−1 cefoxitin (OXOID, UK) or CDMN-selective supplement solution containing 3.2 mg mL−1 moxalactam, 1.2 mg mL−1 norfloxacin, and 50 mg mL−1 cysteine hydrochloride (OXOID, UK) was added to produce TCCFB and TCDMN, respectively. 2.3. Growth of C. difficile in enrichment media A fresh C. difficile ATCC 9689 culture suspension and a three-strain C. difficile cocktail were diluted with peptone water to a concentration of 104 CFU mL−1. TBHI, TCM, TCCFB, and TCDMN were inoculated with C. difficile by adding 1 mL of diluted fresh C. difficile ATCC 9689 culture suspension or three-strain cocktail to 200 mL of enrichment medium, followed by anaerobic incubation at 37 °C for 28 h. C. difficile enriched in TBHI, TCM, TCCFB, and TCDMN was plated on BHI agar plates at suitable dilutions every 2 h for 28 h. The colonies on BHI agar plates were enumerated after anaerobic incubation of BHI plates at 37 °C for 24 h. Enrichment and enumeration of C. difficile in TBHI, TCM, TCCFB, and TCDMN were performed in triplicate. The average growth curve values of C. difficile were entered into GraphPad Prism software v. 4.03 (GraphPad Software, USA) to analyze key growth parameters such as lag time (λ) and maximum growth rate (μ) of C. difficile based on a modified Gompertz growth model. 2.4. Recovery of C. difficile from C. difficile-inoculated chopped beef Chopped beef was purchased from a local market and dispensed into two 225-g specimens in large aseptic plastic bags (Whirl-Pak Bags, USA). To enumerate the inherent bacteria in the chopped beef specimens, 25 g of the remaining chopped beef was homogenized in 225 mL of 0.1% peptone water by using a stomacher (AES Stomacher; AES Chemunex, France). The homogenate was diluted and plated on plate count agar (PCA; OXOID, UK) or BHI agar plates. Aerobic bacteria were enumerated after incubation of PCA plates at 37 °C for 24 h in a conventional incubator. Anaerobic bacteria were determined by the number of colonies on BHI agar plates after anaerobic incubation at 37 °C for 24 h. A fresh C. difficile ATCC 9689 culture suspension and a three-strain C. difficile cocktail were diluted with peptone water to a final concentration of 101 CFU mL−1. Plate counts of diluted C. difficile showed concentrations of 13 CFU mL−1 and 35 CFU mL−1 for diluted C. difficile ATCC 9689 culture suspension and three-strain C. difficile cocktail, respectively. In total, 25 mL each of the diluted C. difficile ATCC 9689 culture suspension and three-strain C. difficile cocktail was added to each 225-g specimen in a large aseptic plastic bag and mixed well. Each inoculated chopped beef specimen was divided into 80 parts, with 2.5-g specimen in each part, and dispensed into aseptic plastic bags (7.5 cm × 12.5 cm;
21
Whirl-Pak Bags, USA). Subsequently, 22.5 mL of TBHI, TCM, TCCFB, or TCDMN was added to 20 chopped beef specimens, each weighing 2.5 g, and mixed well. C. difficile-inoculated chopped beef specimens in TBHI, TCM, TCCFB, and TCDMN were then anaerobically incubated at 37 °C for 2 days. After enrichment, the recovery of C. difficile was screened by streaking the enriched medium on TCCFA supplemented with 7% horse blood. The appearance of gray-colored colonies exhibiting the typical shape of C. difficile after anaerobic incubation at 37 °C for 24 h on TCCFA indicated that the specimen was positive for C. difficile. The gray colonies on TCCFA were streaked on C. difficile-selective agar (CDSA; Becton Dickinson, USA) and anaerobically incubated at 37 °C for 24 h. The recovery of C. difficile from C. difficile-inoculated chopped beef was confirmed by the appearance of a yellow colony with a typical C. difficile shape on CDSA. The recovery frequency was expressed as the number of presumptive positive and confirmed positive C. difficile specimens from C. difficile-inoculated chopped beef per 20 chopped beef specimens in TBHI, TCM, TCCFB, or TCDMN. 3. Results and discussion 3.1. Growth of C. difficile in enrichment media The growth curves of the single strain of C. difficile (ATCC 9689) and those of the three strains of C. difficile (ATCC 9689, ATCC 43593, and ATCC 43601) during anaerobic incubation at 37 °C in TBHI, TCM, TCCFB, and TCDMN are shown in Figs. 1 and 2, respectively. The growth of C. difficile in each enrichment medium was analyzed on the basis of a modified Gompertz model, which allowed the analysis of λ and μ of C. difficile in enrichment medium (Table 1). Plots from the beginning to the exponential-to-stationary phase transition were entered into GraphPad Prism software that was equipped with the modified Gompertz growth model for precise analysis of λ and μ. The growth pattern of C. difficile differed with the contents of the enrichment medium. The order of the μ values for C. difficile in each enrichment media was, from the greatest to the least, in TBHI, TCM, TCCFB, and TCDMN, which reflected that C. difficile grew at a slower pace in selective media than in non-selective media (Table 1). C. difficile in TBHI, TCM, TCCFB, and TCDMN inoculated with the single strain of C. difficile (ATCC 9689) and the three-strain C. difficile cocktail reached the stationary phase at 14, 12, 18, and 20 h and 12, 12, 14, and 14 h, respectively (Figs. 1 and 2). On reaching the stationary phase, the number of C. difficile in TCCFB and TCDMN abruptly decreased. This decrease might be attributed to the adverse effects of D-cycloserine, cefoxitin, moxalactam, or norfloxacin on the survival of C. difficile, despite their resistance to such antibiotics (Figs. 1c and d and. 2c and d). When only the single strain of C. difficile (ATCC 9689) existed in TBHI and TCM, C. difficile recovered and began to grow after 1.607 and 1.727 h, respectively (Table 1). However, the λ values for C. difficile in TBHI and TCM inoculated with the three-strain C. difficile cocktail were 2.825 and 2.473 h, respectively, which were greater than those in media inoculated with the single strain of C. difficile (Table 1). Competition among C. difficile strains was not expected in the enrichment medium with a single strain of C. difficile. However, the effect of competition among different strains of C. difficile on the growth of C. difficile in the enrichment medium containing three different strains of C. difficile could not be ignored, despite the fact that the three different strains belonged to the same species of Clostridium. The delay of λ when three different strains of C. difficile coexisted compared to that for the single strain of C. difficile (ATCC 9689) might be caused by the competition among the different strains of C. difficile (Figs. 1a and b, 2a and b, and Table 1) because λ is generally delayed because of the coexistence of multiple bacterial strains (Smith, 2011). The C. difficile strains used in this study showed varying susceptibilities to D-cycloserine and cefoxitin. The recovery of C. difficile in TCCFB with the single strain of C. difficile (ATCC 9689) was suppressed by either D-cycloserine or cefoxitin, and its λ value was greater than those
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Fig. 1. Growth of Clostridium difficile in enrichment media inoculated with C. difficile ATCC 9689 under anaerobic incubation at 37 °C; curves predicted on the basis of a modified Gompertz equation. (a) TBHI, (b) TCM, (c) TCCFB, and (d) TCDMN.
from TBHI and TCM (Fig. 1 and Table 1). However, the λ value of C. difficile in TCCFB with the three-strain cocktail of C. difficile was the lowest (Fig. 2 and Table 1). This suggests that either C. difficile ATCC 43593 or ATCC 43601 might be more resistant to either D-cycloserine or cefoxitin than C. difficile ATCC 9689. While one or two strains of C. difficile, including C. difficile ATCC 9689, were suppressed by D-cycloserine or cefoxitin in TCCFB, either C. difficile ATCC 43593 or ATCC 43601 might be recovered, resulting in lower λ in TCCFB compared to those from other enrichment media (Table 1). 3.2. Recovery of C. difficile from C. difficile-inoculated beef after enrichment Aerobic and anaerobic plate counts showed the presence of 2.13 × 105 CFU and 1.65 × 105 CFU per 2.5 g of chopped beef for total aerobic and anaerobic bacteria, respectively. As each 2.5-g chopped beef specimen was inoculated with 3 and 9 CFU of C. difficile of the single strain of C. difficile (ATCC 9689) and the three-strain C. difficile cocktail, respectively, the recovery of C. difficile from C. difficile-inoculated chopped beef specimens in different enrichment media was caused by its competition with the inherent bacteria. Thus, the recovery frequency of C. difficile from C. difficile-inoculated chopped beef in enrichment media (Table 2) may be a critical parameter to evaluate the efficiency of enrichment media for the recovery of C. difficile from beef. Despite the presence of C. difficile in all chopped beef specimens, not all specimens were confirmed to be positive for C. difficile, owing to the presence of large numbers of inherent bacteria in chopped beef. The recovery frequency of C. difficile from C. difficile-inoculated chopped beef exhibited wide variation, ranging from 1/20 to 15/20, depending on the enrichment medium. TCCFB and TCDMN are frequently used to selectively enrich C. difficile in meat and clinical samples since C. difficile is resistant to
the antibiotics included in TCCFB and TCDMN (de Boer et al., 2011; Tyrrell, et al., 2013). However, colonies formed by bacteria other than C. difficile were found on TCCFA and CDSA plates (both of which contained D-cycloserine and cefoxitin) after the culture of C. difficileinoculated chopped beef specimens in TBHI, TCM, TCCFB, and TCDMN. This resulted in differences between the number of confirmed positives and presumptive positives (Table 2). Some of the inherent bacteria in chopped beef that were resistant to D-cycloserine, cefoxitin, moxalactam, or norfloxacin grew in TCCFB and TCDMN. Hence, competition between C. difficile and the inherent bacteria in chopped beef that were resistant to the antibiotics in TCCFB and TCDMN was unavoidable. As observed with the growth of C. difficile in the enrichment medium, the growth of C. difficile ATCC 9689 was adversely affected by antibiotics in TCCFB and TCDMN (Fig. 1c and Table 1). The poor frequency of recovery of C. difficile from chopped beef specimens inoculated with the single strain of C. difficile (ATCC 9689) in TCCFB and TCDMN (1/20 and 4/20, respectively) might be attributed to the suppression of the growth of C. difficile ATCC 9689 by antibiotics as well as the growth of inherent bacteria that are resistant to the antibiotics. The frequencies of recovery of C. difficile from chopped beef specimens inoculated with the threestrain C. difficile cocktail in TCCFB and TCDMN (9/20 and 8/20, respectively) were greater than those from chopped beef specimens inoculated with the single strain of C. difficile (ATCC 9689) in the same enrichment medium (Table 2). Additionally, C. difficile showed lower λ and higher μ in specimens inoculated with the three-strain C. difficile cocktail than in those inoculated with a single strain of C. difficile (ATCC 9689) (Table 1). Therefore, the susceptibility of C. difficile to antibiotics included in the selective media might alter the frequency of recovery of C. difficile from C. difficile-inoculated chopped beef specimens after enrichment in selective media. However, the frequencies of recovery of C. difficile from C. difficile-inoculated chopped beef specimens in TCCFB
C. Chai et al. / Journal of Microbiological Methods 109 (2015) 20–24
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Fig. 2. Growth of Clostridium difficile in enrichment media inoculated with a three-strain cocktail of C. difficile (ATCC 9689, ATCC 43593, and ATCC 43601) under anaerobic incubation at 37 °C; curves predicted on the basis of a modified Gompertz equation. (a) TBHI, (b) TCM, (c) TCCFB, and (d) TCDMN.
and TCDMN were lower than those from C. difficile-inoculated chopped beef specimens in TCM, regardless of the number of C. difficile strains present in the chopped beef specimens (Table 2).When C. difficile-inoculated chopped beef specimens were incubated in enrichment media, the bacteria present in the chopped beef, including C. difficile, competed against each other for nutritional resources. The fact that C. difficile grows relatively fast, with a short λ compared to other bacteria might lend it an advantage in bacterial competition (Minton and Clarke, 1989; Sorg and Dineen, 2009). Thus, we speculate that C. difficile ATCC 9689 in TBHI grew rapidly and overcame the presence of large numbers of inherent bacteria, resulting in a recovery frequency of 8/20 from chopped beef specimens inoculated with the single strain of C. difficile (ATCC 9689) (Table 2). However, the recovery frequency from C. difficile-inoculated chopped beef specimens with the three-strain C. difficile cocktail in TBHI was only 1/20 because the competition among the three Clostridium species allowed other bacteria to dominate (Table 2). It is known that C. difficile produces proteases (Poilane et al., 1998). C. difficile present in C. difficile-
inoculated chopped beef specimens enriched in TCM might obtain additional nutrients from the proteolysis of bovine cardiac tissue present in the cooked meat medium, a process similar to that exhibited by other Clostridium spp. grown in cooked meat medium (Willis, 1977). Therefore, in TCM, C. difficile might be more competitive for nutritional resources than the inherent bacteria present in chopped beef specimens, which may explain the observation that the frequencies of recovery of C. difficile from chopped beef specimens in TCM were greater than those in other enrichment media (Table 2). 4. Conclusions The growth and recovery of C. difficile from beef specimens in enrichment broth were largely influenced by the contents of the enrichment broth. The selective media TCCFB and TCDMN are frequently used to selectively enrich C. difficile in food and clinical samples, as C. difficile is resistant to D-cycloserine, cefoxitin (present in TCCFB), moxalactam, and
Table 1 Lag times (λ) and maximum growth rates (μ) of C. difficile in enrichment media, as analyzed using a modified Gompertz growth model. Inoculum
C. difficileATCC 9689
Three-strain cocktail ofC. difficile (ATCC 9689, ATCC 43593, and ATCC 43601)
a 2
R = determination coefficient. b RMSE = root mean square error.
Enrichment medium
TBHI TCM TCCFB TCDMN TBHI TCM TCCFB TCDMN
Parameter estimates λ (h)
μ (log (CFU mL
1.607 1.727 3.449 4.139 2.825 2.473 1.786 3.001
0.847 0.731 0.692 0.567 0.925 0.878 0.826 0.740
Regression analysis −1
)h
−1
)
R2a
RMSEb
Bias
0.998 0.995 0.999 0.996 0.990 0.997 0.998 0.997
0.102 0.164 0.058 0.178 0.270 0.133 0.110 0.154
1.004 1.004 1.002 1.004 1.000 1.004 1.004 1.000
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C. Chai et al. / Journal of Microbiological Methods 109 (2015) 20–24
Table 2 Recovery ratio of Clostridium difficile from C. difficile-inoculated chopped beef specimens after enrichment. Inoculum
Non-selective media
Selective media
TBHI
TCCFB
TCM
TCDMN
Confirmed positive (presumptive positive) C. difficile ATCC 9689 Three-strain cocktail of C. difficile (ATCC 9689, ATCC 43593, and ATCC 43601)
8/20 (10/20) 13/20 (13/20) 1/20 (11/20) 4/20 (5/20) 1/20 (11/20) 15/20 (19/20) 9/20 (11/20) 8/20 (14/20)
norfloxacin (present in TCDMN). However, the growth of C. difficile was adversely affected by antibiotics included in TCCFB and TCDMN. Moreover, we observed that the inherent bacteria in chopped beef specimens were resistant to such antibiotics, and hence grew in TCCFB and TCDMN. The frequency of recovery of C. difficile from C. difficile-inoculated chopped beef specimens after enrichment in TCCFB and TCDMN was equal to or less than 9/20, which reflects the false-negative recovery of C. difficile from C. difficile-inoculated chopped beef specimens in greater than 55% of cases. The frequency of recovery of C. difficile from C. difficile-inoculated chopped beef specimens in TCM was the greatest among all other enrichment media investigated in this study. Proteolysis of the bovine cardiac tissue included in the cooked meat medium by the proteases produced by C. difficile, which provides more nutritional resources to C. difficile, might enable C. difficile to overcome the presence of large number of inherent bacteria in C. difficile-inoculated chopped beef specimens. Therefore, we conclude that TCM is a preferred enrichment medium for the recovery of C. difficile from beef specimens. Acknowledgments This study was supported by the National Research Foundation of Korea (NRF) grant, funded by the Korea government (MEST) (no. 2011-0015547).
References Arroyo, L.G., Rousseau, J., Willey, B.M., Low, D.E., Staempfli, H., McGeer, A., Weese, J.S., 2005. Use of a selective enrichment broth to recover Clostridium difficile from stool swabs stored under different conditions. J. Clin. Microbiol. 43, 5341–5343. Aspinall, S.T., Hutchinson, D.N., 1992. New selective medium for isolating Clostridium difficile from faeces. J. Clin. Pathol. 45, 812–814. de Boer, E., Zwartkruis-Nahuis, A., Heuvelink, A.E., Harmanus, C., Kuijper, E.J., 2011. Prevalence of Clostridium difficile in retailed meat in The Netherlands. Int. J. Food Microbiol. 144, 561–564. Jöbstl, M., Heuberger, S., Indra, A., Nepf, R., Köfer, J., Wagner, M., 2010. Clostridium difficile in raw products of animal origin. Int. J. Food Microbiol. 138, 172–175. Lessa, F.C., Gould, C.V., McDonald, L.C., 2012. Current status of Clostridium difficile infection epidemiology. Clin. Infect. Dis. 55, S65–S70. Levett, P.N., Margaritis-Bassoulis, G., 1985. Enrichment media for isolation of Clostridium difficile from faeces. Eur. J. Clin. Microbiol. 4, 135–136. Minton, N.P., Clarke, D.J., 1989. Clostridia. Springer Science + Business Media, New York. Poilane, I., Karjalainen, T., Barc, M.-C., Bourlioux, P., Collignon, A., 1998. Protease activity of Clostridium difficile strains. Can. J. Microbiol. 44, 157–161. Poutanen, S.M., Simor, A.E., 2004. Clostridium difficile-associated diarrhea in adults. Can. Med. Assoc. J. 171, 51–58. Rupnik, M., Wilcox, M.H., Gerding, D.N., 2009. Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat. Rev. Microbiol. 7, 526–536. Smith, H.L., 2011. Bacterial competition in serial transfer culture. Math. Biosci. 229, 149–159. Songer, J.G., Anderson, M.A., 2006. Clostridium difficile: An important pathogen of food animals. Anaerobe 12, 1–4. Songer, J.G., Trinh, H.T., Killgore, G.E., Thompson, A.D., McDonald, L.C., Limbago, B.M., 2009. Clostridium difficile in retail meat products, USA, 2007. Emerg. Infect. Dis. 15, 819–821. Sorg, J.A., Dineen, S.S., 2009. Laboratory maintenance of Clostridium difficile. Curr Protoc Microbiol. 12, 9A.1.1–9A.1.10. Tyrrell, K.L., Citron, D.M., Leoncio, E.S., Merriam, C.V., Goldstein, E.J.C., 2013. Evaluation of CCFA, CCFA-HT agars and CCMB-TAL broth for recovery of Clostridium difficile from fecal samples. J. Clin. Microbiol. 51, 3094–3096. Willis, A.T., 1977. Anaerobic bacteriology: clinical and laboratory practice. ButterworthHeinemann Limited, London. Zwietering, M.H., Jongenburger, I., Rombouts, F.M., van 't Riet, K., 1990. Modeling of the bacterial growth curve. Appl. Environ. Microbiol. 56, 1875–1881.
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Non-selective and selective enrichment media for the recovery of Clostridium difficile from chopped beef Changhoon Chai, Kyung-Soo Lee, Dayoung Lee, Soyeon Lee, Se-Wook Oh ⁎ Department of Food and Nutrition, Kookmin University, Seoul 136-702, Korea
a r t i c l e
i n f o
Article history: Received 13 October 2014 Received in revised form 1 December 2014 Accepted 1 December 2014 Available online 9 December 2014 Keywords: Antibiotics Beef Clostridium difficile Cooked meat medium Enrichment medium
a b s t r a c t Clostridium difficile exists within the intestines of animals and in meat products. Enrichment of C. difficile in an appropriate medium is necessary for the detection of C. difficile in meat products. Non-selective media (brain heart infusion medium [TBHI] and cooked meat medium containing sodium taurocholate [TCM]) and selective media (cycloserine-cefoxitin-fructose medium [TCCFB] and C. difficile moxalactam-norfloxacin medium containing antibiotics and sodium taurocholate [TCDMN]) can be used to enrich C. difficile. This study aimed to evaluate non-selective and selective enrichment media for the recovery of C. difficile from beef specimens. The efficiency of the enrichment media was investigated on the basis of the recovery frequency of C. difficile from beef specimens inoculated with C. difficile. The beef specimens were inherently contaminated with bacteria (around 104 CFU g−1), and further inoculated with C. difficile (around 100 CFU g−1). The antibiotics in TCCFB and TCDMN adversely affected C. difficile growth. The bacteria inherent to these specimens exhibited resistance to antibiotics and grew during the enrichment of C. difficile-inoculated chopped beef in TCCFB and TCDMN, which hindered the recovery of C. difficile. The frequency of recovery of C. difficile from beef specimens in TCM was higher than that from any other enrichment medium. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Clostridium difficile, a Gram-positive spore-forming anaerobic bacterium, is a causative pathogen of mild diarrhea or life-threatening pseudomembranous colitis (Poutanen and Simor, 2004). The incidence of C. difficile-associated diseases has increased over the past decade (Lessa et al., 2012). C. difficile is found in the intestines of humans and animals (Poutanen and Simor, 2004; Songer and Anderson, 2006) and in meat products (Jöbstl et al., 2010). It may be transmitted to humans via contact with infected humans or animals or by consumption of contaminated food (Jöbstl, et al., 2010; Rupnik et al., 2009). Clinical and environmental samples contain small amounts of C. difficile, and enrichment in an appropriate medium is frequently required to diagnose C. difficile infection and contamination (Arroyo et al., 2005; Levett and Margaritis-Bassoulis, 1985). C. difficile can be enriched in common media used for bacteria, and can be selectively enriched using C. difficile-specific carbohydrates or C. difficile-resistant antibiotics (Arroyo, et al., 2005; Tyrrell et al., 2013). The recovery of C. difficile spores from stool and clinical samples can be improved by incubation in cycloserine-cefoxitin-fructose medium (TCCFB) or C. difficile moxalactam-norfloxacin medium containing sodium taurocholate (TCDMN) (Jöbstl, et al., 2010; Tyrrell, et al., 2013). However, studies using enrichment media for the recovery of C. difficile from meat ⁎ Corresponding author. Tel.: +82 2 910 5778; fax: +82 2 910 5249. E-mail address: [email protected] (S.-W. Oh).
http://dx.doi.org/10.1016/j.mimet.2014.12.001 0167-7012/© 2014 Elsevier B.V. All rights reserved.
products have not yet been reported, despite the public safety concerns regarding C. difficile-associated diseases and the urgent need for research in this area. This study aimed to evaluate non-selective and selective enrichment media for the efficient recovery of C. difficile from beef specimens. In this study, brain heart infusion (TBHI) and cooked meat medium containing sodium taurocholate (TCM) were used as the non-selective enrichment media for C. difficile (Sorg and Dineen, 2009), and TCCFB and TCDMN were used as the selective media (Jöbstl, et al., 2010; Tyrrell, et al., 2013). The growth of C. difficile in TBHI, TCM, TCCFB, and TCDMN was measured, and the growth characteristics in each enrichment medium were analyzed using a modified Gompertz growth model (Zwietering et al., 1990). The efficiency of the enrichment media was evaluated on the basis of the frequency of recovery of C. difficile after incubation of C. difficile-inoculated chopped beef specimens in TBHI, TCM, TCCFB, and TCDMN. 2. Materials and methods 2.1. C. difficile strains and inoculum preparation C. difficile ATCC 9689, ATCC 43593, and ATCC 43601 were obtained from Korean Collection for Type Cultures (KCTC). Prior to inoculation in enrichment medium and chopped beef, each C. difficile strain was separately cultivated in BHI broth (OXOID, UK) at 37 °C for 14 h under anaerobic conditions (80% N2, 10% H2, and 10% CO2) in a Whitley A35
C. Chai et al. / Journal of Microbiological Methods 109 (2015) 20–24
Anaerobic Workstation (Don Whitley Scientific, UK). A three-strain C. difficile cocktail was prepared by mixing an equal volume of each C. difficile culture. Freshly cultivated C. difficile ATCC 9689 suspension and freshly prepared three-strain C. difficile cocktail were plated on BHI agar plates for enumeration and immediately used to inoculate the enrichment media and chopped beef. 2.2. Preparation of enrichment media TBHI and TCM were prepared by adding 10% (w/v) sodium taurocholate solution (Sigma-Aldrich, USA) to sterilized BHI (OXOID, UK) and CM broths (OXOID, UK) to a final concentration of 0.1% (Songer et al., 2009). TCCFB and TCDMN were prepared in accordance with a previously described method (Aspinall and Hutchinson, 1992; Tyrrell, et al., 2013). Proteose peptone (40 g; OXOID., UK), disodium hydrogen phosphate (5 g), potassium dihydrogen phosphate (1 g), magnesium sulfate (0.1 g), sodium chloride (2 g), and fructose (6 g) were dissolved in 910 mL of distilled water. After sterilization, 70 mL of horse blood (Hanil Komed, Korea) and 10 mL of 10% (w/v) sodium taurocholate solution were added. Subsequently, 10 mL of either C. difficile-selective supplement solution containing 25 mg L−1 d-cycloserine and 0.8 mg L−1 cefoxitin (OXOID, UK) or CDMN-selective supplement solution containing 3.2 mg mL−1 moxalactam, 1.2 mg mL−1 norfloxacin, and 50 mg mL−1 cysteine hydrochloride (OXOID, UK) was added to produce TCCFB and TCDMN, respectively. 2.3. Growth of C. difficile in enrichment media A fresh C. difficile ATCC 9689 culture suspension and a three-strain C. difficile cocktail were diluted with peptone water to a concentration of 104 CFU mL−1. TBHI, TCM, TCCFB, and TCDMN were inoculated with C. difficile by adding 1 mL of diluted fresh C. difficile ATCC 9689 culture suspension or three-strain cocktail to 200 mL of enrichment medium, followed by anaerobic incubation at 37 °C for 28 h. C. difficile enriched in TBHI, TCM, TCCFB, and TCDMN was plated on BHI agar plates at suitable dilutions every 2 h for 28 h. The colonies on BHI agar plates were enumerated after anaerobic incubation of BHI plates at 37 °C for 24 h. Enrichment and enumeration of C. difficile in TBHI, TCM, TCCFB, and TCDMN were performed in triplicate. The average growth curve values of C. difficile were entered into GraphPad Prism software v. 4.03 (GraphPad Software, USA) to analyze key growth parameters such as lag time (λ) and maximum growth rate (μ) of C. difficile based on a modified Gompertz growth model. 2.4. Recovery of C. difficile from C. difficile-inoculated chopped beef Chopped beef was purchased from a local market and dispensed into two 225-g specimens in large aseptic plastic bags (Whirl-Pak Bags, USA). To enumerate the inherent bacteria in the chopped beef specimens, 25 g of the remaining chopped beef was homogenized in 225 mL of 0.1% peptone water by using a stomacher (AES Stomacher; AES Chemunex, France). The homogenate was diluted and plated on plate count agar (PCA; OXOID, UK) or BHI agar plates. Aerobic bacteria were enumerated after incubation of PCA plates at 37 °C for 24 h in a conventional incubator. Anaerobic bacteria were determined by the number of colonies on BHI agar plates after anaerobic incubation at 37 °C for 24 h. A fresh C. difficile ATCC 9689 culture suspension and a three-strain C. difficile cocktail were diluted with peptone water to a final concentration of 101 CFU mL−1. Plate counts of diluted C. difficile showed concentrations of 13 CFU mL−1 and 35 CFU mL−1 for diluted C. difficile ATCC 9689 culture suspension and three-strain C. difficile cocktail, respectively. In total, 25 mL each of the diluted C. difficile ATCC 9689 culture suspension and three-strain C. difficile cocktail was added to each 225-g specimen in a large aseptic plastic bag and mixed well. Each inoculated chopped beef specimen was divided into 80 parts, with 2.5-g specimen in each part, and dispensed into aseptic plastic bags (7.5 cm × 12.5 cm;
21
Whirl-Pak Bags, USA). Subsequently, 22.5 mL of TBHI, TCM, TCCFB, or TCDMN was added to 20 chopped beef specimens, each weighing 2.5 g, and mixed well. C. difficile-inoculated chopped beef specimens in TBHI, TCM, TCCFB, and TCDMN were then anaerobically incubated at 37 °C for 2 days. After enrichment, the recovery of C. difficile was screened by streaking the enriched medium on TCCFA supplemented with 7% horse blood. The appearance of gray-colored colonies exhibiting the typical shape of C. difficile after anaerobic incubation at 37 °C for 24 h on TCCFA indicated that the specimen was positive for C. difficile. The gray colonies on TCCFA were streaked on C. difficile-selective agar (CDSA; Becton Dickinson, USA) and anaerobically incubated at 37 °C for 24 h. The recovery of C. difficile from C. difficile-inoculated chopped beef was confirmed by the appearance of a yellow colony with a typical C. difficile shape on CDSA. The recovery frequency was expressed as the number of presumptive positive and confirmed positive C. difficile specimens from C. difficile-inoculated chopped beef per 20 chopped beef specimens in TBHI, TCM, TCCFB, or TCDMN. 3. Results and discussion 3.1. Growth of C. difficile in enrichment media The growth curves of the single strain of C. difficile (ATCC 9689) and those of the three strains of C. difficile (ATCC 9689, ATCC 43593, and ATCC 43601) during anaerobic incubation at 37 °C in TBHI, TCM, TCCFB, and TCDMN are shown in Figs. 1 and 2, respectively. The growth of C. difficile in each enrichment medium was analyzed on the basis of a modified Gompertz model, which allowed the analysis of λ and μ of C. difficile in enrichment medium (Table 1). Plots from the beginning to the exponential-to-stationary phase transition were entered into GraphPad Prism software that was equipped with the modified Gompertz growth model for precise analysis of λ and μ. The growth pattern of C. difficile differed with the contents of the enrichment medium. The order of the μ values for C. difficile in each enrichment media was, from the greatest to the least, in TBHI, TCM, TCCFB, and TCDMN, which reflected that C. difficile grew at a slower pace in selective media than in non-selective media (Table 1). C. difficile in TBHI, TCM, TCCFB, and TCDMN inoculated with the single strain of C. difficile (ATCC 9689) and the three-strain C. difficile cocktail reached the stationary phase at 14, 12, 18, and 20 h and 12, 12, 14, and 14 h, respectively (Figs. 1 and 2). On reaching the stationary phase, the number of C. difficile in TCCFB and TCDMN abruptly decreased. This decrease might be attributed to the adverse effects of D-cycloserine, cefoxitin, moxalactam, or norfloxacin on the survival of C. difficile, despite their resistance to such antibiotics (Figs. 1c and d and. 2c and d). When only the single strain of C. difficile (ATCC 9689) existed in TBHI and TCM, C. difficile recovered and began to grow after 1.607 and 1.727 h, respectively (Table 1). However, the λ values for C. difficile in TBHI and TCM inoculated with the three-strain C. difficile cocktail were 2.825 and 2.473 h, respectively, which were greater than those in media inoculated with the single strain of C. difficile (Table 1). Competition among C. difficile strains was not expected in the enrichment medium with a single strain of C. difficile. However, the effect of competition among different strains of C. difficile on the growth of C. difficile in the enrichment medium containing three different strains of C. difficile could not be ignored, despite the fact that the three different strains belonged to the same species of Clostridium. The delay of λ when three different strains of C. difficile coexisted compared to that for the single strain of C. difficile (ATCC 9689) might be caused by the competition among the different strains of C. difficile (Figs. 1a and b, 2a and b, and Table 1) because λ is generally delayed because of the coexistence of multiple bacterial strains (Smith, 2011). The C. difficile strains used in this study showed varying susceptibilities to D-cycloserine and cefoxitin. The recovery of C. difficile in TCCFB with the single strain of C. difficile (ATCC 9689) was suppressed by either D-cycloserine or cefoxitin, and its λ value was greater than those
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Fig. 1. Growth of Clostridium difficile in enrichment media inoculated with C. difficile ATCC 9689 under anaerobic incubation at 37 °C; curves predicted on the basis of a modified Gompertz equation. (a) TBHI, (b) TCM, (c) TCCFB, and (d) TCDMN.
from TBHI and TCM (Fig. 1 and Table 1). However, the λ value of C. difficile in TCCFB with the three-strain cocktail of C. difficile was the lowest (Fig. 2 and Table 1). This suggests that either C. difficile ATCC 43593 or ATCC 43601 might be more resistant to either D-cycloserine or cefoxitin than C. difficile ATCC 9689. While one or two strains of C. difficile, including C. difficile ATCC 9689, were suppressed by D-cycloserine or cefoxitin in TCCFB, either C. difficile ATCC 43593 or ATCC 43601 might be recovered, resulting in lower λ in TCCFB compared to those from other enrichment media (Table 1). 3.2. Recovery of C. difficile from C. difficile-inoculated beef after enrichment Aerobic and anaerobic plate counts showed the presence of 2.13 × 105 CFU and 1.65 × 105 CFU per 2.5 g of chopped beef for total aerobic and anaerobic bacteria, respectively. As each 2.5-g chopped beef specimen was inoculated with 3 and 9 CFU of C. difficile of the single strain of C. difficile (ATCC 9689) and the three-strain C. difficile cocktail, respectively, the recovery of C. difficile from C. difficile-inoculated chopped beef specimens in different enrichment media was caused by its competition with the inherent bacteria. Thus, the recovery frequency of C. difficile from C. difficile-inoculated chopped beef in enrichment media (Table 2) may be a critical parameter to evaluate the efficiency of enrichment media for the recovery of C. difficile from beef. Despite the presence of C. difficile in all chopped beef specimens, not all specimens were confirmed to be positive for C. difficile, owing to the presence of large numbers of inherent bacteria in chopped beef. The recovery frequency of C. difficile from C. difficile-inoculated chopped beef exhibited wide variation, ranging from 1/20 to 15/20, depending on the enrichment medium. TCCFB and TCDMN are frequently used to selectively enrich C. difficile in meat and clinical samples since C. difficile is resistant to
the antibiotics included in TCCFB and TCDMN (de Boer et al., 2011; Tyrrell, et al., 2013). However, colonies formed by bacteria other than C. difficile were found on TCCFA and CDSA plates (both of which contained D-cycloserine and cefoxitin) after the culture of C. difficileinoculated chopped beef specimens in TBHI, TCM, TCCFB, and TCDMN. This resulted in differences between the number of confirmed positives and presumptive positives (Table 2). Some of the inherent bacteria in chopped beef that were resistant to D-cycloserine, cefoxitin, moxalactam, or norfloxacin grew in TCCFB and TCDMN. Hence, competition between C. difficile and the inherent bacteria in chopped beef that were resistant to the antibiotics in TCCFB and TCDMN was unavoidable. As observed with the growth of C. difficile in the enrichment medium, the growth of C. difficile ATCC 9689 was adversely affected by antibiotics in TCCFB and TCDMN (Fig. 1c and Table 1). The poor frequency of recovery of C. difficile from chopped beef specimens inoculated with the single strain of C. difficile (ATCC 9689) in TCCFB and TCDMN (1/20 and 4/20, respectively) might be attributed to the suppression of the growth of C. difficile ATCC 9689 by antibiotics as well as the growth of inherent bacteria that are resistant to the antibiotics. The frequencies of recovery of C. difficile from chopped beef specimens inoculated with the threestrain C. difficile cocktail in TCCFB and TCDMN (9/20 and 8/20, respectively) were greater than those from chopped beef specimens inoculated with the single strain of C. difficile (ATCC 9689) in the same enrichment medium (Table 2). Additionally, C. difficile showed lower λ and higher μ in specimens inoculated with the three-strain C. difficile cocktail than in those inoculated with a single strain of C. difficile (ATCC 9689) (Table 1). Therefore, the susceptibility of C. difficile to antibiotics included in the selective media might alter the frequency of recovery of C. difficile from C. difficile-inoculated chopped beef specimens after enrichment in selective media. However, the frequencies of recovery of C. difficile from C. difficile-inoculated chopped beef specimens in TCCFB
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Fig. 2. Growth of Clostridium difficile in enrichment media inoculated with a three-strain cocktail of C. difficile (ATCC 9689, ATCC 43593, and ATCC 43601) under anaerobic incubation at 37 °C; curves predicted on the basis of a modified Gompertz equation. (a) TBHI, (b) TCM, (c) TCCFB, and (d) TCDMN.
and TCDMN were lower than those from C. difficile-inoculated chopped beef specimens in TCM, regardless of the number of C. difficile strains present in the chopped beef specimens (Table 2).When C. difficile-inoculated chopped beef specimens were incubated in enrichment media, the bacteria present in the chopped beef, including C. difficile, competed against each other for nutritional resources. The fact that C. difficile grows relatively fast, with a short λ compared to other bacteria might lend it an advantage in bacterial competition (Minton and Clarke, 1989; Sorg and Dineen, 2009). Thus, we speculate that C. difficile ATCC 9689 in TBHI grew rapidly and overcame the presence of large numbers of inherent bacteria, resulting in a recovery frequency of 8/20 from chopped beef specimens inoculated with the single strain of C. difficile (ATCC 9689) (Table 2). However, the recovery frequency from C. difficile-inoculated chopped beef specimens with the three-strain C. difficile cocktail in TBHI was only 1/20 because the competition among the three Clostridium species allowed other bacteria to dominate (Table 2). It is known that C. difficile produces proteases (Poilane et al., 1998). C. difficile present in C. difficile-
inoculated chopped beef specimens enriched in TCM might obtain additional nutrients from the proteolysis of bovine cardiac tissue present in the cooked meat medium, a process similar to that exhibited by other Clostridium spp. grown in cooked meat medium (Willis, 1977). Therefore, in TCM, C. difficile might be more competitive for nutritional resources than the inherent bacteria present in chopped beef specimens, which may explain the observation that the frequencies of recovery of C. difficile from chopped beef specimens in TCM were greater than those in other enrichment media (Table 2). 4. Conclusions The growth and recovery of C. difficile from beef specimens in enrichment broth were largely influenced by the contents of the enrichment broth. The selective media TCCFB and TCDMN are frequently used to selectively enrich C. difficile in food and clinical samples, as C. difficile is resistant to D-cycloserine, cefoxitin (present in TCCFB), moxalactam, and
Table 1 Lag times (λ) and maximum growth rates (μ) of C. difficile in enrichment media, as analyzed using a modified Gompertz growth model. Inoculum
C. difficileATCC 9689
Three-strain cocktail ofC. difficile (ATCC 9689, ATCC 43593, and ATCC 43601)
a 2
R = determination coefficient. b RMSE = root mean square error.
Enrichment medium
TBHI TCM TCCFB TCDMN TBHI TCM TCCFB TCDMN
Parameter estimates λ (h)
μ (log (CFU mL
1.607 1.727 3.449 4.139 2.825 2.473 1.786 3.001
0.847 0.731 0.692 0.567 0.925 0.878 0.826 0.740
Regression analysis −1
)h
−1
)
R2a
RMSEb
Bias
0.998 0.995 0.999 0.996 0.990 0.997 0.998 0.997
0.102 0.164 0.058 0.178 0.270 0.133 0.110 0.154
1.004 1.004 1.002 1.004 1.000 1.004 1.004 1.000
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Table 2 Recovery ratio of Clostridium difficile from C. difficile-inoculated chopped beef specimens after enrichment. Inoculum
Non-selective media
Selective media
TBHI
TCCFB
TCM
TCDMN
Confirmed positive (presumptive positive) C. difficile ATCC 9689 Three-strain cocktail of C. difficile (ATCC 9689, ATCC 43593, and ATCC 43601)
8/20 (10/20) 13/20 (13/20) 1/20 (11/20) 4/20 (5/20) 1/20 (11/20) 15/20 (19/20) 9/20 (11/20) 8/20 (14/20)
norfloxacin (present in TCDMN). However, the growth of C. difficile was adversely affected by antibiotics included in TCCFB and TCDMN. Moreover, we observed that the inherent bacteria in chopped beef specimens were resistant to such antibiotics, and hence grew in TCCFB and TCDMN. The frequency of recovery of C. difficile from C. difficile-inoculated chopped beef specimens after enrichment in TCCFB and TCDMN was equal to or less than 9/20, which reflects the false-negative recovery of C. difficile from C. difficile-inoculated chopped beef specimens in greater than 55% of cases. The frequency of recovery of C. difficile from C. difficile-inoculated chopped beef specimens in TCM was the greatest among all other enrichment media investigated in this study. Proteolysis of the bovine cardiac tissue included in the cooked meat medium by the proteases produced by C. difficile, which provides more nutritional resources to C. difficile, might enable C. difficile to overcome the presence of large number of inherent bacteria in C. difficile-inoculated chopped beef specimens. Therefore, we conclude that TCM is a preferred enrichment medium for the recovery of C. difficile from beef specimens. Acknowledgments This study was supported by the National Research Foundation of Korea (NRF) grant, funded by the Korea government (MEST) (no. 2011-0015547).
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