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Clostridium difficile in ready-to-eat salads, Scotland.

Over the past decade, Clostridium difficile infection has become a prominent cause of healthcare-associated infection. Although C. difficile has been thought of traditionally as a predominantly nosocomial infection, the incidence of community-acquired cases has increased recently, as has the incidence of cases from other healthcare settings such as nursing homes (1). Notably, some evidence has shown that C. difficile may be brought into the healthcare environment by asymptomatic carriers (2). The reported carriage rates of C. difficile in healthy adults have varied from 0% to 3% in Europe to up to 15% in Japan (3). Little is known, however, about the prevalence of C. difficile in the environment and how it may be transmitted to humans.

C. difficile has been found in a variety of environments, including water, soil, animal feces, and foods (4,5); these findings suggest that C. difficile may be transmitted to humans through food, although no foodborne cases have been reported. Because ready-to-eat foods have been implicated in foodborne disease outbreaks associated with Salmonella species (6) and Escherichia coli O157 (7), we examined ready-to-eat salads for the presence of C. difficile.

The Study

We tested 50-g samples from each of 40 packaged ready-to-eat salads purchased from 7 Glasgow supermarkets from May 1 through June 30, 2008, for the presence of C. difficile spores. We essentially used the CDMN (C. difficile, moxalactam, norfloxacin) agar method of Rodriguez-Palacios et al. (4) but also used direct plating and enrichment broth culture. The contents of the 40 salads generally differed, and any salads with the same contents carried different supermarket brands, which eliminated replicate sampling.

Isolates were identified as toxigenic (having genes for toxins A and B) by PCR as previously described (8,9), and ribotypes were identified by PCR (10). The MICs of 6 antimicrobial drugs for these isolates were determined by using E-test strips (AB Biodisk, Solna, Sweden). The following MIC breakpoints were used to define resistance to these drugs: metronidazole, [greater than or equal to]32 [micro]g/mL; vancomycin, [greater than or equal to]16 [micro]g/ mL; cefotaxime, 64 [micro]g/mL; erythromycin, [greater than or equal to]8 [micro]g/mL; moxifl oxacin, [greater than or equal to]8 [micro]g/mL; and clindamycin, [greater than or equal to]8 [micro]g/mL (Clinical and Laboratory Standards Institute, Wayne, PA, USA).

C. difficile spores were detected in 3 (7.5%) of the 40 salad samples after culturing in enrichment broth (Table 1). Thirty-five (87.5%) of the salads were marked as imported from European Union countries; the remaining 5 were from UK suppliers. The 3 contaminated salads were not of UK origin. The 3 isolates were found to be toxinogenic by PCR; 2 were PCR ribotype 017, and 1 was PCR ribotype 001 (Table 1).

The MICs of 6 antimicrobial drugs for each isolate and the resistance profile of each isolate are shown in Table 2. None of the isolates was resistant to vancomycin or metronidazole, and only the 001 isolate was resistant to moxifloxacin and erythromycin. All 3 isolates were resistant or intermediately resistant to clindamycin and cefotaxime; breakpoints for these drugs were highest for the 001 isolate.

Isolates were obtained after being cultured in enrichment broth and not by direct plating, which suggests that spore counts were low (<3.0 CFU/g). The infectious dose required to colonize the healthy human gut is, however, unknown. Isolates were of PCR ribotypes 001 (a common clinical isolate in Scotland [11]) and 017 (a common European PCR ribotype containing isolates that are negative for toxin A and positive for toxin B [12]). No isolate was resistant to vancomycin or metronidazole, which is in accord with findings for other C. difficile isolates found in Scotland (11), but recent studies have highlighted the emergence of increased resistance to metronidazole among C. difficile isolates in England (13).

In general, the PCR ribotype 001 isolate was more drug resistant than the 017 isolates; it was the only isolate resistant to moxifloxacin and erythromycin and had the highest breakpoints to clindamycin and cefotaxime. In a 2005 study in which 271 C. difficile isolates from the UK were examined, all were found to be resistant to cefotaxime (14).

Conclusions

The isolation of these PCR ribotypes from ready-to-eat salads is of concern and highlights the potential risk associated with consuming these salads, particularly since they are not cooked before being consumed. The consumption of these foods by vulnerable groups could possibly lead to C. difficile colonization and an increase in the asymptomatic C. difficile carriage rate among humans, thus increasing the risk for C. difficile transference within the healthcare environment (2). The presence of C. difficile in ready-to-eat salads could result from environmental contamination or transmission by food handlers. Further work is needed to investigate foods as a source of this pathogen and also to assess the role of soil and animals as its reservoirs.

DOI: 10.3201/eid1505.081186

References

(1.) McFarland LV, Beneda HW, Clarridge JE, Raugi GJ. Implications of the changing face of Clostridium difficile disease for health care practitioners. Am J Infect Control. 2007;35:237-53. DOI: 10.1016/ajic.2006.06.004

(2.) Clabots CR, Johnson S, Olson MM, Peterson LR, Gerding DN. Acquisition of Clostridium difficile by hospitalized patients: evidence for colonized new admissions as a source of infection. J Infect Dis. 1992;166:561-7.

(3.) Mulligan ME. Clostridium difficile--its role in intestinal disease. London: Academic Press; 2008. p. 229-56.

(4.) Rodriguez-Palacios A, Staempfli HR, Duffield T, Weese JS. Clostridium difficile in retail ground meat, Canada. Emerg Infect Dis. 2007;13:485-7.

(5.) Al Saif N, Brazier JS. The distribution of Clostridium difficile in the environment of South Wales. J Med Microbiol. 1996;45:133-7.

(6.) Sagoo SK, Little CL, Ward L, Gillespie IA, Mitchell RT. Microbiological study of ready-to-eat salad vegetables from retail establishments uncovers a national outbreak of salmonellosis. J Food Prot. 2003;66:403-9.

(7.) Delaquis P, Bach S, Dinu LD. Behavior of Escherichia coli O157:H7 in leafy vegetables. J Food Prot. 2007;70:1966-74.

(8.) Ward SJ, Douce G, Dougan G, Wren BW. Local and systemic neutralizing antibody responses induced by intranasal immunization with the nontoxic binding domain of toxin A from Clostridium difficile. Infect Immun. 1999;67:5124-32.

(9.) Butcher J. Development of inhibitory analogs of Clostridium difficile toxins as novel therapeutics [dissertation]. Glasgow (UK): Glasgow Caledonian University; 2009.

(10.) Stubbs SL, Brazier JS, O'Neill GL, Duerden BI. PCR targeted to the 16S-23S rRNA gene intergenic spacer region of Clostridium difficile and construction of a library consisting of 116 different PCR ribotypes. J Clin Microbiol. 1999;37:461-3.

(11.) Mutlu E, Wroe AJ, Sanchez-Hurtado K, Brazier JS, Poxton IR. Molecular characterization and antimicrobial susceptibility patterns of Clostridium difficile strains isolated from hospitals in southeast Scotland. J Med Microbiol. 2007;56:921-9. DOI: 10.1099/jmm.0.47176-0

(12.) van den Berg RJ, Claas EC, Oyib DH, Klaassen CH, Dijkshoorn L, Brazier JS, et al. Characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates from outbreaks in different countries by amplified fragment length polymorphism and PCR ribotyping. J Clin Microbiol. 2004;42:1035-41. DOI: 10.1128/JCM.42.3.1035-1041.2004

(13.) Emergence of reduced susceptibility to metranidazole in Clostridium difficile. Weekly Report, News Archives. Health Protection Agency. 2008;2 [cited 2009 Mar 31]. Available from http://www.hpa.org.uk/hpr/archives/2008/news0308.htm#cdiff1

(14.) John R, Brazier JS. Antimicrobial susceptibility of polymerase chain reaction ribotypes of Clostridium difficile commonly isolated from symptomatic hospital patients in the UK. J Hosp Infect. 2005;61:11-4. DOI: 10.1016/j.jhin.2005.01.020

Marwah M. Bakri, Derek J. Brown, John P. Butcher, and Alistair D. Sutherland

Author affiliations: Glasgow Caledonian University, Glasgow, Scotland, UK (M.M. Bakri, J.P. Butcher, A.D. Sutherland); and Scottish Salmonella, Shigella and Clostridium difficile Reference Laboratory, Glasgow (D.J. Brown)

Mrs Makri is a lecturer at Jazan University in Jazan, Saudi Arabia, and a candidate for a PhD in microbiology at Glasgow Caledonian University, Glasgow, Scotland, under the supervision of Dr Sutherland. Her research interest involves the molecular epidemiology of C. difficile.

Address for correspondence: Alistair D. Sutherland, Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Rm C131, Cowcaddens Rd, Glasgow G4 0BA, Scotland, UK; email: a.sutherland@gcal.ac.uk
Table 1. PCR profile for toxins A and B and PCR ribotype of
Clostridium difficile isolates, Scotland, 2008

Sample no./description Toxin A Toxin B PCR ribotype

13/baby leaf spinach -- + 017
24/organic mixed leaf salad -- + 017
35/organic lettuce + + 001

Table 2. Susceptibility of 3 Clostridium difficile isolates to 6
antimicrobial drugs, by source of isolate, Scotland, UK, 2008 *

 Source of isolate

Antimicrobial Baby leaf spinach Organic mixed leaf salad
drug (MIC, [micro]g/mL) (MIC, [micro]g/mL)

Metronidazole S (0.125) S (0.094)
Vancomycin S (0.50) S (0.38)
Moxifloxacin S (0.50) S (0.75)
Clindamycin I (4.0) I (6.0)
Erythromycin S (1.5) S (0.75)
Cefotaxime I (48) R (64)

 Source of isolate

Antimicrobial Organic lettuce
drug (MIC, [micro]g/mL)

Metronidazole S (0.75)
Vancomycin S (1.0)
Moxifloxacin R (256)
Clindamycin R (8.0)
Erythromycin R (192)
Cefotaxime R (256)

* S, sensitive; R, resistant; I, intermediate.
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Article Details
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Title Annotation:DISPATCHES
Author:Bakri, Marwah M.; Brown, Derek J.; Butcher, John P.; Sutherland, Alistair D.
Publication:Emerging Infectious Diseases
Article Type:Report
Geographic Code:4EUUK
Date:May 1, 2009
Words:1538
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