Prevalence of Clostridium difficile in Uncooked Ground Meat Products
from Pittsburgh, Pennsylvania
Scott R. Curry, Jane W. Marsh, Jessica L. Schlackman, and Lee H. Harrison
Infectious Diseases Epidemiology Research Unit, Division of Infectious Diseases, University of Pittsburgh School of Medicine, and Department of Epidemiology, Graduate
School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
The prevalence of Clostridium difficile in retail meat samples has varied widely. The food supply may be a source for C. difficile
infections. A total of 102 ground meat and sausage samples from 3 grocers in Pittsburgh, PA, were cultured for C. difficile. Brand
were positive for C. difficile; both were pork sausage from brand A from the same processing facility (facility A). On subsequent
C. difficile. The isolates recovered were inferred ribotype 078, comprising 6 genotypes. The prevalence of C. difficile in retail
14–16, 18–20, 22, 23). The C. difficile strains recovered in these
are commonly encountered in human outbreaks of C. difficile in-
CDI and the food supply has been made (6, 18). Recent evidence
suggests either that some C. difficile strains are widespread in the
food supply or that laboratory contamination led to overestima-
tion of the prevalence of C. difficile in some studies (13, 21).
We performed a study of C. difficile prevalence in raw retail
ground meats in grocery stores in the Pittsburgh, PA, area. Mul-
tilocus variable-number tandem-repeat analysis (MLVA), a
difficile isolated from food products and to determine the genetic
relatedness between C. difficile isolates recovered from food and
isolates associated with human CDI.
International Conference on the Molecular Biology and Patho-
genesis of the Clostridia [ClosPath 2011], Ames, IA, 28 October
he prevalence of Clostridium difficile contamination of food
products has varied widely, ranging from 0 to 42% (3, 5–10,
MATERIALS AND METHODS
Sampling scheme. We performed a convenience sampling of 102 raw
initial sampling were purchased between 23 February and 28 April 2011.
Items purchased included samples of all available deli counter ground
aging until the time of sampling. Where available, the USDA establish-
ment number identifying the facility of origin for each product was
recorded. Cultures were performed before the sell-by date for each pack-
aged item. Based on the results of the initial sampling, all available brand
Microbiological methods. Broth enrichment cultures were per-
formed for all samples as follows: 10 g meat was aseptically transferred to
100 ml cycloserine (500 mg/liter)-cefoxitin (15.5 mg/liter)-mannitol
broth with 0.1% taurocholate and 0.5% lysozyme (CCMB-TAL) in
120-ml sterile specimen containers (Starplex Scientific, Etobicoke, On-
tario, Canada) and incubated anaerobically at 37°C for 5 days in an an-
aerobic chamber (Coy Labs, Grass Lake, MI). The CCMB-TAL was not
allow for reduction of the liquid medium by exchange with the chamber
atmosphere. Visibly fermented samples were subcultured to prereduced
lin Lakes, NJ) and further subcultured to SBA until pure. Colony mor-
phologies on SBA that were consistent with C. difficile were confirmed
10-g meat sample spiked with 10 ?l of strain CD41 spore stock (?105
CFU) served as a positive control for every 8 samples processed. In addi-
tion, a CCMB-TAL medium negative control of 100 ml was included for
every 8 samples processed.
C. difficile isolates were stored in chopped meat broth (Anaerobe Sys-
tems, Morgan Hill, CA). Genomic DNA was extracted after 48 hours of
0.1% taurocholate (BHIYT) using an automated magnetic bead extrac-
tion platform (NucliSens easyMag; bioMérieux, Durham, NC).
MLVA and tcdC genotyping were performed for all isolates (12). The
tcdC genotype for each isolate was used to infer the ribotype (4). Because
two MLVA tandem-repeat loci (CDR4 and CDR5) are absent in inferred
ference (STRD) was calculated using only MLVA loci CDR6, CDR9,
lished alleles, with the exception that genotype A/A1 has been renamed
site (http://www.pubmlst.org/cdifficile). All isolates with a tcdC genotype
were presumed to be toxigenic.
isolates collected between 2001 and 2009 from CDI patients and asymp-
tomatic carriers at our institution, including 67 isolates originating from
community-acquired, community-onset CDI patients diagnosed be-
tween January and June 2011. Food and UPMC clinical isolates were
considered highly related if the STRD was ?2 (11).
Received 13 March 2012 Accepted 4 April 2012
Published ahead of print 13 April 2012
Address correspondence to Scott R. Curry, email@example.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
June 2012 Volume 78 Number 12 Applied and Environmental Microbiologyp. 4183–4186aem.asm.org