Characterization of Clostridium difficile Isolates from Human Fecal Samples and Retail Meat from Pennsylvania

Abstract

A study was conducted to determine the prevalence of Clostridium difficile and characterize C. difficile isolates from human stool and retail grocery meat samples. Human stool samples (n=317) were obtained from a clinical laboratory and meat samples (n=303) were collected from 8 retail grocery stores from October 2011 through September 2012 from Centre County of Pennsylvania and were examined for C. difficile. C. difficile was isolated from 16.7% of stool samples (n=317) and 6.9%, 11.5%, 14.5%, and 7.8% of beef (n=72), pork (n=78), turkey (n=76), and chicken (n=77) samples, respectively. Six different toxin gene profiles were detected in all human and meat isolates of C. difficile based on the presence or absence of toxin genes tcdA, tcdB, and cdtA and cdtB. Interestingly, 75.6% of the human C. difficile isolates lacked any deletion in the tcdC gene (139-bp), whereas a 39-bp deletion was observed in 61.3% of the C. difficile strains isolated from meat samples. C. difficile from meat samples were more susceptible to clindamycin, moxifloxacin, vancomycin, and metronidazole than C. difficile isolates from human samples. Twenty-five different ribotypes were identified in human and meat C. difficile isolates. In conclusion, significant genotypic and phenotypic differences were observed between human and meat isolates of C. difficile; however, a few C. difficile isolates from meat—in particular ribotypes 078, PA01, PA05, PA16, and PA22 with unique profiles (toxin gene, tcdC gene size and antimicrobial resistance profiles)—were similar to human C. difficile isolates.

Introduction

C lostridium difficile (CD) is a spore-forming, Gram-positive, anaerobic commensal bacteria in the gut (Ananthakrishnan et al., 2010). Frequent use of broad-spectrum antimicrobials in hospital and outpatient settings has resulted in the emergence of antibiotic-resistant CD and has considerably increased the health care risks associated with CD infections (CDI) (Lipsett et al., 1994). It has been reported that around 20% of patients with CDI fail to get cured on antibiotic therapy due to advancement of the infection, relapse, or reinfection, leading to increased antibiotic resistance (Johnson et al., 1999; Dallal et al., 2002).

Virulent strains of CD produce toxins such as enterotoxin toxin A (tcdA), cytotoxin toxin B (tcdB) and binary toxin CD toxin A (cdtA) and CD toxin B (cdtB), tcdA, and tcdB along with negative regulator of toxin A and B (tcdC), and are located on a well-defined genetic element called pathogenic locus (Thelestam et al., 2000). TcdA binds on the apical side of host enterocytes, whereas tcdB binds on the basolateral side of enterocytes. Both of the toxins are proteolytic and are taken into cytoplasm of the cells. TcdC has been reported as a negative regulator of CD toxins on pathogenicity locus, and it has been suggested that any deletion in the tcdC gene may lead to decreased toxigenicity of CD (Jank et al., 2007). In contrast to tcdA and tcdB, both cdtA and cdtB are present outside of PaLoc on the binary toxin locus (Rupnik, 2008). These toxins are cytotoxic but their role is not well understood. Therefore, the presence or absence of the toxins may help us to identify various CD variants. In recent years, ribotyping has been widely used to subtype CD. Many ribotypes have been identified thus far; the most common ribotypes found in humans are 078, 027, 001, and 017 (Fawley et al., 2008; Goorhuis et al., 2008).

Due to an increase in the frequency of CDIs in patients, it is speculated that CD from animal-origin foods can cause CDI in humans. There are reports that show that meat can be a potential source of virulent CD; however, there is no epidemiologic study that establishes a causal relationship of virulent CD strains from meat samples causing CDI in humans (Al Saif and Brazier, 1996). The objectives of this study were as follows: (1) To determine the prevalence of CD in human fecal and retail meat samples, (2) To characterize the phenotype (antibiotic resistance) and genotype (toxin types and ribotypes) of CD, and (3) To identify subtypes of CD that are similar in retail meat and humans.

Materials and Methods

Meat samples

A total of 303 meat samples including ground beef (n=72), ground pork (n=78), ground turkey (n=76), and chicken thighs (n=77) were collected from October 2011 through September 2012 from 8 retail grocery stores in Centre County, PA. Approximately 250 g of each of the 4 varieties of meat samples were collected once a month from each of the 8 retail grocery stores and examined for CD. Meat samples were transported on ice and processed immediately or frozen at −20°C and examined within 3–5 days of collection. CD was isolated from meat samples as described by Rodriguez-Palacios et al. (2007). Briefly, ∼10 g of meat sample was transferred to a stomacher bag containing 50 mL of prereduced C. difficile moxalactam norfloxacin (CDMN) broth supplemented with 5% horse blood, moxalactam, and norfloxacin antibiotics (Oxoid, Basingstoke, UK) and 0.1% sodium taurocholate (Sigma-Aldrich, Inc., St. Louis, MO). The mixture was homogenized in a stomacher for 60 s and incubated anaerobically at 37°C for 10 days. Alcohol shock for spore selection was done by mixing 2 mL thoroughly mixed culture broth and 96% ethanol (1:1 [vol/vol]) for 50 min. After centrifugation (3800×g for 10 min), the sediment was streaked onto on CDMN agar plates incubated anaerobically for 48 h at 37°C.

Human fecal samples

A total of 317 unidentified human fecal samples were obtained from October 2011 through September 2012 from a clinical laboratory in Centre County, PA. Collection of samples was initiated following approval of the Penn State Institutional Board Review and internal review and approval of the Institutional Review Board of the health facility. Fecal sample cups were identified by date. No other data were collected regarding age, sex, occupation, and health status associated with the samples. Fecal samples were processed immediately on being received in the laboratory. CD was isolated as described by Rodriguez-Palacios et al. (2007). Briefly, approximately 0.5–1.0 g of fecal sample was transferred to a stomacher bag containing 1.0–5.0 mL of prereduced Clostridium difficile moxalactam norfloxacin (CDMN) broth and processed as described for meat samples.

Isolation and identification of CD

CD-presumptive-positive colonies were examined for their morphology by Gram stain, and for their l-prolineaminopeptidase activity using proline discs (Pro Disc; Remel, Lenexa, KS). DNA was isolated using a standard phenol/chloroform extraction method (Pospiech and Neumann, 1995) and examined for the tpi gene as described by Lemee et al. (2004). Isolates that were positive for the tpi gene were identified as CD and further examined for CD toxin genes.

Multiplex real-time polymerase chain reaction (PCR) for detection of tcdC CD

Isolates were screened for tcdA, tcdB, binary toxin (cdtA and cdtB) using multiplex real-time PCR as described by Houser et al. (2010). Strain 769 (positive for all four toxins) was obtained from Dr. Songer at the University of Iowa. In addition, ATCC 9689 (ATCC, Manassas, VA) was also used in our study. The size of the tcdC gene was determined using primer sequences and PCR conditions described by Antikainen et al. (2009). The PCR was performed using the Stratagene MX3005 quantitative PCR system (Stratagene, Santa Clara, CA).

Antibiotic susceptibility testing

The antibiotics used for susceptibility testing were selected following consultation with primary care physicians who indicated that clindamycin, moxifloxacin, metronidazole, and vancomycin were the most commonly used antibiotics in a clinical setting in Pennsylvania to treat CDI. The susceptibility of CD isolates from meat and human fecal samples to clindamycin, moxifloxacin, metronidazole, and vancomycin was determined using Etest strips (BioMérieux Inc., Marcy l'Etoile, France) on Brucella blood agar supplemented with hemin and vitamin K, according to the manufacturer's instructions. The plates were incubated for 48 h at 37°C under anaerobic conditions, after which the minimum inhibitory concentration (MIC) values were determined. The MIC breakpoints for clindamycin, vancomycin, metronidazole, and moxifloxacin were compared with those established by the Clinical and Laboratory Standards Institute (CLSI, 2007).

PCR ribotyping of CD isolates

CD isolates previously identified and tested for toxin typing were reinoculated on 5% defibrinated sheep blood agar (Becton, Dickinson and Company, Franklin Lakes, NJ). Plates were incubated at 37°C for 36–48 h under anaerobic conditions. A Chelex resin-based DNA extraction kit (InstaGene Matrix, Bio-Rad, Hercules, CA) was utilized for DNA extraction of all toxin-typed CD isolates. DNA supernatant was collected and stored at −20°C until further use. CD control DNA for ribotypes 001, 017, 027, and 078 was obtained from Drs. Hegarty and Stewart at the Hershey Medical Center, Hershey, PA. Amplification was performed according to the method described previously by Bidet et al. (1999) with a slight modification, initial denaturation of 95°C for 5 min, followed by 35 cycles each consisting of 94°C for 1 min, 57°C for 1 min, and 72°C for 1 min, and final extension at 72°C for 5 min. Ribotype patterns were analyzed with GelCompar II image analysis software (version 5.1; Applied Mathematics, Austin, TX).

Results

Prevalence of CD from human stool samples and retail meat

CD was isolated from 53 of 317 (16.7%) human fecal samples, while CD was isolated from 31 of 303 (10.2%) of meat samples. The isolation rates of CD from ground beef, pork, turkey, and chicken were 6.9, 11.5, 14.5, and 7.8%, respectively.

Toxin gene profiles of CD isolated from humans and meat samples

CD isolates from human fecal and retail meat samples were examined for toxin genes (tcdA, tcdB, cdtA, and cdtB) and tcdC gene size. The combinations of toxin genes were used to assign a profile A–F (Table 1). For example, an isolate that was positive for all tcdA, tcdB, cdtA, and cdtB was assigned a profile A. A total of 6 profiles (A–F) were observed (Table 1). Human isolates belonged to 5 profiles (A–D and F), while meat isolates also belonged to 5 profiles (A, B, and D–F). A total of four profiles including A, B, D, and F were seen in both human and meat CD isolates. The presence of tcdA, tcdB, and cdtA and cdtB toxin genes were observed in 85, 74, and 40% of human CD isolates and 65, 55, and 61% meat CD isolates, respectively (Table 1).

Table 1.

Distribution of Toxin Profiles of Clostridium difficile Isolated from Humans and Retail Meat Samples

	Toxin gene				Meat isolates
Profile	tcdA	tcdB	cdtA & cdtB	Human isolates	Beef	Chicken	Pork	Turkey	Total
A	+	+	+	19	2	4	2	3	11
B	+	+	−	20	1	−	4	1	6
C	+	−	−	4	−	−	−	−	0
D	+	−	+	2	2	−	−	1	3
E	−	−	+	0	−	1	−	4	5
F	−	−	−	8	−	1	3	2	6
Total				53	5	6	9	11	31

	Human isolates							Meat isolates
	Profile							Profile
Toxin gene	A	B	C	D	E	F	Total (%)	A	B	C	D	E	F	Total (%)
tcdA	19	20	4	2	−	−	45 (85)	11	6	−	3	−	−	20 (65)
tcdB	19	20	−	−	−	−	39 (74)	11	6	−	−	−	−	17 (55)
cdtA & cdtB	19	−	−	2	−	−	21(40)	11	−	−	3	−	5	19 (61)

On analysis of the size of the tcdC gene, nearly 75.5% of human CD isolates encoded for the entire 139-bp gene, while only 19.3% of the meat isolates encoded for the 139-bp gene. The 18-bp deletion in tcdC gene was seen in 15% and 19.3% of human and meat isolates, respectively. The 39-bp deletion in the tcdC gene was observed in 9.4% of human CD isolates, while the same deletion was observed in 61.3% of isolates from meat samples (Table 2).

Table 2.

Occurrence of tcdC Gene Deletions in Human and Meat Clostridium difficile Isolates

		Human isolates		Meat isolates
tcdC gene deletion	Profile	Total	%	Beef	Chicken	Pork	Turkey	Total	%
39-bp	A	3	9.4	2	1	2	2	7	61.3
	B	2				4		4
	E	0			1		4	5
	F	0				1	2	3
18-bp	A	4	15		2		1	3	19.3
	B	2						0
	C	1						0
	F	1			1	2		3
0-bp	A	12	75.6		1			1	19.3
	B	16		1			1	2
	C	3						0
	D	2		2			1	3
	F	7						0
		53		5	6	9	11	31

bp, base pairs.

Antimicrobial susceptibility of CD isolates

Resistance to clindamycin and moxifloxacin was observed in 19% and 21% of human CD isolates, respectively. No resistance to vancomycin or metronidazole was observed in humans. Resistance to clindamycin in CD isolates from meat ranged from 0% (beef) to 18% (turkey), while resistance to moxifloxacin ranged from 0% (beef) to 17% (chicken). All CD isolates from meat were 100% susceptible to vancomycin and metronidazole (Table 3). Antimicrobial susceptibilities and the MIC values of human and meat CD isolates to antibiotics are summarized in Table 3.

Table 3.

Antimicrobial Susceptibilities and the Minimum Inhibitory Concentration (MIC) Values of Clostridium difficile Isolates from Humans and Retail Meat

		Clindamycin	Moxifloxacin	Metronidazole	Vancomycin
Human (n=53)	MIC range (μg/mL)	1.5–>256	0.5–>32	0.064–1	0.25–1
	50%	4	1.5	0.25	0.75
	90%	>256	16	1	1
	% Resistance (n)	19 (10)	21 (11)	0 (0)	0 (0)
Beef (n=5)	MIC range (μg/mL)	0.5–1	0.5–1.5	0.032–0.75	0.125–1.5
	50%	0.5	0.5	0.125	0.25
	90%	1	0.75	0.5	1
	% Resistant (n)	0 (0)	0 (0)	0 (0)	0 (0)
Chicken (n=6)	MIC range (μg/mL)	0.50–16	0.25–32	0.032–1	0.50–2
	50%	4	0.25	0.25	0.75
	90%	16	32	1	1.5
	% Resistant (n)	17 (1)	17 (1)	0 (0)	0 (0)
Pork (n=9)	MIC range (μg/mL)	0.75–32	0.25–32	0.032–0.75	0.125–1
	50%	1	4	0.125	0.5
	90%	32	8	0.25	1
	% Resistant (n)	12 (1)	12 (1)	0 (0)	0 (0)
Turkey (n=11)	MIC range (μg/mL)	1–32	0.5–32	0.064–2	0.25–2
	50%	4	4	0.25	0.5
	90%	32	16	0.75	2
	% Resistant (n)	18 (2)	0 (0)	0 (0)	0 (0)

Ribotypes of CD isolated from humans and retail meat samples

CD isolated from human and meat isolates belonged to 25 ribotypes (Table 4). Isolates that were not identical to the known ribotypes (027, 001, 078) were designated from PA01 to PA22. A total of 11 ribotypes (001, PA03, PA04, PA06, PA08, PA10, PA12, PA13, PA15, PA19, and PA21) were unique to human CD isolates, while 3 ribotypes (PA07, PA11, and PA18) were unique to CD isolates from meat. It was observed that 11 of 25 ribotypes (027, 078, PA01, PA02, PA05, PA09, PA14, PA16, PA17, PA20, and PA 22) were common to both human and meat CD isolates (Table 4).

Table 4.

Ribotypes of Clostridium difficile Isolated from Humans and Retail Meat Samples

Ribotype	Isolates (toxin gene profile)
	Human	Total	Beef	Chicken	Pork	Turkey	Total
001	1(C)	1					0
027	1(C), 1(F)	2	2(A)			1(A)	3
078	2(A), 4(B), 1(D), 1(F)	8	2(D)		2(B), 2(F)	2(F)	8
PA01	3(A), 2(B),	5				1(B), 2(E)	3
PA02	2(B)	2			1(B)		1
PA03	2(A)	2					0
PA04	3(B), 1(D), 1(F)	5					0
PA05	1(A), 2(B)	3		1(A)		1(A)	2
PA06	1(A), 2 (F)	3					0
PA07		0		1(A)	1(B)	1(E)	3
PA08	2(A)	2					0
PA09	1(A), 1(B)	2			1(F)		1
PA10	1(C)	1					0
PA11		0		1(E)			1
PA12	2(F)	2					0
PA13	1(B)	1					0
PA14	1(A)	1				1(A), 1(D)	2
PA15	1(F)	1					0
PA16	1(A)	1		1(A)			1
PA17	2(B), 1(C)	3		1(A),1(F)	1(A)		3
PA18		0				1(E)	1
PA19	1(A)	1					0
PA20	1(A), 1(B)	2			1(A)		1
PA21	3(A)	3					0
PA22	2(B)	2	1(B)				1

Of the 11 ribotypes that were from both human and meat isolates, CD ribotypes 078, PA01, PA05, PA16, and PA22 with similar profiles (A, B, D, and F) were found to be present in human stool and meat samples (Table 5). CD ribotype 078 was similar to that isolated from human and ground pork samples. Similarly, CD ribotype PA01 was isolated from turkey and humans. While CD ribotypes PA05 and PA16 were isolated from chicken, human, and turkey samples and ribotype PA22 was isolated from beef and human samples (Table 5).

Table 5.

Characteristics of Clostridium difficile Isolated from Both Humans and Meat Samples

Ribotype	Sample	TcdC gene size	tcdA	tcdB	cdtA & cdtB	Antibiotic resistance	Isolates
078	Human	100	+	+	−	−	1
078	Pork	100	+	+	−	−	3
078	Human	121	−	−	−	−	1
078	Pork	121	−	−	−	−	2
PA01	Human	139	+	+	−	−	1
PA01	Turkey	139	+	+	−	−	1
PA05	Chicken	121	+	+	+	−	1
PA05	Human	121	+	+	+	−	1
PA05	Turkey	121	+	+	+	−	1
PA16	Chicken	121	+	+	+	−	1
PA16	Human	121	+	+	+	−	1
PA22	Beef	139	+	+	−	−	1
PA22	Human	139	+	+	−	−	2

Discussion

CD was isolated from 16.7% of human stool samples obtained from a clinical laboratory in Centre County, PA. In the absence of health history (age, gender, occupation, dietary habits, health status, use of antibiotics, or use of proton pump inhibitors), it is difficult to elaborate on the prevalence rate observed in our study. A recent study conducted in France showed that 38 of 85 (45%) healthy infants from 2 nurseries were asymptomatic carriers of CD, of which 11% harbored toxigenic strains (Rousseau et al., 2012). Rea et al. (2012) observed a CD carriage rate of 1.6% from elderly individuals, 9.5% in outpatient settings, and 21% of patients in short- or long-term care in hospitals. Bengualid et al. (2011) examined individuals for CDI at a community hospital in the Bronx, New York, from 2006 to 2008. Their study showed that 4% of the patients with stools positive for CD were asymptomatic, 7% had community-acquired infection, 57% acquired CDI in the hospital, and 46% of them were patients who had acquired CD from a health care facility prior to admission.

Several studies from Canada, Europe, and the United States have reported isolation of CD in various retail meat samples (Rodriguez-Palacios et al., 2007, 2009; Songer et al., 2009; De Boer et al., 2011). A study conducted in Quebec province of Canada in 2005 over a 10-month period showed that CD was identified in 21% of ground beef and 14.3% of ground veal samples (Rodriguez-Palacios et al., 2007). A Canadian nationwide survey for CD in ground beef showed that CD was isolated from 6.7% of ground beef samples (Rodriguez-Palacios et al., 2009). Weese et al. (2009) reported the overall prevalence of 12% of CD in both ground beef and ground pork retail meat sold in 4 Canadian provinces. Songer et al. (2009) conducted an extensive study in Arizona and showed that 50, 42.9, and 44.4% of ground beef, ground pork, and ground turkey were positive for CD, respectively. In our study, we collected ground beef, ground turkey, chicken thighs, and ground pork from different counties in PA over a period of 12 months. The prevalence of CD in our study is in close agreement with the findings of the Canadian studies (Rodriguez-Palacios et al., 2007, 2009; Weese et al., 2009) but lower than that reported by Songer et al. (2009).

The isolation of CD from retail meat can be influenced by the technique employed for enrichment, isolation, and identification of CD from retail meat. Recently, Limbago et al. (2012) developed a consensus method for culture of CD from meat and then used the method for doing a survey of US retail meats. They examined 1755 retail meat (fresh, nonfrozen ground beef and turkey, whole pork chops and chicken breasts) samples over 12 months during 2010–2011 from 9 FoodNet sites. Using the consensus method, CD was not recovered from the samples examined, but they reported the growth of other Clostridia spp.

Our study showed that ground turkey (14.5%) had the highest prevalence while ground beef (6.9%) had the lowest prevalence of CD. A previous study done in our laboratory by Houser et al. (2012) found CD in 12% of veal calves before slaughter, but were unable to recover CD from veal carcasses following slaughter. On follow-up of the veal carcass from the packing plant to retail outlets, CD was recovered from 6% of ground veal meat. These results suggest that CD may gain access in the meat continuum following breaking of the carcass for retail consumption, and the prevalence may largely depend on the handling, processing, and grinding of meat in retail markets. In a recent study, Kalchayanand et al. (2013) reported that none of the 956 commercially produced beef samples obtained from 8 microbiological monitoring regions of the United States were positive for CD. They were of the opinion that coarsely ground meat chubs are reground, repackaged, and sold as retail ground beef, which would result in postcontamination with spores of CD through the environment.

We examined a total of 84 CD isolates for tcdA, tcdB, binary toxin, and tcdC gene size. Of the human CD isolates, 85% of isolates were determined to be toxigenic (positive for at least 1 of the 3 toxins), while 81% of retail meat CD isolates were toxigenic. Fry et al. (2012) examined 609 swine fecal samples collected from farms in North Carolina and Ohio and found that 85% of CD isolates were positive for toxin gene tcdB and tcdA, and 81% of CD isolates were positive for binary toxin and had a 39-bp deletion in the tcdC gene. Norman et al. (2009) examined 131 CD isolates from swine, of which 93% were positive by PCR for both toxins tcdA and tcdB genes, 98.4% of the isolates harbored a 39-bp deletion in the tcdC gene, and all 131 (100%) of the isolates were positive for the binary toxin gene cdtB. Visser et al. (2012) examined 48 meat samples (ground beef and pork) from 3 major food chains and 3 local meat shops in Winnipeg, Canada. They isolated CD from 6.3% of retail meat samples. CD isolates were detected in two ground beef and one ground pork sample. All three CD isolates were positive for tcdA and tcdB. One beef isolate was positive for binary toxin and had an 18-bp deletion in tcdC. Based on the above-reported studies, it can be inferred that a significantly higher percent of CD isolates from retail meat encode for tcdA, tcdB, and binary toxin. Interestingly, many of the studies have also reported a higher percent of animal and meat isolates with an 18- or 39-bp deletion in the tcdC gene.

Antibiotic resistance to CD treatment is a major problem in CDI patients (Bauer et al., 2009; Shah et al., 2010). Reasons for antibiotic resistance may vary from individual to individual on the basis of age, disease condition, antibiotic use, presence of different strains of CD, and severity of CDI (Owens et al., 2008). Recent studies have demonstrated that antibiotic resistance profiles of CD isolates is quite diverse in different countries (Huang et al., 2009).

We examined CD isolate susceptibility to the four most commonly used antibiotics in clinical settings in Pennsylvania. The human CD isolates were most susceptible to vancomycin (100%), and metronidazole (100%), followed by clindamycin (81%) and moxifloxacin (79%). This observation is consistent with many previous studies on susceptibility of CD to antibiotics (Drudy et al., 2008; Pituch et al., 2011; Tenover et al., 2012; Pirš et al., 2013). The CD isolates from meat showed the highest susceptibility to vancomycin (100%), metronidazole (100%), followed by moxifloxacin (83%–100%) and clindamycin (82%–100%). Based on the percent susceptibility MIC 50% and MIC 90% values, CD isolates from retail meat were more susceptible than human CD isolates to clindamycin, metronidazole, moxifloxacin, and vancomycin. Visser et al. (2012) reported that CD isolates from beef were sensitive to all antimicrobial agents tested, while a CD strain isolated from ground pork was resistant to both clindamycin (resistant at ≥8 μg/mL) and moxifloxacin (resistant at ≥8 μg/mL).

Ribotypes 027 and 078 are among the most commonly found virulent strains accounting for major CDI in humans and have been found in meat samples (Bakker et al., 2010). In our study, a single CD isolate of ribotype 001 was from a human sample. PCR ribotype 027 has been isolated from retail ground beef (Songer et al., 2009) and is associated with human illness (Jhung et al., 2008). In our study, ribotype 027 was present in five samples (two human and three retail meats); however, the human and retail meat samples differed in both toxin gene and tcdC gene deletion profiles. Metcalf et al. (2010) observed that the prevalence of CD in retail pork from four Canadian provinces was 1.8%. They identified three of the seven isolates as ribotype 027 and found that these isolates were indistinguishable from known human pathogenic strains, which is suggestive of the fact that the contaminated pork could be the source of CD in humans.

A total of eight isolates belonged to ribotype 078, of which two sets of isolates (one human and one pork) shared the same toxin gene, tcdC gene deletion, and antimicrobial resistance profiles. Weese et al. (2009) examined chicken legs, thighs, and wings sold in retail outlets in Ontario, Canada. They observed that 12.8% of the chicken samples were positive for CD and all of the isolates belonged to ribotype 078. They stated that this strain from food animals could likely be associated with community-acquired disease in humans. Ribotype 078 has also been isolated from retail pork, turkey, and beef products in the United States (Songer et al., 2009).

We observed unique ribotypes not yet recorded in the ribotype database; these ribotypes were designated as PA#. In this study, we found 10 ribotypes originated exclusively from human samples; 4 were from CD meat samples and 8 ribotypes originated from both human and retail meat samples. Four ribotypes including PA01, PA05, PA16, and PA22 with similar toxin gene, tcdC and antibiotic resistance profiles presented in both human and retail meat samples. Interestingly, shared isolates between human and retail meat with the same ribotype, toxin gene, and tcdC profiles were sensitive to all four antimicrobials used in humans to treat CDI. This suggests that retail meat is less likely to be the source of antimicrobial determinants to vancomycin and metronidazole in human CD isolates.

In summary, the findings of our study corroborate with previously reported investigations (Rupnik, 2007; Weese et al., 2009; Bakker et al., 2010; Metcalf et al., 2010; Hoover and Rodriguez-Palacios, 2013; Quesada Gomez et al., 2013), which suggest that retail meat can be a potential source of CD in humans.

Footnotes

Acknowledgment

This research was funded by United States Department of Agriculture, National Institute of Food and Agriculture (USDA-NIFA) Critical and Emerging Food Safety Issues program grant 2010-03567.

Disclosure Statement

No competing financial interests exist.

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