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Applied and Environmental Microbiology, March 2007, p. 1676-1677, Vol. 73, No. 5
0099-2240/07/$08.00+0     doi:10.1128/AEM.01994-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Quantitative Assessment of the Tetracycline Resistance Gene Pool in Cheese Samples by Real-Time TaqMan PCR{triangledown}

Michele Y. Manuzon,1 Scott E. Hanna,1,{dagger} Hongliang Luo,1 Zhongtang Yu,3 W. James Harper,1 and Hua H. Wang1,2*

Department of Food Science and Technology,1 Department of Microbiology,2 Department of Animal Sciences, The Ohio State University, Columbus, Ohio 432103

Received 22 August 2006/ Accepted 4 January 2007


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ABSTRACT
 
A TaqMan real-time PCR assay was developed to quantify the tetS gene pool present in retail cheeses. This protocol offers a rapid, specific, sensitive, and culture-independent method for assessing antibiotic resistance genes in food samples rich in fats and proteins.


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INTRODUCTION
 
Identification of the main routes of the evolution of antibiotic resistance (AR) and its transmission to humans is essential for effective mitigation. The recent findings of large AR gene pools in commensal bacteria in many ready-to-eat retail products indicates that the food production and processing environment might be involved in AR evolution and that food might have become a direct source of dissemination of various AR gene-containing bacteria to the general public on a daily basis (2, 4, 5, 6). Indeed, up to 107 CFU of antibiotic-resistant (ART) bacteria per gram of food was found in many retail block cheeses and salads examined, with various AR genes detected in at least 10% of the ART isolates (4, 6). Representative AR genes from several food isolates were successfully transmitted to and caused increased resistance in Streptococcus mutans, indicating the mobility and functionality of the AR genes from the food microbiota (6). These studies, however, used conventional plating methods under given incubation conditions and antibiotic concentrations. Thus, the numbers reported represent only a portion of the total ART bacterial load in these foods. The real magnitude of the AR gene reservoir in food ecosystems is yet to be revealed. Here we report the development of a specific, TaqMan-based real-time quantitative PCR (qPCR) assay to detect and quantify the total tetS gene pools in dairy food products. The tetS gene was chosen because it has been found in both retail cheeses (6) and host oral microbiota (3) and may pose a potential risk to consumers.


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Assay development.
 
The tetS-specific primer pair (tetS-realFP, 5'-GTATGTTCATCTTTCTAAG-3', and tetS-realRP, 5'-GCAATAACATCTTTTCAAC-3') and the fluorescence-labeled tetS-specific probe (5' FAM-CCATGTGTCCAGGAGTATCTAC-BHQ3') were designed and synthesized according to published procedures (1). The primer pair results in a 190-bp tetS fragment. The qPCR thermoprofiles consist of 95°C for 30 s, followed by 40 cycles of 95°C for 30 s, 55°C for 45 s, and 72°C for 30 s, on an iCycler (Bio-Rad Laboratories, Hercules, CA).

The 667-bp tetS gene fragment was obtained by PCR using Lactococcus sp. strain CZ-T4 DNA as the template (6) and was used as the real-time PCR standard. The copy number concentration of the original standard was calculated based on the mass concentration and the molecular weight of the tetS gene fragment. Tenfold serial dilutions of the standard were made in sterile water and stored at –80°C until use. A linear standard curve between 101 and 108 gene copies per reaction was established using the tetS standard DNA (r2 > 0.99) (Fig. 1). The standard ranging from 4.86 x 108 to 48 copies per reaction was always run in parallel with the samples.


Figure 1
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  		FIG. 1. Standard curve for determination of tetS gene copy numbers.


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qPCR validation.
 
A tetS+ cheese isolate, Lactococcus lactis GEMCZ-T4, was grown in deMan-Rogosa-Sharpe broth (Becton Dickinson and Company, Sparks, MD) containing 8 µg/ml of tetracycline. A 4-h culture was serially diluted and spiked into a retail shredded-cheese blend, which was previously shown to be free of culturable ART bacteria by a plating method. Both the spiked and unspiked cheese samples were subjected to total DNA extraction by following the method of Yu and Morrison (7) with slight modifications. The DNA was extracted by repeated bead beating in the presence of high concentrations of sodium dodecyl sulfate, NaCl, and EDTA, and was directly used for real-time PCR amplification without the subsequent RNase digestion, proteinase treatment, or column purification steps. The tetS genes were quantified in the DNA samples from both the spiked and unspiked cheese samples by using the specific primers and the TaqMan probe. The tetS gene copy numbers were then compared to the number of Tetr bacteria determined by plate counting on MRS agar with 8 µg/ml tetracycline, which had been incubated anaerobically at 30°C for 48 h. The data presented are the means of at least two replicates of each sample.

Approximately 104 copies of the tetS gene were found in the unspiked cheese matrices by the qPCR assay. The PCR products had the expected sizes and were confirmed by DNA sequencing to be the expected tetS gene amplicons, indicating the presence of DNA templates from dead or unculturable cells. To validate the qPCR assay, the background values were subtracted from the measured tetS gene copy numbers of the spiked samples.

Figure 2 shows the correlation between the copy numbers of the tetS gene as measured by the qPCR and the Tetr bacterial counts as determined by the conventional method with the artificially spiked cheese samples. The reported inoculation levels started at 104 cells, and only the qPCR signals from the inoculated templates, after background subtraction, are shown. The tetS gene copy numbers detected by qPCR are generally higher than the actual plate counts but are within a 10-fold range. This may be attributable to the presence of dead and nonviable cells and/or multiple tetS gene copies in this strain. The results indicate that the qPCR assay can accurately assess the resistance gene pool in cheese samples, and, when the qPCR assay was coupled with the DNA extraction method used in this study, no apparent inhibition to the qPCR reaction was observed.


Figure 2
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  		FIG. 2. Correlation between plate count and real-time PCR in assessing antibiotic resistance genes in cheese samples. Cheese samples were artificially spiked with tetS+ Lactococcus lactis GEMCZ-T4 cells.


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Quantification of tetS gene pools in cheeses.
 
The tetS gene pools in 11 cheese samples purchased from three retail grocery stores were analyzed using the qPCR. Figure 3 shows a positive correlation between the viable counts of Tetr bacteria and the tetS genes present in the samples. The tetS gene copy numbers per gram of cheese sample as determined by the real-time PCR assay were generally 2- to 5-log higher than the plate counts of Tetr bacteria. As pointed out earlier, the difference is likely due to (i) detection of tetS by the qPCR in both culturable and nonculturable cells and (ii) the presence of multiple copies of the tetS gene in the Tetr bacteria.


Figure 3
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  		FIG. 3. Assessment of antibiotic resistance in commercial cheese samples by conventional plate counting and real-time PCR. Values represent the means of results for at least two replicates, and the standard deviations were always less than 10%. A, Colby sample 1; B, Colby sample 2; C, mild cheddar sample 1; D, mild cheddar sample 2; E, mild cheddar sample 3; F, mild cheddar sample 4; G, sharp cheddar; H, extra sharp cheddar sample 1; I, extra sharp cheddar sample 2; J, Monterey Jack; K, mozzarella. Hatched bars, log tetracycline-resistant CFU per g; solid bars, log tetS gene copy number per g.

Overall, the qPCR method coupled with the DNA extraction method offers a distinct advantage over conventional plating methods in assessing the total tetS gene pool directly from foods rich in fat and protein such as cheeses. It may also be useful in assessing the tetS genes present in other types of food samples for risk assessment.


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ACKNOWLEDGMENTS
 
The study was supported by an OSU start-up fund and OARDC seed project OHOA1265 (H. H. Wang) and by the Department of Health Education and Training, U.S. Army Medical Department Center and School (S. E. Hanna).

The views expressed in this article are those of the authors and do not reflect the official policy or position of the United States Army, Department of Defense, or the U.S. Government.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Food Science and Technology, The Ohio State University, Parker FST Building, 2015 Fyffe Ct., Columbus, OH 43210-1007. Phone: (614) 292-0579. Fax: (614) 292-0218. E-mail: wang.707{at}osu.edu. Back

{triangledown} Published ahead of print on 5 January 2007. Back

{dagger} Present address: Department of Defense Veterinary Food Analysis and Diagnostic Laboratory, 2472 Schofield Rd., Fort Sam Houston, San Antonio, TX 78234. Back


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REFERENCES
 
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Applied and Environmental Microbiology, March 2007, p. 1676-1677, Vol. 73, No. 5
0099-2240/07/$08.00+0     doi:10.1128/AEM.01994-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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