Evaluation of diagnostic polymerase chain reaction ( PCR ) for the detection of Escherichia Coli , Staphylococcus aureus and Bacillus cereus in cheese

Polymerase chain reaction (PCR) assay was used to detect pathogenic bacteria in white cheese samples using suitable primers which are based on specific genes. A total of 70 samples of white cheese were collected from different parts of Baghdad for detection of the pathogens, Escherichia coli, Staphylococcus aureus and Bacillus cereus. Cheese was made locally from different sources of milk (buffalo, cows and sheep). Samples were cultured in liquid enrichment medium (Nutrient Broth) and incubated at 37°C for a period of 16-20 h. DNA was extracted by kit extraction and pathogens were diagnosed. Multiplex polymerase chain reaction was used with primers targeting specialized sites of the target gene in one reaction and the results showed the presence of E. coli (87%), S. aureus (20%) and B. cerues (18%). The results of this study revealed that PCR was a rapid and useful tool for detection of pathogenic bacteria in cheese.


INTRODUCTION
The production of cheese from milk is a very ancient process.Cheese manufacturing started about 8000 years ago in the Fertile Crescent between Tigris and Euphrates rivers (Hayaloğlu et al., 2002).The white cheeses is homemade, fresh, soft, and is still mostly made in traditional ways within small unlicensed health workshops, or in some rural homes, according to the specifications of old traditional and the high ratio of salt which works to save them for a long time (Bintsis and Papademas, 2002).The fact that the cheese is rich in nutrients, is a compromise suitable for the growth of microorganisms and reproduction, therefore, the cheese is an important source of the spread of many cases of food poisoning and many types of microbial (Rampling, 1996).Milk products constitute a complex ecosystem of bacteria.Contamination of milk products with pathogenic bacteria is mainly due to processing, handling and unhygienic conditions.This soft white cheese is made of pasteurized or raw milk, it is characterized by a high water content of43.0%and low pH, 5.   1993).Many enteropathogenic microorganisms have been found in milk and dairy products such as cheese, which is usually stored under inadequate temperatures and consumed without any prior thermal treatment.They are frequently associated with outbreaks of food borne diseases.Domestic animals play an important role in causing Escherichia coli infections, mainly cattle and sheep that can be asymptomatic vectors of virulent strains ( Chapmann et al., 1993).
To better control microbial contaminants of food and consequently to reduce foodborne illnesses, rapid and accurate pathogen detection methods are required for effectively monitoring microbial pathogens in food supplies.Traditional detection methods depend upon selective plating combined with immunological and biochemical identification.The negative aspects of these methods are that they are time consuming, laborious, and take several days to complete.In fact, it is impractical to use traditional microbiological methods for high-throughput screening of large number of food samples for the presence of one or more pathogens (Abubakar et al., 2007).PCR is one of the most promising techniques for rapid detection of microorganisms in food.This process has provided increased sensitivity for detection and therefore enhanced the likelihood of detecting bacterial pathogens (Lampel et al., 2000).
The soft white cheese is widely consumed in Iraq, which is marketed and displays in unhealthy way in shops and on the sidewalks.The aim of this study was to evaluate the use of polymerase chain reaction (PCR) in the detection of E. coli, Staphylococcus aureus and Bacillus cereus in soft white cheese.

Samples
This study was carried out from the beginning of September 2013 till the end of January 2014.Seventy white cheese sample were collected randomly from different regions of Baghdad city, which were made locally from different sources of milk (buffalos, cows and sheep), samples were collected using sterile bags and transported to the laboratory for detection of pathogenic bacteria (E.coli, S. aureus, and B. cereus).Weight of 10 g from each sample was homogenized with 90 ml of nutrient broth and incubated at 37°C for 16-20 h with continuous shaking for the purpose of homogenization.

DNA extraction
A volume of 1.5 ml of the post-enriched sample was centrifuged at 14,000 g for 1 min, DNA was extracted using Presto Mini g DNA Bacteria Kit according to manufacturer's instructions (Geneaid, Korea).The extracted DNA was stored −20°C until use.

Agarose gel
After genomic DNA extraction, agarose gel electrophoresis was adopted to confirm the presence and integrity of the extracted DNA. 10 µl portion of the sample was analyzed by electrophoresis in agarose gel (2%), staining with ethidium bromide (Promega, USA), and visualized in UV light.A DNA molecular weight standard 50 bp was analyzed along with the samples (Wang et al., 1997).

PCR Primers
Oligonucleotide primers for the PCR assay (Table 1) were selected based on the published nucleotide sequence of the genes (Wang et al., 1997).

PCR assay
The PCR amplification was performed in a final volume of 25 µl containing 4 µl of DNA template where 1 µl of each primer was added as given in Table 1, together with 8 µl of nuclease free water, 5 µl master mix, and distributed components at a rate of 16 µl each sample; the samples were transported to a thermal cycle using the amplification program consisting of initial denaturation at 94°C for 3 min, 35 cycles with a denaturation at 94°C for 1 min, annealing at 60°C for 45 s and extension at 72°C for 90 s, followed by the final extension at 72°C for 10 min.

RESULTS
The different size of the amplification products allowed rapid and specific discrimination of E. coli, S. aureus and B. cereus.Table 2 shows the results of pathogenic bacteria PCR detection after 24 h of incubation in enrichment media.
PCR results showed contaminated of 61 samples of E. coli (87%), 14 samples of S. aureus (20%) and 13 samples of B. cereus (18%) (Figure 1).Multiplex PCR  2).The mPCR results diverged, some of the cheese samples contain the three types of pathogens under study and some of them did not contain any pathogen and other samples contain only one type of pathogens.

DISCUSSION
The present study attempted to detect S. aureus using specific primers for nuc gene which is responsible for the ability of S. aureus to coagulate plasma and produce a thermostable nuclease (Pinto et al., 2005).Fourteen out of seventy samples were detected to contain S. aureus and revealed the presence of amplified product of the size, 276 bp (Figure 1).The amplification of nuc gene has potential for the rapid diagnosis of S. aureus infection (Brakstad et al., 1992); it is considered the base line in identification and classification of S. aureus.Sixty one (87%) from the total cheese samples was positive for E. coli by amplification of lamB gene which code for maltose transport protein (Figure 1).Other studies have also reported similar finding such as Gonzales et al. (2000) who reported incidence of virulent strains of E. coli in unpasteurized milk cheese.Also, hemolysin BL gene was specific target for detection of B. cereus from cheese sample, thirteen of seventy samples were detected to contain B. cereus and revealed the presence of the amplified product of the size, 185 bp (Figure 1).Kumar et al. (2010) reported that the mPCR was able to detect as low as 10 1 -10 2 of B. cereus organism per ml following overnight enrichment of spiked food samples (vegetable biriyani and milk) in buffered peptone water (BPW), and could also be detected by mPCR in naturally contaminated samples of rice based dishes and milk.This primer sets (Figure 2) shows specificity for the detection of pathogenic genes when used alone or with each.DNA amplification technique is suitable for assessing complex microbial communities (Huyghe et al., 2008); it can be used for the detection and identification of food-borne pathogenic bacteria and environmental microorganisms, for comparative genomic analyses and strain typing.From the foregoing information, we can say that molecular method can characterize the bacteria that contaminate the white cheese depending on amplification of suitable genes.It is considered as fast, high sensitive and specific method when compared with traditional method which is considered easier, but less sensitive, less specific and slower and time consuming (Techathuvanan et al., 2011;Zhang et al., 2011).The molecular assay also revealed results which were equivalent to those obtained with standard methods used in microbiology, however the time required for analysis was reduced from 7-10 to two working days (Amagliani et al., 2007).This study agrees with the study conducted by Holko et al. (2006) on the detection of E. coli in cheeses samples made in the traditional way.Our results show that the above protocol can be used in detection of pathogens in white cheese samples because it is easy and fast with high sensitivity and specificity when compared with traditional methods.

Figure 1 .Figure 2 .
Figure 1.The percentages of microbial species detected in local cheese; the highest percentage of the isolate was E. coli (87%).

Table 1 .
Primer sequences and anticipated sizes of PCR products for E. coli, S. aureus, B. cereus primers used in this study.

Table 2 .
Summary of the results of multiplex PCR assay. No.

of sample Type of milk E. coli S. aureus B. cereus 1
successfully amplified the DNA fragments corresponding in size to each species as follows: E. coli 585 bp, S. aureus 276 bp, B. cereus 185 bp (Figure