Pathogenic bacteria in cheese, raw and pasteurised milk

Abstract Background Foodborne diseases, especially those transmitted by milk and its products, are worldwide problem. Milk is not only a complete food but also a unique medium for activating various bacteria such as Listeria monocytogenes, Staphylococcus aureus and Salmonella typhi. In recent years, numerous bacteria with multiple drug resistance patterns have appeared, and there have been many problems in infection control. Today, ranchers use antibiotics for control of the animal disease, and humans are constantly using animal products containing antibiotics. Objective The purpose of this study was to evaluate the contamination status of raw and pasteurised milk as well as local cheese and to find a rapid Multiplex PCR method for investigation of contamination. Determination of antibiotic resistant isolates is also desirable. Materials and Methods One hundred samples were collected from livestock and retail outlets using culture and molecular methods to identify S. aureus, L. monocytogenes and S. typhi. The antibiotic resistance pattern was determined for the isolates. Results In this study, culture results for 100 samples showed 10% S. aureus isolates while no cases of S. typhi and L. monocytogenes were detected. In real‐time qPCR, S. aureus was isolated in 60% (n = 60) of samples, S. typhi in 53% (n = 53) and L. monocytogenes in 2% (n = 2). The results of sensitivity and specificity of Multiplex PCR for the three studied bacteria indicated general specificity of 72% and sensitivity of 80%. Conclusion Based on the results of this study, it can be concluded that S. typhi, L. monocytogenes and S. aureus are more likely to be detected by real‐time qPCR because of the high sensitivity of this test to culture. Multiplex method was not reliable in this study and cannot be suggested for rapid diagnosis.

the health of the community. Given the importance of milk in human nutrition and general community health, knowledge of its components and the soundness of this important food product is necessary and inevitable (Singhal et al., 2017). Milk and its products contain different nutrients and the pH of milk is equal to 6.8; therefore, it is a good environment for the growth of many microorganisms. Milk has a primary microbial flora when milking cows, but it may be contaminated with other pathogenic microorganisms (Munera-Bedoya et al., 2017). A number of diseases can be transmitted to humans through milk. Microbes that can be transmitted to milk through cattle lead to infection with Staphylococcus aureus, bovine tuberculosis, brucellosis, malaria, streptococcal and salmonellosis infections. Also, some contaminants may be transmitted through milk carriers. Transmission through contaminated soil and equipment can also lead to listeriosis and salmonellosis, as well as Campylobacter-related infections in consumers (Nornberg et al., 2010). The health of raw milk is in the interest of public, and failure provide high quality milk will reduce the level of public health as well as economic health; therefore, due to the importance of this issue, it is vital to quickly and accurately identify raw milk contamination (Abushelaibi et al., 2017). Therefore, it is necessary to study the profile of pathogenic microbes phenotypically.
Considering the importance of milk as an essential nutrient, it is advisable for the research community to identify pathogenic bacteria such as Salmonella spp, S. aureus and Listeria monocytogenes and to study the spread of antibiotic resistance in these pathogens (Marin et al., 2017). Therefore, the current study aimed to evaluate the contamination of raw and pasteurised milk as well as cheese with Salmonella typhi, L.monocytogenes and S.aureus to determine the antibiotic resistance pattern of the isolated bacteria.

Antibiotic susceptibility test
To determine the antibiotic resistance patterns of common milk bacteria, antibiotic susceptibility assay was performed by disk diffusion.

DNA extraction
DNA extraction was performed by boiling method (Oliveira et al., 2014).

Multiplex PCR
For Multiplex PCR, the primers listed in Table 1

RESULTS AND DISCUSSION
Our results showed that only S. aureus was isolated with frequency of 10% (n = 10) by culture method, which was detected in three samples out of a total of 30 cheese samples (11%) as well as seven samples from a total of 35 raw milk samples (22.8%). Also, our results showed two cheeses samples (5.7%) that were positive for L. monocytogenes.  (n = 35), 78% (n = 78) and 2% (n = 2), respectively (Figures 2 and 3). The results of sensitivity and specificity of Multiplex PCR for the three studied bacteria indicated general specificity and sensitivity of 72% and 80%, respectively. Bacterial susceptibility and specificity are listed in cefoxitin, gentamicin and tetracycline but the resistance was observed to ampicillin. In the present study, 100 samples of raw milk, pasteurised milk and cheese were studied for the presence of S. aureus, S. typhi and L. monocytogenes based on culture, biochemical characteristics and amplification of specific genes. Transmission of bacterial species among humans, livestock, farmers and farm workers is increasing dramatically in Europe and many parts of the world. This is of great importance, especially with increasing use of antibiotics, which has increased the resistance to various drugs significantly and has made control and treatment in humans and animals more difficult. Therefore, rising antibiotic resistance is a concern and should be controlled (Patel et al., 2018). Milk is a good environment for S. aureus to grow and produce enterotoxin, especially because enterotoxin retains its biological activity even after pasteurisation. Salmonella spp is one of the most important infectious agents in humans and animals. The crucial role of infected animal populations as a source for preserving and transmitting Salmonella to humans, especially through contaminated food, is quite clear today (Knight-Jones et al., 2016). Fecal entry into milk can also be a main cause of milk contamination with Salmonella; this bacterium can enter raw milk from the outer surface of udder and from equipment, environment, bed and water. L. monocytogenes has been the focus of many researchers due to its high prevalence and mortality rate (Dhanashekar et al., 2012 Sep 1). This bacterium is also responsible for many food poisoning epidemics, especially in industrialised countries. Recently, a stronger association between listeriosis and dairy consumption has been reported compared to other food products, and pasteurised and unpasteurised milk as well as cheese have been identified as a common source of epidemics. L.monocytogenes is able to grow in unpasteurised milk and there is a possibility of increasing number of organisms during storage in milk, storage tanks in farms and silos (Hunt et al., 2012). During the first study conducted in Iran by Jalali and Abedi (2008) regarding L. monocytogenes on various foods, the contamination rate of dairy products was 1.3%, which was consistent with our results (2%). In a study by Rodríguez-Lázaro et al. indicates the high importance of using pre-enrichment and enrichment steps. In general, the present study was conducted due to the significance of food pathogens in raw milk, pasteurised milk and cheese since in some areas, dairy consumption is mostly in the form of raw milk and pasteurisation is done at home by individuals or dairy shops that are responsible for sales. It is necessary to assess these milks for the safety of their microbial quality. On the other hand, although there is some degree of monitoring in pasteurised milk, some substances that do not match the microbial quality of milk have been found in them.

Real-time qPCR test to identify
In this study, the prevalence of S. aureus in raw milk was reported to be 10% by culture method and 60% by real-time qPCR. be concluded that this approach indicates the presence or absence of bacteria in milk. Therefore, we suggest that the following points: 1. Informing and preventing people from consuming raw milk and products produced from it.
2. Awareness of groups at high risk of infection.
3. This information should be provided to food and medicine authorities and the research should be continued by the university to control contamination 4. Studies should be done on a larger scale.

ACKNOWLEDGMENTS
The current study was supported by Ilam university of Medical sciences.

ETHICAL APPROVAL STATEMENT
The current study was approved by ethical committee of Ilam University of medical sciences (ID:981017/104).

CONFLICT OF INTEREST
There is no conflict of interest.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/vms3.604