Co-Occurrence of Staphylococcus aureus and Ochratoxin A in Pasteurized Milk

Pathogens and mycotoxins are serious public health risks for humans and food safety in milk. This study concentrated on detecting Staphylococcus aureus and Ochratoxin A (OTA) in 210 pasteurized milk from ten urban Beijing districts to suggest the co-occurrence of S. aureus with toxin-producing genes and OTA in milk and the possible risk. S. aureus was identified by physiological and biochemical experiments and molecular biology experiments, and enterotoxin genes were identified by PCR. OTA was detected by LC-MS/MS. The study found 29 isolates of S. aureus, of which 17.24% had the sea gene encoding enterotoxin A. OTA was detected in 31 out of 120 samples and the maximum amount of detection was 18.8 μg/kg. The results of this study indicate that when failing to guarantee the cold chain, the presence of S. aureus with enterotoxin genes in milk will present a risk to food safety. Furthermore, the high detection rates and levels of OTA in milk suggest that OTA is a hidden risk. The co-occurrence of S. aureus and OTA in milk is a food safety concern and there is a need to control the occurrence of these two biohazards in milk.


Introduction
Milk is a highly nutritious food containing many macronutrients and micronutrients including proteins, different types of fatty acids and lactose, minerals, antioxidants, and vitamins, that are essential for the growth and maintenance of human health, especially for infants, children, and older adults [1,2]. Therefore, increasing consumption of milk has been observed owing to its high nutritional role in human health throughout the world [3,4]. According to the 2021 China Dairy Quality Report, in 2020, China produced 27.804 million tons of dairy products, and low-temperature milk including pasteurized milk and low-temperature yogurt reached 2.308 million tons, accounting for 8.3%. However, the nutritional richness of milk also makes it susceptible to contamination by microorganisms and toxins [5]. Staphylococcus aureus and Ochratoxin A (OTA) are biohazards, which commonly occur in milk and milk products [6]. Milk is an important food source of foodborne illness due to contamination with S. aureus [7]. Furthermore, mycotoxin contamination in milk is an emerging concern around the globe [8]. Therefore, it makes sense to test for microorganisms and biotoxins in milk. the span of a year to evaluate the enterotoxin genes and the fungal toxin OTA as well as the relationship between enterotoxin genes and enterotoxin. As a result, this study focuses on biological risk factors in milk, including S. aureus and OTA, with a focus on toxins, to suggest the co-occurrence of S. aureus with enterotoxin genes and OTA in milk and the potential risk.

Identification of S. aureus and Detection of Enterotoxin Genes
Forty-seven isolates of presumed S. aureus were isolated in 210 pasteurized milk. Twenty-nine out of 47 isolates were confirmed as S. aureus after coagulase, thermonuclear, biochemical tests and Polymerase Chain Reaction technology. As shown in Figure 1, in lanes 1-5, there is a bright band at 592 bp, which is the nuc gene. S. aureus isolates were further analyzed by PCR for the presence of the sea, seb, sec, sed, and see genes. The most frequently detected gene was seb (7; 24.14%) followed by sec (6; 20.69%), sea (5; 17.24%), sed (4; 13.79%), see (3; 10.34%) ( Table 1).
Another prominent toxin after aflatoxin is OTA, and S. aureus is a common pathogenic bacterium in milk. Pasteurized milk from ten urban districts in Beijing was sampled over the span of a year to evaluate the enterotoxin genes and the fungal toxin OTA as well as the relationship between enterotoxin genes and enterotoxin. As a result, this study focuses on biological risk factors in milk, including S. aureus and OTA, with a focus on toxins, to suggest the co-occurrence of S. aureus with enterotoxin genes and OTA in milk and the potential risk.

Identification of S. aureus and Detection of Enterotoxin Genes
Forty-seven isolates of presumed S. aureus were isolated in 210 pasteurized milk. Twenty-nine out of 47 isolates were confirmed as S. aureus after coagulase, thermonuclear, biochemical tests and Polymerase Chain Reaction technology. As shown in Figure 1, in lanes 1-5, there is a bright band at 592 bp, which is the nuc gene. S. aureus isolates were further analyzed by PCR for the presence of the sea, seb, sec, sed, and see genes. The most frequently detected gene was seb (7; 24.14%) followed by sec (6; 20.69%), sea (5; 17.24%), sed (4; 13.79%), see (3; 10.34%) (     Another prominent toxin after aflatoxin is OTA, and S. aureus is a common pathogenic bacterium in milk. Pasteurized milk from ten urban districts in Beijing was sampled over the span of a year to evaluate the enterotoxin genes and the fungal toxin OTA as well as the relationship between enterotoxin genes and enterotoxin. As a result, this study focuses on biological risk factors in milk, including S. aureus and OTA, with a focus on toxins, to suggest the co-occurrence of S. aureus with enterotoxin genes and OTA in milk and the potential risk.

Identification of S. aureus and Detection of Enterotoxin Genes
Forty-seven isolates of presumed S. aureus were isolated in 210 pasteurized milk. Twenty-nine out of 47 isolates were confirmed as S. aureus after coagulase, thermonuclear, biochemical tests and Polymerase Chain Reaction technology. As shown in Figure 1, in lanes 1-5, there is a bright band at 592 bp, which is the nuc gene. S. aureus isolates were further analyzed by PCR for the presence of the sea, seb, sec, sed, and see genes. The most frequently detected gene was seb (7; 24.14%) followed by sec (6; 20.69%), sea (5; 17.24%), sed (4; 13.79%), see (3; 10.34%) ( Table 1).      The contamination levels of OTA in 120 pasteurized milk were evaluated in this work. The limit of detection (LOD) is 0.015 μg/L and the limit of quantification (LOQ) is 0.049 μg/L. OTA was found in 31 pasteurized milk samples (range 0.11-18.8 μg/L). 25.83% (31/120) of pasteurized milk was contaminated with OTA. 16.13% (5/31) of the samples had a contamination level of more than 10 μg/L ( Table 2). Table S3 has provided detailed detect results for the entire year.

Discussion
Our results show the highest detection rate of seb. Some studies have also examined the classical enterotoxin genes in milk, with the highest detection rate of sed in Bianchi's study at 25% (120/481) [12] and the highest detection rate of see in Grispoldi's study at 47.06% (8/17) [17]. The most common enterotoxins produced by S. aureus isolated from dairy products of bovine or sheep origin were found in the literature to be SEC and SED [30]; the most common enterotoxin produced by S. aureus involved in food poisoning outbreaks was SEA [31]. This is probably related to the differences in the ecological reservoir The contamination levels of OTA in 120 pasteurized milk were evaluated in this work. The limit of detection (LOD) is 0.015 µg/L and the limit of quantification (LOQ) is 0.049 µg/L. OTA was found in 31 pasteurized milk samples (range 0.11-18.8 µg/L). 25.83% (31/120) of pasteurized milk was contaminated with OTA. 16.13% (5/31) of the samples had a contamination level of more than 10 µg/L ( Table 2). Table S3 has provided detailed detect results for the entire year.  In this study, OTA was monitored throughout the year. The results show that the content of OTA detected in winter (October and December) was higher than that in summer (July-September). OTA was not detected from March to May. The content of OTA detected in December reached 18.80 µg/L ( Figure 4). The contamination levels of OTA in 120 pasteurized milk were evaluated in this work. The limit of detection (LOD) is 0.015 μg/L and the limit of quantification (LOQ) is 0.049 μg/L. OTA was found in 31 pasteurized milk samples (range 0.11-18.8 μg/L). 25.83% (31/120) of pasteurized milk was contaminated with OTA. 16.13% (5/31) of the samples had a contamination level of more than 10 μg/L ( Table 2). Table S3 has provided detailed detect results for the entire year.

Discussion
Our results show the highest detection rate of seb. Some studies have also examined the classical enterotoxin genes in milk, with the highest detection rate of sed in Bianchi's study at 25% (120/481) [12] and the highest detection rate of see in Grispoldi's study at 47.06% (8/17) [17]. The most common enterotoxins produced by S. aureus isolated from dairy products of bovine or sheep origin were found in the literature to be SEC and SED [30]; the most common enterotoxin produced by S. aureus involved in food poisoning outbreaks was SEA [31]. This is probably related to the differences in the ecological reservoir

Discussion
Our results show the highest detection rate of seb. Some studies have also examined the classical enterotoxin genes in milk, with the highest detection rate of sed in Bianchi's study at 25% (120/481) [12] and the highest detection rate of see in Grispoldi's study at 47.06% (8/17) [17]. The most common enterotoxins produced by S. aureus isolated from dairy products of bovine or sheep origin were found in the literature to be SEC and SED [30]; the most common enterotoxin produced by S. aureus involved in food poisoning outbreaks was SEA [31]. This is probably related to the differences in the ecological reservoir of S. aureus in different countries and regions of the world [17]. The presence of enterotoxin-producing isolates of S. aureus in pasteurized milk means that failing to guarantee the cold chain could present a food safety risk, particularly if all enterotoxigenic isolates could potentially produce SEA in milk [17]. Research shows that more than half of S. aureus isolates contain at least one gene coding enterotoxin, indicating that milk contaminated with S. aureus is likely to cause food poisoning [32]. Our study results show that 17.24% of S. aureus in pasteurized milk contained sea gene. Therefore, pasteurized milk in these ten urban districts of Beijing may have potential food safety risks.
It is generally accepted that SE production constitutes a risk when S. aureus bacteria exceed a threshold of 10 5 S. aureus CFU/mL of milk during manufacture [33,34]. For example, the production of enterotoxins SEA and SEB are detected in milk when the count of S. aureus exceeds the critical level of 10 5 CFU/mL [35]. Many studies indicate that temperature and pH might also influence the expression of genes that code for the production of enterotoxins [36,37]. A study shows that the production of SEA can usually be detected at 10-45 • C and the yield of SEA increases with the increasing temperature [33,38]. A study indicated that the conditions for SEA production were pH of > 5.0 and aw of > 0.86 at temperatures of > 15 • C [36]. Undissociated lactic acid (1.6 mM compared to 0.2 mM) was reported to be able to increase SEA production of strain cocktails grown in BHI broth [39]. Another study observed sorbic acid stress (0.15%, pH 5) reduced SEA levels using S. aureus Sa17 [40]. In addition, different growth substrates lead to different growth behaviors of bacteria, which can also affect the production of enterotoxins [41]. Therefore, there are a variety of factors that influence the production of toxins.
There are few studies on the detection of OTA in milk because of dietary changes (high concentrate ratios and high feeding levels) that reduce protozoa's capacity for OTA degradation, rumen microbial communities shift, increasing the likelihood that OTA may contaminate milk despite the fact that the rumen microbiota of cattle can degrade OTA [5]. Table 3 shows the detection of OTA in milk in China and abroad. Compared with our results, all are below our maximum detection. Although there are no regulations in other countries of the European Union for OTA in milk, Slovakia sets a limit of 5 µg/kg for milk [42]. Thus, 25.81% of the samples exceeded the Slovak limit for OTA in milk. Mycotoxin-producing strains in feed can multiply and produce toxins during the growth, harvest, and storage of crops. When cows consume contaminated feed, OTA is left in the milk through metabolism [43]. In addition, differences in climate and animal farming systems in different geographical regions may also lead to differences in OTA levels in milk [2]. In addition, different types of milk, such as organic and conventional milk, may also lead to differences in OTA levels due to differences in processing methods and the nutrients contained [44,45]. Some studies have also shown that pasteurized milk is more contaminated in the cold season than in the warm season. A study conducted by Ansari et al. in 2019 on pasteurized cow milk showed that during the cold seasons of the year compared to the warm seasons pasteurized milk samples were more contaminated [46]. Similar results were found in the study of Mokhtari [45]. In the cold season, due to the high humidity in the forage storage area, the possibility of growth of various fungi, including A. flavus and A. ochraceus in the forage and forage, will increase, so the contamination rate of OTA will increase. However, in the warmer season, starting around March, dairy farms have access to fresh feed, reducing the OTA content in milk [2]. At the same time, we noticed that the results for November, January, and February were anomalous compared to the results for October and December. The most important reason for this is that the samples from these three months were produced by cows late to feed of better quality. Although aflatoxins, especially AFM1 are most commonly found in milk and dairy products in many other countries [1], our study found that AFM1 in milk from the Beijing area was well-controlled in the sample. AFM1 was not detected in 120 pasteurized milk. AFM1 was detected in two samples of 360 UHT milk, and the detected amounts were 0.27 µg/kg and 0.10 µg/kg, respectively. In contrast, OTA was detected in 22.22% (80/360) of UHT milk (Table S4.). In 2019, it was reported that the mean value of AFM1 and OTA in pasteurized milk was 0.01286 µg/kg and 0.135 µg/kg, respectively [44], which is consistent with our results. OTA is classified as a Group IIB carcinogen to humans by the International Agency for Research on Cancer [52]. A review of studies on OTA over the past 50 years suggests that the carcinogenicity of OTA may also occur in humans [18]. Although no direct evidence of carcinogenicity to the human body has been found at present, the contamination range of OTA is very wide, the contamination level is very high, and its harm is very great. Therefore, research on the real toxicity of OTA should be paid more attention to by more scholars.
Our research results show that OTA is a hidden risk in milk and it serves as a warning and calls attention to the detection of OTA in milk. Several studies in the last two years have examined the prevalence of OTA in different types of milk. A study conducted in 2016 reported OTA levels ranging from 0.34 to 13 µg/L. The detection rate was 80% (32/40) [5]. The detection levels of OTA in this reference were similar to our results. However, the detection rate was even higher than ours. The OTA detection values of several other papers were relatively small. As a result, OTA in milk is not now garnering more attention than before, and high levels of OTA are still detected in milk samples. Consequently, our work still has warning implications.

Conclusions
This study monitored S. aureus and OTA in pasteurized milk samples in Beijing throughout the year for the first time. The results of this study indicate that when failing to guarantee the cold chain, the presence of S. aureus with enterotoxin genes in milk will present a risk to food safety. Furthermore, the high detection rates and levels of OTA in milk suggest that OTA is a hidden risk. As a result, the findings of this study have some bearing and can be used as a reference point for biological risk factors in milk.

Sampling
From October 2014 to September 2015, a total of 210 pasteurized milk (including 90 copies of Brand A and 120 copies of Brand B) were bought from supermarkets located in 10 urban districts in Beijing, China. ( Figure 5). All samples were delivered at 4 • C and analyzed within 24-48 h. In total, 210 samples of pasteurized milk were detected for S. aureus, and only 120 samples of Brand B were detected for OTA.

Isolation and Detection of S. aureus
We carried out the culture and identification of S. aureus according to the methods described by GB 4789.10-2016. The samples were cultured at 37 °C and 200 rpm for 12 h for 18 h, then crossed on the selective Baird-Parker plate and cultured at 37 °C for 48 h. S. aureus colonies on B-P plates were round, 2-3 mm in diameter, gray or black in color, and surrounded by a turbid zone. The suspected colonies were selected for Gram staining and plasma coagulase test. Gram staining microscopic examination showed that S. aureus was Gram-positive cocci. The experiment on plasma coagulase is as follows. A single suspicious colony was picked from a Baird-Parker plate, inoculated into 5 mLBHI broth, and incubated at 37 °C for 18 h. In the ultra clean table, 0.5 mL of saline was added to the lyophilized rabbit plasma, shaken to dissolve it, then 0.2-0.3 mL of BHI culture was added, shaken well, placed in 37 °C incubator, and observed every half hour for 6 h. The positive result was determined if the volume of clotting or clotting was greater than half of the original volume. The broth culture of the positive plasma coagulase test was also used as the control.
Then the suspected colonies to increase the bacteria and extract the genome were picked out. DNA was extracted with the TIANamp Bacteria DNA Kit (OSR-M502, Tiangen, China). The nuc gene acts as a marker and also the presence of heat resistant nuclease gene (nuc) is strongly associated with the production of enterotoxin and it can be considered an indicator of infection with enterotoxin producer S. aureus [14]. Therefore, the nuc gene was amplified by PCR to identify the S. aureus. PCR was also used to detect the presence of the classic enterotoxin genes sea-see. The PCR reaction was conducted on a C1000 Toucah Thermal Cycler (Bio-Rad, Johannesburg, South Africa). The cycling conditions were initial denaturation at 94 °C for 5 min; followed by 30 cycles of 94 °C for 30 s, 64 °C for 30 s, 72 °C for 60 s, and a final elongation step of 72 °C for 10 min. The PCR products were stored at 4 °C and later separated by 1% agarose gel electrophoresis. D2000 DNA Marker was used. The PCR primers were designed with NCBI according to the nuclease gene sequence, as shown in Table  4.

Isolation and Detection of S. aureus
We carried out the culture and identification of S. aureus according to the methods described by GB 4789.10-2016. The samples were cultured at 37 • C and 200 rpm for 12 h for 18 h, then crossed on the selective Baird-Parker plate and cultured at 37 • C for 48 h. S. aureus colonies on B-P plates were round, 2-3 mm in diameter, gray or black in color, and surrounded by a turbid zone. The suspected colonies were selected for Gram staining and plasma coagulase test. Gram staining microscopic examination showed that S. aureus was Gram-positive cocci. The experiment on plasma coagulase is as follows. A single suspicious colony was picked from a Baird-Parker plate, inoculated into 5 mLBHI broth, and incubated at 37 • C for 18 h. In the ultra clean table, 0.5 mL of saline was added to the lyophilized rabbit plasma, shaken to dissolve it, then 0.2-0.3 mL of BHI culture was added, shaken well, placed in 37 • C incubator, and observed every half hour for 6 h. The positive result was determined if the volume of clotting or clotting was greater than half of the original volume. The broth culture of the positive plasma coagulase test was also used as the control.
Then the suspected colonies to increase the bacteria and extract the genome were picked out. DNA was extracted with the TIANamp Bacteria DNA Kit (OSR-M502, Tiangen, China). The nuc gene acts as a marker and also the presence of heat resistant nuclease gene (nuc) is strongly associated with the production of enterotoxin and it can be considered an indicator of infection with enterotoxin producer S. aureus [14]. Therefore, the nuc gene was amplified by PCR to identify the S. aureus. PCR was also used to detect the presence of the classic enterotoxin genes sea-see. The PCR reaction was conducted on a C1000 Toucah Thermal Cycler (Bio-Rad, Johannesburg, South Africa). The cycling conditions were initial denaturation at 94 • C for 5 min; followed by 30 cycles of 94 • C for 30 s, 64 • C for 30 s, 72 • C for 60 s, and a final elongation step of 72 • C for 10 min. The PCR products were stored at 4 • C and later separated by 1% agarose gel electrophoresis. D2000 DNA Marker was used. The PCR primers were designed with NCBI according to the nuclease gene sequence, as shown in Table 4  We extracted OTA from samples according to the methods described by the GB 5009.96-2016. Pipette 5 mL of fresh milk into 50 mL centrifuge tube, add 20 mL of acetonitrile (84%), add 2 g of anhydrous magnesium sulfate and 1 g of sodium chloride, vortex 2 min, ultrasonic 20 min, and centrifuge at 1650 g for 5 min, 10 mL of the supernatant was evaporated dry in a rotary evaporator at 60 • C. Dissolve with 1 mL of methanol, then add 1 mL of water, mix well, and pass through a 0.22 µm filter membrane, to be analyzed. Each sample was analyzed in triplicate. OTA standard was purchased from J&K Scientific Ltd. Beijing, China.
Following separation, the column effluent was connected to a triple-quadrupole mass spectrometer (Agilent Technologies 6460, CA, U.S.A.) equipped with an ESI source. OTA was detected in positive mode using MRM. Data acquisition and mass spectrometric evaluation were carried out on a Mass Hunter Workstation (Agilent Technologies, Santa Clara, CA, USA). Operating conditions were: Spray Voltage-5500 V, Curtain Gas-30 psi, Temperature-500 • C, Gas 1-30 psi, Gas 2-50 psi, Entrance Potential (EP)-10 V, Collision Energy (CE)-21 eV. De-clustering Potential (DP)-61 V, Exit Potential (CXP)-20 V. The precursor ion was monitored and collision induced dissociation was used to generate product ions. The precursor ion was m/z 404, the product ion was m/z 358 and m/z 238.8, and the ion ratio was 1:1. To evaluate the linearity, five-point calibration curves were constructed to calculate the determination coefficients (R2). The signal-to-noise (S/N) approach was used to estimate the LOD and the LOQ. The gradient OTA standard solution was injected on the liquid chromatograph. The LOD and LOQ were defined based on signal (S)-to-noise (N) ratios of S/N > 3 and S/N > 10, respectively.
LC-MS/MS analysis was done at the Supervision, Inspection & Testing Center for Agricultural Products Quality.

Statistical Analysis
The data were analyzed by SPSS. The significance level was set at p < 0.05, and all experiments were replicated at least three times.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/toxins14100718/s1, Table S1: Detection of Staphylococcus aureus in pasteurized milk. Data from December 2014 to September 2015. Unit is CFU/mL; Table S2: Detection rate of bacteria in pasteurized milk; Table S3: OTA detection level of each district in each month in pasteurized milk. Data from December 2014 to September 2015. "-": not detected; Table S4: Detection of OTA in UHT milk and Pasteurized milk.