How Do You Test Staphylococcus aureus?

Staphylococcus aureus is an important pathogen causing suppurative infection in humans and animals, and it is also one of the common pathogens causing human food poisoning. Staphylococcus aureus is widely distributed in nature, such as air, soil, water, and other environments. This bacteria is also often found on the skin of humans and animals and in the cavities communicating with the external environment. It is reported that the carrying rate of bacteria in normal people can reach 30% to 80%, of which the carrying rate of skin is 8% to 22%, and that of the upper respiratory tracts such as nose and throat is more than 40% or 50%. Thus, food can be contaminated by Staphylococcus aureus via various ways and means, especially through the hands and upper respiratory tract of food processors. As pathogenic Staphylococcus aureus can produce enterotoxins, once it contaminates food and in a suitable temperature environment, it can reproduce and form massive enterotoxins, thus causing food poisoning to consumers.

Food poisoning caused by Staphylococcus aureus is very common in countries all over the world. Especially in North America and Europe, the incidence is higher. In these countries, there are cases of food poisoning caused by Staphylococcus aureus every year, ranking second to third in the number of cases of bacterial food poisoning. The economic loss caused by this is also quite heavy, so at present, worldwide countries have listed Staphylococcus aureus as a legal inspection item for food hygiene.

Over the years, many food microbiology laboratories have been exploring and seeking more accurate and rapid detection methods. By now, the rapid detection methods of Staphylococcus aureus can be classed into three types: rapid detection based on 1) culture, 2) immunology and 3) nucleic acid-based molecular biology.

1. Culture method

1.1 Detection using chromogenic/fluorogenic culture media

Chromogenic/fluorogenic culture media is a new type of medium in which enzymes produced by microorganisms’ own metabolism can react with the corresponding chromogenic substrate to detect microorganisms, reducing the need for pure culture and further biochemical identification of strains.

1.2 Detection using count plates

The existence of Staphylococcus aureus was detected by the reaction of heat-stable nuclease produced by Staphylococcus aureus in the process of culture and chromogenic agent to form a pink ring on the count plate. Count plates consist of two parts, one is a highly selective medium for Staphylococcus aureus, and the other is a reaction tablet containing the chromogenic agent and deoxyribonucleic acid (DNA). These commercial count plates have high specificity and a short detection time.

2. Immunology method

This method uses the specific interaction between antigen and antibody as the theoretical basis for the detection of Staphylococcus aureus. It is characterized by simple operation, strong specificity, and high sensitivity, and is suitable for the detection of a large number of samples.

2.1 ELISA

Enzyme-Linked Immunosorbent Assay (ELISA) is the most commonly used method in immunodiagnosis, and the sandwich ELISA is often applied in the detection of Staphylococcus aureus. This method can detect Staphylococcus aureus enterotoxin within 15 min. However, it also has some defects, for example, the reagents used in the test are high-quality and expensive, and are easily affected by environment, temperature, time, and other conditions.

2.2 IFA

Immunofluorescence assay (IFA) is used to detect Staphylococcus aureus by specific binding of antigen and antibody. The known antibody is conjugated with fluorescent pigment to form a fluorescent marker, and under specific conditions, the sample will react with it, and then the existence of the fluorescent-labeled antigen-antibody complex is observed under a fluorescence microscope. If so, it is proved that the sample contains Staphylococcus aureus. Compared with the traditional method, the detection time of this method is greatly shortened, and the whole process takes 40-50 min.

2.3 RPLA

Reverse passive latex agglutination (RPLA) is based on the principle of an indirect agglutination reaction. The specific antibody is adsorbed on the latex particles and when the antigen and antibody bind specifically, the latex particles agglutination occurs. The degree of agglutination is proportional to the content of bacteria in the sample to be tested. Although RPLA is simple and easy, the sensitized latex is prone to self-agglutination, which affects the sensitivity of the reaction and results in inaccurate detection results.

3. Molecular method

3.1 PCR

This method overcomes the shortcomings of traditional detection methods, and has developed rapidly in recent years, and has been applied in many fields. It was first reported that the PCR method was used to detect Staphylococcus aureus in food in 1991. Wilson et al successfully detected Staphylococcus aureus enterotoxin B and C1 in artificially contaminated dried skimmed milk samples within 8 hours, and the detection limit of target DNA was 1 fg.

3.2 Gene chip

Gene chip technology is a novel technology of reverse solid phase hybridization, which has the characteristics of high throughput, speed, and sensitivity. Its principle is to design an oligonucleotide probe for detecting Staphylococcus aureus and fix it on the nitric acid membrane in a certain way. The target gene is amplified and labeled by primers and hybridized with the gene chip under appropriate conditions, and the existence of Staphylococcus aureus is identified according to the hybridization results. This method has good specificity, high accuracy, and strong maneuverability, and can provide a strong basis for clinical treatment.

3.3 LAMP

In the loop-mediated isothermal amplification (LAMP) technique, four specific primers are designed for 6 regions of the target gene, and strand-displacing DNA polymerase is used to react under isothermal conditions. The amplification could be completed within 1 hour, showing LAMP typical ladder bands, and the amplification results could be judged by naked eye observation. The LAMP does not need expensive instruments and is fast, sensitive, specific, and low cost.

Recently, Lee et al. developed a fast, ultra-sensitive, high-precision POCT device for the detection of the pathogenic bacteria Staphylococcus aureus. The device is based on the “count-on-a-cartridge” (COC) platform, which minimizes complex user interventions and maximizes the detection accuracy with a lower detection limit.

“We combined a high-throughput microfluidic-based immunomagnetic concentration process using magnetic particles (MPs) conjugated with an antibody probe with a cartridge capable of autonomously separating out and capturing a small number of magnetically and fluorescently labeled (using a quorum-based modified autoinducing peptide (mAIP) probe) bacteria from a great number of free unbound MPs within a passively regulated controlled time after concentration.” Professor Lee said. MP-SAab (WHM-G048, CD Bioparticles) and anti-Staphylococcus aureus antibody were used to bind with target bacterial cells. The system is composed of a magnetic concentrator, sensing cartridge, and fluorescent image reader with a built-in counting algorithm. Results can be obtained within a few hours in a user-friendly manner, with high sensitivity and high precision. The analytical performance of this assay is comparable to that of standard plates. According to the global microbiological standards for Staphylococcus aureus, COC analysis shows that the sensitivity is 92.9%, and the specificity is 100%, which is acceptable below 100 CFU/g in the food matrix. This culture-independent, fast, ultra-sensitive, and highly accurate COC analysis method has great potential in places where bacterial monitoring is urgently needed.

References:
1. Lee, W. I., Park, Y., Shrivastava, S., Jung, T., Meeseepong, M., Lee, J., … & Lee, N. E. (2020). A fully integrated bacterial pathogen detection system based on count-on-a-cartridge platform for rapid, ultrasensitive, highly accurate and culture-free assay. Biosensors and Bioelectronics, 152, 112007.
2. Wilson, I. G., Cooper, J. E., & Gilmour, A. (1991). Detection of enterotoxigenic Staphylococcus aureus in dried skimmed milk: use of the polymerase chain reaction for amplification and detection of staphylococcal enterotoxin genes entB and entC1 and the thermonuclease gene nuc. Applied and environmental microbiology, 57(6), 1793-1798.