Advances in the detection of virulence genes of Staphylococcus aureus originate from food

Yingying Zho,Dndn Xi,Pnpn M,Xiofeng Go,Wenyi Kng,Jinfeng Wei,d,*

a National R&D Center for Edible Fungus Processing Technology,Henan University,Kaifeng 475004,China b Joint International Research Laboratory of Food&Medicine Resource Function,Henan Province,Kaifeng 475004,China c Kaifeng Key Laboratory of Functional Components in Health Food,Kaifeng 475004,China d Minsheng College,Henan University,Kaifeng,Henan 475004,China

Keywords:

ABSTRACT

1.Introduction

In recent years,food safety has been widely concerned by countries all over the world,and foodborne diseases caused by pathogens has drawn great attention in scientific research and food industry[1].Staphylococcal food poisoning is a common foodborne disease in the worldwide[2].Staphylococcus aureus(SA)is one of the foodborne pathogenic bacteria and an important pathogen.It is a typical representative of Gram-positive and is widely distributed in nature.S.aureus is a kind of non-flagellar and non-spore forming,and it has catalase and coagulase positive[3].According to the statistics of Centers for Disease Control and Prevention,food poisoning caused by SA is 33%in the United States and 45%in Canada[4].Food safety is a big problem for human being,especially bacteria cause foodborne diseases such as S.aureus,Escherichia coli,etc.This bacteria still seriously threaten human health.Therefore,it is necessary to detect them.

S.aureus secretes multiple toxic proteins.Pathogenic enterotoxins are usually produced when more than 105 CFU/g and cannot be treated by conventional methods[5].Enterotoxin can tolerate high temperature and the genes encoding enterotoxin are sea-ser,seu and sev.The genes sea-see can cause food poisoning by SA[6-8].Hemolysin is an important pathogenic factor of SA,which can be divided into four types α,β,γ and δ.The main pathogenic factor for human beings is α-hemolysin[9].The specific gene in SA nuc is used to encode thermostable nuclease.The enzyme is an important indicator for pathogenicity[10].PVL is one of leukocytokinin by SA produced,and damages the defense barrier and immune response of the body by destroying leukocytes and phagocytes[11].Furthermore,S.aureus also contains other virulence genes,such as disinfectant resistant gene(qacA)[10],plasma coagulase gene(coa,cal)[12,13],toxic shock syndrome toxin-1 gene(tsst-1)[14],etc.

At present,there are traditional and rapid detection methods for S.aureus.Based on the principle of traditional detection and rapid detection methods,this paper summarized their advantages and disadvantages,and provided theoretical basis for rapid and accurate detection of S.aureus.

2.Conventional methods

2.1.Counting detection methods

In traditional detection,S.aureus detection is based on germiculture.According to the national standard GB 4789.10-2016 detect the SA.After enrichment in 7.5%sodium chloride broth,the bacteria are inoculated into Baird-Parker(B-P)plate and blood plate by scribed inoculation,and then gram stain,microscopic examination and plasma coagulase assay.This method is used for qualitative detection the SA.Plate counting and MPN counting are used for quantitative detection the SA in food[15].The paper disk method is based on the reaction of enzymes produced by microorganism metabolism with corresponding chromogenic substrates[16].PetrifilmTMof the USA 3 M Company is regarded as the standard test method of commodity inspection.SN/T 1895-2007 is used for testing[17].Compared with others,the national standard has a series of advantages such as simple operation and no need special apparatus.However,the national standard not rapidly detect pathogenic microorganisms in food because it requires long time and many steps[18].Compared with the national standard method,the paper disk method has short cycle,high sensitivity and simple use,but the cost is high.Tan[19]and Scholer[20]used two methods to detect SA respectively.There is no significant difference detection rate between PetrifilmTMtest and Baird-Parker plate test,and the PetrifilmTMtest is higher sensitivity.

2.2.Chromogenic medium

The principle of chromogenic medium is the same as the paper disk method.It has been used to test microorganisms.Baird-Parker medium is the standard Chromogenic medium in China,but because of its low specificity,other physical and chemical tests need to be supplemented[15,21,22].In Japanese industrial tests,Rabbit Plasma Fibrinogen(RPF)medium was used to detect the coagulasepositive SA,which could cause gray and black precipitation around the bacterial colonies[23].Gu et al.[24]compared the three Chromogenic medium,RPF and CHROMagarTMStaph aureus medium are superior to B-P medium.

The chromogenic medium has specificity,faster than traditional medium and improve accuracy of separate culture.However,there are also some shortcomings.The growth of target bacteria will be affected by the inhibit non-target bacteria substances added to the color medium.Different bacteria may produce the same specific substances in the process of culture,which will result in false positive so that affect the test results.Moreover,the Chromogenic medium need bacteria culture,so the operate cumbersome,and time-consuming and laborious[24-26].

3.Rapid detection methods

3.1.Immunoassay

The basic principle of immunology is the specific combination of antigen and antibody.Microorganisms can be quickly detected by this principle.Immunoassay methods include Enzyme-linked Immunosorbent Assay(ELISA),Enzyme-linked Fluoroimmunoassay(ELFA)and Immunomagnetic Beads Separation,etc.

3.1.1.ELISA

ELISA was first proposed by Swedish scholars Engvall and Perlmann.The way of ELISA has become a new method for the determination of tittle substances[27].Lin et al.[28]provided a new idea for microbial detection by the method of ELISA.Nagaraj[29]and Zhao[30]et al.established double antibody sandwich method to detect SA enterotoxin G and enterotoxin M.Li et al.[31]used cell fusion technology to prepare anti-SEA monoclonal antibodies,its sensitivity of SEA method can be as high as 1.56 ng.The ELISA method is practical,specific and sensitive,and its detection range is at ng and pg levels.It can directly detect enterotoxin in solution and is suitable for qualitative test[32-34].However,the external environment can influence the ELISA detection,and this method has a degree of cross-reaction to similar compounds[33,35].

3.1.2.ELFA

The results of ELFA are expressed by fluorescence intensity,which is more intuitive than ELISA.Hennekinne et al.[36]used this technique to detect SA enterotoxins in 2007.Shen et al.[37]used ELFA to reduce the detection limit enterotoxin A to 0.09 ng/mL.VIDAS system detect SA enterotoxins and the minimum detection value is less than 0.25 ng/mL[33].ELFA is highly sensitive and reduces the detection limit and improves the detection sensitivity[32].However,SA and its enterotoxins are inactivated during food processing,which may lead to incorrect of results[37].

3.1.3.Immunomagnetic separation

Immunomagnetic separation(IMS)is a specific adsorption property of ImpetiCbead.It can be used in the isolation and identification of S.aureus.Deng et al.[38]found that the capture rate of SA ImpetiCbead to target bacteria exceeded 80% when the density of pure bacteria is 10—104CFU/mL.The method also can be used to detect and isolate bacteria quickly.It is no need for the process of increasing bacteria and its instrument is simple[32].However,IMS is expensive and prone to cross-reactions,therefore it need assist detection by other technical methods[3].

3.2.Molecular biology methods

With the rapid development of molecular biology in recent years,the microorganisms detect not limited to traditional methods,but establish multitudinous rapid detection methods of foodborne pathogens at molecular level.

3.2.1.Nucleic acid probe

Nucleic acid probe technology detects microbes based on the principle of complementary base pairing.Firstly applied to detect E.coli[39].Now the technology has been successfully applied to detect SA.DNA probe technique amplifies target bacteria by designing specific primers based on highly conserved nucleic acid sequences[32].Its greatest feature is strong specificity,accuracy and sensitivity.Gao et al.[40]used nuc gene as probe to detect SA in laboratory animals.The sensitivity of this method can reach 1 pg genomic DNA,and the results are consistent with bacteriological tests.Xue[41]and Chen[42]et al.used nucleic acid probes to detect bacterial colonies respectively minimum of 106CFU/mL and 107CFU/mL.The initial level of 10 CFU of SA is detected in the overnight cultures of pre-concentrated food samples.Bacteria need to be enriched before detection,which increases the detection time[3].

3.2.2.Polymerase chain reaction

With the rapid development of molecular biology,Polymerase Chain Reaction(PCR)has become the method which is frequently method for rapid detection of SA.Compared with traditional detection methods,PCR technology is faster,more efficient and sensitive.However,there are also some drawbacks.This technology have to extract DNA,and deal with simple to a large degree impact on detection of PCR.When it is used to detect SA,it cannot distinguish its alive or dead,which may be false positive consequence.Therefore,the results are susceptible to interference[43].There are many methods of PCR technology apply to foodborne microorganisms detection:ordinary PCR,multiplex PCR,reverse transcription PCR(RT-PCR),quantitative real-time PCR(qPCR),etc.Rajeh[44]and Narges[45]used ordinary PCR to detect and identify multidrug resistant bacteria and SA enterotoxins.Sowmya et al.[25]used qPCR to detect SA in food.The detection limits in meat and dairy products respectively are 4.0×10 CFU/mL and 3.5×10 CFU/mL.Wei et al.[46]used multiplex PCR to detect nuc gene.The detection limit is 103CFU/mL,and there is no difference with the results of traditional detection method.Maerle et al.[47]used immunopolymerase chain reaction to detect enterotoxin A and TSST,and the sensitivity of these toxins in SA culture supernatant is no less than 10 pg/mL.Immunocapture polymerase chain reaction(IC-PCR)is a specific and tittle detecting method.Liang[48]and Zhang[49]used IC-PCR to detect many bacteria and bacterial toxins successfully.Dong et al.[50]detect the number of live SA by making qPCR combine with sodium dodecyl sulfate(SDS)and propidium azide(PMA),which makes their result has higher specificity and sensitivity than traditional qPCR.Reddy et al.[51]established a specific IgY-mediated immunocapture PCR-ELISA(ICPCR-ELISA)method to detect enterotoxin A with the sensitivity as 10 pg/mL.

3.2.3.Loop-mediated isothermal amplification

Loop-mediated isothermal amplification(LAMP),initially developed by Japanese scientists Nori et al.[52],is a novel,rapid and high sensitive nucleic acid amplification technology.The sensitivity of LAMP for detecting pure cultured SA is 25 CFU/mL,and 42 CFU/mL for detecting SA in contaminated food[53].The test can be completed within 40-60 min.Sowmya[29]detect SA contains only three steps,which reduces the loss of samples.The sensitivity of LAMP is 102CFU/g,which is more sensitive than PCR 104CFU/g.Sheet[54]established LAMP method with nuc gene to detect SA in dairy cow mastitis successfully.The sensitivity is 0.26 pg DNA of SA and the detection milk containing SA is 9×102CFU/mL.The LAMP method is simple,specific and sensitive,and has good practicability,especially in laboratories with limited resources in developing countries[25].However,due to the limitations of LAMP,it is easy to produce false positive results without strict experimental conditions.

3.2.4.Biosensor

Biosensor is devices which convert the concentration sensitive to biological substances into optical or electrical signals.It are composed of sensing elements and sensor elements[55].It is used to detect SA in food,environment and other substances.The method is easy to implement and does not require any amplification steps[32].Optical biosensor-based adapter biosensor attracted wide attention in pathogen detection due to its sensitivity and selectivity[56].Cao[57]developed a new affinity reagent for the detect SA by retain DNA adapters.Biosensor established by Sara et al.[58],it detect limit is 10 CFU/mL,and obtained results in 10 min,which distinguish SA with E.coli and Staphylococcus epidermidis etc.Huang[59,60]used biosensors to detect SA enterotoxin A and enterotoxin C,they are limited to 8.7 ng/mL and 6 ng/mL.Electrochemical adapter sensors are usually composed of adapters as recognition elements and sensors to generate measurable electrical signals.The electrochemical method has the advantages of rapid reaction,low detection cost,simple operation,easy portability and superiority to optical sensors[61,62].

Biosensors costs are high,the price is relatively expensive,antibodies purification and modification are difficult,so they are not suitable for regular detection of multiple samples in food testing laboratories and industrial departments[63,64].

3.2.5.Flow cytometry

Flow cytometry(FCM)is a method integrates optics,hydrodynamics,electricity and computer technology.It can quantitatively measure and synthetically analyze cells with multiple parameters[20].FCM is used to analyze cell surface markers and rapidly identify SA.It can be used for batch detection with short detection time,but it also requires special instruments[65].Hendrickson et al.[66]used ligand-receptor interaction and wheat germ agglutinin as receptor to detect 106cells/mL in 5 min.

4.Conclusions

Foodborne diseases caused by bacterial contamination seriously threaten human health and food safety.Bacteria rapidly detect has become one of the biggest problems.S.aureus can grow in different conditions and cause food poisoning by secreting enterotoxins[2].There are many methods to detect foodborne microorganisms,such as traditional and rapid detection methods.These methods have their own advantages and disadvantages.Traditional detection do not need to train the technical personnel,its methods are cheap and simple operation,but it is difficult to avoid the process of pathogenic bacteria enrichment and separation,which prolongs the detection time greatly.Rapid detection methods include immunological detection and molecular biological detection.Immunology technology has high specificity,high efficiency,and does not need large instruments and equipment,but false positive results are likely to occur during detection,and the detection speed needs rapidly enriched.Molecular biological methods have developed rapidly in recent years,it has characteristics of specificity and high efficiency.Since the advent of PCR technology,various PCR technologies have been applied to detect foodborne pathogens.qPCR is more sensitive and accurate than ordinary PCR,but its application scope is limited because of its high price and incomplete research.LAMP is a flexible detection method,which requires only simple equipment and is more sensitive than ordinary PCR.Biosensors and FCM can be used to detect SA,but the price is relatively expensive.Therefore,combining the existing principles,methods and technologies,and exploring a rapid,sensitive,stable,simple and inexpensive detection method for detecting foodborne microorganisms are the main development direction in the future.

Acknowledgement

This work was supported by grant(2017YFC1601400)from the Ministry of Science and Technology of the People’s Republic of China,Key Project in Science and Technology of Henan Province(182102410083).