18 Staphylococcus Aureus

18

Staphylococcus Aureus Paolo Moroni, Giuliano Pisoni University of Milan

Paola Cremonesi, Bianca Castiglioni

Institute of Agricultural Biology and Biotechnology

Contents 18.1 Introduction.................................................................................................................................................................... 241 18.1.1 Morphology and Biology.................................................................................................................................. 241 18.1.2 Pathogenesis and Clinical Features.................................................................................................................. 242 18.1.3 Conventional Testing........................................................................................................................................ 243 18.1.4 Molecular Testing............................................................................................................................................. 243 18.1.4.1 Standard PCR.................................................................................................................................. 243 18.1.4.2 Multiplex PCR ................................................................................................................................ 245 18.1.4.3 Real-Time PCR ............................................................................................................................... 246 18.2 Methods.......................................................................................................................................................................... 248 18.2.1 Reagents and Equipment................................................................................................................................... 248 18.2.2 Sample Collection and Preparation.................................................................................................................. 248 18.2.2.1 Sample Collection............................................................................................................................ 248 18.2.2.2 Sample Pretreatment........................................................................................................................ 249 18.2.2.3 DNA Extraction............................................................................................................................... 249 18.2.3 Detection Procedure............................................................................................................................................ 249 18.3 Conclusions and Further Perspectives............................................................................................................................ 250 References.................................................................................................................................................................................. 251

18.1 Introduction 18.1.1 Morphology and Biology Staphylococcus aureus is a Gram-positive coccus, which tends to be arranged in irregular clusters or grape-like clusters when viewed through a microscope and has large, round, golden-yellow colonies, often with hemolysis, when grown on blood agar plates. The golden appearance is the etymological root of the bacteria’s name: the name derived from Greek staphylo (bunch of grapes) and aureus means “golden” in Latin. S. aureus is a facultative anaerobe (fermentative) with the following main features: (i) it can grow at an aw of 0.86 and pH above 4.81 and within the temperature range 7–48°C with an optimum of 35–40°C; (ii) it is catalase positive (meaning that it can produce the enzyme “catalase”) and able to convert hydrogen peroxide (H2O2) to water and oxygen, which makes the catalase test useful to distinguish staphylococci from enterococci and streptococci; (iii) it is oxidase negative; and (iv) it is nonmotile. S. aureus can be differentiated easily from most other staphylococci by its ability to produce coagulase and a heat-stable endonuclease. S. aureus is primarily coagulase-positive (meaning that it can produce the enzyme “coagulase” that causes clot formation) while most other Staphylococcus species are

coagulase-negative. However, while the majority of S. aureus strains are coagulase-positive, some may be atypical and do not produce coagulase enzyme. Members of the S. aureus species produce a number of extracellular compounds including membrane-damaging toxins, epidermolytic toxin, toxic shock syndrome toxin, pyrogenic exotoxin, exoenzymes (coagulase and thermostable nuclease) and staphylococcal enterotoxins (SE). The natural reservoirs for S. aureus are the skin and the mucous membrane of humans and animal. Many staphylococcal species have become adapted to life on particular animal species but, in contrast, S. aureus is present on most marine and terrestrial mammals, and may be present as a nonaggressive member of the normal skin microflora or may be associated with infectivity and disease. Up to 30–50% of the human population are carriers of S. aureus; the body sites most often colonized are the nostrils, throat, hair, and hands. This organism may also be isolated from healthy domestic and food animals as well as being associated with disease, particularly mastitis. S. aureus strains have been classified into biotypes according to their human or animal origin: six different biotypes (human, non-β-hemolytic human, avian, bovine, ovine, and nonspecific) have been identified based on biochemical characteristics.2 241

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Molecular Detection of Foodborne Pathogens

18.1.2 Pathogenesis and Clinical Features Staphylococcal food poisoning (SFP) relies on one single type of virulence factor: the SEs. The SEs are a group of extracellular protein with molecular weights in the range of 27000–30000 Daltons, and similar composition and biological activity. To date, 19 types of staphylococcal SEs have been described.3 On the basis of their antigenicity and mode of action in the host, SEs have been divided into two groups. The members of group 1, classical emetic toxins designated SEA, SEB, SEC1, SECbov, SED, and SEE, are the cause of about 95% of SFP in humans.4 These toxins are resistant to gastrointestinal proteases and after their intestinal absorption, the patient can develop symptoms of food poisoning, depending on the SE amount ingested.4,5 Group 2, includes toxins possibly involved in the remaining 5% of SFP outbreaks, i.e., the recently identified SEs, designated SEG, SEH, SEI, SEJ, SEK, SEL, SEM, SEN, SEO, SEP, SEQ, SER, and SEU,6–12 the emetic activities of which are not currently fully understood (Table 18.1). The symptoms of SFP are abdominal cramps, nausea, vomiting, sometimes followed by diarrhea.6 The onset of symptoms is rapid (from 30 min to 8 h) and spontaneous remission is usually observed after 24 h. Hospitalization is required in approximately 10% of the cases.13 In all cases of SFP, the foodstuff or one of the ingredients was contaminated with a SE-producing S. aureus strain and was exposed, at least for a while, to temperatures that allow S. aureus growth. Most of the time the foodstuff reaches this temperature because of a failure in the refrigeration process, or because a growth-permissive temperature is required during processing (e.g., cheese making). Many different foods can be a good growth medium for S. aureus, and have been implicated in SFP, including milk and cream, cream-filled pastries, butter, ham, cheeses, sausages, canned meat, salads, cooked meals and sandwich fillings. The foods that are most often involved in SFP differ widely from one country to another. In the United Kingdom,

Table 18.1 Major Characteristics of 18 Staphylococcal Enterotoxins (SE) SE Type A B C1 C2 C3 C (bovine) C (sheep) C (goat) D E G

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Reference [119] [120] [121] [122] [123] [124] [124] [124] [125] [126] [127]

SE Type H I J K L M N O P Q R

Reference [128] [127] [129] [130] [131] [7] [7] [7] [132] [133] [134]

U

[135]

for example, 53% of the SFP reported between 1969 and 1990 were due to meat products, meat-based dishes, and especially ham; 22% of the cases were due to poultry, and poultry-based meals, 8% were due to milk products, 7% to fish and shellfish and 3.5% to eggs.14 In France, in a 2-year period (1999–2000), among the cases of SFP in which the food involved had been identified, milk products and especially cheeses were responsible for 32% of the cases, meats for 22%, sausages and pies for 15%, fish and seafood for 11%, eggs and egg products for 11% and poultry for 9.5%.15 In the United States, among the SFP cases reported between 1975 and 1982, 36% were due to red meat, 12.3% to salads, 11.3% to poultry, 5.1% to pastries and only 1.4% to milk products and seafoods. In 17.1% of the cases, the food involved was unknown.16 During a 5-year period (1988–1992), S. aureus caused 5.1% of the food poisoning outbreaks reported in Europe17 and in Italy it caused four of the 233 outbreaks reported. S. aureus has been isolated from several foods: meat and meat products, chicken, milk, and dairy products, fermented food items, vegetables, fish products, etc.14,18 Salted food products, such as ham, have been reported to be responsible for about 24% of all the cases of staphylococcal intoxication.19 Thus, the origins of SFP differ widely among countries; this may be due to differences in the consumption and food habits in each of the countries. In France, for example, the consumption of raw milk cheeses is much higher than in Anglo-Saxon countries. This may explain the relative importance of milk products involved in SFP in France. In any case, the main sources of contamination are humans (handlers contaminate food via manual contact or via the respiratory tract by coughing and sneezing), and contamination occurs after heat treatment of the food. Nevertheless, in foods such as raw meat, sausages, raw milk, and raw milk cheese, contaminations from animal origins are more frequent and due to animal carriage or to infections (e.g., mastitis). Some S. aureus strains, so-called ‘‘endemic strains’’, are present in some processing plants, such as poultry processing lines,20–22 consequently food products may originally become contaminated during or after processing. However, the presence of large numbers of staphylococci is not sufficient cause to incriminate a specific food as the vehicle of food poisoning because not all staphylococci are enterotoxigenic. In addition, demonstration of enteroxigenicity of food isolates is only circumstantial evidence of enterotoxigenic staphylococcal contamination and the potential for causing food poisoning cannot be ascertained without demonstrating the actual presence of the SE in a suspect food. Conversely, neither the absence of S. aureus nor the presence of small numbers of bacteria is complete assurance that the food is safe (because the SE is extremely resistant and may survive processes which kill the bacterial cells). Concerning S. aureus in food, it should be noted that only the enterotoxinogenic strains possess a risk to public health and the criteria applied should prevent the production of SE during processing and the occurrence of SE in the final product. It is generally considered that enterotoxinogenic staphylococci must reach levels of at least 105–106 cfu/g or ml to

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Staphylococcus Aureus

produce detectable amounts of SE and, in Europe, low degree contaminations by S. aureus are tolerated in most foodstuffs (up to 104 cfu/g in raw milk cheeses, in France), as they are not considered a risk for public health.

18.1.3 Conventional Testing Risk assessment in foodstuffs relies on classical microbial detection and quantification of coagulase positive staphylococci on a selective Baird-Parker medium, whose composition is standardized (EN ISO 6888/1 and 2).23,24 The sensitivity of these routine tests is around 102 cfu/g for solid foodstuffs and 10 cfu/g for liquid samples. The different media used for the detection and quantification of S. aureus have been reviewed by Baird and Lee.25 Food testing using microbiological criteria may have limited usefulness for food safety for a number of reasons, including low prevalence of the pathogen, or low diagnostic sensitivity of the testing procedure applied. While the finding of a pathogen in a foodstuff may indicate a problem for public health, necessitating appropriate risk management action; the failure on the other hand to detect a pathogen in a food product does not necessarily mean that the pathogen is absent from that food product, process or food lot.

18.1.4 Molecular Testing Use of DNA-based assays may circumvent some of the problems associated with conventional microbiological procedures. Perhaps the greatest single advantage of DNAbased diagnostic assays is that these methods focus on the unique nucleic acid composition of the bacterial genome rather than on phenotypic expression of products that nucleic acids encode. Therefore, DNA-based identification assays are subject to less variability compared with diagnostic methods based on phenotypic characterization, allowing reliable detection and quantification down to one single nucleic acid target per PCR sample. Moreover, not only the presence of the pathogen but also of the genes encoding for SEs production is important to evaluate as enterotoxins nonproducing strains may also occur. 18.1.4.1 Standard PCR Many polymerase chain reaction (PCR) protocols have been developed for the direct detection or for identification of S. aureus in milk and dairy products.26–33 These PCR methods allow identification of bacteria within hours. However, the sensitivities of PCR assay often vary with different DNA extraction methods, suggesting that the DNA extraction protocols are important for optimization of the assay if it is to be applied to food.34 Consequently, the development of a sample preparation strategy that can effectively sequester high-quality DNA of the pathogenic bacteria from food samples before PCR amplification is needed. Indeed, molecular procedures require highly purified template DNA.35,36 In addition, the PCR-based detection of pathogens is made more difficult when raw material with high

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level of background microflora or complex food matrices are involved.18,37 First, these difficulties could be due to small concentrations of the pathogenic DNA present in a typical sample. Second, various factors affect DNA recovery, including the degree of cellular lysis, binding of DNA to particulate material, and degradation or shearing of DNA. Furthermore, in the case of Gram-positive bacteria such as S. aureus and streptococci, an optimal sample processing method should efficiently lyse resistant bacterial cell walls without damaging target DNA. In addition, direct detection of pathogenic bacteria in food samples38 is hampered by the presence of PCR-inhibitory substances frequently associated with the food matrix itself.39 Particularly in milk, components such as Ca2 + , proteinase, fats, and milk proteins may block DNA and shield it from access by polymerase.40 Breaking down casein and the casein micelle with the enzyme pronase would allow better access to the bacteria for lysis by lysozyme and proteinase K.35 Moreover, cheeses are often regarded as difficult matrices to be assayed by PCR because of the high fat content18,40 which is reported to be one of the major factors reducing the sensitivity of the PCR assay.39 Finally, it is possible that the poor sensitivity of detection of S. aureus is caused by the abundance of DNA extracted from the background microflora of the raw milk acting as inhibiting competitor in PCR reactions. In this case false positive results may occur because of parallel amplification of target genes, such as the regions coding for ribosomal RNA, from closely related species.41 To overcome PCR inhibition problems and to increase the sensitivity of the assay, overnight selective-enrichment was frequently used to increase the level of pathogen detection.31,32,34,38 After the enrichment step, sufficient bacteria were present to allow pathogens to be detected when the original sample had as little as 1 cfu/ml. The higher sensitivity of the assay may be due to the dilution of inhibitory substances in the enrichment broth and the increased number of organisms.32 In milk samples, another effect of enrichment before PCR may be the reduced detection of DNA from nonviable bacteria. However, although the prior enrichment step increases the target concentration, it precludes quantification.36,42,43 The vast number of procedures used for this purpose and articles published focusing on this topic indicate that these problems are still far from being solved.44–49 Therefore, the separation and concentration of foodborne pathogens directly from food samples without culture enrichment represent two of the most important aspects of sample preparation. Methods for separating bacteria directly from a food matrix and then concentrating them depend on several chemical, physical, and biological principles. Numerous methods for isolating bacterial DNA directly from milk have been reported in the literature and involve a wide variety of substances including Chelex-100,28 spin columns,29,32,33 lysozyme and proteinase K,31 diatomaceous earth,50 alkaline extraction,30 and pronase.48,51 Methods that use proprietary reagents such as Insta-Gene Matrix (BioRad) and PrepMan Ultra reagent (Applied Biosystems) were also evaluated. Although these

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reagents were faster and more convenient than other methods evaluated, results were not consistent and reproducible when trying to isolate bacterial DNA from milk.52 The method first described by Allmann et al.48 and modified by Hein et al.51 was one of the most consistent and reproducible system evaluated for isolation of bacteria directly from whole milk samples. Even a buoyant density gradient centrifugation were recently used to separate bacteria from complex food matrices, as well as to remove compounds that inhibit rapid detection methods, such as PCR, and to prevent false-positive results due to DNA originating from dead cells.53 A specific, sensitive, and rapid method to extract DNA directly from the main Gram-positive bacteria (including S. aureus) found in milk and raw cheese samples was also described by Cremonesi and coworkers.54,55 The DNA extraction method is based on the lysing and nuclease-inactivating properties of the chaotropic agent, guanidinium thiocyanate, together with the nucleic acid-binding properties of the silica particles. In absence of PCR inhibitors, the level of sensitivity achieved for S. aureus detection were 10 cfu/ml in milk and 100 cfu/g in cheese. Finally, PCR detection is often hampered by the presence of inflammatory somatic cells. To eliminate this problem, a method that allows the effective separation of bacterial cells from somatic cells in mastitis milk with amino-silica was recently established.56 The authors identified the conditions under which most of the somatic cells were adsorbed and only S. aureus desorbed from amino-silica upon addition of a desorption solution; in this way the procedure effectively eliminated somatic cells in heavily contaminated milk samples, which resulted in improved clarity of the PCR band. The major advantages of PCR lay in the possibility of using only few nanograms of nucleic acid samples, allowing the elimination of culture, rapidity, and easy analysis. Several PCR methods were described for the identification of S. aureus,57,58 and staphylococcal strains harboring copies of enterotoxin genes can be rapidly detected by PCR in food samples.59 In these works, PCR amplification of DNA regions coding for ribosomal RNA (rRNA) was often used because of the presence of hypervariable regions, which facilitates the design of highly specific oligonucleotide probes and common regions for the design of universal probes. Moreover, rDNA is present in many copies, which permits signal enhancement.29,60 In addition to the 16S rDNA gene, the well established standard target for the identification of bacterial species,61 the sequence of the 16S to 23S rDNA spacer region and the 23S rRNA gene have proven useful for identification of S. aureus at the species level.33,41,62 For example, a 23S rDNA-targeted PCR-based system for detection of S. aureus in meat starter cultures and dairy products was developed.41 More recently, using species-specific primers derived from 16S and 23S rDNA, PCR detection was performed with S. aureus isolates. The detection limit of this assay for milk products was 16 cfu/µl, representing a valid diagnostic tool for the detection of milk pathogens in dairy products.63

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Other PCR assay for the detection and identification of this pathogen was established and validated using three conserved genes as the DNA target of S. aureus: fmhA (coding for a factor of unknown function), catalase and femA (coding for a factor essential for methicillin resistance). All the three assay systems showed a detection limit of 100 cells per 20 ml reaction assay.64 The protocols developed could be used for rapid and specific detection of this pathogen in food and environmental samples, especially milk.64 Following the recent publication of the genome sequences of nine S. aureus strains (http://www.ncbi.nlm.nih.gov/ genomes/lproks.cgi), the vicK gene of S. aureus that relates signal transduction of the pathogen was investigated as a target for rapid detection and identification.65 The PCR assay allowed amplification of a 289 bp DNA fragment only from S. aureus and not from other Staphylococcus species and other common bacteria tested. The identification of enterotoxigenic S. aureus strains is usually based on amplification of the coa gene.66–68 Also, the presence of the thermostable nuclease gene (nuc) showed strong correlation with enterotoxin production and it is a marker of food contamination with enterotoxigenic S. aureus.18,69 Further more, Kalorey and coworkers70 characterized genotypically by PCR 37 enterotoxigenic strains of S. aureus isolated from 552 milk samples using not only primers that amplified genes encoding coagulase (coa) or thermonuclease (nuc), but also clumping factor (clfA), enterotoxin A (entA), and the gene segments encoding the immunoglobulin G binding region and the X region of protein A gene (spa). To survey the enterotoxin genotypes for S. aureus strains isolated from food-poisoning cases in Taiwan, PCR primers specific for all SE genes, including SEN, SEO, SEP, SEQ, SER, and SEU, or staphylococcal superantigens genes (SAg), including toxic shock syndrome toxin I (TSST-1) genes, were more recently developed.71 Using these PCR primers the genotypes of 147 S. aureus strains isolated from patients associated with staphylococcal food-poisoning outbreaks occurred during 2001–2003 was assayed. 91.8% of these strains were found positive for one or more SE or SAg genes. To define PCR-based detectability of S. aureus in the early stages of raw milk cheese making, raw milk artificially contaminated by different concentrations of S. aureus FRI 137 strain, harboring nuc, sec, seg, seh, and sei genes was employed in dairy processing resembling traditional raw milk cheese making.36 Samples of milk and curd were PCRanalysed after DNA extraction by targeting all the above genes. The pathogen was detected when the initial contamination was 104 cfu/ml by amplification of nuc and seh genes. 105 and 107 cfu/ml were needed when seg or sei and sec genes were targeted, respectively. Enrichment cultures from raw milk and curd samples provided to increase the detection limit of 1 log on average. Therefore, the direct detection of the pathogen in the raw material and dairy intermediates of production can provide rapid results and highlight the presence of loads of S. aureus potentially representing the risk of intoxication. However, every target gene to be used in the analysis has to be studied in advance in a system similar to

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the real case in order to determine the level of contamination that can be predicted. Often also the consumption of ham or meat containing SE is identified as the cause of illness. Evaluation of the occurrence of most known SE (SEA to SEE; SEG, SEH, SEI, SEM, SEJ, SEN, and SEO) genes, egc (enterotoxin gene cluster containing the following sequence of genes: seo, sem, sei, phient1, phient2, sen, and seg) and tsst1 (toxic shock syndrome toxin 1) gene in both coagulase-positive (CPS) and coagulase-negative (CNS) staphylococcal strains isolated from meat and dairy products were carried out by Blaiotta and coworkers.72 PCR detection methods were used to analysed 109 wild Staphylococcus spp. strains isolated from Napole-type salami, raw water buffalo milk and natural whey cultures used for mozzarella cheese manufacturing, revealing that the occurrence of SE genes in CNS and other non-S. aureus species in these foodstuffs is very rare. To gain insight into the prevalence of S. aureus and its emetic enterotoxins in raw pork and uncooked smoked ham, samples of raw pork, salted meat and ready-for-sale uncooked smoked ham were examined for the prevalence of S. aureus and SEA–SED.60 To this end classical cultural methods were employed as well as molecular biological techniques (PCR) and the results were compared. Fresh meat was contaminated most often. By PCR, 62.2% were identified as being S. aureus positive compared to 57.7% positive samples using the cultural technique. The detection rate decreased significantly during the fabrication process. The pathogen was cultivated from 8.9% of the salted meat samples. Here, 55.6% of the samples reacted positively in the PCR, and finally, in approximately a third of the ready-for-sale smoked hams, S. aureus genes were found. This study clearly shows that the PCR assay is more sensitive than the classical cultural method. Approximately 35% of the staphylococcal strains identified using the PCR technique were enterotoxigenic. Using the SET-RPLA, a percentage of 28.6% enterotoxigenic isolates was ascertained. The detection of SE-genes by PCR is resulted faster and easier to perform than the SET-RPLA. 18.1.4.2 Multiplex PCR The DNA-based identification systems could be targeted to allow for simultaneous rapid screening of a large number of pathogens. For example, rapid and sensitive detection techniques for foodborne pathogens are important to the food industry. In multiplex PCR, multiple pairs of primers specific for different DNA segments are included in the same reaction to enable amplification of multiple target sequences in one assay. In many cases, more than four pairs of PCR primers can be used.73 Primers used in multiplex PCR amplification are chosen to have similar melting temperatures (Tm) as a difference of more than 10°C in the Tm of the two sets of primers may result in differential yields of amplification products,74 and no visible amplification for one or the other target. The major advantage of multiplex PCR over conventional PCR is its cost effectiveness. It reduces the amount of reagents, such as Taq DNA polymerase, used for each diagnosis. Moreover, it requires less preparation and analysis

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time than systems in which several tubes of simplex PCR are used. Phuektes and coworkers32,33 developed a multiplex PCR for detection of S. aureus, Strep. agalactiae, Strep. dysgalactiae, and Strep. uberis that targeted 16S to 23S rRNA spacer regions. However, sensitivity of this multiplex PCR was less than for each individual PCR assay, and enrichment was needed to bring the threshold for detection by multiplex PCR for all bacteria assayed to 1 cfu/ml. A multiplex PCR assay for rapid and simultaneous detection of five foodborne pathogenic bacteria, including S. aureus, was developed by Kim and coworkers.75 Specific primers for multiplex PCR amplification of the Shiga-like toxin (verotoxin type II), femA (cytoplasmic protein), toxR (transmembrane DNA binding protein), iap (invasive associative protein), and invA (invasion protein A) genes were designed to allow simultaneous detection of Escherichia coli O157:H7, S. aureus, Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella, respectively. Finally, a new multiplex PCR-based procedure followed by capillary gel electrophoresis with laser-induced fluorescence detection (multiplex-PCR-CGE-LIF) was approached for the simultaneous detection of S. aureus, Listeria monocytogenes, and Salmonella spp.76 As compared to slab gel electrophoresis, the use of CGE-LIF improved the sensitivity of the multiplex PCR analysis by 10- to 1000-fold, allowing the detection of 2.6 × 103 cfu/ml of S. aureus, 570 cfu/ml of L. monocytogenes, and 790 cfu/ml of Salmonella in artificially inoculated food, without enrichment. Following 6 h of enrichment, as low as 260, 79, and 57 cfu/ml of S. aureus, L. monocytogenes, and Salmonella, respectively, were detected. The multiplex-PCR-CGE-LIF proved a powerful analytical tool to detect various food pathogens simultaneously in a fast, reproducible, and sensitive way. Even for rapid and reliable detection of S. aureus and its enterotoxins in food, a number of multiplex PCR (mPCR)based assays have been reported.33,77–80 In most of these studies, separate reactions are required to identify subsets of these genes. For example, two multiplex PCRs were developed for the detection of enterotoxigenic strains of S. aureus, one multiplex reaction for the simultaneous detection of enterotoxigenic strains type A (entA), type B (entB), and type E (entE) and another for the simultaneous detection of enterotoxigenic strains type C (entC) and type D (entD).79 These two multiplex PCRs were used to determine the presence of enterotoxigenic types for 51 S. aureus strains isolated from meat (sausage, ham, and chorizo) and dairy (powdered milk and cheese) products. Levels of correlation between the presence of genes that code for the production of SE (as determined by PCR) and the expression of these genes (as determined by the indirect enzyme-linked immunosorbent assay, ELISA) were 100% for SEA and SEE, 86% for SEC, 89% for SED, and 47% for SEB. Another multiplex PCR assay was described for the detection and differentiation of enterotoxigenic S. aureus in dairy products.18 In this case, also a solvent extraction procedure was successfully developed for extraction of S. aureus DNA from 10 ml of artificially contaminated skim milk or 20 g

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AU: Figure 18.1 was not cited in the text, is this location correct?

cheddar cheese. Primers targeting the enterotoxin C gene (entC) and thermostable nuclease gene (nuc) were used in the multiplex PCR. DNA was consistently quantified and amplified by uniplex PCR from 10 cfu/ml of S. aureus in skim milk or 10 cfu/20 g cheddar cheese. Even in this case, the sensitivity of the multiplex PCR was lower than for each individual PCR assay, resulting 100 cfu/ml of skim milk and 100 cfu/20 g cheddar cheese. Nevertheless, the developed methodology allows presumptive identification and differentiation of enterotoxigenic S. aureus in less than 6 h. A multiplex PCR for the simultaneous detection of S. aureus 23S rRNA, the coagulase and thermonuclease genes as well as the enterotoxin genes sea, sec, sed, seg, seh, sei, sej, sel was developed81 in order to obtain multiplex amplification products also starting from as little as 1 pg of DNA, corresponding approximately to 10 cfu/ml, showing the excellent specificity and high sensitivity of the assay. This multiplex PCR assay was used to correlate the distribution of genes encoding SEs with the presence of the corresponding SE production in S. aureus isolated from bovine, goat, sheep, and buffalo milk and dairy products.82 A total of 112 strains of S. aureus were tested for SE production by immuno-enzymatic (SEA–SEE) and reversed passive latex agglutination (SEA–SED) methods, while multiplex PCR was applied for SE genes (sea, sec, sed, seg, seh, sei, sej, and sel). Of the total strains studied, 67% were detected to have some SE genes (se), but only 52% produced a detectable amount of the classic antigenic SE types. The bovine isolates frequently had enterotoxin SEA, SED, and SEJ, while SEC and SEL predominated in the goat and sheep strains. The results demonstrated marked enterotoxigenic S. aureus strain variations, in accordance with strain origin and the two methods resulted in different information but concurred on the risk of foodstuff infection by S. aureus (Figure 18.1). 18.1.4.3 Real-Time PCR In recent years, an advanced form of PCR, real-time PCR, has been introduced into clinical microbiology. Compared with conventional PCR, real-time PCR is faster, more suited to high throughput of samples, and can quantitate the nucleic acid concentration. In real-time PCR, amplified products are detected by fluorescence at the moment that they are generated and directly related to the input target quantity, so that quantization is possible. The use of real-time PCR has driven significant changes in the microbial detection procedures. The predominantly phenotype-related methods of culture and antigen detection are being supplanted by the detection, characterization, and quantification of microbial nucleic acids. In comparison with conventional PCR, real-time PCR is more rapid, sensitive, reproducible and reduces the risk of carry-over contamination. The majority of real-time PCR applications in microbiology are for qualitative (resulting in a yes or no answer) detection of a virus, bacterium, fungus or parasite. For this purpose, fluorogenic PCR-based assays have shown promise in the detection of a variety of organisms including bacteria.83–85

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Real-time PCR utilizes the 5′–3′ nuclease activity of Taq DNA polymerase to digest an internal fluorogenic probe labelled with a fluorescent reporter dye and a fluorescent quencher dye.86 During amplification, the probe is hydrolyzed, relieving the quenching of the reporter dye, resulting in an increase in fluorescent intensity. This change in reporter dye fluorescence is quantitative for the PCR product, and under appropriate conditions, for the template. Such methods would be even more useful if they could be fine-tuned to simultaneously detect and quantify a mixture of pathogens in a sample. For example, a multiplex realtime PCR method to simultaneously detect common mastitis pathogens including S. aureus, Strep. agalactiae, and Strep. uberis directly from milk were developed by Gillespie and coworkers.52 This assay is the first to use a multiplex real-time PCR format for detection of pathogens directly from milk. Use of this method eliminates the need for post-PCR analysis by gel electrophoresis. This multiplex real-time PCR assay used information concerning primers for S. aureus and Strep. agalactiae that were shown to be sensitive and specific. Primers and probes for S. aureus were designed within the region used by Reischl et al.87 and Martineau et al.59 The cfb gene encoding the CAMP factor was used as the genetic target for this real-time PCR assay for detection of Strep. agalactiae. A strategy based on 5’ nuclease Triplex PCR was also developed for the rapid detection of nine enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej) of S. aureus.10 This assay was first evaluated using a collection of S. aureus reference strains and then by testing previously characterized S. aureus strains isolated from food. While these assays correctly detected the SE genes in all the reference strains, in tests with field strains there was generally excellent agreement with the results obtained by conventional PCR, except for some strains harboring variant SE genes. The detection limits of these assays were also evaluated using fivefold dilution of recombinant plasmids for each se gene, ranging from 16 to 2000 copies of target se genes in the PCR tube. The development of this method is an improvement that should facilitate epidemiological investigations of staphylococcal food poisoning outbreaks. Two real-time fluorogenic PCR assays were described for the detection of entA, the gene that encodes SEA.88 The assays are useful in detecting and identifying strains of S. aureus that produce SEA and can serve a confirmatory role in determining the presence of SEA in food samples. The assays were tested in two real-time PCR formats, using either dye-labelled DNA probes corresponding to each primer set that are degraded by the 5’ exonuclease activity of Taq polymerase, or a PCR master mix that contains the DNA-binding dye SYBR Green. In both formats the assays have a limit of detection of between one and 13 copies of a S. aureus genome that contains a copy of entA. Neither assay crossreacted with genomic DNA isolated from other strains of S. aureus or other species. Two real-time quantitative PCR (RTQ-PCR) systems using nuc targeted primers, incorporating SYBR-Green I and TaqMan, respectively, have proven specific and suitable for the

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M

1

2

3

4

5

6

7

8

9

M

500 bp

Figure 18.1 Examples of multiplex PCR results for the detection of Staphylococcus aureus strains isolated from raw milk and raw cheese samples. Lanes 1–3, raw bovine milk samples; lane 4, curd, lanes 5–7 raw soft, semi-hard and hard cheese samples; lane 8, positive control; lane 10, negative control. All the samples were positive for 23S rRNA (499 bp), thermonuclease (nuc) (400 bp) and coagulase (coa) (204 bp) genes. The samples in lanes 4 and 5 are nonenterotoxigenic while the other samples contain se genes: lane 1, sample positive for sea gene (180 bp); lane 2, sample positive for sea (180 bp) sej (306 bp) and sed (343 bp) genes; lane 3, sample positive for sec (371 bp) gene; lane 6, sample positive for seg (432 bp) gene; lane 7, sample positive for sel (240 bp) and sec (371 bp) genes; lanes 8, reference strain ATCC 19095 (genes sec, seh, seg, sei, sel). M: 100 bp DNA ladder (Finnzymes).

routine detection and quantification of S. aureus in different food matrices.76 The two real-time PCR approaches improved the sensitivity of conventional PCR by decreasing the detection level to 10 (SYBR-Green I system) and 100 cells (TaqMan). In particular, the SYBR-Green I RTQ-PCR approach established allows the sensitive, automated and quantitative detection of S. aureus for routine analysis at a reasonable cost. A nuc targeted real-time PCR assay was developed to evaluate the risk associated with the enterotoxigenicity of S. aureus in Monte Veronese, a PDO (Protected Designation of Origin) cheese of the Lessinia area in Italy.89 By real-time quantification S. aureus numbers in cheese were found to exceed the limit tolerated by the Italian food legislation in 78% of the instances. Another real-time PCR detection system using a primer set from the sequence of the heat shock protein gene (hsp) and a gene coding for high-temperature-requirement A protein(htrA) was successfully developed to detect 16 S. aureus reference strains and 40 isolates from food-poisoning cases.90 When this primer set was used for the real-time PCR detection of S. aureus in milk and meat samples without the pre-enrichment step, samples with target cell numbers greater than 103 cfu/ml or cfu/g could be detected, indicating the potential quantitative ability of this real-time PCR assay. With a 10 h pre-enrichment step, however, a detection limit of 1 cfu/ml or cfu/g could be obtained. As the classical diagnostic bioassays of SEs as well as the routinely used immunological methods are hampered by several drawbacks regarding sensitivity, specificity, and practicability, sensitive reverse transcription-quantitative PCR

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procedures can also be suitable for the routine detection and quantification of S. aureus and its enterotoxins in food. To analyze the expression of the enterotoxin genes of S. aureus, a reverse transcription real-time PCR was developed.91 Thanks to this assay, various enterotoxin genes were detected, including sea, seg, seh, sei, sen, seo, and sem. When the mRNA detection of the enterotoxin genes was analyzed using a reverse transcriptase PCR, various levels of expression were found depending on the species and enterotoxin gene. Therefore, it is reasonable to suggest that the poisoning risk of S. aureus can be effectively evaluated based on the gene expression at the mRNA level. In addition, a quantitative real-time immuno-PCR (qRT-iPCR) was developed for the detection of SEA and SEB and compared to a commercially available enzyme immunoassay.92 This qRT-iPCR approach was shown to overcome clearly the sensitivity limit of traditional immunological detection procedures for bacterial toxins, as demonstrated in this study for SEs. The development of a stable antibody-DNA conjugate providing a universal signal of amplification offers a versatile as well as a highly sensitive and specific tool for diagnostic and research purposes generally applicable for preformed antibody-antigen complexes. Finally, a quantitative PCR assay was also developed for the diagnosis of udder infections with S. aureus.93 For clinical milk samples, the analytical sensitivity of this assay was 50.7 times and 507 times greater than conventional bacteriology with 100 and 10 µl, respectively. Therefore, this assay, allowing the highly specific detection of S. aureus in bovine milk samples at very low concentrations, might become an important diagnostic tool.

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18.2 Methods 18.2.1 Reagents and Equipment Sample Treatment Reagents Isotonic diluent Alcohol Chlorexidine

Equipment Needed Pad Towels Cotton swabs Aluminum foil Sterile 10 ml tubes Sterile surgical blade Polyethylene stomacher bag

DNA Extraction Protocol Guanidine thiocyanate Sterile 1.5 ml microcentrifuge tubes EDTA Centrifuge for Eppendorf Tris-HCl Timer Triton X-100 Vortex DTT, Dithiothreitol Heat Eppendorf block 65°C Silica (Sigma Aldrich S5631) Pipette tips 1000 µl Absolute ethanol Isopropanol NaCl

Pipette tips 200 µl Disposable gloves

DNA Amplification Accuprime Taq DNA polymerase Sterile 0.2 ml microcentrifuge tubes (Invitrogen 12339-016) Double distilled water Thermal cycler Pipette tips 1000 µl Pipette tips 200 µl DNA molecular size marker 100 bp Pipette tips 20 µl DNA molecular size marker 1 kb Disposable gloves Ethidium bromide Centrifuge for Eppendorf Loading dye Gel electrophoresis apparatus Power supply, Ultra violet source Agarose

18.2.2 Sample Collection and Preparation 18.2.2.1 Sample Collection General consideration. Depending upon the situation, the specimen may be collected from patients, controls, food handlers, animals, and food. The specimens should include the following: • Samples from patients and controls (e.g., serum, stool, vomitus, and urine). • Blood, spleen, intestinal content and liver tissue from fatal cases. • Stool/rectal swabs, throat swabs and exudates/pus from lesions, if any, of food handlers. • Sample of left over suspect food. • Swabs of equipment/utensils with which food was processed. • Samples from animals (e.g., milk). The general principles regarding collection of specimens are:

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• Collect specimens aseptically with a sterile implement and collect in sterile containers. • Collect the specimens of the suspect food at the earliest. • If the food article is solid cut it with a sterile knife and collect 100–200 g of the sample from the centre. • In case of liquids, first thoroughly shake the specimen to mix and then with the help of a sterile tube collect the specimen and shift into a sterile container. • In case of raw meat or poultry, aseptically cut portions of meat/skin and put 100–200 g in a sterile plastic jar and refrigerate the specimen. • To collect the specimen from utensils and/or equipment in which food has been processed, moisten the swab with sterile 0.1% peptone water or buffered distilled water and swab the contact surfaces of the utensils. Then put the swab in an enrichment broth. • Collect aseptically a sample of the water, used for cooking purposes in a quantity ranging from 1–5 l as feasible. • All the specimens should be properly labeled and packed. The following details should accompany the specimens: • Place and address of location where the outbreak occurred. • Symptoms: nausea/vomiting/diarrhoea/fever/uncons ciousness etc. • Date of onset of symptoms. • Date specimens collected. • Method of collection and transportation. • Epidemiological background and the suspect organism. • Condition of the food at the time of collection. The specimen of food collected for analysis should be transported in sterile containers (plastic bag, tubes, etc.) to the laboratory by the most rapid mode available. Perishable food should be kept at 2–8oC. Hot food should be cooled rapidly by putting the containers under cold running water and then held at 0–4oC. Samples should be packed in such a way that there is no spillage during transportation. The receiving laboratory should be pre-informed about the method of transport and anticipated time of receipt in the laboratory. Milk sampling. Monthly bulk milk tank culturing has proven useful in monitoring udder health, particularly with regard to S. aureus.94 The sensitivity of a single bulk tank culture is fairly low, especially when the herd prevalence of S. aureus mastitis is low as well. In other words, often one bulk tank sample will be culture negative for S. aureus even though a herd may have cows infected with this organism. However, bulk tank cultures are highly specific (94%). So, it is rare that a bulk tank culture will be positive when in reality no cows in the herd have staphylococcal mastitis.

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Multiple sampling will improve the sensitivity of bulk tank culturing, particularly with intermittently shedding organisms like S. aureus. Serial testing can be performed by aseptically collecting an agitated bulk tank sample in a sterile container. This procedure can be repeated every other day when the bulk tank contains four milkings. The samples can be frozen immediately after they are obtained and delivered to the testing facility once each month. Milk cultures are the best method to determine if intramammary infection is due to S. aureus. A small (3–5 ml), sterile quarter sample is preferable to a voluminous contaminated composite. Teat ends should be thoroughly scrubbed with an alcohol pad or chlorexidine towels. The fore-milk should be discarded and a mid-stream milk sample obtained in a sterile container. Milk cultures should be immediately chilled to prevent overgrowth of environmental bacteria. If microbiologic procedures are to be delayed, the samples should be frozen. Meat sampling. The wet-dry sampling method95 involves the use of jumbo-head cotton swabs. Samples are collected within an area of 25 cm2 (2.5 × 10 cm). The sampling area is delimited by an autoclaved aluminium foil frame (2.5 ×   10 cm). For each sampling area, a swab is moistened in an isotonic diluent (peptone bacteriological, 1.0 g/l, and sodium chloride, 8.5 g/l) and then rubbed firmly across the sampling area with five strokes. This procedure is then repeated twice, with an angle of approximately 60° each time to ensure an even recovery of bacteria. The procedure is immediately repeated on the same area with a dry swab. Each pair of wet and dry swabs is combined into a single sample in a sterile universal test tube containing 10 ml of isotonic diluent and placed in the refrigerator until microbiological analyses are carried out. Excision-based sampling involves removing a sliver of tissue (2.5 × 10 cm, 3 mm thick) from a meat/skin section. An autoclaved aluminum foil frame (2.5 × 10 cm) is placed over the section, and an initial cut to a depth of approximately 4 mm is made with a sterile surgical blade. The same blade is then used to cut free the tissue sliver from the meat/skin section. Each sample is stored in a single, sealed, polyethylene stomacher bag and placed in the refrigerator for later microbiological analyses. 18.2.2.2 Sample Pretreatment A working method for rapid DNA extraction directly from bovine, ovine, and caprine raw milk and from fresh, soft, semi-hard and hard cheeses samples has been described.54,55 This method is based on the ability of silica-resin to bind DNA in the presence of a high concentration of guanidine thiocyanate chaotropic agent which guarantees an excellent disruption of bacterial cells from the main foodborne pathogens such as Staph. aureus. Milk samples. Dilute 500 µl of milk sample with 500 µl of sterilized saline solution (NaCl 0.9%) and centrifuge for 15 min at 600 × g at 4°C; discard the supernatant containing fat and liquid phase. Repeat the step once. Add to the pellet 50 µl of sterilized saline solution. Resuspend the pellet with vortex (20 sec). The solution will be turbid.

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Raw cheese samples. Dilute 100 mg of cheese sample with 500 µl of lysis buffer (3 M guanidine thiocyanate, 20 mM EDTA, 10 mM Tris–HCl, pH 6.8, 40 mg/l Triton X-100, 10 mg/l DTT) and vortexed for 30 sec to obtain an emulsified solution. 18.2.2.3 DNA Extraction (1) Add 300 µl of lysis buffer and 200 µl of binding solution (40 mg/ml silica from Sigma Aldrich, Milan, Italy, directly suspended in the lysis buffer) to the pellet previously resuspended in 50 µl of saline solution for milk sample or to the emulsified solution for cheese sample. Mix and incubate for 5 min at room temperature. Centrifuge for 30 sec at 450 × g (or 30 sec at 500 × g for cheese sample) and discard the supernatant. (2) Add 200 µl of lysis buffer and resuspend by vortexing (20 sec). Centrifuge for 30 sec at 450 × g (or 30 sec at 500 × g for cheese sample) and discard the supernatant. Repeat this step once. Add 200 µl of washing solution (25% absolute ethanol, 25% isopropanol, 100 mM NaCl, 10 mM Tris-HCl, pH 8) and resuspend by vortexing (20 sec). Centrifuge for 30 sec at 450 × g (or 30 sec at 500 × g for cheese sample) and discard the supernatant. Repeat this step once. (3) Add 200 µl of absolute ethanol solution and resuspend by vortexing (20 sec). Centrifuge for 30 sec at 450 × g (or 30 sec at 500 × g for cheese sample) and discard the supernatant. (4) Vacuum-dry the pellet in an Eppendorf heat block at 56°C for 10 min (or at room temperature for at least 15–20 min). (5) Add 100 µl of elution buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), resuspend the pellet vortexing for 20 sec and incubate for 15 min at 65°C. (6) Centrifuge for 5 min at 450 × g (or 5 min at 500 × g for cheese sample) and transfer the supernatant, containing the DNA, into a clean tube. Note: to increase the DNA yield, a second elution step (with 5 min heating) may be performed. The solution contains pure DNA useful for molecular biology techniques. It can be used immediately or stored at –20°C for up to 6 months.

18.2.3 Detection Procedure In this section, a multiplex-PCR-based protocol is described81 with 11 primer sets to simultaneously identify the species together with two associated virulence marker genes (coagulase and thermostable nuclease genes) and eight of the known SE genes in S. aureus strains isolated from milk and dairy products. All primers used in the study, ranging from 20 to 24-mers, were designed using the Primer3 programme (http://wwwgenome.wi.mit.edu/cgibin/primer/primer3_www.cgi except

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Table 18.2 Primer Identities, Sequences, and Predicted Sizes of PCR Product for the Amplification of Staphylococcus aureus Target Genes Primer Identity

Primer Sequence (5′–3′)

23S-F1200 AGC TGT GGA TTG TCC TTT GG 23S-R1698 TCG CTC GCT CAC CTT AGA AT NUC-F166 AGT TCA GCA AAT GCA TCA CA NUC-R565 TAG CCA AGC CTT GAC GAA CT COA-F2591 CCG CTT CAA CTT CAG CCT AC COA-R2794 TTA GGT GCT ACA GGG GCA AT SEA-F1170 TAA GGA GGT GGT GCC TAT GG SEA-R1349 CAT CGA AAC CAG CCA AAG TT SEC-F97 ACC AGA CCC TAT GCC AGA TG SEC-R467 TCC CAT TAT CAA AGT GGT TTC C SED-F578 TCA ATT CAA AAG AAA TGG CTC A SED-R916 TTT TTC CGC GCT GTA TTT TT SEG-F322 CCA CCT GTT GAA GGA AGA GG SEG-R753 TGC AGA ACC ATC AAA CTC GT SEH-F260 TCA CAT CAT ATG CGA AAG CAG SEH-R722 TCG GAC AAT ATT TTT CTG ATC TTT SEI-F71 CTC AAG GTG ATA TTG GTG TAG G SEI-R637 CAG GCA GTC CAT CTC CTG TA SEJ-F349 GGT TTT CAA TGT TCT GGT GGT SEJ-R654 AAC CAA CGG TTC TTT TGA GG SEL-F158 CAC CAG AAT CAC ACC GCT TA SEL-R397 CTG TTT GAT GCT TGC CAT TG Source: Rosec, J.P. and Gigaud, O., Int. J. Food Microbiol., 77, 61, 70, 2002.

for the SEI forward primer taken from the literature.62 The primer sequences and the PCR product lengths are shown in Table 18.2. (1) In a 200 µl microtube, allocate 50 µl reaction mixture containing 2 µl DNA template, 2 U of AccuPrimeTM Taq DNA polymerase (Invitrogen,), 5 µl of 10 × AccuPrimeTM PCR buffer II containing 2 mM of each dNTPs (Invitrogen), 10 µM of the primer pair 23S-F1200 and 23S-R1698, 20 µM of the primer pairs COA-F2591 and COA-R2794, SEA-F1170 and SEA-R1349, SEC-F97 and SEC-R467, SED-F578 and SED-R916, SEJ-F349 and SEJ-R654, SEL-F158 and SEL-R397, 30 µM of the primer pairs NUCF166 and NUC-R565, SEG-F322 and SEG-R753, 40 µM of the primer pairs SEH-F260 and SEH-R722, SEI-F71 and SEI-R637; with double-distilled water to the final volume of 50 µl. (2) Perform PCR amplification in a thermal cycler GeneAmp PCR System 2700 (Applied Biosystems) with an initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, primer annealing at 56°C for 1 min and extension at 68°C for 1 min, followed by a final extension at 72°C for 7 min. (3) Separate the amplified PCR products by 4% agarose gel electrophoresis (GellyPhor, Euroclone), and

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Amplicon Size (bp)

Target Gene

499

23S rRNA 23S rRNA nuc nuc coa coa sea sea sec sec sed sed seg seg seh seh sei sei sej sej sel sel

400 204 180 371 339 432 463 529 306 240

include a DNA molecular size markers (100-bp and 1-kb DNA ladder; Finnzymes) in each agarose. (4) Stain the gel with ethidium bromide (0.05 µg/µl; Sigma Aldrich), and photograph the gel under ultra violet light using the BioProfile system (Mitsubishi).

18.3 Conclusions and Further Perspectives In conclusion, DNA-based methods to identify S. aureus in food have proved to be more sensitive and rapid than the conventional bacteriological methods. However, all these molecular methods should be standardized to become available for the routine analysis of food or clinical samples in laboratories. These systems are currently used only to confirm the diagnosis of specific bacterial strains. In addition, either a complex PCR with a mixture of large numbers of primers is needed, or a large series of individual PCRs must be run in parallel, or sequentially, to identify different bacterial strains (i.e., different enterotoxigenic S. aureus strains) or different pathogens contained in the same food or clinical sample. Recently, some of these problems were solved by using DNA microarrays. DNA microarrays are a promising diagnostic tool presenting many advantages compared to PCR or classical hybridization-based assays. They can detect tens of thousands of DNA sequences in a single hybridization step,

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allowing not only species determination but also a profiling of virulence factors genes. In the last decade, many DNA microarray have been developed for direct detection of the specific bacterial genes in clinical and food samples. Short oligonucleotide, PCR amplicons, whole genome or genome fragment microarrays have been successfully tested for detection of bacteria.93–112 Genetic variations in 16S rDNA could be utilized for the probe designing used in array identification system. With such a principle, Chiang and coworkers113 have developed a 16S rDNA-based oligonucleotide array system for the rapid diagnosis of the genus and species of bacteria in foods and clinical specimens. By using an array of specific oligonucleotides designed from the variable regions of 16S rDNA sequences, and the reverse hybridization of 16S rDNA amplified products to this array, the specific hybridization patterns for strains of Bacillus spp., E. coli, Salmonella spp., Staphylococcus spp. and Vibrio spp. were established. Another rapid and reliable one-tube microarray based assay was also developed for the simultaneous detection and identification (genetic typing) of almost all known enterotoxin genes of S. aureus.114 This method includes PCR amplification of part of the ent genes with universal primers, followed by analysis of amplicons by hybridization with ent-specific oligonucleotide probes immobilized on the microchip. More recently, Santini and coworkers115 have developed a prototype of medium density gene-segments DNA microarray for detection of the more prominent pathogens causing bloodstream infections, which includes the capture probes for S. aureus. All these DNA hybridisation assays are suitable for S. aureus diagnosis; however faster, cheaper, miniaturized, multianalyte, easier-to-use, and more sensitive approaches are highly desired, especially in the case of decentralized analysis. In this context, electrochemical detection of DNA hybridisation events offers innovative routes.116,117 Recently, the simultaneous detection of different food pathogenic bacteria by means of a disposable electrochemical low density genosensor array was described.118 The analytical method relied on the use of screen-printed arrays of gold electrodes, modified using thiol-tethered oligonucleotide probes. The samples identifying the bacteria of interest were obtained from the corresponding genomic DNAs through PCR amplification. These unmodified PCR products were captured at the electrode interface via sandwich hybridisation with surface-tethered probes and biotinylated signalling probes. The resulting biotinylated hybrids were coupled with a streptavidin–alkaline phosphatase conjugate and then exposed to a α-naphthyl phosphate solution. Differential pulse voltammetry was finally used to detect the α-naphthol signal. The results of these studies demonstrate the usefulness of the microarray assay for the analysis of food pathogens in general and for the analysis of multitoxigenic strains, such as S. aureus strains. Microarrays are not in common use in average laboratories today. However, like any new technology, as more applications are developed for the microarray

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technology, it will become more practical and may well become widely used.

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