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STAPHYLOCOCCUS | Detection by Cultural and Modern Techniques
Detection by Cultural and Modern Techniques J-A Hennekinne, National and European Union Reference Laboratory for Coagulase Positive Staphylococci Including Staphylococcus aureus, French Agency for Food, Environmental and Occupational Health and Safety, Maisons-Alfort, France Y Le Loir, INRA, UMR1253 STLO, Rennes, France; and Agrocampus Ouest, UMR1253 STLO, Rennes, France Ó 2014 Elsevier Ltd. All rights reserved. This article is a revision of the previous edition article by Sita R. Tatini, Reginald Bennett, volume 3, pp 2071–2076, Ó 1999, Elsevier Ltd.
Introduction Staphylococcus aureus is an opportunistic pathogen of humans and warm-blooded animals. Staphylococcus sp. was first described in 1882 by Sir Alexander Ogston who observed the presence of cocci in purulent lesions in humans. These cocci formed grape-like clusters, and Ogston named this organism Staphylococcus (Staphylo means grape in Greek). In 1884, Rosenbach studied this organism in pure culture and named the orange colony-forming coccus, Staphylococcus pyogenes aureus. Soon after, in 1884, Staphylococci entered the history of food poisoning with a large outbreak in Michigan caused by the consumption of a cheese-containing cocci. In 1914, Barber clearly demonstrated that refrigerated stored milk from a mastitic cow caused staphylococcal food poisoning in humans. In 1930, Dack isolated a S. aureus strain from a Christmas cake involved in a food poisoning causing typical symptoms of staphylococcal intoxication. Staphylococcal culture filtrates were injected intravenously to rabbits and ingested by human volunteers, leading to the onset of Staphylococcal food poisoning (SFP), 3 h post ingestion. Dack thus associated food-poisoning outbreak to the presence of a toxin produced by a S. aureus strain. This article, after some general considerations regarding taxonomy, types of strains, reservoir, contamination, and conditions leading to an SFP, will describe the main criteria and identification methods used for S. aureus identification in food samples. The phenotype-based methods will be presented first followed by the molecular-based methods.
Taxonomy To date, nearly 50 species and subspecies have been described in the Staphylococcus genus. Staphylococcus aureus is better known and is frequently involved in the etiology of various toxic infections in humans. Other staphylococcal species nevertheless can cause opportunistic infections. Such infections are often nosocomial, and sometimes life-threatening, and therefore require a rapid and adapted treatment. Among the numerous staphylococcal species and subspecies, only 18 species were found in humans, some of which are associated with infections. The other species are found in animals. Staphylococcal species are generally classified into two groups based on their ability to produce a cell-free coagulase: the coagulase-positive staphylococci (CPS), generally regarded as pathogens, and the coagulase-negative staphylococci (CNS), reportedly less dangerous. Some phenotypic criteria used to distinguish the most frequent CPS species are listed in Table 1. Although some articles reported the
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involvement of CNS in food-poisoning outbreaks, it appears that food isolates of this group do not often carry risk factors, such as enterotoxin genes. Great efforts were dedicated to the rapid and accurate identification of the S. aureus species. Many methods were developed based on the scheme proposed by Kloos and Schleifer in 1975. Several automated identification systems and sensitivity tests are now commercially available.
Conditions Leading to an SFP Outbreak Five conditions are required for the development of an SFP outbreak: a source of enterotoxin-producing staphylococci, a transmission to foodstuff, a foodstuff providing conditions favorable for growth, a permissive temperature during a time lap sufficient for bacterial multiplication and toxinogenesis, and ingestion of toxins in quantity sufficient to trigger the SFP symptoms.
Sources of Enterotoxin-Producing Staphylococci Reservoirs Foodstuff contamination can have a human, animal, or environmental origin. S. aureus is frequently associated to the skin and mucosa of humans and warm-blooded animals, which can be healthy and asymptomatic carriers and are the main reservoirs for S. aureus. S. aureus can also be isolated from the natural environment (soil, sea water and fresh water, dust, air), domestic environment (kitchen, fridge), and hospital environment (surface of furniture, sheets, blanket).
Enterotoxigenic Strains Frequency of staphylococcal enterotoxins A to E (SEA to SEE) by S. aureus strains is highly variable, depending on the publication, on the origin (food origin or another origin), on the tested strains, and on their geographic origin. It appears that the frequency varies as a function of host origin of the strains when they are biotyped (i.e., assigned to a specific host – human, bovine, avian, ovine – according to phenotypic criteria). After various studies, the percentage of strains producing SEA to SEE varies from 30 to 60% for strains of human origin, from 60 to 80% for strains of ovine or caprine origin, and from 0 to 15% for the strains of bovine and avian origin. A study conducted in France on various types of foodstuffs showed that 30.5% of 213 strains tested produced at least one of the five classical enterotoxins (SEA to SEE) with important
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Differentiation criteria among species and subspecies of coagulase-positive (or clumping factor) staphylococci
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Table 1
S. aureus
S. aureus subsp. anaerobius
S. delphini
S. sciuri subsp. S. hyicus S. intermedius S. lugdunensis S. lutrae carnaticus
S. sciuri subsp. rodentium
S. schleiferi subsp. S. schleiferi coagulans
Colony size 6 mm Colony pigmentation Anaerobe growth Aerobe growth Staphylocoagulase Clumping factor Thermonuclease Hemolysis Catalase Modified oxydase Alkaline phosphatase Pyrrolidonyl arylamidase Ornithine decarboxylase Urease b-Glucosidase b-Glucuronidase b-Galactosidase Arginine dihydrolase Acetoin production Nitrate reduction Esculine hydrolysis Resistance to novobiocin
þ þ þ þ þ þ þ þ þ þ d þ þ þ þ
(þ) () þ þ þ þ ND ND ND ND
þ (þ) þ þ þ þ þ ND ND þ ND ND ND þ þ ND
þ þ þ d þ þ þ d d þ þ þ
þ (þ) þ þ d þ d þ þ þ þ d þ d þ
d d þ þ (þ) (þ) þ þ þ d þ þ þ
þ þ þ () þ þ þ ND ND þ ND ND þ þ ND
d (d) þ d () þ þ d þ þ þ þ
d d (d) þ þ () þ þ d þ þ þ þ
þ þ þ þ (þ) þ þ þ (þ) þ þ þ
d þ þ þ þ (þ) þ þ ND ND þ ND ND ND þ þ þ ND
þ d þ ND þ ND ND þ ND ND ND
þ þ (d) ND þ (d) d (d) þ
(þ) þ (þ) ND (d) d (d) (d) þ
d þ (þ)
d þ ND d ND
Acid production (in aerobiosis) from D-Trehalose D-Mannitol D-Mannose D-Turanose D-Xylose D-Cellobiose L-Arabinose
Maltose Saccharose N-Acetylglucosamine Raffinose
þ þ þ þ þ þ þ
ND ND þ þ
(þ) þ ND ND þ þ ND ND
þ þ þ þ
þ (d) þ d () þ þ
þ þ (d) þ þ þ
Symbols: þ, 90% of the strains or more are positive; , 90% of the strains or more are negative; d, 11–89% of the strains are positive; ND, not determined; (), a delayed reaction. Adapted from Kloos, W.E., Bannerman, T.L. 1994. Update on clinical significance of coagulase-negative staphylococci. Clinical Microbiology Review 7, 117–140; Kloos, W.E., Schleifer, K.H. 1986. Genus IV - Staphylococcus Rosenbach 1884. In: Sneath, P.H.A., Mair, N.S., Sharpe, M.E. (Eds.), Bergey's Manual of Systemic Bacteriology, Vol 2. Williams and Wilkins, Baltimore; Schleifer, K.H., 1986. Taxonomy of coagulase-negative staphylococci. In: Mardh, P.-A., Schleifer, K. H. (Ed.), Coagulase-negative Staphylococci. Almquist and Wiksell International. Stockholm, Sweden, pp. 11–26.
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Features
STAPHYLOCOCCUS j Detection by Cultural and Modern Techniques variations depending on the foodstuffs: 12.5% of SE producers for the strains isolated from raw cow milk cheeses, 31.5% for prepared meals, 62.5% goat and sheep raw milk cheeses, and 64% for pastries. In this study, about 60% of food isolates of human origin were enterotoxigenic. Percentages of enterotoxigenic strains indeed vary according to the dominant biotype associated to food category: human biotype in handmade foodstuffs (prepared meals, pastries), and bovine or ovine biotype in cheeses made with cow or ovine raw milk. From 70 to 95% of the S. aureus strains isolated from foodstuffs involved in SFP outbreaks are enterotoxin producers in laboratory conditions (liquid culture in rich medium). Among the strains isolated from SFP outbreaks in France, strains producing SEA, alone or together with SED, are the most prevalent strains. SEB and SEC are rarer and SEE is almost never found. Predominance of SEA is observed in many other countries. Some strains do not produce any detectable SE when tested in laboratory conditions although they were implicated in SFP outbreaks. Such results might be due to the fact that these strains produce SEs that are not detected by the antibodies used for the detection (see Staphylococcus: Detection of Staphylococcal Enterotoxins). Some newly described SEs indeed are not detectable by the commercially available detection kits. Presence of seg to sej genes was sought by polymerase chain reaction (PCR) amplification in S. aureus strains of various origins. These genes are frequently detected, especially seg and sei, which belong to an enterotoxin gene cluster (egc). In some studies, when these genes are sought, the proportion of strains that are potentially SE producers increases significantly. In France, Rosec and Gigaud (2002) showed that 57% of 258 strains isolated from various food were seg, seh, and sei positive and 31% were carrier of these genes only. In Italy, Zecconi et al. (2006) showed that 100% of 50 strains isolated from raw milk of cows suffering from mastitis were seg and sei positive. In Ireland, Smyth et al. (2006) showed that 64% of 157 strains isolated from household fridge were seg and sei positive, whereas only 7% were sea and see positive. PCR techniques detect se genes in the strains but do not allow determining if the SE is actually produced. Only detection of the toxin itself in a suspected foodstuff can demonstrate the involvement of the SE in an SFP.
Contamination Mode Presence of S. aureus in food has two main origins: l
In raw materials from animal origin (meat, milk), contamination can result from a primary contamination. For example, raw milk contamination can be due to the presence of cow suffering from S. aureus mastitis in the herd. Mammalian or avian carcasses can be contaminated during slaughtering and can have different sources: S. aureus carriage on the furs or the feathers, on the udder skin, nares, genital mucosa, and digestive tract; staphylococcal infections (abscesses, superficial infections). l For any other foodstuffs, contamination can have a human origin during food-manufacturing process or in house food preparation. In this case, S. aureus strains can come from
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healthy carriage on skin and mucosa or from staphylococcal infections (infected wounds, sinusitis, pharyngitis, rhinopharyngitis). Contamination can occur either directly during food manipulation, or through respiratory aerosols, which are more abundant during infections of upper airways. Strains of human biotype are highly prevalent in the strains isolated from SFP outbreaks. Strains of ovine biotype or nonhost specific strains can be associated to SFP in France. Environmental contamination (processing plant surfaces) or cross-contamination between foodstuffs is poorly documented as of yet and their incidence remains to be evaluated.
Detection Methods for S. aureus Identification by Conventional Microbiology Selective and Nonselective Media Numerous selective or nonselective media were developed for the isolation of staphylococci. Among the nonselective media, the most commonly used are solid agar media, such as blood agar plates, trypticase-soja agar (TSA), Brain-Heart Infusion agar (BHI agar), Plate count agar plus milk (PCA milk), and their equivalent in liquid media (BHI, TS Broth). Selective media are aimed at inhibiting the growth of bacteria other than S. aureus and at favoring the growth of S. aureus. Selective compounds are high salt concentration (NaCl), which results in low aw like in Chapmann agar, or lithium chloride concentration, glycine and potassium tellurite, and compounds that are included in Baird–Parker (BP) medium and its derivatives (Table 2). If necessary, sulfamethazine can be added to inhibit Proteus spp. growth. Selectivity also can be optimized by adding acriflavine and polymyxine E (colistine) or by applying a temperature above 42 C. One might keep in mind, however, that the more selective the medium is, the more inhibitory it is for stressed S. aureus isolates. Some compounds are useful to differentiate staphylococci colonies (e.g., egg yolk and tellurite in BP, Table 1). BP medium is the medium of choice in food bacteriology because it allows the best recovery of stressed S. aureus cells thanks to sodium pyruvate that activates growth by degrading H2O2 and to the protective effect of egg yolk. However, BP-medium is insufficiently selective when samples are rich in other microbial flora. That is why several variants of BP are proposed (Table 2). Some are judged too selective to allow recovering stressed S. aureus cells and a 1-h preliminary step on BP without selective agents was proposed before the addition of the highly selective medium. The BP medium in which Rabbit Plasma Fibrinogen (RPF-BP) replaces egg yolk is a BP variant used in normalized methods. RPF-BP allows an in situ identification of CPS colonies. Although BP and RPF-BP are the most commonly used media, other media also are used, among which chromogenic media represent a highly convenient way for a rapid identification of S. aureus in various type of samples, including complex food samples (Table 2)
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Table 2
Some selective media used for Staphylococcus aureus detection
Media
Selective agents
Diagnostic criterion
Mannitol-salt Mannitol-salt-acriflavine
Natrium chloride Natrium chloride Acriflavine Potassium tellurite Glycin Lithium chloride Potassium tellurite Glycin Lithium chloride Potassium tellurite Glycin Lithium chloride Natrium chloride Potassium tellurite Incubation at 42 C Potassium thiocyanate Lithium chloride Sodium azide Cycloheximide (antifungal compound) Nalidixic acid Colistin sulfate Potassium tellurite Glycin Lithium chloride
Mannitol fermentation Mannitol fermentation
Baird–Parker
Baird–Parker – Rabbit plasma fibrinogen
Vogel and Johnson modified
4S
KRANEP
Columbia CNA Chromogenic media: l CHROMagar Staph. aureus (Becton Dickinson, Franklin Lakes, United States) l ChromID S. aureus® (Biomérieux, Capronne, France) l Brilliance Staph 24 l 3M™ Petrifilm™ Staph express l
RAPID’Staph Agar
Staphylococcus aureus Detection by Molecular Methods Molecular Characterization of S. aureus Strains The phenotype-based methods, such as phage-typing (lysotype) and capsular serotyping, are abandoned because of their low discriminative power compared with the molecular methods. Among these molecular methods, some methods, like ribotyping, are used to classify the strains at the infraspecies level or to type the strains for epidemiological purposes using pulse-field gel electrophoresis. In 2003, Hennekinne et al. (2003) showed that PFGE was efficient to type S. aureus strains isolated from various foodstuffs.
Characterization of Toxinogenic Strains In the early 1990s, PCR was applied to search specific S. aureus genes in DNA samples obtained directly from foodstuffs and not only in DNA extracted from pure culture. The targeted genes were preferentially those encoding enterotoxins. These latter genes, however, were not always detected in S. aureus food isolates and other S. aureus genes, more widely distributed, such as the nuc gene, encoding thermonuclease, were then chosen (Table 3). These techniques proved a high sensitivity. In 2005, Ikeda et al. (2005) detected by PCR enterotoxin genes sea, seh, seg, and sei in samples of skimmed milk powder involved in an SFP outbreak, although no S. aureus had been found by classical microbiology methods. These results were confirmed by the detection of the SEA and SEH in the milk
Tellurite reduction (black colonies) Clear halo (egg yolk) Coagulase
Tellurite reduction Mannitol fermentation DNAse Tellurite reduction Clear halo (egg yolk) Mannitol fermentation Clear halo (egg yolk) Pigment Pigment Hemolysis Specific colors of the cfu: l Light purple (patented) l
Green (a-glucosidase) Dark blue (patented) l cfu surrounded by a pink halo, nuclease activity l Black l
powder. SEG and SEI were not detectable at that time (no specific detection techniques were available) but possibly were present in the samples. Since 2001, the use of quantitative realtime PCR methods allows evaluating the number of DNA copies present in the samples. This number is correlated linearly with the number of bacteria cells and thus it gives an accurate estimation of the level of S. aureus contamination in the foodstuff. This technique is particularly interesting because food products can be contaminated by S. aureus at some steps of the process and might be inactivated by the treatments applied at some other subsequent steps (providing that DNA has not been degraded: foodstuff treatments can indeed alter bacterial DNA). Goto et al. (2007) showed that the amount of staphylococcal DNA detected in a milk contaminated and then pasteurized at 63 C, 30 min is lower than that detected in the same milk after pasteurization at 72 C, 15 s. These authors hypothesized that part of the DNA is lost due to cell lysis and subsequent DNA hydrolysis during pasteurization. With artificially contaminated milk samples, detection limit is around 600 cfu ml1 in pasteurized milk samples, whereas it is 10 cfu ml1 in raw milk. Detection limits of PCR-based techniques vary from a study to another. Food matrix complexity, fats content, and the presence of potential PCR inhibitors must be taken into account in the choice of proper DNA extraction procedures to obtain a method sufficiently sensitive and reproducible. PCR techniques are not widely used in some foodstuffs because of the lack of standardization criteria.
STAPHYLOCOCCUS j Detection by Cultural and Modern Techniques Table 3
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Some DNA-based techniques for Staphylococcus aureus identification
Gene or sequence
Protein, product, or function
Detection method
16S rRNA 23S rRNA aroA clfA coa femA femB gap hsp60 nuc pnp rpoB Intergenic space rRNA Fragment Sa2052
Ribosomal RNA 16S Ribosomal RNA 23S 5-Enolpyruvylshikimate-3-phosphate synthase Clumping factor A (adhesion factor) Coagulase Cytoplasmic protein FemA Cytoplasmic protein FemB Glyceraldehyde 3-phosphate dehydrogenase Heat shock protein HSP60 Thermonuclease Polynucleotide phosphorylase Subunit b of RNA polymerase Intergenic sequences rRNA 16S-rRNA 23S 2 kb-long EcoRI subfragment resulting from a 44 kb macrorestriction fragment 442 base pairs Sau3A fragment resulting from S. aureus genomic DNA Superoxide dismutase Staphylococcal protein A (two regions detected: region X and region binding to immunoglobulins G) Intergenic sequences tRNA Elongation factor Tu
PCR hybridization PCR hybridization PCR, hybridization PCR PCR PCR multiplex PCR PCR multiplex PCR PCR, PCR-RFLP hybridization hybridization sequencing PCR Hybridization Sequence RS-PCR PCR, hybridization
Fragment Sa442 sodA spa Intergenic space tRNA tuf
Alarçon et al. (2006) described an optimized PCR protocol, for automated quantification in routine. This method is based on a 24-h enrichment step followed by a DNA extraction using DNeasy tissue kit (Qiagen) and a conventional PCR or a qRTPCR using SYBR-Green, which is 10-fold more sensitive than conventional PCR. Goto et al. (2007) advocated washing the cell pellet extracted from milk five times before DNA extraction to obtain reproducible results. Although the number of colony-forming units (cfu) per gram often correlates well with the gene copy number, Hein et al. (2005) observed discrepancies between these figures with gene copy numbers up to 100- or even 1000-fold higher than the estimated cfu per gram in frozen raw milk samples. It is thus difficult to find a clear and systematic correlation between the number of cfu and the gene copy number because some food processes either degrade DNA, kill bacteria, or turn them into a nonculturable state. Although they are highly specific and sensitive, PCR results only allow detection of S. aureus specific genes and genes encoding enterotoxins, but they do not give information about the expression of these genes. In other words, the presence of an SE gene does not mean the presence of the corresponding SE in the foodstuff. In 2011, Cretenet et al. (2011) demonstrated, in a model cheese, that the expression of SE genes can indeed be inhibited in some conditions, that is, the presence of lactic acid bacteria. Recent efforts thus were dedicated to the development of methodologies based on reverse-transcriptase PCR (RT PCR), which enables an estimation of the level of SE gene expression.
Normalized Methods and Alternative Methods Because of sanitary rules and mandatory declaration of foodpoisoning outbreaks, it appears absolutely necessary to use
PCR, hybridization Hybridization PCR PCR PCR, hybridization PCR-RFLP
robust and reliable methods for the detection of CPS and of SE. Such detections rely on normalized methods or on alternative methods. Normalization aims at providing standards, or reference documents, that bring consensual solutions to technical problems in a client–provider perspective. In its international definition, a standard is “a document, established by consensus and approved by a recognized body that provides, for common and repeated use, rules, guidelines or characteristics for activities or their results, aimed at the achievement of the optimum degree of order in a given context.” Usually, standards are heavy to implement for operators, and standard methods may be supplemented by so-called alternative methods with the same response characteristics (this, however, requires a validation against the reference method described in the standard), but implementation will be easier, faster, and cheaper.
Normalized Standards S. aureus is usually isolated from foodstuffs using normalized methods. Reference methods are described in international standards, which are adopted at the European level (NF EN ISO 6888-1, -2, et -3). These are classical techniques of cfu numeration on selective media after direct inoculation of decimal dilutions of food extracts and incubation at 37 C. The standard ISO 6888-3 describes a method of detection and numeration by the technique of the most probable number (MPN) after an enrichment step. The selective medium used in the standard is a modified Giolitti and Cantoni broth whose formula is similar to that of Baird–Parker (BP) broth (see the Alternative Methods section). After enrichment, S. aureus is detected by streaking and incubation on agar selective medium. Such standards are based on two types of agar selective media: BP and RPF-BP.
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Methods using BP medium require a confirmation test of several colonies with characteristic and noncharacteristic aspects, with a coagulase test in vitro. Methods using RPF-BP do not require confirmation test. Whatever the medium used, coagulase-positive colonies are counted and the result is expressed in cfu of CPS (most likely S. aureus) by gram or by milliliter of foodstuff. Fidelity of the methods described in the standards ISO 6888-1 (BP and BP-RPF) in terms of repeatability and reproducibility was evaluated in an interlaboratory test realized with three types of foodstuffs and four levels of S. aureus contamination. The fidelity of the two methods was estimated sufficient. Both methods received an equal status for the numeration of CPS in foodstuffs. RPF-BP is recommended for the detection and numeration of CPS in raw milk cheeses and any other foodstuff with complex microbial flora, which might interfere with the interpretation of BP plates. Regarding PCR detection, no specific standards exist as of yet for S. aureus but a series of standards gathered in the generic name “Food Microbiology – Polymerase Chain Reaction (PCR) for the detection of pathogenic microorganisms in foodstuffs” provide the general principles, especially: l
The standard ISO 22174: General requirements and definitions. l The standard NF EN ISO 20837 for the preparation of samples for qualitative detection by PCR. This standard describes a protocol for DNA extraction from selective broth of enrichment, approved by interlaboratory test for two Gram-negative bacteria. l The standard NF EN ISO 20838 for the amplification and qualitative detection. This standard also describes some tests for the confirmation of the identity of the PCR product obtained and provides remarks on test optimization.
Alternative Methods S. aureus can also be counted in foodstuffs by alternative methods that are commercially available. Such methods are more user friendly and or more rapid than normalized standards. To be used in place of a standardized method for official controls, they must be tested according to a recognized protocol validation, including trials with a reference method and a collaborative study. PetrifilmTM Staph Express (3M) and Rapid Staph Test (BioRad) were validated, in France, by AFNOR in 2003 and in 2005, respectively. The PetrifilmTM Staph Express system includes Petrifilm test containing a selective and differential chromogenic medium for S. aureus plus a confirmation disc allowing observing the DNase activity of colonies. After inoculation and incubation of the test during 24 h at 37 C, S. aureus forms red-purple colonies. If cfu with another color are visualized, the confirmation disk is applied on the test during 3 h at 37 C. Pink halos around the cfu correspond to S. aureus. RapidStaph Test is a Baird–Parker medium optimized for a reading after 24 h incubation (instead of 48 h in the standard ISO 6888-1). Two confirmation tests can be used on characteristic colonies: a rapid latex beads agglutination test (PastorexR Staph-plus, Bio-Rad) or a streak on RPF-BP allowing visualization of CPS cfu after an 18 h incubation at 37 C.
Conclusion Compared with phenotypic methods, molecular methods are based on DNA and are independent of the expression of specific genes in artificial culture conditions (laboratory environment). These traits are relatively stable in nature, compared with phenotypic (biotypes, serotypes, antibiograms). Molecular methods give results independent of potential changes in experimental conditions. Molecular methods do not require in vitro culture and thus allow identifying species that are nonculturable or difficult to grow. Compared with phenotypic methods, molecular methods targeting chromosomal genes allow the identification of all strains of a given species because all bacteria contain DNA. Phenotypic methods remain routinely used because they are pretty fast and do not require expensive equipment and reagents or expertise. With the development of chromogenic media that are increasingly efficient and discriminating, such phenotypic methods have a future in the diagnosis of microbiological hazards in foodstuffs.
See also: Bacillus: Bacillus cereus; Bacillus: Detection of Toxins; Bacillus – Detection by Classical Cultural Techniques; Biochemical and Modern Identification Techniques: Microfloras of Fermented Foods; Ecology of Bacteria and Fungi: Influence of Available Water; Ecology of Bacteria and Fungi in Foods: Influence of Temperature; Ecology of Bacteria and Fungi in Foods: Influence of Redox Potential; Ecology of Bacteria and Fungi in Foods: Effects of pH; Hazard Appraisal (HACCP): The Overall Concept; Hazard Analysis and Critical Control Point (HACCP): Critical Control Points; Hazard Appraisal (HACCP): Involvement of Regulatory Bodies; Hazard Appraisal (HACCP): Establishment of Performance Criteria; Nucleic Acid–Based Assays: Overview; PCR Applications in Food Microbiology; Petrifilm – A Simplified Cultural Technique; Predictive Microbiology and Food Safety; Process Hygiene: Overall Approach to Hygienic Processing; Designing for Hygienic Operation; Process Hygiene: Types of Sterilant; Process Hygiene: Overall Approach to Hygienic Processing; Process Hygiene: Modern Systems of Plant Cleaning; Process Hygiene: Risk and Control of Airborne Contamination; Process Hygiene: Disinfectant Testing; Process Hygiene: Involvement of Regulatory and Advisory Bodies; Process Hygiene: Hygiene in the Catering Industry; Staphylococcus: Staphylococcus aureus; Staphylococcus: Detection of Staphylococcal Enterotoxins; Identification Methods: Introduction; DNA Fingerprinting: Pulsed-Field Gel Electrophoresis for Subtyping of Foodborne Pathogens; Identification Methods: DNA Fingerprinting: Restriction Fragment-Length Polymorphism; Bacteria RiboPrint™: A Realistic Strategy to Address Microbiological Issues outside of the Research Laboratory; Multilocus Sequence Typing of Food Microorganisms; Application of Single Nucleotide Polymorphisms–Based Typing for DNA Fingerprinting of Foodborne Bacteria; Identification Methods and DNA Fingerprinting: Whole Genome Sequencing; Identification Methods: Multilocus Enzyme Electrophoresis; Identification Methods: Chromogenic Agars; Identification Methods: Immunoassay; Identification Methods: DNA Hybridization and DNA Microarrays for Detection and
STAPHYLOCOCCUS j Detection by Cultural and Modern Techniques
Identification of Foodborne Bacterial Pathogens; Identification of Clinical Microorganisms with MALDI-TOF-MS in a Microbiology Laboratory.
Further Reading Akineden, O., Hassan, A.A., Schneider, E., Usleber, E., 2008. Enterotoxigenic properties of Staphylococcus aureus isolated from goats’ milk cheese. International Journal of Food Microbiology 124 (2), 211–216. Alarçon, B., Vicedo, B., Aznar, R., 2006. PCR-based procedures for detection and quantification of Staphylococcus aureus and their application in food. Journal of Applied Microbiology 100 (2), 352–354. Beckers, H.J., Van Leusden, F.M., Bindschedler, O., Guerraz, D., 1984. Evaluation of a pour plate system with rabbit plasma-bovine plasma-agar for the enumeration of Staphylococcus aureus in food. Canadian Journal of Microbiology 30, 470–474. Bergdoll, M.S., 1991. Symposium on microbiology update: old friends and new enemies. Staphylococcus aureus. International Journal AOAC 74, 706–710. Cretenet, M., Nouaille, S., Riviere, J., et al., 2011. Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix. Environmental Microbiology Reports 3 (3), 340–351. De Buyser, M.L., Sutra, L., 2005. Staphylococcus aureus. In: Federighi, M. (Ed.), Bactériologie alimentaire – Compendium d’hygiène des aliments. Economica, Paris, pp. 25–51. De Buyser, M.L., Audinet, N., Delbart, M.O., et al., 1998. Comparison of selective culture media to enumerate coagulase-positive staphylococci in cheeses made from raw milk. Food Microbiology 15, 339–346. De Buyser, M.L., Lombard, B., Schulten, S.M., et al., 2003. Validation of EN ISO standard methods 6888 Part 1 and Part 2: 1999 – Enumeration of coagulasepositive staphylococci in foods. International Journal of Food Microbiology 83, 185–194. Even, S., Leroy, S., Charlier, C., et al., 2010. Low occurrence of risk factors in coagulase negative staphylococci isolated from fermented foodstuffs. International Journal of Food Microbiology 139 (1–2), 87–95. Genigeorgis, C.A., 1989. Present state of knowledge on staphylococcal intoxication. International Journal of Food Microbiology 9, 327–360. Goto, M., Takahashi, H., Segawa, Y., et al., 2007. Real-time PCR method for quantification of Staphylococcus aureus in milk. Journal of Food Protection 70 (1), 90–96.
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Hein, I., Jorgensen, H.J., Loncarevic, S., Wagner, M., 2005. Quantification of Staphylococcus aureus in unpasteurised bovine and caprine milk by real-time PCR. Research in Microbiology 156 (4), 554–563. Hennekinne, J.A., Kerouanton, A., Brisabois, A., De Buyser, M.L., 2003. Discrimination of Staphylococcus aureus biotypes by pulsed-field gel electrophoresis of DNA macro-restriction fragments. Journal of Applied Microbiology 86 (2), 321–329. Ikeda, T., Tamate, N., Yamaguchi, K., Makino, S., 2005. Mass outbreak of food poisoning disease caused by small amounts of staphylococcal enterotoxins A and H. Applied and Environmental Microbiology 71, 2793–2795. Isigidi, B.K., Devriese, L.A., Croegart, T.H., van Hoof, J., 1989. A highly selective twostage isolation method for the enumeration of Staphylococcus aureus in foods. Journal of Applied Bacteriology 66, 379–384. Kerouanton, A., Hennekinne, J.A., Letertre, C., et al., 2007. Characterization of Staphylococcus aureus strains associated with food poisoning outbreaks in France. International Journal of Food Microbiology 115, 369–375. Kloos, W.E., Schleifer, K.H., 1975. Simplified scheme for routine identification of human Staphylococcus species. Journal of Clinical Microbiology 1, 82–88. Le Loir, Y., Gautier, M., 2003. Staphylococcus aureus. In: Le Loir, Y., Gautier, M. (Eds.), Monographie de Microbiologie. Editions Lavoisier Tec&Doc, Paris. Le Loir, Y., Baron, F., Gautier, M., 2003. Staphylococcus aureus and food poisoning. Genetic and Molecular Research 2 (1), 63–76. Lee, Y.D., Moon, B.Y., Park, J.H., Chang, H.I., Kim, W.J., 2007. Expression of enterotoxin genes in Staphylococcus aureus isolates based on mRNA analysis. Journal of Microbiological Biotechnology 17 (3), 461–467. McLauchlin, J., Narayanan, G.L., Mithani, V., O’Neill, G., 2000. The detection of enterotoxins and toxic shock syndrome toxin genes in Staphylococcus aureus by polymerase chain reaction. Journal of Food Protection 63, 479–488. Rosec, J.P., Gigaud, O., 2002. Staphylococcal enterotoxin genes of classical and new types detected by PCR in France. International Journal of Food Microbiology 77, 61–70. Rosec, J.P., Guiraud, J.P., Dalet, C., Richard, N., 1997. Enterotoxin production by staphylococci isolated from foods in France. International Journal of Food Microbiology 35, 213–221. Shimizu, A., Fujita, M., Igarashi, H., et al., 2000. Characterization of Staphylococcus aureus coagulase type VII isolates from Staphylococcal food poisoning outbreaks (1980–1995) in Tokyo, Japan, by pulsed-field gel electrophoresis. Journal of Clinical Microbiology 38, 3746–3749. Smyth, D.S., Kennedy, J., Twohig, J., et al., 2006. Staphylococcus aureus isolates from Irish domestic refrigerators possess novel enterotoxin and enterotoxin-like genes and are clonal in nature. Journal of Food Protection 69 (3), 508–515. Zecconi, A., Cesaris, L., Liandris, E., et al., 2006. Role of several Staphylococcus aureus virulence factors on the inflammatory response in bovine mammary gland. Microbial Pathogens 40, 177–183.
Introduction Staphylococcus aureus is an opportunistic pathogen of humans and warm-blooded animals. Staphylococcus sp. was first described in 1882 by Sir Alexander Ogston who observed the presence of cocci in purulent lesions in humans. These cocci formed grape-like clusters, and Ogston named this organism Staphylococcus (Staphylo means grape in Greek). In 1884, Rosenbach studied this organism in pure culture and named the orange colony-forming coccus, Staphylococcus pyogenes aureus. Soon after, in 1884, Staphylococci entered the history of food poisoning with a large outbreak in Michigan caused by the consumption of a cheese-containing cocci. In 1914, Barber clearly demonstrated that refrigerated stored milk from a mastitic cow caused staphylococcal food poisoning in humans. In 1930, Dack isolated a S. aureus strain from a Christmas cake involved in a food poisoning causing typical symptoms of staphylococcal intoxication. Staphylococcal culture filtrates were injected intravenously to rabbits and ingested by human volunteers, leading to the onset of Staphylococcal food poisoning (SFP), 3 h post ingestion. Dack thus associated food-poisoning outbreak to the presence of a toxin produced by a S. aureus strain. This article, after some general considerations regarding taxonomy, types of strains, reservoir, contamination, and conditions leading to an SFP, will describe the main criteria and identification methods used for S. aureus identification in food samples. The phenotype-based methods will be presented first followed by the molecular-based methods.
Taxonomy To date, nearly 50 species and subspecies have been described in the Staphylococcus genus. Staphylococcus aureus is better known and is frequently involved in the etiology of various toxic infections in humans. Other staphylococcal species nevertheless can cause opportunistic infections. Such infections are often nosocomial, and sometimes life-threatening, and therefore require a rapid and adapted treatment. Among the numerous staphylococcal species and subspecies, only 18 species were found in humans, some of which are associated with infections. The other species are found in animals. Staphylococcal species are generally classified into two groups based on their ability to produce a cell-free coagulase: the coagulase-positive staphylococci (CPS), generally regarded as pathogens, and the coagulase-negative staphylococci (CNS), reportedly less dangerous. Some phenotypic criteria used to distinguish the most frequent CPS species are listed in Table 1. Although some articles reported the
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involvement of CNS in food-poisoning outbreaks, it appears that food isolates of this group do not often carry risk factors, such as enterotoxin genes. Great efforts were dedicated to the rapid and accurate identification of the S. aureus species. Many methods were developed based on the scheme proposed by Kloos and Schleifer in 1975. Several automated identification systems and sensitivity tests are now commercially available.
Conditions Leading to an SFP Outbreak Five conditions are required for the development of an SFP outbreak: a source of enterotoxin-producing staphylococci, a transmission to foodstuff, a foodstuff providing conditions favorable for growth, a permissive temperature during a time lap sufficient for bacterial multiplication and toxinogenesis, and ingestion of toxins in quantity sufficient to trigger the SFP symptoms.
Sources of Enterotoxin-Producing Staphylococci Reservoirs Foodstuff contamination can have a human, animal, or environmental origin. S. aureus is frequently associated to the skin and mucosa of humans and warm-blooded animals, which can be healthy and asymptomatic carriers and are the main reservoirs for S. aureus. S. aureus can also be isolated from the natural environment (soil, sea water and fresh water, dust, air), domestic environment (kitchen, fridge), and hospital environment (surface of furniture, sheets, blanket).
Enterotoxigenic Strains Frequency of staphylococcal enterotoxins A to E (SEA to SEE) by S. aureus strains is highly variable, depending on the publication, on the origin (food origin or another origin), on the tested strains, and on their geographic origin. It appears that the frequency varies as a function of host origin of the strains when they are biotyped (i.e., assigned to a specific host – human, bovine, avian, ovine – according to phenotypic criteria). After various studies, the percentage of strains producing SEA to SEE varies from 30 to 60% for strains of human origin, from 60 to 80% for strains of ovine or caprine origin, and from 0 to 15% for the strains of bovine and avian origin. A study conducted in France on various types of foodstuffs showed that 30.5% of 213 strains tested produced at least one of the five classical enterotoxins (SEA to SEE) with important
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Differentiation criteria among species and subspecies of coagulase-positive (or clumping factor) staphylococci
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Table 1
S. aureus
S. aureus subsp. anaerobius
S. delphini
S. sciuri subsp. S. hyicus S. intermedius S. lugdunensis S. lutrae carnaticus
S. sciuri subsp. rodentium
S. schleiferi subsp. S. schleiferi coagulans
Colony size 6 mm Colony pigmentation Anaerobe growth Aerobe growth Staphylocoagulase Clumping factor Thermonuclease Hemolysis Catalase Modified oxydase Alkaline phosphatase Pyrrolidonyl arylamidase Ornithine decarboxylase Urease b-Glucosidase b-Glucuronidase b-Galactosidase Arginine dihydrolase Acetoin production Nitrate reduction Esculine hydrolysis Resistance to novobiocin
þ þ þ þ þ þ þ þ þ þ d þ þ þ þ
(þ) () þ þ þ þ ND ND ND ND
þ (þ) þ þ þ þ þ ND ND þ ND ND ND þ þ ND
þ þ þ d þ þ þ d d þ þ þ
þ (þ) þ þ d þ d þ þ þ þ d þ d þ
d d þ þ (þ) (þ) þ þ þ d þ þ þ
þ þ þ () þ þ þ ND ND þ ND ND þ þ ND
d (d) þ d () þ þ d þ þ þ þ
d d (d) þ þ () þ þ d þ þ þ þ
þ þ þ þ (þ) þ þ þ (þ) þ þ þ
d þ þ þ þ (þ) þ þ ND ND þ ND ND ND þ þ þ ND
þ d þ ND þ ND ND þ ND ND ND
þ þ (d) ND þ (d) d (d) þ
(þ) þ (þ) ND (d) d (d) (d) þ
d þ (þ)
d þ ND d ND
Acid production (in aerobiosis) from D-Trehalose D-Mannitol D-Mannose D-Turanose D-Xylose D-Cellobiose L-Arabinose
Maltose Saccharose N-Acetylglucosamine Raffinose
þ þ þ þ þ þ þ
ND ND þ þ
(þ) þ ND ND þ þ ND ND
þ þ þ þ
þ (d) þ d () þ þ
þ þ (d) þ þ þ
Symbols: þ, 90% of the strains or more are positive; , 90% of the strains or more are negative; d, 11–89% of the strains are positive; ND, not determined; (), a delayed reaction. Adapted from Kloos, W.E., Bannerman, T.L. 1994. Update on clinical significance of coagulase-negative staphylococci. Clinical Microbiology Review 7, 117–140; Kloos, W.E., Schleifer, K.H. 1986. Genus IV - Staphylococcus Rosenbach 1884. In: Sneath, P.H.A., Mair, N.S., Sharpe, M.E. (Eds.), Bergey's Manual of Systemic Bacteriology, Vol 2. Williams and Wilkins, Baltimore; Schleifer, K.H., 1986. Taxonomy of coagulase-negative staphylococci. In: Mardh, P.-A., Schleifer, K. H. (Ed.), Coagulase-negative Staphylococci. Almquist and Wiksell International. Stockholm, Sweden, pp. 11–26.
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STAPHYLOCOCCUS j Detection by Cultural and Modern Techniques variations depending on the foodstuffs: 12.5% of SE producers for the strains isolated from raw cow milk cheeses, 31.5% for prepared meals, 62.5% goat and sheep raw milk cheeses, and 64% for pastries. In this study, about 60% of food isolates of human origin were enterotoxigenic. Percentages of enterotoxigenic strains indeed vary according to the dominant biotype associated to food category: human biotype in handmade foodstuffs (prepared meals, pastries), and bovine or ovine biotype in cheeses made with cow or ovine raw milk. From 70 to 95% of the S. aureus strains isolated from foodstuffs involved in SFP outbreaks are enterotoxin producers in laboratory conditions (liquid culture in rich medium). Among the strains isolated from SFP outbreaks in France, strains producing SEA, alone or together with SED, are the most prevalent strains. SEB and SEC are rarer and SEE is almost never found. Predominance of SEA is observed in many other countries. Some strains do not produce any detectable SE when tested in laboratory conditions although they were implicated in SFP outbreaks. Such results might be due to the fact that these strains produce SEs that are not detected by the antibodies used for the detection (see Staphylococcus: Detection of Staphylococcal Enterotoxins). Some newly described SEs indeed are not detectable by the commercially available detection kits. Presence of seg to sej genes was sought by polymerase chain reaction (PCR) amplification in S. aureus strains of various origins. These genes are frequently detected, especially seg and sei, which belong to an enterotoxin gene cluster (egc). In some studies, when these genes are sought, the proportion of strains that are potentially SE producers increases significantly. In France, Rosec and Gigaud (2002) showed that 57% of 258 strains isolated from various food were seg, seh, and sei positive and 31% were carrier of these genes only. In Italy, Zecconi et al. (2006) showed that 100% of 50 strains isolated from raw milk of cows suffering from mastitis were seg and sei positive. In Ireland, Smyth et al. (2006) showed that 64% of 157 strains isolated from household fridge were seg and sei positive, whereas only 7% were sea and see positive. PCR techniques detect se genes in the strains but do not allow determining if the SE is actually produced. Only detection of the toxin itself in a suspected foodstuff can demonstrate the involvement of the SE in an SFP.
Contamination Mode Presence of S. aureus in food has two main origins: l
In raw materials from animal origin (meat, milk), contamination can result from a primary contamination. For example, raw milk contamination can be due to the presence of cow suffering from S. aureus mastitis in the herd. Mammalian or avian carcasses can be contaminated during slaughtering and can have different sources: S. aureus carriage on the furs or the feathers, on the udder skin, nares, genital mucosa, and digestive tract; staphylococcal infections (abscesses, superficial infections). l For any other foodstuffs, contamination can have a human origin during food-manufacturing process or in house food preparation. In this case, S. aureus strains can come from
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healthy carriage on skin and mucosa or from staphylococcal infections (infected wounds, sinusitis, pharyngitis, rhinopharyngitis). Contamination can occur either directly during food manipulation, or through respiratory aerosols, which are more abundant during infections of upper airways. Strains of human biotype are highly prevalent in the strains isolated from SFP outbreaks. Strains of ovine biotype or nonhost specific strains can be associated to SFP in France. Environmental contamination (processing plant surfaces) or cross-contamination between foodstuffs is poorly documented as of yet and their incidence remains to be evaluated.
Detection Methods for S. aureus Identification by Conventional Microbiology Selective and Nonselective Media Numerous selective or nonselective media were developed for the isolation of staphylococci. Among the nonselective media, the most commonly used are solid agar media, such as blood agar plates, trypticase-soja agar (TSA), Brain-Heart Infusion agar (BHI agar), Plate count agar plus milk (PCA milk), and their equivalent in liquid media (BHI, TS Broth). Selective media are aimed at inhibiting the growth of bacteria other than S. aureus and at favoring the growth of S. aureus. Selective compounds are high salt concentration (NaCl), which results in low aw like in Chapmann agar, or lithium chloride concentration, glycine and potassium tellurite, and compounds that are included in Baird–Parker (BP) medium and its derivatives (Table 2). If necessary, sulfamethazine can be added to inhibit Proteus spp. growth. Selectivity also can be optimized by adding acriflavine and polymyxine E (colistine) or by applying a temperature above 42 C. One might keep in mind, however, that the more selective the medium is, the more inhibitory it is for stressed S. aureus isolates. Some compounds are useful to differentiate staphylococci colonies (e.g., egg yolk and tellurite in BP, Table 1). BP medium is the medium of choice in food bacteriology because it allows the best recovery of stressed S. aureus cells thanks to sodium pyruvate that activates growth by degrading H2O2 and to the protective effect of egg yolk. However, BP-medium is insufficiently selective when samples are rich in other microbial flora. That is why several variants of BP are proposed (Table 2). Some are judged too selective to allow recovering stressed S. aureus cells and a 1-h preliminary step on BP without selective agents was proposed before the addition of the highly selective medium. The BP medium in which Rabbit Plasma Fibrinogen (RPF-BP) replaces egg yolk is a BP variant used in normalized methods. RPF-BP allows an in situ identification of CPS colonies. Although BP and RPF-BP are the most commonly used media, other media also are used, among which chromogenic media represent a highly convenient way for a rapid identification of S. aureus in various type of samples, including complex food samples (Table 2)
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Table 2
Some selective media used for Staphylococcus aureus detection
Media
Selective agents
Diagnostic criterion
Mannitol-salt Mannitol-salt-acriflavine
Natrium chloride Natrium chloride Acriflavine Potassium tellurite Glycin Lithium chloride Potassium tellurite Glycin Lithium chloride Potassium tellurite Glycin Lithium chloride Natrium chloride Potassium tellurite Incubation at 42 C Potassium thiocyanate Lithium chloride Sodium azide Cycloheximide (antifungal compound) Nalidixic acid Colistin sulfate Potassium tellurite Glycin Lithium chloride
Mannitol fermentation Mannitol fermentation
Baird–Parker
Baird–Parker – Rabbit plasma fibrinogen
Vogel and Johnson modified
4S
KRANEP
Columbia CNA Chromogenic media: l CHROMagar Staph. aureus (Becton Dickinson, Franklin Lakes, United States) l ChromID S. aureus® (Biomérieux, Capronne, France) l Brilliance Staph 24 l 3M™ Petrifilm™ Staph express l
RAPID’Staph Agar
Staphylococcus aureus Detection by Molecular Methods Molecular Characterization of S. aureus Strains The phenotype-based methods, such as phage-typing (lysotype) and capsular serotyping, are abandoned because of their low discriminative power compared with the molecular methods. Among these molecular methods, some methods, like ribotyping, are used to classify the strains at the infraspecies level or to type the strains for epidemiological purposes using pulse-field gel electrophoresis. In 2003, Hennekinne et al. (2003) showed that PFGE was efficient to type S. aureus strains isolated from various foodstuffs.
Characterization of Toxinogenic Strains In the early 1990s, PCR was applied to search specific S. aureus genes in DNA samples obtained directly from foodstuffs and not only in DNA extracted from pure culture. The targeted genes were preferentially those encoding enterotoxins. These latter genes, however, were not always detected in S. aureus food isolates and other S. aureus genes, more widely distributed, such as the nuc gene, encoding thermonuclease, were then chosen (Table 3). These techniques proved a high sensitivity. In 2005, Ikeda et al. (2005) detected by PCR enterotoxin genes sea, seh, seg, and sei in samples of skimmed milk powder involved in an SFP outbreak, although no S. aureus had been found by classical microbiology methods. These results were confirmed by the detection of the SEA and SEH in the milk
Tellurite reduction (black colonies) Clear halo (egg yolk) Coagulase
Tellurite reduction Mannitol fermentation DNAse Tellurite reduction Clear halo (egg yolk) Mannitol fermentation Clear halo (egg yolk) Pigment Pigment Hemolysis Specific colors of the cfu: l Light purple (patented) l
Green (a-glucosidase) Dark blue (patented) l cfu surrounded by a pink halo, nuclease activity l Black l
powder. SEG and SEI were not detectable at that time (no specific detection techniques were available) but possibly were present in the samples. Since 2001, the use of quantitative realtime PCR methods allows evaluating the number of DNA copies present in the samples. This number is correlated linearly with the number of bacteria cells and thus it gives an accurate estimation of the level of S. aureus contamination in the foodstuff. This technique is particularly interesting because food products can be contaminated by S. aureus at some steps of the process and might be inactivated by the treatments applied at some other subsequent steps (providing that DNA has not been degraded: foodstuff treatments can indeed alter bacterial DNA). Goto et al. (2007) showed that the amount of staphylococcal DNA detected in a milk contaminated and then pasteurized at 63 C, 30 min is lower than that detected in the same milk after pasteurization at 72 C, 15 s. These authors hypothesized that part of the DNA is lost due to cell lysis and subsequent DNA hydrolysis during pasteurization. With artificially contaminated milk samples, detection limit is around 600 cfu ml1 in pasteurized milk samples, whereas it is 10 cfu ml1 in raw milk. Detection limits of PCR-based techniques vary from a study to another. Food matrix complexity, fats content, and the presence of potential PCR inhibitors must be taken into account in the choice of proper DNA extraction procedures to obtain a method sufficiently sensitive and reproducible. PCR techniques are not widely used in some foodstuffs because of the lack of standardization criteria.
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Some DNA-based techniques for Staphylococcus aureus identification
Gene or sequence
Protein, product, or function
Detection method
16S rRNA 23S rRNA aroA clfA coa femA femB gap hsp60 nuc pnp rpoB Intergenic space rRNA Fragment Sa2052
Ribosomal RNA 16S Ribosomal RNA 23S 5-Enolpyruvylshikimate-3-phosphate synthase Clumping factor A (adhesion factor) Coagulase Cytoplasmic protein FemA Cytoplasmic protein FemB Glyceraldehyde 3-phosphate dehydrogenase Heat shock protein HSP60 Thermonuclease Polynucleotide phosphorylase Subunit b of RNA polymerase Intergenic sequences rRNA 16S-rRNA 23S 2 kb-long EcoRI subfragment resulting from a 44 kb macrorestriction fragment 442 base pairs Sau3A fragment resulting from S. aureus genomic DNA Superoxide dismutase Staphylococcal protein A (two regions detected: region X and region binding to immunoglobulins G) Intergenic sequences tRNA Elongation factor Tu
PCR hybridization PCR hybridization PCR, hybridization PCR PCR PCR multiplex PCR PCR multiplex PCR PCR, PCR-RFLP hybridization hybridization sequencing PCR Hybridization Sequence RS-PCR PCR, hybridization
Fragment Sa442 sodA spa Intergenic space tRNA tuf
Alarçon et al. (2006) described an optimized PCR protocol, for automated quantification in routine. This method is based on a 24-h enrichment step followed by a DNA extraction using DNeasy tissue kit (Qiagen) and a conventional PCR or a qRTPCR using SYBR-Green, which is 10-fold more sensitive than conventional PCR. Goto et al. (2007) advocated washing the cell pellet extracted from milk five times before DNA extraction to obtain reproducible results. Although the number of colony-forming units (cfu) per gram often correlates well with the gene copy number, Hein et al. (2005) observed discrepancies between these figures with gene copy numbers up to 100- or even 1000-fold higher than the estimated cfu per gram in frozen raw milk samples. It is thus difficult to find a clear and systematic correlation between the number of cfu and the gene copy number because some food processes either degrade DNA, kill bacteria, or turn them into a nonculturable state. Although they are highly specific and sensitive, PCR results only allow detection of S. aureus specific genes and genes encoding enterotoxins, but they do not give information about the expression of these genes. In other words, the presence of an SE gene does not mean the presence of the corresponding SE in the foodstuff. In 2011, Cretenet et al. (2011) demonstrated, in a model cheese, that the expression of SE genes can indeed be inhibited in some conditions, that is, the presence of lactic acid bacteria. Recent efforts thus were dedicated to the development of methodologies based on reverse-transcriptase PCR (RT PCR), which enables an estimation of the level of SE gene expression.
Normalized Methods and Alternative Methods Because of sanitary rules and mandatory declaration of foodpoisoning outbreaks, it appears absolutely necessary to use
PCR, hybridization Hybridization PCR PCR PCR, hybridization PCR-RFLP
robust and reliable methods for the detection of CPS and of SE. Such detections rely on normalized methods or on alternative methods. Normalization aims at providing standards, or reference documents, that bring consensual solutions to technical problems in a client–provider perspective. In its international definition, a standard is “a document, established by consensus and approved by a recognized body that provides, for common and repeated use, rules, guidelines or characteristics for activities or their results, aimed at the achievement of the optimum degree of order in a given context.” Usually, standards are heavy to implement for operators, and standard methods may be supplemented by so-called alternative methods with the same response characteristics (this, however, requires a validation against the reference method described in the standard), but implementation will be easier, faster, and cheaper.
Normalized Standards S. aureus is usually isolated from foodstuffs using normalized methods. Reference methods are described in international standards, which are adopted at the European level (NF EN ISO 6888-1, -2, et -3). These are classical techniques of cfu numeration on selective media after direct inoculation of decimal dilutions of food extracts and incubation at 37 C. The standard ISO 6888-3 describes a method of detection and numeration by the technique of the most probable number (MPN) after an enrichment step. The selective medium used in the standard is a modified Giolitti and Cantoni broth whose formula is similar to that of Baird–Parker (BP) broth (see the Alternative Methods section). After enrichment, S. aureus is detected by streaking and incubation on agar selective medium. Such standards are based on two types of agar selective media: BP and RPF-BP.
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Methods using BP medium require a confirmation test of several colonies with characteristic and noncharacteristic aspects, with a coagulase test in vitro. Methods using RPF-BP do not require confirmation test. Whatever the medium used, coagulase-positive colonies are counted and the result is expressed in cfu of CPS (most likely S. aureus) by gram or by milliliter of foodstuff. Fidelity of the methods described in the standards ISO 6888-1 (BP and BP-RPF) in terms of repeatability and reproducibility was evaluated in an interlaboratory test realized with three types of foodstuffs and four levels of S. aureus contamination. The fidelity of the two methods was estimated sufficient. Both methods received an equal status for the numeration of CPS in foodstuffs. RPF-BP is recommended for the detection and numeration of CPS in raw milk cheeses and any other foodstuff with complex microbial flora, which might interfere with the interpretation of BP plates. Regarding PCR detection, no specific standards exist as of yet for S. aureus but a series of standards gathered in the generic name “Food Microbiology – Polymerase Chain Reaction (PCR) for the detection of pathogenic microorganisms in foodstuffs” provide the general principles, especially: l
The standard ISO 22174: General requirements and definitions. l The standard NF EN ISO 20837 for the preparation of samples for qualitative detection by PCR. This standard describes a protocol for DNA extraction from selective broth of enrichment, approved by interlaboratory test for two Gram-negative bacteria. l The standard NF EN ISO 20838 for the amplification and qualitative detection. This standard also describes some tests for the confirmation of the identity of the PCR product obtained and provides remarks on test optimization.
Alternative Methods S. aureus can also be counted in foodstuffs by alternative methods that are commercially available. Such methods are more user friendly and or more rapid than normalized standards. To be used in place of a standardized method for official controls, they must be tested according to a recognized protocol validation, including trials with a reference method and a collaborative study. PetrifilmTM Staph Express (3M) and Rapid Staph Test (BioRad) were validated, in France, by AFNOR in 2003 and in 2005, respectively. The PetrifilmTM Staph Express system includes Petrifilm test containing a selective and differential chromogenic medium for S. aureus plus a confirmation disc allowing observing the DNase activity of colonies. After inoculation and incubation of the test during 24 h at 37 C, S. aureus forms red-purple colonies. If cfu with another color are visualized, the confirmation disk is applied on the test during 3 h at 37 C. Pink halos around the cfu correspond to S. aureus. RapidStaph Test is a Baird–Parker medium optimized for a reading after 24 h incubation (instead of 48 h in the standard ISO 6888-1). Two confirmation tests can be used on characteristic colonies: a rapid latex beads agglutination test (PastorexR Staph-plus, Bio-Rad) or a streak on RPF-BP allowing visualization of CPS cfu after an 18 h incubation at 37 C.
Conclusion Compared with phenotypic methods, molecular methods are based on DNA and are independent of the expression of specific genes in artificial culture conditions (laboratory environment). These traits are relatively stable in nature, compared with phenotypic (biotypes, serotypes, antibiograms). Molecular methods give results independent of potential changes in experimental conditions. Molecular methods do not require in vitro culture and thus allow identifying species that are nonculturable or difficult to grow. Compared with phenotypic methods, molecular methods targeting chromosomal genes allow the identification of all strains of a given species because all bacteria contain DNA. Phenotypic methods remain routinely used because they are pretty fast and do not require expensive equipment and reagents or expertise. With the development of chromogenic media that are increasingly efficient and discriminating, such phenotypic methods have a future in the diagnosis of microbiological hazards in foodstuffs.
See also: Bacillus: Bacillus cereus; Bacillus: Detection of Toxins; Bacillus – Detection by Classical Cultural Techniques; Biochemical and Modern Identification Techniques: Microfloras of Fermented Foods; Ecology of Bacteria and Fungi: Influence of Available Water; Ecology of Bacteria and Fungi in Foods: Influence of Temperature; Ecology of Bacteria and Fungi in Foods: Influence of Redox Potential; Ecology of Bacteria and Fungi in Foods: Effects of pH; Hazard Appraisal (HACCP): The Overall Concept; Hazard Analysis and Critical Control Point (HACCP): Critical Control Points; Hazard Appraisal (HACCP): Involvement of Regulatory Bodies; Hazard Appraisal (HACCP): Establishment of Performance Criteria; Nucleic Acid–Based Assays: Overview; PCR Applications in Food Microbiology; Petrifilm – A Simplified Cultural Technique; Predictive Microbiology and Food Safety; Process Hygiene: Overall Approach to Hygienic Processing; Designing for Hygienic Operation; Process Hygiene: Types of Sterilant; Process Hygiene: Overall Approach to Hygienic Processing; Process Hygiene: Modern Systems of Plant Cleaning; Process Hygiene: Risk and Control of Airborne Contamination; Process Hygiene: Disinfectant Testing; Process Hygiene: Involvement of Regulatory and Advisory Bodies; Process Hygiene: Hygiene in the Catering Industry; Staphylococcus: Staphylococcus aureus; Staphylococcus: Detection of Staphylococcal Enterotoxins; Identification Methods: Introduction; DNA Fingerprinting: Pulsed-Field Gel Electrophoresis for Subtyping of Foodborne Pathogens; Identification Methods: DNA Fingerprinting: Restriction Fragment-Length Polymorphism; Bacteria RiboPrint™: A Realistic Strategy to Address Microbiological Issues outside of the Research Laboratory; Multilocus Sequence Typing of Food Microorganisms; Application of Single Nucleotide Polymorphisms–Based Typing for DNA Fingerprinting of Foodborne Bacteria; Identification Methods and DNA Fingerprinting: Whole Genome Sequencing; Identification Methods: Multilocus Enzyme Electrophoresis; Identification Methods: Chromogenic Agars; Identification Methods: Immunoassay; Identification Methods: DNA Hybridization and DNA Microarrays for Detection and
STAPHYLOCOCCUS j Detection by Cultural and Modern Techniques
Identification of Foodborne Bacterial Pathogens; Identification of Clinical Microorganisms with MALDI-TOF-MS in a Microbiology Laboratory.
Further Reading Akineden, O., Hassan, A.A., Schneider, E., Usleber, E., 2008. Enterotoxigenic properties of Staphylococcus aureus isolated from goats’ milk cheese. International Journal of Food Microbiology 124 (2), 211–216. Alarçon, B., Vicedo, B., Aznar, R., 2006. PCR-based procedures for detection and quantification of Staphylococcus aureus and their application in food. Journal of Applied Microbiology 100 (2), 352–354. Beckers, H.J., Van Leusden, F.M., Bindschedler, O., Guerraz, D., 1984. Evaluation of a pour plate system with rabbit plasma-bovine plasma-agar for the enumeration of Staphylococcus aureus in food. Canadian Journal of Microbiology 30, 470–474. Bergdoll, M.S., 1991. Symposium on microbiology update: old friends and new enemies. Staphylococcus aureus. International Journal AOAC 74, 706–710. Cretenet, M., Nouaille, S., Riviere, J., et al., 2011. Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix. Environmental Microbiology Reports 3 (3), 340–351. De Buyser, M.L., Sutra, L., 2005. Staphylococcus aureus. In: Federighi, M. (Ed.), Bactériologie alimentaire – Compendium d’hygiène des aliments. Economica, Paris, pp. 25–51. De Buyser, M.L., Audinet, N., Delbart, M.O., et al., 1998. Comparison of selective culture media to enumerate coagulase-positive staphylococci in cheeses made from raw milk. Food Microbiology 15, 339–346. De Buyser, M.L., Lombard, B., Schulten, S.M., et al., 2003. Validation of EN ISO standard methods 6888 Part 1 and Part 2: 1999 – Enumeration of coagulasepositive staphylococci in foods. International Journal of Food Microbiology 83, 185–194. Even, S., Leroy, S., Charlier, C., et al., 2010. Low occurrence of risk factors in coagulase negative staphylococci isolated from fermented foodstuffs. International Journal of Food Microbiology 139 (1–2), 87–95. Genigeorgis, C.A., 1989. Present state of knowledge on staphylococcal intoxication. International Journal of Food Microbiology 9, 327–360. Goto, M., Takahashi, H., Segawa, Y., et al., 2007. Real-time PCR method for quantification of Staphylococcus aureus in milk. Journal of Food Protection 70 (1), 90–96.
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Hein, I., Jorgensen, H.J., Loncarevic, S., Wagner, M., 2005. Quantification of Staphylococcus aureus in unpasteurised bovine and caprine milk by real-time PCR. Research in Microbiology 156 (4), 554–563. Hennekinne, J.A., Kerouanton, A., Brisabois, A., De Buyser, M.L., 2003. Discrimination of Staphylococcus aureus biotypes by pulsed-field gel electrophoresis of DNA macro-restriction fragments. Journal of Applied Microbiology 86 (2), 321–329. Ikeda, T., Tamate, N., Yamaguchi, K., Makino, S., 2005. Mass outbreak of food poisoning disease caused by small amounts of staphylococcal enterotoxins A and H. Applied and Environmental Microbiology 71, 2793–2795. Isigidi, B.K., Devriese, L.A., Croegart, T.H., van Hoof, J., 1989. A highly selective twostage isolation method for the enumeration of Staphylococcus aureus in foods. Journal of Applied Bacteriology 66, 379–384. Kerouanton, A., Hennekinne, J.A., Letertre, C., et al., 2007. Characterization of Staphylococcus aureus strains associated with food poisoning outbreaks in France. International Journal of Food Microbiology 115, 369–375. Kloos, W.E., Schleifer, K.H., 1975. Simplified scheme for routine identification of human Staphylococcus species. Journal of Clinical Microbiology 1, 82–88. Le Loir, Y., Gautier, M., 2003. Staphylococcus aureus. In: Le Loir, Y., Gautier, M. (Eds.), Monographie de Microbiologie. Editions Lavoisier Tec&Doc, Paris. Le Loir, Y., Baron, F., Gautier, M., 2003. Staphylococcus aureus and food poisoning. Genetic and Molecular Research 2 (1), 63–76. Lee, Y.D., Moon, B.Y., Park, J.H., Chang, H.I., Kim, W.J., 2007. Expression of enterotoxin genes in Staphylococcus aureus isolates based on mRNA analysis. Journal of Microbiological Biotechnology 17 (3), 461–467. McLauchlin, J., Narayanan, G.L., Mithani, V., O’Neill, G., 2000. The detection of enterotoxins and toxic shock syndrome toxin genes in Staphylococcus aureus by polymerase chain reaction. Journal of Food Protection 63, 479–488. Rosec, J.P., Gigaud, O., 2002. Staphylococcal enterotoxin genes of classical and new types detected by PCR in France. International Journal of Food Microbiology 77, 61–70. Rosec, J.P., Guiraud, J.P., Dalet, C., Richard, N., 1997. Enterotoxin production by staphylococci isolated from foods in France. International Journal of Food Microbiology 35, 213–221. Shimizu, A., Fujita, M., Igarashi, H., et al., 2000. Characterization of Staphylococcus aureus coagulase type VII isolates from Staphylococcal food poisoning outbreaks (1980–1995) in Tokyo, Japan, by pulsed-field gel electrophoresis. Journal of Clinical Microbiology 38, 3746–3749. Smyth, D.S., Kennedy, J., Twohig, J., et al., 2006. Staphylococcus aureus isolates from Irish domestic refrigerators possess novel enterotoxin and enterotoxin-like genes and are clonal in nature. Journal of Food Protection 69 (3), 508–515. Zecconi, A., Cesaris, L., Liandris, E., et al., 2006. Role of several Staphylococcus aureus virulence factors on the inflammatory response in bovine mammary gland. Microbial Pathogens 40, 177–183.