Coagulase gene polymorphism of Staphylococcus aureus isolates:A study on dairy food products and other foods in Tehran,Iran

Mohmm Mhi Soltn DlllMohmm Rz KhormizhSolmz Agh AmiriAli Akr Soor YrghiRmin Mzhri Nzh Fr

a Division of Food Microbiology,Department of Pathobiology,School of Public Health,Tehran University of Medical Sciences,Tehran,Iran

b Food Microbiology Research Center,Tehran University of Medical Sciences,Tehran,Iran

c Department of Medical Biotechnology,School of Advanced Technologies in Medicine,Iran

d Biosensor Research Center,Endocrinology and Metabolism Cellular and Molecular Science Institute,Tehran University of Medical Sciences,Iran

e Division of Immunology,Department of Pathobiology,School of Public Health,Tehran University of Medical Sciences,Tehran,Iran

Abstract Coagulase is considered as a major determinant factor for the identification of Staphylococcus aureus strains.The 3＇-end coding region of the coagulase (coa) gene contains a series of 81-bp tandem repeats, which differ in the number and location of enzymatic restriction sites among different isolates. coa PCR-RFLP has been used widely to type S.aureus isolates in epidemiological studies.The current study was conducted to investigate the coagulase gene polymorphisms in S.aureus isolated from various food samples using an in house PCR-RFLP method.A total of 100 strains of S.aureus were isolated from food samples.Isolates were typed by PCR-RFLP analysis using NdeI restriction digestion of the coagulase gene PCR products.Results showed that amplification of coagulase genes from S.aureus produced different PCR products.The isolates were grouped into 18 genotypes using RFLP analysis results of the genes.In this study,the S.aureus isolates have been shown to include more than one coagulase genotype,but only had a few coa genotypes predominated.

Keywords: Staphylococcus aureus;PCR-RFLP;Coagulase gene;Food samples

1.Introduction

Staphylococcus aureusis an important food-borne pathogen[1].It is among the most significant pathogens that cause various diseases in humans and animals.In humans,nosocomial and community acquired infections are the most frequently reported problems caused byS.aureus[2,3].The bacterium is one of the most significant pathogens causing intramammary infections(IMI)in dairy ruminants[4].The primary reservoirs forS.aureusare the skin and mucous membranes,especially of the nasopha-ryngeal region of birds and mammals.This microorganism is found in 30%–80%of the human population,thus,unhygienic processing of foods has to be considered as a major risk of contamination [5–7].Staphylococcal food poisoning (SFP) is considered to be one of the leading causes of all food-borne diseases[1].In the last few decades,SFP has been reported as the third cause of food-borne infections in the world[5,8,9].Milks,dairy products and meats,especially in traditional foods,play an important role in SFP;from which,S.aureusstrains have been isolated frequently [1,10,11].Genetic heterogeneity is considerable in natural population ofS.aureus[12].Relatively,many molecular techniques such as random amplified polymorphic DNA (RAPD), ribotyping, multilocus enzyme electrophoresis(MLEE), plasmid profiling and coagulase gene polymorphism have been used for the identification and characterization ofS.aureusin epidemiological studies [13,14].There is no information on genetic diversity ofS.aureusisolated from foods in Iran since the bacterial routine identification is carried on by conventional methods such as Gram staining and catalase,clumping factor, DNase and mannitol fermentation tests [15].However,use of molecular techniques as rapid tools in microbiology research and diagnosis has been increased recently.One of these molecular techniques, PCR-based coagulase genotyping by RFLP analysis(coa-RFLP)of the 3＇end of the gene encoding staphylococcal coagulase has been suggested as a simple and effective method for typingS.aureusisolates in epidemiological studies [16,17].Numerous studies based on the coagulase gene polymorphism have been carried out for genotyping ofS.aureusisolated from bovine mastitic milks and other foods[12,15,18,19].The purpose of the current study was to identifyS.aureussubtypes isolated from food samples,usingcoagene polymorphism profile.

2.Materials and methods

2.1.Bacterial isolates

A total number of 100S.aureusstrains was isolated from food samples(78 isolates from dairy products,16 isolates from meat products and six isolates from other foods).Samples were diluted with normal saline and then homogenized and seeded ontoStaphylococcusselective media (Merck, Germany) and Baird–Parker agar (Merck, Germany).Colonies showing typical aspect of coagulase-positive staphylococci were identified by conventional methods, including Gram staining and catalase, clumping factor, DNase and mannitol fermentation tests.Isolates identified asS.aureuswere frozen in skimmed mild containing 15%(v/v)glycerol at ?20°C until use.

2.2.DNA extraction

DNA was extracted using QIAamp DNA mini kit (Qiagen,Germany),according to the manufacturer’s instructions for Gram-positive bacteria.

2.3.PCR amplification

PCR of thecoagene was carried out using primers COAG2:5＇-CGAGACCAAGATTCAACAAG-3＇and COAG3: 5＇-AAAGAAAACCACTCACATCA-3＇described by Raimundo et al.in 1999 [20].Reactions were prepared in a final volume of 25 μL using HotStarTaq Plus master mix kit (Qiagen,Germany), containing 12.5 μL of HotStarTaq Plus master mix,2.5 μL of 10X buffer,5 μL of RNase-free water,20 pmol of each primer and 3 μL of the template.The amplification program included an initial denaturation step of 3 min at 94°C followed by 30 cycles of 1 min at 94°C, 1 min at 55°C and 1 min at 72°C and a final extension step of 5 min at 72°C.The amplicon size varied in various strains.StaphylococcusepidermidisATCC 12228 andS.aureusCOL were used as controls.

2.4.Restriction enzyme digestion

Generally, 8.5 μL of the PCR product was incubated with 10 U ofNdeI endonuclease enzyme(Fermentas,USA),2.5 μL of restriction buffer and 13 μL of distilled water for 3 h at 37°C.

2.5.Agarose gel electrophoresis

Digested fragments and PCR products were separated in 1%and 2%agarose gels(Gibco,USA),respectively.A 100-bp ladder(Fermentas,USA)was used as a standard molecular marker for the calculation of the sizes of thecoaandNdeI-generatedcoafragments.Gels were visualized under UV light after staining with fluorescent dyes.

2.6.Specificity testing

For the specificity of the primer pair test,the DNA ofS.epidermidisATCC 12228 andS.aureusCOL strain was analyzed.

2.7.Reproducibility testing

For the reproducibility of PCR,5 selected isolates were chosen randomly and tested by twice submitting 3 different PCR products toNdeI digestion.

2.8.Data analysis

The software was used for the size of PCR and RFLP products.Numeric codes were assigned to the PCR genotypes and RFLP patterns.

3.Results and discussion

All isolates produced PCR amplicon with the COAG2 and COAG3 primers (Fig.1).The agarose gel analysis of the digestion products showed ten different sizes, ranging from approximately 500 to 1000 bp.The product sizes of 800, 900 and 850±20 bp were the most frequent sizes,reported for 24,20 and 19%of the isolates,respectively.There was no amplification product of the DNA fromS.epidermidis.As summarized in Table 1,NdeI restriction enzyme digestion of the PCR products generated 18 differentNdeI restriction patterns for all isolates.Except for the products of 500,600 and 1000 bp,amplicons of the same size generated different patterns, with the number of fragment varying from two to three and molecular sizes from approx.80 to 330 bp(Fig.2).Types 9,11 and 14 were the most common patterns and seen in 50%of the isolates(Table 2).Type 1 was the most frequent pattern and reported in 31.25%of meat product samples,while Type 9 was the most frequent pattern in other foods(50%).

Fig.1.PCR products of S.aureus coa coagulase genes isolated from food samples.Lanes 1 and 12,100-bp ladder;Lane 6,S.epidermidis ATCC 12228 negative control; Lane 13, S.aureus COL positive control; Lanes 2–5, 7–11,PCR products.

Table 1 NdeI RFLP patterns and frequency of coagulase genotypes of S.aureus isolated from food samples.

Fig.2.Electrophoretic profile of NdeI restriction fragments of the coa gene PCR products.L to R: Lane 1, 100-bp ladder; Lanes 2, 4, 6 and 8, PCR products;Lanes 2,4,6 and 8,PCR products;Lane 3,PCR-RFLP Type-1 fragments(80-170-330 bp); Lane 9, PCR-RFLP Type-2 fragments (80-250-330 bp); Lanes 5 and 7,PCR-RFLP Type-13 fragments(80-330 bp).

Coagulase protein is a main virulence factor inS.aureus[21].The 3＇end of the coagulase gene contains a series of 81-bp tandem repeats,which is different betweenS.aureusstrains[22].Classification based on the RFLP ofcoagene, has been considered as a simple and accurate method for typingS.aureusisolated from various sources [17,19].Using this method, different genotypes were seen among the studied isolates, which suggest thatS.aureushas a certain degree of heterogeneity in the food samples.The ten different PCR types and the 18NdeI RFLP patterns suggest that the isolatedS.aureusstrains harbor more than one variant of thecoagene.Results also showed that however different genotypes were detected;only a few predominated.These findings were similar to findings published by researchers who tested milk of bovine mastitis in various countries[12,19,21–23]and showed that several coagulase gene types were responsible for the majority of bovine mastitis cases;only some were predominated in each country.El Bayomi et al.by Genotyping using spa PCR-RFLP showed identical restriction banding patterns of MRSA isolates of human and chicken meat origin, indicating the genetic relatedness of the isolates[19].Aslantas et al.typed 80S.aureusisolated from subclinical bovine mastitis milks and distinguished five PCR and nine RFLP types,with two most common genotypes reported for 73.8%of the isolates[22].Guler et al.tested 125S.aureusisolates from bovine clinical mastitis in Turkey and found four PCR products of 1000, 900, 800 and 700 bp, with a 1000 bp product being the predominant product [23].Blaiotta et al.showed in some strains ofStaphylococcus xylosus,Staphylococcus saprophyticus,and Staphylococcus equorum, two catalase genes,katA andkatB, were found [24].Kizerwetter-Swida et al.showedcoagulase-positive staphylococci(CoPS)are opportunistic veterinary pathogens,of whichS.aureus,Staphylococcus delphiniandStaphylococcus intermediuscan be isolated from pigeons.The biochemical identification ofS.delphiniandS.intermediusisolates may be incorrect,because of their phenotypic similarity.A total number of 31 isolates of CoPS was obtained, 15 were identified asS.delphinigroup B, six asS.aureus, four asS.delphinigroup A,three asS.intermediusand three asStaphylococcus schleiferisubsp.coagulans.PFGE restriction patterns ofS.delphinigroup A andS.delphinigroup B form separate clusters, demonstrating their genetic heterogeneity [25].Da Silva et al.tested 33S.aureusfrom milk of goats with clinical and subclinical mastitis,belonging to two regions from Brazil and found 11 genotypes.One predominant type was found in each region in most of the isolates [26].According to Hennekinne et al.Staphylococcal food poisoning(SFP)is one of the most common food-borne diseases caused by the ingestion of staphylococcal enterotoxins(SEs)produced in foods by enterotoxigenicS.aureus[27].Similarly,Moon et al.typedS.aureusisolates from animal and vegetable sources in Korea and distinguished 12 genotypes,varied with the source of the microorganisms.However,only a few genotypes prevailed in each source[28].This distribution might be explained by the coevolution of the hosts and the pathogens,and also differences in reservoirs and imply that the successful transfer of bacteria between bovine mastitis milk,raw meat and vegetables is not a frequent occurrence[22,27].

Table 2 Distribution of NdeI RFLP patterns and frequencies of coagulase based genotypes of S.aureus isolated from food samples.

These characteristics show the necessity of comprehensive studies on epidemiological and ecological profiles of a specific origin before applying food-hygiene control measures.

4.Conclusion

In conclusion, results from the current study showed that however the food samples were contaminated byS.aureusharboring more than one coagulase genotype, only a fewcoagenotypes were predominate.Further studies are needed to determine the common characteristics of the predominate strains.This information can be used to better develop control measures for foods contaminated byS.aureus.

Acknowledgment

This work was supported by a grant from Vice-Chancellor for Research(grant no.5615)of Tehran University of Medical Sciences,Tehran,Iran.