Characterization of Coagulase Positive Staphylococci from Pig Carcasses from Swedish Slaughterhouses

Department of Medical Biochemistry and Microbiology Autumn 2008

Anika Neskovic

Supervisor: Hans Lindmark Department of Microbiology National Food Administration

ABSTRACT

The aim was to characterize 100 coagulase positive staphylococci isolates originating from pig carcasses from Swedish slaughterhouses by biotyping, antibiotic susceptibility testing, typing with pulsed field gel electrophoresis (PFGE) and real-time PCR-screening of the enterotoxin genes sea, sec, seg and sei in order to evaluate the impact on human health. The biotyping classified 56 as non host specific (NHS), 29 as human biotype, five as poultry, one as ovine, one as bovine biotype and eight were unclassified (UCF). Susceptibility testing to 16 antibiotics revealed that 49% of the isolates were resistant to penicillin, which the biotype human dominated among these isolates. The results from the PFGE showed correlation between the biotypes and the pulsotypes obtained with several groups with identical strains. The results from the 47 isolates tested for enterotoxins were that the combination of seg and sei was the most common but sea and sec were also detected. There were slaughterhouses that had certain biotypes and penicillin resistance linked to them.

KEY WORDS

S. aureus, biotyping, enterotoxin, antibiotics, porcine

INTRODUCTION

Staphylococcal food poisoning is a common cause of gastroenteritis worldwide and is one of the most economically important foodborne diseases (3, 21). For example in the United States the medical expenses and losses in productivity is estimated to $1,500,000,000 per year (3). In Spain, Staphylococcus aureus is the second most common pathogen causing outbreaks of food poisoning (1). In Sweden there were three reported outbreaks of staphylococcal food poisoning in 2005 and eight outbreaks in 2006. There are probably a large number of cases not reported, since the disease is not so severe. Food can be contaminated by improper hygiene and high levels of bacteria can be obtained during improper storage temperatures (1).

Staphylococci are a part of the natural skin flora of most mammals and birds. At a given time, 25-30% of the human population is carrying S. aureus. Normally, the bacterium is present on the skin or in the front of the nose. The bacteria can cause serious infections in immune suppressed persons. S. aureus is the most significant human pathogen among the staphylococci (21). The National Food Administration in Sweden has recently discovered that almost a third of pig carcasses from Swedish slaughterhouses carry coagulase positive staphylococcus (15). S. aureus is by far, the species within the genus Staphylococcus that causes disease in humans and is considered an opportunist.

S. aureus is the most common bacterium in the air within a swine barn (5). Pig farmers should wear particle respirators, change clothes and shower before leaving the barn so that vulnerable persons are not exposed (5). Pig farming was recently identified as a risk factor for increased nasal S. aureus carriage (23). Swine slaughter is a process with many risks for contamination of the pork carcass with potentially pathogenic bacteria. The process includes steps where the bacterial number may be reduced (2). S. aureus can be used as an indicator of the general hygiene, including the hygienic status of the equipment in the slaughterhouse (2).

S. aureus is a facultative anaerobic, Gram-positive, catalase-positive coccus that often lies in clusters and is called ”golden staph” due to the yellow colour of the colonies. The bacterium has low demands on moisture and nutritional requirements and can therefore survive and be easily transmitted in hospitals primarily via direct contact, airborne carriage or contact with surfaces like catheters (4). The enzyme catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen. The catalse test is particularly useful in distinguishing staphylococci, which is catalase-positive, from the catalase-negative streptococci and enterococci. Staphylococcus isolates is usually divided into coagulase positive and negative. The coagulase positive isolates are regarded as more virulent and this group includes the species S. aureus, S. hyicus and S. intermedius. Coagulase is an enzyme that converts fibrinogen to fibrin. In the laboratory, it is used to distinguish between different species of Staphylococcus. Free and bound coagulase coats the bacterial cells with fibrin, which shield them from opsonization and phagocytosis.

S. aureus causes food poisoning mainly through the enterotoxins (SE) that are heat stable and consists of short proteins (14). Symptoms which usually occur 1-7 h after ingestion of enterotoxins include vomiting, diarrhoea, headache and blood pressure failure. The clinical signs usually last for approximately 24 h. The epithelial cells in the intestine are affected. The specific emetic mechanism for these enterotoxins is largely unknown, although it is believed that nervcenters in the gut are stimulated by the toxin and signals are transmitted by the vagus and sympathetic nerves to the vomiting center in the brain (21). At least 19 different SEs have so far been documented (21). It is known that about 95% of staphylococcal food poisoning

3 outbreaks are caused by the classical toxins SEA to SEE (19). The remaining 5 % of outbreaks are associated with the newly identified SEs (SEG to SER, SEU and SEV), although the relationship between these new SEs and human food poisoning is not fully known (12, 19). Fifty to seventy percent of the S. aureus strains produce enterotoxins. Strains of the human biotype usually produce enterotoxin whereas others more seldom carry the genes encoding enterotoxins (2).

Pulsed-field gel electrophoresis (PFGE) has been applied extensively to distinguish different strains of S. aureus. The method was developed in 1984 and made it possible to separate DNA fragments with sizes over 10 kb in an agarose gel by a pulsed electric field (7). Biotyping differentiate S. aureus strains into host-specific and none-host-specific biotypes with the four tests; staphylokinase production, β-haemolysin production, bovine plasma coagulation and growth on crystal violet agar. Growth on crystal violet agar gives the three different types A, C or E.

S. aureus is one of the main subjects of studies of antibiotic resistance because it is often multiresistant. Multi-antibiotic resistant Gram-positive cocci represent emerging pathogens especially in the setting of immunocompromised, hospitalized patients (17). Multi-resistant bacteria are an increasing challenge for infection control in hospitals (18). Methicillin-resistant Staphylococcus aureus (MRSA) is a new emerging problem in veterinary medicine. Cases of animal infection and carriage are increasingly reported worldwide (8). Detection of MRSA by appropriate methods should be carried out into antimicrobial resistance programs in veterinary medicine. Penicillin resistance is widespread in animal staphylococci (8, 25, 26). In Dutch slaughterhouses there is a high prevalence of MRSA (5). Levels of commensal bacteria with antimicrobial resistance are higher among pig farmers than among control groups (23). Use of antimicrobials in pets is increasing in Europe. A Danish study on poultry, pigs, cattle and pets showed that pigs consume by far the highest number of “animal daily doses” of antimicrobials (16). Sensitiveness against antibiotics is recorded as minimal inhibitory concentration (MIC). MIC is the lowest concentration of an antimicrobial that will inhibit the visible growth after incubation.

The aim of the present study was to evaluate the pathogenicity of coagulase-positive staphylococci present on Swedish pork meat and evaluate possible contamination sources.

MATERIALS AND METHODS

The samples The coagulase positive staphylococcus isolates were from a base-line study on swine carcasses performed by the National Food Administration in 2004-2005. The samples were taken from the four sites: ham, back, belly and neck. More than 500 samples were taken from ten major slaughterhouses and more than 200 from four small-scale slaughterhouses (15). The present study included the first 100 isolates obtained.

Biotyping The biotyping was made according to Devriese (1984) with the modification that during the production of the fibrinogen plates, the incubation of the substrate was prolonged to 30 min. Briefly, the isolates were examined for production of β-hemolysin, production of staphylokinase, coagulation of bovine plasma and crystal violet growth type. Staphylokinase production, was observed by incubating the isolates on bovine fibrin plates with or without

4 dog serum. The dog serum was used as a plasminogen source. Staphylokinase catalyzes the breakdown of fibrin. Staphylokinase production, is indicated by clearing zones on bovine fibrin plates with dog serum. Beta-haemolysin production on sheep blood agar plates results in broad discoloured zones with sharp edges clearing at 4ºC. Bovine plasma coagulation is tested by adding 0.1 mL of an overnight culture Brain Heart Infusion (BHI) to tubes with diluted (1:3) bovine plasma. Voluminous clots appearing within 6 h were considered positive. The crystal violet spots with a bright or pale yellow colour and yellow spots with blue margins were type A. Blue or violet growth spots with or without an orange tint were type C. White spots or white growth with a blue hue was type E (6).

Antibiotic susceptibility testing Three to five colonies were inoculated in 5 mL cation adjusted Mueller Hinton-broth (National Veterinary Institute, www.sva.se). The tubes were incubated at 37ºC for 4 h to receive a concentration of 108 cfu/mL. Ten µL of the suspension was transferred with a loop to a tube with 10 mL Mueller Hinton broth to receive 105 cfu/mL. The content was vortexed and then poured into a petri dish. With a multipipette, 50 µL was transferred to the wells in the panels VetMICTM GP-mo-A and VetMICTM GP-mo-B (National Veterinary Institute, www.sva.se). The remaining solution was checked for purity by streaking 1 µL on a blood agar plate. The panels and the control plates were incubated at 37ºC for 16 h.

Pulsed field gel electrophoresis (PFGE) The isolates were inoculated on blood agar plates at 37ºC over night. One colony was inoculated in 5 mL brain heart infusion (BHI) and was incubated over night at 37ºC. Seven hundred µL of the suspension was centrifuged at room temperature for 3min at 10,000 g. The supernantant was discarded and the pellet washed with 1 mL TEN-buffer (1 mM Tris-HCl, 10 mM EDTA and 150 mM NaCl; adjusted to pH 7.5). The cells were resuspended in 300 µL EC-buffer (6 mM Tris-HCl, 1 M NaCl, 100 mM EDTA, 0.5% Brij, 0.2% sodium deoxycholate, 0.5% SDS; adjusted to pH 7.0) and 2 µL lysostaphin (1 mg/mL) was added. Subsequently, 300 µL molten 1.2% Seakem Gold Agarose (In vitro Sweden AB, www.invitro.se) dissolved in EC-buffer was mixed with 300 µL bacteria solution and poured into plug moulds. The obtained agarose plugs were incubated in 3 mL EC-buffer in Falcon tubes (50 mL) at 37ºC for 1.5 h. The EC-buffer was replaced with 3 mL TE-buffer (10 mM Tris and 1 mM EDTA; adjusted to pH 7.5) and incubated at 55ºC for at least 1 h. The TE- buffer was replaced with new buffer and the plugs were saved at 4ºC. The following day, plugs were cut into small pieces and pre-incubated in 100 µL 1 x T-buffer (Amersham Biosciences, www.amersham.com). Each sample was incubated with 20 U SmaI (Amersham Biosciences, www.amersham.com) in 25ºC for 3 h. The plugs were casted in to a 1% Agarose NA (GE Healthcare, www.gehealthcare.com) in 0.5 x TBE-buffer (44.6 mM Tris, 44.5 mM boric acid, 10 mM EDTA). The electrophoresis was run with a Biorad CHEF DRIII- instrument (Biorad, www.Biorad.com) using the following conditions; block 1: 5-15 s for 10 h and block 2: 15-60 s for 13 h, voltage was set 6 V/cm and the temperature to 14ºC. The gel was coloured for 25 min in 1 µg/mL ethidium bromide and destained in de-ionized water for 25 min. The gel was photographed in UV-light and the picture was processed in the computer program GelCompar II version 3.5.

Detection of enterotoxin genes Total DNA from selected number of isolates was prepared using the DNeasy® Blood & Tissue Kit (Qiagen, www.qiagen.com) according to the manufacturer´s manual. The master mix contained Power SYBR® Green PCR Master Mix (Applied Biosystems, www.appliedbiosystems.com) and 0.1 µM of both forward and reverse primers. The wells in

5 the 96 well optical reaction plate (Applied Biosystems, www.appliedbiosystems.com) were filled with 20µL mastermix and 5µL DNA template diluted 1:10. The primer sequences and the predicted PCR product sizes are shown in Table 1.

Table 1. Primer sequences and predicted size of the PCR-product. Target gene Oligonucleotide sequence (5´ to 3´) Product size (bp) spa F:TCA AGC ACC AAA AGA GGA AGA 263+/-n x 24 R:GTT TAA CGA CAT GTA CTC CGT TG sea F:GCA GGG AAC AGC TTT AGG C 520 R:GTT CTG TAG AAG TAT GAA ACA CG sec F:CTT GTA TGT AGT GAG GAA TAA CAA 283 R:TGC AGG CAT CAT ATC ATA CCA seg F:CGT CTC CAC CTG TTG AAG G 327 R:CCA AGT GAT TGT CTA TTG TCG sei F:CAA CTC GAA TTT TCA ACA GGT AC 465 R:CAG GCA GTC CAT CTC CTG

The PCR-instrument used was a 7500 Real Time PCR System (Applied Biosystems, www.appliedbiosystems.com). The PCR-program used is shown in Table 2. Table 2. Program used for PCR analysis of the genes spa, sea, sec and seg. Number of cycles Time Temperature (ºC) 1 10min 95 Initial denaturation

42 15s 95 Denaturation 30s1 551 Annealing 45s 72 Elongation

1 15s 95 Dissociation 1min 60 15s 95 1 The annealing time for sei was 1 min and the annealing temperature was 60ºC.

RESULTS

Biotyping Biotyping of 100 coagulase positive staphylococci from slaughtered pigs classified 56 as non host specific, 29 as human biotype, five as poultry, one as ovine and one as bovine biotype (Table 3). Eight isolates were unclassified (UCF) and six of them had growth type E on crystal violet agar, which results in unclassified biotypes. The other two unclassified isolates (345 and 508) displayed combinations of the tests that are not present on the biotyping scheme described by Devriese (1984). The six isolates from slaughterhouse A were all of biotype NHS (Table 4). The isolates of the human biotype were mainly from slaughterhouse J and H.

Table 3. Results from the biotyping. -haemolysin -haemolysin -haemolysin Isolate Date of sampling β violet Crystal Staphylokinase plasma Bovine coagulation Slaughterhouse Biotype Isolate Date of sampling β violet Crystal Staphylokinase plasma Bovine coagulation Slaughterhouse Biotype 78 05-10-04 pos C pos neg B Human 508 29-03-05 neg A neg pos C UCF 80 04-10-04 pos C neg neg C NHS 521 05-04-05 pos A neg neg B NHS 102 12-10-04 pos A pos neg D NHS 523 05-04-05 pos C pos neg B Human 111 18-10-04 pos A pos neg E NHS 527 05-04-05 neg A pos neg D NHS 113 18-10-04 neg C pos neg E Human 528 05-04-05 pos A pos neg D NHS 114 18-10-04 pos A pos neg E NHS 529 05-04-05 neg A pos neg D NHS 115 18-10-04 pos A pos neg E NHS 530 11-04-05 neg C pos neg J Human 125 19-10-04 pos C neg neg F NHS 531 11-04-05 neg C pos neg J Human 136 26-10-04 pos C neg neg G NHS 532 11-04-05 neg C pos neg J Human 235 30-11-04 pos A pos neg A NHS 533 11-04-05 neg C pos neg J Human 275 13-12-04 pos A pos neg E NHS 536 11-04-05 pos C neg pos L Ovine 284 13-12-04 neg C pos neg H Human 537 11-04-05 neg C pos neg L Human 345 25-01-05 neg A neg pos C UCF 539 11-04-05 neg C pos neg L Human 353 31-01-05 pos A neg neg I NHS 540 12-04-05 neg A neg neg H Poultry 357 31-01-05 pos A pos neg E NHS 542 12-04-05 pos C neg neg H NHS 358 31-01-05 pos A pos neg E NHS 544 12-04-05 neg A neg neg H Poultry 359 31-01-05 pos A pos neg E NHS 545 12-04-05 pos A neg pos B Bovine 363 01-02-05 pos A neg neg H NHS 546 12-04-05 pos A neg neg B NHS 364 01-02-05 pos A neg neg H NHS 549 12-04-05 pos A pos neg F NHS 392 14-02-05 neg C pos neg J Human 565 18-04-05 pos A neg neg B NHS 393 14-02-05 neg C pos neg J Human 566 18-04-05 pos C pos neg B Human 394 14-02-05 neg C pos neg J Human 567 18-04-05 pos A neg neg B NHS 409 14-02-05 neg A pos neg D NHS 569 18-04-05 pos C neg neg F NHS 419 21-02-05 pos C pos neg I Human 570 25-04-05 neg C pos neg J Human 420 21-02-05 pos A neg neg I NHS 582 26-04-05 pos C neg neg A NHS 422 22-02-05 neg E pos neg H UCF 599 09-05-05 neg C neg neg H NHS 423 22-02-05 pos A neg neg H NHS 601 10-05-05 neg C pos neg H Human 425 21-02-05 pos C pos neg B Human 603 10-05-05 neg C neg neg B NHS 429 22-02-05 neg C neg neg C NHS 604 10-05-05 neg A pos neg B NHS 430 28-02-05 pos A pos neg A NHS 607 10-05-05 neg A neg neg F Poultry 439 01-03-05 pos A neg neg G NHS 613 16-05-05 neg C pos neg J Human 444 01-03-05 pos A neg neg H NHS 615 17-05-05 pos C neg neg A NHS 446 01-03-05 pos C pos neg H Human 620 16-05-05 pos A neg neg H NHS 448 01-03-05 pos A pos neg B NHS 622 17-05-05 pos A neg neg H NHS 449 01-03-05 pos A pos neg B NHS 623 17-05-05 pos C neg neg H NHS 451 07-03-05 neg A neg neg K Poultry 624 17-05-05 neg E pos neg H UCF 463 08-03-05 pos C neg neg H NHS 625 17-05-05 neg E pos neg H UCF 464 08-03-05 pos A neg neg H NHS 626 17-05-05 neg C pos neg H Human 467 08-03-05 neg A pos neg D NHS 628 16-05-05 pos A neg neg B NHS 469 08-03-05 neg A neg neg D Poultry 629 16-05-05 pos E pos neg B UCF 470 14-03-05 neg C pos neg J Human 631 23-05-05 neg C pos neg E Human 471 14-03-05 pos C neg neg J NHS 633 23-05-05 neg C pos neg E Human 472 14-03-05 neg C pos neg J Human 640 24-05-05 pos E pos neg G UCF 474 14-03-05 pos C neg neg J NHS 641 24-05-05 pos E pos neg G UCF 475 14-03-05 pos C pos neg J Human 642 24-05-05 pos C neg neg G NHS 484 15-03-05 pos A neg neg B NHS 643 23-05-05 neg C pos neg H Human 493 29-03-05 pos A neg neg A NHS 644 23-05-05 neg C pos neg H Human 495 30-03-05 pos A pos neg A NHS 645 23-05-05 neg C pos neg M Human 502 29-03-05 pos C neg neg B NHS 646 23-05-05 pos C neg neg M NHS 503 29-03-05 pos C neg neg B NHS 504 29-03-05 pos A neg neg B NHS

Table 4. The distribution of biotypes within the slaughterhouses. Slaughterhouses Total A B C D E F G H I J K L M NHS 56 6 13 2 6 7 3 3 11 2 2 0 0 1

Human 29 0 4 0 0 3 0 0 6 1 12 0 2 1

UCF 8 0 0 2 0 1 0 2 3 0 0 0 0 0

Poultry 5 0 0 0 1 0 1 0 2 0 0 1 0 0

Ovine 1 0 0 0 0 0 0 0 0 0 0 0 1 0

Bovine 1 0 1 0 0 0 0 0 0 0 0 0 0 0

Total 100 6 18 4 7 11 4 5 22 3 14 1 3 2

Antibiotic sensitivity testing showed that 49% of the isolates were resistant to penicillin (Table 5). The distribution of penicillin resistance in relation to the biotypes is shown in Figure 1. The majority of the NHS was sensitive to penicillin whereas a majority of the isolates with the human biotype and all with UCF biotype were resistant to penicillin. The distribution of penicillin resistant isolates within the slaughterhouses is presented in Figure 2. All isolates from slaughterhouses A and D were penicillin sensitive. A high proportion (91%) of the 22 isolates from slaughterhouse H was resistant to penicillin and 11 of 14 isolates from slaughterhouse J were penicillin resistant. In contrast, only two of the 18 isolates from slaughterhouse B were resistant to penicillin.

Table 5. Distribution of MICs and resistance among the 100 isolates. Bold vertical lines indicate breakpoint for resistance. Percentage Distribution (%) of MIC values (µg/mL) Antibiotic resistant 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 256 Penicillin 49 2 28 10 7 4 49

Cephalothin 2 3 46 37 12 1 1

Oxacillin+2%NaCl 5 6 66 18 5 3 2

Erythromycin 6 53 41 2 4

Chloramphenicol 1 4 76 19 1

Clindamycin 1 53≤ 43 3 1

Tetracycline 1 55≤ 42 2 1

Fusidic Acid 2 6 38 53 1 1 1

Gentamicin 2 40≤ 46 12 2

Kanamycin 4 2 36 46 12 4

Ciprofloxacin 10 68 21 1

Trimethoprim 1 51 46 2 1

Virginamycin 1 6 69 20 4 1

Vancomycin 48≤ 50 2

Streptomycin 1 1≤ 21 43 30 4 1›

Avilamycin 29 38 31 2

45 40 35 30 25 20

isolates 15 10 5 0 NHS Human UCF Poultry Ovine Bovine biotypes

Figure 1. Distribution of penicillin resistance ( ) and penicillin sensitivity ( ) in relation to the biotypes.

isolates 10

0 ABCDEFGH I JKLM slaughterhouses

Figure 2. The distribution of penicillin resistance ( ) and penicillin sensitivity ( ) in the slaughterhouses.

Pulsed-field gel electrophoresis (PFGE) The results from PFGE are shown in dendrograms in Figure 4 and Figure 5. The isolates were divided into 50 different pulsotypes. In figure 5 the isolates are divided in groups A-K after the similarity of the patterns. The largest group (J) contained 15 identical isolates with the human biotype which was penicillin resistant. Group F had four pulsotypes consisting of 14 NHS biotypes and one poultry biotype. Eight of the Group F isolates were penicillin resistant. Six isolates with the UCF biotypes had three pulsotypes (group H) and were penicillin resistant.

Presence of enterotoxin genes Isolates with a similarity index less than 93% to another isolate were screened for the four enterotoxin genes sea, sec, seg and sei. The 47 selected isolates were all positive for spa, the gene encoding protein A, which confirms the classification as coagulase positive staphylococcus and that the DNA was of good quality. The number of isolates positive for 10

sea, sec, seg and sei were 6, 9, 25 and 25, respectively (Table 6). Thirty isolates had at least one enterotoxin gene. The six isolates carrying the sea gene were also penicillin resistant (Figure 3). The presence of enterotoxins is well spread in the dendrogram. Many of the isolates without any of the tested enterotoxin genes displayed also a unique pulsotype.

Table 6. The distribution of the biotypes and their enterotoxin genes. Biotype Isolates sea sec seg sei Isolates positive for se tested NHS 27 1 (4)* 4 (15) 13 (48) 14 (52) 16 (59) Human 13 5 (38) 5 (38) 9 (69) 9 (69) 11 (85) UCF 4 0 (0) 0 (0) 2 (50) 2 (50) 2 (50) Poultry 2 0 (0) 0 (0) 1 (50) 0 (0) 1 (50) Ovine 1 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Total 47 6 (13) 9 (19) 25 (53) 25 (53) 30 (64) * The number in parenthesis denotes percentage

5 isolates

0 sea sec seg sei enterotoxin genes

Figure 3. Distribution of penicillin resistance ( ) and penicillin sensitivity ( ) in isolates with enterotoxin genes.

DISCUSSION

A problem with the biotyping was that some of the frozen original isolates were not pure. They had to be streaked out again on Rabbit Plasma Fibrinogen Agar (RPFLA) plates. Another problem was the production of the fibrin plates. When they were incubated at 37°C for 24 h they became so bright that no clearing could be seen. We changed the production conditions in order to receive better fibrin plates. The biotyping is a relative easy method that does not require much hands-on time. The biotyping has been useful in tracing or estimating the origin of this organism in various food products, in the food industry and also in epidemiological investigations of food-poisoning outbreaks (9). However, several difficulties have been encountered when applying the biotyping method: incompatible results due to differences in test conditions, lack of standardized reagents, problematic reading of haemolysin, bovine plasma coagulation and crystal violet tests observed with some strains. Therefore other typing methods are needed in order to achieve a better discrimination between strains assigned to distinct biotypes e.g. PFGE (9). The PFGE was like an affirmation that the biotyping was correct and to control the heterogeneity of the isolates. When referring to PFGE, a number of restriction enzymes have been tested, but none has been found to be better than SmaI, which was used in the present study. If there are more than two differences in the band pattern, then the two isolates are deemed to be unrelated (25). Genomic DNA finger- printing by PFGE appears to have good discriminatory power and reproducibility (22). A French study on S. aureus from human, animal and food origin showed a high number of

PFGE patterns, like in the present study (9). Consequently, this shows that there is a high degree of genetic variability within the tested isolates. Isolate 540 (poultry) was identical with the NHS isolates in the dendrogram. It was tested twice with PFGE but gave the same result. If the zone of β-hemolysis had been broader then it had been classified as NHS. The biotyping revealed that 29% of the isolates were of the human biotype, which was unexpectedly high. Collin (2004) found that the prevalence of the human biotype among the isolates from chicken carcass was 1%. The differences between the two studies could be due to that pigs may have more contact with humans during rearing and slaughter in comparison to chicken. Slaughterhouses H and J had the highest share of human isolates. These two slaughterhouses are possible sources for contamination of the human biotype.

The fusidic acid on the panel VetMICTM GP-mo-A did not give accurate results. It was therefore analysed on a separate strip. Sometimes there was growth in wells with much higher antibiotic concentration, but not in wells with a lower antibiotic concentration. An advantage with the method is that 16 different antibiotics could be tested at the same time. In almost all studies of this nature, penicillin is associated with the highest frequency of resistant isolates, as was found in a study with Canadian dairy herds with 9.9% of isolates resistant (20). Erskine et al (2002) reported that 46% of the S. aureus isolates from dairy herds in Michigan were resistant to penicillin. In studies concerning European herds, penicillin resistance is more heterogeneous, ranging from only 2% (Norway) to more than 70% (Ireland), with an overall average of around 32% (20). Significant resistance to penicillin (64%) and ampicillin (46%) was reported in a study from Argentina looking at S. aureus from quarters with mastisis (20). The findings from a Swiss study including 142 S. aureus isolates from pig carcasses, found that 25% of the strains were resistant to any of the antimicrobials tested, mostly to penicillin and ampicillin (19). The study by Collin (2004) showed that 14% of the isolates from slaughtered chicken were resistant to penicillin. In the present study 49% of the isolates were penicillin resistant and resistance was more pronounced among the isolates with the human biotype and the unclassified isolates. This could be due to that humans are in more contact with penicillin and therefore are human isolates exposed to a higher selective pressure compared to isolates from animals. Slaughterhouse H and J had the highest percentage of penicillin resistant isolates. The majority of the isolates from slaughterhouse J were of the human biotypes. The pig meat from these slaughterhouses could be contamination sources of penicillin resistant staphylococci. Especially isolates with the human biotypes spread this property.

The advantage of real time PCR is that it amplifies and simultaneously quantifies the targeted DNA molecule. No gel electrophoresis and staining with ethidium bromide are required. The isolates with common pulsotypes were to a higher extent positive for a gene encoding an enterotoxin compared to isolates with an uncommon pulsotype. Consequently, the bigger groups were more toxogenic, presumed that the identical strains in the dendrogram have the same enterotoxin genes. In the present study, 64% of the tested isolates were positive for one or more enterotoxins. This correlates with that 50-70% of the strains produce enterotoxins. In the present study the highest occurence of genes were seg and sei, which was also the case in the study by Collin (2004) and a study on pork and chicken meat in Korea (10). It was demonstrated in a Frensh study that the seg and sei genes are present in S. aureus in a tandem orientation (11). However, in my study isolate 451 carried only seg and isolate 604 had only sei. Although 24 isolates carried both seg and sei and this was also seen in all enterotoxin positive isolates in the study by Collin (2004). Thus, it is very common that the genes seg and sei are present in the same isolate but it is not always the case. The prevalence of seg/sei, seg, sei and sec in pig carcasses from two EU-approved Swiss slaughterhouses were 63%, 31%, 4% and 2%, respectively. The screening included on sea to sed, seg, sei and sej. In contrast, other studies found that the classical enterotoxins, especially 12 sea and sec are more common in pork meat (19). This was also in accordance with the present study. The isolates with sea and sec can be considered as hazard to human health if the pig meat is stored at improper temperatures, where the bacterial number is growing and high amounts of enterotoxins produced. In the study by Collin on chicken meat none of the classical enterotoxin sea, seb, sec, sed were detected. This implicates that S. aureus from pig is more likely to cause food poisoning in comparison to chicken. Interestingly, the six isolates positive for sea were all penicillin resistant. Thus, it can be speculated that sea is linked to penicillin resistance. However, a Polish study on S. aureus from 44 inpatients and 66 carriers saw no correlation between antibiotic resistance and se genes (13).

In the future, all isolates should be tested for enterotoxins for more accurate results on the prevalence of genes and also screening of the other classic enterotoxin genes seb, sed and see should be performed. The classification of the coagulase positive staphylococci; S. aureus, S. intermedius and S hyicus should be carried out by incubation on maltose purple agar under aerobic conditions

ACKNOWLEDGEMENT

A big thank you to my supervisor Hans Lindmark, who was very supportive when I had questions about practical things and about writing. Thank you Ingela Tillander who helped me when I was performing the MIC testing. I also want to thank the other staff that helped me and for the nice atmosphere. My last thank you goes to the National Food Administration for the opportunity for me to perform my diploma work there!

REFERENCES

1. Berrada, H., S. J. Manes and Y. Picó. 2005. Real-time quantitative PCR of Staphylococcus aureus and application in restaurant meals. J. Food. Prot. 69:106-111.

2. Borch, E., T. Nesbakken, and H. Christensen. 1996. Hazard indentification in swine slaughter with respect to foodborne bacteria. Int. J. Food. Microbiol. 30:9-25.

3. Centi-Goga, B. T., M. Karama, P. V. Rossitto, R. A. Morgante and J. S. Cullor. 2003. Enterotoxin production by Staphylococcus aureus isolated from mastitic cows. J. Food. Prot. 66:1693-1696.

4. Clements, M. O., S. P. Watson and S. J. Foster. 1999. Characterization of the major superoxide dismutase of Staphylococcus aureus and its role in starvation survival, stress resistance, and pathogenicity. J. Bacteriol. 181:3898-3903.

5. de Neeling, A. J., M. J. M. van den Broek, E. C. Spalburg, M. G. van Santen- Verheuvel, W. D. C. Dam-Deisz, H. C. Boshuizen, A. W. van den Giessen, E. Duijkeren, and X. W. Huijsdens. 2007. High prevalence of methicillin resistant Staphylococcus aureus in pigs. Vet. Microbiol. 122:366-372.

6. Devriese, L. A. 1984. A simplified system of biotyping Staphylococcus aureus strains isolated from different animal species. J. Appl. Bacteriol. 56:215-220.

7. Ferris, M. M., X Yan, R. C. Habbersett, Y. Shou, C. L. Lemanski, J. H. Jett, T. M Yoshida and B. L. Marrone. 2004. Performance assessment of DNA fragment sizing by high-sensitivity flow cytometry and pulsed-field gel electrophoresis. J. Clin. Microbiol. 42:1965-1976.

8. Guardabassi, L., M. Stegger, and R. Skov. 2007. Retrospective detection of methicillin resistant and susceptible Staphylococcus aureus ST398 in Danish slaughter pigs. Vet. Microbiol. 122:384-386.

9. Hennekinne J. A., A. Kerouanton, A. Brisabois and M. L. Buyser. 2002. Discrimination of Staphylococcus aureus biotypes by pulsed-field gel electrophoresis of DNA macro- restriction fragments. J. Appl. Microbiol. 94:321-329.

10. Hwang, S. Y., S. H. Kim, E. J. Jang, N. H. Kwon, Y. K. Park, H. C. Koo, W. K. Jung, J. M. Kim and Y. H. Park. 2007. Novel multiplex PCR for the detection of the Staphylococcus aureus superantigen and its application to raw meat isolates in Korea. Int. J. Food. Microbiol. 117:99-105.

11. Jarraud, S., G. Gozon, F. Vandenesch, M. Bes, J. Etienne and G. Lina. 1999. Involvement of enterotoxin G and I in staphylococcal toxic shock syndrome and staphylococcal scarlet fever. J. Clin. Mikrobiol. 37:2446-2449.

12. Kwon, N. H., S. H. Kim, K. T. Park, W. K. Bae, J. Y. Kim, J. Y. Lim, J. S. Ahn, K. S. Lyoo, J. M. Kim, W. K. Noh, K. M. Noh, G. A. Bohach and Y. H. Park. 2004. Application of extended single-reaction multiplex polymerase chain reaction for toxin typing of Staphylococcus aureus isolates in South Korea. Int. J. Food. Microbiol. 97:137-145.

13. Lawrynowicz-Pasiorek, M., M. Kochman, K. Piekarska, A. Wyrebiak, E. Potracka, U. Leniak-Chimel and A. Maqdziak. 2006. Enterotoxin genes occurance among S. aureus strains isolated from inpatients and carriers. Med. Dosw. Microbiol. 58:275-81.

14. Le Loir, Y., F. Baron and M. Gaautier. 2003. Staphylococcus aureus and food poisoning. Genet. Mol. Res. 31:63-76.

15. Lindblad, M. 2006. Kartläggning av mikroorganismer på slaktkroppar. Report from national food administration.

16. Lloyd, D. H. 2007. Reservoirs of antimicrobial resistance in pet animals. Clin. Infect. Dis. 45:184-152.

17. Manfredi, R. 2007. Therapeutic perspectives of linezolid in the management of infections due to multiresistant Gram-positive pathogens. Recenti. Prog. Med. 98:143-154.

18. Mikolajczyk, R.T., U. Sagel, R. Bornemann, A. Krämer and M. Kretzschmar. 2007. A statistical method for estimating the proportion of cases resulting from cross-transmission of multi-resistant pathogens in an intensive care unit. J. Hosp. Infect. 65:149-155.

19. Nitzsche, S., C. Zweifel and R. Stephan. 2007. Phemotypic and genotypic traits of Staphylococcus aureus strains isolated from pig. Vet.Microbiol. 120:292-299.

20. Sabour, P. M., J. J. Gill, D. Lepp, J. C. Pacan, R. Ahmed, R Dingwell and K. Leslie. 2004. Molecular typing and distribution of Staphylococcus aureus isolates in eastern Canadian dairy herds. J. Clin. Microbiol. 42:3449-3455. 14

21. Sandel, M. K., and J. L McKillip. 2004. Virulence and recovery of Staphylococcus aureus relevant to the food industry using improvements on traditional approaches. Food. Contr. 15:5-10.

22. Shimizu, A., J. Kawano, C. Yamamoto, O. Kakutani and M. Fujita. 1997. Comparison of pulsed-field gel electrophoresis and phage typing for discriminating poultry strains of Staphylococcus aureus. Am. J. Vet. Res. 58:1412-1416.

23. van Duijkeren, E., R. Ikawaty, M. J. Broekhuizen-Stins, M. D. Jansen, E. C. Spalburg, A. J. de Neeling, J. G. Allaart, A. van Nes, J. A. Wagenaar, and A. C. Fluit. 2008. Transmission of methicillin-resistant Staphylococcus aureus strains between different kinds of pig farms. Vet.Microbiol. 4:383-389.

24. Weller, T.M.A. 2000. Methicillin-resistant Staphylococcus aureus typing methods: which should be the international standard? J. Hosp. Infect. 44:160-172.

25. Werckenthin, C., M. Cardoso, J-L. Martel, and S. Schwarz. 2001. Antimicrobial resistance in staphylococci from animals with particular reference to bovine Staphylococcus aureus, porcine Staphylococcus hycius, and canine Staphylococcus intermedius. 32:341-362.

26. Witte, W., B. Strommenger, C. Stantek, and C. Cuny. 2007. Methicillin-resistant Staphylococcus aureus ST398 in humans and animals, Central Europe. Emerg. Infect. Dis. 13(2):255-258.

APPENDIX

Dice (Opt:1.00%) (Tol 1.0%-3.0%) (H>0.0% S>0.0%) [0.0%-100.0%] iotype slaughterhouse slaughterhouse resistance penicillin smaI smaI isolate b enterotoxin

. 451 K Poultry - G . 469 D Poultry - - . 439 G NHS - G/I . 409 D NHS - G/I . 467 D NHS - - . 529 D NHS - - . 527 D NHS - - . 528 D NHS - G/I . 102 D NHS - G/I . 425 B Human - C/G/I . 475 J Human - C/G/I . 493 A NHS - C/G/I . 463 H NHS X C/G/I . 523 B Human X C/G/I . 549 F NHS - 0 . 419 I Human X 0 . 607 F Poultry X 0 . 449 B NHS - 0 . 569 F NHS - C . 566 B Human - C . 78 B Human - C . 565 B NHS - 0 . 546 B NHS - - . 567 B NHS - 0 . 642 G NHS - - . 484 B NHS - 0 . 521 B NHS - - . 136 G NHS - 0 . 430 A NHS - 0 . 495 A NHS - - . 235 A NHS - - . 448 B NHS - - . 504 B NHS - 0 . 628 B NHS - - . 345 C UCF X 0 . 353 I NHS X 0 . 420 I NHS X 0 . 508 C UCF X 0 . 423 H NHS X - . 444 H NHS X - . 363 H NHS X 0 . 364 H NHS X - . 464 H NHS X - . 540 H Poultry X - . 622 H NHS X - . 620 H NHS X - . 114 E NHS - G/I . 115 E NHS - - . 275 E NHS - - . 111 E NHS - - . 358 E NHS - G/I . 357 E NHS - - . 359 E NHS - - . 615 A NHS - G/I . 623 H NHS X G/I . 80 C NHS - G/I . 125 F NHS - - . 503 B NHS - - . 502 B NHS - - . 599 H NHS - - . 582 A NHS - - . 646 M NHS - - . 641 G UCF X - . 625 H UCF X - . 640 G UCF X - . 422 H UCF X - . 624 H UCF X G/I . 629 E UCF X G/I . 472 J Human X A/G/I . 470 J Human X - . 392 J Human X A/G/I . 394 J Human X - . 393 J Human X - . 613 J Human X - . 603 B NHS X G/I . 471 J NHS - C . 474 J NHS - - . 429 C NHS X A/G/I . 113 E Human X A/G/I . 284 H Human X A/G/I . 530 J Human X - . 532 J Human X A/G/I . 531 J Human X - . 539 L Human X - . 537 L Human X - . 533 J Human X - . 570 J Human X - . 626 H Human X - . 633 E Human X - . 631 E Human X - . 643 H Human X - . 645 M Human X - . 644 H Human X - . 536 L Ovine - 0 . 601 H Human X 0 . 446 H Human X G/I . 604 B NHS - I

Figure 4. Dendrogram based on SmaI-digested DNA from coagulase positive staphylococci from slaughtered pigs.

Group A

Dice (Opt:1.00%) (Tol 1.0%-3.0%) (H>0.0% S>0.0%) [0.0%-100.0%] smaI smaI 90 95 100 enterotoxin* . 451 isolate K slaughterhouse Poultrybiotype resistance penicillin - G . 469 D Poultry - - . 439 G NHS - G/I