Aus dem Department für Nutztiere und öffentliches

Gesundheitswesen in der Veterinärmedizin der

Veterinärmedizinischen Universität Wien

(Departmentsprecher: Univ. Prof. Dr. med.vet. Michael HESS)

Institut für Milchhygiene, Milchtechnologie und Lebensmittelhygiene

Occurrence and molecular epidemiology of Staphylococcus aureus

isolated from individual cows and during processing of a traditional

raw milk cheese in an alpine dairy

DIPLOMARBEIT

zur Erlangung der Würde einer

MAGISTRA MEDICINAE VETERINARIAE

der Veterinärmedizinischen Universität Wien

vorgelegt von

Simone Sagmeister

Wien, im November 2013

2

Supervisor

Dr. med. vet. Beatrix Steßl

Department für Nutztiere und öffentliches Gesundheitswesen in der Veterinärmedizin der

Veterinärmedizinischen Universität Wien,Institut für Milchhygiene, Milchtechnologie und

Lebensmittelwissenschaften

Reviewer

Univ.-Prof. Dr. med. vet. Drillich, Marc

Department für Nutztiere und öffentliches Gesundheitswesen in der Veterinärmedizin der

Veterinärmedizinischen Universität Wien, Bestandsbetreuung bei Wiederkäuern

This work was presented at the 33. Jahrestagung der ÖGHMP; MAY 21-24, 2012; Salzburg, AUSTRIA:

Tracing Staphylococcus aureus from individual cow to raw milk cheese at the end of ripening in an

alpine dairy (Authors: Sagmeister S., Klinger S., Wagner M., Stessl B.).

Furthermore, this work was presented at the Sennereiverband Südtirol with the title:

Nachweis von Staphylococcus aureus während der Schnittkäseherstellung in einer Almsennerei

(Authors: Sagmeister S., Klinger S., Wagner M., Stessl B.).

And finally the work was presented at the 53. Arbeitstagung des Arbeitsgebietes

Lebensmittelhygiene der DVG; SEP 25-28, 2012; Garmisch-Partenkirchen, GERMANY with the title:

Tracing Staphylococcus aureus reservoirs in an alpine dairy (Authors: Sagmeister S., Klinger S.,

Wagner M., Stessl B.).

3

Meiner Familie

4

ACKNOWLEDGEMENTS

I would like to thank:

Univ. Prof. Dr. med. vet. Martin Wagner for facilitating and implementing this diploma thesis

at the Institute of Milk Hygiene,

Dr. med. vet. Beatrix Stessl for her excellent care. Without her knowledge, patience and help

the work would not have come into existence in this form,

Sonja Klinger BSc, who has supported me so great in the laboratory and always had patience,

The employees of the Institute for Milk Hygiene, Milk Technology and Food Hygiene of the

University of Veterinary Medicine Vienna for their helpfulness and friendly cooperation,

have contributed to making this work,

The Südtiroler Sennereiverband and the leaders of alpine dairy for their cooperation.

Benjamin Höller and Ramona Stecher for your willingness to help in correcting the

manuscript and valuable suggestions for improvement,

All my friends who have supported me throughout my study and always had an open ear for

me and my concerns.

My greatest thanks go to my family who has enabled me this study and throughout the years always

supported and encouraged me. Special thank to Mama and Tata.

5

ABBRIVIATIONS ALOA Agar Listeria according to Ottaviani and Agosti API Analytical profile index Aqua dest. Aqua destillata aw Water activity BAM Bacterial analytical manual BHI Brain heart infusion BLEB Buffered Listeria enrichment broth Bp Base-pair BP Baird Parker agar

BPW Buffered Peptone water

BS Brown Swiss °C Degree Celsius Cfu Colony forming unit cm Centimetre CMT California Mastitis test DNA Deoxyribonucleic acid

dNTP's Desoxyribonukleosidtriphosphate E. Escherichia EC European Community EDTA Ethylenediaminetetraacetic acid EFSA European Food Safety Authority EN European norm EU European Union FB Fraser broth FDA U.S. Food and Drug Administration FIG. Figure g Gram(s) G Gravitational acceleration h Hour(s) HFB Half Fraser broth HF Holstein Friesian ISO International Organization for Standardization L. Listeria M Molar mass m Meter(s) mg Milligram(s) min. Minute(s) ml Millilitre(s)

MKTTn Muller-Kauffmann Tetrathionate-Novobiocin broth

6

ABBRIVIATIONS mm Millimetre(s) μl Microlitre(s) N Number (of samples) neg. Negative No Number nr. Number OCLA Oxoid Chromogenic Listeria agar

PALCAM Polymyxin Acriflavin Lithium-Chloride Ceftazidime Aesculin Mannitol agar PCA Plate count agar PCR Polymerase chain reaction PFGE Pulsed field gel electrophoresis pH pH value pos. Positive % Per cent RH Red Holstein

RVS Rappaport-Vassiliadis Soya Peptone broth S. Salmonella SCC Somatic cell count SI Simmental

SM2 Salmonella agar spp. Species pluralis subsp. Subspecies Tab. Table TBE Tris-Borate-Ethylenediaminetetraacetic acid TBC Total bacterial count TG Tyrolean Grey TSA Tryptic Soy agar TSA-Y Tryptic Soy agar plus 6% Yeast U Unit(s) V Volt(s)

VRB Violet Red Bile agar

7

1. INTRODUCTION ................................................................................................................... 10

1.1. BACKGROUND INFORMATION .......................................................................................... 10

1.2. GENERAL ASPECTS OF STAPYLOCOCCUS (S.) AUREUS....................................................... 11

1.2.1. History ............................................................................................. 11

1.2.2. Taxonomy and common characteristics .................................................. 11

1.2.3. Virulence ............................................................................................. 12

1.2.4. Staphylococcal enterotoxins (SEs) ........................................................... 12

1.2.5. Staphylococcal food poisoning (SFP) ....................................................... 13

1.2.6. Bovine Mastitis caused by S. aureus ........................................................ 14

1.2.7. Relevance of S. aureus in cheese ............................................................. 15

1.3. PROCESSING OF SEMI-HARD CHEESE MADE FROM RAW MILK ........................................ 17

1.3.1. Preparation of the milk ............................................................................ 17

1.3.2. Addition of the starter culture ................................................................. 17

1.3.3. Coagulation and cutting ........................................................................... 17

1.3.4. Stirring, heating and syneresis ................................................................. 18

1.3.5. Whey extraction ....................................................................................... 18

1.3.6. Moulding, pressing ................................................................................... 18

1.3.7. Brining ............................................................................................. 18

1.3.8. Ripening ............................................................................................. 19

1.4. LEGAL BASIS ....................................................................................................................... 20

1.4.1. European Commision (EC) regulation No. 853/2004 ............................... 20

1.4.2. Regulation (EC) No. 1441/2007................................................................ 21

1.4.3. Guidelines for good hygiene practice for the milk processing at alpine

dairies ............................................................................................. 23

INDEX

8

1.5. AIM OF THE STUDY ............................................................................................................ 23

2. MATERIALS AND METHODS ................................................................................................. 24

2.1. MATERIALS ........................................................................................................................ 24

2.2. METHODS .......................................................................................................................... 26

2.2.1. Herd characteristics and sampling ....................................................................... 27

2.2.1.1. Herd description-breeds ................................................................................. 27

2.2.1.2. Milking equipment, production and hygiene conditions ............................... 27

2.2.1.3. Sampling ......................................................................................................... 27

2.2.2. Cheese characteristics and sampling details ......................................................... 28

2.2.3. Microbiological investigation of samples ............................................................. 28

2.2.3.1. Investigation of pooled quarter milk samples ................................................ 28

2.2.3.2. Quantitative detection of hygiene indicator bacteria ....................................................... 29

2.2.3.3. Quantitative detection of S. aureus ................................................................................. 30

2.2.3.4. Qualitative detection of Listeria spp. ............................................................................... 30

2.2.3.5. Qualitative detection of Salmonella spp. ......................................................................... 30

2.2.4. Phenotypical confirmation of S. aureus ............................................................... 31

2.2.4.1. Clumping factor ........................................................................................ 31

2.2.4.2. Coagulase ............................................................................................. 31

2.2.4.3. Catalase ............................................................................................. 31

2.2.4.4. Biochemical profiles API ID 32 STAPH ...................................................... 31

2.2.4.5. Enterotoxin ELISA (RIDASCREEN® SET Total A-E) ..................................... 32

2.2.5. Confirmation based on molecular biological techniques ....................................... 32

2.2.5.1. DNA-ISOLATION with CHELEX ® 100 RESIN .............................................. 32

2.2.5.2. DNA-EXTRACTION with NucleoSpin® Tissue Kit ....................................... 33

2.2.5.3. PCR detection of the S. aureus nuc gene .................................................. 33

2.2.5.4. Detection of S. aureus enterotoxin genes ................................................ 34

2.2.5.5. Agarose Gel Electrophoresis .................................................................... 36

9

2.2.5.6. Genotyping using Pulsed Field Gel Electrophoresis (PFGE) ...................... 37

3. RESULTS .............................................................................................................................. 37

3.1. SAMPLE CHARACTERISTICS ............................................................................................... 37

3.1.1. Herd sample characteristics ..................................................................... 38

3.1.1.1. Somatic cell count ........................................................................................... 38

3.1.1.2. California mastitis test .................................................................................... 39

3.1.1.3. S. aureus status of pooled quarter milk samples ............................................ 40

3.1.2. Production sample characteristics ........................................................... 41

3.1.3. Microbiological investigation of samples ................................................ 43

3.1.3.1. Quantitative detection of hygiene indicator bacteria ....................................................... 43

3.1.3.2. Quantitative detection of S. aureus in production samples............................................... 44

3.1.3.3. Qualitative detection of L. monocytogenes and Salmonella spp. ...................................... 44

3.1.4. Variety within phenotypical S. aureus isolate confirmation .................... 44

3.1.5. Enterotoxin PCR ....................................................................................... 45

3.1.6. Enterotoxin ELISA results ......................................................................... 46

3.2. GENOTYPICAL SUBTYPING RESULTS .................................................................................. 47

4. DISCUSSION AND CONCLUSION ............................................................................................... 49

5. EXTENDED SUMMARY ............................................................................................................. 53

6. ZUSAMMENFASSUNG ............................................................................................................. 55

7. REFERENCES ........................................................................................................................... 57

8. APPENDIX ............................................................................................................................... 65

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1. INTRODUCTION

1.1. BACKGROUND INFORMATION Staphylococcus (S.) aureus is mostly prevalent on nasal membranes (anterior nares, nasopharynx)

and the skin of warm-blooded animals. Up to 30-50% of the human populations are carriers

(KLUYTMANS et al., 1997; LE LOIR et al., 2003).

S. aureus is an important bacterial agent having a major impact on human and animal health by

producing a wide range of toxins and is one of the five most common causes of nosocomial infections

(BEREKET et al., 2012). The different toxin types are affecting e.g. the skin, soft tissues, and

respiratory tract.

Furthermore, the agent is responsible for life-threatening diseases as the toxic shock syndrome and

complications during infection due to the development of therapeutic resistance. Methicillin

resistant S. aureus (MRSA) variants, as clonal complex (CC) 398 are worldwide distributed in livestock

(cattle, pigs) and humans (GRAVELAND et al., 2011; KRISHNA AND MILLER, 2012; DOYLE et al., 2012;

GRUMANN et al., 2013).

A further impact on human health is the fact that S. aureus is able to produce heat-stable

enterotoxins during food processing. Therefore, Staphylococcal food poisoning (SFP) is one of the

most prevalent zoonotic diseases (DINGES et al., 2000; BALABAN AND RASOOLY, 2000; LE LOIR et al.,

2003).

This risk is often associated with dairy products made from raw milk (DE BUYSER et al., 2001; OSTYN

et al., 2010). Many routes of S. aureus transmission are possible in the dairy chain (ARCURI et al.,

2010; ROSENGREN et al., 2010; STESSL et al., 2011; figure 1).

A major source of S. aureus contamination of raw milk is when shedded by cows suffering from

subclinical mastitis (AKINEDEN et al., 2001; HEIDINGER et al., 2009; SCHMID et al., 2009; SPANU et

al., 2012). A further way of S. aureus transmission is via mucosa and skin from farmers and food

handlers due to poor hygiene during production (GIAMMANCO et al., 2011; ARGUDIN et al., 2012).

A crucial point in the prevention of transmission of food borne pathogens is an effective cleaning of

milk equipment, because bacteria are surviving in biofilms growing on food residues (FOX et al.,

2005; OLIVER et al., 2005; GUTIÉRREZ et al. 2012).

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Herd level Dairy level Retail level

•Sheeded by cow

•Milking equipment

•Farmer

•Hygiene status of the dairy room

•Biofilms in the alpine dairy equipment

•Insufficient acidification

•Failure of starter culture, heat treatment

•Staff

•Alpine dairy equipment

multifactorial

Figure 1: Possible entrance of S. aureus in the alpine dairy.

1.2. GENERAL ASPECTS OF STAPYLOCOCCUS (S.) AUREUS

1.2.1. History

In 1882 the Scottish surgeon SIR ALEXANDER OGSTON described for the first time a previously

unknown bacteria in the pus from abscesses that looked like grapes and called it Staphylococcus.

Scientific description of the genus and establishment of the species S. aureus were made only 2 years

later in 1884 by ROSENBACH, who isolated the latter pathogen (JOHLER AND STEPHAN, 2010).

1.2.2. Taxonomy and common characteristics

The genus Staphylococcus is assigned to the family Micrococcaceae, which includes also the genera

Micrococcus, Stomatococcus and Planococcus. Actually, Staphylococcus genus currently 37 species

and several subspecies are known structural features of the cell wall and physiological, biochemical

and molecular properties (SCHLEIFER AND BELL, 2009).

In accordance with BERGEY´S MANUAL OF SYSTEMATIC BACTERIOLOGY S. aureus is a gram-positive,

psychrotrophilic, facultative anaerobic, non motile, non sporeforming and catalase-positive coccus,

often occurring in grape-like clusters. The cell wall is resistant to lysozyme but sensitive for

lysostaphin. The bacterium is able to grow in a wide range of temperatures from 10 to 45°C

12

(optimum of 30-37°C) and sodium chloride concentrations up to 25 % (SUTHERLAND et al., 1994).

The typical colony morphology varies in the pigmentation from golden-yellow, yellow, orange to

grey. S. aureus produces an extracellular nuclease protein (thermostable nuclease (TNase)) which is

heat stable (SCHLEIFER AND BELL, 2009).

1.2.3. Virulence

S. aureus is able to produce a wide range of clinical symptoms due to the diversity of virulence

factors (JOHLER AND STEPHAN, 2010). In detail, antibiotic resistance, capsules, coagulase, lipase,

hyaluronidase, protein A, fibronectin-binding protein and multiple toxins are important features

(LARKIN et al., 2009). Some of them are involved in tissue invasion of the host (ORTEGA et al., 2010).

Based on their biological activities virulence factors can be grouped into three functional categories:

adhesins, superantigens and invasins (AKINEDEN et al., 2006; GRUMANN et al., 2013). A selection of

virulence factors are showed in table 1.

Table 1: S. aureus virulence factors (Modified according to AKINEDEN, 2006).

Virulence factors Gene Effect

Clumping factor A clfA Fibrin coagulation

Clumping factor B clfB Fibrin coagulation

Protein A spa Protection against phagocytosis

Coagulase coa Fibrin coagulation

Toxic shock syndrome TSST-1 tst Erythema, shock, fever

Enterotoxins A-U sea-seu Food Poisoning

Exfoliative Toxin A, B eta, etb Toxic-epidemic necrosis

Haemolysin α,β,γ,δ hla,hlb, hlgA, hlgC, hlgB, hld Membrane destruction, hemolysis

Phospholipase C plC Tissue distruction

Staphylokinase sak Destruction of fibrin structures

Thermonuclease nuc Degradation of host nucleid acids

Panton-Valentine leukocidin PLV Leukocyte destruction,tissue necrosis

1.2.4. Staphylococcal enterotoxins (SEs)

S. aureus is a potential human and animal pathogenic organism which is able to produce a great

variability of toxins. To produce Staphylococcal enterotoxins (SEs), S. aureus has to multiply to cell

density of 106 colony forming units per gram (cfu/g) of food (DOYLE et al., 2012; LE LOIR et al., 2003).

SEs are resistant to harsh environmental conditions as heat treatment and low pH, and are still

13

present in food though the organism was inactivated (ORTEGA et al., 2010; HENNEKINNE et al.,

2012).

SEs are regarded as superantigens because they can bind to class II MHC molecules on antigen

presenting cells and stimulate the non-specific T-cell proliferation (LARKIN et al., 2009). The functions

of superantigen and emetic activity exist on separate domains of the protein (DINGES et al., 2000).

Traditionally, SEs have been subdivided into classical (SEA-SEE) and new types (SEG- SElX) (ARGUDIN

et al., 2010; HENNEKINNE et al., 2012; PODKOWIK et al., 2013). If the new SEs play a role in food

borne outbreaks is not yet been clear, because some are reported to have emetic activity but others

have to be tested (BOEREMA et al., 2006). With respect to this, LINA et al. (2004) suggested the term

“staphylococcal enterotoxin-like” (SEI).

Actually, more than 20 SEs are known, thereof SEA and SEB are the best characterized (CHIANG et al.,

2008; HENNEKINNE et al., 2010; PINCHUK et al., 2010). Furthermore, SEA is most responsible for

food poisoning, followed by SED and SEB, but the involvement of other classical SEs has also been

demonstrated (BALABAN AND RASOOLY, 2000; ARGUDIN et al., 2010). In table 2 characteristics are

listed that promote the growth of S. aureus inclusively SE production (BECKER et al., 2007).

Table 2: Qualities that support the growth of S. aureus and enterotoxin production (modified according to BECKER et al., 2007).

Factor Organism growth SE-production

Optimum Range Optimum Range

Temperature 37°C 7-48°C 40-45°C 10-48°C

pH-value 6-7 4-10 7-8 4-9.6

Water activity (aW) 0,99 0,83 -> 0.99 0,98 0,85 -> 0.99

Sodium chloride (%) 0 0-20 0 0-10

1.2.5. Staphylococcal food poisoning (SFP)

The European Food Safety Agency (EFSA) and the European Center of Disease Control (ECDC)

reported for the year 2011 that the third most relevant agents causing food related outbreaks were

toxin producers as Bacillus, Staphylococcus and Clostridium (EFSA and ECDC, 2013). BENNETT et al.

(2013) reported that outbreaks are often associated with errors in food processing and preparation

(93%) and food workers (55%) were the common source for S. aureus outbreaks. To cause symptoms

of staphylococcal intoxication an enterotoxin dose of 100 and 200 ng are sufficient (BENNETT et al.,

2005; SCHELIN et al., 2011). A selection of outbreaks involving SEs is presented in table 3.

14

SE intoxication is characterized by a short incubation period (2 to 6 h) after ingestion of

contaminated food, including symptoms as nausea, vomiting, abdominal cramps and diarrhea (U. S.

FOOD AND DRUG ADMINISTRASTION (FDA), 2013;

http://www.fda.gov/Food/FoodborneIllnessContaminants/CausesOfIllnessBadBugBook/ucm070015.

htm; accessed: 29.09.2013).

Table 3: Food borne SE intoxications reported within the timeframe 2003-2013 (modified according to HENNEKINNE et al., 2012).

Year Country Source Cases

2013 Republic of Korea Fried chicken 9

2009 France Soft cheese 23

2008 Switzerland Raw goat milk 3

2008 Japan Crêpes 74

2008 French district Pasta salad 100

2008 France Carribean meals 47

2008 Germany Pancakes filled with minced chicken 12

2007 Austria Pasteurized milk products 40

2007 Belgium Hamburger 15

2006 Austria Cooked rice, chicken wings 113

2006 France Coco nut pearls (Chinese dessert) 17

2005 India Potato balls > 100

2003 Norway Mashed potato 8

1.2.6. Bovine Mastitis caused by S. aureus

Bovine mastitis causes worldwide enourmous economic losses in the milk production (PEREIRA et al.,

2011). The most relevant bacterial organisms causing contagious mastitis are Streptococcus (Sc.)

agalactiae, Staphylococcus (S.) aureus, Corynebacterium (C.) bovis, Mycoplasma (M.) spp., and

Streptococcus (Sc.) dysgalactiae (FOX and GAY, 1993).

Contagious mastitis bacterial can be divided in two groups: major mastitis pathogens, more virulent

and damaging to the udder (S. aureus, Sc. agalactiae, Sc. uberis, Sc. dysgalactiae, coliforms), and

minor mastitis pathogens as C. bovis and coagulase-negative Staphylococci (REYHER et al., 2012).

Virulence factors involved in the pathogenesis of S. aureus intramammary infection (IMI) are listed in

table 1. S. aureus adheres and invades into mammary epithelial cells, endothelium and fibroblasts,

able to persist intracellularly after the escape from destruction by macrophages (KERRO-DEGO et al.,

2002). Recurrent and therapeutic resistant mastitis is often caused by biofilms in the udder

(MELCHIOR et al., 2006).

15

The main source of S. aureus spread within a dairy herd is contaminated milking equipment. Further

important factors influencing the S. aureus status of a herd are the type of barn, udder preparation,

hygiene during milk production, regular use of California mastitis test (CMT), dry cow treatment,

treatment duration and strategic culling (BARKEMA et al., 2006; BRITTEN, 2012; DUFOUR et al., 2012;

KEEFE, 2012). The sampling of bulk tank milk (BTM) is a helpful method to determine the S. aureus

herd status (FOX AND GAY, 1993; OLDE RIEKERINK et al., 2010).

S. aureus infections of cows are often characterized by high somatic cell counts (SCC), a S. aureus-

positive neighboring quarter, and palpable indurations in the affected quarter.

Furthermore, lesions or hyperkeratotic teat-ends favour udder infections (BHUTTO et al., 2010;

MORET-STALDER et al., 2009). Important factors decreasing the risk of IMI are an adequate teat-end

condition, cleaning the teats with hygienic wipes after premilking, wearing gloves during milking, and

regular udder disinfection (dipping) after milking (DELBES et al., 2006; DUFOUR et al., 2012).

A challenge for mastitis diagnostic in the lab are atypical small colony or coagulase negative variants

of S. aureus which are often overlooked in bacteriological mastitis diagnostic (AKINEDEN et al., 2011;

ATALLA et al., 2011).

1.2.7. Relevance of S. aureus in cheese

S. aureus in milk and dairy products represent 2-6% of bacterial food-borne diseases outbreaks, and

cheese and cheese products are the main vehicles of transmission (DE BUYSER et al., 2001;

LINDQVIST et al., 2002).

The main sources of S. aureus contamination during the processing of cheese are cows suffering from

subclinical mastitis, human cross-contamination during the handling with the raw material, poor

hygiene or manufacturing conditions, and the environment (JORGENSEN et al., 2005b; GIAMMANCO

et al., 2011; JAKOBSEN et al., 2011; ORTOLANI et al., 2010).

Every further step in cheese production increases the growth of S. aureus such as deficient

acidification or high temperature during ripening (HEIDINGER et al., 2009).

S. aureus are able to survive and multiply during raw milk cheese processing. Under normal

conditions S. aureus tends to increase 1.5 to 3 log units (even up to 5 log cycles when weak acidifying

starter cultures are applied to the processed milk) (ZANGERL AND GINZINGER, 2006).

The most sensitive part of the cheese making process present the first 24 hours, in which the growth

of S. aureus increases. After that period the count remains stable or even decreases. Therefore the

16

significant factors in controlling the bacteria are pH, temperature, and starter cultures (DELBES 2006;

LINDQVIST et al., 2002; ALOMAR et al., 2008).

In the processing of cheese, S. aureus can be killed or just stressed, but toxins are heatstable as

described in section 1.2.4. The application of lactic acid starter cultures usually inhibits the growth of

S. aureus. In cheeses processed without the addition of starter cultures an insufficient acidification is

an often observed problem, and the remaining lactose increases the risk of SEs producing S. aureus

growth (LE MARC et al., 2009; ROSENGREN et al., 2010).

In cheese with high water content COENEN (2000) detected 106 – 107 cfu S. aureus /g at the end of

the storing time.

The S. aureus risk of contamination in semi-hard cheese could be minimized by:

the monitoring of somatic cell counts (SCC) and initial S. aureus counts in the supplyied raw

milk,

limiting milk storage time and temperature,

and taking S. aureus process controls at the expected maximum concentration (JAKOB et al.,

2010; http://www.agroscope.admin.ch/publikationen/02121/03151/index.html?lang=en ;

accessed on: 27.09.2013)).

17

1.3. PROCESSING OF SEMI-HARD CHEESE MADE FROM RAW MILK The production of semi-hard raw milk cheese was performed based on following steps (http://www.suedtirolermilch.com/en/south-tyrol-milk/dairy-products/cheese-from-south-tyrol/; accessed: 20.09.2013):

1.3.1. Preparation of the milk An efficient cooling of bulk tank milk at 4° C and good udder and milk hygiene (wearing gloves,

dipping post milking, mastitis - and dry cow treatment, efficient cleaning of the milking equipment)

enables to maintain good quality milk for up to 2 days. At the alpine dairy the milk was stored in

great open bulk tanks with 600 l (figure 2) for 12 and 24 hours.

The fat content in milk should have 2.7 – 3.3 % and in cheese 45% (FDM = fat in dry matter) and has

an especially influence of consistence and flavor on the product. Therefore, the cream (for producing

butter) was removed from the milk surface in the bulk tanks. After this treatment the raw milk was

transferred in a copper vat with a 2.000 l capacity, and was warmed at 31.5°C – 32.5°C (figure 2).

1.3.2. Addition of the starter culture Controlled lactic acid development is very necessary for cheese making. Starter cultures support a

prematuration of milk, improve the coagulation and curd process, and have an important influence

on ripening.

There are two types of starter cultures, mesophilic as mainly present in buttermilk, or thermophilic

yoghurt cultures. Starters differ in their sensitivity to salt, temperature and pH and will continue to

ferment lactose until conditions within the cheese prevent it. The decision on the most appropriate

starter culture depends on traditional knowledge, desired flavor, and the extent of acid development

during manufacturing and in the finished cheese.

At the alpine dairy the starter culture MK 409 (mixed culture of Lactococci and Lactobacillus;

Agroscope, Berne, Switzerland) was applied. The measuremen of the demanded degree of acidity

(Soxhlet-Henkel; SH°) is shown in figure 2. 3.

1.3.3. Coagulation and cutting After correct acidification, the rennet is added to cause the coagulation by casein (optimal

temperature 31.5°C – 32.5°C, 30 min). In the traditional production of semi-hard raw milk cheese,

18

the milk is not heated >40° C to achieve the best yield of cheese loafs, and an excellent flavor from

the highly active milkflora and starter cultures.

During the curdling process a test based on the activity of rennet was performed (a knife was

inserted into the coagulated milk and retracted again (figure 2.4). When the gel was sufficiently

coagulated, the curd was cut with a harp (figure 2.5). The particle size of alpine dairy cheese curd is

very small, comparable to corn or wheat grain.

1.3.4. Stirring, heating and syneresis After cutting the curd, the stirring and heating process is following. Stirring the curd after whey

separation increases the rate of syneresis (rearrangement of casein molecules, results in a tightening

of the casein network) and the whey extraction.

The most important factors influencing the syneresis are the temperature, the decrease of pH after

cutting the curd, and pressure. Rapid decreases of pH and higher curdling temperatures (36°C – 46°C)

have a ppositive influence on whey extraction process (figure 2.6).

1.3.5. Whey extraction Separation of the whey from the curd can play a role in the texture of the cheese, as well as

influencing color and flavor. At this alpine dairy the whey and curd was pumped to a special

container for pressing. During pressing the whey was drained off (figure 2.7 and 2. 8).

1.3.6. Moulding, pressing After pressing the curd in the copper vat the curd was filled in moulds (figure 2.9). After the moulding

the cheese loafs were pressed for at least 6 h. The cheese loafs were turned several times to trigger

the whey extraction. An important fact in cheese manufacturing is that the acidification is ongoing

during mouding (terminal pH 5.15-5.30). Therefore it is important to keep the cheese loafs warm. If

the acidifaction is not well performed, and rest sugar is available for microorganisms, the risk for

pathogen growth is high.

1.3.7. Brining Salt supports the building of a cheese rind including ripening flora, extraction of rest water, and

conservs and flavors the cheese.

After pressing the cheese loafs were transferred into a brine bath for 1 day (figure 2.10). The brine

should be a saturated salt solution (12-15°C, 18 – 20 Baumé degree (Bé), pH 5). Hereafter, the

cheeses were rubbed with dry salt on the surface (figure 2.11).

19

1.3.8. Ripening Ripening transforms the fresh curd to a cheese with desired characteristics and refers to the

biochemical, microbiological, structural, physical and sensory changes that occur during storage.

During ripening lactic acid, proteins and fat are reduced, and salt will be distributed and the texture

of cheese will be changed.

The average ripening time of the alpine semi-hard cheese was three months at 12-15°C and 85 – 95

% humidity (figure 2.12 and figure 2.13).

In-house micro flora of a cheese plant (present in the brine, on the wooden shelves, and in the air of

the ripening room) is essential for the development of a complete and typical smear flora on the

product.

After salting a fast growth of yeasts is important because they make a de-acidification of the surface

and give acid sensitive bacteria the chance to cover the surface completely within days. Therefore,

the smearing of the cheeses was applied at the beginning daily. The smearing solution consisted of

water and whey with a 2% salt content.

Sometimes the surface of old cheeses is mixed into the smearing solution, but this technique involves

a higher risk for undesirable contamination (moulds, Enterococci, Enterobacteria, Pseudomonas,

Listeria spp. etc.). Therefore it is better to smear from the young to the old cheese and change often

the smear liquid.

20

Figure 2: Steps for semi-hard cheese production in the alpine dairy.

Fig. 2.1: Milk storage in open bulk milk tanks; Fig. 2.2: Milk in the cooper milk vat; Fig. 2.3: Determination of acid degree

(SH°); Fig. 2.4: Testing the degree of coagulation; Fig. 2.5: Cutting the curd with a harp; Fig. 2.6: Heating the curd; Fig. 2.7

and Fig. 2.8: Whey removal with a pump; Fig. 2. 9: Filling the mould; Fig.2.10: Cheese in the brine bath; Fig. 2.11.: Smearing

of the cheese; Fig. 2.12: Ripening room; Fig.2.13: Ripened cheese; Fig. 2.14: Transport from the alpine dairy to the farmers.

1.4. LEGAL BASIS

1.4.1. European Commision (EC) regulation No. 853/2004

Specific hygiene rules for the producing of foodstuffs are declared in the European Commision (EC)

regulation 853 (2004).

21

Important facts of the hygiene regulation 853 (2004) are listed below and in table 4:

‘raw milk’ is milk from lactating farm animals that has not been heated > 40°C,

Farm animals, which produce raw milk for human consumption have to be free from

infectious diseases and must be in a good health condition (no signs of enteritis, diarrhea,

fever, mastitis, udder wounds).

Essential is also to follow the withdrawal periods prescribed for medicinal products and to

avoid treatment with unauthorized substances.

Table 4: Somatic cell counts (SCC) and bacterial counts (BC) limits per ml for raw cow milk.

Raw cow milk

Limit Frequency Examination time

BC ≤ 100.000 at least 2/month* at collection

SCC ≤ 400.000 at least 1/month** at collection

Raw cow milk before processing

Limit Frequency Examination time

BC < 300.000 before processing

* Geometric mean over a two-month period, with at least two samples per month.

** Geometric mean over a three-month period, with at least one sample per month.

1.4.2. Regulation (EC) No. 1441/2007

The Regulation (EC) No. 1441/2007 is an amendment of the Regulation (EC) No. 2073/2005 on

special microbiological food process and safety criteria for foodstuffs.

Rules for sampling, testing and prescribed analytical reference methods acc. to International

Organization for Standardization (ISO) are described in (table 5).

22

Table 5: Food safety and process hygiene criteria for cheeses and milk products implemented in the European Regulation EC 1441/2007.

FOOD SAFETY CRITERIA

Food category Microorganisms Sampling plan Limits

Analytic reference method Product stage

n c m M

Cheese, milk powder and whey powder SEs 5 0 not detected in 25 g ELISA test advised by CRL Retail level, during the shelf-line

FOOD HYGIENE CRITERIA

Food category Microorganisms Sampling plan Limits Analytic

Reference method

Product stage Measures n c m M

raw milk cheeses CPS 5 2 104 cfu/g 105cfu/g EN/ISO 6888-2 * at the time of processing where the

number of CPS is expected to be highest

Improving the raw material and process hygiene conditions; In cases of CPS> 105cfu/g, cheese batches have to be tested for SEs

Cheeses made from pasteurized milk CPS 5 2 100 cfu/g 1000 cfu/g EN/ISO 6888-1 or 2

SEs= Staphylococcal enterotoxins; n = number of units comprising the sample; c = number of sample units giving values between m and M; m = the acceptable microbiological level in a sample

unit; M = the level which, when exceeded in one or more samples, would cause the lot to be rejected; ELISA = Enzyme-linked Immunosorbent Assay; CRL=Community reference laboratory;

* If values > 105cfu/g are detected, the cheese batch has to be tested for SEs;

23

1.4.3. Guidelines for good hygiene practice for the milk processing at alpine dairies

The goal of the guidline is the implementation of the EC regulation 852 and 853/2004 in alpine

dairies with difficult topographical circumstances.

In the Guideline pastures are characterized as facilities in alpine regions, only seasonal exploited of

May to October, the continuous grazing period shall be at least 60 days per growing season. Pastures

have to be recognized in Alpine pasture cadastres; normally they are located at altitudes from 800 to

2500 meters. Raw milk is processed without heat treatment to various typical local cheeses, mainly

hard and semi-hard cheese or sour milk and cream cheeses. The cheese processing in alpine dairies is

often difficult due to restricted access to electricity or water (Federal Ministry of Health (2007),

http://www.bmg.gv.at/cms/home/attachments/0/7/4/CH1285/CMS1136888872096/milchverarbeit

ung_auf_almen.pdf; accessed on: 12.07.2013).

1.5. AIM OF THE STUDY The aim of the study was to determine the hygiene status in a small alpine dairy in South Tirol during

the summer production of semi-hard cheese with special focus on the potential pathogen S. aureus.

The dairy herd was a mixed population from different farms and therefore the impact of lactating

cows on the occurrence of S. aureus during the raw milk cheese production was of special interest.

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2. MATERIALS AND METHODS

2.1. MATERIALS Table 6: Equipment and consumables.

Laboratory Equipment

DESIGNATION MANUFACTURER Bunsen burner Georg Becker, Ges.m.b.h.& Co, KG, Vienna, Austria Chef Mapper III Bio-Rad laboratories, Marnes-la-Coquette,France Centrifuge 5424 Eppendorf, Hamburg, Germany Freezer GFL, Burgwedel, Germany Gel Doc imaging system Bio-Rad laboratories, Marnes-la-Coquette,France Gloves m/l MSP medizintechnik GmbH, Vienna, Austria Falcon tubes Greiner, Frickenhausen, Germany Incubator 25° Ehret, Emmendingen, Germany Incubator 30° Ehret, Emmendingen, Germany Incubator 37° Ehret, Emmendingen, Germany Incubator 40° Ehret, Emmendingen, Germany Inoculation loops Sarstedt AG & Co., Nümbrecht, Germany Laboratory scale Sartorius, Göttingen, Germany Maximum recovery Tubes 1,5ml Axyen Inc., Union City, USA Pasteur pipettes Hecht-Assistent, Sondheim, Germany Petri dishes Sterilin Ltd., Newport, UK Pipettes 1 - 10 μl, 10 - 100 μl, 100 - 1000 μl Eppendorf, Hamburg, Germany Pipette tips 1 - 10 μl, 10 - 100 μl, 100 - 1000 μl Biozym, Vienna, Austria Plastic pasteur pipettes 1 - 10 ml Sarstedt AG & Co., Nümbrecht, Germany Power supply Power Pac 1000 Biorad, Vienna, Austria Shaking water bath GFL, Burgwedel, Germany UV-1800 UV-VIS Spectrophotometer SHIMADZU AUSTRIA HANDELS GESMBH, Vienna, Austria Stomacher lab blender Seward Ltd., Worthing, UK Stomacher bags Seward Ltd., Worthing, UK Stomacher filter bags Seward Ltd., Worthing, UK Thermocycler Eppendorf AG, Vienna, Austria Thermomixer compact Eppendorf AG, Vienna, Austria Tubes 0,5ml, 1ml, 1,5ml, 2ml Eppendorf, Hamburg, Germany Vortex IKA ®-Werke GmbH & Co. KG, Staufen, Germany

Chemicals and reagents

DESIGNATION MANUFACTURER Agarose Sigma-Aldrich, Vienna, Austria Aqua bidest Mayerhofer Pharmazeutika, Leonding, Austria Deionized,diethylpyrocarbonate (DEPC) water MBI Fermentas, St. Leon-Rot, Germany Ethanol 96 % Merck KGaA, Darmstadt, Germany

25

Mikrozid Schülke & Mayer, Norderstedt, Germany Nacl Merck KGaA, Darmstadt, Germany Ringer ´s solution Mayerhofer Pharmazeutika, Leonding, Austria Table 6 continued: Equipment and consumables.

Reagents and kits for DNA extraction and gel electrophoresis

DESIGNATION MANUFACTURER Chelex® 100 Resin Bio-Rad Laboratories, Marnes-la-Coquette, France Brome Phenol Blue Gene Ruler 50bp Ladder MBI Fermentas, St. Leon-Rot, Germany Brome Phenol Blue Gene Ruler 100bp Ladder MBI Fermentas, St. Leon-Rot, Germany Brome Phenol Blue Gene Ruler 1kp Ladder MBI Fermentas, St. Leon-Rot, Germany Glycerin Merck KGaA, Darmstadt, Germany Loading Dye Solution MBI Fermentas, St. Leon-Rot, Germany NucleoSpin® Tissue Kit Machery-Nagel, Düren, Germany Sybr Safe Invitrogen, Lofer, Austria TBE-Puffer 10x (Tris-Borat-EDTA-Puffer) Carl Roth, Karlsruhe, Germany Enrichment media

DESIGNATION MANUFACTURER BHI-Y Bouillon: Brain Heart Infusion plus 6 % Yeast Merck KGaA, Darmstadt, Germany Half-Fraser broth Biokar Diagnostics, France Fraser broth Merck KGaA, Darmstadt, Germany Buffered Peptonwater (ISO) Oxoid Ltd., Hampshire, UK Müller-Hinton-Boullion Oxoid Ltd., Hampshire, UK Müller-Kaufmann Tetrathionate-Novobiocin broth (MKTTn) Oxoid Ltd., Hampshire, UK Rappaport-Vassiliadis Soya Peptone (RVS) broth Oxoid Ltd., Hampshire, UK Plating media

DESIGNATION MANUFACTURER Tryptic soy agar plus 0.6% Yeast (TSA-Y) Merck KGaA, Darmstadt, Germany Violet Red Bile Lactose agar (VRBLA) Oxoid Ltd., Hampshire, UK Milk plate count agar with skimmed milk (PCA) Oxoid Ltd., Hampshire, UK Rose-Bengal Chloramphenicol agar (RBCA) Merck KGaA, Darmstadt, Germany Baird Parker (BP) agar Oxoid Ltd., Hampshire, UK Columbia agar plus 5 % sheep blood bioMérieux sa, Marcy l´Etoile, France chromID™ Salmonella agar (SM2) bioMérieux sa, Marcy l´Etoile, France

Aloa agar (Chromogenic Listeria-selective agar acc. to Ottaviani and Agosti) Merck KGaA, Darmstadt, Germany

Xylose-Lysine-Deoxycholate (XLD) agar bioMérieux sa, Marcy l´Etoile, France

PALCAM agar Biokar Diagnostics, Beauvais Cedex, France

Reagents and kits for microbiological confirmation

DESIGNATION MANUFACTURER API ID 32 Staph bioMérieux sa, Marcy l´Etoile, France Catalase test (3% Hydrogen peroxide) Sigma-Aldrich, Vienna, Austria Coagulase plasma Remel Europe, Ltd. Dartford, UK Ridascreen® SET Total A,B,C,D,E R-Biopharm AG, Darmstadt, Germany

26

Table 6 continued: Equipment and consumables.

Material and reagents for cryoconservation

DESIGNATION MANUFACTURER Aqua bidest. Mayrhofer Pharmazeutika, Leonding, Austria Brain Heart Infusion (BHI) Oxoid Ltd., Hampshire, UK Cryo tubes 2 ml Corning, VWR, Vienna, Austria Glycerol Merck KGaA, Darmstadt, Germany

Materials for Polymerase chain reaction (PCR) method

DESIGNATION MANUFACTURER Aqua bidest Mayerhofer Pharmazeutika, Leonding, Austria 10x PCR buffer Invitrogen, Lofer, Austria Magnesium chloride Invitrogen, Lofer, Austria dNTP Mix Thermo Fisher Scientific, Vienna, Austria Platinum® Taq DNA Polymerase Invitrogen, Lofer, Austria PCR Primer Eurofins MWG Operon, Ebersberg, Germany Materials for pulsed-field gel electrophoresis (PFGE)

DESIGNATION MANUFACTURER Aqua dest. Mayrhofer Pharmazeutika, Leonding, Austria Lysozyme Sigma Aldrich, Vienna, Austria Lysostaphin Sigma Aldrich, Vienna, Austria Proteinase K Roche Diagnostics GmbH Applied Science, Vienna, Austria Seakem Gold Agarose Lonza Group Ltd., Basel, Switzerland SmaI MBI Fermentas, St. Leon-Rot, Germany Sodium dodecyl sulfate Sigma Aldrich, Vienna, Austria TBE-Puffer 10x (Tris-Borat-EDTA-Puffer) Carl Roth, Karlsruhe, Germany XbaI MBI Fermentas, St. Leon-Rot, Germany

2.2. METHODS To evaluate the hygiene, pathogen, and potential pathogen (S. aureus) status during the production

of semi-hard cheese, sterile pooled quarter milk samples, bulk tank milk and important samples

during the processing of cheese were investigated during summer 2011. The alpine dairy was located

in the west of South Tyrol (Italy) above 2064 m altitudes. At the beginning of the summer about

1600l – 1700l of raw milk was obtained from 89 lactating cows, resulting in the production of 26 – 28

loafs of cheese.

27

2.2.1. Herd characteristics and sampling

From 15.06.2011 to 11.09.2011 about 89 dairy cows, 22 heifers, three dry cows, 16 calves and 51

pigs were farmed at the dairy. Five persons, including a cheesemaker and the herd management

team were responsible for the animals. Herd management data (owner, day of birth, lactation

number, insemination, California mastitis test (CMT), antibiotic and claw treatment) were recorded

in an accompanying check lists.

2.2.1.1. Herd description-breeds

The cattle grazing on the mountainland in summer 2011 originated from 46 dairy farms from several

small villages in the valley. The most present breed was Brown Swiss (n=71), followed by Simmental

(n=7), and Holstein Friesian (n=4), crossbreeds (n=5), Red Holstein (n=1), and Tyrol Grey (n=1).

2.2.1.2. Milking equipment, production and hygiene conditions

The dairy cows were milked twice per day by three milkers with bucket milker units. The milk

quantities were recorded on a daily base to calculate the milk yield per cow at the end of the

summer. At the beginning of milking the udder was prepared. Therefore the foremilk was discared in

a pre-teatcup, teats were cleaned with alcoholic disposable towels, and checked for the presence of

wounds or hyperkeratosis. After milking the teats were dipped in an iodine solution for disinfection.

The California Mastitis Test (CMT) was performed at least monthly to identify cows with higher

somatic cell counts (SCC) suffering from a milk secretion dysfunction or mastitis. The test is based on

a gel reaction of an anionic surfactant (Schalm solution) with theDNA present in somatic cells in the

milk. An inflammatory process is indicated by increased numbers of somatic cells. The gel reaction is

scored from+ to+++. A score of +++ is indicating a serious mastitis with more than 5,000,000 cells

present in the milk (http://milkquality.wisc.edu/wp-content/uploads/2011/09/california-mastitis-

test-fact-sheet.pdf; accessed on: 01.10.2013). Cows with positive CMT tests were treated with

antibiotics and the milk was discarded.

2.2.1.3. Sampling

At the beginning and end of the summer 2011 the district laboratory (Federation of alpine dairies in

South Tyrol) investigated the bulk milk samples from all alpine dairies for fat and protein content,

SCC, and the concentration of urea.

To determine the hygiene (SCC, aerobic mesophilic counts (AMC), coliforms, yeasts and moulds),

pathogen (L. monocytogenes, Salmonella spp.) and potential pathogen (S. aureus) status during the

28

processing of a semi-hard cheese lot production, sterile pooled quarter milk samples were taken

from each individual cow. Additionally, bulk tank milk and samples from important steps of cheese

processing (whey, curd, cheese after brine, cheese during different stages of ripening; n=104) were

collected. All samples were sent in cooled transport boxes (4°C) to the laboratory of the Institute of

Milk Hygiene (Vetmeduni Vienna) and investigated immeadiately after receiption.

2.2.2. Cheese characteristics and sampling details

The cheese type included in this study was a semi-hard cheese made from raw cow milk with an

average ripening time of three months (date of production: 01.08.2011). Sampling was performed

over a period of three months. Bulk tank milk, vat milk, whey and brine were transferred aseptically

into sterile bottles (250 ml) wearing sterile gloves. Curd and cheese from different processing steps

(after mould filling, during pressing, at different ripening stages and before consumption) were

packed into sterile Stomacher bags (for further sample details see table 7).

Physicochemical parameters (pH, temperature profiles, and water activity (aw)) were recorded during

the cheese production.

Table 7: Samples details during the production of semi-hard cheese.

Sample Processing stage Amount of samples Quantity Bulk tank milk day 1 5 250ml Whey day 1 5 250ml Curd day 1 3 25g Cheese on the press day 1; 2, 4 and 8h after pressing each 3 25g Cheese after brine day 2 3 25g Cheese day 7 3 25g Cheese day 14 3 25g Cheese day 28 3 25g Cheese before consumption 3 25g Brine after production 1 1l Smear after production 1 1l

2.2.3. Microbiological investigation of samples

2.2.3.1. Investigation of pooled quarter milk samples

The milk samples were centrifugated at 4,800 rpm for 5 min, the supernatant was discarded; the cell

pellet was streaked with a three loop inoculation onto Baird Parker and Columbia agar with 5% sheep

blood and incubated at 37°C for 24-48 h. The colonies were classified in typical and atypical. Up to

29

five suspicious colonies for S. aureus were confirmed by clumping factor and tube coagulase test to

detect pathogenic Staphylococci which are able to coagulate plasma. Furthermore, hemolysis,

lecithinase reaction and tellurite utilization were recorded.

2.2.3.2. Quantitative detection of hygiene indicator bacteria

Twenty-five grams of each solid sub sample (A, B, C) were transferred into sterile stomacher filter

bags, diluted in 225 ml of unsupplemented Buffered Peptone Water (BPW), and homogenized in a

Stomacher lab blender for three minutes (sample details table 7). Twenty-five ml of each liquid

sample (milk, whey, brine, and smear) was investigated directly in duplicates. All samples (liquid

samples and initial suspensions of solid matrices) were further serially diluted in ten-fold steps in

Ringer’s solution up to dilution -5. One ml of each step was investigated by pour plate method on

Milk plate count agar with skimmed milk (PCA), Rose-Bengal Chloramphenicol agar (RBCA) and Violet

Red Bile Lactose agar (VRBLA) in duplicates. To quantify the aerobic mesophilic ounts (AMC), PC agar

plates were incubated for 3-4 days at 30°C (ISO 4833 (2003). Yeasts and moulds were enumerated

after 4-7 days on RBCA (incubation temperature 25°C). Coliform counts (CC) were determined on

VRBLA after incubation for 24 h at 30°C (ISO 4832).

Furthermore, 50 ml of all liquid samples was centrifuged at 4800 rpm for 10 min. The pellet was

serially diluted as described before in Ringer´s solution and investigated with the pour plate method

as described before.

The colony forming units per ml or g sample were calculated as follows:

∑ C = Amount of all colonies on all countable agar-plates

n1 = Number of agar-plates counted in the first dilution

n2 = Number of agar-plates

d = Dilution factor of first dilution counted

Only plates (or replicate plates from the same dilution) with 10-300 colonies were counted.

30

2.2.3.3. Quantitative detection of S. aureus

S. aureus counts were determined on Baird Parker Agar according to ISO 6888-1: Horizontal method

for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) -

Part 1: Technique using Baird-Parker agar medium.

Twenty-five gram of each sample was diluted 1:10 in 225 ml Ringer solution. Initial dilutions were

homogenized in a stomacher for 3 min. Subsequently, 333 μl of each sample or initial suspensions (in

case of solid samples) was plated directly on Baird Parker agar plates and subsequently hundred

microliter of dilution was plated onto Baird Parker agar by spatula method. The plating was

performed in duplicates. Plates were incubated at 37°C for 24-48 hours and counted after 48 h.

Typical and atypical colonies from both plates were investigated further according to ISO 6888

(1999). The colonies were streaked on Tryptic Soy Agar plus 6% Yeast. After incubation for 24-48 h at

37°C colonies were confirmed as pure cultures and tested by clumping factor and tube coagulase

test.

2.2.3.4. Qualitative detection of Listeria spp.

Listeria spp. was isolated according to ISO 11290-1 (1996, Amd. 2004). Therefore, twenty-five g (solid

sample) or twenty-five ml (liquid sample) was added to 225 ml Half-Fraser broth, homogenized for

three minutes in a Stomacher lab blender and incubated at 37°C for 24 hours. The primary

enrichment was streaked on PALCAM agar and ALOA agar and the agar plates were incubated at

37°C for 24-48 hours. 100 μl of the primary enrichment (Half-Fraser) was transferred into the sondary

enrichment (Fraser Broth) and incubated at 37°C for 48 h. Subsequently, Fraser broth was streaked

on PALCAM and Agar Listeria acc. to Ottaviani and Agosti (ALOA) agar. Listeria spp. typical colony

morphology is described on PALCAM as black colonies with dark precipitate (esculin hydrolyzis), and

on ALOA as blue-green colonies (ß-D glucosidase cleavage.

L. monocytogenes, L. ivanovii and on some occasions L. seeligeri colonies appear surrounded by a

halo (Phospholipase C activity).

2.2.3.5. Qualitative detection of Salmonella spp.

The isolation of Salmonella spp. was carried out using the standard methodology ISO 6579 (2005).

Twenty-five g (solid sample) or twenty-five ml (liquid sample) of each sample was enriched in 225 ml

buffered Peptone water (BPW), after homogenized for 3 min. The primary enrichments were

31

incubated at 37°C for 18 hours. In the second step 1000 μl were transferred into Muller-Kauffmann

Tetrathionate-Novobiocin broth (MKTTn) (supplemented with novobiocin and iodine-iodide) and 100

μl into Rappaport-Vassiliadis Soya Peptone (RVS). MKTTn was incubated at 37°C for 24 hours and RVS

at 42°C for 24 h. After incubation, both enrichments were streaked on SM2 agar and XLD agar. The

plates were incubated at 37°C for 24-48 h. Salmonella spp.typical colonies are described pale pink to

mauve on SM2 agar (ß-galactosidase reaction) and black on XLD agar (hydrogen-sufide reaction;

xylose,and decarboxylate lysine positive).

2.2.4. Phenotypical confirmation of S. aureus

For the phenotypical identification pure cultures were needed, which were incubated at 37°C for 24

hours on Tryptic soy agar plus 0.6% Yeast (TSA-Y).

2.2.4.1. Clumping factor

Suspicous colonies were suspended in 30 μl of Coagulase Plasma on a sterile object slide. The

reaction was observed for immediate formation of macroscopic precipitates in the form of white

clumps. In cases of no clumping within 10 min, the colonies were further tested in the tube coagulase

test before reporting result as negative.

2.2.4.2. Coagulase

Suspicous colonies were suspended in 8 ml Brain Heart Infusion (BHI) broth and incubated overnight

(18h) at 37°C. Subsequently, 500 μl of Coagulase Plasma and 500 μl of the BHI broth were mixed and

incubated overnight at 37°C. The results were interpretated as follows: positive test result: clot

formation; negative test result: no clot, suspension remained homogenous.

2.2.4.3. Catalase

Suspicious colonies were mixed with one drop of hydrogen peroxide (H2O2). Catalase positive strains

builded bubbles due to the utilization of H2O2 to H2O and O.

2.2.4.4. Biochemical Profiles API ID 32 STAPH

A bacterial suspension of the S. aureus test strains with a turbidity equivalent of 0.5 (McFarland

standard) was prepared. Hereafter, 55 μl of the cell suspension was transferred in each cupule of the

stripes. URE, ADH and ODC were covered with 2 drops of paraffin oil. Then the lid was placed on the

strip and they were incubated at 36°C for 24 hours under aerobic conditions. After incubation a drop

32

of NIT 1 + 2 was added into the cupule 0.0, a drop of VP A + B into the cupule 0.1 and a drop of FB

into the cupules 0.2-0.5. A few minutes later the colour changes were determined.

2.2.4.5. Enterotoxin ELISA (RIDASCREEN® SET Total A-E)

The S. aureus test strains were activated in BHI broth and incubated at 37°C for 24 hours. The

overnight culture was centrifuged at 3.500 x g for 5 min. To avoid that bacterial cells were still

present in the enterotoxin-enzyme linked immunosorbent assay (ELISA), the supernatant was sterile

filtrated and 100 μl of the filtrate per well was used in the assay. Hundred μl each of the filtrate were

added to the wells A to G, and 100 μl of the positive control were added to well H. Liquid was

carefully removed after an incubation time of 1 h at room temperature. The wells were washed three

times with each 250 μl of washing buffer by applying a multichannel pipette. Subsequently, 100 μl of

enzyme conjugate were added to each well, the plate was manually gently agitated and incubated at

room temperature for 1 hour. Then the plates were washed again three times. Hereafter 50 μl of the

substrate and 50 μl of a chromogen indicator were added to each well and incubated in the dark at

room temperature for 30 min. Finally 100 μl of the stop solution were added to each well and the

absorbance was measured at 450 nm in a spectrophotometer.

2.2.5. Confirmation based on molecular biological techniques

Suspect S. aureus colonies were streaked on Tryptic soy agar plates with 6% yeast (TSA-Y) and

incubated at 37°C for 24 h to obtain pure cultures. DNA was extracted applying the Chelex Resin®

extraction protocol (WALSH et al., 1991) or the silica membrane based NucleoSpin® Tissue Kit.

2.2.5.1. DNA-ISOLATION with CHELEX ® 100 RESIN

Chelex® 100 Resin, a chelating agent, removes PCR inhibitors from the bacterial cell and facilitates

cell lysis after heating for 10 min at 100°C. After crude cell degradation polar Resin beads bind polar

cellular compounds. Non-polar nuclear DNA and RNA remains in water solution above chelex

(WALSH et al., 1991).

The first version of DNA extraction was to suspend the whole bacterial material grown on the agar

surface in 1 ml 0.01 M Tris-HCl and centrifugate the cells at 8000xg for 5 min. Hereafter, the

supernatant was discarded and the cell pellet was suspended in 100 μl 0.01 M Tris-HCl followed by

the addition of 400μl Chelex 100 Resin solution. The suspension was vortexed thoroughly, heated at

33

100°C for 10 min and centrifuged at 15,000 x g for 5 sec. Finally 100 μl of the supernatant were

transferred into a Maximum Recovery Tube and stored at -20°C for further molecular analysis.

A second version is applicable for colony confirmation. Therefore, one colony was suspended in 100

μl 0.01 M Tris-HCl, vortexed and 400 μl of the Chelex 100 Resin solution was added. The following

protocol is described in the previous paragraph.

2.2.5.2. DNA-EXTRACTION with NucleoSpin® Tissue Kit

With this method (silica matrix combined with a spin column method), genomic DNA can be

prepared from tissue cells and many other sources. The support protocol for bacteria was used. 1 ml

of bacterial culture was centrifuged for 5 min at 8,000 x g and the supernatant was removed and

transferred in 180 μl Nucleospin buffer plus Lysostaphin (0.2 mg/ml). Hereafter, 25 μl of Proteinase K

was added and incubated overnight at 56 °C in a shaking thermomixer. The day after, samples were

incubated at 70° C for 10 min with 200 μl Buffer B3 (Lysis). To optimize the DNA binding conditions

210 μl ethanol (96 – 100%) was added. By applying the samples to the Nucleospin Tissue Column and

following centrifugation (1 min at 11,000 x g) the DNA was binded to the silica membrane. The silica

membranes in the filters were washed twice (1st wash: 500 μl Buffer BW and 2nd wash: 600 μl

Buffer B5; both followed by centrifugation for 1 min at 11,000 x g). Then the silica membrane was

dried by another centrifugation step (again 1min at 11,000 x g). Finally, 100 μl of elution Buffer BE

(70°C) was used to elude the pure genomic DNA. Finally the DNA was transferred into a maximum

recovery tube and stored at – 20°C.

2.2.5.3. PCR detection of the S. aureus nuc gene

S. aureus was confirmed by a PCR method who amplified the nuc gene, which encodes the

thermostable nuclease of Staphylococcus aureus (BRAKSTAD et al., 1992). The total reaction volume

of each tube was 25 μl, consisting of 20 μl master mix and 5 μl DNA templates (table 8.1-8.3). With

every set of samples, there was a negative control (Aqua dest., Mayrhofer Pharmazeutika, Leonding,

Austria) in order to monitor a possible contamination, a DNA isolation control and a positive controls

(S. aureus NCTC 6571).Table 8.1: Target specific primers.

Primer Sequence nuc A1 5'-GCGATTGATGGTGATACGGTI-3' nuc A2 5'-AGCCAAGCCTTGACGAACTAAAGC-3'

34

Table 8.2: Mastermix for the S. aureus specific nuc gene PCR.

Components Final concentration Stock concentration μL in 1 tube Aqua dest. 11.45 10x puffer 1x 2.5 MgCl2 1.5 mM 50 mM 0.75 nuc A1 400 nM 5000 nM 2 nuc A2 400 nM 5000 nM 2 dNTP`s 200 μM 5000 μM 1 Taq pol (Plat.) 1.5 U 5 U/μl 0.3 Total 20 DNA template 5 Reaction volume 25 Table 8.3: PCR conditions.

Step Temperature Time Initialization 94°C 2 min Denaturation* 94°C 30 sec Annealing* 55°C 30 sec 30 cycles* Elongation* 72°C 1,5 min Final Elongation 72°C 3,5 min

2.2.5.4. Detection of S. aureus enterotoxin genes

The presence of enterotoxin genes (sea-sej) was determined by a multiplex PCR (GONANO et al.,

2009). Primers were combined to obtain two different sets of multiplex PCR: one set contained

primers for sea, seb, sec, sed, and see (PCR 1) and the other one for seg, seh, sei, and sej (PCR 2).The

total reaction volume of 25 μl of one PCR tube contained 20μl of the master mix and 5 μl DNA

template (table 9.1-9.6). Three positive controls were applied in this PCR: DSM 19040, DSM 19041

and DSM 19045.

Table 9.1: Target specific primers PCR 1

Primer Sequence GSEAR 1 5'-GGTTATCAATGTGCGGGTGG-3' GSEAR 2 5'-CGGCACTTTTTTCTCTTCGG-3' GSEBR 1 5'-GTATGGTGGTGTAACTGAGC-3' GSEBR 2 5'-CCAAATAGTGACGAGTTAGG-3' GSECR 1 5'-AGATGAAGTAGTTGATGTGTATGG-3' GSECR 2 5'-CACACTTTTAGAATCAACCG-3' GSEDR 1 5'-CCAATAATAGGAGAAAATAAAAG-3' GSEDR 2 5'-ATTGGTATTTTTTTTCGTTC-3' GSEER 1 5'-AGGTTTTTTCACAGGTCATCC-3' GSEER 2 5'-CTTTTTTTTCTTCGGTCAATC-3'

35

Table 9.2: Mastermix for the PCR 1.

Components Final concentration Stock concentration μL in 1 tube Aqua dest. 12.5 10x puffer 1x 2.5 MgCl2 4 mM 50 mM 2 GSEAR 1 300 nM 50000 nM 0.15 GSEAR 2 300 nM 50000 nM 0.15 GSEBR 1 300 nM 50000 nM 0.15 GSEBR 2 300 nM 50000 nM 0.15 GSECR 1 300 nM 50000 nM 0.15 GSECR 2 300 nM 50000 nM 0.15 GSEDR 1 300 nM 50000 nM 0.15 GSEDR 2 300 nM 50000 nM 0.15 GSEER 1 300 nM 50000 nM 0.15 GSEER 2 300 nM 50000 nM 0.15 dNTP`s 200 μM 5000 μM 1 Taq pol (Plat.) 1 U 5 U/μl 0.5 Total 20 DNA template 5 Reaction volume 25 Table 9.3: PCR conditions PCR1.

Step Temperature Time Initialization 95°C 5 min Denaturation* 94°C 30 sec Annealing* 55°C 30 sec 30 cycles* Elongation* 72°C 1 min Final Elongation 72°C 7 min 4°C ∞ Table 9.4: Target specific primers PCR 2.

Primer Sequence SEG1 5'-TGCTATCGACACACTACAACC-3' SEG2 5'-CCAGATTCAAATGCAGAACC-3' SEH1 5'-CGAAAGCAGAAGATTTACACG-3' SEH2 5'-GACCTTTACTTATTTCGCTGTC-3' SEI1 5'-GACAACAAAACTGTCGAAACTG-3' SEI2 5'-CCATATTCTTTGCCTTTACCAG-3' SEJF 5'-CATCAGAACTGTTGTTCCGCTAG-3' SEJR 5'-CTGAATTTTACCATCAAAGGTAC-3'

36

Table 9.5: Mastermix for the PCR 2.

Components Final concentration Stock concentration μL in 1 tube Aqua dest. 12.9 10x puffer 1x 2.5 MgCl2 4 mM 50 mM 2 SEG1 300 nM 50000 nM 0.15 SEG2 300 nM 50000 nM 0.15 SEH1 200 nM 50000 nM 0.1 SEH2 200 nM 50000 nM 0.1 SEI1 300 nM 50000 nM 0.15 SEI2 300 nM 50000 nM 0.15 SEJF 300 nM 50000 nM 0.15 SEJR 300 nM 50000 nM 0.15 dNTP`s 200 μM 5000 μM 1 Taq pol (Plat.) 1 U 5 U/μl 0.5 Total 20 DNA template 5 Reaction volume 25 Table 9.6: PCR conditions PCR 2.

Step Temperature Time Initialization 95°C 5 min Denaturation* 94°C 30 sec Annealing* 55°C 30 sec 30 cycles* Elongation* 72°C 1 min Final Elongation 72°C 7 min 4°C ∞

2.2.5.5. Agarose Gel Electrophoresis

For a medium-size gel 1.5 g of agarose (Sigma-Aldrich, Vienna, Austria) was mixed with 100 ml of

1xTBE-buffer and melted in a microwave for three minutes. 3.5 μl of SYBR Safe was added. The gel

was transferred into a gel casting platform and gel combs were inserted. After 30 minutes, during

which the gel had hardened, the combs were removed and thr gel was put into the electrophoresis

chamber, which was filled with 1xTBE-buffer. Fife μl of molecular weight standard (50bp-base,

100bp-base, 1kp-base depending on the fragment length) were added into the first and the last well

of every row. 10 μl of each sample, including positive control and negative control, were mixed with

a drop of sample loading buffer (33% glycerine, 0.07% brome phenole blue) and applied into the

wells. 120 V were applied on the gel constantly for 30 min. Finally, the gel was photographed with a

UV light (Gel Doc 2000, Biorad), to visualize PCR products.

37

2.2.5.6. Genotyping using Pulsed Field Gel Electrophoresis (PFGE)

Molecular subtyping of S. aureus was performed applying a SmaI restriction digest according to a

protocol published by MURCHANet al. (2003). S. aureus strains were cultivated on TSA-Y agar and

incubated at 37°C for 24 h. One colony of each test culture was inoculated in 8 ml BHI-broth and

incubated at 37°C for 16 hours. One ml was transferred into a new tube, centrifuged at 8.000 x g for

5 min and the supernatant was discarded. The pellet was suspended twice in cool 1ml PIV buffer and

vortexed at 8.000 x g for 5 min. Thereafter, 0.36 g SeaKem Gold agarose was melted in 30 ml PIV

buffer in the microwave and maintained in a liquid state at 56°C in the water bath. 300 μl of the

agarose were added to 300 μl cell suspensions and mixed and immediately dispensed into the wells

of the plug molds. After 20 min of hardening at room temperature the plugs were put into 5 ml cell

lysis buffer and incubated at 37°C overnight in a shaker water bath. At the next day the buffer was

discarded, the plugs were washed twice with 5 ml of preheated (54°C) ES buffer (3 μl RNAse

(100mg/ml); 55μl lysozyme (10mg/ml; 55 μl lysostaphin (5 mg/ml) and placed in a shaking water bad

for 10 min. Than 25 μl Proteinase K were added to 5 ml ES buffer and the plugs were placed also at

54°C for 2 hours in the water shaker bath. Finally the plugs were washed twice with 10 ml preheated

(54°C) aqua bidest, washed three times with 10 ml preheated 10 x TE buffer and stored at 4°C in TE

buffer. For the restriction digest, the plugs were sliced and the DNA was digested with SmaI (50

U/plug). The plugs were incubated at 25°C (samples) and at 37°C (marker) for four hours. The

emzyme mix was removed and the plugs were equilibrated in 0.5 X TBE buffer. The S. aureus plugs

were placed on a gel comb, including the PFGE global standard Salmonella braenderup strain H9812

as a size marker. 1.5 g SeaKem Gold agarose was melted in 150 ml 0.5 x TBE buffer. The agarose was

poured into the gel casting platform and the comb with the plugs was carefully put into the liquid gel.

The electrophoresis conditions in a Chef DR III system were: 5-15 sec for 7 hours, 15-60 sec for

16hours; 6 volts; included angle 120; cooling at 14°C. The gel was stained with ethidium bromide and

visualized under UV-light.

3. RESULTS

3.1. SAMPLE CHARACTERISTICS A total of 104 samples were collected at the alpine dairy. DNA was isolated from 74 pooled quarter

milk samples and 30 production samples.

38

3.1.1. Herd sample characteristics

Comparing the udder health status from 2010 and 2011 a higher SCC and mastitis prevalence was

noticed. Therefore antibiotic treatment was necessary listed in table 10. During summer 2011 the

amount of cows was changing due to cows suffering from claw problems, therapy resistant mastitis

and some cows went dry.

Table 10: Overall view of used antibiotics.

Name Active substance Pharmaceutical form Animal species

Withdrawal period for cattle (meat and

milk)

Cefatron®L Cefapirin sodium Intramammary suspension Cattle Meat: 10 days

Milk: 96 hours

Cloxalene®Plus Ampicillin + Dicloxacillin Intramammary suspension Cattle Meat: 10 days

Milk: 48 hours

Engemicina®D.D. Oxytetracycline intravenous intramuscular

Cattle, Horse, Sheep,GoatPig,

Dog, Cat

Meat: 27 days Milk: 5 days

Gentagil®Fortius Gentamycin sulfate intramuscular Cattle, Pig Meat: 63 days

Macramid® Lincomycin + Spectomycin intramuscular Cattle, Pig Meat: 20 days Milk: 72 hours

Mamyzin® Penethamate hydriodide intramuscular Cattle Meat: 10 days Milk: 4 days

Neopen® Procain benzylpenicillin + Neomycin intramuscular Cattle, Pig,

Dog, Cat Meat: 56 days

Milk: 5 days

Peracef® Cefoperazone Intramammary suspension Cattle Meat: 4 days

Milk: 108 hours

Repen® Procain

benzylpeniccillin + Dihydrostreptomycin

intramuscular Cattle, Horse, Sheep, Goat, Pig, Dog, Cat

Meat: 74 days Milk: 108 hours

Synulox® Amoxicillin +

Clavulanic acid + Prednisolone

Intramammary suspension Cattle Meat: 10 days

Milk: 48 hours

Vetil® Tylosine intramuscular Cattle Meat: 35 days Milk: 6 days

3.1.1.1. Somatic cell count

At the beginning and at the end of the summer the somatic cell counts (SCC) from all lactating dairy

cows was determined by the Sennereiverband. Most of the cows had at the beginning and end of

summer SCC ranging between 200,000/ml and 1,000,000/ml. At the beginning of the summer the

SCC was on average 301,193/ml. At the end of the summer the SCC increased (466,000/ml on

average; figure 3).

39

Figure 3: SCC distribution in percent of individual cow samples during summer 2011.

3.1.1.2. California mastitis test

Every month a California Mastitis Test (CMT) was perfomed to detect cows with higher SCC (table

11). At the beginning of summer the number of cows with a negative CMT was slightly higher (37%)

than at the end of summer (35 %). The milk from cows suffering from a subclinical or clinical mastitis

was discarded and antibiotic treatment was initialized (table 10, table 11, figure 4).

Figure 4: CMT results from summer 2011.

40

Table 11: Results of the monthly CMT.

Reaction Result 08.07.2011 05.08.2011 10.09.2011 Nr. of cows

(n=89) Nr. of cows

(n=85) Nr. of cows

(n=74)

Negative reaction 33 34 26

Reaction in 1 quarter

+

13

8

10

7

3

2 ++ 2 2 1

+++ 3 1 0

Reaction in 2 quarters

+ / +

16

4

16

7

20

11 + / ++ 7 1 1

+ / +++ 1 2 0 ++ / ++ 3 4 5

++ / +++ 1 1 3 +++ / +++ 0 1 0

Reaction in 3 quarters

+ / + / +

7

0

10

4

8

2 + / + / ++ 1 3 1

+ / ++ / ++ 4 2 4 + / ++ / +++ 1 1 0 ++ / ++ / ++ 1 0 1

Reaction in all quarters

+ / + / + / +

20

1

15

2

17

4 + / + / + / ++ 2 3 1

+ / + / ++ / ++ 1 3 4 + / + / ++ / +++ 2 1 1

+ / + / +++ / +++ 1 1 0 + / ++ / ++ / ++ 4 1 1

+ / ++ / ++ / +++ 0 1 0 ++ / ++ / ++ / ++ 7 0 3 ++ / ++ / ++ / ++ 0 2 0

++ / ++ / ++ / +++ 1 0 2 ++ / ++ / +++ / +++ 0 0 1

+++ / +++ / +++ / +++ 1 1 0

3.1.1.3. S. aureus status of pooled quarter milk samples

A screening analysis of sterile pooled quarter milk samples was performed from 74 cows. The quarter

milk samples were streaked directly and after centrifugation on both, Columbia agar plus 5% sheep

blood (CBA) and Baird Parker agar to compare the sensitivity of detection. A higher amount of S.

aureus positive samples were found by the loop inoculation of the selective agars from the milk

sediment (63.4 %). In further consequence suspicious isolates were divided due to their morphology

on agar into the categories "typical” and “atypical”. In total 104 Staphylococcus spp. isolates,

originating from 65 lactating cows were further investigated. Thereof, 63 isolates were confirmed as

41

S. aureus representing 54.1% of the pooled quarter milk samples. Furthermore, 41 Staphylococcus

spp. isolates were suspected as atypical S. aureus due to weak Lecithinase reactions and weak α-

hemolysis on blood agar. The most prevalent species among the latter atypical isolates were

confirmed by biochemical identification (API ID 32 Staph) as S. xylosus, S. warneri, S. epidermidis, S.

simulans, and Micrococcus spp (table 12).

Table 12: Biochemical results confirmed with API ID 32 STAPH.

Reaction S. aureus S. xylosus S. haemolyticus S. simulans S. epidermidis 1 S. warneri Urease V P N P P P Arginindihydrolase P N P P V V Ornithindecarboxylase N N N N N N Esculin N V N N N N Glucose P P P P P P Fructose P P P P P P Mannose P P N V V N Maltose P P P N P P Lactose P P P P P N Trehalose P P P P N P Mannit P P V V N V Raffinose N N N N N N Nitrat P P P P V V Voges Proskauer P V P N P P ß-Galactosidase N P N P N N Arginin N N N N N N ß-Naphtylphosphat P P N N P N Pyrrolidonylarylamidase V V P P N N Novobiocin N P N N N N Saccharose P P P P P P N-Acetyglucosamin P P P P N N Turranose P V V N P V Arabinose N V N N N N ß-Glucuronidase N P V V N V Ribose N N N N N N Cellobiose N N N N N N

P: positive; N: negative; V: variable.

3.1.2. Production sample characteristics

During the production of semi-hard cheese starting on the 01.08.2011, no deviations from the

processing protocols were evident. The physico-chemical parameters were in accordance with the

critical controlpoints defined in the cheese processing protocols of the Federation of alpine dairies in

South Tyrol (table 12). Interestingly the only deviation parameter was the pH after 2h of acidification

(6.33) which was slightly above the recommended values (5.7-5.9).

42

Table 13: Set point values and critical control points during the processing of alpine semi-hard cheese.

Production steps Parameters Setpoint values Production day 01.08.2011

Values Time

Milk storage

t (h) max 36 h 12 h 24 h temp (°C) max 8° 6.6° 6.6° amount (l) 1110 l 04:00 pH 6.79

Treatment temp (°C) 32°- 35° 34° 06:33 fat content (%) 3 - 3.5 % 3.20%

Preripening temp (°C) 38° 38.1°

Starter culture

pH sour sour 06:56 t (min) 7 - 10 h 9 h acidity (°SH) 38° - 45° 40° quantitiy 4 pipetts / l 2.8 l

Rennet quantity (ml) 18.5 ml / 100l 205.4 ml 07:14 Acidification temp (°C) 31.5° - 32.5° 32° Coaguation t (min) 30 - 40 min 37 min 07:46

Cutting t (min) 10 - 30 min 16 min 07:51 pH 6.61

Stiring t (min) 30 min 36 min Warming

Burning temperature temp (°C) 38° - 46° 43° 08:07 t (min) 30 min 36 min temp (°C) 40° - 46° 38°

Cooling temp (°C) 36° - 46° 38.2° 08:43 t (min) 10 - 40 min 46 min

Filling t (min) 10 - 15 min 10 min 09:30 Weight kg 5.0 - 7.0 kg 4.5 - 6.5 kg

43

Table 13 continued: Set point values and critical control points during the processing of alpine semi-hard cheese.

Production steps Parameters Setpoint values Production day 01.08.2011

Values Time Press t(h) 15 - 20 h 20 h 09:40

Turn

t(min) 10 min 10 min 30 min 30 min 1 h 1 h 2 h 2 h 4 h 4 h cheese temp (°C) 36° pH (2h) 5.7 - 5.9 6.33 cheese temp (°C) 29° pH (4h) 5.56 cheese temp (°C) 27.7° pH(8h) 5.33

Moulding

pH after pressing 5.15 - 5.3 5.07 cheese temp (°C) 19° room temp (°C) 12 - 15° 15.3° dry weight (kg) 112 kg

Brining

t (h max) approx. 24 h approx. 24 h 04:00 next day temp (°C) 12° - 15° 13.3° density(°Be) 18° - 20° 20° NaCl (%) 1.5 - 2 % 1.80% pH 5 5.3

Ripening temp (°C) 12° - 15° 12° - 16° humidity(%) 85 - 95 % 85 - 95 % t(mo) 3 months 4 months

Smear NaCl (%) 2% 2%

3.1.3. Microbiological investigation of samples

3.1.3.1. Quantitative detection of hygiene indicator bacteria

The production samples at each processing stage had aerobic mesophilic counts above 104cfu/ml.

Bulk tank milk samples had the lowest AMC with an average of 7.4x104cfu/ml. During processing the

AMC increased to a maximum of 109cfu/ml (ripened cheese A, B, C before consumption). Whey,

brine and cheese samples during ripening had AMC > 107 cfu/ml or g.

44

All thirty production samples were found to be positive for coliforms. The highest amounts of

coliform counts (CC) were found in the cheeses at the ripening age of 28 d with 7.5x105cfu/g and in

the brine with 3.0x105cfu/ml.

During the ripening of cheese also the growth of moulds and yeasts was steadily increasing with

amounts >104cfu/g for cheeses at day 14 until the end of ripening. The lowest concentrations moulds

and yeasts were found in following samples: bulk milk, whey, curd B and cheese on press 8h A with

<10cfu/ml or g.

3.1.3.2. Quantitative detection of S. aureus in production samples

Twenty-six processing samples (86.7%) were confirmed as S. aureus positive by nuc PCR performed

from Chelex DNA extracted from the whole agar plates. Hundred-two isolates were suspected as

typical or atypical S. aureus. Thereof, 79.4 % isolates were confirmed as S. aureus by nuc PCR. The

lecithinase-positive Staphylocci (LPS) counts ranged <10 cfu (cheese day 28 and before consumption)

to 1.8x108cfu/g during day 14 of ripening. The bulk tank milk was initially contaminated with 1.00

x102 LPS cfu/ml.

3.1.3.3. Qualitative detection of L. monocytogenes and Salmonella spp.

L. monocytogenes and Salmonella spp. was absent in all investigated samples.

3.1.4. Variety within phenotypical S. aureus isolate confirmation

For further characterization a subset of 49 S. aureus isolates from herd level (pooled quarter milk

samples) and 54 S. aureus isolates from production level were selected. A high percentage of S.

aureus isolates was found to be clumping factor (clf) negative. In detail, 71.4 % S. aureus isolates

from the herd level, and 68.5% of the processing level respectively, were tested clf negative.

Furthermore 48 (88.9%) production and 45 (91.8%) herd isolates were coagulase (coa) positive. Six

(11.1%) production and four (8.2%) herd isolates were coa negative. Three S. aureus isolated from

pooled bulk milk were found to be both, clf and coa negative. The latter strains were confirmed by

nuc PCR as S. aureus and showed a classical α/ß hemolysis. Four S. aureus, isolated during the

production of semi-hard cheese before brining and during the first 2h on the press, were atypical in

there morphology. The strains were confirmed nuc positive, but non-hemolytic, clf and coa negative.

45

Table 14: Detailed results of clumping factor and coagulase of S.aureus test strains.

Reaction Production isolates (n=54) Herd isolates (n=49)

clf + coa + 15 13 clf + coa - 2 1 clf - coa + 33 32 clf - coa - 4 3

Figure 5: Detailed results of clumping factor and coagulase of S.aureus test strains represented in percentage.

3.1.5. Enterotoxin PCR

The S. aureus strain subset described in section 3.1.4. was further characterized by enterotoxin PCR

(Appendix Table 1 and 2).

The 49 isolates from the herd level were all positive for Staphylococcal enterotoxin genes (seg). The

isolates showed most often the combination of sed and sej (22.4 %), followed by sea, see, seg

(14.3%), solely sea (12.2%), and sea, sed, see, sej (12.2%). S.aureus strains isolated during production

(n=54) were most often confirmed positive for following enterotoxin gene combinations: sea

(33.3%), sed, sej (13.0%), and sea, sed, seg, sej (9.3%) (figure 6). Ten production isolates were tested

negatively for enterotoxin genes.

46

Figure 6: S. aureus enterotoxin (se) gene combinations from herd and production isolates.

3.1.6. Enterotoxin ELISA results

For the phenotypical characterization two S. aureus herd, six S. aureus production, and three

Staphylococcus spp. isolates were tested. Comparing the genotypical with the phenotypical results,

the isolates with positive S. aureus enterotoxin gene profiles were found to be additionally

enterotoxin C positive (table15).

47

Table 15: Comparison between S. aureus enterotoxin gene PCR and ELISA.

Sample Int. Nr. nuc PCR Enterotoxin PCR ELISA Toxins

Tank milk PR 1 P sea, see, seg A, C, D, E

Whey PR 9 P sea, sed, see, seg, sej A, C, D, E

Curd PR 12 P sea, see, seg, sej A, C, D, E

Cheese on press 2h A PR 33 P sea C

Cheese before brine A PR 72 P sea, see, seg, sej A, C, D, E

Ripened cheese C PR 99 P sea, see, seg, sej A, C, D, E

Ripened cheese C PR 100 P sed, sej C, D

Bulk milk sample 4 SG 1 P sea, sed, see, sej A, C, D, E

Bulk milk sample 62 SG 22 P sea, see, sej A, C, D, E

Cheese 7d C PR 84 N N N

Cheese 14d A PR 86 N N N

Cheese 28d C PR 94 N N N

P: positive; N: negative.

3.2. GENOTYPICAL SUBTYPING RESULTS Genotypical analysis of 49 S. aureus herd isolates applying the restriction enzyme SmaI resulted in

five PFGE types (SA1-SA5) with subtypes (SA1ST1-SA1ST3; SA4 ST1). The 54 S. aureus production

isolates resulted in four PFGE types (SA1-SA4) with subtypes (SA1ST1-SA1ST3; SA4 ST1). The

predominant PFGE type SA1 was tested positive for the enterotoxin genes sea, sed, see, seg, sej. For

further information see figure 7 and 8, and Appendix table 1 and 2.

48

Figure 7: Percentage of S. aureus PFGE types in combination with enterotoxin gene profiles (shown in brackets).

49

Figure 8: Relationship between S. aureus herd and production PFGE types.

4. DISCUSSION AND CONCLUSION S. aureus is a commensal on nasal membranes and the skin of humans and warm-blooded animals.

Up to 30-50% of the human populations are carriers. Some strains possess a high virulence and cause

severe skin and tissue damages, and intoxications due to the production of exotoxins (toxic shock

syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins (DINGENS et al., 2000; KLUYTMANS et al.,

1997; JOHLER AND STEPHAN, 2010; LE LOIR et al., 2003). Major S. aureus reservoirs are the farm

environment, farm animals and also companion and free-ranging cats and dogs. DORADO-GARCÍA et

al. (2013) noticed during a longitudinal survey on veal-calves farms, a higher prevalence of Livestock-

associated methicillin-resistant S. aureus (LA-MRSA) on farms with MRSA carriers. The environmental

contamination played a major part in the transmission of LA-MRSA to farmers and their family.

A special risk of S. aureus transmission into the dairy chain is shedding by ruminants suffering from

subclinical mastitis (AKINEDEN et al., 2001; SPANU et al., 2012). Once present in a dairy herd this

potential pathogen is hardly eradicable using standard mastitis prevention protocols due to difficult

diagnosis, curability, and its contagiousness (ZADOKS et al., 2009).

A crucial point in the prevention of food borne pathogens transmission is effective cow hygiene, with

special focus to udder and milking hygiene. Additionally, barn type and farm environmental hygiene,

fly control and separating heifers from older cows have a major impact on udder health and milk

quality (MC DOUGALL et al., 2009). The potential of S. aureus to survive in biofilms growing on food

residues is extraordinarily high. S. aureus adheres preferable to hydrophobic surfaces at temperature

50

conditions present during the milking and processing of dairy products (GUTIÉRREZ et al. 2012;

PAGEDAR et al., 2010).

The aim of our study was to determine the hygiene and S. aureus status during the processing of

semi-hard cheese made from raw milk on a small alpine dairy in the summer months. There are not

many studies existing which focus on the S. aureus status of mixed dairy herd populations in

mountainous regions. In alpine countries it is tradition to keep dairy cows on the alpine pastures

during summer and produce artesian milk products (GRASSENI, 2012; GRASSENI, 2011; SEGATO et

al., 2010). The risk for subclinical mastitis is considerably higher for cows grazing in a mixed herd at

pastures of alpine dairies due to changes in feeding composition, conflicts in the novel herd

hierarchy, and where routinely applied CMT is lacking (BUSATO et al., 2000).

In our study the hygiene status and the presence/absence of pathogen bacteria (L. monocytogenes,

Salmonella spp., S. aureus) was investigated in a total of 104 milk and cheese processing step

samples.

A high percentage of lactating cows included in this study (>45%) had SCC between 200,000/ml and

1,000,000/ml during grazing in summer 2011, indicating the possibility of subclinical or clinical

mastitis. At the beginning of the summer the average SCC herd status was >300,000 cells/ml. At the

end the SCC increased >400,000 cells/ml. Furthermore, 54% of the pooled quarter milk samples were

found to be S. aureus positive. These findings and the higher antibiotic effort compared to the years

before, indicated a high prevalence of subclinical or not effectively cured mastitis at the home barns.

S. aureus is well described to cause subclinical mastitis, which remains persistent and increases

steadily the milk somatic cell counts (TAPONEN AND PYÖRÄLÄ, 2009). According to OSTERÅS et al.

(2008) treatment before drying-off should reduce the S. aureus prevalence in a dairy herd. Lactating

cows with SCC >400.000/ml milk before drying-off and clinical mastitis in the previous lactation

represent a major risk factor for untreated cows and heifers. Especially, the spread of clonal,

genetically indistinguishable S. aureus strains from cows to heifers is hardly to combat (CASTELANI et

al., 2013; MATYI et al., 2013). Some S. aureus strains are highly prevalent in dairy herds, as genotype

B in the Suisse dairy herds (87%; SYRING et al., 2012). MICHEL et al. (2011) indicated that S. aureus

positive dairy herds have a lower prevalence of other udder pathogens (CNS, Sc. uberis), due to an

omnipresence of the latter contagious organism. We also saw that a predominant S. aureus genotype

(SA1 and subtypes) was present in the majority of the lactating cows and during the processing of

51

alpine semi-hard cheese. This genotype was tested positive for the enterotoxin genes

sea/sed/see/seg/sej in herd and production isolates. Therefore an effective udder hygiene

monitoring program, including treatment of subclinical mastitis during lactation at the home barns is

necessary to avoid a general spread from cow to cow at the alpine dairy (BARLOW et al., 2013).

Effective measures against the S. aureus prevalence are widely discussed. An important key message

is hygiene: wearing gloves, pre-and post-milking teat treatment and disinfection, blanket dry cow

therapy, culling of therapy-resistant animals, housing practices, and having more housing pens for

different cattle groups (heifers, maternity pens) are some of the “non-invasive” suggestions

(DUFOUR et al., 2012; MC DOUGALL et al., 2009).

The increased determination of milk and udder hygiene parameters (SCC, CMT) is necessary

especially in alpine cheese production. Training programs for dairy farmers and alpine dairy staff are

highly needed (WALKENHORST, 2003; http://orgprints.org/2564/2/walkenhorst-2003-

alpenmilch.pdf; accessed on: 27.09.2013).

A further recommendation is the antibiotic therapy of S. aureus subclinical mastitis with e. g.

cefquinome, intra-mammary applied penicillin and pirlimicin, a practical approach with the intent to

improve cure rates (SAINI et al., 2012; SWINKELS et al., 2013). A radical antibiotic treatment has also

its bottleneck. The successful treatment of S. aureus was often associated with an increased number

of new infections with CNS or the development of higher antimicrobial resistant strains.

An additional approach is the often discussed S. aureus vaccination program, with certain

Staphylococcus aureus strains as SP 140 (Startvac®, HIPRA, S.A., Amer, Spain), or bacteriophage

mastitis treatments (DEBERDT et al., 2012; HAN et al., 2012; PEREIRA et al., 2011). The latter

prevention or therapeutically approaches are limited in alpine dairies, where cows of different

breeds, lactation status, and therefore different immune status are combined for a short period in a

barn and on alpine pastures.

The investigated production samples (n=30) included in this study, had AMC >104cfu/ml or g. The

lowest AMC was found in bulk tank milk with 7.4x104cfu/ml. The AMC continually increased during

processing up to 109cfu/g (ripened cheese). We found coliforms in all the production samples

(highest level cheese 28d A with 7.5x105cfu/g). Furthermore the growth of yeasts and moulds was

>104 cfu/g at all product stages. S. aureus was present in 86.7% of the processing samples. The

presence of enterotoxins was highly possible, because values >106cfu/g lecithinase-positive

52

Staphylococci (LPS) were detectable (cheese on press, cheese 7 and 14 days old). Most of the S.

aureus strains isolated in this study were tested positive for at least one enterotoxin gene. The

enterotoxin gene sea was most often detected, followed by sed and sej. The ELISA enterotoxin assay

detected additionally the presence of enterotoxin C. Discrepancies between immunological tests and

PCR results are not uncommon. DORES et al. (2013) found in 75% of samples taken during the

processing of Minas frescal cheese enterotoxin A present, but PCR targeting the enterotoxin genes

(seg) confirmed only one isolate positive. CREMONESI et al. (2007) found 14/33 raw milk cheese

samples positive for seg, but none of the samples harbored enterotoxins.

A higher S. aureus prevalence during the production of farmstead cheeses was also detected in other

studies (67% and 47.3 %; D'AMICO AND DONNELLY, 2010; JAKOBSEN et al., 2011). OPIYO et al. (2013)

recorded poor hygiene practices and microbiological quality in small scale dairies. Most operations

were manual and the process documentations were performed at minimal levels.

The growth and enterotoxin production of S. aureus is highly possible in the early 6h of cheese

production, in cases where no defined starter cultures are applied, or acidification due to starter

cultures is weak (pH>5.8) (DELBES et al., 2006; JAKOBSEN et al., 2011). Therefore we suggest to

determine physicochemical parameters (pH, temperature and aW) during all processing steps even in

alpine dairies. During the study we noticed, that a decrease of free water activity (aW=0.992 in

unripened cheese to 0.947 at the end of ripening) was an efficient barrier against the further

multiplication of S. aureus until the end of ripening (<10 LPS/g cheese at the end of ripening). But

also other parameters (high humidity in the ripening room, preventive bacterial flora and moulds on

the cheese surface) can prevent cheeses from further pathogen contaminations.

53

5. EXTENDED SUMMARY

The present work deals with the hygienic status of an alpine dairy located in South Tyrol. A special

focus was to determine the potential entry of Staphylococcus (S.) aureus during cheese production.

In summer 2011 the udder health, bulk milk tank status and management data were determined.

During the processing of Alpine semi-hard cheese (raw milk cheese) important physicochemical

parameters (pH, temperature profiles, water activity) were supplemented to the production protocol.

A total of 104 samples consisting of pooled quarter milk samples from individual cows (n =74), bulk

tank milk and samples during important processing steps (n=30) were taken. These were

characterized phenotypically and genotypically using standard protocols taking into account cultural,

biochemical and molecular biological aspects (PCR confirmation of the nuc gene). Additionally the S.

aureus isolates were tested for the sea-sej enterotoxin genes by multiplex PCR. The presence of

enterotoxins was determined by an enzyme linked immunosorbent assay (ELISA) including

staphylococcal enterotoxins A-E. To trace possible S. aureus contamination routes, a genotypical

isolate characterization was performed, applying the pulse-field gel electrophoresis (PFGE) method

including a restriction digest with SmaI.

A high percentage of lactating cows included in this study (>45%) had SCC between 200,000/ml and

1,000,000/ml during grazing in summer 2011, indicating the possibility of subclinical or clinical

mastitis. These findings and the higher antibiotic effort compared to the years before, indicated a

high prevalence of subclinical or not effectively cured mastitis at the home barns. About 54.1% of

individual cow samples and 86.7% of production samples were confirmed positive for S. aureus

targeting the nuc gene. The presence of enterotoxins was highly possible, because values >106cfu/g

lecithinase-positive Staphylococci (LPS) were detectable (cheese on press, cheese 7 and 14 days old).

From these samples, 103 isolates (49 from herd and 54 production level) were selected for further

studies. The multiplex-PCR revealed that 90.3% of the S. aureus test strains harbored at least one of

the following enterotoxin genes: sea, sed, see, seg, sei, and sej. Enterotoxin genes sea, sed, sej were

most often present in the S. aureus isolate set. The enterotoxin ELISA detected additionally

enterotoxin C. The PFGE analysis resulted in five, respectively four PFGE types including subtypes, for

the herd and production samples. One dominant S. aureus PFGE type (SA1 and subtypes) was

54

shedded by the majority of the cows and detected also in the cheese before consumption (<10

cfu/g).

The growth and enterotoxin production of S. aureus is highly possible in the early 6h of cheese

production, in cases where no defined starter cultures are applied, or acidification due to starter

cultures is weak (pH>5.8).

Therefore we suggest to determine physicochemical parameters (pH, temperature and aW) during all

processing steps even in alpine dairies. During the study we noticed, that a decrease of free water

activity (aW=0.99 in unripened cheese to 0.94 at the end of ripening) was an efficient barrier against

the further multiplication of S. aureus until the end of ripening (<10 cfu/g cheese at the end of

ripening). But also other parameters (high humidity in the ripening room, preventive bacterial flora

and moulds on the cheese surface) can prevent cheeses from further pathogen contaminations.

The increased determination of milk and udder hygiene parameters (SCC, CMT) is necessary

especially in alpine cheese production. Training programs for dairy farmers and alpine dairy staff are

highly needed. An important key message is hygiene: wearing gloves, pre-and post-milking teat

treatment and disinfection should be routinely applied. Furthermore, blanket dry cow therapy,

culling of therapy-resistant animals, and having more housing pens for different cattle and age

groups are also important measures to prevent udder and potential zoonotic pathogens.

55

6. ZUSAMMENFASSUNG

Die vorliegende Arbeit befasste sich mit dem Hygienestatus einer Alm-Sennerei in Südtirol mit

besonderem Augenmerk auf den potentiellen Eintrag von Staphylococcus (S.) aureus während der

Käseverarbeitung. Im Sommer 2011 wurden die Euter-Gesundheit, der Tankmilch-Status und

Management Daten erhoben. Während der Produktion des Alpkäses (Rohmilch-Schnittkäse) wurde

das Produktions-Protokoll mit wichtigen physikalisch-chemischen Parametern (pH, Temperatur-

Profile, Wasseraktivität) ergänzt. Insgesamt wurden 104 Proben, bestehend aus sterilen

Sammelgemelken (n=74), Tankmilch- und verschiedenen Produktions-Proben (n=30) entnommen.

Diese wurden anhand von Standard Protokollen unter Berücksichtigung kultureller, biochemischer

und molekularbiologischer Aspekte phäno- und genotypisch charakterisiert (PCR-Bestätigung des

nuc-Gens). Desweiteren wurden die S. aureus Isolate mittels Multiplex-PCR auf die Präsenz von

Entertoxin-Genen (sea-sej) untersucht. Die Anwesenheit von Enterotoxinen (A-E) wurde mittels

Enzyme Linked Immunosorbent Assay (ELISA) überprüft. Um die möglichen Kontaminationsrouten

von S. aureus zu beschreiben, wurde eine genotypische Isolatcharakterisierung mittels Pulsfeld-

Gelektrophorese (PFGE) und Restriktionsenzym SmaI durchgeführt.

Ein hoher Prozentsatz der in dieser Studie untersuchten Milchkühe (>45%) hatte somatische

Zellzahlen zwischen 200.000 und 1.000.000/ml im Sammelgemelk, die auf eine mögliche subklinische

oder sogar klinische Mastitis hinwiesen. Diese Feststellung und der höhere antibiotische

Therapieaufwand verglichen mit den Vorjahren, waren ein Hinweis auf nicht vollständig ausgeheilte

Mastitiden in den Herkunftsställen.

54,1% der Sammelgemelks- und Tankmilchproben und 86,7% der Produktions-Proben wurden positiv

auf das S. aureus nuc-Gen getestet. Das Vorhandensein von Enterotoxinen war sehr wahrscheinlich,

da Werte >106cfu/g für Lecithinase-positive Staphylokokken detektiert wurden (Käse während dem

Pressen, 7 Tage und 14 Tage alter Käse).

Von diesen Proben wurden 103 Isolate (49 individuelle und 54 Produktions-Proben) für weitere

Untersuchungen ausgewählt. Die Mulitplex-PCR ergab, dass 90,3% der S. aureus Teststämme

zumindest eines der folgenden Enterotoxin-Gene aufwies: sea, sed, see, seg, sei, und sej, wobei sea,

sed und sej am häufigsten auftraten. Im Enterotoxin-ELISA wurde zusätzlich Enterotoxin C

nachgewiesen. Die PFGE–Analyse ergab bei den Herdenproben vier, bei den Produktionsproben fünf

verschiedene PFGE-Typen inkl. Subtypen. Ein dominanter S. aureus PFGE-Typ (SA1 und Subtypen)

56

wurde von der Mehrheit der Kühe ausgeschieden und auch im Käse vor Verzehr detektiert (<10

cfu/g).

Wenn keine definierten Starterkulturen eingesetzt werden oder keine effektive Ansäuerung (pH>5.8)

stattfindet, ist eine Vermehrung und Enterotoxinbildung durch S. aureus vor allem in den ersten 6h

der Käseproduktion sehr wahrscheinlich.

Aus diesem Grund empfehlen wir, physikalisch-chemische Parameter (pH, Temperatur und

Wasseraktivität (aw)-Wert) während aller Produktionsschritte auch in Almbetrieben zu erheben. In

dieser Studie fiel auf, dass eine Abnahme der freien Wasseraktivität (aW=0.99 in unreifem Käse und

0.94 in reifem Käse) einen effizienten Schutz vor der Vermehrung von S. aureus (<10 cfu/g Käse am

Ende des Reifeprozesses) darstellt. Auch andere Parameter (hohe Luftfeuchtigkeit im Reiferaum,

Präventivflora und oberflächlicher Reifungsschimmel) sind in der Lage, den Käse vor pathogenen

Kontaminationen zu schützen.

Häufigere Erhebung von Milch- und Euter-Hygieneparametern (SCC, CMT) ist besonders in alpinen

Käse-Produktionsstätten wichtig. Trainings-Programme für Milchbauern und Mitarbeitern von

alpinen Molkereibetrieben werden dringend benötigt. Eine bedeutende Kernaussage ist die Hygiene:

das Tragen von Latexhandschuhen, Zitzenbehandlung und – Desinfektion vor und nach dem Melken

sollten routinemäßig angewendet werden. Die Trockenstelltherapie, das Keulen von

therapieresistenten Tieren und die Separierung von verschiedenen Alters- und Tiergruppen sind

ebenfalls wichtige Maßnahmen zur Prevention von Euter- und potentiell lebensmittelpathogenen

Erregern.

57

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EUROPEAN COMMISSION (2007): Commission Regulation (EC) No 1441/2007 of 5 December 2007 amending Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs.Off.J.Eur.Union L 322, 1-18.

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ISO 11290-1 (1996/Amd. 2004): Microbiology of food and animal feeding stuffs-horizontal method for the detection and enumeration of Listeria monocytogenes Part 1: Detection. International Organisation for Standardization. Geneva, Switzerland.

ISO 6888-1 (1999): Microbiology of food and animal feeding stuffs -- Horizontal method for the enumeration of coagulase-positive staphylococci Staphylococcus aureus and other species -- Part 1: Technique using Baird-Parker agar medium. International Organisation for Standardization. Geneva, Switzerland.

ISO 4833 (2003): Microbiology of food and animal feeding stuffs -- Horizontal method for the enumeration of microorganisms -- Colony-count technique at 30 degrees C. International Organisation for Standardization. Geneva, Switzerland.

ISO 6579 (2005): Microbiology of food and animal feeding stuffs -- Horizontal method for the detection of International Organisation for Standardization. Geneva, Switzerland.

ISO 4832 (2006): Microbiology of food and animal feeding stuffs -- Horizontal method for the enumeration of coliforms -- Colony-count technique. International Organisation for Standardization. Geneva, Switzerland

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8. APPENDIX

Table 1: Phenotypical and genotypical characterization of 49 S. aureus herd isolates.

Sample Code Lecithinase Hemolysis Clf Coa +/- PFGE-type Enterotoxin PCR

Pulsotypes

SG 23 pos α/ß neg pos SA5 A neg neg neg E G neg neg neg SG 26 pos α/ß neg pos SA1 A neg neg neg E G neg neg neg SG 22 pos α/ß pos pos SA1 A neg neg neg E neg neg neg J SGS 54 pos α/ß neg pos SA1ST1 A neg neg neg neg neg neg neg J SGS 56 neg α/ß neg pos SA1ST1 A neg neg neg neg neg neg neg J SG 32 neg α/ß neg pos SA1ST3 A neg neg neg neg neg neg neg J SGS 39 pos α/ß neg pos SA4 A neg neg neg neg neg neg neg J SGS 40 pos α/ß neg pos SA4 ST1 A neg neg neg neg neg neg neg J SG 5 pos α/ß pos pos SA1 neg neg neg D neg neg neg neg J SGS 8 pos α/ß pos pos SA1 neg neg neg D neg neg neg neg J SGS 25 pos α/ß pos pos SA1ST1 neg neg neg D neg neg neg neg J SG 17 pos α/ß neg pos SA1ST2 neg neg neg D neg neg neg neg J SGS 37 pos α/ß pos pos SA1ST2 neg neg neg D neg neg neg neg J SG 21 pos α/ß neg pos SA1ST2 neg neg neg D neg neg neg neg J SGS 36 pos α/ß neg pos SA1ST2 neg neg neg D neg neg neg neg J SG 18 pos α/ß pos pos SA1ST2 neg neg neg D neg neg neg neg J SG 19 pos α/ß neg pos SA1ST2 neg neg neg D neg neg neg neg J SGS 38 pos α/ß neg pos SA1ST2 neg neg neg D neg neg neg neg J SGS 35 pos α/ß neg pos SA3 neg neg neg D neg neg neg neg J SGS 57 neg α/ß neg pos SA1ST3 neg neg neg neg neg neg neg neg J SGS 47 neg α/ß neg pos SA2 neg neg neg neg neg neg neg neg neg SGS 48 neg α/ß neg pos SA2 neg neg neg neg neg neg neg neg neg SG 28 neg α/ß neg neg SA2 neg neg neg neg neg neg neg neg neg SGS 9 pos α/ß pos pos SA1 neg neg neg neg neg neg neg neg neg Neg = negative; pos = positive; Clf=clumping factor; coa=coagulase; PFGE-type=Pulsed-field gelelectrophoresis-type;

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Table 1 continued: Phenotypical and genotypical characterization of 49 S. aureus herd isolates.

Sample Code Lecithinase Hemolysis Clf Coa +/- PFGE Enterotoxin genes (A-J)

Pulsotypes SGS 43 pos α/ß neg pos SA1 neg neg neg neg neg neg neg neg neg SGS 33 pos α/ß neg neg SA1ST1 neg neg neg neg neg neg neg neg neg SGS 63 pos α/ß neg pos n. t. A neg neg neg neg neg neg neg neg SGS 53 pos α/ß neg pos SA2 A neg neg neg neg neg neg neg neg SG 8 pos α/ß neg pos SA1 A neg neg neg neg neg neg neg neg SGS 15 pos α/ß neg pos SA1 A neg neg neg neg neg neg neg neg SGS 19 pos α/ß neg neg SA1 A neg neg neg neg neg neg neg neg SGS 28 pos α/ß neg pos SA1ST1 A neg neg neg neg neg neg neg neg SG 16 pos α/ß neg pos SA2 A neg neg D neg neg neg neg J SGS 2 pos α/ß neg pos SA1 A neg neg D neg neg neg neg J SG 30 pos α/ß neg pos SA1ST1 A neg neg D neg neg neg neg J SG 31 pos α/ß neg pos SA1ST1 A neg neg D neg neg neg neg J SGS 1 pos α/ß pos pos SA1ST1 A neg neg D neg neg neg neg J SGS 10 pos α/ß neg pos SA1 A neg neg D E neg neg neg J SG 1 pos α/ß pos pos SA1 A neg neg D E neg neg neg J SG 6 pos α/ß neg pos SA1ST2 A neg neg D E neg neg neg J SGS 7 pos α/ß pos pos SA2 A neg neg D E neg neg neg J SG 20 pos α/ß neg pos SA4 A neg neg D E neg neg neg J SG 36 pos α/ß pos pos SA4 ST1 A neg neg D E neg neg neg J SGS 64 pos α/ß neg pos SA1ST1 A neg neg D neg G neg neg neg SGS 13 pos α/ß neg pos SA1 A neg neg neg E G neg neg neg SGS 18 pos α/ß neg pos SA1 A neg neg neg E G neg neg neg SG 10 pos α/ß pos pos SA1 A neg neg neg E G neg neg neg SGS 27 pos α/ß pos neg SA1 A neg neg neg E G neg neg neg SG 14 pos α/ß pos pos SA1ST2 A neg neg neg E G neg neg neg Neg = negative; pos = positive; Clf=clumping factor; coa=coagulase; PFGE-type=Pulsed-field gelelectrophoresis-type; n. t. =not typable;

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Table2: Phenotypical and genotypical characterization of 54 S. aureus positive production isolates.

Sample Designation Hemolysis Cfl Coa PFGE Enterotoxin genes (A-J) Int. Nr. Pulsotypes

Whey -2 8 α/ß pos pos SA1 A neg neg neg neg neg neg neg neg Whey sediment -4 11 α/ß neg pos SA1 A neg neg neg neg neg neg neg neg Curd A -1 15 α/ß neg pos SA1 A neg neg neg neg neg neg neg neg Curd B -1 16 α/ß neg pos SA1 A neg neg neg neg neg neg neg neg Curd B -1 17 α/ß neg pos SA1 A neg neg neg neg neg neg neg neg Curd B -2 20 α/ß neg pos SA1 A neg neg neg neg neg neg neg neg Curd B -2 21 α/ß pos pos SA1 A neg neg neg neg neg neg neg neg Curd C -1 22 α/ß neg pos SA1 A neg neg neg neg neg neg neg neg Curd C -1 23 α/ß pos pos SA1 A neg neg neg neg neg neg neg neg Cheese on press B 2h -3 45 α/ß neg pos SA1 A neg neg neg neg neg neg neg neg Cheese on press B 2h -4 46 α/ß neg pos SA1 A neg neg neg neg neg neg neg neg Cheese on press C 2h -3 57 α/ß pos pos SA1 A neg neg neg neg neg neg neg neg Cheese on press C 2h -4 58 α/ß neg pos SA1 A neg neg neg neg neg neg neg neg Cheese on press A 8h -4 43 α/ß neg pos SA1 A neg neg neg neg neg neg neg neg Cheese on press A 4h -4 39 α/ß neg pos SA1 A neg neg neg neg neg neg neg neg Cheese on press B 4h -4 52 α/ß neg pos SA1 A neg neg neg neg neg neg neg neg Cheese on press C 4h -4 63 α/ß neg pos SA1 A neg neg neg neg neg neg neg neg Cheese on press A 2h -3 33 α/ß pos neg SA2 A neg neg neg neg neg neg neg neg Curd C -3 30 α/ß neg pos SA1ST1 A neg neg D neg neg neg neg J Cheese on press B 8h -3 54 α/ß neg pos SA1ST2 A neg neg D neg neg neg neg J Whey -2 9 α/ß neg pos SA1 A neg neg D E G neg neg J Cheese on press C 8h -3 67 α/ß neg pos SA1 A neg neg neg E neg neg neg neg Green cheese A -4 73 α/ß pos pos SA1 A neg neg neg E neg neg neg neg Green cheese B -3 76 α/ß neg pos SA1 A neg neg neg E neg neg neg neg Bulk tank milk -1 1 α/ß neg pos SA1 A neg neg neg E G neg neg neg Bulk tank milk sediment -2 4 α/ß pos pos SA4 ST1 A neg neg neg E G neg neg neg Whey -1 6 α/ß pos pos SA1 A neg neg neg E G neg neg J Neg = negative; pos = positive; Clf=clumping factor; coa=coagulase; PFGE-type=Pulsed-field gelelectrophoresis-type;

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Table 2 continued: Phenotypical and genotypical characterization of 54 S. aureus positive production isolates.

Sample Designation Hemolysis Cfl Coa PFGE Enterotoxin genes (A-J) Int. Nr. Pulsotypes

Curd A -1 12 α/ß neg pos SA1 A neg neg neg E G neg neg J Cheese before consumption C v.V. -4 99 α/ß neg pos SA1 A neg neg neg E G neg neg J Cheese on press B 8h -4 55 α/ß neg pos SA1 A neg neg neg E G neg neg J Green cheese A -3 72 α/ß neg pos SA1 A neg neg neg E G neg neg J Cheese on press C 8h -2 65 α/ß neg pos SA1 A neg neg neg E neg neg neg J Curd C -2 26 α/ß neg pos SA1ST2 A neg neg neg neg G neg neg J Cheese on press A 2h -4 35 neg neg neg SA3 A neg neg neg neg neg neg I neg Cheese on press C 4h -2 60 α/ß pos pos SA1ST3 A neg neg neg neg neg neg neg J Cheese on press A 4h -3 37 α/ß neg pos SA1 A neg neg neg neg neg neg neg J Green cheese B -3 75 neg neg neg SA3 neg neg neg D neg neg neg I J Green cheese B -4 77 α/ß neg pos SA1ST1 neg neg neg D neg neg neg neg J Green cheese B -4 78 α/ß neg pos SA1ST2 neg neg neg D neg neg neg neg J Cheese before consumption C v.V. -5 100 α/ß pos pos SA1ST2 neg neg neg D neg neg neg neg J Green cheese A -4 74 α/ß pos pos SA1ST3 neg neg neg D neg neg neg neg J Green cheese A -3 71 α/ß pos neg SA2 neg neg neg D neg neg neg neg J Curd C -2 27 α/ß neg pos SA1ST1 neg neg neg D neg neg neg neg J Cheese before consumption C v.V. -5 101 α/ß pos pos SA1ST2 neg neg neg D neg neg neg neg J Green cheese C -3 79 α/ß neg pos n. t. neg neg neg neg neg neg neg neg neg Bulk tank milk -2 2 α/ß neg pos SA1ST1 neg neg neg neg neg neg neg neg neg Cheese on press C 8h -4 69 α/ß neg pos SA1ST1 neg neg neg neg neg neg neg neg neg Green cheese C -4 80 α/ß neg pos SA1ST1 neg neg neg neg neg neg neg neg neg Green cheese C -4 81 α/ß pos pos SA1ST1 neg neg neg neg neg neg neg neg neg Whey sediment -2 10 α/ß pos pos SA1ST2 neg neg neg neg neg neg neg neg neg Cheese on press A 8h -3 41 α/ß pos pos SA1ST2 neg neg neg neg neg neg neg neg neg Bulk tank milk sediment -2 5 α/ß pos pos SA2 neg neg neg neg neg neg neg neg neg Cheese on press B 4h -3 50 neg neg neg SA3 neg neg neg neg neg neg neg neg neg Cheese on press B 8h -3 53 neg neg neg SA3 neg neg neg neg neg neg neg neg neg Neg = negative; pos = positive; Clf=clumping factor; coa=coagulase; PFGE-type=Pulsedfield gelelectrophoresis-type; n. t.= not typable;