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Review

Identification, typing, ecology and epidemiology of coagulase negative staphylococci associated with ruminants Wannes Vanderhaeghen a,*,1, Sofie Piepers a, Frédéric Leroy b, Els Van Coillie c, Freddy Haesebrouck d, Sarne De Vliegher a a M-team and Mastitis and Milk Quality Research Unit, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium b Research Group of Industrial Microbiology and Food Biotechnology, Faculty of Sciences and Bio-engineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium c Technology and Food Science Unit, Institute for Agricultural and Fisheries Research (ILVO), Brusselsesteenweg 370, 9090 Melle, Belgium d Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium

A R T I C L E

I N F O

Article history: Accepted 3 November 2014 Keywords: Ruminants Dairy cattle Subclinical mastitis Intramammary infections Coagulase negative Staphylococcus spp

A B S T R A C T

Since phenotypic methods to identify coagulase negative staphylococci (CNS) from the milk of ruminants often yield unreliable results, methods for molecular identification based on gene sequencing or fingerprinting techniques have been developed. In addition to culture-based detection of isolates, cultureindependent methods may be of interest. On the basis of molecular studies, the five CNS species commonly causing intramammary infections (IMI) are Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus simulans and Staphylococcus xylosus. Current knowledge suggests that S. chromogenes is a bovine-adapted species, with most cases of IMI due to this bacterium being opportunistic. S. haemolyticus also appears to be an opportunistic pathogen, but this bacterium occupies a variety of habitats, the importance of which as a source of IMI remains to be elucidated. S. xylosus appears to be a versatile species, but little is known of its epidemiology. S. epidermidis is considered to be a humanadapted species and most cases of IMI appear to arise from human sources, but the organism is capable of residing in other habitats. S. simulans typically causes contagious IMI, but opportunistic cases also occur and the ecology of this bacterium requires further study. Further studies of the ecology and epidemiology of CNS as a cause of IMI in cattle are required, along with careful attention to classification of these bacteria and the diseases they cause. © 2014 Elsevier Ltd. All rights reserved.

Introduction Mastitis is the most common and costly disease in the dairy sector worldwide (Halasa et al., 2007; Huijps et al., 2008). It can occur in clinical or subclinical forms, the latter indicated by an increase in somatic cell count. Mastitis typically results from bacterial intramammary infection (IMI), the most common causative agents being staphylococci, streptococci and coliforms (Tenhagen et al., 2006; Bradley et al., 2007). These infectious agents can be grouped on the basis of their impact on udder health, with major pathogens, such as Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and Streptococcus uberis, being distinguished from minor pathogens, such as Corynebacterium spp. and coagulase negative staphylococci (CNS).

* Corresponding author. Tel.: +32 92 647545. E-mail address: [email protected] (W. Vanderhaeghen). 1 Present address: Centre of Expertise on Antimicrobial Consumption and Resistance in Animals (AMCRA vzw), Salisburylaan 133, 9820 Merelbeke, Belgium.

Mastitis pathogens are also differentiated by their main reservoirs and principal mode of transmission within a herd into contagious, environmental and (teat skin-associated) opportunistic pathogens (Pyörälä and Taponen, 2009; Zadoks et al., 2011). The principal source of contagious pathogens is assumed to be the cow or the infected udder, with spread among cows primarily through vectors, such as the milking machine, human hands or flies. Environmental pathogens are assumed to originate from the cow’s environment, contaminating teats mainly between milkings, especially under suboptimal housing conditions. Skin-associated opportunists are pathogens normally residing on the skin that only cause disease under conditions favouring colonisation of the udder. The category of skin-associated opportunists was established principally to classify CNS found in milk, even though sound data on their habitats and main sources was lacking (Pyörälä and Taponen, 2009). Despite their current classification into 48 species and 23 subspecies,1 CNS have long been regarded by mastitis researchers

1

See: http://www.bacterio.net/s/staphylococcus.html (accessed 28 October 2014).

http://dx.doi.org/10.1016/j.tvjl.2014.11.001 1090-0233/© 2014 Elsevier Ltd. All rights reserved.

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as a homogeneous group of little interest. However, CNS are the leading cause of subclinical mastitis on well-managed dairy farms that have controlled contagious pathogens (Bradley, 2002; Pitkälä et al., 2004; Tenhagen et al., 2006; Bradley et al., 2007; Piepers et al., 2007; Schukken et al., 2009). The economic importance of subclinical mastitis is well established (Seegers et al., 2003; Halasa et al., 2007; Huijps et al., 2008) and recent research has focused on the ecology and epidemiology of CNS associated with mastitis. The aim of this review is to assess the progress that has been made in the identification and typing of ruminant-associated CNS and to outline how this has improved our understanding of the prevalence, ecology and epidemiology of CNS species associated with mastitis in dairy cows. In this review, CNS are regarded as pathogens causing IMI and leading to mastitis. However, the pathogenic role of CNS is complex and some CNS species/strains might have a beneficial effect on udder health (Vanderhaeghen et al., 2014).

Identification and typing of coagulase negative staphylococci from ruminants Phenotypic identification CNS constitute the majority of the Staphylococcus genus; only six Staphylococcus (sub)species (S. aureus, Staphylococcus delphini, Staphylococcus intermedius, Staphylococcus lutrae, Staphylococcus pseudintermedius and Staphylococcus schleiferi subsp. coagulans) are typically coagulase positive and two (Staphylococcus hyicus and Staphylococcus agnetis) are coagulase variable. Except for S. aureus and S. hyicus/S. agnetis, coagulase positive staphylococci are rarely isolated from cases of ruminant mastitis. Reliable phenotypic identification within the CNS group is laborious, since it requires a large number of biochemical tests (Kloos and Schleifer, 1975; Devriese et al., 1985; Watts et al., 1991; Thorberg and Brändström, 2000). CNS species-specific data were traditionally obtained using commercial identification kits, such as API Staph ID (bioMérieux) and Staph-Zym (Rosco), although these tests were principally developed for human clinical settings. API Staph ID has been recommended by the American National Mastitis Council for differentiation of CNS (Hogan et al., 1999). However, the typeability (i.e. the ability to obtain a species name) and accuracy (i.e. the ability to obtain a correct species name compared with a gold standard) of these systems for identification of CNS isolates from cows (Thorberg and Brändström, 2000; Taponen et al., 2006, 2008; Capurro et al., 2009; Sampimon et al., 2009b; Park et al., 2011) or small ruminants (Onni et al., 2010, 2012; Koop et al., 2012a) is limited. Using API Staph ID or Staph-Zym, 11–59% unidentified isolates have been reported from milk samples from healthy cows or cows with subclinical or clinical mastitis (Thorberg and Brändström, 2000; Capurro et al., 2009; Sampimon et al., 2009b) and from goat milk (Koop et al., 2012a). The accuracy of API Staph ID was 77%, 76% and 41% compared with conventional biochemical methods (Thorberg and Brändström, 2000), 16S rRNA sequencing (Park et al., 2011) and rpoB sequencing (Sampimon et al., 2009b), respectively. The accuracy of Staph-Zym was 94%, 61% and 31% compared with conventional biochemical methods (Thorberg and Brändström, 2000), tuf sequencing (Capurro et al., 2009) and rpoB sequencing (Sampimon et al., 2009b), respectively. In particular, problems have been encountered in the identification of Staphylococcus chromogenes, Staphylococcus haemolyticus and Staphylococcus simulans, which are common species causing IMI, along with Staphylococcus cohnii, Staphylococcus equorum and Staphylococcus warneri, which less frequently cause IMI (Thorberg and Brändström, 2000; Sampimon et al., 2009b;

Onni et al., 2010; Park et al., 2011; Koop et al., 2012a). Hence, these phenotypic systems should be abandoned for scientific and routine use in bovine CNS species identification.

Molecular identification Sequencing of (housekeeping) genes is considered to be the most reliable method for species identification of bacteria (CLSI 2007; Zadoks and Watts, 2009). The 16S rRNA gene is most universally targeted and several protocols have been established for sequencing the staphylococcal 16S rRNA gene (Boerlin et al., 2003; Becker et al., 2004; Heikens et al., 2005). However, staphylococcal 16S rRNA sequences have a relatively high sequence similarity (mean similarity 97.4%) (Kwok et al., 1999; Shah et al., 2007), making the gene unsuitable for distinguishing closely related species and subspecies (Becker et al., 2004; Taponen et al., 2006). Therefore, sequencing of other genes is preferable and protocols have been validated for hsp60/cpn60/groEL (heat shock protein 60) (Goh et al., 1996; Kwok et al., 1999; Kwok and Chow, 2003), rpoB (β subunit of RNA polymerase) (Drancourt and Raoult, 2002), sodA (superoxide dismutase A) (Poyart et al., 2001), gap (glyceraldehyde-3-phosphate dehydrogenase) (Ghebremedhin et al., 2008; Bergeron et al., 2011), dnaJ (chaperone DnaJ) (Shah et al., 2007) and tuf (elongation factor Tu) (Capurro et al., 2009; Bergeron et al., 2011). Mean interspecific sequence similarities are 77.6% for dnaJ, 81.5% for sodA, 82% for hsp60, 86% for rpoB and 86–97% for tuf (Kwok et al., 1999; Poyart et al., 2001; Drancourt and Raoult, 2002; Kwok and Chow, 2003; Mellmann et al., 2006; Bergeron et al., 2011). rpoB has been shown to be useful for discrimination of staphylococcal subspecies (Mellmann et al., 2006; Bergeron et al., 2011). Genotypic methods used to generate bacterial ‘fingerprints’ that have been optimised for identification of CNS isolates from bovine mastitis include transfer-RNA intergenic spacer PCR (tDNA-PCR) combined with capillary gel electrophoresis (Supré et al., 2009; Koop et al., 2012a), 16S-23S rDNA gene internal transcribed spacer PCR (ITS-PCR) (Bes et al., 2000) and (GTG)5-PCR fingerprinting, a repetitive DNA sequence-based PCR (rep-PCR) technique (Braem et al., 2011). It is important to include isolates originating from both the environment and host in reference libraries generated for these techniques. (GTG)5-PCR has a typeability of 94.7% and an accuracy of 94.3% for identification of CNS (Braem et al., 2011). tDNA-PCR has a typeability of 91% and an accuracy of 99.2% (Supré et al., 2009); an advantage of this technique is the availability of a free software programme (BaseHopper),2 which allows rapid and convenient species identification from a pattern, but reference patterns are specific to the particular equipment and consumables used within a laboratory (Koop et al., 2012a). PCR-restriction fragment length polymorphism (RFLP) applied to the groEL, rrs or gap genes has been used to identify CNS from bovine (Santos et al., 2008; Park et al., 2011), ovine (Onni et al., 2010) and caprine milk (Onni et al., 2012). PCR-RFLP of the 16S rRNA gene is not sufficiently discriminative, in particular for the closely related species S. epidermidis, Staphylococcus caprae and Staphylococcus capitis (Onni et al., 2010). Performance of RFLP-PCR might depend on the restriction enzyme used (Park et al., 2011). Amplified fragment length polymorphism (AFLP) has relatively high discriminatory power and can be used to distinguish most closely related CNS species, with a typeability of 98.4% and accuracy of 99.2%, but is labour intensive and expensive (Taponen et al., 2006, 2007; Piessens et al., 2010). Ribotyping performs well in distinguishing bovine CNS species (Bes et al., 2000; Taponen et al., 2008).

2

See: www.basehopper.be (accessed 28 October 2014).

Please cite this article in press as: Wannes Vanderhaeghen, et al., Identification, typing, ecology and epidemiology of coagulase negative staphylococci associated with ruminants, The Veterinary Journal (2014), doi: 10.1016/j.tvjl.2014.11.001

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Matrix-assisted laser desorption ionisation-time of flight mass spectrometry analysis (MALDI-TOF MS) has been validated for identification of staphylococci from diverse origins (Dubois et al., 2010). Huber et al. (2011) found a perfect correlation of MALDI-TOF MS results with sodA sequencing for three species of methicillinresistant staphylococci from bulk tank milk and the nasal cavity of cows. Tomazi et al. (2014) used MALDI-TOF MS to identify CNS from bovine IMI with groEL PCR-RFLP as the reference method, with a typeability of 95.4%; however, the study design was not optimal, since validation of the groEL PCR-RFLP was based on comparison with biochemical methods (Santos et al., 2008). Biotyper 3.0 software (Bruker Daltonics) has to be used for pattern matching of the raw spectra, so an isolate can only be identified if the appropriate spectrum is recorded in the Biotyper database. In the future, reliable diagnostic identification via whole genome sequencing (WGS) might become the gold standard method for species identification due to the development of cheaper and more accessible next generation sequencing platforms (Köser et al., 2012; Török and Peacock, 2012). Strain typing Strain typing reveals phenotypic and/or genotypic variation at the intra-species level and its usefulness ranges from large scale research of bacterial population structure to small scale infection control or epidemiological studies. This has long been neglected in mastitis due to CNS, but will be indispensable to gain insight into persistence of CNS IMI, sources of infection and routes of transmission. Of the older phenotypic typing methods (Parisi, 1985), only antimicrobial susceptibility testing is still widely applied. Genotyping methods for strain typing vary in taxonomic resolution, discriminatory power and reproducibility (Savelkoul et al., 1999; van Belkum et al., 2007). The cut-off values to consider two isolates as different species or different strains from the same species are critical (Savelkoul et al., 1999; Piessens et al., 2010; Braem et al., 2011). The current reference method for strain typing of staphylococci is pulsed field gel electrophoresis (PFGE); this method has been applied in some studies investigating bovine-associated CNS (Thorberg et al., 2006; Taponen et al., 2008; Gillespie et al., 2009; Rajala-Schultz et al., 2009; Jaglic et al., 2010; Mørk et al., 2012). PFGE is robust and highly discriminatory, but labour intensive and expensive; it is useful mainly for tracking strain variation at a small scale (i.e. quarter, cow or herd level), for example to distinguish pathogenic from unrelated strains. There is a need to develop standardised protocols for strain typing of CNS involved in bovine mastitis, as has been done for S. aureus (Murchan et al., 2003). Other methods that have proven useful for strain typing of CNS include AFLP, rep-PCR and random-amplified polymorphic DNA (RAPD) (Taponen et al., 2006, 2007; Rood et al., 2011; Zong et al., 2011; Piessens et al., 2012). In order to delineate evolutionary relationships between isolates or establish the population structure of a bacterial species, there is a need to examine informative segments of the genome that change less rapidly, such as single nucleotide polymorphisms or sequence variations in housekeeping genes (Witte et al., 2006). Techniques to study such relationships include WGS and multilocus sequence typing (MLST) (Maiden et al., 1998). MLST has been used to delineate the population structure of S. aureus,3 including strains originating from cases of bovine mastitis (Enright et al., 2000; Hata et al., 2010; Sakwinska et al., 2011). MLST protocols have also been optimised for S. epidermidis4 and S. pseudintermedius (Solyman et al., 2013).

3 4

See: http://saureus.mlst.net (accessed 28 October 2014). See: http://sepidermidis.mlst.net (accessed 28 October 2014).

3

Ecology and epidemiology of coagulase negative staphylococci Terminology The ecology of CNS, encompassing the habitats, distribution, population structures, movements and interactions of the different species and strains of CNS, can be distinguished from the epidemiology of CNS, which considers CNS as pathogens in the context of IMI. We propose that the terms ‘host adapted’ (with subcategories such as ‘cow adapted’, ‘sheep adapted’, ‘human adapted’, ‘udder adapted’) and ‘environmental’ should be considered only in an ecological framework to refer to the habitat(s) of species or strains of CNS. The terms ‘contagious’ and ‘opportunistic’ should be used when considering the epidemiology of CNS as pathogens causing IMI. Contagious cases of IMI occur in multiple animals on a farm, can be attributed to a single quarter/cow origin and are spread among cows by means of a vector (e.g. milking machine unit liners). Opportunistic cases of IMI can be attributed to a range of different origins and are not spread among animals. There is a need for comprehensive studies of both the epidemiology and ecology of CNS. Further work is needed to determine the routes of transmission and extramammary sources of CNS, in particular whether milk and udder tissue are habitats for CNS. Species-specific risk factors for CNS IMI have not yet been established. Occurrence of coagulase negative staphylococci in milk Table 1 lists studies that have used genotypic methods to identify CNS from the milk of cattle, sheep and goats. At least 24 CNS species have so far been isolated from bovine milk, the five common species being S. chromogenes, S. haemolyticus, S. epidermidis, S. simulans and Staphylococcus xylosus. S. chromogenes is the most common species, in most studies accounting for more than a quarter of CNS isolates. The prevalence of the other four main species varies substantially (Table 1). S. cohnii, S. warneri, S. hyicus, Staphylococcus sciuri, S. equorum and Staphylococcus hominis feature in some studies, but are generally isolated less frequently. S. epidermidis, S. simulans and S. xylosus are the most prevalent CNS in ovine and caprine milk, while S. caprae is relatively common, and S. chromogenes and S. haemolyticus are less frequent than in bovine milk (Onni et al., 2010, 2012; Koop et al., 2012a, 2012b). Prevalence and incidence of intramammary infections CNS are the most frequent bacteria cultured from milk samples from cattle (Piepers et al., 2007; Pyörälä and Taponen, 2009; Sampimon et al., 2009a; Botrel et al., 2010; De Vliegher et al., 2012; Mørk et al., 2012) and goats (Persson and Olofsson, 2011; Koop et al., 2012b). However, IMI CNS species-specific prevalence or incidence data are scarce. Three studies have reported the prevalence of CNS in quarters or cows from a limited number of farms. Using API Staph for identification of staphylococci in quarter milk samples from heifers from four farms in the USA, Trinidad et al. (1990) identified S. chromogenes in 43.1%, S. hyicus in 24.3%, S. aureus in 19.9%, S. hominis, S. simulans, S. xylosus, S. warneri and S. epidermidis in 0.4– 1.1% and unidentified CNS in 0.4% of quarters. Using tDNA-PCR, the incidence of CNS in monthly quarter milk samples from cohorts of 25 cows on each of three farms in Belgium was 5.8% over 13 months; the incidence of S. chromogenes was 2.7%, whereas the incidence of 12 other species (including one unidentified CNS) was less than 1% (Supré et al., 2011). Using phenotypic identification, the cow level prevalence of S. epidermidis in two Swedish dairy herds on two sampling occasions was 22–31% (Thorberg et al., 2006). Most studies on CNS in mastitis have relied on aerobic culture of bacteria from milk, which might lead to bias arising from the limited resolution of culture-dependent approaches (Dufour et al.,

Please cite this article in press as: Wannes Vanderhaeghen, et al., Identification, typing, ecology and epidemiology of coagulase negative staphylococci associated with ruminants, The Veterinary Journal (2014), doi: 10.1016/j.tvjl.2014.11.001

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Table 1 Summary of recent original studies that presented species-specific numbers of coagulase negative staphylococci isolates identified with molecular identification methods, originating from milk samples of cows, sheep or goats. Origin of isolatesa

Speciesb Total number of isolates

Bovine QMS from six herds QMS taken each month for 13 months from 10 randomly selected cows per herd in six herds QMS taken during 12 samplings within 17 months from 89 cows distributed over three herds QMS from all lactating cows in five herds Milk samples from acute CM Milk samples from SCM QMS of CM and SCM Convenience sample of CNS isolates from the CBMRN (originally isolated from QMS from randomly selected cows with SCM) Caprine HMS from five herds (California, USA), early, mid- and late lactation, and goats of different parity HMS from five herds (The Netherlands), early, mid- and late lactation, and goats of different parity HMS from all goats in 29 herds with a tank milk SCC > 5 × 106/mL in the last three controls Ovine HMS from all sheep in 15 flocks with a tank milk SCC > 5 × 106/mL in the last three controls

S. caprae

S. chromogenes

S. cohnii

S. epidermidis

S. equorum

S. haemolyticus

S. hominis

S. hyicus

S. lugdunensis

263 134

3 –

190 41

– 4

2 16

– 2

16 37

1 5

8 –

– –

179

–

83

20

1

1

11

–

1

–

244 56 98 172 877

– – – – –

76 16 21 63 417

3 – – 5 26

34 4 30 23 34

– – – 10 5

37 8 14 9 71

7 – – 1 3

– 6 1 9 8

– – – – –

115

7

23

6

38

1

–

–

–

–

343

15

119

1

69

–

6

–

–

15

142

19

45

–

53

1

1

–

–

–

226

30

34

–

131

2

10

–

–

–

Origin of isolatesa

Speciesb S. S. S. S. S. saprophyticus sciuri simulans warneri xylosus

Bovine QMS from six herds

–

9c

7

–

24

QMS taken each month for 13 months from 10 randomly selected cows per herd in six herds QMS taken during 12 samplings within 17 months from 89 cows distributed over three herds

4

1

15

3

–

–

3

17

–

28

QMS from all lactating cows in five herds

–

–

63

23

–

Milk samples from acute CM

–

–

14

–

2

Milk samples from SCM

7

–

13

–

4

QMS of CM and SCM

2

3

9

13

15

Convenience sample of CNS isolates from the CBMRN (originally isolated from QMS from randomly selected cows with SCM)

5

8

166

9

92

–

–

23

–

9

–

–

54

3

44

Caprine HMS from five herds (California, USA), early, mid- and late lactation, and goats of different parity HMS from five herds (The Netherlands), early, mid- and late lactation, and goats of different parity

Other

Number of isolates

Identification

Reference

S. capitis S. succinus S. auricularis S. devriesei S. devriesei S. fleurettii S. pasteuri Unidentified S. pasteuri

1 2 3 1 5 6 1 2 1

16S rRNA + rpoB Park et al., 2011 AFLP + rpoB Piessens et al., 2011 tDNA + rpoB Supré et al., 2011

S. lentus Unidentified S. arlettae S. gallinarum S. pseudintermedius Unidentified S. arlettae S. capitis S. fleurettii S. nepalensis S. succinus S. arlettae S. auricularis S. capitis S. gallinarum S. pasteuri S. succinus S. nepalensis S. vitulinus

1 3 2 2 1 2 1 1 5 1 2 8 4 7 5 3 3 2 1

tuf

S. arlettae S. croceolyticus S. gallinarum S. pasteuri S. croceolyticus S. arlettae S. auricularis S. capitis S. gallinarum S. lentus S. rostri

1 4 2 1 4 3 2 3 2 2 1

sodA + PFGE

tuf

Mørk et al., 2012 Persson Waller et al., 2011 Persson Waller et al., 2011

rpoB

Sampimon et al., 2009b

rpoB

Fry et al., 2014

tDNA

Koop et al., 2012a

tDNA

Koop et al., 2012b

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Table 1 Continued Origin of isolatesa

Speciesb S. S. S. S. S. saprophyticus sciuri simulans warneri xylosus

HMS from all goats in 29 herds with a tank milk SCC > 5 × 106/mL in the last three controls

Ovine HMS from all sheep in 15 flocks with a tank milk SCC > 5 × 106/mL in the last three controls

–

–

8

–

6

4

4

Other

Number of isolates

Identification

Reference

4

S. capitisd S. gallinarum S. pasteuri S. devriesei S. rostri

2 1 1 2 1

groEL PCR-RFLP

5

S. saprophyticus S. muscae

2 2

gap and 16S rRNA Onni et al., 2010 PCR-RFLP + gap

Onni et al., 2012

a QMS, quarter milk samples; CM, clinical mastitis; SCM, subclinical mastitis; CBMRN, Canadian Bovine Mastitis Research Network; HMS, half milk samples; SCC, somatic cell count. b The five most prevalent species in each study are indicated in bold. c Subsp. carnaticus. d Subsp. urealyticus.

2012). Using culture-independent methods, a higher microbial diversity and a lower prevalence of CNS have been identified in bovine milk compared with culture-based methods (Kuang et al., 2009; Oikonomou et al., 2012). Using metagenomic pyrosequencing for investigation of bacterial diversity in milk samples from 136 mastitic and 20 healthy dairy cows, 1.8% of 240,524 16S rRNA sequences were classified as Staphylococcus spp. (Oikonomou et al., 2012). A relatively low diversity of staphylococcal species in milk was observed by Hagi et al. (2010). At present, culture-independent methods should be regarded as complementary to culture rather than as the definitive or preferred method for analysis of milk samples. Occurrence of coagulase negative staphylococci in extra-mammary sites Using phenotypic and genotypic (ribotyping) identification, Taponen et al. (2008) identified 15 (sub)species and 10 unknown species clusters of CNS at six extra-mammary sites (udder skin, teat apex > perineum > teat cup liners, teat canal, human hands) on a single dairy cattle farm in Finland. The most frequent species were S. xylosus (teat apex) and S. chromogenes (udder skin), S. arlettae (udder skin), S. saprophyticus (udder skin, teat apex), S. sciuri (all sites) and S. succinus (teat apex). S. xylosus and S. chromogenes were also the two species most frequently identified in milk samples. On six Flemish dairy farms, Piessens et al. (2011) identified 12 CNS species in milk, mostly S. chromogenes, S. haemolyticus, S. epidermidis and S. simulans, whereas at least 23 species were detected in environmental samples (air in the free stall, slatted floors, used sawdust and unused sawdust), mostly S. equorum, S. sciuri, S. haemolyticus, S. fleurettii, S. cohnii and S. simulans. There was considerable variation between herds. Of extramammary CNS, most S. equorum and S. haemolyticus were detected in air samples, most S. sciuri, S. simulans and S. fleurettii were detected in samples from slatted floors and used sawdust, and most S. xylosus isolates were detected in unused sawdust, while S. cohnii was present at all sites. Staphylococci detected by sequencing of 16S rRNA clone libraries and DGGE analysis included S. xylosus, S. arlettae and S. cohnii in samples from the teat canal (Gill et al., 2006), and S. chromogenes, S. haemolyticus, Staphylococcus devriesei, S. hyicus, S. aureus and S. pseudintermedius in samples from the teat apex (Braem et al., 2012). The five main coagulase negative staphylococci causing intramammary infections Staphylococcus chromogenes – High levels of diversity among S. chromogenes from bovine IMI have been detected by AFLP (Taponen et al., 2006) and PFGE (Gillespie et al., 2009; Rajala-Schultz et al.,

2009). Post-milking teat dipping has little effect on S. chromogenes (Quirk et al., 2012). Overall, this suggests that S. chromogenes originates from a nearby extra-mammary source and that most IMI due to this bacterium are opportunistic. S. chromogenes IMI may also be contagious, since some S. chromogenes strains have been found in multiple animals from the same farm (Taponen et al., 2008; Gillespie et al., 2009; Mørk et al., 2012). However, it is not clear whether these observations could be explained by spread through a vector. S. chromogenes was identified in milk samples from 18 cows on three farms in Belgium, but was absent from milking machine unit liners and human skin/gloves, which are considered to be the most important routes of transmission for contagious mastitis (De Visscher et al., 2014). Piessens et al. (2011, 2012) concluded that cows were the main source of S. chromogenes isolated from milk (i.e. that the bacterium is an udder-adapted species), since S. chromogenes was identified more frequently from milk than environmental samples, and several environmental strains had similar AFLP and RAPD profiles to those in milk (suggesting that S. chromogenes was present in the environment as a result of ‘contamination’ from spilled milk). However, Braem et al. (2013) were unable to detect S. chromogenes on the teat apices of these cows by culture or culture-independent methods (although no other body sites were investigated). In other studies, S. chromogenes has been detected in teat canals, on teat skin and other body sites (White et al., 1989; Trinidad et al., 1990; Matthews et al., 1992). S. chromogenes originating from milk had PFGE profiles that corresponded to strains isolated from the teat canal, teat apex, udder skin and perineum, but the direction of transmission could not be determined (Taponen et al., 2008). Thus, it appears that S. chromogenes is a host-adapted species, commonly inhabiting bovine skin, but it is uncertain whether it predominates in any specific region or inhabits internal udder tissue. Staphylococcus haemolyticus – S. haemolyticus appears to be a versatile organism that can occupy diverse habitats. Piessens et al. (2011) detected higher numbers of S. haemolyticus than other CNS in the environment. Although S. haemolyticus also occurred frequently in milk samples from cows on the same farm, relatively few strains were found in both milk and environmental samples (Piessens et al., 2012). S. haemolyticus isolates had the highest diversity of all CNS, indicating that a wide variety of strains is present in different extramammary habitats (Piessens et al., 2012). In other studies, S. haemolyticus has been detected on udder skin (Baba et al., 1980) and teat skin (Devriese and Dekeyser, 1980), and was the most frequent CNS detected on teat apices (Braem et al., 2013). Of note, S. haemolyticus is also one of the more important CNS species in human medicine (Piette and Verschraegen, 2009). The importance of different extra-mammary habitats as reservoirs for

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IMI due to S. haemolyticus is uncertain. On one farm in Norway, similar S. haemolyticus PFGE types were detected in several cows with IMI, but there was insufficient information to determine whether these cases were contagious (Mørk et al., 2012). In an ecological context, S. haemolyticus appears to inhabit a variety of habitats (animal, human and environmental), but it is uncertain whether there are any host or tissue adapted strains. In an epidemiological context, S. haemolyticus appears to act as an opportunistic pathogen (Bexiga et al., 2014). Staphylococcus epidermidis – S. epidermidis is the most important staphylococcal species colonising the human skin (Piette and Verschraegen, 2009) and was initially assumed to be a human adapted species, since it was rarely isolated from bovine teat canals, teat skin or udder skin (Baba et al., 1980; Devriese and Dekeyser, 1980; White et al., 1989; Trinidad et al., 1990; Matthews et al., 1992). However, Braem et al. (2013) detected S. epidermidis on the teat apices of 9.7% of quarters of cows on two Belgian dairy farms. Piessens et al. (2011) identified several strains of S. epidermidis in the dairy environment, many of which could be distinguished from strains found in milk; thus, the presence in the environment of some strains could not necessarily be attributed to spillage of milk (Piessens et al., 2012). This suggests that S. epidermidis may be able to reside in a range of habitats in addition to human skin. However, strain typing by PFGE indicates that most S. epidermidis strains associated with IMI are derived from human contacts (Thorberg et al., 2006; Jaglic et al., 2010). Since S. epidermidis strains detected in IMI in a dairy herd may be clonal, Bexiga et al. (2014) suggested that cow-to-cow transmission is likely. However, when multiple cases of IMI due to S. epidermidis occur in a herd, it is uncertain whether this is due to repeated, opportunistic, transmission of S. epidermidis from a particular human source, or if there is a single introduction from a human point source, followed by contagious transmission through a vector from cow-to-cow. Staphylococcus simulans – Bexiga et al. (2014) observed frequent clustering of S. simulans on dairy farms. Since it is uncertain whether a common source might account for repeated introductions of this bacterium, cow-to-cow transmission appears to be a more plausible explanation than for S. epidermidis. Other studies have found a relatively low diversity among S. simulans strains from bovine milk (Aarestrup et al., 1999; Taponen et al., 2008). In one dairy herd in Finland, S. simulans was the second most frequent species among CNS associated with IMI, but it was uncommon among CNS from extra-mammary udder or body sites. While 2/4 PFGE types among S. simulans from IMI were found in multiple animals, suggesting that it may be contagious, it was not found on the hands of staff working on the farm (Taponen et al., 2008). S. simulans has been detected on the teat apices of one cow in a Belgian dairy herd, but was not detected on the milking machine unit liners or on the skin or gloves of farm workers (De Visscher et al., 2014). In another Belgian dairy herd, S. simulans was present in the milking machine unit liners, but not on teat apices, nor on the skin or gloves of farm workers (De Visscher et al., 2014). S. simulans IMI might also be opportunistic, with different strains being found in different cows in the same herd (Mørk et al., 2012; Piessens et al., 2011, 2012). Whether S. simulans IMI is contagious or opportunistic, it is unclear if there is a major source of infection. In one study, S. simulans IMI could be linked to environmental reservoirs (Piessens et al., 2012). In other studies, S. simulans has been detected in udder sites, including uninfected quarters (Trinidad et al., 1990; Braem et al., 2013). There is a need for further studies to elucidate the ecology of S. simulans and the epidemiology of S. simulans IMI. Staphylococcus xylosus – S. xylosus also appears to be a versatile organism that can occupy diverse habitats. It is the predominant

Staphylococcus spp. in bedding material (Matos et al., 1991) and was detected by Piessens et al. (2011) almost exclusively in environmental samples, particularly unused sawdust. S. xylosus has also been found in teat canals, on teat and udder skin, on other body sites, and on the hands of farm workers (Baba et al., 1980; Devriese and Dekeyser, 1980; White et al., 1989; Gill et al., 2006; Taponen et al., 2008). In contrast, Braem et al. (2013) did not detect any S. xylosus on the teat apices of healthy cows or cows with subclinical mastitis. Thus, it is not clear if all of these habitats can act as a reservoir for S. xylosus IMI and the epidemiological link between S. xylosus IMI and extramammary sources is uncertain.

Conclusions CNS IMI is the focus of increased attention in dairy herds and it has been recognised that CNS are a heterogeneous group of microorganisms, highlighting the need to accurately identify CNS from milk samples. Biochemical methods for identification of CNS have lower typeability and accuracy than molecular methods and therefore phenotypic methods are no longer valid for research into CNS IMI. A variety of molecular techniques are now available for identification of CNS, including gene sequencing and fingerprinting, but not all methods have been validated for identification of CNS from ruminants. More strain typing data are required to improve our understanding of various aspects of CNS IMI. There is a need to harmonise protocols that can act as gold standard methods for strain typing of staphylococci. PFGE has proved to be a valuable technique, but less labour intensive and expensive methods, such as RAPD, are also useful. For evolutionary or population-wide studies of CNS species associated with ruminant udder health, protocols should be established for methods such as MLST. In the future, WGS may replace all other genotypic methods for identification and typing of CNS. Future research should focus on the five major CNS species identified in bovine milk, i.e. S. chromogenes, S. epidermidis, S. haemolyticus, S. simulans and S. xylosus, and S. caprae in sheep and goats. Speciesspecific prevalence data for these CNS in dairy cattle are largely lacking on a herd, cow or quarter level. Although considerable data have been gathered on the occurrence of CNS species at extramammary sites, insights on the ecology and epidemiology of the main CNS species have been limited by partial sampling and the typing methods used in most studies to date. S. chromogenes appears to be a cow-adapted species and S. chromogenes IMI is generally opportunistic. Cases of S. haemolyticus IMI also appear to be mostly opportunistic, but S. haemolyticus seems to be a versatile species that can occupy diverse habitats. S. xylosus also appears to be a versatile organism, but little is known of the epidemiology of S. xylosus IMI. Although S. epidermidis has been considered to be a humanadapted species, it may also occur in other habitats. However, S. epidermidis IMI appears to originate predominantly from human sources. S. simulans manifests as contagious IMI in a herd, though opportunistic S. simulans IMI cases can occur. The ecology of S. simulans and the main sources for S. simulans IMI are still unclear. Overall, if a comprehensive understanding of the ecology and epidemiology of CNS is to be obtained, more ambitious experimental designs are required, using state-of-the-art technology. Moreover, the ecological and epidemiological features of CNS species causing IMI should be clearly differentiated.

Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.

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Please cite this article in press as: Wannes Vanderhaeghen, et al., Identification, typing, ecology and epidemiology of coagulase negative staphylococci associated with ruminants, The Veterinary Journal (2014), doi: 10.1016/j.tvjl.2014.11.001