Antonie van Leeuwenhoek DOI 10.1007/s10482-014-0185-5

ORIGINAL PAPER

Characterization of methicillin-resistant coagulase-negative staphylococci in milk from cows with mastitis in Brazil Nathalia C. C. Silva • Felipe F. Guimara˜es • Marcela de P. Manzi Elena Go´mez-Sanz • Paula Go´mez • Joao P. Arau´jo-Ju´nior • Helio Langoni • Vera L. M. Rall • Carmen Torres

•

Received: 19 January 2014 / Accepted: 22 April 2014 Ó Springer International Publishing Switzerland 2014

Abstract Staphylococci are one of the most prevalent microorganisms in bovine mastitis. Staphylococcus spp. are widespread in the environment, and can infect animals and humans as opportunistic pathogens. The objective of this study was to determine the frequency of methicillin-resistance (MR) among coagulase-negative staphylococci (CoNS) previously obtained from milk of mastitic cows in Brazil and to characterize the antimicrobial resistance phenotype/genotype and the SCCmec type of MRCoNS isolates. Identification of MRCoNS was based on both biochemical and molecular methods. N. C. C. Silva  J. P. Arau´jo-Ju´nior  V. L. M. Rall Department of Microbiology and Immunology, UNESP, Botucatu, SP, Brazil N. C. C. Silva Department of Agri-food Industry, Food and Nutrition– LAN USP, Piracicaba, SP, Brazil F. F. Guimara˜es  M. de P. Manzi  H. Langoni Department of Hygiene Veterinary and Public Health, UNESP, Botucatu, SP, Brazil E. Go´mez-Sanz  P. Go´mez  C. Torres (&) Department of Food and Agriculture, Universidad de La ´ rea de Bioquı´mica y Biologı´a Molecular, Rioja, A Madre de Dios 51, 26006 Logron˜o, Spain e-mail: [email protected] E. Go´mez-Sanz Environmental Genomics and Systems Biology Research Group, Institute of Natural Resource Sciences Zurich University of Applied Sciences (ZHAW), Wa¨denswil, Switzerland

Susceptibility testing for eleven antimicrobials was performed by disk-diffusion agar. Antimicrobial resistance genes and SCCmec were investigated by specific PCRs. Twenty-six MRCoNS were detected (20 % of total CoNS), obtained from 24 animals, and were identified as follows: S. epidermidis (7 isolates), S. chromogenes (7), S. warneri (6), S. hyicus (5) and S. simulans (1). All MRCoNS isolates carried mecA while the mecC gene was not detected in any CoNS. The SCCmec IVa was demonstrated in nine MRCoNS, while the remaining 17 isolates harbored non-typeable SCCmec cassettes. In addition to oxacillin and cefoxitin resistance, MRCoNS showed resistance to tetracycline (n = 7), streptomycin (n = 6), tobramycin (n = 6), and gentamicin (n = 4), and harbored the genes tet(K) (n = 7), str (n = 3), ant(40 ) (n = 6) and aac(60 )aph(200 ) (n = 4), respectively. In addition, seven strains showed intermediate resistance to clindamycin and two to streptomycin, of which two harboured the lnu(B) and lsa(E) genes and two the aad(E) gene, respectively. One isolate presented intermediate erythromycin and clindamycin resistance and harbored an erm(C) gene with an uncommon 89-bp deletion rendering a premature stop codon. MRCoNS can be implicated in mastitis of cows and they constitute a reservoir of resistance genes that can be transferred to other pathogenic bacteria. Keywords Staphylococcus  Coagulase negative  mecA  Antimicrobial resistance  Bovine mastitis

123

Antonie van Leeuwenhoek

Introduction The genus Staphylococcus encloses 47 species and 24 subspecies from which coagulase-negative Staphylococcus (CoNS) are the predominant group (DSMZ, 2012). Although Staphylococcus aureus is the most virulent and relevant species in the genus, methicillinresistant CoNS (MRCoNS) are gaining interest due to its increased detection as causative agents of human and animal infections (Febler et al. 2010; Cui et al. 2013; de Freitas Guimara˜es et al. 2013). CoNS are present on the bodies of cows, and are very common in teat apices (Taponen et al. 2008), but they are also opportunistic pathogens that can cause subclinical or clinical mastitis in cows (Taponen and Pyo¨ra¨la¨ 2009). Several studies have reported the isolation of Staphylococcus spp. from samples of milk of cows with mastitis (Febler et al. 2010; de Freitas Guimara˜es et al. 2013). CoNS can be involved in mastitis infection, resulting on reduced production of milk and decreased quality, causing the most important economic looses in the dairy industry (Febler et al. 2010). Besides, multiresistance in CoNS is worryingly common (Nam et al. 2010; Virdis et al. 2010) since they may act as reservoirs of antimicrobial resistance genes that can be transferred to the more pathogenic S. aureus (Archer and Climo, 1994). CoNS can produce biofilm, which compromises the efficacy of intramammary antibiotic treatment (Virdis et al. 2010). Intercellular adhesion in this process is mediated by polysaccharide intercellular adhesin production (PIA), which is encoded by the locus ica, containing the icaA, icaB, icaC and icaD genes (Ziebuhr et al. 1999). The objective of this study was to characterize MRCoNS isolates previously recovered in milk of cows with mastitis in Brazil (de Freitas Guimara˜es et al. 2013), determining the content in antimicrobial resistance genes and their SCCmec cassette.

Materials and methods Origin of samples A total 128 CoNS isolates were recovered in a previous study from 1,484 milk samples obtained from 518 cows of 11 cow farms of Brazil which showed a positive California-Mastitis-Test (CMT),

123

indicative of clinical or subclinical mastitis. Isolates were identified based on colony morphology, Gram staining, catalase, coagulase and biochemical tests (de Freitas Guimara˜es et al. 2013). Selection criteria for isolates investigated in the present study was based on methicillin resistance profile and the presence of the mecA gene. The MRCoNS included in this study were submitted to molecular identification by amplification and sequencing of the sodA gene (Poyart et al. 2001). Beta-lactam resistance and characterization of the SCCmec cassette Methicillin resistance was verified by susceptibility testing to oxacillin and cefoxitin by disk-diffusion agar according to Clinical and Laboratory Standards Institute recommendations (CLSI, 2012) and subsequent amplification of the mecA gene (CRL-AR, 2009). SCCmec types I to V were investigated in MRCoNS isolates by PCR of the ccr recombinases (1–5) and the mec gene complex type (A to C) as recommended by the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) (Kondo et al. 2007; IWG-SCC, 2009). An additional PCR was performed to differentiate different subtypes of SCCmec IV (a–d) (Zhang et al. 2005). The presence of mecC gene was also tested in our whole collection of 128 CoNS (Cuny et al. 2011). Antimicrobial resistance to non b-lactams and virulence profile Antimicrobial susceptibility testing to tetracycline, erythromycin, clindamycin, gentamicin, tobramycin, trimethoprim-sulphametoxazole, ciprofloxacin, and chloramphenicol was performed by disk-diffusion agar method (CLSI, 2012). Methodology and guidelines for streptomycin were as recommended by the Societe´ Franc¸aise de Microbiologie (www.sfm.asso. fr). Detection of antimicrobial resistance genes [erm(A), erm(B), erm(C), erm(T), erm(Y), msrA, lnu(A), lnu(B), lnu(C), lnu(D), cfr, vga(A), vga(B), vga(C), lsa(B), lsa(E), ant(6)-Ia, ant(9)-I, str, ant(40 ), aac(60 )-aph(200 ), tet(K), tet(M), and tet(L)] was investigated in resistant and intermediate isolates by specific PCRs (Go´mezSanz et al. 2010; Wendlandt et al. 2013). Multiresistance was considered as resistance to at least three

Antonie van Leeuwenhoek

families of antibiotics. The genetic environment of the lincomycin resistance lsa(E) and lnu(B) genes was investigated by PCR mapping in lsa(E)-lnu(B)-positive strains (Lozano et al. 2012). Positive and negative controls from the collection of the University of La Rioja were used in each PCR assay. Presence of the genes contained in the ica operon (icaA, icaB, icaC, icaD) was determined by PCR as previously recommended (Ziebuhr et al. 1999). Sequence of the new erm(C) gene The sequence of the new erm(C) gene was deposited in the EMBL database with the accession number HG970733.

Results Twenty-six of the 128 CoNS isolates recovered in the previous study (de Freitas Guimara˜es et al. 2013) harbored the mecA gene and showed resistance to oxacillin and cefoxitin (20.3 %), and were characterized in the present study. All 128 CoNS (both methicillinsusceptible and methicillin-resistant) showed a negative result by PCR for the mecC gene. The 26 mecA-positive MRCoNS isolates were recovered from 24 animals, which corresponded to 4.6 % of cows with mastitis analyzed in the previous study (24 of 518 mastitic animals). Species identification was as follows (number of isolates): S. epidermidis (7), S. chromogenes (7), S. warneri (6), S. hyicus and (5) and S. simulans (1). The SCCmec IVa was identified in 9 MRCoNS (34.6 %), while the remaining 17 isolates harbored non-typeable SCCmec cassettes. However, two strains exhibited the recombinase genes ccr2 and one strain the ccr1. MRCoNS isolates showed additional resistance to tetracycline (7 isolates), streptomycin (6), tobramycin (6), and gentamicin (4), and harbored the genes: tet(K) (7), str (3), ant(40 )-I (6), and aac(60 )aph(200 ) (4), respectively (Table 1). In addition, seven strains showed intermediate resistance to clindamycin and two to streptomycin, of which two strains harboured the lnu(B) and lsa(E) genes, and two the aad(E) gene, respectively. Both lnu(B)-lsa(E) positive S. chromogenes strains presented the same genetic environment as that previously described in methicillin resistant S. aureus (MRSA) sequence type (ST) 398 isolates in Spain (Lozano et al. 2012).

Interestingly, S. epidermidis strain C6I87 showed an intermediate resistance phenotype to both erythromycin and clindamycin (Table 1). This strain was only positive for the erm(C) gene; however, PCR analysis of this amplicon revealed a slightly smaller fragment than expected (642 bp). Sequencing analysis showed that this strain exhibited a deletion of 89 bp within the erm(C) gene when compared with a functionally active erm(C) prototype from plasmid pUR2940 of MRSA ST398 strain C2940 (GenBank accession number HF583292.1) (Fig. 1a). Sequence comparison analysis of the erm(C) region amplified by PCR and the corresponding region in plasmid pUR2940 revealed that CI6I87 presented a complete conserved functional domain (CD-search) characteristic of S-adenosylmethionine-dependent methyltransferases. The 89-bp deletion implied the presence of a premature stop codon in the reading frame near the 30 end of the ErmC protein (Fig. 1b). None of the lincosamides resistance genes tested yielded positive for the remaining four strains with intermediate clindamycin resistance.

Discussion Bovine mastitis is a real problem for the dairy industry. The antibiotic resistance profile of the bacteria that cause this disease should be studied in order to administer the most adequate treatment. While S. aureus is the most important pathogen and it is broadly investigated, CoNS have been traditionally given little attention despite being considered opportunistic pathogens. Further, they are increasingly detected as causative agents of mastitis in cows. In Brazil, a few studies have detected the presence of CoNS species in milk samples of cows with mastitis (Santos et al. 2010). Low percentage of MRCoNS (4.6 % of cows with mastitis) was detected in the present study, related in all cases to mecA and not to mecC gene. All MRCoNS strains, except for the S. simulans isolate, presented resistance to other antibiotics. Previous studies on bovine milk samples have also reported isolates of S. chromogenes, S. epidermidis, S. hyicus and S. simulans with a mutiresistance profile (Sawant et al. 2009; Sampimon et al. 2011). The SCCmec typing yielded negative results in the majority of strains (65.4 %), which is in accordance

123

Antonie van Leeuwenhoek Table 1 Antimicrobial resistance phenotype and genotype of MRCoNS isolated from milk samples of subclinical and clinical mastitis cows in Brazil Strain

Farm

Species

Resistance phenotype

C6I33

I

S. chromogenes

OXA, FOX

Genes mecA

SCCmec NTa

C6I68

I

S. chromogenes

OXA, FOX

mecA

NT

C6I69

I

S. hyicus

OXA, FOX

mecA

NT

C6I70

I

S. chromogenes

OXA, FOX, CCb, STRb

mecA, lnu(B), lsa(E), aad(E)

NT

C6I7I

I

S. chromogenes

OXA, FOX, CCb, STRb

mecA, lnu(B), lsa(E), aad(E)

NT but ccr2 positive

C6I72

III

S. epidermidis

OXA, FOX

mecA

IVa

C6I73

III

S. epidermidis

OXA, FOX

mecA

IVa

C6I74

III

S. hyicus

OXA, FOX, CCb

mecA

NT

C6I75

III

S. chromogenes

OXA, FOX

mecA

NT

C6I76

III

S. warneri

OXA, FOX

mecA

NT

C6I77 C6I78

III III

S. warneri S. epidermidis

OXA, FOX OXA, FOX, TET

mecA mecA, tet(K)

NT IVa

C6I79

IV

S. hyicus

OXA, FOX, CCb

mecA

NT

C6I80

IV

S. epidermidis

OXA, FOX, TET, GEN, TOB, STR

mecA, tet(K), ant(40 ), aac(60 )-aph(200 )

IVa

C6I8I

IV

S. warneri

OXA, FOX

mecA

NT but ccr2 positive

C6I82

IV

S. chromogenes

OXA, FOX, TET, STR, TOB

mecA, tet(K), ant(40 )

IVa

C6I83

IV

S. warneri

OXA, FOX, TET, STR, GEN, TOB

mecA, tet(K), ant(40 ), aac(6´)aph(200 )

IVa

C6I84

IV

S. epidermidis

OXA, FOX

mecA

NT

C6I85

V

S. chromogenes

OXA, FOX

mecA

NT

C6I86

V

S. warneri

OXA, FOX, TOB, TET, STR, CCb

mecA, str, tet(K), ant(4´)

IVa

b

C6I87

V

S. epidermidis

OXA, FOX, TET, GEN, TOB, STR, CC , ERY

mecA, tet(K), aac(60 )-aph(200 ), ant(40 ), str, erm(C)c

IVa

C6I88

V

S. warneri

OXA, FOX

mecA

NT but ccr1 positive

C6I89

V

S. hyicus

OXA, FOX, STR, CCb

mecA, str

NT

C6I90

V

S. simulans

OXA, FOX

mecA

NT

C6I9I

V

S. hyicus

OXA, FOX

mecA

NT

C6I92

VI

S. epidermidis

OXA, FOX, TET, TOB, GEN

mecA, aac(60 )-aph(200 ), tet(K), ant(40 )

IVa

OXA oxacilin, FOX cefoxitin, STR streptomycin, TET tetracycline, CC clindamycin, ERY erythromycin, TOB tobramycin, GEN gentamicin a

NT non-typeable

b

Intermediate resistance

c

Deletion of 89 bp

with other studies on MRCoNS (Barbier et al. 2010; Zong et al. 2011). These data show the high diversity of SCCmec elements among MRCoNS strains and the need for a broaden SCCmec typing scheme to try to classify this genetic structure in MRCoNS isolates. As observed in this study, the SCCmec IVa is

123

preferentially associated with S. epidermidis (Febler et al. 2010; Zong et al. 2011). Two strains presented intermediate resistance to clindamycin, mediated by the presence of lnu(B) and the recently characterized lsa(E) genes, which confer resistance to lincosamides (Wendlandt et al. 2013).

Antonie van Leeuwenhoek Fig. 1 a Nucleotide sequence comparison of a prototype functionally active macrolide/ lincosamides resistance gene erm(C) (MRSA ST398 plasmid pUR2940, strain C2940 (Genbank accession number HF583292.1) and S. epidermidis strain C6I87. The underlined parts indicate the sequences of the employed primers. b Nucleotide and amino acid comparison analysis of the amplified erm(C) region of C6I87 and the corresponding region in plasmid pUR2940. The conserved functional domain (CD-search) characteristic of S-adenosylmethioninedependent methyltransferases is indicated in gray

123

Antonie van Leeuwenhoek

Lozano et al. (2012) recently described the genetic environment of these two co-located genes for the first time in staphylococci (MRSA ST398) in Spain, and our strains of S. chromogenes revealed to carry a similar structure. In staphylococci, this structure had been only detected in S. aureus and it has been suggested to be acquired from enterococci. The presence of a similar element in CoNS is remarkable and reflects the ability to transfer genetic structures that carry multiple antimicrobial resistance determinants between different bacterial species and genera. Erythromycin is a macrolide antibiotic that blocks the protein synthesis (Spizek and Rezanka 2004); in Staphylococcus, resistance to this antimicrobial is mainly mediated by erythromycin-resistant methylases encoded by erm genes (Weisblum, 1995). Clindamycin belongs to group of lincosamides; it is chemically distinct but has a similar mode of action to macrolides antibiotics. The mechanism of resistance to lincosamides can be also encoded by erm genes (Lina et al., 1999). It is interesting to underline that one S. epidermidis strain exhibited intermediate resistance to erythromycin and clindamycin on the disk test and presented a deletion within the erm(C) gene, which may be responsible for this specific phenotype. The erm(C) gene is generally located on small plasmids and has a major role for resistance to erythromycin (Weisblum, 1995). Our observation suggests that partially deleted erm(C) gene may be still functionally active, what warrants further investigations. Resistance to clindamycin or streptomycin could not be explained by any of the determinants tested in a number of strains, what might suggest the existence of novel mechanisms of resistance among these isolates. Remarkably, this atypical resistance profile to streptomycin was already observed in a former study among S. aureus of the same area (Silva et al. 2013). In Brazil, few studies are available on the prevalence and antimicrobial resistance profile of Staphylococcus spp. in milk of bovine mastitis; however, further surveillance is necessary in order to improve the control and optimize the treatment of mastitis caused by these bacterial species, but also to gain knowledge in the possible risk of transmission of these microorganisms between cows and in-contact humans.

123

Conclusion MRCoNS need special attention because they can be implicated in cow mastitis. Furthermore, MRCoNS may constitute a reservoir of novel and diverse antimicrobial resistance genes and antimicrobial genetic structures that can be transferred to other pathogenic bacteria. Acknowledgments Silva N. C. C. has a fellowship from Capes–Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior, Process–9877-11-8. P. Gomez has a predoctoral fellowship of the Universidad de La Rioja of Spain. Part of this work was financially supported by Project SAF2012-35474 from the Ministerio de Economı´a y Competitividad of Spain and Fondo Europeo de Desarrollo Regional (FEDER).

References Archer GL, Climo MW (1994) Antimicrobial susceptibility of coagulase-negative staphylococci. Antimicrob Agents Chemother 38:2231–2237 Barbier F, Ruppe´ E, Hernandez D, Lebeaux D, Francois P, Felix B, Desprez A, Maiga A, Woerther P, Gaillard K, Jeanrot C, Wolff M, Schrenzel J, Andremont A, Ruimy R (2010) Methicillin-resistant coagulase-negative staphylococci in the community: high homology of SCCmec IVa between Staphylococcus epidermidis and major clones of methicillin-resistant Staphylococcus aureus. J Infect Dis 202:270–281 CLSI. Clinical Laboratory Standards Institute (2012) Performance standards for antimicrobial susceptibility testing, vol 19. National Committee for Clinical Laboratory Standards, Wayne M100-S19 CRL-AR. Community Reference Laboratory for antimicrobial resistance (2009) Multiplex PCR for the detection of the mecA gene and the identification of Staphylococcus aureus National Food Insitute. Technical University of Denmark, Copenhagen Cui L, Wang Y, Li Y, He T, Schwarz S, Ding Y, Shen J, Lv Y (2013) Cfr-mediated linezolid-resistance among methicillin-resistant coagulase-negative staphylococci from infections of humans. PLoS One 8(2):e57096. doi:10.1371/ journal.pone.0057096 Cuny C, Layer F, Strommenger B, Witte W (2011) Rare occurrence of methicillin-resistant Staphylococcus aureus CC130 with a novel mecA homologue in humans in Germany. Plos One 6(9):e24360. doi:10.1371/journal.pone. 0024360.t001 de Freitas Guimara˜es F, No´brega DB, Richini-Pereira VB, Marson PM, de Figueiredo JC, Langoni H (2013) Enterotoxin genes in coagulase-negative and coagulase-positive staphylococci isolated from bovine milk. J Dairy Sci 96:2866–2872

Antonie van Leeuwenhoek DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen). Disponı´vel em (2012) http://old.dsmz.de/ microorganisms/bacterial_nomenclature_info.php?genus= Staphylococcus&show_all_details=1. Acessed 27 Mar 2012 Febler AT, Billerbeck C, Kadlec K, Schwarz S (2010) Identification and characterization of methicillin-resistant coagulase-negative staphylococci from bovine mastitis. J Antimicrob Chemother 65:1576–1582 Go´mez-Sanz E, Torres C, Lozano C, Fernandez-Pe´rez R, Aspiroz C, Ruiz-Larrea F, Zarazaga M (2010) Detection, molecular characterization, and clonal diversity of methicillin-resistant Staphylococcus aureus CC398 and CC97 in Spanish slaughter pigs of different age groups. Food Path Dis 7:1269–1277 IWG-SCC (2009) International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements. Classification of staphylococcal cassette chromosome mec (SCCmec): guidelines for reporting novel SCCmec elements. Antimicrob Agents Chemother 53:4961–4967 Kondo Y, Ito T, Ma XX, Watanabe S, Kreiswirth BN, Etienne J (2007) Combination of multiplex PCRs for Staphylococcal Cassette Chromosome mec type assignment: rapid identification system for mec, ccr, and major differences in junkyard regions. Antimicrob Agents Chemother 51: 264–274 Lina G, Quaglia A, Reverdy ME, Leclercq R, Vandenesch F, Etienne J (1999) Distribution of genes encoding resistance to macrolides, lincosamides, and streptogramins among staphylococci. Antimicrob Agents Chemother 43:1062– 1066 Lozano C, Aspiroz C, Sa´enz Y, Ruiz-Garcı´a M, Royo-Garcı´a G, Go´mez-Sanz E, Ruiz-Larrea F, Zarazaga M, Torres C (2012) Genetic environment and location of the lnu(A) and lnu(B) genes in methicillin-resistant Staphylococcus aureus and other staphylococci of animal and human origin. J Antimicrob Chemother 67:2804–2808 Nam HM, Lim SK, Kim JM, Kang HM, Moon JS, Jang GC, Kim JM, Wee SH, Joo YS, Jung SC (2010) Antimicrobial susceptibility of coagulase-negative Staphylococci isolated from bovine mastitis between 2003 and 2008 in Korea. J Microbiol Biotechnol 20:1446–1449 Poyart C, Quesne G, Boumaila C, Trieu-Cuot P (2001) Rapid and accurate species-level identification of coagulasenegative staphylococci by using the sodA gene as a target. J Clin Microbiol 39:4296–4301 Sampimon OC, Lam TJ, Mevius DJ, Schukken YH, Zadoks RN (2011) Antimicrobial susceptibility of coagulase-negative staphylococci isolated from bovine milk samples. Vet Microbiol 150:173–179

Santos LL, Fogac¸a TF, Pedroso E (2010) Perfil etiolo´gico da mastite bovina na bacia leiteira de Santa Izabel do oeste, Parana´. Ci Anim Bras 11:860–866 Sawant AA, Gillespie BE, Oliver SP (2009) Antimicrobial susceptibility of coagulase-negative Staphylococcus species isolated from bovine milk. Vet Microbiol 134(1–2):73–81 Silva NCC, Guimara˜es FF, Manzi MP, Budri PE, Go´mez-Sanz E, Benito D, Langoni H, Rall VLM, Torres C (2013) Detection, molecular characterization, and clonal diversity of methicillin- susceptible Staphylococcus aureus in milk of cows with mastitis in Brazil. J Dairy Sci 96:6856–6862 Spizek J, Rezanka T (2004) Lincomycin, clindamycin and their applications. Appl Microbiol Biotechnol 64:455–464 Taponen S, Pyo¨ra¨la¨ S (2009) Coagulase-negative staphylococci as cause of bovine mastitis—not so different from Staphylococcus aureus? Vet Microbiol 134:29–36 Taponen S, Bjo¨rkroth J, Pyo¨ra¨la¨ S (2008) Coagulase-negative staphylococci isolated from bovine extramammary sites and intramammary infections in a single dairy herd. J Dairy Res 75:422–429 Virdis S, Scarano C, Cossu F, Spanu V, Spanu C, De Santis EPL (2010) Antibiotic resistance in Staphylococcus aureus and coagulase negative staphylococci isolated from goats with subclinical mastitis. Vet Med Internat 2010:6. doi:10.4061/ 2010/517060. ID 517060 Weisblum B (1995) Erythromycin resistance by ribosome modification. Antimicrob Agents Chemother 39:577–585 Wendlandt S, Lozano C, Kadlec K, Go´mez-Sanz E, Zarazaga M, Torres C, Schwarz S (2013) The enterococcal ABC transporter gene lsa(E) confers combined resistance to lincosamides, pleuromutilins and streptogramin A antibiotics in methicillin-susceptible and methicillin resistant Staphylococcus aureus. J Antimicrob Chemother 268:473–475 Zhang K, McClure JA, Elsayed S, Louie T, Conly JM (2005) Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal cassette chromosome mec types I to V in methicillin-resistant Staphylococcus aureus. J Clinical Microbiol 43:5026–5033 Ziebuhr W, Krimmer V, Rachid S, Loßner I, Gotz F, Hacker J (1999) A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256. Mol Microbiol 32:345–356 Zong Z, Peng C, Lu X (2011) Diversity of SCCmec elements in methicillin-resistant coagulase-negative Staphylococci clinical isolates. Plos One 6(5):e20191. doi:10.1371/ journal.pone.0020191

123