EV0221 Antimicrobial susceptibility of mastitis pathogens isolated from diseased dairy cows across Europe: VetPath monitoring results 25th ECCMID 25 – 28 April 2015 A. de Jong, H. Moyaert, S. Simjee, F. El Garch, S. Haag-Diergarten, U. Klein, C. Ludwig, P. Butty, A. Richard-Mazet, D. Rigaut, J. Thiry, V. Thomas Contact: Copenhagen, Denmark VetPath Study Group, CEESA, Brussels, Belgium [email protected]

Methods VetPath Project • Milk samples were collected in Belgium, Czech Republic, Den- Clinical and Laboratory Standard Institute standards2 using 96 kanamycin and cephalexin / kanamycin which showed i.e. above 32 mg / L were found for tylosin and erythro- l The VetPath project is a pan-European pro- mark, France, Germany, Italy, The Netherlands, Spain and The well TREK plates. slightly higher MIC50 and MIC90 values at 4 mg / L, and mycin (20.2 % resistance), neomycin and kanamycin gramme dedicated to the collection and to United Kingdom. Samples were collected from cows with acute penicillin for which a low MIC50 value of 0.03 mg / L was (no resistance breakpoints) and tetracycline (36.7 % • MIC 50 and MIC90 were calculated and are presented in Table 1 the monitoring of antibiotic susceptibility clinical signs of mastitis, not recently exposed to antimicrobial observed but a MIC90 value of 1 mg / L corresponding resistance). for all antibiotics tested whereas for 5 antibiotics the percent- to 25.0 % of resistance. Resistance to tetracycline was of veterinary pathogens from diseased food- treatment, and were processed according to standard proce- MIC values for S. dysgalactiae were much lower for age resistance was calculated according to CLSI breakpoints low at 5.2 % and for erythromycin 1.0 % of the isolates 50 / 90 producing animals not recently exposed to dures for the isolation and identification of the pathogens at 3 the beta-lactam antibiotics compared to S. uberis. For (Table 2). Only for ceftiofur mastitis-specific breakpoints are were resistant, 0.5 % showed intermediate suscepti- antimicrobials. regional laboratories. Only one isolate per bacterial species per the other antibiotics, MICs were comparable to those available; for the other 4 compounds, CLSI breakpoints set for bility. Neomycin, kanamycin and cephalexin / kanamy- farm was selected. found for S. uberis. Resistance to tetracycline amount- l VetPath is an initiative of, and is coordinated human pathogens are applied. In total 847 isolates were re- cin showed each 1 or 2 isolates with deviating MICs of ed to 56.8 % and 13.7 % of the isolates were resistant by, the Executive Animal Health Study Center • MICs of 16 antibiotics / antibiotic combinations (for E. coli 12) covered, 207 E. coli, 192 S. aureus, 165 coagulase-negative ≥ 128 mg / L. to erythromycin. (CEESA) in Brussels. It has been operational were determined at a central laboratory (LGC; formerly Quotient Staphylococcus spp., 95 S. dysgalactiae and 188 S. uberis. Coagulase-negative Staphylococcus spp. data were since 2002. CEESA’s membership is compo- To determine whether the susceptibility or resistance % Bioresearch, UK) by the broth microdilution according to the similar to the MIC distributions of S. aureus. The an- sed of international pharmaceutical compa- has changed, the MIC values and / or % resistance tibiotics showed MIC values in the range of 0.25 to 50 / 90 nies researching and producing veterinary­ Table 1. In vitro activity (MIC , MIC and MIC ranges in mg/L) of different antibiotics against strains from mastitis in dairy cows in 9 EU countries* 50 observed in VetPath 2002-20064,5 were compared to 50 90 1 mg / L (exception cephalexin and tylosin: 2 mg / L) the present results for 3 major pathogens and for 12 an- medicinal products. and the MIC values varied from 0.12 to 2 mg / L E. coli S. aureus CNS S. uberis S. dysgalactiae 90 tibiotics. MIC values differed at most one step and l Sponsors of the current VetPath project were (cephalexin 4 mg / L). Penicillin resistance was high 50 / 90 (n=207) (n=192) (n=165) (n=188) (n=95) any important shifts haven’t been observed (for MIC (29.1 %); penicillin and cephalonium MIC values 90 the following companies: Bayer Animal Health 50 see Table 3). Similarly, the prevalence of resistance for MIC50 MIC90 MIC Range­ MIC50 MIC90 MIC Range­ MIC50 MIC90 MIC Range­ MIC50 MIC90 MIC Range­ MIC50 MIC90 MIC Range­ were 0.06 mg / l. GmbH, Germany; Boehringer Ingelheim,­ Ger- 5 antibiotics having CLSI breakpoints remained on av- many; CEVA Sante Animale, France;­ Elanco Penicillin - - - 0.03 1 ≤0.004-≥16 0.06 1 ≤0.004-≥16 0.12 0.25 0.008-0.5 0.008 0.008 ≤0.004-0.015 For S. uberis, an MIC50 value of 0.12 to 0.5 mg / L was erage constant (Table 4). These findings suggest that Animal Health, UK; MSD Animal Health, The Cloxacillin - - - 0.25 0.5 0.06-4 0.5 2 0.12-≥16 2 4 0.25-≥16 0.12 0.12 0.06-0.12 observed for penicillin, AMC, cefquinome, cephalexin resistance to mastitis pathogens is neither emerging nor Netherlands; Merial, France; Novartis Animal and cefapirin; MIC90 values were usually one dilution is a progressing phenomenon. Two other ongoing Euro- Health Inc., Switzerland; Vetoquinol S. A., Amoxicillin/ 4 8 0.5-64 0.12 1 0.06-8 0.25 0.5 ≤0.03-2 0.25 0.5 ≤0.03-4 ≤0.03 ≤0.03 ≤0.03-0.06 step higher. For ceftiofur as well as enrofloxacin and pean monitoring projects, GERM-Vet and Résapath,­ 6,7 Clav acid (2:1) France; Virbac, France; Zoetis, Belgium. marbofloxacin MIC50 values were 1 mg / L. High MIC90 have similarly not detected changes in resistance. l The culture collection of VetPath was organi- Cefquinome 0.06 0.12 0.03-≥32 0.5 0.5 0.25-8 0.5 1 0.06-2 0.12 0.25 ≤0.008-0.5 ≤0.008 0.015 ≤0.008-0.03 zed by CEESA, and subsequently from each Table 3. MIC90 values (mg/L) of different antibiotics against bacteria isolated from mastitis in dairy cows recovered 2002-2006 versus 2009-2012 Ceftiofur 0.5 0.5 0.12-≥16 1 1 0.25-8 1 2 0.12-4 1 2 0.06-4 0.06 0.03-0.06 0.06 participating country transmitted to a central E. coli S. aureus S. uberis European laboratory (LGC, Fordham, UK). All Cephalexin 8 16 4-≥64 2 4 0.25-≥64 2 4 0.12-16 0.5 0.5 0.12-1 0.5 0.25-0.5 0.5 2002-06 (n=280) 2009-12 (n=207) 2002-06 (n=250) 2009-12 (n=192) 2002-06 (n=282) 2009-12 (n=188) antimicrobial susceptibility ­determinations Penicillin - - 2 1 0.25 0.25 Cephapirin 16 >32 1-≥64 0.12 0.25 0.06-4 0.12 0.25 ≤0.03-≥64 0.25 0.5 ≤0.03-1 0.06 0.06 ≤0.03-0.06 were performed by LGC. Cloxacillin - - 0.5 0.5 4 4 Amoxicillin/Clavulanic acid 8 8 1 1 0.5 0.5 l The CEESA project manager is Hervé Marion,­ Cephalonium 4 8 1-≥64 0.12 0.25 ≤0.03-4 0.06 0.12 ≤0.03-≥64 ≤0.03 0.06 ≤0.03-0.25 ≤0.03 ≤0.03 ≤0.03-0.06 Cefquinome 0.06 0.12 1 0.5 0.25 0.25 Ceftiofur 0.5 0.5 1 1 2 2 CEESA, Rue Defacqz 1, Bte 8, B-1000 Brussels, Enrofloxacin 0.03 0.06 0.015-≥16 0.12 0.25 0.03-1 0.12 0.5 0.03-2 1 1 0.25-2 0.5 1 0.5-1 Cephalexin 8 16 4 4 0.5 0.5 Belgium. Marbofloxacin 0.03 0.06 0.015-≥16 0.25 0.5 0.12-1 0.25 0.5 0.06-8 1 2 0.12-2 1 1 0.5-2 Cephapirin 32 >32 0.25 0.25 0.5 0.5 For correspondence: Enrofloxacin 0.03 0.06 0.25 0.25 1 1 Tel: +32 2 543 7572 Erythromycin - - - 0.25 0.5 0.12-≥128 0.25 0.5 ≤0.03-≥128 0.06 >64 ≤0.03-≥128 0.06 >64 ≤0.03-≥128 Marbofloxacin 0.06 0.06 0.5 0.5 2 2 e-mail: [email protected] Erythromycin - - 0.5 0.5 16 >64 Tylosin - - - 1 2 0.25-32 2 2 0.25-≥64 0.5 >32 0.12-≥64 0.25 >32 0.12-≥64 Neomycin 2 4 1 1 >64 >64 Tetracycline ≥64 >32 0.5 0.5 64 >32 Kanamycin 4 8 2-≥256 2 4 0.25-≥256 1 2 ≤0.12-64 64 128 1-≥256 32 >128 16-≥256 - : not tested Cephalexin/ 4 8 2-≥128 2 4 0.25-≥128 0.5 1 ≤0.12-≥128 4 4 1-8 4 4 2-8 Table 4. Prevalence of antibiotic resistance (%) in bacteria isolated from mastitis 2002-2006 versus 2009-2012 applying CLSI interpretive criteria* Introduction Kanam (1:10) Antimicrobial resistance of bacterial pathogens is of E. coli S. aureus S. uberis Neomycin 2 4 1-≥128 0.5 1 ≤0.12-≥128 ≤0.12 0.25 ≤0.12-4 64 >64 4-≥128 32 >64 4-≥128 2002-06 (n=280) 2009-12 (n=207) 2002-06 (n=250) 2009-12 (n=192) 2002-06 (n=282) 2009-12 (n=188) concern for the antimicrobial therapy of both humans Amoxicillin/clavulanic acid 1.0 3.9 0.0 0.5 0.0 0.0 Tetracycline 2 >32 1-≥64 0.25 0.5 0.12-≥64 0.25 1 0.12≥64 0.25 >32 0.12-≥64 8 >32 1-≥64 and animals. Harmonized and continuous monitoring Ceftiofur 0.0 1.0 0.0 1.0 0.0 0.0 of antimicrobial susceptibility trends over time is an A dash indicates not tested. Erythromycin - - 1.4 1.0 19.0 20.2 important component of stewardship to ensure long- Penicillin - - 38.0 25.0 0.0 0.0 term antimicrobial efficacy. Tetracycline 16.0 14.5 7.3 5.2 28.7 36.7 Table 2. Prevalence of antibiotic resistance (%) in bacteria isolated from masti- *For CLSI resistance breakpoints see Table 2. A dash indicates no breakpoints are available. The present monitoring study is part of the on-going tis applying CLSI resistance breakpoints* VetPath programme. This programme collects bacte- Results and discussion ria from diseased food animals (cattle, swine, poultry) Antimicrobial Resistance Percentage of resistance** breakpoint For E. coli, amoxicillin / clavulanic acid (AMC), cephalexin and Conclusions References across Europe, employing a protocol with harmonized (mg/L) Ec Sa CNS Su Sd cephalonium showed comparable activity with MIC90 values of The results of this pan-European survey, with 1. Cressier, B., Bissonette, N., (2011). Assessment of an extraction protocol to methods of sampling and bacterial isolation. A central (n=207) (n=192) (n=165) (n=188) (n=95) detect the major mastitis-causing pathogens in bovine milk. J. Dairy Sci 94: 8-16 mg / L. The cephapirin MIC90 value was slightly higher at standardized methods, generally show low MIC 2171-84. laboratory conducts MIC determinations using a pan- >32 mg / L. The ceftiofur MIC value was 0.5 mg / L. The high- 50 Amoxicillin/ Ec, Su, Sd ≥32; 3.9 (3.4) 0.5 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 50 / 90 values and an absence or a low prevalence of 2. Clinical and Laboratory Standards Institute (2013). Approved standard – fourth el of licensed antimicrobials commonly used in veter- clavulanic acid Sa, CNS ≥8 est in vitro activity was observed for one beta-lactam, cefquinome edition. CLSI document VET01-A4. Wayne, PA, USA. inary medicine. (MIC value 0.06 / 0.12 mg / L), and two fluoroquinolones, en- antimicrobial resistance among the major patho- 3. Clinical and Laboratory Standards Institute (2013). Second informational 50 / 90 supplement. CLSI document VET01-S2. Wayne, PA, USA. Ceftiofur ≥ 8 1.0 (0.0) 1.0 (1.6) - 0.0 (1.1) 0.0 (0.0) rofloxacin and marbofloxacin with an MIC value of 0.03 mg / L gens collected from non-treated dairy cows with Bovine mastitis is the most costly disease to the dairy 50 acute mastitis. Exceptions are tetracycline vs. 4. Thomas, V., de Jong, A., Simjee, S., Maher, K., et al. (2012). Antimicrobi- and MIC90 value of 0.06 mg / L. Very few antibiotics have a CLSI al susceptibility of mastitis pathogens isolated from diseased dairy cows industry and antimicrobial therapy is important to con- Erythromycin Sa, CNS ≥ 8; - 1.0 (0.5) 5.5 (2.4) 20.2 (0.5) 13.7 (1.1) nd Su, Sd ≥1 E. coli, S. dysgalactiae and S. uberis, penicil- across Europe: VetPath monitoring results. 22 ECCMID, London, UK. trol mastitis. The economic impact of mastitis includes breakpoint and from Table 2 it can be concluded that resistance 2012. Clin. Microbiol. Infect., 18, Suppl. S3, Abstr. P 1103, 264-265. to ceftiofur and AMC was low. Resistance to tetracycline was lin vs. S. aureus and CNS and erythromycin vs. cost for therapy, discarded milk, reduced milk produc- Penicillin Sa, CNS ≥0.25; - 25.0 (0.0) 29.1 (0.0) 0.0 (35.6) - 5. Thomas, V., de Jong, A., Moyaert, H., Simjee, S., et al. (2015). Antimicrobial moderate at 14.5 %. Of the isolates, 3.4 % showed intermediate S. dysgalactiae and S. uberis. This study under- susceptibility monitoring of mastitis pathogens isolated from acute cases tion and culling and replacement. Five major bacterial Su ≥4 of clinical mastitis in dairy cows across Europe: VetPath results. Intern. J. pathogens of acute mastitis, Escherichia coli, Staphy- susceptibility to AMC, 0.0 % to ceftiofur and 1.4 % to tetracy- scores the urgent need for establishing addi- Antimicrob Agents. In press. Tetracycline Ec, Sa, CNS ≥16; 14.5 (1.4) 5.2 (0.5) 7.3 (0.0) 36.7(0.0) 56.8 (31.6) lococcus aureus, coagulase-negative Staphylococcus cline. For neomycin, although the MIC90 value was 4 mg / L, 5 iso- tional mastitis-specific clinical breakpoints. The 6. GERM-Vet 2011-2012. (2015). Resistenzsituation bei klinisch wichtigen tier- Su, Sd ≥8 lates (1.4 %) were found with an MIC > 64 mg / L. pathogenen Bakterien 2011 / 2012 spp., Streptococcus dysgalactiae and Streptococcus VetPath network, based on quantitative and http://www.bvl.bund.de/resistenzmonitoring.html?nn=1401276 uberis1, were isolated in nine EU countries during 2009 **Ec: Escherichia coli, Sa: Staphylococcus aureus, CNS: coagulase-negative Staphylococcus­ spp., With regard to S. aureus, the antibiotics tested showed compara- representative data, enables to reliably moni- 7. Résapath. (2014). Réseau d’épidémiosurveillance de l’antibiorérsistance des Sd: Streptococcus dysgalactiae and Su: Streptococcus uberis. bacteriés pathogens animals. Bilan 2013. https://www.resapath.anses.fr/ to 2012. The results of their antibiotic susceptibility are A dash indicates no CLSI breakpoint available. ble activity with MIC50 values in the range of 0.12 to 1 mg / L and tor antimicrobial susceptibility of major mastitis resapath_uploadfiles/files/Documents/2013%20RESAPATH%20 presented here. **Figures in parentheses refer to the percentage intermediate resistance. MIC90 values of 0.25 to 2 mg / L with the exception of cephalexin, pathogens across Europe. Rapport%20AnnuelWeb.pdf