Methicillin resistant Staphylococcus aureus (MRSA) in pigs, the Spanish experience M. Concepción Porrero, José-Francisco Fernández- Garayzabal, Ana Mateos and Lucas Domínguez cporrero@visavet.ucm.es Food-borne Zoonoses and Antimicrobial Resistance Unit VISAVET Health Surveillance Centre, UCM Birmingham, UK November 12 th 2013
Staphylococcus aureus Microbiota Host range Clinical infections
β-lactams http://en.wikipedia.org/wiki/file:beta-lactam.svg PBPs penicillin-binding proteins http://commons.wikimedia.org/wiki/file:pbp_catalysis.svg
Methicillin resistant S. aureus Penicillin and cephalosporin resistance http://upload.wikimedia.org/wikipedia/commons/c/c8/meca_resistance.svg
4. Family members 5. Occupational activities 1. Isolates non-typable by PFGE 2. t108/st398 3. tet-r In July 2004, we unexpectedly found MRSA in the preoperative screening cultures of a 6-month-old girl before thoracic surgery. February 2005, 2 new cases of MRSA were identified, one in a pig farmer from a different region and one in the son of a veterinarian who worked mostly with pigs.
http://www.olmix.com/animal-division/pigs
Reference Sampling Detection spa type MLST de Neeling et al., 2007 Nasal swabs 39% pigs; 81%=farms; 100% slaughterhouses t011, t108 and t1254 ST398 Guardabassi et al., 2007 Nasal swabs 2/3 farms t034 (n = 9) and t1793 (n = 1) ST398 van Duijkeren et al., 2008 Nasal swabs 35/310 pigs (11%) t011, t108, t567, t899 and t1939 ST398 Khanna et al., 2008 Nasal and rectal swabs 9/20 farms (45%); 71/285 pigs (24.9%) t034, t571, t002, t067, t653
Baseline study (2008/55/EC) Breeding holdings Dust (at farms) EFSA Journal 2009; 7(11):1376
Different prevalence/ms Mainly ST398 EFSA Journal 2009; 7(11):1376
VAV Network Monitoring Antimicrobial Resistance in bacteria from animal origin in Spain
Monitoring programmes At slaughterhouse Slaughterhouse selection Capacity Geographical distribution Randomly selected batches One sample/batch One batch/farm Samples: skin swabs (ear)
Isolation and identification Swabs MH NaCl 6.5% spa TSB (FOX&ATM) Chromogenic media PVL mecc meca Identification (PCR)
Characterization spa typing Protein A (spa gene) 24pb repeats (insertions, delections, mutation) Short term evolution t127 GCACCAAAA 07 23 21 16 34 33 13 TACATGTCGT t011 GCACCAAAA 08 16 02 25 34 24 25 TACATGTCGT t034 GCACCAAAA 08 16 02 25 02 25 34 24 25 TACATGTCGT t1451 GCACCAAAA 08 16 02 25 34 25 TACATGTCGT http://spa.ridom.de/spatypes.shtml Harmsen et al. 2003
Characterization MLST(Multi Locus Sequence Typing) 7 housekeeping genes Long term evolution Allelic profile ST= Sequence Type ST arcc aroe glpf gmk_ pta_ tpi_ yqil 1 1 1 1 1 1 1 1 5 1 4 1 4 12 1 10 8 3 3 1 1 4 4 3 MLST genes arcc aroe glpf gmk pta tpi yqil Carbamate kinase Shikimate dehydrogenase Glycerol kinase Guanylate kinase Phosphate acetyltransferase Triosephosphate isomerase Acetyl coenzyme A acetyltransferase 398 3 35 19 2 20 26 39 425 18 33 6 20 7 50 48 www.mlst.net Enright et al. 2000
Fattening pigs at slaughterhouses t899 1 2007-2008 t693 t4885 t4884 t4874 t4873 t4872 t4871 t1456 t1451 t1344 t127 t1197 t108 t034 t011 1 1 1 1 1 1 1 1 1 1 1 2 6 18 160/263 (60.8%) Most ST398 122
Breeding pigs at farms (baseline study) 2008 177/361 (49.0%) Most ST398 2 2 1 1 1 3 1 1 1 t011 t034 8 t108 t1197 23 t1255 t127 1 t1344 t1451 t1456 t2329 132 t2330 t4872 t567
Fattening pigs at slaughterhouses 2009 159/276 (57.6%) Most ST398 125 3 17 7 1 1 3 1 1 t011 t034 t108 t1197 t1255 t127 t1451 t4872 t588
Iberian pigs and standard white pigs
150 100 50 0 83% 27 130 32% 25% 34 42 16 14 SWP Ipc Ipm Positive Negative SWP Ipc Ipm t011 97 13 12 t034 1 0 0 t108 16 1 1 t1197 0 1 0 t127 1 0 0 t1344 1 0 0 t1451 5 0 1 t1456 2 0 0 t4871 0 1 0 t4872 1 0 0 t4873 1 0 0 t4874 1 0 0 t4884 1 0 0 t4885 1 0 0 t693 1 0 0 t899 1 0 0 130 16 14
http://tareasccbbpolavide.blogspot.co.uk/2009/12/los-embalses.html
Small ruminants Mastitis: 1995-2009 MSSA; most prevalent ST522, ST133
Wild animals Cervus elaphus Hunted or box-trapped Nares & skin (EGV*nares) N. Navarro Gyps fulvus Capra pyrenaica G. Mentaberre Sus scrofa G. Mentaberre J. Baucells
Natural parks Feeding point Game estates Eurasian griffon vulture Iberian ibex Wild boar Red deer Red deer Iberian ibex Wild boar Eurasian griffon vulture TOTAL Samples 543 369 1594 40 2546 Nasal swabs 269 211 795 40 1315 Skin swabs a 274 154 797 1225 Other b 4 4 ND c 2 2 Tested animals 273 212 817 40 1342 a. Skin swabs were taken from retro-auricular area in red deer and wild boar and from inguinal-mammal area in Iberian ibex; b. Exceptionally, Iberian ibex were sampled on ear (n=2) and retro-auricular skin (n=2); c. ND: not determined
Red deer Iberian ibex Wild boar Eurasian griffon vulture TOTAL Tested animals 273 212 817 40 1342 Positive animals % (CI) 1 0.37 (0.01,2.02) 2 0.94 (0.11,3.37) 7 0.86 (0.35,1.76) 2 5.00 (0.61,16.92) 12 0.89 (0.46,1.56) Isolates 1 2 8 2 13 Nasal swabs 1 3 2 6 Skin swabs 1 1 5 7 Genotype (n) ST398-t011 (1) ST398-t011 (1) ST398-t011 (6) ST398-t011 (2) ST398-t1451 (1) ST1-t127 (2) CI: Exact 95% Fisher's confidence interval P=0.083
Eurasian griffon vulture Iberian ibex Wild boar Red deer
mecc Expanding the presence of Staphylococcus aureus carrying mecc gene: Spanish emergence in wild animals and urban effluents M. Concepción Porrero a, Arancha Valverde a, Pedro Fernández-Llario b, Alberto Díez- Guerrier a, Ana Mateos a, Santiago Lavín c, Rafael Cantón d, José-Francisco Fernández- Garayzabal a, and Lucas Domínguez a, * Swabs/Water samples n=361 isolates Livestock (n=39) Wild animals (n=254) Urban effluents (n=68) Cattle (n=5) Fattening pigs (n=34) Eurasian griffon vulture (n=2) Fallow deer (n=2) Iberian ibex (n=39) Mouflon (n=2) Red deer (n=61) Wild boar (n=148) Urban water (n=68) Identification (PCR)
mecc positives isolates Animal species (Isolates tested) mecc-carrying S. aureus spa type MLST CC AST profile Fallow deer (n=2) 2 t11212 t11212 ST425 ST425 CC425 CC425 PEN -FOX PEN Wild boar (n=148) Urban effluents (n=68) 1 t11212 ST425 CC425 PEN-FOX 1 t843 ST2676 CC130 PEN-ERY *MLST: Multi Locus Sequence Typing. CC: Clonal Complex. AST: Antimicrobial Susceptibility Testing. PEN: Benzylpenicillin. FOX: Cefoxitin.
MRSA vs MSSA n=635 isolates Reservoir Animal Species MRSA MSSA Total meca mecc n % n % n Wild animal Griffon vulture 2 0 2 50,0 2 50,0 4 Fallow deer 0 1 1 100,0 0 0,0 1 Iberian ibex 2 0 2 5,3 36 94,7 38 Mouflon 0 0 0 0,0 2 100,0 2 Red deer 1 0 1 1,7 57 98,3 58 Wild boar 8 1 9 6,2 136 93,8 145 Livestock Cattle 7 0 7 58,3 5 41,7 12 Fattening pigs 178 0 178 86,4 28 13,6 206 Urban water Urban effluent 104 1 105 61,0 67 39,0 172 MRSA: methicillin resistant S. aureus; MSSA: methicillin susceptible S. aureus
Simpson's Index of Diversity Simpson s Index of Diversity and 95% confidence intervals 1,0 0,8 0,6 0,4 0,2 0,0 UW MSSA WA MSSA LA MSSA UW MRSA WA MRSA LA MRSA (Carrico, Silva-Costa et al. 2006) http://darwin.phyloviz.net/comparingpartitions/index.php?link=tool
ST2328 ST5 ST425 ST581 ST1 ST133 ST2671 ST2637 ST1643 ST350 ST522 ST130 ST2681 ST398 ST188 ST2639 ST2640 ST2675 ST15 ST2641 ST2672 ST2673 ST2678 ST2682 ST2729 ST30 ST96 200 150 100 50 MRSA livestock (n=184) 12 10 8 6 4 2 0 MSSA livestock (n=34) 0 ST398 ST1 12 10 8 6 4 2 0 MRSA wild animals (n=15) ST398 ST1 ST425 80 60 40 20 0 MSSA wild animals (n=233) MRSA_mecA MRSA_mecC 80 60 40 20 0 MRSA urban water (n=102) ST125 ST22 ST5 ST1 ST1094 ST72 ST2674 ST398 ST2676 MRSA_mecA MRSA_mecC 30 25 20 15 10 5 0 MSSA urban water (n=67)
MSSA Pigs SCCmec MRSA Pigs?? - Water - Iberian pigs - Other domestic animals MSSA MRSA Wild animals Wild animals MSSA MRSA Urban water Urban water