Methicillin resistant Staphylococcus aureus (MRSA) in pigs, the Spanish experience

Food-borne Zoonoses and Antimicrobial Resistance Unit

VISAVET Health Surveillance Centre, UCM

Birmingham, UK

November 12th 2013

M. Concepción Porrero, José-Francisco Fernández-

Garayzabal, Ana Mateos and Lucas Domínguez

cporrero@visavet.ucm.es

mailto:cporrero@visavet.ucm.es

Staphylococcus aureus

• Microbiota

• Host range

• Clinical infections

http://en.wikipedia.org/wiki/File:Beta-lactam.svg

http://commons.wikimedia.org/wiki/File:PBP_catalysis.svg

PBPs penicillin-binding

proteins

β-lactams

Penicillin and cephalosporin resistance

http://upload.wikimedia.org/wikipedia/commons/c/c8/MecA_Resistance.svg

Methicillin resistant S. aureus

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.

In July 2004, we unexpectedly found MRSA in the

preoperative screening cultures of a 6-month-old girl before

thoracic surgery.

4. Family members

5. Occupational activities

1. Isolates non-typable by PFGE 2. t108/ST398 3. tet-R

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

• Breeding holdings

• Dust (at farms)

EFSA Journal 2009; 7(11):1376

Baseline study (2008/55/EC)

• Different prevalence/MS

• Mainly ST398 EFSA Journal 2009; 7(11):1376

VAV Network

Monitoring Antimicrobial Resistance in bacteria from animal origin in Spain

• At slaughterhouse

• Slaughterhouse selection

– Capacity

– Geographical distribution

• Randomly selected batches • One sample/batch • One batch/farm • Samples: skin swabs (ear)

Monitoring programmes

Swabs

TSB (FOX&ATM)

Chromogenic media

MH NaCl 6.5%

Identification (PCR)

spa

mecA mecC PVL

Isolation and identification

spa typing

• Protein A (spa gene)

• 24pb repeats (insertions, delections, mutation)

• Short term evolution

http://spa.ridom.de/spatypes.shtml

08 16 02 25 34 24 25 GCACCAAAA TACATGTCGT t011

08 16 02 25 GCACCAAAA TACATGTCGT 34 25 t1451

08 16 02 25 02 25 34 24 25 GCACCAAAA TACATGTCGT t034

07 23 21 16 34 33 13 GCACCAAAA TACATGTCGT t127

Characterization

Harmsen et al. 2003

MLST(Multi Locus Sequence Typing) MLST genes

arcC Carbamate kinase

aroE Shikimate

dehydrogenase

glpF Glycerol kinase

gmk Guanylate kinase

pta Phosphate

acetyltransferase

tpi Triosephosphate

isomerase

yqiL Acetyl coenzyme A

acetyltransferase

7 housekeeping genes

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

398 3 35 19 2 20 26 39

425 18 33 6 20 7 50 48

Allelic profile Long term evolution

www.mlst.net

Characterization

Enright et al. 2000

122

1

18

1

1

1

6

2

1

1

1

1

1

1

1

1

t011

t034

t108

t1197

t127

t1344

t1451

t1456

t4871

t4872

t4873

t4874

t4884

t4885

t693

t899 • 2007-2008

• 160/263 (60.8%)

• Most ST398

Fattening pigs at slaughterhouses

• 2008

• 177/361 (49.0%)

• Most ST398

132

1

23

8

2

2 1

1 1

3

1 1

1 t011

t034

t108

t1197

t1255

t127

t1344

t1451

t1456

t2329

t2330

t4872

t567

Breeding pigs at farms (baseline study)

• 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

Fattening pigs at slaughterhouses

Iberian pigs and standard white pigs

130

16 14

27

34 42

0

50

100

150

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

25% 32%

83%

http://tareasccbbpolavide.blogspot.co.uk/2009/12/los-embalses.html

• Mastitis: 1995-2009

• MSSA; most prevalent ST522, ST133

Small ruminants

• Hunted or box-trapped

• Nares & skin (EGV*nares)

Cervus elaphus

N. Navarro

J. Baucells

Gyps fulvus

G. Mentaberre

Capra pyrenaica

G. Mentaberre

Sus scrofa

Wild animals

Red deer Iberian ibex Wild boar Eurasian

griffon vulture TOTAL

Samples 543 369 1594 40 2546

Nasal swabs 269 211 795 40 1315

Skin swabsa 274 154 797 1225

Otherb 4 4

NDc 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

Eurasian griffon vulture

Iberian ibex

Wild boar

Red deer

Natural parks Feeding point Game estates

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)

P=0.083 CI: Exact 95% Fisher's confidence interval

Eurasian griffon vulture Iberian ibex Wild boar Red deer

Livestock (n=39)

Cattle (n=5)

Fattening pigs (n=34)

Wild animals (n=254)

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 effluents (n=68)

Urban water (n=68)

Expanding the presence of Staphylococcus aureus carrying mecC

gene: Spanish emergence in wild animals and urban effluents M. Concepción Porreroa, Arancha Valverdea, Pedro Fernández-Llariob, Alberto Díez-

Guerriera, Ana Mateosa, Santiago Lavínc, Rafael Cantónd, José-Francisco Fernández-

Garayzabala, and Lucas Domíngueza,* mecC

Swabs/Water samples

Identification (PCR)

• n=361 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) 1 t11212 ST425 CC425 PEN-FOX

Urban effluents

(n=68) 1 t843 ST2676 CC130 PEN-ERY

*MLST: Multi Locus Sequence Typing. †CC: Clonal Complex. ‡AST: Antimicrobial Susceptibility Testing.

§PEN: Benzylpenicillin. ¶FOX: Cefoxitin.

mecC positives 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

MRSA vs MSSA • n=635 isolates

UW MSSA WA MSSA LA MSSA UW MRSA WA MRSA LA MRSA

Sim

pso

n´s

In

de

x o

f D

ive

rsity a

nd

95

% c

on

fid

en

ce

in

terv

als

0,0

0,2

0,4

0,6

0,8

1,0

Simpson's Index of Diversity

(Carrico, Silva-Costa et al. 2006) http://darwin.phyloviz.net/ComparingPartitions/index.php?link=Tool

http://darwin.phyloviz.net/ComparingPartitions/index.php?link=Tool

0

50

100

150

200

ST398 ST1

MRSA livestock (n=184)

02468

1012

MSSA livestock (n=34)

02468

1012

ST398 ST1 ST425

MRSA_mecA MRSA_mecC

MRSA wild animals (n=15)

0

20

40

60

80

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

MSSA wild animals (n=233)

0

20

40

60

80

ST125 ST22 ST5 ST1 ST1094 ST72 ST2674 ST398 ST2676

MRSA_mecA MRSA_mecC

MRSA urban water (n=102)

05

1015202530

MSSA urban water (n=67)

SCCmec

MRSA

Wild animals

MSSA

Wild animals

MRSA

Urban water

MSSA

Urban water

- Water - Iberian pigs - Other domestic

animals MSSA

Pigs

MRSA

Pigs ??