Veterinary Susceptibility Testing.
Chris Teale,
Veterinary Laboratories Agency.
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Key Surveillance Objective.
• Provide information of value in formulating
sound policy to limit the development of
resistance in bacteria of animal origin and to
control the spread of antimicrobial
resistance within the animal population and
to man.
Surveillance for Antimicrobial
Resistance at VLA
3 main areas of activity:
Salmonella enterica
Abattoir Surveillance for
Zoonotic and Indicator
Organisms.
Veterinary Clinical Diagnostic
Submissions
Salmonella enterica
• Salmonella isolates are tested against a panel of 16
antimicrobials by disc diffusion test.
• Results are reported annually.
• Isolates originate from veterinary clinical
submissions, the abattoir surveys of animals at the
point of slaughter and from private laboratories
(who are required to submit isolates for testing
under statute - the Zoonoses Order).
Surveillance for Antimicrobial
Resistance in Salmonella.
• Ciprofloxacin MIC is performed on all
isolates that are resistant to nalidixic acid in
the disc diffusion test.
• Ceftriaxone (or other third generation
cephalosporin) MICs are performed on all
isolates that are resistant to either
ceftazidime or cefotaxime.
Surveillance of Veterinary
Clinical Submissions at Regional
Diagnostic Laboratories.
• The VLA has 14 Regional Laboratories that are
strategically located throughout England and
Wales, ensuring coverage of all regions of these
countries.
• Surveillance is achieved through incoming carcase
material and specimens.
• Susceptibility testing is provided as part of the
laboratory diagnostic service. In general, disc
diffusion testing is performed.
Veterinary Clinical Submissions.
• A system has been put in place to collect all
of the susceptibility data from tests
performed at the Regional Laboratories and
this is published annually on the Defra website(http://defraweb/animalh/diseases/zoono
ses/index.htm)
Statistically-based Surveillance at
Abattoirs.
• This is essential to complement clinical
surveillance, in particular for organisms such as
Campylobacter, Enterococcus and E.coli O157,
commensal E.coli, both for prevalence studies and
to assess the antimicrobial resistance of these
organisms.
• Abattoir surveys on pigs, sheep and cattle were
performed in 1999/2000 in Great Britain; these are
currently being repeated.
Harmonisation of Methods.
• Key objective is harmonisation of medical and
veterinary susceptibility testing within the UK.
• Secondary objective is harmonisation at the
international level. A network of all EU veterinary
laboratories is taking this forward within the EU.
• The first joint medical/ veterinary resistance report
covering a range of organisms isolated in 2002 has
been prepared by HPA/VLA.
Disc Diffusion Test.
• Currently used in most UK veterinary laboratories.
• Cut-off zone diameter of 13mm used to
discriminate between sensitive and resistant
strains.
• British Society for Antimicrobial Chemotherapy
method is being adopted at VLA. Automated zone
readers being introduced to allow full
implementation.
Resistance Monitoring at VLA.
• Disc diffusion test.
• Panels of 8 antimicrobials (6 fixed; 2 selected at
each Centre) appropriate for animal species and
Gram-reaction of organism.
• Results (S/R) entered into Access database, which
both generates practitioner report and saves data.
• All databases combined and summary data
produced annually at VLA Shrewsbury.
Disc Diffusion Methods (1)
•
•
•
•
NCCLS
US Standard.
Confluent inoculum
Data for some veterinary pathogens
available
• Mueller-Hinton
• Used in some Scottish laboratories.
Disc Diffusion Methods (2)
• BSAC
• Isosensitest agar
• Semi-confluent inoculum (nudging
colonies)
• UK standard method
• Currently no specific veterinary guidelines
• Harmonisation with medical colleagues
Disc diffusion test -Stokes
method.
Why is zone size determination
useful?
• Early detection of trends in the emergence
of resistance.
• Elucidation of mechanisms of resistance
(and interpretative reading).
• Linkage of zone size to mic data
• Improve Quality Control
• Establishment of robust breakpoints.
The relationship between Zone
Diameter and MIC.
Log
MIC
Zone Diameter
The relationship between Zone
Diameter and MIC.
Log
MIC
Concentration
reached in
animal.
Zone Diameter
The relationship between Zone
Diameter and MIC.
Appropriate Zone Diameter.
Log
MIC
Concentration
reached in
animal.
Zone Diameter
Potential Problems.
•
No breakpoint zone size data available for
some veterinary antimicrobials when
using Isosensitest agar.
• Tissue concentration may differ between
a) Different species
b) Different sites in same animal (eg udder
and serum in dairy cattle)
Total Numbers of Isolates
Examined by VLA.
20000
15000
Salmonella
10000
Others
5000
0
1998 1999 2000 2001 2002 2003
Resistance in E. coli from Pigs, < 1
month old.
90
80
70
Ampicillin
60
Tetracycline
50
Neomycin
40
Apramycin
30
TMP/S
20
Enrofloxacin
10
0
1998
1999
2000
2001
2002
2003
Salmonella spp. -nalidixic acid.
S A L M O N EL L A -N AL IDIX IC AC ID
35
30
25
F re q u e n c y
0
20
20
21
22
23
24
15
25
26
10
5
0
0
5
10
15
Zo n e D ia m e te r ( m m )
20
25
30
Salmonella spp. -ampicillin.
SALMONELLA -AMPICILLIN
25
BSAC breakpoint
R < or = 17mm
20
0
15
14
Frequency
23
24
25
10
26
27
28
29
5
30
0
0
5
10
15
20
-5
Zone diameter (mm)
25
30
35
The relationship between Zone
Diameter and MIC.
Log
MIC
Zone Diameter
Plot of Log MIC against Erythromycin zone sizes for Streptococcus uberis .
Log M.I.C ug/ml
100
10
BSAC Zone
diameter
breakpoint
VLA Zone diameter
breakpoint
1000
False susceptible rate:
BSAC 0
M.I.C (ug/ml)
1
MIC
breakpoint
.
0.1
False resistant rate :
0.01
0
5
10
15
20
25
30
Erythromycin zone size (mm)
35
40
45
Surveillance Outputs.
• “Salmonella in Livestock” publication, produced
annually gives details of resistance in salmonella.
• Annual Antimicrobial Sensitivity Report published on the DEFRA web-site.
• Reports to practitioners -therapeutic panels of
antimicrobials and the expanded salmonella
surveillance sensitivity test.
• Reports of abattoir surveillance (usually
announced at public meetings).
Improving Surveillance Data
Quality.
• Expert rules system
• Harmonisation ring trials.
Expert Rules System (1).
• Alert that an isolate has been recovered to
manager of antimicrobial resistance subprogramme.
• Regional laboratory advised to check result
and bacterial identification.
• Isolate retained in bead culture collection.
Expert Rules System (2).
• Streptococcus spp. – any resistance to
penicillin, ampicillin or amoxycillin/
clavulanate.
• Actinobacillus pleuropneumonia – any
resistance to penicillin, ampicillin, ceftiofur
or enrofloxacin.
• Salmonella enterica – resistance to
enrofloxacin.
Harmonisation Ring Trial
Salmonella enterica.
100% concordance between reference laboratories
for:
•
•
•
•
•
•
Ampicillin
Chloramphenicol
Apramycin (tested at 4/5 laboratories)
Tetracyclines
Trimethoprim/ sulphonamides
Neomycin.
Harmonisation Ring Trial
Salmonella enterica.
• There is a need for further work in some
areas (in particular furazolidone,
streptomycin and cefotaxime).
Harmonisation Ring Trial
Campylobacter spp.
• Three participating laboratories.
• Good concordance for nalidixic acid and
erythromycin.
• Concordance was less good for
ciprofloxacin.
Linkage of resistance genes.
• E.faecium plasmid.
Erythromycin
resistance
gene
Vancomycin
resistance gene
Copper resistance
gene.
Organisms of Particular Concern
in Agriculture from the
Veterinary Perspective.
• Brachyspira hyodysenteriae (Swine
dysentery)
• Multi-resistant Salmonella enterica
• Resistance to third generation
cephalosporins in Salmonella enterica
The Spread of Resistance Genes
and Resistant Organisms.
ANIMALS TO MAN:
• Apramycin/ Gentamicin resistance gene
(AAC(3)IV) in Salmonella Typhimurium.
MAN TO ANIMALS:
• Gentamicin resistance gene (AAC(3)II) in
Salmonella Typhimurium
Joint HPA/ VLA Study on
Resistance in Salmonella
enterica.
• Levels of resistance compared in serotypes
Typhimurium, Enteritidis, Hadar and
Virchow in man and animals in 2000.
The Problem:
• The relative contribution of resistance in
animal salmonella to the overall problem of
resistance in human salmonella strains
remains unquantified.
Methods.
• PHLS breakpoint method on agar
• VLA disc diffusion method
• Kanamycin and neomycin assumed to be
equivalent.
• Nalidixic acid and low-level ciprofloxacin
resistance considered to be directly
comparable.
Salmonella Typhimurium.
• Of the four serotypes examined, resistance was
most common in Salmonella Typhimurium from
animals and man, with definitive phage types 104,
193, 208 and U302 in particular showing
resistance.
• Multi-resistant strains of DT 208 appear to be
associated with pigs; for other DTs the
relationship between animals and man was
complex.
Salmonella Enteritidis, Virchow
and Hadar.
• Substantial differences in the resistance
spectra of a significant proportion of
isolates from humans and food-producing
animals.
• Suggests that food-producing animals in
England and Wales are not the primary
source of these strains.
Overall Levels of Resistance:
Origin
No. of
cultures
Percent drugsensitive
Man
Cattle
Sheep
Pigs
Poultry
13958
1623
166
426
1439
65
79
81
12
57
Main Serotypes in Man and
Animals in 2000:
MAN
•
•
•
•
Enteritidis (8,468)
Typhimurium (2,424)
Hadar (348)
Virchow (309)
ANIMALS
•
•
•
•
Dublin (697)
Typhimurium (602)
Senftenberg (269)
Give (148)
Salmonella Enteritidis
• Cases in man have declined since 1997, though it
remained the most common serotype in 2000.
• 14% of poultry isolates resistant to furazolidone
(67% in France in 1999); <1% human isolates
resistant to furazolidone.
• 5% of animal (poultry) isolates resistant to
nalidixic acid; 11% of human isolates resistant.
S.Virchow
Nalidixic Acid Resistance
% R
• 78% Isolates from
man resistant to one or
more antimicrobials.
• 21% Isolates from
poultry resistant to one
or more
antimicrobials.
60
50
40
30
20
10
0
Series1
Man
Poultry
Resistance to Nalidixic acid in
S.Virchow in France.
• 60.3% of Salmonella Virchow isolates of
poultry origin from France in 1999 were
resistant to nalidixic acid.
• N.B. Data not available for many countries.
Salmonella Hadar
• S. Hadar isolated only from poultry and
man.
• 48% of human isolates and 54% of poultry
isolates resistant to nalidixic acid
• In most cases, resistance detected in poultry
isolates to certain antimicrobials was also
seen in human isolates, to the same
antimicrobials…….BUT
S.Hadar
Trimethoprim Resistance in
S. Hadar.
%R
• 63% of isolates from
man resistant to
Trimethoprim.
• Trimethoprim
resistance not detected
in poultry isolates.
100
50
0
Series1
Man
Poultry
Resistance to Trimethoprim in S.
Hadar in Europe
• Belgium 0% in 1999 and 2000
• France 3.2% of isolates of poultry origin in
1999 resistant to trimethoprim/
sulphonamide.
UK Poultry Consumption 2000
• Home Fed Production 1,512.69
• Imported 355.22 (34 non-EU).
• Exported 173.72
• Total Domestic Usage 1,707.5
(Thousand tonnes)
UK Imports of Whole Fresh of Chilled
Chickens (2000) in Tonnes.
Ita
ly
Be
lg
iu
m
...
Ne
th
er
Fr
an
ce
25,000
20,000
15,000
10,000
5,000
0
UK Imports of Whole Frozen Chickens
(2000) in Tonnes.
Ne
th
e.
..
D
en
m
ar
k
Iri
sh
...
Fr
an
ce
10,000
8,000
6,000
4,000
2,000
0
Imports of Chicken Cuts and
Offal - Fresh or Chilled (Tonnes).
an
y
G
er
m
Be
lg
iu
m
Fr
an
ce
Ne
th
er
...
60,000
50,000
40,000
30,000
20,000
10,000
0
Imports of Frozen Chicken Cuts
and Offal (Tonnes).
Th
ai
la
nd
ar
k
en
m
D
Br
az
il
Fr
an
ce
Ne
th
er
...
80,000
60,000
40,000
20,000
0
Imports of Frozen Chicken
Livers (2000) Tonnes.
50
40
30
20
10
0
Netherlands
France
Irish
Republic
Chicken meat and offal imports -prepared and preserved 2000
(Tonnes)
Thailand
Netherlands
France
Irish Republic
Brazil
Germany
Belgium
Slovenia
Croatia
Denmark
Italy
Cayman Islands
Sweden
Singapore
Chile
Israel
Spain
Hong Kong
Bon Appetit!