Veterinary Susceptibility Testing'

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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.

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Surveillance for Antimicrobial Resistance at VLA
3 main areas of activity
Abattoir Surveillance for Zoonotic and Indicator
Organisms.
Salmonella enterica
Veterinary Clinical Diagnostic Submissions
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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).

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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.

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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.

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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 web-site(http//defraweb/ani
  malh/diseases/zoonoses/index.htm)

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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.

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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.

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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.

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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.

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Disc Diffusion Methods (1)

• NCCLS
• US Standard.
• Confluent inoculum
• Data for some veterinary pathogens available
• Mueller-Hinton
• Used in some Scottish laboratories.

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Disc Diffusion Methods (2)

• BSAC
• Isosensitest agar
• Semi-confluent inoculum (nudging colonies)
• UK standard method
• Currently no specific veterinary guidelines
• Harmonisation with medical colleagues

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Disc diffusion test -Stokes method.
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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.

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The relationship between Zone Diameter and MIC.
Log MIC
Zone Diameter
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The relationship between Zone Diameter and MIC.
Log MIC
Concentration reached in animal.
Zone Diameter
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The relationship between Zone Diameter and MIC.
Appropriate Zone Diameter.
Log MIC
Concentration reached in animal.
Zone Diameter
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Potential Problems.

• No breakpoint zone size data available for some
  veterinary antimicrobials when using Isosensitest
  agar.
• Tissue concentration may differ between
• Different species
• Different sites in same animal (eg udder and
  serum in dairy cattle)

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Total Numbers of Isolates Examined by VLA.
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Resistance in E. coli from Pigs, lt 1 month old.
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Salmonella spp. -ampicillin.
BSAC breakpoint R lt or 17mm
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The relationship between Zone Diameter and MIC.
Log MIC
Zone Diameter
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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).

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Improving Surveillance Data Quality.

• Expert rules system
• Harmonisation ring trials.

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Expert Rules System (1).

• Alert that an isolate has been recovered to
  manager of antimicrobial resistance
  sub-programme.
• Regional laboratory advised to check result and
  bacterial identification.
• Isolate retained in bead culture collection.

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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.

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Harmonisation Ring Trial Salmonella enterica.

• 100 concordance between reference laboratories
  for
• Ampicillin
• Chloramphenicol
• Apramycin (tested at 4/5 laboratories)
• Tetracyclines
• Trimethoprim/ sulphonamides
• Neomycin.

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Harmonisation Ring Trial Salmonella enterica.

• There is a need for further work in some areas
  (in particular furazolidone, streptomycin and
  cefotaxime).

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Harmonisation Ring Trial Campylobacter spp.

• Three participating laboratories.
• Good concordance for nalidixic acid and
  erythromycin.
• Concordance was less good for ciprofloxacin.

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Linkage of resistance genes.

• E.faecium plasmid.

Copper resistance gene.
Erythromycin resistance gene
Vancomycin resistance gene
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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

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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

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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.

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The Problem

• The relative contribution of resistance in animal
  salmonella to the overall problem of resistance
  in human salmonella strains remains unquantified.

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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.

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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.

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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.

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Overall Levels of Resistance
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Main Serotypes in Man and Animals in 2000
MAN
ANIMALS

• Enteritidis (8,468)
• Typhimurium (2,424)
• Hadar (348)
• Virchow (309)

• Dublin (697)
• Typhimurium (602)
• Senftenberg (269)
• Give (148)

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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) lt1 human isolates
  resistant to furazolidone.
• 5 of animal (poultry) isolates resistant to
  nalidixic acid 11 of human isolates resistant.

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S.Virchow

• 78 Isolates from man resistant to one or more
  antimicrobials.
• 21 Isolates from poultry resistant to one or
  more antimicrobials.

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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.

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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.BU
  T

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S.Hadar

• 63 of isolates from man resistant to
  Trimethoprim.
• Trimethoprim resistance not detected in poultry
  isolates.

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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.

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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)

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UK Imports of Whole Fresh of Chilled Chickens
(2000) in Tonnes.
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UK Imports of Whole Frozen Chickens (2000) in
Tonnes.
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Imports of Chicken Cuts and Offal - Fresh or
Chilled (Tonnes).
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Imports of Frozen Chicken Cuts and Offal (Tonnes).
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Imports of Frozen Chicken Livers (2000) Tonnes.
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Chicken meat and offal imports -prepared and
preserved 2000 (Tonnes)
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Bon Appetit!