Veterinary Susceptibility Testing Chris Teale Veterinary Laboratories Agency

Veterinary Susceptibility Testing. Chris Teale, Veterinary Laboratories Agency.

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 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 O 157, 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 13 mm 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.

Resistance in E. coli from Pigs, < 1 month old.

Salmonella spp. -ampicillin. BSAC breakpoint R < or = 17 mm

The relationship between Zone Diameter and MIC. Log MIC Zone Diameter

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 U 302 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 Wales are not the primary source of these strains.

Overall Levels of Resistance:

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

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

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)