1、Veterinary Susceptibility Testing.Chris Teale,Veterinary Laboratories Agency.B B u u r r y y S S t t E E d d m mu u n n d d s s.W Wi in n c c h h e e s s t te e r r.L L a a n n g g f fo o r r d d.S S u u t tt to o n n B B o o n n i in n g g t to o n n.N Ne e w wc c a a s s t tl le e.T T h h i ir r s
2、 s k k.L L u u d d d d i in n g g t to o n n.S S h h r r e e w ws s b b u u r r y y S S t ta a r r c c r r o o s s s s.T T r r u u r r o o.C Ca a r r m ma a r r t th h e e n n.A Ab b e e r r y y s s t tw wy y t th h.P P r r e e s s t to o n n.P P e e n n r r i it th h.L a s s w a d e We y b r i d g
3、e .V.I.C e n t r eV.I.U n i tC e n.V e t L a b sKey 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.Survei
4、llance for AntimicrobialResistance at VLASalmonella entericaVeterinary Clinical DiagnosticSubmissions Abattoir Surveillance for Zoonotic and Indicator Organisms.3 main areas of activity:Salmonella enterica Salmonella isolates are tested against a panel of 16 antimicrobials by disc diffusion test.Res
5、ults 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 Sa
6、lmonella.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 Su
7、bmissions 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 provid
8、ed 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 web-si
9、te(http:/defraweb/animalh/diseases/zoonoses/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 anti
10、microbial 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
11、 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
12、 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 dif
13、fusion 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 Shre
14、wsbury.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 veteri
15、nary guidelines Harmonisation with medical colleaguesDisc 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 C
16、ontrol Establishment of robust breakpoints.The relationship between Zone Diameter and MIC.Zone DiameterLog MICThe relationship between Zone Diameter and MIC.Zone DiameterLog MICConcentration reached in animal.The relationship between Zone Diameter and MIC.Zone DiameterLog MICConcentration reached in
17、 animal.Appropriate Zone Diameter.Potential Problems.No breakpoint zone size data available for some veterinary antimicrobials when using Isosensitest agar.Tissue concentration may differ betweena)Different speciesb)Different sites in same animal(eg udder and serum in dairy cattle)Total Numbers of I
18、solates Examined by VLA.050001000015000200001998 1999 2000 2001 2002 2003SalmonellaOthersResistance in E.coli from Pigs,1 month old.0102030405060708090199819992000200120022003Ampicillin TetracyclineNeomycinApramycinTMP/SEnrofloxacinSalmonella spp.-nalidixic acid.SALMONELL A-NALIDIXIC ACID05101520253
19、035051015202530Zone Diam ete r(mm)Frequency020212223242526Salmonella spp.-ampicillin.SALMONELLA-AMPICILLIN-5051015202505101520253035Zone diameter(mm)Frequency0142324252627282930BSAC breakpoint R or=17mmThe relationship between Zone Diameter and MIC.Zone DiameterLog MICPlot of Log MIC against Erythro
20、mycin zone sizes for Streptococcus uberis.0.010.11101001000051015202530354045Erythromycin zone size(mm)Log M.I.C ug/mlM.I.C(ug/ml)MIC breakpoint.BSAC Zone diameter breakpointVLA Zone diameter breakpointFalse susceptible rate:BSAC 0False resistant rate:Surveillance Outputs.“Salmonella in Livestock”pu
21、blication,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(usual
22、ly 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 sub-programme.Regional laboratory advised to check result and bacterial identifica
23、tion.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
24、.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 are
25、as(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 resistanc
26、e geneVancomycin resistance geneCopper 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 entericaThe Spread of Resis
27、tance 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 TyphimuriumJoint HPA/VLA Study on Resistance in Salmonella enterica.Levels of resistance compared in serotypes T
28、yphimurium,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 Kanamyci
29、n 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 a
30、nd 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 is
31、olates from humans and food-producing animals.Suggests that food-producing animals in England and Wales are not the primary source of these strains.OriginNo.ofculturesPercent drug-sensitiveMan1395865Cattle162379Sheep16681Pigs42612Poultry143957Overall Levels of Resistance:Main Serotypes in Man and An
32、imals in 2000:Enteritidis(8,468)Typhimurium(2,424)Hadar(348)Virchow(309)Dublin(697)Typhimurium(602)Senftenberg(269)Give(148)MANANIMALSSalmonella 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%i
33、n 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.Nalidixic Acid
34、Resistance0102030405060ManPoultry%RSeries1Resistance 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 m
35、an.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.BUTS.Hadar 63%of isolates from man resistant to Trimethoprim.Trimethoprim resistan
36、ce not detected in poultry isolates.Trimethoprim Resistance in S.Hadar.050100ManPoultry%RSeries1Resistance 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 Prod
37、uction 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.05,00010,00015,00020,00025,000FranceNether.BelgiumItalyUK Imports of Whole Frozen Chickens(2000)in Tonnes.02,0004,0006,0008,00010,000F
38、ranceNethe.DenmarkIrish.Imports of Chicken Cuts and Offal-Fresh or Chilled(Tonnes).010,00020,00030,00040,00050,00060,000Nether.FranceBelgiumGermanyImports of Frozen Chicken Cuts and Offal(Tonnes).020,00040,00060,00080,000Nether.BrazilFranceDenmarkThailandImports of Frozen Chicken Livers(2000)Tonnes.01020304050NetherlandsFranceIrishRepublicThailandNetherlandsFranceIrish RepublicBrazilGermanyBelgiumSloveniaCroatiaDenmarkItalyCayman IslandsSwedenSingaporeChileIsraelSpainHong KongChicken meat and offal imports-prepared and preserved 2000(Tonnes)Bon Appetit!
