Expert rules and inexpensive

identification methods

Dr Trevor Winstanley

Department of MicrobiologyRoyal Hallamshire Hospital

Sheffield UK

tgwinstanley@hotmail.com

BSAC web-site

http://www.bsac.org.uk/susceptibility_testing/powerpoint_presentations.cfm

Why identify?n Scientificn Resistant organismsn Recognition of particular pathogensn Evidence of cross-infectionn Distinguish relapse from reinfectionn To match urine and blood culture isolatesn Meaningful epidemiology

n prevalence of species and resistance

Blood isolates 2004-5 %R

AMP CXM CAZ AZT PTZ n

E.coli 56.1 14.2 5.1 4.2 1.5 612

Enterobacter 100 55.5 36.7 32.0 19.5 128

Klebsiellae 100 25.4 9.2 13.8 11.0 283

These three 73.7 22.5 10.2 10.4 6.4 1023

Why identify?

n Taxonomic changesn Detection of intrinsic or inducible

resistancen Prognosisn Species-specific breakpointsn Interpretative reading

BSAC disc diffusion method

E.coli, Proteus mirabilis, Klebsiella spp., Enterobacter spp., Citrobacter spp., Serratia spp., Salmonella spp., Providencia spp.

Species-specific UTI breakpoints Systemic footnotes

“Splitters” vs. “Lumpers”

E.coliS.sonneiShigella spp.P.stuartiiS.typhiSalmonella spp.K.pneumoniaeK.oxytocaE.cloacaeE.aerogenesE.agglomeransHafnia alvei

Serratia spp.P.mirabilisP.vulgarisM.morganiiP.alcaligenesP.rettgeriC.freundiiC.diversusAcinetobacterStenotrophomonasAeromonasPlesiomonas

etc. etc.

Methodsn pH-based reactions

n growth-dependent; 15 - 24 hn Visual detection of bacterial growth

n 18 – 24 hn Enzyme-based reactions

n chromogenic, fluorogenic; 2 - 4 hn Utilisation of C sources

n tetrazolium dye; 4 - 24 hn Detection of volatile fatty acidsn Genetic

n e.g. 16S rRNA sequence analysis

Enterobacteriaceae (n = 30,000)

E.coli

K.pneumoniae

P.mirabilis

E.cloacae

K.oxytoca

Others

M.morganii

Serratia spp.

C.freundii

E.aerogenes

P.vulgaris

90.2% assigned to four species

API-20E (bioMerieux)

n 102 taxan the “gold standard”

BBL Crystal E/NF (Becton Dickinson)

n 30 conventional and chromogenic substrates; 18–20 h

n 38 genera, 104 species

ID Tri-Panel (BD)

n Frozen microtitration platesn Three organisms / platen 30 colorimetric-based substratesn 31 genera; 118 species

Enterotube II (BD)

n 22 genera; 79 species

Roscozym (Rosco)n Chromogenic and conventional enzymatic tests; 4 h

DIATABS (Rosco)

n Individual tabletsn Chromogensn Modified conventional tests

Microgen GN IDn GN A: 12 substrates, 44 species n GN A + B: 24 substrates, 140 species

n includes Xanthomonas spp. and Acinetobacter spp.

GN2 MicroPlate (Biolog/Oxoid)

n MicroLog 1 & 2 – manualn Oxidation of 95 C sources

n tetrazolium dye

n Up to 24 hn 526 species

Micro-ID (Remel)

n Enzymatic and metabolicn 15 reactionsn 4 hours

r/b Enteric Differential System (Remel)

n Two tubes: r/b1 and r/b2

n Cit/Rham expander +/- soranasen 13 genera; 37 species

RapID (Remel)

n Conventional + chromogenic n Electronic databasen RapID ONE

n 19 substrates, 28 genera incl. 60 species, 4 h

n RapID SS/un 11 substrates, 7 genera incl. 2 species, 4 h

Microbact (Oxoid)n Conventional biochemical testsn 12A

n 12 substratesn 15 genera, 34 species but KES

n 12A + 12Bn 24 substratesn 29 genera, 109 speciesn includes non-fermenters

Chromogenic agar

ß-glucosidase

ß-galactosidase

ß-glucuronidase

TDA Indole

CPS ID2/3 (bioMerieux)

Chromagar Orientation (BBL)

Chromogenic UTI (Oxoid)

Uriselect 4 (Bio-Rad)

Chromogenic agar (6570 UTI)

"Coliforms"

TDA +

E.coli

Klebsiella-Enterobacter-Serratia

20.6%

Proteus-Morganella-Providencia

6.9%

72.5%

0

50

100

150

200

250

300

350

400

450

Non LactoseFermenter

"Coliform" fromchild

Gentamicin RE.coli

R Cefpodoxime 2 mg/L

6524 consecutive urines

648 re-examined

0

20

40

60

80

100

120

140

160

180

200

ESBL ESBL + AmpC AmpC Neither

431 R cefpodoxime 2 mg/L

Multipoint inoculation (Mast/Abtek)

ß-galactosidase Indole

Cellobiose Maltose

Inositol Rhamnose

Sorbitol Sucrose

TDA Aesculin

Citrate VP

Urea Lysine

Ornithine

Multipoint inoculation (Mast/Abtek)

Lysine decarboxylase

Ornithine decarboxylase

Glucose

Cellobiose

Indole

Urea

93% of 300 resistant Enterobacteriaceae from urine

Perry et al J.Clin.Path. 1988 41: 1010

Direct urine inoculation

M.morganii+Diffuse brown

K.pneumoniae-+-

E.cloacae+--

E.aerogenes++-

Salmonella sp.Clear

S.marcescensAqua blue

P.vulgaris+Bluish green

P.mirabilis-

C.freundii--+/-

C.diversus+-+

K.oxytoca-++Metallic blue

E.coli+Pink / red

ODCLDCINDCHROMagar

98.7% of 472 isolates Ohkusu J.Clin.Micro. 2000 38 4586

Work smarter, not hardern Rx equivalence

n e.g. cephalothin is predictive of cephradine, cephalexin, cefaclor and cefadroxil

n Marker Rxn e.g. oxacillin, cefoxitin, nalidixic acid,

cefpodoxime

n Cross resistance n e.g. oxacillin resistance predicts ß-lactam

resistance in staphylococci

n Associated resistancen e.g. MRSA, ESBLs

Inherent resistance to Rxn Ampicillin R E.faecalis (probably E.faecium)n Ampicillin S Klebsiella sp.n Nitrofurantoin or colistin S Proteus mirabilisn Colistin S Serratia marcescensn Aztreonam S Gram positive

n Confirm; edit S → R

Exceptional resistance

n Never R à S; confirm and use reference labn S.aureus, streptococci - vancomycin, linezolid,

Synercidn S.pyogenes - penicillinn E.faecalis - ampicillinn Enterobacteriaceae - carbapenemsn Haemophilus, Moraxella, Neisseria - 3 gen cephsn Anaerobes - metronidazole

David Livermore’s green cats

29726E.coli, imipenem

5129Enterobacter, imipenem

7842Klebsiella, imipenem

5664S.pyogenes, penicillin

104195E.faecalis, ampicillin

316610S.aureus, vancomycin

TotalNos. R

PHLS bacteraemias 2001

Addition of comments

n Multi-resistant Gram negativesn MRSA, GISA, GRE, PRPn Alert organisms

n e.g. Str.pyogenes

n Enteric isolates: faecal-oral spreadn Clostridium difficilen Salmonella spp., Campylobacter spp.

Addition of comments

n High frequency mutationn fusidic acid and staphylococcin clindamycin and erythromycin-resistant

staphylococcin some third-generation cephalosporins and

Enterobacteriaceaen S.marcescens / aminoglycosidesn nalidixic acid R coliforms / fluoroquinolones

Interpretative reading

n Test a large panel of appropriate antibiotics

n Identify the organism fullyn Analyse the complete resistance profilen Apply expert knowledge

Published “Expert Rules”

Natural susceptibility to ß-lactams

n Group I n Escherichia coli

Proteus mirabilisSalmonella spp. Shigella spp.

n Group II n Klebsiella spp.

Citrobacter koseriC.amalonaticusE.harmannii

n Group IIIn Enterobacter spp.

Citrobacter freundiin Providencia spp.

Morganella spp. n Proteus vulgarisn Serratia spp.

• Others Yersinia enterocolitica, Ps.aeruginosa, Acinetobacter spp., S.maltophilia

Result given

Validation, correction

Determination of probable

resistance mechanism

Knowledge base

Additional tests if

required

In-vitroresistance phenotype

Interpretative procedure

Klebsiella pneumoniae

SImi/meropenem

SCefepime

SCeftazidime

SCefotaxime

SCefoxitin

SPip-tazobactam

SPiperacillin

RAmpicillin

Klebsiella pneumoniae

Wild type

SImi/meropenem

SCefepime

SCeftazidime

SCefotaxime

SCefoxitin

SPip-tazobactam

SPiperacillin

RAmpicillin

Klebsiella pneumoniae

Wild type

SImi/meropenem

SCefepime

SCeftazidime

SCefotaxime

SCefoxitin

SPip-tazobactam

RPiperacillin

RAmpicillin

Klebsiella pneumoniae

Wild type

SSImi/meropenem

RSCefepime

RSCeftazidime

SSCefotaxime

SSCefoxitin

SSPip-tazobactam

RRPiperacillin

RRAmpicillin

Klebsiella pneumoniae

ESBLWild type

SSImi/meropenem

RSCefepime

RSCeftazidime

SSCefotaxime

SSCefoxitin

SSPip-tazobactam

RRPiperacillin

RRAmpicillin

Klebsiella pneumoniae

ESBLWild type

SSImi/meropenem

RSCefepime

RSCeftazidime

RSCefotaxime

SSCefoxitin

RSPip-tazobactam

RRPiperacillin

RRAmpicillin

Klebsiella pneumoniae

ESBLWild type

RSSImi/meropenem

SRSCefepime

RRSCeftazidime

RRSCefotaxime

SSSCefoxitin

SRSPip-tazobactam

SRRPiperacillin

RRRAmpicillin

Klebsiella pneumoniae

Refer!!ESBLWild type

RSSImi/meropenem

SRSCefepime

RRSCeftazidime

RRSCefotaxime

SSSCefoxitin

SRSPip-tazobactam

SRRPiperacillin

RRRAmpicillin

Klebsiella pneumoniae

Refer!!ESBLWild type

SRSSImi/meropenem

SSRSCefepime

SRRSCeftazidime

RRRSCefotaxime

SSSSCefoxitin

SSRSPip-tazobactam

RSRRPiperacillin

RRRRAmpicillin

Klebsiella pneumoniae

CTX-MRefer!!ESBLWild type

SRSSImi/meropenem

SSRSCefepime

SRRSCeftazidime

RRRSCefotaxime

SSSSCefoxitin

SSRSPip-tazobactam

RSRRPiperacillin

RRRRAmpicillin

Klebsiella pneumoniae

CTX-MRefer!ESBLWild type

SRSSImi/meropenem

RSRSCefepime

RRRSCeftazidime

RRRSCefotaxime

SSSSCefoxitin

RSRSPip-tazobactam

RSRRPiperacillin

RRRRAmpicillin

0.030.030.030.03Meropenem

0.120.120.120.12Imipenem

4844Cefoxitin

128320.120.12Ceftazidime

2640.030.03Ceftriaxone

1210.5Pip + tazobactam

64641281Piperacillin

102425610242Ampicillin

TEM-10+TEM-3+TEM-1+R-

MIC (mg/L) E.coli

Inoculum effect

Queenan et al 2004 J.Clin.Microbiol. 42 269; Bedenic et al 2001 Clin.Microbiol.Infect. 7 626

RSSHV-2 SHV-5 SHV-12

RSTEM-3 TEM-10 SHV-18

SSTEM-1

107106105TAZ TAX FEP PIR POD or AZT

Combination discs (CTX-M-3)

100FEP/CLAV

31TAZ/CLAV

31TAX/CLAVEnterobacter (13)

98FEP/CLAV

74TAZ/CLAV

83TAX/CLAVUnselected (54)

%RxOrganismSensitivity

Cefepime-clavulanate Etests

False positives with K1 K.oxytoca.

Stürenburg et al, J.Antimicrob.Chemother. 2004: 54; 134-8

AmpC

n R to ceftazidime, cefotetan and piperacillin-tazobactam

n ES infers AmpC hyper-productionn suggests testing cefpirome, cefepime,

temocillin and carbapenems

Inducible AmpC

n E.cloacae, C.freundii, M.morganii, Ps.aeruginosa, S.marcescens, Providencia, Hafnia alvei, Aeromonas

Klebsiella ?ESBL

Cefpodoxime

Cefuroxime

Aztreonam

Ceftazidime

Cefotaxime

Gentamicin

K.oxytoca K1

Cefpodoxime

Cefuroxime

Aztreonam

Ceftazidime

Cefotaxime

Gentamicin

Are these results unusual??

SRRRRSRRRRSRSRSSSSRSS

CefalexinCo-amoxiclavAmpicillin

it depends …

6%94%82%120TOTAL:

0222Plasmidic AmpC + ESBL

0848Plasmidic AmpC

0111Chromosomal AmpC +

double ESBL

4111115Chromosomal AmpC + ESBL

2181020Chromosomal AmpC +/- TEM-1

0555ESBL / double ESBL

0121212ESBL (CTX-M)

1454446ESBL (TEM or SVH)

0868K1 Kl.oxytoca

0333SHV-1 / TEM-1,-2

IncorrectCorrect in 1+Correct in 1nGENOTYPE

Winstanley, Parsons, Stürenburg et al JAC 56 292-6 2005

Limitations n Continual need to updaten Organisms with unexpectedly large, or

small, amounts of enzymen Resistance due to impermeability or

effluxn Poorly defined relationship between

resistance mechanism and phenotype e.g. Acinetobacter

Limitations n Novel resistance mechanism producing same

phenotype as known mechanismn Multiple resistance determinants

n multiple aminoglycoside modifying enzymesn multiple TEMs and SHVs

n multiple ESBLs

n ESBLs in presence of AmpCn ß-lactamases + impermeability, porin deficiency,

efflux

Advantages

n Detects anomalous ID and/or ASTn Detects improbable resistance

phenotypesn Detects weakly expressed resistancen Detects rare phenotypesn Deduces results for non-tested agentsn Suggests further agents to be tested

Advantages

n Facilitates study of epidemiology of resistance mechanisms

n Improves clinical interpretationn Improves consistency and qualityn Educationaln Can be universal and genericn Improves antibiotic use