SUPPLEMETAL TESTG Tan Thean Yen
To Supplement Definition: add as a supplement to what seems insufficient "supplement your diet"
Why supplement? urrent methods don t work well Additional information provided by supplemental testing Alternative approaches to current methods More rapid testing results
Why OT supplement? urrent breakpoints are adequate More work onfusing Slower testing results
Three areas to cover 1. inducible resistance v.s. de-repressed resistance 2. Beta-lactamases & beta-lactamase inhibitors 3. Heterogenous populations
Part One DUBLE V.S. DE-REPRESSED
D U T O nducible resistance
nducible resistance De-repressed or resistant D U T O May be OR Always
STAPHYLOO nducible resistance
lindamycin & Erythromycin Macrolide Erythromycin larithromycin Azithromycin Lincosamide lindamycin Lincomycin Streptogramin Quinupristin- Dalfopristin Pristinamycin
E R Y T H R O M Y Erythromycin lindamycin nterpretation S S Organism susceptible to both & L D A M Y R R R S Organism resistant to both May have inducible resistance
E R Y T H R O M Y & L D A M Y
E R Y T H R O M Y & L D A M Y nducible resistance
E R Y T H R O M Y Recommendations for MLS b lindamycin & erythromycin discs Staphylococci: 15-26mm apart Streptococci: 12 mm apart & L D A M Y look for flattening of zone of inhibition
E R Y T H R O M Y & L D A M Y Recommendations for MLS b broth dilution: use combination of erythromycin 4 µg/ml and clindamycin 0.5 µg/ml in a single well growth = inducible resistance OR use disc testing methods, on purity plate.
E R Y T H R O M Y & L D A M Y Other methods Agar dilution Sensitivity = 91%, specificity = 97% Vitek card Sensitivity = 91%, specificity = 100% Lavallee,., Rouleau, D., Gaudreau,., Roger, M., Tsimiklis,., Locas, M.., Gagnon, S., Delorme, J., Labbe, A.. (2010). Performance of an Agar Dilution Method and a Vitek 2 ard for Detection of nducible lindamycin Resistance in Staphylococcus spp.. J. lin. Microbiol. 48: 1354-1357
Part Two BETA-LATAMASES
Beta-lactamases LASS A: extended-spectrum beta-lactamases LASS : amp beta-lactamases LASS B: metallo-beta-lactamases
Plasmid (acquired) G E E A Q U S T O hromosomal (born with it)
amp A M P E Z Y M E S hromosomal Enterobacter spp. & most other Enterobacteriaceae Pseudomonas aeruginosa Plasmid-mediated Klebsiella spp. Salmonella spp. Proteus mirabilis (E. coli)
amp A M P E Z Y M E S chromosomal plasmid genes inducible de-repressed (always on) inducible de-repressed (always on)
AZ A M P MP E Z Y M E S
A M P E Z Y M E S nducible amp resistance
Extended spectrum betalactamases actually a family of related beta-lactamases SHV TEM E S B L three main groups: SHV TEM TX TX usually plasmid-borne
sulbactam Beta lactam Beta lactam inhibitor E S clavulanic acid B L tazobactam
ESBL & beta-lactamase inhibitors E S B L ESBL enzyme breaks down cephalosporin antibiotic
ESBL & beta-lactamase inhibitors E S B L nhibitor binds to ESBL enzyme. Prevents ESBL from breaking down antibiotic. antibiotic
ESBL & beta-lactamase inhibitors E Beta-lactamase inhibitors S B L compete with the beta-lactamase enzyme
ESBL & beta-lactamase inhibitors E S nhibitor > ESBL = susceptible ESBL > nhibitor = resistant B L
Double disk approximation E S B L lavulanic acid cephalosporin
efotaxime E S B L efotaxime & clavulanate
eftazidime + lavulanate M 0.25 E S B L eftazidime M > 32
Other beta-lactamases Beta-lactamase amp MBL KP nhibitor cloxacillin boronic acid EDTA mercaptopropionic acid (MPA) other chelating agents boronic acid
amp Substrate Enzyme nhibitor imipenem Strong inducer.. but generally not broken down by amp
amp Substrate Enzyme nhibitor imipenem strong inducer of amp AD broken down by amp cefoxitin amp cloxacillin
amp cefoxitin = small zone (disc) (by itself) high M (dilution) cefoxitin = larger zone (disc) & cloxacillin lower M (dilution)
Antibiotic 1 eftazidime eftazidime Meropenem 21 mm 21 mm 17 mm Antibiotic 2 eftazidime & clavulanate eftazidime & clavulanate Meropenem & EDTA 27 mm 16 mm 28 mm nterpretation
Part Three HETEROGEEOUS POPULATO
het er o ge ne ous Adjective Diverse in character or content: "a large and heterogeneous collection"
Heterogeneous mostly susceptible small number of resistant strains
STAPHYLOO difficult to detect resistance
Methicillin resistance M E T H L L mediated by the meca gene
Methicillin testing (disc) M E T H L L S. aureus& S. lugdunensis Oxacillin (for S. aureus only) efoxitin (for both) coagulase negative staph efoxitin only
Thymidine-dependent Small olonial Variant S. aureus Kahl B et al. J. lin. Microbiol. 2003;41:410-413
MRSA and SV s M E T H L L slow-growing, atypical phenotype often seen in: cystic fibrosis, foreign-body infections &osteomyelitis susceptibility may be difficult to test best to use pbp2a or meca detection Frank Kipp, Karsten Becker, Georg Peters, and hristof von Eiff. Evaluation of Different Methods To Detect Methicillin Resistance in Small-olony Variants of Staphylococcus aureus. J lin Microbiol. 2004 M arch; 42(3): 1277 1279
other methods M E T H L L detection of pbp2a latex agglutination kits immunochromatographic kits (Binax) detection of meca gene by hybridisation Evigene detection of meca gene by PR BD Diagnostics, epheid, Roche Molecular Diagnostics
Moderately resistant S. aureus M E T H L L don t have the meca gene altered pbp (penicillin binding proteins) rare phenotype cefoxitin (S), oxacillin (R) should respond to drugs like augmentin, cephalexin
Vancomycin V A O M Y large molecule, diffuses slowly in agar no disc diffusion criteria for S. aureus resistance to vancomycin: low-level (intermediate-resistance) high-level (vana mediated) heterogenous (spectrum)
V A O M Y hvsa Heterogenous resistance to vancomycin
hvsa V A O M Y GRD strip Etest macro method Population analysis
SUPPLEMETAL TESTG OR M?
Traditional way Tested result Reported result eftriaxone eftriaxone R efotaxime S efotaxime R eftazidime R eftazidime R efepime S efepime R key-hole effect present ESBL present
Proposed new way Tested result Reported result eftriaxone eftriaxone efotaxime S efotaxime S eftazidime R eftazidime R efepime S efepime S key-hole effect present ESBL present
Why?
For the gene The presence of a resistance gene makes a difference to whether or not a particular antibiotic will work. t s not just the M.
For the gene Look for particular resistance enzymes: amp ESBL MBL Modify the susceptibility according to the presence of resistance enzymes.
For the M... The breakpoint for each antibiotic determines whether patient will respond to that antibiotic. t doesn t matter what the resistance gene is.
FOR AGAST Life is much simpler! s it **really** true? Simple = more consistent lab results s it true for **every** enzyme?
Opinion! hange susceptibility if present inducible clindamycin resistance (blood & bone) Enterobacter spp. and cephalosporin susceptible (blood) Don t know plasmid amp ESBL KP MBL (probably don t change tested result)
onclusions Some resistant organisms may need supplemental methods to detect Susceptibility testing can be complicated Resistance mechanism or tested M? ot yet quite sure.