Difficult or Tricky Antibiotic

Resistance Phenotypes to

Recognize

Dr Koh Tse Hsien

Department of Pathology

Singapore General Hospital

Contents

• Gram-positive

– Staphylococcus aureus with penicillinase production

– Vancomycin Resistant Enterococci (VRE)

– Staphylococcus aureus with reduced susceptibility to

glycopeptides (hVISA, VISA, VRSA)

• Gram-negative

– ampC Cephalosporinases ( AmpC and pAmpC)

– Carbapenemases (IMP, VIM, KPC, and OXA-48)

Mechanisms of Resistance

Target modification

Antibiotic sequestration Enzymatic inactivation

Penicillinase in S. aureus

Penicillinase production

Vancomycin susceptibility in S.

aureus

Vancomycin-intermediate S.

aureus (VISA)

• First described in Japan in 1997

• Intermediate resistant (MIC 4-8 mg/L)

• Hetero-intermediate resistant (MIC <4 mg/L)

• Increased production of cell wall precursors and PBP 2’

• Reduced cross linking in cell wall (less target for vancomycin)

• Lower growth rates and thicker cell walls (vancomycin sequestration)

• Prolonged exposure to vancomycin

MRSA VISA

Micky LEONG (Ms) :: Laboratory Technologist, Electron Microscopy Unit YLLSOM

Vancomycin intermediate

Staphylococcus aureus

Lab detection of VISA

• Not detected by routine disk or automated

methods

• Etest (consistently one twofold dilution

higher)

• Broth microdilution

EUCAST MIC breakpoint (mg/L) Zone diameter breakpoint (mm)

S ≤ R > S ≥ R <

Vancomycin 2 2 - -

CLSI MIC breakpoint (mg/L) Zone diameter breakpoint (mm)

S ≤ I R ≥ S ≥ I R ≤

Vancomycin 2 4-8 16 - - -

1. Glycopeptide MICs are method dependent and should be determined by broth microdilution (reference ISO 20776). S. aureus with vancomycin MIC values of 2 mg/L are on the border of the wild type MIC distribution and there may be an impaired clinical response. The resistant breakpoint has been reduced to 2 mg/L to avoid reporting "GISA" isolates intermediate as serious infections with "GISA" isolates are not treatable with increased doses of vancomycin or teicoplanin.

18) MIC tests should be performed to determine the susceptibility of all isolates of staphylococci to vancomycin. The disk test does not differentiate vancomycin-susceptible isolates of S. aureus from vancomycin-intermediate isolates…The vancomycin 30-μg disk test detects S. aureus isolates containing the vanA vancomycin resistance gene (VRSA). Such isolates will show no zone of inhibition around the disk (zone = 6 mm)…

The clinical significance of vancomycin minimum inhibitory concentration in

Staphylococcus aureus infections: A systematic review and meta-analysis

van Hal et al. CID 2012; 54: 755-71

• High vancomycin MIC (≥1.5 mg/L by Etest) associated with higher mortality rate

• This was driven by BSI with vancomycin MIC of ≥ 2 mg/L by Etest

• Higher vancomycin MIC values (≥1.5 mg/L by Etest) predicted treatment failure

• Etest recommended for all MRSA BSI

How does your lab measure the MIC?

• Etest 0.5-1 dilution higher than broth microdilution (gold standard)

• Automated MIC 1-2 dilutions lower than gold standard

• Most vancomycin outcome studies involved Etest

Vitek versus Etest (92 isolates)

Vitek Etest

Dr Tan TY, Dept of Lab Medicine, Changi General Hospital

(Hetero) hVISA

Lab detection of hVISA

• Population analysis

• Macrodilution Etest (not true MIC)

• Etest GRD

• MHA5T screening plate (5 μg/ml teicoplanin)

• hVISA may predict potential failure of daptomycin therapy

Howden, B. P. et al. 2010. Clin. Microbiol. Rev. 23(1):99-139

FIG. 8. Examples of Etest methodology used to detect hVISA

Vancomycin-resistant S. aureus (VRSA)

• In-vitro transfer of VanA in 1992

• Michigan (June 2002), Pennsylvania (Sept 2002)

• MIC 1024 mg/L, 32 mg/L

• VanA positive

• Plasmid-mediated

• Detected by disk diffusion but may not be detected by automated methods

Tenover, F. C. et al. 2004. Antimicrob. Agents Chemother. 48(1):275-280

FIG. 1. Disk diffusion and Etest analysis of the PA-VRSA isolate on Mueller-Hinton agar

Vancomycin-resistant Enterococci

Vancomycin-resistant Enterococcus

VanA VanB VanC

Vanc MIC

mg/L

64->1000

Resistant

4-1024

Resistant

2-32

Intermediate

Teic MIC

Mg/L

16-512

Resistant

0.5

Susceptible

0.5

Susceptible

Species E. Faecium

E. faecalis

E. Faecium

E. faecalis

E. Gallinarum

E. Casseiflavus

E. flavescens

Genetic

determinant

Acquired Acquired Intrinsic

Transferable Yes Yes No

Van A VRE

VSE

Van B VRE

VRE

VRE Lab Detection

• Disk diffusion-incubate full 24 h, view with transmitted light for hazy zones

• MIC methods

• Screening plates (vancomycin 6 mg/L)

• Multiplex PCR to detect vanA, vanB

Identify all VRE to species level

vanC motile E. gallinarum

Mechanisms of Resistance

inner membrane

outer membrane

periplasmic space

Loss of membrane permeability

Efflux

Enzymatic inactivation

EUCAST MIC breakpoint (mg/L) Zone diameter breakpoint (mm)

S ≤ R > S ≥ R <

Cefotaxime 1 2 20 17

Ceftriaxone 1 2 23 20

Ceftazidime 1 4 22 19

Cefepime 1 4 24 21

CLSI

MIC breakpoint (mg/L) Zone diameter breakpoint (mm)

S ≤ I R ≥ S ≥ I R ≤

Cefotaxime 1 2 4 26 23-25 22

Ceftriaxone 1 2 4 23 20-22 19

Ceftazidime 4 8 16 21 18-20 17

Cefepime 8 16 32 18 15-17 14

EUCAST

1. The cephalosporin breakpoints for Enterobacteriaceae will detect all clinically important resistance mechanisms (including ESBL and plasmid mediated AmpC). Some isolates that produce beta-lactamases are susceptible or intermediate to 3rd or 4th generation cephalosporins with these breakpoints and should be reported as tested, i.e. the presence or absence of an ESBL does not in itself influence the categorisation of susceptibility. In many areas, ESBL detection and characterisation is recommended or mandatory for infection control purposes.

CLSI

(7) Following evaluation of PK-PD properties, limited clinical data, and MIC distributions, revised interpretive criteria for cephalosporins (cefazolin, cefotaxime, ceftazidime, ceftizoxime, and ceftriaxone) and aztreonam were first published in January 2010 (M100-S20) and are listed in this table. Cefazolin interpretive criteria were revised again in June 2010 and are listed below. Cefepime and cefuroxime (parenteral) were also evaluated; however, no change in interpretive criteria was required for the dosages indicated below. When using the current interpretive criteria, routine ESBL testing is no longer necessary before reporting results (ie, it is no longer necessary to edit results for cephalosporins, aztreonam, or penicillins from susceptible to resistant). However, ESBL testing may still be useful for epidemiological or infection control purposes. For laboratories that have not implemented the current interpretive criteria, ESBL testing should be performed as described in Table 2A Supplemental Table 1.

Rationale for change

• PD modeling suggests cephalosporin breakpoints reduced to 1-4 mg/L, possible to obtain serum concentrations of cephalosporins above MIC for the required 40-50% of dosage interval

• Animal experiments show MIC better predictor of outcome than mechanism

• Reports of clinical failure correlated with higher MICs.

AmpC in Gram-negative bacilli

Class C beta-lactamases

• Gene normally found in most Gram negatives except

Klebsiella and Salmonella

• Cephalosporinases

• Not inhibited by clavulanate, tazobactam (except M.

morgannii)

• Resistant to cefoxitin

• Cefepime not generally affected

• Chromosomal

• Generally expressed at low level but are inducible

ESCHAPPM

• Enterobacter cloacae/aerogenes

• Serratia marcescens

• Citrobacter freundii

• Hafnia alvei

• Aeromonas hydrophila/caviae

• Providencia stuartii/rettgeri

• Morganella morganii

Induction vs Stable

Derepression • Induction

– transient switching on of -lactamase

synthesis in response to an inducer (cefoxitin,

imipenem)

• Stable Derepression

– permanent hyperproduction of the -

lactamase independent of an inducer

Chromosomal AmpC in

Enterobacter spp.

Ceftriaxone

Ceftoxitin

Ceftriaxone

Cefoxitin

Augmentin

Aztreonam

K. Pneumoniae

Would you treat this patient with

ceftriaxone?

Ceftriaxone

Cefoxitin

De-repressed mutants

De-repressed mutant

Original strain

Original strain

De-repressed mutant

>32 mg/L-

-1.5 mg/L 1mg/L susceptible-

pAmpC DHA-1

DHA in Klebsiella pneumoniae

CLSI (8) Enterobacter, Citrobacter, and Serratia may develop resistance during prolonged therapy with third-generation cephalosporins as a result of derepression of AmpC β-lactamase. Therefore, isolates that are initially susceptible may become resistant within three to four days after initiation of therapy. Testing of repeat isolates may be warranted.

Enterobacter spp., Citrobacter freundii, Serratia spp. and Morganella morganii. If susceptible in- vitro, the use of monotherapy of cefotaxime should be discouraged, owing to the risk of selection of resistance, or suppress the susceptibility testing result for this agent.

BSAC (EUCAST?)

Inducible plasmid AmpC (DHA) in K. pneumoniae?

CDS method • E. cloacae, E. aerogenes, C. freundii, inducible pAmpC

– High frequency of derepressed mutants (10-5 to 10-6) – Report R to all cephalosporins – Test cefepime and carbapenems – Do not attempt to test other β-lactams

• S. marcescens – No derepressed mutants with ceftazidime, tazocin and

aztreonam – Derepressed mutants with other β-lactams including

cefotaxime.

• H. alvei, P. stuartii, P. rettgeri and M. morganii – Very low mutation rate (10-8) – Test and report accordingly

http://web.med.unsw.edu.au/cdstest/

Carbapenemases in Gram-

negative bacilli

Carbapenemases

• Class A

– KPC

• Class B

– IMP, VIM, NDM

• Class C

• Class D

– OXA-48

EUCAST MIC breakpoint (mg/L) Zone diameter breakpoint (mm)

S ≤ R > S ≥ R <

Ertapenem 0.5 1 25 22

Imipenem 2 8 22 16

Meropenem 2 8 22 16

CLSI MIC breakpoint (mg/L) Zone diameter breakpoint (mm)

S ≤ I R ≥ S ≥ I R ≤

Ertapenem 0.5 1 2 22 19-21 18

Imipenem 1 2 4 23 20-22 19

Meropenem 1 2 4 23 20-22 19

EUCAST

1. The carbapenem breakpoints for Enterobacteriaceae will detect all clinically important resistance mechanisms (including the majority of carbapenemases). Some isolates that produce carbapenemase are categorised as susceptible with these breakpoints and should be reported as tested, i.e. the presence or absence of a carbapenemase does not in itself influence the categorisation of susceptibility. In many areas, carbapenemase detection and characterisation is recommended or mandatory for infection control purposes.

CLSI

24) Following evaluation of PK-PD properties, limited clinical data, and MIC distributions that include recently described carbapenemase-producing strains, revised interpretive criteria for carbapenems were

first published in June 2010 (M100-S20-U) and are listed below. Because of limited treatment options for

infections caused by organisms with carbapenem MICs or zone diameters in the intermediate range,

clinicians may wish to design carbapenem dosage regimens that use maximum recommended doses

and possibly prolonged intravenous infusion regimens, as has been reported in the literature.1 -4 Consultation with an infectious diseases practitioner is recommended for isolates for which the

carbapenem MICs or zone diameter results from disk diffusion testing are in the intermediate or resistant

ranges.

Until laboratories can implement the current interpretive criteria, the modified Hodge test (MHT) should

be performed as described in the updated Table 2A Supplemental Table 3. After implementation of the current interpretive criteria, the MHT does not need to be performed

other than for epidemiological or infection control purposes (refer to Table 2A Supplemental

Table 2).

The following information is provided as background on carbapenemases in Enterobacteriaceae that are

largely responsible for MICs and zone diameters in the new intermediate and resistant ranges, and thus the rationale for setting revised carbapenem breakpoints:

The clinical effectiveness of carbapenem treatment of infections produced by isolates for

which the carbapenem MIC or disk diffusion test results are within the new intermediate (I) range

is uncertain due to lack of controlled clinical studies.

NDM-1

DM23092 IMP+

DM23092 IMP+

Modified Hodge test

pAmpC

pAmpC

pAmpC

OXA-48

Outcomes of infections caused by carbapenemase-

producing K. pneumoniae according to treatment regimen

A combination more than 1 active drug including carbapenem B combination more than 1 active drug excluding carbepenem

C monotherapy with aminoglycoside D monotherapy with carbapenem

E monotherapy with tigecycline

F monotherapy with colistin G inappropriate therapy Tzouvekelis et al. CMR 2012 25: 682-

707

Carbapenemase-producing K.

pneumoniae: (when) might we still

consider treating with carbapenems?

• Carbapenem MIC ≤ 4 mg/L

• High-dose prolonged infusion

• Used in combination with another active

compound

Daikos and Markogiannakis CMID 2011; 17: 1135-1141

My take

• Detection of mechanism may still have role for tertiary care hospitals with resistance problems and complicated patients

• Combination therapy usually including carbapenem for CP-CRE

• Should we be doing more MICs?

• Automated system may not be equivalent to MIC. Etest practical option for most labs