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Screening and detection of plasmid mediated AmpC betalactamases

The capability to detect AmpC is important to improve the clinical management of infections and provide sound epidemiological data. Reduced susceptibility to cefoxitin in the Enterobacteriaceae may be an indicator of AmpC activity, but it should be confirmed by other tests. Laboratories should be able to recognize AmpC derepressed strains and those with plasmid AmpC. Guidelines from the CLSI are not yet available for detection of bacteria with AmpC beta‑lactamases.

DIAGNOSTICA


TYPES OF AmpC BETA‑LACTAMASES Cefepime + Clav. Ceftazidime + Clav.

Boronic acid

Cloxacillin 500µg

Cefoxitin or Imipenem

Cefepime

Chromosomally mediated Amp C (partially derepressed)

No synergism

Synergy with Ceftazidime+Clav. and/or Cefotaxime+Clav.

Synergy with Ceftazidime and/or Cefoxitin

Antagonism with 3rd generation cephalosporins

S zone >26 mm

Plasmid mediated AmpC (or totally derepressed)

No synergism

Synergy

Synergy

S zone >26mm

As above

As above

No antagonism with 3rd generation cephalosporins

Inducible plasmid mediated AmpC ACT‑1, DHA‑1, DHA‑2, CFE‑1, CMY‑13

No synergism

Synergy

Synergy

S zone >26 mm

As above

As above

Antagonism with 3rd generation cephalosporins

Chromosomal *ESAC (E. coli) (not inducible)

No synergism

Synergy

Synergy

As above

As above

No antagonism with 3rd generation cephalosporins

S zone<26 mm MIC 1‑8 µg/ml

*ESAC = Extended spectrum Amp C

SCREENING Derepressed/plasmid AmpC should be suspected when we see: • Resistance to 3rd generation cephalosporins – NOT Cefepime. • Resistance to Cefoxitin (inhibition zone < 16 mm). • No cephalosporin / Clav. synergism. • I / R to Amoxycillin + Clav. • AmpC derepressed Serratia are S to ceftazidime.

CONFIRMATION Apply one Cefotaxime + Clavulanate (CTAX+CL) and one Ceftazidime + Clavulanate (CAZ+CL) Neo‑Sensitabs on an inoculated MH agar plate. In between apply one Boronic Acid Diatabs (BOR) at a distance of approx. 10 mm (edge to edge). If the strain is totally resistant to the Cefalosporins+Clavulanate combination, the distance should be reduced to 5 mm. Apply one Ceftazidime (CAZ) and one Cefoxitin (FOX) Neo‑Sensitabs on an inoculated MH agar plate. In between at a distance of 5‑10 mm edge to edge, apply one Cloxacillin (CLOX) 500 µg Neo‑Sensitabs. Imipenem (IMP) is used to detect the inducible phenotype.

• Providencia, Morganella and Serratia inducible & derepressed may appear S /I to cefoxitin. BOR

10 mm

• Strains producing AAC‑1 beta‑lactamase are susceptible to cefoxitin.

CAZ+ CL

10 mm CTAX+ CL

IMP FOX 15 mm

CAZ CLOX

5‑10 mm

5‑10 mm

Screening and detection of plasmid mediated AmpC betalactamases – version A – 01.2009

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INTERPRETATION A keyhole or ghost zone (synergism) between Boronic Acid and any of Cefotaxime+Clav. or Ceftazidime + Clav. indicates the presence of an AmpC beta‑lactamase.

INDUCIBLE PHENOTYPE The inducible phenotype is identified by a tablet approximation test, using Imipenem or Cefoxitin against 3rd generation cephalosporins (distance 15 mm from edge to edge).

A keyhole or ghost zone between Cloxacillin 500 µg and Ceftazidime and/or Cefoxitin indicates the presence of an AmpC beta‑lactamase.

Distorted zones indicate the presence of an inducible AmpC beta‑lactamase.

Plasmid mediated AmpC differ from chromosomal AmpC in being uninducible (few exceptions). Strains producing inducible plasmid AmpC beta‑lactamases (ACT‑1, DHA‑1, DHA‑2, CFE‑1, CMY‑13) will show antagonism (distorted zone) between Cefoxitin or Imipenem and 3rd generation cephalosporins. Strains of Klebsiella spp, Salmonella spp and P. mirabilis showing synergism with Boronic Acid and/or Cloxacillin 500 µg possess presumptively plasmid mediated AmpC beta‑lactamases. The method cannot distinguish between chromosomal and plasmid mediated AmpC beta‑lactamases in E. coli, but the test is useful to select strains for further analysis. Plasmid mediated are often multiresistant and may show scattered colonies near the edge of the zone of third gen. cephalosporins and aztreonam disks.

Treatment with 3rd generation cephalosporins should be avoided in severe Enterobacter, C. freundii, Serratia and Morganella infections except in UTI, because of risk for selection of cephalosporin‑resistance during therapy. AmpC + ESBL Screening criterion for ESBL presence among AmpC‑producing Enterobacter, C. freundii and Serratia marcescens is Cefepime MIC > 1 ug/ml (inhibition zone< 26 mm). High level expression of AmpC may prevent recognition of an ESBL. Use of Cefepime is more reliable to detect these strains because high AmpC production has little effect on cefepime activity.

Screening and detection of plasmid mediated AmpC betalactamases – version A – 01.2009

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Products mentioned in the brochure including REF no:

Diatabs Product code

Diatabs

10041

Boronic Acid 250 µg

10031

Cloxacillin 500 µg

Neo‑Sensitabs Product code

Original Neo‑Sensitabs

Product code

CLSI potency Neo‑Sensitabs

72312

Ceftazidime+Clav. 30+10 µg

64712

Cefotaxime+Clav. 30 + 10 µg

72212

Ceftazidime 30 µg

64612

Ceftazidime+Clav. 30 + 10 µg

71712

Cefoxitin 60 µg

64012

Ceftazidime 30 µg

74612

Imipenem 15 µg

62912

Cefoxitin 30 µg

71212

Cefepime 30 µg

61212

Imipenem 10 µg

79512

Cefepime+Clav. 30 + 10 µg

63712

Cefepime 30 µg

64812

Cefepime+Clav. 30 + 10 µg

References 1) Xuan Qin et al : Kirby‑Bauer disc approximation to detect inducible third generation cephalosporin resistance in Enterobacteriaceae. Annals Clin Microbiol &Antimicr. 3,1‑6,2004. 2) Tetsuya Yagi et al : Practical methods using Boronic acid compounds for identification of Class C beta‑lactamase producing K. pneumoniae and E. coli. J. Clin. Microbiol 43,2551‑2558,2005. 3) Prof P. Nordmann : Evaluation de tests phenotypiques de detection de cephalosporinases integrant l’utilisation des disques de cloxacilline et d’ acide boronique. Oct 2006.Internal study. 4) Ruppe E et al : First detection of the Ambler Class C 1 AmpC beta‑lactamase in C. freundii by a new simple double‑disk synergy test . J Clin Microbiol 44,4204‑4207,2006. 5) Merlino J : Boronic acid tests for AmpC detection in E. coli and K. pneumoniae isolates. Study at Concord Hospital,Sidney (Australia),nov. 2007. 6) Seok Hoon Jeong et al : Boronic acid disk tests for identification of ESBL production in clinical isolates of Enterobacteriaceae producing chromosomal AmpC beta‑lactamases.Int J Antimicr. Agents 31,467‑471,2008. 7) Adler H et al : Evaluation of a phenotypic method to detect plasmid mediated AmpC beta‑lactamases in Enterobacteriaceae, lacking inducible chromosomal AmpC genes.University of Basel.Congress SGM,Interlaken,June 2008. 8) Mirelis B et al : A simple phenotypic method for the differentiation between acquired and chromosomal AmpC beta‑lactamases in E. coli. Enferm. Infecc Microbiol Clin 24,370‑372, 2006.



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