How can I identify an AmpC‑producing Gram‑negative bacterial isolate?

Identifying AmpC β-Lactamase-Producing Gram-Negative Bacteria

Use cefoxitin disc screening followed by phenotypic confirmation with boronic acid-based disc testing, reserving multiplex PCR for definitive genotypic identification when available.

Screening Approach

Initial Screening Test

• Screen all Gram-negative isolates with cefoxitin (30 μg) disc diffusion as the first-line method 1, 2, 3
• Cefoxitin resistance serves as the primary screening indicator, though it detects only 77.9% of AmpC producers 3
• For optimal sensitivity, screen isolates showing reduced susceptibility to third-generation cephalosporins (ceftazidime and/or cefotaxime) combined with reduced susceptibility to cefoxitin, which achieves 97% sensitivity and 90% specificity 2

Key Organisms to Suspect

Chromosomal AmpC producers include Enterobacter spp., Citrobacter freundii, Serratia marcescens, Morganella morganii, and Providencia spp. 4

• Enterobacter species pose the highest risk because they frequently develop high-level constitutive AmpC expression during therapy with third-generation cephalosporins, even when initially susceptible 4
• Plasmid-mediated AmpC can occur in E. coli and Klebsiella pneumoniae, which do not naturally produce chromosomal AmpC 1, 2

Phenotypic Confirmation Methods

Boronic Acid-Based Testing (Preferred Phenotypic Method)

The disc-based test using boronic acid (specifically phenylboronic acid) shows the best performance as a phenotypic confirmation method 2, 5

• Use 600 μg/mL phenylboronic acid with 30 μg ceftazidime disc for optimal negative predictive value 6
• The combined disc test using cefotetan with boronic acid detects 72% of AmpC producers 1
• Boronic acid at 150 μg/mL detects only 12.5% of cefoxitin-resistant isolates, while 600 μg/mL concentration performs significantly better 6

Alternative Phenotypic Tests

• Cloxacillin-based double disc synergy test detects 56.5% of AmpC producers and is user-friendly for routine laboratories 1
• Three-dimensional extraction method achieves 93.6% detection rate but is more technically demanding 3
• AmpC disc method detects 80.9% of screen-positive isolates and is easier to perform routinely 3

Induction Testing

• Disc approximation test identifies inducible AmpC in isolates sensitive to third-generation cephalosporins, detecting 22.7% of such strains 3
• Inducible AmpC is not found in E. coli and Klebsiella spp., only in organisms with chromosomal AmpC genes 3
• The induction test detects 35.5% of cefoxitin-resistant isolates 1

Genotypic Confirmation (Gold Standard)

Multiplex PCR definitively identifies plasmid-mediated AmpC genes and should be used when available 1, 2, 6

PCR Detection

• Multiplex PCR detects five major plasmid-mediated AmpC gene families: MOX, CIT, DHA, EBC, and FOX 1, 6
• Among cefoxitin-resistant isolates, 18.3-25.8% are positive by multiplex PCR 1, 6
• CIT and MOX are the most commonly detected families, with CIT found in 45.5% of PCR-positive isolates either alone or in combination 6
• CMY-2 (CIT family) is the most prevalent plasmid-mediated AmpC in E. coli, followed by DHA-1 and ACC-1 2

Critical Pitfalls and Caveats

ESBL Co-Production

• 86.1% of AmpC producers also co-produce ESBL enzymes, which can mask each other in phenotypic testing 5
• Pure AmpC production occurs in only 9.2% of isolates 5
• Always perform ESBL screening (ceftazidime/cefotaxime with and without clavulanic acid) in parallel with AmpC testing 1

Resistance Pattern Recognition

AmpC producers demonstrate resistance to all penicillins, cephalosporins, cephamycins, and aztreonam, but are not inhibited by clavulanic acid 4, 3

Prevalence Considerations

• Hospital-acquired infections account for 85.5% (53/62) of AmpC-producing isolates 1
• Community-acquired AmpC producers represent only 12.9% (8/62) of cases 1
• Overall prevalence of plasmid-mediated AmpC in Enterobacteriaceae is approximately 2.6% 2

Recommended Testing Algorithm

1. Screen with cefoxitin disc on all Gram-negative isolates showing third-generation cephalosporin resistance
2. Confirm positive screens with boronic acid disc test (600 μg/mL with ceftazidime)
3. Perform disc approximation test on third-generation cephalosporin-sensitive isolates to detect inducible AmpC
4. Use multiplex PCR when available for definitive identification and epidemiological tracking
5. Always screen for concurrent ESBL production to avoid missing co-producers