2. AmpC
Clinically significant because they confer resistance
• Penicillins,
• Cephalosporins(1st, 2nd and 3rd generation),
• Oxyimino-cephalosporins (e.g., ceftriaxone, cefotaxime, and
ceftazidime),
• Cephamycins (e.g., cefoxitin and cefotetan) and
• Monobactams.
• not affected by the beta lactamase inhibitor like clavulanic acid
4. Chromosomal Amp C Plasmid-based Amp C
expressed constitutively at
a low level
expressed constitutively in
most cases
Enterobacter species,
Citrobacter spp., and
Serratia spp., carry an
inducible Amp C gene
clinical significance as
plasmids transfers both
Amp C and ESBL enzymes
in the same plasmid
5.
6.
7.
8.
9. MECHANISM OF INDUCIBLE AMPC
PUMPS AND PORINS
• presence of porin channels through which β-lactams penetrate and of
efflux pumps, which transport them out of the cell
10. Inducers of AmpC
• Strong inducers : Benzylpenicillin, ampicillin, amoxicillin, and
cephalosporins such as cefazolin and cephalothin
• Weak inducers : Cefotaxime, ceftriaxone, ceftazidime, cefepime,
cefuroxime, piperacillin, and aztreonam
11. Amp C Screening method:
• Cefoxitin disk diffusion method : Cefoxitin 30-ug disc was used.
Isolates with zone diameters less than 18 mm>>>>AmpC producers
12. • Phenyl boronic acid method-PBA(Inhibitor based method)
120 mg of phenyl
boronic acid+ 3ml
Dimethyl
sulfoxide(DMSO)
3ml of distilled
water
20 μl of this liquid
was added on each
cefoxitin(CX) disc
CX PBA and 30 μg
of CX were placed
on the agar at a
distance of 30 mm
a zone difference
>5 mm
AmpC producer
Phenotypic AmpC confirmation methods
14. Phenotypic AmpC confirmation methods
• Cefoxitin Cloxacillin-Double disc synergy test(CC-DDS):12
• This test was based on the inhibitory effect of cloxacillin on AmpC
production. The isolates were inoculated on Mueller Hinton agar.
Cefoxitin/cloxacillin disks (200 μg/ 30 μg) and cefoxitin disk (30 μg)
were used. A difference of 4 mm zone between the two discs was an
indication of AmpC production.
15.
16. Phenotypic AmpC confirmation methods
• Amp C TRIS EDTA disc test:
• The test is based on the use of Tris–EDTA to permeabilize a bacterial cell and
release b-lactamases into the external environment.
• AmpC disks:20 μl of a 1:1 mixture of saline and Tris–EDTA to sterile filter paper
disks and then dried, refrigerated.
• Prior to use, Amp C disks were rehydrated with 20 ml of saline
• Several colonies of each test organism were applied to a disk.
• A 30 μg cefoxitin disk was placed at the inoculated surface of the Mueller– Hinton
agar containing ATCC E coli strain.
• The inoculated Amp C disk was then placed nearly touching the antibiotic disk
with the inoculated disk face coming in contact with the agar surface. The plate
was then inverted and incubated overnight at 350C in ambient air.
• An indentation or a flattening of the zone of inhibition, indicating enzymatic
inactivation of cefoxitin was considered as positive.
17.
18. Phenotypic AmpC confirmation methods
• Disk approximation test (Induction based method):14
• A 0.5 McFarland bacterial suspension was prepared, the surface of
the MHA plate was inoculated with suspension. A 30 μg ceftazidime
disk was placed at the center of the plate then 10 μg imipenem, 30 μg
cefoxitin, and 20/10 μg amoxicillin/clavulanate disks were placed at a
distance of 20 mm from the ceftazidime disk. The plate was inverted
and incubated overnight at 37°C. After overnight incubation, if there
is any obvious blunting or flattening of the zone of inhibition between
the ceftazidime disk and the inducing substrates (imipenem, cefoxitin
and amoxicillin/clavulanate disk) it was considered as a positive result
for AmpC production.
19.
20.
21. Why do we need genotypic methods??
• Phenotypic tests cannot distinguish among the various families of
plasmid-mediated AmpC enzymes
• may also overlook chromosomally determined AmpC β-lactamases
with an extended spectrum
22. GENO TYPIC METHODS
• Vitek 2 MS
• MALDI-TOF
• Multiplex PCR
• Microarray
• Sequencing
• FISH
23. Is the recognition of plasmid-mediated AmpC
enzymes necessary for the average laboratory?
• isolates may appear to be susceptible in vitro to some cephalosporins
and aztreonam yet fail to respond
• isolates producing AmpC β-lactamase are resistant to additional β-
lactams and not susceptible to currently available β-lactamase
inhibitors and have the potential for developing resistance to
carbapenems
• plasmid mediation of AmpC carries the threat of spread to other
organisms within a hospital or geographic region.
• Therapeutic and infection control considerations that recognition is
important
24. TREATMENT
Amikacin Cystitis: 15 mg/kg/dose d IV once. All other infections: 20 mg/kg/dose d IV x 1 dose,
subsequent doses and dosing interval based on pharmacokinetic evaluation
Cefepime* Cystitis: 1 g IV q8h All other infections: 2 g IV q8h, infused over 3 hours*
Ciprofloxacin Ciprofloxacin ESBL-E or AmpC infections: 400 mg IV q8h-q12h OR 500 – 750 mg PO q12h
Ertapenem Ertapenem 1 g IV q24h, infused over 30 minutes
Fosfomycin Fosfomycin Cystitis: 3 g PO x 1 dose
Gentamicin Gentamicin Cystitis: 5 mg/kg/dose d IV once.All other infections: 7 mg/kg/dose d IV x 1
dose, subsequent doses and dosing interval based on pharmacokinetic evaluation
Imipenem-
cilastatin
Cystitis (standard infusion): 500 mg IV q6h, infused over 30 minutes All other ESBL-E or
AmpC-E infections: 500 mg IV q6h, infused over 30 minute
Levofloxacin Levofloxacin 750 mg IV/PO q24h
Meropenem Cystitis (standard infusion): 1 g IV q8h, infused over 30 minutes.All other ESBL-E or AmpC-E
infections: 1-2 g IV q8h, infused over 30 minutes
25. TREATMENT
Nitrofurantoin Nitrofurantoin Cystitis: Macrocrystal/monohydrate (Macrobid®) 100 mg PO q12h Cystitis:
Oral suspension: 50 mg PO q6h
Plazomicin Cystitis: 15 mg/kg d IV x 1 dose All other infections: 15 mg/kg d IV x 1 dose, subsequent
doses and dosing interval based on pharmacokinetic evaluation
Tobramycin Cystitis: 5 mg/kg/dose d IV x 1 dose All other infections: 7 mg/kg/dose d IV x 1 dose;
subsequent doses and dosing interval based on pharmacokinetic evaluation
Septran Cystitis: 160 mg (trimethoprim component) IV/PO q12h Other infections: 8-12 mg/kg/day
(trimethoprim component) IV/PO divided q8-12h (consider maximum dose of 960 mg
trimethoprim component per day)
IDSA Guidance on the Treatment of Antimicrobial-Resistant Gram-Negative Infections: Version 2.0
Published by IDSA, 3/31/2022
Pranita D. Tamma*, Samuel L. Aitken, Robert A. Bonomo, Amy J. Mathers, David van Duin, Cornelius J. Clancy
28. Transmission-based precautions
• applied to the patients with confirmed or suspected communicable
infections
• droplet, airborne, and contact precautions.
• Protective isolation for oncology patients; these patients placed in
rooms with high-efficiency particulate air (HEPA) filtration air and
positive air room pressure relative to the outside
29. REFERENCES
• Dhanashree P, Anuradha B;Phenotypic methods for detection of Amp
C β lactamases in Gram negative clinical isolates of a tertiary care
hospital; Indian Journal of Microbiology Research 2020;7(2):125–129
• Lister, Philip,Wolter, Daniel,Hanson, Nancy; Antibacterial-Resistant
Pseudomonas aeruginosa: Clinical Impact and Complex Regulation of
Chromosomally Encoded Resistance Mechanism ; Clinical
microbiology reviews 2009;582(610);2210.1128/CMR.00040-09
Hinweis der Redaktion
In Gram-negative bacteria, Amp C b-lactamase can be of two types: chromosomal or plasmid mediated.
Chromosomal Amp C genes are expressed constitutively at a low level. Few Enterobacteriaceae like Enterobacter species, Citrobacter spp., and Serratia spp., carry an inducible Amp C gene.7 Plasmid-based Amp C genes are expressed constitutively in most cases. All plasmid-carried Amp C genes have clinical significance as plasmids not only transfer Amp C but also ESBL enzymes in the same plasmid
Wild-type basal expression of ampC. During normal cell wall recycling, 1,6-anhydromuropeptides are removed from the cell wall and transported into the cytoplasm via the AmpG permease. The 1,6anhydromuropeptides are cleaved by AmpD to generate free tripeptides, which are later converted into UDP-MurNAc-pentapeptides. UDPMurNAc-pentapeptide interacts with AmpR bound to the ampR-ampC intergenic region, creating a conformation that represses transcription of ampC. Low basal levels of AmpC are produced, and the enzyme is localized to the periplasmic space. (B)-Lactam induction of ampC expression. Inducing-lactams, such as cefoxitin and imipenem, cross the outer membrane through porins, enter the periplasmic space, and interact with target PBPs. An increase in pools of 1,6-anhydromuropeptides is observed, and AmpD is unable to efficiently process the higher levels of cell wall fragments. The anhydro-MurNAc-peptides (inducing peptides) replace UDP-MurNAc-pentapeptides (suppressing peptides) bound to AmpR, causing a conformational change in the protein. AmpR is converted into a transcriptional activator, ampC is expressed at higher levels, and levels of AmpC increase in the periplasmic space. When the amount of-lactam decreases below "inducing levels," the cytoplasmic pool of anhydroMurNAc-peptides also decreases, and AmpD is able to efficiently cleave these peptides, restoring wild-type ampC expression, as shown in panel A. (C) AmpD-associated derepression of ampC expression. Mutations leading to the inactivation of AmpD or decreased expression of ampD impair the processing of cell wall recycled products and lead to increased levels of anhydro-MurNAc-peptides (inducing peptides) in the cytoplasm. As a result, the binding of inducing peptides to AmpR is favored, AmpR is "locked" in a conformation for transcriptional activation of ampC expression, and high-level constitutive expression of ampC is observed.
Phenyl boronic acid method-PBA(Inhibitor based method)
120 mg of phenyl boronic acid+ 3ml Dimethyl sulfoxide(DMSO) was added to 3ml of distilled water. Then 20 μl of this liquid was added on each cefoxitin(CX) disc. Disk containing 30 μg of CX and another disk containing 30 μg of CX with BA were placed on the agar at a distance of 30 mm. Inoculated plates were incubated overnight at 37°C. An organism demonstrating a zone difference >5 mm was considered as an AmpC producer
