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Introduction of Klebsiella pneumoniae carbapenemase (KPC)

Klebsiella pneumoniae carbapenemase (KPC) is an enzyme produced by bacteria, Klebsiella pneumoniae that makes organism multidrug resistance. This enzyme falls in Ambler class A beta-lactamases.

Types of beta  Lactamases

β-Lactamase can be classified into four molecular classes according to the Ambler scheme.

1. A (Penicillinases, including ESBLs)
2. B (Metallo β- lactamase, MBLs
3. C (AmpC enzymes)
4. D (Oxacillinase)

Importance of Klebsiella pneumoniae carbapenemase (KPC) detection

The importance of KPC detection is due to the following reasons-

1. The emergence of KPCs producing bacteria has become a significant global public health challenge while the optimal treatment remains undefined.
2. This is particularly relevant with Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) or other types of carbapenemase-producing Enterobacteriaceae (CPE) infections because commonly used regimens for empiric antimicrobial treatment do not normally cover multidrug-resistant (MDR)  pathogens.
3. KPC-KP has become one of the most important contemporary pathogens, especially in endemic areas.
4. To provide practical suggestions for physicians dealing with the management of KPC-KP infections in critically ill patients, based on expert opinions.
5. KPC-producing organisms can confer resistance to multiple different antimicrobial classes, including all available β-lactams, fluoroquinolones, and aminoglycosides. As such, infections due to KPCs are associated with high therapeutic failure and mortality rates of at least 50%.
6. The limited number of agents available for the treatment of KPCs presents a tremendous challenge to clinicians.
7. Klebsiella is resistant to multiple antibiotics. This is thought to be a plasmid-mediated property. Longer hospital stays and the performance of invasive procedures are risk factors for the acquisition of these strains.
8. Many isolates are a single sequence type, ST258, and susceptibility is limited to gentamicin, tigecycline, and colistin.

KPC Detection Method

Screening Method

• Antibiotics      Susceptibility
• Imipenem    –  Resistance
• Meropenem – Resistance

Confirmatory Method

Test Requirements 

• Muller-Hinton agar(MHA)
• Antimicrobial disks ( meropenem)
• phenylboronic acid
• EDTA
• Bunsen burner
• Inoculating loop
• Suspected isolate ( bacterial growth)
• Sterile cotton swab sticks
• Densitometer or O.5 McFarland Std.
• Inoculum suspension media (5 ml Tryptone Soya Broth)

Test Procedure

• Inoculum Preparation: Use only pure cultures. Confirm by Gram-staining before starting susceptibility test. Transfer 4-5 similar colonies with a wire, needle, or loop to 5 ml Tryptone Soya. Broth and incubate at 35-37°C
  for 2-8 hours until light to moderate turbidity develops. Compare the inoculum turbidity with that of standard 0.5 McFarland. Dilute the inoculum or incubate further as necessary to attain comparative turbidity. Alternatively, the inoculum can be standardized by another appropriate optical method (0.08 – 0.13 OD turbid suspension at 625 nm).
• Swab the entire agar surface of the plate with standardized inoculum soaked onto the cotton swab.
• Put two meropenem (10 µg) disks with a distance of 20 mm from disk to disk on Muller-Hinton agar as shown above picture.
• Add phenylboronic acid  (10  µl-concentration 300µg/ml) over a disk and EDTA on another disk.
• Incubate the plate overnight at 37°C.

Observation

Observe the zone of inhibition (ZOI).

Result Interpretation of KPC-KP

•  ZOI of meropenem plus phenylboronic acid  is equal and greater than 5 mm that of meropenem plus EDTA:  KPC producer
•  ZOI of meropenem plus phenylboronic acid  is less than 5 mm that of meropenem plus EDTA: Non- KPC producer strain
• Result: KPC producer as shown above image.

Betalactamase detection|ESBL| MBL| KPC |AmpC |Oxacillinase|MRSA|D Zone test positive| iMLSB strain as shown below-

Bibliography

1. https://www.medscape.com/-klebsiella-pneumoniae-carbapenemase-kpc-infections
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3552987/
3. https://www.clinicalmicrobiologyandinfection.com/article/S1198-743X(17)30499-
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC333018/2/
5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3521806
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11. Knothe H, Shah PDP, Krcmery V, Antal M, Mitsuhashi S. Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens. Infection. 1983;11(6):315-7.
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