9- Beta lactam antibiotics and antibiotic assays

Beta lactam antibiotics

•Bactericidal, work on gram positives and negatives (broad spectrum antibiotics)

•Several classes based on structure but all have B-lactam ring, structure mimics D-ala-D-ala peptide which is a substrate for the transpeptidases (PBP)

•Bind tightly to active site to inhibit cell wall synthesis, death by osmotic instability, autolysis

Resistance to B-lactams

•Enzymatic destruction- B-lactamases

•Altered target site- mutational changes in PBP, acquisition of new PBP

•Decreased uptake- efflux pumps, modification to porin channels

Classes of B-lactam antibiotics

•Penicillins- penicillin G/V, methicillin, amoxixillin, cefoxitin

•Cephalosporins- 1st, 2nd, 3rd, 4-5th generation e.g. ampicillin (1st gen)

•Carbapenems- imipenem

•Monobactams- nocardicin A

•(Bacitracin and Glycopeptides)

SCCmec

•Staphylococcal cassette chromosome mec

•Mobile genetic element that incorporates into the chromosome

•Contains the mecA or mecC genes which encode PBP2a which has a low affinity for beta lactam antibiotics

•Confers resistance to methicillin and most B-lactams

Transmission

•Physical skin to skin contact with infected person or carrier

•Contact with MRSA on objects or surfaces- door handles, towels

Laboratory identification

•Screening e.g. before high risk surgery by swabbing nasal passage, groin area

•Culture on MRSA chromogenic agar

•AST, resistant against methicillin, cefoxitin

•PCR- looking for mecA and mecC genes

ESBL

•Extended spectrum beta lactamases

•Over 2000 identified, include ESBL’s AmpC B-lactamases, carbapenemases

•Multi drug resistant bacteria produce multiple B-lactamases

•Hydrolyse the B-lactam ring of antibiotics

Carbapenems mode of action

•B-lactam antibiotics with broad spectrum activity against gram negatives

•Last effective defense aginst K. pneumonia and E. coli

•Enter periplasmic space through outer membrane proteins, inhibit PBP’s and prevent cell wall syntheis which leads to cell lysis

Carbapenem resistance

•Carbapenem resistant enterobacteracieae being reported worldwide

•Intrinsic resistance- S. maltophilia, acinetobacter produces OXA-51 enzymes, non fermenters naturally resistant to ertapenem, serratia and proteus special have low level resistance to impenem

•Acquired resistance through B-lactamases, efflux pumps or mutations to outer membranes

Transmission of carbapenemases

•Carried on mobile genetic element (plasmid)

•Horizontal gene transfer e.g. sharing of plasmid amongst gut flora, can cause resistance to multiple drugs

•Different strains of e.coli may have different resistance genes

•Outbreak seen in different species had the same resistance gene e.g. oxa-48 e.coli, oxa-48 klebsiella

Classes of carbapenems

•Class A- Serine active site, resistance against carbapenems, penicillins, cephalosporins, KPC, endemic in USA, High death rate <50%, MDRO, K.pneumoniae, inhibited by boronic acid

•Class B (metallo-B-lactamases)- Zinc active site, resistance against all B-lactams, e.g. VIM, IMP, both endemic in taiwan and japan, HA MDRO’s, inhibited by EDTA

•Class C - Resistance to penicillins and cephalosporins e.g. CMY, inhibited by cloxacillin

•Class D- Serine active site, resistance to carbapenems, pinicillins, cephalosporins, OXA-48 mainly associated with E.coli and K.pneumoniae, inhibited by avibactum

Screening methods for CPE’s

•AST- Cefpodoxime is the most sensitive in detecting ESBL produciton, inhibition zone measured

•Screening agars e.g. CHROMagar KPC, CARPA SMART

Confirmatory tests

•Phenotypic methods- combination disc test, contain antibiotics alone and in combination with various inhibitors (boronic acid), zone diameters observed, identified according to pattern

•Biochemical methods- Carba NP, blue carba test, B-CARBA test, all detect carbapenem hydrolysis which causes a pH change and changes the colour of the solution

•Lateral flow assays- RESIST-3 detects OXA-48, KPC, NDM from bacterial isolates in 20 minutes

•Molecular methods- PCR with direct faecal swab, 120 alleles in the big 5 families- KPC, NDM, VIM, IMP, OXA-48, family coverage is easy for KPC and NDM and harder for the others as the variants can differ largely

What is an antibiotic assay used for

•Measurement of antibiotic in body fluids or serum

•Ensures the patient is recieving adequate levels of the drug to treat the infection and to prevent toxic levels in blood

•Used to reduce risk of toxicity e.g. nephrotoxic antibiotics

•Used when routes of excretion are impaired, when absorption is uncertain

•Used in neonates with serious infections and those with prolonged therapy for serious infection

•Used in patients that fail to respond to the appropriate therapy

Types of toxicity and sample types

•Antibiotics can cause 8th cranial nerve damage

•Oxotoxicity causes hearing loss

•Nephrotoxicity causes renal damage

•Serum most frequently used, occasionally CSF or urine

Most frequently assayed antibiotics

•Aminoglycoside family e.g. gentamicin (most common)

•Other antibiotics like vancomycin

Through and peak level

•Through level- blood sample taken immediately before dose of antibiotic is administered

•Peak levels- blood taken after 15 mins for IV dose, 30mins-1h after intramuscular dose

•Aminoglycosides are not effective if given orally, absorbed from small intestine to liver via portal vein and inactivated

Example of assay

•Abbott architect

•May be carried out in the clinical chemistry laboratory