Lecture 16 - ESBLs and Ab resistance in TB Flashcards
Horizontal gene transfer
Outbreak of E. coli in Germany in 2011 resulting in over 50 deaths Thought to be hypervirulent due to the increased adsorption of shiga toxin due to the increased adhesive nature and its extensive antibiotic resistance The bla(TEM-1) is normally found in other Enterobacteriaciae
What are beta lactamases?
Enzymes that break down the lactam ring of beta lactam containing antibiotics
Beta lactamases break down peptidoglycan - penicillin binding proteins PBPs
cephalosporins have an oxy amino group to protect the beta lactam ring (to counteract the effect of beta lactamases)
Nearly all Gram negative bacteria have a chromosomally encoded beta lactamase. Enzymes that break down the lactone ring and prevent binding of the antibiotic to the PBP’s involved in peptidoglycan biosynthesis
The first plasmid encoded beta lactamase was identified in the 1960s in Greece and was named after the patient fromwhom it was isolated (Temoniera) bla-TEM1
Classification and nomenclature of beta lactamases
Four main molecular classes based on differences in the active site
Class A beta-lactamases
—characterized by their hydrolytic mechanisms that require an active-site serine at position 70. Include penicillinases and cephalosporinases in the TEM, SHV, and CTX-M-type groups
SHV - contraction of sulfydryl variable
CTX-M - cefotaximase isolated in Munich in the 80’s
Can be inhibited by tazobactam and clavulanate
Class A beta-lactamases with carbapenemase activity include the KPCs
Class B beta-lactamases
— also known as the metallo-beta-lactamases (MBLs), require divalent cations, for their dependence upon zinc for efficient hydrolysis . MBLs can be inhibited by EDTA (an ion chelator), but not by inhibitors such as tazobactam, clavulanate,
Class B beta-lactamases with carbapenemase activity include NDM-1 (new delhi)
Class C beta-lactamases
— Hydrolyzes cephalosporins and cephamycins. E. coli and P, aeruginosa AmpC, CMY-2, FOX-1, MIR-1, P99
Not inhibited by CLA and TZB
Class D beta-lactamases— also referred to as OXA-type enzymes, able to hydrolyzeoxacillin. Variably affected by the beta-lactamase inhibitors clavulanate, sulbactam, or tazobactam.
OXA carbapenemases have been identified inAcinetobacter baumanniiand Enterobacteriaceae (especiallyK. pneumoniae,E. coli, andE. cloacae)
Pathogens can have a mixture of all types of beta lactamases!
Classification and nomenclature of beta lactamases and extended spectrum beta lactamases (ESBLs)
Extended-spectrum β-lactamase (ESBL): a β-lactamase that hydrolyzes oxyimino cephalosporins and monobactams in addition to penicillins and early cephalosporins and that is inhibited by clavulanic acid or tazobactam
Amino acid substitutions in TEM ESBL derivatives. The amino acids listed within the grey bar are those found in the structural gene
of the TEM-1 -lactamase (162). The amino acid numbering is according to the scheme of Ambler et al. (5). Substitutions found in TEM-type
ESBL derivatives are shown under the amino acids of TEM-1. TEM-type variants may contain more than one amino acid substitution. , TEM-2
is not an ESBL but is included in the figure as a derivative of TEM-1. The Gln39Lys substitution does not contribute to the ESBL phenotype, but
a number of ESBLs are derived from TEM-2. , TEM-50 and TEM-68 contain amino acid substitutions that are common to both the ESBL and
the IRT phenotypes. Only the amino acid substitutions that are common to TEM-type ESBLs are shown in this figure.
What is the solution?
New beta lactamase inhibitors?
BLI-489 is a potent inhibitor of all serine β-lactamase classes with low propensity toward resistance development
Tuberculosis
In 2012, 8.6 million people fell ill with TB and 1.3 million died from TB.
Over 95% of TB deaths occur in low- and middle-income countries
In 2012, an estimated 530,000 children became ill with TB and 74,000 HIV-negative children died of TB.
Multi-drug resistant TB (MDR-TB) is present in virtually all countries surveyed.
In 2015, 10.4 million people fell ill with TB and 1.8 million died from TB.
Over 95% of TB deaths occur in low- and middle-income countries
In 2015, an estimated 1million, children became ill with TB and 170,000 HIV-negative children died of TB.
Multi-drug resistant TB (MDR-TB) is present in virtually all countries surveyed with estimates in 2015 of 480,000 cases.
History of treatment of tuberculosis
On November 20, 1944, streptomycin was administered for the first time to a critically ill TB patient
Within months resistant mutants were observed
Many other drugs followed quickly and combination therapy, including isoniazid, was implemented
Combination therapy and DOTS used today
The TB death rate dropped 45% between 1990 and 2012
An estimated 22 million lives saved through use of DOTS
Treatment of tuberculosis
The unique cell wall of mycobacteria is the main target for antibiotics
TB does not stain normally on a gram stain - G+ is purple (as the thick cell wall retains the dye, but TB has thinner peptidoglycan) so acid fast stain used instead
TB has lots of sugars on cell wall - arabinose and galactose. Arabinogalactam and MAGP (massive sticky complex that is linked together)
First-line anti-tuberculous drugs
- ethambutolis EMB or E,
- isoniazidis INH or H,
- pyrazinamideis PZA or Z,
- rifampicinis RMP or R,
- streptomycinis SM or S
Regimen is 2HREZ/4HR3
Treatment - ethambutal and isoniazid
Ethambutol - targets the M genes which are involved in arabinoglactom.
EmbB and EmbA transfer Araf residues to a galactan chain after the initial Araf residues are linked to galactan by arabinofuranosyltransferase, AftA.
Isoniazid (isonicotinohydrazide) -
Involved in inhibiting the synthesis of mycolic acids. Mycolic acids are part of the outer cell surface of G+ and are key to bacterial virulence - they contribute to a property called Cord Factor.
INH is a pro-drug (it binds to NADH that is important in the synthesis of mycolic acid) that is activated by the catalase peroxidase KatG
Mutations in katG show resistance to INH and transformation of INH resistant strains with wild type katG restored susceptibility
How activated INH works exactly is still the subject of debate.
INH inhibits the NADH-dependent enoyl-ACP reductase (inhA)
Made a cosmid library from an INH resistant strain and transformed it into a sensitive strain
Mapping of the cosmid showed that there was one open reading frame (inhA) that had a S94A substitution relative to the wild-type copy.
Mechanism of isoniazid action against InhA is covalent attachment of the activated form of the drug to the nicotinamide ring of NAD bound within the active site of InhA
However, many clinical INH resistant strains don’t have mutations in inhA. One mechanism is to overexpress InhA causing titration of the drug
