The only example of this mechanism is that to β-lactam drugs (peni- cillins, cephalosporins, and carbapenems); however, it is the most successful resistance mechanism of all. It is manifested by the produc- tion of β-lactamases that can hydrolyse and inactivate various mem- bers of this family of antibiotics. The bond that all β-lactamases hy- drolyse is shown by the arrows in Figure 6.1. The same position can be identified in most of the β-lactam antibiotic groups by the arrows shown in Figure 2.1.
The β-lactamase hydrolyses the carbon-nitrogen bond of the β-lactam ring. The integrity of this ring is crucial to the activity of the antibiotic.
In Gram-positive bacteria, the β-lactamase is produced within the cyto- plasm of the cell and is exported through the cell membrane into the surrounding medium. It provides a drug-free blanket around the cell. It also provides protection for other microorganisms in close proximity.
In Gram-negative bacteria, the antibiotic has more difficulty in pene- trating. The β-lactamase is still produced in the cytoplasm; however,
Figure 6.1 Inactivation of amoxicillin with a β-lactamase
HO
N H H2N HO
H2N
OO N
N H
S N O O
HO
COO– H COO–
S
52
CHAPTER 6Mechanisms of antibiotic resistance Figure 6.2 Interaction of amoxicillin with BBBB-lactamases in
Gram-negative bacteria
Porin
Penicillin binding proteins β-lactamase
Outer membrane Periplasmic space Inner membrane Cytoplasm Outside cell Amoxicillin
most of it is exported only as far as the periplasmic space, between the two membranes. It is here that the β-lactamase intercepts the incoming β-lactam drug and destroys it. It is a more directed and more efficient mechanism than that found in Gram-positive bacteria.
It has recently been suggested that there are over 750 β- lactamases found in clinical bacteria. They have conveniently been classified by their molecular structure into four groups. There is no homology between each group but significant homology exists within a group. In all β-lactamases, there is one main active site component;
this can either be a serine residue that provides the catalytic basis for the hydrolysis of penicillins and cephalosporins (Classes A, C, and D) or a metal ion that provides the catalytic basis for hydrolysis, particu- larly for the carbapenems (Class B).
The class A enzymes have been studied in most detail. They com- prise the chromosomal β-lactamases of Gram-positive bacteria and the most common plasmid-encoded β-lactamases. In any study of plasmid-encoded, β-lactamase-conferred resistance performed any- where in the world, at least 75% of all the enzymes will be the class A β-lactamase TEM-1. This enzyme is highly efficient at binding and hydrolizing amoxicillin conferring high-level resistance (MIC
>1,000mg/L). β-lactamase inhibitors were developed specifically to overcome the effects of this enzyme. However, the β-lactamase has been able to mutate to prevent the binding of the inhibitor. The cephalosporins were also exploited to overcome the effect of the TEM-1 β-lactamase. Unfortunately, the TEM molecule has been able to mutate to become an extended-spectrum β-lactamase (ESBL), so that it can bind and hydrolyse the most sophisticated cephalosporins.
There are now about 150 of these ESBL enzymes.
CHAPTER 6Mechanisms of antibiotic resistance
53 The SHV ESBLs, derived from an enzyme closely related to TEM-1,
SHV-1, soon replaced the TEM ESBLs. This is probably because the former were more effective against slow-penetrating cephalosporins, such as ceftazidime, and the SHV enzymes are more effective against the faster penetrating cephalosporins such as ceftriaxone and cefo- taxime. There are about 80 SHV ESBLs. The SHV-2 and, to some extent, SHV-5 ESBLs were very prevalent but now are beginning to be replaced by another group of class A ESBLs, the CTX-M group.
Unlike the previous two, these ESBLs did not derive from mutation of an established plasmid β-lactamase gene but were imported from various species of the Kluyvera genus. There are nearly 100 of these and in the United Kingdom, the most prevalent is CTX-M-15.
The class B enzymes are metallo-β-lactamases. In vitro, they are particularly active against the carbapenems, such as imipenem and meropenem. Although they are usually encoded by the bacterial chromosome, they have to be induced to produce sufficient enzyme levels to confer resistance. Even elevated levels may be insufficient to confer resistance and this type of β-lactamase has to operate in concert with another resistance mechanism, such as reduced perme- ability. An increasing number of the class B β-lactamases have been found to be plasmid-mediated, particularly the IMP and VIM groups, and these are not inducible but are constitutively produced.
The class C β-lactamases are predominantly the chromosomally- encoded β-lactamases of Gram-negative rods. The production of these β-lactamases also has to be induced to produce sufficient enzyme (Fig 6.3). Induction is not a very efficient long-term mecha- nism and the host bacteria are more successful if the repression system is disabled completely. Thus de-repression occurs with a mutation in the repressor gene so that no repressor protein is pro- duced. This is a stable change and can only be reversed with a back mutation.
There are a few class C β-lactamase genes that have migrated onto plasmids, that is, BIL-1 and other members of the CMY-2 group.
When this occurs, only the β-lactamase gene is present, there is no repression system so the gene is expressed constitutively. These are known as ampC β-lactamases. They are difficult to detect because they are usually present in strains that have their own, similar but less effective, class C β-lactamase.
The class D β-lactamases were originally found exclusively plasmid- encoded and predominantly acted against penicillins. However, they have been found to be the part of the chromosomal β-lactamase complement of Acinetobacter spp. The OXA-51-like β-lactamases are the chromosomal enzymes of Acinetobacter baumannii. In Acinetobac- ter spp the class D enzymes can confer resistance to carbapenems (see chapter 7).
54
CHAPTER 6Mechanisms of antibiotic resistance Figure 6.3 Chromosomal class C BBBB-lactamase production in
Gram-negative bacteria
Repressor gene Chromosome
b-lactamase
Repressor
Induction
Interference with repressor protein De-Repression
Mutation in repressor gene
Promotor gene