Hugo W.B., Russel A.D.(ed). Pharmaceutical Microbiology

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though it is worth noting that more than one mechanism may be present at any one

time in a resistant microorganism. This is particularly relevant in terms of the clinical

efficacy of antibiotics. The acquisition of a single resistance mechanism may render a

bacterium microbiologically resistant but therapeutically achievable levels of a drug

may be sufficient to overcome such resistance. The acquisition of a second resistance

mechanism may be necessary to achieve clinical resistance, i.e. the amount of antibiotic

necessary to overcome the resistance mechanism is greater than can be achieved

therapeutically.

Before considering specific mechanisms of resistance for particular classes of

antibiotic it is worth considering potential mechanisms of resistance in bacterial cells.

These are summarized in Fig. 9.1 and specific examples are listed in Table 9.3.

Gram-negative bacteria possess an outer membrane which can act as a barrier

to the penetration of antibiotics. The main route of entry of hydrophilic molecules is

via the porins, which form pores in the outer membrane. Qualitative or quantitative

alterations in these porins can result in the decreased accumulation of antibiotic.

The cytoplasmic (cell) membrane of Gram-positive and Gram-negative bacteria

can also act as an exclusion barrier. Alterations in membrane structure can reduce

penetration or the presence of proteins functioning as efflux pumps can actively

remove antibiotic molecules from the cytoplasm. Bacteria may produce enzymes

which inactivate antibiotics, rendering them ineffective. These may destroy or alter

the antibiotic molecule. Extracellular enzymes will be most effective in inactivating

antibiotics since they will be kept away from their target sites. Gram-negative bacteria

can also produce periplasmic enzymes which will act outside the cytoplasmic membrane.

These mechanisms of resistance rely on reducing or preventing access of antibiotic

to their target sites, but other mechanisms of resistance involving the target sites

themselves can be considered. Alterations in the target site which reduce the binding of

intracellular

Fig. 9.1 Schematic representation of possible mechanisms of resistance in Gram-negative and

Gram-positive bacteria. 1, antibiotic-inactivating enzymes; 2, antibiotic efflux proteins; 3, alteration

or duplication of intracellular targets; 4, alteration of the cell membrane reducing antibiotic uptake;

5, alterations in porins or lipopolysaccharide reducing antibiotic uptake or binding.

Bacterial resistance to antibiotics 185

Table 9.3 Mechanisms of antibiotic resistance

* Depends on chemical nature of drug and on type of organism.

t Penicillin-binding proteins.

antibiotics, but allow the target to retain its normal function, are well known. An

alternative is to bypass the antibiotic-sensitive step by duplicating the target site with

an antibiotic-resistant version. A third related mechanism is to overproduce the target

so that higher antibiotic concentrations are required to exert significant antibacterial

action. In certain species, an enzyme or metabolic pathway may be absent, rendering

the microorganism resistant to antibiotics effective against other bacterial species.

The following sections describe the biochemical mechanisms of resistance to

different classes of antibiotics, with the antibiotics grouped according to their mechanism

of action.

3.1

Inhibitors of nucleic acid synthesis

Antibiotics considered here can be divided into two mechanisms of action: those which

186 Chapter 9

Expression of resistance

Enzymatic inactivation

Enzymatic trapping

Enzymatic modification

Bacterial impermeability*

Antibiotic efflux

Decreased affinity of target

enzymes

Alteration in binding site

Example(s)

Some ^-lactam antibiotics

Chloramphenicol

Some /3-lactam antibiotics

Some aminoglycoside

antibiotics

Some /3-lactam antibiotics

Aminoglycoside antibiotics

Tetracyclines,

chloramphenicol, fusidic acid

Hydrophobic antibiotics:

novobiocin, actinomycin D,

erythromycin, rifampicin

Tetracyclines

/^-Lactam antibiotics

Trimethoprim

Sulphonamides

Streptomycin

Erythromycin

Glycopeptides

Comments

Hydrolysis of the /3-lactam

ring

Conversion to an inactive

compound

Penicillin-binding proteins

Alteration of the molecule by

phosphorylation,

adenylylation or acetylation

Mutational loss of porins

Reduced ability of cells to

take up drugs

Plasmid-mediated decreased

drug accumulation

Difficulty in entering

Gram-negative cells

Energy-dependent efflux of

accumulated drugs

Altered PBPst

Altered dihydroflolate

reductase

Altered dihydropteroate

synthetase

Protein S12 component of

30S ribosomal subunit

determines sensitivity or

resistance

Ribosomes from resistant cells

have lower affinity, resulting

from enzymatic methylation

of adenine in 23S rRNA

Acquired ligase produces

altered peptidoglycan

precursors with lower affinity

inhibit nucleotide metabolism and those which inhibit enzymes involved in nucleic

acid synthesis.

3.1.1 Sulphonamides

Chromosomal and plasmid-mediated resistance to the sulphonamides has been described

(Huovinen et al. 1995).

Two mechanisms of chromosomal resistance have been identified. A mutation of

dihydropteroate synthetase (DHPS) in Strep, pneumoniae produces an altered enzyme

with reduced affinity for sulphonamides. Hyperproduction of p-aminobenzoic acid

(PABA) overcomes the block imposed by inhibition of DHPS. The specific cause of

PABA hyperproduction is unknown, though chromosomal mutation is the probable cause.

Duplication of DHPS, with the second version of the enzyme being resistant to the

sulphonamides, is the cause of plasmid-acquired resistance. Two different enzymes

have been identified, both with lowered affinity for the antibiotic.

3.1.2 Trimethoprim

Trimethoprim is a 2,4-diaminopyrimidine and all three genetic bases of resistance have

been described (Huovinen et al. 1995).

Chromosomal mutations in E. coli result in overproduction of dihydrofolate reductase

(DHFR). Higher concentrations of trimethoprim, which may not be therapeutically

achievable, are therefore required to inhibit nucleotide metabolism. Other mutations

lower the affinity of DHFR for trimethoprim. These two mechanisms of resistance

may coexist in a single strain, effectively increasing the level of resistance to the

antibiotic.

Plasmid- and transposon-mediated resistance is akin to that described for the

sulphonamides, where the sensitive step is bypassed by duplication of the target with a

resistant version. Many different resistant enzymes have been identified thus far.

3.1.3 Quinolones

The quinolones exert their action by binding to DNA gyrase (bacterial topoisomerase

II) and inhibiting its functions. Acquired resistance to the quinolones arises due to

chromosomal mutations in the genes coding for DNA gyrase (Hooper & Wolf son 1993).

The most common mutations arise in the gyrA gene where a single base-pair change

can be sufficient to cause resistance. Levels of resistance can be increased by the presence

of multiple mutations with a region of the gyrA gene known as the quinolone resistance-

determining region. The exact mechanism of resistance is unknown but is thought to

involve a subtle conformational change in DNA gyrase which reduces binding of

quinolones. Mutations in the gyrB gene have also been identified but these lead to

lower levels of resistance. With certain gyrB mutations, bacteria become resistant to

older quinolone analogues such as nalidixic acid, but become hypersusceptible to newer

quinolones such as ciprofloxacin. Mutations in other bacterial topoisomerases have

been identified in Staph, aureus. These are thought to be as important as DNA gyrase

mutations in quinolone resistance in this microorganism.

Bacterial resistance to antibiotics 187

Other chromosomal mutations resulting in quinolone resistance have been found

to decrease permeability of the antimicrobial agent. norB mutants show a decrease in

ompF porin. This is one of the major porins in Gram-negative bacteria. norC mutants

have altered ompF and lipopolysaccharide, though the mutations are not in the ompF

gene itself but appear to occur in a gene or genes whose products regulate OmpF. norC

mutants are less susceptible to some quinolones such as ciprofloxacin but more

susceptible to others.

Resistance to quinolones by efflux has been described in Staph, aureus and Proteus

mirabilis. This gene has been designated nor A in Staph, aureus and is homologous to

membrane transport proteins coupled to the electromotive force. These proteins have

the ability to remove small amounts of quinolone from cells normally and nor A may

have arisen as a result of mutations under selective pressure from quinolone use, resulting

in a transport protein with increased affinity for these agents.

3.1.4 Rifampicin

Rifampicin is the semisynthetic derivative used widely in the UK. Resistance to

rifampicin is primarily due to chromosomal mutations resulting in an altered RNA

polymerase which is less well inhibited by the drug. The mutations tend to be clustered

within short conserved regions of the j3 subunit gene of RNA polymerase. Similar

mutations have been found in all bacterial species studied thus far.

3.2 Inhibitors of protein synthesis

Inhibition of protein synthesis is the antibacterial mechanism shared by most groups of

antibiotics, though the exact action differs.

3.2.1 Aminoglycoside-aminocyclitol group

Three mechanisms of resistance to the aminoglycoside-aminocyclitol (AGAC) group

of antibiotics are recognized (Shaw et al. 1993).

Alteration of the antibiotic molecule is plasmid- or transposon-encoded. Three

classes of enzyme can alter the AGAC molecule. Aminoglycoside adenylyltransferases

(AADs) use adenosine triphosphate (ATP) as a cofactor in modifying certain hydroxyl

groups in the antibiotic molecule by adenylylating them (Fig. 9.2). Aminoglycoside

phosphotransferases (APH) also use ATP to modify certain hydroxyl groups by

phosphorylating them (Fig. 9.2). Aminoglycoside acetyltransferases (AACs) use acetyl

CoA as a cofactor and acetylate susceptible amino groups on the molecule (Fig. 9.2).

These three classes of enzyme have been further subdivided according to which site

on the AGAC molecule is modified. For example, APH(6) phosphorylates the 6-hydroxyl

group on the aminohexose group of streptomycin. Most AGAC antibiotics are susceptible

to more than one modification reaction. Relatively small amounts of the antibiotic are

modified, implying that resistance is determined by the relative rates of drug uptake

and modification. A less efficient modifying enzyme will permit unmodified antibiotic

to reach its ribosomal quantity. A more efficient enzyme, or greater quantities of the

enzyme, will result in resistance.

188 Chapter 9

Fig. 9.2 Modification of AGACs (e.g. kanamycin and amikacin) by resistance enzymes.

A, acetylation (AAC); B, adenylylation (AAD); C, phosphorylation (APH).

AGAC-modifying enzymes are active outside the cytoplasmic membrane, in the

periplasmic space in Gram-negative bacteria and extracellularly in Gram-positives.

Table 9.4 summarizes some of the enzymes involved in AGAC resistance and their

spectrum of activity.

A second mechanism of resistance to the AGACs involves an alteration of the

ribosomal target site. Mutations in the gene coding for ribosomal protein S12 (rpsL in

E. coli) prevent the antibiotics from binding to their target. In mycobacteria, which

possess only one ribosomal RNA operon, mutations in rpoB, coding for 16S rRNA,

also inhibit binding of the drugs.

Acquired resistance due to decreased permeability by mutations affecting membrane

transport have also been reported in other bacteria.

Bacterial resistance to antibiotics 189

Table 9.4 Examples of aminoglycoside-aminocyclitol susceptibility to modifying enzymes

3.2.2 Tetracyclines

Three types of resistance mechanism have also been identified with this class of antibiotic

(Chopra et al. 1992).

Plasmid- or transposon-encoded tetracycline efflux proteins have been described

in a number of bacteria. These efflux proteins are thought to span the cytoplasmic

membrane and are dependent on the proton-motive force for their action. It is thought

that the efflux proteins bind tetracyclines and initiate proton transfer, although no

functional domains have been identified. Eight distinct tetracycline efflux proteins have

been identified thus far.

Plasmid- or transposon-encoded ribosomal protection factors are a second mechan-

ism of resistance to the tetracyclines. These proteins are believed to alter the tetracycline

binding site on the 30S ribosomal subunit, lowering the affinity for the drugs.

OmpF mutations in Gram-negative bacteria such as E. coli (see above) can result

in low level resistance to the tetracyclines by reducing their uptake.

3.2.3 Chloramphenicol

Plasmid- or transposon-encoded chloramphenicol acetyltransferases (CATs) are respon-

sible for resistance by inactivating the antibiotic. CATs convert chloramphenicol to an

acetoxy derivative which fails to bind to the ribosomal target. Several CATs have been

characterized and found to differ in properties such as electrophoretic mobility and

catalytic activity.

Three other mechanisms of chloramphenicol resistance have been described. First,

a transposon-encoded chloramphenicol efflux protein has been identified in E. coli.

Second, some bacterial strains have been found to possess drug-resistant ribosomes,

and third, low level resistance can arise by chromosomal mutations which reduce the

amount of porins and therefore impair uptake. This last mechanism is essentially that

described for the AG AC antibiotics.

190 Chapter 9

Aminoglycoside

Streptomycin

Spectinomycin

Gentamicin

Kanamycin

Tobramycin

Neomycin

Amikacin

Inactivation by

APH(3"), APH(6), AAD(6),

AAD{3")(9)

AAD(3")(9), AAD(9)

APH{2"), AAD(2"), AAC(3),

AAC(2')

APH(3'), APH(2"), AAD<2"),

AAD(4')(4"), AAC(6')

APH(2"), AAD(4')(4"),

AAD(2"), AAC(3), AAC(2'),

AAC(6')

APH(3'), AAD(4')(4"),

AAC(3), AAC(2'), AAC(6')

AAD(4')(4"), AAC(6')

3.2.4

Macrolide, lincosamide and streptogramin (MLS) antibiotics

These three classes of antibiotics are often grouped together because of their similar

mode of action. They share a common mechanism of resistance, but there are some

mechanisms specific to each group (Leclerq & Courvalin 1991).

Plasmid- or transposon-mediated resistance common to the MLS group is due to

RNA methylase genes {ermA, ermB and ermC) which code for the methylation of an

adenine residue in 23 S rRNA. Methylation prevents the drugs from binding to the 50S

ribosomal subunit and confers resistance to all MLS antibiotics.

A gene designated msrA has been identified in Staph, aureus which confers resistance

to macrolides and streptogramins but not to lincosamides. Its function is unknown but

the DNA sequence is homologous to genes coding for known efflux proteins.

Chromosomal mutations in E. coli have been identified as causing macrolide

resistance. eryA alters protein L4 with a concomitant loss of binding to ribosomes.

eryB alters protein L22 with a loss of macrolide binding, though the mutation is not in

the structural gene for L22. eryC mutants are thought to alter the processing of rRNA

and a 30S ribosomal subunit protein, though the precise mechanism of resistance is

unclear. Macrolide resistance in mycobacteria is associated with point mutations in

23S rRNA. Plasmid-mediated inactivation of erythromycin (a 14-membered macrolide)

is common in Gram-negative bacteria and has also been described in some Gram-

positives. The lactone ring is hydrolysed by esterase in Gram-negatives, although

no similar enzymes have been identified in Gram-positives. Erythromycin can also

be phosphorylated, the altered molecule being rendered inactive. Plasmid-mediated

resistance to the lincosamides is common in staphylococci. An enzyme nucleotidylates

the antibiotics at a specific position rendering them inactive. Staphylococcal resistance

to streptogramins is due to inactivation of the antibiotics by plasmid-encoded enzymes.

Streptogramin A is inactivated by an acetyltransferase and streptogramin B by a

hydrolase.

3.2.5 Fusidic acid

Gram-negative bacteria are intrinsically resistant to low levels of fusidic acid (a

steroid) due to exclusion by the outer membrane. Nevertheless, acquired resistance

does occur which has the effect of increasing the level of resistance to the antibiotic.

Acquired resistance also occurs in Gram-positive bacteria normally susceptible to fusidic

acid.

Plasmid-mediated resistance in Gram-positive bacteria is thought to involve

decreased uptake of the drug, although the precise mechanism is unknown. Resistance

to fusidic acid in Gram-negative bacteria is also poorly understood. A CAT-type

enzyme has been identified in resistant strains but no modification or inactivation of

the antibiotic has been observed. It is believed that the CAT forms a tight stoichiometric

complex with the antibiotic, sequestering it and thus rendering it inactive (Davies

1994).

Chromosomal mutations have also been described which produce a modified

translocation factor protein with lowered affinity for fusidic acid.

Bacterial resistance to antibiotics 191

3.2.6 Mupirocin

3.3

3.3.1

Mupirocin is a topical antibiotic that inhibits isoleucyl tRNA synthetase with the

subsequent inhibition of protein synthesis. Mupirocin has become a mainstay in the

treatment of Staph, aureus infection and colonization during hospital outbreaks, and it

is in this organism that acquired resistance has arisen (Gilbart et al. 1993).

High level mupirocin resistance, where strains can be up to 1000 times more resistant

than susceptible strains, is due to the presence of an additional isoleucyl tRNA synthetase

which is resistant to the antibiotic. Resistance is plasmid-encoded, but the resistant

gene differs significantly from the normal susceptible version. The origins of high

level mupirocin resistance are unclear but the low homology with existing genes would

suggest that resistance is acquired from microorganisms other than Staph, aureus. High

level mupirocin resistance is an example of duplication of the target site, the new version

being resistant to the antibiotic. Low level mupirocin resistance results in strains

approximately 50 times more resistant to the antibiotic than susceptible strains. No

extra copies of the isoleucyl tRNA synthetase gene are found, indicating that resistance

has been acquired by mutations in the normal chromosomal gene. Presumably, mupirocin

has less affinity for the altered isoleucyl tRNA synthetase.

Inhibitors of peptidoglycan synthesis

^-Lactams

Acquired resistance to /Mactam antibiotics can occur by three different mechanisms:

inactivation of the antibiotic, alteration of the target site and reduced permeability

(Sanders 1992; Georgopapadakou 1993).

j8-Lactams are inactivated by enzymes called /^-lactamases which hydrolyse the

cyclic amide bond in the antibiotic molecule (Fig. 9.3). Penicillins are converted to

penicilloic acid which is unable to bind to penicillin-binding proteins (PBPs: see Chapter

8). A similar reaction occurs with cephalosporins, except that the cephalosporoic acid

derivative is unstable and tends to break up. A wide variety of/^-lactamases with different

structures and substrate profiles has been identified in recent years and classified

according to the scheme in Table 9.5. Many /3-lactamases are plasmid- or transposon-

Fig. 9.3 Hydrolysis of

/Mactams. Cephalosporoic acid

is unstable (see also Fig. 5.1

and sections 2.1 and 2.2 in

Chapter 5).

192 Chapter 9

Table 9.5 Examples of

b-lactamases

* Based on Bush et al. (1995).

t Plasmid-encoded /3-lactamases (TEM, PSE, OXA, SHV).

encoded but the Group 1 enzymes are mainly chromosomal. The origin of these is

unclear but they may have diverged from existing PBPs. Transfer of these chromosomal

enzymes by conjugation may be possible, but no evidence for this exists. Nevertheless,

some reports indicate that these Group 1 genes may be mobilized into plasmids and

then transferred to other bacteria. Some Group 1 enzymes are constitutive and expressed

at low levels, but in other species these enzymes are inducible by /^-lactams themselves.

Mutations in regulatory genes can lead to constitutive high levels of expression. Such

mutations are of clinical significance since they have led to the development of resistance

to newer /3-lactams previously thought to be resistant to ^-lactamases.

It is worth noting that in Gram-negative organisms, /3-lactamases are found in the

periplasmic space where they inactivate /^-lactams before the antibiotics can bind to

their PBP targets on the cytoplasmic membrane. In Gram-positive organisms, however,

^-lactamases are excreted extracellularly and therefore resistance is very much a

characteristic of the population rather than individual /3-lactamase-producing cells. If

enough enzyme is synthesized, levels of pMactam may be reduced sufficiently to permit

growth of non-pMactamase-producing strains.

Bacterial resistance to antibiotics 193

Group of

enzyme*

2be

2br

Preferred

substrate

Cephalosporin

Penicillins

Penicillins,

cephalosporins

Penicillins,

cephalosporins

monobactams

Penicillins

Penicillins,

carbenicillin

Penicillins,

cloxacillin

Cephalosporin

Penicillins,

cephalosporins,

carbapenems

Most /3-lactams,

including

carbapenems

Penicillins

Inhibited by:

Clavulanic acid

+/-

EDTA

Representative

enzymes

AmpCfrom Gram-negatives

Penicillinases from Gram-positives

TEM-1t,TEM-2, SHV-1

from Gram negatives

TEM-3 to TEM-26

TEM-30 to TEM-36

PSE-1, PSE-3, PSE-4

OXA-1

to OX A-11

Inducible cephalosporinases

from Proteus vulgaris

NMC-Afrom Enterobacter

cloacae, Sme-I from Serratia

marcescens

L1 from Xanthomonas

maltophilia, CcrA from

Bacteroides fragilis

Penicillinase from

Pseudomonas cepacia

A second mechanism of resistance involves alterations in PBPs which affect

binding of /3-lactams. These changes have been found to occur by multiple substitutions

through recombination rather than point mutations. Acquired penicillin resistance in

Strep, pneumoniae is because of such gene mosaics which code for an altered yet

functional PBP with reduced affinity for penicillin. Sections of the susceptible PBP

gene have been replaced by other DNA sequences, presumably via transformation.

Clinically, one of the most important examples of /3-lactam resistance is that found

in methicillin-resistant Staph, aureus (MRSA) strains. These are causing increasing

concern in hospitals, especially because methicillin resistance is often accompanied by

multiple resistance to unrelated antibiotics. Methicillin is resistant to /^-lactamases and

is a mainstay in the treatment of Staph, aureus since over 90% of hospital strains produce

/3-lactamase. Methicillin resistance is due to a novel PBP with low affinity for /3-lactams.

It is capable of functioning when all other PBPs have been inhibited and is sufficient to

catalyse all the reactions necessary for cell growth. Resistance is mediated by the mec

gene, whose origin is unknown. This is an example of resistance by duplication of an

antibiotic target, the new version being resistant to the antibiotic.

A third resistance mechanism is akin to that described for the AGAC antibiotics

and chloramphenicol, whereby changes in the outer membrane porins of Gram-negative

bacteria reduce the penetration of /3-lactams resulting in low levels of resistance.

3.3.2 Glycopeptides

Glycopeptide antibiotics interfere with peptidoglycan synthesis by binding to the D-

alanyl-D-alanine terminus of peptidoglycan precursors. Resistance to glycopeptides

was thought unlikely because the changes in integral structures and functions of the

cell wall and the enzymes involved in its synthesis would render bacteria non-viable.

As is often the case, bacteria have a nasty habit of surprising us!

Acquired resistance to the glycopeptides is transposon-mediated and has so far

been largely confined to the enterococci. This has been a problem clinically because

many of these strains have been resistant to all other antibiotics and were thus effectively

untreatable. Fortunately, the enterococci are not particularly pathogenic and infections

have been confined largely to seriously ill, long-term hospital patients. Two types of

acquired glycopeptide resistance have been described (Woodford et al. 1995). The

VanA phenotype is resistant to vancomycin and teicoplanin, whereas VanB is resistant

ORF1 ORF2 vanR vanS vanH vanA vanX vanY vanZ

IR IR

Transposase Response Dehydrogenase Dipeptidase unknown

regulator ,

lrw

,. „ „ . TcR?

Resolvase HPK Ligase D, D-carboxy-

peptidase

I I I I

Transposition Regulation Required for Accessory

glycopeptide proteins

resistance

Fig. 9.4 Organization of glycopeptide-resistance genes in transposon Tnl546. IR, invested repeats;

HPK, histidine protein kinase; TcR, low level teicoplanin resistance.

194 Chapter 9