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Antibiotic resistance
ANTIBIOTICS
and depends on the ease with which a resistance mechanism can arise. Novel resistance is usually detected in different places in the world at about the same time, implying that antibiotic pressures are similar worldwide and that bacteria have limited means of dealing with the problem of survival. Following the appearance of one mutant resistant progeny, rapidly replicating bacteria can recolonize carrier sites in less than 24 hours. A resistant mutant of Mycobacterium tuberculosis, which replicates once every 24 hours, can recolonize diseased lung within 2 weeks. Once resistance has been selected, organisms transferred from person to person continue to be resistant. When the antibiotic pressure is removed, novel colonizing and infecting flora tend to revert to the sensitive phenotype. Some bacteria containing large resistance plasmids are considered unfit and seem to disappear from the clinical environment more easily than others.
Antibiotic resistance Geoff Scott
In an environment containing vast numbers of micro-organisms saturated with or repeatedly exposed to antibiotics, Darwinian theory predicts the inevitable selection of resistant organisms. Following the introduction of a new antibiotic, resistant strains may be reported within as little as 1 year, and after a latent interval of some years, resistance increases dramatically and the value of the antibiotic for empirical therapy is reduced. Staphylococcus aureus is now almost universally resistant to penicillin – a phenomenon first observed in hospital outbreaks of surgical sepsis in the late 1940s. This organism has shown a remarkable facility to become resistant to every antibiotic introduced, even vancomycin. Reduced sensitivity to vancomycin (vancomycin-intermediate S. aureus) is associated with a thick peptidoglycan cell wall. The complex gene cassette for vancomycin resistance in Enterococcus spp. (vanA) can easily be transferred to S. aureus in vitro and has now been detected in methicillinresistant S. aureus (MRSA), making the organism fully resistant to glycopeptides. It is only a matter of time before strains are seen that are sensitive to only a small number of novel antibiotics in development and perhaps some of the older antibiotics (e.g. tetracycline, chloramphenicol). In contrast, some organisms have not yet acquired resistance despite huge pressures; these include Streptococcus pyogenes, Gram-positive anaerobes such as Clostridium spp. and Peptostreptococcus spp. and Neisseria meningitidis (to penicillin). Almost all anaerobes were considered to be sensitive to metronidazole until recent reports from Spain, France and the USA suggested that resistant Bacteroides fragilis will soon become a serious problem.
Resistance and choice of antibiotics The term ‘antibiotic resistance’ implies that a particular antibiotic is ineffective in a clinical infection. This may be because the organism is inherently resistant to the antibiotic or because it is inaccessible. In vitro, resistance is defined by measuring the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the antibiotic against an organism, under ideal laboratory conditions, using appropriate controls to define the cut-off points between ‘resistant’, ‘intermediate’ and ‘sensitive’. Methods of testing in the laboratory are problematic, however, and any result that does not correlate with clinical experience should be challenged. Low MIC and high MBC imply that an antibiotic is bacteristatic; that is, it inhibits the growth of an organism but is unable to kill it. Even with bactericidal antibiotics, killing in vivo normally requires an intact immune system. Thus, though bacteristatic antibiotics are ineffective in neutropenic sepsis, bactericidal antibiotics may suppress infection only for it to recrudesce when the antibiotic is removed. This observation governs strategies for the management of such infections. Paradoxically, even when an organism is declared resistant to an antibiotic by in vitro tests, it may appear to be effective in clinical use; this may be because MIC/MBC can be exceeded in vivo by giving a sufficiently large dose. This is often seen in lower urinary tract infection and in the treatment of, for example, non-meningeal penicillin-resistant pneumococcal infection with high-dose penicillin.
Ecology Resistance is driven by antibiotic use in human and agricultural/ veterinary practice. Resistance selection occurs by spontaneous mutations occuring at a rate of 10–9–10–6 driven by the presence of antimicrobials. Resistance elements appear in saprophytic bacteria as a result of antibiotic use for growth promotion and protecting crops. When these are eaten, resistance may be transferred to human bacteria that are occasionally pathogenic. The likelihood of finding resistant organisms in the gut is related to the tonnage of antibiotic use in the country in which the individual resides,
Empirical treatment: management of an infection before bacteriology results are available depends on making the correct diagnosis and assessing the antibiotic sensitivities of the suspected organism or organisms from local epidemiological knowledge. Once culture results are known, it is usually another day before in vitro antibiotic sensitivities are available. This is a critical period in the management of an infected patient and explains why, in severe infections, broad-spectrum antibiotics are often chosen initially when the definitive diagnosis is uncertain − and why withholding treatment may be life-threatening. This period is generally more protracted when an organism such as M. tuberculosis is slow to grow in vitro. Delay in the treatment of tuberculous meningitis may lead to permanent neurological damage. Whether the correct antibiotics were chosen initially is known only weeks later.
Geoff Scottt is Consultant in Clinical Microbiology at University College London Hospitals, London, UK. He qualified from the Royal Free Hospital, London, and trained at Northwick Park Hospital, London and University College Hospital. His interests include control of tuberculosis and resistant infections, and changing habits. MEDICINE 33:3
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© 2005 The Medicine Publishing Company Ltd
ANTIBIOTICS
passed from one bacterium to another (even to bacteria of different species) by various mechanisms such as direct transfer by type II pili (Figure 1) and phage transfer. Plasmids reproduce each time the organism divides and can probably be lost as easily as gained, given the correct environment. Transposons are large genetic elements often containing multiple genes necessary to confer phenotypic resistance. They may encode pheromones, which attract bacteria to each other and can be transferred on plasmids, generally being incorporated into the chromosomal DNA of the new host. Free DNA from dead bacteria and even mammalian cells can be incorporated into bacterial chromosomes. A remarkable mechanism of resistance is seen in penicillin-resistant Streptococcus pneumoniae, which have incorporated a mosaic of genes into the chromosome from other resistant α-haemolytic streptococci.
Mechanisms of resistance Resistance may be inherent (e.g. vancomycin against Gramnegative organisms, nitrofurantoin against Proteus spp.) or acquired via genetic elements encoding three fundamental mechanisms: • production of inactivating enzymes • change in the target site • exclusion of the antibiotic from the target site. The latter may occur by restriction of access through porins (only in Gram-negatives) or by active excretion. Antibiotic-inactivating enzymes may be produced in vast excess, surrounding the organism (e.g. β-lactamase from S. aureus), or in limited amounts in the periplasmic space of Gram-negatives. The effect in vivo is similar, but the latter organisms may appear sensitive in vitro. Classically, this is seen in Enterobacterr spp. resistant to extended-spectrum β-lactam antibiotics such as piperacillin and cefotaxime; exposure of the organism to inducers such as penicillin, clavulanate, certain cephalosporins and imipenem may ‘switch on’ the production of chromosomal (AmpC) β-lactamases. The classical TEM (Escherichia coli) and SHV (Klebsiella a spp.) βlactamases (which inactivate ampicillin) have shown a remarkable ability to mutate to extended-spectrum forms (which inactivate cefoxitin and cefotetan) and to inhibitor resistance (not inhibited by clavulanate or tazobactam). CTX-M cefotaximase arose by escape from the chromosome of Kluyvera a spp. and is now widespread in Enterobacteriaceae including E. coli. Other classical inactivating enzymes include chloramphenicol acetyltransferase and aminoglycoside-modifying enzymes.
Problematic resistant bacteria Gram-positive organisms MRSA has replaced methicillin-sensitive S. aureus as the more common cause of hospital-associated sepsis in most parts of the world. The targets of the β-lactam antibiotics are penicillin-binding proteins, carboxypeptidases and transpeptidases, which catalyse bridging of pentapeptide subunits of peptidoglycan. All strains of MRSA already produce penicillinase, but they now have a new penicillin-binding protein (PBP2') that is not inhibited by methicillin and its congeners oxacillin and flucloxacillin. They are resistant to all β-lactam antibiotics. The mecA gene encodes PBP2', but requires expression of several ancillary genes within a transposon for full expression. Thus, some strains with mecA may appear to be sensitive to methicillin in vitro. In addition, a plasmid encodes resistance to a variable number of other antibiotics. Strains with a tendency to spread easily and to predominate in hospitals are termed ‘epidemic MRSA’ (EMRSA). The current epidemic of EMRSA15 or EMRSA16 in the UK started in 1994. In the laboratory, these are detected by their antibiogram, but other typing methods (e.g. pulsed-field gel electrophoresis of chromosomal DNA) are needed to show that two strains are indistinguishable, thus implying that cross-infection has occurred. When a few patients in one or two wards acquire a novel strain, such an outbreak can easily be tracked and then controlled. When EMRSA strains become endemic (Figure 2), more general measures are needed to reduce the risk to patients admitted to the hospital. MRSA may become endemic in nursing homes, creating a pool for novel introduction into hospital; transfer of patients from ward to ward and colonization of staff leads to continued exposure of patients to new strains and helps maintain endemicity. Infection with MRSA is not untreatable. Strains are often (though not predictably) susceptible to gentamicin or other aminoglycosides, rifampicin, co-trimoxazole, chloramphenicol or ciprofloxacin, and sometimes to tetracyclines, macrolides, fusidic acid and pseudomonic acid. (Note that rifampicin and fusidic acid should never be used alone because resistant mutants are selected very rapidly.) MRSA is almost always susceptible to glycopeptides, though strains with reduced sensitivity to vancomycin have been occasionally described in patients with chronic colonization or infection who have been treated for several weeks.1 Some rare strains of S. aureus are dependent on vancomycin to allow them to grow.
Target site change may be a structural alteration preventing binding of an antibiotic, or a mechanism whereby the metabolic pathway that is normally inhibited is bypassed by an alternative one. This is seen in sulphonamide resistance and in MRSA, which has acquired a novel penicillin-binding protein from Staphylococcus scuiri. Important target site changes include topoisomerases II and IV (quinolones), β subunit of DNA-dependent RNA polymerase (rifamycins) and methylation of 23S target (14/15-membered macrolides). Exclusion of antibiotic: porins are protein structures embedded in the outer bilipid membrane of Gram-negative organisms. They control what passes into and out of the cell on the basis of molecular size and charge. Mutations in the genetic elements encoding porins (permeability mutations) may exclude one antibiotic or, more commonly, multiple antibiotics. Pseudomonas aeruginosa a has two outer lipid membranes, produces β-lactamases constitutively, and therefore tends to be more resistant than coliforms. Alternatively, organisms may actively excrete antibiotics, notably tetracyclines, macrolides and quinolones. Efflux pumps may be up-regulated. S. aureus resistant to erythromycin by active excretion remains sensitive to clindamycin, but the more common target site mutation affects susceptibility to both antibiotics.
Acquisition of resistance Direct mutation of chromosomal genes may lead to resistance, and non-fatal mutations are passed to all progeny. Alternatively, small, mobile, circular pieces of DNA termed ‘plasmids’ may be MEDICINE 33:3
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ANTIBIOTICS
Penicillin-resistant S. pneumoniaee strains have shown a gradual phase-shift increase in MIC to penicillin over several years. MIC is about 0.001 mg/litre in sensitive strains and 1 mg/litre in resistant strains. This is achieved by changes in penicillin-binding proteins with lower affinities for β-lactams. In the UK, such strains currently represent about 4% of those causing invasive infection, but in some areas (e.g. Spain) the rate is as high as 50%. These strains are usually resistant to many other useful antibiotics, including cephalosporins, chloramphenicol and erythromycin. Treatment with high-dose penicillin is effective in pneumonia but not in meningitis caused by resistant strains, some of which are sensitive to second-generation and third-generation cephalosporins. The mortality from severe pneumococcal pneumonia remains relatively constant regardless of whether the strain is resistant to penicillin, but meningitis caused by a resistant strain is more likely to be fatal. Splenectomized patients are advised to take life-long low-dose oral penicillin prophylaxis against the rare possibility of overwhelming pneumococcal sepsis. In the future, such prophylaxis may become less effective, but it is difficult to identify an alternative simple and safe regimen.
vanA encodes a structural change in the terminal amino acid of the pentapeptide chain of peptidoglycan, from D-ala to D-lac. This substitution prevents binding of vancomycin, enabling construction of the dipeptide bridges in peptidoglycan to continue. Typing indicates wide heterogeneity of strains, and that carriers usually harbour more than one strain. This implies that the transposon encoding vanA and the accessory genes is promiscuous and easily able to enter the host’s enterococci. These organisms are of low virulence. Infections may respond to simple antibiotics such as amoxicillin if they appear sensitive (Enterococcus faecalis), but are generally resistant to all but a few new antibiotics. Colonization with enterococci is driven by cephalosporins and fluoroquinolones, to which enterococci are constitutively resistant.
Glycopeptide-resistant enterococci: enterococci have become important causes of nosocomial postoperative infection in the USA. They are likely to cause sepsis and pneumonia only in severely ill patients in the UK, and occasionally strains are resistant to vancomycin with (vanA) or without (vanB) teicoplanin. These genes require the action of complex accessory genes for full expression.
Glucose fermenters: in terms of resistance and endemic/epidemic problems in ill, hospitalized patients, Klebsiella, Enterobacterr and Serratia a spp. are more troublesome than E. coli. They produce SHV-type β-lactamases constitutively, and mutations lead to resistance to all β-lactams other than the carbapenems (or occasionally aztreonam). They often lose susceptibility to quinolones and
a
Gram-negative organisms In contrast to resistant Gram-positives, against which old antibiotics or antibiotics in development are occasionally active, some Gram-negative organisms (particularly non-glucose fermenters such as Pseudomonas, Stenotrophomonas and Acinetobacterr spp.), especially in ICUs, are resistant to every useful antibiotic and there are no new agents in development.
b
c
d
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1 DNA can be transferred a on direct contact, b, c via type II pili or d by phages (bacterial viruses). (b and c by courtesy of Zeneca.)
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ANTIBIOTICS
aminoglycosides and become essentially untreatable. Some strains of Salmonella enterica a (particularly typhimurium), and the enteric salmonellae (e.g. S. typhi) have acquired stable broad resistance to all useful antibiotics. These are now pandemic.
one clone resistant to isoniazid that started in 1995 and has led to a resistance rate of 15% in affected areas), and to rifampicin, ethambutol and pyrazinamide in 1% or fewer. Multi-drug-resistant strains account for 1.2%. Strains resistant to all are found throughout the country, but are more commonly seen in London. Most patients can be treated satisfactorily with a standard 6-month regimen, but the results of routine antibiotic sensitivity tests are unknown for 5−16 weeks after sending specimens to the laboratory, and patients with resistant strains need more toxic second-line drugs and prolonged courses of treatment. Rapid liquid culture and detection of genes encoding resistance may improve the speed of laboratory diagnosis.
Non-glucose fermenters: P. aeruginosa has been replaced as a troublesome cause of nosocomial infection by other environmental and skin bacteria such as Acinetobacter baumanii var. calcoaceticus. Some strains are resistant to all available antibiotics, others are sensitive only to carbapenems and some aminoglycosides (e.g. amikacin). In response to long-term antibiotic use over many years, Burkholderia cepacia a tends to replace S. aureus and P. aeruginosa a as colonizing flora in patients with cystic fibrosis. It may also show resistance to many antibiotics and can cause troublesome cross-infection.
Strategies for reducing the impact of resistance Antibiotic resistance is driven by antibiotic use. When antibiotics are superseded and therefore used less, strains resistant to these tend to disappear.
Neisseria spp.: N. gonorrhoeae is interesting in that different strains may exhibit the three mechanisms of resistance to penicillin (reduced permeability, changes in penicillin-binding proteins and β-lactamase production). Each confers a stepwise increase in MIC, and there is no clear cut-off between sensitive and resistant strains. Many strains have become resistant to other oral drugs such as ciprofloxacin, though these are currently rare in the UK. N. meningitidis has been slow to acquire resistance to penicillin, though a few strains isolated recently in Spain have slightly higher MICs than predicted. The value of sulphonamides was largely lost by the 1970s, and though chloramphenicol remains useful, secondgeneration cephalosporins (cefotaxime or ceftriaxone) seem to give the best results in clinical treatment.
In the community: in the UK, more than 80% of human use of antibiotics occurs in the community, mostly for respiratory tract infections. The Standing Medical Advisory Committee, in its report The path of least resistance, made recommendations to reduce inappropriate prescribing.2 • No antibiotics should be given for simple coughs and colds. • Antibiotics should not be routinely prescribed for sore throats, unless there is evidence of streptococcal infection. • Antibiotics are not routinely required for acute otitis media and sinusitis-like symptoms; if given, courses can be limited to 3 days. In addition, 3 days’ treatment should suffice in otherwise healthy women with uncomplicated cystitis. This strategy has been useful in reducing prescribing by GPs, but anxiety has resulted from anecdotal reports of an increase in bacterial respiratory infections.
M. tuberculosis: resistance to first-line antituberculosis agents has been a major problem in the re-emergence of tuberculosis. In some countries (e.g. the states of the former USSR), multi-drugresistant strains (resistant at least to rifampicin and isoniazid) are extremely common, and more so in patients who have been treated previously. In the UK, lone resistance to isoniazid occurs in 5% (except in London, where there is currently an epidemic of
In hospital, antibiotic use can be reduced by several means.
New patients with methicillin-resistant Staphylococcus aureus at University College London Hospitals, 1991–1999
Annual incidence
450 400
Infections
350
Carriers
300 250 200 150 100 50 0
1991
1992
1993
1994
1995
1996
1997
1998
1999
Year 2 MEDICINE 33:3
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Glycopeptide
Sites of action of antibiotics and some resistance mechanisms Porin Glycopeptide too large
Aminoglycosides β-lactams Mutation in porin
Gram- Gram-positive negative Peptidoglycan
Aminoglycosides Macrolides Chloramphenicol
Substrate change
Supercoiled DNA Sulphonamides Trimethoprim
Quinolones
RNA DNA
Periplasmic space (β-lactamase activity)
Folate synthesis
Penicillin-binding proteins
T Topoisomerases
Mutation
Inactivating enzymes β-lactams Active excretion β-lactamases Chloramphenicol acteyltransferase Aminoglycoside-modifying f enzymes
• Routine use of antibiotics for surgical prophylaxis should be reduced to a minimum. • Antibiotics should not be started immediately in all suspected infections. Certain patients (e.g. those with febrile neutropenia or evidence of septicaemia) require urgent antibiotic therapy, but in many other cases (e.g. mild pyrexia postoperatively), it is safe and ultimately preferable to withhold antibiotics until culture results are known or there is clear evidence of bacterial infection. • An alternative is to discontinue antibiotics as soon as information is available suggesting that the problem is not bacterial or has resolved itself. This is termed an ‘antibiotic-stop’ policy, and the aim is to encourage doctors to actively review the need for antibiotics after, say, the second day, given information on cultures and surrogate markers that has by then become available. • Certain antibiotics can be withheld from the hospital formulary. This is the main benefit of an agreed antibiotic policy. The choice of restricted antibiotics may be decided on the basis of cost rather than likely selection of resistance. (These antibiotics must be used occasionally, however, when resistance to all other available drugs has been selected.) • In theory, antibiotics can be rotated such that, for example, predominantly penicillins are used at some times, and cephalosporins or quinolones at others. There is little clear scientific evidence that this has any effect, and major, complicated studies would be needed to determine the effect of change in use on both normal and infecting flora. However, it has been shown that, in a setting of heavy cephalosporin use, discontinuation of use of this class of drugs leads to a reduction in the risk of colonization with glycopeptide-resistant enterococci and Clostridium difficileassociated diarrhoea.
The future Resistance to antibiotics is one of the greatest threats to the success of modern medicine. It has recently become more serious because we can no longer be sure that any antibiotic chosen empirically will work, and because of the emergence of totally resistant bacteria. How to reduce resistance without simply discontinuing use of all antibiotics is a dilemma. We do not know to what degree antibiotic use must be reduced to decrease the selective pressure and allow reversion to sensitivity, nor do we know how to protect the few remaining drugs that can be used to treat resistant infections. Doctors and vets should overcome the view that they have an inalienable right to prescribe empirical antibiotics, and should set targets for reduction of their personal prescribing.
REFERENCES 1 Hiramatsu K, Aritaka N, Hanaki H et al. Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancett 1997; 350: 1670–3. 2 Standing Medical Advisory Committee, Department of Health. The path of least resistance. London: HMSO, 1998. FURTHER READING Baquero F, Blázquez J. Evolution of antibiotic resistance. Trends Ecol Evol 1997; 12: 482–7. Butler C C, Rollnick S, Kinnersley P et al. Reducing antibiotics for respiratory tract symptoms in primary care: consolidating ‘why’ and considering ‘how’. Br J Gen Pracc 1998; 48: 1865–70. Jacobs M R, Felmingham D, Appelbaum P C et al. The Alexander Project 1998–2000. J Antimicrob Chemotherr 2003; 52: 229–46. Lipsitch M, Bergstrom C T, Levin B R. The epidemiology of antibiotic resistance in hospitals: paradoxes and prescriptions. Proc Natl Acad Sci U S A 2000, 97: 1938–43.
Other strategies: development of resistance in human pathogens might be delayed if antibiotics were not used so widely in animal husbandry, particularly for growth promotion. MEDICINE 33:3
Macrolides Quinolones T Tetracyclines
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and depends on the ease with which a resistance mechanism can arise. Novel resistance is usually detected in different places in the world at about the same time, implying that antibiotic pressures are similar worldwide and that bacteria have limited means of dealing with the problem of survival. Following the appearance of one mutant resistant progeny, rapidly replicating bacteria can recolonize carrier sites in less than 24 hours. A resistant mutant of Mycobacterium tuberculosis, which replicates once every 24 hours, can recolonize diseased lung within 2 weeks. Once resistance has been selected, organisms transferred from person to person continue to be resistant. When the antibiotic pressure is removed, novel colonizing and infecting flora tend to revert to the sensitive phenotype. Some bacteria containing large resistance plasmids are considered unfit and seem to disappear from the clinical environment more easily than others.
Antibiotic resistance Geoff Scott
In an environment containing vast numbers of micro-organisms saturated with or repeatedly exposed to antibiotics, Darwinian theory predicts the inevitable selection of resistant organisms. Following the introduction of a new antibiotic, resistant strains may be reported within as little as 1 year, and after a latent interval of some years, resistance increases dramatically and the value of the antibiotic for empirical therapy is reduced. Staphylococcus aureus is now almost universally resistant to penicillin – a phenomenon first observed in hospital outbreaks of surgical sepsis in the late 1940s. This organism has shown a remarkable facility to become resistant to every antibiotic introduced, even vancomycin. Reduced sensitivity to vancomycin (vancomycin-intermediate S. aureus) is associated with a thick peptidoglycan cell wall. The complex gene cassette for vancomycin resistance in Enterococcus spp. (vanA) can easily be transferred to S. aureus in vitro and has now been detected in methicillinresistant S. aureus (MRSA), making the organism fully resistant to glycopeptides. It is only a matter of time before strains are seen that are sensitive to only a small number of novel antibiotics in development and perhaps some of the older antibiotics (e.g. tetracycline, chloramphenicol). In contrast, some organisms have not yet acquired resistance despite huge pressures; these include Streptococcus pyogenes, Gram-positive anaerobes such as Clostridium spp. and Peptostreptococcus spp. and Neisseria meningitidis (to penicillin). Almost all anaerobes were considered to be sensitive to metronidazole until recent reports from Spain, France and the USA suggested that resistant Bacteroides fragilis will soon become a serious problem.
Resistance and choice of antibiotics The term ‘antibiotic resistance’ implies that a particular antibiotic is ineffective in a clinical infection. This may be because the organism is inherently resistant to the antibiotic or because it is inaccessible. In vitro, resistance is defined by measuring the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the antibiotic against an organism, under ideal laboratory conditions, using appropriate controls to define the cut-off points between ‘resistant’, ‘intermediate’ and ‘sensitive’. Methods of testing in the laboratory are problematic, however, and any result that does not correlate with clinical experience should be challenged. Low MIC and high MBC imply that an antibiotic is bacteristatic; that is, it inhibits the growth of an organism but is unable to kill it. Even with bactericidal antibiotics, killing in vivo normally requires an intact immune system. Thus, though bacteristatic antibiotics are ineffective in neutropenic sepsis, bactericidal antibiotics may suppress infection only for it to recrudesce when the antibiotic is removed. This observation governs strategies for the management of such infections. Paradoxically, even when an organism is declared resistant to an antibiotic by in vitro tests, it may appear to be effective in clinical use; this may be because MIC/MBC can be exceeded in vivo by giving a sufficiently large dose. This is often seen in lower urinary tract infection and in the treatment of, for example, non-meningeal penicillin-resistant pneumococcal infection with high-dose penicillin.
Ecology Resistance is driven by antibiotic use in human and agricultural/ veterinary practice. Resistance selection occurs by spontaneous mutations occuring at a rate of 10–9–10–6 driven by the presence of antimicrobials. Resistance elements appear in saprophytic bacteria as a result of antibiotic use for growth promotion and protecting crops. When these are eaten, resistance may be transferred to human bacteria that are occasionally pathogenic. The likelihood of finding resistant organisms in the gut is related to the tonnage of antibiotic use in the country in which the individual resides,
Empirical treatment: management of an infection before bacteriology results are available depends on making the correct diagnosis and assessing the antibiotic sensitivities of the suspected organism or organisms from local epidemiological knowledge. Once culture results are known, it is usually another day before in vitro antibiotic sensitivities are available. This is a critical period in the management of an infected patient and explains why, in severe infections, broad-spectrum antibiotics are often chosen initially when the definitive diagnosis is uncertain − and why withholding treatment may be life-threatening. This period is generally more protracted when an organism such as M. tuberculosis is slow to grow in vitro. Delay in the treatment of tuberculous meningitis may lead to permanent neurological damage. Whether the correct antibiotics were chosen initially is known only weeks later.
Geoff Scottt is Consultant in Clinical Microbiology at University College London Hospitals, London, UK. He qualified from the Royal Free Hospital, London, and trained at Northwick Park Hospital, London and University College Hospital. His interests include control of tuberculosis and resistant infections, and changing habits. MEDICINE 33:3
47
© 2005 The Medicine Publishing Company Ltd
ANTIBIOTICS
passed from one bacterium to another (even to bacteria of different species) by various mechanisms such as direct transfer by type II pili (Figure 1) and phage transfer. Plasmids reproduce each time the organism divides and can probably be lost as easily as gained, given the correct environment. Transposons are large genetic elements often containing multiple genes necessary to confer phenotypic resistance. They may encode pheromones, which attract bacteria to each other and can be transferred on plasmids, generally being incorporated into the chromosomal DNA of the new host. Free DNA from dead bacteria and even mammalian cells can be incorporated into bacterial chromosomes. A remarkable mechanism of resistance is seen in penicillin-resistant Streptococcus pneumoniae, which have incorporated a mosaic of genes into the chromosome from other resistant α-haemolytic streptococci.
Mechanisms of resistance Resistance may be inherent (e.g. vancomycin against Gramnegative organisms, nitrofurantoin against Proteus spp.) or acquired via genetic elements encoding three fundamental mechanisms: • production of inactivating enzymes • change in the target site • exclusion of the antibiotic from the target site. The latter may occur by restriction of access through porins (only in Gram-negatives) or by active excretion. Antibiotic-inactivating enzymes may be produced in vast excess, surrounding the organism (e.g. β-lactamase from S. aureus), or in limited amounts in the periplasmic space of Gram-negatives. The effect in vivo is similar, but the latter organisms may appear sensitive in vitro. Classically, this is seen in Enterobacterr spp. resistant to extended-spectrum β-lactam antibiotics such as piperacillin and cefotaxime; exposure of the organism to inducers such as penicillin, clavulanate, certain cephalosporins and imipenem may ‘switch on’ the production of chromosomal (AmpC) β-lactamases. The classical TEM (Escherichia coli) and SHV (Klebsiella a spp.) βlactamases (which inactivate ampicillin) have shown a remarkable ability to mutate to extended-spectrum forms (which inactivate cefoxitin and cefotetan) and to inhibitor resistance (not inhibited by clavulanate or tazobactam). CTX-M cefotaximase arose by escape from the chromosome of Kluyvera a spp. and is now widespread in Enterobacteriaceae including E. coli. Other classical inactivating enzymes include chloramphenicol acetyltransferase and aminoglycoside-modifying enzymes.
Problematic resistant bacteria Gram-positive organisms MRSA has replaced methicillin-sensitive S. aureus as the more common cause of hospital-associated sepsis in most parts of the world. The targets of the β-lactam antibiotics are penicillin-binding proteins, carboxypeptidases and transpeptidases, which catalyse bridging of pentapeptide subunits of peptidoglycan. All strains of MRSA already produce penicillinase, but they now have a new penicillin-binding protein (PBP2') that is not inhibited by methicillin and its congeners oxacillin and flucloxacillin. They are resistant to all β-lactam antibiotics. The mecA gene encodes PBP2', but requires expression of several ancillary genes within a transposon for full expression. Thus, some strains with mecA may appear to be sensitive to methicillin in vitro. In addition, a plasmid encodes resistance to a variable number of other antibiotics. Strains with a tendency to spread easily and to predominate in hospitals are termed ‘epidemic MRSA’ (EMRSA). The current epidemic of EMRSA15 or EMRSA16 in the UK started in 1994. In the laboratory, these are detected by their antibiogram, but other typing methods (e.g. pulsed-field gel electrophoresis of chromosomal DNA) are needed to show that two strains are indistinguishable, thus implying that cross-infection has occurred. When a few patients in one or two wards acquire a novel strain, such an outbreak can easily be tracked and then controlled. When EMRSA strains become endemic (Figure 2), more general measures are needed to reduce the risk to patients admitted to the hospital. MRSA may become endemic in nursing homes, creating a pool for novel introduction into hospital; transfer of patients from ward to ward and colonization of staff leads to continued exposure of patients to new strains and helps maintain endemicity. Infection with MRSA is not untreatable. Strains are often (though not predictably) susceptible to gentamicin or other aminoglycosides, rifampicin, co-trimoxazole, chloramphenicol or ciprofloxacin, and sometimes to tetracyclines, macrolides, fusidic acid and pseudomonic acid. (Note that rifampicin and fusidic acid should never be used alone because resistant mutants are selected very rapidly.) MRSA is almost always susceptible to glycopeptides, though strains with reduced sensitivity to vancomycin have been occasionally described in patients with chronic colonization or infection who have been treated for several weeks.1 Some rare strains of S. aureus are dependent on vancomycin to allow them to grow.
Target site change may be a structural alteration preventing binding of an antibiotic, or a mechanism whereby the metabolic pathway that is normally inhibited is bypassed by an alternative one. This is seen in sulphonamide resistance and in MRSA, which has acquired a novel penicillin-binding protein from Staphylococcus scuiri. Important target site changes include topoisomerases II and IV (quinolones), β subunit of DNA-dependent RNA polymerase (rifamycins) and methylation of 23S target (14/15-membered macrolides). Exclusion of antibiotic: porins are protein structures embedded in the outer bilipid membrane of Gram-negative organisms. They control what passes into and out of the cell on the basis of molecular size and charge. Mutations in the genetic elements encoding porins (permeability mutations) may exclude one antibiotic or, more commonly, multiple antibiotics. Pseudomonas aeruginosa a has two outer lipid membranes, produces β-lactamases constitutively, and therefore tends to be more resistant than coliforms. Alternatively, organisms may actively excrete antibiotics, notably tetracyclines, macrolides and quinolones. Efflux pumps may be up-regulated. S. aureus resistant to erythromycin by active excretion remains sensitive to clindamycin, but the more common target site mutation affects susceptibility to both antibiotics.
Acquisition of resistance Direct mutation of chromosomal genes may lead to resistance, and non-fatal mutations are passed to all progeny. Alternatively, small, mobile, circular pieces of DNA termed ‘plasmids’ may be MEDICINE 33:3
48
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ANTIBIOTICS
Penicillin-resistant S. pneumoniaee strains have shown a gradual phase-shift increase in MIC to penicillin over several years. MIC is about 0.001 mg/litre in sensitive strains and 1 mg/litre in resistant strains. This is achieved by changes in penicillin-binding proteins with lower affinities for β-lactams. In the UK, such strains currently represent about 4% of those causing invasive infection, but in some areas (e.g. Spain) the rate is as high as 50%. These strains are usually resistant to many other useful antibiotics, including cephalosporins, chloramphenicol and erythromycin. Treatment with high-dose penicillin is effective in pneumonia but not in meningitis caused by resistant strains, some of which are sensitive to second-generation and third-generation cephalosporins. The mortality from severe pneumococcal pneumonia remains relatively constant regardless of whether the strain is resistant to penicillin, but meningitis caused by a resistant strain is more likely to be fatal. Splenectomized patients are advised to take life-long low-dose oral penicillin prophylaxis against the rare possibility of overwhelming pneumococcal sepsis. In the future, such prophylaxis may become less effective, but it is difficult to identify an alternative simple and safe regimen.
vanA encodes a structural change in the terminal amino acid of the pentapeptide chain of peptidoglycan, from D-ala to D-lac. This substitution prevents binding of vancomycin, enabling construction of the dipeptide bridges in peptidoglycan to continue. Typing indicates wide heterogeneity of strains, and that carriers usually harbour more than one strain. This implies that the transposon encoding vanA and the accessory genes is promiscuous and easily able to enter the host’s enterococci. These organisms are of low virulence. Infections may respond to simple antibiotics such as amoxicillin if they appear sensitive (Enterococcus faecalis), but are generally resistant to all but a few new antibiotics. Colonization with enterococci is driven by cephalosporins and fluoroquinolones, to which enterococci are constitutively resistant.
Glycopeptide-resistant enterococci: enterococci have become important causes of nosocomial postoperative infection in the USA. They are likely to cause sepsis and pneumonia only in severely ill patients in the UK, and occasionally strains are resistant to vancomycin with (vanA) or without (vanB) teicoplanin. These genes require the action of complex accessory genes for full expression.
Glucose fermenters: in terms of resistance and endemic/epidemic problems in ill, hospitalized patients, Klebsiella, Enterobacterr and Serratia a spp. are more troublesome than E. coli. They produce SHV-type β-lactamases constitutively, and mutations lead to resistance to all β-lactams other than the carbapenems (or occasionally aztreonam). They often lose susceptibility to quinolones and
a
Gram-negative organisms In contrast to resistant Gram-positives, against which old antibiotics or antibiotics in development are occasionally active, some Gram-negative organisms (particularly non-glucose fermenters such as Pseudomonas, Stenotrophomonas and Acinetobacterr spp.), especially in ICUs, are resistant to every useful antibiotic and there are no new agents in development.
b
c
d
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1 DNA can be transferred a on direct contact, b, c via type II pili or d by phages (bacterial viruses). (b and c by courtesy of Zeneca.)
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ANTIBIOTICS
aminoglycosides and become essentially untreatable. Some strains of Salmonella enterica a (particularly typhimurium), and the enteric salmonellae (e.g. S. typhi) have acquired stable broad resistance to all useful antibiotics. These are now pandemic.
one clone resistant to isoniazid that started in 1995 and has led to a resistance rate of 15% in affected areas), and to rifampicin, ethambutol and pyrazinamide in 1% or fewer. Multi-drug-resistant strains account for 1.2%. Strains resistant to all are found throughout the country, but are more commonly seen in London. Most patients can be treated satisfactorily with a standard 6-month regimen, but the results of routine antibiotic sensitivity tests are unknown for 5−16 weeks after sending specimens to the laboratory, and patients with resistant strains need more toxic second-line drugs and prolonged courses of treatment. Rapid liquid culture and detection of genes encoding resistance may improve the speed of laboratory diagnosis.
Non-glucose fermenters: P. aeruginosa has been replaced as a troublesome cause of nosocomial infection by other environmental and skin bacteria such as Acinetobacter baumanii var. calcoaceticus. Some strains are resistant to all available antibiotics, others are sensitive only to carbapenems and some aminoglycosides (e.g. amikacin). In response to long-term antibiotic use over many years, Burkholderia cepacia a tends to replace S. aureus and P. aeruginosa a as colonizing flora in patients with cystic fibrosis. It may also show resistance to many antibiotics and can cause troublesome cross-infection.
Strategies for reducing the impact of resistance Antibiotic resistance is driven by antibiotic use. When antibiotics are superseded and therefore used less, strains resistant to these tend to disappear.
Neisseria spp.: N. gonorrhoeae is interesting in that different strains may exhibit the three mechanisms of resistance to penicillin (reduced permeability, changes in penicillin-binding proteins and β-lactamase production). Each confers a stepwise increase in MIC, and there is no clear cut-off between sensitive and resistant strains. Many strains have become resistant to other oral drugs such as ciprofloxacin, though these are currently rare in the UK. N. meningitidis has been slow to acquire resistance to penicillin, though a few strains isolated recently in Spain have slightly higher MICs than predicted. The value of sulphonamides was largely lost by the 1970s, and though chloramphenicol remains useful, secondgeneration cephalosporins (cefotaxime or ceftriaxone) seem to give the best results in clinical treatment.
In the community: in the UK, more than 80% of human use of antibiotics occurs in the community, mostly for respiratory tract infections. The Standing Medical Advisory Committee, in its report The path of least resistance, made recommendations to reduce inappropriate prescribing.2 • No antibiotics should be given for simple coughs and colds. • Antibiotics should not be routinely prescribed for sore throats, unless there is evidence of streptococcal infection. • Antibiotics are not routinely required for acute otitis media and sinusitis-like symptoms; if given, courses can be limited to 3 days. In addition, 3 days’ treatment should suffice in otherwise healthy women with uncomplicated cystitis. This strategy has been useful in reducing prescribing by GPs, but anxiety has resulted from anecdotal reports of an increase in bacterial respiratory infections.
M. tuberculosis: resistance to first-line antituberculosis agents has been a major problem in the re-emergence of tuberculosis. In some countries (e.g. the states of the former USSR), multi-drugresistant strains (resistant at least to rifampicin and isoniazid) are extremely common, and more so in patients who have been treated previously. In the UK, lone resistance to isoniazid occurs in 5% (except in London, where there is currently an epidemic of
In hospital, antibiotic use can be reduced by several means.
New patients with methicillin-resistant Staphylococcus aureus at University College London Hospitals, 1991–1999
Annual incidence
450 400
Infections
350
Carriers
300 250 200 150 100 50 0
1991
1992
1993
1994
1995
1996
1997
1998
1999
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Glycopeptide
Sites of action of antibiotics and some resistance mechanisms Porin Glycopeptide too large
Aminoglycosides β-lactams Mutation in porin
Gram- Gram-positive negative Peptidoglycan
Aminoglycosides Macrolides Chloramphenicol
Substrate change
Supercoiled DNA Sulphonamides Trimethoprim
Quinolones
RNA DNA
Periplasmic space (β-lactamase activity)
Folate synthesis
Penicillin-binding proteins
T Topoisomerases
Mutation
Inactivating enzymes β-lactams Active excretion β-lactamases Chloramphenicol acteyltransferase Aminoglycoside-modifying f enzymes
• Routine use of antibiotics for surgical prophylaxis should be reduced to a minimum. • Antibiotics should not be started immediately in all suspected infections. Certain patients (e.g. those with febrile neutropenia or evidence of septicaemia) require urgent antibiotic therapy, but in many other cases (e.g. mild pyrexia postoperatively), it is safe and ultimately preferable to withhold antibiotics until culture results are known or there is clear evidence of bacterial infection. • An alternative is to discontinue antibiotics as soon as information is available suggesting that the problem is not bacterial or has resolved itself. This is termed an ‘antibiotic-stop’ policy, and the aim is to encourage doctors to actively review the need for antibiotics after, say, the second day, given information on cultures and surrogate markers that has by then become available. • Certain antibiotics can be withheld from the hospital formulary. This is the main benefit of an agreed antibiotic policy. The choice of restricted antibiotics may be decided on the basis of cost rather than likely selection of resistance. (These antibiotics must be used occasionally, however, when resistance to all other available drugs has been selected.) • In theory, antibiotics can be rotated such that, for example, predominantly penicillins are used at some times, and cephalosporins or quinolones at others. There is little clear scientific evidence that this has any effect, and major, complicated studies would be needed to determine the effect of change in use on both normal and infecting flora. However, it has been shown that, in a setting of heavy cephalosporin use, discontinuation of use of this class of drugs leads to a reduction in the risk of colonization with glycopeptide-resistant enterococci and Clostridium difficileassociated diarrhoea.
The future Resistance to antibiotics is one of the greatest threats to the success of modern medicine. It has recently become more serious because we can no longer be sure that any antibiotic chosen empirically will work, and because of the emergence of totally resistant bacteria. How to reduce resistance without simply discontinuing use of all antibiotics is a dilemma. We do not know to what degree antibiotic use must be reduced to decrease the selective pressure and allow reversion to sensitivity, nor do we know how to protect the few remaining drugs that can be used to treat resistant infections. Doctors and vets should overcome the view that they have an inalienable right to prescribe empirical antibiotics, and should set targets for reduction of their personal prescribing.
REFERENCES 1 Hiramatsu K, Aritaka N, Hanaki H et al. Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancett 1997; 350: 1670–3. 2 Standing Medical Advisory Committee, Department of Health. The path of least resistance. London: HMSO, 1998. FURTHER READING Baquero F, Blázquez J. Evolution of antibiotic resistance. Trends Ecol Evol 1997; 12: 482–7. Butler C C, Rollnick S, Kinnersley P et al. Reducing antibiotics for respiratory tract symptoms in primary care: consolidating ‘why’ and considering ‘how’. Br J Gen Pracc 1998; 48: 1865–70. Jacobs M R, Felmingham D, Appelbaum P C et al. The Alexander Project 1998–2000. J Antimicrob Chemotherr 2003; 52: 229–46. Lipsitch M, Bergstrom C T, Levin B R. The epidemiology of antibiotic resistance in hospitals: paradoxes and prescriptions. Proc Natl Acad Sci U S A 2000, 97: 1938–43.
Other strategies: development of resistance in human pathogens might be delayed if antibiotics were not used so widely in animal husbandry, particularly for growth promotion. MEDICINE 33:3
Macrolides Quinolones T Tetracyclines
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