JAC Advance Access originally published online on October 26, 2006
Journal of Antimicrobial Chemotherapy 2007 59(1):1-4; doi:10.1093/jac/dkl429
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Leading article |
The potential clinical impact of low-level antibiotic resistance in Staphylococcus aureus
Hospital Saint-Joseph 185 rue Raymond Losserand, 75014 Paris, France
*Tel: +33-1-44-12-36-50; Fax: +-33-1-44-12-36-85; E-mail: fgoldstein{at}hopital-saint-joseph.org
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Abstract |
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 Top Abstract IntroductionWhat is low-level resistance?...Detection of low-level...Clinical impactConclusionsTransparency declarationsReferences |
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Low-level antibiotic resistance in Staphylococcus aureus is a frequently overlooked phenomenon, usually not detected by standard susceptibility testing procedures. It represents a gateway to high-level clinically relevant resistance. Moreover, low-level resistance may be associated with increased virulence, resistance to unrelated compounds and more successful in vivo survival.
Keywords: borderline activity , staphylococci , antimicrobials
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Introduction |
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TopAbstractIntroductionWhat is low-level resistance?...Detection of low-level...Clinical impactConclusionsTransparency declarationsReferences |
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Staphylococcus aureus is one of the most common community- and hospital-acquired pathogens, responsible for a huge array of infections. Antibiotic resistance in S. aureus has become a serious problem in many parts of the world, emphasizing the need to better understand the mechanisms involved in the emergence and spread of resistant strains. Low-level resistance is often underestimated. It is not only a gateway to high-level clinical resistance, but also a gateway to often unsuspected phenomena such as resistance, to unrelated compounds, increased virulence or bacterial adaptation to adverse in vivo conditions. Low-level resistance usually emerges under antibiotic pressure but can also be selected by antiseptics and a variety of non-antimicrobial compounds.
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What is low-level resistance? Where does it start and where does it end? |
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TopAbstractIntroductionWhat is low-level resistance?...Detection of low-level...Clinical impactConclusionsTransparency declarationsReferences |
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There are many definitions established on clinical, pharmacokinetic or genetical backgrounds. The best definition which encompasses all the situations has been suggested by F. Baquero: a low-level resistant organism is an organism with an ‘MIC higher than is common for the susceptible population, devoid of any acquired resistance mechanism’.1 This definition is in full agreement with the cut-off values determined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST): low-level resistance starts at the upper limit of the cut-off values.2 It affects the relationship between the bacteria and an antimicrobial agent to a degree where clinical success is not guaranteed.
Low-level resistance is not linked to the ‘intermediate’ category; however, bacteria with low-level resistance may fall into this category with antibiotics with borderline activity such as ceftazidime or linezolid versus S. aureus.
Another more difficult question is: where does it end? In the absence of any valid definition, I would suggest that low-level resistance ends ... where high-level resistance starts!
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Detection of low-level resistance |
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TopAbstractIntroductionWhat is low-level resistance?...Detection of low-level...Clinical impactConclusionsTransparency declarationsReferences |
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If the cut-off values established on MIC distributions represent an excellent approach, there are situations where standard MICs are unable to detect a mechanism of resistance. In such situations, modified MIC procedures (inoculum size, media, incubation temperature or duration), population analysis profiles or bactericidal studies are sometimes necessary together with the requirement to test reference antibiotics that better detect some resistance mechanisms: lincomycin for clindamycin, teicoplanin for vancomycin [glycopeptide-intermediate S. aureus (GISA)], kanamycin for amikacin.3–5
For small-colony variants (SCV), tests should be carried out as far as possible on Mueller–Hinton agar as supplementation with the nutritional requirements are usually associated with a decrease in the MICs up to normal values and reverse mutants are readily isolated in liquid media.6
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Clinical impact |
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TopAbstractIntroductionWhat is low-level resistance?...Detection of low-level...Clinical impactConclusionsTransparency declarationsReferences |
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Pharmacokinetic/pharmacodynamic reasons
For many antibiotics with a natural borderline activity against some bacteria, a small increase in the MIC will result in a shift from susceptible to intermediate/resistant: this is obvious for antibiotics with a low therapeutic index such as vancomycin, where a small increase in the MIC, above the trough serum level, will readily explain most reported clinical failures.7,8
The very small increase in the MIC of vancomycin, from 1–2 to 2–4 mg/L, remains usually undetected by standard MIC procedures.8 However, as demonstrated by Hiramatsu, when performing a population analysis profile, a vancomycin concentration of 16–32 mg/L is required to inhibit a high density population of GISA, as encountered in vivo in contrast to 3–4 mg/L for fully susceptible strains. Such vancomycin concentrations are unlikely to be attained in most tissues.8
Similar failures have been reported for older compounds such as fusidic acid9 or new antibiotics such as linezolid.10–12
Clinical failures have also been reported for many antibiotics with a poor tissue penetration such as vancomycin or daptomycin, where a minimal increase in the MICs is responsible for a sub-optimal concentration at the site of infection.13–15
Bacteria-producing antibiotic-inactivating enzymes are often associated with clinical failures despite a relatively small increase in the MICs. There are many reports of ampicillin failure in the treatment of penicillinase-producing S. aureus or failure of chloramphenicol in strains producing a chloramphenicol acetyltransferase.
Presence of antibiotic resistance determinants responsible for decreased activity
Presence of a kanamycin resistance determinant encoding AAC (3') III.. Despite a very minor, if any, increase in the MIC of amikacin, the bactericidal and synergistic activities of amikacin (with ß-lactams or glycopeptides) disappear emphasizing the need for routine testing of kanamycin, not amikacin, against all staphylococci when treatment with amikacin is planned.3–5 In a rabbit endocarditis model with isogenic strains, the mean log cfu/g vegetation of S. aureus is significantly higher (8.4 versus 5.5) in the presence of the kanamycin determinant.16
Presence of an MLSB determinant.. The presence of an MLSB determinant, conferring high-level resistance to erythromycin and clindamycin, has only a minor impact on the MIC of quinupristin/dalfopristin or pristinamycin. However, the MBC for the strain harbouring the MLSB determinant will increase by at least 16-fold.
In the rabbit endocarditis model, the log cfu/g vegetation of S. aureus increases from 4.3 to 7.3.17
Increased adherence and virulence
Bacterial adherence is a prerequisite for any colonization or infection: in combination with extracellular factors such as toxins, it will determine the pathogenic capacity of the strain. Increased adherence is a result of the increased expression on the staphylococcal surface of proteins such as fibronectin-binding proteins that facilitate the attachment to fibronectin, fibrinogen or laminin, considered as the host receptors, or on foreign bodies such as catheters. The group of P. Vaudaux has demonstrated in several studies that GISA selected in vivo or in vitro display a more than 100% increase in their adherence to human receptors or foreign bodies. In two independent experiments, they have demonstrated the relationship between adherence and decreased glycopeptide susceptibility. In a first experiment, a GISA selected in vitro on teicoplanin displayed a 100–200% increase in adherence to fibronectin or fibrinogen.18 In a second experience, a fully glycopeptide-susceptible MRSA was introduced into polystyrene cages in a rabbit: after 2 weeks without any antibiotic, when compared by population analysis profiles on vancomycin or teicoplanin agar, the strain recovered from the animals had become a GISA;19 this could help explain why GISA have been recovered mainly in patients with foreign bodies such as catheters.14
The association between GISA and adherence can be explained by the role of several genes such as 
B. This gene is a two-component regulatory system of signal transduction inducible by a vast array of factors such as antibiotics (glycopeptides, ß-lactams, tetracyclines, fluoroquinolones), antiseptics, foreign bodies and a huge body of stress factors. Once induced, B up- or down-regulates more than 250 other genes: among the 198 genes up-regulated, there are genes determining increased MICs of glycopeptides or ß-lactams and increased production of biofilm and of fibronectin-binding proteins.20
Other virulence regulators and particularly the ica, agr and sarA operons are involved.21–24 Biofilms colonize host tissues and a wide variety of medical devices.24 The bacteria embedded in a biofilm are more resistant to phagocytosis and host immune responses and less exposed to antibiotics.24 Hence, the ‘in vivo’ MIC of some antibiotics may increase by a factor of 16x as shown for glycopeptides.25
Increased resistance to unrelated compounds
Very recently, it has been demonstrated that the MIC of daptomycin is higher for GISA than for susceptible strains despite a different mechanism of action or resistance.26,27 It has been attributed to the fact that daptomycin, a very large molecule, has some difficulty in reaching the cellular membrane because of the increased thickness of the cell wall.26,27
Fluoroquinolones often select for low-level resistance mutants that are associated with an overexpression of the norA gene, located on the staphylococcal chromosome: norA encodes a multi-drug efflux pump conferring resistance to unrelated compounds such as antiseptics, biocides and various metabolites.28,29 In fluoroquinolone-resistant mutants of S. aureus, an increase in the MIC of ciprofloxacin from 0.25 to 2 mg/L is associated with a 2- to 16-fold increase in the MIC of various antiseptics.29,30 The clinical impact of such an increase is not known, but it may contribute to reduced antiseptic activity; this may affect the success with which antiseptics eradicate MRSA from our hospitals, especially because the susceptibility of S. aureus to antiseptics is not routinely tested.
Events conferring a growth advantage to bacteria
Increased mutation frequency and MIC levels.. K. Drlica has compared the mutation frequency and MIC levels enabling the selection of resistant mutants in two isogenic S. aureus, one fully susceptible to fluoroquinolones, the other with a parC mutation.31 Despite a very minor increase in the MIC of ciprofloxacin from 0.25 to 0.5 mg/L, the parC mutant yielded a much higher mutation frequency and ciprofloxacin-resistant mutants could be selected in concentrations of up to 8 versus 0.5 mg/L for the fully susceptible strain. This value is called the mutant prevention concentration (MPC). The fact that the MPC has increased to above serum levels, suggests an increased risk of failure. Hence, in this and other studies, the presence of a determinant conferring low-level resistance greatly facilitates the acquisition of a second mechanism conferring high-level resistance.
The increased mutation frequency observed for this and other low-level resistant strains is often attributable to the presence of ‘mutator’ cells, which arise in bacteria under stress conditions.
Decreased fitness-cost.. Resistant mutants are usually considered as less virulent and non-competitive in the absence of antibiotic pressure because of an increased fitness-cost conferring a growth disadvantage. This may not always be true: in a study comparing a low-level rifampicin-resistant S. aureus with the parent strain, it was demonstrated that the mutant had a decreased doubling time (42 versus 55 min) and a higher virulence (100% versus 83% infectivity) when compared with the parent strain.32
In other reports, compensatory mutations have been described, which minimize the genetic burden owing to the increased fitness-cost and conferring to bacteria growth characteristics similar to those of the fully susceptible parent strain.33
Small-colony variants (SCV)
SCV are usually isolated from chronic infections or under antibiotic therapy.34,35 They show an increased capacity to invade cells and remain intracellular under the control of the alternate factor B.35 Because of their relative instability in vivo, they are responsible for relapsing infections. These variants have evolved in order to escape antibiotic pressure and cell-mediated immunity by down-regulating several virulence factors. The most frequent deficiencies concern the electron transport at the cellular membrane and are associated with decreased aminoglycoside susceptibility: these strains require haemin, menadione or polyvitex for normal growth. Other deficiencies such as thymidine requirement have been described (thymine-less mutants).
Because of their delayed growth or even no growth on non-supplemented media, such strains are never isolated in some laboratories; when isolated, these strains are often misidentified because of a lack of pigmentation or negative coagulase tests. Moreover, because of their slow growth, they are often misinterpreted as falsely susceptible to some antibiotics, particularly aminoglycosides, by disc-diffusion susceptibility testing.
There are dozens of reports considering SCV as less virulent, because of their growth disadvantage in vitro and in animal models. However, it has been clearly demonstrated that such strains may have increased virulence: in a study with a haemin-requiring mutant, Vaudaux et al.36 demonstrated that a hemB mutant had a 100% increase in its adherence to fibronectin or fibrinogen-coated coverslips.
SCV are not only more resistant to aminoglycosides but unexpectedly display a low-level resistance to several unrelated new antibiotics such as ceftobiprole, linezolid or moxifloxacin.37 The authors give no explanation for this cross-resistance. Other unexpected cross-resistances may have an explanation: nearly 30 years ago, we published a study on several SCV of S. aureus requiring thiamine for normal growth.6 One of these strains, isolated from a patient treated with co-trimoxazole, had a low-level resistance to aminoglycosides and trimethoprim and grew normally in the presence of thiamine or trimethoprim. When supplemented with either thiamine or trimethoprim, the strain recovered full susceptibility to aminoglycosides. The very likely reasons for the supplementation by thiamine or trimethoprim is attributable to the identical diaminopyrimidine nucleus of both molecules. In the same study, we also isolated menadione-requiring S. aureus. As usual, these SCV had increased aminoglycoside resistance. Similar to the thiamine-requiring strains, the MIC of the aminoglycosides decreased in a proportional manner when the strains were supplemented with menadione. This observation raises a very difficult and unanswered question: to what extent should these strains be supplemented when performing susceptibility testing? What are the most appropriate conditions mimicking in vivo situations?
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Conclusions |
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TopAbstractIntroductionWhat is low-level resistance?...Detection of low-level...Clinical impactConclusionsTransparency declarationsReferences |
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Low-level resistance is not a ‘laboratory curiosity’ but a relatively frequent phenomenon with an often unexpected array of clinical implications. Increased mutation frequencies to higher resistance levels are a gateway to clinical resistance. Moreover, increased virulence, improved in vivo fitness under stress conditions and resistance to unrelated compounds may have major clinical implications.
Hence, a better knowledge of the epidemiology of low-level resistance, an understanding of the mechanisms involved and improved detection techniques for these strains are necessary.
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Transparency declarations |
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TopAbstractIntroductionWhat is low-level resistance?...Detection of low-level...Clinical impactConclusionsTransparency declarationsReferences |
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I declare no conflicting financial interests.
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References |
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TopAbstractIntroductionWhat is low-level resistance?...Detection of low-level...Clinical impactConclusionsTransparency declarationsReferences |
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31
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36
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37
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