ACTAUNIVERSITATISUPSALIENSISUPPSALA2007

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 299

On Fusidic Acid Resistance inStaphylococcus Aureus

TOBIAS NORSTRÖM

ISSN 1651-6214ISBN 978-91-554-6880-4urn:nbn:se:uu:diva-7846

Annie: “The first passage will allow the demon to manifest itself in the flesh”

Ash: “Why the hell would we want to do that?

- Conversation between Annie and Ash, Evil Dead II (1987)

List of publications

This thesis is based on the following paper and manuscripts, referred to in the text by their roman numbers I-V

I. Establishment of a superficial skin infection model in mice by using Staphylococcus aureus and Streptococcus pyogenes.Kugelberg E, Norström T, Petersen TK, Duvold T, Anders-son DI, Hughes D. Antimicrob Agents Chemother. 2005 Aug;49(8):3435-41 Copyright © 2007, the American Society for Microbiology. All rights reserved.

II Structural probing and activity of fusidic acid derivatives against S. aureus and S. pyogenes in vitro and in vivo.

(Manuscript)

III. Activity of aminosterol derivatives of fusidic acid against S.aureus and S. pyogenes in vitro and in vivo.

(Manuscript)

IV. Resistance to fusidic acid can be linked to the Small Colony Variant (SCV) phenotype in Staphylococcus aureus.Norström T, Hughes D.

(Submitted manuscript)

V. Fitness of novel classes of fusidic acid-resistant Staphylococ-cus aureus in vitro and in vivo.Norström T, Frimodt-Møller N, Hughes D.

(Manuscript)

Paper I reprinted with permission granted from ASM.

Contents

List of publications .........................................................................................v

Introduction.....................................................................................................9Overview ....................................................................................................9Bacterial infections.....................................................................................9

Bacterial Infections Caused by Staphylococcus aureus.......................11Antibiotics, discovery and medical applications .................................14Antibiotic resistance ............................................................................15Fusidic acid..........................................................................................18Categorization and development of antibiotic compounds..................21Evaluating compounds with antimicrobial activity .............................24

Current Work............................................................................................27Introduction .........................................................................................27Development of a topical animal model ..............................................27In vitro and in vivo categorization of fusidic acid analogues (manuscript II and III) .........................................................................29Resistance to fusidic acid.....................................................................30

Svensk sammanfattning............................................................................33Acknowledgements ..................................................................................37References ................................................................................................38

Abbreviations

EF-G Elongation Factor G SCV Small Colony Variant FA Fusidic acid KM Kanamycin WHO World Health Organization PBP Penicillin Binding Proteins CF Cystic Fibrosis UTI Urinary Tract Infections SSI Surgical Site Infections LRI Lower Respiratory Infection MSSA Methicillin Sensitive S. AureusMRSA Methicillin Resistant S. Aureusagr Accessory Gene Regulator TSS Toxic Shock Syndrome

9

Introduction

OverviewI will begin my thesis with a brief background on bacterial infections, and the significance of bacterial pathogens. I will present a couple of examples of clinical costs associated with these bacteria, including those of Staphylo-coccus aureus. S. aureus will be the pathogen in focus for the remainder of the thesis. It will be put in a context of specific bacterial diseases, emergence and dissemination of antibiotic resistance and antibiotic development aimed at developing new treatment options, i.e. antibiotic derivatives with in-creased efficacy.

My current work deals with in vitro testing of fusidic acid derivatives, and the development and utilization of a novel topical animal model for assaying the in vivo efficacy of fusidic acid derivatives. I have also investigated the nature of an unknown class of fusidic acid resistance mechanism, and linked this to a particular phenotype often associated with persistent/chronic S. aureus infections.

Bacterial infections Infectious diseases are still, despite the medical advances made during the last century, very much a cause for concern. In 2002, an estimated 15 million deaths were caused by parasitic, viral and bacterial infections (WHO 2004). The majority of these were in the developing world (>90%), and the largest group affected was children below five years of age. Annually, 18% of all deaths among children below five years are caused by diarrheal infections (WHO 2005). Bacterial infections make up roughly 50%, or close to 8 mil-lion, of the reported deaths caused by infectious diseases annually. Table one shows leading causes of deaths caused by bacterial infections listed in de-scending order.

10

Table 1. Deaths caused by bacterial infections listed in descending order.

Type of infection Estimated number of deaths annually

Lower Respiratory Tract infections1 3.9 million Diarrheal infections 1.8 million Tuberculosis 1.6 million Others 2.0 million

Data collected from WHO enlisted regions and are estimates for 2002.

The bias in mortality rates comparing different parts of the world is mainly attributable to differences in adequate health care and to socio-economic factors such as poverty, crowdedness, level of education and health status of a population. Admittedly, much remains to be done in the developed world.

In Europe and the Americas diseases caused by bacterial infections have been of less significance since the introduction of antibiotics and the devel-opment of a functional health-care system. Fewer people today die of infec-tious diseases when compared with the situation just three or four genera-tions ago (McDermott and Rogers 1982). However, re-emerging diseases involving resistant and multi-resistant bacteria are becoming an increasing burden to society. This burden includes the economic costs for prolonged hospital admittance, complications associated with infections difficult to treat and the implementation of strict hospital routines increasing work load.

Urinary Tract Infections (UTI) account for more than 7 million physician visits annually in the US, not including hospital acquired UTI, which ac-count for 1/3 of all nosocomial infections (Talan, Naber et al. 2004). The main causative agent of UTI’s is the enteric bacilli Escherichia coli, respon-sible for more than 85% of UTI in all examined patients (Russo and Johnson 2003). Adding to that the financial cost of other infections caused by E. coli,e.g. pneumonia, SSI (Surgical Site Infections) and sepsis, the annual medical costs for treating UTI in the US alone amount to roughly US$ 1.8 billion (Russo and Johnson 2003).

Lower Respiratory Infection (LRI) is the major cause of death due to bac-terial infections both in the developing and the developed world (WHO 2004). The two most common pathogens associated with LRI are Strepto-coccus pneumoniae and Haemophilus influenzae, together responsible for about half of all LRI worldwide (English 2000). Decreasing the mortality of serious diseases comes with a price tag. Treatment of LRI in U.S. health care amounts to US$12 billion annually. When including absence from work and

1 No distinction is made between bacterial and viral infections in the collected data. Reports have shown however that Streptococcus pneumoniae, Haemophilus influenzae and Staphylo-coccus aureus account for at least half of all LRI worldwide. Scott, J. A. and A. J. Hall (1999). "The value and complications of percutaneous transthoracic lung aspiration for the etiologic diagnosis of community-acquired pneumonia." Chest 116(6): 1716-32, English, M. (2000). "Impact of bacterial pneumonias on world child health." Paediatr Respir Rev 1(1): 21-5.

11

other costs paid for by employers, the combined cost for the US society was an estimated US$112 billion in 1997 (Grossman, Rotschafer et al. 2005).

S. aureus infections, including MSSA and MRSA strains, are increasing rapidly in terms of frequency, treatment failures and mortality, as is the fi-nancial burden associated with counteracting these (Gould 2006). Recent U.S. estimates for MRSA related hospital costs range from US$1.5-4.2 bil-lion annually and losses to the national economy are estimated at US$17-30 billion annually (Noskin, Rubin et al. 2005; Gould 2006). S. aureus infec-tions will be discussed in more detail below, with emphasis on antibiotic resistance in general and fusidic acid resistance in particular.

Bacterial Infections Caused by Staphylococcus aureus

Since being initially readily treatable with a great number of available anti-biotics, S. aureus has re-established itself as the causative agent of difficult-to-treat and destructive infections. The clonal spread of resistant and multi-resistant S. aureus isolates has emphasized the importance of adequate hos-pital routines and antibiotic prescription regimens to counteract the effects of antibiotic resistance development (Johnson, Pearson et al. 2005; Appelbaum 2006).

S. aureus is one of the common naturally occurring cocci in the human host, with an estimated 30-40% of the population acting as carriers (Peacock, de Silva et al. 2001). While often found on the nasal mucosa, it can also be isolated from other moist surfaces such as the axillae and perineum. The vast majority of carriers suffer no ill effects of colonization, but immunocom-promised individuals, or patients recovering from surgery or serious diseases are more susceptible to infections (Foster 2005; van Belkum 2006). Noso-comial infections are the most common type, but community-acquired infec-tions are increasing worldwide (Sibbald, Ziebandt et al. 2006). 16% of all nosocomial infections in the U.S. between 1995-98 were caused by S.aureus. (Rice 2006).

S. aureus is equipped with a vast array of virulence factors, giving it the versatile ability to establish infections in virtually every organ in the human body, causing everything from superficial skin lesions to serious systemic infections such as pneumonia and sepsis (Table 2, p 12) (Lowy 1998; Sib-bald, Ziebandt et al. 2006). The expression of these virulence factors, i.e. superantigens, adhesins, hemolysins etc, is coordinated by a set of accessory regulators. These include, among many, the accessory gene regulator loci (agr), staphylococcal accessory regulator (SarA) and the alternative sigma factor sigmaB. While little is known of the specific external stimuli required to activate these major regulators, it has been shown that they can be use-fully classified on the basis of their dependence for expression on bacterial density. Some accessory regulators are expressed at low cell densities in

12

liquid broth, and others are expressed at high cell densities in liquid broth (Ziebandt, Weber et al. 2001; Ziebandt, Becher et al. 2004). agr is a positive regulator of proteins expressed at high cell densities, e.g. proteases, hemo-lysins and lipases, and negatively regulates low cell density proteins such as immunodominant antigen A. One of the implications is that differences in strain virulence can be attributed to the altered expression patterns of a few regulator proteins (Sibbald, Ziebandt et al. 2006).

Table 2.

Diseases caused by S. aureus

Food-borne diseases Soft tissue infections ImpetigoToxic shock syndrome Septicemia Pneumonia Osteomyelitis Infections associated with prosthetic implants Meningitis

Reported diseases caused by S. aureus in humans

Patient outcome and mortality rates vary depending on the type of infec-tion, and the virulence of the strain. The most serious conditions with the highest mortality rates are the bacteremia cases, with an average death rate of 35%. An outbreak involving a MRSA strain was reported to have an asso-ciated mortality of close to 60% (Romero-Vivas, Rubio et al. 1995; Gould 2005). Pneumonia infections are also difficult to treat, and mortality rates are almost 20%. It was again observed that mortality was higher for MRSA in-fected individuals (Gastmeier, Sohr et al. 2005). TSS (Toxic Shock Syn-drome) is a dramatic symptom of soft tissue infections caused by the secre-tion of superantigens, e.g. TSST(oxin). This triggers a massive immune-response by activating a large proportion of the host’s T-cells, causing them to release the cytokines believed to be the major contributor of TSS symp-toms. (McCormick, Yarwood et al. 2001; Sibbald, Ziebandt et al. 2006). TSS mortality depends on the location of the infection, but is nevertheless associ-ated with severe tissue damage and organ failure. Common uncomplicated skin infections, e.g. impetigo and infected follicles are not associated with high mortality. They remain localized and are medically treated with topical antibiotics if persistent (Cohen 2007).

S. aureus and persistent infections Persistent bacterial infections are characterized by antibiotic treatment fail-ure, long term infections (ranging from months to life long afflictions), and relapsing acute infections interrupting periods of dormancy. S. aureus is frequently associated with persistent infections (other than the asymptomatic

13

colonization of the anterior nares) of soft tissue and bone and joint tissues. The ability to establish these types of infections is attributed to altered regu-lation of accessory regulators affecting virulence, formation of biofilms and switch to the SCV (Small Colony Variant) phenotype (Donlan and Costerton 2002; Goerke and Wolz 2004; Proctor, von Eiff et al. 2006).

S. aureus strains with inactivating mutations in the agr operon are often isolated from CF (Cystic Fibrosis) patients suffering from chronic pulmo-nary infections (Goerke, Campana et al. 2000). This regulatory locus was shown in vitro to negatively affect protein A expression (inhibitor of phago-cytic engulfment) and positively regulate -toxin expression (hemolytic fac-tor). The situation appears however to be more complex in vivo. Virulence factors are often under the influence of several regulatory loci. It has been shown in animal models that -toxin expression is under the influence of agr, sarA and sae (staphylococcal accessory element) suggestive of a com-plex interaction between competing regulatory systems (Goerke, Campana et al. 2000; Goerke, Fluckiger et al. 2001).

Biofilms are defined as polymer matrix enclosed populations of bacteria adhering to a solid surface, exhibiting altered phenotype with respect to growth rate and gene transcription (Donlan and Costerton 2002). In clinical settings, these are problematic complications often associated with surgical or prosthetic implants and plastic catheters. Antibiotic susceptibility is re-duced making treatment difficult, often resulting in treatment failure (Donlan and Costerton 2002; Darouiche 2004; Mack, Becker et al. 2004; Fitzpatrick, Humphreys et al. 2005). Biofilm formation is regulated by quorum sensing (QS) affecting the agr and the luxS loci. Interestingly, contrary to QS sys-tems described in other bacteria these two repress biofilm formation by in-creasing expression of detergent-like peptides (agr), and repressing biofilm exopolysaccaride production (luxS) (Iwatsuki, Yamasaki et al. 2006; Kong, Vuong et al. 2006; Xu, Li et al. 2006).

SCVs have only recently been associated with persistent infections, de-spite descriptions of the phenotype dating back to the early twentieth century (Proctor, von Eiff et al. 2006). This is attributed to difficulties in isolating and categorizing these in clinical samples. The first reported SCV-associated clinical syndrome dates back to 1995 when S. aureus was isolated from pa-tients suffering from persistent and relapsing infections (Proctor, van Langevelde et al. 1995). Since then their significance in persistent infections has become more appreciated as diagnostic techniques and bacterial classifi-cations improve. SCVs are characterized by very slow growth in liquid and on solid media, forming colonies 1/10th the size of normally growing cells. Among clinical isolates two major metabolic defects causing the phenotype are recovered: those that are defective in thymidine metabolism, and those that have a dysfunctional electron transport system (ETS) and are auxotro-phic for ETS precursor molecules, e.g. menadione or hemin (Proctor, von Eiff et al. 2006). Additional phenotypic traits include increased resistance to

14

some antibiotics (Brouillette, Martinez et al. 2004), increased adhesion and persistence in mammalian cells (Vaudaux, Francois et al. 2002), and de-creased expression of exo-proteins and toxins (Moisan, Brouillette et al. 2006).

Antibiotics, discovery and medical applications At around the time of World War II, a new type of antimicrobial drug was introduced into clinical practice, a drug that allowed for quick and simple recovery from bacterial infections, and had relatively harmless side effects (nausea, vomiting, loss of appetite and diarrhea). Isolated from a mould be-longing to the genera Penicillium, it was named penicillin. The antibacterial action of this compound had been discovered some time before, incidentally as a potent inhibitor of S. aureus growth, and described by A. Fleming in his 1929 paper (Fleming 1929). Its implications for medical care were not ap-preciated at the time, and it wasn’t until it was shown in 1940 that penicillin administrated systemically to mice could cure them of lethal streptococcal infections (Chain, Florey et al. 1940), that it became clear that the discovery could have practical uses. The delay in the development of penicillin as an antimicrobial agent was partly due to technical problems in acquiring suffi-cient amounts of the active compound to make control experiments, and partly that testing for systemic in vivo efficacy was not common practice (Rolinson 1998). After the success with penicillin, antibiotic research grew rapidly, and the next couple of years saw the discovery and development of several novel classes of compounds including the tetracyclines, macrolides, aminoglycosides and glycopeptides (Rolinson 1998).

These drugs were immediately put into good use against both gram posi-tive and gram negative bacterial infections, and the “antibiotic era” began. Antibiotic use in health care, combined with improvements in hospital prac-tices and routines, have increased the average life span of the western citizen by as much as ten years (McDermott and Rogers 1982). In a few days, seri-ous bacterial infections could be cured and patient recovery was almost ab-solute, not only lowering mortality rates, but also improving life quality.

From the physicians’ point of view, antibiotics can be divided into broad spectrum and narrow spectrum antibiotics, depending on the range of micro-organisms affected by the drug. Broad spectrum antibiotics e.g. the -lactams and the fluoroquinolones have action against several species of both gram negative and gram positive bacteria, and are used accordingly. Fusidic acid, in contrast, is a typical narrow spectrum antibiotic and is of clinical use only for treatment of S. aureus infections. This has implications for the de-velopment of resistance as will be discussed below.

The mechanisms of action of antibiotics can be classified in principle ac-cording to which cellular processes they inhibit. Basically, there are four major processes targeted by antibiotics: i) cell wall synthesis, ii) protein syn-

15

thesis, iii) nucleic acid synthesis and iv) metabolic pathways. Table 3 sum-marizes these targets with the focus on drugs used to treat S. aureus.

Table 3.Target process Examples of targets

Antibiotic class

Cell wall synthesis PBP, terminal D-ala residues on nascent peptidoglycan chain, cell membrane

-lactams (penicillins and cephalosporins) Glycopeptides (vancomycin)

Daptomycin Protein synthesis 50S subunit,

30S Subunit, Isoleucyl-tRNA Synthetase EF-G

MLS (Macrolides, Lincosamides, Streptogramins) Chloramphenicol Oxazolidinones (linezolid) Tetracyclines, Aminoglycosodes (gentamycin) Mupirocin

Fusidic acid Nucleic acid synthesis Topoisomerse IV and DNA Gyrase RpoB

Fluoroquinolones

Rifampicin Metabolic pathway Biosynthesis of tetrahydrofolic acid

Sulfonamides (trimethoprim)

Antibiotics used for clinical treatment of S. aureus infections.

Antibiotic resistance Resistance mechanisms can in principle be broken down into three different types depending on how they interact with the antibiotic: A-C-T.

A Bacteria can reduce the Activity (A) of the drug. This involves enzy-matically modifying the drug, either by breaking it down (e.g. -lactamases), or by attaching a chemical group onto it (e.g. aminogly-coside acetylation/phosphorylation enzyme AAC(6’)-APH(2”)).

C Reducing the active Concentration (C) of the antibiotic in the bacterial cell. This can be achieved by restricting antibiotic influx (e.g. Porin regulation), by increasing the rate of antibiotic efflux (e.g. mar operon, tetracycline efflux), or by sequestering the antibiotic at a remote loca-tion (e.g. glycopeptides intermediate resistance associated with over-production of murein side chains, GISA, VISA)

16

T Reducing the interaction between the antibiotic and its cellular Target (T). Mechanisms include mutations in genes coding for the target molecule (e.g. fusA mutations); protective functions exerted by exter-nal proteins (e.g. the FusB determinant that binds to EF-G and pro-tects it against fusidic acid interference); and the opening of an alter-native metabolic pathway bypassing the original intended target (e.g. the Van-operon in enterococci, alternative peptidoglycan side chain linkage process, insensitive to vancomycin, VRE)

History and spread of antibiotic resistance Shortly after penicillin was introduced in 1944, resistant variants of bacteria emerged. Prior to the introduction of penicillin, 94% of all examined S.aureus isolates were clinically susceptible to penicillin. Four years later more than 50% of all clinical isolates had become resistant (Livermore 2000). More than anything, this was attributed to an atrociously liberal use of penicillin by physicians and patients alike. By 1960, outbreaks of virulent multi-resistant S. aureus in hospitals had occurred several times. These iso-lates carried additional resistance determinants, often on plasmids and trans-posons, providing the bacteria with resistance against chloramphenicol, erythromycin and streptomycin (Livermore 2000). Today, virtually all strains of S. aureus are resistant to natural penicillins, aminopenicillins and antipseudominal penicillins, (Rice 2006). A large proportion of these strains carry multiple resistance genes. A recently isolated MRSA (named EM-RSA17, E for epidemic) strain had decreased susceptibility against -lactams, aminoglycosides, macrolides, fluoroquinolones, tetracycline, rifam-picin and fusidic acid. The emergence of multi-resistant strains has left hos-pitals with very few drugs that are effective in eradicating S. aureus infec-tions. Vancomycin, a glycopeptide, has been the drug of choice for these multi-resistant types of infections, and its use is restricted to combat emerg-ing resistance. Unfortunately, 1996 saw the first case of increased resistance against vancomycin in a patient in Japan, undergoing long term vancomycin treatment (Rice 2006). Intermediate (GISA, VISA) strains have since then been isolated from hospitals all over the world, and a few high level vanco-mycin resistant (VRSA) strains have been isolated from hospital patients in the USA.

It appears not to be a question of if, but rather a question of when resis-tance to a specific antibiotic emerges. As a function of the selective pressure applied by the clinical environment, mutations or pre-existing resistance genes are selected for, and strains carrying these will subsequently spread. Hospital environments are especially vulnerable to the dissemination of re-sistant clones due to the frequent flux of hospitalized/infected patients, and staff acting as carriers, and an intrinsically high level of use of antibiotics. Broad spectrum antibiotics are used to treat many types of infections and as

17

a consequence, bacteria of many different genera are subject to selection, leading to a rapid selection of resistance (Bjorkman and Andersson 2000; Cizman 2003). Selected genes can spread within species or between closely related species via horizontal gene transfer (HGT) on transmissible genetic elements (Wright 2007), or by clonal expansion of highly virulent resistant strains (Murchan, Aucken et al. 2004; Johnson, Pearson et al. 2005; Smith and Cook 2005). Vectors facilitating HGT of antibiotic resistance include plasmids, integrons, transposons, and phage-mediated transduction. Trans-missible genetic elements very often carry several resistance markers, and multi-drug resistance is thus spread even more rapidly through genetic “hitch-hiking” (Grohmann, Muth et al. 2003; Del Grosso, Camilli et al. 2006; Brenciani, Bacciaglia et al. 2007; Cloeckaert, Praud et al. 2007). Con-sequently, resistance to narrow spectrum antibiotics, or for that matter, anti-biotics with a restricted dosing regimen, will on average take longer to evolve and spread, unless they are co-selected with other resistances.

It was long believed that the cost associated with an antibiotic resistance phenotype would cause the resistant strain to be selected against and weeded out from the population once the antibiotic pressure was removed. However, it has been shown on several occasions that resistance can be easily and quickly stabilized and maintained by compensatory evolution, ameliorating the initial cost of resistance (Bjorkholm, Sjolund et al. 2001; Nagaev, Bjorkman et al. 2001; Andersson 2006). The spread of these genes then be-comes a stochastic process of genetic drift.

MRSAThe appearance of MRSA clones (Methicillin Resistant Staphylococcus aureus) in 1961, and the dissemination of these in health care institutions illustrates the problem of bacterial responses to antibiotics, and the difficul-ties of maintaining the effectiveness of a drug

During the late 1950s and early 1960s, resistance to natural penicillin was so high that the drug was of practically no use for clinical treatments. Resis-tance was also increasing against other classes of antibiotics (Wright 2007). It was discovered at the time that semi-synthetic penicillins could be made from the penicillin core structure 6-APA, and from there new synthetic drugs were created and put into clinical use. One of the first to be marketed was methicillin. Methicillin was effective even against multi resistant strains, including penicillin resistant isolates. However, only one year after its intro-duction, methicillin-resistant clones appeared. This was not caused by -lactamase activity. The bulky side chain on the semi-synthetic penicillins prevents inactivation by -lactamases (Livermore 2000). Methicillin resis-tance was a different type of resistance mechanism altogether. These isolates carried any one of four different resistance cassettes (SCCmec1-4) carrying the mecA gene which codes for an alternative PBP (PBP2a) with a low affin-ity for penicillins (Rolinson 1998; Livermore 2000; Jansen, Beitsma et al.

18

2006). These were named MRSA strains. The SCCmec cassettes are not readily transmissible and genetic analyses have identified 5 major lineages of MRSA strains from which all present MRSA isolates are vertically de-scended. It was suggested that these SCCmec cassettes were present in some S. aureus genomes prior to the introduction of methicillin (Lacey 1984; Kreiswirth, Kornblum et al. 1993).

Clonal expansion of a few strains led to increasing frequencies of MRSA among clinical strains, reaching a maximum of 10-15% in the early 70s. New antibiotic regimens, including the use of the aminoglycoside gen-tamicin, and new hospital hygiene and isolation practices brought the preva-lence of MRSA down to almost 0% by 1980 (Livermore 2000). Gentamicin resistance soon appeared in the form of the bifunctional acetylat-ing/phosphorylating enzyme AAC(6’)-APH(2”) in the transposon Tn4001, located on a conjugative plasmid. The prevalence of aminoglycoside resis-tance increased in clinical S. aureus strains, including MRSA (Byrne, Rouch et al. 1989; Livermore 2000; Udou 2004). Since then, MRSA has again in-creased in frequency, and has remained at a high frequency ever since. By 2004, almost 60% of all S. aureus isolated from ICU (Intensive Care Units) in U.S. hospitals were MRSA (Livermore 2000; Rice 2006). US hospitals as a whole have a prevalence of 35-55%, whereas European countries overall have a prevalence of 20% (ranging from <10% in Scandinavian countries and Holland, up to ~40% in Great Britain, Italy and Greece) (Appelbaum 2006).

Fusidic acid Fusidic acid was first isolated from the fungus Fusidium coccineum(Godtfredsen, Jahnsen et al. 1962) and released onto the market in 1963 where is was used often in combination therapy together with methicillin and penicillin G (Alexander and Hutchinson 1963). The drug was most welcome by the health care authorities, as staphylococcal resistance to most available antibiotics was increasing. It has activity against gram positives in general, and against S. aureus in particular. Fusidic acid is a member of the fusidane antibiotic group, and similar structures with similar activities have been iso-lated from other fungal species: ramycin from Mucor ramannianus(Vanderhaeghe, Vandijck et al. 1965) and fusidane triterpene from Acremo-nium crotocinigenum (Evans, Hedger et al. 2006). The current use of fusidic acid is primarily in the treatment of skin and eye infections, but it is occa-sionally used systemically to treat bone and joint infections and septicaemia (Atkins and Gottlieb 1999; Spelman 1999; Whitby 1999; Darley and MacGowan 2004).

19

Figure 1. The structure of fusidic acid. Essential features for antimicrobial action are marked in red, conserved features in yellow, and interchangeable features in green. Adapted from Duvold et al. (Duvold, Sorensen et al. 2001).

The molecular structure of fusidic acid is steroid like, in that it has the dis-tinct tetracycline ring system, but differs in the sterical conformation of the hexamer rings, i.e. fusidic acid has the chair-boat-chair conformation; refer-ring to how the 6-membered carbon rings are bent. This is a characteristic of the fusidane antibiotic group, and is essential for their function (Duvold, Sorensen et al. 2001). Figure 1 shows a 2-D visualization of fusidic acid with the functionally important side groups marked out. Varying these influences antimicrobial function, and structural probing has been important in unveil-ing the importance of individual side chains for antimicrobial function (Duvold, Sorensen et al. 2001; Duvold, Jorgensen et al. 2002).

The proposed mechanism of action for fusidic acid is by binding to the EF-G molecule when it is in complex with the ribosome during translation. This prevents the release of EF-G from the ribosome after translocation and causes the ribosome to stall. Protein synthesis is thus halted (Nagaev, Bjorkman et al. 2001).

20

Fusidic acid resistance in S. aureusResistance mechanisms to fusidic acid in S. aureus include i) target based resistances, including both target mutation and target protection and ii) an unknown class of resistance mutation(s).

I a Target mutation. It has been shown that point mutations in the fusA gene increase resistance to fusidic acid, usually alter-ing amino acids in the domain III region of EF-G (Johanson and Hughes 1994; Johanson, Aevarsson et al. 1996; Nagaev, Bjorkman et al. 2001). Most of these mutations cause medium level resistance (MIC 4 – 12 µg/ml) but some cause higher level resistance (~30 - >96 µg/ml). On a crystal structure model of EF-G the clustered locations of the resistance muta-tions suggest a binding site for fusidic acid (Laurberg, Kris-tensen et al. 2000). There are reported mutations in domain I and domain V as well, but in response to fusidic acid selec-tion, they constitute the minority. Figure 3 (p 31) shows a crystal structure of EF-G and the location of some resistance mutations.

I b Target protection. Plasmid-borne resistance to fusidic acid has been recognized for over thirty years (Lacey and Grinsted 1972), conferring medium level resistance to fusidic acid (4-8µg/ml). The fusidic acid resistance phenotype is linked to the far1 ORF on the pUB101 plasmid (O'Brien, Price et al. 2002). Continued work by O’Neill et al. concluded that the far1 gene encoded a small cytoplasmic protein that binds to and protects EF-G from fusidic acid interference. The resistance gene was re-named fusB, by analogy with the fusA gene. The expression of fusB is regulated through translational attenuation, and is increased when ribosomal mRNA decoding is slowed down. Two similar proteins (exhibiting c. 45% identity with FusB) have been found to be chromosomally located among some clinical S. aureus and S. intermedius isolates and in S. sapro-phyticus (O'Neill A, McLaws et al. 2007). These FusB homo-logues have been named FusC and FusD respectively, and are believed to protect EF-G from fusidic acid in the same way as FusB.

II Unknown class of resistance mutations. Laboratory selec-tions for fusidic acid resistance in defined laboratory strains routinely give resistant clones mapping outside of the fusAgene at about equal frequency, 10-8 cell-1 generation-1. They

21

are characterized by medium level resistance (4-8 µg/ml) and impaired growth characteristics in liquid broth (Nagaev, Bjorkman et al. 2001).

Resistance in clinical settings As a clinical drug, fusidic acid has been on the European market since 1963, but resistance among clinical strains has stayed at a low level. No clinically relevant cross resistance in S. aureus to any other antibiotics has been re-ported, thus helping to maintain the clinical integrity of the drug (Christiansen 1999; Turnidge 1999).

Recently, the spread of a few epidemic clones has increased the preva-lence of resistance in particular geographical locations (Ravenscroft, Layton et al. 2000; Osterlund, Eden et al. 2002; Tveten, Jenkins et al. 2002; Afset and Maeland 2003; El-Zimaity, Kearns et al. 2004), including a FusR MRSA strain (Andersen, Bergh et al. 1999). These resistant clones started appearing in the mid-90s, in Scandinavia as impetigo-causing variants spreading among children. These clonal outbreaks (as determined by phage typing and PFGE patterns) have in many cases been shown to carry the same resistance determinant, the plasmid-mediated fusB resistance determinant (O'Neill, Larsen et al. 2004), or the related but chromosomally located fusB and fusCgenes (O'Neill A, Larsen et al. 2007; O'Neill A, McLaws et al. 2007).

Prior to the mid 90s, the frequency of fusidic acid resistant clinical strains was low in Scandinavian countries and the UK. Despite the rapid emergence of resistance in vitro, this did not translate into a serious resistance problem in clinical settings, and resistance frequencies remained stable at 1-2% for a long period of time (Shanson 1990). In 1994 reports of increasing prevalence of fusidic acid resistant clinical strains started appearing and by 2007 several epidemic fusidic acid resistance clones have been described (Osterlund, Eden et al. 2002; O'Neill, Larsen et al. 2004; O'Neill A, Larsen et al. 2007). Current resistance frequencies in Scandinavian countries range from 6-8% (SSI 2004; SMI 2006). In other parts of the world resistance levels are mark-edly higher; such that in Kuwait and Greece the incidence of fusidic acid resistant strains in 1998 was c. 23% and 53% respectively. This increase in resistance frequency is mirrored by increased resistances against the other major antibiotics used to treat S. aureus infections and an increased preva-lence of MRSA strains (Samuelsson 2002).

Categorization and development of antibiotic compounds Following the introduction of penicillin into clinical practice, many other types of antibiotics were discovered. Between 1945 and 1960, almost all of the antibiotic classes known today were already described (Wright 2007). However, resistance quickly emerged following the introduction of each of these compounds to the clinical market, and the treatment of some infections

22

became difficult. It became increasingly more tedious and time consuming to hunt for novel naturally produced antibiotics. By coincidence, at the time when resistance to natural antibiotics started to become a real problem, it was discovered that under certain growth conditions penicillin producing fungi could be made to produce the penicillin core structure 6-APA (figure 2), lacking the side chain attached to the nitrogen (Rolinson 1998). Appar-ently, it is the presence of this side chain that gives penicillin and related natural compounds their antimicrobial property; by itself the 6-APA has little antimicrobial effect.

CHCOOH

C(CH3)2

S

NCO

CHNH2CH

CHCOOH

CCH3

S

NCO

CHNH2CH CHR

6-APA 7-APA

CHCOOH

C(CH3)2

S

NCO

CHNH2CH

CHCOOH

CCH3

S

NCO

CHNH2CH CHR

6-APA 7-APA

Figure 2. Core structure of penicillins (6-APA), and cephalosporins (7-APA). Syn-thetic modification of these by introducing novel side groups at specific locations (arrows) created new derivatives with enhanced efficacy, no cross resistance to other penicillins or cephalosporins or new pharmacodynamic properties.

This sparked a new field in antibiotic research; the production of chemically modified antibiotics by the attachment of novel side chains to a core struc-ture. Some of these compounds proved to have novel antibacterial character-istics of their own. Not only could the in vitro and in vivo efficacy of antibi-otics be improved, but the range of bacterial species susceptible to the com-pound could be expanded, making it an antibiotic with a broader spectrum. This not only added clinical value, but increased the financial incentives to produce new variants of existing antibiotic compounds. In addition, bacteria that had gained resistance to the natural antibiotic were often susceptible to the new chemical derivative. This was true for penicillin and the chemically derived antibiotic methicillin. As previously mentioned, this was due to the steric hindrance exerted by the attached side-chain protecting the lactam ring structure from -lactamases. Similar improvements were achieved by modi-fying the related cephalosporin C ring structure 7-APA. No other class of antibiotics has been chemically modified as much as the -lactams have. Figure 3 (p 23) depicts the pharmaceutical industry-engineered structural evolution of penicillin G, and the various chemical variants derived from it.

23

1950

1955

1965

1960

1970

1975

1980

baca

mpi

cilli

n tala

mpi

cilli

nm

ecill

inam

pivm

ecill

inam

nafc

illin

met

hici

llin ox

acill

in

clox

acill

in

flucl

oxac

illin

dicl

oxac

illin

6-A

PA

peni

cilli

n V

peni

cilli

n G

phen

ethi

cilli

n

prop

icill

in

azid

ocill

in

carb

enic

illin

cari

ndac

illin

carf

ecill

in

tem

ocill

in

azlo

cilli

n

apal

cilli

n mez

loci

llin

pipe

raci

llin

amox

ycill

in

cycl

acill

in

epic

illin

ampi

cilli

n

piva

mpi

cilli

nsulb

enic

illin

ticar

cilli

n

1950

1955

1965

1960

1970

1975

1980

baca

mpi

cilli

n tala

mpi

cilli

nm

ecill

inam

pivm

ecill

inam

nafc

illin

met

hici

llin ox

acill

in

clox

acill

in

flucl

oxac

illin

dicl

oxac

illin

6-A

PA

peni

cilli

n V

peni

cilli

n G

phen

ethi

cilli

n

prop

icill

in

azid

ocill

in

carb

enic

illin

cari

ndac

illin

carf

ecill

in

tem

ocill

in

azlo

cilli

n

apal

cilli

n mez

loci

llin

pipe

raci

llin

amox

ycill

in

cycl

acill

in

epic

illin

ampi

cilli

n

piva

mpi

cilli

nsulb

enic

illin

ticar

cilli

n

Figure 3. Chemical laboratory evolution of the 6-APA structure, the structural back-bone of penicillin.

24

Properties other than those affecting the antibiotic-bacteria interaction also became important when developing new derivatives. This was especially true when considering drug pharmacokinetics. These features include the bioavailability, toxicity, metabolic breakdown in the body, distribution in different tissues, and excretion of the drug. The bioavailability of different 6-APA derivatives could vary by orders of magnitude. Comparing seven dif-ferent isoxazolyl penicillins with virtually identical in vitro properties, the extent of serum binding varied between 36% to 98% (Derendorf 1989; Rolinson 1998).

By chemically modifying natural drugs the arsenal available for clinical treatment was greatly increased and provided new drugs that had antimicro-bial activity against strains resistant to older drugs. Almost all classes of antibiotics have been chemically modified, with great success. The fluoro-quinolones are modified derivatives of the original quinolone drug nalidixic acid. The addition of fluoride ions to the core structure greatly improved in vitro and in vivo efficacy and decreased the rate at which clinically relevant levels of resistance to the new drugs emerged (Appelbaum and Hunter 2000).

Evaluating compounds with antimicrobial activity The efficacy of a compound against bacteria is evaluated using in vitro and in vivo methods. These provide information on i) whether there is there an interaction between a compound and its (proposed) target ii) what concentra-tion is needed to inhibit bacterial growth, iii) how growth is inhibited, i.e. are the bacteria being killed, or do they simply stop growing, and iv) how these properties are reflected in an animal model.

In vitro modelsThese experiments are designed to give an estimate of what drug dose is required to inhibit growth; and how this affects bacteria in a concentration-over-time dependant assay, i.e. a killing type experiment. These assays are made in a standardized way to allow for comparison with the literature and other groups.

The concentration at which bacterial growth is inhibited is termed the MIC, short for the “Minimal Inhibitory Concentration”. There are several methods to obtain this estimate, such as the agar diffusion- and the broth dilution methods (Rylander, Brorson et al. 1979). To get this estimate using the broth dilution method, the compound is serially diluted in liquid broth and bacteria are inoculated in each of the various drug concentrations so that a cell density of c. 106 cfu/ml is obtained. The bacteria are incubated ON (~18 hrs), and the inocula are inspected visually for growth. The MIC is set as the lowest concentration at which no visible growth can be observed after the ON incubation. The agar diffusion model relies on measuring the zone of

25

inhibition proximal to discs containing various levels of the compound. The same principle is used when performing the Etest assay. The Etest is made with plastic strip, coated with a concentration gradient of antibiotic on one side, and a calibration scale on the other side. The antibiotic diffuses from the strip into the solid media. MIC read after 18 hours of growth and is the concentration immediately above the point at which the zone of growth inhi-bition intersects the calibrated Etest strip (Brown and Brown 1991).

Time-kill methods are used to test the type of antimicrobial effect a com-pound has on a particular bacterial species. A standardized number of bacte-ria are inoculated in liquid broth (c. 106 cfu/ml) containing the antimicrobial compound. Samples are then drawn at specified time intervals, diluted and plated on non-selective solid media to get an estimate of the remaining num-ber of cfu present in the sample. By varying the concentration in increments of the MIC, a dose/time response can be constructed. On the basis of these tests, antibiotics can classified as either bacteriostatic or bactericidal, they either cause the cell to stop growing without killing it, or they actively kill it. Fusidic acid is a typical bacteriostatic drug, excessive amounts of the drug halt bacterial growth, killing only a relatively small number of cells (Collignon and Turnidge 1999). Penicillin which inhibits the function of the PBP building the cell wall is bactericidal during active growth, causing cells to rupture (Rolinson 1998).

In vivo methods Critical to the evaluation of the clinical relevance of a potential drug is the efficacy it shows in clearing infections in animal models (Craig 1993; George and Rubin 2003). Animal models should meet with certain criteria: i)they should be clinically relevant, ii) experimentally robust, iii) ethically acceptable and iv) convenient to perform. Results should be reliable and reproducible. With this in mind, animal models are designed based on the type of infection, type of bacteria, and type of drug being studied.

S. aureus can be the causative agent of many types of diseases, and there are many animal models using staphylococci reflecting this diversity of in-terests (Akiyama, Kanzaki et al. 1994; Palma, Nozohoor et al. 1996; Tarkowski, Collins et al. 2001; Brouillette and Malouin 2005; Harraghy, Seiler et al. 2006). Some of these models deal with topical skin infections, most notably the burnt skin model, and the suture-wound model (Stieritz, Bondi et al. 1982; Akiyama, Kanzaki et al. 1994; Gisby and Bryant 2000). Both serve the purpose of introducing the infectious agent through a breach in the skin, allowing for infection establishment, and monitoring of treatment success using antibiotics.

Due to intrinsic properties of the infected bacterial host, i.e. distribution of a drug in the body, protein binding vs. free drug concentration, excretion of the drug, different results can be obtained depending on what model sys-tem is used (Collignon and Turnidge 1999; Turnidge 1999; Meagher,

26

Ambrose et al. 2005; Rybak 2006). This is valuable in determining how a drug should be used clinically. Penetration of the drug into various body tissues, metabolic breakdown in the body and free plasma concentration may vary greatly depending on type of administration (oral, intravenous, topical application), drug formula (ointment, tablet etc.) and unique characteristics of the drug (protein binding, half life in the patient) (Meagher, Ambrose et al. 2005; Nightingale 2005). Therefore, models need to be specific and well defined to be clinically relevant.

In summary, animal models represent the in vivo step between in vitrotests and phase 1 clinical trials, where drug effects alone are tested in human volunteers.

27

Current Work

IntroductionMy current projects have involved two separate lines of interests. We have collaborated with LEO Pharma pharmaceuticals in Denmark and developed a superficial skin infection model for use in assaying the efficacy of topically administrated antibiotics (Paper I). This model was later used to measure in vivo efficacy of “classic” fusidic acid derivatives (Manuscript II), and in vivoefficacy of a second, novel, class of aminosterol fusidic acid derivatives (Manuscript III). In vitro characterizations of the “classic” and “aminosterol” fusidic acid derivatives are discussed in manuscript II and III respectively.

The second line of interest deals with the unknown class of fusidic acid resistance mutations, and links these to a subpopulation of S. aureus often associated with persistent clinical infections (Paper IV). Furthermore, on the basis of the observation that in clinical fusidic acid resistant isolates only one of the possible resistance mechanisms appeared to be present, the cumulative in vitro cost and resistance level of these were investigated. Additionally the in vivo cost of first step mutants/plasmid recipients was investigated in an animal model (Paper V).

Development of a topical animal model

Evaluation of published models for testing fusidic acid derivatives Topical infections are today a common class of S. aureus infections receiv-ing treatment with fusidic acid (Spelman 1999). Prior to the publication of our model, there were two models in the literature describing the assessment of antibiotic efficacy against topical infectious agents, The burned skin model (Stieritz, Bondi et al. 1982; Akiyama, Kanzaki et al. 1994), and the suture wound model (Gisby and Bryant 2000).

The burned skin model is a highly invasive procedure causing massive trauma and has ethical issues regarding animal suffering and stress to the animal’s system. We did not evaluate the model ourselves.

The second model, the suture-wound model, can be summarized thus: A dorsal patch of skin on the mouse is shaved clean of fur, and a 2 cm long cut is introduced in the clear area on the anesthetized mouse. This cut penetrates all skin layers. A 1 cm suture thread soaked in a concentrated bacterial sus-pension in placed in the wound, which is subsequently sewn shut using ster-ile thread. Antimicrobial compounds dissolved in cream or ointments are then administered at defined intervals. We experienced this model to be ex-perimentally cumbersome, producing highly variable results, and decided to abandon it (unpublished data). We felt there was a need for a model with an

28

experimental set up that could easily be monitored to assure consistency, and required a minimum of invasive trauma to introduce the infectious agents.

Developing a novel superficial skin infection model for testing fusidic acid derivatives (Paper 1) The infectious agents we wanted to use were S. aureus and Streptococcus pyogenes. The disease spectra of S. pyogenes is somewhat similar to that of S. aureus; skin and soft tissue infections, necrotizing fasciitis, TSS, (Mitchell 2003; Elston 2004; Carapetis, Steer et al. 2005; Young, Aronoff et al. 2005), as well as more specific afflictions such as rheumatic fever (Guilherme, Fae et al. 2005). It would be of clinical significance if the model could be used to test both of these pathogens.

To achieve the conditions during which infections could be established while minimizing the trauma to the animal, we “stripped” the fur and dermal layers of a dorsal patch on the animal using adhesive tape. This routinely required 7-10 runs of stripping before the fur and upper skin layer was re-moved. The tissue damage was monitored by measuring transepidermal wa-ter loss, i.e. a measure of how much water is being lost due to loss of skin integrity. This enabled us to standardize the invasive procedure against a physiologically measurable parameter. Dissolved bacteria were immediately applied to the exposed surface, subsequently set as t=0.

The model was successful in that both S. aureus and S. pyogenes could establish infections lasting for at least four days and these infections were susceptible to fusidic acid (and mupirocin, data not shown) antibiotic treat-ment. The presences of both bacteria were confirmed in histological exami-nations, noting also a strong inflammatory response. Figure 4 shows the recovery of a mouse having received fusidin® treatment twice daily for four days.

Figure 4. A mouse 4 days into the experiment having received fusidic acid treatment twice daily.

29

In vitro and in vivo categorization of fusidic acid analogues (manuscript II and III)

Chemically modifying natural antibiotics, creating synthetic derivatives, is one of the greater achievements in medical research. Second, third and fourth generation antibiotics had either one or several of the following char-acteristics separating them from their progenitors; improved efficacies in vitro and in vivo, no cross-resistance with earlier antibiotics in that class or an increased number of bacteria susceptible to the drug (Rolinson 1998). The success of this has prompted a search for molecules derived from fusidic acid.

The fusidic acid molecule is amendable for change, and several deriva-tives with altered antimicrobial properties have been created (Duvold, Sorensen et al. 2001; Duvold, Jorgensen et al. 2002). Extensive in vitro test-ing has mapped the importance of the various side chains extending from the four-ring structure, and classified the importance of these for microbial func-tion (figure 1, p. 20).

Manuscript II and III discusses the in vitro and in vivo characterization of “classic” and aminosterol derivatives of fusidic acid. The classic derivatives have side chain substitutions at permissive locations. The aminosterol de-rivatives have similar side chains substituted, but in their place are long pro-truding charged molecules.

Broth dilution assays and kill-time experiments distinguished the classic and aminosterol derivatives as two classes with different antimicrobial effi-cacies, and are suggestive of different modes of action. The minimal inhibi-tory concentration was improved upon for one classic derivative against both S. aureus and S. pyogenes. The aminosterols were less effective against S.aureus, but more effective against S. pyogenes. Time-kill experiments con-firmed the bacteriostatic nature of fusidic acid, and similar dynamics were observed for the classic derivative. The aminosterol compounds had a clear bactericidal effect in these experiments. The animal model further separated the two, with the aminosterol derivatives being more efficient in clearing the bacteria.

These results show the potential of fusidic acid derivatives when aiming for improved interaction between the drug and its target, as well as attaching completely novel properties to the molecule. The high protein binding prop-erties of fusidic acid (91-98%) limits its bioavailability in vivo (Collignon and Turnidge 1999; Turnidge 1999), and offers opportunities for further improvements of the structure to remedy this.

30

Resistance to fusidic acid Limiting ourselves to resistance mechanisms described in S. aureus, two known mechanisms of resistance are of clinical significance; target based resistance, including target mutation (fusA mutants) and target protection (fusB-D genes). In paper IV and V we describe the properties of two new resistance classes, fusA-SCV and FusE, and how they might relate to clinical infections and development of resistance.

Selection and categorization of fusA-SCV and FusE-SCV (Paper IV) Selecting for resistance in S. aureus using fusidic acid readily produces fusAmutants. The frequency of these in a bacterial population is at about 10-7-10-8

(Nagaev, Bjorkman et al. 2001). These are mainly located in structural do-main III of EF-G, and cluster in domain interfaces and around the putative binding site of fusidic acid (Johanson and Hughes 1994; Laurberg, Kris-tensen et al. 2000; Nagaev, Bjorkman et al. 2001). Figure 3 (p 32) shows the sterical location of domain III resistance mutations on a crystal structure of EF-G. However, experimental selection identifies a second class of mutants (FusE) mapping outside of fusA appearing at roughly the same frequency (Nagaev, Bjorkman et al. 2001). These differ from the fusA class by very slow growth in vitro, increased resistance to aminoglycosides, and auxotro-phy for hemin, a precursor molecule of the Electron Transport System (ETS). Additionally, the frequency of growth compensation is extremely high (10-6-10-4), upon which wt growth and antibiotic susceptibility levels are often restored. These characteristics coincide with those of the SCV phe-notype, a bacterial subpopulation recently associated with persistent and relapsing infections (Proctor, van Langevelde et al. 1995; Proctor, von Eiff et al. 2006).

Reversing the selection process, i.e. selecting for kanamycin and allowing for slow colonies to appear produces lots of clones fitting the SCV criteria. Screening for fusidic acid among these produced a subset (~1/6th of screened clones) with an additional resistance to fusidic acid. No fusidic acid resis-tance was observed among any of the medium sized or large kanR colonies tested, implying that this feature is exclusive to the SCV class.

Sequencing fusA of clones selected as kanR and screened for fusR re-vealed that about half of these carried fusA-mutations, predominantly in structural domain V of EF-G (figure 3, p. 32), and not in domain III, the domain most frequently modified when selecting with fusidic acid. These were all auxotrophic for hemin, whereas FusE mutants were auxotrophic for hemin or menadione. The increase in resistance to either antibiotic (fus or kan) ranged from medium to high.

To our knowledge, this is the first time clinically relevant cross resistance between fusidic acid and another antibiotic class (aminoglycosides) has been

31

reported. We show this to be an effect of a minimum of two genetic mecha-nisms; one is mutations in fusA, and the other(s) mutations outside of fusA.

A clinically relevant issue is also that by applying a fusidic acid selective pressure, a subpopulation involved in persistent and relapsing infections can be selected for and maintained at least throughout the course of the antibiotic treatment.

Figure 3 shows the location of the domain V mutations found among the fusA-SCVs. Their distinct placement, separate from the classic fusA muta-tions in domain III, suggests the possibility of a novel target-based resistance mechanism.

Figure 5. Ribbon structure of EF-G crystallized from Thermus thermophilus(Hansson, Singh et al. 2005), PDB: 2BM0. Visualization and rendering kindly pro-vided by Christofer Björkelid. Structural domain III and V mutations are marked in red and yellow respectively.

Fitness of novel classes of fusidic acid-resistant Staphylococcus aureus invitro and in vivo (paper V)The fitness cost of FusA-SCVs and FusE-SCVs is large compared to wt, more or much more so than the cost associated with “classic” fusA mutations (domain III) and the presence of the pUB101 plasmid carrying the fusB gene. This is apparent both on solid and in liquid media. We asked whether this is mirrored in an in vivo system, i.e. what would be the significance of these when confronted with a clinical situation? Accordingly, FusA, FusA-SCVs,

32

FusE-SCVs and FusB strains were tested for relative virulence against the parental wt strain in an animal model. In vivo data correlated with in vitrodata except in the case of FusB, the presence of which was associated with a more dramatic decrease in fitness in the animal model. Part of the explana-tion was the observed loss of plasmid, arguably an effect of compatibility issues between the plasmid and the host (Dahlberg and Chao 2003).

Introducing the plasmid into a näive wt strain increased the resistance to fusidic acid from ~0.25 to 6µg/ml. This level of resistance is also observed among clinical strains positive for the fusB gene (O'Neill, Larsen et al. 2004; O'Neill A, Larsen et al. 2007; O'Neill A, McLaws et al. 2007), and is well above the clinical breakpoint values classifying S. aureus as resistant; 1 or 2µg/ml (Collignon and Turnidge 1999). Introducing the plasmid into a strain positive for any of the chromosomal determinants increases the resistance levels in an additive manner. These findings may imply a similar mode of target based resistance for all the resistance determinants, FusA-FusE, i.e. they may each act to lower the probability of interaction between fusidic acid and EF-G on the ribosome. Additive effects on MIC have been ob-served for systems sharing a similar mode of resistance (Lee, Mao et al. 2000). Independent mechanisms of actions, e.g. target alteration in combina-tion with concentration lowering mechanisms, have been shown to act syn-ergistically to increase the MIC when both are present (Lomovskaya, Lee et al. 1999; Oethinger, Kern et al. 2000).

33

Svensk sammanfattning

Antibiotika och bakteriella infectioner Infektioner orsakade av bakterier står för cirka hälften av antalet globala dödsfall orsakade av smittsamma sjukdomar. Enligt ”WHO world report 2004” motsvarar det i absoluta tal drygt 7.5 miljoner människor årligen. Mer än 9 av 10 av dessa återfinns i utvecklingsländer, och den mest drabbade befolkningsgruppen är barn under fem år. Industrialiserade länder har en mycket lägre mortalitet associerad med bakteriella infektioner. De främsta orsakerna till denna skillnad är stora ekonomiska satsningar på sjukvård, en generellt god hälsostatus hos befolkningen, och en regelmässig användning av antibakteriella substanser mot bakteriella infektioner.

Den viktiga roll som antibiotika spelar understryks av de senaste decenni-ernas resistensutveckling och spridning av resistensgener bland vanliga hu-mana patogener. Klinisk klassificering av resistensmönster hos multiresis-tenta stammar försenar lämplig medicinsk behandling med risk för kraftigare symptom och i vissa fall ökad dödlighet som följd. Spridningen av resistens-gener mot kliniska antibiotika har inte mötts upp av forskning och lansering av nya preparat.

Min avhandling diskuterar i) resistensmekanismer hos Staphylococcus aureus riktade mot fusidinsyra, ii) in vitro och in vivo karaktärisering av kemiska fusidinsyrederivat, samt iii) utveckling av en djurmodell för in vivostudier av antibiotikabehandling mot hudinfektioner orsakade av S. aureusoch Streptococcus pyogenes.

Fusidinsyra och S. aureusFusidinsyra är ett naturligt antibiotikum med inhibitorisk effekt på gramposi-tiva celler i allmänhet, och S. aureus i synnerhet. Fusidinsyra har använts kliniskt i Europa sedan början på 60-talet, och under större delen av den perioden har resistensfrekvensen legat på en relativt låg nivå (<2% i Skandi-navien och England), trots snabb resistensutveckling in vitro. De senaste 15 åren vittnar dock om en förändring; en ökad frekvens av epedemiska kloner med fusidinresistens har observerats hos patienter med hudinfektioner orsa-kade av S. aureus. För närvarande är mellan 6-8% av alla kliniska isolat av S. aureus från svenska och danska patienter resistenta mot fusidinsyra.

Verkan och resistensmekanismer Antibiotikumet binder till EF-G när den befinner sig i komplex med riboso-men under translationen. Effekten blir att EF-G fastnar på ribosomen, trans-lationen stannar av, och proteinsyntesen upphör. Beskrivna resistensmeka-nismer i S. aureus inkluderar mutationer i genen som kodar för EF-G, fusA(FusA determinanten), och närvaron av fusB-genen (FusB determinanten, ofta plasmidburen, men den och dess förmodade homolog fusC, återfinns

34

även i kromosomen hos vissa kliniska isolat). Mutationer i fusA förmodas minska affiniteten mellan fusidinsyra och EF-G i komplex med ribosomen. fusB kodar för ett inducerbart protein som har visat sig ha affinitet för EF-G. FusB binder till- och skyddar EF-G mot fusidinsyra

Nuvarande undersökning I avhandlingen beskriver vi hur en undergrupp av fusA-mutationer och kro-mosomala mutationer utanför fusA (resistensdeterminant FusE) medför resi-stens mot både fusidinsyra och aminoglykosider (dessa orsakar felkodning av mRNA under translation genom att binda till A-platsen på 16S rRNA). Detta är det första rapporterade fallet som beskriver klinisk relevant korsre-sistens mellan fusidinsyra och något annat antibiotikum. Vidare så påverkar dessa mutationer cellens fysiologi, och de kännetecknas av väldigt långsam tillväxt, auxotrofi för heme eller menadion och i vissa fall nedsatt hemolytisk förmåga. Dessa egenskaper återfinns hos en subpopulation hos bakterier som kallas för SCV (Small Colony Variants, eng.). Övriga karakteristiska inklu-derar nedsatt respiration, auxotrofi för komponenter i elektrontransportked-jan (thiamine, heme, menadion), minskad virulens generellt men ökad över-levnadsförmåga i däggdjursceller. I samband med att klinisk klassificering av bakterier förbättrats, har SCV i allt högre grad kopplats ihop med kronis-ka infektioner/återfallsinfektioner.

Kategorisering av mutationer som medför korsresistens De fusA-mutationer som medför korsresistens mellan fusidinsyra och ami-noglykosider, samt bildandet av SCV, återfinns främst i strukturdomän V i EF-G. Mutationer i denna enhet har tidigare endast rapporterats hos ett kli-niskt isolat av S. aureus, och i ett fåtal fall i motsvarande gen i andra arter, t.ex. hos Salmonella typhimurium. Selektion för resistens med fusidinsyra generarar främst mutationer i strukturdomän III, såtillvida att de isoleras efter 18-24 timmars tillväxt, då många SCV inte vuxit upp och bildat synliga kolonier. Våra försök visar att fusA-SCV förekommer med samma frekvens som ”klassiska” fusA mutationer. Motsvarande SCV vars mutationer befin-ner sig utanför fusA (FusE determinanten) återfinns även de med ungefär samma frekvens som fusA och fusA-SCV. Däremot så utgör de en minoritet (~15%) av de SCVs som erhålls då selektion sker med en aminoglykosid, och fusidinsyreresistens hos vardera av de aminoglykosidresistenta klonerna undersöks.

Den relativa överlevnadskraften hos FusA, FusA-SCV, FusB och FusE invitro och in vivo jämfört med vildtypen uppskattades genom tillväxtförsök i näringsmedium och i en peritoneal (peritoneum = bukhåla lat.) djurmodell. Kostnaden för resistensdeterminanten in vitro och in vivo var lägst för FusA, och högre för FusA-SCV och FusE. FusB visade sig medföra en marginell kostnad in vitro, men en betydande sådan in vivo. Plasmiden med fusB-genen

35

förlorades i vissa fall under djurförsökets förlopp, något som inte observerats i samma utsträckning in vitro.

FusA, FusB och FusE samverkar troligtvis lokalt för att minska sannolikheten att fusidinsyra binder till EF-GVidare försök med FusB-determinanten visade att tillsammans med FusA, FusA-SCV och FusE ökade resistensen på ett additivt sätt. Införandet av fusB-plasmiden in i vildtypen medförde samma absoluta höjning av mot-ståndskraften som när den fördes in i FusA/FusA-SCV och FusE, oberoende av resistensnivån i mottagarstammen. Liknande resultat har rapporterats när olika resistensmekanismer överlappar och motståndskraften är summan av de två. Detta utesluter inte att liknande överlappningar ligger bakom det observerade förhållandet mellan FusB och FusA+E.

En annan typ av samverkan observeras när icke överlappande resistens-mekanismer kombineras i en cell; då uppstår ofta en synergistisk (multipli-katorisk) resistenseffekt. Så sker t.ex. när resistensmutationer i en gen kom-bineras med ett exportsystem som oberoende av andra geners resistensmuta-tioner aktivt exporterar det aktuella antibiotikumet. Detta talar emot ett sce-nario bestående av icke överlappande resistensmekanismer i S aurues.

Vidare försök ämnar påvisa om den observerade additiva effekten de fac-to är resultatet av resistensmekanismer med överlappande funktioner.

In vitro och in vivo analys av fusidinsyrederivat och utvecklingen av en in vivo modell för antibiotikabehandling av hudinfektioner Parallellt med dessa försök har vi utarbetat en ny djurmodell för uppskatt-ning av antimikrobiella ämnens effektivitet mot hudinfektioner orsakade av S. aureus och Streptococcus pyogenes.

Tidigare beskrivna system inkluderar modeller då den infekterande bakte-rien introduceras till värddjuret genom brännskador eller via stora sår som direkt efter att bakterier ympats i det sys ihop. Vi utvärderade den senare modellen, men fann den alltför experimentellt ohanterlig och beskaffad med alltför stora variationer i resultaten. Med vår modell kan vi med ett mindre invasivt ingrepp introducera de infekterande bakterierna och få dem att eta-blera en infektion. Med hjälp av klistertejp dras pälsen och det övre hudlag-ret av från en dorsalt placerad hudarea om 1x2 cm. Ingreppet kan standardi-seras mot vätskeförlusten från den blottlagda underhuden, en mätbar para-meter, och på så sätt kan närapå identiska förhållanden erhållas inför ymp-ning av bakterier. Vi påvisade etablerade infektioner under modellens varaktighet för både S. aureus och Streptococcus pyogenes.

Denna modell användes när ett antal fusidinsyrederivat analyserades in vi-tro och in vivo för relativ aktivitet jämfört med fusidinsyra. Alla ämnen ana-lyserades för MIC (Minimal Inhibitory Concentration eng.), den koncentra-tion vid vilken bakteriell tillväxt upphör, samt effekten av subletala och leta-la doser av det aktuella ämnet på växande bakterier som en funktion av tid,

36

sk avdödningsexperiment. Lovande kandidater analyserades in vivo för ef-fektivitet mot både S. aureus och Streptococcus pyogenes. Ett ämne visade en förhöjd aktivitet mot båda dessa jämfört med fusidinsyra såväl in vitrosom in vivo. Detta derivat befinner sig för närvarande i kliniska försök. Strukturen och dess associerade antimikrobiella egenskaper kan vid denna tidpunkt inte offentliggöras i enlighet med LEO Pharma praxis.

37

Acknowledgements

While my sphere of gratitude encompasses all of ICM, and some random and non-random locations elsewhere, there are a number of individuals de-serving extra credit.

My deepest gratitude and respect goes to my supervisor and mentor Profes-sor Diarmaid Hughes. Thank you for believing in me and giving me the opportunity to watch science being done at its best. You have not only been great source of scientific inspiration, but also a friend and great collaborator. I’ve always enjoyed our conversations over the years, on or off work, and hope to continue those in the future.

My greatest ally and closest friend Malin Olsson, thank you for coping with me on rainy days and showing your full support in dire times. There is just no way in I could have done this without you. This is our achievement, not mine alone.

My son Malcolm and daughter Flora, for the unconditional love and support you show me when my perception of reality deceives me.

Mum and Dad. Besides the obvious, you are the two most important reasons I begun this hazardous endeavor, for which I will always be grateful. For good or bad, there is nothing I would want to be made different in the past, I will always cherish my childhood.

For showing me that there are more than one path in life, thank you Andreasand Emma, my brother and sister.

No-one can reminisce about one’s childhood without thinking of those dear friends that stuck by you no matter how many apples were wasted or fields burned down. Thank you (in no particular order), Jonas Hultqvist-Sellingand Markus SVEN-BERTO Carlsson.

My close colleagues Linda, Marie, Disa, Patricia and Mira, journey team mates, receivers of random mischief and innocent tricks.

Klas I. Udekwu for teaching me the essence of cynicism.

Mats Pettersson, gamer extraordinaire, prodigy, dude and friend.

Work colleagues, friends and family, if you’re reading this, you know you matter.

38

I would like to thank mrs Dale Tatrom at the Classical Numismatic Group (www.cngcoins.com) and Parviz Ahghari at Pars Coins (www.parscoins.com) for generously granting me permission to use the im-ages of the ancient roman coins decorating the cover of this thesis.

References

Afset, J. E. and J. A. Maeland (2003). "Susceptibility of skin and soft-tissue isolates of Staphylococcus aureus and Streptococcus pyogenes to topical antibiotics: indications of clonal spread of fusidic acid-resistant Staphylococcus aureus." Scand J Infect Dis 35(2): 84-9.

Akiyama, H., H. Kanzaki, et al. (1994). "Staphylococcus aureus infection on experimental croton oil-inflamed skin in mice." J Dermatol Sci 8(1):1-10.

Alexander, W. D. and J. G. Hutchinson (1963). "Staphylococcal septicaemia treated successfully with methicillin, fusidic acid, and penicillin G in combination." Lancet 1: 55-6.

Andersen, B. M., K. Bergh, et al. (1999). "A Norwegian nosocomial out-break of methicillin-resistant Staphylococcus aureus resistant to fusidic acid and susceptible to other antistaphylococcal agents." J Hosp Infect 41(2): 123-32.

Andersson, D. I. (2006). "The biological cost of mutational antibiotic resis-tance: any practical conclusions?" Curr Opin Microbiol 9(5): 461-5.

Appelbaum, P. C. (2006). "MRSA--the tip of the iceberg." Clin Microbiol Infect 12 Suppl 2: 3-10.

Appelbaum, P. C. and P. A. Hunter (2000). "The fluoroquinolone antibacte-rials: past, present and future perspectives." Int J Antimicrob Agents16(1): 5-15.

Atkins, B. and T. Gottlieb (1999). "Fusidic acid in bone and joint infec-tions." Int J Antimicrob Agents 12 Suppl 2: S79-93.

Bjorkholm, B., M. Sjolund, et al. (2001). "Mutation frequency and biological cost of antibiotic resistance in Helicobacter pylori." Proc Natl Acad Sci U S A 98(25): 14607-12.

Bjorkman, J. and D. I. Andersson (2000). "The cost of antibiotic resistance from a bacterial perspective." Drug Resist Updat 3(4): 237-245.

Brenciani, A., A. Bacciaglia, et al. (2007). "Genetic Elements Carrying erm(B) in Streptococcus pyogenes, and Association with tet(M) Tet-racycline Resistance Gene." Antimicrob Agents Chemother.

Brouillette, E. and F. Malouin (2005). "The pathogenesis and control of Staphylococcus aureus-induced mastitis: study models in the mouse." Microbes Infect 7(3): 560-8.

Brouillette, E., A. Martinez, et al. (2004). "Persistence of a Staphylococcusaureus small-colony variant under antibiotic pressure in vivo." FEMS Immunol Med Microbiol 41(1): 35-41.

39

Brown, D. F. and L. Brown (1991). "Evaluation of the E test, a novel method of quantifying antimicrobial activity." J Antimicrob Chemother27(2): 185-90.

Byrne, M. E., D. A. Rouch, et al. (1989). "Nucleotide sequence analysis of IS256 from the Staphylococcus aureus gentamicin-tobramycin-kanamycin-resistance transposon Tn4001." Gene 81(2): 361-7.

Carapetis, J. R., A. C. Steer, et al. (2005). "The global burden of group A streptococcal diseases." Lancet Infect Dis 5(11): 685-94.

Chain, E., H. W. Florey, et al. (1940). "Penicillin as a chemotherapeutic agent. 1940." Lancetii: 226-8.

Christiansen, K. (1999). "Fusidic acid adverse drug reactions." Int J Antim-icrob Agents 12 Suppl 2: S3-9.

Cizman, M. (2003). "The use and resistance to antibiotics in the commu-nity." Int J Antimicrob Agents 21(4): 297-307.

Cloeckaert, A., K. Praud, et al. (2007). "Dissemination of an Extended-Spectrum-{beta}-Lactamase blaTEM-52 Gene-Carrying IncI1 Plas-mid in Various Salmonella enterica Serovars Isolated from Poultry and Humans in Belgium and France, 2001-2005." Antimicrob Agents Chemother.

Cohen, P. R. (2007). "Community-acquired methicillin-resistant Staphylo-coccus aureus skin infections: a review of epidemiology, clinical features, management, and prevention." Int J Dermatol 46(1): 1-11.

Collignon, P. and J. Turnidge (1999). "Fusidic acid in vitro activity." Int J Antimicrob Agents 12 Suppl 2: S45-58.

Craig, W. (1993). "Relevance of animal models for clinical treatment." Eur J Clin Microbiol Infect Dis 12 Suppl 1: S55-7.

Dahlberg, C. and L. Chao (2003). "Amelioration of the cost of conjugative plasmid carriage in Eschericha coli K12." Genetics 165(4): 1641-9.

Darley, E. S. and A. P. MacGowan (2004). "Antibiotic treatment of gram-positive bone and joint infections." J Antimicrob Chemother 53(6): 928-35.

Darouiche, R. O. (2004). "Treatment of infections associated with surgical implants." N Engl J Med 350(14): 1422-9.

Del Grosso, M., R. Camilli, et al. (2006). "The mef(E)-carrying genetic ele-ment (mega) of Streptococcus pneumoniae: insertion sites and asso-ciation with other genetic elements." Antimicrob Agents Chemother50(10): 3361-6.

Derendorf, H. (1989). "Pharmacokinetic evaluation of beta-lactam antibiot-ics." J Antimicrob Chemother 24(3): 407-13.

Donlan, R. M. and J. W. Costerton (2002). "Biofilms: survival mechanisms of clinically relevant microorganisms." Clin Microbiol Rev 15(2):167-93.

Duvold, T., A. Jorgensen, et al. (2002). "17S,20S-Methanofusidic acid, a new potent semi-synthetic fusidane antibiotic." Bioorg Med Chem Lett 12(24): 3569-72.

40

Duvold, T., M. D. Sorensen, et al. (2001). "Synthesis and conformational analysis of fusidic acid side chain derivatives in relation to antibac-terial activity." J Med Chem 44(19): 3125-31.

El-Zimaity, D., A. M. Kearns, et al. (2004). "Survey, characterization and susceptibility to fusidic acid of Staphylococcus aureus in the Car-marthen area." J Antimicrob Chemother 54(2): 441-6.

Elston, D. M. (2004). "Epidemiology and prevention of skin and soft tissue infections." Cutis 73(5 Suppl): 3-7.

English, M. (2000). "Impact of bacterial pneumonias on world child health." Paediatr Respir Rev 1(1): 21-5.

Evans, L., J. N. Hedger, et al. (2006). "An antibacterial hydroxy fusidic acid analogue from Acremonium crotocinigenum." Phytochemistry67(19): 2110-4.

Fitzpatrick, F., H. Humphreys, et al. (2005). "The genetics of staphylococcal biofilm formation--will a greater understanding of pathogenesis lead to better management of device-related infection?" Clin Microbiol Infect 11(12): 967-73.

Fleming, A. (1929). "On the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation of B. influenzae." British Journal of Experimental Pathology 10: 226-32.

Foster, T. J. (2005). "Immune evasion by staphylococci." Nat Rev Microbiol3(12): 948-58.

Gastmeier, P., D. Sohr, et al. (2005). "Mortality risk factors with nosocomial Staphylococcus aureus infections in intensive care units: results from the German Nosocomial Infection Surveillance System (KISS)." Infection 33(2): 50-5.

George, A. and G. Rubin (2003). "A systematic review and meta-analysis of treatments for impetigo." Br J Gen Pract 53(491): 480-7.

Gisby, J. and J. Bryant (2000). "Efficacy of a new cream formulation of mupirocin: comparison with oral and topical agents in experimental skin infections." Antimicrob Agents Chemother 44(2): 255-60.

Godtfredsen, W. O., S. Jahnsen, et al. (1962). "Fusidic acid: a new antibi-otic." Nature 193: 987.

Goerke, C., S. Campana, et al. (2000). "Direct quantitative transcript analysis of the agr regulon of Staphylococcus aureus during human infection in comparison to the expression profile in vitro." Infect Immun68(3): 1304-11.

Goerke, C., U. Fluckiger, et al. (2001). "Impact of the regulatory loci agr, sarA and sae of Staphylococcus aureus on the induction of alpha-toxin during device-related infection resolved by direct quantitative transcript analysis." Mol Microbiol 40(6): 1439-47.

Goerke, C. and C. Wolz (2004). "Regulatory and genomic plasticity of Staphylococcus aureus during persistent colonization and infection." Int J Med Microbiol 294(2-3): 195-202.

Gould, I. M. (2005). "The clinical significance of methicillin-resistant Staphylococcus aureus." J Hosp Infect 61(4): 277-82.

41

Gould, I. M. (2006). "Costs of hospital-acquired methicillin-resistant Staphy-lococcus aureus (MRSA) and its control." Int J Antimicrob Agents28(5): 379-84.

Grohmann, E., G. Muth, et al. (2003). "Conjugative plasmid transfer in gram-positive bacteria." Microbiol Mol Biol Rev 67(2): 277-301, ta-ble of contents.

Grossman, R. F., J. C. Rotschafer, et al. (2005). "Antimicrobial treatment of lower respiratory tract infections in the hospital setting." Am J Med118 Suppl 7A: 29S-38S.

Guilherme, L., K. C. Fae, et al. (2005). "Rheumatic fever: how S. pyogenes-primed peripheral T cells trigger heart valve lesions." Ann N Y Acad Sci 1051: 132-40.

Hansson, S., R. Singh, et al. (2005). "Structural insights into fusidic acid resistance and sensitivity in EF-G." J Mol Biol 348(4): 939-49.

Harraghy, N., S. Seiler, et al. (2006). "Advances in in vitro and in vivo mod-els for studying the staphylococcal factors involved in implant infec-tions." Int J Artif Organs 29(4): 368-78.

Iwatsuki, K., O. Yamasaki, et al. (2006). "Staphylococcal cutaneous infec-tions: invasion, evasion and aggression." J Dermatol Sci 42(3): 203-14.

Jansen, W. T., M. M. Beitsma, et al. (2006). "Novel mobile variants of staphylococcal cassette chromosome mec in Staphylococcus aureus." Antimicrob Agents Chemother 50(6): 2072-8.

Johanson, U., A. Aevarsson, et al. (1996). "The dynamic structure of EF-G studied by fusidic acid resistance and internal revertants." J Mol Biol258(3): 420-32.

Johanson, U. and D. Hughes (1994). "Fusidic acid-resistant mutants define three regions in elongation factor G of Salmonella typhimurium." Gene 143(1): 55-9.

Johnson, A. P., A. Pearson, et al. (2005). "Surveillance and epidemiology of MRSA bacteraemia in the UK." J Antimicrob Chemother 56(3):455-62.

Kong, K. F., C. Vuong, et al. (2006). "Staphylococcus quorum sensing in biofilm formation and infection." Int J Med Microbiol 296(2-3): 133-9.

Kreiswirth, B., J. Kornblum, et al. (1993). "Evidence for a clonal origin of methicillin resistance in Staphylococcus aureus." Science259(5092): 227-30.

Lacey, R. W. (1984). "Antibiotic resistance in Staphylococcus aureus and streptococci." Br Med Bull 40(1): 77-83.

Lacey, R. W. and J. Grinsted (1972). "Linkage of fusidic acid resistance to the penicillinase plasmid in Staphylococcus aureus." J Gen Micro-biol 73(3): 501-8.

Laurberg, M., O. Kristensen, et al. (2000). "Structure of a mutant EF-G re-veals domain III and possibly the fusidic acid binding site." J Mol Biol 303(4): 593-603.

42

Lee, A., W. Mao, et al. (2000). "Interplay between efflux pumps may pro-vide either additive or multiplicative effects on drug resistance." J Bacteriol 182(11): 3142-50.

Livermore, D. M. (2000). "Antibiotic resistance in staphylococci." Int J An-timicrob Agents 16 Suppl 1: S3-10.

Lomovskaya, O., A. Lee, et al. (1999). "Use of a genetic approach to evalu-ate the consequences of inhibition of efflux pumps in Pseudomonas aeruginosa." Antimicrob Agents Chemother 43(6): 1340-6.

Lowy, F. D. (1998). "Staphylococcus aureus infections." N Engl J Med339(8): 520-32.

Mack, D., P. Becker, et al. (2004). "Mechanisms of biofilm formation in Staphylococcus epidermidis and Staphylococcus aureus: functional molecules, regulatory circuits, and adaptive responses." Int J Med Microbiol 294(2-3): 203-12.

McCormick, J. K., J. M. Yarwood, et al. (2001). "Toxic shock syndrome and bacterial superantigens: an update." Annu Rev Microbiol 55: 77-104.

McDermott, W. and D. E. Rogers (1982). "Social ramifications of control of microbial disease." Johns Hopkins Med J 151(6): 302-12.

Meagher, A. K., P. G. Ambrose, et al. (2005). "Pharmacoki-netic/pharmacodynamic profile for tigecycline-a new glycylcycline antimicrobial agent." Diagn Microbiol Infect Dis 52(3): 165-71.

Mitchell, T. J. (2003). "The pathogenesis of streptococcal infections: from tooth decay to meningitis." Nat Rev Microbiol 1(3): 219-30.

Moisan, H., E. Brouillette, et al. (2006). "Transcription of virulence factors in Staphylococcus aureus small-colony variants isolated from cystic fibrosis patients is influenced by SigB." J Bacteriol 188(1): 64-76.

Murchan, S., H. M. Aucken, et al. (2004). "Emergence, spread, and charac-terization of phage variants of epidemic methicillin-resistant Staphy-lococcus aureus 16 in England and Wales." J Clin Microbiol 42(11): 5154-60.

Nagaev, I., J. Bjorkman, et al. (2001). "Biological cost and compensatory evolution in fusidic acid-resistant Staphylococcus aureus." Mol Mi-crobiol 40(2): 433-9.

Nightingale, C. H. (2005). "Future in vitro and animal studies: development of pharmacokinetic and pharmacodynamic efficacy predictors for tissue-based antibiotics." Pharmacotherapy 25(12 Pt 2): 146S-149S.

Noskin, G. A., R. J. Rubin, et al. (2005). "The burden of Staphylococcusaureus infections on hospitals in the United States: an analysis of the 2000 and 2001 Nationwide Inpatient Sample Database." Arch Intern Med 165(15): 1756-61.

O'Brien, F. G., C. Price, et al. (2002). "Genetic characterization of the fusidic acid and cadmium resistance determinants of Staphylococcus aureusplasmid pUB101." J Antimicrob Chemother 50(3): 313-21.

O'Neill A, J., A. R. Larsen, et al. (2007). "Characterization of the Epidemic European Fusidic Acid-Resistant Impetigo Clone (EEFIC) of Staphylococcus aureus." J Clin Microbiol.

43

O'Neill A, J., F. McLaws, et al. (2007). "Genetic basis of resistance to fusi-dic acid in staphylococci." Antimicrob Agents Chemother.

O'Neill, A. J., A. R. Larsen, et al. (2004). "A fusidic acid-resistant epidemic strain of Staphylococcus aureus carries the fusB determinant, whereas fusA mutations are prevalent in other resistant isolates." Antimicrob Agents Chemother 48(9): 3594-7.

Oethinger, M., W. V. Kern, et al. (2000). "Ineffectiveness of topoisomerase mutations in mediating clinically significant fluoroquinolone resis-tance in Escherichia coli in the absence of the AcrAB efflux pump." Antimicrob Agents Chemother 44(1): 10-3.

Osterlund, A., T. Eden, et al. (2002). "Clonal spread among Swedish chil-dren of a Staphylococcus aureus strain resistant to fusidic acid." Scand J Infect Dis 34(10): 729-34.

Palma, M., S. Nozohoor, et al. (1996). "Lack of the extracellular 19-kilodalton fibrinogen-binding protein from Staphylococcus aureusdecreases virulence in experimental wound infection." Infect Immun64(12): 5284-9.

Peacock, S. J., I. de Silva, et al. (2001). "What determines nasal carriage of Staphylococcus aureus?" Trends Microbiol 9(12): 605-10.

Proctor, R. A., P. van Langevelde, et al. (1995). "Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus." Clin Infect Dis 20(1): 95-102.

Proctor, R. A., C. von Eiff, et al. (2006). "Small colony variants: a patho-genic form of bacteria that facilitates persistent and recurrent infec-tions." Nat Rev Microbiol 4(4): 295-305.

Ravenscroft, J. C., A. Layton, et al. (2000). "Observations on high levels of fusidic acid resistant Staphylococcus aureus in Harrogate, North Yorkshire, UK." Clin Exp Dermatol 25(4): 327-30.

Rice, L. B. (2006). "Antimicrobial resistance in gram-positive bacteria." Am J Infect Control 34(5 Suppl 1): S11-9; discussion S64-73.

Rolinson, G. N. (1998). "Forty years of beta-lactam research." J Antimicrob Chemother 41(6): 589-603.

Romero-Vivas, J., M. Rubio, et al. (1995). "Mortality associated with noso-comial bacteremia due to methicillin-resistant Staphylococcus aureus." Clin Infect Dis 21(6): 1417-23.

Russo, T. A. and J. R. Johnson (2003). "Medical and economic impact of extraintestinal infections due to Escherichia coli: focus on an in-creasingly important endemic problem." Microbes Infect 5(5): 449-56.

Rybak, M. J. (2006). "Pharmacodynamics: relation to antimicrobial resis-tance." Am J Infect Control 34(5 Suppl 1): S38-45; discussion S64-73.

Rylander, M., J. E. Brorson, et al. (1979). "Comparison between agar and broth minimum inhibitory concentrations of cefamandole, Cefoxitin, and cefuroxime." Antimicrob Agents Chemother 15(4): 572-9.

44

Samuelsson, S. (2002). "Surveillance of Staphylococcus Infections in Den-mark http://www.ssi.dk/sw1293.asp (2007-03-26)." EPI-NEWS Na-tional Surveillance of Communicable Diseases(10).

Scott, J. A. and A. J. Hall (1999). "The value and complications of percuta-neous transthoracic lung aspiration for the etiologic diagnosis of community-acquired pneumonia." Chest 116(6): 1716-32.

Shanson, D. C. (1990). "Clinical relevance of resistance to fusidic acid in Staphylococcus aureus." J Antimicrob Chemother 25 Suppl B: 15-21.

Sibbald, M. J., A. K. Ziebandt, et al. (2006). "Mapping the pathways to staphylococcal pathogenesis by comparative secretomics." Micro-biol Mol Biol Rev 70(3): 755-88.

SMI (2006). Swedish Institute for Disease Control. Staphylococcus aureus,Statistics on Antimicrobial Resistance 2001-2006 (2007-03-26). http://www.smittskyddsinstitutet.se/in-english/.

Smith, J. M. and G. M. Cook (2005). "A decade of community MRSA in New Zealand." Epidemiol Infect 133(5): 899-904.

Spelman, D. (1999). "Fusidic acid in skin and soft tissue infections." Int J Antimicrob Agents 12 Suppl 2: S59-66.

SSI (2004). Statens Serum Institut, Staphylococcus Reports - Annual report 2004. http://www.ssi.dk/sw3425.asp (2007-03-25).

Stieritz, D. D., A. Bondi, et al. (1982). "A burned mouse model to evaluate anti-Pseudomonas activity of topical agents." J Antimicrob Chemo-ther 9(2): 133-40.

Talan, D. A., K. G. Naber, et al. (2004). "Extended-release ciprofloxacin (Cipro XR) for treatment of urinary tract infections." Int J Antim-icrob Agents 23 Suppl 1: S54-66.

Tarkowski, A., L. V. Collins, et al. (2001). "Model systems: modeling hu-man staphylococcal arthritis and sepsis in the mouse." Trends Mi-crobiol 9(7): 321-6.

Turnidge, J. (1999). "Fusidic acid pharmacology, pharmacokinetics and pharmacodynamics." Int J Antimicrob Agents 12 Suppl 2: S23-34.

Tveten, Y., A. Jenkins, et al. (2002). "A fusidic acid-resistant clone of Staphylococcus aureus associated with impetigo bullosa is spreading in Norway." J Antimicrob Chemother 50(6): 873-6.

Udou, T. (2004). "Dissemination of nosocomial multiple-aminoglycoside-resistant Staphylococcus aureus caused by horizontal transfer of the resistance determinant (aacA/aphD) and clonal spread of resistant strains." Am J Infect Control 32(4): 215-9.

van Belkum, A. (2006). "Staphylococcal colonization and infection: homeo-stasis versus disbalance of human (innate) immunity and bacterial virulence." Curr Opin Infect Dis 19(4): 339-44.

Vanderhaeghe, H., P. Vandijck, et al. (1965). "Identity of Ramycin with Fusidic Acid." Nature 205: 710-1.

Vaudaux, P., P. Francois, et al. (2002). "Increased expression of clumping factor and fibronectin-binding proteins by hemB mutants of Staphy-

45

lococcus aureus expressing small colony variant phenotypes." Infect Immun 70(10): 5428-37.

Whitby, M. (1999). "Fusidic acid in septicaemia and endocarditis." Int J Antimicrob Agents 12 Suppl 2: S17-22.

WHO (2004). The World Health Report 2004 - Changing History. URL: http://www.who.int/whr/2004/en/ (2007-03-25).

WHO (2005). The World Health Report 2005 - Make Every Mother and Child Count. http://www.who.int/whr/2005/en/ (2007-03-25).

Wright, G. D. (2007). "The antibiotic resistome: the nexus of chemical and genetic diversity." Nat Rev Microbiol 5(3): 175-86.

Xu, L., H. Li, et al. (2006). "Role of the luxS quorum-sensing system in biofilm formation and virulence of Staphylococcus epidermidis." In-fect Immun 74(1): 488-96.

Young, M. H., D. M. Aronoff, et al. (2005). "Necrotizing fasciitis: patho-genesis and treatment." Expert Rev Anti Infect Ther 3(2): 279-94.

Ziebandt, A. K., D. Becher, et al. (2004). "The influence of agr and sigmaB in growth phase dependent regulation of virulence factors in Staphy-lococcus aureus." Proteomics 4(10): 3034-47.

Ziebandt, A. K., H. Weber, et al. (2001). "Extracellular proteins of Staphylo-coccus aureus and the role of SarA and sigma B." Proteomics 1(4): 480-93.

Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 299

Editor: The Dean of the Faculty of Science and Technology

A doctoral dissertation from the Faculty of Science andTechnology, Uppsala University, is usually a summary of anumber of papers. A few copies of the complete dissertationare kept at major Swedish research libraries, while thesummary alone is distributed internationally through theseries Digital Comprehensive Summaries of UppsalaDissertations from the Faculty of Science and Technology.(Prior to January, 2005, the series was published under thetitle “Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology”.)

Distribution: publications.uu.seurn:nbn:se:uu:diva-7846

ACTAUNIVERSITATISUPSALIENSISUPPSALA2007