Niels Høiby
email: hoiby@hoibyniels.dk

Thomas Bjarnsholt

Michael Givskov

Søren Molin

Oana Ciofu

Høiby et al: Antibiotic resistance of bacterial biofilms

Keywords:

A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and DNA. Bacterial biofilms cause chronic infections because they show increased tolerance to antibiotics, desinfective chemicals and resist phagocytosis and other components of the defense system of the body. The persistence of e.g. staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infections in cystic fibrosis patients are caused by biofilm growing mucoid strains.

Characteristically, gradients of nutrients and oxygen exsist from the top to the bottom of biofilms and these gradients are associated to decrease of bacterial metabolic activity and increase of doubling times of the bacterial cells and these more or less dormant cells are therefore responsible for some of the tolerance to antibiotics.

Biofilm growth is associated with increased level of mutations and with quorum sensing regulated mechanisms. Conventional resistance mechanisms such as chromosomal beta-lactamase, up-regulated efflux pumps and mutations of antibiotic target molecules in the bacteria also contribute to the survival of biofilms. Biofilm can be prevented by early aggressive antibiotic prophylaxis or therapy and they can be treated by chronic suppresive therapy. A promising strategy may be the use of enzymes which can dissolve the biofilm matrix (e.g. DNase, alginate lyase) and quorum sensing inhibitors, which increases biofilm susceptibility to antibiotics.

Introduction

Biofilm growing bacteria cause chronic infections (1) which are characterized by persisting inflammation and tissue damage (2). Chronic infections, including foreign A c c e p t e d M a n u s c r i p t body infections, are infections which 1) persist in spite of antibiotic therapy and the innate and adaptive immune-and inflammatory response of the host, and 2) which, in contrast to colonization, are characterized by immune response and persisting pathology (Table 1).

The occurrence and achitecture of bacterial biofilms

Foreign body infections are characterized by biofilm growth of the bacteria on the outer and/or inner surface of the foreign body (Table 2). Biofilm growth also occurs on natural surfaces such as teeth (3), heart valves (endocarditis), (4) in the lungs of cystic fibrosis (CF) patients causing chronic bronchopneumonia (2), in the middle ear in patients with persistent otitis media (5), in chronic rhinosinusitis (6), in chronic osteomyelitis and prostetic joint infections (7) (8) (9), in intravenous catheters and stents (10) and in chronic wounds (11) (12)(Fig. 1 A & B). The microbes in biofilms are kept together by a self-produced biopolymeric matrix. The matrix contains polysaccharides, proteins and DNA originating from the microbes and the bacterial consortium can consist of one or more species living in a sociomicrobiological way (1,13) (2,14). The matrix is important since it provides structual stability and protection to the biofilm. The development of bacterial biofilms over time has been intensively studied in vitro by confocal scanning laser microscopy employing green fluorescent protein (gfp)-tagged bacteria. This technique has been combined with advanced in silico image-analysing to produce 3-D images of biofilm (15,16) (17).

As an example, P. aeruginosa produce a mature in vitro biofilm in 5-7 days (Fig. 2).

The development of an in vitro biofilm is initiated by planktonic (freely moving) bacteria which are attached reversible to a surface, which may be conditioned of a layer of e.g. proteins (a pellicle) (3) (18). At this stage, the bacteria A c c e p t e d M a n u s c r i p t are still susceptible to antibiotics and this is in accordance with the success of the peroperative antibiotic prophylaxis for e.g. alloplastic surgery. The next step is irrreversible binding to the surface within the next few hours and multiplication of the bacteria which forms microcolonies on the surface and begin to produce a polymer matrix around the microcolonies (18) . The biofilm grows thickness (up to 50 µm) and under in vitro conditions, mushroom-like or tower-like structures are often observed in the mature biofilm. At that stage, the biofilm show maximum tolerance (= resistance) to antibiotics. Subsequently follows a stage where focal areas of the biofilm dissolves and the liberated bacterial cells can then spread to other location where new biofilms can be formed. This liberation process may be caused by bacteriophage activity within the biofilm (19). The mature biofilm may contain water-filled channels and thereby resemble primitive, multicellular organisms. Motile bacteria can use type-1V pili to mount or climb a biofilm formed by other bacteria and colonize the top of the biofilm resembling a hat (16). Important properties of biofilm growing bacteria are different from those of planktonic growing bacteria and this has significant diagnostic and therapeutic consequenses. The bacteria appear different in biofilm infections since they are located close to each other in aggregates surrounded by the self-produced matrix. In clinical specimens (biopsies, pus, sputum) biofilm can often be recognized by light microscopy although precise identification of all the bacteria within a biofilm can only be done by DNA hybridization techniques and identification of the components of the biofilm matrix require specialized staining techniques (2). Traditional sampling techniques may not be suffucient to culture biofilm growing bacteria sticking to a surface unless the bacteria are released by ultasonic pre-treatment (20). The ordinary culture techniques, however, reveals only the properties of planktonically growing bacteria, and e.g. antibiotic susceptibility A c c e p t e d M a n u s c r i p t testing therefore gives misleading results which do not reflect the increased resistance of the bacteria when living in biofilms. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of antibiotics to biofilm growing bacteria may be up to 100 -1000 fold higher compared to planktonic bacteria (21) (22,23). Methods to test biofilm growing bacteria have therefore been developed but their clinical relevance as regards prediction of clinical successfull therapy awaits confirmation (22,24,25).

Stationary phase physiology, low oxygen concentration and slow growth.

Inspection of environmental as well as in vitro biofilms has revealed that oxygen concentration may be high at the surface but low in the center of biofilms where anaerobic conditions may be present (26). Likewise, growth, protein synthesis and metabolic activity is stratified in biofilms i.e. high level of activity at the surface and low level and slow growth or no growth in the center and this is one of the explanations for the reduced susceptibility of biofilms to antibiotics (27) (28). Very slow in situ growth rates of biofilms of P. aeruginosa have been measured in sputum of CF patients (average doubling time 2-3 hours and presence of a significant number of cells in stationary growth fase) (29). Monotherapy with antibiotics like beta-lactams, which are only active against dividing P. aeruginosa cells, are therefore not very efficent to eradicate biofilm infections (21).

Mutators

The mutation frequency of biofilm-growing bacteria is significantly increased compared to planktonically growing isogenic bacteria (30) and there is an increased horizontal gene transmission in biofilms (31). These physiological conditions may explain why biofilm growing bacteria easily become multi-drug resistant by means A c c e p t e d M a n u s c r i p t of traditional resistance mechanisms against beta-lactam antibiotics, aminoglycosides and fluroquinolones which are detected by routine susceptibility testing in the clinical microbiology laboratory where planktonic bacterial growth is investigated. Thus, bacterial cells in the biofilms may simultaneously produce enzymes that degrade antibiotics, have antibiotic targets of low-affinity and overexpresses efflux pumps which have a broad spectrum of substrates. Achievement of multiple mutations in a bacterial population size of 10 8 -10 10 / ml sputum as is attained under infection of the CF lung (32) imply the presence of a hypermutable bacterial subpopulation and the presence of high percentages of hypermutable P. aeruginosa isolates associated with antibiotic resistance have actually been found in CF patients (33) (34).

The hypermutable phenotype of CF P. aeruginosa isolates is due to alterations in the genes of the DNA repair systems of either the mismatch repair system (MMR) which involves mutS, mutL and uvrD or of the DNA oxidative lesions repair system (GO) which involves mutT, mutY and mutM (35,36). It has been shown that mutations in either of the two systems determine emergence of antibiotic resistant isolates, especially due to selection of isolates expressing multidrug efflux-pumps (36,37). An increased production of endogenous reactive oxygen species and a deficient antioxidant system (38) (39) determine an imbalance between oxidative burden and antioxidant defenses leading to oxidative stress in biofilms. The oxidative stress is considered to cause enhanced mutability in biofilms (30,40) . Recent data suggest that microcolony structures, due to endogenous ocidative stress, are specific sites within the biofilms for enhanced genetic adaptation and evolutionary change (40). In addition, Boles and Singh (41) showed that the endogenous oxidative stress in biofilms promote antibiotic resistance and that addition of anti-oxidants reduced the A c c e p t e d M a n u s c r i p t occurrence of diversity in biofilms. We have previously shown that oxidative stress is linked to the occurrence of hypermutable P. aeruginosa strains in CF patients (34).

In addition to their endogenous oxidative stress, the biofilm-growing bacteria in the CF airways are exposed to reactive oxygen species (ROS) from the activated polymorphonuclear leukocytes (PMN) (34). We have recently shown that the hypoxic environment in the CF sputum is due to the consumption of oxygen by PMNs which liberate reactive oxygen species which can react with the biofilm-embedded bacterial cells (42), thus creating an unique environment with low-oxygen tension filled with reactive oxygen species.

The hypermutability of bacteria in biofilms promotes the emergence of mutations conferring antibiotic resistance which will be selected for by the repeated antibiotic courses administered in order to maintain the lung function of CF patients.

Development of resistance to all classes of antibiotics during the chronic lung infection in CF has been documented (43). Resistance to beta-lactam antibiotics occur due to mutations in regulatory genes of the beta-lactamase production leading to the occurrence of isolates with stable or partially-stable derepressed production of AmpC beta-lactamase (44). Resistance to ciprofloxacin of CF P. aeruginosa isolates was shown to be mediated by mutations in gyrA and alterations in the two efflux systems MexCD-OprJ and MexEF-OprN and resistance to tobramycin was overexpression of the MexXY-OprM multi-drug efflux pump (45) (46). Resistance to colistin was shown to occur due to mutations in the pmr system involved in the LPS structure (47).

A c c e p t e d M a n u s c r i p t

Chromosomal beta-lactamase and biofilm matrix components

The overproduction of chromosomally encoded AmpC cephalosporinase is considered the main mechanism of resistance of CF isolates of P. aeruginosa to lactam antibiotics (48). The most common phenotype of beta-lactamase production in CF isolates is the partially derepressed phenotype with high basal levels of betalactamase that can be induced further to higher levels in the presence of beta-lactam antibiotics (44). The role of this beta-lactamase phenotype is important especially for the resistance to beta-lactam antibiotics acting as strong inducers (carbapenems like imipenem). However, not all beta-lactams are strong inducers and the overexpression of the MexAB-OprM efflux pumps may play, together with beta-lactamases, an important role in the resistance to poor inducers (e.g. piperacillin). Totally derepressed beta-lactamase production is encountered in 2.5% of clinical CF isolates (44) and is responsible for the resistance to both poor and strong inducer beta-lactam antibiotics, independent of the presence of efflux pumps overexpression (49) (50) (51).

We have found an insertion sequence (IS 1669) inactivating the ampD gene in several resistant clinical P. aeruginosa isolates with constitutive high expression of chromosomal beta-lactamase (51) .

The diffusion barrier in biofilms (52) plays a role for biofilm resistance of P. aeruginosa that overproduce beta-lactamase due to the presence in the biofilm matrix of beta-lactamases which will hydrolyze the beta-lactam antibiotics before reaching the bacterial cells (53) (54). Giwercman (55) showed that imipenem and piperacillin were able to induce -lactamase production in P. aeruginosa biofilms. Nichols (56) predicted from mathematical models that the biofilm would not afford protection A c c e p t e d M a n u s c r i p t against diffusion of beta-lactam antibiotics into the bacteria embedded in the biofilm as long as the level of chromosomal beta-lactamase is low. However bacteria expressing high level of chromosomal beta-lactamase growing in biofilms would be exposed to reduced concentration of beta-lactam antibiotics due to accumulation of the enzyme in the polysaccharide matrix. The extracellular -lactamase would inactivate the antibiotic as it penetrates, thereby protecting the deeper-lying cells.

The source of -lactamase in biofilms has been considered to be from a layer of lysed bacteria due to exposure to an antibiotic, with release of defensive enzymes into the extracellular space. We have shown that the source of -lactamase in biofilm may also be the membrane vesicles (MVs) containing beta-lactamase liberated by resistant P. aeruginosa bacteria (57)(58)(54) and we have shown, that high level of free chromosomal beta-lactamase is present in CF sputum (58). We have also shown that strong inducers like imipenem will induce the beta-lactamase through all the bacterial layers while poorer inducers like ceftazidime will influence just the superficial layers of the biofilm, probably due to the inactivation of the antibiotic by beta-lactamase (59, 60)(61)(58) (Figure 3). The protective role played by beta-lactamase in impairing the penetration of betalactams in the biofilm can be seen in Fig. 3A. Treatment with ceftazidime of a biofilm formed by a P. aeruginosa CF strain with stable derepressed levels of betalactamase due to an insertion sequence in ampD (P. aeruginosa ampD -) killed very few bacterial cells (dead bacteria in red) (Figure 3B) in contrast to the complemented strain with low level of beta-lactamase (Fig. 3 C). However, addition of aztreonam improved the efficacy of ceftazidime treatment of the biofilm (Figure 3D, probably because aztreonam acts as a beta-lactamase inhibitor (58). In addition, meropenem, a A c c e p t e d M a n u s c r i p t beta-lactamase stable beta-lactam showed good in vitro efficacy in the treatment of P. aeruginosa biofilms (22) (61). Treatment with ceftazidime of a biofilm formed by the same strain expressing basal levels of beta-lactamase due to complemention with the wild-type ampD (P. aeruginosa ampD+) led to eradication of the biofilm (Figure 3C).

The matrix of the biofilm may also be part of the resistance mechanisms to antibiotics since e.g. sub-MIC concentrations of beta-lactam antibiotics induce increased alginate synthesis in P. aeruginosa biofilms (Fig. 4) (62) (63) and also enhance the biofilm matrix of some slime-producing coagulase-negative staphylococci (64) (65). Originally, it has been thought that tolerance of the biofilms to aminoglycosides was due to a transport limitation due to the binding of these positively charged antibiotics to the negatively charged exopolysaccharide matrix but the repeated dosing of antibiotics during therapy probably leads to saturation of the binding sites (66) (67) (68). As previously discussed, the oxygen limitation and the metabolic rates are probably more important factors which contribute to the tolerance of biofilms to aminoglycosides and ciprofloxacin (69). However, in the respiratory zone of the CF lung with poor access to aminoglycoside aerosols, where the antibiotic concentration is low delayed penetration of the aminoglycosides through thick biofilms may play a role in the tolerance of biofilms to aminoglycosides (2). Recently, it has been shown that administration of DNase and alginate lyase enhanced the activity of tobramycin in biofilms by dissolving the biofilm matrix (70).

Tolerance, adaptive resistance and efflux pumps

Colistin is only active against the non-dividing central part of P. aeruginosa biofilms in vitro (Fig. 5A), whereas the superficial, metabolic active part of the biofilm A c c e p t e d M a n u s c r i p t become tolerant due to upregulation of PmrA-PmrB two-component regulatory systems involved in the adaptive resistance to cationic peptides leading to addition of aminoarabinose to lipid A of LPS (22,71,72). Since the metabolic active surface layer of the biofilm is susceptible to ciprofloxacin (Fig. 5B) in contrast to the dormant central part of the biofilm, combination therapy with this drug and colistin was able to kill all cells in the biofilm in vitro (Fig. 5C) (71). The clinical efficacy has been demonstrated of this combination therapy for the early eradication treatment of P. aeruginosa in CF patients (73).

Tolerance of biofilms to tobramycin is also mediated by low metabolic activity but the high cell density that results in accumulation of extracellular signalling molecules is probably important, as it has been shown that tolerance to tobramycin of P. aeruginosa strain PAO1 biofilm is quorum sensing mediated (Fig. 6)(74) (see below).

In addition, a non-specific mechanism for the tolerance of the metabolic active cells to colistin was shown to be up-regulation of the efflux pump MexAB-OprM (71).

Furthermore, increased efflux pump activity due to mutations has been shown to be a major resistance mechanism against aminoglycoside antibiotics and fluorquinolones in P. aeruginosa from CF patients (46) (45).

High cell density and quorum sensing

Bacteria communicate by means of synthizising and reacting on signal molecules (75)(76)(77)(78). The term QS indicates that this system permits bacteria to sense when a critical number (concentration) of bacteria are present in a limited space in the environment and respond by activating certain genes which then produce e.g. virulence factors such as enzymes or toxins. The QS molecules are small peptides in many Gram-A c c e p t e d M a n u s c r i p t positive bacteria whereas the most well-described QS molecules in Gram-negative bacteria are N-acyl-L-homoserinelactones (AHL) (78). For P. aeruginosa QS regulates the production of virulence factors such as extracellular enzymes and cellular lysins (e.g. rhamnolipid), which are important for the pathogenesis of infections where it functions as a protective shield against phagocytes (79, 80) (81).

QS may also have influence on the development of the biofilm (82) and QS have been shown to determine the tolerance of P. aeruginosa biofilms against antibiotic therapy and against the innate inflammatory response dominated by polymorphonuclear leucocytes (PMNs) (83). The connections between QS and biofilms has been named sociomicrobiology (84).

Quorum sensing inhibitors (QSI)

Much of our knowledge about QS orginate from experiments with QS knock-out mutants and from use of naturally occurring and artificially syntetized QSI compounds (85) (86). Screening for QSI in nature has identified many QSI compounds (87). These naturally occurring QSI compounds can be synthesized and their structure modified and used to inhibit QS in vivo in experimental animal infections (85). Since it has been shown, that bacteria used for experimental animal biofilm infections actually communicate in vivo (88) and also in e.g. CF patients with chronic P. aeruginosa lung infection QSI may be used to treat the infection (89).

Interestingly, some macrolide antibiotics like azithromycin (90) but also other antibiotics like ceftazidime and ciprofloxacin (91) inhibit QS in P. aeruginosa at sub-MIC concentrations leading to inhibition of the virulence of these bacteria although they cannot inhibit their growth at obtainable concentrations in vivo. (94). Most CF patients with chronic P. aeruginosa lung infections are therefore now treated continously with azithromycin ( 95). An expected side-effect has, however, been development of resistance to macrolides in other pathogenic bacteria like S. aureus in CF patients (96). It would therefore be desireable to develop QSI without conventional growth-inhibiting or bacteriocidal activity (78). One example of such naturally occurring QSI is found in garlic extract, which in vitro and in vivo have been able to render otherwise resistant P. aeruginosa biofilms susceptible to antibiotic therapy and to PMN activity and the consequence is eradication of the biofilm both as regards antibiotic therapy and PMN activity which dominates the inflammatory response in CF patients (Fig. 7) (97). According to current knowledge, QSI resistance can only occur due to mutations, which renders the QS deficient bacteria unable to produce virulence factors (98) i.e. the bacteria become non-virulent similar to the result of QSI therapy. If this holds thrue, then resistance problems against conventional antibiotics as we face today will not be a clinical problem.

Foreign body infections constitute an steadily increasing medical problem and comprise e.g. intravenous catheters, intrauterine catheters, naso-laryngeal tubes, stents (Table 2, Fig. 1A), alloplastic materials, hydrocephalus shunts and artificial hearts (99) . If foreign bodies become colonized with biofilm-forming bacteria the result is most often chronic inflammation around the foreign body which either has to be replased or treated with sometimes life-long antibiotic suppressive therapy, although early therapy may sometimes lead to eradication of the condition. Antibiotic coated foreign bodies like e.g. catheters, vascular prostesis have been introduced to prevent biofilm formation and they are quite efficient (100) [START_REF] Høiby | Estimated risk of cross-infection with Pseudomonas aeruginosa in Danish cystic fibrosis patients[END_REF]. It is, however, desirable to

Prophylaxis and treatment of P. aeruginosa biofilms in CF lungsperspectives for other biofilm infections?

The currently used methods for preventing chronic P. aeruginosa biofilms in CF lungs are 1) prevention of cross-infection from other already chronically infected CF patients by isolation techniques and hygiejnic measures (103), 2) early aggressive eradication therapy of intermittent colonization by means of oral ciprofloxacin and nebulized colistin for 3 weeks or even better for 3 months or by using nebulized tobramycin as monotherapy [START_REF] Baumann | Cost of care and clinical condition in paediatric cystic fibrosis patients[END_REF], 3) daily nebulized DNase (Pulmozyme) (105). These 3 methods, which are combined in most CF centers, are successful and costefficient and has completely changed the epidemiology of chronic P. aeruginosa lung infection in CF patients from being very common in CF children to being predominantly a problem for adult patients and no problems of resistance to the antibiotics have been recorded (73,[START_REF] Van Gennip | Augmented effect of early antibiotic therapy in mice with experimental lung infections due to sequentially adapted mucoid strains of Pseudomonas aeruginosa[END_REF][START_REF] Döring | Vaccines and immunotherapy against Pseudomonas aeruginosa[END_REF] . Early aggressive eradication therapy has also been shown to superior in an animal model of P. aeruginosa infection in CF [START_REF] Doring | Antibiotic therapy against Pseudomonas aeruginosa in cystic fibrosis: a European consensus[END_REF]. Furthermore, although vaccines against P. aeruginosa have been developed and undergone clinical trials, they have not been further developed due to the success of the early, aggressive eradication therapy [START_REF] Høiby | Pseudomonas aeruginosa infection in cystic fibrosis. Diagnostic and prognostic significance of Pseudomonas aeruginosa precipitins determined by means of crossed immunoelectrophoresis[END_REF].

The recommended method for treatment of chronic P. aeruginosa biofilm A c c e p t e d M a n u s c r i p t infection is chronic suppressive antibiotic therapy (2)(110) (Fig. 8) which is started when the chronic infection is diagnosed (continuous colonization at the monthly bacteriological examination for 6 months and/or increased level of antibodies against P. aeruginosa (111)(112) . The chronic suppressive therapy consists of daily nebulized colistin or tobramycin for the rest of the patient's life combined with either regular 2-week courses every 3 months of intravenous anti-pseudomonas antibiotics (combination therapy of 2 antibitics: tobramycin or colistin + ceftazidime, or piperacillin/tazobactam, or carbapenem, or aztreonam, or ciprofloxacin) or ad hoc intravenous therapy when clinical deteriorations occur [START_REF] Proesmans | Evaluating the 'Leeds criteria' for Pseudomonas aeruginosa infection in a cystic fibrosis centre[END_REF]. Additionally, DNase is inhaled every day to reduce the viscocity of the DNA-containing sputum [START_REF] Frederiksen | The changing epidemiology of Pseudomonas aeruginosa infection in Danish cystic fibrosis patients, 1974-1995[END_REF], and daily oral azithromycin is given to the patients as mentioned above (95). The chronic suppressive therapy (maintenance therapy) has successfully been able to maintain the pulmonary function or slow the decline of the pulmonary function and prolong the life of the patients for many years (113). The side-effects of the maintenance therapy is high level of conventional resistance mechanisms in the persisting strains and high level of allergy to the beta-lactam antibiotics (114) (43). Similar principles, a) systemic or local prophylactic use of antibiotics to prevent biofilm formation, b) early aggressive eradication therapy to eradicate planktonic growth or early biofilm formation and c) chronic suppressive antibiotic therapy to maintain the function of a inserted foreign body are gradually being introduced in other area of biofilm infection (100) (8) (9). Possibly other approaches taken advantage of the use of QSI may further improve the management of biofilm infections. Characteristically, gradients of nutrients and oxygen exist from the top to the bottom of biofilms and these gradients are associated with decreased bacterial metabolic activity and increased doubling times of the bacterial cells;

it is these more or less dormant cells that are responsible for some of the tolerance to antibiotics. Biofilm growth is associated with an increased level of mutations as well as with quorum-sensing-regulated mechanisms.

Conventional resistance mechanisms such as chromosomal -lactamase, upregulated efflux pumps and mutations in antibiotic target molecules in bacteria also contribute to the survival of biofilms. Biofilms can be prevented by early aggressive antibiotic prophylaxis or therapy and they can be treated by chronic suppressive therapy. A promising strategy may be the use of enzymes that can dissolve the biofilm matrix (e.g. DNase and alginate lyase)

as well as quorum-sensing inhibitors that increase biofilm susceptibility to antibiotics.

Introduction

Biofilm-growing bacteria cause chronic infections [1] characterised by persistent inflammation and tissue damage [2]. Chronic infections, including foreign-body infections, are infections that (i) persist despite antibiotic therapy and the innate and adaptive immune and inflammatory responses of the host and (ii) in contrast to colonisation, are characterised by an immune response and persisting pathology (Table 1).

Occurrence and architecture of bacterial biofilms

Foreign-body infections are characterised by biofilm growth of bacteria on the outer and/or inner surface of the foreign body (Table 2). Biofilm growth also occurs on natural surfaces such as teeth [3], heart valves (endocarditis) [4], in the lungs of cystic fibrosis (CF) patients causing chronic bronchopneumonia [2], in the middle ear in patients with persistent otitis media [5], in chronic rhinosinusitis [6], in chronic osteomyelitis and prosthetic joint infections [7][8][9],

in intravenous (i.v.) catheters and stents [10] and in chronic wounds [11,12] (Fig. 1). The microbes in biofilms are kept together by a self-produced biopolymer matrix. The matrix contains polysaccharides, proteins and DNA originating from the microbes, and the bacterial consortium can consist of one or more species living in sociomicrobiological way [1,2,14,15]. The matrix is important since it provides structural stability and protection to the biofilm. advanced in silico image analysis to produce three-dimensional images of the biofilm [16][17][18]. As an example, Pseudomonas aeruginosa produces a mature in vitro biofilm in 5-7 days (Fig. 2).

Development of an in vitro biofilm is initiated by planktonic (freely moving) bacteria that reversibly attach to a surface, which may be covered by a layer of, for example, proteins (a pellicle) [3,20]. At this stage, the bacteria are still susceptible to antibiotics and this is in accordance with the success of perioperative antibiotic prophylaxis, e.g. for alloplastic surgery. The next step is irreversible binding to the surface within the next few hours and multiplication of the bacteria, which form microcolonies on the surface and begin to produce a polymer matrix around the microcolonies [20]. The biofilm grows in thickness (up to 50 m) and under in vitro conditions mushroom-like or tower-like structures are often observed in the mature biofilm. At that stage, the biofilm shows maximum tolerance (= resistance) to antibiotics.

Subsequently, a stage follows where focal areas of the biofilm dissolve and the liberated bacterial cells can then spread to another location where new biofilms can be formed. This liberation process may be caused by bacteriophage activity within the biofilm [21]. The mature biofilm may contain water-filled channels and thereby resemble primitive, multicellular organisms.

Motile bacteria can use type 1V pili to mount or climb a biofilm formed by other bacteria and colonise the top of the biofilm, resembling a hat [17].

Important properties of biofilm-growing bacteria are different from those of planktonic bacteria and this has significant diagnostic and therapeutic consequences. The bacteria appear different in biofilm infections since they be up to 100-1000-fold higher compared with planktonic bacteria [23][24][25].

Methods to test biofilm-growing bacteria have therefore been developed, but their clinical relevance with regard to prediction of clinically successful therapy awaits confirmation [24,26,27].

Stationary-phase physiology, low oxygen concentration and slow growth

Inspection of environmental as well as in vitro biofilms has revealed that the oxygen concentration may be high at the surface but low in the centre of the biofilm where anaerobic conditions may be present [28]. Likewise, growth, protein synthesis and metabolic activity is stratified in biofilms, i.e. a high level A c c e p t e d M a n u s c r i p t of activity at the surface and a low level and slow or no growth in the centre, and this is one of the explanations for the reduced susceptibility of biofilms to antibiotics [29,30]. Very slow in situ growth rates of P. aeruginosa biofilms have been measured in the sputum of CF patients (average doubling time 2-3 h and the presence of a significant number of cells in stationary growth phase) [31]. Monotherapy with antibiotics such as -lactams, which are only active against dividing P. aeruginosa cells, are therefore not very efficient at eradicating biofilm infections [23].

Mutators

The mutation frequency of biofilm-growing bacteria is significantly increased compared with planktonically growing isogenic bacteria [32] and there is increased horizontal gene transmission in biofilms [33]. These physiological conditions may explain why biofilm-growing bacteria easily become multidrug resistant by means of traditional resistance mechanisms against -lactam antibiotics, aminoglycosides and fluoroquinolones, which are detected by routine susceptibility testing in the clinical microbiology laboratory where planktonic bacterial growth is investigated. Thus, bacterial cells in biofilms may simultaneously produce enzymes that degrade antibiotics, have antibiotic targets of low affinity and overexpress efflux pumps that have a broad range of substrates. Achievement of multiple mutations in a bacterial population size of 10 8 -10 10 /mL of sputum, as attained in infection of the CF lung [34], implies the presence of a hypermutable bacterial subpopulation, and the presence of A c c e p t e d M a n u s c r i p t high percentages of hypermutable P. aeruginosa isolates associated with antibiotic resistance has actually been found in CF patients [35,36].

The hypermutable phenotype of CF P. aeruginosa isolates is due to alterations in genes of the DNA repair systems of either the mismatch repair system (MMR), which involves mutS, mutL and uvrD, or the DNA oxidative lesions repair system (GO), which involves mutT, mutY and mutM [37,38]. It has been shown that mutations in either of the two systems determine the emergence of antibiotic-resistant isolates, especially due to selection of isolates expressing multidrug efflux pumps [38,39]. Increased production of endogenous reactive oxygen species (ROS) and a deficient antioxidant system [40,41] determine an imbalance between oxidative burden and antioxidant defences leading to oxidative stress in biofilms. This oxidative stress is considered to cause enhanced mutability in biofilms [32,42]. Recent data suggest that microcolony structures, due to endogenous oxidative stress, are specific sites within biofilms for enhanced genetic adaptation and evolutionary change [42]. In addition, Boles and Singh [43] showed that the endogenous oxidative stress in biofilms promotes antibiotic resistance and that addition of antioxidants reduced the occurrence of diversity in biofilms.

We have previously shown that oxidative stress is linked to the occurrence of hypermutable P. aeruginosa strains in CF patients [36].

In addition to their endogenous oxidative stress, biofilm-growing bacteria in the CF airways are exposed to ROS from activated polymorphonuclear leukocytes (PMNs) [36]. We have recently shown that the hypoxic A c c e p t e d M a n u s c r i p t environment in CF sputum is due to the consumption of oxygen by PMNs which liberate ROS that can react with the biofilm-embedded bacterial cells [44], thus creating an unique environment with low oxygen tension filled with ROS.

The hypermutability of bacteria in biofilms promotes the emergence of mutations conferring antibiotic resistance, which will be selected for by the repeated antibiotic courses administered in order to maintain the lung function of CF patients. Development of resistance to all classes of antibiotics during chronic lung infection in CF has been documented [45]. Resistance tolactam antibiotics occurs due to mutations in the regulatory genes oflactamase production leading to the occurrence of isolates with stable or partially stable derepressed production of AmpC -lactamase [46]. Resistance to ciprofloxacin of CF P. aeruginosa isolates was shown to be mediated by mutations in gyrA and alterations in two efflux systems (MexCD-OprJ and MexEF-OprN), and resistance to tobramycin was due to overexpression of the MexXY-OprM multidrug efflux pump [47,48]. Resistance to colistin was shown to occur due to mutations in the pmr system involved in the lipopolysaccharide (LPS) structure [49].

Chromosomal -lactamase and biofilm matrix components

Overproduction of chromosomally encoded AmpC cephalosporinase is considered the main mechanism of resistance of CF P. aeruginosa isolates to -lactam antibiotics [50]. The most common -lactamase production phenotype in CF isolates is the partially derepressed phenotype with high basal levels of -lactamase that can be further induced to higher levels in the presence of -lactam antibiotics [46]. The role of this -lactamase phenotype is especially important for resistance to -lactam antibiotics acting as strong inducers (carbapenems such as imipenem). However, not all -lactams are strong inducers and overexpression of the MexAB-OprM efflux pump may, together with -lactamases, play an important role in resistance to poor inducers (e.g. piperacillin). Totally derepressed -lactamase production is encountered in 2.5% of clinical CF isolates [46] and is responsible for resistance both to poor-and strong-inducer -lactam antibiotics, independent of the presence of efflux pump overexpression [51,52]. We have found an insertion sequence (IS1669) inactivating the ampD gene in several resistant clinical P. aeruginosa isolates with constitutive high expression of chromosomal -lactamase [52].

The diffusion barrier in biofilms [53] plays a role in biofilm resistance of P.

aeruginosa that overproduce -lactamase owing to the presence in the biofilm matrix of -lactamases that will hydrolyse the -lactam antibiotics before reaching the bacterial cells [54,55]. Giwercman et al. [56] showed that imipenem and piperacillin were able to induce -lactamase production in P. aeruginosa biofilms. Nichols et al. [57] predicted from mathematical models that the biofilm would not afford protection against diffusion of -lactam antibiotics into the bacteria embedded in the biofilm as long as the level of chromosomal -lactamase was low. However, bacteria expressing a high The source of -lactamase in biofilms has been considered to be from a layer of lysed bacteria owing to exposure to an antibiotic, with release of defensive enzymes into the extracellular space. We have shown that the source oflactamase in biofilm may also be the membrane vesicles containinglactamase liberated by resistant P. aeruginosa bacteria [55,58] and we have

shown that a high level of free chromosomal -lactamase is present in CF sputum [58]. We have also shown that strong inducers such as imipenem will induce the -lactamase through all the bacterial layers, whilst poorer inducers such as ceftazidime will influence just the superficial layers of the biofilm, probably due to inactivation of the antibiotic by -lactamase [58][59][60] (Fig. 3).

The protective role played by -lactamases in impairing the penetration oflactams in the biofilm can be seen in Fig. 3A. Treatment with ceftazidime of a biofilm formed by a P. aeruginosa CF strain with stable derepressed levels of -lactamase due to an insertion sequence in ampD (P. aeruginosa ampD -) killed very few bacterial cells (dead bacteria in red) (Fig. 3B) in contrast to the complemented strain with a low level of -lactamase (P. aeruginosa ampD + ) (Fig. 3C). However, addition of aztreonam improved the efficacy of ceftazidime treatment of the biofilm (Fig. 3D), probably because aztreonam A c c e p t e d M a n u s c r i p t acts as a -lactamase inhibitor [58]. In addition, meropenem, a -lactamasestable -lactam, showed good in vitro efficacy in the treatment of P.

aeruginosa biofilms [24,60]. Treatment with ceftazidime of a biofilm formed by the same strain expressing basal levels of -lactamase due to complementation with the wild-type ampD (P. aeruginosa ampD + ) led to eradication of the biofilm (Fig. 3C).

The matrix of the biofilm may also be part of the resistance mechanism to antibiotics since, for example, sub-MIC concentrations of -lactam antibiotics induce increased alginate synthesis in P. aeruginosa biofilms (Fig. 4) [62,63] and also enhance the biofilm matrix of some slime-producing coagulasenegative staphylococci [64,65]. Originally, it was thought that tolerance of biofilms to aminoglycosides was the result of transport limitation due to the binding of these positively charged antibiotics to the negatively charged exopolysaccharide matrix, but the repeated dosing of antibiotics during therapy probably leads to saturation of the binding sites [66][67][68]. As discussed previously, oxygen limitation and the metabolic rate are probably more important factors contributing to the tolerance of biofilms to aminoglycosides and ciprofloxacin [69]. However, in the respiratory zone of the CF lung with poor access to aminoglycoside aerosols, where the antibiotic concentration is low, delayed penetration of aminoglycosides through thick biofilms may play a role in the tolerance of biofilms to these aminoglycosides [2]. Recently, it has been shown that administration of DNase and alginate lyase enhanced the activity of tobramycin in biofilms by dissolving the biofilm matrix [70].

A c c e p t e d M a n u s c r i p t

Tolerance, adaptive resistance and efflux pumps

Colistin is only antimicrobial active against the non-dividing central part of P.

aeruginosa biofilms in vitro (Fig. 5A), whereas the superficial, metabolically active part of the biofilm becomes tolerant due to upregulation of the PmrA-PmrB two-component regulatory system involved in adaptive resistance to cationic peptides leading to addition of aminoarabinose to lipid A of LPS [24,71,72]. Since the metabolically active surface layer of the biofilm is susceptible to ciprofloxacin (Fig. 5B), in contrast to the dormant central part of the biofilm, combination therapy with ciprofloxacin and colistin was able to kill all cells in the biofilm in vitro (Fig. 5C) [71]. Clinical efficacy has been demonstrated with this combination therapy for the early eradication treatment of P. aeruginosa in CF patients [73].

Tolerance of biofilms to tobramycin is also mediated by low metabolic activity, but the high cell density that results in accumulation of extracellular signalling molecules is probably important, as it has been shown that tolerance to tobramycin of P. aeruginosa strain PAO1 biofilm is quorum-sensing-mediated (Fig. 6) [74] (see below). In addition, a non-specific mechanism for the tolerance of the metabolically active cells to colistin was shown to be upregulation of the MexAB-OprM efflux pump [71]. Furthermore, increased efflux pump activity due to mutations has been shown to be a major resistance mechanism against aminoglycoside antibiotics and fluoroquinolones in P. aeruginosa from CF patients [47,48].

A c c e p t e d M a n u s c r i p t

High cell density and quorum sensing (QS)

Bacteria communicate by means of synthesising and reacting on signal molecules [75][76][77][78]. The term QS indicates that this system allows bacteria to sense when a critical number (concentration) of bacteria are present in a limited space in the environment and respond by activating certain genes that then produce, for example, virulence factors such as enzymes or toxins. The QS molecules are small peptides in many Gram-positive bacteria, whereas the most well described QS molecules in Gram-negative bacteria are N-acyl-L-homoserine lactones [78]. For P. aeruginosa, QS regulates the production of virulence factors such as extracellular enzymes and cellular lysins (e.g. rhamnolipid), which are important for the pathogenesis of infections where it functions as a protective shield against phagocytes [79][80][81]. QS may also influence the development of the biofilm [82] and QS has been shown to determine the tolerance of P. aeruginosa biofilms to antibiotic therapy and to the innate inflammatory response dominated by PMNs [74]. The connection between QS and biofilms has been named sociomicrobiology [83].

Quorum-sensing inhibitors (QSIs)

Much of our knowledge about QS originates from experiments with QS knockout mutants and from the use of naturally occurring and artificially synthesised QSI compounds [84,85]. Screening for QSIs in nature has identified many QSI compounds [86]. These naturally occurring QSI compounds can be synthesised and their structure modified and used to inhibit QS in vivo in experimental animal infections [84]. Since it has been shown that bacteria 15 used for experimental animal biofilm infections actually communicate in vivo [87] and also in, for example, CF patients with chronic P. aeruginosa lung infection, QSIs may be used to treat these infections [88]. Interestingly, some macrolide antibiotics such as azithromycin [89], but also other antibiotics such as ceftazidime and ciprofloxacin [90], inhibit QS in P. aeruginosa at sub-MIC concentrations, leading to inhibition of the virulence of these bacteria even though they cannot inhibit their growth at concentrations obtainable in vivo.

Controlled clinical trials using azithromycin to treat chronic P. aeruginosa lung infection both in CF children and adults have shown significant improvement of their lung function [91][92][93]. Most CF patients with chronic P. aeruginosa lung infections are therefore now treated continuously with azithromycin [94].

An expected side effect, however, has been the development of resistance to macrolides in other pathogenic bacteria such as Staphylococcus aureus in CF patients [95]. It would therefore be desirable to develop QSIs without conventional growth-inhibiting or bactericidal activity [78]. One example of such a naturally occurring QSI is found in garlic extract, which in vitro and in vivo has been able to render otherwise resistant P. aeruginosa biofilms susceptible to antibiotic therapy and to PMN activity and the consequence is eradication of the biofilm both with regard to antibiotic therapy and PMN activity which dominates the inflammatory response in CF patients (Fig. 7) [96]. According to current knowledge, QSI resistance can only occur due to mutations, which render the QS-deficient bacteria unable to produce virulence factors [97], i.e. the bacteria become non-virulent, similar to the result of QSI therapy. If this holds true, then resistance problems against conventional antibiotics as we face today will not be a clinical problem.

A c c e p t e d M a n u s c r i p t 16 Foreign-body infections constitute a steadily increasing medical problem and comprise, for example, infections of i.v. catheters, intrauterine catheters, nasolaryngeal tubes, stents (Table 2; Fig. 1A), alloplastic materials, hydrocephalus shunts and artificial hearts [98]. If foreign bodies become colonised with biofilm-forming bacteria the result is most often chronic inflammation around the foreign body that either has to be replaced or treated with sometimes life-long antibiotic suppressive therapy, although early therapy may sometimes lead to eradication of the condition. Antibiotic-coated foreign bodies, e.g. catheters and vascular prostheses, have been introduced to prevent biofilm formation and they are quite efficient [99,[START_REF] Reid | Biofilms in infectious disease and on medical devices[END_REF]. However, it is desirable to develop other compounds owing to the risk of development of bacterial resistance, and QSIs are strong candidates. QSIs have been shown

to improve synergistically the weak effects of antibiotics and PMNs on biofilmgrowing bacteria in vitro and in vivo in animal experiments, leading to elimination of biofilms (Fig. 7) [79]. These results have led to further development of QSIs as pharmaceutical compounds for patients who are subject to implantation of foreign bodies.

Prophylaxis and treatment of Pseudomonas aeruginosa biofilms in cystic fibrosis lungs: perspectives for other biofilm infections?

The currently used methods for preventing chronic P. aeruginosa biofilms in CF lungs are (i) prevention of cross-infection from other already chronically early aggressive eradication therapy of intermittent colonisation by means of oral ciprofloxacin and nebulised colistin for 3 weeks or, even better, for 3 months or by using nebulised tobramycin as monotherapy [START_REF] Döring | Early intervention and prevention of lung disease in cystic fibrosis: a European consensus[END_REF] and (iii) daily nebulised DNase (Pulmozyme ® ) [START_REF] Frederiksen | Effect of aerosolised rhDNase (Pulmozyme ® ) on pulmonary colonization in patients with cystic fibrosis[END_REF]. These three methods, which are combined in most CF centres, are successful and cost efficient and have completely changed the epidemiology of chronic P. aeruginosa lung infection in CF patients from being very common in CF children to being predominantly a problem for adult patients, and no problems of resistance to the antibiotics have been recorded [73,[START_REF] Baumann | Cost of care and clinical condition in paediatric cystic fibrosis patients[END_REF][START_REF] Frederiksen | The changing epidemiology of Pseudomonas aeruginosa infection in Danish cystic fibrosis patients, 1974-1995[END_REF]. Early aggressive eradication therapy has also been shown to superior in an animal model of P. aeruginosa infection in CF [START_REF] Van Gennip | Augmented effect of early antibiotic therapy in mice with experimental lung infections due to sequentially adapted mucoid strains of Pseudomonas aeruginosa[END_REF]. Furthermore, although vaccines against P. aeruginosa have been developed and have undergone clinical trials, they have not been further developed owing to the success of early aggressive eradication therapy [START_REF] Döring | Vaccines and immunotherapy against Pseudomonas aeruginosa[END_REF].

The recommended method for treatment of chronic P. aeruginosa biofilm infection is chronic suppressive antibiotic therapy [2,[START_REF] Doring | Antibiotic therapy against Pseudomonas aeruginosa in cystic fibrosis: a European consensus[END_REF] (Fig. 8), which is started when the chronic infection is diagnosed (continuous colonisation at the monthly bacteriological examination for 6 months and/or increased level of antibodies against P. aeruginosa) [START_REF] Høiby | Pseudomonas aeruginosa infection in cystic fibrosis. Diagnostic and prognostic significance of Pseudomonas aeruginosa precipitins determined by means of crossed immunoelectrophoresis[END_REF][START_REF] Proesmans | Evaluating the 'Leeds criteria' for Pseudomonas aeruginosa infection in a cystic fibrosis centre[END_REF]. Chronic suppressive therapy consists of daily nebulised colistin or tobramycin for the rest of the patient's life combined with either regular 2-week courses every 3 months of i.v.

antipseudomonal antibiotics (combination therapy of two antibiotics:

tobramycin or colistin + ceftazidime, or piperacillin/tazobactam, or carbapenem, or aztreonam, or ciprofloxacin) or ad hoc i.v. therapy when A c c e p t e d M a n u s c r i p t clinical deterioration occurs [START_REF] Doring | Antibiotic therapy against Pseudomonas aeruginosa in cystic fibrosis: a European consensus[END_REF]. Additionally, DNase is inhaled every day to reduce the viscosity of the DNA-containing sputum [START_REF] Frederiksen | Effect of aerosolised rhDNase (Pulmozyme ® ) on pulmonary colonization in patients with cystic fibrosis[END_REF], and daily oral azithromycin is given to patients as mentioned above [94]. Chronic suppressive therapy (maintenance therapy) has successfully been able to maintain pulmonary function or slow the decline in pulmonary function and prolong the life of patients for many years [START_REF] Frederiksen | Improved survival in the Danish cystic fibrosis centre: results of aggressive treatment[END_REF]. The side effects of maintenance therapy is a high level of conventional resistance mechanisms in the persisting strains and a high level of allergy to -lactam antibiotics [45,112]. Similar principles, e.g. PMNs were stained by 4',6-diamidino-2-phenylindole (DAPI). Reproduced from [11] with permission. aeruginosa biofilm with a combination of ciprofloxacin and colistin.

Pseudomonas aeruginosa PAO1 expressing GFP as a tag was grown as a biofilm in a flow chamber for 4 days. PI was added after Day 4 to monitor continuously the killing of the biofilm by ciprofloxacin and colistin. The image shows the biofilm after 2 days of treatment with 10 g/mL ciprofloxacin and 25 g/mL colistin. Reproduced from [71] with permission.

  Controlled

A

  c c e p t e d M a n u s c r i p t clinical trials using azithromycin to treat the chronic P. aeruginosa lung infection in both CF children and adults have shown significant improvement of their lung function(92) (93)

A

  c c e p t e d M a n u s c r i p t develop other compounds due to the risk of development of bacterial resistance and QSIs are strong candidates. QSI have been shown synergistically to improve the weak effects of antibiotics and PMNs on biofilm growing bacteria in vitro and in vivo in animal experiments leading to elimination of biofilms (Fig. 7) (102). These results have lead to further development of QSI as pharmaceutical compounds for patients who are subject to implantation of foreign bodies.

Funding

  A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t

  Development of bacterial biofilms over time has been intensively studied in vitro by confocal scanning laser microscopy employing green fluorescent protein (GFP)-tagged bacteria. This technique has been combined with A c c e p t e d M a n u s c r i p t

A c c e p t e d M a n u s c r i p t . 6 6

 6 are located close to each other in aggregates surrounded by the selfproduced matrix. In clinical specimens (biopsies, pus, sputum), biofilm can often be recognised by light microscopy, although precise identification of all the bacteria within a biofilm can only be done by DNA hybridisation techniques, and identification of the components of the biofilm matrix require specialised staining techniques[2]. Traditional sampling techniques may not be sufficient to culture biofilm-growing bacteria sticking to a surface unless the bacteria are released by ultrasonic pre-treatment[22]. Ordinary culture techniques, however, reveal only the properties of planktonically growing bacteria and, for example, antibiotic susceptibility testing therefore gives misleading results that do not reflect the increased resistance of the bacteria living in biofilms. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of antibiotics to biofilm-growing bacteria may

  chromosomal -lactamase growing in biofilms would be exposed to a reduced concentration of -lactam antibiotics owing to accumulation of the enzyme in the polysaccharide matrix. The extracellular -lactamase would inactivate the antibiotic as it penetrates, thereby protecting the deeper-lying cells.

A c c e p t e d M a n u s c r i p t . 15

 15

  by isolation techniques and hygienic measures[START_REF] Høiby | Estimated risk of cross-infection with Pseudomonas aeruginosa in Danish cystic fibrosis patients[END_REF], (ii)

  (a) systemic or local prophylactic use of antibiotics to prevent biofilm formation, (b) early aggressive eradication therapy to eradicate planktonic growth or early biofilm formation and (c) chronic suppressive antibiotic therapy to maintain the function of an inserted foreign body, are gradually being introduced in other areas of biofilm infection [8,9,99]. Possibly other approaches taking advantage of the use of QSIs may further improve the management of biofilm infections.

  Koch C, Hjelt K, Pedersen SS, Jensen ET, Jensen T, Lanng S, et al. Retrospective clinical study of hypersensitivity reactions to aztreonam and six other -lactam antibiotics in cystic fibrosis patients receiving multiple treatment courses. Rev Infect Dis 1991;13(Suppl 7):S608-11.

A c c e p t e d M a n u s c r i p t

Fig. 1 .

 1 Fig. 1. (A) Example of a biofilm on a biliary stent, which became the focus of

Fig. 2 .

 2 Fig. 2. Time course of formation of a Pseudomonas aeruginosa biofilm based

Fig. 3 .

 3 Fig. 3. (A) Induction of -lactamase in Pseudomonas aeruginosa biofilm.

Fig. 4 .Fig. 5 .

 45 Fig. 4. Induction of alginate in Pseudomonas aeruginosa biofilms treated with

Fig. 6 .

 6 Fig. 6. Treatment of Pseudomonas aeruginosa biofilm with tobramycin. Wild-

Fig. 7 .

 7 Fig. 7. Four-day-old biofilm formed by green fluorescent protein-tagged

Fig. 8 .

 8 Fig. 8. Gram stain ( 1000) of an explanted lung with a biofilm of

A c c e p t e d M a n u s c r i p t . 1 1 1

 11 Table Some general features of biofilm infections in humans compared with acute planktonic infections and superficial colonisation/normal flora on skin and mucosal membranes. Bold indicates biofilm-

  Fig. 1A.

Figure 1 .

 1 Figure 1. A. Example of a biofilm on a biliary stent which became focus of repeated incidents of sepsis which ultimately was lethal. DNA typning (pulsed field gel electrophorese) showed that it was the same cloneal type of E. coli in the biofilm and in the blood. In spite of relevant antibiotic therapy it was not possible to eradicate the biliary focus on the stent and the second incident of sepsis was lethal. Microphotos: Gram-and methylene blue staining, magnification x 100 and x 1000. Reproduced with permission from Høiby et al. Ugeskrift for Laeger 169:4163-4166;, 2007. B Chronic infected wound with microcolonies of P. aeruginosa surrounded but not penetrated by polymorphonuclear leukocytes. The bacteria are identified by use of fluorescence in situ hybridization (FISH) using a fluorescein-labelled (FITC) peptide nucleic acid (PNA) specific for P. aeruginosa. The polymorphonuclear leukocytes were stained by DAPI. Reproduced from (11) with permission.

A c c e p t e d M a n u s c r i p t

Figure 2 .

 2 Figure 2. Time-course of formation of a P. aeruginosa biofilm based on in vitro

Figure 3B .

 3B Figure 3B. Treatment of P. aeruginosa biofilm with beta-lactams. P. aeruginosa ampD -(levels AmpC beta-lactamase (mU) :1050 basal, 4255 induced) expressing

A c c e

  Figure 3C. Treatment of P. aeruginosa biofilm with beta-lactams. P. aeruginosa

A c c e p t e d M a n u s c r i p tFigure 4

 4 Figure 4 Induction of alginate in P. aeruginosa biofilms treated with sub-MIC

A c c e p t e d M a n u s c r i p tFigure 5A .

 5A Figure 5A. Treatment of P. aeruginosa biofilms with colistin. P. aeruginosa PAO1 expressing green fluorescent protein (gfp) as a tag were grown as biofilms in flowchambers for 4 days. Propidium iodide was added after day 4 to continuosly monitor the killing of the biofilm by colistin. The picture shows the biofilm after 2 days of treatment with colistin 25 µg/ml. Reproduced from(71) with permission.

Figure 5 B

 5 Figure 5 B. Treatment of P. aeruginosa biofilms with ciprofloxacin. P. aeruginosa PAO1 expressing green fluorescent protein (gfp) as a tag was grown as biofilm in a flow-chamber for 4 days and was treated for 2 days with ciprofloxacin 10 µg/ml (A). Propidium iodide was added after day 4 to continuosly monitor the killing of the biofilm by ciprofloxacin. Red staining shows that ciprofloxacin kills the bacteria located at the surface of the biofilm. Reproduced from (71)with permission.

A c c e p t e d M a n u s c r i p tFig. 7 .

 7 Fig. 7. A 4 day old biofilm formed by green fluorescent protein-tagged P. aeruginosa

Table 1 .

 1 Some general features of biofilm infections in humans compared to acute planktonic infections and superficial colonozation/normal flora on skin and mucosal membranes. The bold fonts indicate biofilm specific features.

	Table
	Features of	Necessary	Sufficient	Also found in	Also found in
	biofilm infections	condition for	condition for	acute planktonic	colonization/ normal
		biofilm	biofilm infecions	infections	flora on skin and
		infections			mucosal membranes
	Aggregates of	Yes	Yes	No	No/Yes
	bacteria
	embedded in a
	self-produced
	polymer matrix
	Tolerant to	Yes	Yes	No	No/Yes
	clinical relevant
	PK/PD dosing of
	antibiotics in
	spite of
	susceptibility of
	planktonic cells
	Tolerant to both	Yes	Yes	No	No/Yes -unknown (s-
	innate and				IgA)
	adaptive
	immune reponse
	Inflammation	Yes	No	Yes	No
	Biofilm-specific	No and Yes -	No and Yes -	No	No
	antigens	seldom -e.g.	seldom -e.g.
		P.seudomonas	Pseudomonas
		aeruginosa	aeruginosa
		alginate	alginate
	Antibody	Yes -after some	No	Yes -after some	No
	response	weeks		weeks
	Chronic	Yes	Yes	No	No
	infections
	Foreign body	No	Yes	No but yes the	No
	associated			first day of
	infections			infection
	Located on	No	No	Yes	Yes
	surfaces
	Localized	Yes	No	Yes	Yes
	infetion
	Focus for	Yes	No	Yes	Yes
	spreading or local
	exacerbation

Table 2 .

 2 Natural and pathogenic biofilms on human tissue and foreign bodies.

	Table
	'Organ A' with normal	Connection via	'Organ B' without
	flora	foreign bodies	normal flora
	Skin		blood, peritoneum, middle ear
	Mouth		teeth
	Pharynx		bronchi, lungs,
	Duodenum		bile tract, pancreas
	Urethra		urine bladder
	Vagina		uterus
	'Air in operation room'*		alloplastic, cerebrospinal shunt
	No symptoms		pathologi

*most frequently coagulase negative staphylococci, which occur as biofilms on detached epidermal cells.

Table 2

 2 Natural and pathogenic biofilms on human tissue and foreign bodies

	'Organ A' with normal	Connection via foreign	'Organ B' without normal
	flora	bodies	flora
	Skin		blood, peritoneum, middle
			ear
	Mouth		teeth
	Pharynx		bronchi, lungs
	Duodenum		bile tract, pancreas
	Urethra		urine, bladder
	Vagina		uterus
	'Air in operation room'		alloplastic, cerebrospinal
	a		shunt
	No symptoms		pathology

a Most frequently, coagulase-negative staphylococci, which occur as biofilms on detached epidermal cells.

Funding

None.

Antibiotic resistance of bacterial biofilms

Niels Høiby a,b, *, Thomas Bjarnsholt a,b , Michael Givskov b , Søren Molin c ,


Stephanie K Henderson-Begg

Carmen L Sheppard

Robert C George

David M Livermore

Lucinda M C Hall

Mutation frequency in antibiotic-resistant and -susceptible isolates of Streptococcus pneumoniae

Keywords:

typically involves either mutation or transformation at several well separated chromosomal loci. We postulated that this series of genetic events would be more likely to occur in organisms with deficient DNA repair mechanisms.

Investigation of 27 antibiotic-resistant or -susceptible clinical isolates of S. pneumoniae revealed a broad range of mutation frequencies, but no isolate was as mutable as a mismatch repair (MMR)-deficient laboratory isolate. No correlation was observed between antibiotic resistance and higher mutation frequency. Examination of a further 180 clinical isolates using a newly developed rapid screen method also failed to identify any isolates with a mutation frequency as high as the MMR-deficient control strain. We argue that there is currently no clear evidence of a distinct population of mutators among clinical pneumococci.

A c c e p t e d M a n u s c r i p t

Introduction

Hypermutable bacteria develop spontaneous antibiotic resistance more readily than wild-type organisms [START_REF] Miller | Response of Escherichia coli hypermutators to selection pressure with antimicrobial agents from different classes[END_REF][START_REF] Morosini | Frequency of mutation to rifampin resistance in Streptococcus pneumoniae clinical strains: hexA and hexB polymorphisms do not account for hypermutation[END_REF][START_REF] O'neill | Insertional inactivation of mutS in Staphylococcus aureus reveals potential for elevated mutation frequencies, although the prevalence of mutators in clinical isolates is low[END_REF][START_REF] Tanabe | A conspicuous adaptability to antibiotics in the Escherichia coli mutator strain, dnaQ49[END_REF] and have been identified among clinical isolates of a number of species, including Streptococcus pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Haemophilus influenzae and Neisseria meningitidis [START_REF] Morosini | Frequency of mutation to rifampin resistance in Streptococcus pneumoniae clinical strains: hexA and hexB polymorphisms do not account for hypermutation[END_REF][START_REF] Matic | Highly variable mutation rates in commensal and pathogenic Escherichia coli[END_REF][START_REF] Oliver | High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection[END_REF][START_REF] Richardson | Mutator clones of Neisseria meningitidis in epidemic serogroup A disease[END_REF][START_REF] Watson Me | Hypermutable Haemophilus influenzae with mutations in mutS are found in cystic fibrosis sputum[END_REF]. In several but not all cases, mutators have been found to contain defects in mismatch repair (MMR) pathways [START_REF] Oliver | High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection[END_REF][START_REF] Richardson | Mutator clones of Neisseria meningitidis in epidemic serogroup A disease[END_REF][START_REF] Watson Me | Hypermutable Haemophilus influenzae with mutations in mutS are found in cystic fibrosis sputum[END_REF][START_REF] Prunier | High rate of macrolide resistance in Staphylococcus aureus strains from patients with cystic fibrosis reveals high proportions of hypermutable strains[END_REF]; in addition to increasing the mutation frequency, MMR defects can increase the transformation frequency, further enhancing the potential for dissemination of antibiotic resistance [START_REF] Matic | Structure of recombinants from conjugational crosses between Escherichia coli donor and mismatch-repair deficient Salmonella typhimurium recipients[END_REF][START_REF] Rayssiguier | The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants[END_REF][START_REF] Tiraby | Integration efficiencies of spontaneous mutant alleles of amiA locus in pneumococcal transformation[END_REF][START_REF] Tiraby | Marker discrimination in transformation and mutation of pneumococcus[END_REF].

Multiresistant isolates of S. pneumoniae often exhibit resistance to penicillin, trimethoprim and sulfamethoxazole [START_REF] Fluit | Multi-resistance to antimicrobial agents for the ten most frequently isolated bacterial pathogens[END_REF][START_REF] Jones | The activity of levofloxacin and comparator agents against clinical isolates of Streptococcus pneumoniae collected worldwide during 1999 and 2000[END_REF][START_REF] Roussel-Delvallez | Change of pneumococcal resistance to antibiotics in adults between 1995 and 1997: a study in eight French counties[END_REF][START_REF] Thornsberry | In vitro activity of grepafloxacin and 25 other antimicrobial agents against Streptococcus pneumoniae: correlation with penicillin resistance[END_REF]. Penicillin resistance is acquired through intraspecies or interspecies recombination of penicillin-binding protein (PBP) genes, particularly pbp2b [START_REF] Dowson | Horizontal transfer of penicillin-binding protein genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae[END_REF], trimethoprim resistance through mutation or recombination of the dihydrofolate reductase gene [START_REF] Adrian | Mutations in the dihydrofolate reductase gene of trimethoprim-resistant isolates of Streptococcus pneumoniae[END_REF], and sulfamethoxazole resistance through mutation or recombination of the dihydropteroate synthase gene [START_REF] Maskell | Mechanism of sulfonamide resistance in clinical isolates of Streptococcus pneumoniae[END_REF]. These genes are widely dispersed in the genome so that a minimum of three genetic events is required to attain resistance to all three antimicrobial agents. It therefore seems plausible that acquisition of multiple resistance determinants would occur preferentially within a hypermutable and/or hypertransformable background.

A c c e p t e d M a n u s c r i p t 5 Hypermutable S. pneumoniae have been reported among clinical isolates, but evidence for their association with antibiotic resistance has been somewhat conflicting. Two studies found no association of hypermutability with penicillin resistance or resistance to any agent, whilst another reported mutators only among fluoroquinolone-resistant isolates, although with insufficient numbers of isolates to be sure whether the association was significant [START_REF] Morosini | Frequency of mutation to rifampin resistance in Streptococcus pneumoniae clinical strains: hexA and hexB polymorphisms do not account for hypermutation[END_REF][START_REF] Del Campo | Population structure, antimicrobial resistance, and mutation frequencies of Streptococcus pneumoniae isolates from cystic fibrosis patients[END_REF][START_REF] Gould | Identifying mutator phenotypes among fluoroquinolone-resistant strains of Streptococcus pneumoniae using fluctuation analysis[END_REF].

In the present study, we have validated two methods of screening for mutators among S. pneumoniae and have examined the frequencies of mutation to rifampicin resistance among a collection of antibiotic-resistant and -susceptible clinical isolates.

Material and methods

Bacterial isolates and culture

Bacterial isolates were selected from those submitted to the Health Protection Agency in 2002 as well as from collections previously described [START_REF] Hall | Genetic relatedness within and between serotypes of Streptococcus pneumoniae from the United Kingdom: analysis of multilocus enzyme electrophoresis, pulsedfield gel electrophoresis, and antimicrobial resistance patterns[END_REF][START_REF] Lawrence | Evaluation of serotype prediction by cpsA-cpsB gene polymorphism in Streptococcus pneumoniae[END_REF][START_REF] Lawrence | Evaluation of semiautomated multiplex PCR assay for determination of Streptococcus pneumoniae serotypes and serogroups[END_REF][START_REF] Urwin | Pneumococcal meningitis in the North East Thames Region UK: epidemiology and molecular analysis of isolates[END_REF].

Mutation frequencies were determined for 27 clinical isolates of S. pneumoniae; 11 isolates were fully susceptible, 5 carried only transposon-mediated resistance (i.e. chloramphenicol, erythromycin and tetracycline resistance) and 11 carried resistance to two or more antibiotics, including at least one involving modification of a chromosomal gene (i.e. penicillin, cefotaxime, ciprofloxacin, sulfamethoxazole and trimethoprim resistance) (Table 1). Rapid screens to A c c e p t e d M a n u s c r i p t 6 search for mutators were conducted on a further 180 clinical isolates (63 resistant, 19 susceptible and 98 undetermined). Laboratory strains R6 and hexBdeficient CP1015 [START_REF] Morrison | Isolation and characterization of three new classes of transformation-deficient mutants of Streptococcus pneumoniae that are defective in DNA transport and genetic recombination[END_REF] were used as controls for wild-type and mutator strains, respectively.

Determination of mutation frequency and mutation rate

Mutation frequencies to rifampicin (2 mg/L), ciprofloxacin (2 mg/L) and streptomycin (200 mg/L) resistance were determined as described previously [START_REF] Sk | Effect of subinhibitory concentrations of antibiotics on mutation frequency in Streptococcus pneumoniae[END_REF]. Broths were inoculated with ca. 10 4 colony-forming units from frozen starter cultures. The presence of pre-existing mutants in the inoculum was determined to be highly unlikely based on the known mutant frequency in the starter culture, and use of the median frequency avoided bias from any jackpot cultures. Broths were plated at late logarithmic phase and viable counts were determined for all replicas to avoid discrepancies resulting from autolysis of pneumococci in stationary phase. Mutation rates were calculated using the Jones median estimator, as described by Foster [START_REF] Foster | Methods for determining spontaneous mutation rates[END_REF][START_REF] Jones | Luria-Delbruck fluctuation experiments: design and analysis[END_REF]. The Mann-Whitney U-test was used for statistical analysis of the difference in mutation frequency between resistant and susceptible isolates.

Rapid screen for hypermutability

Overnight growth from a blood agar plate was suspended in brain-heart infusion broth and diluted in a 10 mL volume to an optical density at 620 nm (OD 620 ) of A c c e p t e d M a n u s c r i p t 7 0.1. These broths were then incubated at 37 C until no further increase in OD 620 was detected (ca. 7-8 h), after which nine 10 L spots were placed on a 2 mg/L rifampicin plate and one 10 L spot on a non-selective plate.

Sequence determination

Amplification and sequencing of gene fragments for multilocus sequence typing (MLST) were performed as described by Enright and Spratt [START_REF] Enright | A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease[END_REF].

Results

Comparison of selective markers for mutation frequency experiments

Initial experiments explored rifampicin, streptomycin and ciprofloxacin as selective agents, since resistance to each can be conferred by single point mutations. Other possible agents such as trimethoprim were excluded because many isolates in the collection were already resistant. The frequency of mutation to streptomycin resistance was too low to be reliably measured by our experimental strategy. This probably reflects the small number of nucleotide positions at which point mutation can generate streptomycin resistance. The mutation frequency to ciprofloxacin resistance appeared highly variable in our hands, with frequent jackpot cultures generating large numbers of mutants. For example, the median mutation frequency from five replica broths with isolate PN94/661 ranged from 7.70  10 -9 to 4.19  10 -6 in three separate experiments.

Mutation frequencies to rifampicin resistance were much more reproducible, A c c e p t e d M a n u s c r i p t 8 hence rifampicin was used as the selective marker for this study (further assessment of validity is described below).

Mutation frequency in antibiotic-resistant versus -susceptible isolates

Twenty-seven clinical isolates of S. pneumoniae were investigated, comprising both antibiotic-resistant and -susceptible isolates of serotypes 1, 3, 6B, 9V, 14, 19F and 23F, plus one resistant serotype 15A isolate (antibiotic susceptibilities had been examined previously [START_REF] Maskell | Molecular mechanisms of sulphonamide and trimethoprim resistance in Streptococcus pneumoniae[END_REF]). Resistant and susceptible isolates of serotypes 6B, 9V and 14 were matched where possible on the basis of pulsedfield gel electrophoresis (PFGE) type and multilocus enzyme electrophoresis (MLEE) type as determined previously [START_REF] Hall | Genetic relatedness within and between serotypes of Streptococcus pneumoniae from the United Kingdom: analysis of multilocus enzyme electrophoresis, pulsedfield gel electrophoresis, and antimicrobial resistance patterns[END_REF]; MLST confirmed that these isolates belonged to the Greece 6B -22, Spain 9V -3 and England 14 -9 clonal complexes, respectively.

Median mutation frequencies to rifampicin resistance were determined from five replica cultures of each isolate (Table 1). Mutation frequencies of the 27 isolates approximated to a normal distribution, with a peak in the interval 3.16-9.99  10 -9 (Fig. 1). An apparent second peak comprised isolates for which the median number of mutants from five cultures was zero, giving frequency values less than ca. 1  10 -9 depending on the cell density achieved for different isolates; this is likely to represent a tail in the distribution, spread across a range of frequencies, rather than a genuine peak. Laboratory strain R6 had a mutation frequency (based on 36 cultures) of 8.05  10 -9 , around the middle of the range, whilst the A c c e p t e d M a n u s c r i p t 9 hexB-deficient laboratory strain CP1015 gave a median mutation frequency (30 cultures) of 2.13  10 -7 , higher than any of the clinical isolates. By analogy with other studies, isolates would only be considered hypermutable if the level of mutation was as high as that of a known MMR mutant; hence, none of the clinical isolates were categorised as mutators.

No significant difference in mutation frequency was found between the antibioticresistant and -susceptible isolates overall (determined by Mann-Whitney U-test), regardless of the mode of resistance (chromosomal gene modification or transposon) (Fig. 1). Nor was there a significant difference in mutation frequency between resistant and susceptible representatives of the Spain 9V -3, Greece 6B -22 and England 14 -9 clonal complexes.

Validation of mutation frequency determination

To test the validity of the use of median mutation frequency from five replica cultures in this analysis, six isolates with low, medium or high mutation frequencies were subject to further analysis. Mutation frequencies were determined from 5 to 20 further cultures, using standardised starter cultures for inoculation and monitoring growth to ensure consistent cell density at the time mutants were selected (this is critical due to the rapid autolysis of pneumococci at stationary phase). The combined data sets for each isolate were used to calculate the mutation rate (number of mutations per cell division) by the Jones median estimator [START_REF] Jones | Luria-Delbruck fluctuation experiments: design and analysis[END_REF], which accommodates the plating of only part of a culture, A c c e p t e d M a n u s c r i p t 10 as described by Foster [START_REF] Foster | Methods for determining spontaneous mutation rates[END_REF]. Mutation rates for laboratory strain R6 and hexdeficient CP1015 were also calculated.

Inclusion of additional cultures did, as would be expected from the stochastic nature of mutation, alter the median mutation frequencies somewhat (Table 2), but no isolate would move up or down by more than one category of mutation frequency as defined in Fig. 1. Mutation rates to rifampicin resistance for clinical isolates ranged from 6.0 10 -10 to 1.5  10 -8 ; laboratory strain R6 fell around the middle of this range, whilst known mutator CP1015 had a rate of 2.2  10 -8 , only slightly higher than the most mutable clinical isolate PN02/1325.

Rapid screen for mutators among clinical isolates

As the mutation frequency determinations described above are time consuming to conduct, a rapid method to screen large numbers of clinical isolates for putative mutators was developed. Nine 10 L spots of late log-phase broth cultures inoculated from overnight plates were placed on rifampicin agar. In 12 repeat experiments with the hexB-deficient strain CP1015, a median of 7 colonies per spot was found, with a range of 4 to >12 (Fig. 2). In contrast the wild-type strain R6 gave a median of zero colonies per spot, with a range of 0 to 4 (Fig. 2). Isolates that produced a median of three or more colonies per spot (and confirmed in a repeat test) were subject to full mutation frequency determination. Of 180 clinical isolates examined (63 resistant, 19 susceptible and 98 undetermined), two were potential mutators producing a comparable number A c c e p t e d M a n u s c r i p t 11 of rifampicin-resistant colonies per spot to the hypermutable control. However, the mutation frequencies of these two isolates were determined to be 1.34  10 -8 and 4.99  10 -9 when tested by the standard method, well within the normal range of mutability.

Discussion

In the present study, we have validated the determination of mutation frequency (proportion of mutants in a culture) as an estimation of the amount of mutation occurring by determining mutation rate (number of mutations per cell division) in a proportion of isolates. We have also developed a novel rapid screening method that may be useful to others. Results of the study do not support an association between multiple antibiotic resistance and elevated frequency of mutation. This parallels previous observations that penicillin resistance or resistance to other antimicrobial agents is not associated with hypermutability in pneumococci [START_REF] Morosini | Frequency of mutation to rifampin resistance in Streptococcus pneumoniae clinical strains: hexA and hexB polymorphisms do not account for hypermutation[END_REF][START_REF] Del Campo | Population structure, antimicrobial resistance, and mutation frequencies of Streptococcus pneumoniae isolates from cystic fibrosis patients[END_REF], in contrast to reports in other species.

No frank mutators were detected, either among the 27 isolates for which full mutation frequencies were determined or among a further 180 isolates subject to a rapid detection screen, and only 2/27 (7.4%) had relatively high mutation frequencies. By comparison, two previous investigations had suggested 12.5%

(2/16) and 20% of clinical S. pneumoniae to be hypermutable [START_REF] Morosini | Frequency of mutation to rifampin resistance in Streptococcus pneumoniae clinical strains: hexA and hexB polymorphisms do not account for hypermutation[END_REF][START_REF] Gould | Identifying mutator phenotypes among fluoroquinolone-resistant strains of Streptococcus pneumoniae using fluctuation analysis[END_REF]. However, in neither investigation could the elevated mutation frequency be accounted for by defects in MMR genes as observed in other species [substitutions in HexA and Morosini et al. [START_REF] Morosini | Frequency of mutation to rifampin resistance in Streptococcus pneumoniae clinical strains: hexA and hexB polymorphisms do not account for hypermutation[END_REF] and del Campo et al. [START_REF] Del Campo | Population structure, antimicrobial resistance, and mutation frequencies of Streptococcus pneumoniae isolates from cystic fibrosis patients[END_REF] suggest that there is a bimodal distribution of mutation frequency among pneumococci, with mutators forming a distinct population. We postulate that this finding is due to an anomaly in the presentation of mutation frequency data. These authors have plotted a frequency distribution on a scale that is a mixture of arithmetic intervals and logarithmic intervals so that isolates are grouped in the form 1-2.5, 2.5-5, 5-7.5, 7.5-10, then 10-25, 25-50 etc. Hence, the groups are not equal either on an arithmetic or logarithmic scale. This artefact generates an apparent bimodal distribution due to the lower numbers in the interval 7.5-10 (covering a 1.33-fold range) relative to 10-25 (covering a 2.5-fold range)-a similar distribution was obtained with the data presented here when plotted in the same way. Although this makes no difference to their conclusion regarding resistance, it has led to the widespread reporting of a distinct population of mutators in the species, which appears to be unjustified. Hence, while there are clearly isolates of pneumococci that have mutation frequencies at the high end of the normal spectrum, and these may be enriched in certain situations [START_REF] Del Campo | Population structure, antimicrobial resistance, and mutation frequencies of Streptococcus pneumoniae isolates from cystic fibrosis patients[END_REF], we do not believe that there is yet firm evidence of a genetically or phenotypically distinct mutator population, in contrast to other species [START_REF] Oliver | High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection[END_REF][START_REF] Denamur | High frequency of mutator strains among human uropathogenic Escherichia coli isolates[END_REF].

A c c e p t e d M a n u s c r i p t 13 Our finding does not rule out the possibility that isolates were hypermutable at the time of acquisition of resistance and subsequently reverted to a level of mutability within the normal range because of the long-term disadvantage of elevated mutation rates [START_REF] Boe | The frequency of mutators in populations of Escherichia coli[END_REF][START_REF] Funchain | The consequences of growth of a mutator strain of Escherichia coli as measured by loss of function among multiple gene targets and loss of fitness[END_REF].

A c c e p t e d M a n u s c r i p t 3 ABSTRACT

 3 Development of multiple antibiotic resistance in Streptococcus pneumoniae

A c c e p t e d M a n u s c r i p t 12 HexB

 12 putatively implicated in hypermutability by Gould et al.[START_REF] Gould | Identifying mutator phenotypes among fluoroquinolone-resistant strains of Streptococcus pneumoniae using fluctuation analysis[END_REF] are also found in isolates with normal levels of mutation (Henderson-Begg, Livermore and Hall, unpublished data)].

Fig. 1 .

 1 Fig. 1. Distribution of median mutation frequencies to rifampicin resistance for 27

Fig. 2 .

 2 Fig. 2. Distribution of the median number of rifampicin-resistant colonies per spot

Table 1

 1 Serotype, antibiotic resistance profile and mutation frequency of clinical isolates of Streptococcus pneumoniae Median number of mutants was zero, indicating a mutation frequency of <1 

	Edited Table 1
	Isolate	Serotype Clonal	Antibiotic resistance	Median
			complex a	b	mutation
					frequency
	R6				8.05  10 -9
	CP1015				2.13  10 -7
	86019	1			1.88  10 -8
	R044	1		SUL; TET	6.33  10 -9
	PN02/521 1		TET; PEN; CHL;	<1  10 -9 c
				ERY
	PN94/261 3	ST 233		8.46  10 -9
	PN02/2531 3	Netherlands 3 -	TET; CHL; ERY	8.10  10 -9
			31
	PN94/1413 6B			4.30  10 -9
	PN94/361 6B	TLV Greece 6B -		4.69  10 -9
			22
	PN94/804 6B	SLV Greece 6B -	SUL; TMP; TET	8.06  10 -9
			22
	PN02/1545 6B		TET; ERY	1.39  10 -9
	PN93/1293 6B	Spain 6B -2	SUL; TMP; TET;	<1  10 -9
				PEN; CHL; STR
	PN93/917 9V			1.22  10 -8
	PN94/661 9V	SLV Spain 9V -3		<1  10 -9
	PN94/926 9V	SLV Spain 9V -3 SUL; TMP ; TET;	<1  10 -9
				PEN
	PN93/1802 9V	SLV Spain 9V -3 SUL; TMP; TET;	1.32  10 -8
				PEN

a Clonal complexes recognised by the Pneumococcal Molecular Epidemiology Network; ST, sequence type; SLV, single locus variant; DLV, double locus variant; TLV, triple locus variant. b Resistance to erythromycin (ERY), sulphonamide (SUL), trimethoprim (TMP), chloramphenicol (CHL), penicillin (PEN), tetracycline (TET), streptomycin (STR), ciprofloxacin (CIP) and azithromycin (AZM), as described in [23].

c

Table 2

 2 Comparison of initial median mutation frequency determined from five replica cultures with mutation frequency and mutation rate determined from at least ten

	cultures
	Isolate/strain	Median mutation	Median mutation	Mutation rate
		frequency (5	frequency (no. of	(no. of cultures)
		cultures)	cultures)
	R6 (lab strain)	-	8.05  10 -9 (36)	2.16  10 -9 (36)
	CP1015 (hex	-	2.13  10 -7 (30)	2.24  10 -8 (30)
	mutant)
	PN94/661	<1  10 -9	1.96  10 -9 (25)	5.99  10 -10
				(25)
	PN02/1545	1.39  10 -9	4.13  10 -9 (10)	9.94  10 -10
				(10)
	PN94/361	4.69  10 -9	3.25  10 -9 (20)	8.06  10 -10
				(20)
	PN93/1719	1.40  10 -8	6.61  10 -9 (15)	1.59  10 -9 (15)
	PN02/1325	8.47  10 -8	8.74  10 -8 (10)	1.47  10 -8 (10)
	PN93/132	1.47  10 -7	4.65  10 -8 (10)	6.66  10 -9 (10)

Funding

This work was supported by Barts and The London Charity.

Competing interests

None declared.

Ethical approval

Not required.