ASAIO Journal 2000

Role of Biolms in Antimicrobial Resistance

RODNEY M. DONLAN

Biolms are formed by a spectrum of microorganisms, including pathogens, and provide a means for these organisms to protect themselves against antimicrobial agents. Several mechanisms have been proposed to explain this phenomenon of resistance within biolms, including delayed penetration of the antimicrobial into the biolm extracellular matrix, slowing of growth rate of organisms within the biolm, or other physiologic changes brought about by interaction of the organisms with a surface. The practical implications of biolm formation are that alternative control strategies must be devised both for testing the susceptibility of the organisms within the biolm and treating the established biolm to alter its structure. A number of testing protocols have been developed. Effective treatment strategies will incorporate antimicrobials or other agents that have been demonstrated to penetrate and kill biolm organisms, or treatments that disrupt or target specic components of the biolm matrix. A better understanding of the role of biolms in infection and how in vivo biolms respond to selected treatments requires more study. ASAIO Journal 2000; 46:S47-S52.

clude Staphylococcus aureus (including methycillin-resistant strains),4 8 S. epidermidis,9 15 Escherichia coli,16 19 Pseudomonas aeruginosa,20 25 Burkholderia cepacia,26 Vibrio cholera,27 Candida parapsilosis,28 and C. albicans.29,30 Costerton et al.3 has provided a partial list of human infections where biolms are involved. From several different perspectives, growth within the biolm provides an advantage for microorganisms. Growth on a xed surface provides a constant supply of nutrients through bulk uid mass transfer. As the biolm becomes more established, the component organisms produce an extracellular polymer matrix that binds cells and microcolonies together (and to the substratum) and also inhibits the diffusion of antimicrobials to the cells. This study discusses the mechanisms whereby this process occurs and offers strategies that may effectively control microbial biolms on indwelling medical devices. Mechanisms by which Biolms Confer Antimicrobial Resistance

adhere to submerged surfaces. Costerton et al. dene biolm as matrix-enclosed bacterial populations adherent to each other and/or to surfaces or interfaces. These surfaces may be drinking water pipes, indwelling medical devices, or even human tissues.2,3 The matrix material or slime is primarily polysaccharide in nature1 and is produced by the biolm organisms after they have irreversibly attached to a surface. Figure 1 shows a scanning electron microscopic image of a Staphylococcus biolm on the surface of an indwelling medical device. Despite that the processing required for scanning electron microscopy will dehydrate the extracellular polymer matrix component of biolms, this material can still be visualized in this image in the form of strands or sheets surrounding and encasing the bacterial cells. These extracellular polymers not only act as adhesins, binding the cells to the surface, but also serve to bind cells to one another, prevent or impede the diffusion of antimicrobials to the microcolonies within the biolm, and protect biolm organisms from host defense mechanisms.2 The diversity of microorganisms forming biolms is quite extensive and may include the spectrum of gram-positive and gram-negative bacteria, yeasts, and even lamentous fungi. A number of organisms relevant to public health have been shown to form biolms, either in experimental systems, on indwelling medical devices, or in animal models. These in1

B iolms develop when microorganisms associate with and

Delayed Penetration of the Antimicrobial

For nutrient and antimicrobial molecules to reach microbial cells within biolms, they must diffuse through the biolm matrix or slime produced by and encasing the organisms. This diffusional limitation may be the result of either transport limitation (the inability of the antimicrobial molecules to diffuse through the polymer matrix) or inactivation of the antimicrobial molecule by the matrix material. A number of studies have demonstrated antimicrobial resistance of organisms in biolms due to delayed penetration processes. Suci et al.20 investigated the penetration of ciprooxacin into P. aeruginosa biolms. They demonstrated that penetration of the antibiotic was signicantly impeded by the biolm. Using a type of continuous culture device, they showed that diffusion of 100 g/ml ciprooxacin from the liquid phase of a sterile microbiologic medium to the liquid/solid interface of a sterile surface normally required 40 seconds. Signicantly less drug was transported to the liquid/solid interface of a biolm-containing surface after 21 minutes. Hoyle et al.21 examined the ability of P. aeruginosa mucoid exopolysaccharide (MEP) to bind tobramycin. They found that tobramycin diffusion across the biolm-uid interface and into the biolms was the primary reason that bacterial cells were dispersed from biolms and that these dispersed cells were 15 times more susceptible to tobramycin than were cells in the intact biolms. However Nichols et al.22 found that tobramycin efcacy against biolm cells could not be explained solely by the decreased diffusion into the biolms. They proposed that the effect could also be due to the extremely slow growth rates of bacteria within the depths of the biolm due to depletion of organic nutrients, inorganic ions, and oxygen. DuGuid et al.9 examined the susceptibility S47

From the Biolm Laboratory, Hospital Infections Program, Centers for Disease Control and Prevention, Atlanta, Georgia. Rodney M. Donlan, PhD, Biolm Laboratory, Hospital Infections Program, CDC, Mail Stop C-16, Atlanta, GA 30333.

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DONLAN

Figure 1. Scanning electron micrograph of a staphylococcal biolm on the inner surface of an indwelling medical device. Bar-20 microns. (Photograph by Janice Carr of the Hospital Infections Program of CDC, Atlanta, GA.)

of S. epidermidis to tobramycin. They concluded that organization of the cells within biolms was at least in part responsible for the recalcitrance of these organisms to this antibiotic. Gordon et al.23 investigated three beta lactam (ceftazidime, cefsulodin, piperacillin) and two aminoglycoside (gentamicin and tobramycin) antibiotics for their ability to diffuse through alginate gels. Using both synthetic and bacterially produced alginates, they found that the beta lactams diffused into the matrix much more rapidly than did the aminoglycosides. However, the aminoglycosides initially bound to the alginates, and diffusion increased rapidly after a lag period of 80 100 minutes.

Alteration of the Cellular Growth Rate

An alternative proposed mechanism for resistance of biolm associated cells (sessile organisms) to antimicrobials is that the growth rate of these organisms is signicantly slower than the growth of planktonic (biolm free) cells; therefore, the uptake of the antimicrobial molecules is diminished. A number of studies have addressed this question. Evans et al.16 developed a method of cell culture designed to determine the effect of growth rate apart from other biolm processes. Using this method, they examined the effect of a quaternary ammonium compound (cetrimide) on E. coli biolms. The organisms were most resistant at the slowest growth rates. At growth rates above 0.3 per hour, sessile and planktonic cultures were equally susceptible. Evans et al.17 also examined the effect of ciprooxacin on biolms of P. aeruginosa and E. coli using a similar test method. They found, for P. aeruginosa, that intact biolm cells were more resistant than biolm cells that had been removed from the surface and tested in suspension. However, biolm growth rate per se had a negligible effect on susceptibility. Newly formed daughter cells, those that had recently detached from the biolms, were more susceptible than other populations, indicating that ciprooxacin activity was inuenced by the bacterial cell cycle. DuGuid et al.10 examined the effect of ciprooxacin against S. epidermidis biolms. In all cases, increase in growth rate resulted in an increase in susceptibility. Their results also supported the con-

clusions of Evans et al.17 that there is a profound inuence of the phase of the cell division cycle on the susceptibility of the organisms to antimicrobials. Desai et al.26 studied the susceptibility of Burkholderia cepacia to ciprooxacin and ceftazidime in stationary phase and at different stages of exponential growth. They found that biolm bacteria (sessile cells) increased their resistance by a factor of 150 during the exponential phase; bacteria (both planktonic and biolm) exhibited the highest level of resistance to both antibiotics in the stationary phase. Anwar et al.24 examined the interaction of P. aeruginosa with tobramycin and piperacillin and found that cells in older biolm were signicantly more resistant than those in younger biolms of the same culture. Chuard et al.4 also found that older biolms of S. aureus (4 or 24 hours) were less susceptible than biolms only 2 hours old. This decrease in susceptibility was moderate for eroxacin, higher for oxacillin and vancomycin, and highest for gentamicin. Amorena et al.5 also found that younger (6 hours) S. aureus biolms were more susceptible than older biolms for a number of different antibiotics. Biolm age may be important because with increasing age, there will be greater production of extracellular polymeric substances, leading to reduced nutrient and oxygen penetration into the biolm matrix. All of these studies provide evidence that antimicrobial susceptibility is affected by cell growth rate and in some cases by phase of growth. Gram-negative bacteria respond to environmental stresses such as nutrient limitation by synthesizing sigma factors. Those sigma factors that are under the regulatory control of the rpoS regulon in E. coli in turn regulate the transcription of genes whose products mitigate the effects of stress. Adams and McLean18 studied E. coli biolm formation in chemostats and compared strains with and without the rpoS gene. The rpoS gene is activated during slow growth of these organisms. They found that deletion of the rpoS gene signicantly reduced the ability of E. coli to grow in biolms, while having minimal effect on the same organisms growing planktonically. The rpoS organisms produced biolms with higher cell densities and with a higher number of viable organisms. These results strongly suggest that conditions that elicit a slowing of bacterial growth, such as nutrient limitation or build up of toxic metabolites, are conducive to the formation of biolms, at least for this organism. This would be particularly relevant for organisms within the depths of biolms, where nutrient and oxygen limitation may be acute. A slowdown in growth rate would not be deleterious for cell metabolism and would actually benet the organisms by decreasing antimicrobial uptake.

Other Hypotheses
Dagostino et al.31 speculated that association of bacteria with a surface during biolm formation caused a number of physiologic responses as a result of the repression or induction of genes. They were able to demonstrate that a specic gene was induced by surface associated bacterial cells but that this induction did not occur in liquid media. This nding would support the idea of a tactile receptor in bacteria and could have implications for a wide spectrum of phenotypic characteristics in biolm bacteria, including traits that might confer resistance. Tresse et al.32 examined the susceptibility of agar entrapped E. coli cells to a beta lactam and an aminoglycoside antibiotic under different oxygen tensions. They found that oxygen deciency in the gel layer of the agar contributed to the enhanced resistance of the bacteria to the

BIOFILMS AND ANTIMICROBIAL RESISTANCE Table 1. Selected Testing Protocols for Biolm Antimicrobial Susceptibility Ref. 19 Organism Tested E. coli, P. aeruginosa, S. aureus Test Protocol Biolms grown on pegs under batch, mixed conditions, treated, removed mechanically, and quantied using viable plate counts

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Antibiotics Tested Cefazolin Ciprooxacin Clindamycin Gentamicin Oxacillin Penicillin Vancomycin Cefazolin Cefuzonam Cephalexin Gentamicin Ooxacin Tosuoxacin Cefazolin Cefuroxime Ciprooxacin Erythromycin Gentamicin Novobiocin Penicillin G Phosphomycin Rifampin Tobramycin Vancomycin Fleroxacin Gentamicin Oxacillin Vancomycin Ciprooxacin Quinupristin/dalfopristin Vancomycin Ciprooxacin Latamoxef Cefuroxime Piperacillin Tobramycin Vancomycin

40

S. aureus

Biolms grown on 96 well tissue culture plate under batch, static conditions, treated, then quantied visually

S. aureus

Biolms grown on 96 well tissue culture plate under batch, static conditions, treated, removed mechanically, and quantied using ATP bioluminescence

S. aureus

Biolms grown on polymethylacrylate coverslips coated with bronectin under batch, mixed conditions, treated, removed mechanically, and quantied using viable counts Biolms grown on silicone discs under batch, static conditions, removed mechanically, and quantied by viable counts Biolms grown on cellulose acetate membranes under continuous culture, removed mechanically, and quantied by viable counts Bacteria entrapped in agar disks, treated, removed from disks mechanically, and quantied by optical density Biolms grown on membrane lters installed in a modied Robbins device under owing conditions in a chemostat, lters removed and treated, and then quantied by viable counts.

11 10 41 12

S. epidermidis S. epidermidis E. coli S. aureus P. aeruginosa S. epidermidis

aminoglycoside, but not the beta lactam, and proposed that the effect was due to the lowered uptake of the antibiotic by the oxygen deprived cells. This would be particularly relevant for thicker biolms, where cells within the biolm could become oxygen limited. Practical Implications

Implications for Susceptibility Testing

Because sessile cells within biolms are signicantly more resistant to antimicrobials than are planktonic (suspended) organisms, results generated using standard antimicrobial susceptibility test approaches are invalid as predictors of biolm susceptibility. The National Committee for Clinical Laboratory Standards (NCCLS) does not have an approved method for biolm antimicrobial susceptibility testing at this time. However, several studies have addressed this problem by developing appropriate testing protocols. An ideal susceptibility test would be one that provides results that can predict susceptibility of the organism under in vivo conditions, is reproducible,

provides relatively rapid turnaround of results, and is easy to use. Selected testing protocols that have been used for biolm susceptibility testing are shown in Table 1. Any of these testing methods could be useful in determining the effects of antimicrobial agents against biolms. However, each method has its own strengths and weaknesses in terms of reproducibility, ease of use, and ability to reasonably predict performance of the antimicrobial in vivo. Ceri et al.19 compared planktonic minimal inhibitory concentrations (MIC) with minimal biolm eradication concentrations (MBEC) for three different organisms (E. coli ATCC 25922, P. aeruginosa ATCC 27853, and S. aureus ATCC 29213) for a number of antibiotics. Tables 2 and 3 show their results for P. aeruginosa and S. aureus. This type of data demonstrates the dramatically greater resistance of biolms to antimicrobials, and also shows that certain antibiotics are much more effective than others when used against biolms. For example, their results indicate that ciprooxacin or tobramycin would probably be the antibiotic of choice for P. aeruginosa biolms (Table 2). Gentamicin would be a candidate for control of S. aureus biolms. It is not feasible to

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Table 2. Antibiotic Susceptibility of P. aeruginosa ATCC 27853 as a Planktonic Population (MIC) and as a Biolm Population (MBEC) as Derived by the NCCLS Assay and an Assay with the CBD* Antibiotic Amikacin Aztreonam Ceftazidime Ciprooxacin Gentamicin Imipenem Piperacillin Tobramycin MIC (g/ml) NCCLS Assay 2 2 1 0.25 2 1 2 0.5 MIC (g/ml) Assay with CBD 4 4 2 0.25 4 4 16 1 MBEC (g/ml) A650 16 1,024 1,024 4 128 1,024 1,024 2 MBEC (g/ml) 0 CFU/peg 16 1,024 1,024 4 128 1,024 1,024 2

* Obtained by permission from the American Society for Microbiology. MIC, minimal inhibitory concentration; MBEC, minimal biolm eradication concentration; NCCLS, National Committee for Clinical Laboratory Standards; CBD, CFU, colony forming units. The values were obtained by measuring the turbidity at 650 nm (A650) on a 96 well plate reader. The values were obtained by determination of plate counts.

routinely evaluate antimicrobial efcacy against actual in vivo biolms. However, studies that compare antimicrobial efcacy against biolms developed in both laboratory and animal models could be very useful. Schwank et al.23 evaluated the ability of several different antibiotics to eradicate biolms in both an in vitro system and an animal model. They found that an adherence-positive S. epidermidis strain was much more resistant to the antimicrobial treatment in both the lab and animal models than was an adherence-negative strain. They also showed that there was general agreement between lab and animal studies for the antibiotics tested (amikacin, levooxacin, rifampin, teicoplanin). Rifampin was the most effective drug for both strains tested in both lab and animal systems. Teicoplanin was the least effective.

Implications for Effective Treatment

Microorganisms within biolms are generally resistant to antimicrobials in concentrations normally used for treatment of suspended organisms. Therefore, alternative, novel approaches should be considered as control strategies whenever biolms are suspected as a cause of infection. One approach might be to review the published literature to determine whether certain antibiotics are more effective against biolm associated organisms. Ceri et al.19 found large differences between antibiotics against the same organism. Tobramycin was most effective against E. coli and P. aeruginosa; Gentami-

cin was most effective against S. aureus. Hamilton-Miller and Shah11 found that ciprooxacin and quinupristin/dalfopristin effectively killed sessile S. epidermidis, whereas vancomycin, teicoplanin, and erythromycin were ineffective. Yasuda et al.14 also found clarithromycin to be effective against S. epidermidis biolms. However, in all these studies, levels required for inactivation and eradication of the biolms were higher than required for inactivation of the planktonic organisms. Of course, animal studies and clinical trials would be necessary to validate these treatments. Another approach would be to treat the exposed surfaces of indwelling medical devices with antimicrobial coatings. One example of this kind of treatment was described by Raad et al.15 Central venous catheters were coated with minocycline and rifampin and shown to decrease colonization and biolm formation by gram-positive cocci and reduce catheter related bacteremias mostly caused by S. epidermidis in clinical trials. Enzyme treatments have also been suggested as biolm control strategies. Serratiopeptidase (a proteolytic enzyme) enhanced the activity of ooxacin against sessile P. aeruginosa and S. epidermidis, reducing the MIC and MBC by at least a factor of 2 for each of the strains tested compared to ooxacin alone.34 Johansen et al.35 found that a multicomponent enzyme preparation containing a wide range of carbohydrases was most effective against biolms of different bacteria. Huang et al.36 showed that ultrasound treatment reduced P. aerugi-

Table 3. Antibiotic Susceptibility of S. aureus ATCC 29213 as a Planktonic Population (MIC) and as a Biolm Population (MBEC) Derived by the NCCLS Assay and an Assay with the CBD* Antibiotic Cefazolin Ciprooxacin Clindamycin Gentamicin Oxacillin Penicillin Vancomycin MIC (g/ml) NCCLS assay 0.5 0.25 0.12 0.5 0.12 1 1 MIC (g/ml) Assay with CBD 0.5 0.5 0.25 0.5 0.25 4 1 MBEC (g/ml) A650 1,024 512 128 2 1,024 128 1,024 MBEC (g/ml) 0 CFU/peg 1,024 512 256 2 1,024 128 1,024

* Obtained by permission from the American Society for Microbiology. MIC, minimal inhibitory concentration; MBEC, minimal biolm eradication concentration; NCCLS, National Committee for Clinical Laboratory Standards; CBD, CFU, colony forming units. The values were obtained by measuring the turbidity at 650 nm (A650) on a 96 well plate reader. The values were obtained by determination of plate counts.

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S51 References

nosa biolm levels on stainless steel by 1.5 logs and modestly improved gentamicin efcacy against established biolms. Blenkinsopp et al.37 demonstrated that a low strength electrical eld combined with a low current density could enhance the efcacy of several commercial biocides against P. aeruginosa biolms. They hypothesized that the treatment disrupted the charges on the extracellular polysaccharide matrix, allowing enhanced biocide penetration. Araki and Hosomi38 used a bacteriophage specic for Pseudomonas spp. and demonstrated a reduction in planktonic organisms after addition of the phage to the culture. They suggested that bacteriophage treatment might be effective against biolms. Because phage are highly organism specic, this type of treatment might lend itself to the treatment of clinically relevant biolms, which are commonly pure culture (compared with environmental and industrial biolms).
Conclusions Biolms commonly form in a variety of environments including those relevant to public health. Organisms that grow in a biolm have distinct advantages, including protection from the effects of antimicrobial agents. Several mechanisms have been proposed to explain how biolms convey antimicrobial resistance. A number of studies have shown that either delayed penetration of the antimicrobial molecule or reduced metabolic activity of the organisms within the biolms are the reason for this protective effect. Other studies have suggested that bacteria have a tactile response that will result in phenotypic changes upon encountering a surface. Effective treatment strategies for biolms require that susceptibility testing be performed against biolm organisms. Standard antimicrobial susceptibility testing against planktonic (suspended) organisms is not adequate. A number of biolm susceptibility testing protocols have been published, but these protocols should be evaluated based on reproducibility, ease of use, cost, rapidity, and how well the in vitro results correlate to results obtained in animal studies or clinical trials. Treatments that have been proposed to control or mitigate biolms may rely on specic antimicrobials shown to be more effective in either killing biolm organisms or preventing their adhesion. Other biolm treatments target specic components of the biolms, such as the extracellular polymer matrix. It is clear that standard and accepted procedures for microbiologic control using antimicrobials are inadequate for the treatment of infections where biolms may be involved. A better understanding of the role of biolms in infection is needed. For example, Reid and Bailey39 asked why some patients, colonized by biolms, do not develop symptoms of infection. How does the medical practitioner know whether or not asymptomatic biolm colonization should be treated with an antimicrobial? More studies that investigate how in vivo biolms respond to antimicrobial treatment should be performed to validate laboratory results. Results generated from these types of studies would likely yield more effective treatment strategies. But this outcome will require a greater recognition of the importance of biolms by the medical community at large. A paradigm shift is needed.

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