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European Biopharmaceutical Review

Migrating Microbes

Cystic fibrosis (CF) is the most common life-threatening autosomal recessive disorder in Caucasians. The major cause of morbidity and mortality in CF patients is the progressive pulmonary insufficiency. In CF patients, the lower respiratory airway epithelium is susceptible to bacterial colonisation, most predominantly by Pseudomonas aeruginosa. CF airway isolates are often tested for antibiotic susceptibility, but are rarely eradicated by the antibiotics identified as potentially effective. This is due to the fact that the growth state of P aeruginosa in CF airways is different from that tested under current conventional susceptibility assessment, and most probably represents bacteria in a biofilm rather than planktonic state.

Bacterial biofilms are micro colonies encased in extracellular polysaccharides that result from the adherence of bacteria to surfaces. In a biofilm state, bacteria appear to be very slow growing and are inherently resistant to antimicrobial agents. Therefore, the inability to eradicate P aeruginosa chronic infections may result, in part, from treatment regimens based on minimum inhibitory concentration (MIC) susceptibility testing that does not reflect the true growth state of bacteria in CF patients. Alternatively, biofilm susceptibility in vitro diagnostic test is the only novel assay that provides rapid and reproducible screening of the antibiotic susceptibilities of P aeruginosa biofilm and planktonic cells. As such, this assay is capable of determining more effective antibiotic treatments than those methods currently in use.

MICROBIAL BIOFILM: OVERVIEW

Despite the focus of microbiological research on the culture of pure population in planktonic state, it has become apparent that in natural settings, including clinical environments, bacteria exist predominately within biofilms (1). Biofilms are three-dimensional mosaic consortia of microbes, which accumulate and organise at surfaces within extracellular polymerencased matrices (EPM) or glycocalyx that are predominantly occupied with water channels (see Figure 1) (2). Once the biofilm establishes and matures on surfaces, cells migrate into the bulk fluid phase until they find a new spot to colonise (3). Those metabolically integrated multi-cellular communities are largely regarded as problematic in both industrial and clinical settings. This is due to the fact that biofilms are extremely recalcitrant towards antimicrobial agents and the host’s immune response.



Biofilms have been reported to be 100 to 1,000 times less susceptible than their planktonic counterparts to antimicrobial treatments (4). In humans, the ability of biofilm-associated cells to evade the host’s immune response has been attributed to the fact that not only are the underlying bacteria in the glycocalyx inaccessible to phagocytic cells (5), but also because the polysaccharide component of the glycocalyx matrix blocks complementary protein activation, inhibits chemotaxis, de-granulation of polymorphs and macrophage phagocytosis, as well as depressing the lympho-proliferative response (6,7). Additionally, bacterial exposure to sub-inhibitory antibiotic concentrations induces mucoid phenotypes, which generate thicker biofilms with additional matrix components.

Various mechanisms have been proposed to explain the resistance of biofilms towards aggressive treatment therapies, as no one mechanism by itself serves to adequately explain the long-term chronicity of these communities. Some of those theories included the physiological heterogeneity of the biofilm consortia, the presence of mutant cells that do not undergo programmed cell death (persisters), the over-expression of efflux pump open reading frames, the reaction diffusion limitation of the EPM as well as the predominance of drug-resistant genes (2).

Biofilm-related infections are notoriously hard to eradicate and have been the subject of intense scientific research over the past 30 years (8). Examples of biofilm-associated infections include colonisation of implanted medical devices (central venous catheters, joints prostheses, urinary catheters, pacemakers, mechanical heart valves and implantable cardiac defibrillators), burn wounds, dental caries and the lungs of CF patients (9,10).

CYSTIC FIBROSIS-RELATED CHRONIC INFECTIONS

The failure to eradicate P aeruginosa in CF patients can lead to persistence of multidrug-resistant organisms, and severe clinical signs due to the colonisation of additional environmental organisms, such as Achromobacter xylosoxidans, Stenotrophomonas maltophilia and Burkholderia cepacia (11). Antibiotic therapy, directed by current susceptibility testing, has traditionally been used to treat symptomatic cystic fibrosis patients with chronic infection (12). This treatment may result in clinical improvement and a decrease in only planktonic bacterial burden; however, eradication of infection is quite rare. This is not surprising, since CF chronic airway infections differ significantly from acute bloodstream infections; thus, it seems reasonable that testing strategies should reflect these differences.

CURRENT IN VITRO SUSCEPTIBILITY TESTING: PROS & CONS

Regardless of the recommendations for using the standard susceptibility testing in CF, the clinical utility of standard MIC susceptibility testing has been called into question based on data recently reported by Smith et al (13,14). The MIC measures the actions of antibiotics against planktonic organisms and serves as an important reference in the treatment of many acute infections. However, application of MICs in the treatment of chronic or device-related infections involving bacterial biofilms is often ineffective (15). The response to antibiotic exposure of bacteria growing as biofilms in the clinical laboratory may better predict the response of CF patients to antimicrobial therapy.

Recent research has been performed to examine the susceptibility of a large number of CF clinical isolates of P aeruginosa growing as biofilms and to compare these results with standard MIC determinations (16). Unpublished data by Brown et al also showed that there was a correlation between biofilm antimicrobial resistance and airflow obstruction, as the patients who received antibiotics that were sensitive to P aeruginosa biofilms based on the biofilm susceptibility in vitro diagnostic kit demonstrated significant improvement in respiratory function and had a favourable clinical outcome (17). Evidence of in vitro antibiofilm activity and of different susceptibility patterns by the two methods would support the feasibility of adapting biofilm susceptibility methods to the clinical microbiology laboratory, and opens the way to select more effective antibiotic combinations for CF airway infections than methods in current use.

Current testing does not provide information regarding antibiotic combinations as a treatment option. Nevertheless, physicians commonly use antibiotic combination without any susceptibility test guidance. Biofilm susceptibility in vitro diagnostic test is the only available, easy and reproducible assay for the rapid and reproducible screening of the antibiotic susceptibilities of biofilms (18).

BIOFILM SUSCEPTIBILITY IN VITRO DIAGNOSTIC PANEL

The biofilm susceptibility in vitro diagnostic panel is a Class II in vitro medical device for testing both planktonic and biofilm susceptibility of clinical isolates of P aeruginosa at serum breakpoint levels. Qualitative antimicrobial agent susceptibility information is provided simultaneously for 12 single antibiotics and 35 combination antibiotics. The biofilm susceptibility in vitro diagnostic panel is based on a high-throughput biofilm susceptibility assay, which was part of an internal research at the University of Calgary (18). This high-throughput system consists of a polystyrene lid with 96 downward projecting pegs that can be fitted into a standard 96-well microtiter plate. Through the use of this method, one batch culture apparatus allows single or multiple species biofilms to be tested against an 8 by 12 matrix of controlled variables.

These variables may include growth medium formulations, exposure times, as well as antimicrobials at various concentrations – alone or in combination. This in vitro diagnostic panel can be also used with other disease conditions including medical device related infections, such as urinary catheter infection, peritoneal dialysis associated infections and ventilator associated pneumonia, and other chronic P aeruginosa infections, such as chronic wound or burn infections.

The benefits of using the biofilm susceptibility in vitro diagnostic test – especially with the fact that expanding the scope of this kit to cover other Gram positive and Gram negative bacterial strains as well as fungal infections is currently under development – include: 
  • Reduction in hospital stays 
  • A drop in treatment failure 
  • A reduction in development of resistant bacteria due to the use of inappropriate drugs 
  • The ability to use current antibiotics more effectively 
  • A reduction in the reliance on new antibiotics to treat resistant bacterial strains
We should acknowledge that biofilm infections cause much more damage and many more inflammatory responses due to their continued persistence, therefore it is important to consider the use of the most suitable antimicrobial agent to treat such infections. It is time to take a step forward and adapt the new approach taken in the in vitro susceptibility testing and reinvent the way we run our clinical and industrial microbiology research. As J William Costerton explains, “Those of us in the medical business must think very hard if we are to outmanoeuvre this very old and very successful bacterial life form, and perhaps learn to speak their language, and even enlist them in our never ending fight against disease” (19).

References
  1. Costerton JW, Cheng KJ, Geesey GG, Ladd TI, Nickel JC, Dasgupta M. and Marrie TJ, Bacterial biofilms in nature and disease, Annual Reviews in Microbiology: pp41, 435-465, 1987
  2. Gilbert P, Maira-Litran T, McBain AJ, Rickard AH and Whyte FW, The physiology and collective recalcitrance of microbial biofilm communities, Advanced Microbial Physiology 46: pp202-256, 2002
  3. Aspiras MB, Kazmerzak KM, Kolenbrander PE, McNab R, Hardegen N, Jenkinson HF, Expression of green fluorescent protein in Streptococcus gordonii DL1 and its use as a species-specific marker in coadhesion with Streptococcus oralis 34 in saliva-conditioned biofilms in vitro, App Environ Microbiol 66: pp4,074-4,083, 2000
  4. Gilbert P and McBain AJ, Biofilms: their impact on heath and their recalcitrance toward biocides, American Journal of Infection Control 29: pp252-255, 2001
  5. Bayer AS, Speert DP, Park S, Tu J, Witt M, Nast CC and Norman DC, Functional role of mucoid exopolysaccharide (alginate) in antibiotic-induced and polymorphonuclear leukocyte mediated killing of Pseudomonas aeruginosa, Infect Immun 59: pp302-308, 1991
  6. Wilson M, Bacterial biofilms and human disease, Sci Prog 84: pp235-254, 2001
  7. Donlan RM and Costerton JW, Biofilms: Survival mechanisms of clinically relevant microorganisms, Clin Microbiol Rev 15: pp167-193, 2002
  8. Danese PN and Silhavy TJ, CpxP, a stress-combative member of the Cpx regulon, J Bacteriol 180: pp831-839, 1998
  9. Habash MB, Van der Mei HC, Busscher HJ and Reid G, The effect of water, ascorbic acid, and cranberry derived supplementation on human urine and uropathogen adhesion to silicone rubber, Can J Microbiol 45: pp691-694, 1999
  10. Govan JRW and Deretic V, Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia, Microbiol Rev 60: pp539-574, 1996
  11. Van Devanter D and Van Dalfsen J, How much do Pseudomonas Biofilms contribute to symptoms of pulmonary exacerbation in cystic fibrosis, Pediatr Pulmonol 39: pp504- 506, 2005
  12. Ramsey BW, Management of pulmonary disease in patients with cystic fibrosis, N Eng J Med 335: pp179-188, 2006
  13. Saiman LD, Schidlow and Smith A, Concepts in care: microbiology and infectious disease in cystic fibrosis, Cystic Fibrosis Foundation, Bethesda Md 5, 1994
  14. Smith AL, Fiel SB, Mayer-Hamblett N, Ramsey B and Burns JL, Lack of association between in vitro antibiotic susceptibility testing of Pseudomonas aeruginosa isolates and clinical response to parental antibiotic administration in cystic fibrosis, Chest 123: pp1,495-1,502, 2003
  15. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR and Lappin-Scott HM, Microbial biofilms, Annu Rev Microbiol 49: pp711-745, 1995
  16. Keays T, Ferris W, Vandemheen KL et al, A retrospective analysis of biofilm antibiotic susceptibility testing: a better predictor of clinical response in cystic fibrosis exacerbations, J Cyst Fibros 8: pp122-127, 2009
  17. Brown NE, Zhu J, Salgado J, Tabak J, Rennie R, Turnbull L, Rawal B and Olson ME, Clinical Evaluation of the bioFILM PATM Susceptibility Test in CF Patients Infected with Pseudomonas aeruginosa, unpublished data.
  18. Ceri H, Olson ME, Stremick C, Read RR, Morck D and Buret A, The Calgary biofilm device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms, J Clin Microbiol 37: pp1,771-1,776, 1999
  19. Costerton JW, The Biofilm Primer, Springer Series on Biofilms 1: pp64, 2007


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Amin Omar is Technical Services Manager at Innovotech Inc and has focused his career on investigating biofilms and the role they play in microbial resistance, in addition to the development and testing of novel antimicrobial agents for the treatment of microorganisms in the biofilm state. Amin’s research has centred primarily on elucidating the genetic differences between microorganisms growing at different rates in biofilms in comparison to those within the planktonic state, and determining the implications of these differences with regard to strategies to control biofilms. Email:
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