Introduction

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Biofilms are highly organized communities of microbes made up of one or more species 16 attached to a biotic or abiotic solid surface and often encased in a self-synthesized extracellular matrix (ECM) with varying chemical composition depending on the organism but primarily made up of complex polysaccharides 711 proteins 12 ,13 and nucleic acids. 1417 Microbial biofilm formation is a multistep process 1822 initiated by the presence of a critical number of cells (often sensed by the extraordinary chemical communication ability among individual cells) in the localized niche. 2328 The biofilm life cycle has distinct developmental stages including attachment of planktonic cells to a solid surface, growth of the cells into a mature biofilm community and eventual dispersal of the cells from the microbial community into the surrounding environment.19 The initial adherence to a solid surface is a transient step followed by firm attachment which is promoted by bacterial adhesion molecules. The attached microbial cells multiply in a favorable niche and form a complex three dimensional microbial community with an elaborate chemical communication network. Bacterial cells that make up the biofilm adhere to each other and to the surface with the ECM which often incorporates host derived cellular components. It is estimated that biofilms are composed of 75–95% ECM and only 5–25% bacteria. In a fully matured biofilm the microbial community is encased in the ECM that provides the cells protection from antimicro-bial drugs 2,29,31 and evasion from the immune system of the host. 3235 Under the appropriate conditions the biofilm can persists for a long period of time. Individual cells or group of cells will be eventually detached from the biofilm community and dispersed to the neighboring environments forming other biofilm communities. It is the process of dispersal from an existing biofilm that make them clinically threatening providing a reservoir of microbes capable of initiating recurrent active infection.29,3638

In theory all microbial species ranging from Gram-negative to Gram-positive bacteria, pathogenic yeasts to filamentous fungi and fresh water algae to marine phytoplankton are capable of producing biofilms in the appropriate ecological niches. But most of the biofilm studies have now focused on those organisms causing diseases in man such as Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus mutans, Candida albicans and Aspergillus fumigates (just to name a few) because of their prevalence and clinical importance. The National Institute of Health now estimates as much as 80% of clinical infections in man have biofilm origin. The economic impact due to microbial biofilms in terms of additional cost for the care and treatment of microbial infections for a prolonged period of time is staggering and in the $millions.

Microbial biofilm can be formed by a single species (monomicrobial biofilm) or multiple species (polymicrobial biofilms). Recent advancement in metagenomics and microbiome studies suggests that it is more likely in nature whether it is the human body, the root system of an alpha-alpha plant, a cold lake or a hot spring to have polymicrobial biofilm than monomicrobial biofilm.18,3943 Polymicrobial biofilm producing organisms belong to highly different taxonomic groups, including those belonging to different Kingdoms forming an interkingdom (e.g. fungal-bacterial assemblage) interaction producing biofilm. 44,45 The characteristics of monomicrobial biofilm produced ECM may be different from that of the polymicrobial biofilm and such variations will have profound impact on the susceptibility of the organism(s) to antimicrobial drugs.46,47 Moreover, individual targeted antibiotic treatment may eliminate one species and provide an opportunity for the more virulent one to flourish.

Although many microorganisms of clinical importance are capable of producing biofilms there is significant differences in the ability of even different members of a single Genus in producing biofilm. For example, all Candida species are capable of producing biofilms but there is significant variability in their ability to form mature biofilm. For instance, Candida glabrata and Candida krusei are not prolific producers of dense thick biofilm like C. albicans does. 4852 Nevertheless, all Candida biofilms are known exhibit the hallmark biofilm characteristics such as reduced susceptibility to antifungal drugs, decreased growth rate of the biofilm bound cells, ability to survive under nutrient limiting conditions and presence of an extracellular matrix.

Implications of microbial biofilms in infectious diseases

Formation of biofilms by microbial human pathogens carries important clinical implications in at least four areas related to the treatment and management of infectious diseases: (1) increased tolerance/resistance of the biofilm to antimicrobial drug therapy (2) the ability of cells within the biofilm to withstand or evade host immune defenses (3) biofilm formation on medical devices that can negatively impact the host by causing the failure of the device and/or by serving as a reservoir for future recurrent infection and (4) persistence of certain chronic medical condition by the modulation of the host immune system.

Drug resistance:

Microbial cells in biofilm are often highly tolerant/resistant to antimicrobial drugs and difficult to eradicate with standard antimicrobial therapy. Infact, biofilm cells show as much as a 1000 fold more tolerance/resistance to antimicrobial drugs than their planktonic cell counterparts. Initially, it was believed that the ECM is the mainculprit rendering resistance to the antimicrobial drug by acting as a physical barrier affecting the accessibility of the drug to the target cells. However, recent evidence indicates that limiting the accessibility of the drug to the target cells by ECM acting as a physical barrier may be only a small part of drug resistance mechanisms shown by microbial biofilms.(Figure. 01)

Diagrammatic illustration of the implications of microbial biofilms in infectious diseases.

Fig. 1: Figure. 01

Diagrammatic illustration of the implications of microbial biofilms in infectious diseases.

Horizontal Gene Transfer (HGT) is a frequent mode of intraspecies and interspecies transmission of antimicrobial drug resistance determinants among bacterial cells in biofilms. 5358 HGT is the process by which bacteria can pass genetic material (mobile genetic elements and genomic DNA) from one cell to another horizontally (rather than from the parent to the progeny) by transformation, conjugation or transduction. In transformation bacterial DNA from lysed cells is taken up across their cell membrane by actively growing bacterial cells in a physiological state called competence and incorporates it into their own genome by genetic recombination. Any genetic trait that is advantageous (e.g. antimicrobial drug resistance determinants, ability to utilize alternate nutrient sources, ability to metabolize toxic chemicals) to the survival and fitness of the cell will be retained by natural selection. The newly transformed cell will eventually outgrow the rest of the population under selection pressure and becomes the dominant strain because of natural selection. One of the earliest reported examples of transformation is the classic experiment by Frederick Griffith 59 showing that non-virulent Streptococcus pneumonia became virulent with the addition of cell extract from a virulent strain to a growing S. pneumonia culture. This classic experiment not only paved the way to the discovery of transformation but also provided evidence in support of DNA as the genetic material (Figure. 02).

Scanning electron micrograph of polymicrobial biofilm formed by Pseudomonas aeruginosa and Aspergillus fumigatus.

Fig. 2: Figure. 02

Scanning electron micrograph of polymicrobial biofilm formed by Pseudomonas aeruginosa and Aspergillus fumigatus.

The biofilm was developed on Tissue Culture Thermanox 13 mm coverslips in SD broth in 12-well cell culture dishes at 35˚C for 48 h using A. fumigatus hyphae pregrown for 18 h. The biofilm was washed 3 times with 2 ml each sterile distilled water, fixed for 60 min in 2% glutaraldehyde in 0.1 M sodium cacodylate (NaCac) buffer (pH 7.4), postfixed in 2% osmium tetroxide in NaCac buffer, dehydrated with a graded ethanol series (25-100%) and critical point dried in CO2. The dried specimens were mounted on aluminum stubs with carbon adhesive tabs and sputter coated with gold-palladium. Biofilm was observed and imaged in a FEI XL30 scanning electron microscope (FEI, Hillsboro, OR) at 10 kV. The inset (top right corner) shows F pilus (conjugation tube) formed between two P. aeruginosa cells in 24-h biofilm. Horizontal gene transfer by bacterial conjugation is a more frequent event in microbial biofilms compared to planktonic cultures. Legends: AF, Aspergillus fumigatus hypha; PA, Pseudomonas aeruginosa cells forming biofilm; ECM, extracellular matrix precursor produced by the fungal hyphae.

Similarly, Aspiras et al. 60 have demonstrated that biofilm grown Streptococcus mutans was transformed to erythromycin resistance by the addition of naked DNA and the rates of transformation were 10 to 600 times greater than those observed in cells in planktonic culture. The second HGT mechanism by which the biofilm embedded bacterial cells acquire resistance to antimicrobial drugs depends on their ability to transfer genetic trait from one cell to the other by conjugation. In contrast to transformation, conjugation requires direct physical contact between the donor and the recipient by a conjugation tube called F pilus. The F-pilus made of proteins encoded by the F plasmid in the donor cell contracts drawing the cells closer together and the DNA passes through the conjugation tube to the recipient cell. 61 One of the prerequisites that facilitate conjugational transfer of genetic material is a stable undisturbed environment and close proximity between neighboring cells. The biofilm growth condition is ideal for providing a stable, uninterrupted environment with close proximity to neighboring cells. Dunny et al. 62 demonstrated the conjugational transfer of a tetracycline bearing plasmid in Enterococcus faecalis biofilm at a frequency almost 100 times higher than that obtained in planktonic culture. The close association between microbes of many different species found in naturally occurring (non-laboratory) biofilms would seem to promote the possibility of cross species conjugation. In fact, interspecies conjugation has been observed in the laboratory and in a dual species biofilm consisting of a tetracycline resistant Bacillus subtilis strain and a tetracycline sensitive species Staphylococcus, the drug resistant Bacilluspassed on the resistance trait to Staphylococcus. 63

The third mechanism of HGT is transduction. During the replication of a bacteriophage in the lytic cycle, a bacterial virus is accidently packaged with a piece of bacterial DNA together with the phage genome. When the bacterial DNA carrying phage infects another bacterial cell at least a portion of the viral genomic DNA introduced into the cell is bacterial not viral. The bacterial DNA thus introduced is subsequently incorporated into the bacterial genome either by recombination or by the integration of the bacterial virus into the host genome by lysogeny. If the heterologous DNA thus introduced to the new host bacterial recipient carries antibiotic resistance determinants, then the recipient cell would show resistance/tolerance to antimicrobial drug(s). Reports of transduction within biofilms are not as common as those of transformation or conjugation. However, numerous reports exist of bacterial virus genes being expressed in biofilms. There are also examples of bacterial genes carried by bacteriophages being expressed in the cells of biofilms.

HGT is very rare in planktonic cultures. The persistent biofilm growth provides not only a favorable environment for increased spontaneous mutation but also an increased frequency of HGT. For instance, in S. aureus the frequency (1.9 x 10-4) of HGT is increased by almost 16000 fold compared to the frequency (1 x 10-9) in planktonic cultures. 64 There are several examples of horizontal gene transfer coupled with acquisition of drug resistance under biofilm conditions in a wide variety of biofilm producing organisms. 6568

In addition to the enhanced frequency of spontaneous mutation and HGT within the biofilm community resulting in increased antibiotic resistance/tolerance, the biofilm growth appears to employ some of the classic gene regulation mechanisms for high level resistance/tolerance to antimicrobial drugs. BrlR is a MerR-like transcriptional regulator of multidrug transporters that plays a key role in the high-level drug resistance/tolerance of biofilms of Pseudomonas aeruginosa. Expression of BrlR is biofilm specific. The BrlR binds to its own promoter in the presence of the secondary messenger cyclic-di-GMP and autoinduce its own expression. The increased levels of BrlR in conjunction with another transcriptional regulator called SagS activate the multidrug efflux pump operons mexAB-oprM and mexEF-oprN enhancing the synthesis of the multidrug efflux proteins MexA, and MexE. The increased MexA and MexE levels result in active expulsion of intracellular antimicrobial drug(s) conferring high level resistance/tolerance to biofilm bound cells. 6973

Medical device related infections:

A significant percentage of nosocomial infections are directly related to the implantation of modern medical devices and prosthetics commonly used to either improve the quality of life or to deliver better medical care. 74,75 But the use of these devices on a long term or permanent basis comes with an increased risk for infection. 7582 These medical devices are made of plastic, steel or other type of durable material to which microorganisms attach rapidly with high efficiency. 8385 Because of the long term use of these devices the organisms commonly responsible for medical devices related infections are capable of producing sustainable biofilm. The biofilm community of cells is not only highly resistant to commonly used antimicrobial drugs but also they provide a reservoir for recurrent and at times life threatening bloodstream infections. 36 Moreover, at times the presence of microbial biofilm causes the failure of the device. The most commonly involved medical devices involved in the development of microbial biofilms are intravascular catheters (IVCs), devices used in orthopedic surgery such as hip and knee replacements, metal plates and orthodontics. Studies have also shown that biofilms are able to rapidly recover from mechanical disruption of these devices to reform biofilm within 24 hours. Thus, removal of the device involved followed by aggressive antimicrobial therapy is the only option to clear device-related biofilm-dependent infection.

Biofilm and chronic infection:

Chronic infections are the breeding grounds for microbial biofilm development. In chronic infection (as opposed to active infection) the organism(s) persists for long periods of time (often multiple organisms in the same body site) giving them ample opportunity for mutualistic or synergistic interactions resulting in the formation of polymicrobial biofilm. For instance, in chronically infected cystic fibrosis lungs P. aeruginosa and A. fumigatus that are traditionally antagonistic in nature adapt to each other producing sustainable interaction which often leads to the formation of biofilm involving both species. Obviously, such duel-species microbial growth produces duel-species biofilm whose make up and characteristics could be significantly different, including the production of a mixed microbial ECM. The inflammatory response of the host immune system to the monomicrobial and polymicrobial biofilms could be vastly different indicating clinical implications.

Diabetic chronic wound is another medical condition where microbial biofilm plays a major role in the inability of chronic wound to heal. Chronic wound microbiology is complex and a wide variety of microorganisms are implicated in delaying wound healing. 8694S. aureus (often in combination with the pathogenic yeast C. albicans) is the most common wound bacterium, followed by Enterococcus faecalis, P. aeruginosa, coagulase negative staphylococci and Proteus species. But in all likelihood, they represent only a fraction of microbes involved in chronic wounds. Harrison-Balestra et al. 95 showed that wound-isolated P. aeruginosa displays characteristics of a mature biofilm within 10 hours of in vitro growth suggesting that bacteria in wounds rapidly develop biofilms. Only 6% of acute wounds had microbial biofilms, whereas 60% of chronic wounds exhibited biofilm formation. The polymicrobial nature of wound infections and the possible role of anaerobic bacteria have been emphasized by several investigators. 95,96 Several studies have found a correlation between the presence of multiple bacterial species and non-healing wounds compared to one bacterial species. 96,97

Role of biofilm in modulation of immune response:

The inflammatory response mounted by the immune system of the host against invading pathogens is intended to protect the host and damage the infectious agents. But there are several clinical conditions involving chronic infection where the mounting proinflammatory immune response is detrimental to the host tissue due to the so-called ‘friendly fire’ causing irreversible damage. In most (if not all) of these cases the presence of microbial biofilm is the underlying cause for the misdirected ‘friendly fire’ and the problem is aggravated by the presence of multiple organisms in the biofilm community. 35,98102 Chronically infected cystic fibrosis (CF) lung is a prime example in this case. On the contrary to the rational thinking, most of the lung tissue damage is caused by proinflammatory response of the host immune system and not by the infectious agent(s). Certain chemical component(s) of the ECM produced by the microbial biofilm, including exogenous DNA in the matrix, is directly responsible for the proinflammatory response leading to the lung tissue damage. 103 A second prime example in this case is diabetic chronic wounds often containing microbial biofilm as the root cause of the chronicity of diabetic wounds. The proinflammatory factors produced by the immune system in response to the wound microbial biofilm are thought to be responsible for the wound healing process become fixed in the inflammatory wound healing stage and the body is unable to successfully progress towards the proliferative stage. 98100 The inflammatory response is often unsuccessful in removing the biofilm. On the contrary, it actually increases biofilm survival. A third example of the unintended outcome due to the microbial biofilm mediated modulation of the immune system is periodontitis, a chronic inflammatory condition of the periodontium. Periodontitis is caused by the presence of microbial biofilms formed on teeth called dental plaque. Substances released from the dental plaque such as lipopolysaccharides, antigens and other virulence factors, gain access to the gingival tissue and initiate an inflammatory immune response, leading to the activation of host immune cells. As a result of cellular activation and release of inflammatory mediators such as cytokines, chemokines, arachidonic acid metabolites and proteolytic enzymes collectively contribute to tissue destruction. 6,104,105

Conclusions

  • Microbial biofilm is an emerging clinical problem in infectious diseases. In fact, it is the ‘Achilles Heel’ of antimicrobial therapy and management of infectious diseases in humans because of unprecedented high level antimicrobial drug resistance associated with biofilms.

  • With the increased use of life-saving and quality of life improving medical devices such as catheters, prosthetics and other beautification devices either on a long term or permanent basis, the incidence of microbial biofilm is on the rise and continue to increase in the future causing a formidable clinical problem.

  • Microbial biofilms affect human health in relation to infectious diseases in four major areas, namely, (1) development of increased antibiotic resistance (2) increase in medical device related infections (3) increased persistence of chronic infection and (4) nullify or diminish the host immune function by immunomodulation. A suboptimal treatment and management of infectious diseases due to microbial biofilms may result in increased morbidity and mortality and an increase in healthcare costs.

Future directions

  • Development of antibiofilm drugs directed to one or more of the major stages of biofilm development such as adhesion, maturation and dispersion will be a fruitful area for future biofilm research. By interfering with one or more of the biofilm developmental stage, the spread of recurrent infections due to biofilm-dependent reservoir will be markedly reduced.

  • The lack of effectiveness of antimicrobial treatment as well as the evasion of host immune defense by microbial biofilm is partly due to the presence of ECM. Once the biofilm bound cells are outside the ECM protected environment they are usually vulnerable to antibiotic therapy. However, if they are endowed with acquired resistance similar to those found in drug resistant planktonic cells they could be treated with alternate antimicrobial drugs outside of the ECM. So the key to antibiofilm drug development lies on the ability of the drug to disrupt the ECM. Moreover, ‘ECM busting’ antibiofilm drug will have the ability to disrupt existing biofilm.

  • A major part of the biofilm formation in clinical settings is associated with implantation of temporary and permanent medical devices. So use of bacterial/fungal adhesion resistant material for the construction of such devices will be highly useful in preventing a significant portion of biofilm formation. Additional research in this currently active area will be highly beneficial.