Bacterial Biofilm and the Medical Impact

Abstract

Most pathogenic bacteria species form biofilm as their protective mode of growth, which helps them survive from the bactericidal effect of the antimicrobials or the killing activity of the host immune cells. The bacteria cells’ survivability via biofilm formation creates challenges in the medical field in terms of the device and also disease-related to biofilm. The impact of the bacterial biofilm issue is worsening over time, and the association to the high tolerance to the antimicrobial agents leads to increased morbidity and mortality worldwide. This review will highlight the main characteristics of the biofilm, the issue of biofilm in clinical practice, which also covered the pertinence of the biofilm in clinical practice, device-related biofilm disease, oral disease, and the significant bacterial species involved in the biofilm-related infections. Knowledge about the vital role of bacterial biofilm in related disorders will give new insight into the best approaches and alternative treatments for biofilm-related disease.

1. Introduction

Microbial biofilm is a microscopic entity that significantly affects human health. It is composed of bacterial colonies within a matrix of extracellular polymeric substances, which protect them from environmental stress, shear stress, detergents, antimicrobial agents, and the host’s immune cells. According to the National Institute of Health (NIH), 65% of microbial diseases and 80% of chronic infection is related to biofilm formation [1]. Antibiotics cannot treat several conditions related to biofilm formation due to the high level of biofilm resistance activity. An antibiotic concentration killing effect toward a biofilm might require 1000 times greater than those required to kill the planktonic bacteria cells [2]. In addition, bacterial biofilm causes several diseases in response to both device-related and non-device-related infections. This situation creates challenges for the medical team to provide the best solution or treatment.

Broad heterogeneity of phenotypes developed within a biofilm contributes to the recalcitrance of the sessile bacteria. This condition evolves the bacteria cells inside the biofilm to coordinate and differentiate through the communication system and the releasing of quorum sensing small signaling molecules called autoinducers. Interbacterial communication allows the decision of their density and regulation of the virulence gene expression. This is also the indicator of antibiotic susceptibility profiles of a biofilm. Due to biofilm-cell physiological states, biofilm usually shows high resistance toward most antibiotics. Antibiotics might be effective against the active cells located at the top of the biofilm, in contrast to nutrient-depleted zones at the middle and bottom of the biofilm in which the cell is in the state of dormancy and lack of metabolic activity [3].

The emergence of antibiotic resistance toward biofilm leads to various chronic diseases and is very difficult to treat with efficacy. Most of the recently available antibiotics are not able to resolve the infection. In addition, higher values of minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) used to treat biofilm may result in in-vivo toxicity and other complications. Thus, biofilm formation issues significantly impact human health and the health care industry.

2. Biofilm in clinical practice

A meta-analysis study by Malone et al. (20017) reported the prevalence of biofilms in chronic wounds was 78.2% [4]. This finding supports the clinical assumptions that biofilms appear and are significant in human chronic non-healing wounds. Besides, one of the most prominent clinical-level species is Staphylococcus aureus affecting both hospital-acquired and community-acquired infection. The biofilm production of S. aureus cells isolated from clinical samples shows the association of the biofilm with methicillin and inducible clindamycin resistance [4]. In addition, MRSA strains showed a higher biofilm production than MSSA strains. Suggesting strong biofilm formation increases the possibility of antibiotic resistance and leads to treatment failures in MRSA infections [4].

Other than that, Escherichia coli is reported to lead the urinary tract infection (UTI), contributing to 80 to 90% of all community-acquired and 30 to 50% of all hospital-acquired cases of UTIs [5]. The study of the uropathogenic E. coli revealed a high prevalence of biofilm-forming strains of this group of bacteria that are also highly associated with the multi-drug resistant (MDR) phenotype. Out of 200 E. coli clinical isolates, 62.5% can produce biofilm, with 93% of the isolates showing varied resistance with amoxicillin and co-trimoxazole, followed by gentamycin (87%), cefuroxime (84%), Nalidixic acid (79%), Amoxicillin clavulanic acid (62.5%), Ciprofloxacin (62%), ceftriaxone (55%), Ceftazidime (54%), chloramphenicol (28%), Nitrofurantoin (25.5%) and Imipenem (0.5%) [5].

This finding represents the burden of the biofilm formation issues, which are highly associated with increased antibiotic resistance. In addition, another meta-analysis study concludes that biofilm formation production by microbial species impacts the blood system infection leads to resistance, persistence, and mortality. Staphylococci biofilm producer shows significantly higher prevalence in the resistant strain, whereas Candida species biofilm production highly impacted mortality [6]. High cell density within the biofilm facilitates high rates of horizontal gene transfer between microorganisms through the conjugation process, more frequent within the community inside biofilm than the planktonic bacteria [7].

3. The main characteristic of bacterial biofilm and their resistance to antimicrobial agents

In general, bacterial biofilm shows resistance against antibiotics and human immune systems. The process of biofilm formation initiates with the attachment of the planktonic bacterial cells on the living or non-living surfaces. The attachment will lead to the construction of the micro-colony of the bacteria cells and rise to a three-dimensional structure, followed by biofilm maturation and detachment. The process of biofilm formation until a detachment of the cells is regulated by the cell-to-cell communication known as the quorum-sensing system. Extracellular polymeric substance (EPS) is one of the main components in a biofilm, strengthening the interaction of the microorganism in the biofilm [8]. Typically 65% of the biofilm volume is constituted by the extracellular matrix, partially or mainly composed of polysaccharides, proteins, and nucleic acid [9]. The EPS protects bacteria from environmental stress such as salinity, UV exposure, dehydration, antimicrobial, and phagocytes [10]. Besides, some channels separate the microcolonies inside the biofilm structure to be attached to new niches [1].

There are studies on the resistant mechanism of the bacterial biofilm toward antibiotics. Most of the studies suggest that the production of glycocalyx or EPS matrix and other functions play a prominent role that prevents the penetration of the antimicrobial agents inside the biofilm. Common disinfectant such as chlorine is only 20% or less of the total concentration in the bulk liquid measured inside the biofilm of P. aeruginosa and Klebsiella pneumoniae. Interestingly, a complete equilibrium with the bulk liquid did not reach even after 1 to 2 hour incubation time [11]. Another study also showed the same finding when the biofilm production of P. aeruginosa on a dialysis membrane showed retarded piperacillin diffusion [12].

In contrast, evaluation on Staphylococcus epidermidis biofilm that were grown in the same manner show diffusion of rifampicin and vancomycin across the membrane [13]. Thus, this finding might suggest that inhibition of antibiotic absorption cannot be explained by antimicrobial resistance. Other pathways and mechanisms might be occurring inside the biofilm.

In addition, the difference between thin and thick biofilm formation toward antibiotic resistance has been explored. Penetration of the hydrogen peroxide in the thin biofilm of P. aeruginosa was observed compared to a viscous biofilm, which shows no penetration of that chemical compound inside the biofilm [14, 15]. Interestingly, the penetration of the hydrogen peroxide in the thick biofilm was observed in the mutant strains of P. aeruginosa without katA gene, which is the calatase gene that functions to neutralize the hydrogen peroxide [14].

Furthermore, depletion of the nutrient level inside the biofilm will influence the interaction of the bacteria cells against antimicrobials. Generally, during bacterial growth, the transition from exponential to stationary or no growth leads the bacteria to resistance to antibiotics [3]. Due to low nutrient level and high cell density, the planktonic cell of the bacteria starts to aggregate and initiate attachment and biofilm formation. In the biofilm community, bacteria begin to change their mode to slow-growing. These physiological changes might play a role in the insensitivity of the bacterial cells inside the biofilm toward antibiotics.

Biofilm disease includes device-related infection, chronic infection with the absence of a foreign body, and malfunction of medical devices. Biofilm-related disease or infection is complicated to treat and detect at early stages by microbiological analyses. Thus, characterization of the chemical composition of the EPS might expedite the development of new therapies against biofilm related-infection.

4. Biofilm and device-related infection (DRI)

The emergence of device-related infections is highly associated with biofilm-producing bacteria among critical patients in the intensive-care units. DRI is defined as an infection that occurs in a patient with any device (for example, endotracheal tube, intravascular catheter, or indwelling urinary catheter) for at least 48 hours in use before the onset of infection [16]. Most of the DRI reported in the developed country is led by catheter-related bloodstream infections (CRBSI), followed by catheter-associated urinary tract infections (CAUTI) and ventilator-associated pneumonia (VAP) [17]. In addition, another study of the biofilm formation on or in the medical devices that were examined upon removal from the patients or were tested in animal or laboratory systems. Several medical devices may involve biofilm formation, such as central venous catheters, central venous catheter needleless connectors, contact lenses, mechanical heart valves, pacemakers, peritoneal dialysis catheters, prosthetic joints, tympanostomy tubes, and voice prostheses (Table 1) [18].

Devices	Causative microorganisms	Burden of illness	References
Contact lenses	P. aeruginosa, Staphylococcus aureus, E. coli, Staphylococcus epidermidis, species of Candida spp., Serratia spp., Proteus spp.	Keratitis	Jamal et al. 2018 [1]
Central venous catheter	Pseudomonas aeruginosa, S. aureus, coagulase-negative staphylococci, Klebsiella pneumoniae, E. coli, Acinetobacter baumanii	Bloodstream infections (BSI)	Gahlot et al. 2014 [19]
Urinary catheters	Escherichia coli, Enterococci spp, coagulase negative Staphylococcus, P. aeruginosa, Candida spp., Proteus mirabilis, K. pneumoniae, Morganella morganii.	Urinary tract infection	Nicolle et al. 2015 [20]
Mechanical; heart valves	Streptococcus spp., S. aureus, S. epidermidis, gram-negative Bacillus, Enterococcus, Candida spp. Haemophilus parainfluenzae, Propionibacterium acnes.	Prosthetic valve endocarditis	Jamal et al. 2018, Gomes et al. 2018 [1, 21]
Implantable prosthetic device	S. aureus, S. epidermidis, Streptococcus agalactiae, P. aeruginosa, E. coli, P. acnes, Enterococcus faecalis	Prosthetic joints infection	Benito et al. 2016 [22]
Endotracheal tube	P. aeruginosa, S. aureus, Candida albicans, Streptococcus spp.	Ventilator-associated pneumonia	Fernandez-barat et al. 2016 [23]

Table 1.

Common devices related diseases and the microbial etiology.

Biofilm formation on medical devices is related to the substratum and cell surface properties. For instance, the characters of glass and various metals that are highly charged hydrophilic materials, water pipes, and environmental surfaces are pretty rough or textured. Some materials might be coated with antimicrobial, such as antibiotic-impregnated catheters [24]. The characteristic of the substratum might have a significant effect on the rate of bacterial adhesion and biofilm formation. The rougher and more hydrophobic materials will develop rapid biofilm formation.

Hydrophobicity of both bacteria and material surfaces may influence the adherence capacity of bacterial cells. Hydrophilic material surfaces are usually more resistant to bacterial attachment than hydrophobic materials [25]. Fletcher and Loeb’s (1978) study reported that many marine Pseudomonas sp. are attached to hydrophobic plastics with little or surface charge-free like Teflon, polyethylene, polystyrene, poly (ethylene terephthalate). At the same time, very few are attached to hydrophilic and negatively charged substrata like glass, mica, and oxidized plastics [26]. However, dental plaque formation in the human oral cavity is reported as far less on hydrophobic compared to hydrophilic surfaces, even after nine days without oral hygiene [27]. In addition, another study by Everaert et al. (1997) showed less biofilm formation on hydrophobic silicone rubber voice prosthesis of laryngectomized patients compared to the hydrophilic surfaces after six weeks in the human body [28]. Thus, the role of hydrophobic material surfaces toward rapid biofilm formation is still unclear.

5. Biofilm in chronic infections

Chronic infections are a significant burden to patients and the healthcare systems. Besides, the economy is also impacted and varies depending on chronic infection due to several treatments failure. It is expected that there will be an increase in chronic infection cases in the future due to an aging population concurrent with the rise in lifestyle diseases such as diabetes which is a significant cause of chronic wounds [29]. Bacterial biofilm has been recognized as responsible for most chronic infections, including otitis, diabetic foot ulcer, rhinosinusitis, chronic pneumonia in cystic fibrosis patients, osteomyelitis, and infective endocarditis [30]. These infections affect millions of people each year, with high mortality and morbidity rate as a consequence. The worse issue of biofilm involvement in infection is due to undetectable species responsible as swabs and scrapes of biofilm samples often show culture-negative. This might be due to the strong association of bacteria within the biofilm or their uncultivability. The same problems occur for implant and catheter-related infections; identifying the bacteria has been almost impossible. Up to this date, bacteria species from a biofilm were considered unculturable. In addition, some pathogenic bacteria that cannot grow the culture media are believed to be activated when present in the host system or environment, and later they can initiate infection [31]. The biofilm infection often finalizes as untreatable, leading to the chronic state of bacterial infections. However, chronic infection will lead to an adaptive inflammatory response, characterized by a high level of mononuclear leucocytes and IgG antibodies [32]. In some cases, such as the cystic fibrosis patient suffering chronic lung infection, the inflammatory response shows the chronic response with continued recruitment of polymorphonuclear neutrophils (PMNs) [32]. PMN are the leukocytes critical to the innate immune response against invading pathogens (Table 2).

Diseases	Pathogenesis
Cystic Fibrosis (CF)	P. aeruginosa biofilm induces the infiltration PMNs, subsequent tissue damage, and loss of lung function [33].
Infective endocarditis	Bacterial biofilm diminishes the heart valve function and triggers persistent infection to the circulatory system. Detachment of the biofilm might spread to the other systemic system contributes to kidney, brain, and extremities, particularly risk to emboli [34].
Diabetic foot ulcer	Hyperglycemic conditions cause deleterious effects on the innate immune system associated with altered PMNs, impaired phagocytosis, and bactericidal activity against the infections. Thus, bacterial biofilm in the diabetic foot ulcer implicates the failure of the healing process [35].
Chronic rhinosinusitis	Biofilms contribute to the destruction of the epithelial layer and the absence of cilia and continuous local inflammatory response [36].
Osteomyelitis	Biofilm formation and proliferations lead to an inflammatory bone disorder characterized by increased local cytokines and osteoclastogenesis [37].

Table 2.

Examples of biofilm-related chronic infections and suggestive pathogenesis.

6. Oral diseases

An oral disease associated with bacterial biofilm is periodontal disease. Periodontal disease has been reported by Global Burden of Disease (GBD) 2010 as a global prevalence of 35% for all ages combined and the sixth-most prevalent condition in the world [38]. Initiating biofilm formation at the periodontal area by various pathogenic species of oral bacteria may lead to severe inflammatory disorders that reduce the gum line, bleeding of the gum, and tooth loss. The issue of periodontal disease is not limited to the antibiotic resistance properties of the biofilm but also the aggressive pro-inflammatory response toward the virulence activities of the pathogenic species that reside in the biofilm. In addition, there are associations between periodontal disease and other systemic diseases such as respiratory tract infection, cardiovascular disease, Alzheimer’s disease, gastrointestinal and colon-rectal cancer, diabetes and insulin resistance, and adverse pregnancy outcomes [39]. The association of periodontal disease with systemic disease is possible when the progressive inflammatory activity releases toxins or leakage of microbial products enter the bloodstream thru the blood vessel in the pulp chamber of an infected tooth. This agrees with a meta-analysis of 5 prospective cohort studies (86,092 patients) that indicates that individuals with periodontal disease had 1.14 times higher risk of developing coronary heart disease [40]. Whereas for the case of respiratory tract infection and pneumonia, the lung infection might occur due to the accumulation of the pathogens from saliva or oral cavity at the lower airways. Genetically identical respiratory pathogens isolated from dental plaque and bronchoalveolar lavage fluid from the same patient in the ICU indicate that respiratory pathogens’ significant reservoir might be associated with dental plaque [41].

7. Significant bacterial species related to a biofilm infection

Biofilm-producing bacteria play a significant role in biofilm-related diseases. The biofilm’s high resistance against antimicrobial agents and the host immune system contribute to considerable treatment challenges. Generally, the ability of a microorganism to form biofilms on the human tissue or related medical devices will lead to the association of chronic infection. The most common bacterial species related to biofilm formation in hospital settings are Enterococcus faecalis, S. aureus, S. epidermidis, Streptococcus viridans, E. coli, K. pneumoniae, Proteus mirabilis, Acinetobacter baumannii, and Pseudomonas aeruginosa [42]. These species may originate from the skin of healthcare workers or patients or might be from the surrounding as simple as tap water to which entry ports are exposed or other sources in the environment. For instance, Staphylococcus species mainly colonize humans’ skin and mucous membrane. S. aureus and S. epidermidis are the prominent aetiologic agents for nosocomial infection, surgical site, and bloodstream infection [43, 44]. The persistence of S. aureus biofilm formation is related to antibiotic pressure. This species own the ability to stay in the viable state but is not culturable [45]. Recently, daptomycin has been used as the last resort for treating Gram-positive bacterial infections, including MRSA and Vancomycin-resistant Enterococcus. This is due to its bactericidal activity against these bacteria [46, 47]. Enterococci cause a wide variety of infections in humans, including infection of the endocardium, urinary tract, bloodstream, biliary tract, abdomen, burn wounds, and medical devices [48]. However, the most prevalent is E. faecalis due to its biofilm formation ability and several virulence factors related to the persistence of biofilm formation and heterogeneity in antimicrobial resistance acquiring activity [49].

On the other hand, a study of attributable mortality dan morbidity caused by carbapenem-resistant K. pneumonia showed that 50% of the 391 patients ended with mortality, with 12.2% of the case being bloodstream infections [50]. In addition, K. pneumonia is responsible for many cases of nosocomial infection related to a pyogenic liver abscess or endophthalmitis [51]. Besides that, P. aeruginosa and E. coli are most prevalent for medical device-associated pathogens. P. aeruginosa contributes to 10 to 20% of all nosocomial infections, whereas E.coli contributes to 50% of the infections associated with urinary catheters [52, 53]. At the same time, A. baumannii emerges with significant pathogenicity due to its multi-drug resistant capacity and the ability to form biofilm on several biotic and abiotic surfaces [54]. This species is rapidly spread in the health care facilities and can stay months on the dry surface on insensate objects [55].

8. Conclusion

Biofilm formation is a natural process employed by several bacteria species. This is part of the adaptation process and survival mechanism in response to their environment. Unfortunately, bacterial biofilm formation develops to impact human health and industries. Evolution to adapt toward the surroundings triggered by an antimicrobial substance during a treatment intervention leads the bacteria cell to manage their survival by acquiring the resistant genes thru several pathways and mechanisms. Applying antibiotics to treat bacteria’s biofilm-related infection will lead to another level of resistance activity in the biofilm community as well as toxic effects to the host system. A comprehensive understanding of the biofilm structure organization and the prominent chemical involved might help the researcher elucidate a potent compound or chemical that can degrade or interact with the bacterial biofilm. Alternative methods or therapies other than antibiotics application must be explored to reduce the impact of the bacterial biofilm on human health and the health care industry.