The Association between Biofilm Formation and Antimicrobial Resistance with Possible Ingenious Bio-Remedial Approaches

Abstract

Biofilm has garnered a lot of interest due to concerns in various sectors such as public health, medicine, and the pharmaceutical industry. Biofilm-producing bacteria show a remarkable drug resistance capability, leading to an increase in morbidity and mortality. This results in enormous economic pressure on the healthcare sector. The development of biofilms is a complex phenomenon governed by multiple factors. Several attempts have been made to unravel the events of biofilm formation; and, such efforts have provided insights into the mechanisms to target for the therapy. Owing to the fact that the biofilm-state makes the bacterial pathogens significantly resistant to antibiotics, targeting pathogens within biofilm is indeed a lucrative prospect. The available drugs can be repurposed to eradicate the pathogen, and as a result, ease the antimicrobial treatment burden. Biofilm formers and their infections have also been found in plants, livestock, and humans. The advent of novel strategies such as bioinformatics tools in treating, as well as preventing, biofilm formation has gained a great deal of attention. Development of newfangled anti-biofilm agents, such as silver nanoparticles, may be accomplished through omics approaches such as transcriptomics, metabolomics, and proteomics. Nanoparticles' anti-biofilm properties could help to reduce antimicrobial resistance (AMR). This approach may also be integrated for a better understanding of biofilm biology, guided by mechanistic understanding, virtual screening, and machine learning in silico techniques for discovering small molecules in order to inhibit key biofilm regulators. This stimulated research is a rapidly growing field for applicable control measures to prevent biofilm formation. Therefore, the current article discusses the current understanding of biofilm formation, antibiotic resistance mechanisms in bacterial biofilm, and the novel therapeutic strategies to combat biofilm-mediated infections.

Keywords: AMR; biofilm; biofilm control; extracellular polymeric substances; multidrug resistance; silver nanoparticles.

Figure 1

Stages of biofilm life cycle: initial adhesion (a); initial developed extracellular polymeric substances (EPS) (b); early development of EPS production (c); further development of biofilm EPS for maturation (d); mature biofilm (e); and dispersion of biofilm (f).

Figure 2

Biofilm on human teeth in the form of plaque and/or calcified plaque (calculus): soft plaque at the neck (near gingiva), crown (more towards gingiva), and more permeable dentogingival junction area of human teeth (a–d). Dentogingival junction can be seen shifted towards cemento-enamel junction (a,d), providing more surface for biofilm deposition, exposure of dentine area, and serving as an easy route for passage of bacterial products from biofilm into deep tissues. Hard (calcified) plaque or calculus on artificial surfaces of prosthetic teeth (dental crown or fixed partial denture): more calcified plaque can be seen near the dentogingival junction area, as compared to the crown of prosthetic teeth, and gingival inflammation (gingivitis) of free gingiva may also be seen (e,f). Plaque on early secondary or permanent, as well as primary, teeth (g,h). Calcified or hard plaque on the occlusal area forms mainly in pits, fissures, fossae, and grooves of permanent human teeth (i,j). Change in color of plaque can also be seen, indicating entrapment of minerals from saliva into biofilm and hardening, and soft as well as hard plaque on occlusal, interdental, dentogingival, and cervical areas of permanent human molar teeth (k,l). Very hard plaque of dark yellow, light yellow, or brown in color near the dentogingival, interdental, and cervical areas of permanent human lower anterior teeth (m–p). V-shaped shifting of dentogingival junction and free gingiva away from the neck of the teeth, towards the cemento-enamel junction, can also be seen. (Intraoral and dental pictures were provided by Dr. Mamta of Mamta Dental Clinic, Faridabad, India).

Figure 3

Biofilm-mediated antimicrobial resistance in bacteria: nutrient gradient indicated by color gradient (1), persister cells (2), extracellular DNA (3), intercellular interactions (4), stress response (5), matrix extracellular polysaccharides (6), biofilm cells (7), genetic determinants (8), multidrug efflux pump (9), host surface (10), less active deep layer cells (11), and altered environment (12).

Figure 4

Mechanism of antimicrobial resistance: cell wall modification (a); modification of drug target (b); enzymatic degradation or destruction of antimicrobials (c); and efflux pump (d).

Figure 5

Strategies against biofilm: (a) physical methods of biofilm removal include water jet (1), sound, ultrasonic, electric and other shockwaves (2), and laser and photodynamic therapy (3) to dislodge and disrupt the biofilm in small parts (4) and to break the main biofilm region (5). The host surface or substrate (6) is not usually disturbed or modified in this method. (b) Structural modification and chemistry-dependent methods include techniques to change the texture, pattern, surface and material properties, surface charge, and chemical coating to repel or avoid bacterial cells attaching to the surface (1,2). Additionally, graphene sheets on substrate (3), graphene nanoplatelets (GNP) (6), and chemistry-dependent polyamine-functionalized quantum dots (QDs) are some advanced methods that can be used to inhibit the cell adhesion to substrate (7), and to damage the bacterial cells (4,5) that would provide antimicrobial properties. (c) Small molecules and nanoparticles developed and used for biocompatibility and antimicrobial properties include organic nanoparticles (liposomes and polymers), inorganic nanoparticles (silver, copper, gold, and iron oxide nanoparticles), and specifically designed antimicrobial peptides (AMPs) of 5 to 90 amino acids and with a molecular mass of 1 to 5 kDa. AMPs are capable of disrupting bacterial cell membranes, in addition to having enzymatic and protein activities. Nanoparticles can cause damage to the electron transport chain (1,2), inhibit membrane protein (3), dysfunction of the mitochondria (4), disassembly of ribosome (5), denaturation of proteins (6), oxidative stress by producing ROS (7), inactivation of enzymes (8), damage to DNA (9), damage to cell membrane (10), and degradation of EPS (11).

Figure 6

Synthesis of silver nanoparticles: collection of Azadirachta indica (neem) leaves (a); washing, drying, and grounding of leaves (b); Soxhlet extraction (c); collection of extract (d); preparation of 200 mL 20 mM silver nitrate solution (e); 200 mL deionized water (f); mixing of silver nitrate solution and deionized water (at the time of mixing) (g); incubation at room temperature for 12–18 h (h); change of color of mixture after 12–18 h (i); centrifugation at 1000 rpm for 10 min (j); collection of pellet after discarding the supernatant, washing of pallet with solution of deionized water and ethanol (3:1) for three times, drying, and collection of nanoparticles (k); human dentine-block-based biofilm assay (l–o); cytotoxicity assay (p); SEM analysis (q); and crystal violet-based biofilm assay (r,s).

Figure 7

SEM analyses of biosynthesized silver nanoparticles (a–d).

Figure 8

Boxplot showing colony forming units upon treating Enterococcus faecalis biofilm with antimicrobials. A: control; B: silver nanoparticles 20 µg/mL; C: silver nanoparticles 10 µg/mL; D: silver nanoparticles 20 µg/mL and clove 2500 µg/mL combination; E: silver nanoparticles 10 µg/mL and clove 1700 µg/mL combination; F: silver nanoparticles 5 µg/mL and eugenol 850 µg/mL combination; G: sodium hypochlorite 5%; H: sodium hypochlorite 3; I: chlorhexidine 2%.