1. Introduction

Antibiotics can be used both as prevention and therapy. In terms of current treatments, different strategies include raising dosage concentrations and combining therapy with other antimicrobial agents ^[1][101]. The maturity of the mass of a biofilm should be considered as mature and therefore as less susceptible to treatment ^[2][3][102,103]. This applies to a wide range of species of both facultative aerobic and facultative anaerobic bacteria that form biofilm. These include the classically non-motile Gram-positive Staphylococcus aureus, S. epidermidis, Enterococcus faecium and Gram-negative Acinetobacter baumannii and Klebsiella pneumoniae, as well as the flagellated Gram-negative Pseudomonas aeruginosa and Enterobacter spp. ^[4][5][104,105].

In selecting a suitable antibiotic sufficient biofilm penetration is an important consideration. Hence, tetracyclines, macrolides, rifamycins, lincosamides, quinolones, fusidic acid, oxazolidinones, sulfonamides and nitroimidazole are preferred to glycopeptides, aminoglycosides, polymyxins and β-lactamases as they have the capability to penetrate deeper ^[6][106]. In addition to biofilm age and level of resistance to a given antibiotic, broader considerations for treatment include appropriate duration of antibiotic regimen and dosage optimization ^[7][107].

2. Bacterial resistance

There are several tolerance mechanisms utilized by bacteria that enable them to show resistance and persistence in the face of antibiotic treatment. A growing concern surrounds the fact that biofilms are not only resistant to antibiotics, but frequently also to the host immune response ^[8][108]. In order to combat the thorny problem of antibiotic resistance, suggested solutions include gaining a deeper knowledge of phenotypic and genotypic characteristic features of biofilm ^[9][109]. Mounting evidence indicates that acquiring resistant genes via genetic exchange and through extracellular polymeric substances plays a pivotal role in antibacterial tolerance ^[10][11][110,111].

A specific feature of biofilm is ‘recalcitrance’, a term used to describe its capability to survive in the presence of high doses of antibiotics ^[12][112]. Bacteria within biofilm can exhibit resistance to multiple treatments, even in the presence of high concentrations of bactericidal and bacteriostatic antibiotics and toxic compounds, in stark contrast to their planktonic existence. Noteworthy among various mechanisms by which this complex phenomenon may occur are antibiotic efflux, enzyme activity and reduced permeability. MIC can be used as a quantitative measure of antibiotic resistance; the higher the MIC, the more resistant. Resistance and tolerance each has a potential role in biofilm recalcitrance. Exposure to both bacteriostatic and bactericidal antibiotics can lead to resistance, while it is only the use of bactericidal antibiotics that may result in tolerance ^[12][13][14][112–114].

3. Antibiotic sensitivity tests

The MIC and minimum bactericidal concentration (MBC) are the lowest levels of an antimicrobial agent, typically an antibiotic, required to prevent visible growth upon overnight incubation (i.e., to cause cell stasis) and to kill a particular bacterium, respectively ^[15][115]. Similarly, minimum biofilm inhibitory concentration (MBIC) and MBEC refer to the lowest concentrations needed to achieve inhibition and eradication, respectively ^[16][116]. MIC is much higher for those bacteria that form biofilm compared to those than do not ^[17][117]. This concurs with the observation that biofilms are resistant to antibiotics concentrations up to 1000 × greater than those required to kill free-living bacteria ^[1][101], which signifies a pressing need to use combination therapy instead of monotherapy. The emergence of S. aureus isolates that are resistant to multiple antibiotics is a real concern, especially as it is exaggerated among MRSA strains ^[18][19][118,119].

Performing antibiotic sensitivity tests is necessary to select an appropriate choice and dose of treatment. Determination of MIC and MBEC of bacteria can inform tailored treatments and help to reduce the spread of resistant strains. Staphylococcal isolates from biofilm show a much higher breakpoint for MBEC than for MIC, indicating the importance of applying both biofilm susceptibility tests ^[20][120]. While vancomycin MBEC and MIC of planktonic cells are similar, for biofilm-producing isolates they are markedly different, so from a clinical perspective MBEC is the preferred measure ^[21][121]. Despite the availability of standardized methods to treat biofilm, most successful approaches were determined on planktonic cells. Although MBEC and MBIC values are proposed, this is confounded by limited evidence and complexity of correlation between innate activity towards planktonic cells and those in biofilm ^[22][122].

4. Multi-drug resistance

Resistance to a range of antibiotics has become common among MRSA strains. The formerly frontline β-lactam antibiotic methicillin targets penicillin-binding proteins (PBPs), enzymes that are essential to peptidoglycan synthesis. Yet, due to genetic mutation under the selective pressure imposed by overuse, PBP and PBP2a have become principal resistance factors ^[23][24][123,124]. One study from Nepal showed that the vast majority of multi-drug resistant isolates are MRSA with potential to produce biofilm ^[25][13]. Similarly, all strains of MRSA from nasal carriers possessed the capacity to form a biofilm that showed resistance to multiple antibiotics ^[26][125]. However, another study reported no difference between methicillin-sensitive S. aureus and MRSA strains to form biofilm ^[27][105], implying that there is no direct correlation between the ability of an isolate to form biofilm and its pattern of antibiotic resistance. In some cases, antibiotic therapy may not be completely successful due to low permeability to the biofilm matrix ^[28][35]. When this occurs, either removal of the foreign body, long-term single antibiotic treatment at high dosage and/or combination therapy is advised.

5. Combination therapy

A currently largely successful S. aureus anti-biofilm agent is the glycopeptide antibiotic vancomycin, which acts by interrupting cell wall synthesis ^[29][30][126,127]. Vancomycin is the preferred treatment for MRSA at present, although recently VRSA has been reported. The emergence of these strains, a major public health concern, could be for one or more reasons. Vancomycin is a large compound, which can lead to its weak penetration of biofilm. Additionally, as it inhibits oxygen and nutrient uptake ^[31][128]. In order to address this issue, combination therapy with antibiotics like rifampin and linezolid is proposed ^{[32][33][34][35]}[129–132]. Many tested strains of S. aureus and most of S. epidermidis are susceptible to rifampin, which can penetrate biofilm ^[36][37][133,134]. In keeping with this, rifampin was shown to be the only good candidate for biofilm therapy in isolates with relatively high MBEC for each of vancomycin, rifampin and gentamicin ^[38][135]. The efficacy of rifampin in combination with vancomycin is due to reducing bacterial adhesion ^[39][136]. On a cautionary note, as resistance to rifampin is acquired rapidly it should not be used alone ^[40][137]. The lipopeptide antibiotic daptomycin, an alternative treatment option for MRSA and VRSA, effectively targets biofilm ^[41][138]. Both rifampin and daptomycin can disrupt MRSA biofilm at lower concentrations than that of tigecycline required to eradicate mature biofilm, especially when used in combination. Other antibiotics are able only to prevent cell attachment ^[42][139].

6. Hi-tech delivery of antibiotics

In response to a substantial increase in reports of MRSA and VRSA in recent years, a range of modern medical technologies, such as laser therapy and nanoparticles, have been investigated in attempts to enhance antibiotic efficacy. There are several benefits of harnessing nanoparticles including their high surface area to volume ratio, capacity for drug transportation and antibiotics protection against exposure to pH and enzymes, each of which enhances the efficacy of an administered antibiotic ^[6][43][44][106,140,141]. When gold nanoparticles were used alongside laser therapy to combat resistant strains of S. aureus and P. aeruginosa biofilm viability reduced and, conversely, antibiotic sensitivity increased ^[45][142]. In another study in which gold nanoparticles were conjugated to antibody specific to S. aureus peptidoglycan and activated by exposure to laser, bacterial cell counts were substantially reduced ^[23][123]; potentially, such technology could be used in tandem with antibiotics to boost their efficacy. Continuing research is exploring how to effectively harness enzymes as anti-biofilm agents. Enzymatic degradation is a potentially suitable replacement to using toxic compounds to facilitate antibiotic penetration of biofilm. For example, Mycobacterium proteases have shown promise ^[46][143].