Challenges of antibiotic resistance biofilms and potential combating strategies: a review

Abstract

In this modern era, medicine is facing many alarming challenges. Among different challenges, antibiotics are gaining importance. Recent years have seen unprecedented increase in knowledge and understanding of various factors that are root cause of the spread and development of resistance in microbes against antibiotics. The infection results in the formation of microbial colonies which are termed as biofilms. However, it has been found that a multiple factors contribute in the formation of antimicrobial resistance. Due to higher dose of Minimum Bactericidal Concentration (MBC) as well as of Minimum Inhibitory Concentration (MIC), a large batch of antibiotics available today are of no use as they are ineffective against infections. Therefore, to control infections, there is dire need to adopt alternative treatment for biofilm infection other than antibiotics. This review highlights the latest techniques that are being used to cure the menace of biofilm infections. A wide range of mechanisms has been examined with particular attention towards avenues which can be proved fruitful in the treatment of biofilms. Besides, newer strategies, i.e., matrix centered are also discussed as alternative therapeutic techniques including modulating microbial metabolism, matrix degrading enzyme, photodynamic therapy, natural compounds quorum sensing and nanotechnology which are being used to disrupt extra polymeric substances (EPS) matrix of desired bacterial biofilms.

Introduction

Penicillin was discovered in 1938, after its discovery bacterial infections were controlled by antibiotics successfully (Cranston and Sidebottom 2016). More than 70 years ago, since the discovery of antibacterial drugs, they became essential part of modern healthcare landscapes, which allow the treatment of bacterial infections, which were life threatening previously. The health benefits which are achieved by antibiotics have been threatened by growing antimicrobial resistance, and it is recognized as global crisis (Ventola 2015).

Modern medicine is facing challenges of antibacterial resistance and the effectiveness of antibiotics has been declined owing to rapid dissemination and emerging resistant bacteria. Due to antibacterial resistance related problems, it is estimated that around 0.7 million people die every year (O’Neill 2016). The effects of antibiotics can be resisted by bacteria through different mechanisms including; (a) target site modification, (b) destruction or modification of antibiotic, (c) efflux of antibiotic via efflux transporters, (d) reduce influx of antibiotic by decreased membrane permeability (Munita and Arias 2016).

Acquisition of mutations, caused by the overuse for treatment and prevention of human infection as well as in farming and veterinary medicines, have led to development of bacterial species and strains which are resistant to almost all known antibiotics. Development of new antibiotics occur sparsely because of regulatory scrutiny and high cost. Another question arises regarding the duration that how long bacteria will take to become resistant before adapting to those new antibiotics (Flemming et al. 2016).

A biofilm is microorganisms’ consortium where cells stick to each other and stick to surface: in an extracellular matrix, cells are embedded and protected, which is made of extracellular polymeric substances (Speranza and Corbo 2017). The research on biofilm begun since 1970s, although biofilms existence was first stated by Anton van Leeuwenhoek, after plaque scraped analysis from his own teeth (McCarty et al. 2014). Extra polymeric substances (EPS) act as glue for holding biofilm-bacteria together as well as to protect them against different environmental challenges and host immune system. The ability of bacteria to embed themselves in a matrix of EPS, composed of polysaccharides, humic acids, proteins, and eDNA (Flemming et al. 2016).

Biofilms possess various characteristics, which are significant for survival such as three dimensional structure, adherence to surfaces, adherence to different interfaces, adherence to each other, host defense systems and decrease antimicrobial susceptibility (McCarty et al. 2014). Biofilms persistently attach to biotic and abiotic surfaces which range from human lung or tooth, intestine of cow to submerged rock in a fast moving stream. Cardiac valves, prosthetic medical devices, intrauterine contraceptive devices, catheters, implants, contact lenses, and dental materials are medical devices on which biofilms are colonized (Jolivet and Bonnaure 2014). Within biofilm, the eradication of bacteria is a challenging issue which corresponds to alginate, as a physical barrier as well as remarkable reduction in metabolism of bacteria in mature biofilm (Wood 2016).

The unremitting rise of bacterial pathogens which are highly multidrug-resistant highlights a need for new remedial options. This review explores the developing strategies in the context of avoiding existing mechanisms of resistance. Around the globe, the burden of antimicrobial resistance is an ecological calamity, and it was declared as a global health concern by many regulatory authorities. Therewith the optimism about control of infection that arose after antibiotics discovery has reached to an end and there is a dire need of new infection control strategies. In this review, current challenges, different alternative therapies, and future perspectives are presented to stimulate new technologies to have a battle against different bacterial infection.

Biofilm formation

Among bacterial species there are many differences during the biofilm formation process, and it has been observed in almost all bacterial species (Hughes and Webber 2017). Bacterial appendages like fimbriae, flagella and pili as well as different physical forces like electrostatic interactions and van der Waals forces assist in adhesion of planktonic bacterial cells to surface during early stages of biofilm formation. After attachment to surface, growth of biofilm production proceeds and then maturation continues.

Biofilms are either monolayer or multilayer depends on the interaction between constituent cells and surface. It has been known that sometimes bacteria’s outer surface nature leads to a repulsion. For example, chemical properties of Gram negative bacteria cell wall are mostly negatively charged because of O antigen presence. The multilayer biofilms are mostly formed when repulsive forces of these negatively charged organisms are masked and neutralized by process of divalent cations addition, mutation, EPS synthesis, and down-regulation of genes synthesizing O antigen (Gupta et al. 2016).

There is a distinct growth phase in biofilm as compared to planktonic cells (Lerch et al. 2017). Many microbes form biofilm in response to unfavorable environmental conditions. Many microbial biofilms grow together as a single as well as many pathogen and non-pathogens microbial communities. Biofilm can withstand and challenge environmental stresses like desiccation and starvation, because of their elastic nature and it is effective for survival (Alves et al. 2014).

Mechanism of antibiotic resistance in biofilm

The occurrence of bacteriostatic or bactericidal antimicrobial agent allow the growth of resistant microorganisms at a concentration which usually inhibit the growth. Minimum inhibitory concentration (MIC) is used to measure the resistance in planktonic cultures, it is the lowest concentration at which microorganism’s growth is inhibited by antimicrobial agent. Resistance often leads to mutation or exchange of genetic elements resistant to antibiotics (acquired resistance), however, resistance can be intrinsic which depends on cell’s innate properties and wild-type genes (Blair et al. 2015; Cox and Wright 2013).

The poor penetration of antibacterial into biofilms and intrinsic antibacterial resistance form the two main reasons for resistance of infectious biofilms to antibacterial treatment. Firstly, antibacterial susceptible bacteria are killed in suspension or planktonic mode, while at the outside of biofilm, only biofilm bacteria are reached as well as killed and inner one could not. Whereas, antibiotic-resistant bacteria are neither killed planktonically nor at outside of biofilm. Secondly, a general reason for resistance is poor antimicrobial penetration through a biofilm to its depth. Poor penetration results from reduced antimicrobial diffusion and adsorption on self-produced protective matrix of EPS. Nutrients and metabolic waste products are transported through water channels by EPS matrix. Note that in biofilms pH (approx. 5–9) is lower than physiological one which are outside an infectious biofilm (Fig. 1; Koo et al. 2017; Davies 2003).

Fig. 1.

Intrinsic resistance of antimicrobials and their poor penetration into biofilms; a bacteria which are antimicrobial-susceptible killed planktonically, while antimicrobial-resistant bacteria are neither killed outside of a biofilm nor in a planktonic-mode; b reduced adsorption onto extracellular polymeric substances (EPS) protective matrix or antimicrobials diffusion leads to poor antimicrobials penetration in a biofilm to its depth

Gram-negative bacteria are more resistant to vancomycin and other different antibiotics than Gram-positive cells because of the relative impermeability of the outer membrane of Gram-negative bacteria (Hall and Mah 2017). In the formation of biofilm quorum sensing plays significant regulatory role. It regulates the formation of biofilm in both gram-positive and gram-negative bacteria. The biofilm formed by gram negative bacteria using acyl homoserine lactones as a signal molecule regulated by quorum sensing system. It is consists of signal molecules and their corresponding receptors. For example, in Pseudomonas aeruginosa there are two signaling systems of quorum sensing: rhlI/rhlI and lasl/lasR. Different signal molecules receptors and synthetases are encoded by lasl/rhlI and lasR/rhlR genes, respectively. As the bacterial density increases the secretion of signal molecule increases. After reaching a certain threshold, the signal molecule binds to its corresponding receptor, activates it and it further activates transcriptional regulators relevant to them for synthesis of alginates, extracellular polysaccharides, and toxic factors which leads to a formation of biofilm (Zhao et al. 2020; An et al. 2019).

The Gram-positive bacteria’s biofilm is regulated by quorum sensing system using a signal molecule, i.e., oligopeptides. After modification, two-component sensing protein can recognize it and targets gene expression is regulated through de-phosphorylation and phosphorylation of protein and biofilm formation is further regulated by this. The pathways of quorum sensing system and signaling molecules of different oligopeptide are different in different types of bacteria. For example, in Streptococcus the two-component system is the response regulatory protein and histidine protein kinase, but in Staphylococcus aureus the quorum sensing system is highly conserved (Zhao et al. 2020; McCluskey et al. 2004).

In the presence of a bactericidal antimicrobial agent, the ability of microorganism to survive is their tolerance to an antimicrobial agent, and they neither grow nor die. The tolerance is measured using minimum bactericidal concentration, i.e., the lowest concentration at which bactericidal antimicrobial agent will kill more than 99.9% of cells in culture. Recently, different alternative techniques have been reported to measure the tolerance of microorganisms (Brauner et al. 2016).

Formation of Persister cell has been linked to toxin actions like MazF and RelE from toxin-antitoxin (TA) modules. Persister cells consists of bacteria’s subpopulation which is highly antibiotics tolerant and without undergoing genetic change reach this state. Also, the persister cells number depends on growth stage of bacteria. Since, antibiotics are responsible for killing of most cells and persister cells present in the biofilm are responsible for chronic infections recalcitrance and when the level of antibiotics decreases they remain viable and also repopulate biofilms (Lewis 2010).

Genetic changes arise the resistance mechanisms that inhibit the action of antibiotics, leading to resistance; that is, cells grow where antibiotics are resistant, while persistent cells are dormant and do not grow. In addition, to form persister cells, there appear to be ineffective ways. Like, in the exponential phase, toxin TisB overexpression is effective in inducing a persistence but in the stationary phase it is not effective, suggesting that to enter the persistent state there are many available mechanism for E. coli (Dörr et al. 2010). For the survival of persister cell, low metabolic activity is the main key and reduced metabolic activity has also been linked to increased persistence in ongoing studies conducted with metabolic regulators (Zhang et al. 2012). This suggests that the significantly reduced growth rate associated with bacterial stress response and it is characteristic in the internal structure of biofilm is the main reason for the reduction of biofilms' susceptibility to antibiotics (Wood et al. 2013).

Biofilm resistant to different antimicrobials is multifactorial such as substance delivery, high cell density, persistent cells, an increased resistant mutants number, molecular exchanges, and efflux pump. Biofilm resistance is augmented by several resistance mechanisms such as limited drug uptake. Like, the biofilms produced by resistant or intermediate categories of S. aureus reduced the oxacillin, vancomycin, and cefotaxime penetration (Singh et al. 2010). In many cases, potent eradication activity has been shown by halogenated phenazines HPs against pre-formed Methicillin-resistant Staphylococcus aureus (MRSA) and against human cells it shoes little toxicity. Non‐hemolytic metal (II)‐dependent mechanism action of eradication is eliminated by halogenated phenazines that regulates mechanisms that have been shown to select bacterial cells over a mammalian cell (Cascioferro et al. 2021).

Pathogenicity of biofilm bacteria

There are many factors which attribute the pathogenicity of biofilm forming bacteria. Extracellular molecules are released by biofilms which change gene expression of different virulence factors by the help of quorum sensing. Furthermore, the bacteria in biofilm intensifies the maturation frequency to increase β-lactamase activity, avoid host defenses, exchange plasmids for genes transfer for virulence factors and antibiotic resistance, increase mutation frequency, and enhance efflux pump activity. The extracellular matrix properties also contribute to pathogenicity of biofilm that provide protective barrier along with low immune cell and antibiotics penetration (Shoji and Chen 2020). The minimal inhibitory concentration (MIC) of antibiotics is not effective against bacteria present within biofilm but is established and effective against planktonic bacteria (Khan et al. 2020).

So, antibiotics’ minimal biofilm eradication concentration (MBEC) have to be used in biofilms and also it is up to thousand-fold greater than those of planktonic bacteria (Wolcott and Ehrlich 2008). Biofilms have biochemical and physiologic gradients of nutrients and oxygen that result in depletion of metabolic substances and provides medium for doubling along with cells’ dormancy. This may also contribute biofilms ability to co-occur with other bacteria as well (Shoji and Chen 2020). The exposure to different antimicrobial agents may also induce cell dormancy, which triggers an antitoxin for inactivation of cells, and when antimicrobial stressor is removed, can also cause metabolic dormancy with reversal of dormancy (Malhotra et al. 2019). As many antibiotics target cells which are metabolically active, rapidly dividing cells, may help in resistance of biofilm to antibiotics after exposure to stressors, and also targets slow growth state of dormant cells (Walters et al. 2003; Li et al. 2018). Biofilms cause many persistent infections like dental caries, cystic fibrosis, ocular implant infections, middle ear infections, native valve endocarditis, urinary tract infections, and osteomyelitis. Many pathogens are involved in chronic infections including Streptococcus pneumoniae and Haemophilus influenzae in chronic otitis media, Pseudomonas aeruginosa involved in cystic fibrosis pneumonia, and recurrent urinary tract infections caused by enteropathogenic Escherichia coli are associated with formation of biofilm.

The ability of pathogens significantly increased by biofilms to evade both antibiotics and host defenses. In several infections, they are concerned in clinical manifestation and pathogenesis (Galié et al. 2018). Especially in the Developed World, the number of infections which are caused by biofilms has been estimated between 65% reported by Centers for Disease Control and Prevention and National Institutes of Health reported 80%. Different common infections like catheter infections by Staphylococcus aureus, urinary tract infections by Escherichia coli, and Haemophilus influenza cause child middle-ear infections, gingivitis, and formation of common dental plaque are also caused by biofilms (Bekele et al. 2018). Different biofilm infections caused by P. aeruginosa in cystic fibrosis patients, endocarditis by Staphylococcus aureus that cause mortality and morbidity. Opportunistic pathogenic bacteria like Staphylococcus aureus and Pseudomonas aeruginosa in their host form chronic biofilm-based infections and 8–10% among hospitalized patients are prone to infections.

Under different circumstances like slight changes in pH (either increase or decrease), biofilms can survive as common adjuvant treatments like sodium hypochlorite, hydrogen peroxide, or povidone-iodine partially eradicate Staphylococcus aureus biofilms previously (Ernest et al. 2018). In contrast, in an in-vitro study, the MBEC of acetic acid was determined following 20-min treatment to exceed its threshold of safety, and making it an inappropriate clinical alternative (Tsang et al. 2018). Therefore, the biofilms’ ability to survive in different environments like extreme in pH pose challenge for applicability of different chemical adjuvant treatments. Also, the ability of antibiotics and immune cells to approach a site of infection effectively is limited because of an implant which is a foreign body and lacks a blood supply (Gries and Kielian 2017).

Environmental concerns

There is a growing understanding regarding the role of environment in transmission of different antibiotic-resistant pathogens as well as their evolution. One health perspective is required while addressing the challenge of antibiotic resistance globally, as it includes the connections between animal, human and environment (Fig. 2; Robinson et al. 2016).

Fig. 2.

Spread of antibiotic resistance throughout different ecosystems (Jones-Dias et al. 2016)

Researchers are continuously working in this area but from the literature four main areas are identified which required intensive research including: (a) relative contribution of different antibiotic sources and antibiotic-resistant bacteria present in environment. Quantitative transmission, risks ranking, and risk models are populated by better quantification of contribution from such exposure pathways, sources, and routes of propagation (Pruden et al. 2006). To better assess the importance of different antibiotic-resistant bacteria and antibiotics sources into the environment, natural variability and characteristics of mobile genetic elements and resistant genes, and transfer frequencies and mobilization quantitative knowledge is needed in different environments (Zhu et al. 2017); (b) Role of environment on the evolution of resistance. The relationship between bacterial community’s selection of resistance and exposure of antibiotic is not understood well (Bengtsson-Palme et al. 2018). For the emergence and resistance evolution as priority targets, different attempts to identify environments with high risk should take on a global perspective and not be geographically restricted. In different environments to understand the risk for the resistance emergence, quantitative modelling of different processes of evolution need to be clearly valuable (Manaia 2017); (c) the overall impacts on health of human and animals when expose to environmental-resistant bacteria. The magnitude of direct and indirect health impacts are unclear, which are resulted from environmental exposure to different antibiotic-resistant bacteria (Wuijts et al. 2017). The identification of risk scenarios and critical exposure would help a better directionality understanding behind such bacteria and genes transfer. Extended sample systems covering various regions of the world as well as combining different environmental compartments, selection pressures and time points may contribute to genetic understanding. This can be achieved with better data sharing, but sensitive source tracking tools and high-resolution typing efforts could also be included (Berendonk et al. 2015); (d) the feasibility and efficacy of different social, economic, technological, and behavioral interventions for mitigation of environmental antibiotic resistance. For different types of intervention like industrial discharges management, development of environmental quality standard and acceptable levels of emission for resistant bacteria, selective agents, and methodology needed to define such standards and levels can be useful (Bengtsson-Palme et al. 2018; Larsson et al. 2018). Human health is directly affected by antibiotic-resistant microorganisms presence as is discussed earlier there is a direct link between human and environment. To reduce a risk for selective agents to environment and spread of antibiotic-resistant bacteria, and also risk reduction for transmission back to animals and humans, there are already interventions practices are in place to some extent (Larsson et al. 2018).

Interventions are of both social and technical nature critically. Problems will remain in place without challenges awareness and among those to address them what is at stake. Therefore, for each type of stakeholder strategies are needed to developed as well as evaluated. These include regulatory agencies, media, water, health, agricultural sectors, NGOs, pharma companies, European Union, World Health and Organization, Food and Agriculture Organization, governments, World Organization for Animal Health, and also patients and consumers (Fig. 3; Laxminarayan et al. 2016).

Fig. 3.

An overview of the environmental processes that contribute to the formation and spread of antibiotic resistance and how knowledge gaps are related to it (Larsson et al. 2018)

Health concerns and infections caused by antibiotic-resistant biofilm

Bacterial infections are posing a challenge to life since the human life existence. Over the past centuries, to control human infections new ways have been invented and welcomed with great zeal and for various reasons subsequently been put on hold. As because of increasingly fundamental concern, biofilm development in food industry represents serious public health issue.

Different biofilms also contain bacterial or fungal species which are pathogenic and sometimes just targeting the immunocompromised, (for example, HIV, organ transplant beneficiaries, or oncology patients, etc.). Food intoxication (Bacillus cereus, staphylococcus aureus), gastroenteritis (Escherichia coli, salmonella enterica) and also systematic diseases (Listeria monocytogenes, Escherichia coli) can be caused by these pathogens (Galié et al. 2018).

A secreted peptide antibiotic is produced by Bacillus subtilis, which is known as YIT toxin as well as in biofilm its resistant proteins. In biofilm formation, a mutant was defective which lacks a resistant genes (Kobayashi and Ikemoto 2019). In persister cell and biofilm formation Toxin-Antitoxin (TA) systems are involved and that these systems can be important regulators of switching from planktonic to biofilm life as a response to stress through their secondary messenger, i.e., 3′, 5′-cyclic diguanylic acid (Wang and Wood 2011).

Bacterial biofilms found on factory equipment or food matrixes may arise via infections or intoxications lead to food borne diseases. Biofilms found in different food processing plants can secrete toxins. From there, food matrix can be contaminated which cause individual or multiple intoxications. In each case, human health is at risk because of the biofilms presence in food factory. The amount of risk to human depends on the types of bacterial specie involved. The main sites for the development of biofilm depends on type of factory, and also include milk, water, and other pipeline liquids, tables, reverse osmosis membranes, contact surfaces, pasteurizer plates, packing material, animal carcasses, dispensing tubing, employee gloves, and storage silos for additives and raw materials, etc. (Camargo et al. 2017).

Food security threats

Nowadays, various chemical compounds (hydrogen peroxide, sodium hydroxide solutions, per acetic acid, and sodium hypochlorite) and different physical methods (ultra-sonication and hot steam) are used for controlling the formation of biofilm on working surfaces and also within pipelines in food industries. Disinfection and cleaning of infrastructure is possible based on the processes used in specific industry to get escape from attachment of microbes to surfaces or pipelines. Alternatively, the methodologies like clean in place can help in maintaining a clean surfaces by recirculating liquids or by spraying (Srey et al. 2013).

Though, because of complex nature and unique characteristics of biofilm communities the possibility of physical as well as chemical resistance has been increased, in some cases making elimination very difficult and in favors their persistence. Therefore, instead of elimination, the novel antibacterial approaches have emphasized on prevention of biofilm formation (Gopal et al. 2015).

Biofilms formed in food industry pose a serious health and economic issue. Biofilm existence along surfaces which are used in food manufacturing can cause corrosion on metal surfaces requiring their replacement can lead to financial losses. Furthermore, several bacterial species like Bacillus spp. and Pseudomonas spp. different lipolytic as well as proteolytic enzymes are secreted which produce unpleasant rancid odors and bitter tastes. In such cases, these affected manufacturing batches have to be destroyed and removed (Galié et al. 2018).

Food safety issues

Different stakeholders which belong to academic, food industry, governmental, and international sectors are exceedingly committed in combating the growing epidemic of infections which are resistant to drugs (Roca et al. 2015). Hence, the progress depends on different sectors which are providing well-coordinated efforts to address issues in human health, food, animals, agriculture, and environment (O’Neill 2016).

In agriculture, the widespread utilization of antibiotics for disease prevention and growth promotion has major impact on food safety, production of farm animals, and human health (Watkins et al. 2016; Thanner et al. 2016; WHO, 2014).

Resistance detection and monitoring problems

Although host immune suppression and antibiotic resistance contribute in managing different infection caused by biofilms, but come across another challenge regarding diagnosis of these infections. Clinically, infections are often linked with a signs of pain, swelling, fever, and drainage of surgical site purulent. Moreover, serum biomarkers of infections, including C-reactive protein, erythrocyte sedimentation rate, and leukocytosis are useful for giving generic information about infection whether it is present or not.

However, these biomarkers and clinical signs may not found in chronic infections, as they develop over long period of time. Unlike planktonic bacteria, these biofilms are not easy to detect using different traditional methods. As, in biofilm many bacteria are slow growing and present in metabolically dormant state because of low level of oxygen as well as nutrients within the biofilm which limits the growth of bacteria on culture (Høiby et al. 2010). There are also additional limitation found in different culturing techniques, as in microscopy and histology there is 80 to 90% biofilm detection rate, while in in-vitro bacteria culturing only 30% biofilms are detected (Metso et al. 2014).

For measurement and detection of microbial biofilms several methods have been used by many clinical microbiologists like: the development of online methods of monitoring to follow a growth and removal of deposits, adhesion as well as biofilms formed on surfaces at industrial environment help to reduce cleaning operation cost. Different classical biofilm detection methods like agar plating is not as much effective because of difficulty in culturing of many bacteria forming biofilms. Few pathogens enter into Viable but Non-culturable (VBNC) form having a low metabolic activity. Several screening methods are listed in Table 1 (Shunmugaperumal 2010).

Table. 1.

Different screening methods for antimicrobial and ant biofilm activity of different agents against bacteria producing biofilm (Shunmugaperumal 2010)

Method	Application	Target
Checkerboard Assay Plate	Plate counting of bacteria embedded in biofilm and also Fractional Inhibitory Concentration (FIC) indexes are calculated	To screen antimicrobial activity of different combination of agents
Microtiter plate (MtP)	Measurement of biofilm in response to agent which is produced on the walls of wells	Measures the agent’s effect beside the production of biofilm
Microtiter plate (MtP) Minimal Biofilm Eradication Concentration (MBEC)	Measurement of a biofilm which is remained on the walls of well in response to different agents as well as detecting MBEC of those agents	Measures the agent effect on mature biofilm which is formed on the well’s walls
Vortex and plate counting	Plate counting of bacteria embedded in biofilm as well as detecting the bMBC of agents	To screen the antimicrobial activity of these agents against these bacteria embedded in biofilm
Quantitative PCR	Measurement of specific expression genes of biofilm	To monitor expression of genes of biofilms in response to different agents
Mass spectrometry (MS)	Measurement of exo-enzymes which are located in matrix of biofilm	To monitor expression of proteins in bacteria in response to different agents
Vortex, plate counting, and sonication	Plate counting of bacteria which are embedded in biofilm and also detection of bMBC of agents	To screen the antimicrobial activity of different agents against these bacteria

Culture methods cannot be able to detect to these VBNC cells and under stress conditions such as low temperature, also lead to their survival. These cells can be detected by several methods like PCR amplification (Gião and Keevil 2014). However, at laboratory level few detection methods like qRT-PCR, specific DNA amplification, or agar plating are effective, but not at industrial level because of VBNC cells presence in various biofilms especially in agar plating and expensive equipment’s and reactive. The development of new strategies is of utmost significance for detection of biofilm production in industrial environments (Galié et al. 2018; Fratamico et al. 2009).

Difficulties in biofilm eradication

In industrial and natural environments biofilms are resistant to different antimicrobial agents. Gene expression and structure helps the biofilm forming cells to grow and survive. Also, biofilms are heterogeneous in time as well as in space. There are three different reasons which make eradication of biofilm difficult (Fig. 4; Roy et al. 2018; Verderosa et al. 2019). Firstly, slow or restricted penetration of different antimicrobial agents within biofilm. Secondly, the resistant phenotype which include gene transfer and antimicrobial destroying enzymes. In gene expression, there are unique differences between sessile and planktonic cells which are responsible for physiological alterations during formation of biofilm. Thirdly, altered metabolism and cellular environment, as in a biofilm some cells face limitation of nutrients and live in starved or slow growing stage (Verderosa et al. 2019; Wolfmeier et al. 2018).

Fig. 4.

Three hypothesis for antibiotic resistance in biofilm

As, majority of cells are dormant and are in anoxic condition they have protected phenotype and are not much susceptible to different antimicrobial agents. They are known as persister cells and found in abundance in deep biofilm. Hence, these different parameters make the elimination of biofilm difficult and increase the resistance (Verderosa et al. 2019; Wolfmeier et al. 2018).

Alternative therapies to eradicate biofilms

A successful treatment of infections associated with biofilms is troubled because of increased antibiotics’ resistance in different bacterial communities. Also, different classical antibiotics chemotherapy is incapable to eradicate these bacterial cells completely especially the cells located in central region of biofilm which lead to worsen situation globally. Therefore, novel anti-biofilm agents and alternative strategies are required against antibiotic resistance biofilm communities. Several factors include antibiotics, enzymes, plant extracts, sodium salts, metal nanoparticles, acids, or chitosan derivatives (Fig. 5) have impact on structure of biofilm via numerous mechanisms having different efficiencies (Baek and An 2011; Zaidi et al. 2017; Iannitelli et al. 2011; Misba and Khan 2018; Kolodkin et al. 2012; Munoz et al. 2016; Sun et al. 2013; Zuberi et al. 2017).

Fig. 5.

Alternative approaches against antibiotic-resistant biofilms (modified from Sharma et al. 2019); a natural compound; b nanotechnology; c photodynamic therapy; d quorum sensing; e modulating microbial metabolism; f matrix degrading enzyme, g new advanced innovative elements

Natural compounds

Different strains of bacteria and actinomycetes have been used to synthesize natural compounds, also known as bioactive agents, with specific anti-biofilm properties. However, it has been found that extract (methanolic) of a specie of actinomycetes, associated with corals, reduces the production of biofilm in S. aureus species (Bakkiayaraj and Pandian, 2010). Contrary to this, 4-phenylbutanoic acid (bioactive agent) indicates increased activity against both, gram negative and positive bacterial strains (Nithya et al. 2011). Whereas, extracts from Azadiracta indica and Acacia have shown antimicrobial effect against S. faecalis and S. mutans (Ayrapetyan et al. 2015).

Small molecules that are produced naturally by bacteria including D-amino acids prevent the biofilm formation in E. coli and S. aureus but the dispersion of biofilms (Hochbaum et al. 2011; Kolodkin et al. 2012), thus can be used as anti-biofilm agents. NAC is less active than Tween 80 against mycobacterial biofilms as cell wall of mycobacteria as well as extracellular matrix contains high lipid percentage. It suggests the effectiveness of antibiofilm agent and synergistic drugs effect in treatment of infections caused by mycobacterial communities (Sharma et al. 2019).

Plant extracts

A number of plant species are used as medicine in treatment of various diseases. Antibiotic resistance of different pathogenic bacteria has attracted scientist’s attention towards alternative options and endopharmacology. In a study, plant extracts of five species were analyzed with activity of biofilms and found the inhibition of proprioni bacterium acne biofilm formation due to sub-MIC concentrations of Polygonum cuspidatum (Japanese knotweed), Epimedium brevicornum (rowdy lamb herb), Rhodiola crenulata (arctic root) by 99.2%, 64.8% and 98.5%, respectively (Coenye et al. 2012). However, most promising results were shown by resveratrol by P. cuspidatum, which inhibited 80% biofilm formation at concentration of 0.32% (w/v). Similarly, Icarin from E. brevicornum decreased formation by 40–70% at concentration of 0.01–0.08% (w/v). Salidroside anti-biofilm activity from R. Crenulata (strain dependent) with 0.02–0.25% concentration resulted in reduction of biofilm production for P. acnes LMG 16,711 by 40% and 20% for others (Singh et al. 2012).

Nanotechnology

Nanoparticles are of paramount importance due to their use as an alternative to tackle infections based on multidrug resistance and biofilm (Pelgrift and Friedman 2013). Conventional treatments have many limitations and restrictions which can be covered by their nano-formulations. These formulations possess ability to cross biological barrier. In past years, a variety of nanoparticles have been used as antimicrobial and anti-biofilm metal nanoparticles, green nanoparticles and many other combinations (Baek and An 2011; Sharma et al. 2019). A plethora of reports are there which discuss the elimination of bacterial biofilm communities based on nanoparticles. (Hernández-Sierra et al. 2008; Kulshrestha et al. 2014).

Owing to their massive antibacterial properties, nanoparticles like copper, oxide, silver, zinc and quantum dots can be proved potential against biofilms (Zaidi et al. 2017). Nanoparticles containing reactive oxygen species (ROS) damage cell membranes and cell walls of bacteria by increasing the oxidative stress and enhancing cytoplasmic leakage, respectively, and denaturing the metabolic proteins (Shoji and Chen 2020). It results in altered cell functions and affects physiological processes in bacterial cells (Zaidi et al. 2017). Different in-vivo studies revealed that nanoparticles can be used against a large variety of gram positive and negative bacterial strains with low toxicity of cells and high compatibility (Qayyum and Khan, 2016; Li et al. 2016). It leads to the use of nanoparticles as a promising approach to treat bacterial infections (Khan et al. 2020).

Kulshrestha and his colleagues stated CaF₂-NP’s suppressive effect on genes linked with virulence factors including vicR, ftf, gtfC, comDE and spaP of S. mutans and showed enzymatic activity inhibition associated with cell adhesion, glucan synthesis, acid production, quorum sensing and acid tolerance (Kulshrestha et al. 2016).

Quorum sensing

Quorum sensing (QS) signaling genes were used to control the production of biofilms. A wide variety of inhibitors/compounds can disrupt the QS signaling cascade and can be used as an alternative for the treatment to control biofilm-related infections. Bacterial QS signaling can be disturbed using halogenated furanone extracted from Delisea pulchra (marine algae) (Lonn et al. 2009). Recently, Kaur and his co-workers showed that acyclic diamine (ADM 3) results in better anti-biofilm activity (Kaur et al. 2017). Extracts from garlic, usnic acid, ginseng and azithromycin results in boosting inhibitory actions against fungal and bacterial biofilm formation (Hoffmann et al. 2007; Song et al. 2010). Nitric oxide acts as a signaling molecule which disperses the biofilms in species like P. aeruginosa and increase the efficiency of antimicrobial compounds through c-di-GMP-degrading phosphodiesterase, simulation (Barraud et al. 2009; Sharma et al. 2019).

Photodynamic therapy

Light, photosensitizer and oxygen are three major components of photodynamic therapy. Photosensitizers including pyridyl-porphine, phenothiazine dyes, toluidine blue and malachite green are chemical compounds which are absorbed by the targets, bacteria (Giannelli et al. 2017; Shoji and Chen, 2020).These dyes are functional in oxygen presence and exposure of light having specific wavelength. It results in the synthesis of free radicals and highly reactive oxygen species which inflicts plasma membrane and DNA damage to cell and cause death eventually (Briggs et al. 2018). However, light wavelength range and absorbance spectrum of photosensitizer overlapping results in excitation (Shoji and Chen 2020). After that, photosensitizers transform or convert into an excited state of triplet with long lifetime and transfer energy to oxygen molecule or other biomolecules which depend upon the type of reaction (Fig. 6).

Fig. 6.

Photodynamic processes

Type I (Fig. 6) reaction involves electron transfer from triplet excited state to substrate such as, unsaturated phospholipids membrane, which leads to the hydroxyl radical’s formation (from water) or lipid (from radicals). The produced radicals further react or combine with biomolecules or oxygen to give hydrogen peroxide, in turn leading to either causing peroxidation of lipid or damage cell via production of reactive oxygen species (Briggs et al. 2018). On the other hand, Type II reactions includes transfer of energy from triplet state to a ground state (lower energy) molecular oxygen for producing extremely reactive species, excited single oxygen. It has ability to oxidize biomolecules like lipids and proteins that are present in the cell, and can cause cell death. Both processes can occur in a cell at the same time but Type II mechanism is considered as a major Pathway of APDT. Cells are damaged in two different ways: damage to DNA and disruption of cellular organelles. As DNA is or prime importance in repairing as it holds information for the production or formation of new organelles and materials, it is major cause of death of a cell (Giannelli et al. 2017; Castano et al. 2005). So, major portion of micro-biocidal APDT effect and it can be due to effect on proteins which involve in membrane functions which consequently leaks the cellular structure out of the cell. However, more data and research studies are required to find the efficiency and efficacy of PDT alone and in form of combinations with other antimicrobial therapy.

Modulating microbial metabolism

Improved bacteria targeting in its dormant state, i.e., persister cells, is another potential alternative of targeting a biofilm microorganisms. These cells are more antibiotic resistant and less metabolically active. The microbial sensitivity to antibiotics may improve by stimulation of bacteria to induce a metabolic activity. In one study, it was found that in biofilm state as well as after its dispersal, Methicillin-Susceptible Staphylococcus Aureus (MSSA) strain was tolerant to oxacillin (Fernandez et al. 2016). Though, after the fresh media addition along with different nutrients the bacteria sensitivity increased to oxacillin. And, theoretically, the elimination of bacteria may be improved by increasing a metabolic activity of bacteria which are dormant previously, with co-administration of antibiotics. To avoid deterioration of infection, caution must be taken which can occur because of increased metabolically active bacteria (Chowdhury et al. 2016).

Matrix degrading enzyme

Another promising anti-biofilm strategy is a degradation of biofilm matrix with the help of biofilm matrix degrading enzymes (like Dispersin B (DspB), DNase I, and a-amylase). The penetration of antibiotics will be increased when biofilm structural component degrades, and hence enhance the efficiency of antibiotic. DspB, DNase I, and a-amylase degrade exopolysaccharides, eDNA, and biofilm matrix, respectively, (Sun et al. 2013; Tetz et al. 2009) which inhibit the formation of biofilm as well as degrade the mature biofilms in many microbes like Vibrio cholera, S. aureus, and P. aeruginosa (Kalpana et al. 2012).

As biofilm matrix is composed of proteins, extracellular polysaccharides, and DNA, different studies indicated that biofilm compounds degradation by several enzymes can disrupt the structure of biofilms. Dispersin B targets the exopolysaccharide poly-N-acetylglucosamine (PNAG) of biofilm that is involved in virulence of bacteria which form biofilm (Arciola et al. 2011; Shoji and Chen 2020). It inhibits producing bacteria (Kaplan 2009) and it was shown that in in-vitro studies, biofilms are almost completely eradicate, suggesting it as useful agent for eradication of biofilms independent as well as in conjugation with antibiotics (Darouiche et al. 2009). It may also play a role in prevention of infections. DNase 1 also destabilize a biofilm by degrading an extracellular bacterial DNA (eDNA), although other antimicrobial agents has also shown improve eradication of mature biofilms (Rose et al. 2014; Kaplan 2009; Shoji and Chen 2020).

New advanced innovative elements

Identifying new agents which can inhibit bacterial biofilm but not affect the growth of bacteria could lead to the new anti-toxin strategies development by reducing the selective pressure for resistance of drugs. Thiazoles, thiazolidinone derivatives, and their benzofused systems are widely regarded as the most important nuclei for obtaining molecules having different biological functions, including antidiabetic, anti-inflammatory, antimicrobial, analgesic, antitumor, and anti-HIV (Cascioferro et al. 2019).

A class of 36 new substances 2- (6-phenylimidazo [2, -1-b] [1, 3, 4] thiadiazol-2-yl) -1H-indoles have been well developed and tested for its properties of anti-biofilm against reference strains of Gram-negative bacteria. Indole compounds have been extensively described for their therapeutic abilities as analgesic, antibacterial, antiviral, anti-inflammatory, anticancer agents (Parrino et al. 2017). A new series of derivatives of 1, 2, 4-oxadiazole have been successfully developed and evaluated as new anti-virulence agents. The ability to inhibit the formation of biofilm was tested and compared with Gram-negative as well as Gram-positive pathogens (Parrino et al. 2021). Although, for the imidazo thiadiazole scaffold, there are many biological properties described and its anti-biofilm activity has been reported first time (Schillaci et al. 2017).

Strategies to treat human infections caused by biofilm

Although all strategies are involved in treating infections caused by biofilm. Among those, photodynamic therapy has many applications in preventing infection with wound biofilm. With regard to photochemical and photosensitizer reaction, it is very important that the treatment should be used carefully to kill and stain bacterial cells without affecting the patient's surrounding tissues in the body (Roy et al. 2018). These novel anti-biofilm strategies promise to address the challenge of eradicating infections caused by biofilm. Quorum sensing also plays significant regulatory role. Anti-biofilm molecules interfere with signaling pathways of bacteria in both Gram-negative and Gram-positive bacteria. Anti-biofilm molecules can be any antibiotic, peptide, enzyme, and polyphenols (Roy et al. 2018).

Conclusion and future considerations

Biofilms are microorganism’s communities which are attached to surfaces. The properties of biofilm grown cells are different from those of planktonic cells, and also, show increased resistance to different antimicrobial agents. On the basis of these and previous studies, formation of biofilm can be seen as well-regulated and organized developmental process which results in complex organisms community formation. The antibiotic susceptibility of bacteria is reduced in biofilms because of different reasons like combination of an altered microenvironment, presence of different bacterial persister cells, poor antibiotic penetration, and adaptive responses. Several therapies can be used to eliminate resistance inclduing nanotechnology, quorum sensing, natural compounds and photodynamic therapy.

By disabling the biofilm resistance, the ability of existing antibiotics may be enhanced to clear the infections that are now refractory to available current treatments. There is a need for more work to fully explain the mechanism of antibiotic resistance in biofilms and development of new therapeutic strategies. Also, the studies to further explain how and why bacteria present in biofilms can protect themselves from antimicrobial agents.

Declarations

Conflict of interest

The authors declare no competing financial interest.