Manal Mohammed Alkhulaifi

King Saud University, College of Science, Department of Botany and Microbiology,
Box Office: 55670, 11544 Riyadh, Saudi Arabia.

Abstract

Biofilms are always a major concern in the healthcare field and food industry. The resistant properties of biofilm that allow bacteria to persist are difficult to study. Biofilms are often more resistant to antibiotics than individual planktonic cells. Thus, alternative strategies for managing biofilm formation are needed. Currently, using phages as anti-biofilm agents has been suggested. In this review, some of the diverse strategies, reported in previous studies, for preventing biofilm formation are discussed. Use of phages as anti-biofilm agents can involve phage application prior to biofilm formation, application to biofilms that are already formed, or using phages in association with other mechanisms to physically disrupt the biofilm. The development of novel methods as anti-biofilm agents will add an important dimension to the search for new potent compounds for preventing biofilm-associated infections.

Keywords: Phages, Biofilm, Bacteria, Antibiotics, Resistance.

Introduction

Biofilm
In general, biofilm is an organized multicellular of bacteria, which can be formed either from one or a number of different species and these species live together inside a matrix made of extracellular polymeric substances (EPS) with the capability of attachment to numerous surfaces 1. EPS mainly include polysaccharides, but other biomolecules are also present among which are nucleic acids, lipids, proteins and nucleic acids, which form a scaffold that help the bacteria to stay attached within the biofilm 2, 3. This matrix displays a modified phenotype and regulation of specific drug resistance genes and virulence factors can be observed in bacterial biofilms. Horizontal genetic transfer may occur easily, and therefore facilitating cross- breeding of resistance genes 2, 3, 4, 5. Biofilm is formed in five different stages, Figure (1) shows those five stages 6. 

Fig. 1. Biofilm is formed in five stages, these stages are 1) initial, reversible attachment, 2) Irreversible binding and growth, 3) EPS production and inter communication through quorum sensing, 4) Mature biofilm, and 5) dispersal; essential stage for biofilm dispersion and life cycle (adapted and modified from Mizan and others 2015)7

The complexed composition of the matrices adds an original property to the biofilm which can be the survival ability under extreme conditions, furthermore in addition it enhances the inflow of nutrients, water and signaling molecules which are important accountable for cells communication 8, 9. Furthermore, EPS matrix supplies a barrier between the external environment and the bacteria that prevent antimicrobials from penetration in to the biofilm 10. Biofilms of Salmonella are more resistant to the triclosan antibiotic than Salmonella’s individual planktonic cells 11. Furthermore, the negative charges of the EPS can prevent the antibiotics to achieving the biofilm 12, 13.

Biofilms basically play a fundamental role in infectious diseases. Taking a look at previous literature, it had been proven that 60–70% of most nosocomial infections are directly linked to the clear presence of biofilms14. The most bacteria that is repeatedly associated with medical devices come in particular Staphylococcus epidermidis and S. aureus, followed by P. aeruginosa and a boost of other bacteria that opportunistically infect weakened patients 15-17. Moreover, they can exist as at first glance of medical implants including catheters 18, 19.

Bacteria within biofilms demonstrate both antibiotic and the host defenses resistance 19, additionally they show a decline in the rate of growth, limitation in diffusion and a growth in efflux and enzymes accountable for antimicrobials degradation 20, 21. Generally, the usage of antibiotics to cope with biofilm-related infections doesn’t result in successful cures 12. Many studies confirmed that for biofilm, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were generally higher compared to the planktonic bacterial cells (about from 10 – 1000 times)22-24. As numerous antimicrobials function on actively growing cells which means the antimicrobial function maybe decreased 25. Once bacteria are embedded within a biofilm then all these factors with the altered gene expression and quorum sensing altogether result in the increased resistance against antibiotics 25. The treating biofilm is difficult and challenging which explains why scientific attention was drawn towards it26. Because of this, it is extremely important to find and develop new antimicrobial agents or some other efficient way to a target and destroy biofilm responsible for infections 27, 28.

Studies involving biofilm-phage interaction
Bacteriophages or (phages) in general are viruses that infect bacteria (Figure 2). These viruses were created for targeting the within biofilms 29. They are able to either reside in the bacterial host genome whilst the lysogenic phages do or they can destroy them similar to the lytic phages; which are one of the most suited type for therapeutic model usage. Currently phages are suggested as you are able to alternatives to antibiotics against bacterial infections and are widely explored to minimize the pathogen loads in food products. However, phages may be safer than antibiotics. It is quite simple, simple and fast to isolate them. Their production is inexpensive. Phages are competent against one specific host or host range making them ineffective unlike the natural microflora that exists initially attacked by the biofilm. Phages are green and, until today no serious uncomfortable side effects have been reported 30

Biofilms functions as targets of phage predation has become addressed by numerous studies 31, 32. There exists a lengthy history of phage used in roles that are comparable to those of antibiotics, in addition to ongoing use of phage therapy against bacterial infections 33-35. Biofilms enucleation through phages can involve either phage application ahead of biofilm formation, application to biofilms that are already formed, or using phage impact that’s present in association with other additional mechanisms that may physically disrupt the biofilm.

Fig. 2. Bacteriophage targeting bacteria within biofilm
(Adopted and modified from http://www.nwes-medical.net/news/20151202/ Bacteriophage-therapy-an-alternative-to-antibiotics-An-interview-Professor-Clokie.aspx)

The promising phage Strategies against biofilm such as for instance:

Inhibition of Attachment by phages
Phages are capable to effect on initial adsorption stage of biofilms (or adhered cells). When employing phages that are lytic, as generally could be the case with anti-biofilm phages, then phage infection results in the killing and lysis of bacteria. This likely both impacts biofilms structurally and releases new phage virions that potentially can reach and then infect adjacent bacteria 36. The effect is really a cyclical acquisition and then killing of biofilm bacteria 35. Sillankorvaet al. (2008) reported that single cells followed glass surfaces during 60 minutes, were efficiently inhibited with phage φS1. The cell removal was fast and efficient that result in a biomass reduction of about 90%37.

Inhibition of EPS matrix by Depolymerizing enzymes
It’s been reported that some phages are well effective at penetrating through the EPS matrix by diffusion or because of the presence of phage associated enzymes. It is an undeniable fact a large range of enzymes are able to destroy the biofilm’s EPS matrix. In the case of phages, these enzymes include some which are mainly produced to help in releasing the phages from the host cell and also tailspike proteins that really help in infecting the bacteria within the biofilm, but in general the activity of those enzymes and proteins are strictly localized. However, studies revealed that proteins with activity limited to the virus particle might be released from the lysing cells, and effect the biofilm matrix 38.

Phages can also capable of making depolymerizing enzymes that can degrade the EPS from the genome of the host. Many phage’s genomes also include genes that specialize in producingenzymes effective and functional in breaking down the matrix 29, 39, 40. In many conditions, these enzymes aim for the wall of the bacterial cellthrough the release process from the host cell, but similarly,these enzymesare able to degrade the biofilm EPS. Including the T4 and HK620 phages of E. coli have enzymes that exist on the viral tail, and may have a role in degrading the matrix 39, 40. And yes it was noted that – polysaccharide depolymerase is really an important part of the phage tail and also that – many tail spike proteins have endoglycosidase activity, by breaking down their polysaccharide receptors through hydrolyzation40. It has been reportedthat a phage-induced method of earning the matrix of the biofilm more porous, and therefore helping in the infection processby progeny phage, or a quick infected bacteria reaction can seek to encourage moving away from the focus of infection. Although the presence of polysaccharide depolymerase in phages has been reported. The problem in isolating phages possessing EPS degrading enzymes has led to the re- construction of phages, including the T7 41.

One important point would be to realize that different species of bacteria produce different EPS components. And that is way a depolymerase active contrary to the polysaccharides created by one species of bacteria might not digest that created by other bacteria. However, depolymerases will likely have broader activity than their parentphages among closely related bacteria, since the complexity and the variability in the EPS is below that of the host bacteria. Son, et al, (2010) observed this by comparing the experience of a phage of S.aureus with this specific of the depolymerase so that it produced42. However, neither would affect any bacteria other than Staphylococci, suggesting that multiple depolymerases is likely to be required for targeting mixed biofilms. In which a dynamic depolymerase is liberated, special ―haloes may be observed over the phage plaques formed on bacterial cultures, showing the areas where bacterial polysaccharide has been destroyed. Gutiérrez, et al, (2012) used this approach to detect such activity in two phages infecting S.epidermidis, both which were then confirmed by sequencing to contain genes for pectin lyases43, while Glonti, et al, (2010) identified haloes in cultures of a phage infecting P.aeruginosa and purified a depolymerase protein from the phage44. Yan has classified Phages polysaccharide depolymerasesas endorhamnosidases, alginate lyases, endosialidases and hyaluronidases40.

Pretreatment of catheter using phages
Another important challenges studied in medical care to reduce biofilm formation by S. epidermidis is pre-treating the surfaces of catheter with phages 45. The utilization of phages for the treatment of device-related infections has been the focus of attention since the 20th century. It has been discovered that pretreatment of hydrogel-coated catheters by phage caused the inhibition of S. epidermidis and P. aeruginosa biofilms 45, 46.

Quorum sensing Inhibition (QSI) by phages
One strategy that can be used against biofilm could be the inhibition of Quorum Sensing (QS), that is the cell-to-cell signaling system, this method is in charge of controlling the expression of genes which can be necessary for adding virulence factor, that is responsible for interactions with the host bacteria and also for the regulating the development of the biofilm 47-51. The key intent behind this strategy is not to kill pathogens but to disarm them making them oversensitive to the normal antimicrobial treatments. Furthermore, the QS system is not contributing in any way in mechanisms which can be essential for the bacteria survival, but inhibiting this method won’t be described as a reason behind producing a firm selective pressure suitable enough to cause resistance development 52. Pei & Lamas-Samanamud, (2014) showed that the engineered phage strain T7 that creates the metalloenzymes AiiAlactonase range of action againstsignaling molecules (acyl homoserine lactones) which are mixed up inbacterial quorum sensing is extremely wide that is and these molecules are important for the development of the biofilm53.

Phage Growth within Biofilms
Data collected from experiments indicated that phages do grow well in P. aeruginosa biofilms 54, at least in the primary stages of their development. Two-days-old biofilms. Olson, et al, (1999) reported that out of 17 insensitive strains of P. aeruginosa phages (therefore, planktonic bacterial hosts were used), 8 strains encouraged the same phages growth in the biofilm55. Although they are capable of blocking antibiotics effect within their beginning stages of formation. This finding will follow that ofGupta, et al, (2013)who also stated that the antibiotic resistance begins to appear in the first stages of biofilm formation. Thus, bacteria can be destroyed by phages in cases where antibiotics did have no effect on them56.

Previous studies which helped in explaining the power of to regulate biofilms, Hanlon, et al, (2001) found that phages effecting P. aeruginosa can terminate bacteria in an adultbiofilm and (looking at their sizes) might be diffused through the thickest alginate gel studied. But this activity clearly varied from that of the highly-restricted tailspike proteins57. In the research of Sillankorva, et al, (2004), phages of both P. fluorescens and S. lentus were used andthe effect on the reduction of both single species and mixed biofilms with these agentswas explained. The phages of both of the two hostswere completely sequenced, and clearly it had beenexplained that neitherof these coded for a polysaccharide depolymerase (though the P. fluorescens phage showed which they did encode an endopeptidase)29. Similarly, Doolittle, et al, (1996) reported that the T4 which is E.coli‘s phage doesn’t code for polysaccharide depolymerasedexcept for a restricted tailspike protein, which can only break out from the tail of the phage during the host cell penetration. But nevertheless can spread effectivelythrough a biofilm58.

It is proven by some studies that phages are able to penetrate biofilms even if they are not able to produce polysaccharide depolymerases, but within biofilm, effective infection haven’t been shown in most studies, also some researchers still believe that the existence of EPS-degrading enzymes areextremely important for applications of biofilm 38. A study carried out by Tait, et al, (2002) revealed that using avariety of three phages can entirely destroy a biofilm that’s created from single species, nevertheless in the presence of other bacterial species which were insensitive,this techniquedidn’t have much effect59. A study by Kay, et al, (2011) also demonstrated that the phages efficiency can beworn off in the clear presence of mixed biofilms60. In spite of this, it was reported by Sillankorvaet al, the efficiency could be high in model biofilms even in cases like if an individual bacterial species in the biofilmis targeted by the phage, explaining that phages have the ability of killing a specific type of bacterial host even when itdwells in a mixed organization. In addition, they reported that phages can target an adult biofilm effectively29.

Combining phage with Other Agents
Using phages as mixtures or coupled with antibiotics can completely prevent the development of phage resistance 61. Verma, et al, (2010) recorded that mature biofilms can becomemore adaptable toantibiotics if lytic phages are used62, which fits and will abide by findings that werecurrently reported from some clinical trialsconcerningphage activity63, 64. According to this, using phages and antibiotics in acombined or sequentialmanner has been seen to havethe potential for therapeutic applications. To supporting this, Yilmaz, et al, (2013) indicates that whenever phages coupled with antibiotics were utilized on biofilms of S. aureus these were clearly effected65. Other study suggested that usinga polysaccharide lyase and of DNase enzymes for destroying the matrix, ought to be placed into action alongside with phages 54. Abedon, et al, (2011) also discussed this,although differential diffusion of phagesand co-administered enzymes isregardedas being an issue34. The use of phage can also be joined with physical wounds cleaning. Seth, et al, (2013) used a rabbit ear mold to find that removing damaged tissue or foreign objects from the wound and using phage treatment each of them separately didn’t have any effect in this technique, nevertheless when combining both the result was visible. But, phages could have similar function on biocides and sanitizers used today, but should be applied after the primary cleaning processes, to destroy particular bacterium on the remaining biofilms66. Likewise, GanegamaArachchi, et al, (2013) revealed that using avariety of three different phages could clear Listeria monocytogenes biofilms effectively from steel surfaces. Thus, it should be putin consideration that for treating biofilms temporary by phages, it would be required that the biofilm cells surface be exposed to some disruption prior to phage application67.

Other combinations are also possibleexactly like in the case of biological systems. Liao, et al, (2012) noticed that combining phages with commensal bacteria had synergistic effects in preventing biofilm formation on silicone catheter segments 68, while Zhang & Hu, (2013) observed when using phages coupled with biocide like (chlorine) the effects on filters is increased69. However, further studies have to target on exploring phage activity in the multispecies context, animal models, and in conjunction with other antimicrobials 70. Figure 3 shows the strategies that were used to destroy biofilms within the last few 20 years.

Fig. 3. Phage mediated prevention Biofilm Strategies used within the last few 20 years

Conclusions

Studies which involved interaction between phage and biofilm indicated that phages contain some unique properties and seems promising in biofilms control Different phages have already been used to infect a number of bacterial biofilms. The treatment of biofilms using phages is a complicated process and only strictly lytic phages ought to be used. Like in phage infection of planktonic cells, there are numerous essential steps that require to occur. Phage adsorption to the receptors on the targeted bacteria is the leading part of infection. It is also evident that phages express enzymes which have the ability to of disrupt biofilms. To be able to allow it to be hard to spot, these types of enzymes are induced from the host genome. However, these kinds of applications remain progressing. Thus, at this time to spot the utmost effective strategies of destroying biofilm, they should be speculative in nature. By the time other results are available, new and better strategies will come to light.

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