A REVIEW ON BACTERIAL BIOFILM FORMATION AND DISASSEMBLY

A REVIEW ON BACTERIAL BIOFILM FORMATION AND DISASSEMBLY

Poonam Verma*^{1, 2}, Sanjiv Kumar Maheshwari ¹ and Abhishek Mathur ²

School of Biotechnology, IFTM University ¹, Moradabad, Uttar Pradesh, India

Institute of Transgene Life Sciences (ITLS) ², Dehradun, Uttarakhand, India

ABSTRACT: The icaADBC gene was first identified in Staphylococcus epidermidis, and is also present in Staphylococcus aureus and other Staphylococcal species. PIA is produced by the gene products encoded by the icaADBC operon. Asymptomatically colonized patients and health care workers are the major sources of MRSA in the hospital environment. MRSA-infected patients in burns units are particularly problematic because the big surface area of denuded skin can produce a large inoculum of organisms that can be easily transmitted to other patients via the hands of health care workers. Extensive skin lesions also result in heavy shedders of MRSA. Clinical isolates of Staphylococcus aureus can express the icaADBC-encoded polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/ PNAG). The icaADBC dependent and independent pathways will be stimulated using different chemicals and level of biofilm formation as well as PIA/PNAG level will be assayed. Besides, proteomics and transcriptomics analysis will be performed to get insights in the interaction of various factors of the pathways involved in the biofilm formation in wild type as well as mutant strains. The biofilm development in MRSA is ica independent and involves a protein adhesin(s) regulated by SarA and agr, whereas SarA-regulated PIA/PNAG plays a more important role in MSSA biofilm development in ica dependent pathway. This will lead to the establishment of a comprehensive interactome of biofilm formation.

icaADBC operon, PIA/PNAG, Biofilm, MRSA, MSSA, PIA

INTRODUCTION: Bacterial biofilms are important topic interest for hospital related infections. Clinical isolates of Staphylococcus aureus plays an important role for the attachment on artificial surface i.e. implanted biomedical devices causing bacterial biofilms.

Bacterial biofilms mostly caused by Staphylococcal aureus or Staphylococcus epidermidis results in colonization on biomedical implants and subsequent sepsis. However biofilm formation is not restricted to Staphylococcus species.

The phenomenon has been observed in different other bacterial species, including Streptococcus gordonii ¹, Escherichia coli ², Pseudomonas fluorescens ³ and Pseudomonasaeruginosa etc. Bacterial biofilms can be defined as an assemblage of microorganisms which can attach to any type of surfaces and subsequently get covered by an extracellular polysaccharide matrix.

Henrici was the first to observe bacterial biofilm formation in 1933 as he found that water bacteria doesn’t grow in a free floating form but attached to a submerged surface. Bacterial biofilm formation is a two-step process involving attachment of bacteria to substrate surface followed by formation of multiple layers of biofilm due to cell-cell adhesion ^4-6 is shown in Figure 1.

FIGURE 1: MODEL OF BIOFILM FORMATION

Bacterial teichoic acids play important role in initial step of biofilm formation or colonization on medical devices like- Artificial surfaces. Molecular mechanisms underlying the bacterial biofilm formation is complex and variations exist in among different strains. Since the last two decades, various surface proteins, extracellular proteins, capsular polysaccharides, adhesin (PSA) and autolysins are found to be involved in regulation of biofilm development. Different bacterial mutants have been collected for the last two decades and the functions of the genes have been identified as listed in Table 1.

TABLE 1: FUNCTION OF THE MUTANT GENES INVOLVED IN BIOFILM FORMATION OF DEPENDENT AND INDEPENDENT PATHWAYS OF S. AUREUS STRAINS

Broadly the mechanisms of bacterial biofilm formation can be classified into two groups i.e. the biofilm formation via icaADBC dependent pathway and icaADBC independent pathways shown in Figure 2.

FIGURE 2: REPRESENTATION OF PROTEINS INVOLVED IN icaADBC DEPENDENT PATHWAY AND ICAADBC INDEPENDENT PATHWAYS

The icaADBC locus is found to be the major factor responsible for the bacterial biofilm formation via icaADBC independent pathway. The icaADBC locus is involved in formation of extracellular polysaccharide adhesion termed polysaccharide intercellular adhesion (PIA) or polymeric N-acetyl glucosamine (PNAG).

Both S. aureus and S. epidermidis which are gram-positive cocci bacteria have capacity to attach with biomedical surfaces for the development of biofilm phenomenon. Both of the species forms biofilm in 2 steps i.e. cell-cell adhesion due to ica operon and have capacity to produce PIA and develop biofilm in vitro condition enzymes, that having a linkage UDP-N-acetylglucosamine in vitro condition ⁷.

The high level of morbidity and mortality caused by Staphylococcus found in  human being is due to the  high frequency of infection by both S. aureus and S. epidermidis strains with the help of ica gene locus and it is found that all Staphylococcus species have icaADBC locus that have capacity to developed biofilm formation in vitro conditions.

icaADBC-encoded polysaccharide intercellular adhesin or poly-N-acetylglucosamine (PIA/PNAG) enzymes contributed important role for production of biofilm phenomenon in anaerobic conditions in both Staphylococcus aureus and Staphylococcus epidermidis, thus icaADBC-encoded (PIA/PNAG) enzymes promoted the production of the molecular cross-talk of biofilm process and they as a virulence factor in an anaerobic environment condition in vivo (natural medium) ⁸. The exact role of poly-N-acetylglucosamine (PNAG) was identified in arlRS mutant who showed that PNAG participated in both process of primary attachment and gathering of biofilm formation. The authors usedHussain-Hastings-White modified medium [HHWm] for determining of those factors that decrease the biofilm formation process in presence of mutagenesis and systematic disruption S. aureus biofilm strain. Biofilm formation is done with the help of the arlRS mutant then do not shows any changes, if icaADBC gene are present in this process, that report shows exopolysaccharide PNAG is not participated in biofilm formation  in HHW medium ⁹.

One of the major factors identified in bacterial biofilm formation is the SarA protein. This protein is found to play an important role in biofilm development.  SarA protein also known as msa protein and it is necessary for controlled the virulence factors and showed SarA expression. Reduced msa gene expression resulted in reduced SarA gene expression in biofilm development steps ¹⁰. Also the msa mutant is found to form a weak and unstable biofilm. The molecular mechanisms underlying the bacterial biofilm formation is complex and some independent regulators and environmental factors are play important role in development of assembly of S. aureus strain in nature. Both msa andSarA genes are play important role as a regulator of biofilm formation process of S. aureus strain ^11-13.

Another representative member involved in icaADBC independent pathway is the Bap proteinbap protein present in Staphylococcus aureus surface contributed in biofilm development process and recognized the new genes that involved in biofilm development. All staphylococcus stains have Bap protein, having capacity to formed high adhesion power for formation of biofilm process.

In this result, authors concluded the relationship between BAP–PIA-PNSG and find out Bap⁺and Bap^— proteins bybothstrains produced the PIA-PNSG enzyme. Bap protein is involved in biofilm formation on artificial medium providing a role of attachment ¹⁴. One another interesting is the agr protein of Staphylococcus aureus which participate in quorum-sensing system and icaADBC mediated biofilm forming pathways as well as that protein improved the virulence factors expression power in auto-inducing peptides (AIPs) or glucose depletion triggers detachment ¹⁵.

Although icaADBC dependent pathway consist of the major mechanisms of bacterial biofilm formation, however parallel mechanisms do exist for bacterial biofilm formation. In clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) strain, environmental factors do not always contributed for the increment of the biofilm formation process,  however it is major mechanism of bacterial biofilm formation in clinical isolates  and MRSA biofilm formation due to added of glucose in medium then it showed  icaADBC independent pathway ¹⁶.

Thus, although biofilm formation in clinical isolates of the biofilm formation is independent phenomenon of ica operon as suggested by the authors however on BHI medium it is not essential. This result concluded that the regulatory mechanisms controlled the biofilm phenotype phenomenon of clinical staphylococcus species. In this study authors concluded that the regulatory mechanisms controlled the biofilm phenotype phenomenon of clinical isolates staphylococci species. Thus there is an inherent difference within the MRSA and MSSA. While the biofilm development in MSSA is icaADBC dependent, in MRSA it is independent due to presence of other factors and the environment such as acidic growth medium and glucose. It was interesting to look at the MRSA ¹⁷.

Two proteins fnbA and fnbB proteins were found to reduce the biofilm formation in MRSA strain however these mutants have no effect in MSSA biofilm formation thus these proteins could be having important role in icaADBC independent pathway. It was gradually found that the primary attachment of the biofilm is not mediated by FnBP but encouraged the level of intracellular communication.

The independent pathway of biofilm formation is encouraged by FnBP and is completely dependent on SarA. Thus both fnbA and fnbB proteins have potential role in independent pathway and possibly with the known ligand binding activities of these multi-factorial.

fnbA and fnbB are actually the extracellular matrix proteins which participated in intercellular accumulation and biofilm formation in  S. aureus. Fibronectin protein also has an important role to play in S. aureus biofilm formation. The virulence of the extracellular matrix formation is dependent on the extracellular matrix protein like fibronectin. There are also other reports that MRSA strain from clinical samples that have capacity to between a proteineous and an exopolysaccharidic biofilm matrix, depending on environmental conditions ¹⁸.

An FnBP mutant shows more biofilm production in clinical isolated samples like- implanted catheter than PIA/PNAG-deficient mutant. It is concluded that clinical samples like- colonization in a murine foreign-body infection of catheters naturally formed biofilm by S. aureus stain due to FnBPs factor than PIA/PNAG. It will be interesting to discover the natural strains of bacteria those have capacity to form biofilm in matrix and interchange between polysaccharide and a protein-based biofilm. Various other protein factors were also reported to be participating in ica independent S. aureus biofilm formation pathway like atl protein ¹⁹ atl protein important in the early events of the FnBP-dependent S. aureus biofilm phenotype for autolysin.

Mutant agr locus protein participated in the increment of FnBP-dependent biofilm formation, whether the SarA mutation, which encourage the protease production and closed to their FnBP-mediated biofilm development function. Again authors analyzed the atl regulation role; whenever atlR are enhanced the autolysin process and atlR::Tcr mutation in BH1CC enhanced biofilm-forming capacity. In this study authors concluded the role of atlR, agr, SarA, and sigB proteins in S. aureus biofilm formation as well as atl protein participated in initial attachment and cDNA release during the initial stages of ica-independent, FnBP-mediated biofilm development. Atl play primary attachment on substance with the help of protein lysis through the cell lysis, cDNA release, and cell accumulation. FnBPs played important role in initial biofilm formation and maturation ^{17, 20}.

Another set of interesting gene is hfq expression in low fluid shear culture in gram positive organism. In this result authors concluded that presence of S. aureus strain in low-fluid-shear environment and it’s proved the formation of biofilm/colonization phenotype due to virulence characteristics i.e. hfq gene as well as shows the influence key factors between colonization during the initial host-pathogen interaction ²¹. In this work, it is concluded that fluid-shear culture environment supports S. aureus presentation and showed the independent biofilm formation pathway.

In this work, it is concluded that the persisting nosocomial bacteria are present in liquid fluid of human body and formed the colonization in that due to forming of independent biofilm forming pathway. Biofilms development is a multi-factorial involving polysaccharide, protein, and DNA components, which is maintained by various regulating factors. In Staphylococcus aureus MW2 strain, Rsp is represent as a repressor in biofilm formation steps and shows attachment and biofilm formation process by fnbA factor ²². Clinical isolates of Staphylococcus aureus strain showed the biofilm phenotypes supported by the major cell wall autolysin and the fibronectin-binding proteins or the icaADBC-encoded polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG).

Thus, S. aureus biofilm formation and regulation both arevery complex process due to contribution of multiple components in this phenomenon, which are included to the polysaccharide, proteins, and extracellular DNA. There are also findings that an AraC/XylS factors are family regulator as well as Rsp gene behave like a repressed biofilm formation in Staphylococcus aureus MW2 strain.

Biofilm formation process in methicillin-susceptible S. aureus (MSSA) strain is dependent on PIA/PNAG but methicillin-resistant isolate (MRSA) express an atl/FnBP-mediated biofilm phenotype showed a relationship between susceptibility to β-lactam antibiotics and biofilm process, whether MRSA changed the biofilm phenotype and attenuates virulence process in Staphylococcus aureus device- associated infections.

S. aureus is one of the leading cause of nosocomial infections, reported worldwide which shows  many roles like- antibiotic resistance, enzyme and toxin production, biofilm forming and immune evasion capability.

Authors data showed that clinical isolates of MSSA are more likely to form a PNAG-dependent biofilm than MRSA isolates, which produce an atl/FnBP-dependent biofilm, explained the methicillin susceptibility influences biofilm expression roles ²³.

Staphylococcus aureus strain is a leading cause of nosocomial chronic infection and widely associated in biofilm formation pathway on biotic and abiotic surfaces with the help of bap-mediated matrix protein, which stops the cellular internalization through binding to host receptor. Biofilm forming organisms also showed resistance against antibiotics/antiseptics due to formation of   biofilm matrix, composed of exopolysaccharides, proteins, nucleic acids and lipids and participated as a defense structure protecting bacteria against the host immune system and antimicrobial therapy.In this research-work authors concluded that bap protein is used as a biofilm forming factors participated in the interaction of S. aureus with host cells and showed that bap protein helps in adhesion with surfaces but prevents the entry of S. aureus into epithelial cells.

Authors found specific dual roles of bap protein first is attachment of persistent infections, and second is promoting the adhesion of S. aureus to epithelial cells of the mammary gland by impairing the bacterial internalization through the interaction and impairing host cell invasion ²⁴. Authors have divided bovine mastitis S. aureus isolates into 3 groups on the basis of genetic elements, these are important for biofilm formation i.e. group 1 (ica+, bap+), group 2 (ica+, bap negative), and group 3 (ica negative, bap negative) respectively. The bovine mastitis disease appeared due to biofilm formation in S. aureus strains and bap is the most important gene for that process of biofilm formation ²⁵.

Bacterial biofilms are therefore to be dependent on icaADBC dependent and independent pathway. Therefore there could be the possibility of cross talk between the two pathways. Certain other protein factors are also found to have an important role in biofilm formation. Many types of regulatory pathway controlled by regulatory molecules, like- DNA binding protein SarA, regulatory RNAIII effector molecule of the agr system of growth process.

Increasing and decreasing regulating power is controlled by SarZ gene, i.e. a member of SarA/MarR family of transcriptional regulators. SarZ is a regulatory molecule of the regulatory phase and its increases to virulence factors that necessary for the biofilm development process of S. aureus infections. SarZ also shown the transcriptional and phenotypic analyses process in the S. aureus (MRSA and MSSA) both strains. However introduced mutation in SarZ gene is decreased agr transcription pathway. SarZ gene controlled the sarA and agr genes expression of virulence factors in biofilm formation phenomenon, like- hla and sspA ²⁶.

Some other protein factors such as cid and lrg genes present in S. aureus bacterium and show opposing effects on the control of murein activity and lysis during the biofilm development in cells grown in planktonic culture. Similarly Staphylococcus aureus have also specific surface protein (SasC) factor which participate in the cell aggregation and biofilm production due to colonization and help in accumulation in pathogenic bacteria ²⁷.

PSMs (biofilm maturation factor) also participate as a specific gene in biofilm maturation steps, characteristic development, channel-containing biofilm structure, and dissemination, control of biofilm expansion and biofilm detachment in vivo condition of S. aureus. Biofilm structure is dependent on biofilm maturation factor (PSM) expression of local differences, which is further proceeding by quorum-sensing (agr) activity.

It has been also concluded that intercellular adhesion (ica) gene promoted by Aap and SasG genes of staphylococcus species are also known as zinc zipper, in which Zn²⁺ ion is present in between self-association events stretches of B repeats on opposing Aap or SasG molecules. Studies on the molecular cross-talk of biofilm forming mechanisms of bacteria are not well known by scientists. Biofilm formation mechanisms are not well known due to deficiency of knowledge about biofilm structure, attachment, maturation and detachment process. It is also found in author’s result; biofilm development process controlled by agr negative gene however agr mutant strains have capacity to formed thick biofilm.

The following experiments will be interesting to perform: preparation of recombinant strains of Staphylococcus aureus expressing full length and domain of proteins involved in icaADBC dependent biofilm forming pathway; preparation of recombinant strains of Staphylococcus aureus expressing full length and domain of proteins involved in icaADBC independent biofilm pathway and comparative investigations in the level of proteomics and transcriptomics of different recombinant strains to describe the possible interactions between proteins of icaADBC dependent and icaADBC independent pathway.

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    How to cite this article:

    Verma P, Maheshwari SK and Mathur A: A review on Bacterial Biofilm Formation and Disassembly. Int J Pharm Sci Res 2013: 4(8); 2900-2906. doi: 10.13040/IJPSR. 0975-8232.4(8).2900-06

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