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 ~ Introduction
 ~ Conclusions
 ~ Acknowledgement
 ~  References
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  Table of Contents  
REVIEW ARTICLE
Year : 2014  |  Volume : 32  |  Issue : 2  |  Page : 112-123
 

Revamping the role of biofilm regulating operons in device-associated Staphylococci and Pseudomonas aeruginosa


1 Department of Studies in Microbiology, University of Mysore, Mysore, Karnataka, India
2 Department of Microbiology, JSS Medical College, Sri Shivarathreeshwara Nagara, Mysore, Karnataka, India

Date of Submission08-May-2013
Date of Acceptance22-Oct-2013
Date of Web Publication2-Apr-2014

Correspondence Address:
Shubha Gopal
Department of Studies in Microbiology, University of Mysore, Mysore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.129766

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 ~ Abstract 

Extensive use of indwelling devices in modern medicine has revoked higher incidence of device associated infections and most of these devices provide an ideal surface for microbial attachment to form strong biofilms. These obnoxious biofilms are responsible for persistent infections, longer hospitalization and high mortality rate. Gene regulations in bacteria play a significant role in survival, colonization and pathogenesis. Operons being a part of gene regulatory network favour cell colonization and biofilm formation in various pathogens. This review explains the functional role of various operons in biofilm expression and regulation observed in device-associated pathogens such as Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa.


Keywords: Biofilm, operon, implant, Pseudomonas, Staphylococcus


How to cite this article:
Halebeedu PP, Kumar GV, Gopal S. Revamping the role of biofilm regulating operons in device-associated Staphylococci and Pseudomonas aeruginosa. Indian J Med Microbiol 2014;32:112-23

How to cite this URL:
Halebeedu PP, Kumar GV, Gopal S. Revamping the role of biofilm regulating operons in device-associated Staphylococci and Pseudomonas aeruginosa. Indian J Med Microbiol [serial online] 2014 [cited 2019 Dec 12];32:112-23. Available from: http://www.ijmm.org/text.asp?2014/32/2/112/129766



 ~ Introduction Top


Biofilm is a conglomerate aggregation of microorganisms which are irreversibly associated with a surface and enclosed in a hydrated matrix. [1] They are made up of single or mixed species population and differ from planktonic forms by showing reduced growth, increased drug resistance and adaptability. The fundamental unit of a biofilm is a microcolony, wherein close contact of cells provides a perfect environment for the creation of nutrient gradients, genetic exchange and signalling. [2] Channels present in biofilm favour exchange of water, bacterial waste, nutrients, enzymes, metabolites and oxygen. Biofilm-related infections may be caused by a single monopolized species or a mixture of species. The seeding of biofilm on any device is triggered by microorganisms present on the skin of the host, cross-contamination of healthcare workers, tap water to which entry ports are exposed, or other sources within the local environment. [3] The occurrence of a biofilm on a medical device may also hamper the function of the device itself. [4] This, in turn, can cause weakening of both health and quality of life of an individual. Increasing use of medical devices in healthcare systems is always associated with a definitive risk of bacterial infections which is commonly termed as "foreign body-related bacterial infections" (FBRI). [5] FBRIs comprise bloodstream infections, orthopaedic implant-associated infections and catheter-associated infections. Organisms observed on these devices are either Gram-positive or Gram-negative bacteria or yeasts or a mixture of all. These pathogens are either commensals on skin or are nosocomial in origin. Various studies on the incidence of device associated pathogens have revealed that biofilms primarily consist of Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa.[5] Voluminous work has been carried out in understanding the molecular mechanisms involved in biofilm formation. Operons, being one of the major molecular circuits in biofilm expression, have been well studied in these pathogens. Operons are the clusters of coregulated genes with related functions found commonly in bacterial genomes. The series of genes in an operon are transcribed as a single mRNA and consists of an upstream promoter and a downstream terminator. [6] Genes transcribed in an operon are shown to be functionally related and are often engaged in regulating the same metabolic pathway. [7] Extensive research on biofilm forming pathogens has opened new avenues in exploring the role of these operons in biofilm expression and regulation. The current review is focused on various operons and their functions which are reported in device-associated pathogens such as S. aureus, S. epidermidis and P. aeruginosa. These operons are also regulated by various transcription and global regulators. A short description on well-reported transcriptional and global regulators of Staphylococcus and Pseudomonas aeruginosa biofilms have been explained in this review.

Operons of Staphylococcal biofilms

Staphylococci
are commonly found on indwelling medical devices such as central venous catheters, needleless connectors, endotracheal tubes, intrauterine devices, mechanical heart valves, pacemakers, peritoneal dialysis catheters, prosthetic joints, tympanostomy tubes, urinary catheters and voice prostheses. [8] After inserting indwelling devices into the patients, they are rapidly coated with host-derived extracellular matrix components and plasma proteins which can function as adhesion molecules for Staphylococcal attachment. [8] One of the reasons for catheter and implant infections to occur frequently is the poor vascularization at implantation site. The inability of the innate and adaptive defence mechanisms to reach these sites makes it difficult for the host to fight infection. [8] In regions devoid of circulation, Staphylococci are free to grow, spread and form a resistant biofilm structure. [8] Among the Staphylococcal species that appear in the list of the leading aetiological agents, Staphylococcus aureus and S. epidermidis are, respectively, at the first and the second positions. There are a certain number of Coagulase-negative Staphylococci (CoNS) that are emerging as new pathogens, such as S. hominis, S. haemolyticus, S. capitis and S. warneri.[8] With reference to S. aureus and S. epidermidis, the regulation of biofilms by operons and their known functions are explained below. [Table 1] gives an overview of biofilm regulating operons, encoded proteins and their functions.
Table 1: Genes of Staphylococcal biofi lm operons, encoding proteins and their functions

Click here to view


Intercellular adhesin operon (icaADBC)

Production of the extracellular polysaccharide, termed 'poly-N-acetylglucosamine (PNAG)' in S. aureus and 'polysaccharide intercellular adhesin (PIA)' in S. epidermidis is the well-known mechanism observed in Staphylococcal biofilms. [9] PNAG/PIA is produced by enzymes regulated by the ica operon [9] and this operon is frequently observed in isolates obtained from indwelling devices rather than the carriage strains. [10] High incidence (nearly 50%) of ica positive S. epidermidis isolates has been reported in intensive care unit isolates. [11] In contrast, the observed tendency of ica operon in S. aureus clinical isolates was 100%. [12] These data suggest that the presence of ica operon in clinical and device-associated Staphylococci can trigger biofilm formation and thus it has become an essential biofilm regulatory component.

The ica gene locus consists of icaADBC genes. [1],[13] [Figure 1]a and all these genes are regulated by icaR[9],[14] and the teicoplanin-associated locus regulator, tcaR.[14] PIA is composed of β-1,6-linked N-acetylglucosamine residues (80-85%) and an anionic fraction with a lower content of non-N-acetylated D-glucosaminyl residues that contains phosphate and ester-linked succinate (15-20%). [15] Upregulation of ica gene locus is influenced by environmental conditions, such as glucose, ethanol, high osmolarity, high temperature, anaerobiosis, sub-inhibitory concentrations of tetracycline or quinupristin-dalfopristin. [16] tagO gene involved in the production of wall teichoic acids has also been reported to regulate the expression of ica loci. [17] Besides, transcriptional regulators such as alternative sigma factor B (SigB), [16] Staphylococcal accessory regulator A (SarA) [16] and Staphylococcal accessory regulator X (SarX) [18] have been reported to influence biofilm production by controlling the expression of icaADBC operon. An outlook on the function of these transcriptional and global regulators in icaADBC expression is explained in [Table 2]. The occurrence of phase variation due to alternating insertion and excision of insertion sequence 256 (IS256) to ica operon has shown to elicit PIA-independent proteinaceous biofilms. [10],[19] Thus, a thorough understanding of these regulators, IS256 elements and environmental factors involved in ica operon expression and regulation is very much essential to know more about Staphylococcal biofilms.
Table 2: Regulators of Staphylococcal and Pseudomonas aeruginosa biofi lm operons

Click here to view
Figure 1: Biofi lm-regulating operons in Staphylococci (a) icaADBC operon (b) dltABCD operon (c) cidABC operon (d) psmâ operon (e) agrACDB
operon


Click here to view


D- alanine poly (phosphoribitol) ligase operon (dltABCD)

The role of teichoic acids, highly charged cell wall polymers in biofilm formation have been reported in Staphylococci.[20] Teichoic acids being a major component of bacterial cell wall help in the attachment of cell surface proteins and favour biofilm development. [20] In Staphylococci, two classes of teichoic acids have been reported; peptidoglycan-bound wall teichoic acids and membrane-anchored lipoteichoic acids. [17] The operon dltABCD programs the synthesis of peptidoglycan-bound wall teichoic acids with the release of four functional proteins responsible for the esterification of teichoic acids with D-alanine [Figure 1]b. Isolates with mutations in the dltABCD operon specifically in dltA has shown to increase net negative charge on the cell surface and unable to colonize on glass or polystyrene surface. [20] They also failed to produce D-alanine esters in its teichoic acids and did not show confluent growth on U-bottom plates when stained with safranin. [20]. The expression of dltABCD operon is repressed by high concentration of Na + and moderate concentrations of Mg 2+ and Ca 2+ . [21] Moreover, the ArlRS two component system negatively regulates the transcription of dlt operon in presence of high concentration of Mg 2+ ions. [21] These facts strongly support the functionality of teichoic acid's negative charge in bacterial non-adherence and reduced biofilm formation. Thus, dltABCD operon regulates the expression of biofilms in Staphylococci by generating a net positive charge on the surface. Any mutation in dltABCD operon will lead to reduced biofilm production due to the generation of net negative charge on bacterial cell wall.

Holin-like protein (cidABC) operon

The role of bacterial cell death with the release of extracellular DNA has been implicated in the establishment of biofilms in various organisms. [22] Regulated bacterial cell death and lysis in S. aureus is favoured by an enzyme murein hydrolase. [22] This enzyme targets cleavage of cell wall peptidoglycans and has shown to take part in cell growth, cell division, separation of daughter cells, peptidoglycan recycling and regulated cell lysis. [22] The regulation of murein hydrolase activity is favoured by cid operon which acts as an effector by encoding holin-like proteins. [23] Advanced research on cid operon has shown their importance in biofilm development. [23] cid operon consists of three genes cidA, cidB, cidC and a regulator cidR [Figure 1]c. This operon is positively regulated by the transcriptional factor cidR which is a part of this operon. A study on cidA mutant (lysis-defective mutant) showed loosely compacted cells with decreased adherence in both static and flow-cell biofilm systems. [23] This study further helped scientists to look into the role of extracellular DNA released during cell lysis for the development of biofilms. Treatment of wild type S. aureus with DNAse showed increased destabilization. [24] But a similar treatment on cidA mutants had a minimal effect. [23] cidA mutants have also shown the reduced biofilm formation even in animal models. [22] The expression of cid operon is regulated by LytSR two component regulatory system. [24] Its role on cid operon expression has been explained in [Table 2]. These data opine that the cid operon-mediated cell lysis coupled with the release of extracellular DNA has an essential role in maintaining the structural integrity of Staphylococcal biofilms.

Phenol-soluble Modulin β operon

PSM belongs to a class of short peptides which function as surfactants and pro-inflammatory molecules in Staphylococci.[25] They are subdivided into three classes namely PSM-α consisting of ~ 20 amino acids and PSM-β consisting of ~ 40-45 amino acids and PSM-γ (encoded by hld gene) is a delta toxin. [1],[26] During the maturation of biofilm, β-type PSM peptides are encoded by psmβ0 operon [27] [Figure 1]d. The role of PSM-β in biofilm maturation was first observed in S. epidermidis using an isogenic psmβ mutant. [27] This mutant expressed a dense and extended biofilm in flow cell systems when compared to its wild type. [27] This study showed that PSM-β can favour the detachment of biofilms by disrupting the non-covalent molecular interactions within the biofilm.

Accessory Gene Regulator (agrACDB) operon

Agr is considered as a global regulator in Staphylococcal virulence and quorum sensing mechanism in Staphylococcus aureus.[28],[29] The agr genetic locus is nearly 3 kb in size and comprises of diverse transcription units. [30] This locus is activated by two promoters P2 and P3 [Figure 1]e. The P2 programs the expression of agrACDB operon and an autoinducing ligand. [30] Whereas, P3 promoter directs the expression of RNAIII molecules, which, in turn, regulate the expression of virulence and quorum-sensing genes. [30]

Agr system has been reported to negatively regulate the expression of microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) which are involved in biofilm initiation. [1],[30] These molecules are highly expressed during colonization and once it is achieved, agr down-regulates MSCRAMMs expression. [30] Furthermore, the dependence of agr operon in biofilm expression is mainly observed by tight regulation of PSM expression. [31] With the aid of green fluorescent protein expression, a study showed that agr-favoured biofilm detachment by upregulating the expression of psmβ0 operon. [29] In the same study, agr mutants formed compact biofilms in in vitro conditions when compared to its wild type. [29] Thus, agr system negatively regulates the expression of biofilm by positively regulating the expression of psmβ operon.

Operons of Pseudomonas aeruginosa biofilm

Pseudomonas aeruginosa
is a major nosocomial pathogen which causes life-threatening infections in immunocompromised individuals and patients suffering from respiratory illness. P. aeruginosa is responsible for ventilator-associated pneumonia with high mortality when compared to other pathogens. [32] It is also a dominating pathogen in cystic fibrosis patients leading to chronic infections followed by death. The formation of biofilm by P. aeruginosa can prolong the disease status in cystic fibrosis patients and favours the growth of diverse microorganisms leading to secondary infections. [33] P. aeruginosa is also involved in urinary tract infections with severe complications by forming irreversible biofilms on urinary catheters. [34] It is frequently reported as a major pathogen in ventilator-assisted devices coronary stents and endotracheal tubes. [35] These reports infer that the pathogen P. aeruginosa has a notable impact in device associated infections through biofilm formation. The production of extracellular polymeric substance (EPS) serves as a backbone for biofilm establishment and plays a vital role in initial attachment, cell-cell interactions, tolerance and exchange of genetic materials. [33] P. aeruginosa EPS matrix primarily consists of alginate, polysaccharides (psl and pel), proteins, cyclic-di-GMP-regulated adhesin A protein (cdrA), cup fimbria, type IV pili, lectins and extracellular DNA (eDNA). Different operons have been discovered for the expression and regulation of these EPS components. [33] Here we describe few of the well-known operons involved in EPS matrix and biofilm production in P. aeruginosa. Biofilm regulating operons in P. aeruginosa, its encoded proteins and their functions are explained in [Table 3].
Table 3: Genes of P. aeruginosa biofi lm operons, encoding proteins and their functions

Click here to view


Alginate operon

Alginate is an acetylated polymer made up of high molecular weight non-repetitive monomers of β-1,4 linked L-Glucuronic and D-Mannuronic acids. [36] The alginate operon is made up of twelve genes [Figure 2]a. Momentous work has been carried out in understanding the role of this exopolysaccharide alginate in P. aeruginosa biofilm formation. A defined architecture and resistance to tobramycin was reported in alginate over producing strain. [37] Another report has shown that super mucoid P. aeruginosa strain failed to attach properly and formed thicker biofilm with an outsized unmitigated mushroom like microcolonies. [38] These reports have shown that, the regulation of various genes in alginate operon has a considerable role in alginate production and biofilm regulation. Post-transcriptional regulation of membrane anchoring protein Alg44 and the glycosyl transferase Alg8 together plays a vital role in polymerization leading to alginate production. [39] Attenuated total reflection/Fourier transform-infrared spectrometry (ATR/FT-IR) and scanning confocal laser microscopy (SCLM) have revealed that alginate is not a major component of P. aeruginosa biofilm and also for the interfacial adhesion or growth. [40] In the same study, in vitro mutagenesis of algD and algJ, expressed an improper alginate and an alginate without O-acetylation respectively. These mutants failed to form three-dimensional structures but showed flat biofilms similar to non-mucoid strains. These observations proved that alginate with O-acetylation can promote the structural stability of biofilms. However, it also suggests that the alginate may not be an essential structural component of P. aeruginosa biofilm. [40] Apart from alginate, polysaccharides were also observed in biofilms of non-mucoid strains. [41] These polysaccharides were later identified as pellicle polysaccharide and a polysaccharide synthesized at the polysaccharide synthesis loci.
Figure 2: Biofi lm-regulating operons in Pseudomonas aeruginosa (a) Alginate operon (b) pel operon (c) psl operon (d) cdrAB operon (e) cupA
operon


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Pellicle polysaccharide operon

The ability of P. aeruginosa to form biofilm at the air-liquid interface in static cultures suggested the role of a special pellicle polysaccharide in biofilm formation. It was first reported in PA14 strain by screening its transposon library for pellicle-deficient mutants. [42] This study disclosed the existence and functioning of a seven gene operon pel in PA14 strain [42] [Figure 2]b. To understand the role of pel operon in biofilm production, mutants lacking few pel genes were evaluated for biofilm initiation and integrity. This study revealed that Pel did not significantly affect biofilm initiation in PA14 pel mutants. Instead it disturbed the colony morphology and the ability of these cells to bind congo red. [42] A similar study comprehended the role of Pel in PAK strain which is known to show biofilm initiation using type IV pili. Mutational analysis of pel genes as well as pili associated genes in this strain showed failure to initiate biofilm. [42] Thus, pel locus is considered as an essential component of biofilm regulation in PA14 and PAK strains. The expression of pel loci is regulated by a secondary messenger molecule c-di-GMP and a transcription factor FleQ. [43] Another study has shown that the allosteric binding of c-di-GMP to pelD activates Pel synthesis. [44] These data suggest that pel operon is regulated by multiple factors and the expression of Pel is essential for biofilm formation.

Polysaccharide Synthesis Locus Operon

The role of psl operon in P. aeruginosa biofilm formation was discovered by conducting mutagenesis studies on PAO1 and PA14 strains. The disruption of alginate operon in these strains, were earlier thought to reduce biofilm formation. But interestingly biofilms were observed in these strains with rich polysaccharide matrix even in the absence of alginate. Identification of psl operon by three independent research groups confirmed the presence of 15 co-transcribing genes (pslA to pslO) [Figure 2]c and displayed an essential role in biofilm regulation. [45] The mannose-rich polysaccharide Psl expressed by psl operon is reported to be a highly conserved in numerous P. aeruginosa strains. [46] Cell-to-cell interactions and cell-to-surface interactions mediated by this polysaccharide is very much essential for the formation of biofilm matrix and its maintenance. [47] Helical symmetry of Psl has been explored and this has shown to favour cell-cell and cell-surface interactions. [47] Lectin staining to localize Psl polysaccharide in mushroom-shaped biofilms showed its localisation at the periphery. [47] This confirmed that Psl accumulates at the periphery of microcolonies in mushroom shaped biofilms. [47] The expression of Psl polysaccharide is regulated by two sensor kinases: Lost adherence Sensor (LadS) and Regulator of Exopolysaccharide and type III Secretion (RetS). [48] Besides, it is also regulated by c-di-GMP. [43] A novel protein cyclic di-guanylate monophosphate regulator A (CdrA) expressed by cdrAB gene complex is reported to crosslink with Psl and this displayed elevated biofilm matrix production [49] With all these findings we can infer that in P. aeruginosa Psl polysaccharide is an essential component of EPS matrix and its expression can favour biofilm formation in non-mucoid strains.

Cyclic di-guanylate monophosphate regulator operon

Research on extracellular matrix expansion has revealed that cyclic diguanylate monophosphate (c-di-GMP) positively regulates production of matrix components at the transcriptional and allosteric level. [50] It is now known that higher concentrations of intracellular c-di-GMP triggers expression of biofilms in liquid medium and at lower levels they promote motility and planktonic lifestyle. [50] The sum total of genes induced in response to elevated levels of c-di-GMP was comparatively low in P. aeruginosa and most of these genes belonged to the pel and psl operons. [51] However, further investigation revealed that two genes termed as PA4625 and PA4624 encoded two unique proteins which enhanced EPS matrix expansion and biofilm formation. [50] These genes exhibited a two-partner secretion system (TPS) and expressed a large concealed adhesin protein with transporter function. [50] They were termed as cyclic diguanylate-regulated TPS partner A (cdrA) and cyclic di-guanylate-regulated TPS partner B (cdrB), respectively and both were contiguous to form a single operon [50] [Figure 2]d. CdrA is dependent on the presence of Psl polysaccharide and aggregation can be disrupted by adding mannose. [50] CdrB functions as a transporter of CdrA and this was confirmed by mutational analysis, wherein CdrB mutants failed to show auto-aggregation of cells and biofilm formation. [50] Thus cdrAB operon with two important genes is very much essential for proper functioning and expansion of EPS matrix,

Chaperone Usher Pathway A operon

Extracellular appendages such as pili or fimbriae in Gram-negative bacteria can play a pivotal role in attachment, cell mobilization, protein transport, genetic exchange and invasion. [51] Based on the biosynthetic pathways these pili are classified into five types such as chaperone-usher pili (CU pili), type IV pili, type III secretion needle pili and type IV secretion pili. [51] Among these, CU pili have gained much attention due to their complex assembly mechanisms. [52] The assemblage of CU pili is favoured by a chaperone synthesized at the periplasmic region and by a porous outer-membrane protein termed as usher. [52] Chaperons prevent polymerization of pilus in the periplasm, favour its folding and direct them towards the usher. [52] Usher as a molecular motor facilitates the assembly of chaperons to pilus subunits and secretes the pili through the usher pore. [52] Mutational studies have shown that the initiation of biofilm and its maturation in P. aeruginosa requires the expression of type IV pili and flagella on its cell surface. [53] These factors thwart the repulsive forces exerted on a surface, favouring the formation of biofilms. [53] However, Tn5 mutational analysis in P. aeruginosa strain revealed the presence of new adhesion factors other than pili and flagella. [54] Among these mutants few of them showed reduced expression of proteins similar to periplasmic chaperons exclusively observed in chaperon/usher pathways expressed by cupA, cupB, cupC and cupD gene clusters. [55] cupA mutant displayed reduced biofilm formation in the absence of type IV pili and further investigation disclosed the presence of cupA1, cupA2, cupA3, cupA4 and cupA5 genes [55] [Figure 2]e. In order to identify whether all the genes of cupA operon are involved in biofilm formation, loss of function mutations in cupA2 and cupA3 has been carried out. This study validated the essential role of cupA3 in biofilm expression. [55] A transcriptional regulator termed as MvaT encoded by mvaT gene which was first reported in P. mevalonii, downregulates the expression of cupA operon and blocks the expression of biofilm. [55] This regulator also displayed flaunted virulence gene expression in P. aeruginosa.[55] Besides, upregulation of cupA operon in a phase variable manner is carried out by cupA gene regulator (cgr) comprising four genes cgrA, cgrB, cgrC and a global regulator of anaerobic gene expression (Anr) which is present upstream to cupA operon. [56] The presence of anaerobic environment in P. aeruginosa triggers higher expression of biofilm aided by Anr gene regulation. [56] These data suggest that the expression of cupA operon requires multiple factors and have a tremendous impact on biofilm expression when compared to other cup operons.

Biofilms and gene expression-message from transcriptome analysis

Transcriptome analysis plays a key role in understanding the expression of various genes at different stages of biofilm formation. It also helps us in identifying novel genes involved in biofilm regulation. Currently transcriptome analysis is being carried out either by microarray or by RNA sequencing technology. [57] Whole genome transcriptome profiling of S. aureus UAMS -1 strain showed altered expression of 580 genes during biofilm maturation. [58] When expression was compared in biofilm and planktonic state, nearly 48 genes showed enhanced expression and 84 genes showed reduced expression in biofilm than their planktonic counterpart. [58] Moreover, the expression of icaD showed consistent upregulation and Spa was extremely downregulated in S. aureus UAMS -1 biofilms. [58] Similar studies on S. epidermidis 1457 (non-producer of biofilm) and S. epidermidis RP62A (strong producer of biofilm) have shown that nearly 12% of genes expressed in varied fashion and 6% of genes showed equal up and downregulation during biofilm formation. [27] agr expression was downregulated and SarA expression was consistent with biofilm formation in S. epidermidis RP62A. [27] These data suggest that S. aureus and S. epidermidis show differential expression of genes during biofilm formation.

In P. aeruginosa transcriptome analysis has been carried out in different growth phases. Transcriptome of planktonic cultures obtained at logarithmic and stationary phase have been compared with the developing and confluent biofilm transcriptome. [59] Nearly 19.4% of the PAO1 genome showed differential expression of genes in planktonic cultures (10.5% genes were up regulated and 8.9% were down-regulated). [59] When the logarithmic phase transcriptome of planktonic culture was compared with 8 h developing biofilm transcriptome, only 3.1% of the genome showed differential expression. (0.8% of genes were up-regulated and 2.3% of genes were down-regulated). [59] However, when the transcriptome of stationary phase planktonic culture was compared with the transcriptome of confluent biofilms ~ 14.3% of the genes were differentially expressed. [59] Comparative transcriptome analysis between developing and confluent biofilms also revealed considerable variation in gene expression with 15.5% differential expression. [59] Furthermore, transcriptome of developing and confluent biofilm were found to be related to the transcriptome of logarithmic and stationary phase cultures. Genes encoding transport proteins and transcriptional regulators were also found to be upregulated in developing and confluent biofilms of P. aeruginosa PAO1 strain. [59]

Quantitative and qualitative RNA sequence data analysis also revealed the existence of more than 3000 transcriptional start sites and the expression of small RNAs in P. aeruginosa.[60] Thus, transcriptome analysis of P. aeruginosa biofilms has helped us in understanding the rate of genes and small RNA expression at different phases of biofilm growth. RNA sequence-based transcriptome analysis has also improved our understanding of gene expression in biofilms.


 ~ Conclusions Top


Global incidences of biofilm-associated infections are increasing at an alarming rate due to the excessive usage of medical implants. In association with host derived conditioning factors and hydrophobic, hydrophilic and electrostatic interactions, these implants enhance the growth of pathogens in patients leading to persistent infections with increased drug resistance. Operons provide an idealistic model to understand various environmental and nutritional factors impeding or promoting the expression of biofilms. Pathogens such as S. aureus, S. epidermidis and P. aeruginosa are repeatedly shown to form biofilms on indwelling medical devices and caused dreadful long-term infections with increased drug resistance. Operons in these organisms demonstrate diverse function with differential expression. However, all these operons are either positively or negatively regulated by various genes, two component regulators, transcriptional factors and global regulators. More importantly, transcriptome and RNA sequence analysis of biofilms disclosed differential gene expression, existence of novel genes and transcription start sites. Thus, operons and its associated regulators depict an intricate molecular regulation with a profound impact on biofilm production and gene expression. With all these understandings and vast knowledge in the genetic regulation of biofilms, it is still unclear how these pathogens have evolved to exhibit enhanced drug tolerance and resistance. Spurred research in this context is the need of the hour to improve health status in our society.[61]


 ~ Acknowledgement Top


Authors thank Indian Council of Medical Research for providing Senior Research Fellowship to PHP (ICMR award letter No. 80/763/2012-ECD-I dated 02.04.2013) and University Grants Commission for the Major Research Project to SG (UGC order F.No. 39-201/2010 (S.R) dated 27.12.2010)

 
 ~ References Top

1.Otto M. Staphylococcal biofilms. Curr Top Microbiol Immunol 2008;322:207-28.  Back to cited text no. 1
[PUBMED]    
2.Davey ME, O'Toole GA. Microbial biofilms: From ecology to molecular genetics. Microbiol Mol Biol Rev 2000;64:847-67.  Back to cited text no. 2
    
3.Safdar N, Kluger DM, Maki DG. A review of risk factors for catheter-related bloodstream infection caused by percutaneously inserted, noncuffed central venous catheters-Implications for preventive strategies. Medicine 2002;81:466-79.  Back to cited text no. 3
    
4.Davis LE, Cook G, Costerton JW. Biofilm on ventriculoperitoneal shunt tubing as a cause of treatment failure in coccidioidal meningitis. Emerg Infect Dis 2002;8:376-9.  Back to cited text no. 4
    
5.von Eiff C, Jansen B, Kohnen W, Becker K. Infections Associated with Medical Devices: Pathogenesis, Management and Prophylaxis. Drugs 2005;65:179-214.  Back to cited text no. 5
    
6.Brouwer RW, Kuipers OP, van Hijum S. The relative value of operon predictions. Brief Bioinform 2008;9:367-75.  Back to cited text no. 6
    
7.Okuda S, Kawashima S, Kobayashi K, Ogasawara N, Kanehisa M, Goto S. Characterization of relationships between transcriptional units and operon structures in Bacillus subtilis and Escherichia coli. BMC Genomics 2007;8:48.  Back to cited text no. 7
    
8.Hooper S, Percival S, Cochrane C, Williams D. Biofilms and Implication in Medical Devices in Humans and Animals. In: Percival S, Knottenbelt D, Cochrane C, editors. Biofilms and Veterinary Medicine. Springer Series on Biofilms. Berlin, Heidelberg: Springer; 2011. p. 191-203.  Back to cited text no. 8
    
9.Heilmann C, Schweitzer O, Gerke C, Vanittanakom N, Mack D, Götz F. Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis. Mol Microbiol 1996;20:1083-91.  Back to cited text no. 9
    
10.Ziebuhr W, Heilmann C, Götz F, Meyer P, Wilms K, Straube E, et al. Detection of the intercellular adhesion gene cluster (ica) and phase variation in Staphylococcus epidermidis blood culture strains and mucosal isolates. Infect Immun 1997;65:890-6.  Back to cited text no. 10
    
11.Fitzpatrick F, Humphreys H, Smyth E, Kennedy CA, O'Gara JP. Environmental regulation of biofilm formation in intensive care unit isolates of Staphylococcus epidermidis. J Hosp Infect 2002;52:212-8.  Back to cited text no. 11
    
12.Fowler VG, Fey PD, Reller LB, Chamis AL, Corey GR, Rupp ME. The intercellular adhesin locus ica is present in clinical isolates of Staphylococcus aureus from bacteremic patients with infected and uninfected prosthetic joints. Med Microbiol Immun 2001;189:127-31.  Back to cited text no. 12
    
13.Rohde H, Frankenberger S, Zahringer U, Mack D. Structure, function and contribution of polysaccharide intercellular adhesin to Staphylococcus epidermidis biofilm formation and pathogenesis of biomaterial-associated infections. Eur J Cell Biol 2010;89:103-11.  Back to cited text no. 13
    
14.Vuong C, Otto M. Staphylococcus epidermidis infections. Microbes Infect 2002;4:481-9.  Back to cited text no. 14
    
15.Mack D, Fischer W, Krokotsch A, Leopold K, Hartmann R, Egge H, et al. The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear beta-1,6-linked glucosaminoglycan: Purification and structural analysis. J Bacteriol 1996;178:175-83.  Back to cited text no. 15
    
16.Fitzpatrick F, Humphreys H, O'Gara JP. The genetics of staphylococcal biofilm formation-will a greater understanding of pathogenesis lead to better management of device-related infection? Clin Microbiol Infect 2005;11:967-73.  Back to cited text no. 16
    
17.Holland LM, Conlon B, O'Gara JP. Mutation of tagO reveals an essential role for wall teichoic acids in Staphylococcus epidermidis biofilm development. Microbiol-SGM 2011;157:408-18.  Back to cited text no. 17
    
18.Rowe SE, Mahon V, Smith SG, O'Gara JP. A novel role for SarX in Staphylococcus epidermidis biofilm regulation. Microbiol-SGM 2011;157:1042-9.  Back to cited text no. 18
    
19.Ziebuhr W, Krimmer V, Rachid S, Lößner I, Götz F, Hacker J. A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: Evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256. Mol Microbiol 1999;32:345-56.  Back to cited text no. 19
    
20.Gross M, Cramton SE, Götz F, Peschel A. Key Role of Teichoic Acid Net Charge in Staphylococcus aureus Colonization of Artificial Surfaces. Infect Immun 2001;69:3423-6.  Back to cited text no. 20
    
21.Koprivnjak T, Mlakar V, Swanson L, Fournier B, Peschel A, Weiss JP. Cation-Induced Transcriptional Regulation of the dlt Operon of Staphylococcus aureus. J Bacteriol 2006;188:3622-30.  Back to cited text no. 21
    
22.Rice KC, Bayles KW. Molecular Control of Bacterial Death and Lysis. Microbiol Mol Biol Rev 2008;72:85-109.  Back to cited text no. 22
    
23.Rice KC, Mann EE, Endres JL, Weiss EC, Cassat JE, Smeltzer MS, et al. The cidA murein hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus aureus. Proc Natl Acad Sci U S A 2007;104:8113-8.  Back to cited text no. 23
    
24.Sharma-Kuinkel BK, Mann EE, Ahn JS, Kuechenmeister LJ, Dunman PM, Bayles KW. The Staphylococcus aureus LytSR Two-Component Regulatory System Affects Biofilm Formation. J Bacteriol 2009;191:4767-75.  Back to cited text no. 24
    
25.Mehlin C, Headley CM, Klebanoff SJ. An Inflammatory Polypeptide Complex from Staphylococcus epidermidis: Isolation and Characterization. J Exp Med 1999;189:907-18.  Back to cited text no. 25
    
26.Otto M. Staphylococcal Infections: Mechanisms of Biofilm Maturation and Detachment as Critical Determinants of Pathogenicity. Annu Rev Med 2013;64:175-88.  Back to cited text no. 26
[PUBMED]    
27.Yao Y, Sturdevant DE, Otto M. Genomewide Analysis of Gene Expression in Staphylococcus epidermidis Biofilms: Insights into the Pathophysiology of S. epidermidis Biofilms and the Role of Phenol-Soluble Modulins in Formation of Biofilms. J Infect Dis 2005;191:289-98.  Back to cited text no. 27
    
28.Bronner S, Monteil H, Prevost G. Regulation of virulence determinants in Staphylococcus aureus: Complexity and applications. FEMS Microbiol Rev 2004;28:183-200.  Back to cited text no. 28
    
29.Yarwood JM, Bartels DJ, Volper EM, Greenberg EP. Quorum sensing in Staphylococcus aureus biofilms. J Bacteriol 2004;186:1838-50.  Back to cited text no. 29
    
30.Novick RP. Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol Microbiol 2003;48:1429-49.  Back to cited text no. 30
[PUBMED]    
31.Wang R, Braughton KR, Kretschmer D, Bach TH, Queck SY, Li M, et al. Identification of novel cytolytic peptides as key virulence determinants for community-associated MRSA. Nat Med 2007;13:1510-4.  Back to cited text no. 31
    
32.Guidelines for the Management of Adults with Hospital-acquired, Ventilator-associated, and Healthcare-associated Pneumonia. Am J Respir Crit Care Med 2005;171:388-416.  Back to cited text no. 32
    
33.Harmsen M, Yang LA, Pamp SJ, Tolker-Nielsen T. An update on Pseudomonas aeruginosa biofilm formation, tolerance, and dispersal. FEMS Immunol Med Microbiol 2010;59:253-68.  Back to cited text no. 33
    
34.Mittal R, Aggarwal S, Sharma S, Chhibber S, Harjai K. Urinary tract infections caused by Pseudomonas aeruginosa: A minireview. J Infect Public Health 2009;2:101-11.  Back to cited text no. 34
    
35.Lynch AS, Robertson GT. Bacterial and Fungal Biofilm Infections. Annu Rev Med 2008;59:415-28.  Back to cited text no. 35
    
36.Ramsey DM, Wozniak DJ. Understanding the control of Pseudomonas aeruginosa alginate synthesis and the prospects for management of chronic infections in cystic fibrosis. Mol Microbiol 2005;56:309-22.  Back to cited text no. 36
    
37.Hentzer M, Teitzel GM, Balzer GJ, Heydorn A, Molin S, Givskov M, et al. Alginate Overproduction Affects Pseudomonas aeruginosa Biofilm Structure and Function. J Bacteriol 2001;183:5395-401.  Back to cited text no. 37
    
38.Hay ID, Gatland K, Campisano A, Jordens JZ, Rehm BH. Impact of Alginate Overproduction on Attachment and Biofilm Architecture of a Supermucoid Pseudomonas aeruginosa Strain. Appl Environ Microb 2009;75:6022-5.  Back to cited text no. 38
    
39.Oglesby LL, Jain S, Ohman DE. Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization. Microbiology 2008;154:1605-15.  Back to cited text no. 39
    
40.Nivens DE, Ohman DE, Williams J, Franklin MJ. Role of alginate and its O acetylation in formation of Pseudomonas aeruginosa microcolonies and biofilms. J Bacteriol 2001;183:1047-57.  Back to cited text no. 40
    
41.Wozniak DJ, Wyckoff TJ, Starkey M, Keyser R, Azadi P, O'Toole GA, et al. Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms. Proc Natl Acad Sci U S A 2003;100:7907-12.  Back to cited text no. 41
    
42.Friedman L, Kolter R. Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms. Mol Microbiol 2004;51:675-90.  Back to cited text no. 42
    
43.Hickman JW, Harwood CS. Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor. Mol Microbiol 2008;69:376-89.  Back to cited text no. 43
    
44.Lee VT, Matewish JM, Kessler JL, Hyodo M, Hayakawa Y, Lory S. A cyclic-di-GMP receptor required for bacterial exopolysaccharide production. Mol Microbiol 2007;65:1474-84.  Back to cited text no. 44
    
45.Matsukawa M, Greenberg EP. Putative exopolysaccharide synthesis genes influence Pseudomonas aeruginosa biofilm development. J Bacteriol 2004;186:4449-56.  Back to cited text no. 45
    
46.Ryder C, Byrd M, Wozniak DJ. Role of polysaccharides in Pseudomonas aeruginosa biofilm development. Curr Opin Microbiol 2007;10:644-8.  Back to cited text no. 46
    
47.Ma LM, Conover M, Lu HP, Parsek MR, Bayles K, Wozniak DJ. Assembly and Development of the Pseudomonas aeruginosa Biofilm Matrix. PLoS Pathog 2009;5:e1000354.  Back to cited text no. 47
    
48.Ventre I, Goodman AL, Vallet-Gely I, Vasseur P, Soscia C, Molin S, et al. Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes. Proc Natl Acad Sci U S A 2006;103:171-6.  Back to cited text no. 48
    
49.Borlee BR, Goldman AD, Murakami K, Samudrala R, Wozniak DJ, Parsek MR. Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix. Mol Microbiol 2010;75:827-42.  Back to cited text no. 49
    
50.Hickman JW, Tifrea DF, Harwood CS. A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels. Proc Natl Acad Sci U S A 2005;102:14422-7.  Back to cited text no. 50
    
51.Fronzes R, Remaut H, Waksman G. Architectures and biogenesis of non-flagellar protein appendages in Gram-negative bacteria. EMBO J 2008;27:2271-80.  Back to cited text no. 51
    
52.Waksman G, Hultgren SJ. Structural biology of the chaperone-usher pathway of pilus biogenesis. Nat Rev Microbiol 2009;7:765-74.  Back to cited text no. 52
    
53.O'Toole GA, Kolter R. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 1998;30:295-304.  Back to cited text no. 53
    
54.Vallet I, Olson JW, Lory S, Lazdunski A, Filloux A. The chaperone/usher pathways of Pseudomonas aeruginosa: Identification of fimbrial gene clusters (cup) and their involvement in biofilm formation. Proc Natl Acad Sci U S A 2001;98:6911-6.  Back to cited text no. 54
    
55.Vallet I, Diggle SP, Stacey RE, Cámara M, Ventre I, Lory S, et al. Biofilm Formation in Pseudomonas aeruginosa: Fimbrial cup Gene Clusters Are Controlled by the Transcriptional Regulator MvaT. J Bacteriol 2004;186:2880-90.  Back to cited text no. 55
    
56.Vallet-Gely I, Sharp JS, Dove SL. Local and Global Regulators Linking Anaerobiosis to cupA Fimbrial Gene Expression in Pseudomonas aeruginosa. J Bacteriol 2007;189:8667-76.  Back to cited text no. 56
    
57.Morozova O, Hirst M, Marra MA. Applications of New Sequencing Technologies for Transcriptome Analysis. Annu Rev Genomics Hum Genet 2009;10:135-51.  Back to cited text no. 57
    
58.Beenken KE, Dunman PM, McAleese F, Macapagal D, Murphy E, Projan SJ, et al. Global gene expression in Staphylococcus aureus biofilms. J Bacteriol 2004;186:4665-84.  Back to cited text no. 58
    
59.Waite RD, Papakonstantinopoulou A, Littler E, Curtis MA. Transcriptome analysis of Pseudomonas aeruginosa growth: Comparison of gene expression in planktonic cultures and developing and mature biofilms. J Bacteriol 2005;187:6571-6.  Back to cited text no. 59
    
60.Dotsch A, Eckweiler D, Schniederjans M, Zimmermann A, Jensen V, Scharfe M, et al. The Pseudomonas aeruginosa Transcriptome in Planktonic Cultures and Static Biofilms Using RNA Sequencing. PLOS One 2012;7:e31092.  Back to cited text no. 60
    
61.Irie Y, Starkey M, Edwards AN, Wozniak DJ, Romeo T, Parsek MR. Pseudomonas aeruginosa biofilm matrix polysaccharide Psl is regulated transcriptionally by RpoS and post-transcriptionally by RsmA. Mol Microbiol 2010;78:158-72.  Back to cited text no. 61
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]

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