Quantification Of Enterococci In Biofilm Growth Biology Essay

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There are a myriad of systems available for detailing the formation of static bacterial biofilms. As antibiotic resistance within biofilms has emerged as an increasing problem thus there is a growing interest in the ability to accurately quantify bacterial numbers in biofilm for a plethora of reasons such as drug susceptibility testing. Thus the intrinsic value inherent to this study correlates to a precise evaluation of three surrogate assay pertaining to the quantification of biofilms formed by Enterococci in 96-well microtiter plates after time optimisation was achieved for the various assay techniques: the Crystal Violet (CV) assay, the Alamar Blue (AB) assay and the XTT assay.

From the genus Enterococcus, the species Enterococcus faecalis (E.faecalis) was chosen for biofilm creation as it is a common noscomial infection with a strong survival ability which allows for an intrinsic adaption to antimicrobial treatment within a broad range of conditions. Enterococci have been noted to survive in conditions that include a temperature range of 10°c to 45 °c or even at 60°c for 30minutes, apH of up to 9.6 and salinity of up to 6.5% NaCl (Daniels V, 2008; Shepard and Gilmore, 2002 and Witte et al., 1999).

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The utilization of E.faecalis isolates will allow in theory and thus in practice the creation of a biofilm which is far more difficult to eradicate than E.faecalis in planktonic form. Hence such an undertaking can be merited as scientifically relevant, if one examines the estimated total worldwide anti-microbial usage which is deemed to be 100,000 to 200,000 tons per year. This unquestionably applies strong selective pressure on bacteria to become resistant to a myriad of conventionally used anti-microbials thus there is an inherent need for an accurate ability to quantify bacterial numbers present in biofilms in order to determine if anti-microbials designed to eradicate biofilm populations are indeed effective (Daniels V, 2008; da Costa et al., 2007).

The hypothesis inherent to this project addresses "A comparison of multiple assay techniques for the quantification of Enterococci in biofilm growth in 96-well microtiter plates". Intellectual conceptions of this hypothesize directly correlates to work carried out by Victoria Daniels during her PhD study entitled "Antibiotic Resistant Enterococci in Irish Waters: Molecular Epidemiology and Hydrological Control 2008". Biofilm production will take place in a 96-well microtiter plate under normal conditions, consequently the estimated quantification of CFU's present in the various biofilms will highlight definitive although limited variations between the myriad of assays utilized.

Introduction

1.1 Pathogenicity

A pathogen is an infectious biological agent that causes disease or illness to its host. The term is primarily used for agents that disrupt the normal physiology of a multi-cellular animal or plant.

Pathogen- organism with a demonstrated capacity to cause disease

Virulence- relative degree of pathogenicity

The factors by which viruses achieve pathogenicity are referred to as the virulence factors; hence virulence is a measure of a pathogens disease causing capacity (Nicklin et al 2002).

Enterococci entry into the host organism is accommodated for the most part by the environment and displays direct correlation to dissemination of antibiotic resistant strains in water which is consumed by the relevant host organisms. Thus water has been recognised as a primary reservoir for the transmission of many food and waterborne human enteropathogens (Daniels V, 2008).

The study of virulence factor genes will require discussion of homologous genes and proteins. Homology among genes or proteins reflects evolution by divergence from a common ancestor. When two homologous genes in different species have the same function, they are known as orthologs; when two genes in the same or different species have different functions they are known as paralogues. (Nicklin et al 2002)

1.1.1 Noscomial Infection

Noscomial infection pertains to infections that occur in hospitalized patients that are receiving antibiotic treatment or chemotherapeutics that has consequently disrupted the normal gut micro-flora ratio in the large intestine. Infections that are deemed to be noscomial if they principally occur 48hours post entry within the health care unity (hospital) or within a 30 day window post discharge.

1.2 Enterococcus

The genus Enterococcus is comprised of motile bacteria that are ubiquitous in nature and are natural inhabitants of the oral cavity, intestinal microflora and female genitalia of both humans and animal (Mohamed and Hung, 2007). The genus Enterococcus consist of lactic acid bacteria of the phylum Firmicutes. Pertaining to the class of Bacilli, Enterococci are determined to be gram-positive cocci, that frequently exsist as short chains or in pairs (diplocooci) (Gilmore MS et al., 2002). Their fermentative ability is enhanced by the lack of a Kerb's Cycle Respiratory chain (Shnakar et al., 2002).The two most prevalent species responsible for human enterococcal infections in the intestines are Enterococcus faecalis (E.faecalis) and Enterococcus faecium (E.faecium).

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1.2.1 Enterococcus faecalis

E.faecalis is a non-motile, facultative anaerobic micro-organism displaying both pathogenic and commensal properties (non-harmful co-existing state) and is believed to be responsible for 80-90% of human enterococcal infections (Jones et al., 1994). Exhibiting commensal properties like related species inherent to the genus Enterococcus, E.faecalis can induce life treating infection in immune-compromised humans, where it is normally ascertained from an exogenous source especially prevalent within the noscomial setting where pathogenicity will be enhanced in E.faecalis by its inherently natural high levels of antibiotic resistance.

1.2.2 Enterococcus faecium

E.faecalis is believed to account for 10-20% of human enterococcal infections (Jones et al., 1994). Located in the genus Enterococcus, it is a gram positive bacterium, group D alpha hemolitic or nonhemollitic, present in both a pathogenic and commensal state (Ryan and Ray., 2004). Within an enhanced pathogenic state antibiotic-resistant E.faecalis can be regarded as VRE (Vancomycin-resistant enterococcus).

1.3 Antibiotic resistance

Multi-drug resistance is attributed to a large proportion of the Enterococci genome consisting of mobile genetic elements (Mohamed and Hung, 2007). Having been once regarded as of minor consequence to infectious disease, enterococci's notoriety has increased exponentially in the past two decades to a state where Enterococci represent a leading noscomial pathogen that routinely presents a considerable therapeutic challenge (Shepard and Gilmore 2002). The epidemiological spread of antibiotic resistance Enterococci related disease displays direct correlation to the widespread usage of antibiotics in farming practices thus enhancing virulence by applying a strong evolutionary selective pressure (Witte et al., 1999).

Current treatment is antibiotic based with a combination of ampicillin or amoxycillin plus gentamicin being the preferential choice. Identification of E.faecalis or E.faecium or their toxins should not lead to the reflex prescription of metronidazole or teicoplanin. The current use of antibiotics is unilaterally viewed as unsustainable due to Enterococci's continued antibiotic resistance thus there is the need to understand with greater authority the pathogenicity of Enterococci in both planktonic and biofilm form.

1.3.1 Mobile genetic elements (MGE's)

Mobile Genetic Elements (MGE's) of a genome relates to a specific type of DNA that possesses the innate ability to exhibit movement within the genome. The Mobilome is the total collective term of all the MGE's in the genome.

These mobile genetic elements are putatively responsible for the acquisition by Enterococci of an extensive array of genes involved in antimicrobial resistance, virulence, host interaction and the production of surface structures (Mohamed and Hung, 2007).

1.3.2 Lateral and Horizontal gene transfer

Lateral Gene Transfer (LGT) and Horizontal Gene Transfer (HGT) are terms used to describe the acquisition by an organism of genetic material from another organism and the subsequent incorporation of that genetic material without being the offspring of that organism (Insert Citation).

Such gene transfer contrasts with vertical inheritance which forms the core of the neo-Darwinist belief in the central role of reproductive isolation between species in evolution (Koonin et al., 2001).

The gene transfer methods in question will confer a selective advantage on the recipient organism and can be classified into distinct categories of acquisition of new genes. Acquisition of paralogs of existing genes and xenologous gene displacement are common place. Xenologous gene displacement is whereby a gene is displaced by a horizontal transferred ortholog from another lineage (xenolog) (Koonin et al., 2001).

1.3.3 β- lactams

There is general consensus in the scientific community that enterococci have displayed an intrinsic low level resistance to the bacterial effects of β-lactam antibiotics. Enterococci are approximately 100 fold less susceptible to β-lactam than streptococci however enterococci displaying resistance patterns have developed unique mechanisms of resistance. Such β-lactam resistant isolates have increased in incidence and exhibit greater levels of resistance (Shepard and Gilmore 2002).

Isolates of E.faecalis continue to display susceptibility to the bacteriostatic effects of therapeutic concentrations of β-lactams however most E.faecium isolates are 4 to 16 fold less susceptible (Mc Donald et al., 1997).

Indeed 83% of clinical isolates of E.faecium demonstrating resistance produce increased levels of an alternative penicillin-binding protein (PBP's) or PBP's in combination with unique amino acid substitutions that vastly decreases affinity with regards benzylpenicillin (Shepard and Gilmore 2002).

Also isolates of E.faecalis have been distinguished that produce β-lactamase, allowing genetic evidence to imply that β-lactamase production is ascertained as a result of the acquisition of the Staphylococcus aureus β-lactamase operon. This would suggest that Enterococci possess a character trait allowing for the exchange of resistant determinants with other gram-positive bacteria most likely via lateral or horizontal gene transfer (Shepard and Gilmore 2002).

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1.3.4 Aminoglycosides

In adequately addressing enterococcal infections, synergistic combination therapies became standard practice. R.C Moellering and his colleagues demonstrated a low level of intrinsic resistance to aminoglycosides is achieved through the ability of the enterococcal cell wall to curtail up-regulation of the drug (Moellering et al., 1971).

Acquired in addition to the intrinsic mechanisms of low level resistance, enterococci exhibit resistance to aminoglycosides via genes conferring strong levels of resistance. Streptomycin represents the main class of anti-biotic utilized in combination therapy. Resistance to Streptomycin by Enterococci directly correlates to two main resistance mechanisms.

Isolated strains of Enterococci displaying resistance to Streptomycin are mediated by single mutations within a protein of the 30S ribosomal subunit, the target of aminoglycosidase activity (Shepard and Gilmore 2002).

In addition nine-genes that encode enzymes targeting eight different aminoglycosides have been highlighted. The up-regualtion of one of two specific aminoglycoside-modifying enzymes [ANT (6')- Ia or ANT(3'')-Ia ] will orchestrate resistance to Streptomycin

1.3.5 Glycopeptides

The utilization of glycopeptide vancomycin as a treatment method for enterococcal and other serious gram-positive infections is a direct consequence of vastly increased virulence and multi-drug resistance among pathogenic bacterial communities.

Glycopeptides inhibit cell wall synthesis by forming complexes with the peptidyl-D-alanyl-D-alanine (D-Ala-D-Ala) termini of peptidoglycan precursors at the cell surface. This acts by preventing incorporation of the blocked precursor formation associated with enhanced strength of the cell wall (Shepard and Gilmore 2002).

By the start of 2003, five phenotypes of glycopeptides resistant enterococci had been accurately identified, each exhibiting distinctive specifities and inherent resistance characteristics to vancomycin and teicoplanin.

The glycopeptides resistance directly correlates from the synthesis of alternate peptidoglycan precursors with reduced affinity for vancomycin and teicoplanin (Shepard and Gilmore 2002).

1.4 Enterococci genome

JCVI CMR in recent times sequenced the genome of Enterococci faecalis V583 and published the findings under the work entitled "Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis" (Paulsen et al., 2003) and more recently examined "Proteomic Analysis of the Enterococcus faecalis V583 Strain and Clinical Isolate V309 under Vancomycin Treatment" (Wang et al., 2010). The Genome of E.Faecalis is now understood to consist of greater than 25% of exogenous acquired DNA. The primary chromosome is 3,218,031 bp long with 72, 64 and 19 open reading frames present. The G+ C content in relation to the primary chromosome is 37.5% and is estimated to 34% for the other three plasmids respective (Shnakar et al., 2002).

Understanding virulence determinants in Enterococci, will address how strong selective evolution pressure has acquired or aided development of vacomycin resistant Enterococci (E.faecalis) strains. A conserved domain in the form of a pathogenicity island inherent to the genome of E.faecalis contains a gene 150kbp long that is assumed to be responsible for virulence

1.4.1Conserved domains

Conservation relates to change which has occurred at specific positions on amino acid sequences via reassortment. Thus conserved domains are the functional modules of proteins that remain invariable (unchanged) despite various other reassortment changes which may have occurred on that protein sequence.

A domain is a discrete portion of a protein assumed to fold independently of the rest of the protein due its essential function.

1.4.2 Pathogenicity islands (PAI's)

Pathogenicity islands (PAI's) are regarded as a distinct class of genomic islands acquired primarily via (LGT) or (HGT), which are incorporated within the genome of the pathogenic micro-organism. Designated as occupying relatively large genomic regions from 10-200kb they encode specific genes correlating or orchestrating virulence. PAI's may be deemed as discrete genetic units flanked by direct repeats, insertion sequences or tRNA genes which are sites for recombination into the DNA (Shnakar et al., 2002).

Pertaining to the sequenced E.faecalis V583, Paulsen and his colleague's investigations have highlighted a PAI on the genome of E.faecalis. The region in question consists of a gene, 150kbp in length which displays a lower G+C content the rest of the genome and encodes genes that aid the bacteria in host infection, including genes for a toxin that punctures cell walls and genes foe molecules that aid E.faecalis adhering to surfaces (Shnakar et al., 2002).

1.5 Pathogenicity Genes (Antibiotic resistant determinants)

The sequencing of the genome of E.faecalis has heavily skewed scientific thinking toward several confirmed and putative virulence factors that play a role in pathogenesis in particular the" esp" gene that has demonstrated to be part of a large confirmed PAI harbouring multiple virulence factors in E.faecalis and E.faecium (Shnakar et al., 2002). Nathan Shankar and his colleagues screened for the presence of the "esp" by PCR in E.faecalis isolates. This work purposes that the "esp" gene may serve as a marker for the presence of the PAI of interest and ultimately as a means of identifying virulent lineages of Enterococci (Shnakar et al., 2002)

1.6 Biofilm

Biofilm is a population of cells attached irreversibly on various biotic and abiotic surfaces, and encased in a hydrated matrix of exopolymeric substances, proteins, polysaccharides and nucleic acids (Costerton, 2001). Biofilm formation is a complex developmental process involving attachment and immobilisation on a surface, cell-to-cell interaction, and micro-colony formation, formation of a confluent biofilm and the development of a three-dimensional biofilm structure. Their formation occurs in response to a variety of cues, including high cell density, nutrient deprivation and physical environmental stresses (O'Toole et al., 2000). Bacteria in a biofilm display differing behavioural patterns in comparison to their planktonic bacteria (Mohamed and Hung, 2007).

Biofilms are notoriously difficult to eradicate and are a source of many chronic infections. According to the National Institutes of Health, biofilms are medically important, accounting for over 80 % of microbial infections in the body (Lewis, 2001). Bacteria in biofilms colonize a wide variety of medical devices, such as catheters, artificial cardiac pacemakers, prosthetic heart valves and orthopaedic appliances, and are associated with several human diseases, such as native valve endocarditis, burn wound infections, chronic otitis media with effusion and cystic fibrosis (Costerton et al., 1999). Enterococci in biofilm form are more highly resistant to antibiotics than planktonically growing enterococci. A mature biofilm can tolerate antibiotics at concentrations of 10-1000 times more than are required to kill planktonic bacteria thus the potential impact of biofilm formation is highly significant.

Within biofilm the regulation of bacterial gene expression in response to cell population density is accomplished through the production of extracellular signal molecules called autoinducers in a process called quorum sensing (Miller and Bassler, 2001).

1.6.1Quorum Sensing

Biofilm production is known to be regulated by quorum sensing systems in several bacterial pathogens. In the board understanding of the term Quorum sensing correlates to a decision making process utilized by decentralized groups to orchestrate behavioural patterns. Quorum sensing represents a significant aspect in co-ordinating gene expression dependent on a myriad of factors but most notably local density of their population. Bacteria that utilize quorum sensing most frequently create and thus secrete signalling molecules (autoinducers or pheromones). The bacteria in question consist of a receptor that detects the signalling molecule (inducer). Upon the inducer binding to the receptor, activation of transcription of certain genes occurs including those specifically for inducer (Camilli and Bassler 2006; http://en.citizendium.org)

1.6.2 Biofilm Stage Development

FIG 1.0:

Gram Staining

The gram staining technique is named after its inventor, a Danish bacteriologist, Hans Christian Gram who devised the gram stain protocol in 1882 and had his findings published in 1884. The main stain utilized in the procedure is Crystal Violet (CV). The test is still utilised to this day as the initial test performed for the purpose of identifying bacteria.

The test mechanics are centred on the fact that gram positive micro-organisms have a higher peptidoglycan and somewhat lower lipid content than gram positive bacteria thus bacteria that retain the CV/iodine complex will display a purple brown appearance after microscopy examination and can be referred to as gram positive or gram non-negative.

Catalyse test

FIG 2.0:

Crystal Violet (CV) assay

FIG 3.0:

IUPAC name

4-[(4-dimethylaminophenyl)-phenyl-methyl]-N,N-dimethyl-aniline

Invitrogen© Alamar Blue assay

The AB assay fundamentally works as a cell health indicator. The mechanism by which the AB assay operates is dictated by live cells maintain a reducing environment inside the cytosol of the cell. Thus resazurin (the active ingredient of the AB assay), is a non-toxic, cell permeable compound that can be observed as a blue colour which is virtually non-fluorescent. Upon entry of the cells inherent to the reaction, resazurin is reduced to resorufin, a compound that displays a definitive red colour and is highly fluorescent (FIG 4.0), thus viable cells will continuously convert resazurin to resorufin subsequently increasing the colour of the media surrounding the cells and the overall fluorescence (Invitrogen© Alamar Blue S.O.P).

Fig 4.0:Resazurin reduction to resorufin (Invitrogen© Alamar Blue S.O.P).

Biotium© XTT assay

Cell proliferation and viability assays are routinely utilized within the laboratory. The lineage of the modern XTT assay can be directly traced back to the non-radioactive, colorimetric assay system utilizing XTT that has been denoted to (Scudiero, P.A et al., 1988) who first published a description of the procedure protocol. This procedure has been subsequently manipulated to increase applicability and repeatability with the assay now utilised for spectrophotometric quantification of cell growth and viability . The assay mechanics are fundamentally centred on the cleavage of the yellow tetrazolium salt XTT to form an orange formazon dye by metabolic active cells (Gerlier and Thomasset 1986) as displayed in FIG 5.0.

The XTT assay has a broad spectrum with regard applicability, with regards to bacteria the XTT is reduced by the enzymatic action of the respiratory chain centred in the cytoplasmic membrane which contrasts to the XTT mode of action in yeast where tetrazolium salts are reduced by mitochondrial dehydrogenases (McCluskey et al., 2005; Berridge et al., 1996)

1.12 Optical Density(OD) absorbance

1.13 Spectrophotometer absorbance

2.0. Scientific Paper

A comparison of multiple assay techniques for the quantification of Enterococci in biofilm grown in 96-well microtiter plates.

Cliff Gilligan.

Centre for Molecular Bioscience, University of Ulster, Cromore Road, Coleraine, County Londonderry BT52 1SA, Northern Ireland.

Abstract

In this investigation, three surrogate assays pertaining to the quantification of biofilms formed by Enterococci in 96-well microtiter plates were evaluated after time optimization was achieved for the various assay techniques: the Crystal Violet (CV) assay, the XTT assay and the Alamar Blue (AB) Assay. From the genus Enterococcus the species Enterococcus faecalis (E.faecalis) was utilized as the primary protagonist organism inherent to this investigation. A synopsis of this investigation would reveal in general, the three assays displayed a broad applicability reinforced with a high repeatability for the majority of isolates. Subsequently the estimated quantification of CFU's present in the various biofilms highlights definitive although limited variations between the myriad of assays utilized. Consequently the data inherent to this investigation clearly indicates that some assays are less suitable with regards quantifaction of Enterococci in biofilm form.

Keywords: Biofilm quantification; Crystal Violet; Alamar Blue; XTT; 96-well microtiter plate

Introduction

Biofilm may be considered as a population of cells attached irreversibly on various biotic and abiotic surface planes, and encased in a hydrated matrix of exopolymeric substances, proteins, polysaccharides and nucleic acids (Costerton, 2001). Hence one may state, biofilm creation is a multifaceted developmental procedure including an attachment phase and a separate immobilisation phase on a surface, cell-to-cell interaction, and micro-colony formation, thus in turn establishment of a confluent biofilm occurs and finally the creation of a three-dimensional biofilm arrangement.

Biofilm formation occurs as a responsive action to a myriad of cues, including high cell density, nutrient deprivation and physical environmental stresses orchestrated by quorum sensing (Ghannour and O'Toole, 2004 and O'Toole et al., 2000).

Biofilms are notoriously hard to eliminate and are a source of numerous debilitating infections. Bacteria in biofilms inhabit a wide yet unique variety of medical devices, such as likes of catheters, artificial cardiac pacemakers, prosthetic heart valves and orthopedic appliances, and are associated with several nosocomial infections (Costerton et al., 1999). Bacteria in a biofilm will display variance with regard behavioural models that contrast to their planktonic state and this is true for Enterococci (Mohamed and Hung, 2007).

Enterococci that have achieved a biofilm structure are additional highly resistant to a vast array of antibiotics than planktonically growing enterococci. A mature biofilm can abide antibiotics at concentrations of 10-1000 times more than are required to kill planktonic bacteria thus the possible impact of biofilm formation is highly significant with this statement particular true with regards enterococci.

The genus Enterococcus is comprised of motile bacteria that are ubiquitous in nature and are natural inhabitants of the oral cavity, intestinal microflora and female genitalia of both humans and animal (Mohamed and Hung, 2007). The genus Enterococcus consist of lactic acid bacteria of the phylum Firmicutes. Pertaining to the class of Bacilli, Enterococci are determined to be gram-positive cocci, that frequently exist as short chains or in pairs (diplocooci) (Gilmore MS et al., 2002). Their fermentative ability is enhanced by the lack of a Kerb's Cycle Respiratory chain (Shnakar et al., 2002).

The two most prevalent species responsible for human enterococcal infections in the intestines are Enterococcus faecalis (E.faecalis) and Enterococcus faecium (E.faecium).

E.faecalis is a non-motile, facultative anaerobic micro-organism displaying both pathogenic and commensal properties (non-harmful co-existing state) and is believed to be responsible for 80-90% of human enterococcal infections (Jones et al., 1994). Exhibiting commensal properties like related species inherent to the genus Enterococcus, E.faecalis can induce life treating infection in immune-compromised humans, where it is normally ascertained from an exogenous source especially prevalent within the noscomial setting where pathogenicity will be enhanced in E.faecalis by its inherently natural high levels of antibiotic resistance. Within an enhanced pathogenic state antibiotic-resistant E.faecalis can be regarded as VRE (Vancomycin-resistant enterococcus).

E.faecium is believed to account for 10-20% of human enterococcal infections (Jones et al., 1994). Positioned in the genus Enterococcus, it is a gram positive bacterium, group D alpha hemolitic or nonhemollitic, present in both a pathogenic and commensal state (Ryan and Ray., 2004).

The last two decades has witnessed the introduction of a vast array of model systems for the in vitro analysis of biofilm formation and development (McLean et al., 2004). Conventional plating is the means by which quantification of biofilm biomass is achieved with regards the vast majority of these systems, with conventional plating being perceived by current scientific thinking as quite labour intensive and slow (Costerton, 2001).

Thus numerous surrogate assay techniques for biofilm quantification in 96-well plates have become more common place within the laboratory setting. These techniques can be divided into three means of analysis: firstly the biofilm biomass assay (centred around quantification of matrix and both live and dead cells), secondly viability assays (centred around quantification of what are perceived as live (viable) cells and finally matrix quantification assays (which work through precise dye staining methods for the cell matrix components).

The first denoted description of a Crystal Violet (CV) protocol has been credited to Christensen et al., 1985 and since this publication the protocol has been manipulated to deliver increased accuracy which equates to accurate biofilm biomass quantification in the entire well network (Stepanovic et al., 2000). CV is somewhat flawed in evaluating numbers of live cells to dead cells as both including the matrix are stained in the CV protocol.

In order to accurately distinguish between live and dead cells, quantification assay techniques have been developed that address the metabolic activity of viable cells. A myriad of stains utilize tetrazolium salts, including 5-cyano-2, 3-ditolyltetrazolium chloride (ctc) and 2,3-bis (2-methoxyl-4-nitro-5-sulfopheryl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) (Gabrielson et al., 2002; McCloskey et al., 2005).

The Invitrogen© Alamar Blue (AB) Assay is fundamentally centred on the reduction of resazurin by metabolically active cells. This blue compound produced during the procedure is actively reduced to pink resorufin, which is fluorescent (O'Brien et al., 2000). Although primarily utilized in viability assays for mammalian cells, the AB assay has been extensively utilized in antibiotic resistance testing of various bacteria to a myriad of antibiotics including: β- lactams, Aminoglycosides and Glycopeptides (Palomino et al., 2002)

The Biotium© XTT Assay is centred on the reduction of the XTT dye (a tetrazolium derivative) to formazon which is scientifically proven to be water soluble (Roehm et al., 1991). The amount of water soluble product generated from XTT is proportional to the number of living cells in the sample.

This investigation examines three surrogate methods utilized for the quantification of enterococci, a biofilm biomass assay and two separate viability assays. After optimisation was established for all three assays in question, the applicability of all three assays for the quantification of enterococci in biofilm form grown in 96-well plates was evaluated.

2.2. Materials and Methods

2.2.1 Strains and methods

The E.faecalis strains utilized in this investigation had previously been obtained from a defined catchment area in County Monaghan; Ireland including seven farms and three water sampling sites (The Monaghan Kiosk, Doogary and Knockronagham) and had previously underwent genus and species identification and antibiotic resistance analysis as outlined in the protocol by (Daniels V, 2009) as part of her PhD study. Working from information inherent to this PhD study six E.faecalis isolates which displayed antibiotic resistance: MF06618; MW1058; MW02050; ST1065; MW02072 and MW02047 were chosen along with three randomly selected isolates MW02022; MW03036 and MW02102.

All strains were transferred to Tryptone Soy Broth (TSA; QIAGEN United Kingdom) plates with 20% w/v Glycerol (QIAGEN United Kingdom) and cultured aerobically at 37.5°c for 24 to 48 hours. The TSA was supplemented with 8µg ml-1 vanconmycin (QIAGEN United Kingdom) which was below the minimum inhibitory concentration (MIC) required for vanconmycin to inhibit biofilm development.

2.3. Isolate identification

2.3.1 Gram Staining

Adhering to standard operational procedure (SOP) established within the laboratory, an enterococci specimen sample was collected on a blunt cooled inoculation loop. A medium to large droplet of sterile water was transferred onto a cover plate. The specimen was then mixed into the water droplet until the sample turned cloudy. The sample was then allowed to air dry or the Bunsen drying method was applied.

When dry, CV was added until the microscope slide was covered and then it was allowed to rest for 30-55seconds before it was then washed under a running tap. The excess water was washed off with iodine and then left for 30-45 seconds. The microscope slide was then in turn washed again under gentle running water before undergoing an alcohol wash for 10seconds before a subsequent wash with tap water occurred. Safranin (QIAGEN United Kingdom) was utilized as a counter stain for 2-3minutes before the microscope slide underwent a final wash, drain and blot dry before microscopy examination determined isolate identification. Each of the nine enterococci isolates underwent the gram staining procedure.

2.3.2 Catalyse Test

The catalyse test was performed to determine if the catalyse enzyme was present in the various enterococci isolates. A drop of 3% hydrogen peroxide was placed onto a clean microscope slide. An isolated colony was extracted with an inoculating loop. The loop carrying some of the isolate was placed into the drop of hydrogen peroxide. The microscope slide was observed for evolution of bubbles which would have represented a positive reaction. The reaction however was negative as expected under procedure guidelines. A catalyse test was performed on each enterococci isolate under investigation in turn with a negative reaction being determined for all nine enterococci isolates.

2.4. Biofilm formation

An inoculation dip for each of the nine cultured isolates occurred in universal tubes containing 3-5ml of nutrient broth culture (QIAGEN United Kingdom) and then each tube was grown to the stationary phase via aerobic incubation at 37.5°c for 24hours, after which time biofilm growth was assessed and the results recorded. 100µl of each culture medium was transferred via pipette to a fresh universal tube containing 9.9mls of nutrient broth culture creating a dilution culture of 1:100. Subsequently six extractions of 100 µl was made from each of the nine 1:100 dilution cultures and then added to each well of the 96-well plate in a pre-determined manner. The 96-well plate was incubated at 37.5°c for 24 to 48hours.

2.5 Measurement of spectrophotometer absorbance and optical density (OD)

Spectrophotometer absorbance signals for the AB and XTT assays were measured through utilization of a FLUOstar omega multi-label microtiter plate reader (BMG LABTECH Offenburg, Germany) and the OD for the CV assay was recorded using a veraMAX absorbance reader (VeraMax, Molecular Devices, USA) operational with a broad spectrum band pass range.

2.6 Biofilm Quantification

2.6.1 Crystal Violet (CV) Assay

The initial step entailed establishing four small trays (labeled A to D) with the last three each containing 1 to2 inches of deionised water. Planktonic bacteria was removed from the microtiter plate by vigorously shaking the plate out over the waste tray(labeled A). Then the plate underwent a washing procedure as it was submerged in the first water tray (labeled B) before it was rapidly shook out over the waste tray (labeled A).

An addition of 125µl of 0.1% crystal violet solution was made to each well and the staining process was allowed to occur for a 10 minute period at room temperature.

The CV solution was removed in two steps: firstly by shaking out the microtiter plate over the waste tray (labeled A) and secondly by removing excess CV solution by washing the plate successively in each of the next two water trays (labeled C and D) and once again using the waste tray (labeled A) as a collection point for as much liquid as possible after each wash.

This rigorous washing and shaking was designed as a means of removing all CV solution that was utilized in staining adherent bacteria attached to the 96-well plate surface.

The microtiter plate was then inverted and tap dried on paper towels removing any remaining excess. The plate was then allowed to air dry and was then suitable for storage at room temperature for at least several weeks.

An addition of 200µl of 95% ethanol (QIAGEN United Kingdom) was made to each stained well. The dye was allowed to solubilise after covered incubation for 10 to 15 minutes at room temperature. Subsequently the contents of each well was mixed by pipetting and then 125 µl of the CV/ethanol solution was transferred from each well to afresh well in a optically clear flat bottom 96-well plate. The OD absorbance was measured at a wavelength of 500 to 600nm after 2, 5, 19, 24 and 48hours. The assay was performed in triplicate.

2.6.2 Invitrogen© Alamar Blue Assay

The Invitrogen© Alamar Blue (AB) Assay is a widely available commercial resazurin solution kit. Stock solutions were refrigerated for storage and removed from storage one hour prior to use. 100 µl of AB reagent was added directly to the biofilm growth in the nutrient broth culture medium present in the wells of the 96-well plate. The quantity of AB added represented 10% of the sample volume. An incubation period of 4hours was adhered to at 37.5°c with spectrophotometer absorbance readings established at 570 nm after 1, 2, 3 and 4hour intervals. This assay was again carried out in triplicate.

2.6.3 XTT Assay

The XTT assay was kept in refrigerated storage at -70°c prior to use thus one bottle of XTT solution and one vial of activation reagent were thawed. Pertaining to one 96-well plate, 25 µl of activation reagent was mixed with 5ml of XTT solution to derive activated XTT solution. 50 µl of activated XTT solution was

added to each well and then incubated for 24hours with spectrophotometer absorbance readings taken at 550nm after 2, 5, 19 and 24 hour time intervals with time optimization expected to occur after 5hours.

2.7 Statistical Analysis

PASW statistical analysis was carried out via SPSS 17. Software (SPSS, Chicago, Il, USA)

2.8 Results and Discussion

The results inherent to this investigation pertain essentially to three aspects of use with regards the three surrogate assays utilized in assessing the quantification of biofilms. Firstly conditions to establish the optimal cell proliferation time for each surrogate assay was defined. Secondly these optimal procedural conditions were adhered to during biofilm growth of the nine enterococci isolates thus establishing their applicability. Finally the quantification of cells in the biofilms was established at optimal cell proliferation time conditions by obtaining their various spectrophotometer absorbance and OD absorbance readings thus allowing for a determination to be made as to which assay is most suitable for the quantification of enterococci cells in biofilms. The means by which biofilm strength was ascertained entailed plotting the biofilm growth against the incubation time.

A standard means of biofilm growth from statistical analysis indicates:

[Using the criteria of Joannis-Cassan et al., 2007]

0 to 0.5 represents a "weak" biofilm

0.5 to 1.0 represents a "moderate" biofilm

1.0 to 3.5 represents varying degrees of "strong" biofilm

2.8.1 Quantification of biofilm biomass using the CV assay

In order to determine the time optimization required for a CV assay, it was thus necessary to carry out a complete CV assay which allowed for the average OD absorbance readings for three assays (as the experiment was carried out in triplicate) to be ascertained after 2, 5, 19 and 24 hours. The average OD absorbance readings (Which represent biofilm growth) were plotted against the five time interval periods as displayed in Fig 7.0.

FIG 7.0 Biofilm Growth after 2,5,19, 24 and 48 hours: Vertical index (Biofilm Growth/strength) Horizontal Index (Various Enterococci isolates)

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Thus a determination was made (as displayed in Fig 8.0.) as to which time interval represented optimization with regards time. The time interval of 24hours clearly represented the optimal time for cell growth

Fig 8.0 Biofilm growth after 24hours: (Time optimization for the CV assay) Vertical index (Biofilm Growth/strength) Horizontal Index (Various Enterococci isolates)

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A general examination of the applicability and repeatability of this assay indicates a high applicability and repeatability ratio as only minor differences were recorded between the three assays (based on a 63-well reaction used during each assay). The assay was also determined to be quite rigorous as a standard error of mean (SEM) was determined to be less than 5%.

2.8.2 Quantification of biofilms using the Invitrogen© Alamar Blue assay

In the resazurin based AB assay an incubation period of 4hours was expected to optimal for all nine isolates with regard to biofilm growth (cell proliferation/cell viability). In order to establish time optimisation an AB assay was performed with spectrophotometer absorbance readings ascertained at 570nm after 1, 2, 3 and 4hour time periods. The average absorbance readings were obtained as the assay was carried out in triplicate.

Thus the average spectrophotometer absorbance readings (which represent

Biofilm growth/strength) were established after the four time intervals as displayed in Fig 9.0.

Fig 9.0 Biofilm Growth after 1, 2, 3 and 4hours: Vertical index (Biofilm Growth/strength) Horizontal Index (Various Enterococci isolates)

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The time period of 4hours clearly represents the optimal time for cell proliferation/cell viability with the biofilm biomass as represented in Fig10.0

Fig 10.0 Biofilm growth after 4hours: (Time optimization for the AB assay) Vertical index (Biofilm Growth/strength) Horizontal Index (Various Enterococci isolates)

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A general investigation of the applicability and repeatability of this assay indicates it is high in applicability and repeatability as a low standard of deviation was observed the three assays performed (based on a 63-well reaction used during each assay). Thus this assay can be deemed quite rigorous as a SEM of less than 5% was observed.

2.8.3 Quantification of biofilms using the Biotium© XTT assay

To establish time optimization for the quantification of biofilms using the XTT assay, a XTT assay was initiated with spectrophotometer absorbance readings taken at 550nm after 2, 5, 19 and 24hours. An incubation time of 5hours was expected to be optimal for all isolates with regard to biofilm growth (cell proliferation/cell viability).

The average absorbance readings were obtained as the assay was again carried out in triplicate. Fig 11.0 represents the average spectrophotometer absorbance readings (which indicate cell growth/strength) within the four time intervals relevant to the assay.

Fig 11.0 Biofilm Growth after 2,5,19, 24 hours: Vertical index (Biofilm Growth/strength) Horizontal Index (Various Enterococci isolates)

The time interval of 5hours represents the optimal time for cellular biofilm growth (cell proliferation/cell viability) for the majority of isolates with the exception of the isolates MF06018 AND MW02022. Thus Fig 12.0 cellular biofilm growth during optimized conditions for the XXT assay

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Fig 12.0 Biofilm growth after 5hours: (Time optimization for the XTT assay) Vertical index (Biofilm Growth/strength) Horizontal Index (Various Enterococci isolates)

A general examination of the applicability and repeatability of the XTT assay indicates it is high with regards applicability and repeatability as a low standard of deviation was recorded between the three various assays performed (based on a 63-well reaction used during each assay). Also this assay can be regarded as quite rigorous as a SEM of less than 5% was recorded.

2.9 Conclusion

This investigation was centred on an examination of three surrogate assays that may represent an alternative to the traditional plate count method for high throughput quantification of bacteria in this case enterococci isolates present in microbial biofilms grown in 96-well microtiter plates.

The three assays in question; the CV assay, the Invitrogen© AB assay and the Biotium© XTT assay were optimized with regard to time and contrasted and compared.

Time optimisation for the CV assay was determined to be 24hours, 4hours for the AB assay and 5hours for the XTT assay. All these assays yielded definitively different numbers of biofilm cells during what was established as time optimisation as displayed in figures 8.0, 10.0 and 12.0.

The CV assay displayed the greatest cellular biomass growth during optimal conditions. This was expected as because the CV assay acts upon the dispersion phase of biofilm development and the assay dye interacts via binding with negatively charged surface molecules (which may be alive or dead) and polysaccharides in the extracellular matrix. Thus the CV assay is less sensitive in theory than its counterparts under investigation. Therefore while this assay is highly applicable to a vast spectrum of micro-organisms, is easily, relatively cheap in comparison to the others, is deemed straightforward and can be deemed to a certain extent the industry standard of the three assays examined. The major flaw inherent to this assay is in its inability to differentiate between living and dead cells. Therefore one can state with the utmost confidence that this should not be utilized for drug susceptibility testing of biofilms.

With regards the viability assays, the AB assay proved highly consistent with optimal biofilm growth observed after 4hours in all isolates in the 1.4 to 2.1 strength range. However despite the XTT assay being the most expensive of the three assays to operate and being quite time intensive, it displayed no added value for the quantification of bacterial cells. In fact, questions have to be raised with regards to the accuracy of the XTT assay as it displayed vastly differing readings for the nine isolates at the optimised time interval. Indeed two of the strains MW02072 and MW02047 displayed readings similar to those observed in the CV assay, which in theory should have occurred as the CV assay did not differentiate between living and dead cells unlike the XTT assay which was based on cell viability.

A comparison then must be drawn between the two viability assays: the AB assay and the XTT assay. In theory the XTT assay is the less efficient of the two assays as the minimal number of bacterial cells required ascertaining a detectable signal is higher than in the AB assay.

Thus this study proposes that the AB assay, which displayed excellent applicability and repeatability for the quantification of Enterococci in biofilm, is the most suitable of the three assay techniques examined. I t should also be noted that the AB assay was extremely consistent in its active readings and can relay absorbance readings which distinguish between live and dead cells.