Antibiotic resistance has emerged as an increasing problem, thus there is growing interest in ecological investigations of studies detailing the flow of bacteria which display antibiotic resistance through the environment and food chain. The potential role the environment plays in the dissemination of antibiotic resistance has become a major concern.
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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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.
E.faecalis 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). Within an enhanced pathogenic state antibiotic-resistant E.faecalis can be regarded as VRE (Vancomycin-resistant enterococcus).
Enterococci have been identified as an indicator organism in identification of sources of contamination. Although there is much concern surrounding the possibility of faecal bacteria and pathogens in water, the central concern now is of the presence of antibiotic resistance bacterium in water sources, which has taken precedence over the former. The selection of Enterococci as an indicator organism in such an examination is due to their survival ability which allows intrinsically adaptation to antimicrobial treatment within a board range of conditions. Enterococci have been noted to survive in conditions that include a temperature range of 10ï‚°C to 45ï‚°C or even at 60 ï‚°C for 30minutes, a pH of up to 9.6 and salinity of up to 6.5% NaCl (Daniels V, 2008; Shepard and Gilmore, 2002; Witte et al., 1999).
1.2 Pathogenicity Genes (Virulence determinants)
Virulence and 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 virulence thus there is the need to understand with greater authority the pathogenicity of Enterococci in both planktonic and biofilm form
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).
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. 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).
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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 (Koonin et al., 2001).
There is general consensus in the scientific community that enterococci have displayed an intrinsic low level resistance to the bacterial effects of Î²-lactam antibiotics. Enterococci are approximately 100 fold less susceptible to Î²-lactam than streptococci however enterococci displaying resistance patterns have developed unique mechanisms of resistance. Such Î²-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 Î²-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 Î²-lactamase, allowing genetic evidence to imply that Î²-lactamase production is ascertained as a result of the acquisition of the Staphylococcus aureus Î²-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).
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. 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 (Shepard and Gilmore 2002).
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).
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.
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 for molecules that aid E.faecalis adhering to surfaces (Shnakar et al., 2002).
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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)
Biofilm may be considered as 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 creation is a multifaceted developmental procedure including an attachment phase and an separate immobilisation phase on a surface, cell-to-cell interaction, and micro-colony formation, establishment of a confluent biofilm and the creation of a three-dimensional biofilm arrangement. Their formation occurs in reply to a myraid of cues, including high cell density, nutrient deprivation and physical environmental stresses (O'Toole et al., 2000). Bacteria in a biofilm will display variance with regard behavioural models in contrast to their planktonic state (Mohamed and Hung, 2007).
Biofilms are notoriously hard to eliminate and are a source of a myriad of chronic infections. According to the National Institutes of Health, biofilms are medically significant, accounting for somewhere over 80 % of all microbial infections in the body (Lewis, 2001). Bacteria in biofilms inhabit a wide yet unique variety of medical devices, such as catheters, artificial cardiac pacemakers, prosthetic heart valves and orthopaedic appliances, and are linked to a large extent with a myriad of 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 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.
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).
1.5 Biofilm Assays
The Crystal Violet assay is a relatively basic, high throughput method adhered to in ascertaining biofilm quantification in the entire microtiter well complement. After biofilm formation is achieved by inoculating cells directly into sterile microtiter plates filled with 100Âµl of appropriate medium per well, the assay entails separating planktonic bacteria present in the wells from their counterparts in biofilm form.
This will be achieved by a vigorous shaking removing all non-adherent bacteria. The use of the Crystal Violet stain allows for absorbance detection to be established by measuring the absorbance value of each stained 96 well on a micro-plate reader after 16, 40 and 64 hours at 620nm (Merritt et al., 2005).
Cell health can be determined by multiple methods and essentially the InvitrogenÂ© Alamar Blue Assay is a cell viability reagent. Plasma membrane integrity, DNA sysntheis, DNA content, enzyme activity, presence of ATP and cellular reducing conditions are known indicators of cell viability and cell death. Alamar blue cell viability reagent acts as a cell health monitor and thus indicator by utilizing the power of living cells to quantitatively measure then proliferation of various human and animal cell lines, bacteria, plant and fungi allowing the operation to ascertain relative cytotoxicity od agents within various chemical classes (InvitrogenÂ© Alamar Blue cell viability Reagent S.O.P)
The BiotiumÂ© XTT cell viability assay kit presents a simple method for resolving the number of cell life, utilizing standard microplate absorbance readers. Determination of live cell numbers is frequently adhered to as a means of assessing the rate of cell proliferation and to screen cytotoxic agents. XTT is a tetrazolium derivative, it is based on the activity of mitochondria enzymes in live cells that reduce XTT and are inactivated shortly after cell death (BiotiumÂ© XTT Cell Viability assay Kit S.O.P).
Hypothesis and purpose of proposed investigation
The creation of a biofilm with selected Enterococci isolates will allow in theory and thus in practice the transfer of antibiotic resistance genes providing a unique insight into drug susceptibility of bacterial biofilms. Hence such an undertaking can be merited as scientifically relevant to this discipline of study, if one examines the estimated total world wide anti-microbial usage which is deemed to be 100,000 to 200,000 tones per year. This unquestionably applies strong selective pressure on bacteria to become resistant to a myriad of conventionally used anti-microbials (Daniels V, 2008; da Costa et al., 2007).
The hypothesize inherent to this project, is that biofilm development could enhance antibiotic resistance in Enterococci. 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. A myriad of techniques such as Crystal Violet assays, a novel Alamar Blue microplate assay with microplate reading and XTT assays including Antibiotic Resistance Analysis (ARA) via the disc diffusion method will be utilized to examine antibiotic drug susceptibility in co-ordination with biofilm viability and biofilm tolerance of antibiotics and thus a determination can be made as to which assay provides the best means of assessing Enterococci biofilm antibiotic resistance.