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Enterococcus Faecalis and Enterococcus faecium are the most common species of bacteria cultured from humans. They exist normally in the intestinal flora of both humans and animals and although often they are non-threatening, they have the potential to cause serious nosocomial infections. One of the most common causes of infection is by indwelling medical devices where Enterococci acts as part of a mixed infection usually.Enterococci have developed resistance against antibiotics and bacterocides making them highly pathogenic to immuno comprised individuals.This is usually due to the development of a biofilm.In this study we will look at the effects of biofilm formation on Enterococci and methods which can be used to prevent biofilms.We will be particularly focusing on the use of bioactive glass as a coating on biomedical devices.
1.1 General introduction:
Enterococci are one of the leading causes of nosocomial infections and they play a vital role in the initial beginning and persistence of antimicrobial resistance (Cetyankaya et al., 2000, Hancock et al., 2000, Kuhn et al., 2000 and Witte et al., 1999).They occur naturally in the oral cavity, normal intestinal microflora and the female genital tract of both humans and animals. Their main areas of infection are the urinary tract, bloodstream, intra-abdominal and pelvic regions, as well as surgical sites and the central nervous system (Mohamed & Huang, 2007).
What especially aids the resistance of the enterococci species is their ability to form biofilms,which is a major factor in their ability to evade the immune system. They are particularly dangerous in the case of biomedical devices such as catheters, artificial cardiac pacemakers, prosthetic heart valves and bone grafts (Mohamed & Huang, 2007). In this study, particular attention will be focused on bioactive glass used in biomedical devices and its effects on biofilm formation.
1.2 Taxonomy and pathogenesis:
Enterococci was initially thought to be part of the Streptococci group however, they were separated from this group in 1984 by studies on DNA-DNA and DNA-rRNA hybridization reactions (Schelifer & Kilpper-Balz, 1984). The separation of these groups was further confirmed by 16S rRNA analysis (Ludwig et al., 1985), which demonstrated that other gram positive cocci such as Lactococci differed from Enterococci. Enterococci belong to the firmicute group, with low G+C content. By phylogeny, the closet relation to Enterococci is the genus Vagococcus and following this are the genera Carnobactrium, Acrococcus, Tetragenococcus, Aerococcus and Abiotrophia. The linkages which were associated with the Enterococci in the past such as the Streptococci and the Lactococci are more distantly related then these groups (Devriese et al, 2006). Check this - sentence is unclear
1.3 Relation to phenotypic characteristics:
Enterococci show a large number of phenotypic characteristics which are shared among those in the group. This is highly useful information as regards general identification of a bacterium. However there are also exceptions to this e.g. E. cecorum is carboxyphilic and is not resistant to drying unlike most Enterococci.
The E. gallinarum group contain a van C gene which allows for partial resistance to Vancomycin and other glycopeptides antibiotics. Recent studies have shown that some strains of E .faecum have also now become resistant to vancomycin, with an average of 50% showing resistance (Se et al, 2009).
One of the main habitats of Enterococci is the gut of mammals and birds, including humans and domestic animals. If they are found outside the gut it is generally taken as an indication of faecal pollution.
Host Species variation:
The most common Enterococci in humans and animals are E. faecalis and E. faecium, with E.faecalis being more common. E. cecorum is common in the bacterial flora of poultry and pigs (Devriese et al., 2006), while E. hirae is commonly observed in the porcine gut as well as in poultry, cattle, dogs and cats. There are some groups which still have not been well characterized i.e. E. avium species.
Other body sites:
Other habitats of Enterococci include the vagina and throat of humans; however this is not common, with only 20% of individuals being positive. In a hospital setting, most isolates are E. faecalis and E. faecium, both conferring resistance recently to vancomycin by Van A and Van B phenotypes (Sood et al., 2008).
1.5 Age variation:
In animals, Enterococci make up the major flora of the intestine in the first few days of life. It is noticeable that E. faecalis in particular largely dominates over other species in babies less than 1 week old (Noble, 1999).
This decline is also noticeable in other species such as chickens, where E. faecalis and E. faecium begin to decrease in numbers over time (Devriese et al., 2006).
1.6 Pathogenesis of Enterococci:
Enterococci cause infection by colonizing a host's tissue and resisting the host's immune defences. Enterococci colonize hosts by attachment to intestinal and urinary tract epithelial cells by adhesions which exist on the bacterial surface. Growth conditions effect the expression of these bacterial adhesions. In addition, this attachment can be solidified by a host cell itself i.e. renal tubular cells in vitro have been found to produce an aggregation substance which aids E. faecalis in attaching to the cells. This material is protein based and aids in binding the donor and recipient bacteria to facilitate plasmid transfer.
In order to kill Enterococci in vitro, polymorphonuclear leucocytes (PMNLs) need to be in the presence of serum complement proteins and they are enhanced by anti-enterococcal antibodies.
It was discovered that Enterococci themselves produce a number of factors which are thought to be linked to pathological changes in the host post-infection. Certain elements produced by E. faecalis are chemotactic for PMNLs in vitro i.e. sex pheromones and plasmid-encoded pheromone inhibitors.
It is thought that this may, in fact, be an associative cause of inflammatory response within the host cell. Some E. faecalis also produce a hemolysin which is plasmid encoded, which in turn increases the severity of the infection. Enterococci are also capable of promoting platelet aggregation and tissue factor-dependent fibrin production, which is involved in enterococcal endocarditis (Johnson, 1994).
1.7 Infections caused by Enterococci in humans:
Enterococci cause 5-15% of bacterial endocarditis. Most isolates obtained are E. faecalis , also other enterococcal species (Garvey & Neu, 1978; Moellering et al, 1974) from human intestines were sent to the centre of disease control, among these were E. avium,E. casseliflavus,E. durans,E. faecalis and E. faecium (Facklam & Collins,1989). The average age for this disease appears to be 65 (Koenig & Kaye, 1961). However, it is also seen to occur at times in children and very rarely in infants (Macaulay, 1954). Males suffer more with this disease and tend to be older than their female counterparts with endocarditis. Genitoururinary and billary portals were associated risk factors with endocarditis. In the study carried out by Mandell, 50% of the men tested had genitourinary portals i.e. genitourinary instrumentation as well as 38% of the women, including abortion or instrumentation (Mandell et al., 1970). This disease was also common among drug addicts, it was estimated that 5-10% of cases were caused by Enterococci. Unlike Staphylococcal endocarditis in the addicts, aortic and mitral valves are usually involved with Enterococci (Reiner et al., 1976).
In recent studies carried out on vancomycin resistant endocarditis (VRE) it was found that the mitral valve was less involved than the aortic valve (Stevens & Edmond, 2005; Mcdonald et al., 2005).
Included risk factors for endocarditis are urinary tract infections and instrumentation.
Urinary tract infections
Noscomial infections are the most common infections caused by Enterococci, these are generally associated with catheterization or instrumentation. Cystitis and pyelonephritis can occur with occasional cases of prostatis and perinephric abscesses.
An investigation carried out on elderly men found that that the microbiology of bacteriuria in both in-patients and out-patients mainly identified as Enterococcus as the most common uropathogen and it was isolated in 22.5% of the patients.Of these cases, 45% were catheter related, showing the high correlation between the two (Cornia et al., 2006).
Studies have shown that urinary tract infections in children are generally associated with enterococcal urinary tract abnormalities and other similar complications (Bitsori et al., 2005). This suggests that diagnosis of UTIs caused by Enterococcus spp. show an abnormality of the uninary tract which has not yet been discovered.Â
There are a few common sources of enterococcal bacteremia, these include the urinary tract, intra-abdominal foci, intravascular catheters and wounds. In the case of catheters there are high incidences in femoral locations. Enterococcal bacteremia which is community-acquired is more associated with endocarditis (up to 36% of cases) than noscomial bacteremia (0.8%). Nosocomial bacteremia can come from many sources. These include polymicrobial bacteremias which include Enterococci and other flora associated with the bowel. Surgical sites and burn wound infections make up the other sources. Blood cultures are carried out to assess the presence of Enterococci in the blood (Chatterjee et al., 2007).
Intra-abdominal and pelvic infections
Intra-abdominal infections include biliary tract infection, intra-abdominal abscess, peritonitis,endometritis and salpingitis. Enterococci are usually part of a mixed group of bacteria which infect these areas. Anti microbial therapy is usually recommended to treat these forms of mixed infections. If blood culture results are positive for Enterococci, anti enterococcal bactericidal activity is advised. In immunocomprised patients such as AIDS victims or cancer sufferers, there is a higher risk of treatment failure and deaths (Chatterjee et al., 2007).
1.8 Virulence genes associated with Enterococci:
A study was carried out in Brazil on virulence determinants in a collection of E. faecalis strains which were isolated from different clinical settings. These included 50 from the urine of patients with UTIs, 25 from purulent sources which included abdominal secretion, secretion of renal fluid, bone fragments, splenic aspirate and peritoneal fluid. Others included 19 samples from rectal swabs where patients had E. faecalis but there was no enterococcal infection present.
From this study it was evident that virulence factors varied, depending on the clinical source. Most of the strains found were common inhabitants of humans except in the case of the cylB and cylM genes. It is evident from Table 1.0 that over 50% of the clinical strains showed the presence of efaA and eep gene markers. The esp gene was also notable in this study, however compared to other studies carried out (Creti et al., 2004; Eaton & Gasson., 2001; Franz et al., 2001) it appears that the esp gene was a fraction higher then reported in these papers.
Table 1.0:Frequency of genetic virulence markers amongÂ E. faecalisÂ strains isolated from different clinical sources
Virulence Â markers*
No. (%)Â E. faecalisÂ strains harbouring virulence genes distributed according to clinical source
(nÂ = 50)
Purulent exudates (nÂ = 26)
Rectal swab (nÂ = 19)
(nÂ = 95)
1 ( 3.8)
0 ( 0.0)
0 ( 0.0)
1 ( 3.8)
1 ( 5.3)
9 ( 9.5)
N*agg, Aggregation substance;Â cylA, activation of cytolysin (haemolysin/bacteriocin);Â cylB, transport of cytolysin;Â cylM, post-translation modification of cytolysin;Â eep,Â enhancedÂ expression of pheromone;Â efaA,Â E. faecalisÂ endocarditis antigen A;Â enlA, enterolysin A belonging to class III bacteriocins;Â esp,Â enterococcalÂ surfaceÂ protein;Â gelE, extracellular metalloendopeptidase produced byÂ E. faecalis.
(Bittencourt de Marques & Suzart,2004)
It was found that gelE and aggA genes were evident in 45/% and 37%, of samples, respectively whereas the other virulence factors were detected in far less quantities (<17%). The gene which encoded enterolysin A was also investigated. It was found in low concentrations below 9.5% among clinical strains. It was mainly found in urinary strains rather than the other groups of isolates.
It was found that the genes which make up the cytolysin operon (cylaA, cylB, cylM) ,which were present in all isolates studied were present in comparable proportions except in purulent fluids. The genes themselves are involved in activation, secretion and post-translational modification of cytolysin. This data differs from studies carried out by other groups which examined strains of E. faecalis isolated from endocarditis, bacteraemia and faeces patients (Eaton & Gasson, 2001; Franz et al., 2001; Archimbaud et al., 2002; Creti et al., 2004).
From table 1.0 it was postulated that, due to the high incidence of virulence genes in urinary samples, these genes contributed to the bacterial colonization and growth in developing a urinary tract infection (Bittencourt de Marques & Suzart, 2004). The role of many of these virulence genes is still unknown and further studies are currently being carried out to investigate them.
1.9 Biomedical devices commonly inhabited by Enterococci:
In order to put an implant in the body it must firstly meet two specific criteria: (1.) it must have specific mechanical properties to replace the functions of the defective body tissues or organs and (2.) it must be accepted by the host.
It is recognized that there are 4 major host responses to an implant, these are:
The material itself releases toxic compounds which kill surrounding tissue.
The material isn't toxic, however it is gradually degraded and replaced by the surrounding tissue which is undergoing repair.
The material is non-toxic, however it cannot be degraded by the host which reacts by encapsulation of the implant.
The material is non-toxic, but highly involved with the surrounding tissues, forming chemical bonds which stabilize the implant.
Bioactive glass, dense hydroxylapatite ceramics and bioactive composites are part of the final group (Vaudaux et al, 2000).
Central Venous catheters:
Through the use of scanning and transmission electron microscopy it has been proven that almost all indwelling venous catheters are colonized by bacteria embedded in biofilm matrices. The most commonly isolated bacteria include S. epidermidis, S. aureus, P. aeruginosa and E. faecalis (Maki, 1994).
The origin of such bacteria is generally from the patients own microflora, from health care personnel or contaminated I.V fluids. They can also gain access to the catheter by external migration from the skin or internally from the catheter port or hub. Sometimes colonization may occur as soon as 24 hours after the device as been implanted. It may be as a result of the host producing conditioning films which include platelets, plasma and tissue proteins (Maki & Mermel, 1998). A study carried out by Raad et al., (1993) found that the extent of biofilm formation and the location depended on how long the catheter was in place. It was found that short term biofilms, less than ten days, formed greater biofilms on the external surface, whereas long term biofilms, which were 30 days or more, formed biofilms on the inner lumen of the catheter. Another possible growth factor studied was the nature of the fluid given through central venous catheters. It was found that gram positive bacteria e.g. S. epidermidis and S. aureus did not grow sufficiently in intravenous fluids while gram negative bacteria such as P.aerguinosa, Klebsiellaspp and the Enterococci species did.
Mechanical heart valves:
Bacteria may attach to and develop biofilms on parts of mechanical heart valves and surrounding heart tissues which leads to a condition known as prosthetic valve endocarditis. Enterococci are one of the main micro organisms involved in this condition as well as S. aureus and S. epidermidis. These bacteria may come from the skin or other medical devices such as central venous catheters or dental work. By implanting the device, tissue damage occurs, which leads to the accumulation of circulating platelets and fibrin in this area. This also encourages microorganisms to accumulate here as there is a rich source of nutrition with the accumulated products. Biofilms generally develop on the surrounding tissues or the sewing cuff fabric used to attach the device to the tissue than on the actual valve (Ilingworth et al, 1998).
These devices may easily acquire biofilms on the inner or outer surfaces of the device. The most common organisms include E. faecalis, E. coli, P. aeruginosa and S. epidermidis. The longer the catheter is in place, the more likely an organism will develop a biofilm, resulting in a urinary tract infection. According to Brisset et al., (1996) the hydrophobicity of both the organisms and surfaces play a major role in adhesion to the catheter. Catheters with both hydrophilic and hydrophobic areas have the widest variety of organisms colonizing there. Other factors which increase bacterial attachment included calcium and magnesium ions, an increase in the pH of the urine and ionic strength.
In standard procedures today either an autograft or an allograft is used in 90% of cases. Autografts are ideal as they already possess all the necessary characteristics for new bone growth, mainly osetoconductivity (tissue supports the attachment of osteoblasts and osteoprogenitor cells which promote growth), osteogenicity (when the osetoblasts aid the production of minerals to calcify the collagen matrix that forms the substrate for the new bone) and osteoinductivity (the ability of the bone graft to encourage non differential stem cells or osteoprogenitor cells to transform into osteoblasts). In this case, tissue is taken from the patient, usually from the iliac crest and placed at the site of injury. Allografts are the alternative to autografts, where bone is taken from cadavers or donors. These are highly advantageous as there isn't a risk of short supply as there is if one is taking bone from the actual patient. However allografts have their own associated problems, although they are treated by tissue freezing, freeze drying, gamma radiation, ethylene oxide etc. there is still a risk of disease transmission infection, with incidents of HIV occurring as well as Hepatitis B and C (Boyce et al., 1999, Tomford, 1995 & Conrad et al,1995). In autografts the risk of infection tends to increase with the number of people working on the bone transplant during transplant harvesting as there is a higher MIC count. A study was carried out of 303 bone transplantations which revealed that there were 21 cases of infection with Enterococci accounting for 56% of the mixed infections (Pruss et al., 2003).
2.Introduction to biofilms:
2.1 Structure of biofilms:
A biofilm is made up of a population of cells which are attached irreversibly onto various surfaces which may be biotic or abiotic. The biofilm itself is encased in a matrix which is heavily hydrated and made up of exopolymeric substances, polysaccharides, proteins and nucleic acids (Costerton, 2001). Bacteria which are embedded in biofilms act differently to free living bacteria as they work in a unit communicating by quorum sensing. This is achieved through the production of extracellular signal molecules called autoinducers (Miller & Bassler, 2001). Bacteria in biofilms colonize a wide variety of medical devices as previously mentioned. Enterococci which exist in biofilms are highly resistant to many antibiotics in comparison to free living Enterococci, making their impact on patients far more destructive as they are difficult to treat.
2.2 Biofilm Formation:
Initially when the medical device is placed within the patient the organic molecules in tissue fluid form a conditioning layer on the device. Enterococci, along with other micro organisms immediately form an aggregate on the conditioning film. (Kuchna & O'Toole, 2000; Bos et al., 1999). With the continued adhesion of different microorganisms, a multilayered complex is formed. The colony of microorganisms attaches firmly to the device and is surrounded with a glue-like slime matrix which is self-produced. This is made up of exopolysaccaharide and some lipids, proteins and nucleic acids. Once the colony has attached, the process is irreversible. The biofilm itself has an irregular terrain with many individual colonies of non-uniform shape and finger-like columns which are surrounded by channels filled with fluid containing nutrients, enzymes and waste products (Marshall, 1994). Depending on the conditions in which a biofilm is formed, it's cellular morphology and matrix content differs (Kumamoto, 2002). The biofilm adhesion to the conditioning film depends solely on its strength of attachment. Studies have shown that human blood aids the attachment of gram negative and gram positive bacteria (Murga et al., 2001). Biofilms generally form on unhealthy living tissue and dying tissues such as burns, wounds and skin ulcers (Serralta et al., 2001). Bone fragments and exposed bone are susceptible tissues for biofilm formation (Gristina et al.,1990). Biomedical devices are particularly susceptible to biofilm formation as they are generally wet or moist surfaces which are favourable for micro organisms. Biofilms which form on bone cement use the surface of the bone graft to spread into adjacent tissues and form new colonies of microorganisms which are protected by biofilms (Gristina & Costerton, 1985). It is found that the older the biofilm is, the more dangerous it becomes providing more protection and resistance from antimicrobials and biocides (Bos et al., 1999;Van de Belt et al., 2000).
2.3 Factors which aid biofilm formation:
A major influence on biofilm formation is the availability of nutrients such as serum, iron, C02, glucose, osmolarity, pH and temperature. Biofilm production is controlled by carbohydrate metabolism among most gram negative bacteria including E. faecalis (Pillai et al., 2004). A study carried out using TSB medium with glucose (1%) and without glucose proved this theory, as the glucose appeared to enhance the biofilm production in comparison with the TSB without the glucose (Baldassarri et al., 2001). Another study found that increasing the concentration of glucose from 0.2% to 0.5%, increased the production of biofilm (Kristich et al., 2004). It is thought that a regulator which is glucose-dependent may be directly or indirectly involved in control of fsr and fsr mediates catabolite control of biofilm production by the downstream proteases; gelatinase,serine and protease (Pillai et al., 2004).
There has been a recent report on the involvement of surface proteins with Enterococci biofilm formation in a higher glucose concentration (Tendolkar et al., 2004). Two strains of E. faecalis were examined: esp positive FA2-2 (pESPF) and OGIRF (pESPF) which produces a thicker and fuller biofilm than controls: esp negative FA2-2 (pAT28) and OGIRF (pAT28). A glucose concentration of greater than 0.5% was used to achieve this result. An interesting discovery was made involving the transcriptional regulator, bopD (bopABCD operon), which showed it had the same sequence as a variety of proteins responsible for the regulation of maltose metabolism, which is vital for biofilm production (Hufnagel et al., 2004).
Studies on osmolarity using E. faecalis showed that an osmostality of 2-3% sodium chloride in a medium totally destroys the process of biofilm formation, however, not the bacteria. This lead to the theory that E. faecalis monitors the conditions before forming a biofilm. Studies were also carried out on different media using E. faecalis, it was found that after 6-8 hours biofilm aggregation slowed down when using TSB M17 and M9YE. Other media proved to be even more effective as the density of the biofilm decreased after 4 hours using Todd-Hewitt yeast extract and brain heart infusion (Kristich et al., 2004). These show that certain factors will influence the development of long term biofilms while others will lead to short term biofilm formation.
A study was also carried out looking at the effect of human serum on E. faecalis biofilm formation. It was found that when 10% human serum was added to the culture medium, this increased the adhesion properties of E. faecalis ATCC 29212 to silicone and glass surfaces, much like those used in medical devices (Gallardo-Moreno et al., 2002). Maccrophages were looked at in another study and the ability of biofilm producing E. faecalis isolates to survive within them compared to non-biofilm producing isolates. The isolates that expressed esp were found to survive within rat perperitoneal macrophages (>24hr) for a longer length of time then esp-negative isolates (Baldassari et al., 2004). It is obvious from these studies that environmental signals play a major role in the regulation of biofilm formation from initiation to the maturation of biofilms.
Studies have been carried out on E.faecalis which have identified an esp homologue (Eaton & Gasson, 2002). Toledo-Arana and colleagues have found that 93.5% of E.faecalis esp-positive isolates have shown biofilm formation on an abiotic surface while none of the esp-negative strains have done so (Toledo et al., 2001). Further information was found in this study regarding the activation of the esp gene. It was found that when the esp gene was inactivated in two mutant stains of E.faecalis but not in a third this resulted in poor biofilm production. This gave evidence that the esp gene is involved in biofilm formation however there are other factors also to consider. Other studies by different groups were also carried out on the esp gene. It was found that two esp-deficient E.faecalis strains,FA2-2 and OGIRF, increased their amounts of biofilm after successful introduction and expression of the esp gene (Tendolkar et al., 2004). A group lead by Tendolkar carried out further studies a year later, they found that the expression of Esp in two different heterologous hosts, E.Faecium and Lactococcus lactis had no effect on biofilm production, suggesting that factors which are unique to each bacterial species play a role in the surface protein's enhancement in biofilm development (Tendolkar et al., 2005). Contradicting studies were carried out by another group in 2004, a study was carried out on 74 esp-positive isolates which all produced biofilms and 89 esp-negative isolated, where 77 produced biofilms. Among the positive species that did produce biofilms, 69% were strong, 46% were medium while 30% were weak producers of biofilm. It was observed that none of the 12 non biofilm producing producers were esp-positive. It was indicated by the authors that esp is not necessary for biofilm formation however there is a strong link between the gene and biofilm formation (Mohamed et al., 2004).
The gelatinase (GelE) of E.faecalis is an extracellular zinc metallprotease that can cause hydrolysis of gelatine, casein and collagen. Studies carried out found that gelatinase and serine protease (SprE) are in an operon, gelE-SprE, which is regulated by a quorum sensing system which is encoded by the fsr locus (Qin et al., 2001). Two gelE mutants of E.faecalis OGIRF TX5128, a gelE insertion mutant (GelE-,SprE-) and TX5264,a non-polar deletion mutant(GelE-,SprE+) showed a 46% and 37% decrease in biofilm production relative to wild type OGIRF (Mohamed et al., 2004). These results show that gelE rather than serine protease is important for biofilm formation. Further studies were done by another group by cloning gelE into a plasmid, PMSP3614 which was under control by a nicin-inducible promoter which allowed expression of gelE in E.faecalis JH2.It was observed that there was a partial restoration of biofilm formation by this strain which suggests that gelE is involved in enhancing biofilm formation (Kristich et al., 2004). It appears although genetic manipulation studies have proven that gelatinase is involved In biofilm formation and is essential to it, epidemiological studies have not supported this among tests carried out on E.faecalis.
Table:Genetic determinants involved in E.faecalis biofilm formation