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Enterococci are gram positive, catalase/oxidase negative, non-spore forming, facultative anaerobic bacteria which when visualised under the light microscope exist singly, as diplococci or in short chains. Despite naturally occurring in the lower gastrointestinal (GI) tract, oral cavity and the skin Enterococci has rapidly emerged from what used to be described as a harmless, medically unimportant microorganism to the third most common nosocomial pathogen with the potential to cause life threatening infections (Fisher and Phillips 2009). There are currently more than twenty known species, however according to the UK Health Protection Agency (HPA) the two most common isolated from almost 80% of clinical samples are E.faecalis and E.faecium (Health Protection Agency 2009).
Enterococci are quite rugged and when outside the mammalian body can survive harsh conditions therefore easily transmitted on inanimate surfaces. Despite their optimum growth temperature being 35°c the majority of species are able to grow between 10 and 45°c (Tendolkar, Baghdayan and Shanker 2003). In addition they can grow in up to pH9.6, in saline conditions (6.5% NaCl) and can hydrolyse esculin in the presence of 40% bile salts (Sood et al 2008). The ability to survive such conditions explains why it has become such a problem in healthcare settings. It can be easily transferred to toilet seats and door handles used by multiple patients, many of which are likely to be immunocompromised therefore susceptible to bacterial infection meaning hospitals can act as a 'hub'. As more strains become resistant to antibiotics, detergents and other cleaning agent's even the strict hospital cleaning and sanitizing regimes may not be sufficient to kill them. This further hinders the problem allowing transmission to and from surgical instruments and medical personnel (Fisher and Phillips 2009).
Their presence has also been noted in foodstuffs, in particular those derived from animal origin such as milk, cheese, sausage and minced beef which has been partially attributed to their ability withstand heating at 60°c for 30 minutes (Foulquie et al 2006). For years Enterococci have been used in the food industry, both in the manufacturing process and as probiotics (Gupta and Garg 2009). Kuhn et al (2003) reported that agricultural crops treated with pig manure tested 100% positive for Enterococci, whilst those grown in the absence of animal fertilisers reduced numbers by approximately 77% suggesting transmission by food products. However one should be aware that not all strains isolated from foodstuffs possess the virulent nature which can lead to disease.
Enterococci are associated with a wide range of clinical manifestations including Bacteraemia, surgical site and soft issue wound infections, Urinary Tract Infections (UTI), neonatal sepsis, endocarditis, intra-abdominal/pelvic infections and meningitis (Butler 2006).
2. Virulence Factors
Virulence factors can be defined as molecules expressed or secreted by pathogenic bacteria to facilitate host colonization, entry into cells, evasion of the host immune system and the obtaining of nutrients essential for growth and survival (Giridhara- Upadhyaya, Ravikumar and Umapathy 2009). In Enterococci some virulence genes are located on a 150kb pathogenicity island (PAI) first described by Huyche, Spiegel and Gilmore (1991), whilst others can be found on bacterial plasmids. Over the last decade much research has been carried out on the virulent properties of Enterococci however many of the mechanisms are still incompletely defined. Some of the key factors are described below.
2.1. Enterococcal Surface Protein (ESP)
ESP is a 200kDa cell wall anchored protein found predominantly in clinical isolates of E.faecalis. It is thought to enhance adhesion and colonization of specific host tissues as well as playing a role in the evasion of the host immune response (Foulquie et al 2006). Shanker et al (2001) demonstrated the role of ESP in the adhesion and colonization of uroepithilial cells in ascending urinary tract infection (UTI). Other studies suggest ESP has an important role in biofilm formation (Heikens, Bonten and Willems 2007).
2.2. Aggregation Substance (AS)
AS is a 137 kDa surface glycoprotein which when expressed promotes clumping of bacterial cells. The gene is located on a plasmid regulated by a pheromone secreted from cells which do not possess the plasmid themselves (Hallgren et al 2009). This promotes conjugative plasmid transfer to plasmid free cells via pili, which in addition have been shown to play a role in biofilm formation (Hendrickx et al 2008). AS also promotes adherence to eukaryotic cells including renal epithelium, enterocytes and neutrophils (Koch et al 2004). Aggregation of cells increases the hydrophobicity of the surface which has been suggested to assist the bacteria in the evasion of the host immune system (Fisher and Phillips 2009). A recent study has also demonstrated the importance of AS in the manifestation of endocarditis (Chuang et al 2009).
Haemolysin is a cytolytic toxin encoded on a plasmid regulated by a two stage quorum sensing mechanism. It Î²-haemolytic properties are capable of lysing human, horse and rabbit erythrocytes (Giridhara- Upadhyaya, Ravikumar and Umapathy 2009). It has also believed to act as a bacteriocin by inhibiting the growth of other closely related gram positive microorganisms giving it a competitive advantage in the body (Coburn et al 2004).
The degredative enzyme Hyaluronidase depolymerises hyaluronic acid, an important component of human connective tissue facilitating the organism's movement through the host tissue. The metalloendopeptidase gelatinase hydrolyses cellular components such as gelatine, haemoglobin and other peptides along with serine protease in order to obtain nutrients essential for growth and survival (Fisher and Phillips 2009). A recent study has also demonstrated gelatinase's ability to cleave C3 proteins suggesting a role in evasion of the host immune response by inactivation of the complement system (Park et al 2008). The genes encoding gelatinase and serine protease are closely located and controlled by the same quorum sensing mechanism. An accumulation of an auto inducing peptide (AIP) secreted by Enterococci during exponential growth leads to transcriptional events on the separate fsr locus subsequently leading to expression of the hydrolytic enzymes in question to harbour more nutrients (Pillai et al 2002).
Other virulence factors include capsular polysaccharides, extracellular superoxide (Fisher and Phillips 2009) and collagen adhesion (Cna) (Nallapareddy, Singh and Murray 2008).
3. Host defence mechanisms
With the emergence of multiple resistance Enterococci as the third most common nosocomial pathogen it is surprising that very few studies have been published and that little is known about the host defence mechanisms. Studies have shown that generally only hospitalized patients, and therefore usually immunocompromised suffer symptomatic infection and normal healthy individuals are able to evade (Koch et al 2004). Much of the work carried out has been on the innate immune response which is commonly considered to be the first line of defence against invading pathogens.
Both gram positive and negative organisms display pattern associated molecular patterns (PAMPS) which are recognised by pattern recognition receptors (PRR) on the surface of immune cells. A study by Giebelen et al (2009) suggested peritoneal macrophages are the first line of innate defence against Enterococcal infection. Mice who did not have the macrophages exhibited higher levels of bacterial colonization than those who did have them. It was also pointed out that in dealing with the pathogen the macrophages prevented a massive inflammatory response as levels of cytokines/chemokines and neutrophils infiltration were significantly higher in the macrophage free group. The data from this study is somewhat limited in the fact that peritoneal macrophages are localized in the peritoneal cavity thus circulating macrophages may behave differently. Other studies have demonstrated the ability of some Enterococci to survive inside macrophages potentially using them as a vehicle for entry into other tissues (Sussmuth et al 2000), however this again may be due to immunocompromised patients already having a suppressed immune response.
Another study has demonstrated the importance of Toll like receptor 2 (TLR2) and the MyD88 adaptor protein in the recognition of PAMPs and subsequent recruitment of neutrophils (Leendertse et al 2008). Leendertse et al (2009) demonstrated the importance of neutrophils in removal of the pathogen by showing that neutropenic mice showed a greater level of colonization and increased time to pathogen clearance. This is further evident in Willems et al (2009) that demonstrated patients who have undergone an acute phase response, and thus are unable to efficiently recruit neutrophils are more susceptible to infection.
The data available is fairly limited and doesn't allow reliable conclusions to be drawn regarding host defence therefore further studies are required to gain a better understanding of the mechanisms.
The majority of UTI's, sort tissue wounds and intra-abdominal infections are classed as uncomplicated and therefore can usually be treated by administration of a single antibiotic, preferably ampicillin due to its lower effective dose requirement compared with penicillin (Butler 2006). These Î²-lactam antibiotics competitively bind and inhibit transpeptidase enzymes responsible for cross linking the bacterial cell wall peptidoglycan chains ultimately causing cell lysis. Î²-lactamase producing Enterococci possessing resistance to conventional beta lactam antibiotics can usually be treated with the glycopeptide Vancomycin (Cunha 2006). Nitrofurantoin is also effective in the treatment of lower UTIs. It is suggested that in the reduced form Nitrofurantoin causes damage to bacterial DNA thus inhibiting protein synthesis (Sandegren et al 2008).
Complicated infections usually require multi-drug (combination) therapy. Failure of a first line beta-lactam alone in the treatment of bacteraemia requires combination therapy with an aminoglycoside such as gentomycin or streptomycin, which binds to cytosolic ribosomes inhibiting protein synthesis (Champney 2001). A similar regime is often adopted for treatment of endocarditis (Westphal, Plicht and Naber 2009).
The narrowing spectrum of effective drugs due to the emergence of resistant strains, in particular Vancomycin resistance Enterococci (VRE) makes it difficult to effectively treat infected individuals. Despite a recent decline (Health Protection Agency Reports 2009) the incidence of VRE in healthcare settings has grown enormously in the last twenty years and has been attributed to both the use of avoparcin (a similar compound to Vancomycin) containing animal feeds and widespread use of Vancomycin, especially in the USA where evidently it has become increasingly easy to obtain such drugs (Nailor and Sobel 2009). Resistance is conferred by modification of the D-Ala-D-Ala moiety of the N-acetylglucosamine/N-acetylmuramic acid peptides of the peptidoglycan cell wall component. Instead the terminal Alanine is replaced by a Lactate to form D-Ala-D-Lac. Despite weakening the cell wall it results in a 1000 fold decrease in the efficacy of Vancomycin (Altun et al 2008). The three identified resistance variants are designated Van A, Van B and Van C. The Van B and C variants confer resistance to Vancomycin, but not the similar compound Teicoplanin which therefore can be substituted in some infections. Van A is the most clinically significant being resistant to both Vancomycin and Teicoplanin (Zheng et al 2009).
4.4.Recent developments in antibiotic treatment
Novel antibiotic development is very much an active research area and several new compounds have recently become available. Linezolid is an up and coming synthetic antibiotic belonging to the group of oxazolidinones and is effective in the treatment of complicated skin infections caused by VRE (Smith et al 2005). To prevent a repeat of the resistance problem associated with Vancomycin it is usually recommended as a last resort, however in recent years several resistant strains have been reported (Kainer et al 2007). The lipopeptide daptomycin binds to the outer cell membrane triggering depolarization. The outflow of K+ ions leads to a loss in membrane potential and consequently cell death (Silverman, Perlmutter and Shapiro 2003). It is predominantly used for skin infections including those caused by VRE (Poutsiaka et al 2007). Tigecycline is a recently approved glycylcycline effective against several strains of Enterococci including both E.faecalis and E.faecium . Similar to many other antibiotics it binds to the ribosome inhibiting protein synthesis (Yemisen et al 2009).
One of the key recent antibiotics is the semi-synthetic lipoglycopeptide Oritavancin shown to be rapidly effective against VRE. Although it has not been definitively defined several potential mechanisms of action have been proposed including inhibition of cell wall synthesis, disruption of membrane potential and inhibition of RNA synthesis (Belley et al 2009). Another recent study suggested Oritavancin inhibits transpeptidation of peptidoglycan (Patti et al 2009). Due to lack of clinical trial data the drugs approval request was rejected pending further studies therefore it could be several years before it becomes available.
Once considered a harmless organism naturally present in the body of mammals, Enterococci has rapidly become a multiple resistant nosocomial pathogen with the ability to cause life threatening infections. Several virulence factors have been identified on a pathogenicity island in addition to several others located on plasmids acquired from other virulent gram positive organisms. Further studies are required to determine the host defence mechanisms to gain a better understanding and help develop new treatment regimes. Most uncomplicated infections can be easily treated with simple Î²-lactam antibiotics. Those of a more serious nature however are becoming problematic due to the emergence of multiple drug resistant strains. Again a more definitive understanding of the mechanisms of action of treatments will assist in the development of new ones.
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