In this paper, we analyze human pathogenesis, especially urinary tract infections, endocarditis, necrosis, bacteremia and other blood and skin disorders that are caused by bacterial infections. Here the focus is on enterococcal infections, how such infections spread and what can be done to prevent their transmission. In this context, we discuss the characteristics features of the different strains of Enterococci, with the common E. faecium and E. faecalis types although we mention properties of other Enterococci as well.
In the first introductory section we take a look at the types of Enterococci, the different strains that cause different infections in humans as well as animals, where they reside, how they spread and multiply and the various resistances that they have developed to antibiotics. The role of antibiotics in preventing and controlling enterococcal infections has also been discussed highlighting the challenges that the medical profession faces with the development and growth of increasing numbers of antibiotic resistant strains. In this regard we discuss the special case of VRE or Vancomycin Resistant Enterococci.
In the second and final section of our paper we discuss case studies of vancomycin, ampicillin, penicillin and other types of antibiotic resistance as found in isolates collected and reported all over the world. For our purposes we have discussed cases from Korea, India, Poland, and Turkey and have also emphasized on the growing need to educate health care workers to use strict precautionary methods to prevent spread of VRE and other enterococcal infection via contacts in the hospitals and emergency or intensive care units.
Our analysis is divided into two parts – one of which is descriptive giving an account of enterococcal infection and the various diseases associated with it. Here we discuss the different strains of Enterococci and the types of genes required for encoding. We also discuss the epidemiology and habitat, prevalence and occurrence of Enterococci in general. In the second part we discuss specific cases reported by researchers all over the world on the alarming increase in enterococcal infections and the grave challenges posed by such infections given the fact that enterococcal strains are becoming increasingly resistant to antibiotics and no options for treatment for enterococcal infections remain really available.
Around the end of the 19th century, McCallum and Hastings (1899) addressed Enterococci pathogenesis by isolating such an organism from a case of acute endocarditis. This isolated strain showed a resistance to desiccation for a heating of up to 60°C, and also showed resistance to antibiotics, chloroform and carbolic acid. This initial variety became one of the most infectious and prominent strains among nosocomial pathogens and after E. coli, Enterococcus account for 12% of all cases of bacterial infections (Gilmore, 2002).
Among these infections by Enterococcus, Enterococcus faecalis comprise of 80% of cases and the infections include urinary tract infections, bacteremia, endocarditis and intra-abdominal infections. However Enterococci are the leading cause for surgical wound infection and nosocomial bacteremia. A leading cause for concern is that Enterococci are increasingly becoming resistant to all standard antibiotics and therapies (Gilmore, 2002). Thus the problem of nosocomial infection is compounded by multiple antibiotic resistances. As we mentioned most enterococcal infections are caused by Enterococcus faecalis which are more likely to express overt traits of virulence and also more likely to retain sensitivity to at least one effective antibiotic. The remaining (nearly 20%) of infections are caused by Enterococcus faecium, a species which is devoid of overt pathogenic traits but are the most resistant and even likely to be resistant to all antibiotics (Huycke et al, 1998).
The presence of VRE or Vancomycin Resistant Enterococci in the blood stream has been associated with increased mortality and patients with enterococcal bacteremia have been found twice as likely to die when the infection causing bacteria was resistant to vancomycin. Vancomycin represents the last resort to the treatment of bacterial infection and is often given as the last available therapeutic. For these reasons, a resistance to vancomycin poses a serious challenge to the therapeutic interventions for enterococcal infections (Hancock and Gilmore, 2000). According to Huycke et al (1998), effective control of enterococcal infections will require 1. better understanding of the interaction between Enterococci, the hospital environment, and humans, 2) prudent antibiotic use, 3) better contact isolation in hospitals and other patient care environments, and 4) improved surveillance (Huycke et al, 1998).
According to recent studies reporting on data from last 3 years, E. faecium showed a general trend of increasing resistance towards both ampicillin and vancomycin. However E. faecalis remains the most frequent enterococcal isolate. The over representation of E. faecalis among the two strains of enterococcus is that E. faecalis in contrast with E. faecium is of more abundant variety in nature and is found mainly in the human gastrointestinal tract (Andrew and Mitchell, 1997). In any instance the number of E. faecalis can be 100 times higher than E. faecium variety of enterococcus.
Enterococci inhabit the bowels and are found in the intestinal regions of all animals. Enterococci are commonly found in sewage and in untreated surface water. In humans the typical reading found in human stool is up to 108 CFU per gm. Huycke et al write that the predominant variety inhabiting human intestine varies in different regions in Europe, US and Far East. Although there are 14 or more enterococcal species, only E. faecalis and E. faecium colonize and infect humans in significant numbers and infections to other enterococcal species are rarely seen (Devreise et al, 1993, also cited in Huycke et al, 1998).
Enterococci are capable of surviving extreme conditions and tolerate temperatures 10°C through 45°C with tolerance for acidic and alkaline, hypotonic and hypertonic conditions. They are intrinsically resistant to several antibiotics although Penicillin, ampicillin, piperacillin, imipenem, and vancomycin are among few antibiotics showing inhibitory functions against E. faecalis. As far as intrinsic resistance is concerned, it is highly based on chromosomal genes unlike acquired resistance and virulence traits which are usually transposon or plasmid encoded (Andrew and Mitchell, 1997).
Huycke et al (1998) further note that Enterococci have been recognized to cause nosocomial bacteremia, surgical wound infection and urinary tract infection. Usually two types of Enterococci can cause such infections - those originating from the patients' native flora that are not likely to have any resistance beyond that which is intrinsic to the genus and this is unlikely to spread from bed to bed; and those isolates that possess multiple antibiotic resistance traits and capable of nosocomial transmission. The multiple drug resistant Enterococci (MDR), those strains with significant resistance to two or more antibiotics often with the inclusion of vancomycin, used as a last resort is an increasing therapeutic challenge and has been instrumental in highlighting the role of nosocomial pathogens. The spread of antibiotic resistant strains of Enterococci to the extent that Enterococci are resistant to all standard therapies show the vulnerability of our medical facilities and the great challenges posed by this post-antibiotic era. This has been discussed in several studies and continues to remain a matter of controversy.
There are several phenotypes of vancomycin-resistant enterococci; VanA (showing resistance to vancomycin and teicoplanin) and VanB (showing resistance to vancomycin only) are the two most common varieties. In the US, most studies reveal that VanA account for 60% and VanB for 40% of vancomycin resistant enterococci. (VRE) isolates. The two VanA and VanB types are isolated from clinical, food or veterinary samples of bacteria (Gilmore, 2002). However antibiotic resistant genes can be transferred and one such example Staphylococcus aureus has been rendered vancomycin resistant through transfer of resistance from E. faecalis on skin of mice. The mechanism of resistance as seen in Staphylococcus aureus does not seem to involve VanA and VanB genes.
Enterococcal strains have been showing increased resistance to antimicrobial agents in the past few years and drugs that were once considered completely effective in the treatment of Enterococci like penicillin, amino glycosides, and vancomycin are no longer effective in treatment of resistant Enterococci. The treatment and removal of Enterococci infections that are resistant to one or a combination of antibiotics pose a clinical challenge to all researchers and practitioners of the medical and health care profession. Structurally, Enterococci are Gram-positive, facultative, anaerobic organisms and show similar morphology to streptococci group D. However, Enterococci have different nuclei acid hybridizations and in 1984 were grouped in a separate class of bacteria. The E. faecium has been found to be resistant to several antibiotics (Andrew and Mitchell, 1997). Within the human gastro-intestinal tract, Enterococci are found in significant numbers and these bacteria also inhabit the skin, oropharyngeal and vaginal secretions and also in the perineal area.
The enterococcal infections are found in the urinary tracts, in intra-abdominal abscesses, in the blood and account for 165 of all urinary tract infections and 8% of all bacteremias. Endocarditis, meningitis, respiratory infections and neonatal sepsis can also be due to enterococcal, although they are less commonly affected by Enterococci and may have other causes. Enterococci have been considered to be the prime reason for the spread of nosocomial infections and can spread by direct or indirect contact. Control measures in hospitals have to be in practice as enterococcal infections can be spread by patients and through medical equipment or hospital staff. Rapid spreading and development of Enterococci can lead to development of multidrug-resistant strains of Enterococci (Gilmore, 2002). Most researchers suggest that control measures for restricting the spread of Enterococci have to be continuous and should involve a multidisciplinary task force including hospital epidemiologists, pharmacists, therapeutic committee members, hospital and medical staff and infection control committee members. We have given a chart on the different isolates of Enterococci obtained.
In this section, we aimed to give a general introduction to Enterococci and the prevalence of E. faecalis and E. faecium bacterial strains. We also discussed the importance of vancomycin resistant Enterococci or VRE in medical research as vancomycin seems to be the last and strongest available alternative and the most effective for treatment of enterococcal infection. Considering this, any vancomycin resistance is extremely important clinically as this throws a new challenge in eradicating and treatment of enterococcal infections that are not only increasing in distribution and numbers but by showing resistance to nearly all antibiotics, are almost impossible to treat. We now move on to more detailed discussions of Enterococci and multidrug resistant strains as also different types of resistance to antibiotics, prevalence of enterococcal infections, case studies and the various phenotypes of vancomycin resistant Enterococci that include VanA and VanB among other varieties.
Enterococci are associated with diseases including infection of the blood stream that is bacteremia, diseases of the heart valves or endocarditis and diseases of the brain or meningitis that can occur in severely ill patients and these strains of bacteria frequently colonize open wounds, surgical wounds, urinary tracts, intestinal regions and skin ulcers. In these conditions, bacteria usually develop from lesions. Enterococci are the most antibiotic resistant bacterial strain although minor infections can be treated using tetracyclines, penicillin or ciprofloxacin. However, only strong penicillin such as ampicillin and vancomycin given by injections are effective against severe bacterial infections such as endocarditis or meningitis. Serious infections require the administration of simultaneous doses of several strong antibiotics.
The antibiotics were mainly considered as potentially effective for treatment of bacterial infections and until recently the resistant strains of Enterococci was never a challenge. However recent studies have shown that many enterococcal species are completely resistant to standard therapeutic antibiotics and this has posed a serious challenge to the medical profession in combating bacterial infection. This was first discovered in 1986 when the first vancomycin resistant Enterococcus was discovered in France and the following year another strain was isolated in the UK. Resistance to vancomycin in Enterococci is not intrinsic but acquired by genetic material passed on from other bacterial strains that are vancomycin resistant but may not cause human infection (Dunny et al.1991).
Vancomycin resistant Enterococci are also resistant towards teicoplanin, another type of antibiotic and teicoplanin resistant Enterococci also similarly show a resistance towards vancomycin thus resistance towards vancomycin and teicoplanin seems to be related. In UK and other European regions vancomycin type antibiotics are given to animals and certain strains of Enterococci found in animals is quite indistinguishable form that found in humans. Usually, speculations of humans acquiring bacterial infection from contact or eating meat of animals have been quite strong. These bacteria acquired from animals make way into the human gut and resides in the intestines and can be found in the stool upon examination (Hancock and Gilmore, 2000). Sometimes, VRE can spread from the intestines and cause infections in other parts of the body.
VRE or vancomycin resistant strains of Enterococci are generally seen in patients who have been exposed to hospital environments for a long time, have received antibiotics such as vancomycin, teicoplanin and cephalosporin, have had surgical operations or have been fed by naso-gastric tubes. VRE are also seen in individuals who have never been hospitalized or who have been given only few doses of antibiotics. Vancomycin resistant strains of bacteria do not cause any special type of illness and may have common features with vancomycin susceptible strains of bacteria. VRE is not usually seen in normal healthy people and chances of acquiring them in out of hospital settings are rare provided a level of social hygiene is maintained (Hancock and Gilmore, 1998).
Recent studies have shown increasing cases of enterococcal infections even outside the hospital environment, especially in European countries. In the United States, enterococcal infections remain concentrated in hospital settings. Vancomycin sensitive Enterococci as well as Vancomycin Resistant Enterococci can both cause a common range of infections including urinary, heart and brain diseases. VRE infections in hospitals are usually reported from patients who had surgical operations, renal dialysis, transplants and patients admitted to intensive care units (Hancock and Gilmore, 2000). The cases of VRE reported in hospitals has been increasing rapidly in the last few decades and this has been bolstered by the fact that bacterial strains are showing an increased resistance over the past few years. This has been reported in different parts of the world as we will discuss in the following sections.
According to recent statistic, in the United States alone, Enterococci cause 110000 urinary tract infections, with 25000 cases of bacteremia infections, 40000 wound infections and 1100 cases of endocarditis. However the National Nosocomial Infections Surveillance System data suggest there has been little or no change in the percentage of enterococcal infections in the last two decades. Since enterococcal infections are generally accompanied by severe co morbid illnesses, enterococcal infection deaths are difficult to ascertain. From 7% to 50% of deaths, enterococcal sepsis is seen although the death risk associated with antibiotic-resistant enterococcal bacteremia is many times more than when infected with susceptible enterococcal bacteremia. infection with MDR (multiple drug resistant) Enterococci occur worldwide and in the United States alone, percentage of nosocomial infections caused by vancomycin resistant Enterococci increased 20 times from 1989 through 1993.
The Surveillance Network (TSN) database permits the assessment of resistance profiles by compilations from more than 100 clinical laboratories in the US. It detects emergence of resistance profiles and identify mechanisms in resistant strains that can pose a public health threat. There is an increased resistance of E. faecium to vancomycin and ampicillin although in contrast the E. faecalis resistance to ampicillin and vancomycin is quite uncommon. Of the two strains, although E. faecium is more resistant to vancomycin and ampicillin, E. faecalis is more commonly found, a point we have already mentioned. Since both the strains E. faecalis and E. faecium are considered together, several important points of differences in the two strains are overlooked. Huycke et al (1998) contend that vancomycin and ampicillin resistance can provide selective advantage for the species faecium and not for the species faecalis. The relative absence of these resistances in E. faecalis may be due to a momentary lack of penetrance and equilibration of traits. According to researchers in the field, due to the internal differences of the two different species of Enterococci, any meaningful research on enterococcal resistance must include species identification (Huycke et al 1998, Hancock and Gilmore, 2000)
Nearly all patients who are infected or colonized with multiple drug resistant Enterococci are given treatments using clindamycin, ciprofloxacin, cephalosporin, aztreonam, amino-glycoside and metronidazole which are more often used for MDR infections than vancomycin. The conditions associated or related to or causing MDR infections have been identified as prolonged hospitalization, high severity of illnesses, intra-abdominal surgery, renal insufficiency, and exposure to contaminated surfaces and objects in hospitals or patient care areas.
The importance of vancomycin resistance in bacterial species has been a subject of considerable research in recent times. Two types of vancomycin resistance have been identified. The first type is intrinsic resistance and isolates of Enterococcus gallinarum and E. casseliflavus/ E. flavescens all demonstrate an inherent low level resistance to vancomycin that is intrinsic. The second type is acquired resistance in which enterococcal species become resistant to vancomycin by acquisition of certain genetic information from some other organism. This acquired resistance to vancomycin is seen in E. faecium and E. faecalis and has also been seen in E. raffinosus, E. avium and E. durans and other enterococcal species.
Genes of the categories VanA, VanB, VanC, VanD and VanE all contribute to vancomycin resistance in Enterococci. Among all the different species, E. faecium is the most frequently isolated species of VRE found in hospitals and produces high vancomycin and teicoplanin inhibitory concentrations with isolate typically containing VanA genes. A lower level resistance to vancomycin is seen in VanB containing isolate and shows much lower levels of vancomycin resistance and little or no resistance to teicoplanin. VanD containing isolates of E. faecium show moderate resistance to vancomycin and teicoplanin and shows similar properties as VanE containing isolate of E. faecalis. Thus acquired resistance is seen in E. faecium and E. faecalis, the two most commonly occurring strains and also seen in E. raffinosus, E. avium and E. durans and other enterococcal species. Intrinsic resistance to vancomycin is seen in E. gallinarum, E. casseliflavus and E. flavescens and all these isolates contain VanC genes. Thus presence of intrinsic resistance or acquired resistance is indicated by the presence of VanC or VanA / VanB genes.
Knowledge of the type of resistance as also knowledge of different characteristic of various species of Enterococci is important for effective infection control. VanA and VanB genes as they are acquired can be transferable and spread through different organisms, VanC genes represent intrinsic resistance and characteristic are not transferable and thus have not been commonly associated with serious infections or mass outbreak of diseases. Among structural differences, E. faecium and E. faecalis, the two common varieties are non-motile whereas E. gallinarum and E. casseliflavus and E.flavescens are generally motile. Isolates of E. casseliflavus and E. flavescens have a yellow pigment and presence of pigments and motility distinguishes the various species of Enterococci. VanA and VanB isolates are also distinguished from VanC isolates by properties of intrinsic or acquired resistance as also by susceptibility to antibiotics.
Screening for vancomycin resistant strains can be done in a number of ways. Peri-rectal or anal swabs are used or stool specimens are used directly. One of the methods of isolation is using bile esculin azide agar plates containing 6 µg/ml of vancomycin. Any black colonies noted should be identified as enterococcus and this can be confirmed further by using an MIC (minimum inhibitory concentrations) test method before confirming VRE colonization (Sundsfjord et al, 2004). To check whether the species is vancomycin resistant, a suspension of the organism can be isolated on a brain heart agar (BHIA) plate containing 6 µg/ml of vancomycin. The NCCLS (National Committee for Clinical Laboratory Standards) recommends performing an MIC vancomycin resistance test and pigment productions and motility tests to distinguish VanC inherent resistance in species and VanA and VanB type acquired resistance (Gilmore, 2002). Usually screening for VRE is dependent on facilities available in the health care setting concerned. At certain healthcare facilities, infection control personnel screen newly admitted or high risk patients identified as patients with urinary infections, or oncology/ surgery patients and patients in intensive care units. These patients are identified to be more vulnerable and at greater risk of VRE infection and colonization.
Due to the rapid development and increase of vancomycin resistant enterococcal strains, treatment of bacterial infections caused by Enterococci has become increasingly problematic. Firstly due to resistance acquired to antibiotics, the range of antibiotics that can be effective for treatment is limited. It is difficult to predict whether the bacteria will be susceptible to any antibiotic or will show resistance towards it. Secondly, available antibiotics are not as varied different strains of Enterococci which show that medical research has not been able to keep up with the pace of enterococcal growth and variations. Thus effective treatment for enterococcal infections are not just delayed, they may seem to be completely unavailable. Research is under way to develop new strains of antibiotics that can be effective in treatment of enterococcal infections. Many individuals who may have enterococcal colonization in their gut may not need antibiotics and can show spontaneous recovery.
It has been recommended that vancomycin, teicoplanin and cephalosporin should be restrictively given only to individuals who are suffering from severe enterococcal infections to maximize the effectiveness of these drugs. Limited use of vancomycin type antibiotics prevent colonization of Enterococci in animals and spread to humans. Proper cleanliness, especially in hospitals settings, with through washing and sterilization of medical equipment have been considered necessary to prevent spreading of VRE infections. All VRE infected patients are treated in separate rooms and hospital staff members wear aprons and gloves while taking care of these infected patients. When such patients are discharged from hospitals, special care is taken to remove their used linen and dispose any clinical waste or personal content that can spread the infection.
As we mentioned, the two types of resistance showed by Enterococci are intrinsic and acquired. We discuss intrinsic and acquired resistance in more detail here. Intrinsic resistance is mediated chromosomally and is controlled by genes, intrinsic resistance is non transferable. Acquired resistance is mediated by plasmids and transposons.
Intrinsic resistance is seen in Enterococci that tend to show a very low level resistance to many antibiotics that are generally used for Gram positive bacterial infections. Enterococci show a lower level resistance to beta-lactams due to the production of penicillin-binding proteins showing lower affinities. Thus ampicillin and penicillin G are more effective than beta-lactams for treatment of Enterococci. Intrinsic resistance in Enterococci generally tends to show a tolerance for beta-lactams. The minimum inhibitory concentrations (MICs) shown by streptococci may be up to 100 times lower than for Enterococci, thus Enterococci seem to be generally more resistant and hardy that other bacterial species. Enterococci are more intrinsically resistant to cephalosporins than towards ampicillin or penicillins, so cephalosporins are usually ineffective for enterococcal treatments. E. faecium seems to have high intrinsic resistance and show a very high resistance to beta-lactams than the other species whereas E. faecalis has a higher level of resistance to amino glycosides than the other species.
Amino-glycosides have a decreased ability to penetrate the outer cell envelope of Enterococci and thus there is a low level intrinsic resistance towards amino glycosides in certain bacteria. Penetration of the antibiotics is necessary as amino glycosides can only act intracellularly and with decreased penetration, the action of such antibiotics becomes difficult. Certain cell-wall active antibiotics such as penicillin, carbapenems or glycopeptides are effective for bactericidal activity as in cases of bacteremia, endocarditis and meningitis. Apart from the more common antibiotics, Enterococci are also susceptible to flouro-quinolones but are not susceptible to sulfamethoxazole or trimethoprim showing intrinsic resistance to these compounds as well. Antibiotic clindamycin is considered generally ineffective against enterococcal colonizations. Usually antibiotics that are used for bacterial infections other than Gram-positive infections and also amino glycosides have only limited efficacy for treatment of Enterococci. Intrinsic resistance in Enterococci thus varies from a low level to high level and Enterococci can be intrinsically resistant to various kinds of drugs such as amino glycosides, sulfamethoaxole, beta-lactams, cephalosporins and penicillin. However, as we shall discuss, resistance to vancomycin or penicillin is usually acquired.
Intrinsic resistance is genetically programmed and chromosomally mediated. However acquired resistance is mediated by plasmids or by transposons. Thus acquired resistance is transferable and resistance can be transferred to other Enterococci species or to streptococci or staphylococci. Acquired resistance results in higher level resistance than intrinsic resistance and among the common varieties of resistance is penicillin-acquired resistance which occurs due to changes in the penicillin binding proteins and affinity of these proteins decreases. Aminoglycoside-acquired resistance develops due to aminoglycoside-modifying enzymes that decrease the drug's ability to bind with ribosomes that are essential to its proper action on the Enterococci.
Some bacterial species showing a high level gentamicin resistance produces beta-lactams and resistance towards aminoglycosides and gentamicin occur due to the same plasmids. Acquired high level resistance was first seen towards the antibiotic streptomycin. When endocarditis infections were treated with gentamicin and penicillin, relapses of the infection occurred showing a high level gentamicin resistance (HLGR) with MIC of 2000 µg/mL. High level gentamicin resistance (HLGR) is strong acquired resistance that has become a problem for hospital staff as gentamicin is no more considered effective against enterococcal infections. However certain Enterococci strains which show low streptomycin resistance are capable of possessing high level gentamicin resistance.
VRE or Vancomycin Resistant Enterococci, is the most common and widely discussed subtypes of acquired resistance. This acquired resistance against vancomycin can result in serious infections and can even prove to be fatal. Different varieties of this resistance is seen which is mediated by plasmids or transposons. Vancomycin resistance occurs when proteins are synthesized by the resistant Enterococci and this can be VanA, VanB and VanC varieties. VanA and VanB phenotypes are found in E. faecium but are also seen in E. faecalis. VanA phenotype is highly resistant to vancomycin and teicoplanin, whereas VanB shows moderate to high level resistance to vancomycin but is susceptible to teicoplanin. VanC shows a low level resistance to vancomycin and does not show any teicoplanin resistance found mainly in E. gallinarum and E. casseliflavus. The proteins synthesized by the Enterococci produce resistance by acting as ligases and alter cell wall characteristics that stop being permeable to vancomycin.
The mechanisms of resistance, however can be biochemical or genetic. Glycopeptide resistance occurs due to the presence of peptidoglycan synthesis that synthesizes low-affinity precursors and also allows elimination of precursors normally produced by the host. As for the genetic mechanism of resistance, the six glycopeptide resistance genotypes in Enterococci are vanA, vanB, vanC, vanD, vanE, and vanG (Lee, et al. 2004). The vanA glycopeptide resistance is characterized by acquired and inducible resistance to both vancomycin and teicoplanin and is mediated by Tn1546 or similar transposons. The vanB glycopeptide resistance is characterized by acquired and induced resistance to vancomycin of various levels but not resistance to teicoplanin. The vanB determinants are located in enterococcal chromosome and can be transferred with transfer DNA associations. The alleles of vanB genes are vanB1, vanB2, and vanB3. Among these the vanB2 gene is the most widespread and the gene cluster is associated with Tn5382 and Tn1549.
The vanB1 gene cluster is found in the composite transposon Tn1547. The vanD gene cluster is located in the chromosome and four strains D1-D4 have been identified. This is characterized by the presence of a vanY carboxypeptidase which is sensitive to penicillin and vanX that has no dipeptidase activity (Lee et al. 2004). The vanE resistance gene in E.faecalis is resistant to low levels of vancomycin and is susceptible to teicoplanin. VanE is a ligase and has 55% similarity to vanC ligates and the vanE operon has a vanXY bifunctional enzyme. The vanG phenotype is resistant to lower levels of vancomycin and susceptible to teicoplanin. The vanG operon leads to production of peptidoglycan precursors. Unlike glycopeptide resistance, oxazolidinone resistance is developed by bacteria by changing the ribosomal target site. This is especially characterized by linezolid resistance and is usually seen in prolonged courses of therapeutic intervention.
In the last few years there has been increased resistance to vancomycin with many resistant strains also found to be resistant to penicillins and aminoglycosides. Hospital intensive care units have seen nearly a 15% rise in case of VRE infections in a span of last five years. VRE has been thought to be related to the increased usage of vancomycin on development of Methicilin-resistant Staphylococcus aureus (MRSA) as well as on other gram positive organisms that showed beta-lactam resistance. The NNISS (National Nosocomial Infections Surveillance System) has reported a 20 times increase of vancomycin resistant strains of Enterococci from 1989 to 1993 and is now also seen in Methicilin-resistant strains.
Considerable concern has been expressed about the emergence of Vancomycin-resistant Enterococci (VRE) as the application of Vancomycin has been an important mode of treatment of Enterococcal infections. Furthermore, as antibiotic resistance is encoded by mobile genetic elements such as plasmids, the possibility that Vancomycin resistance might be rapidly disseminated from VRE to Methicilin-resistant Staphylococcus aureus (MRSA) is a major concern. MRSA is one of the principle causes of serious nosocomial infections and Vancomycin currently is the main antibiotic available to treat such infections. Vancomycin resistance has now begun to emerge worldwide among MRSA. Recent studies have shown that this trend continues and there is no decrease in infections by vancomycin resistant strains nor is there any significant development of vancomycin susceptible strains.
As far as the prevalence and effects of vancomycin resistant enterococcal infections are concerned, 7.8% of enterococcal strains isolated from intra-abdominal regions have been found to be vancomycin resistant. 4.1% such resistant strains have been isolated from skin areas with 3.8% blood isolates. Some older studies report that the mortality rates for enterococcal bacteremia can be up to 68% and overall 17.2% of patients with Enterococci in their blood can die with high mortality rates for VRE groups rather than non-VRE groups.
The results obtained from hospitals regarding the exact number of VRE cases vary according to the size of the hospital. As reported by Flores et al (1996), hospitals fewer than 200 beds had no reported cases of VRE while in hospitals with more than 500 beds, vancomycin resistance was found in more than 3.6% of Enterococci cases. Of the reported 32 cases of VRE isolates, 20 showed high-level resistance to vancomycin and teicoplanin, which is the VanA variety and 10 showed moderate to high resistance to vancomycin but some susceptibility to teicoplanin which represents the characteristics of VanB phenotype. In one study cited by Flores et al (1996) 105 VRE were isolated from 31 hospitals in 14 states of the US and the distribution of species in the 105 cases was found as follows: 82 of these was of type Enterococcus faecium, 8 E. faecalis, 5 E. gallinarum, 3 E. casseliflavus, 1 E raffinosus. The 105 resistant isolates had 71 VanA phenotypes, 26 VanB phenotypes, 5 VanC and 3 unknown varieties. Also certain varieties of phenotypes were found to be distributed according to geographical locations and of the VanA and VanB varieties, VanB was found to be more dispersed geographically. VanB has a lower level of resistance and may not be easily detected, especially in microbiology laboratories.
For enterococcal infections the typical treatment is giving penicillin or ampicillin and vancomycin is given as an alternative to penicillin allergic patients or in cases of non beta lactamase mediated penicillin resistance. Usually these should be combined with aminoglycosides in order to facilitate bactericidal activity. However when organisms become resistant to aminoglycosides or penicillin, it is a medical and treatment problem. 55% of E. faecalis and 33% of E. faecium have been reported to show combined resistance to penicillin and ampicillin. So in most cases vancomycin remains the only alternative to treatment of Enterococci but this may prove ineffective in a patient who has been infected with VRE strains. Treatment of vancomycin resistant Enterococci becomes even more difficult when there is a concomitant resistance to beta-lactam and aminoglycoside as well, often known as the condition of MDR or multidrug resistance. When HLGR or high level gentamicin resistance is present along with vancomycin resistance, any effective bactericidal combination for treatment becomes nearly unavailable (Hancock and Gilmore, 2000).
However some susceptibility tests with streptomycin may be carried out to check its effectiveness in treatment of these resistant strains. Sometimes however single drug therapy or administration of ampicillin, vancomycin or teicoplanin has been found to be effective in endocarditis and meningitis, however there may be chances of relapse. Enterococcal endocarditis is caused by bacterial organisms that are highly resistant to aminoglycoside but are susceptible to ampicillin and treatment is more effective when ampicillin is administered as a continuous infusion rather than given as intermittent intramuscular injections. High level resistance to penicillin along with VRE as seen in E. faecium can also be very difficult case for treatment. However, although E. faecium show high levels of resistance to ampicillin, vancomycin, penicillin and aminoglycosides, ciprofloxacin combined with ampicillin and novobiocin can be used as treatment against E. faecium. Ciprofloxacin can develop resistance in bacteria when used alone and has moderate effectiveness against most resistant strains.
Studies on vancomycin and penicillin resistant strains have shown that both these drugs have moderate activity against resistant organisms. In the presence of vancomycin resistance, penicillin may show an increased affinity for penicillin binding proteins. Moderate effectiveness in the treatment of endocarditis using combinations of antibiotics such as gentamicin, vancomycin and ampicillin has been found. Endocarditis is caused by vancomycin and ampicillin resistant E faecium. Many recent studies have found that when vancomycin is given in combination with ampicillin, treatment may be effective even when administered on individually ampicillin resistant and vancomycin resistant strains. Rifampin shows inhibitory activity in treatment of enterococcal infections but may be antagonistic when used with beta-lactams. In a review of different cases reported by Flores et al., bacteriologic cure could be achieved by administering a combination of rifampin, ciprofloxacin and gentamicin on E. faecium.
Bacteriological cure of vancomycin resistant Enterococci has also been achieved within a combination of three or four drugs including ampicillin, imipenem, gentamicin and vancomycin. VRE is a health hazard and the cause of nosocomial infections. It poses serious problems and challenges to the health care system. Incidence and reports of vancomycin resistant Enterococci have been increasing quite rapidly but treatment options available for these multidrug resistant organisms are clearly falling behind and becoming outdated and unavailable. VRE is highly adaptable and develops quick resistance to nearly all kinds of antibiotics making it indispensable to develop adequate transmission control and preventative measures if not treatment to prevent occurrence and spread of these bacterial strains.
Prevention of vancomycin resistant Enterococci is one of the foremost concerns among health care professional given the fact that most treatment or cure methods might prove ineffective. Since the treatment of multidrug resistant enterococcal infections remains controversial, measures can be taken to prevent further transmission of these infections. Usually survival periods of these bacteria on surfaces are quite high and this allows transmission through contact of surfaces quite a possibility. Despite a five second wash of hands, 20% of the organisms will remain so health workers are advised to clean their hands for 30 seconds after treatment of patients with VRE. As a protective measure gloves are worn and changed by hospital staff before moving on to other patients.
Certain commonly used medical equipments like stethoscopes, blood glucose monitors, rectal thermometers, weighing scales, and other devices can become contaminated with these bacteria and should be properly sterilized and cleaned after use. Flores et al report an outbreak of E. faecium VanA type of Enterococci that was spread by electronic thermometers which may become contaminated even if proper probe sheaths are used. Thus such instrument should be given only for one patient if strict disinfection measures are to be followed. Although the patients attacked by Enterococci can show apparent recovery, these organisms might remain in the digestive tract for more than a year, so patients with previous history of VRE infections should always be isolated first to see whether there is any relapse of VRE colonization.
From an analysis of vancomycin resistant Enterococci, the different strains and characteristics of resistant strains, we discussed on prevention and treatment of VRE infection. in the following section we will give an account of vancomycin resistance and enterococcal infections as discussed in case studies and recent research from different regions of the world.
Boost et al (2004) tried to estimate the rate of carriage of vancomycin resistant Enterococci (VRE) in hospitalized patients studied in a hospital in Hong Kong. Rectal swabs were collected from all the patients who were admitted to intensive care units and along with this stool specimens were also collected. Stool specimens were also collected from healthy individuals. These specimens were then further cultured to isolate strains of Enterococci. All the isolates were identified and the MIC or minimum inhibitory concentrations for vancomycin were also determined along with susceptibility and resistance to other kinds of antibiotics.
According to the study, 125 isolates of Enterococci were obtained with 75 of them being E. faecalis and 35 of them E. faecium. 11 strains of E. gallinarum and 2 strains of E. casseliflavus were also isolated. All strains were found to show intermediate and moderate levels of resistance towards vancomycin and no strain showed very high levels of vancomycin resistance. High level gentamicin resistance was found in 37% of cases and high level streptomycin resistance was found in 46% of strains with some resistance to other drugs including the flouroquinolones also seen. The authors claim that VRE colonization is low in Hong Kong and this may be attributable to low levels of vancomycin use. Boost et al (2004) suggest that since high levels of aminoglycoside resistance and flouroquinolone resistance are common, continuous monitoring to check a spread of VRE infections should be recommended.
Semedo et al. (2003) investigated the occurrence of virulence traits including cytolysin, adhesions and hydrolytic enzymes in 164 collected enetrococci mainly found in clinical and food isolates. They found up to 15 cyl genotypes and widespread occurrence of cyl operon. The authors reported a significant association of this virulent trait with clinical isolates. The paper also emphasizes the role of gelatinase, lipase and DNase as virulence determinants. Since no statistical association was found between virulence clusters and species allocation, according to the authors, it can be suggested that virulence determinants are a common trait within the entire genus Enterococcus. Semedo et al claim that clinical strains of the bacteria seem to be associated with high virulence potential whereas food, commensal and environmental strains have fewer virulence determinants in them. In another study on virulence determinants, Eaton and Gasson (2001) report that clinical E. faecalis strains had more virulence determinants than did food strains and ‘all of the E. faecalis strains tested possessed multiple determinants’. The authors also report that ‘E. faecium strains were generally free of virulence determinants’ (2001, p.1628).
In another study giving reports on VE strains in Korea, Seo et al (2005), compare characteristics of VRE isolates from humans, poultry and pigs. They discuss that since 1989 there has been a rapid increase in the incidence of enterococcal bacteremia and endocarditis and all Vancomycin Resistant Enterococci (VRE) have emerged as an important nosocomial pathogen. Avoparcin has been used on animals for infections associated with VRE. According to this study, a multiplex polymerase chain reaction (PCR) method was established to detect and differentiate resistant types of Enterococci which helped in amplifying and classifying the van genes A, B, C and D that encode all the vancomycin resistant elements and properties. The authors used this method to investigate the types of VRE in two types of farms where avoparcin has been used and where avoparcin has not been used.
1091 animal feces samples were collected from 70 pig farms and 32 poultry farms and a total number of 425 Enterococci were isolated from these fecal samples. Among these 425 isolates, 6 of these showed high level vancomycin resistance with Minimal Inhibitory Concentration, MIC of 64-256mug/ml. Out of these 6 high level VRE, 3 were isolated from poultry farms that had used avoparcin and 3 that did not use the antibiotic. All the 6 high level VRE showed the presence of VanA gene. 67 of the isolates showed a low level vancomycin resistance and this was associated with the presence of VanC gene. The authors also conducted a comparative analysis to find genetic relatedness between the high level VRE of six animal isolates and 31 human isolates that were obtained. Animal isolates did not show much common with human isolates of bacterial strains, according to this study although similarities between human VRE isolates were seen.
In a study performed in South India, Prakash et al (2005) claim that E. faecium and E. faecalis constitute 90% of the clinical isolates of Enterococci, the leading cause of nosocomial infections. However, they suggest that despite the preponderance of E. faecium and E. faecalis, there has been am alarming increase in other resistant enterococcal species and these have showed considerable resistance to beta-lactams and glycopeptides. The authors reiterate that instead of grouping all Enterococci in one category, the proper grouping and categorization of Enterococci at the species level. To report new and unusual resistant strains of Enterococci, the authors conducted the study in a hospital care unit for 2 years. Isolates of Enterococci were collected from the clinical samples along with thorough antimicrobial susceptibility testing. Species validation was done and results analyzed. According to this study there was a prevalence of atypical species of Enterococci which was neither E. faecalis nor E. faecium and of the 46 isolates , 15 were E. gallinarum (comprising 6.2%), 10 of these were E. avium (making 4.1%), 6 were E. raffinosus (2.5%), 6 were E. hirae (2.5%), 4 E. mundtii (1.7%), and 3 were E. casseliflavus-including the two atypical isolates (1.2%), and 2 E. durans (comprising of 0.8%). 5% or 12 isolates were confirmed as E. faecalis, E. faecium and E. casseliflavus.
The authors note that 'antimicrobial susceptibility testing depicted the emergence of high-level aminoglycoside and beta-lactam resistance' and this was found among different species apart from intrinsic vancomycin resistant varieties, while all the species that were obtained in these tests were susceptible to linezolid and teicoplanin. Prakash et al (2005) conclude that their study shows emergence of multidrug-resistance among the more uncommon and unusual species of Enterococci and this poses a serious therapeutic challenge for medical practitioners. The authors' reiterate that precise identification of enterococcal strains at the species level is absolutely necessary to have any access to species specific antimicrobial resistance as also knowing the epidemiological patterns to determine which drug is effective for which strain of enterococcal and which drug is ineffective. Only identification of exact species and the related susceptibility and resistances can give a comprehensive picture of future therapeutic efficacy.
Takeuchi and colleagues (2005) discuss an interesting case of transfer of gentamicin and erythromycin resistant traits. They examined the drug resistance and transferability of resistance in 218 Enterococcus faecium clinical isolates which were obtained from in-patients of a Japanese university hospital for a period of 9 years from 1990 through 1999. These findings indicated that among 218, 161 or 73.9% isolates were drug resistant to at least one drug and among these 58.2% or 127 were resistant to two or more drugs. E. faecium which is vancomycin resistant Enterococci was not isolated from these patients. The authors used broth and filter mating to study the transferability of drug-resistance to E. faecium strains.
6 of the 48 gentamicin resistance Enterococci and 50 of the 101 erythrocin resistance traits were transferred by filter mating. The gentamicin resistant traits of 5 isolates and the erythromycin resistant traits of four isolates were transferred to recipient strains. The authors report that each of the 4 erythromycin resistant trans-conjugants which were obtained by broth mating harbored a large conjugative plasmid. These plasmids showed no homology with the well-characterized enterococcal conjugative plasmid. The erythromycin resistant traits which were transferred by filter mating were actually transferred from a chromosome of donor strain to different sites within the pheromone responsive plasmid of the recipient strain and this suggests that the erythromycin resistance trait is encoded on a conjugative transposon (Takeuchi et al., 2005).
The antibiotic properties of oxazolidinone antimicrobial linezolid have been considered as effective for the treatment of infections caused by Gram-positive bacteria including the vancomycin resistant Enterococci (VRE). Krawczyk et al (2004) reported the first case of isolation of a linezolid resistant vancomycin resistant Enterococcus faecium strain in Poland from a hematological unit in a hospital. According to the authors, PCR-RFLP analysis of rDNA and allele-specific PCR of the domain V region of 23S ribosomal RNA gene demonstrated the presence of G2576U mutation which was previously associated with linezolid resistance.
Coleri et al (2004) studied 23 antibiotic resistant clinical isolates of Enterococcus spp, that caused infection in a hospital in Turkey to determine the antibiotic resistance patterns and the resistance plasmids involved. The biochemical and physiological identification test were applied by the Vitek system and results of protein profiles were compared. According to the study, of the 23 isolates, 20 were identified as E. faecalis and 2 were E. faecium whereas 1 was E. gallinarum. 24 different kinds of antibiotics belonging to 10 different groups were used in the susceptibility tests. In 10 of 23 enterococcal isolates, multiple antibiotic resistance was found and overall resistance to the used antibiotics was 47.3% and the low level resistance recorded was at 16.65%.
Among the isolates tested, 8.7% demonstrated a high level gentamicin resistance (HLGR), 17.4% demonstrated high level streptomycin resistance and 43.5% showed high level penicillin resistance. 34.8% of Enterococci showed high level vancomycin resistance and 60.9% exhibited low level resistance to vancomycin and teicoplanin. The plasmid sizes were seen to range form 2.08 to 56.15 kb. Two different plasmids with molecular sizes of 33.49 and 13.6 kb were seen in experiments where antibiotic acriflavine was used. In this case, the first plasmid determined glycopeptides and penicillin resistance and the second plasmid determined either glycopeptide or penicillin resistance as found in two different strains of E faecalis. A 22.58 kb plasmid showing and determining kanamycin resistance was also detected in an E. faecium strain. After treatments with antibiotics were done, there was an elimination of protein bands as seen in certain isolates of E. faecium and E. faecalis. Coleri et al conclude that their study indicates the increase of antibiotic resistant Enterococci especially of the VRE variety in hospital isolates.
In a study on antimicrobial resistance shown by enterococcal strains isolated from university hospitals in Brazil, Maschieto et al (2004) reports the results of antimicrobial resistance seen in Enterococci isolated from the intestinal tracts of patients. The identification of strains was at the species level and was performed by suing the conventional biochemical tests API 20 Strep (bioMerieux) and polymerase chain reactions assay. The species distribution reported was 34% E. faecium, 335 E. faecalis, 23.7% E. gallinarum, 5.2% E. casseliflavus. 1% E. avium and 1% E. hirae. The intrinsic resistance to vancomycin was characterized by the presence of VanC genes and was found in E. gallinarum and E. casseliflavus.
According to the authors, the high prevalence of VanC phenotype Enterococci is important because these species have been reported as causing a wide variety of diseases. The authors did not find vancomycin resistant E. faecium or E. faecalis and none of the isolates of these species was a beta-lactamase producer as well. Maschieto et al also report in the study that 13.4% clinical isolates showed multi-resistance patterns defined by resistance to three classes of antibiotics plus resistance to at least one aminoglycoside that is either gentamicin or streptomycin or both. The resistance to several antibiotics is shown by enterococcal strains as also seen in this study and Maschieto and his colleagues express concern on this because of the consequent decrease in therapeutic options available for treatment of infections caused by resistant Enterococci.
In a relevant study on the transfer of enterococcal infections in hospital set up via the hands of health care workers, Duckro et al (2005) conducted a systematic study to determine the exact roles of contaminated hospital environment and patients skin carriage in the spread of vancomycin resistant Enterococci. The authors mention that the transfer of VRE via the hands of health care workers in hospitals is generally assumed but not definitely known. This study was done to determine the frequency of VRE transmission from sites in the environment or on patients' skin to the clean environmental or skin sites via the contaminated hands of health care workers that can happen during routine daily care. For their method, the authors cultured sites on the intact skin of 22 patients which were colonized by VRE and also cultured sites in patients' rooms before and after they received routine care by 98 health care workers. The sites touched by the health care workers were recorded. Cultures were obtained from hands of health care workers and also from gloves obtained before and after care.
All the isolates that were obtained from sites went through pulsed-field gel electrophoresis. According to the authors, a transfer of infection was noted to have occurred when a culture-negative side becomes positive with VRE pulsotype after being touched by a health care worker (HCW) who had the same pulsotype on his or her hands or gloves and who had earlier touched a colonized or contaminated site. The results indicated were as follows - the health care workers touched 151 negative sites and after they touched the sites tested positive for VRE. 10.6% of these negative sites became positive for VRE after contacts. The number of times that contact with a site led to a transfer was found to be highest for antecubital fossae (flexor fossae tumors) and blood pressure cuffs. The authors concluded that there is a 10.6% chance of vancomycin-resistant Enterococci to be transferred from contaminated sites in the environment or on patients' intact skin to the clean sites via the hands or gloves of healthcare workers. From this they suggest that decontaminating the environment and patients' intact skin may be an important infection control measure for controlling of the spread of VRE.
Going from studies on prevention of the spread and transmission of enterococcal infections, if we turn our attention to the treatment of diseases and the recent medical research that is going into developing new drugs for enterococcal infections, we have the following report. Torres-Viera and Dembry (2004) reviews recent publications on the new antimicrobial drugs that are used for the treatment of vancomycin resistant Enterococci. Newer drugs against vancomycin resistant Enterococci are becoming available or will be available soon and these drugs are under constant review. Quinupristin-dalfopristin, a streptogramin, and linezolid, which is an oxazolidinone, have been considered as effective for treatment of VRE. However the authors claim that although these drugs are safe and effective they are bacteriostatic against Enterococci. In their study they have described bacterial isolates which are resistant either to streptogramin or to linezolid. Daptomycin which is a lipopeptide antimicrobial also has good in-vitro bactericidal activity against Enterococci however the authors claim that since very limited clinical data exist regarding the treatment of any serious enterococcal infections using this particular compound, it cannot be elaborately discussed.
Ramoplanin is another glycolipodepsipeptide antimicrobial used in clinical trials and it is tested for its initial administrations for the first time although it is not systematically absorbed after oral administration but it can be effective for the prevention of bloodstream infection in patients colonized with vancomycin resistant enterococcal strains. The effectiveness of Ramoplanin is being currently evaluated. Oritavancin and dalbavancin - these are both glycopeptides and tigecycline which is a monocycline derivative are being evaluated in different phase trials, yet they are not yet commercially available. The authors summarize that the treatment of vancomycin resistant Enterococci is problematic although these new drugs Daptomycin, Ramoplanin and linezolid variations are giving some hope that treatment of vancomycin resistant Enterococci may not be ultimately impossible. The authors conclude that the 'rational use of antibiotics, strict guidelines for the use of new compounds, and adherence to infection control practices continue to be essential components of the management of vancomycin-resistant Enterococci colonization and infection' (Torres-Viera and Dembry, 2004, p.541).
In this section we discussed several aspects of enterococcal infections, occurrence, prevalence and spread. We also gave examples citing case studies reported by several researchers in the field. All the reports, on enterococcal and VRE infections seem to indicate the fact that enterococcal infections are on the rise and with increasing resistance to nearly all therapeutic methods available, medical research needs to do some serious thinking on how to counteract these different stronger strains of bacteria. Intrinsic resistance is generally low level resistance but is seen in E. faecium as well. Intrinsic resistance is non-transferable as it is programmed and chromosomally mediated. Acquired resistance is completely transferable, its transmission is rapid and widespread, and all vancomycin and ampicillin and penicillin resistances are usually acquired resistance.
The studies discussed here are varied with comparison of enterococcal stains obtained from humans, poultry and pig isolates and the preponderance of unusual strains of Enterococci in isolates obtained from hospitals in India. Among the preventative and treatment methods, several researchers discussed here emphasize the need to identify the different strains of Enterococci as each enterococcal strain seems to have not only different characteristics but also different resistances and show different susceptibility levels or resistant levels to different antibiotics. Identification of different strains and isolation of the right kind of strains and treatment of each strain differently is required for proper and effective treatment of enterococcal infections.
Thus we can say the first major step to correct eradication of enterococcal infections is identification and differentiation of the different enterococcal species. The second step involves prevention of enterococcal transmission by taking extra care in hospital environments and by maintaining cleanliness and decontamination of all medical equipment and devices such as gloves. Proper washing of hands by hospital workers, as also complete disposal of bed linen and other personal belongings of the patients should be maintained with complete and separate treatment of the patient in hospital environment. The treatment of enterococcal infections include applications of vancomycin as a final therapeutic intervention , or for vancomycin resistant strains, a combination of strong antibiotics as several studies have indicated that a combination of antimicrobial drugs can be more effective than just one type. Finally the need to develop medical research to the extent that it can counter resistant bacterial strains and discover drugs stronger than vancomycin to outsmart the very sturdy and resistant Enterococci has also been consistently mentioned in medical journals and research articles.
We began this dissertation essay with an introductory note on the development of resistant enterococcal strains since its discovery from endocarditis in the end of the 19th century. The enterococcal strain isolated not only showed strong survival properties but also from t
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