REVIEW ARTICLE
By
Prof. Dr.
Kamal Fahmy
INTRODUCTION
Since the discovery of penicillin G and its first use in 1944 (91), the war has not been stopped between bacteria and man. Bacteria have developed mechanisms to resist the action of penicillin and have been succeeded and man has developed new antibiotics to which resistance has been also occurred after a short period of successful use of each developed anti-bacterial agent.
In 1944, nearly all Staphylococcus aureus isolates were susceptible to penicillin, by 1950 the percentage of susceptible isolates decreased to 50% due to appearance of penicillinase-producing strains of Staphylococcus aureus. For these strains penicillinase-resistant penicillins including methicillin were introduced, but victory was brief and methicillin-resistant strains were rapidly developed (91).
In the last years, we have been forced to limit our therapeutic options in face of these insidious enemies. Microbial populations especially gram-positive bacteria have developed resistance to most of the traditionally used antibiotics leaving some few options including vancomycin which has been considered the last weapon to be used against multi-resistant gram-positive bacteria (13).
Vancomycin is the first member of the glycopeptide group of antibiotics in discovery and also still the main clinically used glycopeptide antibiotic worldwide followed by teicoplanin. Vancomycin was discovered during a study done on a soil sample taken from jungles. The soil sample was found to contain a microorganism, Streptomyces orientalis, which produced a substance found to show anti-bacterial activity against gram-positive bacteria, this substance was later called vancomycin (69).
The use of vancomycin for treatment of gram-positive bacterial infections has been limited to special situations including resistance or allergy to other favored common antibiotics (69).
This limited use was due to many reasons, including the relatively high degree of adverse reactions that include skin rash (red man syndrome), thrombophlebitis, ototoxicity (nerve deafness), and nephrotoxicity. So high care has been necessary for using vancomycin (27,135).
Another reason for limited use of vancomycin is the relatively non-simple administration, as it requires slow intravenous method of administration. This is because vancomycin is poorly absorbed from the intestine and so can not given by oral route except in case of use for treatment of antibiotic-associated pseudo-membranous colitis where it should be given by oral route (27,120).
Lastly, but the most important reason for limited use of vancomycin is the fear that its wide use may encourage the emergence of resistance to this valuable drug (156).
This limited use of vancomycin may actually account to the markedly delayed appearance of resistance to it compared to other newly introduced antibiotics. Vancomycin showed uniform activity against gram-positive bacteria for more than 30 years following its introduction in 1956 (81).
But in recent years, some reports from different parts of the world showed emergence of vancomycin-resistant strains particularly among enterococci. This emergence was first reported in 1988 (148), which was so, the year of arrival of a new term in clinical microbiology named: vancomycin-resistant enterococci (VRE).
Moreover and more recently, emergence of vancomycin-non-susceptible staphylococcal strains (including both intermediate and resistant bacteria) was reported among both of S.aureus (13,79,144) and coagulase negative staphylococci (58,132), which has caused considerable concern in the last few years and again created a new term in medical microbiology named: vancomycin-resistant staphylococci.
Actually, the resistance to vancomycin, in most cases, is usually associated with cross-resistance to the other glycopeptide; teicoplanin and so these bacteria may be more accurately described as glycopeptide-resistant bacteria. However the term of vancomycin-resistant bacteria has been more commonly used to describe this condition in general (60,102).
Anyhow, The recent emergence of resistance to glycopeptides antibiotics among gram-positive bacteria, actually, represents a new medical challenge as vancomycin has long been considered the last active agent against the multi-resistant gram-positive bacteria. Great efforts are now done in the field of medical research to improve our knowledge and understanding of vancomycin resistance. This article reviews the major aspects of vancomycin resistance of gram-positive bacteria, mainly in enterococci and staphylococci.
Mechanism of Antibacterial Action of Vancomycin
Vancomycin and the other glycopeptide, teicoplanin, are narrow spectrum bactericidal agents that are active against gram-positive bacteria including staphylococci, streptococci, enterococci, Bacillus anthracis, Corynebacterium diphtheria and clostridia. They are not effective against gram-negative bacteria or mycobacteria (12,69).
Glycopeptides, in general, are large in size and have high molecular weights and so can not pass through the outer membrane of gram-negative bacteria to reach the target site. Thus, all gram-negative bacteria are resistant to glycopeptides including vancomycin while nearly all gram-positive bacteria are susceptible (120).
Glycopeptides have a bactericidal action, which is mediated by inhibition of bacterial cell wall, which results in bacterial cells surrounded only by the fragile cytoplasmic membranes. As the osmotic pressure inside the bacterial cells is usually higher than the surrounding environment, the bacterial cell will rapidly take up fluid, swell and explode (27).
The specific target for the antibacterial action of glycopeptides, in the bacterial cell wall, is the peptidoglycan layer. It is clear from the name given for this layer that it is composed of two components: polysaccharides (glycan) and polypeptides (peptido) (27).
The polysaccharides in the peptidoglycan layer are alternating N-acetylglucosamine and N-acetylmuramic acid, while the polypeptides are present in the form of side chains attached to N-acetylmuramic acid (27).
For synthesis of the peptidoglycan layer, two molecules of D-alanine are joined by a ligase enzyme to form D-alanyl- D-alanine (D-Ala-D-Ala) dipeptide which is then added to N-acetylmuramyl tripeptide to form N-acetylmuramyl pentapeptide. Through the terminal D-alanyl- D-alanine, cross bridging occurs between the pentapeptide side chains (Transpeptidation) which is accompanied by binding of N-acetylmuramic acid to N-acetylglucosamine (Transglycosylation). These two processes of transpeptidation and transglycosylation result in polymerization of peptidoglycan layer as a single giant molecule (12).
Vancomycin and teicoplanin produce their anti-bacterial action by binding with high affinity to the D-Ala-D-Ala termini of the N-acetylmuramyl pentapeptide molecules leading to prevention of transpeptidation and transglycosylation and so inhibition of peptidoglycan synthesis (12,120).
Mechanisms of Bacterial Resistance to Vancomycin
Mechanisms of Resistance to Vancomycin in Enterococci
VanA Vancomycin Resistance
This is the most common type of vancomycin-resistant enterococcal strains. Enterococci belonging to this type show high-level resistance to vancomycin (MIC ≥ 64 ug/ml) with low levels of resistance to teicoplanin (16-512 ug/ml). Most VanA enterococci are isolates of Enterococcus faecium followed by E. faecalis, but this type may also rarely detected in E. avium, E. casseliflavus, E. durans, E. gallinarum and E. raffinosus (100).
VanA resistance has been found to be under the control of a gene cluster located on a transposon designated Tn1546. This transposon usually resides on a plasmid but may also reside on the bacterial chromosome (10). The resistance genes can be transmitted through conjugation to different species of enterococci (26) and even to other genera including staphylococci (108).
VanA cluster includes the following genes: vanR, vanS, vanH, vanA and vanX genes (160). Expression of these genes of VanA cluster results in synthesis of peptidoglycan precursors terminating in a depsipeptide D-alanyl-D-lactate (D-Ala-D-Lac) instead of the usual D-Ala-D-Ala terminus. Vancomycin binds to D-Ala-D-Lac with markedly lower affinity than it does to the normal dipeptide product (30,116).
VanA protein is a ligase enzyme of altered substrate specificity, which produce the depsipeptide D-Ala-D-Lac (29)
VanH protein is a D-hydroxy acid dehydrogenase enzyme, which reduces pyruvate to D-lactate and so creates a pool of D-lactate for use in the above reaction of VanA ligase (8).
VanX protein is a D, D-dipeptidase enzyme lacking activity against D-Ala-D-Lac. This enzyme cleaves D-Ala-D-Ala produced by the ordinary ligase to minimize the competing synthesis of normal pentapeptide (121,164).
Also, VanA cluster carries two other essential regulatory genes designated vanS and vanR, which regulate the transcription of the above mentioned vanH, vanA and vanX genes. The vanS gene is termed so because its protein product is a sensor protein that is membrane-associated and detects the presence of vancomycin through an unknown mechanism (5). When the sensor protein is activated by exposure to vancomycin, the active product of the sensor protein signals the cytoplasmic VanR protein (the response regulator) which acts as transcriptional activator which activates a promoter that lies between vanS and vanH genes. Then the activated promoter activates co-transcription of the main glycopeptide resistance genes: vanH, vanA, and vanX genes (6).
So, vanA gene is not able alone to produce vancomycin resistance, which is produced by the combined action of the three enzymes that are encoded by the three main resistance genes: vanH, vanA and vanX. In turn, these main resistance genes are under the control of additional and also essential two regulatory genes: vanR and vanS (5).
The transposon Tn1546 also carries two other genes, vanY and vanZ located downstream to the VanA cluster of genes. These two genes may contribute to but are not essential for glycopeptide resistance. VanY protein is a D, D-carboxypeptidase and it is associated with cytoplasmic membrane and its production is inducible by vancomycin which suggest that its transcription may be regulated with the VanA cluster. It cleaves the terminal D-Ala from any normal membrane-bound peptidoglycan precursors that may have been escaped the action of the VanA cluster of genes. Its action contributes to the level of resistance to the glycopeptide antibiotics (9). VanZ protein increases the MICs of teicoplanin but not of vancomycin, through mechanisms that have not been clarified (7).
VanB Vancomycin Resistance
This is the second common type of vancomycin-resistant enterococcal strains. Enterococci belonging to this phenotype show low level resistance to vancomycin (MIC > 8 ug/ml) while show sensitivity to teicoplanin (MIC ≤ 8 ug/ml). VanB resistance is induced following exposure to vancomycin only not to teicoplanin while the previously described VanA resistance is induced after exposure to either vancomycin or teicoplanin. This phenotype is usually detected among E. faecalis followed by E. faecium but it may be also rarely detected among other enterococcal species (100).
As with VanA class, this VanB resistance is controlled with a gene cluster (VanB gene and related genes). The gene cluster of VanB class is located on a transposon termed Tn1547. This transposon usually resides on the bacterial chromosome but rarely on a plasmid (118). Genes responsible for VanB type of vancomycin resistance may be transmitted by conjugation to different species of enterococci (74) and also to genera other than enterococci, which was recorded in an isolate of Streptococcus bovis (117).
The protein product of vanB gene is an abnormal ligase (VanB) that is structurally related to VanA ligase (76% amino acid homology). VanB ligase also favors production of abnormal peptidoglycan precursors with pentapeptide that terminates in the depsipeptide D-Ala-D-Lac (57).
Genes of VanB-type resistance that correspond to those of VanA-type resistance are designated vanRB, vanSB, vanHB, vanXB, and vanYB. They show the same activities as in VanA class but in contrast to VanS protein of the VanA class; the VanSB protein does not recognize teicoplanin, which explains the inability of this glycopeptide to induce VanB resistance. Bacteria with VanB resistance show no corresponding gene to vanZ of VanA type-resistant bacteria (11).
Recent studies reported that vanB gene shows DNA sequence heterogeneity that allows subtyping of vanB gene into 3 subtypes: vanB -1, vanB-2 and vanB-3 (43, 113). With vanB-2 the most prevalent type (160)
VanC Vancomycin Resistance
On contrast to both VanA and VanB resistance, which are acquired and transferable types, VanC type of resistance is an intrinsic constitutive property that was detected in Enterococcus gallinarum, Enterococcus casseliflavus and Enterococcus flavescens. This form of resistance shows low-level resistance to vancomycin (MIC of 8-32 ug/ml) and sensitivity to teicoplanin (<8>256 ug/ml) than normally expected and also resulting in resistance to teicoplanin (50).
VanD Vancomycin Resistance
VanD type of resistance was first described after recognition of a new resistance gene designated vanD in 1997. The strain carrying this resistance gene was E. faecium strain and showed MIC for vancomycin and teicoplanin of 64 and 4 ug/ml respectively (114).
More studies showed that vanD gene is a ligase gene that found on partial sequencing to be distinct from but similar to VanA and VanB genes and the amino acid sequence of VanD showed 67% identity to those of VanA. The vanD gene was found to be located on chromosome and is not transferable to other enterococci (111).
In more recent studies, VanD-type-resistant enterococci have been found to be constitutively resistant to vancomycin by nearly exclusive synthesis of peptidoglycan precursors terminating in D-Ala-D-Lac. The vanD gene has been identified and found to encode for a ligase similar to that of VanA and VanB types. Also, other genes of VanD cluster that correspond to those of VanA-type were identified and designated vanHD that encodes for a dehydrogenase, vanXD that encodes for a dipeptidase, vanRD that is a regulator gene and vanSD that encodes for a sensor protein (45).
VanE Vancomycin Resistance
More recently, a new vancomycin resistance gene designated vanE has been identified in E. faecalis BM4405, which has been resistant to low levels of vancomycin (MIC: 16 ug/ml) and susceptible to teicoplanin (MIC: 0.5 ug/ml) (59).
This new resistance type has similarities to the intrinsic VanC type of resistance. The amino acid sequence of VanE protein has greater identity to VanC (55%) than to VanA (45%) and VanB (43%) or VanD (44%) (59).
Vancomycin-Dependent Enterococci
Not only vancomycin resistance but also vancomycin dependence has been observed in some strains of VanA and VanB types of resistance to vancomycin in enterococci. Vancomycin-dependent enterococci have been isolated from apparently culture-negative clinical samples by plating them on vancomycin-containing agar, such as that used for isolation of campylobacter or gonococci (46).
A possible explanation of this phenomenon is that these organisms may turn off their normal production of D-Ala-D-Ala and then can grow only if a substitute structure is made. With most VanA and VanB types of resistance, this occurs only in presence of vancomycin, which induces synthesis of VanH dehydrogenase and VanA or VanB ligase that make D-Ala-D-Lac (102).
The reason for the turning off the synthesis of D-Ala-D-Ala by the bacterial cell may be that as long as vancomycin is present, D-Ala-D-Ala is not necessary for cell wall synthesis and even it is destroyed by the action of VanX. Once the vancomycin is removed, D-Ala-D-Lac is no longer synthesized and the cell stops to grow or replicate, as it needs to either D-Ala-D-Ala or D-Ala-D-Lac (46,102).
Reversion to vancomycin independence has been observed which may be explained by either a mutation that leads to constitutive production of D-Ala-D-Lac or turning on the usual mechanism for synthesis of D-Ala-D-Ala (46,103).
Mechanisms of Resistance to Vancomycin in Staphylococci
Early reports have been demonstrated the presence of phenomenon of tolerance to vancomycin among some strains of S. aureus especially those that are methicillin-resistant. Tolerance has been explained failure of treatment with vancomycin of infections caused by these bacteria inspite of the detected susceptibility according to the MIC results. Moreover, some studies have shown increased mortality in patients with bacteremia or endocarditis due to these tolerant S.aureus strains inspite of vancomycin therapy (97,119).
Moreover, in the last years, some studies have been reported the emergence of vancomycin-resistant strains in staphylococcal clinical isolates that, by definition, show in-vitro MICs of >4 ug/ml. This has been reported in both S. aureus and coagulase negative staphylococci (125,132).
This emergence was significantly marked in hospitals that had been endemic with strains of methicillin-resistant staphylococci and followed the policy of empirical use of glycopeptides. Extensive use of glycopeptides (vancomycin or teicoplanin) allowed selection of resistant strains from both of S. aureus and coagulase negative staphylococci, mainly among S. epidermidis followed by S. hemolyticus, S. hominis and S. warneri (35,58,125).
This view is supported by the laboratory finding that glycopeptide-resistant mutants of S.aureus have been experimentally selected by gradually increasing the levels of vancomycin present during in-vitro growth (131).
Also, this view is supported by the observation that heteroresistance to vancomycin is a common phenomenon among staphylococci (60,159). The presence of this heteroresistance may allow selection of resistant strains in vivo when infections caused by these bacteria are treated with vancomycin.
The presence of vancomycin heteroresistance phenomenon in staphylococci may arise the need to modify the methods used for detection of vancomycin resistance in the clinical microbiology laboratory. The inoculum tested should be sufficiently large to compensate for the frequency of expression.
The resistance of staphylococci to vancomycin has been found to be reversible under laboratory conditions. In an experiment to prove this phenotypic reversion, S. aureus isolates showing resistance to vancomycin were repeatedly cultivated on non-selective media. After 15 days of passages, vancomycin and teicoplanin-susceptible strains were obtained from each isolate. Moreover several obtained strains lost the subpopulations with vancomycin resistance. This reversion of vancomycin-resistance phenotype may explain the difficulty in isolating vancomycin-resistant clinical isolates of staphylococci from patients who do not respond to vancomycin therapy and possibly may account for some of the difficulties in identification of such isolates in the clinical microbiology laboratory (25).
In a case study, primary testing of clinically isolated S. epidermidis strain showed resistance to vancomycin. As the result in such situations should be confirmed, the test was repeated from the isolate subculture and the surprising result was that the strain showed sensitivity to vancomycin even on more repeated tests. In such conditions, the question arises if the first primary test was in error and so, the isolation from the same patient was repeated and the tests were done again. The results were the same as with the previous isolation, which indicated that the primary susceptibility test was not in error but the problem was in an elusive phenotype (49).
These findings may put a base for a new modification in cultivation of staphylococci in the clinical laboratory. It may be better to use a medium containing a low level of vancomycin as well as the routine media to ensure that vancomycin-resistant isolates can be maintained on the medium with low level vancomycin. Also, we should consider the primary results of vancomycin susceptibility tests and not the results of those done from subcultures.
The mechanism of resistance of staphylococci to vancomycin is still not clear. But transfer of the VanA gene cluster from enterococci to Staphylococcus aureus has been demonstrated in the laboratory by in vitro experiment and also on the skin of mice. This finding has raised concern about the possibility of occurrence of such mechanism of genetic transfer in clinical isolates of staphylococci, which often co-colonize wound infection sites with enterococci (108). However, there is no study showed the presence any of Van genes clusters in clinical isolates of staphylococci and so such mechanism can not be proved up till now (60).
Some current observations may lead to clarification of the possible mechanism(s) of staphylococcal resistance to vancomycin. From these observations, it has been observed that vancomycin-resistant staphylococci in presence of vancomycin in a liquid medium form aggregates of bacterial cells. The drug is then become undetectable in the medium followed by disintegration of the bacterial aggregates into single cells with the same appearance of cells grown in antibiotic-free medium. Examination of the bacterial aggregates by electron microscope showed the production of large amount of extracellular material, which had staining properties similar to that of the cell wall material. Vancomycin could be recovered from the cell wall material in biologically active form. These findings may suggest that these bacteria may resist the action of vancomycin through the ability to sequester the antibiotic molecules in a bound form to an extra material of cell wall (64,131,132).
In a recent study, 16 clinical isolates of vancomycin-resistant S.aureus from 7 countries were subjected to serial daily passages in drug-free medium. After 10-84 days of these passages, strains susceptible to vancomycin (MICs: < name="Epidemiology">Epidemiology of Vancomycin-Resistant Bacteria
Prevalence of Vancomycin-Resistant Bacteria
Prevalence of vancomycin-resistant enterococci increased dramatically in last years. Different studies have shown that the proportion of vancomycin-resistant strains among isolated enterococci rose gradually to reach an average percentage near to 25% in the last years, the initial rise was observed in intensive care units followed by general hospital wards (33, 73,127).
In the recent years, the average percentage of methicillin-resistant staphylococci in different communities is around 50 % of total hospital staphylococcal infections (58,60). However, the vancomycin-resistant strains of staphylococci are still of low prevalence. These currently low-prevalent strains are actually of great importance as they were isolated from different countries in the world (58,80,137,144,152) and evidences accumulate suggesting that there is great possibility to be more common by time because the number of reports about these vancomycin-resistant strains increases year after year. Moreover, the reported heteroresistance to vancomycin among staphylococci may allow selection of more resistant strains by time (60).
The prevalence rates of vancomycin-resistant S. aureus differ widely between different studies, for instance these prevalence rates ranged in Japanese studies from 1.3 up to 20%. This discrepancy may be attributed to the current controversy that surrounds the methods of detection of these strains with heteroresistance to vancomycin (152).
In an epidemiological hospital study done through one year, it was found that among 535 subjects with coagulase negative bacteremia, 20 subjects had a glycopeptide-resistant strain (19 strains were resistant to teicoplanin and 1 was resistant to both teicoplanin and vancomycin). The isolated resistant strains were mainly from intensive care units followed by medical and then surgical wards. Mortality rates were 25% among cases infected with resistant strains and 18% among other control cases infected with the same type of infections but caused by vancomycin-susceptible coagulase negative staphylococci which was significant difference (142).
Actually, mortality rates are becoming very high among patients infected with vancomycin-resistant gram-positive bacteria especially cases with bacteremia and/or endocarditis in which choice of treatment regimen with bactericidal activity is critical for success. For instance in a series of 125 episodes of enterococcal bacteremia, death occurred in 17.6% and in 8% of deaths, the fatal outcome was considered directly related to bacteremia (67).
Origin of Vancomycin-Resistant Bacteria
The origin of vancomycin resistance in gram-positive bacteria appears to be not the same in all emerged strains. This may be suggested from absence of uniform mechanism for vancomycin resistance between enterococci and staphylococci and also presence of different genotypes and subtypes for resistance among enterococci, which suggest that Van clusters have acquired by enterococci on multiple occasions followed by horizontal dissemination particularly of VanA and VanB (160).
Moreover, characterization of staphylococci that have been emerged in different parts of the world by pulsed field gel electrophoresis (PFGE) analysis showed non-identical pattern between isolates from different countries which suggested that these isolates do not represent one clone that has been disseminated (145).
Nosocomial Origin
It has been suggested that most vancomycin-resistant enterococci originate in hospitals and then spread to the community (61). Also most vancomycin-resistant strains of staphylococci appear to have originated in hospitals under the selective pressure of vancomycin use in these hospitals. These strains caused outbreaks of infection in some of these hospitals (80,144)
However, some studies showed that glycopeptide-resistant enterococci might be isolated from outpatients with no history of hospitalization, which has been suggested that hospital is not the single origin for glycopeptide-resistant enterococci (66).
Human Colonization Origin
Enterococci may be part of the normal flora of the intestine, mouth, urethra and vagina (103) and it was reported that vancomycin-resistant enterococci may be isolated from sewage and this may represent a source for these resistant bacteria to the community (147).
Once acquired, colonization by vancomycin-resistant enterococci can last for years serving as a reservoir for potential infection of the colonized patient and also to other subjects (73).
Selection of Resistant Strains
Many of the patients, with vancomycin-resistant gram-positive bacteria, have been previously exposed to vancomycin or teicoplanin therapy. This was reported in all of enterococci (72), S. aureus (144) and coagulase negative staphylococci (17,58). Exposure to vancomycin, either for treatment or for prophylaxis, may exert selective pressure, which results in increase of undetectable levels of pre-existing vancomycin-resistant bacteria to detectable levels. In such a case, vancomycin administration does not cause vancomycin resistance to develop and also does not increase the possibility of exogenous transmission of such bacteria to the patient but increases the likelihood of detection of vancomycin-resistant bacteria (73).
Early experimental studies showed that staphylococci cultivation in presence of sub-inhibitory glycopeptide concentrations could induce, under laboratory conditions, selection for clones for which the glycopeptides MICs are increased (15).
Also, stepwise and single-step exposure to teicoplanin and/or vancomycin has allowed mutant clones for which glycopeptide MICs are increased to be obtained from susceptible coagulase negative staphylococci (17,85).
Recent experimental studies done by bacterial populations analysis showed that glycopeptide resistance may be expressed heterogeneously in both S. aureus (1,77) and coagulase negative staphylococci (17,18). Heterogeneous resistance to vancomycin is defined as that observed in strains for which the conventional vancomycin MICs are <4> 8 ug of vancomycin per ml (78).
This heterogeneous resistance may result in detection of susceptibility of the organism to vancomycin in-vitro by the conventional MIC dilution method while in-vivo selected resistant subpopulations may be responsible for failure of therapy with vancomycin (1,18,78).
Non-Human Origin
Non-human origin of these bacteria has been suspected because vancomycin-resistant enterococci were isolated from farm animals (including duck, chicken, turkey, pig, pony and dog) and from each of five uncooked chickens bought from retail outlets (14).
These findings may suggest that vancomycin-resistant enterococci may pass from animals to man via the food routes especially in light of the reported findings that some human isolates are not distinguishable from isolates from non-human sources (14).
Avoparcin
Avoparcin is a glycopeptide not used for humans but may be used for animals as a growth-promoting factor. Glycopeptide-resistant enterococci were isolated from farms using this glycopeptide agent while no such bacterial isolates were detected in farms not using this agent and this finding may suggest that the use of avoparcin may represent a selective pressure for emergence of glycopeptide-resistant enterococci colonizing farm animals. These animals may be an important reservoir of glycopeptide-resistant enterococci for humans and even may be the origin or the source for these bacteria (88,140).
Avoparcin was used in Europe but not in the USA and this may explain a reported observation that colonization of healthy people with vancomycin-resistant enterococci is absent in the USA and endemic among humans and animals in Europe. From The European animal reservoir, vancomycin-resistant enterococci and resistance genes have spread to humans. This spread confirmed absence of boundaries between animals and humans, between healthy and diseased subjects, between countries and probably between continents (21,127).
The link between use of avoparcin and emergence of glycopeptide-resistant enterococci has been further confirmed by a study done in Netherlands and showed marked decrease in prevalence of colonization of healthy subjects with vancomycin-resistant enterococci after stopping use of this agent (149).
Avoparcin was used as a feed additive agent in New Zealand for poultry production from 1977 until June 2000. A recent study has been done to evaluate the effect of its use on prevalence of vancomycin-resistant enterococci. This study showed that these bacteria were only isolated from poultry fecal samples taken from farms where avoparcin was in use or previously used. The study showed that patterns of pulsed field gel electrophoresis (PFGE) of digested DNA of the poultry isolates of vancomycin-resistant enterococci were identical or closely related to human isolates of these bacteria in the same areas and molecular characterization showed identical transposons (Tn1546) in both human and poultry isolates (95).
Transmission of Vancomycin-Resistant Bacteria
Vancomycin-resistant enterococci have great ability for transmission. This ability has allowed these strains to cause several hospital outbreaks in different countries (103). In case of vancomycin-resistant staphylococci, the condition differs because these bacteria, in most cases, have been isolated from individual patients usually previously treated with vancomycin for long periods. There is no evidence for epidemiological spread of the recently emerged vancomycin-resistant staphylococcal strains except in very rarely recorded hospital transmission (35,60,142).
De novo emergence of vancomycin resistance in enterococci through genetic mutation induced by exposure to vancomycin has been found to be rare in any individual patient. Accordingly, the marked increase in vancomycin-resistant enterococci may be attributed mainly to rapid cross-transmission, which has occurred mainly in hospitals where these bacteria represent major causative agents of nosocomial infections (103).
Hospital environmental contamination has been found to be an important factor in transmission of vancomycin-resistant enterococci. This environmental contamination may be so marked in case of a patient with intestinal vancomycin-resistant enterococcal colonization and having diarrhea, which may result in hospital outbreaks (24,63,72,103).
Such situation has been commonly occurred in hospitals when a patient develops antibiotic-associated diarrhea caused by C. difficile and then treated with oral vancomycin which may allow for selection of intestinal vancomycin-resistant enterococci which find a rapid route for spread in the hospital through the already present diarrhea. This serious situation was the basis for the current recommendation to avoid vancomycin as a primary treatment of antibiotic-associated diarrhea (73).
Enterococci were found to have the ability to survive on a variety of hospital environmental surfaces for variable periods of time ranging from 1 hour up to 7 days. These findings suggested that the hospital environment is a possible reservoir for nosocomial infections with vancomycin-resistant enterococci (139).
In an outbreak of vancomycin-resistant E. faecium in medical-surgical ICU, the epidemic strain was isolated repeatedly from the rectal probe handles of three electronic thermometers. Disposable probe sheaths for protection of probes from contamination were in routine use in this unit but proved to be not satisfactory for this purpose (92).
These findings make necessary to improve the cleaning and decontaminating procedures to be satisfactory to avoid the possible role of hospital environmental contamination in spread of vancomycin-resistant bacteria.
Predisposing Risk Factors for Infection with Vancomycin-Resistant Bacteria
The most important predisposing risk factor for infections caused by vancomycin-resistant bacteria is the overuse or misuse of antibiotics. In patients colonized with low numbers of glycopeptide-resistant gram-positive bacteria, antibiotics may allow overgrowth of these bacteria with increased risk of infection and dissemination (73).
Not only vancomycin which select for colonization or infection with vancomycin-resistant bacteria but also some other antibiotics including broad-spectrum cephalosporins especially third generation members which show high biliary concentrations that kill most bacteria in the upper gastrointestinal tract with negligible activity against enterococci in general. The antibiotics with activity against anaerobic bacteria especially metronidazole and clindamycin may be also responsible for selection of vancomycin-resistant enterococci (73).
Other risk factors also include proximity to affected patients, care by colonized staff, use of invasive procedures, hospitalization in the previous year, number and length of stays in the hospital and also the transfer between hospitals (142,143).
For vancomycin-resistant enterococcal infections, colonization with vancomycin-resistant enterococci represents an important risk factor for different types of infections caused by these bacteria. From the colonization site, infection may occur in the form of urinary tract infections, surgical wound infections, peritonitis, bacteremia and endocarditis. This occurs more commonly in presence of a foreign material such as prosthetic heart valves and endovascular devices, joint prostheses and cannulas used in dialysis patients (103).
For vancomycin-resistant staphylococcal infections, risk factors include ongoing or recent dialysis, presence of foreign prosthetic material and recurrent infections with methicillin-resistant staphylococci. Many published reports suggest that vancomycin-resistant staphylococci appear to have developed from pre-existing methicillin-resistant strains that infected the patients in the months before detection of the vancomycin-resistant strains. These reports show similar pulsed field gel electrophoresis (PFGE) patterns between the early isolated methicillin-resistant strains and the newly developed vancomycin-resistant strains (60).
Laboratory Methods for Detection of Vancomycin Resistance
In the current years, clinical microbiology laboratories worldwide must be highly alert to detect glycopeptide-resistant strains of gram-positive bacteria in clinical specimens. This may allow early preventive and control measures to be taken to prevent spread of such strains. Definite criteria for interpretation of tests for glycopeptide susceptibility are markedly essential in this aspect. Also the choice of suitable method is of special importance in this field.
Criteria for Interpretation of Glycopeptide Antibiotics Susceptibility Tests
The criteria for determination of resistance to either vancomycin or teicoplanin by both disk diffusion and MIC methods are shown in the following table according to the national committee for clinical laboratory standards (104,105)
Organism
Antibiotic
Susceptible
Intermediate
Resistant
Zone D.* (mm)
MIC
ug/ml
Zone D.* (mm)
MIC
ug/ml
Zone D.* (mm)
MIC
ug/ml
Enterococci
Vancomycin
Teicoplanin
≥ 17
≥ 14
≤ 4
≤ 8
15-16
11-13
8-16
16
≤ 14
≤ 10
≥ 32
≥ 32
Other gram -positive bacteria
Vancomycin
Teicoplanin
≥ 12
≥ 14
≤ 4
≤ 8
10-11
11-13
8-16
16
≤ 9
≤ 10
≥ 32
≥32
* Zone diameters when using 30 ug disks on Muller-Hinton agar and after exactly 24 h of incubation (104,105).
According to the criteria of the national committee for clinical laboratory standards (NCCLS) the disk diffusion method for vancomycin shows interpretive criteria that differ between enterococci and other gram-positive bacteria. While no differences in these interpretive criteria are found between these two groups of bacteria when testing vancomycin by MIC method or testing teicoplanin by either disk diffusion or MIC methods (104,105).
However, there are some differences between the current American NCCLS interpretive criteria and the recommendations made to define categories of susceptibility to glycopeptides in some other countries. The British Society for Antimicrobial Chemotherapy has recommended the use of a single breakpoint concentration of 4 ug/ml to define resistance to both vancomycin and teicoplanin (163).
Also, in France, the “ Comite de l’Antibiogramme de la Societe Francaise de Microbiologie” has recommended MIC of ≤4 to define susceptibility of both vancomycin and teicoplanin, together with a zone diameter of >17 mm around 30-ug-disk for each of them (136).
Also, the Swedish Reference Group for Antibiotics recommends a single MIC breakpoint concentrations of 4 ug/ml for both vancomycin and teicoplanin and also recommends zone diameter of >16 mm for susceptibility to both glycopeptide antibiotics (83).
In contrast with criteria of NCCLS, those of British, French and Swedish recommendations make no distinction between tests performed on enterococci and those performed on other gram-positive bacteria (83,104,105, 136,163).
In the United States, the term “vancomycin-resistant S. aureus” is reserved for isolates with MICs >32 ug/ml. But outside the United States, This term may be used to refer to S. aureus with MICs >8 ug/ml which means classification of the isolates to either susceptible or resistant to vancomycin with no intermediate category. Some researchers prefer to use the term “non-susceptible isolates” to describe both the resistant and intermediate groups together (60).
Disk Diffusion Method
In all studies, the criteria of disk diffusion method for both vancomycin and teicoplanin show small difference between zones of inhibition of susceptible and resistant bacteria when compared to other antibiotics. This finding may be the cause that made the disk diffusion method of low discriminative power for detection of all levels of glycopeptide resistance, and this finding may be explained by the observation that glycopeptide antibiotics diffuse poorly through agar. The diffusion coefficients in Muller-Hinton agar are 0.72 mm²/h and 0.47 mm²/h for vancomycin and teicoplanin respectively (32).
Disk diffusion method is effective for detecting high level resistance to glycopeptide antibiotics (MICs > 256 ug/ml). At this level of resistance no zones of inhibition (diameter, 6 mm) occur around 30-ug-disk (161).
However, the diffusion method may be not so effective for detection of lower levels of resistance to glycopeptides when using NCCLS criteria (105) and different studies showed that the disk diffusion methods may show false susceptibility when compared to the standard MIC method. This finding was found in all of glycopeptide-resistant enterococci (128), S. aureus (144) and coagulase negative staphylococci (17,58).
E Test
A variation of the traditional disk diffusion test is the more recently introduced E test. The advantage of this test over disk diffusion is that it can determine the MIC for the tested organism. The E test consists of a plastic strip that contains a continuous gradient of antibiotic. So, the zone of inhibition occurs in the form of ellipse and the MIC is then read at the point where this ellipse touches the E-test strip (28).
Using E-test strip for determination of MIC of vancomycin for vancomycin susceptible organisms shows slightly higher MICs than when determination is done by agar dilution or broth microdilution methods but do not result in false evaluation of the test organism as resistant (128).
For teicoplanin, the MICs determined with the E test was found to be correlated well with agar dilution method for a collection of clinical and reference isolates of staphylococci and enterococci (153).
However, another study have indicated that E test shows lower teicoplanin MICs for coagulase negative staphylococci than does agar dilution which may lead to false susceptibility results (96).
Agar Dilution MIC Method
Agar dilution MIC method has been the standard method against which the other methods to detect glycopeptide resistance are evaluated. The method has the advantage of being based on agar dilution and so not affected by many of the problems associated with the diffusion methods. Also, it has the advantage of ability to use multipoint inoculator to allow multiple isolates to be tested on a single plate (104).
Vancomycin Agar Screening Method
An agar plate containing a single breakpoint concentration of vancomycin or teicoplanin may be used as a rapid screening method for glycopeptide non-susceptible bacteria. However, the screening concentration should be chosen carefully in order to maximize the sensitivity and specificity of the test because the selection of this concentration to be just equal to the MIC may lead to decrease in the specificity of the test especially at high inocula. It is recommended for breakpoint screening to use vancomycin concentration of 6 ug/ml in agar. An isolate should be examined by spotting 10 ul of a 0.5 McFarland concentration of this isolate onto the surface of a plate of brain heart infusion agar (rather than Muller-Hinton agar) and the test should be read after exactly 24 h. of incubation (104,141).
This method has the advantage of rapidly examining large number of isolates for vancomycin resistance because many isolates may be spotted easily on the same plate.
Broth Dilution MIC Method
Broth dilution method may be done in tubes and then called macrodilution method or done in microtitration plate and then called microdilution method. Both methods were found to correlate well with the reference agar dilution method (86).
Broth microdilution performed in cation-adjusted Muller-Hinton broth and read after a full 24 hours, is the method recommended by the NCCLS (104), and it has replaced agar dilution method as the reference standard method in some studies (87,146).
Automated Systems
As with the disk diffusion method, the automated systems, such as Vitek systems, show some difficulty in detecting resistance to glycopeptides especially if the level of resistance is low and so some false susceptibility results are reported by these systems (157).
These difficulties with the Vitek automated systems in this aspect made many reports to consider these automated systems unacceptable methods for evaluation of susceptibility of bacteria to vancomycin (165).
However, the problem with these automated systems may be in the software, which may be in need to some adjustments or alterations to pick up all levels of resistance to vancomycin (158).
Determination of MBC and Time Kill Tests for Detection of Vancomycin-Tolerant Staphylococci
Determination of MBC and time kill tests may be essential to detect strains of staphylococci that are tolerant to vancomycin inspite of results of MIC tests which by definition show susceptibility. Tolerance has been defined, in general, as an MBC:MIC ratio of >32. Also, tolerance may be defined in time-kill studies as kill of <99.9% name="treatment">Treatment of Vancomycin-Resistant Gram-Positive Bacterial Infections
The traditional treatment for gram-positive bacterial infections has been a synergistic combination of a cell wall active agent such as a β-lactam and an aminoglycoside (27).
But gram-positive bacteria gradually showed increasing resistance to β-lactams by production of β-lactamase enzyme and also increasing resistance to aminoglycosides by production of aminoglycosides-modifying enzymes (84).
Emergence and spread of resistance among gram-positive bacteria to the traditional antibiotics led to gradual increase of vancomycin use as the last weapon for treatment of infections caused by multi-resistant strains of gram-positive bacteria including enterococci, staphylococci and Clostridium difficile (166).
Vancomycin has been used for this purpose either singly or in combination with an aminoglycoside, as both drugs together show effective synergism both in vitro and in vivo. This synergism is based on an increased intracellular penetration of the aminoglycoside due to the effect of the glycopeptide on the bacterial cell wall (41,76).
However, emergence of vancomycin-resistant gram-positive bacteria in the last years has created a real therapeutic problem for infections caused by these bacteria as these bacteria are already resistant to most or even all other antibiotics (27,100). This problem led to occurrence of many types of infections that could not be treated with any available drug and resulted in increased morbidity and mortality due to these infections (67,142).
Moreover, when certain agent shows in vitro activity against some isolates of these bacteria, this agent may be only bacteriostatic and of uncertain efficacy in vivo or may be associated with high degree of toxicity (100).
So, this problem is still under condensed investigations and trails to create new weapons for these bacteria or at least to put a certain schedule of the current limited options to face this problem as possible.
Teicoplanin
Teicoplanin is the other glycopeptide to which many strains of vancomycin-resistant enterococci are susceptible (all phenotypes of vancomycin-resistant enterococci except VanA). However, in-vivo resistance had been found to develop to teicoplanin in a VanB strain, which has made this therapeutic option of limited use in these cases (75).
Combination of a Glycopeptide and a β-Lactam
A combination of a glycopeptide and a β-lactam was tried and found to show accepted efficiency against some strains of vancomycin-resistant enterococci that although show resistance to each of the two agents alone, are still inhibited by a combination of them (102).
Actually, the effect of this combination is antagonism when used against susceptible enterococci but when used for vancomycin-resistant strains, some of these strains may show synergistic effect with unknown mechanism (102).
However, this combination is still of limited use as the strains of vancomycin-resistant enterococci which show susceptibility to it are markedly few. Moreover, even these few strains may show subpopulations that are resistant to this combination and so can be preferentially selected after exposure to these combined drugs (103).
Combination of a glycopeptide and a β-lactam was also tried to decrease the emergence of vancomycin resistance among methicillin-resistant S. aureus and coagulase negative staphylococci. A combination of vancomycin and oxacillin, each in a sub-MIC concentration, has been found to show marked in-vitro synergism and efficacy against these bacteria without in-vitro selection of vancomycin-resistant strains (47). But clinical use of this combination for methicillin-resistant staphylococcal infections is still to be evaluated.
However, the laboratory can test for efficacy of this combination by comparing the results of sensitivity tests of the isolate to the selected β-lactam antibiotic by either diffusion methods or dilution MIC methods with and without added vancomycin to the used agar or broth (103).
New Aminoglycosides
Arbekacin is a new semi-synthetic aminoglycoside. It has been found to show excellent activity against a wide variety of bacteria that produce aminoglycoside-modifying enzymes. The combination of arbekacin and ampicillin demonstrated inhibitory activity against vancomycin-resistant enterococci and may prove useful for treatment of infections caused by these bacteria (84)
New Cephalosporins
A new experimental cephalosporin designated RWJ-54428 has been found to show a good activity against resistant gram-positive bacteria including vancomycin-resistant strains. In a mouse model of pyelonephritis due to Enterococcus faecalis, this new cephalosporin showed a bactericidal effect with tolerable doses. However, clinical evaluation of this new agent is still underway (68).
New Fluoroquinolones
Fluoroquinolones group (ofloxacin, norfloxacin, ciprofloxacin and enoxacin) has only moderate activity against enterococci. A bactericidal effect of such antibiotics on enterococci is inoculum dependent and may be seen only at concentrations unreached in serum of patients in clinical use (126). Their use against enterococci is so, limited to urinary tract infections (101).
Newer fluoroquinolones, such as clinafloxacin, showed greater activity against gram-positive bacteria including enterococci but vancomycin-resistant enterococci remained among the least susceptible gram-positive bacteria to these antibiotics (31).
In a recent study, experimental fluoroquinolones showed promising results in this aspect. In this study, moxifloxacin and garenoxacin (BMS-284756) were used for treatment of experimental meningitis induced in rabbit by inoculation with vancomycin-tolerant Streptococcus pneumonia strain. These experimental fluoroquinolones showed successful treatment of that condition and proved to be effective in killing and eradication of the causative bacteria (123). However, use of these new experimental fluoroquinolones in treatment of vancomycin-resistant enterococci and staphylococci is still to be evaluated.
Lipopeptides:
Daptomycin (LY146032) is an acidic lipopeptide antibiotic that has good activity against gram-positive bacteria and is similar in spectrum of activity to vancomycin but differs in the mechanism of action. It acts by interference with cell membrane transport mechanisms resulting in rapid bactericidal activity. It has shown promising results in vitro against all species of enterococci including vancomycin-resistant strains and there was no cross-resistance with vancomycin or teicoplanin (130).
Also it showed good activity against the newly emerged vancomycin-resistant S. aureus strains (Mu-50 and 992) in experimental studies (2).
Inspite of these promising in-vitro results, early clinical trials were disappointing as these trials showed ineffective therapy of gram-positive endocarditis and bacteremia by this agent which have been explained by very high (> 90%) plasma protein binding (124). Trials with higher but not toxic doses in humans are still under evaluation (90).
New Glycopeptide Agents
A great effort has been done in the recent years to develop new agents within the glycopeptide group of antibiotics that may show good activity against gram-positive bacteria that show non susceptibility to the current glycopeptides (vancomycin and teicoplanin). This current intensive research may allow for clinical use, some new antibiotics that can be described as, second-generation glycopeptides (94).
The second-generation glycopeptides have the same mechanism of action of those of first generation (vancomycin and teicoplanin) which means that they act by binding to the D-Ala-D-Ala terminus of the N-acetylmuramyl pentapeptide molecules leading to prevention of transpeptidation and transglycosylation and so inhibition of peptidoglycan synthesis. However, these second-generation glycopeptides may differ from first-generation glycopeptides in the ability to bind to the substitutions to D-Ala-D-Ala terminus, which are produced by vancomycin-resistant bacteria (3,94).
Ramoplanin is a newly developed glycopeptide that showed high activity against vancomycin-resistant gram-positive bacteria in general (39,130).
Limitations to use of this glycopeptide antibiotic was put when further studies showed that addition of human serum resulted in fourfold increase in the MIC which suggested the need for high dose for successful treatment. This dose was found to be toxic for systemic use. So, ramoplanin was only suggested to use for local actions such as clearance of glycopeptide-resistant enterococci from the gastrointestinal tract (82), and eradication of C. difficile from the intestine without a risk of colonization by glycopeptide-resistant enterococci (16).
Another newly developed semi-synthetic glycopeptide is that designated LY333328 (now, called oritavancin). It is an investigational N-alkyl semi-synthetic derivative of the naturally occurring glycopeptide LY264826 (40).
Oritavancin is one of the most active agents against vancomycin-resistant enterococci including the most resistant strains of VanA type. Also, it has been found to show good activity against vancomycin-resistant staphylococci (94,99,112).
Oritavancin activity is affected by binding to serum proteins and this affection should be compensated by increase of the dose of the drug. The data are still lacking regarding to the pharmacokinetics in humans and the degree of toxicity (98).
These data should be available to determine if it is possible to achieve the desired concentrations of oritavancin in humans without causing serious side effects. Clinical trials are currently done for this purpose (90)
Mannopeptimycins are new group of glycopeptides antibiotics. Varepsilon is one member of this group and it has been found in a recent study to show good activity against vancomycin-resistant enterococci. This new group showed rapid inhibition of peptidoglycan synthesis and had no effect on cell membrane, DNA, RNA, or protein synthesis suggesting a primary action through inhibition of bacterial cell wall biosynthesis (133)
It is clear that the group of glycopeptides can offer new treatment possibilities for vancomycin-resistant gram-positive bacteria, but further work is still needed to produce a satisfactory bactericidal glycopeptide that is effective and nontoxic in systemic use and lacks cross-resistance with the currently in-use glycopeptides, vancomycin and teicoplanin.
Oxazolidinones
The oxazolidinones are new class of antibiotics with good activity against enterococci and are different from any other class. The mechanism of activity is through inhibition of protein synthesis leading to a bacteriostatic effect (54,89,129).
Linezolid is an agent in this group. It has been found to be of excellent activity against multi-antibiotic-resistant enterococci (4,22). Linezolid has been recently approved as a therapeutic option for vancomycin-resistant enterococci on the basis of in vitro susceptibility and clinical efficiency from multicenter, pharmaceutical company-sponsored clinical trials (4,90,109).
In a recent case study, linezolid was given to a patient suffering from cerebrospinal meningitis caused by Enterococcus faecium that was found to be resistant to vancomycin. The patient was cured when received linezolid intravenously for 4 weeks, which led to complete eradication of the causative agent. This suggested that linezolid is safe and effective therapy for vancomycin-resistant enterococcal meningitis (138).
In another recent study, linezolid has been found to decrease the emergence of vancomycin resistance in S.aureus when given in combination with vancomycin but with no detected synergism (70). Also it has been found in another study to be effective, when given alone, against S. aureus including strains which are methicillin-resistant (109) and also against vancomycin-resistant strains (60). These findings may make linezolid a suitable alternative to vancomycin for treatment of these strains.
Another novel oxazolidinone compound designated PH-027 has been demonstrated a potent antibacterial activity comparable to or better than that of linezolid against gram-positive bacteria and seems to be a good possible therapeutic option for vancomycin-resistant bacteria in the near future (115).
Streptogramins
From this group, quinupristin-dalfopristin (RP59500, Synercid) has been recently approved as a therapeutic drug for vancomycin-resistant-enterococci. This drug is a mixture of two compounds with synergistic activity against gram-positive cocci including enterococci. This drug acts by interference with bacterial protein synthesis that results in a bacteriostatic effect. It has been found to be active against vancomycin-resistant E. faecium but is not active against E. faecalis (90)
It has also been proved that quinupristin-dalfopristin is active against all strains of S. aureus including methicillin-resistant strains (91,109) which may allow to add this drug to the limited number of therapeutic options for treatment of these bacteria.
Comparative studies between the two recently approved antibiotics for vancomycin-resistant gram-positive bacteria (linezolid and quinupristin-dalfopristin) are still needed.
Prevention and Control of the emerging Vancomycin-resistant gram-positive bacteria
Reduction of Vancomycin Use
Vancomycin use has been reported as a major risk factor for colonization and infection with vancomycin-resistant gram-positive bacteria (58,72).
So, vancomycin use should be limited only to the appropriate conditions. These conditions are as following (34):
1. Treatment of serious infections due to β-lactam-resistant gram-positive microorganisms.
2. Treatment of serious infections with gram-positive microorganisms in patients with β-lactam hypersensitivity.
3. When treatment of antibiotic associated colitis with metronidazole fails.
4. Prophylaxis against gram-positive bacterial endocarditis following certain procedures which are known to be associated with high risk.
5. Prophylaxis in a high-risk surgery such as that involving implantation of prosthetic devices. In such case a single dose given immediately before surgery is sufficient unless the surgery lasts more than 6 h., in which case the dose should be repeated, only for another dose.
Vancomycin use in hospitals was found to be markedly increased in the period from 1980 to 1990 which may explain the appearance of resistance to this important antimicrobial agent by its use in many situations which are actually not in high need to it (93).
The situations at which the use of vancomycin should be stopped to avoid emergence of more resistant bacteria are as follows (34): -
1. Routine surgical prophylaxis.
2. Empirical therapy for febrile patients.
3. Treatment in response to a single blood culture positive for coagulase negative staphylococci, if other blood cultures drawn in the same time frame are negative which means that if contamination with skin flora is likely.
4. Empirical use for patients whose cultures are negative for β -lactam-resistant gram-positive bacteria.
5. Prophylaxis of infection or colonization of vascular catheters.
6. Selective decontamination of the digestive tract.
7. Eradication of MRSA colonization.
8. Primary treatment of antibiotic associated colitis.
9. Routine prophylaxis in very low-birth-weight infants.
10. Routine prophylaxis in patients on continuous ambulatory peritoneal dialysis or hemodialysis.
11. Use of vancomycin solution for topical application or irrigation.
Educational Programs
Continuous educational programs including educational sessions are needed for all members of hospital staff including consulting physicians, resident physicians, medical students, pharmacy personnel, nurses, laboratory personnel and other direct patient caregivers. The education programs should include informations about the extent and epidemiology of vancomycin-resistant bacteria and the potential impact of this problem on the outcome of patient care (34).
Early Detection by Appropriate Laboratory Methods in Hospitals Where Vancomycin-Resistant Bacteria have not been Detected
Early detection of patients colonized or infected with vancomycin-resistant bacteria is an essential component of any hospital program designed to prevent nosocomial infections with vancomycin-resistant bacteria (23).
So, the microbiology laboratory is considered the first line of defense in any hospital against the spread of vancomycin-resistant bacteria. The microbiology laboratories in hospitals should be able to use the appropriate methods for prompt and accurate detection of first isolates of these resistant bacteria to allow early prevention of transmission. The prevention and control measures will be more difficult, if the detection is delayed (34).
Prevention of Transmission of Vancomycin-Resistant Bacteria
The following precautions are recommended to prevent patient-to-patient transmission of vancomycin-resistant bacteria (63): -
1. The patients infected or colonized with vancomycin-resistant bacteria should be isolated in special rooms.
2. Gloves and gowns should be used when entering rooms of patients infected or colonized with vancomycin-resistant bacteria.
3. Gloves and gowns should be removed before leaving rooms of patients and the hands should be washed immediately with an antiseptic soap.
4. After removal of gloves and gowns and hand washing, it is important to avoid contact with environmental surfaces potentially contaminated (e.g. doorknobs) in the rooms of patients.
In addition to these isolation precautions, the instruments such as stethoscopes, sphygmomanometers or rectal thermometers should be used for a single patient or a group of patients infected or colonized with vancomycin-resistant bacteria. Otherwise, these instruments may transmit infection with these resistant bacteria to other patients and even may cause a hospital outbreak (92). If such devices are to be used for other patients, they should be adequately cleaned and disinfected (151).
To remove a patient from isolation, he should give negative results for at least three cultures, one week at least apart, from multiple body sites including stool, rectal swab, perineal area, axilla, and umbilicus. Also, specimens should be taken from wounds, catheters or colostomy sites if present (23).
For hospitals with endemic vancomycin-resistant bacteria, more additional recommendations should be followed (34): -
1. Control efforts should be initially focused in ICUs. Such units may serve as a reservoir for vancomycin-resistant bacteria, from which these bacteria spread to other parts of the hospitals when the patients are well enough to be transferred.
2. Every effort should be done to minimize movements of health care givers between patients who are positive and those who are negative for vancomycin-resistant bacteria.
3. Hospital personnel should be examined carefully for chronic skin and nail problems. Hand and rectal swabs cultures should be performed for all hospital personnel routinely. Personnel who prove to be carriers of vancomycin-resistant bacteria should be removed from care of patients who are negative for vancomycin-resistant bacteria until their carrier state has been eradicated.
4. Adequate procedures should be applied for the routine care, cleaning and disinfection of environmental elements in the hospital (e.g. bedrails, charts, doorknobs … etc). Cultures from hospital environmental elements should be routinely done to assess the efficacy of hospital policies and procedures in this aspect.
5. Representative vancomycin-resistant bacteria should be sent to reference laboratories for typing by PFGE and other suitable typing methods to help in precise definition of reservoir and determination of methods of transmission, which allow the control programs to be more successful.
Eradication of Vancomycin-Resistant Enterococcal Colonization of Gastro-Intestinal Tract
One of the major problems with control of vancomycin-resistant enterococci is the spread of colonization with these bacteria in healthy populations that may represent a main non-easily controlled source for infections with these bacteria. In many outbreaks caused by these bacteria, the number of infections was low while colonization showed extensive spread among normal healthy populations. This make the current problem even more difficult in control than the previous problem of MRSA as no colonization spread among healthy subjects has been occurred with outbreaks caused by that bacteria. Colonization with vancomycin-resistant enterococci occurs in the general open populations with no definite increased risk groups, which make colonization of these bacteria difficult to be controlled (20).
There has been an interest in eradication of vancomycin-resistant enterococci when present as a commensal in the gastrointestinal tract to decrease the subsequent risk of infection in the individual patient and to reduce the reservoir of these bacteria in the community (55).
Some studies showed good results when oral bacitacin had been used for this purpose with few relapses (36,110). Also, some other studies suggested successful use of ramoplanin for the same aim (82,90). Successful eradication of colonization with vancomycin-resistant enterococci was also noted in another study using a combination of rifampicin and doxycycline, with negative rectal swabs at 1 and 6 months (44).
However, many other studies have shown some conflicting disappointing results. For instance some studies used a combination therapy of bacitracin plus doxycycline, the results of which showed that this combined therapy may initially clear vancomycin-resistant enterococci from stools of treated patients but with longer follow up, no significant difference occurs between treated and untreated patients. The results of these studies show also that vancomycin-resistant enterococci may be undetectable in feces of the treated patients for only the period during which the antibiotics are given for them (154,155).
New approaches may prove useful for this purpose including administration of probiotic agents such as Bacillus coagulans, which showed promising results for this purpose in experimental animals when given orally (48). However, it is clear that more research is still highly needed to reach to an effective regimen for eradication of vancomycin-resistant enterococci from the gastrointestinal tract of colonized patients, which represents an important requirement for successful prevention and control of these bacteria.
Lastly, we should mention that strict application of the previously outlined, currently possible, measures for prevention and control of these emerging vancomycin-resistant gram-positive bacteria is the cornerstone to overcome this new medical problem. This problem which created a new situation of infections caused by bacteria with no any available active therapeutic option which resulted in increased morbidity and mortality due to these infections in the last years.
We should stress on strict control of hospital infections to change the idea that hospitals are infectious places that may act as vehicles for transmission of the most serious and even fatal infections. This is a very important item especially in light of the current estimations that at any one time a fifth of all inpatients in a hospital have an infection, half of which have acquired in the hospital (33,63).
We are also in urgent need to develop new therapeutic agents against all microorganisms in general and specifically against vancomycin-resistant gram-positive bacteria, otherwise we will be in front of these armed enemies without active weapons. Any developed new active anti-bacterial agent should be then used carefully, avoiding overuse or misuse, which may again push us to the same problem. The problem may be even worse if bacteria pushed man to start of what may be described as “post-antibiotic phase” a time when hospitals will be colonized with bacteria resistant to all available antimicrobial agents and therefore man returns to the pre-penicillin age.
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As with VanA class, this VanB resistance is controlled with a gene cluster (VanB gene and related genes). The gene cluster of VanB class is located on a transposon termed Tn1547. This transposon usually resides on the bacterial chromosome but rarely on a plasmid (118). Genes responsible for VanB type of vancomycin resistance may be transmitted by conjugation to different species of enterococci (74) and also to genera other than enterococci, which was recorded in an isolate of Streptococcus bovis (117).
The protein product of vanB gene is an abnormal ligase (VanB) that is structurally related to VanA ligase (76% amino acid homology). VanB ligase also favors production of abnormal peptidoglycan precursors with pentapeptide that terminates in the depsipeptide D-Ala-D-Lac (57).
Genes of VanB-type resistance that correspond to those of VanA-type resistance are designated vanRB, vanSB, vanHB, vanXB, and vanYB. They show the same activities as in VanA class but in contrast to VanS protein of the VanA class; the VanSB protein does not recognize teicoplanin, which explains the inability of this glycopeptide to induce VanB resistance. Bacteria with VanB resistance show no corresponding gene to vanZ of VanA type-resistant bacteria (11).
Recent studies reported that vanB gene shows DNA sequence heterogeneity that allows subtyping of vanB gene into 3 subtypes: vanB -1, vanB-2 and vanB-3 (43, 113). With vanB-2 the most prevalent type (160)
VanC Vancomycin Resistance
On contrast to both VanA and VanB resistance, which are acquired and transferable types, VanC type of resistance is an intrinsic constitutive property that was detected in Enterococcus gallinarum, Enterococcus casseliflavus and Enterococcus flavescens. This form of resistance shows low-level resistance to vancomycin (MIC of 8-32 ug/ml) and sensitivity to teicoplanin (<8>256 ug/ml) than normally expected and also resulting in resistance to teicoplanin (50).
VanD Vancomycin Resistance
VanD type of resistance was first described after recognition of a new resistance gene designated vanD in 1997. The strain carrying this resistance gene was E. faecium strain and showed MIC for vancomycin and teicoplanin of 64 and 4 ug/ml respectively (114).
More studies showed that vanD gene is a ligase gene that found on partial sequencing to be distinct from but similar to VanA and VanB genes and the amino acid sequence of VanD showed 67% identity to those of VanA. The vanD gene was found to be located on chromosome and is not transferable to other enterococci (111).
In more recent studies, VanD-type-resistant enterococci have been found to be constitutively resistant to vancomycin by nearly exclusive synthesis of peptidoglycan precursors terminating in D-Ala-D-Lac. The vanD gene has been identified and found to encode for a ligase similar to that of VanA and VanB types. Also, other genes of VanD cluster that correspond to those of VanA-type were identified and designated vanHD that encodes for a dehydrogenase, vanXD that encodes for a dipeptidase, vanRD that is a regulator gene and vanSD that encodes for a sensor protein (45).
VanE Vancomycin Resistance
More recently, a new vancomycin resistance gene designated vanE has been identified in E. faecalis BM4405, which has been resistant to low levels of vancomycin (MIC: 16 ug/ml) and susceptible to teicoplanin (MIC: 0.5 ug/ml) (59).
This new resistance type has similarities to the intrinsic VanC type of resistance. The amino acid sequence of VanE protein has greater identity to VanC (55%) than to VanA (45%) and VanB (43%) or VanD (44%) (59).
Vancomycin-Dependent Enterococci
Not only vancomycin resistance but also vancomycin dependence has been observed in some strains of VanA and VanB types of resistance to vancomycin in enterococci. Vancomycin-dependent enterococci have been isolated from apparently culture-negative clinical samples by plating them on vancomycin-containing agar, such as that used for isolation of campylobacter or gonococci (46).
A possible explanation of this phenomenon is that these organisms may turn off their normal production of D-Ala-D-Ala and then can grow only if a substitute structure is made. With most VanA and VanB types of resistance, this occurs only in presence of vancomycin, which induces synthesis of VanH dehydrogenase and VanA or VanB ligase that make D-Ala-D-Lac (102).
The reason for the turning off the synthesis of D-Ala-D-Ala by the bacterial cell may be that as long as vancomycin is present, D-Ala-D-Ala is not necessary for cell wall synthesis and even it is destroyed by the action of VanX. Once the vancomycin is removed, D-Ala-D-Lac is no longer synthesized and the cell stops to grow or replicate, as it needs to either D-Ala-D-Ala or D-Ala-D-Lac (46,102).
Reversion to vancomycin independence has been observed which may be explained by either a mutation that leads to constitutive production of D-Ala-D-Lac or turning on the usual mechanism for synthesis of D-Ala-D-Ala (46,103).
Mechanisms of Resistance to Vancomycin in Staphylococci
Early reports have been demonstrated the presence of phenomenon of tolerance to vancomycin among some strains of S. aureus especially those that are methicillin-resistant. Tolerance has been explained failure of treatment with vancomycin of infections caused by these bacteria inspite of the detected susceptibility according to the MIC results. Moreover, some studies have shown increased mortality in patients with bacteremia or endocarditis due to these tolerant S.aureus strains inspite of vancomycin therapy (97,119).
Moreover, in the last years, some studies have been reported the emergence of vancomycin-resistant strains in staphylococcal clinical isolates that, by definition, show in-vitro MICs of >4 ug/ml. This has been reported in both S. aureus and coagulase negative staphylococci (125,132).
This emergence was significantly marked in hospitals that had been endemic with strains of methicillin-resistant staphylococci and followed the policy of empirical use of glycopeptides. Extensive use of glycopeptides (vancomycin or teicoplanin) allowed selection of resistant strains from both of S. aureus and coagulase negative staphylococci, mainly among S. epidermidis followed by S. hemolyticus, S. hominis and S. warneri (35,58,125).
This view is supported by the laboratory finding that glycopeptide-resistant mutants of S.aureus have been experimentally selected by gradually increasing the levels of vancomycin present during in-vitro growth (131).
Also, this view is supported by the observation that heteroresistance to vancomycin is a common phenomenon among staphylococci (60,159). The presence of this heteroresistance may allow selection of resistant strains in vivo when infections caused by these bacteria are treated with vancomycin.
The presence of vancomycin heteroresistance phenomenon in staphylococci may arise the need to modify the methods used for detection of vancomycin resistance in the clinical microbiology laboratory. The inoculum tested should be sufficiently large to compensate for the frequency of expression.
The resistance of staphylococci to vancomycin has been found to be reversible under laboratory conditions. In an experiment to prove this phenotypic reversion, S. aureus isolates showing resistance to vancomycin were repeatedly cultivated on non-selective media. After 15 days of passages, vancomycin and teicoplanin-susceptible strains were obtained from each isolate. Moreover several obtained strains lost the subpopulations with vancomycin resistance. This reversion of vancomycin-resistance phenotype may explain the difficulty in isolating vancomycin-resistant clinical isolates of staphylococci from patients who do not respond to vancomycin therapy and possibly may account for some of the difficulties in identification of such isolates in the clinical microbiology laboratory (25).
In a case study, primary testing of clinically isolated S. epidermidis strain showed resistance to vancomycin. As the result in such situations should be confirmed, the test was repeated from the isolate subculture and the surprising result was that the strain showed sensitivity to vancomycin even on more repeated tests. In such conditions, the question arises if the first primary test was in error and so, the isolation from the same patient was repeated and the tests were done again. The results were the same as with the previous isolation, which indicated that the primary susceptibility test was not in error but the problem was in an elusive phenotype (49).
These findings may put a base for a new modification in cultivation of staphylococci in the clinical laboratory. It may be better to use a medium containing a low level of vancomycin as well as the routine media to ensure that vancomycin-resistant isolates can be maintained on the medium with low level vancomycin. Also, we should consider the primary results of vancomycin susceptibility tests and not the results of those done from subcultures.
The mechanism of resistance of staphylococci to vancomycin is still not clear. But transfer of the VanA gene cluster from enterococci to Staphylococcus aureus has been demonstrated in the laboratory by in vitro experiment and also on the skin of mice. This finding has raised concern about the possibility of occurrence of such mechanism of genetic transfer in clinical isolates of staphylococci, which often co-colonize wound infection sites with enterococci (108). However, there is no study showed the presence any of Van genes clusters in clinical isolates of staphylococci and so such mechanism can not be proved up till now (60).
Some current observations may lead to clarification of the possible mechanism(s) of staphylococcal resistance to vancomycin. From these observations, it has been observed that vancomycin-resistant staphylococci in presence of vancomycin in a liquid medium form aggregates of bacterial cells. The drug is then become undetectable in the medium followed by disintegration of the bacterial aggregates into single cells with the same appearance of cells grown in antibiotic-free medium. Examination of the bacterial aggregates by electron microscope showed the production of large amount of extracellular material, which had staining properties similar to that of the cell wall material. Vancomycin could be recovered from the cell wall material in biologically active form. These findings may suggest that these bacteria may resist the action of vancomycin through the ability to sequester the antibiotic molecules in a bound form to an extra material of cell wall (64,131,132).
In a recent study, 16 clinical isolates of vancomycin-resistant S.aureus from 7 countries were subjected to serial daily passages in drug-free medium. After 10-84 days of these passages, strains susceptible to vancomycin (MICs: < name="Epidemiology">Epidemiology of Vancomycin-Resistant Bacteria
Prevalence of Vancomycin-Resistant Bacteria
Prevalence of vancomycin-resistant enterococci increased dramatically in last years. Different studies have shown that the proportion of vancomycin-resistant strains among isolated enterococci rose gradually to reach an average percentage near to 25% in the last years, the initial rise was observed in intensive care units followed by general hospital wards (33, 73,127).
In the recent years, the average percentage of methicillin-resistant staphylococci in different communities is around 50 % of total hospital staphylococcal infections (58,60). However, the vancomycin-resistant strains of staphylococci are still of low prevalence. These currently low-prevalent strains are actually of great importance as they were isolated from different countries in the world (58,80,137,144,152) and evidences accumulate suggesting that there is great possibility to be more common by time because the number of reports about these vancomycin-resistant strains increases year after year. Moreover, the reported heteroresistance to vancomycin among staphylococci may allow selection of more resistant strains by time (60).
The prevalence rates of vancomycin-resistant S. aureus differ widely between different studies, for instance these prevalence rates ranged in Japanese studies from 1.3 up to 20%. This discrepancy may be attributed to the current controversy that surrounds the methods of detection of these strains with heteroresistance to vancomycin (152).
In an epidemiological hospital study done through one year, it was found that among 535 subjects with coagulase negative bacteremia, 20 subjects had a glycopeptide-resistant strain (19 strains were resistant to teicoplanin and 1 was resistant to both teicoplanin and vancomycin). The isolated resistant strains were mainly from intensive care units followed by medical and then surgical wards. Mortality rates were 25% among cases infected with resistant strains and 18% among other control cases infected with the same type of infections but caused by vancomycin-susceptible coagulase negative staphylococci which was significant difference (142).
Actually, mortality rates are becoming very high among patients infected with vancomycin-resistant gram-positive bacteria especially cases with bacteremia and/or endocarditis in which choice of treatment regimen with bactericidal activity is critical for success. For instance in a series of 125 episodes of enterococcal bacteremia, death occurred in 17.6% and in 8% of deaths, the fatal outcome was considered directly related to bacteremia (67).
Origin of Vancomycin-Resistant Bacteria
The origin of vancomycin resistance in gram-positive bacteria appears to be not the same in all emerged strains. This may be suggested from absence of uniform mechanism for vancomycin resistance between enterococci and staphylococci and also presence of different genotypes and subtypes for resistance among enterococci, which suggest that Van clusters have acquired by enterococci on multiple occasions followed by horizontal dissemination particularly of VanA and VanB (160).
Moreover, characterization of staphylococci that have been emerged in different parts of the world by pulsed field gel electrophoresis (PFGE) analysis showed non-identical pattern between isolates from different countries which suggested that these isolates do not represent one clone that has been disseminated (145).
Nosocomial Origin
It has been suggested that most vancomycin-resistant enterococci originate in hospitals and then spread to the community (61). Also most vancomycin-resistant strains of staphylococci appear to have originated in hospitals under the selective pressure of vancomycin use in these hospitals. These strains caused outbreaks of infection in some of these hospitals (80,144)
However, some studies showed that glycopeptide-resistant enterococci might be isolated from outpatients with no history of hospitalization, which has been suggested that hospital is not the single origin for glycopeptide-resistant enterococci (66).
Human Colonization Origin
Enterococci may be part of the normal flora of the intestine, mouth, urethra and vagina (103) and it was reported that vancomycin-resistant enterococci may be isolated from sewage and this may represent a source for these resistant bacteria to the community (147).
Once acquired, colonization by vancomycin-resistant enterococci can last for years serving as a reservoir for potential infection of the colonized patient and also to other subjects (73).
Selection of Resistant Strains
Many of the patients, with vancomycin-resistant gram-positive bacteria, have been previously exposed to vancomycin or teicoplanin therapy. This was reported in all of enterococci (72), S. aureus (144) and coagulase negative staphylococci (17,58). Exposure to vancomycin, either for treatment or for prophylaxis, may exert selective pressure, which results in increase of undetectable levels of pre-existing vancomycin-resistant bacteria to detectable levels. In such a case, vancomycin administration does not cause vancomycin resistance to develop and also does not increase the possibility of exogenous transmission of such bacteria to the patient but increases the likelihood of detection of vancomycin-resistant bacteria (73).
Early experimental studies showed that staphylococci cultivation in presence of sub-inhibitory glycopeptide concentrations could induce, under laboratory conditions, selection for clones for which the glycopeptides MICs are increased (15).
Also, stepwise and single-step exposure to teicoplanin and/or vancomycin has allowed mutant clones for which glycopeptide MICs are increased to be obtained from susceptible coagulase negative staphylococci (17,85).
Recent experimental studies done by bacterial populations analysis showed that glycopeptide resistance may be expressed heterogeneously in both S. aureus (1,77) and coagulase negative staphylococci (17,18). Heterogeneous resistance to vancomycin is defined as that observed in strains for which the conventional vancomycin MICs are <4> 8 ug of vancomycin per ml (78).
This heterogeneous resistance may result in detection of susceptibility of the organism to vancomycin in-vitro by the conventional MIC dilution method while in-vivo selected resistant subpopulations may be responsible for failure of therapy with vancomycin (1,18,78).
Non-Human Origin
Non-human origin of these bacteria has been suspected because vancomycin-resistant enterococci were isolated from farm animals (including duck, chicken, turkey, pig, pony and dog) and from each of five uncooked chickens bought from retail outlets (14).
These findings may suggest that vancomycin-resistant enterococci may pass from animals to man via the food routes especially in light of the reported findings that some human isolates are not distinguishable from isolates from non-human sources (14).
Avoparcin
Avoparcin is a glycopeptide not used for humans but may be used for animals as a growth-promoting factor. Glycopeptide-resistant enterococci were isolated from farms using this glycopeptide agent while no such bacterial isolates were detected in farms not using this agent and this finding may suggest that the use of avoparcin may represent a selective pressure for emergence of glycopeptide-resistant enterococci colonizing farm animals. These animals may be an important reservoir of glycopeptide-resistant enterococci for humans and even may be the origin or the source for these bacteria (88,140).
Avoparcin was used in Europe but not in the USA and this may explain a reported observation that colonization of healthy people with vancomycin-resistant enterococci is absent in the USA and endemic among humans and animals in Europe. From The European animal reservoir, vancomycin-resistant enterococci and resistance genes have spread to humans. This spread confirmed absence of boundaries between animals and humans, between healthy and diseased subjects, between countries and probably between continents (21,127).
The link between use of avoparcin and emergence of glycopeptide-resistant enterococci has been further confirmed by a study done in Netherlands and showed marked decrease in prevalence of colonization of healthy subjects with vancomycin-resistant enterococci after stopping use of this agent (149).
Avoparcin was used as a feed additive agent in New Zealand for poultry production from 1977 until June 2000. A recent study has been done to evaluate the effect of its use on prevalence of vancomycin-resistant enterococci. This study showed that these bacteria were only isolated from poultry fecal samples taken from farms where avoparcin was in use or previously used. The study showed that patterns of pulsed field gel electrophoresis (PFGE) of digested DNA of the poultry isolates of vancomycin-resistant enterococci were identical or closely related to human isolates of these bacteria in the same areas and molecular characterization showed identical transposons (Tn1546) in both human and poultry isolates (95).
Transmission of Vancomycin-Resistant Bacteria
Vancomycin-resistant enterococci have great ability for transmission. This ability has allowed these strains to cause several hospital outbreaks in different countries (103). In case of vancomycin-resistant staphylococci, the condition differs because these bacteria, in most cases, have been isolated from individual patients usually previously treated with vancomycin for long periods. There is no evidence for epidemiological spread of the recently emerged vancomycin-resistant staphylococcal strains except in very rarely recorded hospital transmission (35,60,142).
De novo emergence of vancomycin resistance in enterococci through genetic mutation induced by exposure to vancomycin has been found to be rare in any individual patient. Accordingly, the marked increase in vancomycin-resistant enterococci may be attributed mainly to rapid cross-transmission, which has occurred mainly in hospitals where these bacteria represent major causative agents of nosocomial infections (103).
Hospital environmental contamination has been found to be an important factor in transmission of vancomycin-resistant enterococci. This environmental contamination may be so marked in case of a patient with intestinal vancomycin-resistant enterococcal colonization and having diarrhea, which may result in hospital outbreaks (24,63,72,103).
Such situation has been commonly occurred in hospitals when a patient develops antibiotic-associated diarrhea caused by C. difficile and then treated with oral vancomycin which may allow for selection of intestinal vancomycin-resistant enterococci which find a rapid route for spread in the hospital through the already present diarrhea. This serious situation was the basis for the current recommendation to avoid vancomycin as a primary treatment of antibiotic-associated diarrhea (73).
Enterococci were found to have the ability to survive on a variety of hospital environmental surfaces for variable periods of time ranging from 1 hour up to 7 days. These findings suggested that the hospital environment is a possible reservoir for nosocomial infections with vancomycin-resistant enterococci (139).
In an outbreak of vancomycin-resistant E. faecium in medical-surgical ICU, the epidemic strain was isolated repeatedly from the rectal probe handles of three electronic thermometers. Disposable probe sheaths for protection of probes from contamination were in routine use in this unit but proved to be not satisfactory for this purpose (92).
These findings make necessary to improve the cleaning and decontaminating procedures to be satisfactory to avoid the possible role of hospital environmental contamination in spread of vancomycin-resistant bacteria.
Predisposing Risk Factors for Infection with Vancomycin-Resistant Bacteria
The most important predisposing risk factor for infections caused by vancomycin-resistant bacteria is the overuse or misuse of antibiotics. In patients colonized with low numbers of glycopeptide-resistant gram-positive bacteria, antibiotics may allow overgrowth of these bacteria with increased risk of infection and dissemination (73).
Not only vancomycin which select for colonization or infection with vancomycin-resistant bacteria but also some other antibiotics including broad-spectrum cephalosporins especially third generation members which show high biliary concentrations that kill most bacteria in the upper gastrointestinal tract with negligible activity against enterococci in general. The antibiotics with activity against anaerobic bacteria especially metronidazole and clindamycin may be also responsible for selection of vancomycin-resistant enterococci (73).
Other risk factors also include proximity to affected patients, care by colonized staff, use of invasive procedures, hospitalization in the previous year, number and length of stays in the hospital and also the transfer between hospitals (142,143).
For vancomycin-resistant enterococcal infections, colonization with vancomycin-resistant enterococci represents an important risk factor for different types of infections caused by these bacteria. From the colonization site, infection may occur in the form of urinary tract infections, surgical wound infections, peritonitis, bacteremia and endocarditis. This occurs more commonly in presence of a foreign material such as prosthetic heart valves and endovascular devices, joint prostheses and cannulas used in dialysis patients (103).
For vancomycin-resistant staphylococcal infections, risk factors include ongoing or recent dialysis, presence of foreign prosthetic material and recurrent infections with methicillin-resistant staphylococci. Many published reports suggest that vancomycin-resistant staphylococci appear to have developed from pre-existing methicillin-resistant strains that infected the patients in the months before detection of the vancomycin-resistant strains. These reports show similar pulsed field gel electrophoresis (PFGE) patterns between the early isolated methicillin-resistant strains and the newly developed vancomycin-resistant strains (60).
Laboratory Methods for Detection of Vancomycin Resistance
In the current years, clinical microbiology laboratories worldwide must be highly alert to detect glycopeptide-resistant strains of gram-positive bacteria in clinical specimens. This may allow early preventive and control measures to be taken to prevent spread of such strains. Definite criteria for interpretation of tests for glycopeptide susceptibility are markedly essential in this aspect. Also the choice of suitable method is of special importance in this field.
Criteria for Interpretation of Glycopeptide Antibiotics Susceptibility Tests
The criteria for determination of resistance to either vancomycin or teicoplanin by both disk diffusion and MIC methods are shown in the following table according to the national committee for clinical laboratory standards (104,105)
Organism
Antibiotic
Susceptible
Intermediate
Resistant
Zone D.* (mm)
MIC
ug/ml
Zone D.* (mm)
MIC
ug/ml
Zone D.* (mm)
MIC
ug/ml
Enterococci
Vancomycin
Teicoplanin
≥ 17
≥ 14
≤ 4
≤ 8
15-16
11-13
8-16
16
≤ 14
≤ 10
≥ 32
≥ 32
Other gram -positive bacteria
Vancomycin
Teicoplanin
≥ 12
≥ 14
≤ 4
≤ 8
10-11
11-13
8-16
16
≤ 9
≤ 10
≥ 32
≥32
* Zone diameters when using 30 ug disks on Muller-Hinton agar and after exactly 24 h of incubation (104,105).
According to the criteria of the national committee for clinical laboratory standards (NCCLS) the disk diffusion method for vancomycin shows interpretive criteria that differ between enterococci and other gram-positive bacteria. While no differences in these interpretive criteria are found between these two groups of bacteria when testing vancomycin by MIC method or testing teicoplanin by either disk diffusion or MIC methods (104,105).
However, there are some differences between the current American NCCLS interpretive criteria and the recommendations made to define categories of susceptibility to glycopeptides in some other countries. The British Society for Antimicrobial Chemotherapy has recommended the use of a single breakpoint concentration of 4 ug/ml to define resistance to both vancomycin and teicoplanin (163).
Also, in France, the “ Comite de l’Antibiogramme de la Societe Francaise de Microbiologie” has recommended MIC of ≤4 to define susceptibility of both vancomycin and teicoplanin, together with a zone diameter of >17 mm around 30-ug-disk for each of them (136).
Also, the Swedish Reference Group for Antibiotics recommends a single MIC breakpoint concentrations of 4 ug/ml for both vancomycin and teicoplanin and also recommends zone diameter of >16 mm for susceptibility to both glycopeptide antibiotics (83).
In contrast with criteria of NCCLS, those of British, French and Swedish recommendations make no distinction between tests performed on enterococci and those performed on other gram-positive bacteria (83,104,105, 136,163).
In the United States, the term “vancomycin-resistant S. aureus” is reserved for isolates with MICs >32 ug/ml. But outside the United States, This term may be used to refer to S. aureus with MICs >8 ug/ml which means classification of the isolates to either susceptible or resistant to vancomycin with no intermediate category. Some researchers prefer to use the term “non-susceptible isolates” to describe both the resistant and intermediate groups together (60).
Disk Diffusion Method
In all studies, the criteria of disk diffusion method for both vancomycin and teicoplanin show small difference between zones of inhibition of susceptible and resistant bacteria when compared to other antibiotics. This finding may be the cause that made the disk diffusion method of low discriminative power for detection of all levels of glycopeptide resistance, and this finding may be explained by the observation that glycopeptide antibiotics diffuse poorly through agar. The diffusion coefficients in Muller-Hinton agar are 0.72 mm²/h and 0.47 mm²/h for vancomycin and teicoplanin respectively (32).
Disk diffusion method is effective for detecting high level resistance to glycopeptide antibiotics (MICs > 256 ug/ml). At this level of resistance no zones of inhibition (diameter, 6 mm) occur around 30-ug-disk (161).
However, the diffusion method may be not so effective for detection of lower levels of resistance to glycopeptides when using NCCLS criteria (105) and different studies showed that the disk diffusion methods may show false susceptibility when compared to the standard MIC method. This finding was found in all of glycopeptide-resistant enterococci (128), S. aureus (144) and coagulase negative staphylococci (17,58).
E Test
A variation of the traditional disk diffusion test is the more recently introduced E test. The advantage of this test over disk diffusion is that it can determine the MIC for the tested organism. The E test consists of a plastic strip that contains a continuous gradient of antibiotic. So, the zone of inhibition occurs in the form of ellipse and the MIC is then read at the point where this ellipse touches the E-test strip (28).
Using E-test strip for determination of MIC of vancomycin for vancomycin susceptible organisms shows slightly higher MICs than when determination is done by agar dilution or broth microdilution methods but do not result in false evaluation of the test organism as resistant (128).
For teicoplanin, the MICs determined with the E test was found to be correlated well with agar dilution method for a collection of clinical and reference isolates of staphylococci and enterococci (153).
However, another study have indicated that E test shows lower teicoplanin MICs for coagulase negative staphylococci than does agar dilution which may lead to false susceptibility results (96).
Agar Dilution MIC Method
Agar dilution MIC method has been the standard method against which the other methods to detect glycopeptide resistance are evaluated. The method has the advantage of being based on agar dilution and so not affected by many of the problems associated with the diffusion methods. Also, it has the advantage of ability to use multipoint inoculator to allow multiple isolates to be tested on a single plate (104).
Vancomycin Agar Screening Method
An agar plate containing a single breakpoint concentration of vancomycin or teicoplanin may be used as a rapid screening method for glycopeptide non-susceptible bacteria. However, the screening concentration should be chosen carefully in order to maximize the sensitivity and specificity of the test because the selection of this concentration to be just equal to the MIC may lead to decrease in the specificity of the test especially at high inocula. It is recommended for breakpoint screening to use vancomycin concentration of 6 ug/ml in agar. An isolate should be examined by spotting 10 ul of a 0.5 McFarland concentration of this isolate onto the surface of a plate of brain heart infusion agar (rather than Muller-Hinton agar) and the test should be read after exactly 24 h. of incubation (104,141).
This method has the advantage of rapidly examining large number of isolates for vancomycin resistance because many isolates may be spotted easily on the same plate.
Broth Dilution MIC Method
Broth dilution method may be done in tubes and then called macrodilution method or done in microtitration plate and then called microdilution method. Both methods were found to correlate well with the reference agar dilution method (86).
Broth microdilution performed in cation-adjusted Muller-Hinton broth and read after a full 24 hours, is the method recommended by the NCCLS (104), and it has replaced agar dilution method as the reference standard method in some studies (87,146).
Automated Systems
As with the disk diffusion method, the automated systems, such as Vitek systems, show some difficulty in detecting resistance to glycopeptides especially if the level of resistance is low and so some false susceptibility results are reported by these systems (157).
These difficulties with the Vitek automated systems in this aspect made many reports to consider these automated systems unacceptable methods for evaluation of susceptibility of bacteria to vancomycin (165).
However, the problem with these automated systems may be in the software, which may be in need to some adjustments or alterations to pick up all levels of resistance to vancomycin (158).
Determination of MBC and Time Kill Tests for Detection of Vancomycin-Tolerant Staphylococci
Determination of MBC and time kill tests may be essential to detect strains of staphylococci that are tolerant to vancomycin inspite of results of MIC tests which by definition show susceptibility. Tolerance has been defined, in general, as an MBC:MIC ratio of >32. Also, tolerance may be defined in time-kill studies as kill of <99.9% name="treatment">Treatment of Vancomycin-Resistant Gram-Positive Bacterial Infections
The traditional treatment for gram-positive bacterial infections has been a synergistic combination of a cell wall active agent such as a β-lactam and an aminoglycoside (27).
But gram-positive bacteria gradually showed increasing resistance to β-lactams by production of β-lactamase enzyme and also increasing resistance to aminoglycosides by production of aminoglycosides-modifying enzymes (84).
Emergence and spread of resistance among gram-positive bacteria to the traditional antibiotics led to gradual increase of vancomycin use as the last weapon for treatment of infections caused by multi-resistant strains of gram-positive bacteria including enterococci, staphylococci and Clostridium difficile (166).
Vancomycin has been used for this purpose either singly or in combination with an aminoglycoside, as both drugs together show effective synergism both in vitro and in vivo. This synergism is based on an increased intracellular penetration of the aminoglycoside due to the effect of the glycopeptide on the bacterial cell wall (41,76).
However, emergence of vancomycin-resistant gram-positive bacteria in the last years has created a real therapeutic problem for infections caused by these bacteria as these bacteria are already resistant to most or even all other antibiotics (27,100). This problem led to occurrence of many types of infections that could not be treated with any available drug and resulted in increased morbidity and mortality due to these infections (67,142).
Moreover, when certain agent shows in vitro activity against some isolates of these bacteria, this agent may be only bacteriostatic and of uncertain efficacy in vivo or may be associated with high degree of toxicity (100).
So, this problem is still under condensed investigations and trails to create new weapons for these bacteria or at least to put a certain schedule of the current limited options to face this problem as possible.
Teicoplanin
Teicoplanin is the other glycopeptide to which many strains of vancomycin-resistant enterococci are susceptible (all phenotypes of vancomycin-resistant enterococci except VanA). However, in-vivo resistance had been found to develop to teicoplanin in a VanB strain, which has made this therapeutic option of limited use in these cases (75).
Combination of a Glycopeptide and a β-Lactam
A combination of a glycopeptide and a β-lactam was tried and found to show accepted efficiency against some strains of vancomycin-resistant enterococci that although show resistance to each of the two agents alone, are still inhibited by a combination of them (102).
Actually, the effect of this combination is antagonism when used against susceptible enterococci but when used for vancomycin-resistant strains, some of these strains may show synergistic effect with unknown mechanism (102).
However, this combination is still of limited use as the strains of vancomycin-resistant enterococci which show susceptibility to it are markedly few. Moreover, even these few strains may show subpopulations that are resistant to this combination and so can be preferentially selected after exposure to these combined drugs (103).
Combination of a glycopeptide and a β-lactam was also tried to decrease the emergence of vancomycin resistance among methicillin-resistant S. aureus and coagulase negative staphylococci. A combination of vancomycin and oxacillin, each in a sub-MIC concentration, has been found to show marked in-vitro synergism and efficacy against these bacteria without in-vitro selection of vancomycin-resistant strains (47). But clinical use of this combination for methicillin-resistant staphylococcal infections is still to be evaluated.
However, the laboratory can test for efficacy of this combination by comparing the results of sensitivity tests of the isolate to the selected β-lactam antibiotic by either diffusion methods or dilution MIC methods with and without added vancomycin to the used agar or broth (103).
New Aminoglycosides
Arbekacin is a new semi-synthetic aminoglycoside. It has been found to show excellent activity against a wide variety of bacteria that produce aminoglycoside-modifying enzymes. The combination of arbekacin and ampicillin demonstrated inhibitory activity against vancomycin-resistant enterococci and may prove useful for treatment of infections caused by these bacteria (84)
New Cephalosporins
A new experimental cephalosporin designated RWJ-54428 has been found to show a good activity against resistant gram-positive bacteria including vancomycin-resistant strains. In a mouse model of pyelonephritis due to Enterococcus faecalis, this new cephalosporin showed a bactericidal effect with tolerable doses. However, clinical evaluation of this new agent is still underway (68).
New Fluoroquinolones
Fluoroquinolones group (ofloxacin, norfloxacin, ciprofloxacin and enoxacin) has only moderate activity against enterococci. A bactericidal effect of such antibiotics on enterococci is inoculum dependent and may be seen only at concentrations unreached in serum of patients in clinical use (126). Their use against enterococci is so, limited to urinary tract infections (101).
Newer fluoroquinolones, such as clinafloxacin, showed greater activity against gram-positive bacteria including enterococci but vancomycin-resistant enterococci remained among the least susceptible gram-positive bacteria to these antibiotics (31).
In a recent study, experimental fluoroquinolones showed promising results in this aspect. In this study, moxifloxacin and garenoxacin (BMS-284756) were used for treatment of experimental meningitis induced in rabbit by inoculation with vancomycin-tolerant Streptococcus pneumonia strain. These experimental fluoroquinolones showed successful treatment of that condition and proved to be effective in killing and eradication of the causative bacteria (123). However, use of these new experimental fluoroquinolones in treatment of vancomycin-resistant enterococci and staphylococci is still to be evaluated.
Lipopeptides:
Daptomycin (LY146032) is an acidic lipopeptide antibiotic that has good activity against gram-positive bacteria and is similar in spectrum of activity to vancomycin but differs in the mechanism of action. It acts by interference with cell membrane transport mechanisms resulting in rapid bactericidal activity. It has shown promising results in vitro against all species of enterococci including vancomycin-resistant strains and there was no cross-resistance with vancomycin or teicoplanin (130).
Also it showed good activity against the newly emerged vancomycin-resistant S. aureus strains (Mu-50 and 992) in experimental studies (2).
Inspite of these promising in-vitro results, early clinical trials were disappointing as these trials showed ineffective therapy of gram-positive endocarditis and bacteremia by this agent which have been explained by very high (> 90%) plasma protein binding (124). Trials with higher but not toxic doses in humans are still under evaluation (90).
New Glycopeptide Agents
A great effort has been done in the recent years to develop new agents within the glycopeptide group of antibiotics that may show good activity against gram-positive bacteria that show non susceptibility to the current glycopeptides (vancomycin and teicoplanin). This current intensive research may allow for clinical use, some new antibiotics that can be described as, second-generation glycopeptides (94).
The second-generation glycopeptides have the same mechanism of action of those of first generation (vancomycin and teicoplanin) which means that they act by binding to the D-Ala-D-Ala terminus of the N-acetylmuramyl pentapeptide molecules leading to prevention of transpeptidation and transglycosylation and so inhibition of peptidoglycan synthesis. However, these second-generation glycopeptides may differ from first-generation glycopeptides in the ability to bind to the substitutions to D-Ala-D-Ala terminus, which are produced by vancomycin-resistant bacteria (3,94).
Ramoplanin is a newly developed glycopeptide that showed high activity against vancomycin-resistant gram-positive bacteria in general (39,130).
Limitations to use of this glycopeptide antibiotic was put when further studies showed that addition of human serum resulted in fourfold increase in the MIC which suggested the need for high dose for successful treatment. This dose was found to be toxic for systemic use. So, ramoplanin was only suggested to use for local actions such as clearance of glycopeptide-resistant enterococci from the gastrointestinal tract (82), and eradication of C. difficile from the intestine without a risk of colonization by glycopeptide-resistant enterococci (16).
Another newly developed semi-synthetic glycopeptide is that designated LY333328 (now, called oritavancin). It is an investigational N-alkyl semi-synthetic derivative of the naturally occurring glycopeptide LY264826 (40).
Oritavancin is one of the most active agents against vancomycin-resistant enterococci including the most resistant strains of VanA type. Also, it has been found to show good activity against vancomycin-resistant staphylococci (94,99,112).
Oritavancin activity is affected by binding to serum proteins and this affection should be compensated by increase of the dose of the drug. The data are still lacking regarding to the pharmacokinetics in humans and the degree of toxicity (98).
These data should be available to determine if it is possible to achieve the desired concentrations of oritavancin in humans without causing serious side effects. Clinical trials are currently done for this purpose (90)
Mannopeptimycins are new group of glycopeptides antibiotics. Varepsilon is one member of this group and it has been found in a recent study to show good activity against vancomycin-resistant enterococci. This new group showed rapid inhibition of peptidoglycan synthesis and had no effect on cell membrane, DNA, RNA, or protein synthesis suggesting a primary action through inhibition of bacterial cell wall biosynthesis (133)
It is clear that the group of glycopeptides can offer new treatment possibilities for vancomycin-resistant gram-positive bacteria, but further work is still needed to produce a satisfactory bactericidal glycopeptide that is effective and nontoxic in systemic use and lacks cross-resistance with the currently in-use glycopeptides, vancomycin and teicoplanin.
Oxazolidinones
The oxazolidinones are new class of antibiotics with good activity against enterococci and are different from any other class. The mechanism of activity is through inhibition of protein synthesis leading to a bacteriostatic effect (54,89,129).
Linezolid is an agent in this group. It has been found to be of excellent activity against multi-antibiotic-resistant enterococci (4,22). Linezolid has been recently approved as a therapeutic option for vancomycin-resistant enterococci on the basis of in vitro susceptibility and clinical efficiency from multicenter, pharmaceutical company-sponsored clinical trials (4,90,109).
In a recent case study, linezolid was given to a patient suffering from cerebrospinal meningitis caused by Enterococcus faecium that was found to be resistant to vancomycin. The patient was cured when received linezolid intravenously for 4 weeks, which led to complete eradication of the causative agent. This suggested that linezolid is safe and effective therapy for vancomycin-resistant enterococcal meningitis (138).
In another recent study, linezolid has been found to decrease the emergence of vancomycin resistance in S.aureus when given in combination with vancomycin but with no detected synergism (70). Also it has been found in another study to be effective, when given alone, against S. aureus including strains which are methicillin-resistant (109) and also against vancomycin-resistant strains (60). These findings may make linezolid a suitable alternative to vancomycin for treatment of these strains.
Another novel oxazolidinone compound designated PH-027 has been demonstrated a potent antibacterial activity comparable to or better than that of linezolid against gram-positive bacteria and seems to be a good possible therapeutic option for vancomycin-resistant bacteria in the near future (115).
Streptogramins
From this group, quinupristin-dalfopristin (RP59500, Synercid) has been recently approved as a therapeutic drug for vancomycin-resistant-enterococci. This drug is a mixture of two compounds with synergistic activity against gram-positive cocci including enterococci. This drug acts by interference with bacterial protein synthesis that results in a bacteriostatic effect. It has been found to be active against vancomycin-resistant E. faecium but is not active against E. faecalis (90)
It has also been proved that quinupristin-dalfopristin is active against all strains of S. aureus including methicillin-resistant strains (91,109) which may allow to add this drug to the limited number of therapeutic options for treatment of these bacteria.
Comparative studies between the two recently approved antibiotics for vancomycin-resistant gram-positive bacteria (linezolid and quinupristin-dalfopristin) are still needed.
Prevention and Control of the emerging Vancomycin-resistant gram-positive bacteria
Reduction of Vancomycin Use
Vancomycin use has been reported as a major risk factor for colonization and infection with vancomycin-resistant gram-positive bacteria (58,72).
So, vancomycin use should be limited only to the appropriate conditions. These conditions are as following (34):
1. Treatment of serious infections due to β-lactam-resistant gram-positive microorganisms.
2. Treatment of serious infections with gram-positive microorganisms in patients with β-lactam hypersensitivity.
3. When treatment of antibiotic associated colitis with metronidazole fails.
4. Prophylaxis against gram-positive bacterial endocarditis following certain procedures which are known to be associated with high risk.
5. Prophylaxis in a high-risk surgery such as that involving implantation of prosthetic devices. In such case a single dose given immediately before surgery is sufficient unless the surgery lasts more than 6 h., in which case the dose should be repeated, only for another dose.
Vancomycin use in hospitals was found to be markedly increased in the period from 1980 to 1990 which may explain the appearance of resistance to this important antimicrobial agent by its use in many situations which are actually not in high need to it (93).
The situations at which the use of vancomycin should be stopped to avoid emergence of more resistant bacteria are as follows (34): -
1. Routine surgical prophylaxis.
2. Empirical therapy for febrile patients.
3. Treatment in response to a single blood culture positive for coagulase negative staphylococci, if other blood cultures drawn in the same time frame are negative which means that if contamination with skin flora is likely.
4. Empirical use for patients whose cultures are negative for β -lactam-resistant gram-positive bacteria.
5. Prophylaxis of infection or colonization of vascular catheters.
6. Selective decontamination of the digestive tract.
7. Eradication of MRSA colonization.
8. Primary treatment of antibiotic associated colitis.
9. Routine prophylaxis in very low-birth-weight infants.
10. Routine prophylaxis in patients on continuous ambulatory peritoneal dialysis or hemodialysis.
11. Use of vancomycin solution for topical application or irrigation.
Educational Programs
Continuous educational programs including educational sessions are needed for all members of hospital staff including consulting physicians, resident physicians, medical students, pharmacy personnel, nurses, laboratory personnel and other direct patient caregivers. The education programs should include informations about the extent and epidemiology of vancomycin-resistant bacteria and the potential impact of this problem on the outcome of patient care (34).
Early Detection by Appropriate Laboratory Methods in Hospitals Where Vancomycin-Resistant Bacteria have not been Detected
Early detection of patients colonized or infected with vancomycin-resistant bacteria is an essential component of any hospital program designed to prevent nosocomial infections with vancomycin-resistant bacteria (23).
So, the microbiology laboratory is considered the first line of defense in any hospital against the spread of vancomycin-resistant bacteria. The microbiology laboratories in hospitals should be able to use the appropriate methods for prompt and accurate detection of first isolates of these resistant bacteria to allow early prevention of transmission. The prevention and control measures will be more difficult, if the detection is delayed (34).
Prevention of Transmission of Vancomycin-Resistant Bacteria
The following precautions are recommended to prevent patient-to-patient transmission of vancomycin-resistant bacteria (63): -
1. The patients infected or colonized with vancomycin-resistant bacteria should be isolated in special rooms.
2. Gloves and gowns should be used when entering rooms of patients infected or colonized with vancomycin-resistant bacteria.
3. Gloves and gowns should be removed before leaving rooms of patients and the hands should be washed immediately with an antiseptic soap.
4. After removal of gloves and gowns and hand washing, it is important to avoid contact with environmental surfaces potentially contaminated (e.g. doorknobs) in the rooms of patients.
In addition to these isolation precautions, the instruments such as stethoscopes, sphygmomanometers or rectal thermometers should be used for a single patient or a group of patients infected or colonized with vancomycin-resistant bacteria. Otherwise, these instruments may transmit infection with these resistant bacteria to other patients and even may cause a hospital outbreak (92). If such devices are to be used for other patients, they should be adequately cleaned and disinfected (151).
To remove a patient from isolation, he should give negative results for at least three cultures, one week at least apart, from multiple body sites including stool, rectal swab, perineal area, axilla, and umbilicus. Also, specimens should be taken from wounds, catheters or colostomy sites if present (23).
For hospitals with endemic vancomycin-resistant bacteria, more additional recommendations should be followed (34): -
1. Control efforts should be initially focused in ICUs. Such units may serve as a reservoir for vancomycin-resistant bacteria, from which these bacteria spread to other parts of the hospitals when the patients are well enough to be transferred.
2. Every effort should be done to minimize movements of health care givers between patients who are positive and those who are negative for vancomycin-resistant bacteria.
3. Hospital personnel should be examined carefully for chronic skin and nail problems. Hand and rectal swabs cultures should be performed for all hospital personnel routinely. Personnel who prove to be carriers of vancomycin-resistant bacteria should be removed from care of patients who are negative for vancomycin-resistant bacteria until their carrier state has been eradicated.
4. Adequate procedures should be applied for the routine care, cleaning and disinfection of environmental elements in the hospital (e.g. bedrails, charts, doorknobs … etc). Cultures from hospital environmental elements should be routinely done to assess the efficacy of hospital policies and procedures in this aspect.
5. Representative vancomycin-resistant bacteria should be sent to reference laboratories for typing by PFGE and other suitable typing methods to help in precise definition of reservoir and determination of methods of transmission, which allow the control programs to be more successful.
Eradication of Vancomycin-Resistant Enterococcal Colonization of Gastro-Intestinal Tract
One of the major problems with control of vancomycin-resistant enterococci is the spread of colonization with these bacteria in healthy populations that may represent a main non-easily controlled source for infections with these bacteria. In many outbreaks caused by these bacteria, the number of infections was low while colonization showed extensive spread among normal healthy populations. This make the current problem even more difficult in control than the previous problem of MRSA as no colonization spread among healthy subjects has been occurred with outbreaks caused by that bacteria. Colonization with vancomycin-resistant enterococci occurs in the general open populations with no definite increased risk groups, which make colonization of these bacteria difficult to be controlled (20).
There has been an interest in eradication of vancomycin-resistant enterococci when present as a commensal in the gastrointestinal tract to decrease the subsequent risk of infection in the individual patient and to reduce the reservoir of these bacteria in the community (55).
Some studies showed good results when oral bacitacin had been used for this purpose with few relapses (36,110). Also, some other studies suggested successful use of ramoplanin for the same aim (82,90). Successful eradication of colonization with vancomycin-resistant enterococci was also noted in another study using a combination of rifampicin and doxycycline, with negative rectal swabs at 1 and 6 months (44).
However, many other studies have shown some conflicting disappointing results. For instance some studies used a combination therapy of bacitracin plus doxycycline, the results of which showed that this combined therapy may initially clear vancomycin-resistant enterococci from stools of treated patients but with longer follow up, no significant difference occurs between treated and untreated patients. The results of these studies show also that vancomycin-resistant enterococci may be undetectable in feces of the treated patients for only the period during which the antibiotics are given for them (154,155).
New approaches may prove useful for this purpose including administration of probiotic agents such as Bacillus coagulans, which showed promising results for this purpose in experimental animals when given orally (48). However, it is clear that more research is still highly needed to reach to an effective regimen for eradication of vancomycin-resistant enterococci from the gastrointestinal tract of colonized patients, which represents an important requirement for successful prevention and control of these bacteria.
Lastly, we should mention that strict application of the previously outlined, currently possible, measures for prevention and control of these emerging vancomycin-resistant gram-positive bacteria is the cornerstone to overcome this new medical problem. This problem which created a new situation of infections caused by bacteria with no any available active therapeutic option which resulted in increased morbidity and mortality due to these infections in the last years.
We should stress on strict control of hospital infections to change the idea that hospitals are infectious places that may act as vehicles for transmission of the most serious and even fatal infections. This is a very important item especially in light of the current estimations that at any one time a fifth of all inpatients in a hospital have an infection, half of which have acquired in the hospital (33,63).
We are also in urgent need to develop new therapeutic agents against all microorganisms in general and specifically against vancomycin-resistant gram-positive bacteria, otherwise we will be in front of these armed enemies without active weapons. Any developed new active anti-bacterial agent should be then used carefully, avoiding overuse or misuse, which may again push us to the same problem. The problem may be even worse if bacteria pushed man to start of what may be described as “post-antibiotic phase” a time when hospitals will be colonized with bacteria resistant to all available antimicrobial agents and therefore man returns to the pre-penicillin age.
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