Chapter One Gram-Positive Organisms as Human Pathogens
Chapter Two Antibiotics against Gram-positive cocci: Mechanism of action
Chapter Three Mechanisms of Antibiotic Resistance
Chapter Four Antimicrobial Susceptibility Testing
Antimicrobial resistance remains, more than ever, a key issue for medical microbiology. The development of antibiotic resistance by bacteria is an evolutionary inevitability, a convincing demonstration of their ability to adapt to adverse environmental conditions. Some Gram-positive organisms are extremely adaptable and rapidly develop resistance, whereas others have not developed good strategies to overcome antibiotics. Staphylococci and enterococci, in particular are associated with clinically relevant resistance. The epithet of superbugs, if one can define these as bacterial pathogens resistant to almost all clinically available agents, can be truly applied to resistant strains of Gram-positive species, especially to methicillin-resistant Staphylococcus aureus (MRSA) and to glycopeptide- or vancomycin- resistant enterococci (GRE or VRE).
Gram-positive organisms are the most common bacterial pathogens that cause diseases in humans, with staphylococci, enterococci and streptococci occurring most frequently(Metzger et al., 2009). Although Staphylococci, streptococci and enterococci predominate, other organisms also occur, such as micrococci, anaerobe cocci and other rare cocci in in polymicrobial infections, and immunocompromised hosts (Metzger et al., 2009).
Modern lifestyles have significantly changed the incidence, prevalence and, to some degree, the presentation of infections caused by Gram-positive organisms. During the evolution of humankind, pneumococcal pneumonia, scarlet fever, tetanus and diphtheria were among the most common fatal infections. Anthrax and botulism became important disorders associated with ranching, farming and food storage, and listeriosis is closely linked to the processing of milk, fish and meat. During the Second World War, clostridial gas gangrene was a very common cause of death in German field hospitals, as penicillin was available only to the Allied troops. The introduction of penicillin and subsequently other antimicrobial agents, as well as effective vaccine programs, has dramatically changed the interaction between microbes and humans (Metzger et al., 2009).
In terms of antimicrobial susceptibility, some Gram-positive organisms have remained sensitive to most antimicrobials, whereas others, including staphylococci, and enterococci, have developed clinically relevant resistance (Metzger et al., 2009).
Staphylococci are responsible for a plethora of medical problems, including skin and soft-tissue infections (SSTIs), surgical site infections (SSIs), endocarditis and bacteraemia. An increasing number of infections
are related to medical developments, including the use of joint prostheses, immunosuppressants and catheters (Casey et al., 2007).
Staphylococcus aureus was first documented as a human pathogen in the 19th century. Today, it is the most common single pathogen in human medicine causing serious, invasive infections such as soft tissue infections, endocarditis, osteomyelitis, bacteremia, septic arthritis, and nosocomial pneumonia (Drew, 2007, Metzger et al., 2009).
S.aureus is a major cause of many serious hospital- and community-acquired infections. It is also the most common cause of hospital-acquired bacteraemia and is associated with significant morbidity and mortality rates of up to 64%, varying with the infection site and the susceptibility of the particular strain. This pathogenicity reflects its ability to produce a variety of toxins, to attach firmly to prosthetic material by production of a glycocalyx and to an extraordinary ability to develop antimicrobial resistance (Casey et al., 2007).
Coagulase-negative staphylococci (CoNS) are important components of the resident human skin flora. However, the association of these weakly virulent microorganisms with various infections has now been clearly elucidated. CoNS are now recognised as causative agents of post-operative endophthalmitis, bacteremia, endocarditis, shunt-related central nervous system infections, pneumonia, osteomyelitis and wound infection. Staphylococcus saprophyticus is also a frequent cause of urinary tract infection in young women, and is sometimes a cause of bacteremia in hospitalized patients (Casey et al., 2007).
The frequency with which the CoNS are implicated in nosocomial bacteraemia is increasing. CoNS are responsible for the largest proportion of central venous catheter-related bloodstream infections in inpatients. This is concurrent with advances in medical practice, including the increased use of indwelling medical devices such as vascular catheters, prosthetic joints, vascular grafts, prosthetic heart valves and peritoneal catheters as well as with the number of immunocompromised patients (Casey et al., 2007) . CoNS also are currently the most common bloodstream infection treated in neonatal and pediatric intensive care units and have significant impact on patients mortality and morbidity (Venkatesh et al., 2006).
I) 2. Enterococci
Enterococci are a part of the normal human faecal flora, and were originally classified as enteric Gram-positive cocci belonging to the genus Streptococcus. During the mid 1980s, studies involving fatty acid composition, nucleic acid hybridization and comparative oligonucleotide
cataloguing of 16s RNA led to the acceptance that the enterococci were sufficiently different from other streptococci to merit their own genus (Sood et al., 2008).
Enterococci have traditionally been regarded as low grade pathogens, however, they have emerged as an increasingly important cause of nosocomial infections in the last decade. Although about a dozen enterococcus species have been identified, only two are responsible for the majority of human infections, which are Enterococcus faecalis and E. faecium (Sood et al., 2008).
A major reason why these organisms survive in hospital environment is the intrinsic resistance to several commonly used antibiotics and, perhaps more importantly, their ability to acquire resistance to all currently available antibiotics, either by mutation or by receipt of foreign genetic material through the transfer of plasmids and transposons (Sood et al., 2008).
Enterococcus faecalis differs in many ways from Enterococcus faecium. It is considered a primary pathogen in urinary tract and mixed intra-abdominal infections, including those of the biliary tract, and is usually sensitive to aminopenicillins. E. faecium, on the other hand, shows resistant patterns similar to MRSA, yet is considered to be of low intrinsic virulence. More than 90% of vancomycin-resistant enterococci (VRE) are E. faecium, and in many hospitals in the U.S., the majority of E. faecium are VRE. Subsequently, it commonly contributes to secondary infections in severely ill individuals who have been previously exposed to multiple antibiotics (Metzger et al., 2009). It is also worth noting that many studies have documented high mortality rates attributable to enterococcal bacteraemia, ranging from 42% to 68% (Sood et al., 2008).
Nevertheless, enterococci are not a common cause of bacteremia in healthy children or late-onset sepsis in neonates. More commonly, enterococcal bacteremia is a nosocomial infection. Enterococci usually cause late-onset neonatal sepsis and bacteremia in compromised, seriously ill children such as patients in hematology/oncology and intensive care units and those with intravascular catheters (Butler, 2006).
Streptococci are a diverse group of pathogens, including Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus viridans, S. milleri, anaerobic streptococci and Streptococcus pneumonia (Metzger et al., 2009).
There have been several attempts in history to classify streptococci based on haemolytic reactions, antigens, and other phenotypic characteristics. Streptococci of clinical importance were classified based on their action on blood-containing agars. Some streptococci (β-hemolytic) can lyse blood cells and cause complete clearing of blood in the vicinity of their growth. Other strains cause no change in blood agar (γ- or nonhemolytic), while the remainder of the streptococci (α- hemolytic) reduce hemoglobin and cause a greenish discoloration of the agar. In 1937, Sherman, classified the streptococci into the pyogenic, viridans, enterococcal, and lactic divisions, based on phenotypic traits . By the mid-1980s, the enterococcal streptococci and the “dairy” or “lactic” streptococci were moved into new genuses. Later studies gave rise to updated classification schemes based on 16S ribosomal RNA sequences and other molecular information. It became apparent that the hemolytic reactions, Lancefield antigens, and other phenotypic characteristics relied on in the past were not always accurate predictors of genetic relatedness among strains. These characteristics are, however, still useful to clinical laboratorians for the presumptive identification of many commonly encountered streptococci (Bisno and Ruoff, 2005).
Streptococcus spp. have tended to remain susceptible to many antibacterials with which they have been challenged. Streptococcus pyogenes, in particular, remains almost universally susceptible to penicillin. Streptococcus pneumoniae, however, has demonstrated a much greater propensity to develop resistance. Apart from its obvious role in community-acquired pneumonia, S. pneumoniae may also be responsible for otitis media, meningitis, nosocomial pneumonia, acute exacerbations of chronic obstructive pulmonary disease and bacteraemia (Amyes, 2007).
Aside from S. pneumoniae, other pathogenic streptococci include: S.milleri which can cause purulent infections of the skin and the gastrointestinal tract, S. agalactiae which is commonly found in the genital tract of women and may therefore cause life-threatening infections in the newborn and S. viridans which are commensals in the oral cavity and typically associated with bloodstream infections (Metzger et al., 2009) .
The development of antibiotic resistance by bacteria is an evolutionary inevitability, a convincing demonstration of their ability to adapt to adverse environmental conditions. Human kind has been using antibiotics for less than 70 years but, despite the technological innovation, we are constantly being challenged by the ready adaptability of the bacterial pathogens that we naively sought to conquer and eradicate. This ‘arms race’ has resulted in bacteria that are resistant to all antibacterial classes. Antibiotic resistance remains, more than ever, a key issue for medical microbiology (Woodford, 2005). In this section, the most commonly used antibiotics against Gram-positive pathogens, their mechanism of action, bacterial resistance and clinical applications are reviewed, with special emphasis on staphylococci.
The β-lactams are the most widely used antibacterial class. Since the introduction of penicillin in the 1940s, the class has been developed and expanded to provide a continuing flow of agents with enhanced activity against bacteria resistant to preceding members (Woodford, 2005).
β-lactams are bactericidal agents that act by binding to ‘‘penicillin-binding proteins’’, which are proteins involved in cell-wall synthesis (Gold and Pillai, 2009).
The cell wall of Gram-positive bacteria is formed of a thick layer of peptidoglycan that protects against osmotic rupture. The basic subunit of the peptidoglycan component is a disaccharide monomer of N -acetylglucosamine (NAG) and N -acetylmuramic (NAM) pentapeptide. Peptidoglycan is composed of long polysaccharide chains of NAG and NAM pentapeptide. The pentapeptide consists of amino acid residues alternating between L- and D-stereoisomers and terminating in D-alanyl-D-alanine. A stem peptide of variable length and composition is attached to the third amino acid of this pentapeptide. Pentapeptides are then joined with stem peptides to form a cross-link between polysaccharide chains. This reaction is catalyzed by a transpeptidase. This transpeptidation reaction is sensitive to inhibition by β-lactams. The penicillin-sensitive reactions are catalyzed by a family of closely related proteins, penicillin-binding proteins (PBPs). β-Lactam antibiotics produce their lethal effect on bacteria by inactivation of multiple PBPs simultaneously, and thus inhibiting cell wall synthesis. The inhibition of PBPs also leads to disruption of a crucial event probably at the time of cell division. This disturbed morphogenesis is hypothesized to initiate cell death (Chambers, 2005) .
Resistance to β-lactam drugs in staphylococci is mediated by two major mechanisms. The first is a highly prevalent narrow-spectrum β-lactamase that confers resistance to penicillins exclusive of the penicillinase-resistant penicillins dicloxacillin, oxacillin, and nafcillin. Most cephalosporins and the carbapenems are not greatly hydrolyzed by this enzyme, and β-lactamase inhibitors can protect otherwise vulnerable penicillins (Gold and Pillai, 2009). The other major mechanism of resistance is the presence of the penicillin-binding protein PBP2a, mediated by the mecA gene that confers resistance to all current β-lactams creating the MRSA phenotype (Gold and Pillai, 2009).
The adverse drug reactions caused by β-lactams include a number of class effects that occur to varying degrees after exposure to these agents and several effects that are particularly prominent with specific agents. The former can include various hypersensitivity reactions ranging from urticaria to allergic interstitial nephritis to anaphylaxis; rash; drug fever; serum sickness; gastrointestinal toxicity and hematologic toxicity. Most β -lactams have relatively limited drug interactions (Gold and Pillai, 2009).
When penicillin was first introduced in 1944, over 94% of Staphylococcus aureus isolates were susceptible; by 1950 half were resistant. By 1960 many hospitals had outbreaks of virulent multi-resistant S. aureus (Livermore, 2000). Most current staphylococcal isolates can produce a narrow-spectrum β -lactamase that inactivates penicillins (other than nafcillin, oxacillin, and dicloxacillin) (Gold and Pillai, 2009). Resistance to penicillin among S aureus isolates is estimated to be 99% in some medical centres (Gold and Pillai, 2009). Production of penicillinase can be plasmid-mediated, but chromosomal integration of its gene is frequent (Livermore, 2000). Against the relatively rare penicillin-susceptible staphylococci, penicillin (or ampicillin or amoxicillin) may be used for treatment of infections.
Methicillin was introduced in the early 1960s to combat hospital strains of penicillinase-producing S. aureus, it being resistant to hydrolysis by these enzymes (Woodford, 2005). Methicillin, nafcillin and the oxacillins, all have bulky 6' acyl groups replacing the 6' phenylacetyl group in benzylpenicillin. These groups can sterically hinder attack on the β-lactam ring, thus allowing activity to be retained against penicillinase-positive staphylococci (Livermore, 2000).
Since staphylococci secrete their β -lactamase into the extracellular milieu, protecting the entire bacterial population, there was little or no subsequent selection of strains with more potent β –lactamase variants. This is in contrast to Gram-negative bacteria, which retain β -lactamases within the layers of their cell wall so that variant strains that produce more, or more-potent (extended-spectrum or inhibitor-resistant) enzymes have been selected (Livermore, 2000).
Oxacillin and nafcillin are the most frequently used parenteral drugs in this group, and dicloxacillin is the major oral formulation. These drugs are the drugs of choice for treating serious infections caused by MSSA, and methicillin-sensitive CoNS (Gold and Pillai, 2009).
However, resistance to methicillin was noted shortly after its introduction. MRSA and methicillin-resistant coagulase-negative staphylococci (MRCoNS) are all too familiar in today’s hospitals (Woodford, 2005). Whereas normal staphylococci employ three penicillin-binding proteins, PBPs 1, 2, and 3, to catalyse cross-linking of peptidoglycan, Methicillin-resistant strains have an additional component, PBP 2' or 2a, which has low affinity for β-lactams. MRSA consequently are resistant to all β-lactams. The mecA gene encoding for PBP 2' is carried by large sections of chromosomally inserted DNA, which have no homologues in methicillin-susceptible strains. These inserts have been termed staphylococcal cassette chromosome mec (SSC mec), of which three variants are known (Livermore, 2000). Regulation of mecA expression is complex. Some MRSA have homogeneous resistance to β-lactams, with all the cells in a population expressing resistance; others have heterogeneous resistance, with resistance only expressed by a small minority of the cell population (Livermore, 2000).
The β-lactam– β -lactamase inhibitor combinations (amoxicillin-clavulanate, ticarcillin-clavulanate ; ampicillin-sulbactam and piperacillin-tazobactam) are other treatment options for penicillinase-producing methicillin-susceptible staphylococci. They all have good activity against MSSA, but not MRSA, and are active against anaerobes and gram-negative bacilli to varying degrees, making them appropriate choices for the treatment of polymicrobial infections including MSSA (Gold and Pillai, 2009).
Cephalosporins are the most frequently prescribed antibiotic medications and have a long record of efficacy and relative safety (Gold and Pillai, 2009). However, they are active only against methicillin-susceptible strains and not against methicillin-resistant staphylococci.
Methicillin-resistant staphylococci are resistant to all other penicillins, carbapenems, cephems and β-lactam/β-lactamase inhibitor combinations and results for these drugs should be reported as resistant, regardless of the in vitro susceptibility results (Clinical and Laboratory Standards Institute, 2007).
Cephalosporins are generally grouped into generations based on antimicrobial spectrum of activity. The first-generation drugs include the parenteral agent cefazolin and the oral drug cephalexin. These drugs have good activity against methicillin-susceptible staphylococci and Streptococcus pyogenes, but only limited activity against gram-negative bacilli. Some second-generation drugs, such as cefuroxime, maintain good antistaphylococcal activity with somewhat greater gram-negative activity. The third-generation drugs have even broader gram-negative spectrum and several are active against methicillin-susceptible staphylococci. The more recently developed fourth-generation drug has very broad gram-negative activity and maintains clinically useful activity against methicillin-susceptible staphylococci. The first-generation cephalosporins are drugs of choice for the treatment of methicillin-susceptible staphylococci infections in patients who are unable to tolerate antistaphylococcal penicillins. In the absence of a severe penicillin allergy, such as anaphylaxis (because of concerns for cross-reactivity), they are favored over use of vancomycin in this setting (Gold and Pillai, 2009) .
Carbapenems are broad-spectrum β-lactam antibiotics with activity against anaerobes, gram-negative bacteria, and gram-positive bacteria, including MSSA. The most commonly used carbapenems are imipenem and meropenem However, currently approved carbapenems lack clinically useful activity against methicillin-resistant staphylococci (Gold and Pillai, 2009).
The monobactam drugs, e.g. aztreonam have activity against aerobic Gram-negative bacilli, but lack clinically relevant activity against staphylococci (Gold and Pillai, 2009).
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Figure(II-1): Chemical structure of three clinically important β-lactams: A) benzylpenicillin, B)oxacillin, C)Cefazolin and D)Cephalexin
Vancomycin (Figure(II.2)), the first glycopeptide antibiotic, was first isolated in the mid-1950s from a strain of the actinomycete Amycolatopsis orientalis during a screening program promoted by Eli Lilly to isolate antistaphylococcal drugs effective against the serious infections caused by penicillinase-producing Staphylococcus aureus strains (Biavasco et al., 2000). It became a drug of high clinical interest in the late 1970s following the emergence, especially in hospital-associated infections of compromised patients, of highly and multiply resistant, but vancomycin-susceptible, gram-positive pathogens (Biavasco et al., 2000).
Vancomycin is not absorbed from the gastrointestinal tract in clinically relevant concentrations, and so it must be used as a parenteral agent for systemic infections (Gold and Pillai, 2009).
Teicoplanin (Figure(II.3)), the second glycopeptide discovered, was first obtained in the late 1970s in the Lepetit research laboratories by the fermentation of Actinoplanes teichomyceticus and became commercially available in Europe in the late 1980s (Biavasco et al., 2000). Teicoplanin was first introduced in Egypt in 2008 under the brand name Targocid.
Despite the introduction of several new antimicrobial agents active against Gram-positive pathogens in the recent years, vancomycin remains the golden standard for treating a variety of healthcare-associated infections caused by Gram-positive pathogens, particularly staphylococci and enterococci (Eliopoulos, 2003, Tenover and McDonald, 2005, Drew, 2007)
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Figure (II.2): Structure of vancomycin
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Figure(II.3): Structure of teicoplanin
Vancomycin is a slowly bactericidal drug that acts by binding to the terminus of the peptidoglycan precursor, the building block for the bacterial cell wall (Gold and Pillai, 2009). The synthesis of peptidoglycan in the production of bacterial cell walls requires several steps. In the cytoplasm, a racemase converts L-alanine to D-alanine (D-Ala), and then 2 molecules of D-Ala are joined by a ligase, creating the dipeptide D-Ala-D-Ala, which is then added to uracil diphosphate– N -acetylmuramyl-tripeptide to form uracil diphosphate – N -acetylmuramyl - pentapeptide. Uracil diphosphate – N -acetylmuramyl - pentapeptide is bound to the undecaprenol lipid carrier, which, after the addition of GlcNAc from uracil diphosphate–GlcNAc, allows translocation of the precursors to the outer surface of the cytoplasmic membrane. N -acetylmuramyl-pentapeptide is then incorporated into nascent peptidoglycan by transglycosylation and allows the formation of cross-bridges by transpeptidation (Courvalin, 2006).
Vancomycin binds with high affinity to the D-Ala-D-Ala C-terminus of the pentapeptide, thus blocking the addition of late precursors by transglycosylation to the nascent peptidoglycan chain and preventing subsequent cross-linking by transpeptidation (Courvalin, 2006).
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Figure (II.4):Peptidoglycan biosynthesis and mechanism of action of vancomycin (Courvalin, 2006)
Binding of the antibiotic to the C-terminal d-Ala–d-Ala of late peptidoglycan precursors prevents reactions catalyzed by transglycosylases, transpeptidases, and the D,D-carboxypeptidases. Ddl= D-Ala:D-Ala ligase; MurF, a synthetase protein; UDP, uracil diphosphate.
Vancomycin is considered by many to be the mainstay for the treatment of invasive infections caused by multidrug-resistant Staphylococci, in part because of extensive published experience in the treatment of serious, invasive infections and a favorable safety profile (Drew, 2007). But, unfortunately, there is ample clinical experience to indicate that vancomycin is a less than optimal drug for the treatment of either staphylococcal or enterococcal infections. For example, recently, patients with MRSA bacteremia who participated in a randomized trial and were treated with vancomycin had a clinical success rate of only 31.8% (Food and drug administration, 2006). Several drug-related factors may contribute to such poor outcomes within susceptible-organisms, for example, vancomycin’s microbiologic activity may be compromised in the presence of biofilms produced by some S. aureus organisms and isolates from patients receiving long-term vancomycin therapy for bacteremia usually show a loss of an accessory gene-regulator function in MRSA which contributes to treatment failure (Drew, 2007).
Acquired bacterial resistance to glycopeptides antibiotics was first reported in 1986, in coagulase-negative staphylococci. However, the attention soon deviated to focus on enterococci, primarily because of the relatively infrequent occurrence of glycopeptide-resistant Staphylococci in clinical practice. Vancomycin-resistant enterococci went on to become major nosocomial pathogens, particularly in the USA. Only recently have glycopeptides-resistant staphylococci come back to the fore, following a report of the dissemination of Staphylococcus aureus strains resistant to vancomycin in Japanese hospitals (Biavasco et al., 2000).
Vancomycin resistance in enterococci occurs mainly by target modification. VanA-type resistance is the most frequently encountered type of glycopeptides resistance in enterococci, and is mediated by a transposon which reduces pyruvate to d-Lac, and by the VanA ligase, which catalyzes the formation of an ester bond between d-Ala and d-Lac. The resulting d-Ala-d-Lac replaces the d-Ala-d-Ala dipeptide in peptidoglycan synthesis, a substitution that decreases the affinity of the molecule for glycopeptides (Courvalin, 2006). It was initially feared that S. aureus would acquire the van genes that code for vancomycin resistance in Enterococcus species
(Srinivasan et al., 2002). Under laboratory conditions, it was possible to transfer the VanA resistance genes responsible for vancomycin-resistance in enterococci to Staphylococcus aureus (Noble et al., 1992) . However, glycopeptide resistance of enterococcal origin has not spread naturally to Staphylococcus aureus (Hiramatsu, 1998, Biavasco et al., 2000). In Staphylococci, the mechanism of glycopeptides resistance seems to be the development of a thickened and abnormally disorganized cell wall that essentially traps vancomycin (Gold and Pillai, 2009) (Figure(II.5)). It has been also demonstrated that cell wall synthesis and turnover are upregulated in VRSA isolates, leading to thicker and more-disorganized cell walls. In order to exert an effect, vancomycin must reach the cytoplasmic membrane and bind with nascent cell wall precursors, thereby inhibiting their incorporation into the growing cell wall. It has been proposed that the thicker, disorganized cell walls can actually trap vancomycin at the periphery of the cell, thereby blocking its action. In fact, it has been shown that vancomycin can be recovered intact from the cell walls of VISA and VRSA isolates, indicating that the antibiotic is not being inactivated but merely sequestered by the bacteria (Srinivasan et al., 2002).
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Figure(II.5): Proposed model for the capture of vancomycin in the cell wall of VRSA organisms (Srinivasan et al., 2002)
In the United States, vancomycin has maintained 100% susceptibility in vitro against S. aureus in several surveillance studies (Drew, 2007). However, there were several case reports of escalated vancomycin MIC among isolates and even of vancomycin-resistant clinical isolates (Drew, 2007). The CDC has also reported a few cases of clinical VRSA isolates(Centers For Disease Control and Prevention, 2002a, Centers for Disease Control and Prevention, 2002b, Centers for Disease Control and Prevention, 2004).
While clinical VRSA isolates remain rare in the United States, there is a concern because under selective pressure from vancomycin, a series of sequential mutations can be selected and the organism transis from full susceptibility to hetero-VISA(hVISA) to VISA (Gold and Pillai, 2009).
In Egypt, less information is available about the rates of vancomycin-resistance in Gram positive cocci. However, published data indicate that situation in Egypt is much worse. In a recent report analyzing nosocomial blood stream infection in intensive care units at Assiut University Hospitals, vancomycin-resistance among Gram-positive isolates was 9.9% (Ahmed et al., 2009). In another report describing Gram-positive infections among cancer patients in the National Cancer Institute in Cairo, 15.5 % of S.aureus isolates and 11% of CoNS isolates were vancomycin-resistant. 3.5% of S.aureus and 1.4% of CoNS isolates were vancomycin-intermediate (Ashour and el-Sharif, 2007).
II.3.3 Macrolides and lincosamides
The macrolides-lincosamide-streptogramin (MLS) antibiotics currently in clinical use for Gram-positive infections include several macrolides (erythromycin, clarithromycin, and the azalide azithromycin), the lincosamide drug clindamycin, and streptogramin B antibiotic quinupristin, which is combined with the streptogramin A antibiotic dalfopristin in the drug Synercid (Gold and Pillai, 2009). Quinopristin-dalfopristin will be discussed in a separate section.
The MLS group of drugs are bacteriostatic agents that share a common mechanism of action, i.e., they bind to the bacterial ribosome and block protein elongation (Gold and Pillai, 2009).
Macrolide resistance is Staphylococci was recently reported to be very high, and thus, currently available macrolides are rarely if ever appropriate empiric agents (Gold and Pillai, 2009). Although community acquired strains are more susceptible to erythromycin than are nosocomially acquired strains, their susceptibilities are also poor (Drew, 2007). Therefore, erythromycin has only a limited use in the treatment of multidrug resistant staphylococcal infections (Drew, 2007).
Clindamycin on the other hand is still widely used to treat staphylococcal infections. Some authors even suggest that clindamycin probably has efficacy similar to that of vancomycin (Anstead et al., 2007). It has specific use in patients with β-lactam hypersensitivity, in the treatment of infections caused by isolates resistant to alternative agents, and in the treatment of staphylococcal toxin-mediated disease (Gold and Pillai, 2009). However, most nosocomial MRSA isolates are resistant to clindamycin, but, most community-acquired isolates still retain susceptibility(Anstead et al., 2007). Reports of clinadmycin efficacy against community acquired MRSA in children have also been described (Martinez-Aguilar et al., 2003).
Nevertheless, the use of clindamycin has several potential drawbacks. First is the phenomenon of inducible clindamycin resistance, that will be discussed later. Clindamycin should not be used to treat serious staphylococcal infections if the isolate is resistant to erythromycin unless the D-test is performed. A second drawback is its short half-life, necessitating three or four times daily dosing, which may decrease patient compliance (Anstead et al., 2007). Accordingly, knowledge of local resistance patterns and use of the D-zone test for erythromycin-resistant isolates, should serve as important guides in the thoughtful treatment with clindamycin (Metzger et al., 2009).
Macrolide-resistant isolates of S. aureus, coagulase-negative Staphylococcus spp., and β-hemolytic streptococci may express constitutive or inducible resistance to clindamycin (methylation of the 23S rRNA encoded by the erm gene; also referred to as MLSB (macrolide, lincosamide, and type B streptogramin resistance) or may be resistant only to macrolides (efflux mechanism encoded by the msrA gene in staphylococci or a mef gene in streptococci) (Clinical and Laboratory Standards Institute, 2006b, Clinical and Laboratory Standards Institute, 2007).
The erm -mediated resistance may be constitutively or inducibly expressed. Constitutive expression results in cross-resistance to all macrolides, lincosamides, and type B streptogramins (MLSBC phenotype). However, in inducible resistance, erm -mediated macrolide resistance is induced by erythromycin, but not by clindamycin or streptogramin B antibiotics, which retain in vitro activity. In isolates that are resistant to erythromycin, inducible resistance to clindamycin should be detected in by the disk approximation test ‘‘D-test’’. Isolates that show inducible resistance to clindamycin may develop resistance to that drug on treatment (Gold and Pillai, 2009). Hence, CLSI currently suggests performing the D-test on all erythromycin-resistant staphylococcal isolates (Clinical and Laboratory Standards Institute, 2007). After erm -encoded methylation, the second most common mechanism of resistance to macrolides in staphylococci is the Msr ATP-binding cassette pumps that confer resistance to 14-membered ring macrolides (eg, erythromycin and clarithromycin) and type B streptogramins(Saiman et al., 2003).
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II.3.4 Trimethoprim/sulfamethoxazole ( TMP-SMX)
Both trimethoprim and sulfonamides inhibit enzymes in the bacterial folate biosynthesis pathway (Figure(II.7)). The rationale for the combination of trimethoprim with sulfamethoxazole is that each component blocks a different step in the folate biosynthetic pathway. Formation of dihydrofolic acid from para-aminobenzoic acid is inhibited by sulfamethoxazole, whereas trimethoprim blocks dihydrofolate reductase, which inhibits the formation of tetrahydrofolic acid from dihydrofolic acid. By inhibiting synthesis of tetrahydrofolic acid, the metabolically active form of folic acid, trimethoprim -sulfamethoxazole inhibits bacterial thymidine synthesis(Figure(II.8)). This sequential inhibition in the folic acid pathway appears to account for the antibacterial synergism displayed by the two drugs, which often have a bactericidal effect (Grim et al., 2005).
The compound is active against most Enterobacteriaceae, Haemophilus influenzae, Chlamydia trachomatis, Nocardia asteroides, and many strains of streptococci and staphylococci, among others (Grim et al., 2005).
Community-acquired MRSA is frequently susceptible to trimethoprim/ sulfamethoxazole (Drew, 2007, Metzger et al., 2009). However, recent clinical efficacy data for the use of TMP-SMX in patients with serious Staphylococcal infections are sparse(Drew, 2007). In a randomized trial published in 1992, vancomycin and trimethoprim-sulfamethoxazole were compared in a variety of S. aureus infections in intravenous drug users. Among the 101 patients evaluated, infections were due to MRSA in 47% and 65% were bacteremic. Forty-three received trimethoprim-sulfamethoxazole and fifty-eight received vancomycin. The overall cure rates were 98% (57/58) and 86% (37/43) of vancomycin- and trimethoprim-sulfamethoxazole–treated patients, respectively. Failure occurred mostly in patients with tricuspid valve endocarditis and only in those with methicillin-sensitive S.aureus. All patients with MRSA infections in both groups were considered cured. The authors concluded that although vancomycin is generally superior to trimethoprim-sulfamethoxazole, trimethoprim-sulfamethoxazole can still be considered an alternative to vancomycin therapy in some cases of MRSA infections (Markowitz et al., 1992).
Only a small number of other reports are available and present anecdotal uses of TMP-SMX for treatment of staphylococcal infections(Grim et al., 2005). Shams et al. reported a case of a patient with persistent bacteremia caused by a MRSA infected left-ventricular assist device in which trimethoprim–sulfamethoxazole treatment was successful after vancomycin, linezolid, and quinoprustin-dalfopristin all failed (Shams et al., 2005). Efficacy of trimethoprim/sulfamethoxazole in patients with infections due to VISA has been difficult to determine because of previous and concomitant therapies administered to these patients (Drew, 2007). However, in pediatric patients, TMP-SMX is still likely to be effective against community-acquired MRSA and can be considered an alternative regimen (Marcinak and Frank, 2003).The drug was successfully used to treat pediatric MRSA infections resistant to clindamycin and erythromycin (Frank et al., 2002). Another report describes an infant with sepsis secondary to MRSA endocarditis and meningitis. The patient was first given vancomycin monotherapy, and after 48 hours of treatment, cultures were still positive. Rifampin was added, and cultures remained positive another 72 hours later. At that time, vancomycin and rifampin were discontinued, and TMP-SMX was started. Blood and cerebrospinal fluid cultures were thereafter negative, but the patient died 2 weeks later due to worsening heart failure. Postmortem blood cultures were negative (Tamer and Bray, 1982). Although published clinical experience is scarce and some physicians might hesitate to use TMP-SMX for serious infections without more experience, but mild-to-moderate infections can still be managed this way (Marcinak and Frank, 2003). Use of trimethoprim–sulfamethoxazole in neonates is potentially contraindicated owing to exacerbation of hyperbilirubinemia (free bilirubin) and resulting kernicterus (Marcinak and Frank, 2006).
Potential advantages to the use of TMP-SMX include convenient dosing (i.e. twice/day), availability of both intravenous and oral dosage forms, cidal activity, low cost and retention of activity in vitro against S. aureus despite almost three decades of use(Anstead et al., 2007, Drew, 2007). Liabilities of the drug are a high rate of hypersensitivity reactions, myelosuppression, and electrolyte disturbances (Anstead et al., 2007).
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TMP-SMX resistance has markedly increased among Gram-negative bacteria, limiting the use of this drug against infections caused by these organisms. However, TMP-SMX resistance among most Gram-positive bacteria, and MRSA in particular, has remained relatively low; therefore, this drug may continue to be used in management of infections caused by such bacteria (Shams et al., 2005). Nevertheless,the reported rate of TMP-SMX resistance in S.aureus is highly variable. Resistance in the United States ranges from 0–74%,and worldwide resistance ranges from 8-100%(Grim et al., 2005).
Bacterial resistance to TMP-SMX may develop independently of TMP and SMX, but this has not been fully elucidated. Sulfonamide resistance among S. aureus may be attributed to two mechanisms. Most resistance is likely due to chromosomally mediated overproduction of para -aminobenzoic acid. A sulfonamide-resistant plasmid has also been described in S. aureus, although the mechanism of resistance is unclear. Resistance to TMP also may be chromosomally or plasmid mediated. A single amino acid substitution, Phe98 to Tyr98 in dihydrofolate reductase, is thought to be the molecular origin of TMP resistance in S. aureus. It is hypothesized that this amino acid alteration leads to a particular dihydrofolate reductase (type S1) that results in decreased affinity for S.aureus. Exogenous thymidine will render TMP-SMZ inactive, because it bypasses the double biosynthetic blockade (Grim et al., 2005, Proctor, 2008).
In short, in an era of increasing community-associated MRSA, trimethoprim/sulfamethoxazole may still be an attractive therapeutic option, especially in skin and soft tissue infections contingent on local susceptibility patterns(Gold and Pillai, 2009).
Figure(II.7): Chemical structure of trimethoprim (left) and sulfamethoxazole (right)
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Figure(II.8): Mechanism of action of TMP-SMX
DHFR = dihydrofolate reductase ; DHPS=dihydropteroate synthase; dTMP= deoxythymidine 5'-phosphate; dUMP=deoxyuridine monophosphate; Nuc=thermonuclease; NupC= pyrimidine nucleoside transport protein; ABA= para-aminobenzoic acid; THF= tetrahydrofolate (Proctor, 2008) .
Rifampin (Fig(II.9)) is a potent bactericidal antibiotic that inhibits chain initiation of bacterial DNA-dependent RNA polymerase(Anstead et al., 2007, Gold and Pillai, 2009). It is available in oral and iv preparations(Anstead et al., 2007)
Rifampin still demonstrates potent in vitro activity against S. aureus and resistance to rifampin is usually rare among Staphylococcal isolates(Anstead et al., 2007, Drew, 2007, Gold and Pillai, 2009). However, recent in vitro reports demonstrate limited activity of rifampin against VISA(Drew, 2007). Unfortunately, when rifampin is used against S. aureus as monotherapy, resistance occurs rapidly via a one-step target-site mutation in the RNA polymerase. Rifampin is thus often used as part of combination therapy (usually with vancomycin) to treat some cases of serious invasive staphylococcal infections (Anstead et al., 2007, Drew, 2007). In a prospective study to evaluate the efficacy of rifampicin as an adjunct therapy in burn cases with MRSA septicaemia not responding well to vancomycin, the combination showed excellent therapeutic results. Fourteen MRSA septicaemic patients with burns who did not respond to therapeutic doses of vancomycin within 5–6 days in spite of in vitro susceptibility of isolates to vancomycin, were treated with rifampicin as an adjunct therapy for 5 days. Institution of rifampicin, as an adjunct to vancomycin therapy, showed a dramatic clinical response. Thirteen patients recovered and one died who had 70% deep burns and blood cultures revealed a multi-resistant Acinetobacter in addition to MRSA (Gang et al., 1999). In another experimental study in a rabbit model, the combination of rifampin with either quinopristin-dalfopristin or vancomycin was significantly more effective against staphylococcal infections than monotherapy of either drugs alone (Saleh-Mghir et al., 2002). In addition, treatment with rifampin for adults or pediatric patients, administered either orally or parenterally, in combination with a β-lactam or vancomycin is the currently recommended treatment for staphylococcal prosthetic valve endocarditis, including those caused by S aureus and S.epidermidis (Gold and Pillai, 2009).
Resistance to rifampin occurs mainly by alteration of the target site, the RNA polymerase (Wehrli, 1983). Various mutations can arise within rpoB gene(which encodes the β-subunit of RNA polymerase) that confer different levels of resistance to rifampicin. These mutations are readily selected in vitro after a single selection step(O'Neill et al., 2001).
Rifampin is associated with a wide spectrum of side effects and clinicians must also be cognizant of the potential risk for drug-drug interactions caused by rifampin-associated induction of the hepatic cytochrome P-450 system(Gold and Pillai, 2009).
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Figure(II.9): Chemical structure of rifampin
Nalidixic acid was the first quinolone antibacterial agent licensed for use by the FDA. Since introduction of nalidixic acid in the 1960s, subsequent generations of fluoroquinolones have been discovered. Fluorination of quinolone compounds resulted in the introduction of norfloxacin in 1986 and ciprofloxacin in 1987, followed by other second-generation fluoroquinolones, including levofloxacin. Additional modifications resulted in third and fourth-generation fluoroquinolones, that include gatifloxacin and moxifloxacin among others(Committee on Infectious Diseases, 2006).
The first fluoroquinolones introduced into clinical use were largely developed for treatment of infections due to Gram-negative bacteria, although ciprofloxacin had limited activity against some Gram-positive bacteria. Subsequently developed and marketed fluoroquinolones—including levofloxacin, sparfloxacin, grepafloxacin, trovafloxacin, gatifloxacin, and moxifloxacin—have had increased activity against Gram-positive cocci and have received regulatory approvals for treatment of patients with various infections with these organisms(Hooper, 2002).
Some fluoroquinolones are no longer available, and others are of limited use clinically. Currently, ciprofloxacin, levofloxacin, gatifloxacin, and moxifloxacin are the most widely used fluoroquinolones(Committee on Infectious Diseases, 2006). Current FDA-approved clinical uses of these fluoroquinolones are numerous, and include but are not limited to, urinary tract infections, skin and skin-structure infections, pneumonia, bone and joint infections and typhoid fever (Andriole, 2005). However, they do not include bacteremia.
Fluoroquinolones are bactericidal agents that inhibit bacterial DNA gyrase and topoisomerase IV(Gold and Pillai, 2009). Both enzymes are necessary for DNA replication; DNA gyrase acting by altering DNA supercoiling; and topoisomerase IV acting to separate interlocked DNA strands to allow segregation of daughter chromosomes into daughter cells. Both enzymes are composed of two types of subunits, called GyrA and GyrB in DNA gyrase, and ParC and ParE in topoisomerase IV. GyrA is similar in structure to ParC, and GyrB is similar in structure to ParE. Fluoroquinolones interact with complexes of each enzyme in DNA to trap the complex such that it serves as a barrier to the progression of the DNA replication enzyme complex along the DNA, thereby blocking DNA replication. Subsequent events, involve generation of an irreparable DNA break triggered by this block, ultimately resulting in bacterial-cell death (Hooper, 2002).
Significant differences exist in the in vitro activity of various fluoroquinolones against Staphylococci. In general, MICs to newer fluoroquinolones (e.g., moxifloxacin, gatifloxacin, and levofloxacin) are lower than those seen for older fluoroquinolones (e.g., ciprofloxacin). In
contrast to MSSA, however, the in vitro potency of fluoroquinolones against MRSA is significantly reduced. Fluoroquinolones have also demonstrated high resistance rates against VISA and VRSA. Therefore, fluoroquinolones are not routinely recommended in suspected or documented MRSA infections (Drew, 2007). Fluoroquinolone resistance in enterococci has been surveyed less extensively but is substantial, especially among strains of Enterococcus faecium, which are now commonly vancomycin-resistant(Hooper, 2002).
Bacterial resistance to fluoroquinolones occurs by changes in the bacterial target enzymes DNA gyrase and topoisomerase IV, which reduce drug binding, and by action of native bacterial membrane pumps (multidrug resistance efflux pumps) that remove drug from the cell. In the first case, resistance occurs due to mutations in aminoacids, particularly those in certain regions of each enzyme subunit called the quinolone-resistance-determining-region (QRDR), which make the enzyme less sensitive to inhibition by fluoroquinolones. In staphylococci, single mutations seem to be sufficient to cause clinical resistance. In the second case, mutations can result in an increased transcription of the structural gene for the pump. In both cases, quinolone exposure selects for spontaneous mutants that are present in large bacterial populations, and which contain chromosomal mutations that alter the target protein or increase the level of pump expression. Fluoroquinolones have also been shown to increase the rates of bacterial mutation in the laboratory due to their ability to damage bacterial DNA and trigger the error-prone SOS DNA repair system. The extent to which the mutagenic properties of fluoroquinolones in bacteria contribute to the emergence of fluoroquinolone resistance in clinical settings is not known (Hooper, 2002).
Side effects that may be associated with this class include gastrointestinal distress, neuropsychiatric changes, cardiac conduction disturbances, abnormalities in liver enzymes, hypoglycemia or hyperglycemia, and drug rashes. They can be also associated with tendonitis and tendon rupture, with increased risk among older patients (greater than 60); recipients of corticosteroids; or patients status-post kidney, heart, or lung transplant(Gold and Pillai, 2009).
Besides the above-mentioned side effects, fluoroquinolones were found to affect cartilage in juvenile animals, resulting in an irreversible arthropathy. This finding was consistent in all flouroquinolones, and hence fluoroquinolone use in patients under 18 years is prohibited except in certain conditions that do not include bacteremia. The only permitted fluoroquinolone is ciprofloxacin, and is licensed by the FDA for use in children only in complicated urinary tract infections, pyelonephritis, and postexposure treatment for inhalation anthrax(Committee on Infectious Diseases, 2006).
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Figure(II.10): Chemical structure of: A) ciprofloxacin, B)levofloxacin, c)gatifloxacin and d)moxifloxacin
The first discovered aminoglycoside "Streptomycin" dates back as 1944. Several aminoglycosides have been discovered since then, that include kanamycin (1958), gentamicin (1962), tobramycin (1967) and amikacin (1972) among others. They are basically a large group of bacterial secondary metabolites, derived from several bacterial genera, that include Streptomyces, Micromonospora and Bacillus (Davies, 2007).
Aminoglycosides are bactericidal agents that act by disrupting bacterial protein synthesis (Gold and Pillai, 2009).
They are multifunctional hydrophilic sugars that possess several amino and hydroxy functionalities. The amine moieties are mostly protonated in biological media; hence,these antibiotics can be considered polycationic species. Since they are polycationic, they show a high binding affinity for nucleic acids, mainly for certain portions of RNAs and specifically for the prokaryotic rRNA. Different classes of aminoglycoside antibiotics bind to different sites on the rRNA, depending on the structural complementarity between the two. For example, neomycin, paromomycin, gentamicin, and kanamycin are believed to bind to the A-site on the 16S rRNA in bacteria . Four bases, in the rRNA A-site are responsible for interacting with tRNA. The binding of these aminoglycosides to the A-site in the decoding region (i.e., the site of codon and anticodon recognition) interferes with the accurate recognition of cognate tRNA by rRNA during translation. These interactions are also thought to interfere with the translocation of tRNA from the A-site to the peptidyl-tRNA site "P-site"(Kotra et al., 2000).
Resistance to aminoglycosides is widely reported. But, even though the binding site for aminoglycosides is in rRNA, resistance to aminoglycosides is not manifested by alteration of this target in general. This is in part due to the fact that the function of the rRNA is central in the protein biosynthetic process and that this function is so well preserved across genera that it cannot be impaired by the possibility of such structural alteration. The most common mechanism for clinical resistance to aminoglycosides is their structural modification by specific enzymes expressed in resistant organisms. The binding of the modified aminoglycoside antibiotics to their target sites is thus compromised. There are three classes of these enzymes: aminoglycoside phosphotransferases (APHs), aminoglycoside nucleotidyltransferases (ANTs), and aminoglycoside acetyltransferases (AACs). Gentamicin C, for example is susceptible to at least five or six modifying enzymes. The structural modifications of aminoglycosides result in a severe reduction of the ability of the modified antibiotic to bind to the target RNA due to unfavorable steric and/or electrostatic interactions (Kotra et al., 2000).
Other mechanisms of resistance include: reduced uptake, mutational or enzymatic modification of 16SrRNA and mutational modification of ribosomal proteins (Davies and Wright, 1997).
Class-specific side effects include nephrotoxicity and ototoxicity(Gold and Pillai, 2009).
Aminoglycosides (specifically gentamicin (Figure(II.11)) demonstrate favorable activity in vitro against S.aureus. In contrast, activity in vitro against VISA is poor (Drew, 2007).
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Like rifampin, use as monotherapy for invasive staphylococcal infections should be avoided(Drew, 2007). Published guidelines in the US and the UK describe the role of gentamicin in the treatment of staphylococcal endocarditis(Elliott et al., 2004, Baddour et al., 2005). There is however no evidence that the use of aminoglycosides with glycopeptides improves outcome in MRSA bacteraemia or endocarditis, and using aminoglycosides with vancomycin should be avoided, where possible, because of the risk of increased toxicity (Gemmell et al., 2006). Therefore, aminoglycosides are generally reserved for use in combination therapy (most commonly with β-lactams) for the treatment of select invasive staphylococcal infections(Drew, 2007). S aureus endocarditis is the treatment indication for which the addition of an aminoglycoside is most often considered(Gold and Pillai, 2009).
Figure(II.11): Structure of gentamicin
Gentamicin is a mixture of isomeric aminoglycoside antibiotics (gentamicin C1, gentamicin C1A, and gentamicin C2) produced by Micromonospora purpurea or M. echinospora, for gentamicin C1: R1=R2=CH3, for gentamicin C1A: R1=R2=H, for gentamicin C2: R1=CH3, R2=H
II.3.8 New agents
Some relatively new antibiotics have received FDA approval and are being used in clinical practice in the US and several other countries. However, these antibiotics have not been introduced to the Egyptian market it. The most important of which are: Streptogramins, e.g. Quinupristin/dalfopristin, Oxazolidinones, e.g. linezolid, Lipo(glyco)peptides, e.g. daptomycin and Glycylcyclines, e.g. tigecycline.
Streptogramins are macrocyclic lactone peptolide antibiotics produced by streptomycetes. They are classified as A and B compounds according to their basic primary structure. Compounds of the A group are polyunsaturated cyclic macrolactones, whereas compounds of the B group are cyclic hexadepsipeptides. A and B compounds are bacteriostatic when used separately, but in combination they are bactericidal particularly against Gram-positive bacteria, more potent, and may be active even when there is resistance to 1 component (El Solh and Allignet, 1998, Eliopoulos, 2003). The mixtures of A and B compounds act by binding the 50S ribosomal subunit resulting in an irreversible inhibition of bacterial protein synthesis. A compounds inactivate the donor and acceptor sites of the peptidyltransferase catalytic center and inhibit the two first steps of peptide chain elongation. They provoke a lasting conformational alteration of the 50S ribosomal subunit and increase its affinity for B compounds .The B compounds cause inhibition of peptide bond formation and induce the premature dissociation of incomplete protein chains. The synergic effect of this drug combination appears to result from the fact that it targets both early and late stages of protein synthesis (El Solh and Allignet, 1998, Aksoy and Unal, 2008).
Quinupristin/dalfopristin (Synercid®), the first streptogramin to receive FDA approval, is a fixed mixture of two streptogramin antibiotics quinupristin (a streptogramin B) and dalfopristin (a streptogramin A) at a ratio of 30:70, which together achieve bactericidal activity (Figure(II.12)). Its FDA approval is mainly for the treatment of serious or life-threatening infections associated with vancomycin-resistant E. faecium bacteremia and complicated skin infections caused by MSSA and group A streptococci, but several other clinical applications are being explored (Metzger et al., 2009). However, it is almost always inactive against Enterococcus faecalis where an efflux pump conferring resistance to dalfopristin appears to be intrinsic in this species (Eliopoulos, 2003).
Quinupristin/dalfopristin was previously reported to have a rapid bactericidal mode of action against staphylococci and streptococci and a bacteriostatic activity versus enterococci, especially VRE strains (Jones et al., 1998). When tested against 28,000 clinical Gram-positive cocci isolates, it was effective against 99.0- 99.99% of S.aureus isolates, 98.0 - 100.0% of coagulase-negative Staphylococci isolates, and 87.0 to 92.0% of E. faecium isolates (Jones et al., 1998). In another study involving 63 centres in five countries, it proved successful in treatment of MRSA infections failing conventional therapy (Drew et al., 2000). However, success rates for bacteremia of unknown source were below the population mean (Drew et al., 2000). In a multicenter retrospective study on the use of quinupristin/dalfopristin in pediatric patients with serious Gram-positive infections who had no further therapy options because of resistance to, failure on or intolerance to standard antibiotic treatment, quinupristin/dalfopristin demonstrated apparent efficacy in some patients and was reasonably well-tolerated. The authors concluded that quinupristin/dalfopristin is a reasonable therapeutic options for the treatment for pediatric patients with serious Gram-positive infections, especially in those unable to receive alternative therapy(Loeffler et al., 2002).\
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