Resistant Gram-positive Pathogens Research Articles

Collateral damage and what the future might hold. The need to balance prudent antibiotic utilization and stewardship with effective patient management

Summary Increased severity of illness among hospitalised patients and an ageing population have led to an increased incidence of hospital acquired infections and represent a significant challenge to the clinician in terms of managing infections. The collateral damage which can occur with antibiotic therapy is also an important consideration when initiating empirical antibiotic therapy, particularly in patients who are seriously ill or immunocom-promised. Collateral damage is the term used to describe the adverse ecological effects of antibiotic therapy, such as the selection of drug-resistant organisms, and the adverse events associated with antibiotic therapy such as Clostridium difficile disease. Antibiotic use and ineffective infection control have been implicated in the development and spread of resistant Gram-positive and Gram-negative bacterial pathogens which are associated with increased mortality and morbidity, prolonged hospitalisation and increased costs. Carbapenem consumption and mechanical ventilation have been linked to colonisation or infection with problematic organisms including methicillin-resistant Staphylococcus oureus, Pseudomonas aeruginosa and Stenotrophomonas maltophilia , while cephalosporin use has been associated with evolution of infections due to vancomycin-resistant enterococci (VRE) and Gram-negative bacilli producing extended-spectrum β-lactamases (ESBL), and to colonisation or superinfection with Clostridium difficile. The safety profile of antibiotics must also be taken into consideration when selecting therapy, and single broad-spectrum agents may provide excellent coverage with a low risk of adverse events. The use of single agents may be associated with lower costs, improved ease of administration and fewer drug-drug interactions. However, in an environment of increasing resistance, initial aggressive therapy may be required to avoid excessive mortality and morbidity. Ideally antibiotic therapy should be directed by culture and knowledge of local susceptibility patterns. Before culture results are available therapy may need to be initiated empirically to cover the likely pathogens. In neutropenic patients with fever the current guidelines recommend the use of empirical therapy at the onset of fever for all patients. Where no aetiology is identified, antibiotic therapy should continue for at least 2 weeks while aggressive attempts are made to define the source of fever. When the aetiology of infection has been identified, therapy should be adjusted to provide optimal treatment with the best safety profile and lowest cost. The principal of avoiding collateral damage provides a useful framework for selecting antibiotics for empirical therapy in today's changing environment.

Prevalence of Mastitis Pathogens and Their Resistance Against Antimicrobial Agents in Dairy Cows in Brandenburg, Germany

The primary objective of this study was to determine management practices concerning mastitis in Brandenburg, Germany, the prevalence of mastitis pathogens in dairy cows, and their resistance to selected antimicrobial agents. A further objective was to study the potential effect of parity and stage of lactation on the resistance of Staphylococcus aureus isolates against ampicillin. Milk samples for microbiological culture were collected from 4 groups of clinically healthy cows (first lactation, >1 lactation, >50 d in milk, and >250 d in milk; 8 cows/group) in 80 dairy herds. Resistance of gram-positive pathogens against 6 antimicrobial agents was tested using the broth microdilution method. Mastitis pathogens were isolated from 26.4% of the milk samples. Coagulase-negative staphylococci (CNS, 9.1% of quarters) and Corynebacterium bovis (7.3%) were the pathogens most frequently isolated. Among the major pathogens, Staph. aureus (5.7%) and Streptococcus uberis (1.0%) had the highest prevalence. Streptococcus agalactiae was isolated in samples from 29% of the herds. Although the prevalence of most pathogens was higher in older cows, the prevalence of CNS was higher in primiparous cows. Results of the mastitis control questionnaire showed that cows with clinical mastitis were transferred to a sick cow pen in 70% of the herds. Cephalosporins were the drug of first choice for treatment of clinical mastitis cases followed by fixed combinations of antimicrobial agents, β-lactamase–resistant penicillins, and penicillin. Most farmers treated cows 3 to 4 times per case. Cloxacillin, alone or in combination, and penicillin were most often used for dry-cow therapy. Antimicrobial resistance of the pathogens was within the range of other reports. Resistance of Staph. aureus to ampicillin increased significantly during the first lactation. Further research is required to determine the factors that lead to the selection of Staph. aureus strains that are resistant to ampicillin during the first lactation.

Design, Synthesis, and Biological Evaluation of 4-Alkyliden-beta Lactams: New Products with Promising Antibiotic Activity Against Resistant Bacteria

The design, synthesis, and antibacterial activity of 4-alkyliden-azetidin-2-ones as new antimicrobial agents against multidrug-resistant pathogens is reported. 4-Alkyliden-azetidin-2-ones were easily obtained using an original protocol starting from 4-acetoxy-azetidinones and diazoesters. Parent compounds were further elaborated to obtain a small library of 4-alkylidene derivatives. A molecular modeling approach using GRID descriptors based on the concept of VRS identified attractive drug candidates and contributed to the rationalization of functional group effects in QSARs. The in vitro antibacterial activity of the new agents was evaluated against 43 recent clinical isolates of antibiotic-susceptible and -resistant Gram-positive and Gram-negative pathogens by determining their minimum inhibitory concentrations (MICs). The most active compound showed MIC values ranging from 0.25 to 32 mg/L against some of the bacterial species tested. Interestingly, some compounds demonstrated similar activity against methicillin-susceptible and -resistant strains of Staphylococcus aureus suggesting possible alternative mechanisms of action of these agents, supported by citotoxicity and preliminary scanning electron microscopy studies.

Antibiotics for treatment of resistant gram-positive coccal infections

Vancomycin is considered the workhorse for the treatment of most drug-resistant gram-positive bacterial infections. However, concerns have been raised regarding the increasing rates of vancomycin-resistant enterococci and the clinical shortcomings of vancomycin in the treatment of invasive Staphylococcus aureus infections. Resources have been committed to the development of antimicrobial agents with activity against these organisms. This review will focus on the newer antibacterial agents that have been developed for the treatment of resistant gram-positive pathogens. Included in this review are the agents: quinupristin-dalfopristin, linezolid, daptomycin, telithromycin, and tigecycline.

Novel Mechanism of Antibiotic Resistance Originating in Vancomycin-Intermediate Staphylococcus aureus

As an aggressive pathogen, Staphylococcus aureus poses a significant public health threat and is becoming increasingly resistant to currently available antibiotics, including vancomycin, the drug of last resort for gram-positive bacterial infections. S. aureus with intermediate levels of resistance to vancomycin (vancomycin-intermediate S. aureus [VISA]) was first identified in 1996. The resistance mechanism of VISA, however, has not yet been clarified. We have previously shown that cell wall thickening is a common feature of VISA, and we have proposed that a thickened cell wall is a phenotypic determinant for vancomycin resistance in VISA (L. Cui, X. Ma, K. Sato, et al., J. Clin. Microbiol. 41:5-14, 2003). Here we show the occurrence of an anomalous diffusion of vancomycin through the VISA cell wall, which is caused by clogging of the cell wall with vancomycin itself. A series of experiments demonstrates that the thickened cell wall of VISA could protect ongoing peptidoglycan biosynthesis in the cytoplasmic membrane from vancomycin inhibition, allowing the cells to continue producing nascent cell wall peptidoglycan and thus making the cells resistant to vancomycin. We conclude that the cooperative effect of the clogging and cell wall thickening enables VISA to prevent vancomycin from reaching its true target in the cytoplasmic membrane, exhibiting a new class of antibiotic resistance in gram-positive pathogens.

Daptomycin (DAP) Use in Patients with Hematologic (HEM) Cancer History; Post-Marketing Experience in a Registry.

Background: DAP is a novel lipopeptide antibiotic, approved for complicated skin and skin structure infections, with potent in vitro activity against many resistant Gram-positive organisms. Registration studies often limit or exclude patients with severe illness. Antibiotic resistant Gram-positive pathogens are frequently encountered in compromised patient populations; however, minimal information is available pre-approval in these patients.Methods: Cubicin® Outcomes Registry and Experience (CORE) is a Phase IV retrospective observational chart review (45 institutions) to quantitate characteristics and clinical outcomes of patients (pts) receiving DAP. A standard case report form including demographic, disease state, clinical, and microbiologic data was completed by each investigator. Cure, failure, and improvement were assessed using standard definitions. Nonevaluable pts had insufficient data available to determine clinical outcome. Of the 1160 pts in CORE, 56 were reported with a history of HEM cancer. Eighteen (32%) were nonevaluable; 6 of those expired. This report describes the 38 pts with an assigned outcome.Results: Fifty-five percent of pts were female, 63% were 31 to 65 years of age, and 76% of infections developed in a hospital setting. Common comorbidities included cardiovascular disease (24%), immunosuppression (18%), and transplant (11%). The most common infections were; bacteremia catheter-related (53%), skin and skin structure (34%), and bacteremia noncatheter-related (18%). Seventy-one percent (27/38) of pts were culture positive before DAP with the most common pathogens being Enterococcus sp. (52%, of which 71% were VRE), coagulase-negative staphylococci (41%), and S. aureus (19%, of which 80% were MRSA). A high percentage of pts (92%) received antibiotics (abx) prior to DAP. The most frequent initial dosing regimens were 4mg/kg QD (50%), and 6mg/kg QD (29%). The mean duration of DAP therapy was 13.6 days (range 2–44). Dose (P=0.3) and duration (P=0.58) did not vary by primary pathogen. Twenty-nine (76%) pts received concomitant abx therapy, most commonly with fluoroquinolones (31%), metronidazole (28%) or cephalosporins (24%). The mean time to clinical response was 2.4 days (n=28) and unaffected by primary pathogen (P=0.43) or the influence of concomitant abx therapy (P=0.55). All pts were reported as cured (66%) or improved (34%).Conclusion: The data from the CORE registry demonstrates that DAP shows early clinical response and clinical cure for HEM patients where catheter associated infections and VRE are prevalent. These data provide preliminary data on the use of DAP in this compromised patient population. This information should lead to further prospective studies in cancer patients with DAP.

Agents for the treatment of multidrug-resistant gram-positive endocarditis

Several newer agents with activity against multidrug- resistant gram-positive pathogens are available. These agents have in vitro and clinical data supporting their utility in the treatment of infections caused by pathogens such as methicillin-resistant staphylococci and vancomycin-resistant enterococci. Daptomycin appears to be rapidly bactericidal, and linezolid and quinupristin/dalfopristin also are cidal against staphylococci. Although the agents have several properties that are attractive for use in endocarditis, clinical data are limited. Further investigation with each agent and combination therapy are warranted before definitive recommendations can be made.

Gram-Positive Resistance: Pathogens, Implications, and Treatment Options: Insights from the Society of Infectious Diseases Pharmacists

Despite the advent of new antibiotics, resistance in gram-positive pathogens, including staphylococci and enterococci, continues to increase. This is evident with the recent emergence of vancomycin-resistant Staphylococcus aureus . Newer treatment agents are available, including quinupristin-dalfopristin, linezolid, and daptomycin. In addition, investigational agents are being explored. Clinical trials have been conducted for various infections, such as skin and skin structure infections, pneumonia, and bloodstream infections. Antibacterial activity, site of infection, and potential for adverse effects must be taken into account when making decisions regarding therapy.

Daptomycin for treating infected diabetic foot ulcers: evidence from a randomized, controlled trial comparing daptomycin with vancomycin or semi-synthetic penicillins for complicated skin and skin-structure infections

The predominant pathogens causing diabetic foot infections are Gram-positive cocci, many of which are now resistant to commonly prescribed antibiotics. Daptomycin is a new agent that is active against most Gram-positive pathogens. To compare the effectiveness of daptomycin against semi-synthetic penicillins or vancomycin, we analysed the subset of diabetic patients with an infected ulcer enrolled in two randomized, controlled investigator-blind trials of patients with complicated skin and soft-tissue infections presumptively caused by Gram-positive organisms. Patients with a diabetic ulcer infection were prospectively stratified to ensure they were equally represented in the treatment groups, then randomized to either daptomycin [4 mg/kg every 24 h intravenously (iv)] or a pre-selected comparator (vancomycin or a semi-synthetic penicillin) for 7-14 days. Among 133 patients with a diabetic ulcer infection, 103 were clinically evaluable; 47 received daptomycin and 56 received a comparator. Most infections were monomicrobial, and Staphylococcus aureus was the predominant pathogen. Success rates for patients treated with daptomycin or the comparators were not statistically different for clinical (66% versus 70%, respectively; 95% CI, -14.4, 21.8) or microbiological (overall or by pathogen) outcomes. Both treatments were generally well tolerated, with most adverse events of mild to moderate severity. The clinical and microbiological efficacy and safety of daptomycin were similar to those of commonly used comparator antibiotics for treating infected diabetic foot ulcers caused by Gram-positive pathogens. Daptomycin should be considered for treating these infections, especially those caused by resistant Gram-positive pathogens.

Pharmacokinetics of Oritavancin in Plasma and Skin Blister Fluid following Administration of a 200-Milligram Dose for 3 Days or a Single 800-Milligram Dose

Oritavancin is a novel glycopeptide currently being developed for the treatment of complicated skin and skin structure infections (cSSSI), including those caused by multidrug resistant gram-positive pathogens. The disposition of oritavancin in skin structures was investigated using a cantharide-induced blister fluid model. Seventeen healthy male subjects received oritavancin, but only 16 subjects were evaluated after one subject discontinued study drug. Each subject (eight per dose group) received 200 mg of oritavancin once a day for 3 days (group A) or 800 mg as one single dose (group B). Group A plasma samples and exudates from blister fluid were collected on days 3, 4, 7, 9, and 12 and on days 3, 4, 7, and 9, respectively. Group B samples and exudates were collected on days 1, 2, 5, 7, and 10 and on days 1, 2, 5, and 7, respectively. Drug concentrations were determined using a liquid chromatography-tandem mass spectrometry assay and, subsequently, pharmacokinetic analysis was performed. Differences between treatment groups in ratios for area under the concentration-time curve for blister fluid and plasma (AUC(blister fluid)/AUC(plasma) ratios) were evaluated using a t test (alpha = 0.05). Mean maximum concentration of drug in plasma or blister fluid was approximately 8-fold and 11-fold higher in plasma than in blister fluid following the 200- or 800-mg doses of oritavancin, respectively. Mean AUC(blister fluid)/AUC(plasma) ratios at 24 h were 0.190 (standard deviation [SD], 0.052) and 0.182 (SD, 0.062) for groups A and B, respectively (P = 0.791). To place these results in a clinical context, mean drug concentrations in blister fluid exceed the oritavancin MIC at which 90% of strains are inhibited of Staphylococcus aureus (2 microg/ml) by approximately 2- to 5.5-fold at 12 h and 1.5- to 3-fold at 24 h following administration of both dosing regimens. These results support the potential use of oritavancin for the treatment of cSSSI.

In vitro activity of daptomycin against isolates of Staphylococcus aureus and enterococci: first results from Turkey

There has been a steady rise in the prevalence of resistant Gram-positive pathogens and concerns about the clinical effectiveness of glycopeptides in treating infections due to Staphylococcus aureus. Daptomycin is a novel lipopeptide antimicrobial agent with activity against Gram-positive organisms, including multi-resistant strains. It is licensed in the USA and Europe for the treatment of complicated skin and soft tissue infections caused by Gram-positive organisms at a dose of 4 mg/kg once daily. It has also been licensed in the USA for the treatment of S. aureus bacteraemia and right-sided endocarditis at 6 mg/kg once daily. It is a safe and well-tolerated antibiotic, particularly at the current dosing regimen. Antimicrobial resistance, whilst being increasingly reported, still remains relatively rare. Further studies are required to determine the role of daptomycin for the treatment of osteomyelitis and septic arthritis, as well as its use in combination therapy.

Susceptibility to quinupristin-dalfopristin and linezolid in 839 clinical isolates of Gram-positive cocci from Norway.

A total of 839 clinical isolates of Gram-positive cocci from Norway including Staphylococcus aureus (n = 214), coagulase negative Staphylococcus spp. (n = 100), Streptococcus pyogenes (n = 99), Streptococcus agalactiae (n = 80), Streptococcus pneumoniae (n = 127), Streptococcus spp. viridans group (n = 70), Enterococcus faecalis (n = 75), and Enterococcus faecium (n = 74), were tested by E-test for susceptibility to a range of antimicrobials including the novel antibiotics quinupristin-dalfopristin and linezolid. Subgroups of oxacillin resistant S. aureus and coagulase negative Staphylococcus spp., penicillin non-susceptible S. pneumoniae and vancomycin resistant Enterococcus spp. were specifically included as they are the intended targets for these new drugs. All isolates were susceptible to linezolid (MIC5o and MIC9o 0.25-2.0 mg/l, MIC range 0.12-2 mg/l). Staphylococcal and streptococcal isolates were also susceptible to quinupristin-dalfopristin except for some intermediately susceptible viridans group isolates (MIC54, and MIC90 0.25-2 mg/l, MIC range 0.125-2 mg/l). Enterococcus faecium (MIC90 = 4.0 mg/l) and Enterococcus faecalis (MIC50 = 8.0 mg/l, MIC90 > or = 32 mg/l) were less susceptible to this substance. There was no linkage between reduced susceptibility to linezolid or quinupristin-dalfopristin and resistance to other classes of antimicrobials. The study demonstrated a high prevalence of in vitro susceptibility to linezolid and quinupristin-dalfopristin, which is necessary for their use in the treatment of infections with resistant Gram-positive pathogens. The results were used to evaluate the appropriateness of breakpoints and to define a baseline for monitoring possible future emergence of resistance to quinupristin-dalfopristin and linezolid in Norway.

The status of antimicrobial resistance in Taiwan among Gram-positive pathogens: the Taiwan Surveillance of Antimicrobial Resistance (TSAR) programme, 2000

In the Taiwan Surveillance of Antimicrobial Resistance programme, isolates were collected from 21 hospitals over a 3-month period in 2000 (TSAR II) and rates of resistance in Gram-positive pathogens were determined. Resistance rates were high including oxacillin resistance in Staphylococcus aureus (60%) and coagulase-negative staphylococci (80%), high-level gentamicin resistance (HLGR) in Enterococcus faecalis (60%) and penicillin non-susceptibility in Streptococcus pneumoniae (69%). Oxacillin resistance had increased from 1998 (TSAR I) and may be spreading into outpatient settings. In contrast, less than 2% enterococci were vancomycin-resistant. No linezolid resistance was found in either staphylococci or enterococci.

A review of linezolid: the first oxazolidinone antibiotic

Resistance of Gram-positive bacterial pathogens, such as Staphylococcus aureus and Enterococcus faecium, to existing antibiotics continues to increase, and new antibiotics with activity against these pathogens are in demand. Linezolid (Zyvox®, Pharmacia and Upjohn) is the first agent of a new class of antibiotics called the oxazolidinones. Linezolid possesses excellent microbial activity against a wide variety of Gram-positive pathogens including those resistant to methicillin and vancomycin (Vancocin®, Eli Lilly). Linezolid is available for intravenous and oral administration and possesses excellent bioavailability. It exhibits good penetration into pulmonary, as well as skin and related structure tissues, and does not require dosage adjustment in hepatic or renal dysfunction. Linezolid is generally well-tolerated, with the predominant adverse effect manifesting as a duration dependent, reversible thrombocytopenia. Linezolid possesses monoamine oxidase inhibitor activity and caution is warranted with coadministration of adrenergic or seritonergic medications. Clinical trials conducted with linezolid in skin and structure infections, lower respiratory tract infections and vancomycin-resistant enterococcal infections demonstrate that linezolid is an effective therapy. Recent data suggest that linezolid may be superior to vancomycin for the treatment of infections caused by methicillin-resistant S. aureus. Linezolid is an excellent and promising new antibiotic for the treatment of resistant Gram-positive pathogens.

Synthesis and structure-activity relationships of 5-amino-6-fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-8-methylquinolonecarboxylic acid antibacterials having fluorinated 7-[(3R)-3-(1-aminocyclopropan-1-yl)pyrrolidin-1-yl] substituents.

A series of novel 5-amino-6-fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-8-methylquinolones bearing fluorinated (3R)-3-(1-aminocyclopropan-1-yl)pyrrolidin-1-yl substituents at the C-7 position (2-4) was synthesized to obtain potent drugs for infections caused by Gram-positive pathogens, which include resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae (PRSP), and vancomycin-resistant enterococci (VRE). These fluorinated compounds 2-4 exhibited potent antibacterial activity comparable with that of a compound bearing a non-fluorinated (3R)-3-(1-aminocyclopropan-1-yl)pyrrolidine moiety at the C-7 position (1) and had at least 4 times more potent activity against representative Gram-positive bacteria than ciprofloxacin (CPFX), gatifloxacin (GFLX), or moxifloxacin (MFLX). Among them, the 7-[(3S,4R)-4-(1-aminocyclopropan-1-yl)-3-fluoropyrrolidin-1-yl] derivative 3 (=DQ-113), which showed favorable profiles in preliminary toxicological and nonclinical pharmcokinetic studies, exhibited potent antibacterial activity against clinically isolated resistant Gram-positive pathogens.

Molecular analysis of streptogramin resistance in enterococci

AbstractThe new semi-synthetic streptogramin antibiotic combination quinupristin/dalfopristin (Synercid®) is a promising alternative for a treatment of infections with multiple resistant gram-positive pathogens, e.g. glycopeptide- and multi-resistant Enterococcus faecium. Streptogramins consist of two unrelated compounds, a streptogramin A and B, which act synergistically when given in combination. Mechanisms conferring resistance against both components are essential for resistance against the combination in E. faecium. In this species resistance to streptogramin A compounds is mediated via related acetyltransferases VatD and VatE. Resistance against streptogramins B is either encoded by the widespread ermB gene cluster conferring resistance to macrolide-lincosamide-streptogramin B antibiotics or via expression of the vgbA gene, which encodes a staphylococcal-type lactonase. E. faecalis is intrinsically resistant to streptogramins.Due to a wide use of streptogramins (virginiamycins S/M) in commercial animal farming a reservoir of streptogramin-resistant E. faecium isolates had already been selected. Determinants for streptogramin resistance are localized on plasmids that can be transferred into an E. faecium recipient both in vitro in filter-matings and in vivo in the digestive tracts of rats. Hybridization and sequencing experiments revealed a linkage of resistance determinants for streptogramins A and B on definite plasmid fragments.

Oxazolidinones: Clinical pharmacology and use in the treatment of infections caused by resistant gram-positive pathogens

During the last decade, gram-positive bacteria have emerged as important nosocomial and community-acquired pathogens. Development of antimicrobial resistance has complicated the therapeutic management of patients with infections caused by gram-positive pathogens and, in many cases, therapeutic alternatives are limited. The oxazolidinones, a novel class of antimicrobials, show potent activity against gram-positive organisms, including those with multiple-drug resistance. Linezolid, the only agent in this class currently approved for clinical use, shows favorable pharmacokinetic and toxicologic profiles and offers considerable therapeutic promise. Phase III clinical trials with linezolid have shown efficacy in patients with pneumonia and skin and soft tissue infections. Compassionate use of linezolid in both adult and pediatric patients also has shown efficacy in patients with methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococcal infections. Currently, the primary role of linezolid appears to be in the treatment of patients with infections caused by drug-resistant pathogens. Future development of oxazolidinone analogues with enhanced activity against gram-negative pathogens and mycobacteria may further expand the utility of these novel compounds. Copyright © 2001 by W.B. Saunders Company

Molecular Mechanisms of Antibiotic Resistance in Gram-Positive Pathogens.

The widespread and uncontrolled use of antibiotics, both for human consumption and animal feed, has encouraged the development of drug resistance in a variety of pathogenic bacteria. Gram-positive species employ resistance mechanisms which include the modification of the antibiotic structure, mutagenesis of key amino acids in the macromolecular targets of specific chemotherapeutics, or drug efflux from the cell, among others. These three main mechanisms have been identified in resistance profiles for systems involved in protein biosynthesis, nucleic acid replication, and bacterial cell wall generation. This work will review how Gram-positive bacteria have manipulated all three classes of targets in the generation of resistance. Upcoming and recently approved antibacterials for human consumption will also be highlighted.

Comparative In vitro activities of daptomycin and vancomycin against resistant gram-positive pathogens.

The in vitro activity of daptomycin against 224 current gram-positive clinical isolates including vancomycin-resistant Enterococcus faecium (VREF), methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus spp. (MRSS), and penicillin-resistant Streptococcus pneumoniae (PRSP) was evaluated. The MICs at which 90% of isolates are inhibited for daptomycin and vancomycin, respectively, were as follows: MRSA, 1 and 2 microg/ml; MRSS, 1 and 4 microg/ml; PRSP, 1 and 0.5 microg/ml; and VREF, 2 and >64 microg/ml. Daptomycin was bactericidal against 82% of 17 VREF isolates. The antibacterial activity of daptomycin was strongly dependent on the calcium concentration of the medium. Daptomycin was active against all gram-positive cocci tested.

Enzymes of vancomycin resistance: the structure of d-alanine– d-lactate ligase of naturally resistant Leuconostoc mesenteroides

Background: The bacterial cell wall and the enzymes that synthesize it are targets of glycopeptide antibiotics (vancomycins and teicoplanins) and β-lactams (penicillins and cephalosporins). Biosynthesis of cell wall peptidoglycan requires a crosslinking of peptidyl moieties on adjacent glycan strands. The d-alanine– d-alanine transpeptidase, which catalyzes this crosslinking, is the target of β-lactam antibiotics. Glycopeptides, in contrast, do not inhibit an enzyme, but bind directly to d-alanine– d-alanine and prevent subsequent crosslinking by the transpeptidase. Clinical resistance to vancomycin in enterococcal pathogens has been traced to altered ligases producing d-alanine– d-lactate rather than d-alanine– d-alanine. Results: The structure of a d-alanine– d-lactate ligase has been determined by multiple anomalous dispersion (MAD) phasing to 2.4 Å resolution. Co-crystallization of the Leuconostoc mesenteroides LmDdl2 ligase with ATP and a di- d-methylphosphinate produced ADP and a phosphinophosphate analog of the reaction intermediate of cell wall peptidoglycan biosynthesis. Comparison of this d-alanine– d-lactate ligase with the known structure of DdlB d-alanine– d-alanine ligase, a wild-type enzyme that does not provide vancomycin resistance, reveals alterations in the size and hydrophobicity of the site for d-lactate binding (subsite 2). A decrease was noted in the ability of the ligase to hydrogen bond a substrate molecule entering subsite 2. Conclusions: Structural differences at subsite 2 of the d-alanine– d-lactate ligase help explain a substrate specificity shift ( d-alanine to d-lactate) leading to remodeled cell wall peptidoglycan and vancomycin resistance in Gram-positive pathogens.