Antagonistic Effect of Colistin on Vancomycin Activity against Methicillin-Resistant Staphylococcus aureus in In Vitro and In Vivo Studies.

As concerns arise that the vancomycin MIC of methicillin-resistant Staphylococcus aureus (MRSA) could be increased by concurrent colistin administration, we evaluated the effect of colistin on vancomycin efficacy against MRSA via in vitro and in vivo studies. Among MRSA blood isolates collected in a tertiary-care hospital, we selected representative strains from community-associated MRSA strains (CA-MRSA; ST72-MRSA-SCCmec IV) and hospital-acquired MRSA strains (HA-MRSA; ST5-MRSA-SCCmec II). USA CA-MRSA (USA300), HA-MRSA (USA100), N315 (New York/Japan clone), and a MRSA standard strain (ATCC 43300) were used for comparison. We performed checkerboard assays to identify changes in the vancomycin MIC of MRSA following colistin exposure and evaluated the effect of a vancomycin-colistin combination using time-kill assays. We also assessed the in vivo antagonistic effect by administering vancomycin, colistin, and a combination of these two in a neutropenic murine thigh infection model. In the checkerboard assays, vancomycin MICs of all MRSA strains except N315 were increased by from 0.25 to 0.75 µg/ml following colistin exposure. However, the time-kill assays indicated antagonism only against ST5-MRSA and USA100, when the vancomycin concentration was twice the MIC. In the murine thigh infection model with ST5-MRSA and USA100, vancomycin monotherapy reduced the number of CFU/muscle >1 log10 compared to a combination treatment after 24 h in ST5-MRSA, indicating an antagonistic effect of colistin on vancomycin treatment. This study suggests that exposure to colistin may reduce the susceptibility to vancomycin of certain MRSA strains. Combination therapy with vancomycin and colistin for multidrug-resistant pathogens might result in treatment failure for concurrent MRSA infection.

Modulation of Staphylococcus aureus Response to Antimicrobials by the Candida albicans Quorum Sensing Molecule Farnesol.

In microbial biofilms, microorganisms utilize secreted signaling chemical molecules to coordinate their collective behavior. Farnesol is a quorum sensing molecule secreted by the fungal species Candida albicans and shown to play a central physiological role during fungal biofilm growth. Our pervious in vitro and in vivo studies characterized an intricate interaction between C. albicans and the bacterial pathogen Staphylococcus aureus, as these species coexist in biofilm. In this study, we aimed to investigate the impact of farnesol on S. aureus survival, biofilm formation, and response to antimicrobials. The results demonstrated that in the presence of exogenously supplemented farnesol or farnesol secreted by C. albicans in biofilm, S. aureus exhibited significantly enhanced tolerance to antimicrobials. By using gene expression studies, S. aureus mutant strains, and chemical inhibitors, the mechanism for the enhanced tolerance was attributed to upregulation of drug efflux pumps. Importantly, we showed that sequential exposure of S. aureus to farnesol generated a phenotype of high resistance to antimicrobials. Based on the presence of intracellular reactive oxygen species upon farnesol exposure, we hypothesize that antimicrobial tolerance in S. aureus may be mediated by farnesol-induced oxidative stress triggering the upregulation of efflux pumps, as part of a general stress response system. Hence, in mixed biofilms, C. albicans may influence the pathogenicity of S. aureus through acquisition of a drug-tolerant phenotype, with important therapeutic implications. Understanding interspecies signaling in polymicrobial biofilms and the specific drug resistance responses to secreted molecules may lead to the identification of novel targets for drug development.

Extracellular DNA impedes the transport of vancomycin in Staphylococcus epidermidis biofilms preexposed to subinhibitory concentrations of vancomycin.

Staphylococcus epidermidis biofilm formation is responsible for the persistence of orthopedic implant infections. Previous studies have shown that exposure of S. epidermidis biofilms to sub-MICs of antibiotics induced an increased level of biofilm persistence. BODIPY FL-vancomycin (a fluorescent vancomycin conjugate) and confocal microscopy were used to show that the penetration of vancomycin through sub-MIC-vancomycin-treated S. epidermidis biofilms was impeded compared to that of control, untreated biofilms. Further experiments showed an increase in the extracellular DNA (eDNA) concentration in biofilms preexposed to sub-MIC vancomycin, suggesting a potential role for eDNA in the hindrance of vancomycin activity. Exogenously added, S. epidermidis DNA increased the planktonic vancomycin MIC and protected biofilm cells from lethal vancomycin concentrations. Finally, isothermal titration calorimetry (ITC) revealed that the binding constant of DNA and vancomycin was 100-fold higher than the previously reported binding constant of vancomycin and its intended cellular d-Ala-d-Ala peptide target. This study provides an explanation of the eDNA-based mechanism of antibiotic tolerance in sub-MIC-vancomycin-treated S. epidermidis biofilms, which might be an important factor for the persistence of biofilm infections.

The effect of 2,3-dihydroxybenzoic acid and tempol in prevention of vancomycin-induced nephrotoxicity in rats.

One of the major adverse effects of vancomycin (VAN) is nephrotoxicity, which the mechanism is not fully understood. However, there is some evidence that oxidative injury could be involved in its pathogenesis. In this study, we examined two antioxidants 4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl (tempol) a superoxide dismutase mimetic and 2,3-dihydroxybenzoic acid (DHB) an iron chelator in VAN-induced nephrotoxicity in rats. DHB at doses of 50 and 100 mg/kg and tempol at doses of 7.5, 15 and 30 mg/kg were administered subcutaneously to rats 30 min prior to intraperitoneal injection of 200 mg/kg VAN. Drug administrations were done every 12 h for 7 days. In animals which received only VAN, the activity of urinary gamma-glutamyl-transferase (GGT) decreased and the activity of lactate dehydrogenase (LDH) in urine increased significantly compared to controls. Serum urea and creatinine (Cr) concentrations and the weight of animals' kidneys increased and body weights were decreased significantly in this group compared to controls. DHB at both doses normalized the GGT activity, but only at the higher dose restore the LDH activity. Both doses of DHB ameliorated the rise in serum urea and Cr concentrations and improved the changes in kidney and body weights significantly. Tempol did not show any beneficial effects at all. There were marked pathologic changes in tubules of kidneys of VAN treated animals. The tissue injury was prevented by both doses of DHB and there was almost no sign of tubular injury in 100 mg/kg treated group. Tempol in any doses could not prevent the tissue injury and there were significant differences in tissue injury in all tempol treated rats with controls. It seems that VAN-induced nephrotoxicity is at least partly due to free radical formation. Hydroxyl radicals might play a major role in VAN-induced nephrotoxicity, since an iron chelator (DHB) could reverse the adverse effects. However, production of other radicals such as superoxide is also probable.

[Combined effect of vancomycin or teicoplanin plus a beta-lactam antibiotic in mouse infection models caused by beta-lactam antibiotec-induced vancomycin resistant MRSA (BIVR)].

The concomitant use of low concentration beta-lactam antibiotics antagonizes the activity of vancomycin against some strains of MRSA. These strains, called beta-lactam antibiotic-induced vancomycin resistant MRSA (BIVR), have been increasing for a number of years. We previously reported that the combination of VCM and ceftizoxime displayed this antagonism not only in vitro, but also in vivo, in a systemic infection caused by BIVR in mice. In the present study, we validated the antagonism in combinations of VCM with other beta-lactams, i.e., flomoxef (FMOX), ampicillin (AMPC), azthreonam (AZT) and imipenem/cilastatin (IPM/CS), in systemic infections and pneumoniain in mice. The survival rate of the mice with systemic infections caused by BIVR treated with combinations of FMOX, AMPC, AZT, and IPM/CS with VCM were significantly lower than with VCM monotherapy, and the number of residual viable cells in the kidneys of mice treated with combinations of FMOX and IPM/CS with VCM were significantly higher than with VCM monotherapy The number of residual viable cells in the lungs of mice with pneumonia caused by BIVR treated with the combination of IPM/CS and VCM was significantly higher than with VCM monotherapy. On the other hand, the survival rate with combination therapy consistings IPM/CS plus teicoplanin (T EIC) was significantly higher, and the number of residual viable cells in the kidney was significantly lower, than with TEIC monotherapy alone. In the mice with pneumonia, the number of residual viable cells in the lung after combination therapy with IPM/CS and TEIC was significantly lower than with TEIC monotherapy. Combination therapy with beta-lactams plus VCM showed antagonistic in models of systemic infection and pulmonary infection caused by BIVR, whereas combination therapy consisting of a beta-lactam plus TEIC had a synergistic effect in the same models, even though VCM and TEIC are member of the same glycopeptide antibiotic class.

Protective effects of different antioxidants and amrinone on vancomycin-induced nephrotoxicity.

We have studied the effects of three antioxidants and amrinone, an inotropic agent, against vancomycin-induced nephrotoxicity in rats by investigating renal function and morphology. Thirty adult female Sprague Dawley rats (168-234 g) were divided into six groups. A saline-treated group served as control. The other five groups were treated for 7 days with vancomycin alone or in combination with alpha-lipoic acid, Ginkgo biloba extract 761, melatonin or amrinone. On day 8, all the rats were sacrificed by decapitation, kidney tissues were excised immediately and blood and kidney samples were collected. Blood urea and creatinine, kidney tissue malondialdehyde levels, and kidney superoxide dismutase and glutathione (GSH) peroxidase activities were measured. The kidneys were also examined for histological changes. Vancomycin administration led to increased urea, creatinine and malondialdehyde levels and decreased superoxide dismutase and GSH peroxidase activities. Co-administration of alpha-lipoic acid, Ginkgo biloba extract, melatonin or amrinone with vancomycin prevented the increases in the urea, creatinine and melondialdehyde levels and also resulted in higher superoxide dismutase and GSH peroxidase activities. The antioxidants and AMR improved the renal pathology compared to rats treated with vancomycin alone (P<0.05). These results indicate that the three antioxidants and amrinone have potential protective effects against vancomycin-induced nephrotoxicity, which might in part be due to inhibition of free oxygen radical production. Amrinone was the most effective drug as judged on the basis of the pathological findings.

Individualized vancomycin dosing in obese patients: a two-sample measurement approach improves target attainment.

STUDY OBJECTIVE: To compare the likelihood of vancomycin target trough concentration attainment based on infectious diagnosis and/or minimum inhibitory concentration for the organism by using a two-sample approach versus the prior institutional standard of a trough-only approach in obese patients receiving vancomycin. DESIGN: Preintervention and postintervention study. SETTING: Community acute care hospital. PATIENTS: One hundred fifty hospitalized adults with a body mass index (BMI) of 30 kg/m(2) or greater and treated with vancomycin for at least 48 hours between July 2013 and March 2014 were evaluated to compare the frequency of steady-state therapeutic trough concentration attainment between two approaches: a trough-only dosing method (preintervention group [75 patients]) and a dosing strategy measuring two vancomycin serum concentrations during the elimination phase (peak and trough) to calculate pharmacokinetic parameters and individualize the maintenance regimen (postintervention group [75 patients]). Data for the preintervention group were retrospectively retrieved from a 4-month period for patients admitted between July and October 2013, prior to implementation of the two-point vancomycin dosing protocol. Initial vancomycin empiric dose selection for both groups utilized the same population-based pharmacokinetic equations. MEASUREMENTS AND MAIN RESULTS: Median (5th-95th percentile) age, weight, and BMI were 59 (34-80) years, 105 (79.8-164) kg, and 34.5 (30.0-55.1) kg/m(2) , respectively, for all patients. The percentages of initial therapeutic trough concentrations achieved in the preintervention and postintervention groups were 32.0% and 42.7%, respectively (p=0.117). For patients with a second trough measurement, 31.0% in the preintervention group and 65.2% in the postintervention group were within the therapeutic range (p=0.024). CONCLUSION: Measurement of two serum vancomycin concentrations significantly improves subsequent target trough concentration attainment in the obese population.

Vancomycin-resistant enterococci.

Enterococci have been recognized as an important cause of nosocomial infections for almost 20 years and as a cause of endocarditis for almost a century. While long known for their capacity of displaying multiple antibiotic resistant traits, the extent to which this could occur was not fully appreciated until the emergence of enterococci with acquired resistance to vancomycin; this resistance has been particularly problematic because it often occurs in the uncommon subset of enterococci that are also highly resistant to ampicillin-a combination with devastating therapeutic consequences. The observation that vancomycin resistance can be transferred to and expressed in other gram-positive organisms, for which vancomycin is often considered the primary therapeutic alternative, is a chilling reminder of just how close we may be to a wide array of potentially untreatable "killer" microbes.

In vitro evaluation of clindamycin in combination with oxacillin, rifampin, or vancomycin against Staphylococcus aureus.

In a study of antibiotic combinations of clindamycin with rifampin, oxacillin, or vancomycin using the time kill-curve method, the combination of clindamycin and rifampin were sometimes synergistic (5 of 15 times), otherwise indifferent and always enhanced killing of fifteen tested Staphylococcus aureus isolates. In contrast, vancomycin and clindamycin or oxacillin and clindamycin were either indifferent or antagonistic (approximately 50%). Vancomycin alone, however, was generally as effective as the combinations of clindamycin and rifampin.

Intrinsically vancomycin-resistant gram-positive organisms: clinical relevance and implications for infection control.

Intrinsic resistance to vancomycin in gram-positive bacteria presumably predates acquired vancomycin resistance in enterococci but it has only recently generated interest. Intrinsically resistant enterococci possessing the vanC gene and the non-enterococcal genera Leuconostoc, Lactobacillus, Pediococcus and Erysipelothrix are known to cause human infection. This review examines the available data on their identification, resistance mechanisms, epidemiology, clinical infections and antimicrobial susceptibility. Intrinsically vancomycin-resistant gram-positives are usually opportunistic pathogens. Although serious infections may occur, treatment options remain available. No additional infection control measures for the intrinsically resistant genera appear justified with currently available evidence, although vigilance should be maintained to detect future changes in susceptibility patterns.

Prevalence of colonization with vancomycin-resistant enterococci in various population groups in Berlin, Germany.

In order to prevent the spread of vancomycin-resistant enterococci (VRE), the epidemiology of this micro-organism must be defined. The prevalence of colonization with VRE in various population groups in Berlin was investigated and the risk factors associated with VRE colonization assessed. In a cross-sectional study, rectal swabs were taken from seven population groups (healthy students, outpatients, home nursing patients, normal care and critical care patients of a community hospital and university hospital). Every one completed a questionnaire (age, gender, previous hospital stays, antibiotic therapy). Rectal swabs were examined for the presence of normal gut flora and VRE. All VRE isolates were typed by pulsed-field gel electrophoresis (PFGE). VRE colonization prevalence ranged from 0.9% (students) to 4.2% (nursing-home patients) in non-hospitalized subjects; in hospitalized patients prevalence ranged from 1.8% (regular care ward of a community hospital) to 16.3% (ICU patients of a university hospital). Location (university hospital, OR = 3.5) and age (> or = 60 years, OR = 2.2) were independent risk factors for VRE colonization. Within one population group, isolates with identical PFGE patterns were found in up to three people; one strain was found in four subjects belonging to different groups. Our findings suggest that VRE are imported from the community into hospitals with subsequent spread within the institution.

Nosocomial infections and nosocomial pneumonia.

Nosocomial infections are a major source of revenue loss, morbidity, and even mortality to surgical patients. This review presents current issues regarding nosocomial infections and nosocomial pneumonias. This study is a literature review that presents material on nosocomial infections in general and details regarding Clostridium difficile and vancomycin-resistant enterococcus infections. Nosocomial infections, including pneumonias, are serious medical complications, and prevention by strict adherence to barrier precaution is the most important means of protecting the patient from hospital-acquired bacterial flora.

[Bacteremia due to vancomycin-resistant enterococci in neutropenic cancer patients]. / Bacteriemia por enterococos resistentes a vancomicina en los pacientes neutropénicos con cáncer.

BACKGROUND: The aim of this study was to determine the incidence, clinical characteristics and outcome of vancomycin-resistant enterococcal bacteremia. PATIENTS AND METHODS: We included all cases of enterococcal bacteremia in neutropenic cancer patients documented between January 1986 and December 1995 in a 1,000-bed university hospital, where a prospective surveillance of all cases of bacteremia is regularly done. Molecular typing was performed on all vancomycin-resistant strains with the analysis of chromosomic DNA by macrorestriction. RESULTS: Seventeen cases of enterococcal bacteremia were documented. Seven (41%) were caused by vancomycin-resistant strains (E. faecium 3 and E. gallinarum 4), six of which occurred in the last 5 years of the study period. The average age of patients was 43 years (18-69) and most of them had acute leukemia. Eighty percent of these patients had received vancomycin and/or cephalosporins within 2 weeks prior to bacteremia. Previous administration of antibiotics was more frequent in patients with bacteremia caused by vancomycin-resistant enterococci than in those with bacteremia caused by susceptible strains (86% vs 30%; p < 0.05). The mean number of previous antibiotics (2.4 vs 0.8; p < 0.05) as well as days of treatment (13.6 vs 4.3; p = 0.05) were also higher among patients with resistant enterococcal bacteremia. The overall mortality was 57%. CONCLUSIONS: This study shows the emergence of sporadic cases of bacteremia caused by vancomycin-resistant enterococci in neutropenic cancer patients in our area. This fact seems to be related with the previous administration of antibiotics and advice that a rational use of these agents is needed.

Staphylococcus epidermidis extracted slime inhibits the antimicrobial action of glycopeptide antibiotics.

Certain strains of Staphylococcus epidermidis produce a mucoid slime that appears to be an important virulence factor. After crude slime was isolated from selected strains of S. epidermidis, phenol and chloroform were used to remove proteins and lipids. The remaining extract contained a polysaccharide that was seen on SDS gels stained with Stains-all. This extract was an inhibitor of the antimicrobial action of vancomycin, raising the minimum inhibitory concentration (MIC) to vancomycin in all 18 isolates of S. epidermidis. A dose-response curve was seen between the amount of extract added and the degree of resistance, as measured by both MIC and growth curves. A similar effect was noted with MICs of organisms to teicoplanin. Addition of the extract did not change the MIC to LY146032, although a modest effect on growth rate was observed. The extract did not raise the MIC to clindamycin, rifampin, and cefazolin. The extract reversed the synergism seen between vancomycin and gentamicin in the 5 strains tested in time-kill studies. The interference by slime of the antimicrobial effect of vancomycin and teicoplanin may explain why these antibiotics are sometimes ineffective in eradicating foreign body infections due to slime-producing coagulase-negative staphylococci.

Inhibition of antibiotic activity in vitro by synthetic melanin.

Synthetic melanin (100 to 1,000 mug/ml) markedly inhibited the in vitro activity of aminoglycosides and tetracyclines but did not affect the activity of betalactam antibiotics, erythromycin, or clindamycin.

Antagonism of teicoplanin and vancomycin activity by chocolatized Mueller-Hinton agar.

Chocolatized (80 degrees C, 13 min) Mueller-Hinton agar antagonized the inhibitory activities of teicoplanin and vancomycin against reference strains of Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Staphylococcus aureus, and Enterococcus faecalis. This antagonism was due to heat-exposed sheep erythrocytes, sheep hemoglobin, and the supernatant fluid from lysed sheep erythrocytes, but not to sheep serum. Neither water-soluble cholesterol, bovine albumin, bovine serum, hematin, hemin nor egg yolk suspension antagonized teicoplanin and vancomycin.

Influence of fosfomycin and tobramycin on vancomycin-induced nephrotoxicity.

Since combinations of fosfomycin and vancomycin or tobramycin and vancomycin could be of advantage in the therapy of staphylococcal infections, we studied renal tolerance of both combinations. The experimental animal was the rat and the parameters of nephrotoxicity were cyturia and enzymuria. The experiments showed that fosfomycin at dosages of 50 and 250 mg/kg protected against nephrotoxicity caused by vancomycin (dose: 50 mg/kg), whereas the administration of both tobramycin (dose: 2.5 mg/kg) and vancomycin (dose: 50 mg/kg) resulted in an increase of cyturia and enzymuria. However, repeated dosing of vancomycin (single dose: 50 mg/kg) led to renal accumulation when combined with fosfomycin (single dose: 250 mg/kg); renal vancomycin concentrations were lower. This study suggests similarities in the renal handling of vancomycin and aminoglycosides and demonstrates the possibility of reducing drug-associated nephrotoxicity.

Comparison of the in vitro activities of teicoplanin and vancomycin against Clostridium difficile and their interactions with cholestyramine.

The in vitro activity of teicoplanin was compared with that of vancomycin against fecal isolates of Clostridium difficile. All strains were susceptible to both antibiotics, but teicoplanin was fourfold more active than vancomycin. Cholestyramine was found to bind teicoplanin almost completely, reducing its activity to nondetectable levels.

Measurement of levels of aminoglycosides and vancomycin in serum in the presence of new beta-lactam antibiotics.

We evaluated the effect of new beta-lactam antibiotics (azlocillin, mezlocillin, piperacillin, cefotaxime, moxalactam, and cefoperazone) on assays for aminoglycosides and vancomycin. These antibiotics produced no interference in an immunoassay for gentamicin and tobramycin. The new penicillins produced no interference in a bioassay of amikacin and vancomycin with penicillinase incorporated into the assay agar. Bioassay in the presence of the cephalosporins required predigestion with cephalosporinase. We describe a method for accurate bioassay in the presence of the available cephalosporins, with the exception of moxalactam.

Reversal by a specific peptide (diacetyl-alpha gamma-L-diaminobutyryl-D-alanyl-D-alanine) of vancomycin inhibition in intact bacteria and cell-free preparations.

Vancomycin inhibited the growth of Bacillus megaterium, Staphylococcus aureus and Micrococcus lysodeikticus, and in cell-free preparations from B. megaterium it inhibited the formation of mucopeptide and enhanced the accumulation of the lipid intermediate in the biosynthetic pathway. All these inhibitory processes were reversed by the presence of a synthetic peptide analogous to un-cross-linked mucopeptide side chains, namely diacetyl-l-diaminobutyryl-d-alanyl-d-alanine. A considerable amount of vancomycin was found in recovering cells, whether recovery was caused by peptide or took place naturally because a low initial concentration of antibiotic was used. In cell-free preparations pretreated with vancomycin, continued inhibition of mucopeptide synthesis depended on the presence of cell-wall material. This inhibition was also reversible by added peptide.