Guidelines for the control and prevention of meticillin-resistant Staphylococcus aureus (MRSA) in healthcare facilities.

Meticillin-resistant Staphylococcus aureus (MRSA) remains endemic in many UK hospitals. Specific guidelines for control and prevention are justified because MRSA causes serious illness and results in significant additional healthcare costs. Guidelines were drafted by a multi-disciplinary group and these have been finalised following extensive consultation. The recommendations have been graded according to the strength of evidence. Surveillance of MRSA should be undertaken in a systematic way and should be fed back routinely to healthcare staff. The inappropriate or unnecessary use of antibiotics should be avoided, and this will also reduce the likelihood of the emergence and spread of strains with reduced susceptibility to glycopeptides, i.e. vancomycin-intermediate S. aureus/glycopeptide-intermediate S. aureus (VISA/GISA) and vancomycin-resistant S. aureus (VRSA). Screening for MRSA carriage in selected patients and clinical areas should be performed according to locally agreed criteria based upon assessment of the risks and consequences of transmission and infection. Nasal and skin decolonization should be considered in certain categories of patients. The general principles of infection control should be adopted for patients with MRSA, including patient isolation and the appropriate cleaning and decontamination of clinical areas. Inadequate staffing, especially amongst nurses, contributes to the increased prevalence of MRSA. Laboratories should notify the relevant national authorities if VISA/GISA or VRSA isolates are identified.

The effect of pharmacokinetics on the bactericidal activity of ciprofloxacin and sparfloxacin against Streptococcus pneumoniae and the emergence of resistance.

The pharmacokinetics of ciprofloxacin and sparfloxacin were simulated in vitro and the effects of pharmacodynamic parameters on bactericidal activity and the emergence of quinolone resistance were examined for Streptococcus pneumoniae. Simulated serum concentrations of ciprofloxacin 500 mg bd were more rapidly bactericidal than sparfloxacin 200 mg bd, despite lower values for the key pharmacodynamic parameters (AUC/MIC and C(max)/MIC). This was possibly related to the slower oral absorption of sparfloxacin, which delayed achievement of the MIC compared with ciprofloxacin. In addition, sparfloxacin was shown to have similar bactericidal activity to ciprofloxacin when tested at the same concentrations, despite its four-fold better potency in MIC terms. The emergence of resistance following exposure to ciprofloxacin appeared to be dependent on the C(max)/MIC ratio and the AUC above the MIC, but not the AUC/MIC ratio. Resistance (at least four-fold increase in MIC) developed when the C(max)/MIC ratio was less than four or the AUC above the MIC was less than 10, and the resulting cultures regrew fully. In contrast, pneumococci with a two- to four-fold increase in sparfloxacin MIC were selected in the presence of serum concentrations of sparfloxacin despite a C(max)/MIC ratio higher than 12, but these isolates remained clinically susceptible by breakpoint MIC and their growth was inhibited by repeated dosage of sparfloxacin. Nevertheless, the selection of pneumococci with reduced susceptibility, and the possibility of further mutation to highly resistant strains supports the use of quinolones that rapidly eradicate pneumococci at conventional doses and achieve concentrations, in both serum and tissues, which exceed at least 4 x MIC.

In vitro activities of oral beta-lactams at concentrations achieved in humans against penicillin-susceptible and -resistant pneumococci and potential to select resistance.

The beta-lactam susceptibilities of 65 strains of Streptococcus pneumoniae for which penicillin MICs covered a broad range were assessed. The order of potency was amoxicillin (AMX) = amoxicillin-clavulanate (AMC) > penicillin G > cefpodoxime (CPO) > cefuroxime (CXM) > cefprozil > cefaclor > loracarbef > cefixime. No decrease in susceptibility was seen following repeated subculture of two penicillin-susceptible strains of S. pneumoniae in AMX, AMC, cefaclor, or loracarbef, whereas repeated exposure to CPO and CXM resulted in 4- to 32-fold decreases in susceptibility for both strains. When one of these strains was exposed to concentrations of CPO, CXM, AMX, and AMC achieved in the serum of humans following the administration of an oral dose, all agents were rapidly bactericidal, with no decrease in susceptibility up to 72 h. This was consistent with antibiotic concentrations exceeding the MICs for 100% of the dosing interval. For a penicillin-resistant strain, MICs were exceeded for 29% of the 12-h dosing interval for 500 mg of AMX, 42% of the interval for AMC with 875 mg of AMX and 125 mg of clavulanate (875/125 mg of AMC) 21% of the interval for 500 mg of CXM, and 0% of the interval for 200 mg of CPO. Consequently, only 875/125 mg of AMC produced a sustained bactericidal effect. A four- to eightfold reduction in susceptibility to CPO and CXM and cross-resistance with cefotaxime, but not penicillin or AMC, were selected following exposure to simulated serum CPO and CXM concentrations. In addition, AMX and AMC were the only agents which consistently produced a >99% reduction in bacterial numbers in time-kill studies using concentrations of antibiotic achieved in middle ear fluid for all three strains of penicillin-resistant S. pneumoniae tested.

Oxygen scavenging, NADH oxidase and metronidazole resistance in Helicobacter pylori.

Failure of triple-therapy regimes to eradicate Helicobacter pylori from the stomach is thought to be due to the occurrence of a metronidazole-resistant bacterial population. Exposure of metronidazole-resistant (MtzR) strains of H. pylori to an anaerobic environment causes the activation of metronidazole and the loss of resistance. Using metronidazole-sensitive (MtzS) clinical isolates, we selected mutants conferring resistance to metronidazole, which were used to investigate the effect of bacterial cell density upon the activation of metronidazole. The addition of metronidazole, at a final concentration of 10 mg/L, to MtzR cultures of a bacterial cell density >1 x 10(6) cfu/mL, caused a loss in viability. No loss in viability, however, occurred upon addition of metronidazole to MtzR cultures of a cell density of <1 x 10(6) cfu/mL. MtzS cultures lost viability irrespective of the initial cell density, indicating that oxygen scavenging at the site of metronidazole reduction may occur in these cultures. The ability of MtzS wild types, MtzR isogenic mutants and MtzR wild types to scavenge oxygen from the intracellular environment was investigated. H. pylori cultures contained NADH and NADPH oxidase activity. NADPH oxidase activity was always more than double the NADH oxidase activity. MtzR mutants possessed approximately one-third the NADH oxidase activity found in their respective MtzS parent wild types. MtzR wild types possessed a low NADH oxidase level similar to that found in the MtzR mutants. We propose that metronidazole resistance may be mediated through an inability of MtzR strains to remove oxygen from the site of metronidazole reduction, thereby preventing metronidazole activation.

Population Development of Heterodera glycines and Soybean Yield in Soybean-maize Rotations Following Introduction into a Noninfested Field.

An 11-year field study was initiated in 1979 to monitor population development of Heterodera glycines. Fifty cysts of a race 5 population were introduced into plots in a field with no history of soybean production and that had been in sod for 20 years. Soybean cultivars either susceptible or resistant to H. glycines were grown either in monoculture or rotated with maize in a 2-year rotation. During the first 5 years, resistant cultivars with the Peking source of resistance were planted. After year 5, monocuhure of Peking resistance resulted in 18 cysts/250 cm(3) of soil, whereas populations resulting from the continuous cropping of susceptible soybean resulted in 45 cysts/250 cm(3). Some plots in all treatments, including control plots, were contaminated at the end of year 5. Crop rotation delayed population development of H. glycines. During years 6 through 11 cv. Fayette (PI88.788 source of resistance) was planted. In year 6 numbers of cysts declined to 1/250 cm(3) of soil in the treatment consisting of monocultured Fayette. At the end of year 10, cysts were below the detection level in all treatments in which Fayette was planted. Yield of susceptible soybean in monoculture with or without H. glycines infestation was lower beginning in year 6 when compared to yield of soybean grown in rotation and remained lower throughout the duration of the experiment except for 1987 (year 9). Yields of susceptible and resistant soybean were different each year except for drought years in 1980 and 1988. From 1979 to 1982 differences in yield were due to lower yield potential of resistant cultivars. Except for the drought year, yield of cv. Fayette was greater than susceptible Williams 82 during years 6 through 11.

The interaction of nitroaromatic drugs with aminothiols.

The effect of cysteamine and glutathione addition on the redox behaviour of metronidazole, chloramphenicol, M&B 4998, nitrofurazone, and nifuroxime has been studied by electrochemical techniques. The presence of thiol influences the redox behaviour of the nitro compound in a number of ways. In aqueous media, the single-step nitro/hydroxylamine reduction shows a decrease in current and a shift to more positive potentials, which is assigned to the thiol acting as the reducing agent, but only after the formation of the nitro radical anion. In addition, the reversible RNO/RNHOH couple is greatly diminished or removed. In a dimethylformamide/H2O solvent, the nitro radical anion can be selectively generated. The effect of thiol addition on the stability of the radical anion is strongly dependent on the drug, the identity of the thiol, and the concentration of the supporting electrolyte. The presence of thiol can result in an increase or a decrease in the lifetime of the radical with no apparent correlation with the redox couple of the nitro compound, or can act as an oxidizing agent and regenerate the original nitro compound. These disparate routes by which thiol can modify the redox characteristics of nitro compounds suggest that the traditional role of thiol as a radical scavenger needs to be extended.

The influence of microaerophilia and anaerobiosis on metronidazole uptake in Helicobacter pylori.

Resistance of Helicobacter pylori to metronidazole during therapy for gastroduodenal ulcers is claimed to be responsible for failure to eradicate the pathogen and thus the disease. Resistance to metronidazole and other nitroimidazoles is rare and documented only for anaerobes; the mechanism of resistance in typical microaerophiles, like Helicobacter, is not known. We have studied metronidazole uptake using high performance liquid chromatography in metronidazole sensitive and resistant strains of H. pylori under conditions of microaerophilia and in anaerobiosis. The uptake of metronidazole was faster in sensitive strains than resistant ones and was also increased in anaerobiosis. Drug uptake and the rate of cell kill was found to be dependent upon the relative oxygen tension of the environment and the cell density, both of which determine the redox conditions of the media. We suggest that resistance displayed in microaerophilia, but which disappears in anaerobiosis, may not involve futile cycling nor the induction of superoxide dismutase and catalase. We further propose that resistant organisms may have alterations in the pattern of pyruvate metabolism as documented for anaerobic bacteria and protozoa and that resistance in microaerophilia may involve the relative efficiencies of detoxifying oxygen in susceptible and resistant strains of H. pylori.

Redox potential and oxygen concentration as factors in the susceptibility of Helicobacter pylori to nitroheterocyclic drugs.

Metronidazole sensitive (MtzS) and resistant (MtzR) strains of Helicobacter pylori were tested for their sensitivities to eleven nitroheterocyclic drugs of known reduction potential under a wide range of environmental conditions. Under microaerophilic conditions, MtzR strains were sensitive to all the 2-nitroimidazoles, resistant to every 5-nitroimidazole, and slightly sensitive to the nitrothiazole, niridazole. MtzS strains were sensitive to all the nitroimidazoles except for 4(5)-nitroimidazole which has the lowest redox potential of all the drugs investigated. MtzS strains displayed the greatest sensitivity towards niridazole and satranidazole, the latter having the highest redox potential of the 5-nitroimidazoles. High and low oxygen tensions had no effect on the activity of the drugs but periods of anaerobic incubation caused resistant strains to become sensitive to the 5-nitroimidazoles. Superoxide dismutase and catalase were not induced by metronidazole and enzyme levels showed no correlation with resistance patterns. The results show that futile cycling does not occur in H. pylori and that the mechanism of action of the nitroimidazoles is related to their redox potential. Anaerobiosis abolishes resistance to metronidazole which suggests that it may be mediated through the activation of anaerobic metabolic pathways which function less under microaerophilic conditions or not at all.

Electrochemical studies of tirapazamine: generation of the one-electron reduction product.

The electrochemical properties of the benzotriazine di-N-oxide, tirapazamine (SR4233), and the mono- and zero-N-oxides, SR4317 and SR4330 respectively, have been investigated in dimethylformamide and acetonitrile. The voltammetry of tirapazamine is complicated, with up to 6 reduction steps being identified, depending on the solvent. Both SR4317 and SR4330 show two reduction steps. The first reduction of all three compounds is a reversible or quasi-reversible step, which is assigned to a 1-electron addition. Cyclic voltammetric studies show that the anion radical product is stable, although the tirapazamine 1-electron addition product shows a tendency to participate in a chemical following reaction. Subsequent reduction steps are all highly irreversible in nature. The 2nd electron transfer of SR4317 results in the formation of the free base, SR4330, which is identified voltammetrically. Comparison is made with the voltammetric behaviour of quinoline and quinoline-oxide.

Evidence for the direct interaction of reduced metronidazole derivatives with DNA bases.

The electrochemical behaviour of the bioreductive redox active nitroimidazole drug metronidazole has been examined in the presence and absence of the DNA bases using three electrochemical techniques, all of which indicate the capacity for interaction between reduced products and DNA bases. The 4-electron metronidazole (RNO2) metronidazole-hydroxylamine (RNHOH) couple in an aqueous medium shows a positive shift in reduction potential upon addition of thymine, adenine and guanine, but a negative shift for cytosine. Interpretation of these results for an irreversible process is, however, inconclusive. In dimethylformamide/H2O the presence of DNA base on the one-electron addition product, the nitro radical anion, was examined by cyclic voltammetry. All except guanine resulted in interaction with the metronidazole nitro radical anion (RNO2-), as measured by the decrease in the return-to-forward peak current ratio, in the following order of increasing reactivity: cytosine, adenine and thymine (at a metronidazole: base ratio of 1:1). The increase in the stability of the radical anion by increasing the pH of the dimethylformamide/H2O medium resulted in a decreased reaction with thymine.

The reactivity of chloramphenicol reduction products with DNA bases.

PURPOSE: The interaction between the constituent bases of deoxyribonucleic acid and the reduction products of the nitro-aromatic compound chloramphenicol and its nitroso derivative have been studied using an electrochemical system. METHODS AND MATERIALS: The changes to the voltammetry of chloramphenicol and nitrosochloramphenicol upon addition of adenine, cytosine, guanine, and thymine at various concentrations have been measured. The biological implications of reductive activation of both chloramphenicol and nitrosochloramphenicol were examined using a phi X174 double transfection technique which measures biologically relevant deoxyribonucleic acid damage. RESULTS: Measurement of the voltammetric response of chloramphenicol shows that the most noticeable change upon base addition is a decrease in the lifetime of the nitro radical anion in the following order of decreasing activity: adenine, thymine, and cytosine. No effect was observed with guanine. The reversible 2-electron nitrosochloramphenicol-hydroxychloramphenicol couple showed no interaction on the voltammetric timescale, although binding of the hydroxylamine to guanine was observed. Interaction of the azo derivative, formed as a consequence of further reduction plus chemical reaction of nitrosochloramphenicol was observed. Biological studies showed that no significant effect on deoxyribonucleic acid by chloramphenicol or nitrosochloramphenicol was observed under oxic conditions. Controlled reduction of nitrosochloramphenicol to the hydroxylamine gave considerably less damage than when nitrosochloramphenicol or chloramphenicol was completely reduced. CONCLUSION: The chloramphenicol nitro radical anion reacts selectively with the bases of deoxyribonucleic acid. Reduction products of nitrosochloramphenicol beyond the 2-electron hydroxylamine are highly reactive to deoxyribonucleic acid.

Studies on DNA damage and induction of SOS repair by novel multifunctional bioreducible compounds. II. A metronidazole adduct of a ruthenium-arene compound.

A new transition metal complex of the 5-nitroimidazole, metronidazole (1-beta-hydroxyethyl-2-methyl-5-nitroimidazole), has been prepared and its potential use as a hypoxic cell cytotoxic agent examined. The preparation of the complex [(eta6-C6H6)RuCl2(metronidazole)] is described together with its characterization using standard spectroscopic techniques. Electrochemical investigations showed that coordination to the metal centre had not altered the electron affinity of the metronidazole, but kinetic studies using the cyclic voltametric mode demonstrated that the one-electron addition product, the nitro radical anion, had a decreased lifetime, with a half-life of 7.75 and 11.9 s for the coordinated and free metronidazole ligand respectively. Biological studies employed viscosity measurements, DNA SOS repair capacity and a transfection assay to examine the effect on DNA. Conductance studies were also employed to determine the influence on intact Escherichia coli growth rates. The ruthenium-metronidazole complex showed greater activity than metronidazole aerobically, but a higher differential activity under hypoxic reduction conditions, due to activation of the NO2 group. Results with intact cells suggested a greater selective cytotoxicity with metronidazole coordinated to ruthenium than attained with the free ligand.

Repair of damage induced by SR 4233.

The benzotriazine di-N-oxide, SR 4233, was electrolytically reduced at constant potential at pH 4.0 at a reduction rate of 5%/hr under N2 in the presence of phi X174 DNA. During the reduction process, the biological infectivity of the bacterial phage was measured by a double transfection technique, either into the wild-type Escherichia coli strain, or into a series of seven mutants with specific, known defects in their capacity to repair DNA. The survival of phi X174 was evaluated as an index of drug damage and from this we conclude that SR 4233 induces pH-dependent DNA damage in E coli, which is recognized and repaired primarily by the uvrC gene product and by the exonuclease III and endonuclease III gene products. These gene products act primarily upon and are responsible for the recognition of strand breaks and repair of oxidized and fragmented pyrimidine products, indicating that SR 4233 induces strand breaks in DNA resulting from oxidative damage to pyrimidines. As damage is maximized at acid pH, we further propose that the damage mechanism is a process of electron transfer from pyrimidine nucleotides in DNA (i.e., oxidation) to the protonated benzotriazine di-N-oxide one-electron radical anion.

The interaction of reduced metronidazole with DNA bases and nucleosides.

The electrochemical behavior of the 1-electron couple for the bioreductive drug metronidazole has been examined in the presence and absence of the biological target molecules, DNA bases, and nucleosides, including uracil and uridine. Using cyclic voltammetry as the investigation technique, the change in return-to-forward peak current ratio, ipr/ipf, from the control, recorded in the absence of target, was measured as a function of scan rate and biological target concentration. All target molecules, except adenosine and guanine, resulted in interaction with RNO2.-, as measured by the decrease in the ipr/ipf ratio in the following order of increasing reactivity: adenine, guanosine, thymine, uracil, uridine, and thymidine (at a metronidazole:target ratio of 1:1). No decrease in ipr/ipf was observed with cytosine or cytidine until ratios of 1:20 and 1:30, respectively, were attained. An approximately linear relationship was found between the percentage change in the CV response and log[target] allowing us to determine the sensitivity of RNO2.- to the concentration of the target species. The implication for the biological action of metronidazole and other nitro-heterocyclic drugs is discussed.

Electrochemical characteristics of nitroheterocyclic compounds of biological interest. VIII. Stability of nitro radical anions from cyclic voltammetric studies.

The stability of the one electron addition product of four biologically important nitroheterocyclic compounds has been examined electrochemically. Using cyclic voltammetry the tendency of the nitro radical anion to undergo disproportionation was studied by two methods of analysis. The first was based on determining the voltammetric time-constant required for half of the reduction product, RNO2-., to react further. The second concerned the minimum volume of dimethylformamide which had to be added to the aqueous electrolytic medium to give a specific cyclic voltammetric response. Both methods were found to compare well with the results obtained for RNO2-. stabilities using a theoretically derived procedure for a second order reaction following a charge-transfer step. The use of these alternative approaches for quantifying the reactivity of reduction products is discussed. The time-constant method in particular may be useful in studying complex reaction pathways.

A comparison of the relative activities of 8 radiosensitizers in the SOS chromotest.

Misonidazole, and RSU 1069 and 6 of its analogues are all reported to show increased cytotoxicity towards hypoxic cells compared to oxic cells. DNA is considered to be the target through which these drugs exert their cytotoxic activity. Therefore we monitored induction of the SOS response in uvrABC excinuclease proficient and deficient strains of E. coli, under oxic and hypoxic conditions, as an indirect method of assessing the activity of these drugs towards DNA in a biological system. This was done using the SOS chromotest which utilizes E. coli strains which possess a sfiA::lacZ fusion allowing induction of the SOS response to be monitored by assaying beta-galactosidase activity. All of the drugs tested here show some induction of the SOS response in both uvrABC excinuclease proficient and deficient strains. Data shown here suggests that the uvrABC excinuclease is important in the production of a SOS induction signal from RSU 1069-induced DNA lesions and that RSU 1069 may act as a crosslinking agent. The data also shows that SOS induction activity and toxicity do not necessarily correlate and that production of a SOS induction signal may occur via a different pathway for RSU 1069 than for its analogues.