Factors modulating the pH at which calcium and magnesium phosphates precipitate from human urine.

The factors controlling the rate at which crystalline bacterial biofilms develop on indwelling bladder catheters are poorly understood. It is known that normally the pH of voided urine (pHv) is lower than the pH at which calcium and magnesium phosphates come out of urine solution (pHn). In patients who develop infections with urease producing bacteria, however, the pHv rises above the pHn and precipitation of the phosphates occurs in the urine and the biofilm. The aim of this study was to examine ways of manipulating the pHn of urine so that more of its calcium and magnesium remain in solution under alkaline conditions. The experimental data show that pHn can be elevated by decreasing the calcium, magnesium and phosphate concentrations. Increasing the fluid intake of a human subject so that the urinary calcium fell from 120 mg/l to 25 mg/l, for example, resulted in the pHn increasing from 6.48 to 8.22. The addition of citrate to urine also produced a rise in the pHn. The daily consumption of 500 ml of fresh orange juice increased urinary citrate concentrations from 0.35 to around 1.21 mg/ml and the pHn rose from 7.24 to 8.2. The pHn of urine is thus a highly variable parameter. It can be manipulated by controlling the urinary concentrations of magnesium, calcium, phosphate and citrate ions. We suggest that increasing fluid intake with citrate containing drinks would reduce the extent of encrustation on catheters in patients infected with urease producing bacteria.

Pulmonary Balantidium coli infection in a leukemic patient.

A 59-year-old woman suffering from chronic lymphocytic leukemia developed pulmonary lesions; bronchoalveolar lavage was performed for possible systemic fungal infection. However, direct microscopic analysis revealed ciliated protozoa identified as Balantidium coli. B. coli is the only known pathogenic ciliate, and is usually associated with intestinal infection in areas associated with pig rearing. On very rare occasions the organisms may invade extra-intestinal organs, in this case the lungs of an immunocompromised patient. This case is unusual as balantidiasis is rare in Europe, the patient had no obvious contact with pigs, and there was no history of diarrhea prior to pulmonary colonization. Metronidazole was rapidly administered, and the condition improved after 24-48 hr.

The antibacterial activity of vancomycin towards Staphylococcus aureus under aerobic and anaerobic conditions.

AIMS: To investigate the antibacterial efficacy of vancomycin towards Staphylococcus aureus under aerobic and anaerobic conditions, and to assess the influence of oxygen on the duration of the post-antibiotic effect (PAE) after exposure to vancomycin. METHODS AND RESULTS: Culture-based techniques and flow cytometric measurements of 5-cyano-2,3-ditolyl tetrazolium chloride (an indicator of redox activity) and the membrane potential-sensitive fluorophore Sytox Green, were used to test four staphylococcal strains. The MICs for all strains, and the duration of PAE, were similar whether tested with or without oxygen. However, a fivefold logarithmic reduction in cell counts was observed in 10-15 h aerobically, depending on strain, compared with longer than 60 h in an anaerobic environment. Flow cytometric data correlated well with counts of colony-forming units under both aerobic and anaerobic conditions. CONCLUSIONS: The death rate of Staph. aureus exposed to vancomycin was greater in the presence of oxygen, although MIC values and PAE durations were similar whether tested aerobically or anaerobically. Also, flow cytometry provided a rapid and sensitive alternative to plate counts for the assessment of antibiotics in oxygen-free conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study underlines the need for further anaerobic testing using different strain/antibiotic combinations, the results of which will have clinical significance due to the anaerobic nature of some sites of infection.

Triclosan and antibiotic resistance in Staphylococcus aureus.

Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) is an antimicrobial agent used in hygiene products, plastics and kitchenware, and for treating methicillin-resistant Staphylococcus aureus (MRSA) outbreaks. S. aureus strains with low-level resistance to triclosan have emerged. It has been claimed that strains with decreased susceptibility to biocides may also be less susceptible to antibiotics. We tested the susceptibility of S. aureus clinical isolates to triclosan and several antibiotics. Triclosan MICs ranged between 0.025 and 1 mg/L. Some, but not all, strains were resistant to several antibiotics and showed low-level triclosan resistance. S. aureus mutants with enhanced resistance to triclosan (< or =1 mg/L) were isolated. In several cases this resistance was stably inherited in the absence of triclosan. These mutants were not more resistant than the parent strain to several antibiotics. Changes in triclosan MICs associated with the acquisition of a plasmid encoding mupirocin resistance were not observed, suggesting that the triclosan/mupirocin co-resistance seen in a previous study was not the result of a single resistance gene or separate genes on the same plasmid. The continuous exposure of a triclosan-sensitive S. aureus strain to sub-MIC concentrations of triclosan for 1 month did not result in decreased susceptibility to triclosan or to several antibiotics tested. Triclosan-induced potassium leakage and bactericidal effects on a triclosan-sensitive strain, a resistant strain and a strain selected for increased resistance were compared with those of non-growing organisms, exponentially growing organisms and organisms in the stationary phase. No significant differences between the strains were observed under these conditions despite their different MICs. Biocides have multiple target sites and so MICs often do not correlate with bactericidal activities. The ability of S. aureus to develop resistance to triclosan and the current view that triclosan may have a specific target in Escherichia coli, namely enoyl reductase, underline the need for more research on the mechanisms of action and resistance.

Fluorescence monitoring of antibiotic-induced bacterial damage using flow cytometry.

BACKGROUND: Conventional techniques used to assess bactericidal activities of antibodies are time-consuming; flow cytometry has been used as a rapid alternative. In this study, the membrane potential-sensitive fluorescent probes bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)) and Sytox Green, the redox dye cyano-2,3-ditolyl tetrazolium chloride (CTC), and the Baclite viability test kit were used to assess the effects of ceftazidime, ampicillin, and vancomycin on clinical isolates of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, respectively. METHODS: Bacterial cultures were grown to early exponential phase, at which point the antibiotics were added at their breakpoint values, and incubation was allowed to continue. At timed intervals, samples were stained and flow cytometric analysis was performed on a Skatron Argus 100 arc-lamp based dual-parameter flow cytometer. RESULTS: All the dyes successfully identified antibiotic-induced damage in the three strains, although different fluorescence responses between the dyes were observed. DiBAC4(3) and Sytox Green overestimated numbers of nonviable bacteria relative to loss of viability as judged by plate counts. CTC, a measure of respiratory activity, revealed antibiotic-induced population heterogeneity illus trated by the development of several subpopulations. The "live" component of the viability kit identified two populations corresponding to viable and nonviable organisms, whereas the "dead" component only revealed single populations, the fluorescence intensity of which increased with antibiotic exposure. CONCLUSIONS: Flow cytometry provides a rapid and sensitive technique for the evaluation of the antibacterial activities of antibiotics. The use of a range of fluorophores specific for different cellular characteristics may be beneficial, bearing in mind the different fluorescence responses observed among the dyes used here.

Antibiotic and biocide resistance in methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococcus.

Concern has been growing regarding the potential of antibiotic and disinfectant co-resistance in clinically important bacteria. In this study, the susceptibilities of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) to chlorhexidine (CHX), the quaternary ammonium compounds cetylpyridinium chloride (CPC) and benzalkonium chloride (BC), triclosan, dibromopropamidine isethionate (DBPI) and triclocarban were compared. MRSA exhibited low-level resistance to CHX and the QACs, with MICs of 1.5 to 3-fold (CHX), and 2 to 4-fold (QACs) higher than MSSA. However, the MIC values for MRSA ranged between 0.025 (the MIC of MSSA) and 1 microg/mL with triclosan, and between <5 (the MIC of MSSA) and 75 microg/mL with DPBI. Nevertheless, these strains remain relatively sensitive to most of these antimicrobial agents. The bactericidal efficacy of CHX, CPC and DBPI (with the exception of one strain) correlated with their MIC value. This was not observed using triclosan; MRSA and MSSA strains were equally susceptible to its killing effect, regardless of MIC. The permeabilizing agent, ethylenediamine tetraacetic acid (EDTA) was unable to potentiate the antibacterial activities of the biocides against any of the strains tested. Attempts to select for staphylococcal strains with increased resistance to triclosan, CPC or CHX, using disc diffusion, step-wise broth, or repeated exposure/recovery technique, were only partially successful, and resistance was found to be unstable. The susceptibilities of vancomycin-resistant enterococcus (VRE) and vancomycin-sensitive enterococcus (VSE) to the biocides were also compared and found to be similar both in terms of MIC testing and time-kill studies.

Flow cytometric assessment of the postantibiotic effect of methicillin on Staphylococcus aureus.

The postantibiotic effect (PAE) following a 2-h exposure of Staphylococcus aureus NCTC 6571 to methicillin (5x the MIC) was investigated with fluorescent probes, 5-cyano-2,3-di-4-tolyl tetrazolium chloride (CTC), an indicator of respiratory activity, and the membrane potential-sensitive compound bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. Counts of the numbers of CFU on solid agar correlated well with information gained from the CTC and DiBAC4(3) fluorescence intensity distributions obtained by flow cytometry and revealed that the postantibiotic effect was 3.1 h. Due to the capacity of flow cytometry to provide information on the heterogeneity of a bacterial population, both fluorescent probes identified the emergence of an active subpopulation 4 h after removal of the methicillin, indicating the recovery of a small percentage of the population. After removal of the methicillin and resuspension of the cells in methicillin-free medium, a further decrease in the respiratory activity and the membrane integrity of the population was observed, although the CFU counts hardly varied, indicating continued antibiotic-induced damage. Also, CTC fluorescence measurements identified numerous subpopulations during the PAE period; this suggests that the PAE is complex, with individual organisms exhibiting various degrees of recovery. Flow cytometry thus provides a rapid and sensitive alternative to traditional techniques that have been used to study PAE, with the added advantage that physiological changes can be detected as they arise.

Oxygen uptake and antioxidant responses of the free-living diplomonad Hexamita sp.

The free-living anaerobic flagellate Hexamita sp. was observed to actively consume O2 with a K(m) O2 of 13 microM. Oxygen consumption increased linearly with O2 tension up to a threshold level of 100 microM, above which it was inhibited. Oxygen uptake was supported by a number of substrates but probably not coupled to energy conservation as cytochromes could not be detected spectro-photometrically. In addition, inhibitors specific for respiratory chain components did not significantly affect O2 uptake. Respiration was however, partially inhibited by flavoprotein and iron-sulfur protein inhibitors. NAD(P)H supported O2 consumption was measured in both particulate and soluble fractions; this activity was partially inhibited by quinacrine. A chemosensory response was observed in cells exposed to air, however no response was observed in the presence of superoxide dismutase plus catalase. Catalase and nonspecific peroxidase activity could not be detected, but superoxide dismutase plus catalase. Catalase and nonspecific peroxidase activity could not be detected, but superoxide dismutase activity was present. Superoxide dismutase was sensitive to NaN3, and H2O2 but not KCN, suggesting a Fe prosthetic group. Flow cytometric analysis revealed that thiol levels in live cells were depleted in the presence of t-butyl H2O2. The observed NADPH-driven glutathione reductase activity is believed to recycle oxidized thiols in order to re-establish reduced thiol levels in the cell. The corresponding thiol cycling enzyme glutathione peroxidase could not be detected. The ability to withstand high O2 tensions (100 microM) would enable Hexamita to spend short periods in a wider range of habitats. Prolonged exposure to O2 tensions higher than 100 microM leads to irreversible damage and cell death.

A flow cytometric study of antibiotic-induced damage and evaluation as a rapid antibiotic susceptibility test for methicillin-resistant Staphylococcus aureus.

Flow cytometry using the anionic membrane potential-sensitive fluorescent probe, bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)), enabled assessment of antibiotic-induced membrane perturbation in five clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and two antibiotic-sensitive reference strains, NCTC 6571 and 8325-4, after establishment of steady-state growth in liquid cultures inoculated from single colonies. Flow cytometric indications of the enhanced DiBAC4(3) uptake after treatment with vancomycin at 0.1, 1, 4 and 10 x MIC showed excellent comparison with viability losses quantified as cfu on solid agar in MRSA isolate QC. The antibiotic susceptibility patterns to benzylpenicillin, methicillin and vancomycin for all isolates used in this study could be determined in 2-4 h from an overnight plate culture. This technique thus provides a rapid and reproducible antibiotic sensitivity test which may be applicable in routine clinical practice.