Phenotypic Resistance to Disinfectants and Antibiotics in Methicillin-Resistant Staphylococcus aureus Strains Isolated from Pigs.

The aim of this research was to study the phenotypic resistances to disinfectants and antibiotics in strains of methicillin-resistant Staphylococcus aureus (MRSA) obtained from Canary black pigs. Analyses were performed on 54 strains of MRSA, isolated in Canary black pigs from the province of Tenerife (Spain); all of them carried the mecA gene. The strains were isolated by means of nasal swab samples of healthy pigs, collected under veterinarian supervision. Bactericidal activity of antiseptics and disinfectants was tested by means of the dilution-neutralization method. Susceptibility to the disinfectants glutaraldehyde, peracetic acid and silver nitrate was assessed, as well as to the antiseptics chlorhexidine, benzalkonium chloride and povidone iodine. Susceptibility to a wide array of antibiotics representing the main groups was determined by means of the disc diffusion method. All the strains demonstrated susceptibility to the disinfectants tested at the recommended concentration, and even to dilutions equal to or lesser than 1/16. The most effective antiseptic and disinfectant were, respectively, chlorhexidine and silver nitrate. With regard to the antibiotics, the strains proved to be multiresistant. All presented phenotypic resistance to the ß-lactam antibiotics ampicillin, penicillin and cefoxitin, as well as to numerous aminoglycosides, tetracycline and trimethoprim-sulfamethoxazole. It was also observed that 61.1% of the strains were carriers of plasmids. Our results underline that in the strains such as MRSA, which show multiple resistances to antibiotics, the antiseptics and disinfectants show great efficacy. Moreover, as other authors also suggest, for the treatment and prevention of infections caused by MRSA, the use of ß-lactam and aminoglycoside antibiotics may be less effective.

Study of the hormetic effect of disinfectants chlorhexidine, povidone iodine and benzalkonium chloride.

The study of the dose-response relationship of disinfectants is of great importance in treating infection, the objective being to use concentrations above the minimal bactericidal concentration (MBC). Below these concentrations, the bacteriostatic or bactericidal effect may be insufficient. Moreover, at low concentrations, a hormetic effect may be observed, producing a stimulation of growth instead of inhibitory action. Hormesis is not well known in the context of antimicrobial substances. This study investigates the possible existence of a hormetic effect in three commonly used antiseptics-chlorhexidine digluconate, povidone iodine and benzalkonium chloride-on strains of reference of Staphylococcus aureus and Pseudomonas aeruginosa. Growth curves were determined for different concentrations of the disinfectants. The variables studied-concentration of disinfectant, instantaneous growth rate and number of generations-were analysed using linear, quadratic and cubic models to adjust for the variables. The three disinfectants tested show a significant hormetic effect with P. aeruginosa and a less significant effect with S. aureus. These findings point to a dose-response effect that is not linear at low concentrations, while hormetic effects observed at some low concentrations result in greater bacterial growth. In infected wounds, materials or surfaces where microorganisms may occupy zones of difficult access for a disinfectant, the hormetic effect may have important consequences.

Specificity of the neutralizers as the cause of errors in evaluating disinfectant efficacy: an assessment of triclosan.

UNLABELLED: One of the most commonly used methods for assessing disinfectants in vitro is the dilution-neutralization test. Although it is known that using an effective neutralizer is essential for obtaining reliable results, in the case of disinfectants like triclosan, whose neutralization is more complicated, an adequate neutralizer is not always used. Here, we compare the efficacy of different neutralizers described previously, and others that have been modified, against several antiseptic formulations of triclosan. The strains of reference used were Pseudomonas aeruginosa, Enterococcus faecium, Staphylococcus aureus and Escherichia coli. The solutions of antiseptic were triclosan 2% aqueous solution, and three alcoholic solutions containing 2, 0·068 and 0·5% triclosan, respectively. In the dilution-neutralization test, six neutralizers with different degrees of complexity were assayed. Our results show that the antiseptic solutions studied are effective against the strains of reference, with the exception of triclosan 2% aqueous solution against Ps. aeruginosa. Most neutralizers proved ineffective, depending on the antiseptic solution and the bacterial species, particularly in the case of Staph. aureus. These findings confirm a need to test every neutralizer specifically for each disinfectant, at the maximum concentration used, and for every specific bacterial strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The choice of an adequate neutralizing agent is essential for obtaining precise results in the evaluation of disinfectants using the dilution-neutralization method. In this study, we show that the neutralization of triclosan and some of its commercial solutions is not a simple task, requiring complex neutralizers. Moreover, the action of the neutralizer is shown to depend on the micro-organism tested. Specificity in view of the disinfectant and the micro-organism calls for performing the neutralizing assay in each particular case of application of the dilution-neutralization method. We also demonstrate that some of the neutralizers described for triclosan in previous studies are not sufficiently effective.

Effect of upper- and lower-limb exercise training on circulating soluble adhesion molecules, hs-CRP and stress proteins in patients with intermittent claudication.

OBJECTIVES: To investigate the effects of exercise training on levels of circulating biomarkers associated with the progression of atherosclerosis and risk of cardiovascular events in patients with intermittent claudication. METHODS: Circulating levels of soluble adhesion molecules (sVCAM-1, sICAM-1, sE-selectin), high sensitivity C-reactive protein (hs-CRP) and stress proteins (Hsp60 and Hsp70) in patients randomised to a 24-week programme of arm- or leg-cranking exercise were compared with those in usual care controls. RESULTS: Arm and leg exercise similarly improved lower-limb aerobic exercise capacity (20% vs 19%, respectively; P<0.001) and maximum walking distance (30% vs 35%, respectively; P<0.001). Improvements in training limb-specific peak oxygen consumption were attenuated for patients in the highest vs lowest quartile for circulating sVCAM-1 levels at baseline (3% vs 25% respectively, P<0.001). Although circulating hs-CRP levels tended to be lower in the arm-cranking group (-1.55 [95% CI: -1.06 to -2.26]mgl(-1)), exercise training had no effect on circulating levels of soluble adhesion molecules or stress proteins. CONCLUSIONS: These findings suggest that high levels of circulating sVCAM-1 are associated with an attenuated exercise training response and that arm-cranking exercise may provide an effective stimulus for evoking systemic anti-inflammatory adaptations in patients with intermittent claudication.

Analysis of purified gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and tandem mass spectrometry.

The stress protein gp96 exhibits a number of immunological activities, the majority of studies into which have used gp96 purified from a variety of tissues. On the basis of 1-D gel electrophoresis, the purity of these preparations has been reported to range between 70% and 99%. This study analyzed gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and liquid chromatography electrospray ionization tandem mass spectrometry (MS-MS). The procedure for purifying gp96 was reproducible, as similar protein profiles were observed in replicate gels of gp96 preparations. The purity of the preparations was typically around 70%, with minor co-purified proteins of varying molecular weights and mobilities being present. Dominant bands at 95-100 kDa in preparations from Wistar rats and C57BL/6 mice were identified as gp96 by ECL Western blotting. Multiple bands having similar, yet distinct molecular weights and differing pI mobility on ECL Western blots were confirmed as being gp96 in preparations from Wistar rats using MS-MS. The most striking feature of the 2-D gel analysis was the presence of additional dominant bands at 55 kDa in preparations from Wistar rats, and at 75-90 kDa in preparations from C57BL/6 mice. These were identified as gp96 by ECL Western blotting and, in the case of preparations from Wistar rats, by MS-MS. Although the lower molecular weight, gp96-related molecules might be partially degraded gp96, their reproducible presence, definition and characteristics suggest that they are alternative, species-specific isoforms of the molecule. A 55 kDa protein which exhibited a lower pI value than gp96 was present in all preparations and this was identified as calreticulin, another putative immunoregulatory molecule. This study confirms the reproducibility of the gp96 purification protocol and reveals the presence of multiple gp96 isoforms, some of which likely result from post-translational modifications such as differential glycosylation and phosphorylation.

Levels of HSP70 in HIV(+) patients in different viroimmunological states.

The aim of the study was to establish an association between the viroimmunological status of HIV positive patients and their levels of the 70-kD heat shock protein (Hsp70). The longitudinal retrospective case study involved 61 patients at the University Hospital in Granada (Spain) from 1999 to 2002. Twenty-five patients were viroimmunologically stable, while the other 36 patients had suffered virological failure. A minimum of three blood samples were taken at intervals of at least 3 months for the patients who were stable virologically and immunologically, whereas four samples were taken for the virological failure group: two previous to the onset of virological failure, a third corresponding to the time of virological failure, and a fourth at least 3 months after remission of virological failure. Blood samples were also obtained from 20 healthy control subjects; Hsp70 levels in all were determined by enzyme immunoassay. The mean concentration of Hsp70 was 145.4 ng/ml in the HIV-infected patients as opposed to 72.1 ng/ml in the controls. While the viroimmunologically stable group showed levels similar to those of the controls (66.5 ng/ml), the mean value of Hsp70 in the virological failure group was nearly four times as high (249.1 ng/ml), yet this difference was not statistically significant. The patients treated with reverse transcriptase inhibitors were found to have significantly higher levels of Hsp70 than the other subjects. The transformed variable Hsp70/CD4(+) presents less variability than the Hsp70 value itself, giving a higher degree of statistical significance, and may be considered a useful parameter for diagnostic, prognostic, and therapeutic management of HIV positive patients.

Evaluation of microbiological and physicochemical indicators for wastewater treatment.

The quality control of wastewater treatments was monitored using selected novel and classical physicochemical and microbiological indicators, and the associations of the treatments with the effluents was analyzed. The microbiological indicators monitored were heterotrophic plate count (HPC), total coliforms (TC), fecal coliforms (FC), fecal streptococci (FS), sulfite-reducing clostridia (SRC), Pseudomonas aeruginosa, and Salmonella spp. The stages of wastewater treatment also were evaluated through determination of ammonia; biological oxygen demand (BOD(5)); chemical oxygen demand (COD); chloride; conductivity; suspended dissolved and total solids; fats; nitrate, nitrite, and total nitrogen; pH; phosphate and total phosphorus. Additional indicators included the Escherichia coli growth inhibition (IGEC) bioassay for assessing whole effluent toxicity, spectral determinations between wavelengths (lambda) 190-650 nm, and total (TP) and soluble (SP) protein contents. Of the more common physicochemical parameters, only BOD(5), COD, suspended and total solids, and fats showed a statistically significant reduction between raw water and effluent; for the microbiological indicators, significant reduction was seen only for HPC, FC, and Ps. aeruginosa. We suggest that determinations of Ps. aeruginosa be commonly used as an indicator of wastewater quality. Spectral analysis--most notably the values of absorbance at 225, 255, and 295 nm-revealed a statistically significant correlation with several physicochemical parameters. Statistical analysis of SP and TP values showed them to be good indicators of contamination. The quantitative study of Salmonella spp. and the results of the IGEC bioassay show the need for close control of infectious and toxic risks in wastewater and effluents.

Efficacy of some neutralizers in suspension tests determining the activity of disinfectants.

The ability of six mixtures to neutralize glutaraldehyde, o-phthalaldehyde and peracetic acid was tested using four reference strains: Pseudomonas aeruginosa CIP A22, Escherichia coli CIP 54127, Staphylococcus aureus CIP 53154, and Enterococcus faecium CIP 5855. Glutaraldehyde was the hardest to neutralize, and peracetic acid the easiest. The most effective mixture was Tween 80 with sodium bisulphate, sodium thioglycolate, lecithin and cysteine, and the least effective was Tween 80, lecithin and histidine. The efficacy of the neutralizers may indicate a propensity loss of activity from interfering substances when disinfectants are used in practice.