Prolonged treatment of Salmonella enterica serovar Typhimurium with commercial disinfectants selects for multiple antibiotic resistance, increased efflux and reduced invasiveness

Objectives: To study how disinfectants affect antimicrobial susceptibility and phenotype of Salmonella enterica serovar Typhimurium SL1344. Methods: Wild-type strain SL1344 and its isogenic gyrA mutant were passaged daily for 7 days in subinhibitory concentrations, and separately for 16 days in gradually increasing concentrations of a quaternary ammonium disinfectant containing formaldehyde and glutaraldehyde (QACFG), an oxidizing compound blend (OXC), a phenolic tar acids-based disinfectant (TOP) and triclosan. The MICs of antimicrobials and antibiotics for populations and representative isolates and the proportion of cells resistant to the MICs for the wild-type were determined. Expression of acrB gene, growth at 37 degrees C and invasiveness of populations in Caco-2 intestinal epithelial cells were assessed. Results: QACFG and triclosan showed the highest selectivity for variants with reduced susceptibility to chloramphenicol, tetracycline, ampicillin, acriflavine and triclosan. Populations treated with the above biocides had reduced invasiveness in Caco-2 cells, and altered growth kinetics. Resistance to disinfectants was observed only after exposure to gradually increasing concentrations of triclosan, accompanied with a 2000-fold increase in its MIC. Growth in OXC and TOP did not affect the MICs of antibiotics, but resulted in the appearance of a proportion of cells resistant to the MIC of acriflavine and triclosan for the wild-type. Randomly selected stable variants from all populations, except the one treated with TOP, over-expressed acrB. Conclusions: In vitro exposure to QACFG and triclosan selects for Salmonella Typhimurium cells with reduced susceptibility to several antibiotics. This is associated with overexpression of AcrAB efflux pump, but accompanied with reduced invasiveness.

Anionic metabolic profiling of urine from antibiotic-treated rats by capillary electrophoresis–mass spectrometry

A recently developed capillary electrophoresis (CE)-negative-ionisation mass spectrometry (MS) method was used to profile anionic metabolites in a microbial-host co-metabolism study. Urine samples from rats receiving antibiotics (penicillin G and streptomycin sulfate) for 0, 4, or 8 days were analysed. A quality control sample was measured repeatedly to monitor the performance of the applied CE-MS method. After peak alignment, relative standard deviations (RSDs) for migration time of five representative compounds were below 0.4 %, whereas RSDs for peak area were 7.9–13.5 %. Using univariate and principal component analysis of obtained urinary metabolic profiles, groups of rats receiving different antibiotic treatment could be distinguished based on 17 discriminatory compounds, of which 15 were downregulated and 2 were upregulated upon treatment. Eleven compounds remained down- or upregulated after discontinuation of the antibiotics administration, whereas a recovery effect was observed for others. Based on accurate mass, nine compounds were putatively identified; these included the microbial-mammalian co-metabolites hippuric acid and indoxyl sulfate. Some discriminatory compounds were also observed by other analytical techniques, but CE-MS uniquely revealed ten metabolites modulated by antibiotic exposure, including aconitic acid and an oxocholic acid. This clearly demonstrates the added value of CE-MS for nontargeted profiling of small anionic metabolites in biological samples.

Messages about antibiotic resistance in different newspaper genres

Poorer people are more likely to use antibiotics; inappropriate antibiotic use causes resistance, and health campaigns attempt to change behaviour through education. However, fuelled by the media, the public think antibiotic resistance is outside their control. Differences in the attribution of blame for antibiotic resistance in two genres of UK newspapers, targeting distinct socioeconomic groups, were examined using a mixed methods approach. Firstly, depiction of blame was categorised as either external to the lay public (outside their control) or internal (lay person accountable) and subjected to a chi-square test. Secondly, using critical discourse analysis, we examined the portrayal of the main agents through newspaper language. Data from 597 articles (307 broadsheets) analysed revealed a significant association between newspaper genre and attribution of blame for antibiotic resistance. While both newspaper types blamed antibiotic resistance predominantly on factors external to the lay public, broadsheets were more likely to acknowledge internal factors than tabloids. Tabloids provided a more skewed representation, exposing readers to inaccurate explanations about antibiotic resistance. They highlighted ineptitude in health professionals, victimising patients and blaming others, while broadsheets used less emotive language. Pharmacists should take special care to communicate the importance of appropriate antibiotic use against this backdrop of distortion.

Hydrophilic interaction chromatography–mass spectrometry for anionic metabolic profiling of urine from antibiotic-treated rats

Hydrophilic interaction chromatography–mass spectrometry (HILIC–MS) was used for anionic metabolic profiling of urine from antibiotic-treated rats to study microbial–host co-metabolism. Rats were treated with the antibiotics penicillin G and streptomycin sulfate for four or eight days and compared to a control group. Urine samples were collected at day zero, four and eight, and analyzed by HILIC–MS. Multivariate data analysis was applied to the urinary metabolic profiles to identify biochemical variation between the treatment groups. Principal component analysis found a clear distinction between those animals receiving antibiotics and the control animals, with twenty-nine discriminatory compounds of which twenty were down-regulated and nine up-regulated upon treatment. In the treatment group receiving antibiotics for four days, a recovery effect was observed for seven compounds after cessation of antibiotic administration. Thirteen discriminatory compounds could be putatively identified based on their accurate mass, including aconitic acid, benzenediol sulfate, ferulic acid sulfate, hippuric acid, indoxyl sulfate, penicillin G, phenol and vanillin 4-sulfate. The rat urine samples had previously been analyzed by capillary electrophoresis (CE) with MS detection and proton nuclear magnetic resonance (1H NMR) spectroscopy. Using CE–MS and 1H NMR spectroscopy seventeen and twenty-five discriminatory compounds were found, respectively. Both hippuric acid and indoxyl sulfate were detected across all three platforms. Additionally, eight compounds were observed with both HILIC–MS and CE–MS. Overall, HILIC–MS appears to be highly complementary to CE–MS and 1H NMR spectroscopy, identifying additional compounds that discriminate the urine samples from antibiotic-treated and control rats.

Array based detection of antibiotic resistance genes in Gram negative bacteria isolated from retail poultry meat in the UK and Ireland

The use of antibiotics in birds and animals intended for human consumption within the European Union (EU) and elsewhere has been subject to regulation prohibiting the use of antimicrobials as growth promoters and the use of last resort antibiotics in an attempt to reduce the spread of multi-resistant Gram negative bacteria. Given the inexorable spread of antibiotic resistance there is an increasing need for improved monitoring of our food. Using selective media, Gram negative bacteria were isolated from retail chicken of UK-Intensively reared (n = 27), Irish-Intensively reared (n = 19) and UK-Free range (n = 30) origin and subjected to an oligonucleotide based array system for the detection of 47 clinically relevant antibiotic resistance genes (ARGs) and two integrase genes. High incidences of β-lactamase genes were noted in all sample types, acc (67%), cmy (80%), fox (55%) and tem (40%) while chloramphenicol resistant determinants were detected in bacteria from the UK poultry portions and were absent in bacteria from the Irish samples. Denaturing Gradient Gel Electrophoresis (DGGE) was used to qualitatively analyse the Gram negative population in the samples and showed the expected diversity based on band stabbing and DNA sequencing. The array system proved to be a quick method for the detection of antibiotic resistance gene (ARG) burden within a mixed Gram negative bacterial population.

Structural characterization of the interaction of the polyene antibiotic Amphotericin B with DODAB bicelles and vesicles

Amphotericin B (AmB) is widely used in the treatment of systemic fungal infections, despite its toxic effects. Nephrotoxicity, ascribed as the most serious toxic effect, has been related to the state of aggregation of the antibiotic. In search of the increase in AmB antifungal activity associated with low toxicity, several AmB-amphiphile formulations have been proposed. This work focuses on the structural characterization of a specific AmB formulation: AmB associated with sonicated dioctadecyl dimethylammonium bromide (DODAB) aggregates. Here, it was confirmed that sonicated DODAB dispersion is constituted by DODAB bicelles, and that monomeric AmB is much more soluble in bicelles than in DODAB vesicles. A new optical parameter is proposed for the estimation of the relative amount of amphiphile-bound monomeric AmB. With theoretical simulations of the spectra of spin labels incorporated in DODAB bicelles it was possible to prove that monomeric AmB binds preferentially to lipids located at the edges of DODAB bicelles, rigidifying them, and decreasing the polarity of the region. That special binding of monomeric AmB along the borders of bicelles, where the lipids are highly disorganized, could be used in the formulation of other carriers for the antibiotic, including mixtures of natural lipids which are known to form bicelles. (C) 2011 Elsevier B.V. All rights reserved.

Polymeric electro-mechanic devices applied to antibiotic-controlled release

Polymeric electroactive blends formed by electropolymerized aniline inside a non-conductive polyacrylamide porous matrix were already shown as suitable materials for the electrocontrolled release of model compounds like safranin. In this paper the intermolecular interactions between the two components of the blend are put in evidence by Raman spectroscopy measurements. Also, in situ optical microscopy was used to follow changes occurring in the polyaniline/polyacrylamide blend during pyrocathecol violet release tests. These two sets of experiments show the possibility of controlling electrochemically the release of both, safranin (a cation) and pyrocathecol violet (an anion) and allow to infer a release mechanism based on the electromechanical properties of the blends explaining the dependence of the release kinetics on the applied potential. Tetracycline release curves for different potentials and pHs are shown and the obtained profiles are in agreement with those expected for a device acting as an electrochemically driven pump due to the artificial muscle properties of the conducting phase of the blends. (c) 2007 Elsevier B.V. All rights reserved.

The Isolation and Characterization of Multiply Antibiotic Resistant Strains of Fish Pathogenic Flavobacterium Species

Since the development of the first antibiotics in the 1940’s, there has been widespread overuse in both clinical and agricultural applications. Antibiotic resistance has become a significant problem as a result of subsequent dissemination of antibiotics into the environment, and multiply-resistant strains of bacteria are now a major pathogenic threat. In this study eight separate strains of Flavobacterium responsible for recent disease outbreaks in fish hatcheries throughout Maine were collected and analyzed. All eight strains were found to be resistant to high levels of a number of different antibiotics, including those used for aquaculture as well as human chemotherapeutic applications. Flavobacterium isolates were also shown phenotypically to transfer antibiotic resistance determinants using a conjugation mating system in which Flavobacterium was the donor and Escherichia coli DH5- alpha was the recipient. This experiment suggests that it may be possible for Flavobacterium strains to transfer their multiple antibiotic resistance determinants to human pathogenic bacterial strains. Importantly, none of the hatcheries from which the Flavobacterium isolates were obtained had ever used antibiotics to treat their fish stock. It is possible that there is another selective agent responsible for the development of antibiotic resistance in the absence of antibiotic pressure. Mercury is one possible candidate, as all of the strains tested were resistant to mercuric chloride and it is known that genes encoding antibiotic resistance can be carried on the same mobile genetic elements that encode for mercury resistance. Preliminary data also suggest that the majority of the Flavobacterium isolates contain genes for mercuric ion reduction, which would confirm the mercury resistance genotype.

Salmoninae Commensal Bacteria Show a Positive Correlation Between Antibiotic Resistance and Mercury Resistance Profiles

Bacterial isolates from natural sites with high toxic and heavy metal contamination more frequently contain determinants for resistance to antimicrobials. Natural strains were isolated from the ingesta and external slime of Salmo salar (Linnaeus, 1758) and Salvelinusjontinalis (Mitchell, 1814). Fish specimens were acquired from Casco Bay hatcheries, Casco, ME where there is no history of antibiotic use. Seventy-nine bacterial strains, including many well-documented salmonid commensals (an association from which the fish derives no benefit), were identified using 165 rRNA gene sequencing. Mercury resistant isolates were selected for initially on 25μM HgCI2. Strains were then grown at 20-24°C on Trypticase Soy Agar (TSA) plates containing 0-1000μM HgCl2 or 0-130μM Phenyl Mercuric Acetate (PMA). Mercury in the hatchery feed water due to ubiquitous non-point source deposition has selected for the mercury resistance observed in bacterial strains. Antibiotic resistance determinations, as measured by Minimum Inhibitory Concentration MIC) assays were performed on the 79 bacterial isolates using Sensititrel antimicrobial susceptibility panels. A positive linear correlation between the mercury (pMA and HgCl2) MIC's and antibiotic resistance for all observed strains was demonstrated. Conjugation experiments with Pseudomonas, Aeromonas, and Azomonas donors confirmed phenotypic transfer of penicillin and cephem resistances to Escherichia coli DH5a recipients. Conjugation experiments with Pseudomonas donors showed minimal transfer of tetracycline and minoglycoside resistances to Escherichia coli DH5a recipients. Our study suggests that the accumulation of antimicrobial resistances observed in these natural bacterial populations may be due to the indirect selective pressure exerted by environmental mercury.

Bottled mineral water as a potential source of antibiotic resistant bacteria

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Antibiotic treatment schemes for very severe community-acquired pneumonia in children: a randomized clinical study

Objective. To compare clinical response to initial empiric treatment with oxacillin plus ceftriaxone and amoxicillin plus clavulanic acid in hospitalized children diagnosed with very severe community-acquired pneumonia (CAP).Methods. A prospective randomized clinical study was conducted among children 2 months to 5 years old with a diagnosis of very severe CAP in the pediatric ward of São Paulo State University Hospital in Botucatu, São Paulo, Brazil, from April 2007 to May 2008. Patients were randomly divided into two groups by type of treatment: an oxacillin/ceftriaxone group (OCG, n = 48) and an amoxicillin/clavulanic acid group (ACG, n = 56). Analyzed outcomes were: time to clinical improvement (fever and tachypnea), time on oxygen therapy, length of stay in hospital, need to widen antimicrobial spectrum, and complications (including pleural effusion).Results. The two groups did not differ statistically for age, sex, symptom duration before admission, or previous antibiotic treatment. Time to improve tachypnea was less among ACG patients than OCG patients (4.8 +/- 2.2 versus 5.8 +/- 2.4 days respectively; P = 0.028), as was length of hospital stay (11.0 +/- 6.2 versus 14.4 +/- 4.5 days respectively; P = 0.002). There were no statistically significant differences between the two groups for fever improvement time, time on oxygen therapy, need to widen antimicrobial spectrum, or frequency of pleural effusion.Conclusions. Both treatment plans are effective in treating very severe CAP in 2-monthto 5-year-old hospitalized children. The only analyzed outcome that favored amoxicillin/clavulanic acid treatment was time required to improve tachypnea.

The controlled release of antibiotic by hydroxyapatite: Anionic collagen composites

Major problems with the treatment of osteomyelitis are associated with poor antibiotic distribution at the site of infection due to limited blood circulation to the skeletal tissue. Improved treatment procedures have been used in drug delivery systems that include bioceramics and natural and synthetic polymers. This work reports the development of anionic collagen:hydroxyapatite composite paste for sustained antibiotic release. Antibiotic release by the composite was characterized by two steps. In the first, 15.0 +/- 4.9% was released in the first 5 h (n = 53) by a normal Fick diffusion mechanism. In the second step, only 16.8 +/- 2.2% was released after 7 days. In conclusion, hydroxyapatite:anionic collagen composite can be an efficient support for sustained antibiotic release in the treatment of osteomyelitis because most of the antibiotic release may be associated with composite bioresorption, thus permitting antibiotic release throughout the healing process. Hydroxyapatite:anionic collagen paste showed good biocompatibility associated with bone tissue growth with material still being observed after 60 days from the time of implants.

Evaluation of chitosan gel as antibiotic and photosensitizer delivery

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)