Ester Fusté

Class 1 integrons in environmental and clinical isolates of Pseudomonas aeruginosa

The aims of this study were to ascertain the presence and spread of class 1 integrons amongst environmental and clinical isolates of Pseudomonas aeruginosa and to characterise their variable regions. A total of 76 isolates (56 clinical and 20 environmental) were studied. The presence of plasmids was explored, and polymerase chain reaction (PCR) was used for integron detection. All amplicons were sequenced. PCR detected class 1 integrons in 26 of the 56 clinical isolates; environmental isolates were integron-free. No plasmids were found, thus all the integrons found are possibly on the chromosome. Most isolates presented one amplicon, except PA110514 and PA116136, which showed two PCR products each. Variable regions revealed that 18 strains carried only one gene involved in aminoglycoside resistance, whereas in 3 strains gene cassettes were not found. The most prevalent cassettes amongst isolates were those encoding aminoglycoside adenyltransferase B (aadB). Several of the strains had acquired the same or a highly similar cassette array as those detected in geographically distant P. aeruginosa. This finding suggests that contact with bacterial reservoirs contributes to the evolution of this pathogen towards multiresistance. Empty structures found may represent a reservoir increasing the capacity to adapt to the environment. However, these integrons are not retained when the selective pressure disappears. It is hypothesised that integrons containing gene cassettes are crucial vehicles for the rapid horizontal transfer of resistance. If this is so, reduced use of antibiotics may lead to a significant decrease in the carriage of integrons amongst P. aeruginosa strains.

An overview of antimicrobial peptides and the latest advances in their development

ABSTRACT Introduction: The recent dramatic increase in the incidence of antimicrobial resistance has been recognized by organizations such as the United Nations and World Health Organization as well as the governments of the USA and several European countries. A relatively new weapon in the fight against severe infections caused by multi-drug resistant bacteria is antimicrobial peptides (AMPs). These include colistin, currently regarded as the last line of antimicrobial therapy against multi-drug resistant microorganisms. Areas covered: Here, the authors provide an overview of the current research on AMPs. The focus is AMPs currently being developed for the treatment of recalcitrant bacterial infections, the synergies of AMPs and antibiotics, and the activity of AMPs against biofilm. This review also includes a brief introduction into the use of AMPs in infections caused by Mycobacterium, fungi, and parasites. Expert opinion: In research into new antimicrobials, AMPs are gaining increasing attention. While many are natural and are produced by a wide variety of organisms, others are being newly designed and chemically synthesized in the laboratory to achieve novel antimicrobial agents. The same strategy to fight infections in nature is thus being effectively exploited to safeguard human and animal health.

Killing effect of nanoencapsulated colistin sulfate on Pseudomonas aeruginosa from cystic fibrosis patients

Pseudomonas aeruginosa frequently infects the respiratory tract of cystic fibrosis (CF) patients. Multidrug-resistant phenotypes and high capacity to form stable biofilms are common. Recent studies have described the emergence of colistin-resistant isolates in CF patients treated with long-term inhaled colistin. The use of nanoparticles containing antimicrobials can contribute to overcome drug resistance mechanisms. The aim of this study was to explore antimicrobial activity of nanoencapsulated colistin (SLN-NLC) versus free colistin against P. aeruginosa clinical isolates from CF patients and to investigate their efficacy in biofilm eradication. Susceptibility of planktonic bacteria to antimicrobials was examined by using the broth microdilution method and growth curve assay. Minimal biofilm eradication concentration (MBEC) and biofilm prevention concentration (BPC) were determined to assess antimicrobial susceptibility of sessile bacteria. We used atomic force microscopy (AFM) to visualize treated and untreated biofilms and to determine surface roughness and other relevant parameters. Colistin nanoparticles had the same antimicrobial activity as free drug against planktonic bacteria. However, nanoencapsulated colistin was much more efficient in the eradication of biofilms than free colistin. Thus, these formulations have to be considered as a good alternative therapeutic option to treat P. aeruginosa infections.

Carbapenem-resistance mechanisms of multidrug-resistant Pseudomonas aeruginosa

Clonal dissemination of multidrug-resistant Pseudomonas aeruginosa (MDRPA) is a major concern worldwide. The aim of this study was to explore the mechanisms leading to the carbapenem resistance of an MDRPA clone. Isolates were obtained from a surgical wound, sputum, urine and a blood culture. Pulsed-field gel electrophoresis (PFGE) showed high genomic homogeneity of these isolates and confirmed the circulation of an endemic clone belonging to serotype O4. Outer membrane protein (OMP) bands were visualized by SDS-PAGE, meropenem accumulation was measured in a bioassay and integrons were detected by PCR. Efflux pumps were studied for several antimicrobial agents and synergic combinations thereof in the presence or absence of both carbonyl cyanide m-chlorophenylhydrazone (CCCP) and Phe-Arg-β-naphthylamide (PAβN) at final concentrations of 10 and 40 mg l(-1), respectively. On OMP electrophoretic profiles, MDRPA showed a reduction of outer membrane porin D (OprD) and PCR demonstrated the presence of a class 1 integron with a cassette encoding aminoglycoside adenyltransferase B (aadB). Meropenem accumulation was slightly higher in bacilli than in the filamentous cells that formed in the presence of antibiotics. Overexpression of the efflux pump MexAB-OprM and a functional MexXY-OprM were detected in all isolates.

Role of TolC in Klebsiella oxytoca resistance to antibiotics

OBJECTIVES The Gram-negative human pathogen Klebsiella oxytoca is often resistant to several antibiotics such as fluoroquinolones, erythromycin, tetracycline, chloramphenicol and others. The aim of this study was to look at the mechanisms leading to this resistance and particularly the role of TolC and efflux mechanisms in determining resistance. METHODS Ciprofloxacin accumulation was measured spectrofluorometrically. Growth inhibition assays were performed in the presence or absence of carbonyl cyanide m-chlorophenylhydrazone (10 mg/L, final concentration). The genome of K. oxytoca was analysed for the existence of loci encoding tolC by PCR using primers for the Enterobacter aerogenes tolC gene and subsequently sequenced. A plasmid named pUC18TolC was constructed and inserted into Escherichia coli C600tolC,Tn5, and the function of TolC was analysed. The structure modelling was performed using the Modeller program. RESULTS The existence of the AcrAB efflux mechanism was demonstrated in the species, and a TolC-like protein, a channel-forming protein at the external membrane that allows the extrusion of antibiotics by the AcrAB efflux pump, was cloned, sequenced and a model proposed. CONCLUSIONS K. oxytoca express a functional TolC that lacks a fragment of six amino acids characteristic of the external loops of TolC in E. coli. This makes this species resistant to a few colicins.

Synergistic Antipseudomonal Effects of Synthetic Peptide AMP38 and Carbapenems.

The aim was to explore the antimicrobial activity of a synthetic peptide (AMP38) and its synergy with imipenem against imipenem-resistant Pseudomonas aeruginosa. The main mechanism of imipenem resistance is the loss or alteration of protein OprD. Time-kill and minimal biofilm eradication concentration (MBEC) determinations were carried out by using clinical imipenem-resistant strains. AMP38 was markedly synergistic with imipenem when determined in imipenem-resistant P. aeruginosa. MBEC obtained for the combination of AMP38 and imipenem was of 62.5 μg/mL, whereas the MBEC of each antimicrobial separately was 500 μg/mL. AMP38 should be regarded as a promising antimicrobial to fight MDR P. aeruginosa infections. Moreover, killing effect and antibiofilm activity of AMP38 plus imipenem was much higher than that of colistin plus imipenem.

Synergism between Outer Membrane Proteins and Antimicrobials

ABSTRACT Antibiotic-resistant bacteria are becoming one of the most important problems in health care because of the number of resistant strains and the paucity of new effective antimicrobials. Since antibiotic-resistant bacteria will continue to increase, it is necessary to look for new alternative strategies to fight against them. It is generally accepted that Gram-negative bacteria are intrinsically less susceptible than Gram-positive bacteria to antimicrobials. The main reason is that Gram-negative bacteria are surrounded by a permeability barrier known as the outer membrane (OM). Hydrophilic solutes most often cross the OM through water-filled channels formed by a particular family of proteins known as porins. This work explores the possibility of using exogenous porins to lower the required amounts of antibiotics (ampicillin, ciprofloxacin, cefotaxime, clindamycin, erythromycin, and tetracycline). Porins had a bactericidal effect on Escherichia coli cultures, mainly in the logarithmic phase of growth, when combined with low antibiotic concentrations. The use of different antibiotic-porin mixtures showed a bactericidal effect greater than those of antibiotics and porins when used separately. It was possible to observe different behaviors according to the antibiotic type used.

Free and Nanoencapsulated Tobramycin: Effects on Planktonic and Biofilm Forms of Pseudomonas

Cystic fibrosis (CF) is a genetic disorder in which frequent pulmonary infections develop secondarily. One of the major pulmonary pathogens colonizing the respiratory tract of CF patients and causing chronic airway infections is Pseudomonas aeruginosa. Although tobramycin was initially effective against P. aeruginosa, tobramycin-resistant strains have emerged. Among the strategies for overcoming resistance to tobramycin and other antibiotics is encapsulation of the drugs in nanoparticles. In this study, we explored the antimicrobial activity of nanoencapsulated tobramycin, both in solid lipid nanoparticles (SLN) and in nanostructured lipid carriers (NLC), against clinical isolates of P. aeruginosa obtained from CF patients. We also investigated the efficacy of these formulations in biofilm eradication. In both experiments, the activities of SLN and NLC were compared with that of free tobramycin. The susceptibility of planktonic bacteria was determined using the broth microdilution method and by plotting bacterial growth. The minimal biofilm eradication concentration (MBEC) was determined to assess the efficacy of the different tobramycin formulations against biofilms. The activity of tobramycin-loaded SLN was less than that of either tobramycin-loaded NLC or free tobramycin. The minimum inhibitory concentration (MIC) and MBEC of nanoencapsulated tobramycin were slightly lower (1–2 logs) than the corresponding values of the free drug when determined in tobramycin-susceptible isolates. However, in tobramycin-resistant strains, the MIC and MBEC did not differ between either encapsulated form and free tobramycin. Our results demonstrate the efficacy of nanoencapsulated formulations in killing susceptible P. aeruginosa from CF and from other patients.

New and old tools to evaluate new antimicrobial peptides

The emergence of antimicrobial resistance due to the overuse of antimicrobials together with the existence of naturally untreatable infections well demonstrates the need for new instruments to fight microbes. Antimicrobial peptides (AMPs) are a promising family of molecules in this regard, because they abundantly occur in nature and the results of preliminary studies of their clinical potential have been encouraging. However, further progress will benefit from the standardization of research methods to assess the antimicrobial properties of AMPs. Here we review the diverse methods used to study the antimicrobial power of AMPs and recommend a pathway to explore new molecules. The use of new methodologies to quantitatively evaluate the physical effect on bacterial biofilms such as force spectroscopy and surface cell damage evaluation, constitute novel approaches to study new AMPs.

Identification and characterization of a cell wall porin from Gordonia jacobaea

Gordonia jacobaea is a bacterium belonging to the mycolata group characterized by its ability to produce carotenoids. Mycolic acids in the cell wall contribute to reducing the permeability of their envelopes requiring the presence of channel-forming proteins to allow the exchange of hydrophilic molecules with the surrounding medium. Identification and purification of the channel-forming proteins was accomplished by SDS-PAGE, Mass spectrometry and Mass peptide fingerprinting and the channel-forming activity was studied by reconstitution in lipid bilayers. Here, we describe for the first time the presence of a cell-wall protein from G. jacobaea with channel-forming activity. Our results suggest that this protein bears a low similarity to other hypothetical proteins from the genus Gordonia of uncharacterized functions. The channel has an average single-channel conductance of 800 pS in 1 M KCl, is moderately anion-selective, and does not show any voltage dependence for voltages between +100 and -100 mV. The channel characteristics suggest that this protein could be of relevance in the import and export of negatively charged molecules across the cell wall. This could contribute to design treatments for mycobacterial infections, as well as being of interest in biotechnology applications.