Teresa Vinuesa

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.

Clinical and antimicrobial evaluation of a mouthwash and toothpaste for xerostomia: a randomized, double-blind, crossover study.

OBJECTIVES Specific toothpaste and mouthwash for xerostomia based on triclosan, fluoride, and mineral salts were studied in order to evaluate their efficacy in improving the quality of life as well as their in vitro antimicrobial action. MATERIALS AND METHODS 30 patients with dry mouth and hyposalivation confirmed by sialometry were included in a randomized, double-blind, crossover study, during two weeks for each experimental phase. Dryness and oral comfort, difficulty in speaking, chewing, swallowing and using dental prostheses, as well as taste and sleep disorders were evaluated. Values of minimal biocidal and minimal inhibitory concentrations of both products against microorganisms that often cause oral disorders in these patients were determined and compared. RESULTS The use of the mouthwash and toothpaste studied improved the dry mouth symptoms, whilst no significant differences when compared with the salivary enzyme substitutes were observed. The test products showed, in general, a higher biocidal and inhibitory action than the control products, with particularly noteworthy action against cariogenic bacteria. CONCLUSIONS The use of a toothpaste and mouthwash based on triclosan, fluoride, gingival revitalizers and mineral salts, improves the quality of life of patients suffering xerostomia.

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.

Characterization of the antimicrobial susceptibility of fungi responsible for onychomycosis in Spain

Due to the increase of choices relative to antifungals, there is a need to improve the standardization of in vitro methods used to determine the antifungal susceptibility of fungal pathogens. Our study evaluated the in vitro susceptibility of filamentous fungi isolated from patients with toenail onychomycosis against itraconazole, ciclopirox, eberconazole, fluconazole and terbinafine. The minimum inhibitory concentration (MIC) of these antifungal agents was determined with 100 isolates, including dermatophytes (70 strains) and non-dermatophyte molds (30 strains). The susceptibility of fungal isolates was measured by using a technique modified for dermatophytes (0.5 × 10(3)-0.5 × 10(4) conidia/ml as inocula) which followed the procedures described by the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST) and the Clinical and Laboratory Standard Institute (CLSI M38-A). MIC ranges were 0.016-8.0 μg/ml for itraconazole, ciclopirox and eberconazole, 0.063-32.0 μg/ml for fluonazole, and 0.004-2.0 μg/ml for terbinafine. In vitro susceptibility tests indicated that eberconazole has a broad antimicrobial profile, including dermatophytes, as well as other filamentous fungi. Terbinafine was active (0.016-0.250 μg/ml) against dermatophytes.

Oral Microbiota, Periodontal Status, and Osteoporosis in Postmenopausal Females.

BACKGROUND Alterations in hormone levels during menopause decrease bone density and may worsen oral health, favoring the growth of periodontal pathogens, whose detection could improve the diagnosis of periodontitis. The aim of this study is to detect and quantify the main periodontal pathogens in the oral microbiota of postmenopausal females and to explore the relationship between clinical and periodontal parameters. METHODS This was an observational cross-sectional study of 76 postmenopausal females. Dental examinations and sampling for microbiologic evaluation were performed, and a history of osteoporosis/osteopenia was collected. Real-time polymerase chain reaction was used for detecting and quantifying Fusobacterium nucleatum (Fn), Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Campylobacter rectus (Cr), and Tannerella forsythia (Tf). The results obtained were subjected to statistical analyses. Statistical significance was defined as P <0.05. RESULTS Periodontitis was detected in 77.1% of females with osteoporosis/osteopenia (P >0.05). A significant correlation was found between osteoporosis and missing teeth. T. forsythia and C. rectus were detected in 100% of the samples, Fn and Pg in 98.7%, and Aa in 73.7%. CONCLUSIONS Osteoporosis did not influence the prevalence of periodontitis among postmenopausal females. The presence of periodontopathogenic bacteria was not sufficient to confirm disease. A preventive maintenance program for postmenopausal females, particularly osteoporotic females, who are at greater risk of tooth loss, could minimize the potential effects of bone loss on periodontal tissues.

Atomic force microscopy visualization of injuries in Enterococcus faecalis surface caused by Er,Cr:YSGG and diode lasers

Aim: To visualize by Atomic Force Microscopy the alterations induced on Enterococcus. faecalis surface after treatment with 2 types of laser: Erbium chromium:yttrium-scandium-gallium-garnet (Er,Cr:YSGG) laser and Diode laser. Material and Methods: Bacterial suspensions from overnight cultures of E. faecalis were irradiated during 30 seconds with the laser-lights at 1 W and 2 W of power, leaving one untreated sample as control. Surface alterations on treated E. faecalis were visualized by Atomic Force Microscopy (AFM) and its surface roughness determined. Results: AFM imaging showed that at high potency of laser both cell morphology and surface roughness resulted altered, and that several cell lysis signs were easily visualized. Surface roughness clearly increase after the treatment with Er,Cr:YSGG at 2W of power, while the other treatments gave similar values of surface roughness. The effect of lasers on bacterial surfaces visualized by AFM revealed drastic alterations. Conclusions: AFM is a good tool to evaluate surface injuries after laser treatment; and could constitute a measure of antimicrobial effect that can complete data obtained by determination of microbial viability. Key words:Atomic force microscopy, Er,Cr:YSGG laser, diode laser, Enterococcus faecalis, surface roughness.