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.

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.

Abstract: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.

The aims of this study were to ascertain the presence and spread of class 1 integrons among 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 among 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 among P. aeruginosa strains.

Objective Despite its use was abandoned several decades ago, the polycationic peptide colistin has become the last hope to treat severe infections caused by multidrug-resistant Gram-negative bacteria. Thus, the development of colistin resistance may seriously compromise the efficacy of treatment. Moreover, colistin has high toxicity being dose dependent. A potentially effective strategy to avoid resistance may be to combine colistin with other antimicrobials. This may help in the rescue of old antimicrobials and in reducing toxic undesired effects. Methods Antimicrobial susceptibility determination, efflux machinery function measurements in different conditions and measurement of inhibition of the extrusion by colistin were performed. Moreover, modifications of anisotropy of the membranes by using fluorescent dyes was accomplished. Results Sub-inhibitory concentrations of colistin have a synergistic effect with several antimicrobials that act intracellularly (targeting protein synthesis and DNA replication). This effect was demonstrated through the uptake increases of acridine orange. in Pseudomonas aeruginosa, Escherichia coli and Acinetobacter baumanii but also in an intrinsically colistin-resistant species as Serratia marcescens . Measurements of the anisotropy of bacterial membranes, as a measure of membrane fluidity, showed significant changes indicative of colistin activity. Conclusion The alterations in the cellular efflux machinery that resulted in higher intracellular concentrations of acridine orange, and likely of other antimicrobials combined with data of membrane fluidity and measured synergism in vitro allow us to envisage the use of these combinations to fight infections caused by multidrug–resistant bacteria.

Microbial biofilms are involved in almost all infectious pathologies of the oral cavity. This has led to the search for novel therapies specifically aimed at biofilm elimination. In this study, we used atomic force microscopy (AFM) to visualize injuries and to determine surface roughness, as well as confocal laser scanning microscopy (CLSM) to enumerate live and dead bacterial cells, to determine the effects of photodynamic therapy (PDT) on Enterococcus faecalis biofilms. The AFM images showed that PDT consisting of methylene blue and a 670-nm diode laser (output power 280 mW during 30 s) or toluidine blue and a 628-nm LED light (output power 1000 mW during 30 s) induced severe damage, including cell lysis, to E. faecalis biofilms, with the former also causing an important increase in surface roughness. These observations were confirmed by the increase in dead cells determined using CLSM. Our results highlight the potential of PDT as a promising method to achieve successful oral disinfection.

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-bnaphthylamide (PAbN) 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.

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.