Veronica Lazăr

Hybrid magnetite nanoparticles/Rosmarinus officinalis essential oil nanobiosystem with antibiofilm activity

Biofilms formed by fungal organisms are associated with drastically enhanced resistance against most antimicrobial agents, contributing to the persistence of the fungi despite antifungal therapy. The purpose of this study is to combine the unique properties of nanoparticles with the antimicrobial activity of the Rosmarinus officinalis essential oil in order to obtain a nanobiosystem that could be pelliculised on the surface of catheter pieces, in order to obtain an improved resistance to microbial colonization and biofilm development by Candida albicans and C. tropicalis clinical strains. The R. officinalis essential oils were extracted in a Neo-Clevenger type apparatus, and its chemical composition was settled by GC-MS analysis. Functionalized magnetite nanoparticles of up to 20 nm size had been synthesized by precipitation method adapted for microwave conditions, with oleic acid as surfactant. The catheter pieces were coated with suspended core/shell nanoparticles (Fe3O4/oleic acid:CHCl3), by applying a magnetic field on nanofluid, while the CHCl3 diluted essential oil was applied by adsorption in a secondary covering treatment. The fungal adherence ability was investigated in six multiwell plates, in which there have been placed catheters pieces with and without hybrid nanoparticles/essential oil nanobiosystem pellicle, by using culture-based methods and confocal laser scanning microscopy (CLSM). The R. officinalis essential oil coated nanoparticles strongly inhibited the adherence ability and biofilm development of the C. albicans and C. tropicalis tested strains to the catheter surface, as shown by viable cell counts and CLSM examination. Due to the important implications of Candida spp. in human pathogenesis, especially in prosthetic devices related infections and the emergence of antifungal tolerance/resistance, using the new core/shell/coated shell based on essential oil of R. officinalis to inhibit the fungal adherence could be of a great interest for the biomedical field, opening new directions for the design of film-coated surfaces with antibiofilm properties.

Efficient transmission of IncFIIY and IncL plasmids and Klebsiella pneumoniae ST101 clone producing OXA-48, NDM-1 or OXA-181 in Bucharest hospitals

Carbapenemase-producing Enterobacteriaceae are increasingly reported, with NDM-1 (class B) and OXA-48 (class D) being the most widespread enzymes worldwide [1]. In Romania, a country with a high rate of carbapenemase-producing enterobacterial isolates (http://ecdc.europa.eu/en/healthtopics/antimicrobial_resistance/ database/Pages/database.aspx), molecular epidemiological data are scarce and are limited to certain regions of the country [2]. We aimed to elucidate the epidemiology and to provide detailedmolecular data (genetic platforms, plasmid types and clones) for carbapenemaseproducing Enterobacteriaceae from two hospitals in Bucharest, Romania. A total of 21 carbapenemase-producing Enterobacteriaceae isolates (14 Klebsiella pneumoniae, 6 Enterobacter cloacae complex and 1 Escherichia coli) recovered from patients hospitalised in different units of two central hospitals between March and December 2012 were characterised (Table 1). They corresponded to all carbapenemase-producing isolates identified among multidrug-resistant Enterobacteriaceae resistant to ertapenem [according to Clinical and Laboratory Standards Institute (CLSI) 2012 guidelines]. Identification of isolates and antibiotic susceptibility testing were performed by automated (VITEK II, MicroScan) and standard methods (hsp60 genotyping for E. cloacae, disk diffusion) (http:// www.eucast.org). Carbapenemase production and identification of bla genes (blaNDM, blaVIM, blaIMP, blaKPC, blaOXA-48-like, blaCTX-M-1 group) were performed as described previously [3] and the genetic context of blaNDM-1, blaOXA-48 and blaOXA-181 was determined by PCR and sequencing (Supplementary Table S1). The relationship among the isolates was established by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) for E. coli (http:// mlst.warwick.ac.uk/mlst/) as well as for representative K. pneumoniae (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html) and Enterobacter hormaechei (http://pubmlst.org/ecloacae) strains. Conjugation experiments were performed on isolates representing variable clones or plasmids by broth/solid mating as described previously [3]. Plasmid content was characterised by PCR-based replicon typing (PBRT) and replicon sequence typing (RST) (http://pubmlst.org/ plasmid/), S1 nuclease PFGE and hybridisation (blaNDM-1, blaOXA-48, blaOXA-181, blaCTX-M-15, rep probes) as reported previously [3]. Conserved regions of the prototype pOXA-48 plasmid (repA, traU, parA; GenBank accession no. JN626286) were characterised by PCR and sequencing for representative OXA-48and OXA-181-producing strains, and relatedness among OXA-48-encoding plasmids was evaluated by restriction fragment length polymorphism (RFLP) using EcoRI as restriction enzyme [4]. Eight isolates of different Enterobacteriaceae produced NDM-1 (six E. hormaechei, one K. pneumoniae and one E. coli), eleven K. pneumoniae produced OXA-48 and two K. pneumoniae produced both NDM-1 and OXA-181 (differing from OXA-48 in four amino acid changes, whilst the corresponding bla genes are 94% homologous), and most of them encoded also CTX-M-15 (Table 1). These data, together with previous reports, support that the Balkan region might be an important reservoir of these carbapenemases [2]. NDM-1-producers were assigned to the worldwide spread ST74 or ST171 E. hormaechei (http://pubmlst.org/ecloacae) or ST101 K. pneumoniae clones and the apparently sporadic ST5 E. coli clone (http://mlst.warwick.ac.uk/mlst/). The blaNDM-1 gene in all bacterial species was located within conjugative 100 kb IncFIIY plasmids (Y4:A-:B42) containing a genetic platform (ISCR14/6–rmtC– ΔISAba125–blaNDM-1–bleMBL–trpF–groEL) identical to those identified in different Enterobacteriaceae species (K. pneumoniae, K. oxytoca, E. cloacae), suggesting efficient transmission in the nosocomial setting [5]. When detected, blaCTX-M-15 was located within IncR plasmids. Isolates producing NDM-1 and OXA-181 also belonged to identical ST101 K. pneumoniae. The blaNDM-1 gene was chromosomally located embedded in an identical genetic platform to that described above (ISCR14/6–rmtC–ΔISAba125–blaNDM-1–bleMBL–trpF–groEL), suggesting a common origin. ISEcp1 was found upstream of the blaOXA-181 gene and also upstream of blaCTX-M-15 in an IncL plasmid carrying repA, traU and parA genes identical to those from pOXA48-like plasmids [4]. Thus, we report for the first time the acquisition of ISEcp1–blaOXA-181 by a pOXA-48-like IncL plasmid, instead of previously described locations (7.6 kb ColE, GenBank accession no. JN205800; 84 kb IncT, GenBank accession no. JQ996150; 60 kb IncN, GenBank accession no. KM660724). FIIK1 and FII2 were also detected in these isolates. Most OXA-48-producing isolates belonged to K. pneumoniae ST101 and sporadically to ST395 K. pneumoniae, both sequence types commonly linked to the intercontinental dissemination of blaOXA-48 and previously described in Romania [1,2].We observed that in the region analysed, blaOXA-48 was located in a Tn1999.2 transposon variant (upstream IS1999 sequence disrupted by IS1) within closely related 50– 60 kb pOXA-48-like conjugative IncL plasmids, which constitute the most common vehicles for blaOXA-48 (Tn1999.2, IncL) described worldwide [1,4]. These isolates simultaneously carried IncR (where blaCTX-M-15 was located) and IncFIIK2 replicons (Table 1). The association of ST101 K. pneumoniaewith different carbapenemases suggests extended dissemination of this clone in Bucharest and elsewhere in Romania [2], and the differences in the plasmidome of isolates producing NDM-1 (IncFIIY, IncR), NDM-1 and OXA-181 (IncL, IncFIIK1, IncFII2) or OXA-48 (IncL, IncFIIK2, IncR) enzymes (Table 1) highlight its ability to acquire variable genetic platforms. This study provides a detailed characterisation of carbapenemaseproducing Enterobacteriaceae from Bucharest (Romania) at the molecular level (genetic platforms, plasmids, clones) and additional evidence that the Balkan region constitutes an important reservoir

Development and Sequential Analysis of a New Multi-Agent, Anti-Acne Formulation Based on Plant-Derived Antimicrobial and Anti-Inflammatory Compounds.

The antibacterial and anti-inflammatory potential of natural, plant-derived compounds has been reported in many studies. Emerging evidence indicates that plant-derived essential oils and/or their major compounds may represent a plausible alternative treatment for acne, a prevalent skin disorder in both adolescent and adult populations. Therefore, the purpose of this study was to develop and subsequently analyze the antimicrobial activity of a new multi-agent, synergic formulation based on plant-derived antimicrobial compounds (i.e., eugenol, β-pinene, eucalyptol, and limonene) and anti-inflammatory agents for potential use in the topical treatment of acne and other skin infections. The optimal antimicrobial combinations selected in this study were eugenol/β-pinene/salicylic acid and eugenol/β-pinene/2-phenoxyethanol/potassium sorbate. The possible mechanisms of action revealed by flow cytometry were cellular permeabilization and inhibition of efflux pumps activity induced by concentrations corresponding to sub-minimal inhibitory (sub-MIC) values. The most active antimicrobial combination represented by salycilic acid/eugenol/β-pinene/2-phenoxyethanol/potassium sorbate was included in a cream base, which demonstrated thermodynamic stability and optimum microbiological characteristics.

Antibiotic Resistance and Virulence Phenotypes of Recent Bacterial Strains Isolated from Urinary Tract Infections in Elderly Patients with Prostatic Disease

Acute bacterial prostatitis is one of the frequent complications of urinary tract infection (UTI). From the approximately 10% of men having prostatitis, 7% experience a bacterial prostatitis. The purpose of this study was to investigate the prevalence of uropathogens associated with UTIs in older patients with benign prostatic hyperplasia and to assess their susceptibility to commonly prescribed antibiotics as well as the relationships between microbial virulence and resistance features. Uropathogenic Escherichia coli was found to be the most frequent bacterial strain isolated from patients with benign prostatic hyperplasia, followed by Enterococcus spp., Enterobacter spp., Klebsiella spp., Proteus spp., Pseudomonas aeruginosa, and Serratia marcescens. Increased resistance rates to tetracyclines, quinolones, and sulfonamides were registered. Besides their resistance profiles, the uropathogenic isolates produced various virulence factors with possible implications in the pathogenesis process. The great majority of the uropathogenic isolates revealed a high capacity to adhere to HEp-2 cell monolayer in vitro, mostly exhibiting a localized adherence pattern. Differences in the repertoire of soluble virulence factors that can affect bacterial growth and persistence within the urinary tract were detected. The Gram-negative strains produced pore-forming toxins—such as hemolysins, lecithinases, and lipases—proteases, siderophore-like molecules resulted from the esculin hydrolysis and amylases, while Enterococcus sp. strains were positive only for caseinase and esculin hydrolase. Our study demonstrates that necessity of investigating the etiology and local resistance patterns of uropathogenic organisms, which is crucial for determining appropriate empirical antibiotic treatment in elderly patients with UTI, while establishing correlations between resistance and virulence profiles could provide valuable input about the clinical evolution and recurrence rates of UTI.

Antimicrobial and cytotoxic activity of graphene-based perioceuticals

Abstract Globally, periodontitis is one of the most common chronic bacterial infections in the adult population. It is estimated that about 50% of those affected have gingivitis (inflammation and bleeding gums with swelling, reddening, exudate, and change to the normal insertion contour). Current therapeutic strategies are costly, selective, and limited in time. Different approaches have been employed for controlling oral biofilm including prevention of its formation and destruction of the cell biofilm. However, its control requires bacteriostatic agents and antibiofilm strategies for inhibition of initial adherence. In the context of the increased rates of antibiotic resistance, the discovery of active agents against bacteria embedded in biofilm represents an important control strategy for chronic oral diseases; natural products derived from medicinal plants have been proven to be an abundant source of pharmacologically active compounds. Volatile oils, which are products of secondary plant metabolism, are valuable resources for pharmacognostic studies and the development of new therapeutic antiinfectives. This study investigated the application of graphene as a transport and release system for clove oil.

11 – Fate of antibiotics during water treatment: impact on antimicrobial resistance in environmental and clinical strains

Wastewater treatment plants (WWTPs) provide appropriate conditions for the selection of antibiotic resistance (AR), as well as for the exchange of AR genes between pathogenic and environmental bacteria, and further dissemination among members of the endogenous microbial community, due to the presence of antibiotics, high organic content, as well as high microbial density and diversity. The purpose of this chapter is to highlight some aspects regarding the origin, transfer, and accumulation of antibiotic resistance genes in the WWTPs aquatic environment.

18 – Microbial biofilms from the aquatic ecosystems and water quality

In nature, bacteria generally exists in the biofilm state, consisting cells attached to each other and also to a surface, embedded in a protective matrix. The aquatic biofilms are complex and dynamic communities developed on all exposed interfaces of the respective ecosystems, with a considerable microbial biodiversity, composed of different species of bacteria, diatoms, and protozoa. Their structure is dependent on the species composition of the respective area microbiota, their microbial metabolism, and environmental conditions. Drinking water distribution systems are continuously exposed to a variable water flow, containing biodegradable organic substances and microorganisms, which originate either from the natural biofilms or from some incidents appeared in the distribution network. A major concern for water retailers is to maintain the quality of drinking water from the treatment plant to the tap water. The purpose of this chapter is to give an overview on the characteristics of microbial biofilms found in the aquatic ecosystems with a special focus on their both negative and beneficial roles for the water treatment and quality.