Versatile Polypeptide-Functionalized Plasmonic Paper as Synergistic Biocompatible and Antimicrobial Nanoplatform.

Nowadays, thanks to nanotechnological progress, which itself guides us more and more closely toward not only the efficient design of innovative nanomaterials or nanostructures, but to the improvement of their functionality, we benefit from an important asset in the battle against pathogenic illnesses. Herein, we report a versatile biocompatible plasmonic nanoplatform based on a Whatman paper incorporating positively-charged gold nanospherical particles via the immersion approach. The morphological characterization of the as-engineered-plasmonic paper was examined by SEM (scanning electron microscopy) and HRTEM (high-resolution transmission electron microscopy) investigations, while its surface chemical modification with a synthetic polypeptide, specifically RRWHRWWRR-NH2 (P2), was proved by monitoring the plasmonic response of loaded gold nanospheres and the emission signal of P2 via fluorescence spectroscopy. The as-functionalized plasmonic paper is non-cytotoxic towards BJ fibroblast human cells at bactericidal concentrations. Finally, the antimicrobial activity of the P2-functionalized plasmonic paper on both planktonic bacteria and biofilms was tested against two reference strains: Gram-positive Bacteria, i.e., Staphylococcus aureus and the Gram-negative Bacteria, i.e., Escherichia coli, determining microbial inhibition of up to 100% for planktonic bacteria. In line with the above presented nanoplatform's proper design, followed by their functionalization with active antimicrobial peptides, new roads can be open for determining antibiotic-free treatments against different relevant pathogens.

European external quality assessments for identification, molecular typing and characterization of Staphylococcus aureus.

Objectives: We present the results of two European external quality assessments (EQAs) conducted in 2014 and 2016 under the auspices of the Study Group on Staphylococci and Staphylococcal Infections of ESCMID. The objective was to assess the performance of participating centres in characterizing Staphylococcus aureus using their standard in-house phenotypic and genotypic protocols. Methods: A total of 11 well-characterized blindly coded S. aureus (n = 9), Staphylococcus argenteus (n = 1) and Staphylococcus capitis (n = 1) strains were distributed to participants for analysis. Species identification, MIC determination, antimicrobial susceptibility testing, antimicrobial resistance and toxin gene detection and molecular typing including spa typing, SCCmec typing and MLST were performed. Results: Thirteen laboratories from 12 European countries participated in one EQA or both EQAs. Despite considerable diversity in the methods employed, good concordance (90%-100%) with expected results was obtained. Discrepancies were observed for: (i) identification of the S. argenteus strain; (ii) phenotypic detection of low-level resistance to oxacillin in the mecC-positive strain; (iii) phenotypic detection of the inducible MLSB strain; and (iv) WGS-based detection of some resistance and toxin genes. Conclusions: Overall, good concordance (90%-100%) with expected results was observed. In some instances, the accurate detection of resistance and toxin genes from WGS data proved problematic, highlighting the need for validated and internationally agreed-on bioinformatics pipelines before such techniques are implemented routinely by microbiology laboratories. We strongly recommend all national reference laboratories and laboratories acting as referral centres to participate in such EQA initiatives.

Comparative evaluation of aggressiveness traits in staphylococcal strains from severe infections versus nasopharyngeal carriage.

Despite their commensal status, staphylococci can become problematic pathogens expressing multiple and redundant virulence factors. This study aimed to evaluate aggressiveness markers comparatively in staphylococcal strains isolated from severe infections versus asymptomatic carriage in order to identify clinically relevant bacterial traits that could easily be detected in clinical practice and could be suggestive for particular host-pathogen interactions such as cyto-adhesion or biofilm formation, ultimately orienting the clinical decision-making process. We have used in vitro phenotypic methods to assess adhesion to and invasion of eukaryotic cells, biofilm development, and expression of soluble virulence factors in 92 Staphylococcus spp. strains. The adhesion index, invasion capacity, biofilm formation and expression of soluble factors did not differ significantly between clinical and commensal strains. The major bacterial traits we found to be significantly more prevalent in clinical staphylococci were the aggregative adhesion pattern (P = 0.012), cluster adhesion (P = 0.001) and tetrad morphology (P = 0.018). The aggregative adhesion pattern was correlated with higher cyto-adhesion (P < 0.001), higher invasion capacity (P = 0.003) and lower Carmeli scores (P = 0.002). Three major bacterial traits, namely tetrad morphology, aggregative adhesion pattern, and resistance to methicillin (acronym: TAM), can be used to compute an aggressiveness score (SAS) predictive of the staphylococcal strain's virulence and capacity to initiate and develop a biofilm-driven chronic infectious process versus a fulminant acute infection, in a susceptible host.

HOST-PATHOGEN INTERACTION IN INFECTIONS DUE TO STAPHYLOCOCCUS AUREUS. STAPHYLOCOCCUS AUREUS VIRULENCE FACTORS.

Staphylococcus aureus (S. aureus) is a major cause of hospital-acquired (HA-SA) and community-acquired (CA-SA) infections worldwide. It is isolated from many human body sites, from animals and from foods, from the environment. Pathogenesis is caused by many virulence factors and antibiotic resistance. The host immune system tries to keep under control this pathogen, but many virulence factors produced by this under the regulatory systems control attack the immune system. The epidemiology of S. aureus is in permanent dynamic and is changing quickly. Therefore, it is necessary to monitor infections, to find new effective molecules and vaccines against this pathogen. In order to reduce this problem which affects public health as a whole, the search for new therapeutic alternatives must be associated with policies to control antibiotic use. Community-acquired and hospital-acquired infections epidemiological surveillance guided by scientific studies should be constant habits among health professionals and hospitals.

Phenotypic and genotypic characterization of Staphylococcus aureus strains isolated from a familial foodborne outbreak.

Although Staphylococcus aureus is frequently reported among the common causative agents of foodborne diseases in Europe, very little is known about the strains involved in staphylococcal food-poisoning outbreaks in our region. Therefore, the aim of this study was to characterize the staphylococcal strains recovered from an autochthonous food-poisoning familial outbreak through phenotypic and genotypic methods. Ten S. aureus strains from food and human sources, submitted to the reference laboratory, were tested for susceptibility to 18 antibiotics by disk diffusion and production of enterotoxins A, B, C, D using a reversed passive latex-agglutination assay, and further analyzed by multiplex PCR-based assays for the detection of sea, seb, sec, sed, see, seh, sei, sej, sem, and sen genes. Phage typing, pulsed-field gel electrophoresis (PFGE) and spa typing were performed for evaluating the clonal relatedness of the isolates. Isolates from stool samples and food displayed a similar antibiotic resistance profile, produced enterotoxin B, were PCR-positive for seb, sei and sem genes, and revealed an indistinguishable SmaI macrorestriction pattern at PFGE analysis, suggesting that incriminated food was most likely the source of this food poisoning outbreak. The isolate which expressed a different antibiotic susceptibility profile and tested negative at the screening for enterotoxin production carried seh gene and was discriminated by a nine-band different PFGE profile from the rest. Combined phenotypic and genotypic profiles by multiple typing are necessary to explore key features of epidemic strains and start to lead to a better understanding of the local epidemiology of infections due to toxigenic S. aureus strains.

Antimicrobial activity of copper and silver nanofilms on nosocomial bacterial species.

Contaminated surfaces are possible vehicles in infection transmission. It is known that both Copper (Cu) and Silver (Ag) efficiently inactivate microbes by direct contact. Aiming at using these metals for benefitting from their antimicrobial effect, but to avoid subsequent toxic effects, we evaluated the antimicrobial activity of nanometric thin Silver and Copper films covering less expensive materials. Using a modified version of the Japan Industrial Standard JIS Z 2801:2000, we demonstrated the antimicrobial activity of the surfaces covered with metal ions nanofilms on microorganisms possibly involved in nosocomial infections and on Bacillus anthracis, bacteria with possible implication in bioterrorist attacks. Copper covered surfaces proved to have better antimicrobial activity than Silver surfaces. Silver covered surfaces showed better activity on Gram negative bacteria than on Gram positive cocci. Going deeper with studies on antimicrobial effects using new methods with better direct and/or functional discriminatory capacity is needed in order to provide additional information on the mechanisms of Silver and Copper nanofilms antimicrobial activity.

Antimicrobial activity of plant essential oils against bacterial and fungal species involved in food poisoning and/or food decay.

The currative properties of aromatic and medicinal plants have been recognized since ancient times and, more recently, the antimicrobial activity of plant essential oils has been used in several applications, including food preservation. The purpose of this study was to create directly comparable, quantitative data on the antimicrobial activity of some plant essential oils prepared in the National Institute of Research-Development for Chemistry and Petrochemistry, Bucharest to be used for the further development of food packaging technology, based on their antibacterial and antifungal activity. The essential oils extracted from thyme (Thymus vulgaris L.), basil (Ocimum basilicum L.), coriander (Coriandrum sativum L.), rosemary (Rosmarinus officinalis L.), sage (Salvia officinalis L.), fennel (Foeniculum vulgare L.), spearmint (Mentha spicata L.) and carraway (Carum carvi L.) were investigated for their antimicrobial activity against eleven different bacterial and three fungal strains belonging to species reported to be involved in food poisoning and/or food decay: S. aureus ATCC 25923, S. aureus ATCC 6538, S. aureus ATCC 25913, E. coli ATCC 25922, E. coli ATCC 35218, Salmonella enterica serovar Enteritidis Cantacuzino Institute Culture Collection (CICC) 10878, Listeria monocytogenes ATCC 19112, Bacillus cereus CIP 5127, Bacillus cereus ATCC 11778, Candida albicans ATCC 10231, Aspergillus niger ATCC 16404, Penicillium spp. CICC 251 and two E. coli and Salmonella enterica serovar Enteritidis clinical isolates. The majority of the tested essential oils exibited considerable inhibitory capacity against all the organisms tested, as supported by growth inhibition zone diameters, MICs and MBC's. Thyme, coriander and basil oils proved the best antibacterial activity, while thyme and spearmint oils better inhibited the fungal species.

[Evaluation of antibiotic resistance in the frame of the surveillance system for nosocomial infections. Strong and weak points]. / Evaluarea rezistentei la antibiotice în cadrul sistemului de supraveghere a infectiilor nosocomiale. Puncte forte si puncte slabe.

Since 2005 a sentinel system for surveillance of nosocomial diseases has been introduced in Romania which had, among other objectives, the evaluation of antibiotic resistance. The surveillance methodology was shared annually, the number of participants varying between 12 and 40 hospitals. During 2005-2008 the Reference Laboratory for Nosocomial Infections and Antibiotic Resistance in the "Cantacusino" NIRDMI received 1481 bacterial strains, comprising 531 S. aureus, 486 Pseudomonas aeruginosa, 439 enterobacteria and 25 enterococci strains. The resistance percents widely differred for some species, especially regarding the type of hospital unit that sent the strains (ex., Pseudomonas aeruginosa isolated form patients with burns). A great variability was noted concerning the manner in which nominalized hospitals responded to the solicitations in the methodology that was shared. especially regarding participation to a national bank for bacterial strains. Only for 5 out of the 40 hospitals that participated along the 4 years in the sentinel programme the annual comparative evaluations of antibiotic resistance were achieved. for a small number of microorganisms that underwent surveillance (S. aureus, E. coli). Among the strong points of the system we can point out: unity in methodology; working protocols for microbiological investigation given to all the participants; special forms for reporting. Among the weak points, we consider: modification in the number of participant hospitals during the program: unequal participation of hospitals, with unwanted effects on the sample representativity of analysed microbial strains; difficulties in stocking and processing laboratory data. In order to increase the quality of data provided, we consider the following as useful: harmonization of the objectives regarding integrated surveillance of nosocomial infections and antibiotic resistance in hospital environment, correlated with the ECDC demands and recommendations; inclusion in the system of sanitary units that fit the needs of the program fir microbiological identification and investigation of nosocomial infections; intensification of the training activities of persons involved in the program, regardless of the level of responsibility and the acknowledgement of each person's responsibilities in nosocomial infection monitoring; external and internal control implementation in the laboratories: selection of aspects concerning the emergence and spread of antibiotic resistance that come out during surveillance in order to be studied using molecular methods for the emphasizing of mechanisms and causes, in view of implementing measures for prevention and control; evaluation of the necessity for molecular investigation in view of identifying resistant bacterial clones in the hospitals in Romania etc.

[Antimicrobial resistance surveillance: from the bottle plunged into the ocean to a critical evaluation of the methods]. / Supravegherea rezistentei la antibiotice: de la sticla aruncata în ocean la o evaluare critica a metodelor.

According to the resolution adopted by the Member States in the "Microbial threat" Conference, Copenhaga, 1988. antimicrobial resistance surveillance is one of the four pillars of the control strategy of this increasingly serious public health problem, coming together with the antimicrobial consumption surveillance, intersectoral actions and antibiotics prudent use promotion. Seventeen years ago, O'Brien and col. were describing the current degree of the antimicrobial resistance surveillance as follow: "There are no reliable data in this area--simply fragments of information and anecdotes that we use to draw an overall picture" (Diagnostic Microbiology and Infectious Diseases, 1992, 15.53S-60S). Ten years later, Morris and Masterton were showing that there were reports underlying the big volume of activity delivered in the field of antimicrobial resistance surveillance during that time, but there were no major changes in respect of the data quality (JAC,. 2002, 49, 7-10). According to the WHO definition, surveillance, as continuous and systematic process of data collection, data analysis and data reporting, is reaching it's scope only if it is able to provide information valuable for action. Though it is by excellence a type of surveillance based on the microbiology laboratory activity, antimicrobial resistance surveillance is not coming to be useful according to the WHO and EUCAST (European Committee for Antimicrobial Susceptibility Testing) acception without using the epidemiology methods in order to make possible the adoption of a clear strategy, starting from the definition of the type of information that we want to obtain. Pending on the scope and taking into account the preexisting premises for setting up a surveillance network, we need to select the appropriate surveillance methods, in respect of data and strains collection and storage, data reporting, appropriate denominators (population categories. admission days, patient days etc.), data stratifying etc. In Romania there are few data on antimicrobial resistance surveillance which could resist to a critical evaluation of representativity, reporting to adequate denominators, stratifying methodologies which would allow to follow trends, comparing data by wards, hospitals, counties, intercomparing data with other countries etc. Contacting the European Antimicrobial Resistance Surveillance System in 2001 was an initial modality to decrease the huge gap existing by that time, but could not remain the unique solution to develop in this direction. On the other hand, participating in the European Antimicrobial Resistance Surveillance System is enforcing the involvement of all implicated professional categories, improving logistic and interdisciplinary collaboration, in order to set up a systematic surveillance. We are supporting the initiative of a critical evaluation of the existing situation, as of setting up a surveillance strategy in accordance with the targetted goals, starting from the recent recommendations of WHO and ESCMID Antimicrobial Resistance Surveillance Study Group. This initiative could contribute, together with the participation in the European antimicrobial resistance surveillance program, to the efficient turning account of the resources and uncoordinated and sometimes redundant efforts of different working groups including prestigious microbiology, infectious diseases and epidemiology specialists.

[Tricophyton rubrum in a case of tinea unguium phenotypic mycological diagnostic algorithm and its limits]. / Tricophyton rubrum într-un caz de tinea unguium--algoritmul de diagnostic micologic fenotipic si limitele acestuia.

Trichophyton rubrum is the most common dermatophyte isolated from human dermatophytosis. We present the mycological diagnosis algorithm and results in an onychomycosis case--tinea unguium. Biological samples have been inoculated on both usual and special culture media and incubated at both 30 degrees C and 37 degrees C for a period of fourteen days. Based on the macroscopic and microscopic characters and on the physiological and biochemical tests results, we identified a Trichophyton rubrum granular type dermatophyte. We are discussing phenotypic mycological diagnosis limits and molecular diagnostic benefits for rapid setting up of fungal therapy in order to avoid chronic mycosis and their complications.

[Trends of antimicrobial resistance in microbial strains isolated from invasive infections in Romania. Experience of participation in EARSS (European Antimicrobial Resistance Surveillance System), 2002-2008]. / Tendintele rezistentei la antibiotice a izolatelor din infectii invazive în România. Experienta participarii în Sistemul European de Supraveghere a Rezistentei la Antibiotice (EARSS) în perioada 2002-2008.

EARSS (European Antimicrobial Resistance Surveillance System) is the biggest antimicrobial resistance surveillance project in the world financed from public finds, aiming to provide validated and comparable official data on antimicrobial resistance of invasive microbial strains (isolated from blood and CSF), belonging to 6 indicator bacterial species, i.e.: S. aureus, E. coli, E. faecium/faecalis, Str. pneumoniae, Ps. aeruginosa, K. pneumoniae. Romania reported data to EARSS since 2002 so far. Though the number of participating laboratories increased progressively from 12 to 35, the number of hospitals which reported for EARSS. as the number of strains included in the data base remained steady and relatively low. This issue is related to the particular position of Romania in the European context, in respect of the very low number of blood cultures performed in hospitals. Our paper is presenting the trends of antimicrobial resistance in the indicator strains in the 2002-2008 interval. During the 2002-2008 interval, Romania reported to EARSS a total number of 1276 bacterial strains, distributed by species as follows: 513 S aureus, 369 E. coli, 128 Streptococcus pneumoniae, 127 Enterococcus spp.. 71 Klebsiella pneumoniae, 68 Pseudomonas aeruginosa. Klebsiella pneumoniae and Pseudomonas aeruginosa were reported, according to the EARSS protocol, only for the 2005-2008 interval. It is difficult to describe trends, specially in Enterococcus, Streptococcus pneumonaie and the 2 species collected only since 2005, because of the low number of isolates, but there are several results that are supporting us to claim that antimicrobial resistance in invasive isolates is a real problem in Romanian hospitals, like in other Central, Southern and South Eastern European countries: more than 25% of S. aureus strains resistant to methicilline, with more than 50% in some years, high aminoglycozides resistance in more than 70-80% of Enterococcus faecium invasive strains, more than 80% of strains resistant to 3rd generation cephalosporines etc.

Short tryptophan- and arginine-rich peptide shows efficacy against clinical methicillin-resistant Staphylococcus aureus strains isolated from skin and soft tissue infections.

In recent years methicillin-resistant Staphylococcus aureus has posed a challenge in treating skin and soft tissue infections. Finding new antimicrobial agents has therefore become imperative. We evaluated the in vitro antimicrobial activity of a synthetic peptide, P6, against multidrug resistant clinical strains of Staphylococcus aureus isolated from skin and soft tissue infections. The P6 antimicrobial effect was evaluated in vitro by determining MIC/MBC, the ratio of live/dead cells and the effects induced at membrane level. The therapeutic efficiency was determined against human skin cells. P6 inhibited growth for all strains between 8 and 16 mg/L and killed all bacterial strains at 16 mg/L. The therapeutic potential was found to be 30 and 15 in the presence of BSA. We showed that P6 localizes at membrane level, where it acts slowly, by depolarizing it and affecting its integrity. P6 can be considered a good candidate for use as an antimicrobial agent in topical applications.

Usefulness of complex bacteriological and serological analysis in patients with spondyloarthritis.

Spondyloarthritis (SpA) is a group of associated chronic systemic inflammatory immune-mediated rheumatic diseases affecting axial and peripheral joints and entheses. The aim of the present study was to identify what parameters are useful to determine in order to better understand the correlation between the disease activity/severity and the microbiological results/immune status against intestinal and/or urogenital pathogens. Microorganisms known to trigger SpA, including Klebsiella spp., Yersinia spp., Salmonella spp., Campylobacter spp. and Chlamydia spp., were analyzed in various specimens (stool, urine, synovial fluid and serum) collected from 27 randomly selected SpA patients and 26 healthy controls using a combined direct and indirect approach relying on conventional culture technique and nucleic acid-based assays together with serological testing by ELISA. Although Escherichia coli derived from phylogroup A prevailed in the gut microflora of the patients and controls, differences were observed regarding the representatives of the other phylogroups with a higher prevalence of E.coli members of phylogenetic group B1 in the stool specimens of patients. Antibodies against the targeted species were detected in SpA patients and controls, and the serological profiles of the former were more diverse and complex. In conclusion, the detection of anti-bacterial antibodies combined with other specific laboratory investigations should be more extensively used to monitor SpA patients in association with their symptoms and in order to determine and administer more effective therapeutics.

Poly(3-hydroxybutyrate) Modified by Nanocellulose and Plasma Treatment for Packaging Applications.

In this work, a new eco-friendly method for the treatment of poly(3-hydroxybutyrate) (PHB) as a candidate for food packaging applications is proposed. Poly(3-hydroxybutyrate) was modified by bacterial cellulose nanofibers (BC) using a melt compounding technique and by plasma treatment or zinc oxide (ZnO) nanoparticle plasma coating for better properties and antibacterial activity. Plasma treatment preserved the thermal stability, crystallinity and melting behavior of PHB‒BC nanocomposites, regardless of the amount of BC nanofibers. However, a remarkable increase of stiffness and strength and an increase of the antibacterial activity were noted. After the plasma treatment, the storage modulus of PHB having 2 wt % BC increases by 19% at room temperature and by 43% at 100 °C. The tensile strength increases as well by 21%. In addition, plasma treatment also inhibits the growth of Staphylococcus aureus and Escherichia coli by 44% and 63%, respectively. The ZnO plasma coating led to important changes in the thermal and mechanical behavior of PHB‒BC nanocomposite as well as in the surface structure and morphology. Strong chemical bonding of the metal nanoparticles on PHB surface following ZnO plasma coating was highlighted by infrared spectroscopy. Moreover, the presence of a continuous layer of self-aggregated ZnO nanoparticles was demonstrated by scanning electron microscopy, ZnO plasma treatment completely inhibiting growth of Staphylococcus aureus. A plasma-treated PHB‒BC nanocomposite is proposed as a green solution for the food packaging industry.

Bacteriophage-driven inhibition of biofilm formation in Staphylococcus strains from patients attending a Romanian reference center for infectious diseases.

The increasing burden of invasive biofilm-related staphylococcal infections has led to a dire need for new agents to prevent biofilm formation. Bacteriophages may hypothetically alter a biofilm through several mechanisms, including induction of depolymerizing enzymes and lysis of persistent bacteria. We have assessed the influence of commercially available bacteriophage cocktails on Staphylococcus spp. clinical strains viability and biofilm formation. We analyzed 83 staphylococcal strains from patients consecutively admitted to a Romanian infection reference center from October 2014 through May 2015; the strains were characterized by phenotypic and genetic tools for their resistance and virulence features and for their phyliation. Experiments were performed in triplicate. Methicillin-susceptible strains were significantly more susceptible to all tested phages: 1.7-fold higher susceptibility for PYO, 1.4-fold for INTESTI, 2.9-fold for PHAGYO, 2.7-fold for PHAGESTI and 3.9-fold for STAPHYLOCOCCAL; t030 strains were significantly more susceptible to PYO and INTESTI compared with t127 strains. We identified a significant decrease in biofilm formation in the presence of both low and high PYO and INTESTI concentrations (P < 0.001). In conclusion, Staphylococcus strains from Romania displayed fairly good susceptibility to commercially available bacteriophages. We have also ascertained there is phage-driven in vitro inhibition of biofilm formation, the results potentially impacting prevention of prosthetic infections.

Microbiological and immunological study of staphylococcus vaccine effects in periodontitis.

The aim of this study was to evaluate the immunomodulatory effect of the staphylococcal vaccine inoculated subcutaneously in 15 patients with chronic periodontitis. Bacteriological investigation of samples collected from the periodontal pocket for aerobic and anaerobic microorganisms was performed by classic bacteriological procedures before and after vaccination. The following immune system parameters were evaluated: C reactive protein (CRP), serum level of C3 complement fraction, IgG, IgA, and IgM by immunodiffusion, PMN granulocytes ROS release after in vitro stimulation with opsonized zymosan (OZ) and Concanavalin A (ConA) by chemiluminescence assay and lymphocytes sets and subsets by flow-cytometry immunophenotyping. The microbiological investigations revealed high frequency of Staphylococcus spp isolation and the presence of the most common anaerobe agents incriminated in human periodontitis like Fusobacterium, Porphyromonas, Peptostreptococcus, Veillonella spp and the reduction of this flora in the periodontal pocket after therapy. The immunological parameters quantification showed the absence of CRP, normal values of C3, IgG, IgA, IgM in the majority of cases. All patients presented normal values of lymphocytes sets and subsets. Significant increase of PMN respiratory burst after ConA stimulation was observed before vaccination which turned to normal values after therapy and a low ROS level both before and after therapy suggesting PMN Fc receptors dysfunction in this group of patients. The data presented in our study suggest an immunomodulatory effect of staphylococcal vaccine therapy in periodontitis and high frequency of Staphylococcus spp recovering from the periodontal pocket of investigated subjects.

Carbapenemase-Producing Klebsiella pneumoniae in Romania: A Six-Month Survey.

This study presents the first characterization of carbapenem-non-susceptible Klebsiella pneumoniae isolates by means of a structured six-month survey performed in Romania as part of an Europe-wide investigation. Klebsiella pneumoniae clinical isolates from different anatomical sites were tested for antibiotic susceptibility by phenotypic methods and confirmed by PCR for the presence of four carbapenemase genes. Genome macrorestriction fingerprinting with XbaI was used to analyze the relatedness of carbapenemase-producing Klebsiella pneumoniae isolates collected from eight hospitals. Among 75 non-susceptible isolates, 65 were carbapenemase producers. The most frequently identified genotype was OXA-48 (n = 51 isolates), eight isolates were positive for blaNDM-1 gene, four had the blaKPC-2 gene, whereas two were positive for blaVIM-1. The analysis of PFGE profiles of OXA-48 and NDM-1 producing K. pneumoniae suggests inter-hospitals and regional transmission of epidemic clones. This study presents the first description of K. pneumoniae strains harbouring blaKPC-2 and blaVIM-1 genes in Romania. The results of this study highlight the urgent need for the strengthening of hospital infection control measures in Romania in order to curb the further spread of the antibiotic resistance.