Sequencing and Partial Molecular Characterization of BAB-TMP, the Babeș Strain of the Fixed Rabies Virus Adapted for Multiplication in Cell Lines.

The rabies virus is a major zoonosis that causes severe nervous disease in humans, leading to paralysis and death. The world's second anti-rabies center was established in 1888 by Victor Babeș, in Bucharest, where an eponymous strain of rabies was isolated and used to develop a method for immunization. The Babeș strain of the rabies virus was used for over 100 years in Romania to produce a rabies vaccine for human use, based on animal nerve tissue, thus having a proven history of prophylactic use. The present study aimed to sequence the whole genome of the Babeș strain and to explore its genetic relationships with other vaccine strains as well as to characterize its relevant molecular traits. After being adapted for multiplication in cell lines and designated BAB-TMP, 99% of the viral genome was sequenced. The overall organization of the genome is similar to that of other rabies vaccine strains. Phylogenetic analysis indicated that the BAB-TMP strain is closely related to the Russian RV-97 vaccine strain, and both seem to have a common ancestor. The nucleoprotein gene of the investigated genome was the most conserved, and the glycoprotein showed several unique amino acid substitutions within the major antigenic sites and linear epitopes.

Genome characterisation of norovirus GII.P17-GII.17 detected during a large gastroenteritis outbreak in Romania in 2021.

Norovirus (NoV) is one of the leading causes of acute gastroenteritis worldwide. Genotype GII.P17-G.II.17 emerged in Asia between 2013 and 2015 and transiently replaced the GII.4 Sydney 2012 variant circulating at that time. We present the genome characterisation of a GII.P17-GII.17 strain causing a large outbreak in Romania in 2021. Our study shows that the 2021 strain belongs to a novel cluster of genotype GII.17, different from the two previously recognised P.17 clusters. Distinctive substitutions in predicted conformational epitopes of VP1 were identified for this new cluster. Also, our phylogenetic analysis showed the existence of another P.17 cluster grouping strains from France and Canada.

Characterization of Carbapenemase-Producing Klebsiella pneumoniae Isolates from Two Romanian Hospitals Co-Presenting Resistance and Heteroresistance to Colistin.

Klebsiella pneumoniae is a notorious human pathogen involved in healthcare-associated infections. The worldwide expansion of infections induced by colistin-resistant and carbapenemase-producing Enterobacterales (CPE) isolates has been increasingly reported. This study aims to analyze the phenotypic and molecular profiles of 10 colistin-resistant (CR) isolates and 2 pairs of colistin-heteroresistant (ChR) (parental and the corresponding resistant mutants) isolates of K. pneumoniae CPE sourced from two hospitals. The phenotypes of strains in the selected collection had been previously characterized. Antimicrobial susceptibility testing was performed using a Vitek 2 Compact system (BioMérieux SA, Marcy l'Etoile, France), the disc diffusion method, and broth microdilution (BMD) for colistin. Whole-genome sequencing (WGS) did not uncover evidence of any mobile colistin resistance (mcr) genes, although the mgrB gene of seven isolates appeared to be disrupted by insertion sequences (ISKpn25 or ISKpn26). Possible deleterious missense mutations were found in phoP (L4F), phoQ (Q426L, L26Q, L224Q, Q317K), pmrB (R256G, P95L, T157P, V352E), and crrB (P151S) genes. The identified isolates belonged to the following clonal lineages: ST101 (n = 6), ST147 (n = 5), ST258 (n = 2), and ST307 (n = 1). All strains harbored IncF plasmids. OXA-48 producers carried IncL and IncR plasmids, while one blaNDM-1 genome was found to harbor IncC plasmids. Ceftazidime-avibactam remains a therapeutic option for KPC-2 and OXA-48 producers. Resistance to meropenem-vaborbactam has emerged in some blakPC-2-carrying isolates. Our study demonstrates that the results of WGS can provide essential evidence for the surveillance of antimicrobial resistance.

Cellular and Molecular Targets for Non-Invasive, Non-Pharmacological Therapeutic/Rehabilitative Interventions in Acute Ischemic Stroke.

BACKGROUND: Cerebral circulation delivers the blood flow to the brain through a dedicated network of sanguine vessels. A healthy human brain can regulate cerebral blood flow (CBF) according to any physiological or pathological challenges. The brain is protected by its self-regulatory mechanisms, which are dependent on neuronal and support cellular populations, including endothelial ones, as well as metabolic, and even myogenic factors. OBJECTIVES: Accumulating data suggest that "non-pharmacological" approaches might provide new opportunities for stroke therapy, such as electro-/acupuncture, hyperbaric oxygen therapy, hypothermia/cooling, photobiomodulation, therapeutic gases, transcranial direct current stimulations, or transcranial magnetic stimulations. We reviewed the recent data on the mechanisms and clinical implications of these non-pharmaceutical treatments. METHODS: To present the state-of-the-art for currently available non-invasive, non-pharmacological-related interventions in acute ischemic stroke, we accomplished this synthetic and systematic literature review based on the Preferred Reporting Items for Systematic Principles Reviews and Meta-Analyses (PRISMA). RESULTS: The initial number of obtained articles was 313. After fulfilling the five steps in the filtering/selection methodology, 54 fully eligible papers were selected for synthetic review. We enhanced our documentation with other bibliographic resources connected to our subject, identified in the literature within a non-standardized search, to fill the knowledge gaps. Fifteen clinical trials were also identified. DISCUSSION: Non-invasive, non-pharmacological therapeutic/rehabilitative interventions for acute ischemic stroke are mainly holistic therapies. Therefore, most of them are not yet routinely used in clinical practice, despite some possible beneficial effects, which have yet to be supplementarily proven in more related studies. Moreover, few of the identified clinical trials are already completed and most do not have final results. CONCLUSIONS: This review synthesizes the current findings on acute ischemic stroke therapeutic/rehabilitative interventions, described as non-invasive and non-pharmacological.

Non-invasive, non-pharmacological/bio-technological interventions towards neurorestoration upshot after ischemic stroke, in adults-systematic, synthetic, literature review.

Considering its marked life-threatening and (not seldom: severe and/or permanent) disabling, potential, plus the overall medico-psycho-socio-economic tough burden it represents for the affected persons, their families and the community, the cerebrovascular accident (CVA)-including with the, by far more frequent, ischemic type-is subject to considerable scientific research efforts that aim (if possible) at eliminating the stroke induced lesions, and consist, as well, in ambitious-but still poorly transferable into medical practice-goals such as brain neuroregeneration and/or repair, within related corollary/upshot of neurorestoration. We have conducted, in this respect, a systematic and synthetic literature review, following the "Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)" concept. Accordingly, we have interrogated five internationally renowned medical data bases: Elsevier, NCBI/PubMed, NCBI/PMC, PEDro, and ISI Web of Knowledge/Science (the last one to check whether the initially identified articles are published in ISI indexed journals), based on a large (details in the body text) number of most appropriate, to our knowledge, key word combinations/"syntaxes"-used contextually-and subsequently fulfilling the related, on five steps, filtering/selection methodology. We have thereby selected 114 fully eligible (of which contributive: 83-see further) papers; at the same time, additionally, we have enhanced our documentation-basically, but not exclusively, for the introductive part of this work (see further)-with bibliographic resources, overall connected to our subject, identified in the literature within a non-standardized search. It appears that the opportunity window for morph-functional recovery after stroke is larger than previously thought, actually being considered that brain neurorestoration/repair could occur, and therefore be expected, in later stages than in earlier ones, although, in this context, the number of cases possibly benefitting (for instance after physical and/or cognitive rehabilitation-including with magnetic or direct current transcranial stimulation) is quite small and with more or less conflicting, related outcomes, in the literature. Moreover, applying especially high intense, solicitating, rehabilitation interventions, in early stages post (including ischemic) stroke could even worsen the functional evolution. Accordingly, for clarifications and validation of more unitary points of view, continuing and boosting research efforts in this complex, interdisciplinary domain, is necessary. Until finding (if ever) effective modalities to cure the lesions of the central nervous system (CNS)-including post ischemic stroke-it is reasonable and recommendable-based on rigorous methodologies-the avail of combined ways: physiatric, pharmacologic, possibly also bio-technologic. On a different note, but however connected to our subject: periodic related systematic, synthetic literature reviews reappraisals are warranted and welcome.

A Snapshot of the Genetic Diversity of Salmonella Enteritidis Population Involved in Human Infections in Romania Taken in the European Epidemiological Context.

In the absence of consistent national molecular typing data to enhance the surveillance of Salmonella Enteritidis, it was considered useful to collect baseline information on the genetic diversity and antibiotic susceptibility of strains isolated in Romania between January 2016 and April 2020 and compare them to strains described in major international outbreaks of the same period. A collection of 245 clinical isolates were genotyped by a standardised multiple-locus variable-number of tandem repeats analysis (MLVA) 5-loci protocol and screened for antimicrobial resistance against 15 compounds. Twenty strains were further subjected to whole genome sequencing (WGS) and compared to epidemiologically relevant high-throughput sequencing data available in European databases. Twenty-seven MLVA genotypes were identified, of which three, commonly reported in Europe between 2016-2020, covered 72% of the collection. Antibiotic resistance was detected in 30% of the strains, with resistance to nalidixic acid and ciprofloxacin as the most common phenotype, and also associated with two prevalent MLVA clones. WGS-derived multilocus sequence typing (MLST) revealed a single sequence type (ST11) further resolved into 10 core-genome MLST complex types. The minimum spanning tree constructed from the cgMLST data clustered Romanian and international strains, which shared more than 95% of the core genes, revealing links with a contemporaneous multi-country outbreak. This study could be regarded as a forerunner to the advent of using this integrative approach in the public health practice at a national level and thus contribute to the concerted actions at a European level to stop outbreaks.

Solvatochromic Study of Two Carbanion Monosubstituted 4-Tolyl-1,2,4-triazol-1-ium Phenacylids in Binary Hydroxyl Solvent Mixtures.

Two 4-tolyl-1,2,4-triazol-1-ium methylids, namely 4-tolyl-1,2,4-triazol-1-ium-phenacylid and 4-tolyl-1,2,4-triazol-1-ium-4'-nitro-phenacylid, are studied from solvatochromic point of view in binary solvent mixtures of water with ethanol and water with methanol. The contributions (expressed in percent) of the universal and specific interactions are separated from the spectral shifts recorded in the visible range for each composition of the binary solvent mixture. The essential role of the orientation and induction interactions in the studied solutions was demonstrated. Based on the statistic cell model of the binary solvent mixture solutions, the difference between the formation energies of ylid-water and ylid-alcohol complexes is estimated. The composition of the ylid's first solvation shell was also established using the model of the binary solvent mixture solutions. The results obtained from the statistical cell model were compared with those obtained by using the Suppan's model, resulting a good agreement.

Optimized Synthesis of New N-Mustards Based on 2-Mercaptobenzoxazole Derivatives with Antitumor Activity.

New di-(ß-chloroethyl)-amides of some acids derived from 2-mercaptobenzoxazole were prepared by reaction of the corresponding pivalic mixed anhydrides with di-(ß-chloroethyl)-amine. A study regarding the optimization of the chemical reactions was made for the case of di-(ß-chloroethyl)-amines. The quantum chemical analysis by Spartan'14 was made in order to establish the most stable configuration of the ground electronic states for the obtained chemical structures and some physico-chemical parameters of N-mustards reported in this paper. Mercaptobenzoxazoles substituted in the side chain with the cytotoxic group show antitumor activity and they inhibit Ehrlich Ascites in an appreciable proportion compared to the drug I.O.B.-82, as our studies evidenced.

The seventh pandemic of cholera in Europe revisited by microbial genomics.

In 1970, the seventh pandemic of cholera (7 P) reached both Africa and Europe. Between 1970 and 2011, several European countries reported cholera outbreaks of a few to more than 2,000 cases. We report here a whole-genome analysis of 1,324 7 P V. cholerae El Tor (7 PET) isolates, including 172 from autochthonous sporadic or outbreak cholera cases occurring between 1970 and 2011 in Europe, providing insight into the spatial and temporal spread of this pathogen across Europe. In this work, we show that the 7 PET lineage was introduced at least eight times into two main regions: Eastern and Southern Europe. Greater recurrence of the disease was observed in Eastern Europe, where it persisted until 2011. It was introduced into this region from Southern Asia, often circulating regionally in the countries bordering the Black Sea, and in the Middle East before reaching Eastern Africa on several occasions. In Southern Europe, the disease was mostly seen in individual countries during the 1970s and was imported from North and West Africa, except in 1994, when cholera was imported into Albania and Italy from the Black Sea region. These results shed light on the geographic course of cholera during the seventh pandemic and highlight the role of humans in its global dissemination.

Molecular Typing of Escherichia coli O157 Isolates from Romanian Human Cases.

Verocytotoxin-producing Escherichia coli (VTEC) of serogroup O157 are among the most important causes of severe cases of foodborne disease and outbreaks worldwide. As little is known about the characteristic of these strains in Romania, we aimed to provide reference information on the virulence gene content, phylogenetic background, and genetic diversity of 7 autochthonous O157 strains collected during 2016 and 2017 from epidemiologically non-related cases. These strains were typed by a combination of phenotypic and molecular methods routinely used by the national reference laboratory. Additionally, 4 of them were subjected to whole-genome sequencing (WGS), and public web-based tools were used to extract information on virulence gene profiles, multilocus sequence types (MLST), and single nucleotide polymorphism (SNP)-based phylogenetic relatedness. Molecular typing provided evidence of the circulation of a polyclonal population while distinguishing a cluster of non-sorbitol-fermenting, glucuronidase-negative, phylogenetic group E, MLST 1804 strains, representing lineage II and clade 7, which harbored vtx2c, eae-gamma, and ehxA genes. A good correlation between the routine typing methods and WGS data was observed. However, SNP-based genotyping provided a higher resolution in depicting the relationships between the O157:H7 strains than that provided by Pulse-field gel electrophoresis. This study should be a catalyst for improved laboratory-based surveillance of autochthonous VTEC.

Molecular characterisation of human Shiga toxin-producing Escherichia coli O26 strains: results of an outbreak investigation, Romania, February to August 2016.

IntroductionAt the beginning of 2016, an increase in paediatric haemolytic uremic syndrome (HUS) cases was observed in Romania. The microbiological investigations allowed isolation of Shiga toxin-producing Escherichia coli (STEC) O26 as the causative agent from most cases. Methods: An enhanced national surveillance of HUS and severe diarrhoea was established across the country following the identification of the first cases and was carried out until August 2016. A total of 15 strains were isolated from 10 HUS and five diarrhoea cases. Strains were characterised by virulence markers (i.e. stx type/subtype, eae, ehxA genes), phylogroup, genetic relatedness and clonality using PCR-based assays, PFGE and multilocus sequence typing (MLST). The first six strains were further characterised by whole genome sequencing (WGS). Results: Five PCR-defined genotypes were distinguished. All strains from HUS cases harboured stx2a and eae, with or without stx1a, while strains from diarrhoea cases carried exclusively stx1a and eae genes. PFGE resolved strains into multiple pulsotypes, compatible with a certain geographic segregation of the cases, and strains were assigned to phylogroup B1 and sequence type (ST) 21. WGS confirmed the results of conventional molecular methods, brought evidence of O26:H11 serotype, and complemented the virulence profiles. Discussion/conclusion: This first description of STEC O26 strains from cases in Romania showed that the isolates belonged to a diverse population. The virulence content of most strains highlighted a high risk for severe outcome in infected patients. Improving the national surveillance strategy for STEC infections in Romania needs to be further considered.

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.

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.

Molecular characterization of bacteremic Escherichia coli isolates in Romania.

The increasing prevalence of invasive infections caused by antibiotic resistant Escherichia coli strains in Romanian patients, already mentioned in the European reports, requires better knowledge of their specific traits. Thus, a set of 38 E. coli blood isolates, collected between 2010 and 2012 at one of the local hospitals participating into the European Antimicrobial Resistance Surveillance Network, was investigated retrospectively with respect to the phylogenetic origin, extraintestinal virulence-associated markers (i.e. fimH, papC, papG alleles, sfa/foc, afa/dra, hly, cnf1, sat, iucC, fyuA, ibeA), and beta-lactamase encoding genes (i.e. bla CTX-M, bla TEM, and bla SHV alleles). The isolates with extended-spectrum beta-lactamase (ESBL) phenotypes were further characterized using PCR-based replicon typing and multilocus sequencing typing. For ST131 members, pulsed-field gel electrophoresis (PFGE) and PCR-based detection of fimH30 allele were performed. Overall, the isolates were more likely members of the major phylogenetic group A (53 %) and to a lesser extent of groups B2 (29 %), D (10 %), and B1 (8 %). All but three of the virulence markers sought (i.e. papGI, hly, cnf1) were detected with prevalence ranging from 3 % (i.e. ibeA, papGIII) to 87 % (fimH). As expected, the most complex genotypes (four to seven virulence markers) defined the isolates derived from phylogenetic groups B2 and D. ESBL producers were bla CTX-M-15-positive, mostly of phylogroup A (67 %), harboured IncF multireplicon plasmids, and belonged to six sequence types (i.e. ST10, ST131, ST167, ST410, ST540, ST1275). Members of ST10 clonal complex (i.e. ST10, ST167) were the most common. The ST131 isolates belonged to H30 subclone and displayed 74 % similarity at PFGE analysis.

ANTIMICROBIAL RESISTANCE AND GENETIC DIVERSITY OF SHIGELLA SONNEI ISOLATES FROM ROMANIA.

Although not endemic in the European Union and European Economic Area, shigellosis is included among the priority food- and waterborne diseases carefully surveyed by the European Centre for Disease Prevention and Control. From 2010 to 2012, 1018 cumulated confirmed shigellosis cases were reported to the European Surveillance System by Romania. This retrospective study aimed to provide insights into the antibiotic resistance and genetic diversity of a set of Shigella sonnei isolates recovered during that period. A total of 59 S. sonnei isolates were subjected to antimicrobial susceptibility testing (ampicillin, cefotaxim, chloramphenicol, streptomycin, gentamicin, kanamycin, tetracycline, sulphonamides compound, trimethoprim, trimethoprim-sulfamethoxazole, nalidixic acid, and ciprofloxacin), biotyping, and molecular characterization of resistance to third-generation cephalosporins, fluoroquinolones, and integron content. Pulsed-field gel electrophoresis (PFGE) was performed in order to assess the genetic relatedness of the isolates. Thirty-eight (64%) of the studied isolates displayed multidrug-resistant (MDR) phenotypes, the most common resistance profile comprising resistance to ampicillin, streptomycin, sulphonamides compound, trimethoprim, and trimethoprim-sulfamethoxazole. Resistance to cefotaxim was detected in a biotype g blaCTX-M-15 and a biotype e blaCMY-2-positive isolate, respectively. Resistance to ciprofloxacin and/or nalidixic acid was detected in three MDR isolates and was due to known mutations in gyrA gene leading to aminoacid substitutions (i.e. Ser83Leu, Asp87Tyr, Asp87Gly). Either class 1 or class 2 integrons were identified in 10 isolates. Comparisons of XbaI PFGE patterns of S. sonnei isolates revealed 9 clonal groups and 6 unique patterns. The genotyping results suggested that dissemination of clonal groups of S. sonnei may have persisted over the years in Romania.

MECHANISMS OF RESISTANCE TO CIPROFLOXACIN AND GENETIC DIVERSITY OF ESCHERICHIA COLI STRAINS ORIGINATING FROM URINE CULTURES PERFORMED FOR ROMANIAN ADULTS.

Urinary tract infections (UTI) with Escherichia coli are among the most common infections presenting in general practice. Fluoroquinolones (FQs) are relied on for their empirical therapy but recent reports indicate a concerning increase in the percentage of FQ-resistant E. coli isolates in many countries, including Romania. Sixty E. coli strains with ciprofloxacin resistance and cephalosporin susceptibility isolated from urine specimens of non-hospitalized patients during a five-month period (October 2014 - February 2015) were further analyzed to determine the molecular basis of FQ resistance (i.e. mutations in chromosomal gyrA, gyrB, parC genes and presence of plasmid-borne qnrA, qnrB, qnrS, and aac(6'-Ib-cr genes), the phylogenetic background (i.e. phylogenetic groups A, B1, B2, C, D, E, F or clade I), O25b/ST131 status, and genetic relatedness inferred from the XbaI pulsed-field gel electrophoresis (PFGE) profiles as a measure of isolate-specific genetic composition. The PCR-based phylotyping showed that most strains were assigned to non-B2 phylogenetic groups (i.e. group A/21 strains, group B1/14 strains, group B2/10 strains, group C/8 strains, group D/3 strains, group F/4 strains). Already described chromosomal mutations associated to FQ resistance were found, the strains being double gyrA mutants (i.e. Ser83Leu, Asp87Asn) with one or two parC mutations (e.g. Ala56Thr, Ser80Ile, Glu84Gly). Seven percent of the strains harboured plasmid-borne genes qnrS1 (2 strains) and aac(6'-Ib-cr (2 strains). Based on the PCR results, 15% of the strains were members of the O25b/ST131 clone and possessed the gyrA/parC allele combination which is considered as hallmark of H30 subclone. PFGE genotyping revealed a genetically diverse population of FQ-resistant E. coli. ST131 strains displayed more homogeneous PFGE profiles than non-ST131. The ST131 cluster extended to 77.74% similarity versus 60% overall. These findings underscore the need for ongoing surveillance to capture the complexity of the emerging population of FQ-resistant strains disseminated across our community.

MOLECULAR CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS STRAINS ISOLATED FROM INFECTIVE ENDOCARDITIS.

Infective endocarditis (IE) is an infection of the heart endothelium and valves and is frequently a consequence of a sanguine flow turbulence and injury of endocardium. Recent studies revealed an increase of Staphylococcus aureus strains involved in IE, but no evident correlations between the genetic background of this bacterium and IE involvement of certain strains have been found yet. In this study we analyzed the virulence profile, including adhesins, exotoxins, superantigens and biofilm determinants, along with agr type detection, for S. aureus strains isolated from IE, versus non-IE originating strains. We performed also bacterial typing (SCCmec typing, spa-typing and MLST typing), in order to compare our strains with international databases repositories. Although the study was carried out on a reduced number of isolates, our observations confirm the previous works, showing that no major differences were observed between the genetic backgrounds of the two groups of strains analyzed. Notably, the added value of this study was optimization of two new multiplex PCR protocols, and the enrichment of international databases with three new spa-types, three new MLST alleles and four new MLST sequence types.

Reverse-vaccinology strategy for designing T-cell epitope candidates for Staphylococcus aureus endocarditis vaccine.

Staphylococcus aureus is an opportunistic pathogen causing various inflammatory diseases from skin and tissue local infections, to serious life threatening infections including endocarditis. Experimental models for endocarditis demonstrated that virulence factors of S. aureus, that are very important in infection of heart vegetations, are surface proteins which promote bacterial adherence. Until now, efforts to develop effective vaccines against S. aureus were unsuccessful, partly due to the fact that different vaccine formulations have targeted mainly B-cell immunity. Reverse vaccinology is applied here, in order to identify potential vaccine epitope candidates. The basic epitopes prediction strategy relied on detection of a common antigenic 9-mer epitope meant to be able to stimulate both the B-cell and T-cell mediated immunity. Ten surface exposed proteins were chosen for antigenicity testing. Using a web-based system, five T-cell epitopes corresponding to fibronectin binding protein A (FDFTLSNNV and YVDGYIETI), collagen adhesin (FSINYKTKI), serine-rich adhesin for platelets (LTFDSTNNT) and elastin binding protein (FAMDKSHPE) were selected as potential vaccine candidates. Epitopes sequences were found to be conserved among the different S. aureus genomes screened from NCBI GenBank. In vitro and in vivo immunological tests will be performed in order to validate the suitability of the epitopes for vaccine development.

Q fever endocarditis in Romania: the first cases confirmed by direct sequencing.

Infective endocarditis (IE) is a serious, life-threatening disease with highly variable clinical signs, making its diagnostic a real challenge. A diagnosis is readily made if blood cultures are positive, but in 2.5 to 31% of all infective endocarditis cases, routine blood cultures are negative. In such situations, alternative diagnostic approaches are necessary. Coxiella burnetii and Bartonella spp. are the etiological agents of blood culture-negative endocarditis (BCNE) most frequently identified by serology. The purpose of this study is to investigate the usefulness of molecular assays, as complementary methods to the conventional serologic methods for the rapid confirmatory diagnostic of Q fever endocarditis in patients with BCNE. Currently, detection of C. burnetii by culture or an antiphase I IgG antibody titers >800 represents a major Duke criterion for defining IE, while a titers of >800 for IgG antibodies to either B. henselae or B. quintana is used for the diagnosis of endocarditis due to Bartonella spp. We used indirect immunofluorescence assays for the detection of IgG titers for C. burnetii, B. henselae and B. quintana in 57 serum samples from patients with clinical suspicion of IE. Thirty three samples originated from BCNE patients, whereas 24 were tested before obtaining the blood cultures results, which finally were positive. The results of serologic testing showed that nine out of 33 BCNE cases exhibited antiphase I C. burnetii IgG antibody titer >800, whereas none has IgG for B. henselae or B. quintana. Subsequently, we used nested-PCR assay for the amplification of C. burnetii DNA in the nine positive serum samples, and we obtained positive PCR results for all analyzed cases. Afterwards we used the DNA sequencing of amplicons for the repetitive element associated to htpAB gene to confirm the results of nested-PCR. The results of sequencing allowed us to confirm that C. burnetii is the causative microorganism responsible for BCNE. In conclusion, the nested PCR amplification followed by direct sequencing is a reliable and accurate method when applied to serum samples, and it may be used as an additional test to the serological methods for the confirmatory diagnosis of BCNE cases determined by C. burnetii.