Screening of Molecular Virulence Markers in Staphylococcus aureus and Pseudomonas aeruginosa Strains Isolated from Clinical Infections

Staphylococcus (S.) aureus and Pseudomonas (Ps.) aeruginosa are two of the most frequently opportunistic pathogens isolated in nosocomial infections, responsible for severe infections in immunocompromised hosts. The frequent emergence of antibiotic-resistant S. aureus and Ps. aeruginosa strains has determined the development of new strategies in order to elucidate the different mechanisms used by these bacteria at different stages of the infectious process, providing the scientists with new procedures for preventing, or at least improving, the control of S. aureus and Ps. aeruginosa infections. The purpose of this study was to characterize the molecular markers of virulence in S. aureus and Ps. aeruginosa strains isolated from different clinical specimens. We used multiplex and uniplex PCR assays to detect the genes encoding different cell-wall associated and extracellular virulence factors, in order to evaluate potential associations between the presence of putative virulence genes and the outcome of infections caused by these bacteria. Our results demonstrate that all the studied S. aureus and Ps. aeruginosa strains synthesize the majority of the investigated virulence determinants, probably responsible for different types of infections

Increased risk of chikungunya infection in travellers to Thailand during ongoing outbreak in tourist areas : cases imported to Europe and the Middle East, early 2019

We report nine travellers with confirmed chikungunya virus infection, returning from tourist areas of Thailand to Sweden, Switzerland, the United Kingdom, Romania, Israel and France, diagnosed in January and February 2019. These sentinel tourists support the intensification of chikungunya virus circulation in Thailand and highlight the potential for importation to areas at risk of local transmission

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

First Detection and Molecular Characterization of Usutu Virus in <i>Culex pipiens</i> Mosquitoes Collected in Romania

Usutu virus (USUV) is an emergent arbovirus in Europe causing mortality in bird populations. Similar to West Nile virus (WNV), USUV is maintained in sylvatic cycles between mosquito vectors and bird reservoirs. Spillover events may result in human neurological infection cases. Apart from indirect evidence provided by a recent serological study in wild birds, the circulation of USUV in Romania was not assessed. We aimed to detect and molecular characterize USUV circulating in mosquito vectors collected in South-Eastern Romania—a well-known WNV endemic region—during four transmission seasons. Mosquitoes were collected from Bucharest metropolitan area and Danube Delta, pooled, and screened by real-time RT-PCR for USUV. Partial genomic sequences were obtained and used for phylogeny. USUV was detected in Culex pipiens s.l. female mosquitoes collected in Bucharest, in 2019. The virus belonged to Europe 2 lineage, sub-lineage EU2-A. Phylogenetic analysis revealed high similarity with isolates infecting mosquito vectors, birds, and humans in Europe starting with 2009, all sharing common origin in Northern Italy. To our knowledge, this is the first study characterizing a strain of USUV circulating in Romania

West Nile virus lineage 2 in Romania, 2015–2016: co-circulation and strain replacement

Abstract Background West Nile virus (WNV) is endemic in southeastern Romania and, after the unprecedented urban epidemic in Bucharest in 1996 caused by lineage 1 WNV, cases of West Nile fever have been recorded every year. Furthermore, a new outbreak occurred in 2010, this time produced by a lineage 2 WNV belonging to the Eastern European clade (Volgograd 2007-like strain), which was detected in humans and mosquitoes in the following years. Results We report here, for the first time, the emergence, in 2015, of lineage 2 WNV belonging to the monophyletic Central/Southern European group of strains which replaced in 2016, the previously endemized lineage 2 WNV Volgograd 2007-like strain in mosquito populations. The emerged WNV strain harbors H249P (NS3 protein) and I159T (E glycoprotein) substitutions, which have been previously associated in other studies with neurovirulence and efficient vector transmission. Conclusions In 2016, both early amplification of the emerged WNV and complete replacement in mosquito populations of the previously endemized WNV occurred in southeastern Romania. These events were associated with a significant outbreak of severe West Nile neuroinvasive disease in humans

Original Contributions to the Chemical Composition, Microbicidal, Virulence-Arresting and Antibiotic-Enhancing Activity of Essential Oils from Four Coniferous Species

This study aimed to establish the essential oil (EO) composition from young shoots of Picea abies, Larix decidua, Pseudotsuga menziesii, and Pinus nigra harvested from Romania and evaluate their antimicrobial and anti-virulence activity, as well as potential synergies with currently used antibiotics. The samples’ EO average content varied between 0.62% and 1.02% (mL/100 g plant). The mono- and sesquiterpene hydrocarbons were dominant in the composition of the studied EOs. The antimicrobial activity revealed that the minimum inhibitory concentration (MIC) values for the tested EOs and some pure compounds known for their antimicrobial activity ranged from 6.25 to 100 µL/mL. The most intensive antimicrobial effect was obtained for the Pinus nigra EO, which exhibited the best synergistic effect with some antibiotics against Staphylococcus aureus strains (i.e., oxacillin, tetracycline, erythromycin and gentamycin). The subinhibitory concentrations (sMIC) of the coniferous EOs inhibited the expression of soluble virulence factors (DN-ase, lipase, lecithinase, hemolysins, caseinase and siderophore-like), their efficiency being similar to that of the tested pure compounds, and inhibited the rhl gene expression in Pseudomonas aeruginosa, suggesting their virulence-arresting drug potential

Distribution of Insecticide Resistance Genetic Markers in the West Nile Virus Vector Culex pipiens from South-Eastern Romania

Culex pipiens pipiens and Culex pipiens molestus mosquitoes are the vectors of West Nile virus in south-eastern Romania, an area of intense circulation and human transmission of this virus. The level of insecticide resistance for the mosquito populations in the region has not been previously assessed. Culex pipiens mosquitoes collected between 2018 and 2019 in south-eastern Romania from different habitats were subjected to biotype identification by real-time PCR. Substitutions causing resistance to organophosphates and carbamates (F290V and G119S in acetylcholinesterase 1) and to pyrethroids (L1014F in voltage gated Na+ channel) were screened by PCR or sequencing. Substitutions F290V and G119S were detected at very low frequencies and only in heterozygous state in Culex pipiens molestus biotype specimens collected in urban areas. The molestus biotype population analysed was entirely homozygous for L1014F, and high frequencies of this substitution were also found for pipiens biotype and hybrid mosquitoes collected in urban and in intensive agriculture areas. Reducing the selective pressure by limiting the use of pyrethroid insecticides only for regions where it is absolutely necessary and monitoring L1014F mutation should be taken into consideration when implementing vector control strategies

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 bla(NDM-1) gene, four had the bla(KPC-2) gene, whereas two were positive for bla(VIM-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