Antimicrobial Resistance and the Prevalence of the Panton-Valentine Leukocidin Gene among Clinical Isolates of Staphylococcus aureus in Lithuania.

This study aimed to determine resistance to antimicrobials of Staphylococcus aureus strains isolated from clinical specimens in Lithuanian hospitals and to identify the genes conferring resistance and virulence. The study was carried out from June 2019 to September 2021. S. aureus strains were isolated from skin, soft tissues, blood, lower respiratory tract, urine and other specimens. Antibiotic susceptibility testing was performed using the disc diffusion method according to EUCAST guidelines. All isolates were analyzed for detection of the ermA, ermC, mecA, mecC, tetK, tetM, and lukF-PV genes by multiplex real-time PCR. The 16S rRNA coding sequence was applied as an internal PCR control. Altogether, 745 S. aureus strains were analyzed. Antimicrobial susceptibility testing revealed that all isolates were susceptible to rifampin and vancomycin. Of the 745 strains, 94.8% were susceptible to tetracycline, 94.5% to clindamycin, and 88.3% to erythromycin. The lowest susceptibility rate was found for penicillin (25.8%). Six percent of the tested strains were methicillin-resistant S. aureus (MRSA). The majority of methicillin-resistant strains were isolated from skin and soft tissues (73.3%), with a smaller portion isolated from blood (17.8%) and respiratory tract (8.9%). The ermC gene was detected in 41.1% of erythromycin-resistant S. aureus strains, whereas ermA was detected in 32.2% of erythromycin-resistant S. aureus strains. 69.2% of tetracycline-resistant S. aureus strains had tetK gene, and 28.2% had tetM gene. 7.3% of S. aureus isolates harbored lukF-PV gene. The frequency of the pvl gene detection was significantly higher in MRSA isolates than in methicillin-susceptible S. aureus isolates (p < 0.0001).

Phylogenetic characterisation of tick-borne encephalitis virus from Lithuania.

The Baltic states are the region in Europe where tick-borne encephalitis (TBE) is most endemic. The highest notification rate of TBE cases is reported in Lithuania, where the incidence of TBE has significantly increased since 1992. A recent study reported 0.4% prevalence of TBE virus (TBEV) in the two most common tick species distributed in Lithuania, Ixodes ricinus and Dermacentor reticulatus, with the existence of endemic foci confirmed in seven out of Lithuania's ten counties. However, until now, no comprehensive data on molecular characterisation and phylogenetic analysis have been available for the circulating TBEV strains. The aim of this study was to analyse TBEV strains derived from I. ricinus and D. reticulatus ticks collected from Lithuania and provide a genotypic characterisation of viruses based on sequence analysis of partial E protein and NS3 genes. The 54 nucleotide sequences obtained were compared with 81 TBEV strains selected from the NCBI database. Phylogenetic analysis of the partial E and NS3 gene sequences derived from 34 Lithuanian TBEV isolates revealed that these were specific to Lithuania, and all belonged to the European subtype, with a maximum identity to the Neudoerfl reference strain (GenBank accession no. U27495) of 98.7% and 97.4%, respectively. The TBEV strains showed significant regional genetic diversity. The detected TBEV genotypes were not specific to the tick species. However, genetic differences were observed between strains from different locations, while strains from the same location showed a high similarity.

Osteochondral Repair and Electromechanical Evaluation of Custom 3D Scaffold Microstructured by Direct Laser Writing Lithography.

OBJECTIVE: The objective of this study was to assess a novel 3D microstructured scaffold seeded with allogeneic chondrocytes (cells) in a rabbit osteochondral defect model. DESIGN: Direct laser writing lithography in pre-polymers was employed to fabricate custom silicon-zirconium containing hybrid organic-inorganic (HOI) polymer SZ2080 scaffolds of a predefined morphology. Hexagon-pored HOI scaffolds were seeded with chondrocytes (cells), and tissue-engineered cartilage biocompatibility, potency, efficacy, and shelf-life in vitro was assessed by morphological, ELISA (enzyme-linked immunosorbent assay) and PCR (polymerase chain reaction) analysis. Osteochondral defect was created in the weight-bearing area of medial femoral condyle for in vivo study. Polymerized fibrin was added to every defect of 5 experimental groups. Cartilage repair was analyzed after 6 months using macroscopical (Oswestry Arthroscopy Score [OAS]), histological, and electromechanical quantitative potential (QP) scores. Collagen scaffold (CS) was used as a positive comparator for in vitro and in vivo studies. RESULTS: Type II collagen gene upregulation and protein secretion was maintained up to 8 days in seeded HOI. In vivo analysis revealed improvement in all scaffold treatment groups. For the first time, electromechanical properties of a cellular-based scaffold were analyzed in a preclinical study. Cell addition did not enhance OAS but improved histological and QP scores in HOI groups. CONCLUSIONS: HOI material is biocompatible for up to 8 days in vitro and is supportive of cartilage formation at 6 months in vivo. Electromechanical measurement offers a reliable quality assessment of repaired cartilage.

Lauryl Gallate Activity and Streptococcus mutans: Its Effects on Biofilm Formation, Acidogenicity and Gene Expression.

Streptococcus mutans bacterium is implicated in the pathogenesis of dental caries due to the production of biofilm and organic acids from dietary sucrose. Despite the availability of various means of prophylaxis, caries still has a high worldwide prevalence. Therefore, it is important to find new pharmaceuticals to inhibit S. mutans biofilm formation and acidogenicity. The aim of the current study was to evaluate the activity of lauryl gallate (dodecyl gallate) against S. mutans acidogenicity, the expression of biofilm-associated genes, and biofilm development on solid surfaces (polystyrene, glass). The biofilm quantities produced by S. mutans bacteria were assessed using colorimetric and optical profilometry techniques. Acidogenicity was evaluated by measuring the pH of the biofilm growth medium with microelectrode. Assessment of the expression of gene coding for glucan-binding protein B (gbpB), glucosyltranferases B, -C, -D (gtfB, -C, -D), and the F-ATPase ß subunit of F1 protein (atpD) was carried out using a quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The results demonstrate the capacity of lauryl gallate to significantly inhibit S. mutans acidogenicity and biofilm development on solid surfaces, in a dose-dependent manner, compared to untreated bacteria (p < 0.05). The highest activity of lauryl gallate occurred at a concentration of 98.98 µM, at which it suppressed biofilm formation by 100% and lowered pH levels by 98%. The effect of lauryl gallate treatment on gene expression changes, as demonstrated by our RT-qPCR data, was limited to the gtfD gene only, was a significant (48%) decrease in gene expression, obtained for the biofilm-producing bacteria, while a 300% increase in fold change for the same gene occurred in the planktonic cells. It is important to note that in previous studies we showed a broader effect of related derivatives. However, a similar magnitude of difference in effects between biofilm and planktonic cells for the atpD gene was obtained after treatment with octyl gallate and reverse magnitude for the same gene after treatment with ethyl gallate. Therefore, to ascertain the possible direct or indirect effects of lauryl gallate, as well as octyl gallate and ethyl gallate, more research is needed to examine the effects on the amount of enzymes and on the enzymatic activity of the products of the affected genes that are involved in the production and maintenance of biofilm by S. mutans.

New record of Rickettsia vini in Ixodes lividus ticks from Lithuania.

Ixodes lividus Koch, 1844 (Ixodida: Ixodidae) ticks are parasites specific to the sand martin (Riparia riparia). The distribution range of I. lividus covers Europe and Asia and they are found wherever sand martins nest. Borrelia burgdorferi sensu stricto, Borrelia garinii and Rickettsia vini have recently been detected in I. lividus ticks in Central Europe. The aim of this study was to investigate the presence of Rickettsia spp. (Rickettsiales: Rickettsiaceae) and Borrelia spp. (Spirochaetales: Spirochaetaceae) in I. lividus ticks in Lithuania. A total of 2622 non-engorged I. lividus from two different colonies comprising 40 sand martin nests were collected in Lithuania in 2013 and 2015. Rickettsial DNA was detected in 106 of the 117 pooled larvae samples, in all examined seven nymphs, four females and one male specimen. Sequence analysis of partial gltA, ompA and htrA genes showed 99-100 % identity with the corresponding R. vini sequences deposited in GenBank. None of I. lividus tested was infected with Borrelia burgdorferi sensu lato. This is the first time that the presence of R. vini has been reported in Lithuania.

Prevalence and diversity of Bartonella species in small rodents from coastal and continental areas.

Worldwide, Bartonella infections are known to inflict a wide range of mammals and, within rodents alone, more than 20 Bartonella species have been detected. There is, however, a lack of studies on the presence of Bartonella spp. in rodents in the Baltic region. We analysed 580 individuals belonging to eight small rodent species trapped in coastal and continental areas of Lithuania during 2015-2016. The presence of Bartonella DNA was examined by real-time PCR targeting the ssrA gene. The molecular characterization of the bacteria strains was based on sequence analysis of two housekeeping genes (rpoB, groEL) and the intergenic spacer region (ITS). For the rodents overall, the prevalence of Bartonella spp. was 54.8%, while the prevalence figures for each of the individual species were 8.3% in M. musculus, 15.8% in A. agrarius, 33.3% in M. arvalis, 42.4% in M. glareolus, 53.4% in M. oeconomus, 57.5% in M. minutus, 79.6% in A. flavicollis to 80% in M. agrestis. Sequence analysis revealed that the Bartonella strains belonged to the B. grahamii, B. taylorii, B. rochalimae, B. tribocorum, B. coopersplainsensis and B. doshiae genogroups. The highest Bartonella infection rates and the highest species diversity were both detected in rodents captured in the coastal area. To our knowledge, these are the first reports of the presence of B. coopersplainsensis, B. doshiae and B. tribocorum in Lithuania.

Prevalence and co-infection with tick-borne Anaplasma phagocytophilum and Babesia spp. in red deer (Cervus elaphus) and roe deer (Capreolus capreolus) in Southern Norway.

Anaplasma phagocytophilum and Babesia spp. are causative agents of tick-borne infections that are increasingly considered as a threat to animal and public health. To assess the role of cervids in the maintenance of zoonotic pathogens in Norway, we investigated the prevalence of A. phagocytophilum and Babesia spp. in free-ranging roe deer and red deer. Initial screening of spleen samples of 104 animals by multiplex real-time PCR targeting the major surface protein (msp2) gene and 18S rRNA revealed the presence of A. phagocytophilum infection in 81.1% red deer (Cervus elaphus) and 88.1% roe deer (Capreolus capreolus), and Babesia spp. parasites in 64.9% red deer and 83.6% roe deer, respectively. Co-infections were found in 62.2% red deer and 79.9% roe deer. Nested PCR and sequence analysis of partial msp4 and 18S rRNA genes were performed for molecular characterization of A. phagocytophilum strains and Babesia species. A total of eleven A. phagocytophilum msp4 gene sequence variants were identified: five different variants were 100% identical to corresponding A. phagocytophilum sequences deposited in the GenBank database, while other six sequence variants had unique nucleotide polymorphisms. Sequence analysis of the 18S rRNA gene demonstrated the presence of multiple Babesia species, including Babesia capreoli, Babesia divergens, Babesia venatorum and Babesia odocoilei/Babesia cf. odocoilei. This study is the first report demonstrating the prevalence and molecular characterization of A. phagocytophilum strains and Babesia species in roe deer and red deer in Norway. The high infection and co-infection rates with A. phagocytophilum and Babesia spp. in red deer and roe deer suggest that these cervids may play an important role in the transmission of single and multiple pathogens.

Tetramethylpyrazine-Inducible Promoter Region from Rhodococcus jostii TMP1.

An inducible promoter region, PTTMP (tetramethylpyrazine [TTMP]), has been identified upstream of the tpdABC operon, which contains the genes required for the initial degradation of 2,3,5,6-tetramethylpyrazine in Rhodococcus jostii TMP1 bacteria. In this work, the promoter region was fused with the gene for the enhanced green fluorescent protein (EGFP) to investigate the activity of PTTMP by measuring the fluorescence of bacteria. The highest promoter activity was observed when bacteria were grown in a nutrient broth (NB) medium supplemented with 5 mM 2,3,5,6-tetramethylpyrazine for 48 h. Using a primer extension reaction, two transcriptional start sites for tpdA were identified, and the putative −35 and −10 promoter motifs were determined. The minimal promoter along with two 15 bp long direct repeats and two 7 bp inverted sequences were identified. Also, the influence of the promoter elements on the activity of PTTMP were determined using site-directed mutagenesis. Furthermore, PTTMP was shown to be induced by pyrazine derivatives containing methyl groups in the 2- and 5-positions of the heterocyclic ring, in the presence of the LuxR family transcriptional activator TpdR.

Zanamivir Diminishes Lung Damage in Influenza A Virus-infected Mice by Inhibiting Nitric Oxide Production.

BACKGROUND/AIM: Severe pulmonary influenza A virus (IAV) infection causes lung inflammation and expression of inducible nitric oxide synthase (iNOS), leading to overproduction of nitric oxide (NO). We studied whether zanamivir reduces pulmonary inflammation through inhibition of NO production in mice. MATERIALS AND METHODS: We treated IAV-infected mice daily with intranasal zanamivir. Controls were infected and either placebo-treated or untreated, or not infected and placebo-treated. Mice were weighed daily. After euthanasia on day 3, lungs were excised and bronchoalveolar lavage was performed and fluid nitrite concentration was determined. Lungs were analyzed microscopically. iNOS and IAV RNA levels in lungs were assessed using quantitative reverse transcription-polymerase chain reaction (RT-qPCR). RESULTS: Mice undergoing zanamivir treatment had less weight loss, viral replication, and lung damage, as well as significant reductions of local NO and iNOS mRNA synthesis (p<0.05). CONCLUSION: Zanamivir is associated with an anti-inflammatory effect mediated through inhibition of NO production in IAV-infected mice.

A case of successfully treated relapsing peritoneal dialysis-associated peritonitis caused by Gordonia bronchialis in a farmer.

Gordonia species are aerobic, weakly acid-fast, Gram-positive pathogens that rarely cause human infections, usually in immunocompromised patients. It is uncommon bacilli in cases of peritoneal dialysis-related peritonitis. The small number of infections with Gordonia species reported for humans may be stipulated by the difficulty in identifying the organism using conventional techniques. Careful review of Gram stains and modified-acid-fast stains should be done, so that confusion with other actinomycetes is minimized, pending the genotypic identification. Here we report a case that was caused by Gordonia bronchialis and thus required different considerations of treatment.

Modified Nucleotides as Substrates of Terminal Deoxynucleotidyl Transferase.

The synthesis of novel modified nucleotides and their incorporation into DNA sequences opens many possibilities to change the chemical properties of oligonucleotides (ONs), and, therefore, broaden the field of practical applications of modified DNA. The chemical synthesis of nucleotide derivatives, including ones bearing thio-, hydrazino-, cyano- and carboxy groups as well as 2-pyridone nucleobase-containing nucleotides was carried out. The prepared compounds were tested as substrates of terminal deoxynucleotidyl transferase (TdT). The nucleotides containing N4-aminocytosine, 4-thiouracil as well as 2-pyridone, 4-chloro- and 4-bromo-2-pyridone as a nucleobase were accepted by TdT, thus allowing enzymatic synthesis of 3'-terminally modified ONs. The successful UV-induced cross-linking of 4-thiouracil-containing ONs to TdT was carried out. Enzymatic post-synthetic 3'-modification of ONs with various photo- and chemically-reactive groups opens novel possibilities for future applications, especially in analysis of the mechanisms of polymerases and the development of photo-labels, sensors, and self-assembling structures.

ß-Glucan from Saccharomyces cerevisiae Induces IFN-γ Production In Vivo in BALB/c Mice.

AIM: ß-Glucan is one of the most abundant polymers in nature and has been established as an immunomodulator. This compound has notable physiological effects on mammalian immune systems, including anti-tumor and anti-infective activities and can activate the immune response. It is considered that the immune-stimulating activities of ß-glucan can depend on physicochemical parameters, such as molecular size. Saccharomyces cerevisiae, also known as baker's yeast, is a frequently used source of ß-glucan. The aim of the experiments was to investigate how different Saccharomyces cerevisiae ß-glucan preparations with different molecular size affect interferon-gamma (IFN-γ) production in BALB/c mice. MATERIALS AND METHODS: In vivo and in vitro BALB/c mouse models were used for the investigations. Different ß-glucan preparations were orally administrated in the in vivo experiments. IFN-γ production in BALB/c mice was analyzed by enzyme-linked immunosorbent assay and measuring interferon-γ RNA concentration. RESULTS: The results showed that orally-administered ß-glucan from S. cerevisiae enhanced IFN-γ production in BALB/c mice in the in vivo model, but not by mouse leukocytes in vitro. Moreover, water-soluble ß-glucan enhanced IFN-γ production more effectively than did particulate ß-glucan. CONCLUSION: IFN-γ plays an important role in immunity against viral and bacterial infections. Our experiments have shown that ß-glucan preparations enhance IFN-γ production in BALB/c mice and can be potentially used for immune system stimulation in mammals. Current results may be used to develop soluble ß-glucan nutritional supplements.

Preclinical study of SZ2080 material 3D microstructured scaffolds for cartilage tissue engineering made by femtosecond direct laser writing lithography.

Over the last decade DLW employing ultrafast pulsed lasers has become a well-established technique for the creation of custom-made free-form three-dimensional (3D) microscaffolds out of a variety of materials ranging from proteins to biocompatible glasses. Its potential applications for manufacturing a patient's specific scaffold seem unlimited in terms of spatial resolution and geometry complexity. However, despite few exceptions in which live cells or primitive organisms were encapsulated into a polymer matrix, no demonstration of an in vivo study case of scaffolds generated with the use of such a method was performed. Here, we report a preclinical study of 3D artificial microstructured scaffolds out of hybrid organic-inorganic (HOI) material SZ2080 fabricated using the DLW technique. The created 2.1 × 2.1 × 0.21 mm(3) membrane constructs are tested both in vitro by growing isolated allogeneic rabbit chondrocytes (Cho) and in vivo by implanting them into rabbit organisms for one, three and six months. An ex vivo histological examination shows that certain pore geometry and the pre-growing of Cho prior to implantation significantly improves the performance of the created 3D scaffolds. The achieved biocompatibility is comparable to the commercially available collagen membranes. The successful outcome of this study supports the idea that hexagonal-pore-shaped HOI microstructured scaffolds in combination with Cho seeding may be successfully implemented for cartilage tissue engineering.

Development of a multiple-run high-resolution melting assay for Salmonella spp. genotyping HRM application for Salmonella spp. subtyping.

A quick and robust Salmonella spp. differentiation method based on high-resolution DNA melting (HRM) was developed. DNA samples from 134 Salmonella spp. strains and 20 serotypes were tested. Each serotype was represented by at least 2 strains. All raw data were derived on the Rotor-Gene 65H0-100 system using the designed 8 primer pairs. The reference samples for HRM error evaluation between runs were applied. Raw data error minimization and fluorescence normalization between runs were carried out by application of the proposed calculations. The data analysis showed that repetitive sequence targets are much more informative than the nonrepetitive ones. The method possesses a high potential and can be adopted for further subtyping analyses.