Rotavirus vaccination started in Slovenia in 2007 on a voluntarily basis. The vaccination rate is relatively low (up to 27%) and no increasing trend is observed. We present rotavirus genotype distribution among children hospitalized for rotavirus gastroenteritis in Slovenia. Eight consecutive rotavirus seasons were followed, from 2005/06 to 2012/13, and 113 strains of the most common rotavirus genotypes were randomly selected for molecular characterization of rotavirus VP7 and VP4 (VP8(∗)) genome segments. During the vaccine introduction period, from 2007 to 2013, rotavirus genotype prevalences changed, with G1P[8] decreasing from 74.1% to 8.7% between 2007/08 and 2010/11 seasons, replaced by G4P[8] and G2P[4], with up to 52.0% prevalence. Comparable analysis of VP7 and VP8(∗) genome fragments within G1P[8] genotype lineages revealed considerable differences for rotavirus strains circulating before and during the vaccination period. The G1P[8] rotavirus strains from the pre-vaccination period clustered in a phylogenetic tree within Rotarix®-like VP7 and VP8(∗) lineages. However, since 2007, the majority of G1P[8] strains have shifted to distant genetic lineages with lower nucleotide (88.1-94.0% for VP7 and 86.6-91.1% for VP8(∗)) and amino acid (93.8-95.2% for VP7 and 85.3-94.6% for VP8(∗)) identities to the vaccine Rotarix® strain. This change also resulted in a different deduced amino acid profile at the major VP7 and VP8(∗) antigenic epitopes.

Contamination of cell cultures is the most common problem encountered in cell culture laboratories. Besides the secondary cell contaminations often occurring in the cell laboratories, the contaminations originating from donor animal or human tissue are equally as common, but usually harder to recognize and as such require special attention. The present study describes the detection of porcine adenovirus (PAdV), strain PAdV-SVN1 in cultures of normal porcine urothelial (NPU) cells isolated from urinary bladders of domestic pigs. NPU cell cultures were evaluated by light microscopy (LM), polymerase chain reaction (PCR), and additionally assessed by transmission electron microscopy (TEM). Characteristic ultrastructure of virions revealed the infection with adenovirus. The adenoviral contamination was further identified by the sequence analysis, which showed the highest similarity to recently described PAdV strain PAdV-WI. Additionally, the cell ultrastructural analysis confirmed the life-cycle characteristic for adenoviruses. To closely mimic the in vivo situation, the majority of research on in vitro models uses cell cultures isolated from human or animal tissue and their subsequent passages. Since the donor tissue could be a potential source of contamination, the microbiological screening of the excised tissue and harvested cell cultures is highly recommended.

Viruses are the most common causative agents of inflammation in the oral cavity and throat region. Most respiratory tract infections are self-limiting and require no specific treatment. However, patients often use different self-medication therapies that can treat both the symptoms and the cause. Throat lozenges with a fixed combination of benzydamine hydrochloride and cetypiridinium chloride (BH/CPC) have been shown to provide effective symptomatic relief for sore throat, but their effect on viruses has not been investigated to date. The antiseptic, cetylpyridinium chloride (CPC), has already been described as a successful bactericide. In addition, there are some studies suggesting its efficacy against certain enveloped viruses. Thus, the aim of our study was to examine the virucidal activity of CPC and a combination of BH/CPC as a free active substance or as lozenge on SARS-CoV-2 in vitro. Under in-laboratory simulated conditions of lozenge administration, we incubated SARS-CoV-2 with three different concentrations of each of the active substances, CPC, free BH/CPC or BH/CPC, as a lozenge suspension for 1 min, 5 min and 15 min of contact time. Infective viral particles were detected in cell cultures and the viral titre was calculated accordingly. Our results show that all active substances in high-concentration suspensions, as well as a medium concentration of the BH/CPC combination, exhibited a 4-log reduction in viral titre. Additionally, the highest concentration of BH/CPC as a lozenge had a faster virucidal effect compared to CPC as a free active substance alone, since a contact time as short as 1 min reduced the initial virus concentration by more than 4-log. This study demonstrates the effective strong virucidal effect of the lozenge, with the possibility of viral load reduction in the oral cavity and, consequently, reduced risk of viral transmission.

Enterococcus faecalis, responsible for a majority of human and nosocomial enterococcal infections, is intrinsically resistant to aminoglycoside antibiotics (such as gentamicin, GEN), which must be used in a combined therapy to be effective. Nitroxoline (NTX) is an old antibiotic, underused for decades, but rediscovered now in an era of growing antibiotic resistance. In this in vitro study, the types of interactions between NTX and GEN on 29 E. faecalis strains were analyzed with an aim to find synergistic antimicrobial and antiadhesion combinations. Transmission electron microscopy (TEM) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to analyze changes in cell morphology and bacterial proteome after monotreatments and combined treatments. The results showed the synergistic effect for six combinations on eight strains, including the ATCC29212, and an additive effect for most strains. Combinations causing a complete inhibition of adhesion were established. Cell membrane integrity was affected by NTX, while combined NTX/GEN treatment caused dramatic changes in cell morphology. Upregulation of the expression of many proteins was established, with some emerging only after combined treatment. The results strongly imply that NTX has the potential for use in combined therapy with GEN against enterococci and it could further provide a substantial contribution to an ongoing fight against antimicrobial resistance and nosocomial infections.

Astrocytes, an abundant type of glial cells, are the key cells providing homeostasis in the central nervous system. Due to their susceptibility to infection, combined with high resilience to virus-induced cell death, astrocytes are now considered one of the principal types of cells, responsible for virus retention and dissemination within the brain. Autophagy plays an important role in elimination of intracellular components and in maintaining cellular homeostasis and is also intertwined with the life cycle of viruses. The physiological significance of autophagy in astrocytes, in connection with the life cycle and transmission of viruses, remains poorly investigated. In the present study, we investigated flavivirus-induced modulation of autophagy in human astrocytes by monitoring a tandem fluorescent-tagged LC3 probe (mRFP-EGFP-LC3) with confocal and super-resolution fluorescence microscopy. Astrocytes were infected with tick-borne encephalitis virus (TBEV) or West Nile virus (WNV), both pathogenic flaviviruses, and with mosquito-only flavivirus (MOF), which is considered non-pathogenic. The results revealed that human astrocytes are susceptible to infection with TBEV, WNV and to a much lower extent also to MOF. Infection and replication rates of TBEV and WNV are paralleled by increased rate of autophagy, whereas autophagosome maturation and the size of autophagic compartments are not affected. Modulation of autophagy by rapamycin and wortmannin does not influence TBEV and WNV replication rate, whereas bafilomycin A1 attenuates their replication and infectivity. In human astrocytes infected with MOF, the low infectivity and the lack of efficient replication of this flavivirus are mirrored by the absence of an autophagic response.

Background: Enterococcus faecalis is a major cause of complicated urinary tract infections (UTIs), characterized by intrinsic resistance and pronounced biofilm formation. Nitroxoline (NTX), a metal-chelating uroantiseptic, accumulates in urine and exhibits antibiofilm activity. Hydroquinone (HQ), the active urinary metabolite of arbutin-containing herbal preparations, is also excreted into urine and may contribute to antimicrobial activity in situ. This study investigated the antimicrobial and antibiofilm effects of NTX and HQ, individually and in combination, against uropathogenic E. faecalis isolates. Methods: Minimum inhibitory (MIC), bactericidal (MBC), and anti-adhesion (MAC) concentrations were determined using broth microdilution. Interaction was assessed by the checkerboard method and expressed as the fractional inhibitory concentration index (FICI). Biofilm inhibition was quantified by colony-forming unit (CFU) enumeration following exposure to subinhibitory concentrations. Ultrastructural alterations of E. faecalis following exposure to NTX and HQ were examined by transmission electron microscopy (TEM). Results: NTX demonstrated MIC values ranging from 0.002–0.016 mg/mL (MIC50/MIC90: 0.004/0.008 mg/mL), while HQ exhibited MIC values of 0.78–1.56 mg/mL (MIC50/MIC90: 0.78/1.56 mg/mL). Synergistic interactions (FICI ≤ 0.5) were observed in selected isolates, with up to eightfold and sixteenfold reductions in NTX and HQ concentrations, respectively. Additive effects predominated in the remaining isolates without antagonism. The combination achieved 3–5 log₁₀ reductions in adherent bacterial counts compared to untreated controls and up to 4 log₁₀ reductions compared to single-agent exposure. In several strains, complete inhibition of adhesion was observed. TEM analysis revealed marked envelope disruption, cytoplasmic condensation, and structural collapse following combined treatment. Conclusions: Given that both NTX and HQ are active within the urinary environment, their combination may represent a pharmacologically relevant strategy targeting both bacterial growth and early biofilm establishment in enterococcal UTIs. These findings support further in vivo and pharmacokinetic investigations to evaluate the clinical applicability of this combination.