Bacteria of the genus Cutibacterium are Gram-positive commensals and opportunistic pathogens that represent a major challenge in the diagnosis and treatment of implant-associated infections (IAIs). This study provides insight into the distribution of different sequence types (STs) of C. acnes, and the presence of virulence factors (VFs) in 64 Cutibacterium spp. isolates from suspected or confirmed IAIs obtained during routine microbiological diagnostics. Fifty-three C. acnes, six C. avidum, four C. granulosum, and one C. namnetense isolate, collected from different anatomical sites, were included in our study. Using whole-genome sequencing and a single-locus sequencing typing scheme, we successfully characterized all C. acnes strains and revealed the substantial diversity of STs, with the discovery of six previously unidentified STs. Phylotype IA₁, previously associated with both healthy skin microbiome and infections, was the most prevalent, with ST A1 being the most common. Some minor differences in STs’ distribution were observed in correlation with anatomical location and association with infection. A genomic analysis of 40 investigated VFs among 64 selected strains showed no significant differences between different STs, anatomical sites, or infection-related and infection undetermined/unlikely groups of strains. Most differences in VF distribution were found between strains of different Cutibacterium spp., subspecies, and phylotypes, with CAMP factors, biofilm-related VFs, lipases, and heat shock proteins identified in all analyzed Cutibacterium spp.

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.

The effective identification of bacterial and fungal isolates is essential for microbiological monitoring in environments like speleotherapeutic caves. This study compares MALDI-TOF MS and the OmniLog ID System, two high-throughput culture-based identification methods. MALDI-TOF MS identified 80.0% of bacterial isolates to the species level, while the OmniLog ID System identified 92.9%. However, species-level matches between the methods were only 48.8%, revealing considerable discrepancies. For discrepant results, MALDI-TOF MS matched molecular identification at the genus level in 90.5% of cases, while the OmniLog ID System matched only in 28.6%, demonstrating MALDI-TOF MS’s superiority. The OmniLog ID System had difficulties identifying genera from the order Micrococcales. Fungal identification success with MALDI-TOF MS was 30.6% at the species level, potentially improvable with a customised spectral library, compared to the OmniLog ID System’s 16.7%. Metagenomic approaches detected around 100 times more microbial taxa than culture-based methods, highlighting human-associated microorganisms, especially Staphylococcus spp. In addition to Staphylococcus spp. and Micrococcus spp. as indicators of cave anthropisation, metagenomics revealed another indicator, Cutibacterium acnes. This study advocates a multi-method approach combining MALDI-TOF MS, the OmniLog ID System, culture-based, and metagenomic analyses for comprehensive microbial identification. Metagenomic sampling on nitrocellulose filters provided superior read quality and microbial representation over liquid sampling, making it preferable for cave air sample collection.

Campylobacter concisus is recognized as a potential pathogen in gastrointestinal diseases, particularly in patients with chronic intestinal diseases. This study investigates the genomic characteristics, phylogenetic distribution, virulence factors, resistance genes and presence of plasmids in C. concisus isolates from Slovenian patients with community-acquired infectious diarrhoea. Prospectively collected isolates were analysed using whole-genome sequencing (WGS). WGS analysis revealed substantial genetic diversity among isolates, with distinct differences observed between two genomospecies (GS1, GS2). GS1 isolates had smaller genomes, lower GC content, and fewer coding regions than GS2 isolates. Multilocus sequence typing confirmed a high degree of genetic diversity, with most isolates belonging to novel sequence types. Plasmids, including pSma1 and pICON, were more prevalent in GS2 isolates. The virulence factors Zot and Exo9 toxins were detected in both genomospecies, with Zot predominantly found in GS1 and Exo9 in GS2. The T6 secretion system was prevalent in both groups, whereas the T4SS was less frequently observed. The combination of the T6SS, plasmids, and toxins suggests a complex mechanism of pathogenicity. This study highlights the high genetic diversity of C. concisus and provides new insights into its genomic features and virulence factors. The presence of plasmids and secretion systems, particularly the T6SS, underscores the potential of C. concisus for adaptation and pathogenicity.

This study aimed to employ advanced bioinformatics and modern sequencing approaches to solve a diagnostic problem of persistent Campylobacter spp. molecular detection yet negative culture results from four consecutive stool samples of a previously healthy patient with newly diagnosed selective IgA deficiency with prolonged diarrhoea METHODS: Metagenomic next-generation sequencing (mNGS) based on short-paired end reads with basic bioinformatic read classification analysis was used at first. Due to confusing results, advanced bioinformatics involving contigs construction and classification, reference genome mappings and reads filtering with BBSplit, additionally coupled with metagenomic long-reads sequencing and Full-length 16S rRNA metabarcoding were employed to further elucidate the results. Virulence factors were analysed using the Prokka Genome Annotation tool. Modified classical bacteriology methods were finally used for further clarification. Short-pair end reads analysis identified several Campylobacter species in all four samples. After advanced bioinformatic approaches were applied, candidatus C. infans was suspected as the putative pathogen. This result was further supported by metagenomic long-reads sequencing and Full-length 16S rRNA metabarcoding. Nevertheless, after modifying the culture conditions based on mNGS results, a mixed culture of candidatus C. infans and C.ureolyticus was obtained. Sequencing of the mixed culture resulted in an 87.48 % and 73.47 % genome coverage of candidatus C. infans and C. ureolyticus, respectively. In the candidatus C. infans genome more virulence factors hits were found than in the C. ureolyticus genome thus supporting the first as the most probable cause of symptoms. This study shows the pivotal role and strengths of mNGS in unravelling an unusual case of diarrhoea and demonstrates how mNGS can guide established microbiological methods to improve on current limitations. However, it also emphasises the need for careful interpretation of sequencing data, particularly for closely related bacterial species from clinical samples that are known to support complex microbial communities.