Carbapenemase and extended-spectrum beta-lactamase-producing bacteria in waters originating from a single landfill in Slovenia.

Groundwater, rainwater, and leachate associated with a single landfill were analysed to detect extended-spectrum beta-lactamase (ESBL)-producing and carbapenemase (CP)-producing bacteria. After cultivation on three commercial selective-differential media, 240 bacterial isolates were obtained and identified by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Isolates from clinically relevant species were further genotyped by enterobacterial repetitive intergenic consensus polymerase chain reaction, and tested for antibiotic susceptibility and presence of CPs and ESBL enzymes. Two ESBL-producing isolates and two isolates producing CPs were detected in rainwater, groundwater, and leachate: Klebsiella oxytoca complex with the gene for the ESBL enzyme CTX-M-1 and the gene for the CP OXA-48, Serratia fonticola with the gene for the ESBL enzyme FONA-2, and Pseudomonas aeruginosa with the gene coding Verona integron-encoded Metallo-beta-lactamases (VIM) metallo-beta-lactamase. Our study indicates that bacteria with ESBL and CP genes can be present in landfill-associated waters.

Different sampling strategies for optimal detection of the overall genetic diversity of methicillin-resistant Staphylococcus aureus.

Surveillance schemes for methicillin-resistant Staphylococcus aureus (MRSA) are widely established at the national and international levels. Due to the simple standardization of the protocol, mainly isolates from bloodstream infections are used. However, the limitations of this simple surveillance system are well described. We conducted a comprehensive analysis of MRSA isolates in a large Slovenian region over 5 years to identify the optimal sample group for assessing the overall MRSA diversity. At the same time, this study provides to date non-available molecular characterization of Slovenian MRSA isolates. A total of 306 MRSA isolates from various sources were sequenced and phenotypically tested for resistance. The isolates exhibited significant molecular diversity, encompassing 30 multi locus sequence type (MLST) sequence types (STs), 39 ST-SCCmec genetic lineages, 49 spa types, and 29 antibiotic resistance profiles. Furthermore, the isolate pool comprised 57 resistance genes, representing 22 resistance mechanisms, and 96 virulence genes. While bloodstream isolates, commonly used in surveillance, provided insights into frequently detected clones, they overlooked majority of clones and important virulence and resistance genes. Blood culture isolates detected 21.3% spa types, 24.1% resistance phenotypes, and 28.2% MLST-SCCmec profiles. In contrast, strains from soft tissues demonstrated superior genomic diversity capture, with 65.3% spa types, 58.6% resistance phenotypes, and 71.8% MLST-SCCmec profiles. These strains also encompassed 100.0% of virulence and 82.5% of resistance genes, making them better candidates for inclusion in surveillance programs. This study highlights the limitations of relying solely on bloodstream isolates in MRSA surveillance and suggests incorporating strains from soft tissues to obtain a more comprehensive understanding of the epidemiology of MRSA.IMPORTANCEIn this study, we investigated the diversity of methicillin-resistant Staphylococcus aureus (MRSA), a bacterium that can cause infections that are difficult to treat due to its resistance to antimicrobial agents. Currently, surveillance programs for MRSA mainly rely on isolates from bloodstream infections, employing a standardized protocol. However, this study highlights the limitations of this approach and introduces a more comprehensive method. The main goal was to determine which group of samples is best suited to understand the overall diversity of MRSA and to provide, for the first time, molecular characterization of Slovenian MRSA isolates. Our results suggest that including MRSA strains from soft tissue infections rather than just blood infections provides a more accurate and comprehensive view of bacterial diversity and characteristics. This insight is valuable for improving the effectiveness of surveillance programs and for developing strategies to better manage MRSA infections.

Description of Komagataeibacter melaceti sp. nov. and Komagataeibacter melomenusus sp. nov. Isolated from Apple Cider Vinegar.

Two novel strains AV382 and AV436 were isolated from a submerged industrial bioreactor for production of apple cider vinegar in Kopivnik (Slovenia). Both strains showed very high (≥98.2%) 16S rRNA gene sequence similarities with Komagataeibacter species, but lower 16S-23S rRNA gene internal transcribed spacer (ITS). The highest similarity of the 16S-23S rRNA gene ITS of AV382 was to Komagataeibacter kakiaceti LMG 26206T (91.6%), of AV436 to Komagataeibacter xylinus LMG 1515T (93.9%). The analysis of genome sequences confirmed that AV382 is the most closely related to K. kakiaceti (ANIb 88.2%) and AV436 to K. xylinus (ANIb 91.6%). Genome to genome distance calculations exhibit for both strains ≤47.3% similarity to all type strains of the genus Komagataeibacter. The strain AV382 can be differentiated from its closest relatives K. kakiaceti and Komagataeibacter saccharivorans by its ability to form 2-keto and 5-keto-D-gluconic acids from glucose, incapability to grow in the presence of 30% glucose, formation of C19:0 cyclo ω8c fatty acid and tolerance of up to 5% acetic acid in the presence of ethanol. The strain AV436 can be differentiated from its closest relatives K. xylinus, Komagataeibacter sucrofermentans, and Komagataeibacter nataicola by its ability to form 5-keto-D-gluconic acid, growth on 1-propanol, efficient synthesis of cellulose, and tolerance to up to 5% acetic acid in the presence ethanol. The major fatty acid of both strains is C18:1ω7c. Based on a combination of phenotypic, chemotaxonomic and phylogenetic features, the strains AV382T and AV436T represent novel species of the genus Komagataeibacter, for which the names Komagataeibactermelaceti sp. nov. and Komagataeibacter melomenusus are proposed, respectively. The type strain of Komagataeibacter melaceti is AV382T (= ZIM B1054T = LMG 31303T = CCM 8958T) and of Komagataeibacter melomenusus AV436T (= ZIM B1056T = LMG 31304T = CCM 8959T).