Objectives: This study presents the standardized protocols developed by the Clinical-Based Human Microbiome Research and Development Project (cHMP) in the Republic of Korea.Methods: It addresses clinical metadata collection, specimen handling, DNA extraction, sequencing methods, and quality control measures for microbiome research.Results: The cHMP involves collecting samples from healthy individuals and patients across various body sites, including the gastrointestinal tract, oral cavity, respiratory system, urogenital tract, and skin. These standardized procedures ensure consistent data quality through controlled specimen collection, storage, transportation, DNA extraction, and sequencing. Sequencing encompasses both amplicon and whole metagenome methods, followed by stringent quality checks. The protocols conform to international guidelines, ensuring that the data generated are both reliable and comparable across microbiome studies.Conclusion: The cHMP underscores the importance of methodological standardization in enhancing data integrity, reproducibility, and advancing microbiome-based research with potential applications for improving human health outcomes.

Vancomycin-variable enterococci (VVE), though genetically containing van genes, are phenotypically sensitive to vancomycin. If VVE is undetected or does not grow on the vancomycin-resistant enterococci (VRE) selection medium, or both, it can acquire resistance upon exposure to vancomycin. This characteristic is clinically important for the treatment and prevention of VRE. This study aims to analyze the prevalence and characteristics of VVE in Korea through the Global Antibiotic Resistance Surveillance System (Kor-GLASS) and emphasize the importance of VVE. A total of 3342 enterococcal bloodstream isolates were collected through the Kor-GLASS between 2017 and 2022. Antibiotic susceptibility testing, van gene detection, and multilocus sequence typing were conducted with all the isolates. The trends in the domestic prevalence of VVE were analyzed and compared with global prevalence data. Among the isolates, 197 (5.9%), including 124 Enterococcus faecium and 73 E. faecalis, were identified as VVE. While the VRE incidence has declined in Korea since 2020, the VVE incidence is significantly rising. In Korea, only the vanA gene has been detected in both VRE and VVE, and no other van gene variants have been identified. Most of these isolates belong to CC17 (91.3%), with ST17, ST817, and ST80 as the predominant types. We have shown that continuous surveillance is essential in Korea due to the persistently high prevalence of VRE and the potential evolution of VVE into VRE. Consequently, it is critical to evaluate Enterococcus species isolated from domestic clinical settings for their phenotypic vancomycin resistance and the molecular detection of van genes, irrespective of the strain.

Bacterial two-component regulatory systems (TCRS) are associated with the expression of virulence factors and antibiotic susceptibility. In Staphylococcus aureus, 16 TCRS types have been identified. The histidine kinase/response regulator SAV1321/SAV1322 in the S. aureus shares considerable homology with the TCRS DesKR in Bacillus subtilis. However, a function for the SAV1322 locus has not yet been assigned. Deletion of the SAV1322 locus in S. aureus results in reduced growth when cultured under low (25 °C) and high (46 °C) temperature conditions. The sav1322 deletion mutant is more tolerant to oxidative stress in vitro and is less pathogenic in a murine infection model when compared with wild-type parent strain Mu50. Furthermore, the sav1322 mutant exhibits lower MICs for gentimicin, tetracyclines and glycopeptides, increased autolysis, and a thinner cell wall when compared with the wild-type strain. Microarray and proteomic analyses show that the expression of cell-wall-associated genes glmS and murZ are lower, and the expression of heat shock and stress-related genes (hrcA, ctsR, dnaK, dnaJ, grpE, clpB, and clpC) are higher in the sav1322 mutant when compared with the wild-type strain. In addition, the sav1322 mutant displays altered expression of proteins involved in carbohydrate/energy metabolism, cell wall metabolism, and stress or heat shock response, as well as other metabolic processes including lipid metabolism, amino acid biosynthesis, purine or pyrimidine metabolism, transcription, and protein biosynthesis. The S. aureus SAV1322 locus plays a pronounced role in temperature adaptation, antibiotic resistance, and virulence by regulating a wide range of genes and proteins involved in metabolism and stress tolerance.

Jung Wook Kim, Ph.D. and Kwang Jun Lee, Ph.D.. Ann Lab Med 2023;43:355-63. https://doi.org/10.3343/alm.2023.43.4.355

Objectives: Linezolid resistance among Enterococcus species poses a growing clinical and public health concern, especially due to the dissemination of transferable resistance genes, such as optrA. This study aimed to evaluate the prevalence of linezolid resistance and to characterize the molecular epidemiology and genetic contexts of optrA-positive linezolid-resistant Enterococcus (LRE) isolates from clinical and animal sources in South Korea. Methods: A total of 2156 Enterococcus isolates, collected through nationwide surveillance from hospitalized patients and healthy livestock (pigs, cattle, and chickens) between 2017 and 2019, were retrospectively analyzed. Phenotypic susceptibility testing, optrA gene screening, and whole-genome sequencing were performed to investigate genetic environments and phylogenetic relationships. Results: The prevalence of linezolid resistance was 0.2% in clinical isolates, 3.3% in pigs, 4.3% in cattle, and 1.4% in chickens. optrA-positive linezolid-resistant isolates were less frequent, with rates of 0.1%, 1.4%, 0.9%, and 1.0%, respectively. Multilocus sequence typing identified sequence types (STs) 330 and ST476 in E. faecalis from humans, with no shared STs between human and livestock isolates. The optrA gene was located either chromosomally, frequently associated with transposon Tn6674, or on multidrug resistance plasmids. Notably, optrA variants exhibited host-specific distribution patterns. Phylogenetic analysis demonstrated considerable genomic diversity, and Korean ST476 isolates were genetically related to international strains reported from livestock, poultry products, and wild birds, suggesting potential global dissemination. Conclusions: This study provides a comprehensive, nationally representative assessment of linezolid resistance in South Korea. The findings highlight the zoonotic potential and possible international dissemination of optrA-carrying ST476 lineages, underscoring the need for integrated One Health surveillance to monitor and control the spread of transferable resistance genes.

Carbapenem-resistant Enterobacterales (CRE) constitute an urgent global health threat, and microbiota-based strategies are increasingly being sought as alternatives to conventional antibiotics. To identify a probiotic candidate with potent anti-CRE activity, we isolated human-derived Lactiplantibacillus plantarum LP1 and performed integrated phenotypic and genomic analyses. L. plantarum LP1 displayed outstanding gastrointestinal tolerance (relative survival/proliferation rate of 113.22%) and high adhesion to Caco-2 cells (99.01%), outperforming the reference strain L. plantarum ATCC 14917, while cell-free supernatants completely inhibited CRE growth and removed up to 82.31 ± 2.24% of preformed biofilms. Hybrid whole-genome sequencing produced a complete 3.31 Mb circular chromosome assembled as a single contig, comprising 3,109 coding sequences and more than 500 genes absent from the reference strain L. plantarum ATCC 14917, including 2,977 clusters of orthologous groups (COG)-classified genes (216 related to carbohydrate transport and metabolism) and 53 carbohydrate-active enzymes, consistent with the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of carbohydrate metabolic pathways. BAGEL4 analysis identified a class IIb plantaricin gene cluster, supporting a bacteriocin-mediated mechanism of CRE inhibition. Collectively, these findings demonstrate that L. plantarum LP1 possesses strain-specific expansion of metabolic and antimicrobial functions that underpin its strong anti-CRE activity and highlight its potential as a next-generation probiotic.