Marius Surleac

The gut microbiome influences host health, affecting gastrointestinal, metabolic, immune, cardiovascular, and neurological functions. A balanced microbiome is associated with favourable health outcomes. However, excessive antibiotic use and dietary habits can disrupt this ecosystem, leading to dysbiosis and affecting body homeostasis. We present the first comprehensive metagenomic analysis of the gut microbiome in a healthy Romanian cohort. With no prior high-resolution profiling on this population, characterized by high antibiotic consumption, this cohort contributes to understanding microbiome variation in European populations. We report microbiome features consistent with other European populations, including well-defined community configurations, and provide new insights into how these relate to within-phylum diversity. We observe an enrichment of Enterobacteriaceae , a pattern that may be shaped by population-level exposures, including antibiotic use. The analysis of antimicrobial resistance genes, contextualized with data from other cohorts and the European Centre for Disease Prevention and Control, showed an increased prevalence of genes linked to beta-lactams, macrolides, and quinolones—antibiotics commonly used in this population. Finally, we investigate the relationship between the microbial profile and the systemic immune responses, inferred from correlations with in vitro cytokine production. Notably, we identify a potential immune-priming role for Collinsella species and a link between the Prevotella enterotype and the cytokine production capacity. Importance: This first comprehensive study of the healthy gut microbiome in a Romanian cohort contributes to a baseline for the microbiome and resistome composition of this population. While universally accepted definitions of “healthy” microbiomes, or baseline resistomes, remain lacking, such data help contextualise future studies and support the monitoring of dynamics. The Enterobacteriaceae abundance suggests a microbiome composition potentially influenced by antimicrobial consumption, a relevant pattern in a region with a high burden of nosocomial infections. In addition, the prevalence of antimicrobial resistance genes, and the concordance with commonly used antibiotics in the community reinforces the need to address antibiotic use in public health strategies. Although links between the gut microbiome and host immunity are not fully understood, our findings are consistent with a role for microbiome composition in immune-related traits. The association between the Prevotella enterotype and cytokine balance may provide a basis for further investigation of enterotype-specific immune characteristics.

Background/Objectives: This study aims to characterize antibiotic resistance (AR) and virulence markers in Salmonella spp. isolated from Romanian outpatients’ stool samples. Methods: In 2019, community-acquired Salmonella strains were collected and identified using MALDI-TOF mass spectrometry, antibiotic susceptibility profiles have been determined with the MicroScan system, and soluble virulence factors were evaluated using specific culture media, while biofilm formation was quantified in 96-well plates. Molecular analysis targeted resistance genes for β-lactams (e.g., bla_TEM and bla_SHV); tetracyclines (e.g., tet(A)); sulphonamides; and quinolones, as well as virulence genes (e.g., invA, spvC, pldA, and held). Whole-genome sequencing (WGS) was performed on 19 selected isolates. A silver nanoparticles (AgNPsol) alternative to conventional antibiotics was tested for effectiveness against multidrug-resistant (MDR) isolates. Results: From the total of 309 Salmonella isolates (65.05% from children under 4 years of age) belonging to four subtypes and four serovars, 27.86% showed resistance to at least one antibiotic, most frequently to tetracycline, ampicillin, and piperacillin. The strains frequently expressed haemolysin (67%), aesculinase (65%), and gelatinase (62%). Resistance to trimethoprim-sulfamethoxazole was encoded by the sul1 gene in 44.83% of the strains and to tetracyclines by the tet(A) gene (59.52%). The ESBL genes bla_TEM, bla_SHV, and bla_CTX-M were detected by PCR in 16.18%, 2.91%, and 0.65% of the strains, respectively. Additionally, 98.63% of the strains carried the invA marker, with notable positive associations between bla_SHV, qnrB, and sul1 with spvC. Conclusions: The present findings revealed significant patterns in Salmonella isolates, subtypes, serovars, AR, and virulence, emphasising the need for continuous surveillance of Salmonella infections. Additionally, the potential of AgNPs as an alternative treatment option was demonstrated, particularly for paediatric S. enterica infections.

Motivation: Antimicrobial resistance is increasingly recognized as one of the most significant global health threats, with profound implications for human, animal, and environmental health. Genome analysis represents a very useful tool that provides accurate and reproducible results allowing for the advancement of knowledge regarding antimicrobial resistance diagnosis, therapeutics, surveillance, transmission, and evolution. However, due to increasing complexity of bacterial genome analysis and computational power required for genomic approaches, there is a continuous need for comprehensive, user-friendly tools for data analysis. We developed Pangenome and Genomic Analysis Suite (PeGAS), to address some of these challenges by offering an all-in-one pipeline that performs a range of analyses. Results: PeGAS integrates key genomic analysis features of bacteria whole genome sequencing, including the prediction of antimicrobial resistance profiles, sorted by various categories of antibiotics, VF detection, and plasmid replicon assignment. The pipeline also performs pangenome analysis, multilocus sequence typing, genome assembly quality control (by reporting statistics such as GC content, contig length, the number of contigs, as well as variation from certain GC thresholds) providing a comprehensive genomic overview. PeGAS also offers the ability to restart seamlessly from any sporadic interruptions that might occur during long or resource-intensive runs. Availability and implementation: PeGAS is available at: https://github.com/liviurotiul/PeGAS

Influenza viruses continue to be an important public health threat. Vaccination is the most effective measure to control the influenza virus circulation. However, these viruses are continuously evolving through antigenic drift/shift, and thus the vaccine efficiency is affected. The aim of this study was to characterize the viral strains circulating in Romania, in a population with declining vaccination coverage, during the last three cold seasons by evaluating the hemagglutinin antigenic relatedness to the vaccine strains. All the available sequences collected between August 2021 and June 2024 were analyzed by using phylogenetic analysis and the P_epitope model to predict vaccine efficacy. The results showed that the 2021/2022 influenza season was dominated by the circulation of highly diverse clades of A(H3N2) viruses with high mutational divergence as compared to the vaccine strain, which might contribute to the reduction in vaccine efficacy. During the 2022/2023 influenza season, both influenza A and B viruses were reported, with few antigenic site mutations. The 2023/2024 influenza season was dominated by the circulation of influenza A viruses: A/H1N1pdm09 clade 6B.1A.5a.2a and A/H3N2 clade 2a.3a.1. The clade 2a.3a.1 also showed high variability when compared to the vaccine strain, presumably leading to reduced vaccine efficacy. This study illustrates the high diversity of influenza viruses circulating in a population with low vaccination coverage during the previous cold seasons. The viral diversity impacted vaccine efficacy, hence the need for public health programs to increase vaccine uptake and improve vaccine formulation in order to limit viral transmission.

Background/Objectives: Graft-versus-host disease (GVHD) is a severe complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) resulting from histocompatibility differences between donor and host cells leading to inflammation, tissue damage, and compromised patient outcome. Extracorporeal photopheresis (ECP) is considered as a second-line treatment administered to patients with GVHD who do not respond to corticosteroid treatment or who experience a relapse after an initial response and are therefore classified as steroid refractory (SR). The aim of this study is to evaluate the clinical response rates in both pediatric and adult patients with acute (a) or chronic (c) GVHD and to assess the effectiveness of ECP using the real-world data from a single center. Methods: We performed a retrospective study on 30 patients, including 11 pediatric and 19 adult patients who were treated with ECP as a second-, third-, or fourth-line therapy for (a) and (c) GVHD, alongside corticosteroids and other immunomodulatory medications. The median time from aGVHD onset to ECP was 11.5 days (range: 3 days–9 months), while for cGVHD, the median time was 90 days (range: 2 days–9 months). Results: The overall response rate (ORR) in the aGVHD patient population was 60% with a median of 9 procedures (range: 2–20). For cGVHD patients, the ORR was 70% after a median of 23.5 ECP procedures (range: 8–43). Most patients had skin involvement, with ECP achieving an ORR of 81.8% in aGVHD and 77.7% in cGVHD cases. Conclusions: ECP is a beneficial therapy for patients with (a) and (c) GVHD who have not responded to corticosteroids and other forms of immunosuppressive therapy. Specifically, ECP demonstrated efficacy in improving skin and oral symptoms and permitted reductions in or the elimination of their corticosteroid usage. The study found that extending the duration of ECP treatment was associated with better outcomes, and no detectable complications were observed over a 38-week period.

Background: This study provides a comprehensive analysis of Acinetobacter baumannii in aquatic environments and fish microbiota by integrating culture-dependent methods, 16S metagenomics, and antibiotic resistance profiling. Methods: A total of 83 A. baumannii isolates were recovered using culture-dependent methods from intra-hospital infections (IHI) and wastewater (WW) and surface water (SW) samples from two southern Romanian cities in August 2022. The antibiotic susceptibility was screened using disc diffusion, microdilution, PCR, and Whole Genome Sequencing assays. Results: The highest microbial load in the analyzed samples was found in Glina, Bucharest, for both WW and SW samples across all investigated phenotypes. For Bucharest isolates, the resistance levels corresponded to fluoroquinolones > aminoglycosides > β-lactam antibiotics. In contrast, A. baumannii from upstream SW samples in Târgoviște showed the highest resistance to aminoglycosides. The bla_OXA-23 gene was frequently detected in IHI, WW, and SW isolates in Bucharest, but was absent in Târgoviște. Molecular phylogeny revealed the presence of ST10 in Târgoviște isolates and ST2 in Bucharest isolates, while other minor STs were not specifically correlated with a sampling point. Using 16S rRNA sequencing, significant differences in microbial populations between the two locations was identified. The low abundance of Alphaproteobacteria and Actinobacteria in both locations suggests environmental pressures or contamination events. Conclusions: These findings indicate significant fecal contamination and potential public health risks, emphasizing the need for improved water quality monitoring and management.

Background: This study provides a comprehensive analysis of Acinetobacter baumannii in aquatic environments and fish microbiota by integrating culture-dependent methods, 16S metagenomics, and antibiotic resistance profiling. Methods: A total of 83 A. baumannii isolates were recovered using culture-dependent methods from intra-hospital infections (IHI), wastewater (WW) and surface water (SW) samples from two southern Romanian cities in august 2022. The antibiotic susceptibility was screened using disc diffusion, microdilution, PCR and Whole Genome Sequencing assays. Results: The highest microbial load in the analyzed samples was found in Glina, Bucharest for both WW and SW samples across all investigated phenotypes. For Bucharest isolates, the resistance levels corresponded to fluoroquinolones > aminoglycosides > β-lactam antibiotics. In contrast, A. baumannii from upstream SW samples in Targoviste showed the highest resistance to aminoglycosides. The blaOXA-23 gene was frequently detected in IHI, WW, and SW isolates in Bucharest but was absent in Targoviste. Molecular phylogeny revealed presence of ST10 in Târgoviște isolates and ST2 in Bucharest isolates, while other minor STs were not specifically correlated with a sampling point. Using 16S rRNA sequencing, significant differences in microbial populations between the two locations was identified. The low abundance of Alphaproteobacteria and Actinobacteria in both locations suggests environmental pressures or contamination events. Conclusions: These findings indicate significant fecal contamination and potential public health risks, emphasizing the need for improved water quality monitoring and management.

The emerging opportunistic fungal pathogen Candida auris raises significant concerns for public health due to its outbreak potential, the associated high mortality, increased resistance to antifungals, challenging identification to species level, since commonly used diagnostic methods can confuse this fungus with other Candida spp. The present outbreak report describes probably some of the first Candida auris cases in Romania, providing clinical and epidemiological data, and also whole genome sequencing data. The cases were identified in three hospitals in Bucharest during the first eight months of 2022.

Background: Romania is one of the European countries reporting very high antimicrobial resistance (AMR) rates and consumption of antimicrobials. We aimed to characterize the AMR profiles and clonality of 304 multi-drug resistant (MDR) Acinetobacter baumannii (Ab) and Pseudomonas aeruginosa (Pa) strains isolated during two consecutive years (2018 and 2019) from hospital settings, hospital collecting sewage tanks and the receiving wastewater treatment plants (WWTPs) located in the main geographical regions of Romania. Methods: The strains were isolated on chromogenic media and identified by MALDI-TOF-MS. Antibiotic susceptibility testing and confirmation of ESBL- and CP- producing phenotypes and genotypes were performed. The genetic characterization also included horizontal gene transfer experiments, whole-genome sequencing (WGS), assembling, annotation and characterization. Results: Both clinical and aquatic isolates exhibited high MDR rates, especially the Ab strains isolated from nosocomial infections and hospital effluents. The phenotypic resistance profiles and MDR rates have largely varied by sampling point and geographic location. The highest MDR rates in the aquatic isolates were recorded in Galați WWTP, followed by Bucharest. The Ab strains harbored mostly bla_OXA-23, bla_OXA-24, bla_SHV, bla_TEM and bla_GES, while Pa strains bla_IMP, bla_VIM, bla_NDM, bla_VEB, bla_GES and bla_TEM, with high variations depending on the geographical zone and the sampling point. The WGS analysis revealed the presence of antibiotic resistance genes (ARGs) to other antibiotic classes, such as aminoglycosides, tetracyclines, sulphonamides, fosfomycin, phenicols, trimethoprim-sulfamethoxazole as well as class 1 integrons. The molecular analyses highlighted: (i) The presence of epidemic clones such as ST2 for Ab and ST233 and ST357 for Pa; (ii) The relatedness between clinical and hospital wastewater strains and (iii) The possible dissemination of clinical Ab belonging to ST2 (also proved in the conjugation assays for bla_OXA-23 or bla_OXA-72 genes), ST79 and ST492 and of Pa strains belonging to ST357, ST640 and ST621 in the wastewaters. Conclusion: Our study reveals the presence of CP-producing Ab and Pa in all sampling points and the clonal dissemination of clinical Ab ST2 strains in the wastewaters. The prevalent clones were correlated with the presence of class 1 integrons, suggesting that these isolates could be a significant reservoir of ARGs, being able to persist in the environment.