Klebsiella pneumoniae is a notorious human pathogen involved in healthcare-associated infections. The worldwide expansion of infections induced by colistin-resistant and carbapenemase-producing Enterobacterales (CPE) isolates has been increasingly reported. This study aims to analyze the phenotypic and molecular profiles of 10 colistin-resistant (CR) isolates and 2 pairs of colistin-heteroresistant (ChR) (parental and the corresponding resistant mutants) isolates of K. pneumoniae CPE sourced from two hospitals. The phenotypes of strains in the selected collection had been previously characterized. Antimicrobial susceptibility testing was performed using a Vitek 2 Compact system (BioMérieux SA, Marcy l’Etoile, France), the disc diffusion method, and broth microdilution (BMD) for colistin. Whole-genome sequencing (WGS) did not uncover evidence of any mobile colistin resistance (mcr) genes, although the mgrB gene of seven isolates appeared to be disrupted by insertion sequences (ISKpn25 or ISKpn26). Possible deleterious missense mutations were found in phoP (L4F), phoQ (Q426L, L26Q, L224Q, Q317K), pmrB (R256G, P95L, T157P, V352E), and crrB (P151S) genes. The identified isolates belonged to the following clonal lineages: ST101 (n = 6), ST147 (n = 5), ST258 (n = 2), and ST307 (n = 1). All strains harbored IncF plasmids. OXA-48 producers carried IncL and IncR plasmids, while one bla_NDM-1 genome was found to harbor IncC plasmids. Ceftazidime–avibactam remains a therapeutic option for KPC-2 and OXA-48 producers. Resistance to meropenem–vaborbactam has emerged in some bla_kPC-2-carrying isolates. Our study demonstrates that the results of WGS can provide essential evidence for the surveillance of antimicrobial resistance.

Because little is known about the integrons which constitute an important means of spreading resistance in bacteria circulating in Romania, this study aimed to detect antibiotic resistance gene cassettes embedded in integrons in a convenient collection of 60 ciprofloxacin-resistant Escherichia coli isolates of various phylogroups, associated with community-acquired urinary tract infections. Characterization of the integrons was accomplished by PCR, restriction fragment length polymorphism typing, and DNA sequencing of each identified type. More than half of the tested E. coli strains were positive for integrons of class 1 (31 strains) or 2 (1 strain). These strains derived more frequently from phylogenetic groups A (15 of 21 strains), B1 (10 of 14 strains), and F (3 of 4 strains), respectively. While 20 strains carried class 1 integrons which could be assigned to nine types, eleven strains carried integrons that lacked the 3’-end conserved segment. The attempts made to characterize the gene cassettes located within the variable region of the various integrons identified in this study revealed the presence of genes encoding resistance to trimethoprim, aminoglycosides, beta-lactams or chloramphenicol. The evidence of transferable resistance determinants already established in the autochthonous E. coli strains highlights the need for improved control of resistance-carrying bacteria.

The zoonotic Shiga toxin-producing Escherichia coli (STEC) group is unanimously regarded as exceptionally hazardous for humans. This study aimed to provide a genomic perspective on the STEC recovered sporadically from humans and have a foundation of internationally comparable data. Fifty clinical STEC isolates, representing the culture-confirmed infections reported by the STEC Reference Laboratory between 2016 and 2023, were subjected to whole-genome sequencing (WGS) analysis and sequences were interpreted using both commercial and public free bioinformatics tools. The WGS analysis revealed a genetically diverse population of STEC dominated by non-O157 serogroups commonly reported in human STEC infections in the European Union. The O26:H11 strains of ST21 lineage played a major role in the clinical disease resulting in hospitalisation and cases of paediatric HUS in Romania surpassing the O157:H7 strains. The latter were all clade 7 and mostly ST1804. Notably, among the Romanian isolates was a stx2a-harbouring cryptic clade I strain associated with a HUS case, stx2f- and stx2e-positive strains, and hybrid strains displaying a mixture of intestinal and extraintestinal virulence genes were found. As a clearer picture emerges of the STEC strains responsible for infections in Romania, further surveillance efforts are needed to uncover their prevalence, sources, and reservoirs.

In the absence of consistent national molecular typing data to enhance the surveillance of Salmonella Enteritidis, it was considered useful to collect baseline information on the genetic diversity and antibiotic susceptibility of strains isolated in Romania between January 2016 and April 2020 and compare them to strains described in major international outbreaks of the same period. A collection of 245 clinical isolates were genotyped by a standardised multiple-locus variable-number of tandem repeats analysis (MLVA) 5-loci protocol and screened for antimicrobial resistance against 15 compounds. Twenty strains were further subjected to whole genome sequencing (WGS) and compared to epidemiologically relevant high-throughput sequencing data available in European databases. Twenty-seven MLVA genotypes were identified, of which three, commonly reported in Europe between 2016–2020, covered 72% of the collection. Antibiotic resistance was detected in 30% of the strains, with resistance to nalidixic acid and ciprofloxacin as the most common phenotype, and also associated with two prevalent MLVA clones. WGS-derived multilocus sequence typing (MLST) revealed a single sequence type (ST11) further resolved into 10 core-genome MLST complex types. The minimum spanning tree constructed from the cgMLST data clustered Romanian and international strains, which shared more than 95% of the core genes, revealing links with a contemporaneous multi-country outbreak. This study could be regarded as a forerunner to the advent of using this integrative approach in the public health practice at a national level and thus contribute to the concerted actions at a European level to stop outbreaks.

Introduction. Application of next-generation sequencing technology generated a massive amount of information on the gut microbiome composition used to understand its role in the healthy state and in various diseases. We aimed to provide information on the gut microbiota composition of Romanian subjects diagnosed with ankylosing spondylitis, an immune-mediated arthropathy linked to a genetic predisposition and gut dysbiosis. Methods. Stool samples collected from 25 patients with ankylosing spondylitis and 16 healthy controls were investigated using high-throughput DNA sequencing of 16S rRNA amplicons from seven different hypervariable regions and Ion Torrent PGM instrument. Microbial composition of metagenomic data was analyzed with QIIME software and differential abundance analysis of taxa encompassed linear discriminant analysis effect size (LEfSe). Results. Overall, 14 phyla, 114 families, 114 genera, and 275 species were identified across the 41 samples, the aggregated data revealing as most abundant the phyla Bacteroidetes, Firmicutes, and Proteobacteria, the families Bacteroidaceae, Prevotellaceae, and Ruminococcaceae, the genera Bacteroides, Prevotella, and Faecalibacterium, and Prevotella copri species. Using various cutoffs for abundance and prevalence, core taxonomic members were identified which in general were shared between the patients and controls. However, evidence was gained that the diversity in the microbiomes from the former cohort was lower than for controls and that certain taxa had significantly different abundance between the two groups. Conclusion. This study allowed an informative high-throughput 16S rRNA profiling of the gut microbiota needed to identify microbiome signatures of risk in the autochthonous population with AS.

"Introduction: Shiga toxin-producing Escherichia coli (STEC) can cause severe human infections especially in the pediatric population who are at risk of progressing to life threatening systemic complications such as hemolytic uremic syndrome (HUS). In Romania, enhanced monitoring of STEC-associated HUS was implemented in year 2016 after a cluster of infections caused by STEC O26:H11 serotype heightened public awareness of their consequences. Objectives: This study describes the microbiological investigation initiated to identify a STEC-associated HUS pediatric case and determine additional infections among his household contacts. Methods: Fecal specimens collected from a small child hospitalized with HUS and his asymptomatic parents and brother were investigated using a culture-based approach which targeted STEC isolation, identification, and typing. PCR-based assays for key virulence genes (i.e., stx1 and stx2 types/subtypes, eae, and ehxA) were combined with traditional methods such as serogroup determination by slide agglutination. Also, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used for strain genotyping. Results: STEC was identified only in the stool samples of the siblings, one who developed HUS and the other who remained asymptomatic. Both strains belonged to O26 E. coli serogroup and displayed an identical virulence gene profile (presence of stx2a, eae, and ehxA genes). Molecular typing by PFGE showed the STEC strains as highly related genetically and MLST assigned them to sequence type ST21 lineage. Conclusion: A family cluster of O26 STEC infections was detected by using a culture- based molecular approach. Further analysis at whole genome sequencing resolution level is needed for a detailed characterization of such pathogens of significant public health importance. Keywords: HUS, Shiga toxin-producing Escherichia coli, virulence genes, PCR, molecular typing"