Integrated analysis of patient networks and plasmid genomes reveals a regional, multi-species outbreak of carbapenemase-producing Enterobacterales carrying both blaIMP and mcr-9 genes.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) are challenging in healthcare, with resistance to multiple classes of antibiotics. This study describes the emergence of IMP-encoding CPE amongst diverse Enterobacterales species between 2016 and 2019 across a London regional network. METHODS: We performed a network analysis of patient pathways, using electronic health records, to identify contacts between IMP-encoding CPE positive patients. Genomes of IMP-encoding CPE isolates were overlayed with patient contacts to imply potential transmission events. RESULTS: Genomic analysis of 84 Enterobacterales isolates revealed diverse species (predominantly Klebsiella spp, Enterobacter spp, E. coli); 86% (72/84) harboured an IncHI2 plasmid carrying blaIMP and colistin resistance gene mcr-9 (68/72). Phylogenetic analysis of IncHI2 plasmids identified three lineages showing significant association with patient contacts and movements between four hospital sites and across medical specialities, which was missed on initial investigations. CONCLUSIONS: Combined, our patient network and plasmid analyses demonstrate an interspecies, plasmid-mediated outbreak of blaIMPCPE, which remained unidentified during standard investigations. With DNA sequencing and multi-modal data incorporation, the outbreak investigation approach proposed here provides a framework for real-time identification of key factors causing pathogen spread. Plasmid-level outbreak analysis reveals that resistance spread may be wider than suspected, allowing more interventions to stop transmission within hospital networks.

Molecular Epidemiology of Group B Streptococci in Lithuania Identifies Multi-Drug Resistant Clones and Sporadic ST1 Serotypes Ia and Ib.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a leading cause of neonatal infections. Yet, detailed assessment of the genotypic and phenotypic factors associated with GBS carriage, mother-to-baby transmission, and GBS infection in neonates and adults is lacking. Understanding the distribution of GBS genotypes, including the predominance of different serotypes, antimicrobial resistance (AMR) genes, and virulence factors, is likely to help to prevent GBS diseases, as well as inform estimates of the efficacy of future GBS vaccines. To this end, we set out to characterise GBS isolates collected from pregnant and non-pregnant women in Kaunas region in Lithuania. Whole genome sequences of 42 GBS isolates were analysed to determine multi-locus sequence typing (MLST), the presence of acquired AMR and surface protein genes, and the phylogenetic relatedness of isolates. We identified serotypes Ia (42.9%, 18/42), III (33.3%, 14/42), V (21.4%, 9/42), and a single isolate of serotype Ib. Genomic analyses revealed high diversity among the isolates, with 18 sequence types (STs) identified, including three novel STs. 85.7% (36/42) of isolates carried at least one AMR gene: tetM or tetO (35/42), ermB or lsaC (8/42) and ant6-Ia and aph3-III (2/42). This study represents the first genomic analysis of GBS isolated from women in Lithuania and contributes to an improved understanding of the global spread of GBS genotypes and phenotypes, laying the foundations for future GBS surveillance in Lithuania.

A New Sugar for an Old Phage: a c-di-GMP-Dependent Polysaccharide Pathway Sensitizes Escherichia coli for Bacteriophage Infection.

Bacteriophages are ubiquitous parasites of bacteria and major drivers of bacterial ecology and evolution. Despite an ever-growing interest in their biotechnological and therapeutic applications, detailed knowledge of the molecular mechanisms underlying phage-host interactions remains scarce. Here, we show that bacteriophage N4 exploits a novel surface glycan (NGR) as a receptor to infect its host Escherichia coli. We demonstrate that this process is regulated by the second messenger c-di-GMP and that N4 infection is specifically stimulated by the diguanylate cyclase DgcJ, while the phosphodiesterase PdeL effectively protects E. coli from N4-mediated killing. PdeL-mediated protection requires its catalytic activity to reduce c-di-GMP and includes a secondary role as a transcriptional repressor. We demonstrate that PdeL binds to and represses the promoter of the wec operon, which encodes components of the enterobacterial common antigen (ECA) exopolysaccharide pathway. However, only the acetylglucosamine epimerase WecB but none of the other ECA components is required for N4 infection. Based on this, we postulate that NGR is an N-acetylmannosamine-based carbohydrate polymer that is produced and exported to the cell surface of E. coli in a c-di-GMP-dependent manner, where it serves as a receptor for N4. This novel carbohydrate pathway is conserved in E. coli and other bacterial pathogens, serves as the primary receptor for various bacteriophages, and is induced at elevated temperature and by specific amino acid-based nutrients. These studies provide an entry point into understanding how bacteria use specific regulatory mechanisms to balance costs and benefits of highly conserved surface structures. IMPORTANCE Because bacterial surface glycans are in direct contact with the environment they can provide essential protective functions during infections or against competing bacteria. But such structures are also "Achilles' heels" since they can serve as primary receptors for bacteriophages. Bacteria thus need to carefully control the exposure of conserved surface glycans to balance costs and benefits. Here, we identify a novel exopolysaccharide that is widely conserved in E. coli and is used by N4 and related bacteriophages as primary receptor. We demonstrate that the synthesis of NGR (N4 glycan receptor) is tightly controlled by the second messenger c-di-GMP in a highly specific manner and by a single diguanylate cyclase. These studies provide an example of how bacteria can alleviate the strong selective pressure imposed on them by bacteriophages entering through conserved surface structures by carefully regulating their synthesis and secretion.

Molecular Characterization of Uropathogenic Escherichia coli Reveals Emergence of Drug Resistant O15, O22 and O25 Serogroups.

Background and Objectives: Uropathogenic Escherichia coli (UPEC) are common pathogens causing urinary tract infections (UTIs). We aimed to investigate the relationship among clinical manifestation, serogroups, phylogenetic groups, and antimicrobial resistance among UPEC. Materials and Methods: One-hundred Escherichia coli isolates recovered from urine and ureteral scrapings were used for the study. The prevalence of antimicrobial resistance was determined by using European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations. E. coli serogroups associated with UTI, as well as phylogenetic diversity were analyzed using multiplex PCR reactions. Results: Eighty-seven strains (87%) were isolated from females, while 13 (13%) from males. A high frequency of resistance to cephalosporins (43%) and fluoroquinolones (31%) was observed. Among UTI-associated serogroups O15 (32.8%), O22 (23.4%), and O25 (15.6%) were dominant and demonstrated elevated resistance rates. The E. coli phylogenetic group B2 was most common. These observations extended to pregnant patients with asymptomatic bacteriuria. Conclusions: Due to high rates of resistance, strategies using empirical therapy of second-generation cephalosporins and fluoroquinolones should be reconsidered in this population.

New in vitro model evaluating antiseptics' efficacy in biofilm-associated Staphylococcus aureus prosthetic vascular graft infection.

PURPOSE: To develop a new in vitro model of prosthetic vascular graft infection (PVGI) and evaluate antimicrobial and biofilm-disrupting efficacy of 0.1% octenidine dihydrochloride, 10% povidone-iodine and 0.02% chlorhexidine digluconate against biofilm-producing Staphylococcus aureus (S. aureus). METHODOLOGY: The effect of antiseptics on the microscopic integrity and antimicrobial effect on S. aureus biofilms was tested by growing biofilms on glass coverslips, in the modified Lubbock chronic wound pathogenic biofilm (LCWPB) model and on the surface of vascular grafts using qualitive and quantitative methods as well as by scanning electron microscopy (SEM). RESULTS: Chlorhexidine worked best on destroying the integrity of S. aureus biofilms (P=0.002). In the LCWPB model, octenidine and povidone-iodine eradicated all S. aureus colonies (from 1.79 × 109 c.f.u. ml-1 to 0). In the newly developed PVGI model, the grafts were successfully colonized with biofilms as seen in SEM images. All antiseptics demonstrated significant antimicrobial efficacy, decreasing colony counts by seven orders of magnitude (P=0.002). Octenidine was superior to povidone-iodine (P=0.009) and chlorhexidine (P=0.041). CONCLUSION: We implemented an innovative in vitro model on S. aureus biofilms grown in different settings, including a clinically challenging situation of PVGI. The strongest antimicrobial activity against S. aureus biofilms, grown on prosthetic vascular grafts, was showed by 0.1% octenidine dihydrochloride. We suggest that combinational therapy of antiseptics between chlorhexidine with either povidone-iodine or octenidine dihydrochloride should be tested in further experiments. Despite the need of further studies, our findings of these in vitro experiments will assist the management of vascular graft infection in clinical cases.