Dissemination of extensively drug-resistant NDM-producing Providencia stuartii in Europe linked to patients transferred from Ukraine, March 2022 to March 2023.

BackgroundThe war in Ukraine led to migration of Ukrainian people. Early 2022, several European national surveillance systems detected multidrug-resistant (MDR) bacteria related to Ukrainian patients.AimTo investigate the genomic epidemiology of New Delhi metallo-ß-lactamase (NDM)-producing Providencia stuartii from Ukrainian patients among European countries.MethodsWhole-genome sequencing of 66 isolates sampled in 2022-2023 in 10 European countries enabled whole-genome multilocus sequence typing (wgMLST), identification of resistance genes, replicons, and plasmid reconstructions. Five bla NDM-1-carrying-P. stuartii isolates underwent antimicrobial susceptibility testing (AST). Transferability to Escherichia coli of a bla NDM-1-carrying plasmid from a patient strain was assessed. Epidemiological characteristics of patients with NDM-producing P. stuartii were gathered by questionnaire.ResultswgMLST of the 66 isolates revealed two genetic clusters unrelated to Ukraine and three linked to Ukrainian patients. Of these three, two comprised bla NDM-1-carrying-P. stuartii and the third bla NDM-5-carrying-P. stuartii. The bla NDM-1 clusters (PstCluster-001, n = 22 isolates; PstCluster-002, n = 8 isolates) comprised strains from seven and four countries, respectively. The bla NDM-5 cluster (PstCluster-003) included 13 isolates from six countries. PstCluster-001 and PstCluster-002 isolates carried an MDR plasmid harbouring bla NDM-1, bla OXA-10, bla CMY-16, rmtC and armA, which was transferrable in vitro and, for some Ukrainian patients, shared by other Enterobacterales. AST revealed PstCluster-001 isolates to be extensively drug-resistant (XDR), but susceptible to cefiderocol and aztreonam-avibactam. Patients with data on age (n = 41) were 19-74 years old; of 49 with information on sex, 38 were male.ConclusionXDR P. stuartii were introduced into European countries, requiring increased awareness and precautions when treating patients from conflict-affected areas.

Genomic Characterization and Phylogenetic Analysis of Linezolid-Resistant Enterococcus from the Nostrils of Healthy Hosts Identifies Zoonotic Transmission.

Linezolid resistance in Enterococcus spp. is increasingly considered critically important and a public health threat which mandates the need to understand their genomic contents and dissemination patterns. Here, we used whole-genome sequencing to characterize the resistome, virulome and mobile genetic elements of nine linezolid-resistant (LZDR) enterococci (seven optrA-E. faecalis, one poxtA-E. faecium and one optrA-E. casseliflavus) previously obtained from the nares of healthy dogs, pigs, pig farmers and tracheal samples of nestling storks in Spain. Also, the relatedness of the isolates with publicly available genomes was accessed by core-genome single nucleotide polymorphism (SNP) analysis. The optrA gene of the E. faecalis and E. casseliflavus isolates was located downstream of the fexA gene. The optrA gene in the E. casseliflavus isolate was carried in a plasmid (pURX4962), while those in the seven E. faecalis isolates were chromosomally located. The OptrA proteins were mostly variants of wild type (DP-2: Y176D/T481P; RDK: I104R/Y176D/E256K; DD-3: Y176D/G393D; and EDD: K3E/Y176D/G393D), except two that were wild type (one E. faecalis and one E. casseliflavus). The poxtA gene in the E. faecium isolate was found alone within its contig. The cfrD was upstream of ermB gene in the E. casseliflavus isolate and flanked by ISNCY and IS1216. All the LZDR enterococci carried plasmid rep genes (2-3) containing tetracycline, chloramphenicol and aminoglycoside resistance genes. All isolates except E. casseliflavus carried at least one intact prophage, of which E. faecalis-ST330 (X4957) from a pig carried the highest (n = 5). Tn6260 was associated with lnuG in E. faecalis-ST330 while Tn554 was with fexA in E. feaecalis-ST59 isolates. All except E. casseliflavus (n = 0) carried at least two metal resistance genes (MRGs), of which poxtA-carrying E. faecium-ST1739 isolate contained the most (arsA, copA, fief, ziaA, znuA, zosA, zupT, and zur). SNP-based analyses identified closely related optrA-E. faecalis isolates from a pig and a pig farmer on the same farm (SNP = 4). Moreover, optrA- carrying E. faecalis-ST32, -ST59, and -ST474 isolates from pigs were related to those previously described from humans (sick and healthy) and cattle in Spain, Belgium, and Switzerland (SNP range 43-86). These findings strongly suggest the transmission of LZDR-E. faecalis between a pig and a pig farmer and potential inter-country dissemination. These highlight the need to strengthen molecular surveillance of LZDR enterococci in all ecological niches and body parts to direct appropriate control strategies.

Seagrass-mediated rhizosphere redox gradients are linked with ammonium accumulation driven by diazotrophs.

Seagrasses can enhance nutrient mobilization in their rhizosphere via complex interactions with sediment redox conditions and microbial populations. Yet, limited knowledge exists on how seagrass-derived rhizosphere dynamics affect nitrogen cycling. Using optode and gel-sampler-based chemical imaging, we show that radial O2 loss (ROL) from rhizomes and roots leads to the formation of redox gradients around below-ground tissues of seagrass (Zostera marina), which are co-localized with regions of high ammonium concentrations in the rhizosphere. Combining such chemical imaging with fine-scale sampling for microbial community and gene expression analyses indicated that multiple biogeochemical pathways and microbial players can lead to high ammonium concentration within the oxidized regions of the seagrass rhizosphere. Symbiotic N2-fixing bacteria (Bradyrhizobium) were particularly abundant and expressed the diazotroph functional marker gene nifH in Z. marina rhizosphere areas with high ammonium concentrations. Such an association between Z. marina and Bradyrhizobium can facilitate ammonium mobilization, the preferred nitrogen source for seagrasses, enhancing seagrass productivity within nitrogen-limited environments. ROL also caused strong gradients of sulfide at anoxic/oxic interfaces in rhizosphere areas, where we found enhanced nifH transcription by sulfate-reducing bacteria. Furthermore, we found a high abundance of methylotrophic and sulfide-oxidizing bacteria in rhizosphere areas, where O2 was released from seagrass rhizomes and roots. These bacteria could play a beneficial role for the plants in terms of their methane and sulfide oxidation, as well as their formation of growth factors and phytohormones. ROL from below-ground tissues of seagrass, thus, seems crucial for ammonium production in the rhizosphere via stimulation of multiple diazotrophic associations. IMPORTANCE: Seagrasses are important marine habitats providing several ecosystem services in coastal waters worldwide, such as enhancing marine biodiversity and mitigating climate change through efficient carbon sequestration. Notably, the fitness of seagrasses is affected by plant-microbe interactions. However, these microscale interactions are challenging to study and large knowledge gaps prevail. Our study shows that redox microgradients in the rhizosphere of seagrass select for a unique microbial community that can enhance the ammonium availability for seagrass. We provide first experimental evidence that Rhizobia, including the symbiotic N2-fixing bacteria Bradyrhizobium, can contribute to the bacterial ammonium production in the seagrass rhizosphere. The release of O2 from rhizomes and roots also caused gradients of sulfide in rhizosphere areas with enhanced nifH transcription by sulfate-reducing bacteria. O2 release from seagrass root systems thus seems crucial for ammonium production in the rhizosphere via stimulation of multiple diazotrophic associations.

Gonorrhoea on the rise in Denmark since 2022: distinct clones drive increase in heterosexual individuals.

A surge in gonorrhoea in Denmark has occurred since 2022, a 46% increase from 2021. National surveillance, leveraging mandatory reporting and epidemiological data, highlights three distinct clades linked to heterosexual transmission. Despite the rise, these exhibit high susceptibility, contrasting MSM-associated strains. Geographical hotspots and age-specific patterns further illuminate transmission dynamics. The combination of genomic and epidemiological data provides novel insights into the evolving landscape of gonorrhoea, indicating potential shifts in infection dynamics and transmissibility.

Biofilm formation and cell plasticity drive diazotrophy in an anoxygenic phototrophic bacterium.

IMPORTANCE: The contribution of non-cyanobacterial diazotrophs (NCDs) to total N2 fixation in the marine water column is unknown, but their importance is likely constrained by the limited availability of dissolved organic matter and low O2 conditions. Light could support N2 fixation and growth by NCDs, yet no examples from bacterioplankton exist. In this study, we show that the phototrophic NCD, Rhodopseudomonas sp. BAL398, which is a member of the diazotrophic community in the surface waters of the Baltic Sea, can utilize light. Our study highlights the significance of biofilm formation for utilizing light and fixing N2 under oxic conditions and the role of cell plasticity in regulating these processes. Our findings have implications for the general understanding of the ecology and importance of NCDs in marine waters.

Increase in invasive group A streptococcal infections and emergence of novel, rapidly expanding sub-lineage of the virulent Streptococcus pyogenes M1 clone, Denmark, 2023.

A highly virulent sub-lineage of the Streptococcus pyogenes M1 clone has been rapidly expanding throughout Denmark since late 2022 and now accounts for 30% of the new invasive group A streptococcal infections. We aimed to investigate whether a shift in variant composition can account for the high incidence rates observed over winter 2022/23, or if these are better explained by the impact of COVID-19-related restrictions on population immunity and carriage of group A Streptococcus.

Chemotaxis may assist marine heterotrophic bacterial diazotrophs to find microzones suitable for N2 fixation in the pelagic ocean.

Heterotrophic bacterial diazotrophs (HBDs) are ubiquitous in the pelagic ocean, where they have been predicted to carry out the anaerobic process of nitrogen fixation within low-oxygen microenvironments associated with marine pelagic particles. However, the mechanisms enabling particle colonization by HBDs are unknown. We hypothesized that HBDs use chemotaxis to locate and colonize suitable microenvironments, and showed that a cultivated marine HBD is chemotactic toward amino acids and phytoplankton-derived DOM. Using an in situ chemotaxis assay, we also discovered that diverse HBDs at a coastal site are motile and chemotactic toward DOM from various phytoplankton taxa and, indeed, that the proportion of diazotrophs was up to seven times higher among the motile fraction of the bacterial community compared to the bulk seawater community. Finally, three of four HBD isolates and 16 of 17 HBD metagenome assembled genomes, recovered from major ocean basins and locations along the Australian coast, each encoded >85% of proteins affiliated with the bacterial chemotaxis pathway. These results document the widespread capacity for chemotaxis in diverse and globally relevant marine HBDs. We suggest that HBDs could use chemotaxis to seek out and colonize low-oxygen microenvironments suitable for nitrogen fixation, such as those formed on marine particles. Chemotaxis in HBDs could therefore affect marine nitrogen and carbon biogeochemistry by facilitating nitrogen fixation within otherwise oxic waters, while also altering particle degradation and the efficiency of the biological pump.

Sinking Trichodesmium fixes nitrogen in the dark ocean.

The photosynthetic cyanobacterium Trichodesmium is widely distributed in the surface low latitude ocean where it contributes significantly to N2 fixation and primary productivity. Previous studies found nifH genes and intact Trichodesmium colonies in the sunlight-deprived meso- and bathypelagic layers of the ocean (200-4000 m depth). Yet, the ability of Trichodesmium to fix N2 in the dark ocean has not been explored. We performed 15N2 incubations in sediment traps at 170, 270 and 1000 m at two locations in the South Pacific. Sinking Trichodesmium colonies fixed N2 at similar rates than previously observed in the surface ocean (36-214 fmol N cell-1 d-1). This activity accounted for 40 ± 28% of the bulk N2 fixation rates measured in the traps, indicating that other diazotrophs were also active in the mesopelagic zone. Accordingly, cDNA nifH amplicon sequencing revealed that while Trichodesmium accounted for most of the expressed nifH genes in the traps, other diazotrophs such as Chlorobium and Deltaproteobacteria were also active. Laboratory experiments simulating mesopelagic conditions confirmed that increasing hydrostatic pressure and decreasing temperature reduced but did not completely inhibit N2 fixation in Trichodesmium. Finally, using a cell metabolism model we predict that Trichodesmium uses photosynthesis-derived stored carbon to sustain N2 fixation while sinking into the mesopelagic. We conclude that sinking Trichodesmium provides ammonium, dissolved organic matter and biomass to mesopelagic prokaryotes.

A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction.

The CRISPR (clustered regularly interspaced short palindromic repeats) system protects archaea and bacteria by eliminating nucleic acid invaders in a crRNA-guided manner. The Sulfolobus islandicus type III-B Cmr-α system targets invading nucleic acid at both RNA and DNA levels and DNA targeting relies on the directional transcription of the protospacer in vivo. To gain further insight into the involved mechanism, we purified a native effector complex of III-B Cmr-α from S. islandicus and characterized it in vitro. Cmr-α cleaved RNAs complementary to crRNA present in the complex and its ssDNA destruction activity was activated by target RNA. The ssDNA cleavage required mismatches between the 5΄-tag of crRNA and the 3΄-flanking region of target RNA. An invader plasmid assay showed that mutation either in the histidine-aspartate acid (HD) domain (a quadruple mutation) or in the GGDD motif of the Cmr-2α protein resulted in attenuation of the DNA interference in vivo. However, double mutation of the HD motif only abolished the DNase activity in vitro. Furthermore, the activated Cmr-α binary complex functioned as a highly active DNase to destroy a large excess DNA substrate, which could provide a powerful means to rapidly degrade replicating viral DNA.

Genetic determinants of PAM-dependent DNA targeting and pre-crRNA processing in Sulfolobus islandicus.

Bacteria and Archaea encode clustered, regularly interspaced, short palindromic repeat (CRISPR) systems to confer adaptive immunity to invasive viruses and plasmids. Recent studies of CRISPR systems revealed that diverse CRISPR-associated (Cas) interference modules often coexist in different organisms but functions of cas genes have not been dissected in any of these systems. The crenarchaeon Sulfolobus islandicus encodes three distinct CRISPR interference modules, including a type IA system and two type IIIB systems: Cmr-α and Cmr-ß. To study the genetic determinants of protospacer-adjacent motif (PAM)-dependent DNA targeting activity and mature CRISPR RNA (crRNA) production in this organism, mutants deleting individual genes of the type IA system or removing each of other Cas modules were constructed. Characterization of these mutants revealed that Cas7, Cas5, Cas6, Cas3' and Cas3" are essential for PAM-dependent DNA targeting activity, whereas Csa5, along with all other Cas modules, is dispensable for the targeting in the crenarchaeon. Cas6 is implicated as the only enzyme for pre-crRNA processing and the crRNA maturation is independent of the DNA targeting activity. Importantly, we show that Cas7 and Cas5 are essential for stabilizing the processing intermediates and mature crRNAs, respectively, and that depleting the helicase or nuclease domain of Cas3 leads to the accumulation of processing intermediates. This demonstrates that in addition to Cas6, other Cas proteins of an archaeal type IA system also contribute to crRNA processing.