Integrated molecular, phenotypic and epidemiological surveillance of antimicrobial resistance in Neisseria gonorrhoeae in Germany.

Numbers of infections with Neisseria gonorrhoeae are among the top three sexually transmitted infections (STI) worldwide. In addition, the emergence and spread of antimicrobial resistance (AMR) in Neisseria gonorrhoeae pose an important public-health issue. The integration of genomic, phenotypic and epidemiological data to monitor Neisseria gonorrhoeae fosters our understanding of the emergence and spread of AMR in Neisseria gonorrhoeae and helps to inform therapy guidelines and intervention strategies. Thus, the Gonococcal resistance surveillance (Go-Surv-AMR) was implemented at the Robert Koch Institute in Germany in 2021 to obtain molecular, phenotypic and epidemiological data on Neisseria gonorrhoeae isolated in Germany. Here, we describe the structure and aims of Go-Surv-AMR. Furthermore, we point out future directions of Go-Surv-AMR to improve the integrated genomic surveillance of Neisseria gonorrhoeae. In this context we discuss current and prospective sequencing approaches and the information derived from their application. Moreover, we highlight the importance of combining phenotypic and WGS data to monitor the evolution of AMR in Neisseria gonorrhoeae in Germany. The implementation and constant development of techniques and tools to improve the genomic surveillance of Neisseria gonorrhoeae will be important in coming years.

Potato Wart Isolates from Europe and North America Form Distinct Clusters of Genetic Variation.

We have extended previously published sets of simple sequence repeat markers for Synchytrium endobioticum, selected to be polymorphic for the German-standard isolates of pathotypes P1, P2, P6, P8, and P18. These markers also complement the extensive published information on DNA polymorphisms for the mitogenomes of Synchytrium endobioticum. This extended set of 35 markers representing 73 alleles differentiated 51 isolates from Europe and North America into three large, well-separated clusters and subclusters using dendrogram analysis, principal coordinates analysis (PCoA), and population substructure analysis using STRUCTURE 2.3.4 software. This suggests a limited number of introgressions of the wart disease pathogen into current potato growing areas, followed by recombination and admixture of populations through human activities. The new markers extend the published marker sets and are useful tools for future analyses of population structure and dynamics in Synchytrium endobioticum, which are necessary to understand the biology of the interaction between the pathogen and its potato host and to develop future control strategies.

First Description of a Temperate Bacteriophage (vB_FhiM_KIRK) of Francisella hispaniensis Strain 3523.

Here we present the characterization of a Francisella bacteriophage (vB_FhiM_KIRK) including the morphology, the genome sequence and the induction of the prophage. The prophage sequence (FhaGI-1) has previously been identified in F. hispaniensis strain 3523. UV radiation induced the prophage to assemble phage particles consisting of an icosahedral head (~52 nm in diameter), a tail of up to 97 nm in length and a mean width of 9 nm. The double stranded genome of vB_FhiM_KIRK contains 51 open reading frames and is 34,259 bp in length. The genotypic and phylogenetic analysis indicated that this phage seems to belong to the Myoviridae family of bacteriophages. Under the conditions tested here, host cell (Francisella hispaniensis 3523) lysis activity of KIRK was very low, and the phage particles seem to be defective for infecting new bacterial cells. Nevertheless, recombinant KIRK DNA was able to integrate site-specifically into the genome of different Francisella species after DNA transformation.

Construction of a New Phage Integration Vector pFIV-Val for Use in Different Francisella Species.

We recently identified and described a putative prophage on the genomic island FhaGI-1 located within the genome of Francisella hispaniensis AS02-814 (F. tularensis subsp. novicida-like 3523). In this study, we constructed two variants of a Francisella phage integration vector, called pFIV1-Val and pFIV2-Val (Francisella Integration Vector-tRNAVal-specific), using the attL/R-sites and the site-specific integrase (FN3523_1033) of FhaGI-1, a chloramphenicol resistance cassette and a sacB gene for counter selection of transformants against the vector backbone. We inserted the respective sites and genes into vector pUC57-Kana to allow for propagation in Escherichia coli. The constructs generated a circular episomal form in E. coli which could be used to transform Francisella spp. where FIV-Val stably integrated site specifically into the tRNAVal gene of the genome, whereas pUC57-Kana is lost due to counter selection. Functionality of the new vector was demonstrated by the successfully complementation of a Francisella mutant strain. The vectors were stable in vitro and during host-cell infection without selective pressure. Thus, the vectors can be applied as a further genetic tool in Francisella research, expanding the present genetic tools by an integrative element. This new element is suitable to perform long-term experiments with different Francisella species.

Functional Analysis of the Alternative Sigma-28 Factor FliA and Its Anti-Sigma Factor FlgM of the Nonflagellated Legionella Species L. oakridgensis.

Legionella oakridgensis causes Legionnaires' disease but is known to be less virulent than Legionella pneumophilaL. oakridgensis is one of the Legionella species that is nonflagellated. The genes of the flagellar regulon are absent, except those encoding the alternative sigma-28 factor (FliA) and its anti-sigma-28 factor (FlgM). Similar to L. oakridgensis, Legionella adelaidensis and Legionella londiniensis, located in the same phylogenetic clade, have no flagellar regulon, although both are positive for fliA and flgM Here, we investigated the role and function of both genes to better understand the role of FliA, the positive regulator of flagellin expression, in nonflagellated strains. We demonstrated that the FliA gene of L. oakridgensis encodes a functional sigma-28 factor that enables the transcription start from the sigma-28-dependent promoter site. The investigations have shown that FliA is necessary for full fitness of L. oakridgensis Interestingly, expression of FliA-dependent genes depends on the growth phase and temperature, as already shown for L. pneumophila strains that are flagellated. In addition, we demonstrated that FlgM is a negative regulator of FliA-dependent gene expression. FlgM seems to be degraded in a growth-phase- and temperature-dependent manner, instead of being exported into the medium as reported for most bacteria. The degradation of FlgM leads to an increase of FliA activity.IMPORTANCE A less virulent Legionella species, L. oakridgensis, causes Legionnaires' disease and is known to not have flagella, even though L. oakridgensis has the regulator of flagellin expression (FliA). This protein has been shown to be involved in the expression of virulence factors. Thus, the strain was chosen for use in this investigation to search for FliA target genes and to identify putative virulence factors of L. oakridgensis One of the five major target genes of FliA identified here encodes the anti-FliA sigma factor FlgM. Interestingly, in contrast to most homologs in other bacteria, FlgM in L. oakridgensis seems not to be transported from the cell so that FliA gets activated. In L. oakridgensis, FlgM seems to be degraded by protease activities.

Growth-related Metabolism of the Carbon Storage Poly-3-hydroxybutyrate in Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaires disease, has a biphasic life cycle with a switch from a replicative to a transmissive phenotype. During the replicative phase, the bacteria grow within host cells in Legionella-containing vacuoles. During the transmissive phenotype and the postexponential (PE) growth phase, the pathogens express virulence factors, become flagellated, and leave the Legionella-containing vacuoles. Using (13)C labeling experiments, we now show that, under in vitro conditions, serine is mainly metabolized during the replicative phase for the biosynthesis of some amino acids and for energy generation. During the PE phase, these carbon fluxes are reduced, and glucose also serves as an additional carbon substrate to feed the biosynthesis of poly-3-hydroxybuyrate (PHB), an essential carbon source for transmissive L. pneumophila. Whole-cell FTIR analysis and comparative isotopologue profiling further reveal that a putative 3-ketothiolase (Lpp1788) and a PHB polymerase (Lpp0650), but not enzymes of the crotonyl-CoA pathway (Lpp0931-0933) are involved in PHB metabolism during the PE phase. However, the data also reflect that additional bypassing reactions for PHB synthesis exist in agreement with in vivo competition assays using Acanthamoeba castellannii or human macrophage-like U937 cells as host cells. The data suggest that substrate usage and PHB metabolism are coordinated during the life cycle of the pathogen.

Identification and characterization of episomal forms of integrative genomic islands in the genus Francisella.

Recently, we identified a putative prophage on a genomic island (GI) within the genome sequence of Francisella hispaniensis isolate AS0-814 (Francisella tularensis subsp. novicida-like 3523) by the analysis of the CRISPR-Cas systems of Francisella. Various spacer DNAs within the CRISPR region of different F. tularensis subsp. novicida strains were found to be homologous to the putative prophage (Schunder et al., 2013, Int. J. Med. Microbiol. 303:51-60). Now we identified the GI (FhaGI-1) as a mobile element which is able to form a circular episomal structure. The circular episomal form of FhaGI-1 is generated by F. hispaniensis, and the excision of the island is an integrase-dependent and site-specific process. Furthermore, we could demonstrate that the excision of the island is also possible in other bacterial species (Escherichia coli). In addition, we could show that a genetically generated small variant of the island is also functional and, after its electroporation into strain F. tularensis subsp. holarctica LVS, the GI was stable and site-specifically integrated into the genome of the transformants. The integrase is sufficient for the integration and excision of the small variant into and from the DNA backbone, respectively. Thus, the element may be suitable to be used as a genetic tool in F. tularensis research. Furthermore, we identified the tRNA(Val) gene of Francisella as an integration site for GIs. Genomic island FphGI-1 was identified in Francisella philomiragia ATCC 25016. We were not able to detect the episomal form of this GI, probably due to a mutated attR site. However, we could demonstrate that integrative GIs are present in Francisella and that they may allow horizontal gene transfer between different Francisella species.