Klebsiella oxytoca inhibits Salmonella infection through multiple microbiota-context-dependent mechanisms.

The Klebsiella oxytoca species complex is part of the human microbiome, especially during infancy and childhood. K. oxytoca species complex strains can produce enterotoxins, namely, tilimycin and tilivalline, while also contributing to colonization resistance (CR). The relationship between these seemingly contradictory roles is not well understood. Here, by coupling ex vivo assays with CRISPR-mutagenesis and various mouse models, we show that K. oxytoca provides CR against Salmonella Typhimurium. In vitro, the antimicrobial activity against various Salmonella strains depended on tilimycin production and was induced by various simple carbohydrates. In vivo, CR against Salmonella depended on toxin production in germ-free mice, while it was largely toxin-independent in mice with residual microbiota. This was linked to the relative levels of toxin-inducing carbohydrates in vivo. Finally, dulcitol utilization was essential for toxin-independent CR in gnotobiotic mice. Together, this demonstrates that nutrient availability is key to both toxin-dependent and substrate-driven competition between K. oxytoca and Salmonella.

Enteroaggregative Escherichia coli: Frequent, yet underdiagnosed pathotype among E. coli O111 strains isolated from children with gastrointestinal disorders in the Czech Republic.

Enteroaggregative Escherichia coli (EAEC) strains including those of serogroup O111 are important causes of diarrhea in children. In the Czech Republic, no information is available on the etiological role of EAEC in pediatric diarrhea due to the lack of their targeted surveillance. To fill this gap, we determined the proportion of EAEC among E. coli O111 isolates from children with gastrointestinal disorders ≤ 2 years of age submitted to the National Reference Laboratory for E. coli and Shigella during 2013-2022. EAEC accounted for 177 of 384 (46.1 %) E. coli O111 isolates, being the second most frequent E. coli O111 pathotype. Most of them (75.7 %) were typical EAEC that carried aggR, usually with aaiC and aatA marker genes; the remaining 24.3 % were atypical EAEC that lacked aggR but carried aaiC and/or aatA. Whole genome sequencing of 11 typical and two atypical EAEC O111 strains demonstrated differences in serotypes, sequence types (ST), virulence gene profiles, and the core genomes between these two groups. Typical EAEC O111:H21/ST40 strains resembled by their virulence profiles including the presence of the aggregative adherence fimbriae V (AAF/V)-encoding cluster to such strains from other countries and clustered with them in the core genome multilocus sequence typing (cgMLST). Atypical EAEC O111:H12/ST10 strains lacked virulence genes of typical EAEC and differed from them in cgMLST. All tested EAEC O111 strains displayed stacked-brick aggregative adherence to human intestinal epithelial cells. The AAF/V-encoding cluster was located on a plasmid of 95,749 bp or 93,286 bp (pAAO111) which also carried aggR, aap, aar, sepA, and aat cluster. EAEC O111 strains were resistant to antibiotics, in particular to aminopenicillins and cephalosporins; 88.3 % produced AmpC ß-lactamase, and 4.1 % extended spectrum ß-lactamase. We conclude that EAEC are frequent among E. coli O111 strains isolated from children with gastrointestinal disorders in the Czech Republic. To reliably assess the etiological role of EAEC in pediatric diarrhea, a serotype-independent, PCR-based pathotype surveillance system needs to be implemented in the future.

Genomic surveillance of STEC/EHEC infections in Germany 2020 to 2022 permits insight into virulence gene profiles and novel O-antigen gene clusters.

Shiga toxin-producing E. coli (STEC), including the subgroup of enterohemorrhagic E. coli (EHEC), are important bacterial pathogens which cause diarrhea and the severe clinical manifestation hemolytic uremic syndrome (HUS). Genomic surveillance of STEC/EHEC is a state-of-the-art tool to identify infection clusters and to extract markers of circulating clinical strains, such as their virulence and resistance profile for risk assessment and implementation of infection prevention measures. The aim of the study was characterization of the clinical STEC population in Germany for establishment of a reference data set. To that end, from 2020 to 2022 1257 STEC isolates, including 39 of known HUS association, were analyzed and lead to a classification of 30.4 % into 129 infection clusters. Major serogroups in all clinical STEC analyzed were O26, O146, O91, O157, O103, and O145; and in HUS-associated strains were O26, O145, O157, O111, and O80. stx1 was less frequently and stx2 or a combination of stx, eaeA and ehxA were more frequently found in HUS-associated strains. Predominant stx gene subtypes in all STEC strains were stx1a (24 %) and stx2a (21 %) and in HUS-associated strains were mainly stx2a (69 %) and the combination of stx1a and stx2a (12.8 %). Furthermore, two novel O-antigen gene clusters (RKI6 and RKI7) and strains of serovars O45:H2 and O80:H2 showing multidrug resistance were detected. In conclusion, the implemented surveillance tools now allow to comprehensively define the population of clinical STEC strains including those associated with the severe disease manifestation HUS reaching a new surveillance level in Germany.

Tools and mechanisms of vacuolar escape leading to host egress in Legionella pneumophila infection: Emphasis on bacterial phospholipases.

The phenomenon of host cell escape exhibited by intracellular pathogens is a remarkably versatile occurrence, capable of unfolding through lytic or non-lytic pathways. Among these pathogens, the bacterium Legionella pneumophila stands out, having adopted a diverse spectrum of strategies to disengage from their host cells. A pivotal juncture that predates most of these host cell escape modalities is the initial escape from the intracellular compartment. This critical step is increasingly supported by evidence suggesting the involvement of several secreted pathogen effectors, including lytic proteins. In this intricate landscape, L. pneumophila emerges as a focal point for research, particularly concerning secreted phospholipases. While nestled within its replicative vacuole, the bacterium deftly employs both its type II (Lsp) and type IVB (Dot/Icm) secretion systems to convey phospholipases into either the phagosomal lumen or the host cell cytoplasm. Its repertoire encompasses numerous phospholipases A (PLA), including three enzymes-PlaA, PlaC, and PlaD-bearing the GDSL motif. Additionally, there are 11 patatin-like phospholipases A as well as PlaB. Furthermore, the bacterium harbors three extracellular phospholipases C (PLCs) and one phospholipase D. Within this comprehensive review, we undertake an exploration of the pivotal role played by phospholipases in the broader context of phagosomal and host cell egress. Moreover, we embark on a detailed journey to unravel the established and potential functions of the secreted phospholipases of L. pneumophila in orchestrating this indispensable process.

Structure-function relationships underpin disulfide loop cleavage-dependent activation of Legionella pneumophila lysophospholipase A PlaA.

Legionella pneumophila, the causative agent of a life-threatening pneumonia, intracellularly replicates in a specialized compartment in lung macrophages, the Legionella-containing vacuole (LCV). Secreted proteins of the pathogen govern important steps in the intracellular life cycle including bacterial egress. Among these is the type II secreted PlaA which, together with PlaC and PlaD, belongs to the GDSL phospholipase family found in L. pneumophila. PlaA shows lysophospholipase A (LPLA) activity which increases after secretion and subsequent processing by the zinc metalloproteinase ProA within a disulfide loop. Activity of PlaA contributes to the destabilization of the LCV in the absence of the type IVB-secreted effector SdhA. We here present the 3D structure of PlaA which shows a typical α/ß-hydrolase fold and reveals that the uncleaved disulfide loop forms a lid structure covering the catalytic triad S30/D278/H282. This leads to reduction of substrate access before activation; however, the catalytic site gets more accessible when the disulfide loop is processed. After structural modeling, a similar activation process is suggested for the GDSL hydrolase PlaC, but not for PlaD. Furthermore, the size of the PlaA substrate-binding site indicated preference toward phospholipids comprising ~16 carbon fatty acid residues which was verified by lipid hydrolysis, suggesting a molecular ruler mechanism. Indeed, mutational analysis changed the substrate profile with respect to fatty acid chain length. In conclusion, our analysis revealed the structural basis for the regulated activation and substrate preference of PlaA.

Salmonella enterica in farm environments in the Ashanti Region of Ghana.

BACKGROUND: Salmonella enterica are important foodborne pathogens and the third leading cause of death among diarrheal infections worldwide. This cross-sectional study investigated the frequency of antibiotic-resistant Salmonella enterica in commercial and smallholder farm environments in the Ashanti Region of Ghana. A total of 1490 environmental samples, comprising 800 (53.7%) soil (from poultry, pigs, sheep, goats and cattle farms), 409 (27.4%) pooled poultry fecal and 281 (18.9%) dust (from poultry farms) samples, were collected from 30 commercial and 64 smallholder farms. All samples were processed using standard culture methods. Isolates were identified by biochemical methods and confirmed using the VITEK 2 System. Antibiotic susceptibility testing was carried out by disk diffusion following the EUCAST guidelines. Serotyping was performed using the Kauffman White Le Minor Scheme. RESULTS: The overall Salmonella frequency was 6.0% (n/N = 90/1490); the frequency varied according to the type of sample collected and included: 8.9% for dust (n/N = 25/281), 6.5% for soil (n/N = 52/800) and 3.2% for pooled poultry fecal samples (n/N = 13/409). Salmonella was also recovered from commercial farm environments (8.6%, n/N = 68/793) than from smallholder farms (3.2%, n/N = 22/697) (PR = 2.7, CI: 1.7 - 4.4). Thirty-four different Salmonella serovars were identified, the two most common being Rubislaw (27.8%, n/N = 25/90) and Tamale (12.2%, n/N = 11/90). Serovar diversity was highest in strains from soil samples (70.6%, n/N = 24/34) compared to those found in the dust (35.2%, n/N = 12/34) and in fecal samples (29.4%, n/N = 10/34). Salmonella frequency was much higher in the rainy season (8.4%, n/N = 85/1007) than in the dry season (1.0%, n/N = 5/483) (PR = 8.4, 95% CI: 3.3 - 20.0). Approximately 14.4% (n/N = 13/90) of the isolates were resistant to at least one of the tested antimicrobials, with 84.6% (n/N = 11/13) being resistant to multiple antibiotics. All Salmonella Kentucky (n = 5) were resistant to ciprofloxacin. CONCLUSION: This study showed that farm environments represent an important reservoir for antibiotic-resistant Salmonella, which warrants monitoring and good husbandry practices, especially in commercial farms during the rainy season, to control the spread of this pathogen.

O-Antigen Diversification Masks Identification of Highly Pathogenic Shiga Toxin-Producing Escherichia coli O104:H4-Like Strains.

Shiga toxin-producing Escherichia coli (STEC) can give rise to a range of clinical outcomes from diarrhea to the life-threatening systemic condition hemolytic-uremic syndrome (HUS). Although STEC O157:H7 is the serotype most frequently associated with HUS, a major outbreak of HUS occurred in 2011 in Germany and was caused by a rare serotype, STEC O104:H4. Prior to 2011 and since the outbreak, STEC O104:H4 strains have only rarely been associated with human infections. From 2012 to 2020, intensified STEC surveillance was performed in Germany where the subtyping of ~8,000 clinical isolates by molecular methods, including whole-genome sequencing, was carried out. A rare STEC serotype, O181:H4, associated with HUS was identified, and like the STEC O104:H4 outbreak strain, this strain belongs to sequence type 678 (ST678). Genomic and virulence comparisons revealed that the two strains are phylogenetically related and differ principally in the gene cluster encoding their respective lipopolysaccharide O-antigens but exhibit similar virulence phenotypes. In addition, five other serotypes belonging to ST678 from human clinical infection, such as OX13:H4, O127:H4, OgN-RKI9:H4, O131:H4, and O69:H4, were identified from diverse locations worldwide. IMPORTANCE Our data suggest that the high-virulence ensemble of the STEC O104:H4 outbreak strain remains a global threat because genomically similar strains cause disease worldwide but that the horizontal acquisition of O-antigen gene clusters has diversified the O-antigens of strains belonging to ST678. Thus, the identification of these highly pathogenic strains is masked by diverse and rare O-antigens, thereby confounding the interpretation of their potential risk.

Large Multicountry Outbreak of Invasive Listeriosis by a Listeria monocytogenes ST394 Clone Linked to Smoked Rainbow Trout, 2020 to 2021.

Whole-genome sequencing (WGS) has revolutionized surveillance of infectious diseases. Disease outbreaks can now be detected with high precision, and correct attribution of infection sources has been improved. Listeriosis, caused by the bacterium Listeria monocytogenes, is a foodborne disease with a high case fatality rate and a large proportion of outbreak-related cases. Timely recognition of listeriosis outbreaks and precise allocation of food sources are important to prevent further infections and to promote public health. We report the WGS-based identification of a large multinational listeriosis outbreak with 55 cases that affected Germany, Austria, Denmark, and Switzerland during 2020 and 2021. Clinical isolates formed a highly clonal cluster (called Ny9) based on core genome multilocus sequence typing (cgMLST). Routine and ad hoc investigations of food samples identified L. monocytogenes isolates from smoked rainbow trout filets from a Danish producer grouping with the Ny9 cluster. Patient interviews confirmed consumption of rainbow trout as the most likely infection source. The Ny9 cluster was caused by a MLST sequence type (ST) ST394 clone belonging to molecular serogroup IIa, forming a distinct clade within molecular serogroup IIa strains. Analysis of the Ny9 genome revealed clpY, dgcB, and recQ inactivating mutations, but phenotypic characterization of several virulence-associated traits of a representative Ny9 isolate showed that the outbreak strain had the same pathogenic potential as other serogroup IIa strains. Our report demonstrates that international food trade can cause multicountry outbreaks that necessitate cross-border outbreak collaboration. It also corroborates the relevance of ready-to-eat smoked fish products as causes for listeriosis. IMPORTANCE Listeriosis is a severe infectious disease in humans and characterized by an exceptionally high case fatality rate. The disease is transmitted through consumption of food contaminated by the bacterium Listeria monocytogenes. Outbreaks of listeriosis often occur but can be recognized and stopped through implementation of whole-genome sequencing-based pathogen surveillance systems. We here describe the detection and management of a large listeriosis outbreak in Germany and three neighboring countries. This outbreak was caused by rainbow trout filet, which was contaminated by a L. monocytogenes clone belonging to sequence type ST394. This work further expands our knowledge on the genetic diversity and transmission routes of an important foodborne pathogen.

Multicenter Preparedness Exercise Enables Rapid Development of Cluster-Specific PCR-Based Screening Assays from Bacterial Genomic Data.

PCR-based screening assays targeting strain-specific genetic markers allow the timely detection and specific differentiation of bacterial strains. Especially in situations where an infection cluster occurs, fast assay development is crucial for supporting targeted control measures. However, the turnaround times (TATs) for assay setup may be high due to insufficient knowledge about screening assay methods, workflows, and software tools. Here, two blind-coded and quality-controlled ring trials were performed in which five German laboratories established PCR-based screening assays from genomic data that specifically target selected bacterial clusters within two bacterial monospecies sample panels. While the first ring trial was conducted without a time limit to train the participants and assess assay feasibility, in the second ring trial, a challenging time limit of 2 weeks was set to force fast assay development as soon as genomic data were available. During both ring trials, we detected high interlaboratory variability regarding the screening assay methods and targets, the TATs for assay setup, and the number of screening assays. The participants designed between one and four assays per cluster that targeted cluster-specific unique genetic sequences, genes, or single nucleotide variants using conventional PCRs, high-resolution melting assays, or TaqMan PCRs. Assays were established within the 2-week time limit, with TATs ranging from 4 to 13 days. TaqMan probe delivery times strongly influenced TATs. In summary, we demonstrate that a specific exercise improved the preparedness to develop functional cluster-specific PCR-based screening assays from bacterial genomic data. Furthermore, the parallel development of several assays enhances assay availability.

[Assessment of available and currently applied typing methods including genome-based methods for zoonotic pathogens with a focus on Salmonella enterica]. / Bestandsaufnahme der verfügbaren und aktuell eingesetzten Typisierungsmethoden einschließlich genombasierter Verfahren von Zoonoseerregern am Beispiel von Salmonella enterica.

BACKGROUND: In recent years, whole genome sequencing (WGS) in combination with bioinformatic analyses has become state of the art in evaluating the pathogenicity/resistance potential and relatedness of bacteria. WGS analysis thus represents a central tool in the investigation of the resistance and virulence potential of pathogens, as well as their dissemination via outbreak clusters and transmission chains within the framework of molecular epidemiology. In order to gain an overview of the available genotypic and phenotypic methods used for pathogen typing of Salmonella and Shiga toxin-producing and enterohemorrhagic Escherichia coli (STEC/EHEC) in Germany at state and federal level, along with the availability of WGS-based typing and corresponding analytical methods, a survey of laboratories was conducted. METHODS: An electronic survey of laboratories working for public health protection and consumer health protection was conducted from February to June 2020. RESULTS AND CONCLUSION: The results of the survey showed that many of the participating laboratories provide a wide range of phenotypic and molecular methods. Molecular typing is most commonly used for species identification of Salmonella. In many cases, WGS-based methods have already been established at federal and state institutions or are in the process of being established. The Illumina sequencing technology is the most widely used technology. The survey confirms the importance of molecular biology and whole genome typing technologies for laboratories in the diagnosis of bacterial zoonotic pathogens.

[Assessment of available and currently applied typing methods of zoonotic pathogens using the example of Shiga toxin-producing and enterohemorrhagic Escherichia coli (STEC/EHEC)]. / Bestandsaufnahme der verfügbaren und aktuell eingesetzten Typisierungsmethoden von zoonotischen Erregern am Beispiel von Shiga Toxin-bildenden bzw. enterohämorrhagischen Escherichia coli (STEC/EHEC).

INTRODUCTION: In order to improve patient care and to increase food safety within the framework of One Health, the project "Integrated Genomic Surveillance of Zoonotic Agents (IGS-Zoo)" aims to develop concepts for a genomic surveillance of Shiga toxin(Stx)-producing and enterohemorrhagic Escherichia coli (STEC/EHEC) in Germany. METHODS: An online survey was conducted to assess the currently available and applied STEC/EHEC typing methods in the federal laboratories of veterinary regulation, food control, and public health service. RESULTS: Twenty-six questionnaires from 33 participants were evaluated with regard to STEC/EHEC. The number of STEC/EHEC-suspected samples that the laboratories process per year ranges between 10 and 3500, and out of these they obtain between 3 and 1000 pathogenic isolates. Currently the most frequently used typing method is the determination of Stx- and intimin-coding genes using polymerase chain reaction (PCR). Whole genome sequencing (WGS) is currently used by eight federal state laboratories, and nine are planning to implement it in the future. The most common obstacle for further typing of STEC/EHEC is that isolation from sample material is often unsuccessful despite apparent PCR detection of the stx genes. DISCUSSION: The results of the survey should facilitate the integration of the analysis methods developed in the project and emphasize the target groups' individual needs for corresponding training concepts.

Invasive listeriosis outbreaks and salmon products: a genomic, epidemiological study.

Invasive listeriosis, caused by Listeria (L.) monocytogenes, is a severe foodborne infection, especially for immunocompromised individuals. The aim of our investigation was the identification and analysis of listeriosis outbreaks in Germany with smoked and graved salmon products as the most likely source of infection using whole-genome sequencing (WGS) and patient interviews. In a national surveillance programme, WGS was used for subtyping and core genome multi locus sequence typing (cgMLST) for cluster detection of L. monocytogenes isolates from listeriosis cases as well as food and environmental samples in Germany. Patient interviews were conducted to complement the molecular typing. We identified 22 independent listeriosis outbreaks occurring between 2010 and 2021 that were most likely associated with the consumption of smoked and graved salmon products. In Germany, 228 cases were identified, of 50 deaths (22%) reported 17 were confirmed to have died from listeriosis. Many of these 22 outbreaks were cross-border outbreaks with further cases in other countries. This report shows that smoked and graved salmon products contaminated with L. monocytogenes pose a serious risk for listeriosis infection in Germany. Interdisciplinary efforts including WGS and epidemiological investigations were essential to identifying the source of infection. Uncooked salmon products are high-risk foods frequently contaminated with L. monocytogenes. In order to minimize the risk of infection for consumers, food producers need to improve hygiene measures and reduce the entry of pathogens into food processing. Furthermore, susceptible individuals should be better informed of the risk of acquiring listeriosis from consuming smoked and graved salmon products.

Third generation cephalosporin resistance in clinical non-typhoidal Salmonella enterica in Germany and emergence of blaCTX-M-harbouring pESI plasmids.

Non-typhoidal Salmonella enterica is an important gastrointestinal pathogen causing a considerable burden of disease. Resistance to third generation cephalosporins poses a serious threat for treatment of severe infections. In this study occurrence, phylogenetic relationship, and mechanisms of third generation cephalosporin resistance were investigated for clinical non-typhoidal S. enterica isolates in Germany. From 2017 to 2019, we detected 168 unique clinical S. enterica isolates with phenotypic resistance to third generation cephalosporins in a nation-wide surveillance. Compared to previous years, we observed a significant (P=0.0002) and consistent increase in resistant isolates from 0.41 % in 2005 to 1.71 % in 2019. In total, 34 different serovars were identified, most often S. Infantis (n=41; 24.4 %), S. Typhimurium (n=27; 16.1 %), S. Kentucky (n=21; 12.5 %), and S. Derby (n=17; 10.1 %). Whole genome analyses revealed extended-spectrum ß-lactamase (ESBL) genes as main cause for third generation cephalosporin resistance, and most prevalent were blaCTX-M-1 (n=55), blaCTX-M-14 (n=25), and blaCTX-M-65 (n=23). There was no strict correlation between serovar, phylogenetic lineage, and ESBL type but some serovar/ESBL gene combinations were detected frequently, such as blaCTX-M-1 and blaCTX-M-65 in S. Infantis or blaCTX-M-14b in S. Kentucky. The ESBL genes were mainly located on plasmids, including IncI, IncA/C variants, emerging pESI variants, and a novel blaCTX-M-1harbouring plasmid. We conclude that third generation cephalosporin resistance is on the rise among clinical S. enterica isolates in Germany, and occurrence in various S. enterica serovars is most probably due to multiple acquisition events of plasmids.

Ongoing High Incidence and Case-Fatality Rates for Invasive Listeriosis, Germany, 2010-2019.

We used 10 years of surveillance data to describe listeriosis frequency in Germany. Altogether, 5,576 cases were reported, 91% not pregnancy associated; case counts increased over time. Case-fatality rate was 13% in non-pregnancy-associated cases, most in adults ≥65 years of age. Detecting, investigating, and ending outbreaks might have the greatest effect on incidence.

Quorum sensing governs a transmissive Legionella subpopulation at the pathogen vacuole periphery.

The Gram-negative bacterium Legionella pneumophila is the causative agent of Legionnaires' disease and replicates in amoebae and macrophages within a distinct compartment, the Legionella-containing vacuole (LCV). The facultative intracellular pathogen switches between a replicative, non-virulent and a non-replicating, virulent/transmissive phase. Here, we show on a single-cell level that at late stages of infection, individual motile (PflaA -GFP-positive) and virulent (PralF - and PsidC -GFP-positive) L. pneumophila emerge in the cluster of non-growing bacteria within an LCV. Comparative proteomics of PflaA -GFP-positive and PflaA -GFP-negative L. pneumophila subpopulations reveals distinct proteomes with flagellar proteins or cell division proteins being preferentially produced by the former or the latter, respectively. Toward the end of an infection cycle (˜ 48 h), the PflaA -GFP-positive L. pneumophila subpopulation emerges at the cluster periphery, predominantly escapes the LCV, and spreads from the bursting host cell. These processes are mediated by the Legionella quorum sensing (Lqs) system. Thus, quorum sensing regulates the emergence of a subpopulation of transmissive L. pneumophila at the LCV periphery, and phenotypic heterogeneity underlies the intravacuolar bi-phasic life cycle of L. pneumophila.

NAD(H)-mediated tetramerization controls the activity of Legionella pneumophila phospholipase PlaB.

The virulence factor PlaB promotes lung colonization, tissue destruction, and intracellular replication of Legionella pneumophila, the causative agent of Legionnaires' disease. It is a highly active phospholipase exposed at the bacterial surface and shows an extraordinary activation mechanism by tetramer deoligomerization. To unravel the molecular basis for enzyme activation and localization, we determined the crystal structure of PlaB in its tetrameric form. We found that the tetramer is a dimer of identical dimers, and a monomer consists of an N-terminal α/ß-hydrolase domain expanded by two noncanonical two-stranded ß-sheets, ß-6/ß-7 and ß-9/ß-10. The C-terminal domain reveals a fold displaying a bilobed ß-sandwich with a hook structure required for dimer formation and structural complementation of the enzymatic domain in the neighboring monomer. This highlights the dimer as the active form. Δß-9/ß-10 mutants showed a decrease in the tetrameric fraction and altered activity profiles. The variant also revealed restricted binding to membranes resulting in mislocalization and bacterial lysis. Unexpectedly, we observed eight NAD(H) molecules at the dimer/dimer interface, suggesting that these molecules stabilize the tetramer and hence lead to enzyme inactivation. Indeed, addition of NAD(H) increased the fraction of the tetramer and concomitantly reduced activity. Together, these data reveal structural elements and an unprecedented NAD(H)-mediated tetramerization mechanism required for spatial and enzymatic control of a phospholipase virulence factor. The allosteric regulatory process identified here is suited to fine tune PlaB in a way that protects Legionella pneumophila from self-inflicted lysis while ensuring its activity at the pathogen-host interface.

Complete Genome Sequences of Three Clinical Listeria monocytogenes Sequence Type 8 Strains from Recent German Listeriosis Outbreaks.

We report here the closed genome sequences of three clinical Listeria monocytogenes strains of multilocus sequence typing (MLST) sequence type 8 (ST8). These strains are representatives of three separate listeriosis outbreak clusters (Alpha1, Pi4, and Sigma1) that affected Germany between 2012 and 2020.

Genome-wide insights into population structure and host specificity of Campylobacter jejuni.

The zoonotic pathogen Campylobacter jejuni is among the leading causes of foodborne diseases worldwide. While C. jejuni colonises many wild animals and livestock, persistence mechanisms enabling the bacterium to adapt to host species' guts are not fully understood. In order to identify putative determinants influencing host preferences of distinct lineages, bootstrapping based on stratified random sampling combined with a k-mer-based genome-wide association was conducted on 490 genomes from diverse origins in Germany and Canada. We show a strong association of both the core and the accessory genome characteristics with distinct host animal species, indicating multiple adaptive trajectories defining the evolution of C. jejuni lifestyle preferences in different ecosystems. Here, we demonstrate that adaptation towards a specific host niche ecology is most likely a long evolutionary and multifactorial process, expressed by gene absence or presence and allele variations of core genes. Several host-specific allelic variants from different phylogenetic backgrounds, including dnaE, rpoB, ftsX or pycB play important roles for genome maintenance and metabolic pathways. Thus, variants of genes important for C. jejuni to cope with specific ecological niches or hosts may be useful markers for both surveillance and future pathogen intervention strategies.

Toward an Integrated Genome-Based Surveillance of Salmonella enterica in Germany.

Despite extensive monitoring programs and preventative measures, Salmonella spp. continue to cause tens of thousands human infections per year, as well as many regional and international food-borne outbreaks, that are of great importance for public health and cause significant socio-economic costs. In Germany, salmonellosis is the second most common cause of bacterial diarrhea in humans and is associated with high hospitalization rates. Whole-genome sequencing (WGS) combined with data analysis is a high throughput technology with an unprecedented discriminatory power, which is particularly well suited for targeted pathogen monitoring, rapid cluster detection and assignment of possible infection sources. However, an effective implementation of WGS methods for large-scale microbial pathogen detection and surveillance has been hampered by the lack of standardized methods, uniform quality criteria and strategies for data sharing, all of which are essential for a successful interpretation of sequencing data from different sources. To overcome these challenges, the national GenoSalmSurv project aims to establish a working model for an integrated genome-based surveillance system of Salmonella spp. in Germany, based on a decentralized data analysis. Backbone of the model is the harmonization of laboratory procedures and sequencing protocols, the implementation of open-source bioinformatics tools for data analysis at each institution and the establishment of routine practices for cross-sectoral data sharing for a uniform result interpretation. With this model, we present a working solution for cross-sector interpretation of sequencing data from different sources (such as human, veterinarian, food, feed and environmental) and outline how a decentralized data analysis can contribute to a uniform cluster detection and facilitate outbreak investigations.