Biology subjects

Flieger, A.

Publications and source records attributed to Flieger, A..

12 recordsLinked to original sources

bioRxiv2026

Dual species interactions shield Campylobacter against multiple antibiotics

BackgroundCampylobacter is a major food-borne pathogen causing diarrhea. In severe cases, treatment typically involves fluoroquinolones (FQ) and macrolides. However, high proportions of FQ resistance (FQR) have made macrolides the remaining first-line treatment option. As part of the Campylobacter surveillance program in Germany, we investigated multidrug resistance (MDR) in clinical Campylobacter samples collected 2010 to 2022. MethodsA total of 6,980 samples was single-colony purified and analyzed for antimicrobial resistance phenotypes. Genome sequencing was performed for 2,912 samples, and antimicrobial resistance genes were mapped. Cultures showing MDR were further analyzed for contaminating DNA and studied using scanning electron microscopy (SEM). FindingsWe found that 453 (6%) Campylobacter samples were resistant to both FQ and macrolides. Two of the C. jejuni samples were resistant to antibiotics from ten different classes. Genome analysis revealed that these samples, despite being derived from single colonies, contained >10% Enterococcus DNA reads. SEM confirmed the presence of coccoid bacteria interspersed with spiral-shaped Campylobacter. Additional culture-based purification resulted in pure C. jejuni isolates that retained FQR but lost macrolide resistance. We further showed that presence of MDR Enterococcus spp. in the mixed samples protected C. jejuni from above-MIC (minimum inhibitory concentration) of several ribosome-targeting antimicrobials whereas pure Campylobacter were susceptible. These findings revealed that Campylobacter may gain antimicrobial resistance advantages through close interactions with Enterococcus spp. InterpretationMDR phenotypes in Campylobacter cultures may arise through close, dual-species interactions with other highly resistant intestinal bacteria, such as MDR Enterococcus spp. and may have practical implications for antibiotic treatment. FundingThis study was funded by the German Ministry of Health dedicated to the National Reference Centre for Salmonella and other Bacterial Enteric Pathogens.

microbiology↗

bioRxiv2025

A pbpB1 mutation causing β-lactam resistance in clinical Listeria monocytogenes isolates

ObjectivesListeriosis is a severe foodborne infection and associated with high mortality. Treatment is based on ampicillin, amoxicillin or penicillin, often combined with gentamicin, but meropenem is also used occasionally. {beta}-lactam resistant Listeria monocytogenes isolates are infrequently described but the mechanism of resistance is not known. MethodsA clinical L. monocytogenes isolate with suspected {beta}-lactam resistance was collected from a German listeriosis patient. Resistance profiling, whole genome sequencing, comparative genomics and genetic experiments were used to identify the causative DNA polymorphism. Spontaneous ampicillin resistant suppressors of L. monocytogenes reference strain EGD-e were selected and their genomes sequenced. ResultsA W428R substitution near the active site of penicillin binding protein B1 (PBP B1) was identified as the cause of ampicillin, amoxicillin and meropenem resistance. Further clinical L. monocytogenes isolates with similar resistance profiles were found by searching the genome database of the German consultant laboratory for Listeria for pbpB1 W428R-positive isolates. Furthermore, the same mutation was selected for in strain EGD-e during cultivation in the presence of ampicillin. ConclusionsL. monocytogenes can develop {beta}-lactam resistance by a specific substitution in PBP B1, likely selected for during {beta}-lactam treatment. Antibiotic susceptibility testing should be considered an important part of adequate listeriosis therapy.

microbiology↗

bioRxiv2024

Discovery of a synthetic small molecule targeting the central regulator of Salmonella pathogenicity

The enteric pathogen Salmonella enterica serovar Typhimurium relies on the activity of effector proteins to invade, replicate, and disseminate into host epithelial cells and other tissues, thereby causing disease. Secretion and injection of effector proteins into host cells is mediated by dedicated secretion systems, which hence represent major virulence determinants. Here, we report the identification of a synthetic small molecule with drug-like properties, C26, which suppresses the secretion of effector proteins, and consequently hinders bacterial invasion of eukaryotic cells. C26 binds to and inhibits HilD, the transcriptional regulator of the major secretion systems. While sharing the same binding pocket as the previously described long-chain fatty acid ligands, C26 inhibits HilD with a unique binding mode and a distinct mechanism. We provide evidence for target engagement within infected eukaryotic cells and present analogs with improved potency and suitability as scaffolds to develop anti-virulence agents against Salmonella infections in humans and animals.

microbiology↗

bioRxiv2024

High density genomic surveillance and risk profiling of clinical Listeria monocytogenes subtypes in Germany, 2018-2021

AO_SCPLOWBSTRACTC_SCPLOWFoodborne infections represent a significant public health concern, particularly when outbreaks affect many individuals over prolonged time. Systematic collection of pathogen isolates from infected patients, whole genome sequencing and phylogenetic analyses allow recognition and termination of outbreaks after source identification and risk profiling of abundant lineages. We here present a multi-dimensional analysis of >1,800 genome sequences from clinical L. monocytogenes isolates collected in Germany between 2018-2021. These isolates covered 62% of all notified cases and belonged to 188 infection clusters. 42% of these clusters were active for >12 months, 60% generated cases cross-regionally, including 11 multinational clusters. 37% of the clusters were caused by sequence type (ST) ST6, ST8 and ST1 clones and for selected clusters, we provide further epidemiological and genetic information. Frequencies of materno-fetal and brain infections allowed risk profiling of the most abundant STs, differentiating ST1 as hyper- and ST8, ST14, ST29 as well as ST155 as hypovirulent from ST6 with average virulence potential. Hepatocyte infection experiments confirmed these virulence differences. Inactivating mutations were found in several virulence and house-keeping genes, particularly in hypovirulent STs. Our work supports prioritization of clusters for epidemiological investigations and reinforces the need to analyse the mechanisms underlying hyper- and hypovirulence.

microbiology↗

bioRxiv2023

Rapid enzymatic detection of Shigatoxin-producing E. coli using fluorescence-labeled oligonucleotide substrates

Shigatoxin-producing E. coli (STEC) are important human pathogens causing disease ranging from diarrhea to severe hemolytic uremic syndrome. As STEC are transmitted via animals, food, and water, and may produce large outbreaks, their timely and qualified detection including isolate recovery is of high importance, but challenging and labor-intense. Thus, the availability of an easy-to-perform rapid test would be a tremendous advance. Since the common feature and major virulence factor of otherwise multifaceted STEC is the Shiga toxin (Stx), we developed a detection method for Stx, specifically for its catalytic RNA-N-glycosidase activity targeting the Sarcin Ricin Loop (SRL) of 28S ribosomal RNA. To this end, synthetic ssDNA substrates mimicking the SRL were designed and linked to a fluorophore and quencher pair, which conferred a fluorescence signal after cleavage by Stx. Optimal results using bacterial culture supernatants or single colonies were achieved for substrate StxSense 4 following 30 to 60 minutes incubation. Importantly, different Stx1 and Stx2 subtypes, diverse STEC serotypes, and Shigella were detected. In conclusion, the assay offers rapid and facile detection of STEC based on a real-time readout for Stx activity. Therefore, it may improve STEC risk evaluation, therapy decisions, outbreak and source detection, and simplify research for antimicrobials.

microbiology↗

bioRxiv2023

Structure-function relationships underpin disulfide loop cleavage-dependent activation of Legionella pneumophila lysophosholipase 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 at residue E266/L267 located 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 /{beta} 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 both substrate access and membrane interaction before activation; however, the catalytic and membrane interaction site gets more accessible when the disulfide loop is processed. After structural modelling, 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 towards 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.

biochemistry↗

bioRxiv2022

Recent O-antigen diversification masks highly pathogenic STEC O104:H4

Shiga toxin-producing E. 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 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 (ST) 678. 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. ImportanceOur data suggest the high virulence ensemble of the STEC O104:H4 outbreak strain remains a global threat because genomically similar strains cause disease worldwide, but that horizontal acquisition of O-antigen gene clusters has diversified the O-antigens of strains belonging to ST678. Thus, identification of these highly pathogenic strains is masked by diverse and rare O-antigens, thereby confounding the interpretation of their potential risk.

microbiology↗

bioRxiv2021

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.

genomics↗

bioRxiv2020

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

The virulence factor and phospholipase PlaB promotes lung colonization, tissue destruction, and intracellular replication of Legionella pneumophila, the causative agent of Legionnaires disease. It is 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 phospholipase /{beta}-hydrolase domain augmented by two non-canonical two-stranded {beta}-sheets, {beta}6/{beta}7 and {beta}9/{beta}10. The C- terminal domain reveals a novel fold displaying a bilobed {beta}-sandwich with a hook structure that is required for dimer formation and complementation of the phospholipase domain in the neighboring monomer. 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 tetrameric form and concomitantly reduced activity. {beta}9/{beta}10 mutants revealed a decrease in the tetrameric fraction, altered activity profiles, and mislocalization. Protein variants lacking the hook or strands {beta}6/{beta}7 were unaffected in terms of localization but lost their activity, and lid mutants changed substrate specificity. 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 regulatory process identified is ideally suited to fine tune PlaB in a way that protects L. pneumophila from self-inflicted lysis while ensuring its activity at the pathogen-host interface.

microbiology↗

bioRxiv2020

Population structure-guided profiling of antibiotic resistance patterns in clinical Listeria monocytogenes isolates from Germany identifies pbpB3 alleles associated with low levels of cephalosporin resistance

AO_SCPLOWBSTRACTC_SCPLOWCase numbers of listeriosis have been increasing in Germany and the European Union during the last decade. In addition reports on the occurrence of antibiotic resistance in Listeria monocytogenes in clinical and environmental isolates are accumulating. The susceptibility towards 14 antibiotics was tested in a selection of clinical L. monocytogenes isolates to get a more precise picture of the development and manifestation of antibiotic resistance in the L. monocytogenes population. Based on the population structure determined by core genome multi locus sequence typing (cgMLST) 544 out of 1,220 sequenced strains collected in Germany between 2009 and 2019 were selected to cover the phylogenetic diversity observed in the clinical L. monocytogenes population. All isolates tested were susceptible towards ampicillin, penicillin and co-trimoxazole - the most relevant antibiotics in the treatment of listeriosis. Resistance to daptomycin and ciprofloxacin was observed in 493 (91%) and in 71 (13%) of 544 isolates, respectively. While all tested strains showed resistance towards ceftriaxone, the minimal inhibitory concentrations (MIC) observed varied widely between 4 mg/L up to >128 mg/L. An allelic variation of the penicillin binding protein gene pbpB3 could be identified as the cause of this difference in ceftriaxone resistance levels. This study is the first population structure-guided analysis of antimicrobial resistance in recent clinical isolates and confirms the importance of penicillin binding protein B3 (PBP B3) for the high level of intrinsic cephalosporin resistance of L. monocytogenes on a population-wide scale.

microbiology↗

bioRxiv2019

Can genome-based analysis be the new gold-standard for routine Salmonella serotyping?

Salmonella enterica is the second most reported bacterial cause of food-borne infections in Europe. Therefore molecular surveillance activities based on pathogen subtyping are an important measure of controlling Salmonellosis by public health agencies. In Germany, at the federal level, this work is carried out by the National Reference Center for Salmonella and other Bacterial Enteric Pathogens (NRC). With rise of next generation sequencing techniques, the NRC has introduced whole-genome-based typing methods for S. enterica in 2016. In this study we report on the feasibility of genome-based in silico serotyping in the German setting using raw sequence reads. We found that SeqSero and seven gene MLST showed 98% and 95% concordance, respectively, with classical serotyping for the here evaluated serotypes, including the most common German serotypes S. Enteritidis and S. Typhimurium as well as less frequently found serotypes. The level of concordance increased to >99% when the results of both in silico methods were combined. However, both tools exhibited misidentification of monophasic variants, in particular monophasic S. Typhimurium and therefore need to be fine-tuned for reliable detection of this epidemiologically important variant. We conclude that with adjustments Salmonella genome-based serotyping might become the new gold standard.

microbiology↗

bioRxiv2019

A patatin-like phospholipase is crucial for gametocyte induction in the malaria parasite Plasmodium falciparum

Patatin-like phospholipases (PNPLAs) are highly conserved enzymes of prokaryotic and eukaryotic organisms with major roles in lipid homeostasis. The genome of the malaria parasite Plasmodium falciparum encodes four putative PNPLAs with predicted functions during phospholipid degradation. We here investigated the role of one of the plasmodial PNPLAs, a putative PLA2 termed PNPLA1, during blood stage replication and gametocyte development. PNPLA1 is present in the asexual and sexual blood stages and here localizes to the cytoplasm. PNPLA1-deficiency due to gene disruption or conditional gene-knockdown had no effect on erythrocytic replication, gametocyte maturation and gametogenesis. However, blood stage parasites lacking PNPLA1 were severely impaired in gametocyte induction, while PNPLA1 overexpression promotes gametocyte formation. The loss of PNPLA1 further leads to transcriptional down-regulation of genes related to gametocytogenesis, including the gene encoding the sexual commitment regulator AP2-G. Additionally, lipidomics of PNPLA1-deficient asexual blood stage parasites revealed overall increased levels of major phospholipids, including phosphatidylcholine (PC), which is a substrate of PLA2. Because PC synthesis is pivotal for erythrocytic replication, while the reduced availability of PC precursors drives the parasite into gametocytogenesis, we hypothesize that the high PC levels due to PNPLA1-deficiency prevent the blood stage parasites from entering the sexual pathway.

cell biology↗