Biology subjects

Leptihn, S.

Publications and source records attributed to Leptihn, S..

12 recordsLinked to original sources

bioRxiv2026

Sequential Use of Two Capsule-Targeting Klebsiella Phages Reveals Order-Dependent Efficacy and Distinct Resistance Pathways in vitro and in vivo

Bacteriophage cocktails are widely assumed to improve treatment of multidrug-resistant Klebsiella pneumoniae, yet many therapeutic phages target capsular polysaccharides (CPS), potentially promoting antagonism and shared resistance. Here, we investigated how receptor usage, resistance evolution, and dosing order influence the activity of two K1-specific phages, Loop and Spear, against a hypervirulent ST23 strain. Using in vitro killing assays and Bliss analysis, we show that a 1:1 Loop and Spear cocktail did not improve bacterial suppression compared to Loop alone and instead exhibits multiplicity-of-infection-dependent antagonism, consistent with competition for a shared CPS receptor. Sequential dosing revealed strong order dependence: treatment with Spear followed by Loop qualitatively altered growth trajectories in a subset of cultures, whereas the reverse order or repeated dosing, provided little additional benefit. Resistance profiling showed that mutants resistant to both phages predominantly carried mutations in capsule synthesis or export genes, whereas Spear specific resistance was associated with mutations in fkpA, encoding a periplasmic chaperone for outer membrane protein biogenesis. Adsorption assays confirmed that capsule associated mutations abolished Loop attachment, while {Delta}fkpA mutants retained Loop binding, supporting CPS to be a primary receptor with Spear additionally requiring an FkpA-dependent secondary receptor. In a Galleria mellonella infection model, a capsule-mutant resistant isolate showed reduced virulence, and only sequential therapy with Spear followed by Loop improved survival beyond monotherapy. These findings show that receptor sharing can render phage cocktails antagonistic and highlight sequential, order-aware regimens as a strategy to exploit resistance-virulence trade-offs while limiting the emergence of double-resistant mutants.

microbiology↗

bioRxiv2026

TattleTail: A Pyocin Prediction Tool

Tailocins are phage tail-like bacteriocins (PTLBs) thought to be remnants of prophages that have lost the ability to package viral genomes while retaining the ability to kill closely-related bacterial strains, thereby mediating bacterial competition. Tailocins produced by Pseudomonas aeruginosa are referred to as pyocins. Apart from their contribution to ecological fitness, they also have the potential to be harnessed as highly-specific antimicrobials to treat antibiotic resistant bacterial infections. Although pyocins lack the genetic components to package viral genomes, pyocin-encoding gene clusters share a high degree of genetic homology to phage tail genes, attributed to their shared ancestry. This poses a significant annotation-based challenge, as current prophage prediction tools, which rely on phage homology for prediction, can misclassify pyocins or tailocins as prophages. Pyocins unknowingly being misannotated as prophages is not only a bioinformatic issue, but can certainly confound experiments examining bacterial competition and prophage induction, if the experimental setup is based on this unintentional misannotation. In this study, we present "TattleTail", the first version of a bioinformatic tool designed to accurately identify tailocins in genome sequences, with a focus on identifying phage tail-derived pyocin-encoding gene clusters in P. aeruginosa in its first iteration. The tool leverages conserved pyocin gene cluster markers and accounts for the absence of canonical phage features, such as capsid, terminase and integrase genes, thereby distinguishing pyocins from intact and cryptic prophages. Validation in P. aeruginosa and non-P. aeruginosa genomes confirmed the presence of pyocin regions in all P. aeruginosa genomes, while none were detected in any non-P. aeruginosa genomes. Notably, TattleTail enabled the identification of representative pyocin-encoding gene clusters in clinical P. aeruginosa isolates. The identified pyocins in the clinical isolates were induced using mitomycin C, visualized via transmission electron microscopy, processed via tangential flow filtration and demonstrated bactericidal activity, thereby confirming TattleTail predictions. TattleTail aims to complement existing prophage prediction tools during genomic analyses involving phage-derived elements in bacterial genomes, allowing more accurate identification of these elements facilitated by robust discrimination between prophages and tailocins.

bioinformatics↗

bioRxiv2022

In vitro and in vivo evaluation of the biofilm-degrading Pseudomonas phage Motto, as a candidate for phage therapy

Infections caused by Pseudomonas aeruginosa are becoming increasingly difficult to treat due to the emergence of strains that have acquired multidrug resistance. Therefore, phage therapy has gained attention as an alternative to the treatment of pseudomonal infections. Phages are not only bactericidal but occasionally show activity against biofilm as well. Here, we describe the Pseudomonas phage Motto which has the ability to clear P. aeruginosa infections in an animal model and also exhibited biofilm-degrading properties. The phage has substantial antibiofilm activity against strong biofilm-producing isolates (n = 10), with at least a 2-fold reduction within 24 hours. To demonstrate the safety of using phage Motto, we performed cytotoxicity studies with human cell lines (HEK 293 and RAW 264.7 macrophages). Using a previously established in vivo model, we demonstrated the efficacy of Motto in C. elegans, with a 90% survival rate when treated with the phage at an MOI of 10. ImportancePhages are often evaluated mainly on their ability to kill bacterial hosts. One important aspect, however, is often neglected: Their ability to degrade biofilms. Not all phages are able to disintegrate biofilms. While phages can kill planktonic cells, it also has often been observed that phages are not able to infect those that are embedded in biofilms. Phage Motto appears to have highly efficient enzymes that degrade biofilms, and might therefore be a highly valuable therapeutic candidate.

microbiology↗

bioRxiv2022

From motor protein to toxin: Mutations in the zonula occludens toxin (Zot) of Vibrio cholerae phage CTXΦ suggest a loss of phage assembly function

Prophages, i.e. dormant viruses residing in bacterial cells, are not just passive passengers in the bacterial host. Several prophage-encoded genes have been shown to be contributors to bacterial virulence by mediating antimicrobial resistance or by providing toxins. Other prophage genes exhibit beneficial effects on the host by modulating e.g. motility or biofilm formation. In this study, we used an in vivo phage assembly assay and tested an extensive array of single point mutations or their combinations found in Zot, the zonula occludens toxin encoded by the Vibrio cholerae phage CTX{phi}. The assay makes use of the highly homologous Zot-like protein g1p of the filamentous Coliphage M13, a motor protein that mediates the trans-envelope assembly and secretion of filamentous phages. We also measured the in vitro ATP hydrolysis of purified proteins, and quantified virus production in V. cholerae mediated by Zot or the Zot-like protein of the two Vibrio phages CTX{phi} and VFJ{phi}. In addition, we investigated sequence variations of the Walker motifs in Vibrio species using bioinformatics method, and revealed the molecular basis of ATP binding using molecular docking and molecular dynamics simulation based on the structure predicted by AlphaFold2. Our data indicates that g1p proteins in Vibrio can easily accumulate deleterious mutations and likely lose the ability to efficiently hydrolyse ATP, while the CTX{phi} Zot was further exapted to now act as an auxiliary toxin during the infection by Vibrio cholerae.

microbiology↗

bioRxiv2022

Phage-antibiotic combinations to control Pseudomonas aeruginosa-Candida two-species biofilms

Phage-antibiotic combinations to treat bacterial infections are gaining increased attention due to the synergistic effects often observed when applying both components together. This synergy has also been observed for bacteria embedded in biofilms as many phages are capable of degrading the heterogeneous material, often preventing antibiotic compounds from reaching the embedded bacteria. Most studies however focus on a single pathogen, although in many clinical cases multiple species are present at the site of infection. The aim of this study was to investigate the anti-biofilm activity of phage-antibiotic/antifungal combinations on single- and dual-species biofilms formed by the bacterium P. aeruginosa and the fungal pathogen Candida albicans, two microorganisms commonly found together in cystic fibrosis patients. The Pseudomonas phage Motto in combination with ciprofloxacin had significant anti-biofilm activity in disintegrating 24-hour-old pseudomonal biofilms. Also, other antibiotic combinations, such as cefotaxime, gentamicin, meropenem and tetracycline showed substantial effects on biofilms. We then compared biofilms formed by P. aeruginosa alone with the dual-species biofilms formed by bacteria and C. albicans. Here, we found that the phage together with the antifungal fluconazole was active against 6-hour-old dual-species biofilms but showed only negligible activity against 24-hour-old biofilms. Similarly, the combination of antibacterial compounds together with the phage showed no synergistic effects on biofilms formed by P. aeruginosa and C. albicans. This study lays the first foundation for potential therapeutic approaches to treat infections caused by bacteria and fungi using phage-antibiotic combinations.

microbiology↗

bioRxiv2022

A Lysozyme Murein Hydrolase with Broad-spectrum Antibacterial Activity from Enterobacter Phage myPSH1140

Bacteriophages and bacteriophage-derived peptidoglycan hydrolases (endolysins) present promising alternatives for the treatment of infections caused by multi-drug resistant Gram-negative and Gram-positive pathogens. In this study, Gp105, a putative lysozyme murein hydrolase from Enterobacter phage myPSH1140 was characterized in silico, in vitro as well as in vivo using the purified protein. Gp105 contains a T4-type lysozyme-like domain (IPR001165) and belongs to Glycoside hydrolase family 24 (IPR002196). The putative endolysin indeed had strong antibacterial activity against Gram-negative pathogens including E. cloacae, K. pneumoniae, P. aeruginosa, S. marcescens, Citrobacter sp. and A. baumannii. Also, an in vitro peptidoglycan hydrolysis assay showed strong activity against purified peptidoglycans. This study demonstrates the potential of Gp105 to be used as an antibacterial protein to combat Gram-negative pathogens.

microbiology↗

bioRxiv2022

A multiwell-plate Caenorhabditis elegans assay for assessing the therapeutic potential of Bacteriophages against Clinical Pathogens

In order to establish phage therapy as a standard clinical treatment for bacterial infections, testing of every phage to ensure the suitability and safety of the biological compound is required. While some issues have been addressed over recent years, standard and easy-to-use animal models to test phages are still rare. Testing of phages in highly suitable mammalian models such as mice is subjected to strict ethical regulations, while insect larvae such as the Galleria mellonella model suffers from batch-to-batch variations and requires manual operator skills to inject bacteria, resulting in unreliable experimental outcomes. A much simpler model is the nematode Caenorhabditis elegans which feeds on bacteria, a fast growing and easy to handle organism which can be used in high-throughput screening. In this study, two clinical bacterial strains of Escherichia coli, one Klebsiella pneumoniae and one Enterobacter cloacae strain were tested on the model system together with lytic bacteriophages that we isolated previously. We developed a liquid-based assay, in which the efficiency of phage treatment was evaluated using a scoring system based on microscopy and counting of the nematodes, allowing increasing statistical significance compared to other assays such as larvae or mice. Our work demonstrates the potential to use Caenorhabditis elegans to test the virulence of strains of Klebsiella pneumoniae, Enterobacter cloacae and EHEC/ EPEC as well as the efficacy of bacteriophages to treat or prevent infections, allowing a more reliable evaluation for the clinical therapeutic potential of lytic phages. ImportanceValidating the efficacy and safety of phages prior to clinical application is crucial to see phage therapy in practice. Current animal models include mice and insect larvae, which pose ethical or technical challenges. This study examined the use of the nematode model organism, C. elegans as a quick, reliable and simple alternative for testing phages. The data shows that all the four tested bacteriophages can eliminate bacterial pathogens and protect the nematode from infections. Survival rates of the nematodes increased from <20% in the infection group to >90% in the phage treatment group. Even the nematodes with poly-microbial infections recovered during phage cocktail treatment. The use of C. elegans as a simple whole-animal infection model is a rapid and robust way to study the efficacy of phages before testing them on more complex model animals such as mice.

microbiology↗

bioRxiv2021

Correlation of mitochondrial TOM core complex stop-and-go and open-closed channel dynamics

The role of lateral diffusion of proteins in the membrane in the context of function has not been examined extensively. Here, we explore the relationship between protein lateral diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC). Optical ion flux sensing through single TOM-CC molecules shows that TOM-CC can occupy three ion permeability states. Whereas freely diffusing TOM-CC molecules are preferentially found in a high permeability state, physical tethering to an agarose support causes the channels to transition to intermediate and low permeability states. This data shows that combinatorial opening and closing of the two pores of TOM-CC correlates with lateral protein diffusion in the membrane plane, and that the complex has mechanosensitive-like properties. This is the first demonstration of {beta}-barrel protein mechanosensitivity, and has direct conceptual consequences for the understanding of the process of mitochondrial protein import. Our approach provides a novel tool to simultaneously study the interplay of membrane protein diffusion and channel dynamics.

biophysics↗

bioRxiv2021

Phage Resistance Mechanisms Increase Colistin Sensitivity in Acinetobacter baumannii

Few emergency-use antibiotics remain for the treatment of multidrug-resistant bacterial infections. Infections with resistant bacteria are becoming increasingly common. Phage therapy has reemerged as a promising strategy to treat such infections, as microbial viruses are not affected by bacterial resistance to antimicrobial compounds. However, phage therapy is impeded by rapid emergence of phage-resistant bacteria during therapy. In this work, we studied phage-resistance of colistin sensitive and resistant A. baumannii strains. Using whole genome sequencing, we determined that phage resistant strains displayed mutations in genes that alter the architecture of the bacterial envelope. In contrast to previous studies where phage-escape mutants showed decreased binding of phages to the bacterial envelope, we obtained several not uninfectable isolates that allowed similar phage adsorption compared to the susceptible strain. When phage-resistant bacteria emerged in the absence of antibiotics, we observed that the colistin resistance levels often decreased, while the antibiotic resistance mechanism per se remained unaltered. In particular the two mutated genes that conveyed phage resistance, a putative amylovoran-biosynthesis and a lipo-oligosaccharide (LOS) biosynthesis gene, impact colistin resistance as the mutations increased sensitivity to the antibiotic.

microbiology↗

bioRxiv2020

Characterisation of bacteriophage JD419, a Staphylococcal phage with an unusual morphology and broad host range

As an antimicrobial therapy, therapeutic phages, also known as "Phage therapy" are able to inactivate multi-drug resistant bacteria such as methicillin and vancomycin resistant S. aureus and thus present a possible treatment for infections that are otherwise incurable. In this paper, we present a novel phage called JD419, which has a remarkably wide host-range. The virulent phage JD419 exhibits an elongated capsid and was able to infect and lyse 83 of all 129 tested clinical strains (64.3%) of multi-drug resistant S. aureus including MRSA. To evaluate the potential as a therapeutic phage, we tested the ability of phage JD419 to remain infectious after treatment exceeding physiological pH or temperature. The lytic activity of the phage was retained at pH values of 6.0-8.0 and at temperatures below 50{degrees}C. As phages sometimes contain virulence genes, we sequenced the complete genome of JD419. The 45509 bp genome contains a predicted 65 ORFs, none of which show homology to any known virulence or antibiotic resistance genes. Our study illustrates that Staphylococcus phage JD419 has the potential to be used for diagnostic, prophylaxic and therapeutic purposes.

microbiology↗

bioRxiv2020

A Biological Inventory of Prophages in A. baumannii Genomes Reveal Distinct Distributions in Classes, Length and Genomic Positions

Acinetobacter baumannii is of major clinical importance as the bacterial pathogen often causes hospital acquired infections, further complicated by the high prevalence of antibiotic resistant strains. Aside from natural tolerance to certain antibiotic classes, resistance is often acquired by the exchange of genetic information via conjugation but also by the high natural competence exhibited by A. baumannii. In addition, bacteriophages are able to introduce resistance genes but also toxins and virulence factors via phage mediated transduction. In this work, we analysed the complete genomes of 177 A. baumannii strains for the occurrence of prophages, and analysed their taxonomy, size and positions of insertion. Among all the prophages that were detected, Siphoviridae and Myoviridae were the two most commonly found families, while the average genome size was determined as 3.98 Mbp. Our data shows the wide variation in the number of prophages in A. baumannii genomes and the prevalence of certain prophages within strains that are most "successful" or potentially beneficial to the host. Our study also revealed that only two specific sites of insertion within the genome of the host bacterium are being used, with few exceptions only. Lastly, we analysed the existence of genes that are encoded in the prophages, which confer antimicrobial resistance (AMR). Several phages carry AMR genes, including OXA-23 and NDM-1, illustrating the importance of lysogenic phages in the acquisition of resistance genes.

bioinformatics↗

bioRxiv2020

BacAnt: the combination annotation server of antibiotic resistance gene, insertion sequences, integron, transposable elements for bacterial DNA sequences

Whole genome sequencing (WGS) of bacteria has become a routine method in diagnostic laboratories. One of the clinically most useful advantages of WGS is the ability to predict antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) in bacterial sequences. This allows comprehensive investigations of such genetic features but can also be used for epidemiological studies. A plethora of software programs have been developed for the detailed annotation of bacterial DNA sequences, such as RAST (Rapid Annotation using Subsystem Technology), Resfinder, ISfinder, INTEGRALL and The Transposon Registry. Unfortunately, to this day, a reliable annotation tool of the combination of ARGs and MGEs is not available, and the generation of genbank files requires much manual input. Here, we present a new webserver which allows the annotation of ARGs, integrons, and transposable elements at the same time. The pipeline generates genbank files automatically, which are compatible with easyfig for comparative genomic analysis. Our BacAnt code and standalone software package are available at https://github.com/xthua/bacant with an accompanying web application at http://bacant.net. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/284273v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@262a07org.highwire.dtl.DTLVardef@1914001org.highwire.dtl.DTLVardef@101089corg.highwire.dtl.DTLVardef@dc5807_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗