Biology subjects

Makarewicz, O.

Publications and source records attributed to Makarewicz, O..

8 recordsLinked to original sources

bioRxiv2025

Galleria mellonella model for studying Gram-positive bacterial implant biofilms

Implant-associated biofilm infections, particularly those caused by Staphylococcus aureus and Enterococcus faecalis, present significant challenges in clinical settings, often necessitating surgical removal. This study investigates the potential of the invertebrate model Galleria mellonella for evaluating biofilm formation of S. aureus and E. faecalis clinical isolates, the leading causes of implant-associated infections. Utilizing expanded polytetrafluoroethylene (ePTFE) sutures as a surrogate for cardiac implants, we employed two biofilm formation methodologies reflecting the two main routes of implant infections: in vivo biofilm development within larvae mimicking hematogenous spread and pre-formed biofilm transplantation mimicking contamination during surgery. Scanning electron microscopy revealed complex biofilm structures for the biofilm formed inside of the larvae on implant, closely mimicking clinical conditions. Antibiotic treatments with vancomycin and rifampicin demonstrated significant reductions in bacterial biofilms, proving highly effective. The study highlights the G. mellonella models potential for preclinical biofilm research, offering a cost-effective and ethical alternative to vertebrate models while providing valuable insights into biofilm-related infections and their treatment.

microbiology↗

bioRxiv2024

Synergistic cross-kingdom host cell damage between Candida albicans and Enterococcus faecalis

The fungus Candida albicans and the Gram-positive bacterium Enterococcus faecalis share mucosal niches in the human body. As opportunistic pathogens, both are found to expand population size during dysbiosis, and can cause severe systemic infections in susceptible individuals. Here, we show that the presence of C. albicans results in increased host cell damage by E. faecalis. Furthermore, E. faecalis aggravates oropharyngeal candidiasis in mice. Increased damage is mediated by enterococcal cytolysin, and involves both physical interaction and altered glucose availability. Physical interaction promotes accumulation of bacteria on host cells, facilitating contact of cytolysin with host cells. Glucose depletion by the metabolic activity of the fungus sensitized host cells to cytolysin. This work illustrates how a complex interplay between fungi and bacteria can result in detrimental consequences for the host.

microbiology↗

bioRxiv2024

AbrB antirepressor AbbA is a competitive inhibitor of AbrB-phyC interaction

In Bacillus species, the interaction between the repressor AbrB and the antirepressor AbbA is vital for regulating gene expression. Our study reveals that AbrB binds DNA cooperatively the promoter of the phyC gene through multiple tetramers, forming a complex regulatory mechanism. AbbA disrupts AbrBs DNA binding by competing for its DNA-binding sites, as shown by surface plasmon resonance (SPR) and gel shift assays. Circular dichroism (CD) confirmed that AbbA does not bind directly to the phyC promoter but mimics DNA to interfere with AbrB. Small-angle X-ray scattering (SAXS) data suggest that AbbA resembles a deformed DNA double helix. Our results indicate that AbbA binds to AbrBs DNA-binding sites located with the N-terminal domain causing AbrB displacement. The interaction exhibits negative cooperativity, with both high- and low-affinity binding sites, as evidenced by Scatchard plots and kinetic studies. Our findings suggest that AbbA effectively mimics DNA to displace AbrB, activating transition-state genes. This research enhances our understanding of bacterial gene regulation and provides insights into the complex mechanisms controlling transcription in Bacillus species.

molecular biology↗

bioRxiv2024

Diversification of Pseudomonas aeruginosa biofilm populations under repeated phage exposures decreases the efficacy of the treatment

Phage therapy has been proposed as a therapeutic alternative to antibiotics for treatment of chronic, biofilm-related P. aeruginosa infections. To get a deeper insight into the complex biofilm-phage interactions, we investigated in the present study the effect of three successive exposures to lytic phages of biofilms formed by the reference strains PAO1 and PA14 as well as of two sequential clinical P. aeruginosa isolates from the sputum of a patient with cystic fibrosis (CF). The Calgary device was employed as biofilm model and the efficacy of phage treatment was evaluated by measurements of the biomass stained with crystal violet (CV) and of the cell density of the biofilm bacterial population (CFU/ml) after each of the three phage exposures. The genetic alterations of P. aeruginosa isolates from biofilms exposed to phages were investigated by whole genome sequencing. We show here that the anti-biofilm efficacy of the phage treatment decreased rapidly with repeated applications of lytic phages on P. aeruginosa strains with different genetic background. Although we observed the maintenance of a small subpopulation of sensitive cells after repeated phage treatments, a fast recruitment of mechanisms involved in the persistence of biofilms to the phage attack occurred, mainly by mutations causing alterations of the phage receptors. However, mutations causing phage tolerant phenotypes such as alginate-hyperproducing mutants were also observed. In conclusion, a decreased anti-biofilm effect occurred after repeated exposure to lytic phages of P. aeruginosa biofilms due to recruitment of different resistance and tolerance mechanisms.

microbiology↗

bioRxiv2024

Temperate bacteriophage induced in Pseudomonas aeruginosa biofilms can modulate bacteriophage and antibiotic resistance

Given the high levels of resistance in Gram-negative bacteria, phage therapy is garnering increasing attention. In Germany, a clinical study is already underway investigating a phage cocktail for the treatment of Pseudomonas aeruginosa in cystic fibrosis (CF) patients. In our study, we examined susceptibility to virulent phages and the PF1-like prophage and antimicrobial profiles and of P. aeruginosa isolates from a local cystic fibrosis cohort to identify correlations and lysogenic conversion of the prophegs. Consistent with other studies, prophage Pf4 is the most prevalent in this cohort and is activated in the absence of other influences during biofilm formation. These phages can be transferred to other strains that do not contain Pf1-like prophages, thereby influencing the dynamics of bacterial populations in the CF lung. This also rapidly leads to the emergence of a subpopulation resistant to the virulent phages, potentially complicating phage therapy. However, this subset also becomes more susceptible to most antibiotics commonly used in CF, which could be a useful treatment strategy. Interestingly, this bacterial subset lost its susceptibility to colistin, an important inhaled antibiotic in CF, which could lead to treatment failure. Our research highlights both the difficulties and potential strategies to improve treatment options for CF patients. Author summaryWe investigated 51 P. aeruginosa isolates obtained from CF patients for the presence of PF1-like prophages and characterized their susceptibility prior and after lysogenig conversion of the prophages to three virulent phages. Our study revealed that the temperate phage Pf4 is the most prominent PF1-like prophage in this cohort, undergoing lysogenic conversion during biofilm formation. The virions identified in the biofilm supernatants are superinfective and transferable to other prophage-free P. aeruginosa isolates, shaping population dynamics in the CF lung. Prophage reactivation results in the survival of a sub-population with reduced susceptibility to virulent phages, posing a potential challenge for phage therapy. However, this sub-population exhibited restored susceptibility to most CF-relevant antibiotics, presenting an intriguing therapeutic opportunity. Targeted prophage reactivation could sensitize multidrug-resistant P. aeruginosa in CF patients, enhancing or even restoring antimicrobial efficacy. Notably, this sub-population also showed a loss of colistin susceptibility, which may lead to treatment failure when colistin is used as an inhaled antipseudomonal antibiotic. Our findings highlight both significant challenges and potential therapeutic opportunities for optimizing the treatment of CF patients. However, these findings are preliminary and require further investigation, particularly regarding the possibility of lysogenic conversion in other prophages (such as Siphoviridae) and how these interplays with resistance to virulent phages. Further studies are ongoing and will enhance our understanding of the role of prophages in the pathophysiology of CF.

microbiology↗

bioRxiv2023

Bacteriophage-mediated decolonization of Enterobacteriaceae in a novel Galleria mellonella gut colonization model

PurposeGalleria mellonella larvae have emerged as an invertebrate model for studying bacterial pathogenesis and novel therapeutic options due to ethical concerns associated with the use of mammalian models such as mice. The benefits of using G. mellonella larvae include a less complex microbiome in the gut, making it suitable for gut colonization studies. The intestinal colonization of Klebsiella pneumoniae (Kp) and Escherichia coli (Ec), two of the most antibiotic-resistant bacteria on the World Health Organizations (WHO) priority list, plays a key role in the spread of antibiotic resistance. Bacteriophage therapy is emerging as a promising alternative for antibiotic-resistant bacteria due to its ability to specifically target and infect bacterial hosts, making it suitable for gut decontamination. This study aimed to establish a novel Enterobacteriaceae G. mellonella larvae gut colonization model and compare the efficacy of conventional antibiotic treatment with a one-time phage cocktail in decolonizing the gut. ApproachLarvae were force-fed with different concentrations of bacterial doses of K. pneumoniae and E. coli at 0 h, 24 h, and 48 h, followed by survival monitoring at 24 h intervals. After 48 h and 120 h of the last force feed, the colony forming unit (CFU) count in the gut was evaluated. After successful colonization, larvae were one-time force-fed with either a 107 PFU/larvae bacteriophage cocktail or with ciprofloxacin 4 mg/L or meropenem 2 mg/L. After 24 h post phage feeding, CFU counts were determined. Main findingsThree bacterial doses of 106 CFU/larvae led to a stable gut colonization in the larvae gut regardless of the K. pneumoniae and E. coli strains. Bacteriophage force-feeding reduced bacterial colonization by 4 log10 CFU/larvae whereas antibiotic treatment led to a 2 log10 CFU/larvae reduction compared to the control. The novel alternative G. mellonella model for gut colonization studies can be used for proof-of-concept studies, reducing or even obviating the number of follow-up experiments in vertebrate models.

microbiology↗

bioRxiv2021

Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore's adaptive sequencing

Metagenomic sequencing is promising for clinical applications to study microbial composition concerning disease or patient outcomes. Alterations of the vaginal microbiome are associated with adverse pregnancy outcomes, like preterm premature rupture of membranes and preterm birth. Methodologically these samples often have to deal with low relative amounts of prokaryotic DNA and high amounts of host DNA (> 90%), decreasing the overall microbial resolution. Nanopores adaptive sampling method offers selective DNA depletion or target enrichment to directly reject or accept DNA molecules during sequencing without specialized sample preparation. Here, we demonstrate how selective human host depletion resulted in a 1.70 fold ({+/-} 0.27 fold) increase in total sequencing depth, providing higher taxonomic profiling sensitivity. At the same time, the microbial composition remains consistent with the control experiments. The complete removal of all human host sequences is not yet possible and should be considered as an ethical approval statement might still be necessary. Adaptive sampling increased microbial sequencing yield in all 15 sequenced clinical routine vaginal samples, making it a valuable tool for clinical surveillance and medical-based research, which can be used in addition to other host depletion methods before sequencing.

molecular biology↗

bioRxiv2020

What the Phage: A scalable workflow for the identification and analysis of phage sequences

Phages are among the most abundant and diverse biological entities on earth. Phage prediction from sequence data is a crucial first step to understanding their impact on the environment. A variety of bacteriophage prediction tools have been developed over the years. They differ in algorithmic approach, results, and ease of use. We, therefore, developed "What the Phage" (WtP), an easy-to-use and parallel multitool approach for phage prediction combined with an annotation and classification downstream strategy, thus, supporting the users decision-making process by summarizing the results of the different prediction tools in charts and tables. WtP is reproducible and scales to thousands of datasets through a workflow manager (Nextflow). WtP is freely available under a GPL-3.0 license (https://github.com/replikation/What_the_Phage).

bioinformatics↗