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7.4
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Microorganisms
Special Issues
Contemporary Perspectives on Bacterial Virulence Factors
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Contemporary Perspectives on Bacterial Virulence Factors

A special issue of Microorganisms (ISSN 2076-2607). This special issue belongs to the section "Molecular Microbiology and Immunology".

Deadline for manuscript submissions: 15 May 2025 | Viewed by 4827

Special Issue Editors

Dr. Matthew Jackson

E-Mail Website
Guest Editor
School of Medicine, Wayne State University, Detroit, MI, USA
Interests: bacterial virulence
Dr. Kevin Theis

E-Mail Website
Guest Editor
Department of Biochemistry, Microbiology & Immunology, Wayne State University, Detroit, MI 48201, USA
Interests: pathogen; host–microbe interactions; medical microbiology
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Bacterial pathogens are classified using a variety of criteria, with one of them being their capacity to produce virulence factors (VFs) that enable them to cause damage to a mammalian host. VFs are classified according to their capacity to colonize specific body sites, resist phagocytic killing, induce an aberrant immune response, or incapacitate eukaryotic cells through cytolytic and enzymatic mechanisms. Just as original microbe hunters characterized the causative agents of infectious diseases using Koch’s postulates as guideposts, modern microbiologists have endeavored to define the criteria used to characterize a bacterial VF. Sequence homologies and in vitro methodologies to characterize the factor’s mechanism of action are currently used to identify new VFs. We suggest that VF characterization has become subject to reasoning errors and encourage considering the ecological view of their origins to overcome anthropocentric bias. In this Special Issue, we will initiate a conversation by first challenging VF characterization using examples of two well-studied bacterial pathogens (Staphylococcus aureus and Pseudomonas aeruginosa). Subsequent articles will provide arguments supporting state-of-the-art processes for VF designation and characterization.

Dr. Matthew Jackson
Dr. Kevin Theis
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Microorganisms is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • bacterial pathogens
  • virulence factors
  • immune response
  • staphylococcus aureus
  • pseudomonas aeruginosa

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Published Papers (3 papers)

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Research

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29 pages, 7062 KiB  
Article
Gram Negative Biofilms: Structural and Functional Responses to Destruction by Antibiotic-Loaded Mixed Polymeric Micelles
by Tsvetozara Damyanova, Rumena Stancheva, Milena N. Leseva, Petya A. Dimitrova, Tsvetelina Paunova-Krasteva, Dayana Borisova, Katya Kamenova, Petar D. Petrov, Ralitsa Veleva, Ivelina Zhivkova, Tanya Topouzova-Hristova, Emi Haladjova and Stoyanka Stoitsova
Microorganisms 2024, 12(12), 2670; https://doi.org/10.3390/microorganisms12122670 - 23 Dec 2024
Viewed by 408
Abstract
Biofilms are a well-known multifactorial virulence factor with a pivotal role in chronic bacterial infections. Their pathogenicity is determined by the combination of strain-specific mechanisms of virulence and the biofilm extracellular matrix (ECM) protecting the bacteria from the host immune defense and the [...] Read more.
Biofilms are a well-known multifactorial virulence factor with a pivotal role in chronic bacterial infections. Their pathogenicity is determined by the combination of strain-specific mechanisms of virulence and the biofilm extracellular matrix (ECM) protecting the bacteria from the host immune defense and the action of antibacterials. The successful antibiofilm agents should combine antibacterial activity and good biocompatibility with the capacity to penetrate through the ECM. The objective of the study is the elaboration of biofilm-ECM-destructive drug delivery systems: mixed polymeric micelles (MPMs) based on a cationic poly(2-(dimethylamino)ethyl methacrylate)-b-poly(ε-caprolactone)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA35-b-PCL70-b-PDMAEMA35) and a non-ionic poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO100-b-PPO65-b-PEO100) triblock copolymers, loaded with ciprofloxacin or azithromycin. The MPMs were applied on 24 h pre-formed biofilms of Escherichia coli and Pseudomonas aeruginosa (laboratory strains and clinical isolates). The results showed that the MPMs were able to destruct the biofilms, and the viability experiments supported drug delivery. The biofilm response to the MPMs loaded with the two antibiotics revealed two distinct patterns of action. These were registered on the level of both bacterial cell-structural alterations (demonstrated by scanning electron microscopy) and the interaction with host tissues (ex vivo biofilm infection model on skin samples with tests on nitric oxide and interleukin (IL)-17A production). Full article
(This article belongs to the Special Issue Contemporary Perspectives on Bacterial Virulence Factors)
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Figure 1

Figure 1
<p>(<b>a</b>) Hydrodynamic diameter, D<sub>h</sub>, and (<b>b</b>) ζ-potential variations as a function of the micellar concentration of SCPMs and MPMs based on PDMAEMA<sub>35</sub>-PCL<sub>70</sub>-PDMAEMA<sub>35</sub> and Pluronic F127 triblock copolymers. (<b>c</b>) Size distribution curves (<b>d</b>) DLS correlation functions and (<b>e</b>) representative AFM micrograph of MPMs prepared from PDMAEMA<sub>35</sub>-PCL<sub>70</sub>-PDMAEMA<sub>35</sub> and Pluronic F127 triblock copolymers at a molar ratio of 1:1 in the concentration range of 1 to 0.125 mg mL<sup>−1</sup>. The PDI values ranged in the 0.11–0.19 interval. All DLS measurements were performed at 25 °C.</p> Full article ">Figure 2
<p>Variations of encapsulation efficiency (<b>a</b>) and drug loading content (<b>b</b>) as a function of the composition of SCPMs and MPMs based on PDMAEMA<sub>35</sub>-PCL<sub>70</sub>-PDMAEMA<sub>35</sub> and Pluronic F127 triblock copolymers. The loading was performed at polymer-to-drug mass ratio of 10:1.</p> Full article ">Figure 3
<p>Hydrodynamic diameter, Dh, (<b>a</b>,<b>c</b>,<b>e</b>) and ζ potential (<b>b</b>,<b>d</b>,<b>f</b>) of empty or loaded with antibiotics MPMs based on PDMAEMA<sub>35</sub>-PCL<sub>70</sub>-PDMAEMA<sub>35</sub> and Pluronic F127 triblock copolymers in the concentration range of 1 to 0.125 mg mL<sup>−1</sup>. Measurements were performed at 25 °C at pH 7. Each data point represents the arithmetic mean ± SD of three separate experiments.</p> Full article ">Figure 3 Cont.
<p>Hydrodynamic diameter, Dh, (<b>a</b>,<b>c</b>,<b>e</b>) and ζ potential (<b>b</b>,<b>d</b>,<b>f</b>) of empty or loaded with antibiotics MPMs based on PDMAEMA<sub>35</sub>-PCL<sub>70</sub>-PDMAEMA<sub>35</sub> and Pluronic F127 triblock copolymers in the concentration range of 1 to 0.125 mg mL<sup>−1</sup>. Measurements were performed at 25 °C at pH 7. Each data point represents the arithmetic mean ± SD of three separate experiments.</p> Full article ">Figure 4
<p>Drug release profiles of MPMs based on PDMAEMA<sub>35</sub>-PCL<sub>70</sub>-PDMAEMA<sub>35</sub> and Pluronic F127 triblock copolymers, prepared at a 10:1 polymer-to-drug mass ratio, determined by HPLC. MPMs were formed at molar ratios of 3:1 (<b>a</b>), 1:1 (<b>b</b>), and 1:3 (<b>c</b>). The release was performed at 37 °C in phosphate buffer pH 7.4. Each data point represents the arithmetic mean ± SD of three separate experiments.</p> Full article ">Figure 5
<p>Cytotoxicity of the SCPMs and MPMs based on PDMAEMA<sub>35</sub>-PCL<sub>70</sub>-PDMAEMA<sub>35</sub> and Pluronic F127 triblock copolymers loaded with CF (<b>a</b>) or AZ (<b>b</b>) at a 10:1 polymer-to-drug mass ratio. The micelles were applied for 4 h in concentrations of 0.5, 0.25, and 0.125 mg mL<sup>−1</sup> onto confluent cultured HaCaT. The results are presented as percentage of the control—cells cultivated parallelly in DMEM. The data are the means of four repeats and are presented as the mean ± SD. Differences between control (DMEM) and treated with micelles cells are accepted as statistically significant (*) when <span class="html-italic">p</span> &lt; 0.05 and (**) when <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 6
<p>Reduction of the biomass of mature 24 h biofilms as a result of treatment for 4 or 24 h with 0.25 mg mL<sup>−1</sup> of empty or antibiotics-loaded MPMs based on PDMAEMA<sub>35</sub>-PCL<sub>70</sub>-PDMAEMA<sub>35</sub> and Pluronic F127 triblock copolymers. The results were calculated as percentage of the biofilm at the start of each experiment. (<b>a</b>) <span class="html-italic">E. coli</span> 25922; (<b>b</b>) <span class="html-italic">P. aeruginosa</span> PAO1. Results for biofilms treated with dH<sub>2</sub>O are included since the micelles were dispersed in dH<sub>2</sub>O. Each data point represents the mean ± SD of six repeats.</p> Full article ">Figure 7
<p>Viability of the biofilms after treatment for 24 h with 0.25 mg mL<sup>−1</sup> of empty or antibiotics-loaded MPMs based on PDMAEMA<sub>35</sub>-PCL<sub>70</sub>-PDMAEMA<sub>35</sub> and Pluronic F127 triblock copolymers. Viability was estimated by the reduction of resazurin using the Alamar Blue reagent (Invitrogen). The results were calculated as percentage of the untreated control (biofilm cultivated parallelly in M63 medium in the absence of the tested agents). dH<sub>2</sub>O bars are included to show the effect of treatment with dH<sub>2</sub>O alone_ the medium in which the micelles were dispersed. (<b>a</b>) <span class="html-italic">E. coli</span> 25922; (<b>b</b>) <span class="html-italic">P. aeruginosa</span> PAO1. Each data point represents the mean ± SD of six repeats. <span class="html-italic">p</span> &lt; 0.05 (*); <span class="html-italic">p</span> &lt; 0.001 (***), ANOVA test.</p> Full article ">Figure 8
<p>Reduction of biofilms of pathogenic strains of <span class="html-italic">E. coli</span> treated with empty or antibiotic-loaded MPMs 3:1 (<b>a</b>) and of <span class="html-italic">P. aeruginosa</span> treated with empty or antibiotic-loaded MPMs 1:1 (<b>b</b>). The results were calculated as percentage of the “0” controls, i.e., the amount of biofilms of the strains before the start of the treatments. Each data point represents the mean ± SD of six repeats.</p> Full article ">Figure 9
<p>Scanning electron microscopy of biofilms of <span class="html-italic">E. coli</span> 25922 (<b>A</b>–<b>H</b>) and <span class="html-italic">P. aeruginosa</span> PAO1 (<b>I</b>–<b>P</b>). Arrows: white—infolds of the cell wall; yellow—outer membrane vesicles; red—tunneling nanotubules. (<b>A</b>) <span class="html-italic">E. coli</span> 48 h control biofilm; (<b>B</b>,<b>E</b>,<b>F</b>,<b>F1</b>) <span class="html-italic">E. coli</span> 24 h biofilm treated for a further 24 h with empty MPMs 3:1; yellow asterisk mark slimy covering of cells in some areas of the treated biofilm. (<b>G</b>,<b>G1</b>) <span class="html-italic">E. coli</span> 24 h biofilm treated for a further 24 h with CF-loaded MPMs 3:1; (<b>H</b>,<b>H1</b>) <span class="html-italic">E. coli</span> 24 h biofilm treated for a further 24 h with AZ-loaded MPMs 3:1. (<b>I</b>,<b>M</b>) <span class="html-italic">P. aeruginosa</span> 48 h control biofilm; (<b>J</b>,<b>N</b>) <span class="html-italic">P. aeruginosa</span> 24 h biofilm treated for a further 24 h with empty MPMs 1:1; (<b>K</b>,<b>O</b>) <span class="html-italic">P. aeruginosa</span> 24 h biofilm treated for a further 24 h with CF-loaded MPMs 1:1; (<b>L</b>,<b>P</b>,<b>P1</b>) <span class="html-italic">P. aeruginosa</span> 24 h biofilm treated for a further 24 h with AZ-loaded MPMs 1:1; white asterisks, cells with extensively blebbed surfaces.</p> Full article ">Figure 10
<p>Histological sections of skin explants infected with <span class="html-italic">P. aeruginosa</span> PAO1 biofilm. (<b>A</b>) Untreated 24 h ex vivo biofilm. (<b>B</b>,<b>C</b>) Mature 24 h biofilms on skin explants were treated for 24 h with 0.25 mg mL<sup>−1</sup> of MPMs 1:1 loaded with CF (<b>B</b>) or AZ (<b>C</b>). Bar = 10 µm.</p> Full article ">Figure 11
<p>Effect of MPMs loaded with CF or AZ on NO (<b>a</b>) and IL-17A (<b>b</b>) production in ex vivo murine skin explant <span class="html-italic">P. aeruginosa</span> PAO1 biofilm model. Murine skin explants were infected with <span class="html-italic">P. aeruginosa</span> for 24 h for the development of biofilm. Afterwards the skin explants were treated with 50 µL of either 0.5 or 0.25 mg mL<sup>−1</sup> MPMs loaded with CF or AZ. Control samples, infected or uninfected with <span class="html-italic">P. aeruginosa</span> biofilm, were treated in parallel with either PBS or dH<sub>2</sub>O (the solvent for the MPM samples). Data represents mean ± SD from 3 samples/group * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 when comparing the biofilm groups to the control PBS one, ANOVA test; ## <span class="html-italic">p</span> &lt; 0.05 when comparing the non-biofilm groups to the control PBS one, ANOVA test.</p> Full article ">

Review

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18 pages, 1431 KiB  
Review
Immunosenescence: How Aging Increases Susceptibility to Bacterial Infections and Virulence Factors
by Nikolaos Theodorakis, Georgios Feretzakis, Christos Hitas, Magdalini Kreouzi, Sofia Kalantzi, Aikaterini Spyridaki, Zoi Kollia, Vassilios S. Verykios and Maria Nikolaou
Microorganisms 2024, 12(10), 2052; https://doi.org/10.3390/microorganisms12102052 - 11 Oct 2024
Viewed by 1360
Abstract
The process of aging leads to a progressive decline in the immune system function, known as immunosenescence, which compromises both innate and adaptive responses. This includes impairments in phagocytosis and decreased production, activation, and function of T- and B-lymphocytes, among other effects. Bacteria [...] Read more.
The process of aging leads to a progressive decline in the immune system function, known as immunosenescence, which compromises both innate and adaptive responses. This includes impairments in phagocytosis and decreased production, activation, and function of T- and B-lymphocytes, among other effects. Bacteria exploit immunosenescence by using various virulence factors to evade the host’s defenses, leading to severe and often life-threatening infections. This manuscript explores the complex relationship between immunosenescence and bacterial virulence, focusing on the underlying mechanisms that increase vulnerability to bacterial infections in the elderly. Additionally, it discusses how machine learning methods can provide accurate modeling of interactions between the weakened immune system and bacterial virulence mechanisms, guiding the development of personalized interventions. The development of vaccines, novel antibiotics, and antivirulence therapies for multidrug-resistant bacteria, as well as the investigation of potential immune-boosting therapies, are promising strategies in this field. Future research should focus on how machine learning approaches can be integrated with immunological, microbiological, and clinical data to develop personalized interventions that improve outcomes for bacterial infections in the growing elderly population. Full article
(This article belongs to the Special Issue Contemporary Perspectives on Bacterial Virulence Factors)
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Figure 1

Figure 1
<p>Summary of the components of the immune system.</p> Full article ">Figure 2
<p>Key features of immunosenescence.</p> Full article ">
16 pages, 320 KiB  
Review
The Epistemology of Bacterial Virulence Factor Characterization
by Matthew Jackson, Susan Vineberg and Kevin R. Theis
Microorganisms 2024, 12(7), 1272; https://doi.org/10.3390/microorganisms12071272 - 22 Jun 2024
Viewed by 2640
Abstract
The field of microbial pathogenesis seeks to identify the agents and mechanisms responsible for disease causation. Since Robert Koch introduced postulates that were used to guide the characterization of microbial pathogens, technological advances have substantially increased the capacity to rapidly identify a causative [...] Read more.
The field of microbial pathogenesis seeks to identify the agents and mechanisms responsible for disease causation. Since Robert Koch introduced postulates that were used to guide the characterization of microbial pathogens, technological advances have substantially increased the capacity to rapidly identify a causative infectious agent. Research efforts currently focus on causation at the molecular level with a search for virulence factors (VFs) that contribute to different stages of the infectious process. We note that the quest to identify and characterize VFs sometimes lacks scientific rigor, and this suggests a need to examine the epistemology of VF characterization. We took this premise as an opportunity to explore the epistemology of VF characterization. In this perspective, we discuss how the characterization of various gene products that evolved to facilitate bacterial survival in the broader environment have potentially been prematurely mischaracterized as VFs that contribute to pathogenesis in the context of human biology. Examples of the reasoning that can affect misinterpretation, or at least a premature assignment of mechanistic causation, are provided. Our aim is to refine the categorization of VFs by emphasizing a broader biological view of their origin. Full article
(This article belongs to the Special Issue Contemporary Perspectives on Bacterial Virulence Factors)
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