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Selective Bacteriocins: A Promising Treatment for Staphylococcus aureus Skin Infections Reveals Insights into Resistant Mutants, Vancomycin Resistance, and Cell Wall Alterations (Félix Jaumaux & al, Antibiotics, May 23, 2023)
The emergence of antibiotic-resistant S. aureus has become a major public health concern, necessitating the discovery of new antimicrobial compounds. Given that the skin microbiome plays a critical role in the host defence against pathogens, the development of therapies that target the interactions between commensal bacteria and pathogens in the skin microbiome offers a promising approach. Here, we report the discovery of two bacteriocins, cerein 7B and cerein B4080, that selectively inhibit S. aureus without affecting S. epidermidis, a commensal bacterium on the skin. Our study revealed that exposure of S. aureus to these bacteriocins resulted in mutations in the walK/R two-component system, leading to a thickening of the cell wall visible by transmission electron microscopy and subsequent decreased sensitivity to vancomycin. Our findings prompt a nuanced discussion of the potential of those bacteriocins for selective targeting of S. aureus on the skin, given the emergence of resistance and co-resistance with vancomycin. The idea put forward implies that by preserving commensal bacteria, selective compounds could limit the emergence of resistance in pathogenic cells by promoting competition with remaining commensal bacteria, ultimately reducing chronical infections and limiting the spread of antibiotic resistance.
Figure: Transmission electron microscopy (TEM) images of S. aureus cells for investigating cellular characteristics. (a) depicts wild type cells during the stationary growth phase, displaying typical behaviour in terms of cell division and cell wall thickness. (b) shows TEM images of isolated mutant B cells, which exhibit increased cell wall thickness compared to the wild type cells. (c) highlights abnormal cell divisions in the mutant cells.
In this study, we assessed the potential of bacteriocins and their in vitro synergistic effects in combination with anti-tuberculosis drugs against Mycobacterium tuberculosis. We evaluated the in vitro activity of chemically synthesized bacteriocins in combination with rifampicin (RIF), ofloxacin, and moxifloxacin against the reference M. tuberculosis H37Rv and a clinical-resistant strain. We first screened the bacteriocin PARAGEN collection and found active bacteriocins. We then determined their minimal inhibitory concentration (MIC), minimal bactericidal concentration, and their fractional inhibitory index by the checkerboard microdilution assay. Remarkably, we identified four bacteriocins with interesting antimycobacterial activity alone and in combinations with RIF, ofloxacin, and moxifloxacin, with significant reduction of the MIC that showed impressive synergistic effects against the susceptible and resistant clinical strains. In conclusion, our preliminary results show promising bacteriocins candidate used in a synergistic combination with anti-tuberculosis drugs and emphasize the need for combined therapy as a new strategy to enhance the activity of existing drugs, which may confer very promising therapeutic benefits against M. tuberculosis.
Figure: Schematic representation of MIC and MBC assay. Series of two-fold dilutions of LncZ used to determine the MIC (range 100–3.12 mg/mL). The figure shows the reduction of the resazurin dye from purple to pink. The lowest concentration (MIC) at which no growth was seen is 25 mg/mL. MBC has been determined from MIC by reculturing broth dilutions wells that inhibit growth at MIC, 2 · MIC and 4 · MIC (blue wells at and above the MIC) to agar plates for CFU counting. MBC, minimal bactericidal concentration.
Microbes’ broad physiological activities are pillars of our planet’s ecological evolution. Balanced microbiomes are essential for all living organisms on the planet, including humans. The huge potential of microbe physiology has been used empirically by humankind for centuries for food and beverage processing and in many other industries. Due to environmental issues, energy and chemical production faces the challenge of shifting from an oil-based to a bio-based industry mobilizing microbial biotechnology. This requires the development of new technologies based on synthetic biology to control industrial microbiomes. Here we briefly describe natural microbiomes to draw parallels with industrial biobased production environments. We suggest that bacteriocins a.k.a. anti-microbial peptides can potentially become elements of an industrial genetic firewall to stabilise and protect these environments.
Antibiotics have changed human health and revolutionised medical practice since the Second World War. Today, the use of antibiotics is increasingly limited by the rise of antimicrobial-resistant strains. Additionally, broad-spectrum antibiotic activity is not adapted to maintaining a balanced microbiome essential for human health. Targeted antimicrobials could overcome these two drawbacks. Although the rational design of targeted antimicrobial molecules presents a formidable challenge, in nature, targeted genetically encoded killing molecules are used by microbes in their natural ecosystems. The use of a synthetic biology approach allows the harnessing of these natural functions. In this commentary article we illustrate the potential of applying synthetic biology towards bacteriocins to design a new generation of antimicrobials.
The continuous emergence of microbial resistance to our antibiotic arsenal is widely becoming recognized as an imminent threat to global human health. Bacteriocins are antimicrobial peptides currently under consideration as real alternatives or complements to common antibiotics. These peptides have been much studied, novel bacteriocins are regularly reported and several genomic databases on these peptides are currently updated. Despite this, to our knowledge, a physical collection of bacteriocins that would allow testing and comparing them for different applications does not exist. Rapid advances in synthetic biology in combination with cell-free protein synthesis technologies offer great potential for fast protein production. Based on the amino acid sequences of the mature peptide available in different databases, we have built a bacteriocin gene library, called PARAGEN 1.0, containing all the genetic elements required for in vitro cell-free peptide synthesis.
Using PARAGEN 1.0 and a commercial kit for cell-free protein synthesis we have produced 164 different bacteriocins. Of the bacteriocins synthesized, 54% have shown antimicrobial activity against at least one of the indicator strains tested, including Gram-positive and Gram-negative bacteria representing commonly used lab strains, industrially relevant microorganisms, and known pathogens. This bacteriocin collection represents a streamlined pipeline for selection, production, and screening of bacteriocins as well as a reservoir of ready-to-use antimicrobials against virtually any class of relevant bacteria.
Streptococcus salivarius, a commensal bacterium dwelling in our gut, uses a secreted signaling peptide or pheromone to communicate with siblings in order to coordinate secretion of bacteriocins (bacteria killers used for predation) and acquisition of foreign DNA (competence for natural transformation) at the population scale, a strategy to optimize the survival rate.
Dr. J. Mignolet, R&D project Manager at Syngulon and visitor researcher at the LIBST (Pascal Hols’ lab, BGM), unearths new pheromones able to disconnect these two processes, specifically driving the exclusive production of bacteriocins (collaboration with UCLouvain).
This discovery might be considered for medical applications. Administration of activating peptides in mouth or gut should mobilize bacteriocins on demand in the endogenous S. salivarius population of the human microbiome and clear mucosa from specific pathogenic bacteria.
This figure adapted from Mignolet et al, 2018, shed light on the different paths to activate bacteriocin production in S. salivarius. In blue, the previously known cell-cell communication ComRS system couples predation (bacteriocin genes in red) to foreign DNA acquisition (comX gene in green). In pink/purple the newly described ScuR/SarF duo exclusively triggers bacteriocin secretion via the addition of a synthetic peptide.
Under the specter of the resurgence of pathogens due to the propagation of antimicrobial resistance (AMR) genes, innovative bacteria-killer strategies are needed. In Syngulon, we are currently developing bacteriocin-based solutions to tackle this problem and bacterial contamination in general. In a review recently published in Trends in Microbiology, we summarize the benefits of bacteriocins compared to antibiotics for personalized human applications, and we specifically emphasize their easy large-scale production and bioengineering. We also highlight how we could stimulate bacteriocin-producing bacteria of our microbiota to exploit their arsenal “on-site” and reshape endogenous bacterial populations (in collaboration with UCLouvain).
This figure from from Hols et al., 2019 highlights the four modes of bacteriocin production. In addition to the classical fermentation process, new developments in chemical synthesis allow an economically sustainable production of bacteriocins. Moreover, bacteriocins can be produced by synthetic biology techniques such as in vitro transcription-translation systems. Finally, bacteriocin-producing bacteria of our microbiota might be stimulated to secrete bacteriocins and treat gut infection.
This figure also depicts the bioflexibility of natural bacteriocins that might be engineered to provide variant molecules with more interesting properties. For instance, next-generation bacteriocins could be developed that are more resistant to proteases, are more efficient, possess a different prey spectrum, or could be a chimera of several bacteriocins.
Bacteriocins are known to play a role in bacterial communication and ecology. For example, the gut and oral cavity are parts of the human body that accommodate thousands of different bacterial species. These bacteria, often beneficial for human health, are continuously in a stressful environment and compete for food and space. When he was researcher in Prof. Pascal Hols lab (UCL/LIBST), Dr. Johann Mignolet (now R&D Project Manager of Syngulon) demonstrated that Streptococcus salivarius, a commensal human gut bacterium, uses a communication pheromone to concomitantly trigger two responses: the ability to modify its genome via the acquisition of “foreign” DNA and the production of potent bacteriocins. These toxins or non-transformable variants of S. Salivarius could be used for medical purposes to kill harmful multi-resistant superbugs such as Staphylococcus aureus and several streptococci.
This figure adapted from Mignolet et al, 2018, shows the different transcriptional cascades that trigger competence entry and expression of bacteriocin–encoding genes in four different streptococci models: S. salivarius, S. thermophilus, S. mutans and pneumoniae. Specifically interesting is that the BlpRH/BlpC bacteriocin regulatory system is missing or incomplete in S. Salivarius. The boxes show systems shared between species. Large continuous arrows depict transcriptional control, and dashed arrows display protein translation. Small continuous arrows indicate protein/peptide/phosphate motion.
The bacterium-killing assay below demonstrates addition of a pheromone inducing bacteriocin production, which leads to an inhibitory effect on surrounding bacteria.