Antimicrobial resistance (AMR) results from acquiring mutations or genes that allow microbes to withstand antimicrobials. These genetic modifications can also cause non-selected changes in other cell phenotypes, such as fitness and susceptibility to antibiotic classes different from the one under selection. These collateral effects of AMR acquisition could be exploited to improve treatments. This is increasingly important in light of AMR as a major global health problem, associated with 4.71 million deaths worldwide in 2021, with 1.14 million deaths directly attributable to AMR. Additionally, the lack of development of novel antimicrobials active against resistant infections continues to compound the crisis. Even if new antimicrobials are developed, resistance will most likely emerge through adaptive evolution. Therefore, it is imperative to explore strategies to maintain the efficacy of new and existing antibiotics and limit the acquisition of AMR to continue to treat infections successfully. Understanding the impact of resistance acquisition on the bacterial phenotype, including collateral effects, has the potential to improve therapeutic outcomes. In this perspective, we will summarize the collateral effects of resistance acquisition, including fitness costs, collateral susceptibility networks, mutant selection windows and the probability of establishment of bacterial populations, and explore how these may be exploited to enhance the effectiveness of existing antibiotics. We will also discuss how challenges such as genetic background, polymicrobial infections and the environment are current barriers to the translation of these principles into the clinic.
Rumen protozoa are an integral component of the ruminant foregut microbiome, with a profound influence on feed digestion, microbial ecosystem dynamics and enteric methane emissions. Although they represent only a small proportion of species richness, protozoa contribute significantly to microbial biomass and functions in fibre degradation, volatile fatty acid production, microbial protein turnover, pH modulation and hydrogen-mediated methanogenesis. In addition to highlighting their importance to rumen function, this review summarizes classical and contemporary methodologies for enumerating, identifying and characterizing rumen protozoa. These methodologies include microscopy, fluorescent staining, flow cytometry and molecular approaches such as 18S rRNA, ITS and 28S rRNA amplicon sequencing. All major rRNA primers widely used over the last two decades are catalogued and their coverage is evaluated against current databases, highlighting the superior taxonomic resolution provided by ITS-based markers. The integration of single-cell sequencing and long-read platforms with multi-omics approaches (metagenomics, metatranscriptomics and metaproteomics) and advanced bioinformatic tools enables genome-resolved and functionally informed analyses of rumen protozoa. Methodological advances are integrated with functional insights to elucidate the contributions of protozoa to rumen ecology, feed efficiency and methane output. Key research gaps are also identified, including the need for improved microscopy techniques, automated image analysis, optimized cryopreservation protocols and robust culturing systems. This synthesis provides a comprehensive resource for researchers in microbial ecology, animal nutrition and environmental science, enabling precise protozoal profiling and informing strategies to enhance livestock productivity while mitigating environmental impacts.
PhrS ( Pseudomonas Hfq-binding RNA S) is a dual-function small RNA of Pseudomonas aeruginosa . The base-pairing small regulatory RNA PhrS-R positively regulates pyocyanin biosynthesis. PhrS-R stimulates the production of the Pseudomonas quinolone signal (PQS) by indirectly activating the translation of pqsR mRNA, encoding the quorum sensing regulator PqsR (MvfR), and by inhibiting the synthesis of the anthranilate degradation regulator AntR. Furthermore, PhrS-R was shown to promote biofilm formation, swarming motility and CRISPR-Cas immunity. In addition, PhrS encodes a 37 amino acid long and conserved small protein (PhrS-P), the function of which remained unknown. Here, we provide evidence that PhrS-P is an inner membrane protein which does not contribute to the known physiological roles of its RNA counterpart. RNA-Seq-based transcriptome analyses revealed that multiple aminoglycoside resistance genes, including those of the AmgR regulon, are differentially expressed depending on PhrS-P. AmgR is the response regulator of the AmgRS two-component system (TCS), which affects aminoglycoside and zinc resistance in P. aeruginosa . A PhrS-P-deficient mutant strain exhibited reduced fitness when exposed to sub-inhibitory concentrations of the aminoglycosides tobramycin and amikacin as well as zinc. Based on our findings, we present a working model wherein PhrS-P positively regulates the AmgRS TCS activity through an unknown mechanism, leading to increased bacterial fitness in the presence of aminoglycosides.
Members of the Chromobacterium genus – instantly recognizable for their vivid purple pigment – have played a disproportionately influential role in shaping quorum sensing research. The discovery that violacein production in the model species Chromobacterium subtsugae is governed by an N -acyl l -homoserine lactone (AHL)-based LuxI/LuxR signalling system ignited a wave of breakthroughs in the field. With its genetic simplicity and visually striking phenotype, Chromobacterium emerged as a powerful and accessible model, enabling sensitive detection of AHLs across diverse bacteria and opening the door to fundamental insights into signalling promiscuity, interspecies communication and the molecular logic of quorum sensing pathways. In this review, we highlight both the foundational discoveries and ongoing contributions of the Chromobacterium genus, arguing that it has been central not only to the origins of AHL-based quorum sensing research but also to the field’s expansion towards increasingly sophisticated, community-level perspectives that continue to define its trajectory today – and into the future.
The mycotoxin deoxynivalenol (DON) contamination in animal feed has been reported to reduce the growth performance of animals and compromise their immune system, especially pigs. Bacterial detoxification is considered a promising strategy to mitigate its harmful effects. We previously identified a bacterial isolate ( Bacillus sp. LS100) from the chicken gut with the ability to detoxify DON. To explore its potential application in swine production, the current study was focused on in vitro characterization of Bacillus sp. LS100 with regard to its capacity to detoxify DON. The bacterium was stable in keeping its activity to fully detoxify DON when subcultured for 12 passages under anaerobic conditions. The bacterial culture was successfully scaled up and stored for at least 1 month without losing any activity to detoxify DON. In addition, it was able to fully detoxify DON in mouldy corn with a diluted medium in an anaerobic atmosphere. While the bacterium exhibited a certain degree of tolerance to oxygen, it was sensitive to low pH and bile salts, including pig gastric fluid. In the presence of digesta from the pig jejunum or ileum, the bacteria retained near-full or full detoxification activity of DON after 24 or 48 h incubation under anaerobic conditions. To protect Bacillus sp. LS100 from the detrimental effects of gastric acid and bile salt, the bacterium was encapsulated anaerobically. The encapsulated bacteria stored in wet beads at 22 °C exhibited greater stability and retained detoxification activity longer than those stored in dry beads. After exposure to simulated gastric fluid for 2 h, the bacterium in the wet beads retained the DON detoxification activity. The current study suggests that, when adequately protected, Bacillus sp. LS100 holds promise as a viable strategy for DON removal in the pig gut.
