To develop countermeasures against phage-resistant bacteria without antibiotics, a detailed phenotypic characterization of phage-resistant Escherichia coli BW25113 was performed. Phage susceptibility testing of E. coli BW25113 deletion mutants involved in lipopolysaccharide (LPS) synthesis revealed that the first glucose residue of the LPS outer core was essential for infection by phage S127. From E. coli BW25113 cells that survived S127 exposure, four phage-resistant strains were isolated and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the phage-resistant strains had LPS with a smaller molecular mass compared with that of the E. coli BW25113 parental strain. Fluorescence microscopy after BacLight staining, along with comparisons of viable counts on non-selective versus selective media, indicated increased membrane permeability in the resistant strains, resulting in heightened sensitivity to monocaprin, a natural non-ionic surfactant. Furthermore, upon elevating the culture temperature from 30°C to 37°C, the resistant strains exhibited increased Congo Red binding and autoaggregation, which was not observed in the parental strain. Viability assays revealed that both the phage-resistant strains and deep rough mutants, such as ΔhldE and ΔwaaG, did not grow at 46°C. Notably, regrowth after phage S127 treatment at 37°C was significantly delayed in the E. coli BW25113 parental strain grown at 46°C than in that grown at 37°C. E. coli populations that became phage resistant owing to truncated LPS chains were highly sensitive to hydrophobic antibacterial substances and high temperatures, suggesting that these could be critical factors for controlling phage-resistant bacteria.
Importance: The application of phages in agriculture and food-producing environments often faces challenges in the control of phage-resistant bacteria. To effectively address this issue, a deeper understanding of the unique phenotypes associated with phage resistance is warranted. Few studies have suppressed the regrowth of phage-resistant populations without using antibiotics, based on detailed phenotypic characterization. Here, we report that the phage-resistant Escherichia coli population selected by lytic phage S127 was sensitive to elevated temperature and decreased viability at 46°C. Furthermore, Congo Red binding and autoaggregation, which have been reported to exhibit unique behaviors in E. coli deep rough mutants, were dependent on high culture temperature. Our findings highlight a novel, exploitable phenotype of phage resistance in host bacteria that could be applied to the biocontrol of phage resistance in foodborne pathogens without the use of antibiotics in practical settings.
Poultry house fine particulate matter (PM2.5) poses significant respiratory risks to poultry by penetrating deep into the lung and triggering inflammatory cascades. In this study, 21- to 28-day-old broilers were exposed to total suspended particulates enriched in PM2.5 (2 mg/m³, 2 h/day) to investigate pulmonary injury and gut-lung axis perturbations. PM2.5 exposure induced collapse of the hexagonal lobular architecture, elevated pulmonary expression of IL-1β, IL-2, IL-6, IL-8, and IL-10, and activated NF-κB signaling. Concurrently, cecal microbiota α-diversity increased while the community shifted toward pro-inflammatory taxa (Alistipes, Rikenellaceae) and away from SCFA-producing species (Bacteroides uniformis, Parabacteroides). Oral supplementation of B. uniformis restored its abundance, replenished acetate and propionate levels, and attenuated lung injury by reducing APC activation (CD40, CCL4) and Th1 polarization (T-bet, IFN-γ, IL-18), while promoting regulatory T cell markers (FoxP3). Dietary sodium propionate supplementation in feed (0.4%) similarly mitigated pulmonary inflammation and Th1 skewing, albeit without enhancing Treg responses. These findings demonstrate that PM2.5-induced lung damage is intricately linked to gut dysbiosis and SCFA depletion and that restoration of B. uniformis or its metabolite propionate can recalibrate the gut-lung axis to suppress innate and adaptive inflammatory pathways. This work highlights microbiota- and metabolite-based interventions as promising strategies to protect poultry respiratory health and performance under air-polluted conditions.IMPORTANCEThis study reveals that poultry house-derived PM2.5 not only causes direct lung inflammation but also perturbs the gut-lung axis by depleting beneficial SCFA-producing bacteria. The resulting gut dysbiosis amplifies respiratory immune injury, highlighting a previously underappreciated systemic effect of airborne pollutants in livestock environments. Our findings suggest that microbiota- and metabolite-targeted dietary strategies can mitigate air pollution-induced health risks in poultry. This work provides new insights into the broader ecological and agricultural consequences of PM2.5 exposure and supports sustainable, non-antibiotic interventions to enhance animal welfare and productivity under deteriorating air quality conditions.
Despite the increasing number of reports on hypervirulent and extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae infections, data on the distribution of these pathogens in the community are limited. To address this knowledge gap, we investigated the carriage rates of K. pneumoniae complex in the stools of community-dwelling individuals in Japan. From 627 stool samples submitted to a commercial diagnostic laboratory, 407 Klebsiella strains were identified from 368 samples, corresponding to a colonization rate of 58.7%. Based on whole-genome sequencing, K. pneumoniae was the most prevalent species (n = 218, 53.6%), followed by Klebsiella variicola (n = 137, 33.7%). The detection rate of K. variicola was higher than previously reported in studies from other Asian countries. The overall distribution of sequence types (STs) was similar to those observed in previous studies of clinical isolates. However, hypervirulent K. pneumoniae clones, specifically ST23-K1 and ST412-K57, and ESBL-producing strains were rare, each accounting for less than 1% of the strains. These findings suggest that, while carriage of K. pneumoniae complex species is common in the community, healthcare settings may represent a more significant reservoir of hypervirulent and ESBL-producing K. pneumoniae strains in this epidemiological setting.IMPORTANCEKlebsiella pneumoniae complex species are bacteria that can cause serious infections, especially in hospital settings. Some types have become more dangerous because they are resistant to antibiotics or highly virulent. To better understand where these harmful clones come from, this study looked for Klebsiella species in healthy people living in the community in Japan. The results showed that these bacteria are commonly found in the gut, particularly K. pneumoniae and K. variicola. While some strains with traits linked to antibiotic resistance or severe infections were identified, they were rare. These findings suggest that most people carry Klebsiella strains as commensals and that the more dangerous forms of Klebsiella are likely spreading mainly in healthcare settings.
Despite the promise of phages as antibiotic alternatives, their efficacy is often undermined by the rapid emergence of bacterial resistance. Phage-derived enzymes, particularly depolymerases, offer a compelling strategy to overcome this limitation and enhance antibacterial therapy. Focusing on Vibrio pathogens, the major threats to global aquaculture, our bioinformatic analysis revealed that 79.4% of cultured and 46.2% of uncultured Vibrio phages encode putative depolymerases, underscoring a vast but underexploited antibacterial resource. We further isolated and characterized VnaP, a depolymerase-encoding phage (novel genus, Caudovircetes) that forms distinctive halo plaques indicative of depolymerase activity. Genome analysis identified ORF193, encoding a novel polysaccharide depolymerase lacking sequence or structural homology to any characterized depolymerases. Heterologously expressed Dep193 efficiently degraded Vibrio surface polysaccharides and exhibited potent antibiofilm activity. While Dep193 exhibits modest standalone antibacterial activity, its synergistic combination with VnaP significantly enhances bacterial clearance and delays resistance emergence across multiple Vibrio species. As the first biochemically validated Vibrio phage depolymerase, Dep193 broadens the known diversity of these enzymes and establishes an effective strategy for Vibrio control in aquaculture.IMPORTANCEThe rapid emergence of antibiotic-resistant Vibrio strains threatens global aquaculture sustainability, necessitating alternative antimicrobial strategies. This study identifies and characterizes Dep193, a novel phage-encoded depolymerase with polysaccharide-degrading and antibiofilm activities that enhances phage therapy efficacy through a previously unreported mechanism. The Dep193-phage VnaP combination exhibits broad-spectrum activity against multiple Vibrio species, demonstrating strong potential as a therapeutic strategy for aquaculture. Notably, Dep193 lacks any recognizable functional domains found in characterized depolymerases, representing the first validated member of a novel evolutionary clade. These findings expand the known diversity of phage depolymerases and provide a promising avenue for the targeted control of Vibrio infections in aquaculture.
In this work, we investigated the chemical process underlying the interplay between two rice sheath pathogens: Pseudomonas fuscovaginae, the causal agent of sheath brown rot, and Rhizoctonia solani AG 1-IA, which causes sheath blight. Specifically, we studied the fate of the bacterial cyclic lipopeptides (CLiPs) syringotoxin and fuscopeptin in this interaction. Both compounds exhibit potent antifungal activity against R. solani and induce phytotoxic effects. Ultra-performance liquid chromatography-tandem mass spectrometry analysis demonstrated that R. solani AG 1-IA likely secretes two distinct enzymes: an esterase that hydrolyzes the ester bond in syringotoxin, producing a linear derivative, and a protease that cleaves the glycine-alanine bond within the peptide backbone of fuscopeptin. The degradation products lack antifungal and phytotoxic activities, nullifying the competitive advantage of P. fuscovaginae. These enzymatic effects showed increased activity at 28°C. In contrast, R. solani AG 2-2 did not degrade CLiPs. Further analysis with a broader range of R. solani isolates revealed that CLiP degradation is a common trait among AG 1-IA isolates. This study provides the first evidence that R. solani AG 1-IA actively neutralizes the antifungal and phytotoxic activities of P. fuscovaginae through targeted enzymatic degradation.IMPORTANCERice is a global staple crop that is susceptible to various pathogens, including Pseudomonas fuscovaginae, causing sheath brown rot, and Rhizoctonia solani AG 1-IA, which causes sheath blight. Notably, P. fuscovaginae primarily occurs at lower temperatures, whereas R. solani AG 1-IA is more prevalent under warmer conditions. Previous research demonstrated that P. fuscovaginae produces higher levels of the virulence-associated cyclic lipopeptides (CLiPs) syringotoxin and fuscopeptin at 18°C, potentially explaining its pathogenicity on rice plants grown at high altitudes. Conversely, R. solani AG 1-IA, which is sensitive to these CLiPs, was found to secrete CLiP-degrading enzymes, with degradation activity enhanced at 28°C. When combined with the reduced CLiP production by P. fuscovaginae at higher temperatures, this enzymatic degradation may confer a competitive advantage to R. solani in warmer environments. The absence of reports documenting the co-occurrence of both pathogens in field conditions may, at least in part, be explained by this temperature-dependent antagonism.
The ability of Listeria to show reduced susceptibility to sanitizers commonly used in fresh produce packing and processing environments continues to be mentioned as a concern. We assessed the survival of 501 produce-associated Listeria isolates (328 Listeria monocytogenes [LM] and 173 Listeria spp. [LS]) after 30 s of exposure to benzalkonium chloride (BC, 300 ppm) and peroxyacetic acid (PAA, 80 ppm). A subset of 108 isolates was also exposed to sodium hypochlorite (NaOCl, 500 ppm) for 30 s. Isolates showed a range of log reductions, including 2.76-5.73 log for BC, 0.15-6.16 log for PAA, and 1.34-7.02 log for NaOCl; the variance of log reductions was significantly lower for BC compared to PAA and NaOCl. Cluster analysis on log reduction data identified four clusters, including one cluster of five LM isolates that showed reduced susceptibility to all three sanitizers. Log reductions of LS were significantly lower than LM after exposure to PAA, indicating reduced PAA susceptibility among LS. Whole genome sequence (WGS)-based characterization of all isolates revealed that the presence of known BC resistance genes (i.e., bcrABC, mdrL, and sugE1/2) was not significantly associated with log reductions to BC, and the presence of stress survival islet SSI-2 was not significantly associated with log reductions to PAA and NaOCl. Genome-wide association studies did not reveal any association of pangenome genes with phenotypic sanitizer susceptibility but identified several SNPs in core genes as associated with sanitizer susceptibility.IMPORTANCEDespite frequently stated concerns about LM and LS with reduced susceptibility to sanitizers (which could facilitate persistence and increase risk of product contamination), there are limited data available on Listeria susceptibility to sanitizers used in produce packing and processing environments at their recommended use-level concentrations. Importantly, our data showed that reduced sanitizer susceptibility of Listeria is not linked to the presence of any previously reported sanitizer resistance genes. However, we identified a group of five LM isolates that showed reduced susceptibility to all three sanitizers tested; these isolates represented lineages I, II, and III. Combined, these data suggest that there are no distinct "sanitizer-resistant" clonal Listeria groups and that WGS data may not be particularly valuable for predicting sanitizer susceptibility at use-level concentrations. Moreover, the high variability of log reductions observed across all three sanitizers highlights the importance of considering log reduction variability, in addition to average log reduction, when assessing different sanitizers.
To mitigate bacterial contamination in underground farmland, a comprehensive understanding of the transport and adhesion mechanisms of phytopathogenic bacteria in porous media is crucial for safeguarding soil and groundwater. This study aims to elucidate the effects of Pseudomonas amygdali pv. tabaci 6605 flagella (wild type, ΔfliC strain) and their glycosylation (Δfgt1 and Δfgt2 strains) on bacterial transport and deposition in sandy porous media through a combination of experimental observations and numerical simulations. Flagella play a key role in bacterial transport and deposition dynamics through its surface properties. Their intrinsic hydrophobicity enhances bacterial adhesion and promotes deposition onto sandy grains while simultaneously limiting transport through the porous medium. However, glycosylation of flagellin introduces hydrophilic glycans, which counteract this effect by increasing the overall hydrophilicity of the bacterial surface. As a result, glycosylated flagella facilitate bacterial mobility and improve recovery in the effluent while reducing retention within the sand matrix. These findings highlight the critical influence of flagellar biochemical modifications on bacterial behavior in porous environments. They provide valuable insights for understanding and managing microbial contamination in subsurface systems.IMPORTANCEThis work, conducted using homogeneous laboratory sand, could be extended to other types of abiotic media found in natural environments, such as clay, heterogeneous sands, and soils. Our study highlights the impact of flagellar glycosylation on bacterial behavior, an essential factor for assessing the risk posed by phytopathogenic bacteria in agricultural settings and for developing effective soil bioremediation strategies. Moreover, this study provides valuable insights into the mechanisms governing bacterial transport and deposition at the macroscopic (column) scale under dynamic flow conditions. Investigating unsaturated flow conditions, which better approximate real field scenarios, may further our understanding of bacterial interactions at air-solid-water interfaces. Future research should explore bacterial movement across different spatial scales. In particular, pore-scale experiments can provide direct evidence of processes such as attachment and motility. This could significantly enhance our understanding of microbial dynamics in complex environments.
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