Frontiers in Cellular and Infection Microbiology最新文献

Introduction: Ceftazidime-avibactam represents a crucial therapeutic option for managing infections attributable to carbapenem-resistant P. aeruginosa. Nonetheless, the emergence of resistance to ceftazidime-avibactam in P. aeruginosa presents a significant challenge for clinical anti-infective therapy. This study primarily elucidates the mechanisms by which P. aeruginosa transitions from drug sensitivity to resistance during ceftazidime-avibactam treatment, ultimately resulting in therapeutic failure.

Methods: The susceptibility testing was performed using the broth microdilution method, conjugation experiment was performed via the filter mating method, the genetic surroundings of bla _KPC-249 and comparison of plasmids structures was performed using short/long-read genome sequencing and analysis method, the resistance of transconjugant carried the bla _KPC-249 to ceftazidime-avibactam was performed via molecular cloning method.

Results: For P. aeruginosa isolates from a patient, the minimum inhibitory concentrations (MICs) of ceftazidime-avibactam (CAZ-AVI) were determined as follows: isolates from sputum and bronchoalveolar lavage fluid both exhibited an MIC of 2 mg/L without blaKPC. In comparison, the blood-isolated strain P. aeruginosa PAE045 showed a significantly elevated MIC of >128 mg/L against CAZ-AVI. Plasmid conjugation experiments results demonstrated that the plasmid harboring the bla _KPC-249 gene could be successfully transferred to the recipient strain PAO1 ^rifR (rifampicin-resistant P. aeruginosa PAO1). Third-generation sequencing results revealed that the bla _KPC-249 gene was located on a plasmid with an approximate size of 37 kb. Compared with the wild-type e bla _KPC-2 gene, the bla _KPC-249 gene had two additional amino acid residues in its encoded protein: threonine (Thr, T) at position 182 and serine (Ser, S) at position 183. Furthermore, the upstream and downstream regions of the bla _KPC-249 gene were flanked by the insertion sequences ISKpn6 and ISKpn27, respectively.

Discussion: These mobile genetic elements may play a role in the capture and dissemination of the bla _KPC-249 gene. The bla _KPC-249 gene is identified as a novel mutant variant of the bla _KPC gene family, which mediates the resistance of P. aeruginosa to the antimicrobial agent ceftazidime-avibactam.

Background: Ubiquitination is a posttranslational modification that affects protein function, stability, and localization and is thereby balancing protein homeostasis. During infection, ubiquitination is crucial in regulating host cell signaling pathways in pathogen recognition, clearance and mounting an efficient immune response. S. aureus is an opportunistic pathogen that is able to invade and multiply within both phagocytic and non-phagocytic mammalian cells depending on virulence factor expression of the respective S. aureus strain. Selective autophagy serves as a host defense mechanism to combat intracellular bacterial persistence by targeting and degrading intracellular pathogens. However, S. aureus can subvert autophagosomal degradation and exploit these organelles for intracellular replication.

Results: We examined the role of the E3 ligase S-phase kinase-associated protein 2 (SKP2), a component of the SKP1-Cullin1-F-box (SCF) - complex, during S. aureus infection in alveolar epithelial and in macrophage-like cells. Upon S. aureus infection, we demonstrate increased SKP2 abundance through acetylation-induced stabilization and translocation into the cytoplasm. Cytoplasmic SKP2 modulated autophagy induction. By downregulation of SKP2, the level of the autophagy marker LC3-II was elevated which was accompanied by increased survival of intracellular S. aureus. Conversely, SKP2 overexpression in host cells reduced LC3-II levels followed by reduced intracellular bacteria.

Conclusion: These findings underscore that SKP2 is an important regulator of autophagosome formation, preventing excessive autophagy from being exploited by S. aureus. In conclusion, our findings reveal novel molecular mechanisms involved in the interaction between host cells and S. aureus providing potential approaches for targeted therapeutic intervention.

Background: Porcine reproductive and respiratory syndrome (PRRS) is a widely prevalent disease of reproductive failure of pregnant pigs and respiratory syndromes in pigs of different ages, especially in piglets. The etiological agents include PRRS virus (PRRSV) genotypes 1 (PRRSV-1) and PRRSV-2, whereas their clinical symptoms are similar and hard to differentiate. It is necessary to establish accurate and reliable methods for differential detection of PRRSV-1 and PRRSV-2.

Methods: Two pairs of specific primers and probes were designed basing on the PRRSV-1 and PRRSV-2 ORF6 gene. The reaction conditions and procedures of the duplex crystal digital PCR (cdPCR) were optimized. The specificity, sensitivity, and repeatability of the developed assay were evaluated. The application of the developed assay was assessed by testing 2,185 clinical tissue samples.

Results: The results indicated that the concentration of the templates and their Ct values had good linear relationship with R² of 0.998. This method could specifically detect PRRSV-1 and PRRSV-2, without cross-reaction with other swine viruses. The limits of detection (LODs) of the assay were 4.507 copies/reaction and 5.607 copies/reaction for PRRSV-1 and PRRSV-2, respectively, which was approximately 30 times more sensitive than that of the duplex real-time quantitative PCR (qPCR). The repeatability test showed that the intra- and inter-assay coefficients of variation (CVs) were 0.74%-0.93% and 0.63%-1.62%, respectively. This method was validated by testing 2,185 clinical samples from Guangxi Province in South China, and the positivity rates of PRRSV-1 and PRRSV-2 were 2.20% (48/2,185) and 23.43% (512/2,185), respectively. The coincidence rates of the developed assay with the qPCR assay recommended by the World Organisation of Animal Health (WOAH) were 99.73% and 99.73%, respectively, while with the duplex qPCR developed in this study were 99.82% and 99.77%, respectively.

Conclusions: These results indicated that a rapid and accurate duplex cdPCR method with high sensitivity and excellent specificity had been successfully developed for the differential detection of PRRSV-1 and PRRSV-2.

Skin wound infections are common and clinically challenging. Conventional antibiotic therapies are increasingly ineffective because of escalating bacterial resistance, highlighting the urgent need for alternative treatment strategies. Antibacterial hydrogels, multifunctional polymeric materials that integrate moisturizing, drug delivery, controlled release, and wound-healing properties, have emerged as highly promising candidates for managing infected wounds. Based on their underlying antimicrobial mechanisms, these systems can be broadly classified into three main categories: chemical, physical, and biological antibacterial hydrogels, which achieve bactericidal efficacy through drug release, physical disruption, or modulation of the host microenvironment and immune responses, respectively. Of tremendous significance is the advent of stimuli-responsive intelligent hydrogels, which provides new opportunities for achieving precise and efficient antibacterial therapy. This review systematically summarizes the material selection, design strategies, and representative advances in antibacterial hydrogels, with particular emphasis on their core mechanisms, strengths, and limitations, aiming to offer theoretical foundations and research perspectives for the rational optimization and clinical translation of next-generation antibacterial hydrogels.

Nontargeted metagenomic surveillance of the poultry enteric virome reveals underrecognized threats to poultry health and productivity in intensive production systems. In South Asia, avian rotavirus A (AvRV-A) and avian orthoreovirus (ARV) are frequently detected in broilers by conventional diagnostics, whereas chicken megrivirus genotype C (ChMeV-C) is often identified through metagenomic surveillance. Often present in both clinical disease and coinfections, these viruses may impair gut function, immune responses, and growth performance, yet their genomic diversity and evolutionary dynamics in poultry remain poorly characterized. Here, we report complete genomes of AvRV-A, ARV, and ChMeV-C strains co-detected via nontargeted metagenomic next-generation sequencing (ntNGS) in a pooled cloacal sample comprising 150 commercial broiler chickens (19 and 33 days old) collected from three commercial farms in Kamrup Rural District, Assam, Northeast India. Despite routine vaccination, all three flocks experienced > 10% mortality, poor weight gain, and postmortem lesions including pale kidneys and hepatomegaly. Phylogenetic analyses revealed segmental clustering in ARV and AvRV-A consistent with reassortment-driven divergence, though not supported by detectable recombination, while ChMeV-C clustered within a distinct C1 sublineage, suggesting intercontinental lineage connectivity and highlighting the need to expand regional genomic baseline data. We also identified nonsynonymous single nucleotide polymorphisms in several key viral proteins, including RNA-dependent RNA polymerases (VP1 of AvRV-A, λB of ARV, and 3D of ChMeV-C), capsid proteins (VP2 and VP7 of AvRV-A, λA and σB of ARV, and VP0 and VP1 of ChMeV-C), and replication-associated nonstructural proteins. These findings expand the genomic baseline for poultry enteric viruses in South Asia, reveal novel polymorphic signatures, and underscore the value of ntNGS-based metagenomic surveillance in virus detection, diversity monitoring, and informing vaccine and biosecurity strategies.

The prevalence of mupirocin resistance in MRSA severely limits therapeutic options for skin and soft tissue infections. This study aimed to evaluate the potential synergistic activity between mupirocin and protocatechuic acid ethyl ester (EDHB) through in vitro and in vivo investigations. Clinical S. aureus isolates were characterized for antibiotic resistance profiles and molecular features via antimicrobial susceptibility testing, MLST, and spa typing. For MRSA isolates, checkerboard and time-kill assays were performed to assess in vitro synergy. The potential interference of EDHB with bacterial membrane integrity and efflux pumps was investigated using propidium iodide and ethidium bromide, respectively. The disk diffusion method was applied to test the retained antimicrobial activity of mupirocin and EDHB in ointment formulations. A murine dermal wound model was established to evaluate in vivo efficacy by topical application of mupirocin and EDHB, alone or in combination, on infected wounds. EDHB alone exhibited limited activity but synergistically reduced mupirocin MICs by 4-8-fold in most strains. Checkerboard analysis revealed synergistic or partial synergistic interactions against MRSA. Time-kill curves further indicated that combining these two drugs can effectively inhibit the planktonic S. aureus. EDHB rapidly disrupts cytoplasmic membrane integrity via concentration-dependent propidium iodide influx, independent of norA/mepA efflux pump modulation. The enhanced antibacterial activity of mupirocin and EDHB was sustained in ointment formulations, resulting in superior therapeutic outcomes with combination therapy compared to monotherapy. EDHB acts as a membrane-disrupting adjuvant that synergizes with mupirocin against MuR-MRSA, offering a promising strategy to combat recalcitrant S. aureus infections through localized combination therapy.

The oral-gut axis represents a critical bidirectional pathway linking oral microbiota to systemic health. Dysbiosis of the oral microbiome, driven by pathogens like Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus species, and Helicobacter pylori, disrupts gut ecology via direct translocation, metabolite signaling (e.g., TMAO, SCFAs), and immune crosstalk (e.g., Th17). This leads to gut barrier dysfunction, systemic inflammation, and metabolic disturbances, contributing to diverse diseases beyond the oral cavity. Evidence supports causal links with conditions including rheumatoid arthritis, cardiovascular diseases, neurodegenerative disorders, metabolic syndrome, and gastrointestinal cancers. Emerging diagnostic tools exploit these oral pathogens as biomarkers for non-invasive disease detection. Therapeutic strategies, such as probiotics, dietary interventions, and periodontal therapy, target this axis to restore microbial homeostasis and ameliorate systemic inflammation. Future research must focus on longitudinal human studies and multi-omics approaches to elucidate mechanistic details and develop effective clinical interventions for preventing and managing systemic diseases linked to oral-gut microbial dysbiosis.

Objective: To characterize the systemic effects of high-fat diet (HFD)-induced obesity across different ages, explore the microbiota-related obesity endotype using 16S rRNA sequencing, and identify key microbial genera as candidate markers for longitudinal monitoring and future interventional validation.

Materials and methods: Male C57BL/6J mice were randomly assigned to a standard chow diet (SCD) or HFD group, maintained until 4, 12 and 18 months of age as the young, middle-aged and old groups, respectively, at which time animals were euthanized. Systemic effects were evaluated by measuring body weight, Lee's index, glucose-lipid metabolism, liver function, and blood oxygen levels, coupled with behavioral tests for mood and cognitive performance. Blood samples were collected to quantify LPS and Aβ1-42 levels using ELISA. Oral and fecal samples were collected for 16S rRNA sequencing to analyze microbiota diversity and community structure. Differential genera were identified by LEfSe, and those consistently altered in both oral and gut samples were operationally designated as marker genera. Targeted metabolomics was performed to analyze short-chain fatty acids (SCFAs). Correlations were evaluated using Spearman analysis.

Results: Compared with SCD, HFD mice showed systemic alterations across all age groups, including progressive obesity, elevated blood lipids and liver enzymes, accompanied by reduced blood oxygen, increased Aβ1-42 and LPS levels, increased anxiety-/depression-like behaviors, and impaired spatial memory. HFD significantly remodeled the alpha/beta-diversity and community structure of oral and gut microbiota, inducing stable enrichment of Romboutsia_B and depletion of beneficial genera (Bifidobacterium, Akkermansia, and Muribaculum). The abundance of Romboutsia_B positively correlated with obesity, blood lipids, liver enzyme levels, hypoxia, and inflammatory markers, but negatively correlated with multiple cognitive-behavioral parameters. Functional prediction and SCFA further profiling indicated that HFD enhanced lipid metabolism and environmental adaptation pathways, while reducing polysaccharide degradation and vitamin metabolism.

Conclusions: Long-term HFD is associated with systemic remodeling of the oral-gut-liver-brain axis across ages. Romboutsia_B, a pro-inflammatory-associated genus stably enriched in the oral and gut across all age groups, holds potential as a noninvasive microbial biomarker and candidate target for future intervention studies for obesity and its liver-brain comorbidities.

Coccidiosis, caused by Eimeria parasites, is a major threat to global poultry production, and increasing restrictions on conventional anticoccidial drugs highlight the need for safer, more sustainable alternatives. Progress has been hindered by the lack of rapid, sensitive, and animal-sparing in vitro assays for quantifying parasite replication and drug efficacy. This study reports the development of a novel bioluminescent platform for anticoccidial screening based on a genetically modified Eimeria tenella line expressing NanoLuc luciferase (EtNluc). Parasite-associated bioluminescence enabled rapid and quantitative monitoring of intracellular development, allowing the tracking of different replication phases through schizont formation and merozoite release. Time course analysis showed minimal changes in relative light units (RLU) between 2 and 24 hours post infection (hpi), followed by a marked increase between 24 and 72 hpi, consistent with parasite replication. Among the tested multiplicities of infection (MOI), 4:1 exhibited the fastest growth, described by a linear model (slope = 2908 RLU/h, R² = 0.84). A same-well repeated-measure analysis (2 and 72 hpi) confirmed the dose-dependent replication, with mean slopes of 2052.85, 765.07 and 523.63 RLU/h, respectively, supporting the selection of the MOI 4:1 for anticoccidial screening. These experimental conditions were used to evaluate the anticoccidial efficacy of commercial anticoccidial drugs (salinomycin and robenidine) and natural compounds (thyme and oregano essential oils, thymol, and carvacrol) under two experimental designs: short pre-incubation of sporozoites, and continuous exposure throughout intracellular development. Pre-incubation with commercial anticoccidials reduced invasion approximately to 65% for salinomycin and 44% for robenidine, whereas the essential oils and their bioactive constituents inhibited invasion by 30-55%, and reduced the replication slope to 33-60% of control values. Continuous exposure significantly impaired intracellular development for all treatments, reducing replication to 10-30% of controls, providing additional evidence that plant-derived compounds can complement commercial anticoccidials for integrated strategy for coccidiosis control in chickens. Overall, the EtNluc bioluminescent system provided a rapid, sensitive, and scalable method for quantifying E. tenella growth, suitable for in vitro anticoccidial screening, supporting the characterization of novel anticoccidial while reducing reliance on animal experimentation.