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PubMed2025

Targeted next-generation sequencing for drug-resistant tuberculosis diagnosis: implementation considerations for bacterial load, regimen selection and diagnostic algorithm placement.

INTRODUCTION: Early and accurate diagnosis of drug-resistant tuberculosis (DR-TB) is essential for improving treatment outcomes. Phenotypic drug susceptibility testing (pDST) is comprehensive but slow, while rapid molecular assays provide resistance information for a limited number of drugs. Targeted next-generation sequencing (tNGS) offers the potential for broad and rapid resistance detection, but its integration into diagnostic algorithms has been hindered by uncertainty about its placement within existing workflows. METHODS: This study evaluated the extent to which two tNGS solutions-Deeplex Myc-TB (GenoScreen) and TB Drug Resistance Test (Oxford Nanopore Technologies, ONT)-provided interpretable drug resistance results that could inform regimen design, in comparison to other WHO-recommended molecular assays and pDST. Data were collected from three high-burden DR-TB settings under the Seq&Treat study. Sequencing success rates and drug resistance detection were analysed based on: (1) the initial Xpert MTB/RIF result (very low, low, medium, high), (2) resistance results for drugs in WHO-recommended regimens and (3) performance relative to other WHO-endorsed assays. The potential impact of different algorithms on the estimates was also considered. Key factors influencing successful tNGS adoption within diagnostic pathways were identified, leveraging insights from the Seq&Treat diagnostic accuracy study. RESULTS: Sequencing success rates were 88.5% (GenoScreen) and 93.1% (ONT) across 763 samples. While tNGS provided complete resistance data for 73%-86% of drugs in recommended regimens, pDST achieved 92%-93%. Both tNGS solutions matched or exceeded the sensitivity of WHO-recommended molecular assays. CONCLUSIONS: This study highlights the critical role of tNGS as a centralised tool for comprehensive drug resistance testing to inform DR-TB treatment decisions following initial screening assays. By complementing existing molecular tests with tNGS, diagnostic workflows can be optimised to ensure timely and comprehensive resistance detection. These findings support policy updates to integrate tNGS into global TB diagnostic algorithms. TRIAL REGISTRATION NUMBER: NCT04239326.

Humans↗

PubMed2026

Oral cavity as a reservoir of respiratory pathogens: microbial dynamics in patients receiving mechanical ventilation.

OBJECTIVE: To investigate the prevalence, co-occurrence, and temporal dynamics of Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, and Pseudomonas aeruginosa in oral and tracheal samples from critically ill patients receiving mechanical ventilation. METHODS: This observational analytical study included 21 adult patients receiving mechanical ventilation who were admitted to an intensive care unit. Oral and tracheal samples were collected within the first 24 h after intubation (Day 0) and again after 48 h (Day 2). Bacterial genomic deoxyribonucleic acid was extracted and analyzed using quantitative realtime polymerase chain reaction. Microbial detection was evaluated qualitatively based on the presence or absence of each microorganism and quantitatively using relative bacterial load analysis. Statistical analyses assessed correlations between oral and tracheal colonization patterns and temporal changes in bacterial load. RESULTS: More than 43% of oral and tracheal samples tested positive for at least one investigated pathogen at both collection time points, indicating persistent microbial colonization during mechanical ventilation. Significant positive correlations for Staphylococcus aureus detection were observed between oral and tracheal samples on Day 0 (p=0.0028) and Day 2 (p=0.0021), suggesting a possible association between oral colonization and lower airway microbial presence. Streptococcus pneumoniae detected in initial oral samples showed a significant correlation with tracheal detection after 48 h (p=0.03). Quantitative analyses demonstrated no significant changes in bacterial load over time for most microorganisms. However, Klebsiella pneumoniae showed a significant increase in tracheal bacterial load between Day 0 and Day 2 (p=0.04), with higher tracheal loads than oral samples on Day 2 (p=0.01). CONCLUSION: The oral cavity may serve as an important reservoir of respiratory pathogens in patients receiving mechanical ventilation. The observed correlations between oral and tracheal colonization and pathogen-specific colonization patterns reinforce the relevance of oral health management in critical care settings. In particular, the increase in tracheal Klebsiella pneumoniae load suggests that lower airway proliferation may occur during mechanical ventilation independently of simultaneous increases in oral bacterial load.

Humans↗

PubMed2026

High within-herd prevalence and assessment of production impacts of Coxiella burnetii in postpartum cattle from a Scottish dairy herd.

Coxiella burnetii is endemic in Great Britain dairy herds but detailed investigations of C. burnetii epidemiology and impacts on livestock production in this region are scarce. The objectives of this cross-sectional study were to first determine the within-herd prevalence of C. burnetii on a commercial Scottish dairy farm with prior detection of C. burnetii and second assess the relationship between pathogen load and individual health and performance traits. Vaginal swabs were collected from cows up to seven days postpartum. DNA extracts were tested for C. burnetii by IS1111 qPCR assay. Generalized linear models were used to estimate the relationships between bacterial load and health and performance traits. A total of 313 swabs were collected between 15 December 2022 and 06 July 2023, of which 301 (96.1%) had detectable C. burnetii DNA with Ct ≤ 40. The Ct values observed (12.9-38.7) corresponded to estimated genome equivalents per reaction of < 1 to ∼2.5 million genomes. Higher bacterial loads were seen in primiparous compared to multiparous cows. Higher bacterial loads were also more likely in cows that had experienced one or more negative reproductive outcomes, including abortion, premature delivery, prolonged gestation, stillbirth and dystocia. The direction of causation underlying the observed associations and representativeness of findings from this single herd require further investigation. This study provides insights into the within-herd dynamics of C. burnetii and evidence generated through sampling and diagnostic testing approaches that can inform future work to characterize the epidemiology and impacts of this infection in Great Britain dairy cattle and globally.

Cattle↗

PubMed2025

Absolute quantification of the living skin microbiome overcomes relic-DNA bias and reveals specific patterns across volunteers.

BACKGROUND: As the first line of defense against external pathogens, the skin and its resident microbiota are responsible for protection and eubiosis. Innovations in DNA sequencing have significantly increased our knowledge of the skin microbiome. However, current characterizations do not discriminate between DNA from live cells and remnant DNA from dead organisms (relic DNA), resulting in a combined readout of all microorganisms that were and are currently present on the skin rather than the actual living population of the microbiome. Additionally, most methods lack the capability for absolute quantification of the microbial load on the skin, complicating the extrapolation of clinically relevant information. RESULTS: Here, we integrated relic-DNA depletion with shotgun metagenomics and bacterial load determination to quantify live bacterial cell abundances across different skin sites. Though we discovered up to 90% of microbial DNA from the skin to be relic DNA, we saw no significant effect of this on the relative abundances of taxa determined by shotgun sequencing. Relic-DNA depletion prior to sequencing strengthened underlying patterns between microbiomes across volunteers and reduced intraindividual similarity. We determined the absolute abundance and the fraction of population alive for several common skin taxa across body sites and found taxa-specific differential abundance of live bacteria across regions to be different from estimates generated by total DNA (live + dead) sequencing. CONCLUSIONS: Our results reveal the significant bias relic DNA has on the quantification of low biomass samples like the skin. The reduced intraindividual similarity across samples following relic-DNA depletion highlights the bias introduced by traditional (total DNA) sequencing in diversity comparisons across samples. The divergent levels of cell viability measured across different skin sites, along with the inconsistencies in taxa differential abundance determined by total vs live cell DNA sequencing, suggest an important hypothesis for certain sites being susceptible to pathogen infection. Overall, our study demonstrates a characterization of the skin microbiome that overcomes relic-DNA bias to provide a baseline for live microbiota that will further improve mechanistic studies of infection, disease progression, and the design of therapies for the skin. Video Abstract.

Humans↗

PubMed1976

Effects of age and cold on pulmonary bacterial clearance in the young pig.

Young pigs (1, 6, 18, or 26 days of age) were exposed to an aerosol of a nonpathogenic strain of Escherichia coli and then held for a 3-hour clearance period in either a thermoneutral (32 C environmental temperature at 1 or 6 days of age, 24 C at 18 or 26 days of age) or a cold (6 C) environment. Pigs were then killed and pulmonary bacterial clearance was determined. There appeared to be an age dependent improvement in the capability of the pig to clear its lungs of the bacterial load. Cold appeared to inhibit pulmonary bacterial clearance, but this inhibitory effect became progressively less as the pigs became older. In a supplementary experiment conducted to assess the effect of age-within-litter (2 and 14 days of age) on pulmonary bacterial clearance, it was confirmed that young pigs were less able to clear nonpathogenic E coli from the lungs.

Age Factors↗

PubMed2025

Transitions in lung microbiota landscape associate with distinct patterns of pneumonia progression.

The precise microbial determinants driving clinical outcomes in severe pneumonia are unknown. Competing ecological forces produce dynamic microbiota states in health and disease, and a more thorough understanding of these states has the potential to improve pneumonia therapy. Here, we leverage a large collection of bronchoscopic samples from patients with suspected pneumonia to determine lung microbial ecosystem dynamics throughout the course of pneumonia. We combine 16S rRNA gene, metagenomic, and metatranscriptomic sequencing with bacterial-load quantification to reveal clinically relevant drivers of pneumonia progression. Microbiota states are predictive of pneumonia subtypes and exhibit differential stability and pneumonia therapy response. Disruptive forces, such as aspiration, are associated with cohesive changes in gene expression and microbial community structure. In summary, we show that host and microbiota landscapes change in unison with clinical phenotypes and that microbiota state dynamics reflect pneumonia progression. We suggest that distinct pathways of lung microbial community succession mediate pneumonia progression.

Humans↗

PubMed2025

The efficacy of a filtered handheld far-ultraviolet disinfection device for decontamination of high-touch surfaces in healthcare settings: a genomic bacterial analysis.

BACKGROUND AND OBJECTIVES: Enhanced environmental disinfection is linked to reduced hospital-acquired infection rates. In this study, we aimed to evaluate the efficacy of an emerging disinfection technology, a filtered far-UV-C handheld (FFUHH) device, for reducing bacterial loads on high-touch surfaces in shared clinical workrooms, and to isolate, identify and characterize clinically significant environmental pathogens. METHODS: We compared samples from high-touch items (dictation device, mouse, armchair, desk, and keyboard) before and after FFUHH treatment. Samples were collected weekly: contact plates for colony counts and swabs before and after intervention on standardized adjacent areas for each surface, respectively. The swabs were enriched and cultured on selective media to isolate pathogens. Environmental samples, as well as clinical samples collected from patients during the study period, were validated using MALDI-TOF and whole genome sequencing. RESULTS: Among the 440 collected plates (220 before and 220 after treatment), the highest mean colony count pre-treatment was detected from armchairs, and the lowest from keyboards. The mean reduction of colony-forming units ranged 53% and 83% and was statistically significant (P < 0.05) across all surfaces except for the keyboard. We characterized multidrug-resistant Staphylococcus epidermidis ST5 and ST16 strains, a carbapenem-resistant Acinetobacter baumannii, and a Klebsiella pneumoniae genetically related to a clinical isolate with a rare sequence type not previously detected in our institution. CONCLUSION: The FFUHH effectively reduced the microbial burden on high-touch surfaces. It can offer an advantage for surface disinfection and an alternative to routinely used biocides.

Humans↗

PubMed2026

Mycobacterium tuberculosis Rv0158 negatively regulates the cGAS-STING pathway mediated type I IFN production and enhances intracellular survival.

BACKGROUND: Type I interferons (IFN) play an important role in the host defense against Mycobacterium tuberculosis (M. tb) infection and disease pathogenesis. Although M. tb has evolved several mechanisms to evade host immune surveillance, the mechanism used to regulate type I IFN expression remains unclear. METHODS: In this study, genome-wide high-throughput loss-of-function screening was performed to screen M. tb determinants that regulate the Type I IFN pathway, and the role for M. tb Rv0158 in inhibiting type I IFN responses was identified in vitro and in vivo. RESULTS: The M. tb coding protein Rv0158 was identified among many transposon (Tn) insertion mutants, which increased the expression of IFN-β and some pro-inflammatory cytokines. The results suggested that Rv0158 is associated with reduced STING protein levels and suppression of cGAS-STING-mediated innate immune responses, suggesting that Rv0158 may indirectly facilitate STING degradation or modulate its stability through host-interacting partners. Rv0158 also down-regulated the transcription of interferon-stimulated genes (ISGs) and increased the bacterial load in mice. CONCLUSION: Overall, our finding identified a new bacterial factor Rv0158, these results reveal an important role for M. tb Rv0158 in inhibiting Type I IFN responses, which improves our understanding of the immune evasion mechanisms of M. tb.

Immune escape↗

PubMed1978

Antibodies to phospholipids in experimental nocardiosis.

Live Nocardia asteroides injected intraperitoneally into guinea pigs produced antibodies to phospholipids of Nocardia whereas antibodies were not detected in animals injected with heat-killed N. asteroides. The antibody titer was found to be related with the degree of infection; a significant decrease was noted after sulfadiazine treatment suggesting that antibodies were produced in response to an increasing bacterial load that occurred as the infection progressed.

Animals↗

PubMed2026

Genome-wide Identification and Expression Profiling Reveal the Galectin Gene Family Diversity and their Possible Role in Antibacterial Mucosal Immunity in Japanese Flounder (Paralichthys olivaceus).

Galectins are a family of proteins that bind specifically to β-galactosides. Their importance in innate immunity of mammals has been well-documented. However, the systematic identification and characterization of galectin gene family remain limited in teleost. In this study, we identified 13 galectin genes (lgals2, lgals2a, lgals2b, lgals3, lgals3a, lgals3b, lgals4, lgals8, lgals8a, lgals9, grp, grp-b, grp-c) from Paralichthys olivaceus genome and analyzed their tissue expressions and expressions in response to Gram-negative and Gram-positive bacterial infections in mucosal tissues (gills, intestine and skin). The P. olivaceus galections were classified into three distinct types based on carbohydrate recognition domains (CRDs). Phylogenetic and syntenic analyses revealed that these galectins are closely related to their counterparts in turbot and zebrafish. Moreover, the transcripts of the 13 galectins were widespread across all tested tissues of healthy fish and regulated following challenge with Vibrio anguillarum or Streptococcus iniae in mucosal tissues, indicating their involvement in P. olivaceus immune response to bacterial infections. The lgals2a was significantly upregulated in the three mucosal tissues by either bacterial infection, whereas lgals9 and grp were basically downregulated in these tissues by either infection. On the other hand, the lgals3b and lgals4 exhibited a bacteria-specific responsive expression as they were upregulated by V. anguillarum whereas remained stable upon S. iniae infection in the gills. We also observed a positive correlation between expression level and bacterial load for the upregulated galectin genes and a negative correlation for the downregulated galectin genes. These results suggest a functional divergence among galectin members in mucosal immunity against bacterial infection in P. olivaceus.

Animals↗

PubMed1977

Reticuloendothelial phagocytic response to bacterial challenge after traumatic shock.

Resistance to intravenous (IV) and intraperitoneal (IP) bacterial challenge during periods of reticuloendothelial (RE) depression following trauma as well as the influence of bacteremia on RE phagocytosis were studied. The experimental shock model utilized was the anesthetized (2 mg/100 g sodium pentobarbital) male rat subjected to nonlethal Noble-Collip drum trauma. During post-traumatic RE depression (60 min after injury) rats were challenged IV or IP with Escherichia coli (1.02 X 10(10)). The clearance half-time of the bacterial load injected intravenously in controls was 1.23 +/- 0.10 min. In contrast, the half-time was 3.62 +/- 0.69 min after sublethal trauma (p less than 0.005) and associated with prolonged blood bacterial retention. Pulmonary localization of E. coli administered either IV or IP was elevated in traumatized rats. Comparison of routes of bacterial challenge with respect to blood levels of viable bacteria suggested lower host bacterial resistance to the IP injection as opposed to the IV route of administration. Production of experimental bacteremia in normal rats resulted in a 39% depression (p less than 0.01) of RE test colloid clearance rate accompanied by a 49% increase (p less than 0.01) in pulmonary colloid localization. The data suggest that depressed systemic RE clearance capacity following trauma may decrease systemic resistance to septicemia, and that severe bacteremia may further undermine the functional state of the reticuloendothelial system.

Animals↗

PubMed2026

Arginine-substituted Mastoparan-C derivatives combat dual bacterial pathogens: in vitro mechanistic insights and in vivo efficacy in polymicrobial wounds.

UNLABELLED: The synergistic interactions in multi-pathogen infections compromise wound healing and limit therapeutic efficacy. In this study, we designed and synthesized arginine-substituted derivatives of the antimicrobial peptide Mastoparan-C (MP-C). Among them, Arg²MP-C and Arg4.11.12MP-C exhibited potent, broad-spectrum activity against both Escherichia coli and Staphylococcus aureus. Their enhanced antibacterial activity is associated with increased positive charge and optimized hydrophobicity. Mechanistically, both peptides employ a dual-target strategy, disrupting bacterial membranes and binding genomic DNA; Arg²MP-C acted most rapidly against the E. coli envelope, while Arg4.11.12MP-C caused the strongest membrane damage to S. aureus. In a murine polymicrobial wound model, Arg²MP-C treatment nearly achieved complete wound closure by day 10, significantly reduced bacterial loads, and promoted tissue regeneration. This study demonstrates that arginine engineering can yield peptides with potent, multi-mechanistic action, identifying Arg²MP-C as a promising candidate for combating polymicrobial wound infections. IMPORTANCE: Wounds infected with multiple bacterial species are notoriously difficult to treat, often leading to poor healing and limited effectiveness of existing therapies. In this study, we developed new antimicrobial peptides by introducing arginine substitutions into a natural peptide called Mastoparan-C. Two of our engineered peptides, Arg²MP-C and Arg4.11.12MP-C, showed potent activity against two common wound pathogens, Escherichia coli and Staphylococcus aureus. These peptides work through a dual mechanism: disrupting bacterial membranes and binding to bacterial DNA. In a mouse model of mixed-infection wounds, treatment with Arg²MP-C led to nearly complete wound closure by day 10, drastically reduced bacterial counts, and promoted tissue repair. Our findings highlight arginine engineering as a promising strategy to create next‑generation antimicrobial agents that can effectively combat complex polymicrobial wound infections, addressing a critical unmet need in clinical wound care.

Animals↗

PubMed1976

[Comparison of the efficacy of 3 methods of testing for the sterility of eye ointments with antibiotics].

Efficiency of 3 methods for testing sterility of antibiotic ophthalmic ointments was compared. During storage of antibiotic ophthalmic ointments infected with bacteria some "self-sterilization" takes place mainly due to death of the antibiotic sensitive bacteria. The results of determination of the ointment non-sterility depends on the level of their bacterial loading, sensitivity of the cultures to the antibiotic and time of the culture contact with the antibiotic in the ointment before its testing. The method of the ointment solution in isopropylmiristate filtration through a membrane filter followed by washing of the filter from the antibiotic and its placing into a thioglycol medium proved to be most effective for determination of bacterial admixtures in antibiotic ointments.

Anti-Bacterial Agents↗

PubMed1979

Ecological effects of antibiotic production by dermatophyte fungi.

Antibiotic production by dermatophyte fungi has been demonstrated in vivo in the lesions of patients with dermatomycoses. Patients infected with antibiotic-producing strains more frequently carried cocci resistant to penicillin and other antibiotics than did patients infected with non-producer strains. The total bacterial load was less in lesions caused by producer fungi. In vitro studies demonstrated the selection of penicillin-resistant S. aureus from mixed populations of resistant and sensitive cells.

Anti-Bacterial Agents↗

PubMed2026

The diagnostic potential of combined quantitative polymerase chain reaction and next-generation sequencing using the same primers for periprosthetic joint infection.

Next-generation sequencing (NGS) enables the detection of specific pathogens unidentifiable by conventional cultures, but its application in orthopedics remains inconsistent due to background contamination and irreproducible findings. This study evaluated the diagnostic performance of a novel workflow combining broad-range 16S rRNA gene quantitative PCR (qPCR) screening with downstream NGS, focusing on bacterial biomass thresholds. The qPCR assay demonstrated excellent intrarater reliability, with an intraclass correlation coefficient (ICC) of 0.961 (95% confidence interval, 0.881 to 0.997). Based on serially diluted positive controls, a quantitative threshold of 10⁵ CFU/mL was established as the minimum concentration required for the consistent detection of fastidious taxa, such as Escherichia coli. When evaluated against conventional cultures using 95 sonicate fluid and 276 pre/intraoperative tissue samples, the qPCR assay achieved a sensitivity of 80% and a specificity of 72%. Subsequent NGS sequencing of 26 clinical samples and 9 controls showed concordance in 4 of 6 culture-positive infected cases with NGS taxonomy, whereas the remaining discrepancies were likely attributable to culture-based phenotypic misidentification. Notably, among the qPCR-positive cases, three were culture-negative, including two hip prosthesis loosening cases exhibiting polymicrobial profiles, and one post-traumatic osteoarthritis case harboring low-level Staphylococcus. Crucially, this post-traumatic patient developed delayed periprosthetic joint infection (PJI) 2 years post-surgery, with cultures identifying Staphylococcus previously detected by the initial NGS analysis. Integrating qPCR screening with targeted NGS effectively refines pathogen identification, filters environmental artifacts, and overcomes the diagnostic limitations of culture-negative infections in orthopedic practice.IMPORTANCENext-generation sequencing (NGS) enables the detection of specific pathogens in clinical samples that are not identifiable by conventional methods. However, NGS applications in orthopedics have not been quantitatively evaluated, and findings have been inconsistent owing to contaminants and the presence of non-credible causative organisms. These factors primarily stem from the failure to evaluate low-biomass samples and the absence of proper controls, such as negative controls or mock community DNA samples. This study demonstrates that interpreting results from low-biomass samples requires careful consideration because NGS relies on relative bacterial abundances; distinguishing likely pathogens from contaminants is particularly challenging when bacterial loads are low. We demonstrated that combining NGS with quantitative PCR (qPCR) and applying a Cq cutoff can reduce false positives.

Humans↗

PubMed2026

A posttranslational modification of fimbriae drives pathogenicity in Klebsiella pneumoniae.

Antimicrobial resistance is a severe public health burden. Especially concerning are multidrug resistant (MDR) infections, which restrict treatment options and significantly increase mortality risk. A major cause of MDR infections worldwide is carbapenem-resistant Klebsiella pneumoniae (CRKp). The predominant CRKp sequence type worldwide is ST258. However, the factors underlying ST258's epidemic success are not well defined. Genomic analyses of clinical isolates of CRKp have found that the two-component regulatory system CrrAB is a genomic feature of ST258, suggesting that it may contribute to its global dominance. Despite this, the molecular details underpinning CrrAB's contribution to ST258 Kp biology and pathogenicity are poorly understood. We used RNA-sequencing to identify the regulon of CrrA and found that CrrAB induces the expression of a gene, encoding Crr-regulated fimbriae modifying protein (CfmP), that is essential for pathogenesis driven by this two-component system. We performed mass spectrometry analyses of fimbriae purified from Kp expressing or lacking cfmP and found that CfmP induces a novel oxidation to a histidine residue in the major pilin subunit of fimbriae, FimA. We demonstrate that this oxidation significantly increases host cell adhesion and high bacterial loads within the host. CrrAB also drives high antibiotic resistance in CRKp. Thus, our results place CrrAB at the intersection of pathogenicity and antibiotic resistance supporting its function as an important regulatory system driving the global dominance of ST258.

Klebsiella pneumoniae↗

PubMed2026

Chimeric vaccine based on Iraqi HLA alleles against a predominant local Escherichia coli phylogroup.

INTRODUCTION: Escherichia coli remains amongst the most globally important pathogens implicated in severe clinical manifestations. The progressive rise in multidrug-resistant strains highlights the urgent need for new vaccines. Therefore, this study was designed to develop a new multi-epitope vaccine containing the most conserved epitopes across E. coli pathotypes. Consequently, the study aimed to investigate the immunoadjuvant role of faecal microbiota transplantation in enhancing vaccine efficacy. METHODS: Eighteen of the most conserved B-cell and T-cell epitopes of FimH, LptD, and BamA proteins were selected and included in a single construct. During the epitope selection process, HLA alleles predominant in the Iraqi population, as reported in previous studies, were used as criteria for selecting T-cell epitopes. The chimeric protein was expressed in BL21 E. coli and purified using affinity chromatography. Vaccine cross-protective immunity and protection were tested in in vivo experiments. Different formulations were used in the experimental evaluation: three doses of 100 μg of purified chimeric protein, injected intraperitoneally alone or encapsulated in PLGA nanoparticles, after faecal microbiota transplantation with and without gut microbiota modulation mediated by a cocktail of antibiotics. IgG1, IL-4, INF-γ, and NLRP3 levels were measured at 30 and 75 days after the first immunisation dose. Immunised mice were challenged with the local B2 UPEC phylogroup, and protection efficacy was considered 48 h later. Finally, the histological effects of the different chimeric protein formulations on the liver were assessed. RESULTS: All vaccine formulations except those after faecal microbiota transplantation without gut microbiota modulation induce significant increases in IgG1, IL-4, and INF-γ levels at different times. Only vaccination after faecal microbiota transplantation with gut microbiota modulation elicited robust NLRP3 levels at 30 and 75 days after, and this was linked to the highest reduction in bladder bacterial load by 813-fold compared to the other formulations, as well as the mildest effect on liver histological changes. DISCUSSION: These results demonstrated that the chimeric vaccine provides preliminary protection against a local B2 UPEC isolate. Furthermore, modulating gut microbiota via faecal transplantation markedly enhances the immunogenicity and protective efficacy of vaccination, suggesting its adjuvanticity.

Animals↗

PubMed2026

Association Between the Root Canal Microbiome and Apical Lesion Size: An Observational Shotgun Metagenomic Study.

AIM: The aim was to characterize the taxonomic and functional composition of the microbiome involved in primary endodontic infections and to evaluate their association with the periapical lesion size using shotgun metagenomic sequencing. METHODOLOGY: Samples from primary root canal infections diagnosed with apical periodontitis were analysed with shotgun sequencing. Samples were classified according to the lesion size as small (< 3 mm) or large (> 7 mm). The bacterial DNA copies in each group were quantified by qPCR. Taxonomic and functional annotations were made using Bracken/Kraken2 and HUMAnN3 software. Species richness, Shannon, Simpson and Pielou indices were used to measure alpha diversity. The similarity of the bacterial communities between study groups was evaluated by Principal Coordinate Analysis based on Bray-Curtis distances. The ALDEx2 package was used to infer the differences between species, and the edgeR package for KEGG pathways. For all statistical analyses, p < 0.05 was considered as significant. RESULTS: A total of 49 samples were analysed, 27 with small lesions and 22 with large lesions. Species richness and Shannon indices showed differences between both groups, whereas no differences were seen according to Simpson and Pielou indices. A different community composition (PERMANOVA, p = 0.0019) was observed between the two groups. Three species were significantly enriched in the large lesion samples, Filifactor alocis, Lachnospiraceae bacterium oral taxon 500 and Olsenella uli, while three others were enriched in small lesion samples, Acinetobacter baumannii, Acinetobacter pittii and Cutibacterium acnes. Functionally, benzoate, flavonoid and steroid degradation, the sphingolipid signalling pathway and proteasome function were enriched in samples with large lesions. Monoterpenoid biosynthesis, phospholipase D signalling, the sulphur relay system and staurosporine biosynthesis were enriched in small lesions. CONCLUSIONS: Teeth with large periapical lesions harbour greater bacterial loads and exhibit a more diverse microbial community than those with small lesions. Differences in species-level taxonomic composition were observed between both groups. Functionally, large lesions are enriched in pathways associated with immune evasion and pro-inflammatory activity, whereas small lesions are characterized by pathways related to apoptosis, metabolic adaptation and anti-inflammatory processes. These findings suggest that lesion severity is also shaped by the functional potential of the microbiome to modulate host inflammation.

Humans↗