Bacteria belonging to the family Enterobacteriaceae occur as commensals or pathogens in humans. The increasing prevalence of multidrug-resistant bacteria has raised global concern. Following the reclassification of Enterobacteriaceae species into the order Enterobacterales in 2016. However, no quantification method has been reported that is designed and validated in the context of the current order-level taxonomy of Enterobacterales. Therefore, we aimed to develop a novel reverse transcription quantitative PCR (RT-qPCR) system for the quantification of Enterobacterales based on 23S rRNA. The system was used to quantify Enterobacterales species, and its applicability was evaluated using fecal samples from 99 patients with gastrointestinal cancer and 10 healthy volunteers. Furthermore, we performed a proof-of-concept evaluation of its utility for rapid assessment of antimicrobial susceptibility in carbapenem-resistant Enterobacterales (CRE). The system exhibited high analytical sensitivity and enabled quantification of diverse Enterobacterales spp., including newly classified species, with theoretical detection limits of 10[3] cells/g feces and 1 cell/mL blood. In healthy volunteers, intestinal Enterobacterales were dominated by Escherichia coli and Enterobacter, whereas preoperative patients with gastrointestinal cancer exhibited greater diversity. In vitro, viable CRE cell counts determined by the RT-qPCR system 2 h after antibiotic treatment correlated well with culture-based susceptibility results obtained after 24 h, suggesting the feasibility of rapid antimicrobial susceptibility assessment within 6 h. The developed 23S rRNA RT-qPCR system provides a sensitive approach for quantifying Enterobacterales within the current taxonomic framework and may facilitate the rapid assessment of antimicrobial activity against CRE. CLINICAL TRIAL REGISTRATION: There is no clinical trial registration.

B. parapertussis is one of the etiologic agents of whooping cough and its incidence has increased following the COVID-19 pandemic. Since current pertussis vaccines fail to induce protection against B. parapertussis, the course of infection critically relies on the innate immune response. However, in the absence of opsonic antibodies, B. parapertussis fails to activate resting neutrophils, thereby preventing bactericidal responses. We here investigated whether neutrophil priming with gamma-interferon (IFN-γ), the main proinflammatory cytokine present during infection, changes this outcome. We observed that IFN-γ- priming of neutrophils did not enhance extracellular nor intracellular bactericidal mechanisms against B. parapertussis. Human neutrophils remain basically unresponsive to this pathogen despite IFN-γ priming, and this lack of activation proved independent of adenylate cyclase toxin (CyaA), the immunomodulatory toxin produced by B. parapertussis. Collectively, our findings demonstrate that B. parapertussis does not trigger neutrophil activation, irrespective of the inflammatory context. This ability to evade a key arm of the innate immune response likely constitutes a critical pathogenic trait that, particularly in the absence of cross-protection conferred by current pertussis vaccines, underlies persistent infections and the continued circulation of the pathogen.

As companion animals live in close contact with humans, the presence of antimicrobial-resistant organisms in these animals is an important issue in veterinary medicine from a One Health perspective. In this study, we isolated a total of 385 Escherichia coli strains from fecal samples collected from 218 companion dogs and 167 companion cats that visited veterinary clinics in Tokyo, Japan, from 2021 to 2024. Antimicrobial resistance to third-generation cephalosporins and fluoroquinolones increased significantly with host age, even though fecal abnormalities were low relevance. A total of 60 isolates were resistant to at least one third-generation cephalosporin, and whole-genome analysis was performed to further characterize these isolates. The isolates were classified as CTX-M gene-carrying or AmpC-associated isolates, including those carrying acquired AmpC genes and those harboring chromosomal ampC promoter mutations. The age-associated increase was more pronounced among CTX-M gene-carrying isolates than among AmpC-associated isolates. CTX-M gene-carrying isolates frequently exhibited concurrent levofloxacin resistance and were predominantly represented by ST131. Core-genome multilocus sequence typing revealed that these CTX-M gene-carrying isolates were phylogenetically similar to previously reported isolates of both human and animal origin. These findings suggest that the age-associated increase in resistance is related to the expansion of specific CTX-M gene-carrying lineages rather than to a uniform increase across all cephalosporin-resistance mechanisms. Our results support the need for continuous investigation within a One Health framework and further epidemiological and genomic surveillance.

Streptococcus sobrinus is a member of the Streptococcus mutans group and is a cariogenic bacterium similar to S. mutans. One of the characteristics of these bacteria is bacitracin resistance, and BceAB (MbrAB), an ABC transporter of S. mutans, has been reported to be involved in bacitracin resistance. However, the factors responsible for bacitracin resistance in S. sobrinus are unknown. In this study, 11 S. sobrinus strains were isolated from the oral cavity, and their bacitracin susceptibility was assessed. The results showed that 9 strains were low-susceptible to bacitracin (MIC = 128-512 µg/mL), whereas 2 strains were highly susceptible (MIC = 32 µg/mL). Whole-genome analysis of all strains identified genes encoding an ABC transporter (designated as BceAB) and its adjacent two-component system (BceRS), which showed high homology with the BceAB-BceRS locus in S. mutans. We successfully generated bceAB-deletion strains from bacitracin-low-susceptibility strains (HSB4 and HSB10) and a bacitracin-high-susceptibility strain (HSB8). Upon verifying their bacitracin susceptibility, the deletion strains derived from the bacitracin-low-susceptibility strains exhibited increased susceptibility compared to the wild-type strains, whereas the deletion strain of HSB8 showed no change in susceptibility. Quantitative RT-PCR revealed that, in the low-susceptibility HSB5 strain, bacitracin induced bceAB expression in a dose-dependent manner, whereas no such induction was observed in the high-susceptibility HSB7 strain. Comparison of the amino acid sequences of the BceAB-BceRS region among representative clinical isolates revealed that only BceS contained a mutation at the 179th amino acid residue (E179K) specific to the bacitracin-high-susceptibility strain. Based on these results, BceAB was concluded to be an intrinsic factor for bacitracin resistance, and BceRS may be associated with bacitracin-induced expression of BceAB in S. sobrinus.

BteA is a cytotoxic effector translocated into host cells via the type III secretion system (T3SS) of Bordetella. The T3SS gatekeeper protein BopN controls BteA translocation, yet the BteA region mediating this regulation remains unknown. Here, we demonstrate that BteA interacts with BopN through its C-terminal region, and this interaction is required for efficient translocation into host cells. Using a CyaA-based translocation assay, we showed that BteA comprising the N-terminal 448 amino acids (BteA-N448) and BteA with a deletion of amino acids 249-448 (BteA-∆249-448) were translocated in a BopN-dependent manner, whereas BteA comprising the N-terminal 248 amino acids (BteA-N248) was translocated independently of BopN. Pull-down assays confirmed that BteA-N448 and BteA-Δ249-448 physically interact with BopN, whereas BteA-N248 does not. ColabFold-based structure prediction identified candidate interface residues, and subsequent mutagenesis revealed that simultaneous alanine substitution of aspartate 263 (D263) and phenylalanine 340 (F340) abolished BopN-dependent translocation. Our results demonstrated that the amino acid residues D263 and F340 in BteA play important roles in BopN-mediated translocation control. Furthermore, the C-terminal 14-amino-acid region of BteA was strongly suggested to be involved in this regulation. The present study provides new insights into the effector-gatekeeper network that controls T3SS-mediated pathogenicity in Bordetella.

The mechanisms underlying seasonal patterns in influenza epidemics have long been an enigma. Our laboratory study revealed that ambient ozone significantly inactivates airborne influenza viruses synergistically with higher relative humidity and temperature. This led us to hypothesize that natural seasonal fluctuations in these ambient factors enhance or mitigate the influenza-transmission risk by altering the efficiency of airborne virus inactivation, thereby contributing to the continuation or subsiding of influenza epidemics. To test the hypothesis, we examined datasets covering 5 years of annual environmental ozone concentrations, relative humidity, and temperature, and reported influenza case numbers in two cities in Japan, Sendai and Naha, which exhibit typical temperate and subtropical climate patterns, respectively; based on the findings of our previous study and actual ambient environmental data, we estimated the influenza airborne-transmission risk in these cities and compared it with observed epidemics. In both cities, the persistence of winter epidemics was strongly associated with low ozone concentration (< 35 ppb), and their subsiding in spring was associated with increases in ozone (≧ 35 ppb) and relative humidity (≧ 50%), even at lower temperature (< 20°C). The summer outbreak in Naha was not suppressed solely under high temperatures (25°C-30°C) and relative humidity (≧ 70%), but was associated with a subsequent increase in ozone (≧ 35 ppb). A similar phenomenon was recognized during the 2009 influenza pandemic. These results were consistent with our hypothesis: influenza epidemic patterns corresponded with the influenza transmission risk estimated from the synergistic inactivation patterns of airborne viruses by ambient ozone, relative humidity, and temperature.

To characterize variation in the milk microbiota of clinically healthy dairy cows, milk samples were collected repeatedly from four cows throughout lactation and analyzed by 16S rRNA gene amplicon sequencing. Alpha-diversity analysis showed no significant effects of parity, lactation stage, udder quarter position, or the interaction between lactation stage and position. Although sample-level PERMANOVA indicated significant associations of beta diversity with cow identity and parity, these associations were not supported when the repeated-measures structure was appropriately accounted for. Restricted-permutation PERMANOVA showed no significant effects of lactation stage, udder quarter position, or their interaction, and exact permutation testing at the individual-cow level showed no significant effect of parity. Ordination and hierarchical clustering analyzes nevertheless revealed substantial variation in bacterial community composition among individual samples. Hierarchical clustering based on genus-level relative abundance separated most samples into two major groups characterized by relatively high and low diversity. The high-diversity group showed a relatively high abundance of the class Clostridia, whereas the low-diversity group was characterized by a predominance of Gammaproteobacteria, particularly Escherichia/Shigella in some samples. Overall, the milk microbiota of healthy dairy cows showed substantial sample-to-sample variation, but the present data did not provide clear evidence that parity, lactation stage, or udder quarter position systematically influenced microbial diversity. These findings provide baseline information on variation in the milk microbiota of healthy dairy cows and highlight the importance of accounting for repeated measurements and individual variation in studies of the bovine milk microbiota.

Exopolysaccharides (EPS) produced by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 have been reported to exert immunomodulatory effects, primarily in antiviral responses; however, their role in antibacterial host defense remains unclear. Here, we investigated whether EPS enhance macrophage-mediated killing of Streptococcus pyogenes, a facultative intracellular pathogen, using an in vitro co-culture system with NK-like cells. EPS alone did not enhance the intracellular bactericidal activity of macrophages. In contrast, EPS significantly increased bacterial killing when macrophages were co-cultured with NK-like cells. Mechanistically, EPS induced interleukin (IL)-12 expression in macrophages, while interferon (IFN)-γ production was not detected when macrophages or NK-like cells were cultured separately, but was significantly induced in the co-culture system. Neutralization of IL-12 or IFN-γ significantly abrogated the EPS-mediated enhancement of bactericidal activity, indicating that both cytokines are required for this effect. These findings support a model in which EPS stimulate macrophages to produce IL-12, leading to NK cell activation and subsequent IFN-γ-dependent enhancement of macrophage bactericidal function. Our results reveal a previously uncharacterized role of probiotic-derived EPS in promoting antibacterial immunity through macrophage-NK cell crosstalk.

Many mammalian cells restrict viral replication by utilizing various host restriction factors. We recently demonstrated that CCHC-type zinc-finger-containing protein 3 (ZCCHC3) suppresses human immunodeficiency virus type 1 (HIV-1) replication through multiple mechanisms. We also revealed that single-nucleotide polymorphisms in human ZCCHC3 affect its antiviral function; however, whether similar genetic and functional diversity is present in other species remains unknown. In this study, we investigated the genetic and functional diversity of ZCCHC3 in cynomolgus macaques, a critical animal model for HIV-1-related research. Sequencing analysis of eight independent ZCCHC3 clones per animal revealed substantial amino acid diversity among cynomolgus macaques. We selected 12 representative variants and examined their antiviral activity against several retroviral vectors derived from HIV-1, simian immunodeficiency virus, feline immunodeficiency virus, and murine leukemia virus. Moreover, using replication-competent HIV-1, we showed that selected cynomolgus macaque ZCCHC3 variants can affect both viral production and viral infectivity. These results suggest that the genetic and functional diversity of ZCCHC3 is not limited to humans and underscore the importance of considering ZCCHC3 variation in cynomolgus macaques when using them as animal models for HIV-1-related research.

Staphylococcus aureus is responsible for a wide range of pyogenic infections, food poisoning, and allergic inflammation. S. aureus produces a very wide variety of proteinaceous exotoxins, which are classified by structure as β-barrel pore-forming toxin, oligonucleotide/oligosaccharide-binding (OB)-fold/β-grasp proteins, and triple-helix bundle-containing proteins. We previously reported that three staphylococcal exotoxins belonging to the first two groups activate basophils and mast cells, which are innate immune cells that play a central role in allergic inflammation. In this study, we focused on a group of triple-helix bundle-containing toxins and examined their ability to activate murine bone marrow-derived mast cells and basophils. Extracellular complement-binding protein (Ecb), previously known as a complement inhibitor, was found to induce IL-4 and IL-6 production in basophils but not that of IL-6 and IL-13 in mast cells. Ecb did not induce IL-4 production in murine splenocytes or CD4[+] T cells, suggesting that it is a basophil-specific activator. Ecb-induced IL-4 expression in basophils in the absence of serum. The C3-binding-deficient mutant of Ecb also activated basophils, indicating that Ecb activates basophils independently of its complement-inhibiting activity. Dasatinib, a Src kinase inhibitor, prevented Ecb-induced IL-4 production in basophils, suggesting that the activation required a Src family kinase. These findings reveal a novel function of Ecb, that is, the activation of basophils to produce IL-4 in an IgE-independent manner, and suggest a dual Ecb contribution to immune evasion-by interfering with complement activation and skewing Th2 immunity-and a role in the development of allergic inflammation-by inducing IL-4 production.

Salmonella enterica serovar Typhimurium (STm) is a leading cause of diarrheal disease and efficiently colonizes the gut lumen despite microbiota-mediated colonization resistance. Emerging evidence indicates that STm does not simply evade or overcome these barriers, but instead exploits host immune responses and microbiota-derived metabolic activities to create a permissive intestinal niche. In this review, we discuss recent advances in our understanding of STm gut colonization, with a focus on how host antimicrobial factors and microbiota-derived metabolites are repurposed to promote pathogen growth, motility, and invasion. We highlight findings showing that the antimicrobial lectin RegIIIβ, together with microbiota-dependent metabolites such as fatty acid-derived cues and polyamines, can unexpectedly facilitate STm colonization. Together, these studies support a model in which host defense programs and microbial metabolism are redirected to promote pathogen fitness, revealing potential therapeutic opportunities for controlling enteric infections.

In this review, we introduce a promising strain for studying pathogenesis of nontuberculous mycobacterial pulmonary disease (NTM-PD), Mycobacterium intracellulare M.i.198. M.i.198 was isolated from a patient with progressive Mycobacterium avium-intracellulare complex pulmonary disease (MAC-PD) in whom surgical operation was required to suppress disease progression. M.i.198 shows progressive illness in infected mice in terms of bacterial load, inflammation, and by high-dose inoculation, mortality as well. Although M.i.198 is resistant to clarithromycin, the pathogenicity of M.i.198 in mice indicates the high potency for use to evaluate drug efficacies in vivo. Furthermore, M.i.198 can be genetically manipulated, which is quite different from other MAC strains, especially those of M. avium subsp. hominissuis. We herein report the bacteriological findings, experimental virulence and functional genomic features of M.i.198 with a small review of MAC strains used for animal models of MAC-PD.

Toxigenic Corynebacterium ulcerans, a zoonotic pathogen, was isolated repeatedly from a cat in an in-house group in Japan. Although the cat was cured once by antibiotic treatment, clonal isolates of the original pathogenic strain were recovered repeatedly during a 2-year course. Clonal isolates of those isolates were also obtained from other asymptomatic cats in the same shelter, suggesting that environmental circulation of the pathogen had occurred. Care should be taken to monitor and cure the animals if any of their groupmates test positive for the pathogen, even if asymptomatic.

Chlamydia trachomatis (C. trachomatis) is a strictly parasitic pathogen that heavily relies on host cells for generating energy, acquiring nutrients, and evading immune responses. Mitochondrial dynamics-the balance of fusion and fission-are integral to cellular functions, including the maintenance of homeostasis, the regulation of metabolic processes, and the modulation of host innate immune pathways. Accordingly, C. trachomatis can specifically change the host mitochondrial dynamics to promote its intracellular replication. Mitochondrial fragmentation has been observed during the later phases of C. trachomatis infection; Nevertheless, the exact mechanisms remain poorly defined. The research aimed to determine the effect of the C. trachomatis secretory protein pORF5 in this process. In stable pORF5-expressing Hela cells, we employed confocal microscopy to analyze mitochondrial morphology and Western blotting to measure the expression of key mitochondrial dynamics proteins. Finally, immunofluorescence was used to monitor Drp1 mitochondrial translocation, and the effects of a pathway inhibitor on mitochondrial fission were evaluated. We observed that the plasmid-encoded protein pORF5 can induce mitochondrial fission. Mechanistically, this process is dependent on the activation of the ERK/Drp1 signaling axis, which indicates the crucial importance of this pathway and its effect on pORF5-induced mitochondrial fragmentation.

A C-type lectin with specificity for N-acetyl-d-glucosamine was purified from the cutaneous mucus of chub mackerel (Scomber japonicus) by affinity chromatography. The lectin comprises 159 amino acid residues and contains an unusual substitution within the conserved Ca[2+]-binding motif, in which Trp-Asn-Asp is replaced by Trp-Ser-Asp. Reverse transcription-PCR analysis demonstrated that the lectin gene is specifically expressed in the skin, and immunohistochemistry localized the protein to the outer epidermal layer, predominantly in squamous cells. Agglutinating activity was observed against L. garvieae, but not against any of the other bacterial species tested. An approximately 25-kDa bacterial protein was identified as a potential ligand, although its identity remains unresolved. Collectively, these findings indicate that this lectin (SjCL) functions as a mucosal immune molecule and may contribute to the first line of defense against bacterial pathogens in teleost fish.