Takeaki Wajima

Horizontal gene transfer in Haemophilus spp. is associated with antimicrobial resistance development. Recently, a relationship between quinolone resistance and this transfer has been reported. This study aimed to investigate the mechanisms underlying quinolone resistance spread by focusing on both homogeneous and heterogeneous transfer to H. influenzae. Quinolone-resistant strains of three Haemophilus spp., H. influenzae, H. haemolyticus, and H. parainfluenzae, were used as resistant donors. The H. influenzae laboratory strain Rd and the highly transformable strains 2017-22B and 2018-Y41 were used as recipients. Horizontal transfer assays were performed using genomic DNA from resistant donors or resulting transformants. Horizontal transfer assays demonstrated that quinolone resistance was transferred from the genomic DNA of H. influenzae and H. haemolyticus to all recipient strains. In contrast, resistance from H. parainfluenzae genomic DNA was transferred only to the strains with higher transformability. In all cases, transfer efficiency was higher when DNA from the transformants was used than when DNA from the original strain was used. Notably, genomic DNA from transformants obtained by transferring resistance from H. parainfluenzae to highly transformable H. influenzae strains transformed Rd into a quinolone-resistant variant. Furthermore, no significant differences in growth were observed between the parent strains and transformants. Our findings suggest that highly transformable strains, owing to their enhanced transformation capacity and retained fitness, may facilitate or amplify the dissemination of quinolone resistance under experimental conditions.

Spore-forming bacteria are widely distributed in the environment and present a major challenge in healthcare settings due to their resistance to alcohol-based disinfectants. Understanding this resistance is essential for infection control education, especially in medical fields. Here, we propose a simple, low-cost laboratory exercise that combines hand hygiene training with a visual demonstration of microbial persistence using soil as a source of spore-forming bacteria. In this exercise, students imprint their hands on agar plates under three conditions: unwashed, soap-washed, and alcohol-disinfected. After 48 h of incubation, the plates reveal clear differences in microbial growth, highlighting the limited efficacy of alcohol-based sanitizers against spores. Gram staining of colonies provides presumptive evidence of the presence of spore-forming bacteria. This exercise addresses a common misconception among healthcare trainees—that alcohol sanitizers provide complete protection—and emphasizes the importance of proper handwashing with soap and water. Its simplicity, safety, and strong visual impact make it an effective and adaptable tool for microbiology education and healthcare professional training.

The M protein is an essential virulence factor of Streptococcus pyogenes , or group A streptococcus (GAS), one of the most common and dangerous human pathogens. Molecular and functional characterization of M protein variants and their interactions with host components is crucial for understanding streptococcal pathogenesis and vaccine development. The M3 protein is produced by the prevalent emm 3 GAS serotype, which is frequently associated with severe invasive diseases. Here, we structurally and biochemically characterize the interaction of M3 with human collagens. High-resolution structures of the N-terminal M3 domain in the free state as well as bound to a collagen peptide derived from the Collagen Ligands Collection reveal a novel T-shaped protein fold that presents binding sites complementing the characteristic topology of collagen triple helices. The structure of the M3/collagen peptide complex explains how emm 3 GAS and related streptococci, such as Streptococcus dysgalactiae subsp. equisimilis , can target collagens to enable colonization of various tissues. In line with this, we demonstrate that the M3/collagen interaction promotes enhanced biofilm formation of emm 3 GAS in an emm type-specific manner, which can be inhibited with the recombinant N-terminal M3 domain. Further, emm 3 GAS are shown to colocalize with collagen in tissue biopsies from patients with necrotizing soft tissue infections, where GAS biofilms are common. This observation is reproduced in infected organotypic skin models. Together, these data provide detailed molecular insights into an important streptococcal virulence mechanism with implications for the understanding of invasive infections, strategies for treating biofilm and M-protein-based vaccine design.

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Sulfamethoxazole-trimethoprim (SXT) is an important empirical treatment agent against various bacterial infections. In this study, we aimed to elucidate the mechanisms underlying SXT resistance in Haemophilus influenzae clinical isolates from Japan, as information on such resistance remains limited. A total of 79 H. influenzae clinical isolates collected in 2018 and 2022 were analyzed. The SXT resistance rates were 38.7% in 2018 and 35.3% in 2022. Multilocus sequence typing analysis revealed that ST422 was the most common sequence type (36.7%), followed by ST107 (26.7%). Horizontal transfer assays using the genomic DNA or PCR-amplified fragments revealed that SXT resistance was transferred to the susceptible isolates via genomic DNA and PCR-amplified folA fragments, indicating that FolA mediates SXT resistance in H. influenzae . Site-directed mutagenesis revealed that the substitution of isoleucine at position 95 in FolA was associated with SXT resistance. All SXT-resistant isolates had an amino acid substitution at position 95 in FolA: leucine in 26 of the 30 strains, valine in 3 strains, and glycine in 1 strain. Our findings demonstrate that SXT resistance in H. influenzae was prevalent and can spread via horizontal transfer. Furthermore, an amino acid substitution at position 95 of FolA played a key role in conferring resistance.

Antimicrobial resistance (AMR) is a serious global concern. AMR pathogens are found in hospitals and communities. Haemophilus influenzae is a common pathogen associated with community-acquired infections. H. influenzae infections are usually treated with β-lactams, macrolides, and quinolones. However, the drug-resistant strains have emerged. The resistance mechanisms of H. influenzae are complex but are roughly characterized by the acquisition of a mutation in antimicrobial-targeting genes and exogenous resistant genes. Generally, the former cannot be transferred horizontally to a susceptible strain. However, several studies have demonstrated that, in the case of H. influenzae, both the former and the latter can be transferred horizontally. In this review, we provide an overview of the bacterial features and antimicrobial resistance of H. influenzae. We also summarize the unique and ingenious antimicrobial resistance mechanisms used by this pathogen based on the findings of recent studies. These are expected to facilitate the understanding of AMR pathogens in the community and develop strategies to combat infections.

Haemophilus influenzae presents significant concerns regarding antimicrobial resistance. The circumstances surrounding H. influenzae have been changing owing to changes in antimicrobial usage. In the present study, to determine the current situation of H. influenzae, the antimicrobial susceptibility trends were investigated. In total, 21 clinical isolates were analyzed. Antimicrobial susceptibility measured using the broth dilution method was compared with that reported in previous studies at the same hospital. Quinolone low-susceptible isolates were further characterized by multi-locus sequence typing. Upon comparing the susceptibility data in 2022 with those in the past 15 years, the number of β-lactamase nonproducing ampicillin-resistant isolates was decreased. Regarding recent changes, β-lactam-susceptible isolates were found to have gradually increased every year. However, β-lactamase-producing isolates did not decrease. In particular, the ratio of β-lactamase-producing amoxicillin and clavulanic acid-resistant isolates in 2022 was the highest among all the years studied. Moreover, quinolone low-susceptible isolates were still present, suggesting that these isolates could have been indigenized in the community. Furthermore, a β-lactamase-producing amoxicillin and clavulanic acid-resistant isolate was found to have emerged among the quinolone low-susceptibility isolates. At first glance, these findings indicate that antimicrobial resistance has decreased among H. influenzae in clinical settings. However, β-lactamase-producing isolates and quinolone low-susceptible isolates have remained at a constant rate in the community.

Introduction. Reports of β-lactamase-producing Haemophilus influenzae are increasing worldwide. Aim. This study aimed to elucidate the molecular characteristics and evolution of β-lactamase-producing H. influenzae. Methodology. A total of 159 clinical isolates were characterized using multi-locus sequence typing. Antimicrobial resistance genes and integrative and conjugative element (ICE) types were identified through PCR and DNA sequencing. The genetic structure of ICE was further investigated using whole-genome sequencing. Results. Out of 159 clinical isolates, 20.8% (n=33) were β-lactamase producers. Thirteen sequence types (STs) were identified. ST 103, 155, 165 and 388 have been identified in previous studies, suggesting that strains with these STs tend to acquire the β-lactamase gene bla _TEM-1. Among β-lactamase producers, 66.7% (n=22) of bla _TEM-1 were located on ICE. The ICEs could be classified into two groups based on their sequence (types I and II). Among these strains, 2017-Y3 harboured a macrolide resistance gene, mef (A/E), in ICE. A comparative analysis of the ICE region of this strain and those from other countries suggested that each isolate was derived from ICE type I or II. These regions, including mef (A/E), were similar to those of Tn6822, which is commonly found in Streptococcus. Conclusions. This study revealed several STs associated with the acquisition of β-lactamase genes on ICEs. Additionally, ICE evolution involved the acquisition of exogenous genes. The accumulation of resistance genes in ICE raises concerns regarding the emergence of multidrug-resistant H. influenzae.

Reports of novel species of α-hemolytic Streptococcus have increased recently. However, limited information exists regarding the pathogenicity of these species, with the exception of Streptococcus pneumoniae and Streptococcus pseudopneumoniae . In this study, a quinolone-resistant α- Streptococcus strain, MTG105, was isolated from the sputum of a patient with pneumonia. This strain was first identified as S. pneumoniae at the hospital laboratory; however, it exhibited unique genetic features upon further analysis. Digital DNA-DNA hybridization and average nucleotide identity based on BLAST values from whole-genome sequencing revealed MTG105 to be a novel species closely related to S. pseudopneumoniae . Although MTG105 carried two copies of the pneumolysin gene, similar to S. pseudopneumoniae , this isolate exhibited susceptibility to optochin under both aerobic and 5% CO ₂ conditions. Notably, no biochemical features could be used to definitively identify this species. In an infection assay using organotypic lung tissue models, MTG105 induced epithelial damage comparable to that of S. pneumoniae and S. pseudopneumoniae , possibly suggesting its potential as a pathogenic α- Streptococcus . The natural transformation abilities of Streptococcus species facilitate their exchange of genes within the same genus, resulting in the existence of species with increasingly more diverse genome structures. Therefore, the identification of this species highlights the importance of monitoring the emergence of novel species exhibiting virulence and/or multidrug resistance. This isolate was proposed as a novel species, designated Streptococcus nakanoensis sp. nov. The type strain was MTG 105 ^T (= JCM 35953 ^T = CCUG 76894 ^T ). IMPORTANCE: The genus Streptococcus encompasses a wide range of bacteria with more than 60 species. Recently, there has been a notable increase in reports of novel species of α- Streptococcus based on genomic analysis data. However, limited information exists regarding the pathogenicity of these species. In this study, a quinolone-resistant α-hemolytic Streptococcus strain, MTG105, was isolated from a patient with pneumonia. Genetic analysis revealed that this species was a novel species closely related to S. pseudopneumoniae . In an infection assay using organotypic lung tissue models, MTG105 induced epithelial damage comparable to that caused by S. pneumoniae and S. pseudopneumoniae , strongly suggesting its potential as a pathogenic α- Streptococcus . The natural transformation abilities of Streptococcus species facilitate gene exchange within the same genus, leading to the emergence of species with increasingly diverse genome structures. Therefore, the identification of this species underscores the importance of monitoring the emergence of novel species exhibiting virulence and/or multidrug resistance.