Emi Tanaka

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.

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.

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.

In 2019, a high-level quinolone-resistant Haemophilus haemolyticus strain (levofloxacin MIC = 16 mg/L) was isolated from a paediatric patient. In this study, we aimed to determine whether the quinolone resistance of H. haemolyticus could be transferred to Haemophilus influenzae and to identify the mechanism underlying the high-level quinolone resistance of H. haemolyticus. A horizontal gene transfer assay to H. influenzae was performed using genomic DNA or PCR-amplified quinolone-targeting genes from the high-level quinolone-resistant H. haemolyticus 2019-19 strain. The amino acids responsible for conferring quinolone resistance were identified through site-directed mutagenesis. By adding the genomic DNA of H. haemolyticus 2019-19, resistant colonies were obtained on agar plates containing quinolones. Notably, H. influenzae grown on levofloxacin agar showed the same level of resistance as H. haemolyticus. Sequencing analysis showed that gyrA, parC and parE of H. influenzae were replaced by those of H. haemolyticus, suggesting that horizontal transfer occurred between the two strains. When the quinolone-targeting gene fragments were added sequentially, the addition of parE, as well as gyrA and parC, contributed to high-level resistance. In particular, amino acid substitutions at both the 439th and 502nd residues of ParE were associated with high-level resistance. These findings indicate that quinolone resistance can be transferred between species and that amino acid substitutions at the 439th and 502nd residues of ParE, in addition to amino acid substitutions in both GyrA and ParC, contribute to high-level quinolone resistance.

Quinolone-resistant bacteria are known to emerge via the accumulation of mutations in a stepwise manner. Recent studies reported the emergence of quinolone low-susceptible Haemophilus influenzae ST422 isolates harbouring two relevant mutations, although ST422 isolates harbouring one mutation were never identified. To investigate if GyrA and ParC from quinolone low-susceptible isolates can be transferred horizontally and simultaneously to susceptible isolates. Genomic DNA was extracted from an H. influenzae isolate harbouring amino acid substitutions in both gyrA and parC and mixed with clinical isolates. The emergence of resistant isolates was compared, and WGS analysis was performed. By adding the genomic DNA harbouring both mutated gyrA and parC, resistant bacteria exhibiting recombination at gyrA only or both gyrA and parC loci were obtained on nalidixic acid and pipemidic acid plates, and the frequency was found to increase with the amount of DNA. Recombination events in gyrA only and in both gyrA and parC occurred with at least 1 and 1–100 ng of DNA, respectively. The genome sequence of a representative strain showed recombination events throughout the genome. The MIC of quinolone for the resulting strains was found to be similar to that of the donor. Although the recombination efficacy was different among the various strains, all strains used in this study obtained multiple genes simultaneously. These findings indicate that H. influenzae can simultaneously obtain more than two mutated genes. This mechanism of horizontal transfer could be an alternative pathway for attaining quinolone resistance.

The presence of Haemophilus influenzae strains with low susceptibility to quinolones has been reported worldwide. However, the emergence and dissemination mechanisms remain unclear.