This study is the first to demonstrate that mutations in the chromosomal- amp C attenuator region are responsible for the emergence of cefazolin resistance in Escherichia coli strains. The resistance was maintained even after culturing E. coli without cefazolin. This study highlights one of the key factors contributing to the increase in the number of cefazolin-resistant E. coli strains, which can pose a considerable challenge for treating common infections, such as urinary tract infections.

Purpose: Cefmetazole (CMZ), a cephamycin antibiotic, is primarily used as a definitive therapy for Extended Spectrum β-Lactamase (ESBL)-producing Escherichia coli infections. However, the mechanism of CMZ resistance in E. coli is still unknown. To elucidate the resistance mechanism and to determine combined drugs for prevention of resistance acquisition. Methods: Clinical isolates of 14 ESBL-producing E. coli and non-producing 12 isolates were used in in vitro testing of CMZ resistance acquisition. After 10-day of CMZ exposure (1st subculture), these strains were incubated in an antibacterial-free medium for 14-day. These strains were again exposed to CMZ for 10-day (2nd subculture) and confirmed for changes in MIC. For each strain detected after 1st subculture, each mRNA expression level of porin, chromosomal ampC, and drug-efflux pump was measured using real-time RT-PCR. Relebactam (REL) has the potency to recover antimicrobial activity against carbapenem-resistant Enterobacterales that has porin deficiency. REL was added to the CMZ dilution series, and MIC changes and those of porin were confirmed. Results: Of these 26 strains, 15 strains (57.7%) acquired resistance after 1st subculture, but after passage culture on the antibacterial-free medium, 11 strains recovered susceptibility. These 11 strains showed resistance after 2nd subculture. The expression levels of ompF and ompC were significantly decreased in these strains (P< 0.05). When REL was added, all strains suppressed resistance acquisition after 1st subculture. The mechanism was the activation of ompF. Conclusion: Our results showed that the mRNA expression levels of genes encoding porin were decreased in the strains that acquired resistance due to CMZ exposure, and that ompF and ompC in particular were thought to be involved in the acquisition of resistance. The CMZ acquisition of resistance was also suppressed by the concomitant use of REL and actually suppressed the decrease in mRNA expression in ompF. It was confirmed that porin reactivated by REL.

Although the development of disinfection technologies with novel mechanisms has stagnated, we demonstrate the bactericidal effects and mechanisms of high-speed nanodroplet generation technology. The first development of this technology in 2017 gushes out a water droplet of 10 nm in size at 50 m/s; however, the target surface does not become completely wet. Nanodroplets were exposed to biofilm models of Staphylococcus aureus , Pseudomonas aeruginosa , Escherichia coli , and Serratia marcescens . This phenomenon was verified when the nanodroplets collide with the surface of the bacteria at an impact pressure of ~75 MPa. S. aureus was exposed to nanodroplets for 30 seconds at 75 MPa, which exploded the bacterial body and completely sterilized. Eighteen MPa damaged the bacterial surface, causing peptidoglycan leakage. S. aureus was repaired and survives in this state. In contrast, in Gram-negative bacteria, nanodroplets with 18 MPa penetrated some biofilm-forming bacteria but did not hit all of them, and the viable count was not significantly reduced. Although all three bacterial species were completely sterilized at 75 MPa, the disinfectant effect was affected by the biomass of the biofilm formed. In summary, our findings prove that nanodroplets at 18 MPa on the bacterial surface were ineffective in killing bacteria, whereas at 75 MPa, all four bacterial species were completely sterilized. The disinfection mechanism involved a high-velocity collision of nanodroplets with the bacteria, physically destroying them. Our results showed that disinfection using this technology could be an innovative method that is completely different from existing disinfection techniques. IMPORTANCE: Although existing disinfection techniques demonstrate bactericidal effects through chemical reactions, concerns regarding human toxicity and environmental contamination have been raised. To the best of our knowledge, this study is the first in the world to reveal that the use of this technology, with nanodroplets of less than 100 nm, can destroy and sterilize bacterial cells by colliding with biofilm-forming bacteria at 75 MPa. Furthermore, because this technology uses only water, it can solve the problems of human toxicity and environmental contamination caused by existing disinfection techniques. Because of its minimal water usage, it can be employed for sanitation worldwide without being limited to specific regions. Our report proposes an unprecedented physical disinfection approach that utilizes a high-speed nanodroplet generation technology.

Objective: Many fluoroquinolones, such as ciprofloxacin, are used clinically. We investigated the relationship between resistance acquisition and exposure duration in each drug through the exposure of fluoroquinolone to Escherichia coli clinical isolates in vitro. Methods: Eleven E. coli clinical isolates were exposed to each fluoroquinolone, ie, ciprofloxacin, levofloxacin, sitafloxacin, garenoxacin, and lascufloxacin, with the concentration of the mutant selection window for 5 days; these procedures were repeated 5-times. In addition, the DNA sequence in the quinolone-resistance determining region (QRDR) and the expression level in the drug efflux pump acrA were analyzed to determine the resistance mechanism. Results: Although resistant strains were not detected after 5 to 10 days of exposure to fluoroquinolone, after 25 days of exposure to ciprofloxacin and levofloxacin, 100% and 45% of isolates acquired resistance, respectively. Due to 25 days of exposure to sitafloxacin, garenoxacin, and lascufloxacin, MIC measurement was elevated 2- to 4096-fold for those of the parental strain, and the cross-resistance rate to levofloxacin was 72%, 54%, and 27%, respectively. In strains with high fluoroquinolone resistance, acrA overexpression was observed in addition to QRDR mutation. Conclusion: In our findings, fluoroquinolone resistance was not observed in the E. coli strain after 5- to 10-days of exposure. However, resistance acquisition was detected frequently after 15- to 25-days of exposure. Among fluoroquinolones, lascufloxacn had the least impact on the resistance acquisition in E. coli. Plain Language Summary: The need to combat quinolone-resistant E. coli is urgent. The resistance acquisition tendency of E. coli varies according to the drug used. After 25 days of exposure, 45% of E. coli showed levofloxacin resistance. Lascufloxacin may have the lowest influence on resistance acquisition to regular E. coli.

Postbiotics are increasingly incorporated into functional foods and supplements due to their potential health benefits, particularly immune modulation. However, the mechanisms by which these products influence antiviral immunity remain incompletely understood. Type I interferons, especially interferon-α (IFN-α), are central mediators of early antiviral defense, acting primarily through the activation of plasmacytoid dendritic cells (pDCs). Five commercially available postbiotic products containing heat-killed bacterial strains were evaluated for their ability to stimulate pDCs and induce IFN-α production. Bacterial uptake by pDCs was analyzed using confocal microscopy with Z-stack imaging, and IFN-α levels were quantified by ELISA. Among the tested strains, only Lactococcus lactis strain Plasma (LC-Plasma) demonstrated significant internalization by pDCs and induced measurable IFN-α production (73.8 ± 2.5 pg/mL) at the recommended daily dose. This effect was not observed with other strains, even at higher bacterial loads (up to 1 × 10¹¹ cells). Z-stack imaging confirmed that LC-Plasma was actively phagocytosed by pDCs, whereas other strains, such as L. paracasei MCC1849, adhered to the cell surface without internalization. The pDC concentration used in the assay approximated physiological levels in human blood. Notably, the IFN-α level induced by LC-Plasma exceeded that reported in the serum of hospitalized COVID-19 patients. L. lactis strain Plasma uniquely activates pDCs and induces IFN-α production under physiologically relevant conditions, distinguishing it from other postbiotic strains. These findings suggest that LC-Plasma may serve as a functional postbiotic with the potential to enhance antiviral immunity and mitigate disease severity.