Takumi Sato

Objective: The emergence of resistance to ceftolozane/tazobactam (CTLZ/TAZ) in Pseudomonas aeruginosa poses a significant clinical challenge. This study investigates the mechanisms underlying CTLZ/TAZ resistance in a clinical isolate and examines the role of prior antibiotic exposure. Methods: A TAZ/CTLZ-resistant P. aeruginosa strain (Pa-R) was isolated from a patient with a lung abscess after six weeks of antibiotic therapy. Genetic relatedness between Pa-R and a pre-treatment susceptible strain (Pa-S) was assessed using PCR-based open reading frame typing (POT). Carbapenemase genes were detected via multiplex PCR. Sequencing and expression analyses of ampC, ftsI, oprD, and mex family genes were conducted. RNA interference targeting mexJK was performed to validate its role in resistance. In vitro antimicrobial exposure assays were conducted to evaluate resistance acquisition and the impact of prior ceftazidime (CAZ) exposure. Results: Pa-R and Pa-S were genetically identical and lacked carbapenemase genes. No mutations or overexpression of ampC were observed; however, mexJK expression was approximately 30-fold higher in Pa-R. RNA interference using mexJK-specific dsRNA restored CTLZ/TAZ susceptibility, confirming that high MexJK-OprM expression is the mechanism of resistance. In vitro assays demonstrated that CTLZ/TAZ alone did not induce resistance, whereas prior CAZ exposure led to CTLZ/TAZ resistance accompanied by mexJK overexpression. Conclusion: This study demonstrated that overexpression of the MexJK-OprM efflux pump is a key mechanism underlying TAZ/CTLZ resistance. In addition, prior administration of CAZ contributed to the acquisition of resistance, highlighting the need to consider the risk of resistance acquisition when switching antibiotics. In such cases, the choice of carbapenems should be considered. Plain Language Summary: Ceftolozane/tazobactam -resistant P. aeruginosa strain was isolated from a patient with a lung abscess after six weeks of antibiotic therapy.Overexpression of the MexJK-OprM efflux pump is a key mechanism underlying ceftolozane/tazobactam resistance.The prior administration of ceftazidime contributed to the acquisition of ceftolozane/tazobactam resistance.

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.

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.

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.

Trimethoprim-/sulfamethoxazole-resistant small colony variants (SCVs) of Staphylococcus aureus , which are selected by use of trimethoprim/sulfamethoxazole, are involved in intractable biofilm-forming infection. This study aimed to determine the biofilm formation ability in trimethoprim-/sulfamethoxazole-resistant SCVs of S. aureus and investigate the bactericidal activity of differential antimicrobial agents to its biofilm-forming S. aureus . Between 32 S. aureus wild type (WT) and 32 SCVs selected from its WT, the amount of formed biofilm was compared. Vancomycin, daptomycin, rifampicin, and minocycline were exposed to biofilm-forming S. aureus to determine viable bacterial counts and its susceptibility. The biofilm-producing quantify of SCVs was approximately twice that formed by its WT. Vancomycin and daptomycin reduce 4 logs the bacterial counts of biofilm-forming WT at 24 hours, but did not affect SCVs. Rifampicin and minocycline considerably decreased both WT and SCVs; however, both bacterial counts recovered to an initial number 48 hours later. These survival strains showed resistance to each drug, and rpoB mutation or tet38 mRNA overexpression was confirmed.