The Drug-Specific Propensity Regarding the Acquisition of Fluoroquinolone Resistance in Escherichia coli: An in vitro Challenge and DNA Mutation Analysis.

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.

Cefmetazole Resistance Mechanism for Escherichia Coli Including ESBL-Producing Strains.

Purpose: Cefmetazole (CMZ), a cephamycin antibiotic, is primarily used as a definitive therapy for Extended Spectrum ß-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.

The Risk of Emerging Resistance to Trimethoprim/Sulfamethoxazole in Staphylococcus aureus.

Objective: Due to the spread of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), the demand for trimethoprim/sulfamethoxazole (SXT) is increasing in the world. It is not clear whether the resistant strain emerges by overuse of SXT. We investigated here the emergent risk of the SXT-resistant mutant in S. aureus by an in vitro SXT exposure experiment. Methods: A total of 40 S. aureus clinical isolates (20 MSSA and 20 MRSA isolates) were exposed to sub-MIC of SXT for consecutive days, and MIC of SXT was determined every day. In addition, the dfrB DNA sequencing was performed to detect the mutation in the SXT-resistant strain. Results: The SXT-resistant strain began to emerge on the eighth day and accounted for 45% (18/40 clinical isolates) after 14 days. Moreover, one half of these resistant strains showed F98Y mutation in DfrB to retain SXT-resistance without selective pressure. Conclusion: The emergent risk was SXT exposure of 14 days or more.

Overproduction of Chromosomal ampC ß-Lactamase Gene Maintains Resistance to Cefazolin in Escherichia coli Isolates.

Cefazolin, an active in vitro agent against Escherichia coli, is used to treat urinary and biliary tract infections. Cefazolin is used widely as an antibiotic, and the increase in the emergence of cefazolin-resistant E. coli in many countries is a major concern. We investigated the changes in the susceptibility of E. coli clinical isolates to cefazolin following exposure. A total of 88.9% (16/18 strains) of the strains acquired resistance to cefazolin. All strains with an MIC to cefazolin of 2 µg/mL became resistant. The expression of chromosomal ampC (c-ampC) increased up to 209.1-fold in the resistant strains. Moreover, 11 of the 16 E. coli strains (68.8%) that acquired cefazolin resistance maintained the resistant phenotype after subculture in cefazolin-free medium. Therefore, the acquisition and maintenance of cefazolin resistance in E. coli strains were associated with the overexpression of c-ampC. Mutations in the c-ampC attenuator regions are likely to be maintained and are one of the key factors contributing to the increase in the number of cefazolin-resistant E. coli worldwide. IMPORTANCE This study is the first to demonstrate that mutations in the chromosomal-ampC 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.

Comparison of Pharmaceutical Characteristics between Brand-Name Meropenem and Its Generics.

This study aimed to provide comparative information of pharmaceutical properties, including particle morphology and distribution uniformity, solubility, presence of residual solvent and insoluble particles, and antimicrobial activities, between brand-name meropenem (Mepem®, BNM) and its six generic products (GPs A-F) marketed in China. Particles of GP-A and -C in dry powder had similar diameters of BNM, while other GPs were larger. Only BNM and GP-A were completely dissolved within 100 s in the lab condition. No insoluble particles >25 µm in diameter were detected in BNM and GP-E. Regarding stability of GPs solutions evaluated by concentration of open-ring metabolites at 6 h and 8 h, BNM showed the lowest open-ringed metabolite concentrates. Residual solvent of acetone detected in one GP showed the maximum value, while ethanol and ethyl acetate were detected both in product E and product F. The concordance rates (%) of minimum inhibitory concentration (MIC) of each generic compared to BNM were 89.5, 85, 87.5, 88, 88.5, and 86.5, respectively, although no significant difference was reached in MIC. Pharmaceutical characteristic differences between the BNM and GPs identified in this study could provide insights into understanding the deviations in the drug manufacturing processes of generic drugs.

In vitro Tolerability of Biofilm-Forming Trimethoprim-/Sulfamethoxazole-Resistant Small Colony Variants of Staphylococcus aureus Against Various Antimicrobial Agents.

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.

Mutant selection window of disinfectants for Staphylococcus aureus and Pseudomonas aeruginosa.

OBJECTIVES: The aim of this study was to determine the mutant selection window (MSW) of various disinfectants against Staphylococcus aureus and Pseudomonas aeruginosa clinical isolates to determine the tendency of these strains to acquire resistance to disinfectants. METHODS: A total of 60 S. aureus isolates [30 methicillin-resistant S. aureus (MRSA) and 30 methicillin-susceptible S. aureus (MSSA)] and 30 P. aeruginosa, including 2 multidrug-resistant P. aeruginosa (MDRP), were collected in Japan. The minimum inhibitory concentrations (MICs) and mutant prevention concentrations (MPCs) of disinfectants, including sodium hypochlorite (NaOCl), against these strains were established to determine the MSW. RESULTS: The MSW50, MSW80 and MSW100 for sodium hypochlorite against S. aureus and P. aeruginosa were 4×, 8× and 16× MIC, respectively. Strains surviving in the sodium hypochlorite MSW remained at a concentration of ≤0.3% (≤3072µg/mL). CONCLUSIONS: This is the first evaluation of the bactericidal activity against S. aureus and P. aeruginosa strains surviving in the MSW of disinfectants. Environmental disinfection at low concentrations of sodium hypochlorite does not kill micro-organisms. Proper use of sodium hypochlorite shows a bactericidal effect against various pathogenic micro-organisms and is inexpensive, making it frequently used globally.

An α-Lipoic acid derivative, and anti-ROS agent, prevents the acquisition of multi-drug resistance in clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most common causes of nosocomial infections, and its multi-drug resistance has been a serious problem worldwide. The aim of this study was to evaluate whether exposure to piperacillin and reactive oxygen species (ROS) could lead to multi-drug resistance for clinical isolates of P. aeruginosa. The inhibition of this acquired resistance by the anti-ROS agent was also examined. In vitro inducement of multi-drug resistance was performed against 20 clinical isolates. These strains were incubated for 24 h and transferred 5 times after being exposed to 1 mM H2O2 (ROS) in addition to a sub-MIC of piperacillin by the agar dilution method. Each MIC of piperacillin and levofloxacin was determined. As the mechanism of levofloxacin resistance, mutation of QRDR was investigated. The expression level of genes encoding efflux pumps; mexA, mexY, mexC, and D2 porin; oprD were determined by real-time PCR. Multi-resistance to both piperacillin and levofloxacin was induced with 4 of 20 strains (20%). No amino acid change was confirmed in QRDR. These strains showed overexpression of mexA, mexY, mexC, and another one showed decrease of oprD expression. Resistance development in 4 strains was inhibited by the same method including the anti-ROS agent, sodium zinc histidine dithiooctanamide (DHL-His-Zn). In conclusion, stimulation by ROS promoted acquisition of multi-drug resistance in 20% of isolates of P. aeruginosa, and DHL-His-Zn completely inhibited this acquisition of resistance. Therefore, this anti-ROS agent may be useful to assist antimicrobial chemotherapy by preventing multi-drug resistance.

Screening method for trimethoprim/sulfamethoxazole-resistant small colony variants of Staphylococcus aureus.

OBJECTIVES: Trimethoprim/sulfamethoxazole (SXT) is used to treat Staphylococcus aureus infections. However, the effect of treatment with SXT is sometimes not sufficient and there are patients whose treatment has to be prolonged. There are few reports of isolated strains of SXT-resistant S. aureus, but it is possible that some resistant strains cannot be detected by current testing methods We have therefore developed a tool to identify these resistant strains. METHODS: The mutant selection window (MSW) of SXT for 40 clinical isolates of S. aureus, including 20 methicillin-resistant S. aureus (MRSA), was determined. The optimum concentration of SXT and thymidine in agar for detecting SXT-resistant small colony variants (SCVs) of S. aureus was investigated. RESULTS: The MSW50 and MSW90 of SXT, presented as a multiple of the minimum inhibitory concentration (MIC), were 16× MIC and >256× MIC, respectively. SCVs were detected within the MSW in 32 (80%) of the 40 clinical isolates studied. To maintain the morphology of SCVs, the most suitable concentrations of SXT and thymidine for screening were 4mg/L and 0.01µg/mL, respectively. All 32 SCVs were resistant to SXT (MIC >32mg/L). The sensitivity and specificity of this screening method was 100% and 88.9%, respectively. CONCLUSIONS: SXT-resistant SCVs are not usually detected by routine laboratory tests performed in hospitals. However, the screening test described here can easily distinguish SXT-resistant SCVs among S. aureus isolated from specimens. This newly developed screening test could become an important tool to prevent inappropriate use of SXT.

Mutant selection window of four quinolone antibiotics against clinical isolates of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis.

Community-acquired pneumonia and otitis media are caused by several bacterial species, including Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. For the treatment of these diseases, various quinolones are frequently used. We determined the mutant prevention concentration (MPC) of four quinolones, levofloxacin, sitafloxacin, tosufloxacin, and garenoxacin, using 92 clinical isolates and evaluated each mutant selection window (MSW). Furthermore, the DNA sequence of the quinolone resistance-determining region (QRDR) for the resistant mutant selected based on the MSW was determined. The MIC90 and MPC90 of levofloxacin were 0.781 µg/mL and 6.250 µg/mL for S. pneumoniae and 0.049 µg/mL and 1.563 µg/mL for M. catarrhalis and were higher than those for the other three quinolones. In addition, 5 strains of 30 S. pneumoniae (16.7%) selected based on the MSW of levofloxacin acquired resistance to only levofloxacin. In these 5 strains, a mutation of gyrA and/or parC was detected. In this study, no resistant mutant was selected in the MSW of any of the other three quinolones. On the other hand, clinical isolates of H. influenzae showed no resistance by all quinolone exposure. Finally, The MIC value and the mutation status in the QRDR did not change after 14 passages in antibiotic-free medium. In conclusion, our findings suggest that the increased use of levofloxacin may contribute to the increased quinolone-resistance of S. pneumoniae and M. catarrhalis.

Antimicrobial efficacy of combined clarithromycin plus daptomycin against biofilms-formed methicillin-resistant Staphylococcus aureus on titanium medical devices.

In vitro efficacy of combined eradication therapy with clarithromycin and daptomycin against biofilm-formed methicillin-resistant Staphylococcus aureus on the orthopedic titanium devices was evaluated. The bactericidal effect of this antibiotic was investigated by a re-culture test, the scanning electron microscopy, and fluorescence microscopy using a double-staining dyes. Clarithromycin decreased the amount to half in 24 h. Although MRSA biofilms were not eradicated with clarithromycin or daptomycin alone, clarithromycin combined with daptomycin was useful to sterilize titanium devices within 72 h. This in vitro study showed that combined treatment with clarithromycin plus daptomycin is useful to eradicate staphylococcal biofilms formed on orthopedic devices.

[Epidemiological study of influenza-like illness under enhanced surveillance in Saku, Nagano Prefecture, outpatients during the 2008-09 influenza season].

Many influenza-like illness (ILI) outpatients visit healthcare facilities such as internal medicine and pediatric clinics every year. In Japan, however, ILI is reported only by sentinel healthcare facilities. We studied the number of ILI subjects visiting sentinel and non-sentinel healthcare facilities during the 2008-09 season in Saku, Nagano prefecture, obtaining the numbers of cases from sentinel and non-sentinel facilities. Most ILI subjects visited internal and pediatric facilities, and some visited otorhinolaryngological clinics not included as sentinel sites. We also estimated the total number of influenza cases based on data from sentinel facilities and total surveyed facilities, including non-sentinel. We divided facilities into hospitals with pediatrics, pediatric clinics, internal medicine and pediatric clinics, hospitals and clinics with internal medicine but no pediatrics, and otorhinolaryngological clinics. Estimated sentinel-site ILI cases was 2862, including 1020 for hospitals with pediatrics and 1,674 for clinics with internal medicine and pediatrics. The estimated number of ILI cases from total facilities surveyed was significantly lower, at 503 for hospitals with pediatrics, and 741 for clinics with internal medicine and pediatrics. Estimated ILI cases from categories not including sentinel sites were 967 for hospitals and clinics with internal medicine but no pediatrics, and 71 for otorhinolaryngological clinics. The estimated number of total ILI cases differed by 18.5%, depending on facility categories. This indicates that more detailed analysis is needed to accurately estimate ILI cases.

[Epidemiological study of health impact on influenza outpatients during 2008-2009 influenza season, Shonai, Yamagata].

To make up for the lack of data on influenza-like illness (ILI), we studied patterns among 6,828 ILI patients seen at 34 healthcare facilities during the 2008-2009 influenza season in Shonai, Yamagata, Japan. Healthcare facilities were classified into 1) hospitals, 2) pediatric clinics, 3) internal medicine and pediatric clinics, 4) internal medicine clinics, and 5) others. The majority went to hospitals, but the highest percentage in all outpatient visits was 13.7% seen at pediatric clinics during the peak epidemiological week. Based on estimated incidence and hospitalization data for pandemic (H1N1) 2009 from Ministry of Health, Labour and Welfare, Japan, we estimated the number of ILI patients at 59,600-89,400 and the number hospitalized for pandemic (H1N1) 2009 at 895-2,240 in Shonai. If those with ILI follow the same consultation patterns as outpatients in the 2008/09 influenza season, this indicates an estimated 23,800-35,700 with ILI will go to hospitals and 20,040-30,060 to pediatric clinics during the H1N1 pandemic. This in turns means that an urgent need will arise for appropriate measures reducing this potentially huge burden during pandemic (H1N1) 2009 outbreak in Japan.

Magnetic nanoparticle-supported crown ethers.

Magnetic nanoparticle (MNP)-supported crown ethers were successfully prepared and evaluated as catalysts for solid-liquid phase-transfer reactions; the catalytic activities of the MNP-supported crown ethers were not inferior to those of non-supported crown ethers; after the reactions, the catalysts could be readily separated using an external magnet and reused without significant loss of catalytic efficiency.

Magnetically separable phase-transfer catalysts.

Magnetic nanoparticles-supported quaternary ammonium and phosphonium salts were prepared and evaluated as phase-transfer catalysts. Some of them exhibited good activities that were comparable to that of tetra-n-butylammonium iodide. The catalysts were readily separated using an external magnet and reusable without significant loss of their catalytic efficiency.

Detection of Legionella pneumophila serogroup 7 strain from bathwater samples in a Japanese hospital.

Hospital-acquired legionellosis is one of the serious problems in nosocomial infection. For risk assessment of nosocomial Legionella infection, we surveyed samples from bathrooms for public use in three hospitals and two nursing homes to determine whether Legionella pneumophila was present. A total of 70 hot bathwater samples and samples wiped from bathtubs were collected at 1-h intervals. Fifteen shower-water and 15 inner-head samples were obtained at the start of a bath. Water samples were cultured using the Legionella spp. selective medium, and discrimination between L. pneumophila and other Legionella spp. was performed by PCR analysis. L. pneumophila serogroup 7 was detected in 1 bathwater and 1 wiped sample, both of which were collected 1 h after daily use from the same bathtub in a hospital. However, L. pneumophila SG7 was not detected in any other samples. Furthermore, the concentrations of free residual chlorine in most bath- and shower-water samples were lower than 0.1 mg/l. These results suggest that L. pneumophila has become a potential pathogen for nosocomial infections in public-type hospital baths. From the point of view of an infection-control program, it might be advisable to hold the concentration of free residual chlorine at 0.2-0.4 mg/l, which is generally required for public baths in Japan.

Synthesis of a novel photoresponsive axially chiral phosphine ligand containing an arylazo group and its application to palladium-catalyzed asymmetric allylic alkylation.

Novel photoresponsive axially chiral monophosphine ligands containing azobenzene moiety were prepared and applied to a palladium-catalyzed allylic alkylation. The reaction of rac-1,3-diphenyl-2-propenyl acetate gave the alkylated products with up to 90% enantiomeric excess. The ligand exhibited a trans to cis photoisomerization upon irradiation with UV light.

Exo-selective asymmetric Diels-Alder reaction of acrylate ester.

Diels-Alder reaction of 2-acryloyloxy-2'-(2-pyridylazo)-1,1'-binaphthyl with cyclopentadiene in the presence of an equimolar amount of tin(IV) chloride showed unusual exo-selectivity with a high degree of diastereofacial differentiation.

Asymmetric alpha-alkylation of phenylacetates using 2-alkylamino-2'-hydroxy-1,1'-binaphthyls as chiral auxiliaries.

alpha-Alkylation of 2-phenylacetate ester of 2-alkylamino-2'-hydroxy-1,1'-binaphthyls with various alkyl iodides proceeded with good stereoselectivities. An intramolecular hydrogen bonding between N-H group and the carbonyl oxygen seemed to play an important role for asymmetric induction. The auxiliary was also applicable to Diels-Alder reaction of an acrylate.