In Vivo Pharmacodynamic Study of Cefiderocol, a Novel Parenteral Siderophore Cephalosporin, in Murine Thigh and Lung Infection Models.

The pharmacokinetic (PK) and pharmacodynamic (PD) parameters which correlated with the in vivo efficacy of cefiderocol were evaluated using neutropenic murine thigh and lung infection models in which the infections were caused by a variety of Gram-negative bacilli. The dose fractionation study using the thigh infection model in which the infection was caused by Pseudomonas aeruginosa showed that the cumulative percentage of a 24-h period that the free drug concentration in plasma exceeds the MIC (%fT>MIC) rather than the free peak level divided by the MIC (fCmax/MIC) and the area under the free concentration-time curve over 24 h divided by the MIC (fAUC/MIC) was the PK/PD parameter that best correlated with efficacy. The study with multiple carbapenem-resistant strains revealed that the %fT>MIC determined in iron-depleted cation-adjusted Mueller-Hinton broth (ID-CAMHB) better reflected the in vivo efficacy of cefiderocol than the %fT>MIC determined in cation-adjusted Mueller-Hinton broth (CAMHB). The mean %fT>MIC of cefiderocol required for a 1-log10 reduction against 10 strains of Enterobacteriaceae and 3 strains of Pseudomonas aeruginosa in the thigh infection models were 73.3% and 77.2%, respectively. The mean %fT>MIC for Enterobacteriaceae, P. aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia in the lung infection model were 64.4%, 70.3%, 88.1%, and 53.9%, respectively. These results indicate that cefiderocol has potent efficacy against Gram-negative bacilli, including carbapenem-resistant strains, irrespective of the bacterial species, in neutropenic thigh and lung infection models and that the in vivo efficacy correlated with the in vitro MIC under iron-deficient conditions.

Stability and low induction propensity of cefiderocol against chromosomal AmpC ß-lactamases of Pseudomonas aeruginosa and Enterobacter cloacae.

Objectives: The siderophore cephalosporin cefiderocol possesses in vitro activity against MDR Gram-negative bacteria. The stability of cefiderocol against serine- and metallo-type carbapenemases has been reported previously, but little is known about how cefiderocol interacts with chromosomal AmpC ß-lactamases. We investigated a number of features of cefiderocol, namely antibacterial activity against AmpC overproducers, stability against AmpC ß-lactamases and propensity for AmpC induction using Pseudomonas aeruginosa and Enterobacter cloacae. Methods: MICs were determined by broth microdilution according to CLSI guidelines. The MIC of cefiderocol was determined in iron-depleted CAMHB. Hydrolysis of the antibiotics was determined by monitoring the changes in the absorbance in the presence of AmpC ß-lactamase, and AmpC induction was evaluated by double disc diffusion and nitrocefin degradation assays. Results: The MICs of ceftazidime and cefepime for PAO1 increased 4- to 16-fold with inactivation of either ampD or dacB, whereas cefiderocol MICs were little affected by these inactivations (<2-fold increase). Cefiderocol has 17- and 740-fold lower affinity (higher Ki) to AmpCs of P. aeruginosa SR24-12 and E. cloacae P99, respectively, compared with ceftazidime. Both disc diffusion and nitrocefin degradation assays indicated that cefiderocol did not induce AmpC ß-lactamases of P. aeruginosa PAO1 and ATCC 27853 and E. cloacae ATCC 13047, whereas imipenem did. Conclusions: Cefiderocol showed in vitro activity against the AmpC-overproducing strains, low affinity for chromosomal AmpC ß-lactamases, and a low propensity of temporal induction of AmpC ß-lactamases of P. aeruginosa and E. cloacae. These features relating to chromosomal AmpC could explain the potent antibacterial activity of cefiderocol against drug-resistant strains producing AmpC ß-lactamases.

IMP-27, a Unique Metallo-ß-Lactamase Identified in Geographically Distinct Isolates of Proteus mirabilis.

A novel metallo-ß-lactamase gene, blaIMP-27, was identified in unrelated Proteus mirabilis isolates from two geographically distinct locations in the United States. Both isolates harbor blaIMP-27 as part of the first gene cassette in a class 2 integron. Antimicrobial susceptibility testing indicated susceptibility to aztreonam, piperacillin-tazobactam, and ceftazidime but resistance to ertapenem. However, hydrolysis assays indicated that ceftazidime was a substrate for IMP-27.

In Vitro Antimicrobial Activity of a Siderophore Cephalosporin, S-649266, against Enterobacteriaceae Clinical Isolates, Including Carbapenem-Resistant Strains.

S-649266 is a novel siderophore cephalosporin antibiotic with a catechol moiety on the 3-position side chain. Two sets of clinical isolate collections were used to evaluate the antimicrobial activity of S-649266 against Enterobacteriaceae. These sets included 617 global isolates collected between 2009 and 2011 and 233 ß-lactamase-identified isolates, including 47 KPC-, 49 NDM-, 12 VIM-, and 8 IMP-producers. The MIC90 values of S-649266 against the first set of Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, Enterobacter aerogenes, and Enterobacter cloacae isolates were all ≤1 µg/ml, and there were only 8 isolates (1.3%) among these 617 clinical isolates with MIC values of ≥8 µg/ml. In the second set, the MIC values of S-649266 were ≤4 µg/ml against 109 strains among 116 KPC-producing and class B (metallo) carbapenemase-producing strains. In addition, S-649266 showed MIC values of ≤2 µg/ml against each of the 13 strains that produced other types of carbapenemases such as SME, NMC, and OXA-48. The mechanisms of the decreased susceptibility of 7 class B carbapenemase-producing strains with MIC values of ≥16 µg/ml are uncertain. This is the first report to demonstrate that S-649266, a novel siderophore cephalosporin, has significant antimicrobial activity against Enterobacteriaceae, including strains that produce carbapenemases such as KPC and NDM-1.

Stability of Novel Siderophore Cephalosporin S-649266 against Clinically Relevant Carbapenemases.

To better understand the antibacterial activity of S-649266 against carbapenemase producers, its stability against clinically relevant carbapenemases was investigated. The catalytic efficiencies (kcat/Km) of IMP-1, VIM-2, and L1 for S-649266 were 0.0048, 0.0050, and 0.024 µM(-1) s(-1), respectively, which were more than 260-fold lower than that for meropenem. Only slight hydrolysis of S-649266 against KPC-3 was observed. NDM-1 hydrolyzed meropenem 3-fold faster than S-649266 at 200 µM.

Comparison of the risk of acquiring in vitro resistance to doripenem and tazobactam/piperacillin by CTX-M-15-producing Escherichia coli.

To compare the risk of acquiring in vitro resistance between doripenem and tazobactam/piperacillin by CTX-M-15-producing Escherichia coli, the in vitro frequency of resistance was determined. Four strains carrying multiple ß-lactamases such as blaOXA-1 or blaCTX-M-27 as well as blaCTX-M-15 and blaTEM-1 were used. No resistant colonies appeared on doripenem-containing plates, whereas resistant colonies were obtained from three of four test strains against tazobactam/piperacillin using agar plate containing 8- to 16-fold MIC of each drug. These three acquired tazobactam/piperacillin-resistant strains were not cross-resistant to doripenem, and they showed 1.9- to 3.1-fold higher piperacillin-hydrolysis activity compared to those of each parent strain. The change of each ß-lactamase mRNA expression measured by real-time PCR varied among three resistant strains. One of three tazobactam/piperacillin-resistant strains with less susceptibility to ceftazidime overexpressed both blaCTX-M-15 and blaTEM-1, and the other two strains showed higher mRNA expression of either blaTEM-1 or blaOXA-1. These results demonstrate that multiple ß-lactamases carried by CTX-M-15-producing E. coli contributed to the resistance to tazobactam/piperacillin. On the other hand, these resistant strains maintained susceptibility to doripenem. The risk of acquiring in vitro resistance to doripenem by CTX-M-15-producing E. coli seems to be lower than that to tazobactam/piperacillin.

Prevalence and molecular characterization of CTX-M extended-spectrum ß-lactamase-producing Escherichia coli from 2000 to 2010 in Japan.

The prevalence of extended-spectrum ß-lactamase (ESBL) in Enterobacteriaceae has been increasing worldwide. The aims of this study were to determine the prevalence of ESBLs among clinical isolates of Escherichia coli obtained from 2000 to 2010 in Japan, and to characterize the sequence type (ST) and antimicrobial susceptibility of the bla(CTX-M)-carrying strains. The genes for ß-lactamases were determined by conventional PCR and sequencing, and the antimicrobial susceptibility test was performed by the broth microdilution method. Among the 948 strains, 35 were judged as ESBL-positive strains. The positive rates ranged from 0.6% to 3.9% until 2008, but surged to 10.3% in 2010. Thirty-three of them carried bla(CTX-M), but all were negative for ESBL-type bla(TEM) and bla(SHV). bla(CTX-M-14) was the most prevalent (18/33) among bla(CTX-M)-carrying strains, followed by bla(CTX-M-15) (7/33) of which five were isolated in 2008 and 2010. Additionally, bla(CTX-M-27) appeared in 2010 for the first time in this study and accounted for more than a third of the bla(CTX-M)-carrying strains. From the MLST analysis, ST131 known as a world pandemic clone, has been predominantly isolated since 2006. The major types of ESBLs carried by ST131 strains clearly shifted from bla(CTX-M-14) to bla(CTX-M-15) and/or bla(CTX-M-27) between 2006 and 2010. Most of these isolates were still susceptible to doripenem, latamoxef (moxalactam), flomoxef and cefmetazole. Our results suggest that a change of the dominant type of ESBL among Enterobacteriaceae is currently in progress in Japan, and therefore further periodic surveillance is needed.

Development of an in vitro compound screening system that replicate the in vivo spine phenotype of idiopathic ASD model mice.

Autism Spectrum Disorder (ASD) is a developmental condition characterized by core symptoms including social difficulties, repetitive behaviors, and sensory abnormalities. Aberrant morphology of dendritic spines within the cortex has been documented in genetic disorders associated with ASD and ASD-like traits. We hypothesized that compounds that ameliorate abnormalities in spine dynamics might have the potential to ameliorate core symptoms of ASD. Because the morphology of the spine is influenced by signal inputs from other neurons and various molecular interactions, conventional single-molecule targeted drug discovery methods may not suffice in identifying compounds capable of ameliorating spine morphology abnormalities. In this study, we focused on spine phenotypes in the cortex using BTBR T + Itpr3 tf /J (BTBR) mice, which have been used as a model for idiopathic ASD in various studies. We established an in vitro compound screening system using primary cultured neurons from BTBR mice to faithfully represent the spine phenotype. The compound library mainly comprised substances with known target molecules and established safety profiles, including those approved or validated through human safety studies. Following screening of this specialized library containing 181 compounds, we identified 15 confirmed hit compounds. The molecular targets of these hit compounds were largely focused on the 5-hydroxytryptamine receptor (5-HTR). Furthermore, both 5-HT1AR agonist and 5-HT3R antagonist were common functional profiles in hit compounds. Vortioxetine, possessing dual attributes as a 5-HT1AR agonist and 5-HT3R antagonist, was administered to BTBR mice once daily for a period of 7 days. This intervention not only ameliorated their spine phenotype but also alleviated their social behavior abnormality. These results of vortioxetine supports the usefulness of a spine phenotype-based assay system as a potent drug discovery platform targeting ASD core symptoms.

The multidrug efflux pump MdtEF protects against nitrosative damage during the anaerobic respiration in Escherichia coli.

Drug efflux represents an important protection mechanism in bacteria to withstand antibiotics and environmental toxic substances. Efflux genes constitute 6-18% of all transporters in bacterial genomes, yet the expression and functions of only a handful of them have been studied. Among the 20 efflux genes encoded in the Escherichia coli K-12 genome, only the AcrAB-TolC system is constitutively expressed. The expression, activities, and physiological functions of the remaining efflux genes are poorly understood. In this study we identified a dramatic up-regulation of an additional efflux pump, MdtEF, under the anaerobic growth condition of E. coli, which is independent of antibiotic exposure. We found that expression of MdtEF is up-regulated more than 20-fold under anaerobic conditions by the global transcription factor ArcA, resulting in increased efflux activity and enhanced drug tolerance in anaerobically grown E. coli. Cells lacking mdtEF display a significantly decreased survival rate under the condition of anaerobic respiration of nitrate. Deletion of the genes responsible for the biosynthesis of indole, tnaAB, or replacing nitrate with fumarate as the terminal electron acceptor during the anaerobic respiration restores the decreased survival of ΔmdtEF cells. Moreover, ΔmdtEF cells are susceptible to indole nitrosative derivatives, a class of toxic byproducts formed and accumulated within E. coli when the bacterium respires nitrate under anaerobic conditions. Taken together, we conclude that the multidrug efflux pump MdtEF is up-regulated during the anaerobic physiology of E. coli to protect the bacterium from nitrosative damage through expelling the nitrosyl indole derivatives out of the cells.

Roles of Salmonella multidrug efflux pumps in tigecycline resistance.

OBJECTIVES: salmonella enterica strains exhibiting decreased susceptibility to tigecycline have been reported. In this study, we sought to elucidate the roles of Salmonella multidrug efflux pumps and AcrAB regulators in tigecycline resistance. METHODS: we examined the involvement of multidrug efflux pumps and AcrAB regulators in resistance to tigecycline and other glycylcyclines by determining the MICs of the drugs for Salmonella multidrug efflux pump and AcrAB regulator-overproducing or -deleted strains. Strains of S. enterica serovar Typhimurium derived from the wild-type strain ATCC 14028s were used in this study. RESULTS: a plasmid carrying the tet gene conferred resistance to 9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline ('DMG-DMDOT') minocycline, doxycycline and tetracycline, but does not affect tigecycline resistance. Deletion of acrAB resulted in strains with significantly increased susceptibility to tigecycline and other glycylcyclines. Plasmids carrying the acrAB or acrEF gene restored increased susceptibility of the acrAB-deleted mutant to all tested compounds. Deletion of ramA, a positive regulator of acrAB, slightly increased susceptibility to tigecycline. Overexpression of ramA and deletion of ramR, a repressor of ramA, resulted in decreased susceptibility to all tested compounds. This phenotype, modulated by ramA or ramR, was not observed in the acrB-deleted background. CONCLUSIONS: AcrAB and AcrEF confer resistance to tigecycline and tetracycline derivatives in Salmonella. RamA and RamR are also involved in resistance to tigecycline in an AcrAB-dependent manner.

TolC dependency of multidrug efflux systems in Salmonella enterica serovar Typhimurium.

OBJECTIVES: TolC is a major outer membrane channel and it plays an important role in the excretion of a wide range of molecules, including antibiotics. A recent study has shown that Salmonella enterica serovar Typhimurium has nine functional drug efflux pumps; however, the TolC dependency of these efflux pumps remains to be studied in detail. The aim of this study was to investigate the TolC dependency of multidrug efflux pumps in this organism. METHODS: All genes encoding the drug efflux systems were cloned into the pUC118 vector. Constructed plasmids were transformed into DeltaacrB and DeltatolC mutants of S. enterica serovar Typhimurium ATCC 14028s, and then the drug susceptibilities of these transformants were determined. RESULTS: Plasmids carrying the acrAB, acrD, acrEF, mdsAB, mdtABC, emrAB or macAB genes did not confer resistance to the tolC mutant, whereas they conferred drug resistance to the acrB mutant. Only three plasmids carrying mdsABC, mdfA or mdtK conferred resistance to the tolC mutant. CONCLUSIONS: TolC is required for the function of seven drug efflux systems (AcrAB, AcrD, AcrEF, MdsAB, MdtABC, EmrAB and MacAB) in S. enterica serovar Typhimurium.

Mechanism of resistance and antibacterial susceptibility in extended-spectrum ß-lactamase phenotype Klebsiella pneumoniae and Klebsiella oxytoca isolated between 2000 and 2010 in Japan.

Clinical isolates of Klebsiella pneumoniae and Klebsiella oxytoca collected from 20 Japanese medical facilities between 2000 and 2010 were analysed to evaluate the mechanisms of resistance and antibacterial susceptibilities to 14 antimicrobials. Overall, eight of 484 (1.6%) K. pneumoniae and 19 of 359 (5.3%) K. oxytoca were determined to be extended-spectrum ß-lactamase (ESBL) phenotype isolates, and the identified ESBLs amongst the K. pneumoniae isolates were CTX-M-2, -3, -14 and -15, and SHV-12. In contrast, overproduction of chromosomal ß-lactamase OXY-2, which was due to a distinct mutation at the - 10 promoter region of this gene, conferred the ESBL phenotype to all the K. oxytoca isolates except one. Based on the Clinical and Laboratory Standards Institute breakpoints, all the ESBL phenotype K. pneumoniae were susceptible to doripenem, flomoxef, moxalactam (latamoxef), cefmetazole and tazobactam/piperacillin, whereas the ESBL phenotype K. oxytoca were susceptible to ceftazidime and ceftibuten in addition to the above, with the exception of tazobactam/piperacillin. Amongst the oral antimicrobials, ceftibuten was relatively effective against both ESBL phenotype Klebsiella species compared with levofloxacin and amoxicillin/clavulanic acid.

AcrB, AcrD, and MdtABC multidrug efflux systems are involved in enterobactin export in Escherichia coli.

Escherichia coli produces the iron-chelating compound enterobactin to enable growth under iron-limiting conditions. After biosynthesis, enterobactin is released from the cell. However, the enterobactin export system is not fully understood. Previous studies have suggested that the outer membrane channel TolC is involved in enterobactin export. There are several multidrug efflux transporters belonging to resistance-nodulation-cell division (RND) family that require interaction with TolC to function. Therefore, several RND transporters may be responsible for enterobactin export. In this study, we investigated whether RND transporters are involved in enterobactin export using deletion mutants of multidrug transporters in E. coli. Single deletions of acrB, acrD, mdtABC, acrEF, or mdtEF did not affect the ability of E. coli to excrete enterobactin, whereas deletion of tolC did affect enterobactin export. We found that multiple deletion of acrB, acrD, and mdtABC resulted in a significant decrease in enterobactin export and that plasmids carrying the acrAB, acrD, or mdtABC genes restored the decrease in enterobactin export exhibited by the ΔacrB acrD mdtABC mutant. These results indicate that AcrB, AcrD, and MdtABC are required for the secretion of enterobactin.