In vitro efficacy of humanized regimen of flomoxef against extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae.

This study compared the efficacy of flomoxef with other ß-lactam antibiotics against extended-spectrum ß-lactamases (ESBL)-producing bacteria of clinical relevance. First, the prevalence and ß-lactamase genotypes of ESBL-producing strains among Escherichia coli and Klebsiella pneumoniae isolates collected in Japan from 2004 to 2018 were investigated. High MIC90 values (>64 µg/mL) of ceftriaxone, cefepime, and ceftazidime and low MIC90 values (≤0.06-2 µg/mL) of flomoxef, cefmetazole, and meropenem against both species were observed. Second, a chemostat model was used to analyze the efficacy of humanized regimens of three oxacephem/cephamycin antibiotics (flomoxef, cefmetazole, cefoxitin) and two other antibiotics (meropenem and piperacillin/tazobactam) in suppressing the growth of five ESBL-producing E. coli and two K. pneumoniae strains. Flomoxef, piperacillin/tazobactam, and meropenem showed good bactericidal effects with >4 log10 CFU/mL reduction without bacterial regrowth at 24 h even when the MIC of test isolates was >MIC90. Cefmetazole and cefoxitin resulted in regrowth of test isolates with MIC ≥MIC90 at 24 h. Cefmetazole, cefoxitin, flomoxef, and meropenem showed increased MICs for regrown samples. A clear relationship between the proportion of time that the free drug concentration exceeded the MIC (%fT>MIC) and antibiotic efficacy was found for flomoxef, cefoxitin, and cefmetazole, and flomoxef had the highest %fT>MIC, whereas discrepancies between Clinical and Laboratory Standards Institute breakpoint and bactericidal activity were observed for cefmetazole. Flomoxef was effective in preventing the growth of all ESBL-producing strains, even those with an MIC eight times the MIC90. Thus, flomoxef may be a good alternative to meropenem in context of carbapenems sparing stewardship.

Staphylococcus aureus Penicillin-Binding Protein 2 Can Use Depsi-Lipid II Derived from Vancomycin-Resistant Strains for Cell Wall Synthesis.

Vancomycin-resistant Staphylococcus aureus (S. aureus) (VRSA) uses depsipeptide-containing modified cell-wall precursors for the biosynthesis of peptidoglycan. Transglycosylase is responsible for the polymerization of the peptidoglycan, and the penicillin-binding protein 2 (PBP2) plays a major role in the polymerization among several transglycosylases of wild-type S. aureus. However, it is unclear whether VRSA processes the depsipeptide-containing peptidoglycan precursor by using PBP2. Here, we describe the total synthesis of depsi-lipid I, a cell-wall precursor of VRSA. By using this chemistry, we prepared a depsi-lipid II analogue as substrate for a cell-free transglycosylation system. The reconstituted system revealed that the PBP2 of S. aureus is able to process a depsi-lipid II intermediate as efficiently as its normal substrate. Moreover, the system was successfully used to demonstrate the difference in the mode of action of the two antibiotics moenomycin and vancomycin.

Elucidation of the active conformation of vancomycin dimers with antibacterial activity against vancomycin-resistant bacteria.

Covalently linked vancomycin dimers have attracted a great deal of attention among researchers because of their enhanced antibacterial activity against vancomycin-resistant strains. However, the lack of a clear insight into the mechanisms of action of these dimers hampers rational optimization of their antibacterial potency. Here, we describe the synthesis and antibacterial activity of novel vancomycin dimers with a constrained molecular conformation achieved by two tethers between vancomycin units. Conformational restriction is a useful strategy for studying the relationship between the molecular topology and biological activity of compounds. In this study, two vancomycin units were linked at three distinct positions of the glycopeptide (vancosamine residue (V), C terminus (C), and N terminus (N)) to form two types of novel vancomycin cyclic dimers. Active NC-VV-linked dimers with a stable conformation as indicated by molecular mechanics calculations selectively suppressed the peptidoglycan polymerization reaction of vancomycin-resistant Staphylococcus aureus in vitro. In addition, double-disk diffusion tests indicated that the antibacterial activity of these dimers against vancomycin-resistant enterococci might arise from the inhibition of enzymes responsible for peptidoglycan polymerization. These findings provide a new insight into the biological targets of vancomycin dimers and the conformational requirements for efficient antibacterial activity against vancomycin-resistant strains.

Mode of action of Van-M-02, a novel glycopeptide inhibitor of peptidoglycan synthesis, in vancomycin-resistant bacteria.

Van-M-02, a novel glycopeptide, was revealed to exert potent activities against Gram-positive bacteria, including vancomycin-resistant enterococci (VRE) and vancomycin-resistant Staphylococcus aureus (VRSA). A crude assay system was then used to study the mode of action of Van-M-02 as a peptidoglycan synthesis model of both vancomycin-susceptible and -resistant strains. The results suggested that Van-M-02 inhibits the synthesis of lipid intermediates irrespective of their termini. This inhibitory activity may contribute to the anti-VRE and anti-VRSA activities observed.

Colorimetric dimethyl sulfide sensor using Rhodovulum sulfidophilum cells based on intrinsic pigment conversion by CrtA.

A colorimetric whole-cell sensor for dimethyl sulfide (DMS) was constructed based on the in vivo conversion of intrinsic pigments in response to the analyte. In a marine bacterium, Rhodovulum sulfidophilum, carotenoids are synthesized via the spheroidene pathway. In this pathway, demethylspheroidene, a yellow carotenoid, is converted to spheroidene under catalysis of O-methyltransferase. Spheroidene monooxygenase (CrtA) catalyzes the terminal step of the pathway and converts spheroidene to spheroidenone, a red carotenoid. Here, the CrtA gene in R. sulfidophilum was removed and then reintroduced downstream of the DMS dehydrogenase gene promoter. Using this whole-cell sensor, 3 muM DMS or dimethyl sulfoxide can be detected without adding any color-forming reagent. The ratio of the red spheroidenone to total carotenoids increased, as the DMS concentration was raised to 0.3 mM. Comparison of the signal to the background color indicated a shift in the color coordinate from a yellow to a red hue. An intense signal was obtained with 1-day incubation at a high cell density when sensor cells at the exponential growth phase were used. These results show that the genetically engineered R. sulfidophilum cells can be used to monitor the quality of marine aquacultural environments by the naked eye.

Whole-cell arsenite biosensor using photosynthetic bacterium Rhodovulum sulfidophilum. Rhodovulum sulfidophilum as an arsenite biosensor.

An arsenite biosensor plasmid was constructed in Escherichia coli by inserting the operator/promoter region of the ars operon and the arsR gene from E. coli and the crtA gene, which is responsible for carotenoid synthesis in the photosynthetic bacterium, Rhodovulum sulfidophilum, into the broad-host-range plasmid vector, pRK415. The biosensor plasmid, pSENSE-As, was introduced into a crtA-deleted mutant strain of R. sulfidophilum (CDM2), which is yellow in culture due to its content of spheroiden (SE) and demethylspheroidene (DMSE). CDM2 containing pSENSE-As changed from yellow to red by the addition of arsenite, which caused enzymatic transformation of SE and DMSE to spheroidenone (SO) and demethylspheroidenone (DMSO). Reverse transcriptase PCR analysis showed that the color change depended on transcription of the crtA gene in pSENSE-As. The color change could be clearly recognized with the naked eye at 5 microg/l arsenite. The biosensor strain did not respond to other metals except for bismuth and antimony, which caused significant accumulation of SO and DMSO in the cells at 60 and 600 microg/l, respectively. This biosensor indicates the presence of arsenite with a bacterial color change without the need to add a special reagent or substrate for color development, enabling this pollutant to be monitored in samples by the naked eye in sunlight, even where electricity is not available.

Unusual accumulation of demethylspheroidene in anaerobic-phototrophic growth of crtA-deleted mutants of Rhodovulum sulfidophilum.

Rhodovulum sulfidophilum produces carotenoids in the spheroidene pathway. Spheroidene monooxygenase, CrtA, catalyzes the conversion of spheroidene to spheroidenone. crtA-deleted mutants of R. sulfidophilum did not produce spheroidenone and demethylspheroidenone. In these mutants, the ratio of demethylspheroidene to spheroidene increased with exposure to light. One mutant exhibiting a spheroidene-predominant phenotype did not grow under anaerobic-light conditions and was devoid of bacteriochlorophyll a, even under semiaerobic-light conditions There was no difference in the growth of the mutants under aerobic-dark conditions. These data suggest that demethylspheroidene is important for photosynthesis in R. sulfidophilum.

Comparative study of the N-terminal hydrophilic region in Streptomyces lividans and E. coli FtsY.

Streptomyces lividans FtsY (SlFtsY) was cloned and overexpressed in Escherichia coli. Analysis of the amino acid (aa) sequence showed a concentration of hydrophilic aa's in the N-terminal half region of SlFtsY as observed in that of E. coli FtsY (EcFtsY). However, the length of the hydrophilic region was shorter in SlFtsY than in EcFtsY. Overexpression of SlFtsY in E. coli resulted in growth suppression as in the case of the overexpression of EcFtsY, while growth suppression as a result of the overexpression of the C-terminal half region of SlFtsY was limited. This result suggests that the N-terminal hydrophilic region of SlFtsY, regardless of its short length, would behave like its counterpart region of EcFtsY in E. coli.