Fluorescein-di-β-d-galactopyranoside (FDG), a fluorogenic compound, is hydrolyzed by β-galactosidase in the cytoplasm of Escherichia coli to produce a fluorescent dye, fluorescein. We found that both FDG and fluorescein were substrates of efflux pumps, and have developed a new method to evaluate efflux-inhibitory activities in E. coli using FDG and a microfluidic channel device. We used E. coli MG1655 wild-type, ΔacrB (ΔB), ΔtolC (ΔC) and ΔacrBΔtolC (ΔBC) harboring plasmids carrying the mexAB-oprM (pABM) or mexXY-oprM (pXYM) genes of Pseudomonas aeruginosa. Two inhibitors, MexB-specific pyridopyrimidine (D13-9001) and non-specific Phe-Arg-β-naphthylamide (PAβN) were evaluated. The effects of inhibitors on pumps were observed using the microfluidic channel device under a fluorescence microscope. AcrAB-TolC and analogous pumps effectively prevented FDG influx in wild-type cells, resulting in no fluorescence. In contrast, ΔB or ΔC easily imported and hydrolyzed FDG to fluorescein, which was exported by residual pumps in ΔB. Consequently, fluorescent medium in ΔB and fluorescent cells of ΔC and ΔBC were observed in the microfluidic channels. D13-9001 substantially increased fluorescent cell number in ΔBC/pABM but not in ΔBC/pXYM. PAβN increased medium fluorescence in all strains, especially in the pump deletion mutants, and caused fluorescein accumulation to disappear in ΔC. The checkerboard method revealed that D13-9001 acts synergistically with aztreonam, ciprofloxacin, and erythromycin only against the MexAB-OprM producer (ΔBC/pABM), and PAβN acts synergistically, especially with erythromycin, in all strains including the pump deletion mutants. The results obtained from PAβN were similar to the results from membrane permeabilizer, polymyxin B or polymyxin B nonapeptide by concentration. The new method clarified that D13-9001 specifically inhibited MexAB-OprM in contrast to PAβN, which appeared to be a substrate of the pumps and permeabilized the membranes in E. coli.

A highly cephem-resistant Escherichia coli strain, FP1546, isolated from the fecal flora of laboratory dogs previously administered 1-lactam antibiotics was found to produce a P-lactamase, FEC-1, of 48-kilodalton size and pl 8.2. FEC-1 hydrolyzed cefuroxime, cefotaxime, cefmenoxime, and ceftriaxone, as well as the enzymatically less-stable antibiotics cephaloridine, cefotiam, and cefpiramide. Of the oxyimino-cephalosporins, ceftizoxime was fairly stable to FEC-1. FEC-1 differed notably from chromosomal E. coli cephalosporinase, especially in its broad-spectrum substrate profile and its high inhibition by clavulanic acid, sulbactam, and imipenem. A conjugation study revealed that FEC-1 was encoded by a 74-megadalton plasmid, pFCX1. This may be the first instance of a plasmid-mediated oxyimino-cephalosporinase from E. coli.Over 30 plasmid-mediated f-lactamases (mostly penicillinases) have been found in gram-negative bacteria and classified according to characteristics such as substrate specificity, isoelectric point, and molecular weight. Common are the TEM, OXA, SHV, and PSE types (10,(17)(18)(19)24), with the TEM type of penicillinase being the most frequently isolated plasmid P-lactamase from such gram-negative bacteria as members of the family Enterobacteriaceae, Pseudomonas aeruginosa, Haemophilus influenzae, and Neisseria gonorrhoeae. On the other hand, there have been only two reports of plasmid-mediated cephalosporinases, from Proteus mirabilis (2) and Achromobacter spp. (14).We evaluated the possible resistance mechanisms of oxyimino-cephalosporin-resistant Escherichia coli isolated from the fecal flora of laboratory dogs, and we identified a 3-lactamase which hydrolyzes oxyimino-cephalosporins such as cefuroxime, cefotaxime, cefmenoxime, and ceftriaxone in addition to cephaloridine. The physiological properties of this enzyme were quite distinct from those of chromosomal cephalosporinases from E. coli (20,23). We confirmed that this enzyme was plasmid mediated and had properties similar to those of oxyimino-cephalosporinase type I (7). MATERIALS AND METHODSBacterial strains. E. coli FP1546 was isolated from the fecal flora of a laboratory dog being used for pharmacokinetic studies of P-lactam antibiotics. The nalidixic acidresistant derivative of E. coli CSH2 (metB F-) was kindly provided by T. Yokota of Juntendo University.Antibiotics. Commercially available cephaloridine, cephalothin, cefamandole, cefotiam, cefmetazole, cefsulodin, cefuroxime, cefotaxime, cefmenoxime, ceftriaxone, ceftazidime, cefoperazone, cefpiramide, cefoxitin, moxalactam, and imipenem were used. Cefazolin and ceftizoxime were from Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan. * Corresponding author.Clavulanic acid, sulbactam, and nitrocefin were synthesized in our laboratories.Susceptibility testing. Antibacterial activity of test antibiotics was determined by the agar dilution method. Hundredfold dilutions of overnight cultures in Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.) were inoculated with a multipoint replic...

Specific pathogen-free Mongolian gerbils were infected orally with Helicobacter pylori to establish a new small animal model of severe gastritis H. pylori was recovered by culture from both antrum and body over a 16-week period after a single inoculation. The number of H. pylori colonising the antrum was about 100-fold higher than in the body, and this was consistent throughout the experiment. Histological examination showed that all animals developed severe inflammation with infiltration of polymorphonuclear leucocytes and mononuclear cells into the lamina propria and submucosa of the antrum from 4 weeks after infection. From 8 weeks after infection, multifocal lymphoid follicles appeared in the lamina propria and submucosa, and micro-erosions were also observed in the epithelial layer. At 16 weeks after infection, ulceration with disruption of the lamina muscularis mucosae was observed in the antral mucosa. To determine whether H. pylori caused gastritis or not, infected gerbils were treated with amoxycillin. After the treatment, gastritis could not be seen in the gastric mucosa. Therefore, the Mongolian gerbil is a useful small animal model to study the pathogenesis of H. pylori in gastric ulceration and severe gastritis and to assess anti-H. pylori treatment.

(−)-Epigallocatechin-3-O-gallate (EGCG) has useful antiviral, antimicrobial, antitoxin, and antitumor properties. Previously, Mori et al. (2008) found that addition of long acyl chains (C16–18) to EGCG enhanced its anti-influenza virus activity up to 44-fold. The chemical stability of EGCG against oxidative degradation was also enhanced by acylation. We further evaluated the in vitro activity spectrum of the EGCG derivatives against a wide range of bacteria and fungi. A series of EGCG O-acyl derivatives were synthesized by lipase-catalyzed transesterification. These derivatives exhibited several-fold higher activities than EGCG, particularly against Gram-positive organisms. Antifungal MICs of the derivatives were also two to fourfold lower than those of EGCG. The activities of the EGCG derivatives against Gram-negative bacteria were not distinguishable from those of EGCG. Among the derivatives evaluated, MICs of dioctanoate and palmitate (C16) for 17 Staphylococcus aureus strains were 4–32 μg/ml, although MIC of EGCG for these 17 strains was ≥128 μg/ml. C16 demonstrated rapid bactericidal activity against methicillin-resistant S. aureus (MRSA) ATCC43300 at ≥16 μg/ml. The enhanced activity of C16 against S. aureus was supported by its increased membrane-permeabilizing activity determined by increased SYTOX Green uptake. The EGCG derivatives were exported in Escherichia coli using the efflux pump AcrAB–TolC. The tolC deletion mutant exhibited higher sensitivity to EGCG and the derivatives than wild-type. Addition of long alkyl chains to EGCG significantly enhanced its activities against several bacteria and fungi, particularly against S. aureus including MRSA. C16 might potentially become under specified circumstances an alternative or supplement to antibiotics and disinfectants in the future.