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Although it is well known that the morphology of Gram-negative rods changes on exposure to antibiotics, the morphology of antibiotic-resistant bacteria in the absence of antibiotics has not been widely investigated. Here, we studied the morphologies of 10 antibiotic-resistant strains of Escherichia coli and used bioinformatics tools to classify the resistant cells under light microscopy in the absence of antibiotics. The antibiotic-resistant strains showed differences in morphology from the sensitive parental strain, and the differences were most prominent in the quinolone-and β-lactam-resistant bacteria. A cluster analysis revealed increased proportions of fatter or shorter cells in the antibiotic-resistant strains. A correlation analysis of morphological features and gene expression suggested that genes related to energy metabolism and antibiotic resistance were highly correlated with the morphological characteristics of the resistant strains. Our newly proposed deep learning method for single-cell classification achieved a high level of performance in classifying quinolone-and β-lactam-resistant strains.

Changes in expression levels of drug efflux pump genes, mexB and mexY, and porin gene oprD in Pseudomonas aeruginosa were investigated in this study. Fifty-five multidrug-resistant P. aeruginosa (MDRP) strains were compared with 26 drug-sensitive strains and 21 strains resistant to a single antibiotic. The effect of the efflux inhibitor Phe-Arg-β-naphthylamide on drug susceptibility was determined, and gene expression was quantified using real-time quantitative real-time reverse transcription polymerase chain reaction. In addition, the levels of metallo-β-lactamase (MBL) and 6′-N-aminoglycoside acetyltransferase [AAC(6′)-Iae] were investigated. Efflux pump inhibitor treatment increased the sensitivity to ciprofloxacin, aztreonam, and imipenem in 71%, 73%, and 29% of MDRPs, respectively. MBL and AAC(6′)-Iae were detected in 38 (69%) and 34 (62%) MDRP strains, respectively. Meanwhile, 76% of MDRP strains exhibited more than 8-fold higher mexY expression than the reference strain PAO1. Furthermore, 69% of MDRP strains expressed oprD at levels less than 0.01-fold of those in PAO1. These findings indicated that efflux pump inhibitors in combination with ciprofloxacin or aztreonam might aid in treating MDRP infections.

Bile represses Salmonella enterica serovar Typhimurium (S. Typhimurium) intestinal cell invasion, but it remains unclear which bile components and mechanisms are implicated. Previous studies reported that bile inhibits the RamR binding to the ramA promoter, resulting in ramA increased transcription, and that ramA overexpression is associated to decreased expression of type III secretion system 1 (TTSS-1) invasion genes and to impaired intestinal cell invasiveness in S. Typhimurium. In this study, we assessed the possible involvement of the ramRA multidrug efflux regulatory locus and individual bile salts in the bile-mediated repression of S. Typhimurium invasion, using Caco-2 intestinal epithelial cells and S. Typhimurium strain ATCC 14028s. Our results indicate that (i) major primary bile salts, chenodeoxycholate and its conjugated-derivative salts, cholate, and deoxycholate, activate ramA transcription in a RamR-dependent manner, and (ii) it results in repression of hilA, encoding the master activator of TTSS-1 genes, and as a consequence in the repression of cellular invasiveness. On the other hand, crude ox bile extract and cholate were also shown to repress the transcription of hilA independently of RamR, and to inhibit cell invasion independently of ramRA. Altogether, these data suggest that bile-mediated repression of S. Typhimurium invasion occurs through pleiotropic effects involving partly ramRA, as well as other unknown regulatory pathways. Bile components other than the bile salts used in this study might also participate in this phenomenon.

Gut microbiota has been reported to be closely related to host energy metabolism and immunity, and thus influence the development and progression of various human diseases. To date, the gut microbial metabolites such as short-chain fatty acids, defensins, cathelicidins, and lactoferrin in feces have been investigated as biomarkers associated with various disease conditions. In this review, we introduce intestinal and fecal pH, which is relatively easy and rapid to measure compared to the composition of the gut microbiota and its metabolites. In particular, this review presents the distribution of pH in the human body, its role and clinical significance, and various factors that affect intestinal and fecal pH, including the gut microbiota and its metabolites.

Fatty acids salts exert bactericidal and bacteriostatic effects that inhibit bacterial growth and survival. However, bacteria can overcome these effects and adapt to their environment. Bacterial efflux systems are associated with resistance to different toxic compounds. Here, several bacterial efflux systems were examined to determine their influence on fatty acid salt resistance in Escherichia coli. Both acrAB and tolC E. coli deletion strains were susceptible to fatty acid salts, while plasmids carrying acrAB, acrEF, mdtABC, or emrAB conferred drug resistance to the ΔacrAB mutant, which indicated complementary roles for these multidrug efflux pumps. Our data exemplify the importance of bacterial efflux systems in E. coli resistance to fatty acid salts.

The emergence of bacteria that are resistant to antibiotics is common in areas where antibiotics are used widely. The current standard procedure for detecting bacterial drug resistance is based on bacterial growth under antibiotic treatments. Here we describe the morphological changes in enoxacin-resistant Escherichia coli cells and the computational method used to identify these resistant cells in transmission electron microscopy (TEM) images without using antibiotics. Our approach was to create patches from TEM images of enoxacin-sensitive and enoxacin-resistant E. coli strains, use a convolutional neural network for patch classification, and identify the strains on the basis of the classification results. The proposed method was highly accurate in classifying cells, achieving an accuracy rate of 0.94. Using a gradient-weighted class activation mapping to visualize the region of interest, enoxacin-resistant and enoxacin-sensitive cells were characterized by comparing differences in the envelope. Moreover, Pearson’s correlation coefficients suggested that four genes, including lpp, the gene encoding the major outer membrane lipoprotein, were strongly associated with the image features of enoxacin-resistant cells.

The prototype fexA gene confers combined resistance to chloramphenicol and florfenicol. However, fexA variants mediating resistance only to chloramphenicol have been identified, such as in the case of a Staphylococcus aureus isolate recovered from poultry meat illegally imported to Germany. The effects of the individual mutations detected in the fexA sequence of this isolate were investigated in this study. A total of 11 fexA variants, including prototype fexA and variants containing the different previously described mutations either alone or in different combinations, were generated by on-chip gene synthesis and site-directed mutagenesis. The constructs were inserted into a shuttle vector and transformed into three recipient strains (Escherichia coli, Staphylococcus aureus, and Salmonella Typhimurium). Subsequently, minimal inhibitory concentrations (MIC) of florfenicol and chloramphenicol were determined. In addition, protein modeling was used to predict the structural effects of the mutations. The lack of florfenicol-resistance mediating properties of the fexA variants could be attributed to the presence of a C110T and/or G98C mutation. Transformants carrying fexA variants containing either of these mutations, or both, showed a reduction of florfenicol MICs compared to those transformants carrying prototype fexA or any of the other variants. The significance of these mutations was supported by the generated protein models, indicating a substitution toward more voluminous amino-acids in the substrate-binding site of FexA. The remaining mutations, A391G and C961A, did not result in lower florfenicol-resistance compared to prototype fexA.

Fatty acids salts exhibit bacteriostatic and bactericidal effects to inhibit bacterial growth and survival. Bacteria adapt to their environment to overcome these antibacterial effects through undefined mechanisms. In Gram-negative bacteria, drug efflux systems are associated with resistance to various substances. Studies have identified multiple drug efflux systems in Salmonella enterica. The aim of this study was to investigate whether drug efflux systems contribute to fatty acid salts resistance in S. enterica. We used deletion and overexpressing strains of S. enterica for drug efflux transporters. Susceptibility to fatty acid salts was determined by measuring minimum inhibitory concentrations and performing growth assays. Our findings revealed that acrAB, acrEF, emrAB and tolC in S. enterica contribute resistance to fatty acid salts. Furthermore, EmrAB, which is known to function with TolC, contributes to the fatty acid salts resistance of S. enterica in a TolC-independent manner. This study revealed that drug efflux systems confer fatty acid satls resistance to S. enterica. Notably, although EmrAB is normally associated with antimicrobial resistance in a TolC-dependent manner, it was found to be involved in fatty acid salts resistance in a TolC-independent manner, indicating that the utilization of TolC by EmrAB is substrate dependent in S. enterica.

Multidrug efflux pumps are inner membrane transporters that export multiple antibiotics from the inside to the outside of bacterial cells, contributing to bacterial multidrug resistance (MDR). Postgenomic analysis has demonstrated that numerous multidrug efflux pumps exist in bacteria. Also, the co-crystal structural analysis of multidrug efflux pumps revealed the drug recognition and export mechanisms, and the inhibitory mechanisms of the pumps. A single multidrug efflux pump can export multiple antibiotics; hence, developing efflux pump inhibitors is crucial in overcoming infectious diseases caused by multidrug-resistant bacteria. This review article describes the role of multidrug efflux pumps in MDR, and their physiological functions and inhibitory mechanisms.

Indole is a signal molecule derived from the conversion of tryptophan, and it is present in bacterial respiratory gas. Besides influencing bacterial growth, indole exhibits effects on human health, including a positive effect on inflammation and protection against pathogens. However, a high fecal indole concentration (FIC) can suggest an unbalanced gut flora or the presence of certain pathogens. To analyze the indole produced by bacteria, its collection and detection is required. Traditional methods usually require centrifugation of liquid bacterial culture medium and subsequent extraction of indole from the medium or partial purification of indole from fecal samples (e.g., by distillation or extraction). In this study, we demonstrate the possibility of identifying gas contents directly from bacteria, and we distinguish the difference in species and their genetics without the need to centrifuge or extract. Using an absorbent sheet placed above a liquid culture, we were able to collect gas content directly from bacteria. Gas chromatography-mass spectrometry (GC-MS) was used for the analysis. The GC-MS results showed a clear peak attributed to indole for wild-type Escherichia coli cells (MG1655 and MC4100 strains), whereas the indole peak was absent in the chromatograms of cells where proteins, part of the indole production pathway from tryptophan (TnaA and TnaB), were not expressed (by using tnaAB-deleted cells). The indole observed was measured to be present in a low nmol-range. This method can distinguish whether the bacterial genome contains the tnaAB gene or not and can be used to collect gas compounds from bacterial cultures quickly and easily. This method is useful for other goals and future research, such as for measurements in restrooms, for food-handling facilities, and for various applications in medical settings.

Multidrug transporters possess several drug binding sites. Here the authors describe a transport path specific for planar aromatic cations in the E. coli multi-drug transporter AcrB.

Summary: Despite being fundamentally important and having direct therapeutic implications, the functional genomics of the clinical isolates of multidrug-resistant (MDR) pathogens is often impeded by the lack of genome-editing tools. Here, we report the establishment of a highly efficient, in situ genome-editing technique applicable in clinical and environmental isolates of the prototypic MDR pathogen P. aeruginosa by harnessing the endogenous type I-F CRISPR-Cas systems. Using this approach, we generate various reverse mutations in an epidemic MDR genotype, PA154197, and identify underlying resistance mechanisms that involve the extensive synergy among three different resistance determinants. Screening a series of “ancestor” mutant lines uncovers the remarkable sensitivity of the MDR line PA154197 to a class of small, cationic peptidomimetics, which sensitize PA154197 cells to antibiotics by perturbing outer-membrane permeability. These studies provide a framework for molecular genetics and anti-resistance drug discovery for clinically isolated MDR pathogens. : Xu et al. develop an efficient genome-editing technique in genetically recalcitrant clinical and environmental P. aeruginosa strains by exploiting their endogenous type I-F CRISPR-Cas system. Employing the technique, they identify underlying resistance mechanisms of an epidemic MDR genotype, PA154197, efficiently and develop anti-resistance strategies based on collateral sensitivity. Keywords: multidrug resistance, Pseudomonas aeruginosa, native CRISPR-Cas system, collateral sensitivity, cationic peptidomimetics, genome editing, multidrug efflux pump, anti-resistance drug discovery

Golgins are a family of Golgi-localized long coiled-coil proteins. The major golgin function is thought to be the tethering of vesicles, membranes, and cytoskeletal elements to the Golgi. We previously showed that knockdown of one of the longest golgins, Giantin, altered the glycosylation patterns of cell surfaces and the kinetics of cargo transport, suggesting that Giantin maintains correct glycosylation through slowing down transport within the Golgi. Giantin knockdown also altered the sizes and numbers of mini Golgi stacks generated by microtubule de-polymerization, suggesting that it maintains the independence of individual Golgi stacks. Therefore, it is presumed that Golgi stacks lose their independence following Giantin knockdown, allowing easier and possibly increased transport among stacks and abnormal glycosylation. To gain structural insights into the independence of Golgi stacks, we herein performed electron tomography and 3D modeling of Golgi stacks in Giantin knockdown cells. Compared with control cells, Giantin-knockdown cells had fewer and smaller fenestrae within each cisterna. This was supported by data showing that the diffusion rate of Golgi membrane proteins is faster in Giantin-knockdown Golgi, indicating that Giantin knockdown structurally and functionally increases connectivity among Golgi cisternae and stacks. This increased connectivity suggests that contrary to the cis-golgin tether model, Giantin instead inhibits the tether and fusion of nearby Golgi cisternae and stacks, resulting in transport difficulties between stacks that may enable the correct glycosylation of proteins and lipids passing through the Golgi.

The rise in multidrug resistance (MDR) is one of the greatest threats to human health worldwide. MDR in bacterial pathogens is a major challenge in healthcare, as bacterial infections are becoming untreatable by commercially available antibiotics. One of the main causes of MDR is the over-expression of intrinsic and acquired multidrug efflux pumps, belonging to the resistance-nodulation-division (RND) superfamily, which can efflux a wide range of structurally different antibiotics. Besides over-expression, however, recent amino acid substitutions within the pumps themselves—causing an increased drug efflux efficiency—are causing additional worry. In this review, we take a closer look at clinically, environmentally and laboratory-evolved Gram-negative bacterial strains and their decreased drug sensitivity as a result of mutations directly in the RND-type pumps themselves (from Escherichia coli, Salmonella enterica, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Acinetobacter baumannii and Legionella pneumophila). We also focus on the evolution of the efflux pumps by comparing hundreds of efflux pumps to determine where conservation is concentrated and where differences in amino acids can shed light on the broad and even broadening drug recognition. Knowledge of conservation, as well as of novel gain-of-function efflux pump mutations, is essential for the development of novel antibiotics and efflux pump inhibitors.

The overexpression of RND-type exporters is one of the main causes of multidrug resistance (MDR) in Gram-negative pathogens. In RND transporters, such as Escherichia coli's main efflux pump AcrB, drug efflux occurs in the porter domain, while protons flow through the transmembrane domain: remote conformational coupling. At the border of a transmembrane helix (TM8) and subdomain PC2, there is a loop which makes a hoisting movement by a random-coil-to-α-helix change, and opens and closes a drug channel entrance. This loop is supposed to play a key role in the allosteric conformational coupling between the transmembrane and porter domain. Here we show the results of a series of flexibility loop-mutants of AcrB. We determined the crystal structure of a three amino acid truncated loop mutant, which is still a functional transporter, and show that the short α-helix between Cβ15 and the loop unwinds to a random coil in the access and binding monomers and in the extrusion monomer it makes a partially stretched coil-to-helix change. The loop has undergone compensatory conformational changes and still facilitates the opening and closing of the channel. In addition, more flexible mutated loops (proline mutated and significantly elongated) can still function during export. The flexibility in this region is however limited, as an even more truncated mutant (six amino acid deletion) becomes mostly inactive. We found that the hoisting-loop is a highly flexible hinge that enables the conformational energy transmission passively.

We previously showed that berberine attenuates MexXY efflux-dependent aminoglycoside resistance in Pseudomonas aeruginosa. Here, we aimed to synthesize berberine derivatives with higher MexXY inhibitory activities. We synthesized 11 berberine derivatives, of which 13-(2-methylbenzyl) berberine (13-o-MBB) but not its regiomers showed the most promising MexXY inhibitory activity. 13-o-MBB reduced the minimum inhibitory concentrations (MICs) of various aminoglycosides 4- to 128 fold for a highly multidrug resistant P. aeruginosa strain. Moreover, 13-o-MBB significantly reduced the MICs of gentamicin and amikacin in Achromobacter xylosoxidans and Burkholderia cepacia. The fractional inhibitory concentration indices indicated that 13-o-MBB acted synergistically with aminoglycosides in only MexXY-positive P. aeruginosa strains. Time-kill curves showed that 13-o-MBB or higher concentrations of berberine increased the bactericidal activity of gentamicin by inhibiting MexXY in P. aeruginosa. Our findings indicate that 13-o-MBB inhibits MexXY-dependent aminoglycoside drug resistance more strongly than berberine and that 13-o-MBB is a useful inhibitor of aminoglycoside drug resistance due to MexXY.

The emergence and spread of multidrug-resistant P. aeruginosa infections is of great concern, as very few agents are effective against strains of this species. Methanolic extracts from the Coptidis Rhizoma (the rhizomes of Coptis japonica var. major Satake) or Phellodendri Cortex (the bark of Phellodendron chinense Schneider) markedly reduced resistance to anti-pseudomonal aminoglycosides (e.g. amikacin) in multidrug-resistant P. aeruginosa strains. Berberine, the most abundant benzylisoquinoline alkaloid in the two extracts, reduced aminoglycoside resistance of P. aeruginosa via a mechanism that required the MexXY multidrug efflux system; berberine also reduced aminoglycoside MICs in Achromobacter xylosoxidans and Burkholderia cepacia, two species that harbor intrinsic multidrug efflux systems very similar to the MexXY. Furthermore this compound inhibited MexXY-dependent antibiotic resistance of other classes including cephalosporins (cefepime), macrolides (erythromycin), and lincosamides (lincomycin) demonstrated using a pseudomonad lacking the 4 other major Mex pumps. Although phenylalanine-arginine beta-naphthylamide (PAβN), a well-known efflux inhibitor, antagonized aminoglycoside in a MexXY-dependent manner, a lower concentration of berberine was sufficient to reduce amikacin resistance of P. aeruginosa in the presence of PAβN. Moreover, berberine enhanced the synergistic effects of amikacin and piperacillin (and vice versa) in multidrug-resistant P. aeruginosa strains. Thus, berberine appears to be a novel type inhibitor of the MexXY-dependent aminoglycoside efflux in P. aeruginosa. As aminoglycosides are molecules of choice to treat severe infections the clinical impact is potentially important.

The recent global increase in the prevalence of antibiotic-resistant bacteria and lack of development of new therapeutic agents emphasize the importance of selecting appropriate antimicrobials for the treatment of infections. However, to date, the development of completely accelerated drug susceptibility testing methods has not been achieved despite the availability of a rapid identification method. We proposed an innovative rapid method for drug susceptibility testing for Pseudomonas aeruginosa that provides results within 3 h. The drug susceptibility testing microfluidic (DSTM) device was prepared using soft lithography. It consisted of five sets of four microfluidic channels sharing one inlet slot, and the four channels are gathered in a small area, permitting simultaneous microscopic observation. Antimicrobials were pre-introduced into each channel and dried before use. Bacterial suspensions in cation-adjusted Mueller-Hinton broth were introduced from the inlet slot and incubated for 3 h. Susceptibilities were microscopically evaluated on the basis of differences in cell numbers and shapes between drug-treated and control cells, using dedicated software. The results of 101 clinically isolated strains of P. aeruginosa obtained using the DSTM method strongly correlated with results obtained using the ordinary microbroth dilution method. Ciprofloxacin, meropenem, ceftazidime, and piperacillin caused elongation in susceptible cells, while meropenem also induced spheroplast and bulge formation. Morphological observation could alternatively be used to determine the susceptibility of P. aeruginosa to these drugs, although amikacin had little effect on cell shape. The rapid determination of bacterial drug susceptibility using the DSTM method could also be applicable to other pathogenic species, and it could easily be introduced into clinical laboratories without the need for expensive instrumentation.

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.

Streptococcus pneumoniae, a Gram-positive bacterium, is a major cause of invasive infection-related diseases such as pneumonia and sepsis. In blood, erythrocytes are considered to be an important factor for bacterial growth, as they contain abundant nutrients. However, the relationship between S. pneumoniae and erythrocytes remains unclear. We analyzed interactions between S. pneumoniae and erythrocytes, and found that iron ion present in human erythrocytes supported the growth of Staphylococcus aureus, another major Gram-positive sepsis pathogen, while it partially inhibited pneumococcal growth by generating free radicals. S. pneumoniae cells incubated with human erythrocytes or blood were subjected to scanning electron and confocal fluorescence microscopic analyses, which showed that the bacterial cells adhered to and invaded human erythrocytes. In addition, S. pneumoniae cells were found associated with human erythrocytes in cultures of blood from patients with an invasive pneumococcal infection. Erythrocyte invasion assays indicated that LPXTG motif-containing pneumococcal proteins, erythrocyte lipid rafts, and erythrocyte actin remodeling are all involved in the invasion mechanism. In a neutrophil killing assay, the viability of S. pneumoniae co-incubated with erythrocytes was higher than that without erythrocytes. Also, H2O2 killing of S. pneumoniae was nearly completely ineffective in the presence of erythrocytes. These results indicate that even when S. pneumoniae organisms are partially killed by iron ion-induced free radicals, they can still invade erythrocytes. Furthermore, in the presence of erythrocytes, S. pneumoniae can more effectively evade antibiotics, neutrophil phagocytosis, and H2O2 killing.

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.

We investigated the characteristics of pharmaceutical concentrations and antimicrobial activities in river water from the Tone River system in Gunma Prefecture. The mean concentrations of diphenhydramine, clarithromycin, carbamazepine, and bezafibrate in the midstream of the Tone River were 8.6, 29, 3.8, and 8.1 ng/L, respectively. Their concentrations were nearly half of those in the midstream of the Ayase River, the main water source of which is wastewater. Seasonal variations in pharmaceutical concentrations were high in winter and low in late spring and autumn. This variation depended on the flow rate of the river water, which in turn depended on the rainfall in the upstream area. Except for bezafibrate, the pharmaceutical concentrations in river water did not change after 5 days of incubation at 30°C, indicating that biochemical degradation during the hot summer season was minimal. A comparison of the concentrations between the sampling locations revealed that the pharmaceutical load was proportional to basin population, and the annual fluxes of pharmaceuticals from Gunma Prefecture were estimated to be 98, 210, 28, and 53 kg/year, respectively. Disc diffusion assay of some samples of Tone River water extracts revealed inhibition zones owing to their antimicrobial activity. However, no relationship was observed between the diameter of the inhibition zone and clarithromycin concentration in the river water. These results suggest that the antimicrobial activities of the river samples were not dependent on clarithromycin. We are currently investigating the pollution and drug-resistant bacteria present in the Tone River in detail. [ABSTRACT FROM AUTHOR]

Bile represses Salmonella enterica serovar Typhimurium (S. Typhimurium) intestinal cell invasion, but it remains unclear which bile components and mechanisms are implicated. Previous studies reported that bile inhibits the RamR binding to the ramA promoter, resulting in ramA increased transcription, and that ramA overexpression is associated to decreased expression of type III secretion system1 (TTSS-1) invasion genes and to impaired intestinal cell invasiveness in S. Typhimurium. In this study, we assessed the possible involvement of the ramRA multidrug efflux regulatory locus and individual bile salts in the bile-mediated repression of S. Typhimuriuminvasion, using Caco-2 intestinal epithelial cells and S. Typhimurium strain ATCC 14028s. Our results indicate that (i) major primary bile salts, chenodeoxycholate and its conjugated-derivative salts, cholate, and deoxycholate, activate ramA transcription in a RamR-dependent manner, and (ii) it results in repression of hilA, encoding themaster activator of TTSS-1 genes, and as a consequence in the repression of cellular invasiveness. On the other hand, crude ox bile extract and cholate were also shown to repress the transcription of hilA independently of RamR, and to inhibit cell invasion independently of ramRA. Altogether, these data suggest that bile-mediated repression of S. Typhimurium invasion occurs through pleiotropic effects involving partly ramRA, as well as other unknown regulatory pathways. Bile components other than the bile salts used in this study might also participate in this phenomenon. [ABSTRACT FROM AUTHOR]