Bacteroides fragilis is the most common anaerobe isolated from clinical infections, and in this report we demonstrate a characteristic of the species that is critical to their success as an opportunistic pathogen. Among the Bacteroides spp. in the gut, B. fragilis has the unique ability of efficiently harvesting complex N-linked glycans from the glycoproteins common to serum and serous fluid. This activity is mediated by an outer membrane protein complex designated as Don. Using the abundant serum glycoprotein transferrin as a model, it has been shown that B. fragilis alone can rapidly and efficiently deglycosylate this protein in vitro and that transferrin glycans can provide the sole source of carbon and energy for growth in defined media. We then showed that transferrin deglycosylation occurs in vivo when B. fragilis is propagated in the rat tissue cage model of extraintestinal growth, and that this ability provides a competitive advantage in vivo over strains lacking the don locus.

Bacteroides is a major component of the human gut microbiota which has a broad impact on the development and physiology of its host and a potential role in a wide range of disease syndromes. The predominance of this genus is due in large part to expansion of paralogous gene clusters, termed polysaccharide utilization loci (PULs), dedicated to the uptake and catabolism of hostderived and dietary polysaccharides. The nutritive value and availability of polysaccharides in the gut vary greatly; thus, their utilization is hierarchical and strictly controlled. A typical PUL includes regulatory genes that induce PUL expression in response to the presence of specific glycan substrates. However, the existence of additional regulatory mechanisms has been predicted to explain phenomena such as hierarchical control and catabolite repression. In this report, a previously unknown layer of regulatory control was discovered in Bacteroides fragilis. Exploratory transcriptome sequencing (RNA-seq) analysis revealed the presence of cis-encoded antisense small RNAs (sRNAs) associated with 15 (30%) of the B. fragilis PULs. A model system using the Don (degradation of N-glycans) PUL showed that the donS sRNA negatively regulated Don expression at the transcriptional level, resulting in a decrease in N-glycan utilization. Additional studies performed with other Bacteroides species indicated that this regulatory mechanism is highly conserved and, interestingly, that the regulated PULs appear to be closely linked to the utilization of host-derived glycans rather than dietary plant polysaccharides. The findings described here demonstrate a global control mechanism underlying known PUL regulatory circuits and provide insight into regulation of Bacteroides physiology. IMPORTANCEThe human gut is colonized by a dense microbiota which is essential to the health and normal development of the host. A key to gut homeostasis is the preservation of a stable, diverse microbiota. Bacteroides is a dominant genus in the gut, and the ability of Bacteroides species to efficiently compete for a wide range of glycan energy sources is a crucial advantage for colonization. Glycan utilization is mediated by a large number of polysaccharide utilization loci (PULs) which are regulated by substrate induction. In this report, a novel family of antisense sRNAs is described whose members repress gene expression in a distinct subset of PULs. This repression downregulates PUL expression in the presence of energy sources that are more readily utilized such as glucose, thereby allowing efficient glycan utilization. T he human gut microbiota plays diverse roles in the normal functioning of host physiology and under pathological conditions. These activities include but are not limited to the extraction of energy in undigested dietary components, normal development of the intestinal tract and the immune system, and the onset of obesity and diabetes (1-5). Species of Bacteroidetes constitute a numerically predominate phylum in the human gut, and the genus Bacteroid...

Efflux pumps in Gram-negative bacteria such as Pseudomonas aeruginosa provide intrinsic antimicrobial resistance by facilitating the extrusion of a wide range of antimicrobials. Approaches for combating efflux-mediated multidrug resistance involve, in part, developing indirect antimicrobial agents capable of inhibiting efflux, thus rescuing the activity of antimicrobials previously rendered inactive by efflux. Herein, TXA09155 is presented as a novel efflux pump inhibitor (EPI) formed by conformationally constraining our previously reported EPI TXA01182. TXA09155 demonstrates strong potentiation in combination with multiple antibiotics with efflux liabilities against wild-type and multidrug-resistant (MDR) P. aeruginosa. At 6.25 µg/mL, TXA09155, showed ≥8-fold potentiation of levofloxacin, moxifloxacin, doxycycline, minocycline, cefpirome, chloramphenicol, and cotrimoxazole. Several biophysical and genetic studies rule out membrane disruption and support efflux inhibition as the mechanism of action (MOA) of TXA09155. TXA09155 was determined to lower the frequency of resistance (FoR) to levofloxacin and enhance the killing kinetics of moxifloxacin. Most importantly, TXA09155 outperformed the levofloxacin-potentiation activity of EPIs TXA01182 and MC-04,124 against a CDC/FDA panel of MDR clinical isolates of P. aeruginosa. TXA09155 possesses favorable physiochemical and ADME properties that warrant its optimization and further development.

The ability to rescue the activity of antimicrobials that are no longer effective against bacterial pathogens such as Pseudomonas aeruginosa is an attractive strategy to combat antimicrobial drug resistance. Herein, novel efflux pump inhibitors (EPIs) demonstrating strong potentiation in combination with levofloxacin against wild-type P. aeruginosa ATCC 27853 are presented. A structure activity relationship of aryl substituted heterocyclic carboxamides containing a pentane diamine side chain is described. Out of several classes of fused heterocyclic carboxamides, aryl indole carboxamide compound 6j (TXA01182) at 6.25 µg/mL showed 8-fold potentiation of levofloxacin. TXA01182 was found to have equally synergistic activities with other antimicrobial classes (monobactam, fluoroquinolones, sulfonamide and tetracyclines) against P. aeruginosa. Several biophysical and genetic studies rule out membrane disruption and support efflux inhibition as the mechanism of action (MOA) of TXA01182. TXA01182 was determined to lower the frequency of resistance (FoR) of the partner antimicrobials and enhance the killing kinetics of levofloxacin. Furthermore, TXA01182 demonstrated a synergistic effect with levofloxacin against several multidrug resistant P. aeruginosa clinical isolates.

No abstract

The purpose of the study was to establish a mathematical model for correlating the combination of ultrasonography and noncontrast helical computerized tomography (NCHCT) with the total energy of Holmium laser lithotripsy.In this study, from March 2013 to February 2014, 180 patients with single urinary calculus were examined using ultrasonography and NCHCT before Holmium laser lithotripsy. The calculus location and size, acoustic shadowing (AS) level, twinkling artifact intensity (TAI), and CT value were all documented. The total energy of lithotripsy (TEL) and the calculus composition were also recorded postoperatively. Data were analyzed using Spearman's rank correlation coefficient, with the SPSS 17.0 software package. Multiple linear regression was also used for further statistical analysis.A significant difference in the TEL was observed between renal calculi and ureteral calculi (r = –0.565, P < 0.001), and there was a strong correlation between the calculus size and the TEL (r = 0.675, P < 0.001). The difference in the TEL between the calculi with and without AS was highly significant (r = 0.325, P < 0.001). The CT value of the calculi was significantly correlated with the TEL (r = 0.386, P < 0.001). A correlation between the TAI and TEL was also observed (r = 0.391, P < 0.001). Multiple linear regression analysis revealed that the location, size, and TAI of the calculi were related to the TEL, and the location and size were statistically significant predictors (adjusted r2 = 0.498, P < 0.001).A mathematical model correlating the combination of ultrasonography and NCHCT with TEL was established; this model may provide a foundation to guide the use of energy in Holmium laser lithotripsy. The TEL can be estimated by the location, size, and TAI of the calculus.

MreB is a cytoskeleton protein present in rod-shaped bacteria that is both essential for bacterial cell division and highly conserved. Because most Gram (-) bacteria require MreB for cell division, chromosome segregation, cell wall morphogenesis, and cell polarity, it is an attractive target for antibacterial drug discovery. As MreB modulation is not associated with the activity of antibiotics in clinical use, acquired resistance to MreB inhibitors is also unlikely. Compounds, such as A22 and CBR-4830, are known to disrupt MreB function by inhibition of ATPase activity. However, the toxicity of these compounds has hindered efforts to assess the in vivo e cacy of these MreB inhibitors. The present study further examines the structure-activity of analogs related to CBR-4830 as it relates to relative antibiotic activity and improved drug properties. These data reveal that certain analogs have enhanced antibiotic activity. In addition, we evaluated several representative analogs (9, 10, 14, 26, and 31) for their abilities to target puri ed E. coli MreB (EcMreB) and inhibit its ATPase activity. Except for 14, all these analogs were more potent than CBR-4830 as inhibitors of the ATPase activity of EcMreB with corresponding IC 50 values ranging from 6.3 ± 2.0 to 29.4 ± 8.8 uM.condensed with ethyl formate to provide 2-(hydroxymethylene)cyclopentan-1-one. This intermediate was reacted with the appropriate diazonium salt to give the desired hydrazineylidene intermediates, which were converted under acidic condition to the 1,4-dihydrocyclopenta[b]indole-3(2H)-ones, 11a as well as a mixture of 12a and 13a. The mixture of 12a and 13a was resolved by formation of their N-Boc derivatives (12b and 13b), chromatographic separation, followed by removal of their Boc groups.The impact of removing two methylene groups and opening the tetrahydrocyclohexyl ring of CBR-4830 on relative antimicrobial activity was also evaluated. The varied 2-(1-aminoethyl)indoles 14-21 were prepared as outlined in Scheme 3.The Weinreb amide derivatives 14b-21b were formed from the various 2-carboxyindole derivatives and then converted using methyllithium to the methyl ketones 14c-21c. Reductive amination of 14c-21c using ammonium acetate and sodium cyanoborohydride provide 14-21.The in uence of mono-or dimethylation on the amino group as well as methylation of the 1-position of 1-(5-bromo-1-methyl-1H-indol-2-yl)ethan-1-amine was examined. Compounds 22, 23, and 24 were synthesized as outlined in Scheme 4.Reductive amination was performed using N-methylamine or N,N-dimethylamine, followed by reduction with sodium cyanoborohydride was used to prepare 22 or 23. Methylation of 14c to form the 1methylindole derivative 24a was accomplished using potassium carbonate in DMF and methyl iodide.Intermediate 24a was then converted 24 using ammonium acetate and sodium cyanoborohydride.We also examined the impact on activity of having the primary amine of 1-(5-bromo-1-methyl-1H-indol-2yl)ethan-1-amine attached to a primary carbon as opposed to a secondary carbon. We s...

MreB is a cytoskeleton protein present in rod-shaped bacteria that is both essential for bacterial cell division and highly conserved. Because most Gram (-) bacteria require MreB for cell division, chromosome segregation, cell wall morphogenesis, and cell polarity, it is an attractive target for antibacterial drug discovery. As MreB modulation is not associated with the activity of antibiotics in clinical use, acquired resistance to MreB inhibitors is also unlikely. Compounds, such as A22 and CBR-4830, are known to disrupt MreB function by inhibition of ATPase activity. However, the toxicity of these compounds has hindered efforts to assess the in vivo efficacy of these MreB inhibitors. The present study further examines the structure-activity of analogs related to CBR-4830 as it relates to relative antibiotic activity and improved drug properties. These data reveal that certain analogs have enhanced antibiotic activity. In addition, we evaluated several representative analogs (9, 10, 14, 26, and 31) for their abilities to target purified E. coli MreB (EcMreB) and inhibit its ATPase activity. Except for 14, all these analogs were more potent than CBR-4830 as inhibitors of the ATPase activity of EcMreB with corresponding IC50 values ranging from 6.3 ± 2.0 to 29.4 ± 8.8 uM.