Glutamine promotes antibiotic uptake to kill multidrug-resistant uropathogenic bacteria.

The prevalence of multidrug-resistant bacteria has been increasing rapidly worldwide, a trend that poses great risk to human and animal health and creates urgent need for pharmaceutical and nonpharmaceutical approaches to stop the spread of disease due to antimicrobial resistance. Here, we found that alanine, aspartate, and glutamate metabolism was inactivated, and glutamine was repressed in multidrug-resistant uropathogenic Escherichia coli using a comparative metabolomics approach. Exogenous glutamine promoted ß-lactam­, aminoglycoside-, quinolone-, and tetracycline-induced killing of uropathogenic E. coli and potentiated ampicillin to eliminate multidrug-resistant Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella peneumoniae, Edwardsiella tarda, Vibrio alginolyticus, and Vibrio parahaemolyticus. Glutamine-potentiated ampicillin-mediated killing was effective against biofilms of these bacteria in a mouse urinary tract infection model and against systemic infection caused by E. coli, P. aeruginosa, A. baumannii, or K. peneumoniae in a mouse model. Exogenous glutamine stimulated influx of ampicillin, leading to the accumulation of intracellular antibiotic concentrations that exceeded the amount tolerated by the multidrug-resistant bacteria. Furthermore, we demonstrated that exogenous glutamine promoted the biosynthesis of nucleosides including inosine, which in turn interacted with CpxA/CpxR and up-regulated OmpF. We validated the physiological relevance of the mechanism by showing that loss of purF, purH, cpxA, or ompF elevated antibiotic resistance in antibiotic-sensitive strains. In addition, glutamine retarded the development of ampicillin resistance. These results may facilitate future development of effective approaches for preventing or managing chronic, multidrug-resistant bacterial infections, bacterial persistence, and difficult-to-treat bacterial biofilms.

Analysis of virulence and immunogenic factors in Aeromonas hydrophila: Towards the development of live vaccines.

Aeromonas hydrophila, a bacterium that is widespread in aquatic environments, is responsible for causing haemorrhagic disease in both aquatic and terrestrial species. With the purpose of developing a live vaccine, herein we have investigated nine strains of A. hydrophila (Ah-01 to Ah-09) isolated from diseased fish. A study of virulence factors that contribute to pathogenicity and immunogenicity in the host Cyprinus carpio suggests that the presence of ß-hly, act and fla genes contribute to pathogenesis: strains Ah-01, Ah-02 and Ah-03 (ß-hly+ /act+ /fla+ genotype) were highly pathogenic to C. carpio, whereas Ah-05 and Ah-06 (ß-hly- /act- /fla- genotype) showed weak pathogenicity. Accordingly, Ah-02 and Ah-03 were selected to prepare inactivated vaccines, whereas Ah-05 and Ah-06 were chosen as live vaccines. Ah-06 live vaccine was found to have the best protective efficacy, with a protective rate of about 85%, whereas rates of other vaccines were significantly lower, in the range 37%-59%. In addition, DNA vaccines based on genes altA, aha and omp showed immune protection rates of 25%, 37.5% and 75%, respectively. Our data demonstrate that the ß-hly- /act- /fla- /altA+ /aha+ /omp+ genotype has weak pathogenicity and high immunogenicity, and provide a simple and effective way to screen for live A. hydrophila vaccines.

Molecular characteristics, pathogenicity and medication regimen of Aeromonas hydrophila isolated from common carp (Cyprinus carpio L.).

Aeromonas hydrophila causes disease in fish known as Motile Aeromonas Septicemia (MAS), also named as bacterial hemorrhagic septicemia. In this study, a pathogenic A. hydrophila strain was isolated from common carp Cyprinus carpio L., which were suffering from severe hemorrhagic septicemia. According to the phylogenetic analysis derived from 16S rDNA sequence, the isolate formed a single branch in the A. hydrophila group, named AhHN1. Artificial infection results indicated that AhHN1 showed strong pathogenicity in C. carpio and the LD50 was 1.38 × 106 CFU/fish, the clinical symptoms and pathological features of infected fish were similar to those observed in natural infections. The antimicrobial susceptibility testing revealed that AhHN1 resistance to more than 13 kinds of antimicrobial agents. However, the AhHN1 strain exhibited an extremely sensitivity to enrofloxacin, the in vitro activities of enrofloxacin were subsequently investigated and drug selection window (MSW) was 0.0016-0.0125 µg/ml. Pharmacokinetics data showed that plasma concentration of enrofloxacin was 0.0016, 0.0148 and 0.0282 µg/ml at 24 hr after orally administered with 2.5, 5 and 10 mg/kg enrofloxacin. Moreover, dosing once a day of 2.5, 5 and 10 mg/kg enrofloxacin, which the relative protection ratio (RPS) was amounted to 33.3, 66.7, and 83.3%, respectively. Therefore, 5 mg/kg enrofloxacin was considered to be the rational regimen for controlling AhHN1 infection in C. carpio in the countries where the use of enrofloxacin is permitted in aquaculture. The aim of this study was to establish a scientific medication regimen for the prevention and therapy of the mutidrug-resistant A. hydrophila infection.

Sulfonic acid-functionalized polyallylamine (sevelamer) as an efficient reusable strong solid acid catalyst for the synthesis of xanthenes derivatives.

Sevelamer (polyallyamine resin)-supported sulfonic acid (S-SO3H) has been prepared from the reaction of sevelamer with chlorosulfonic acid and characterized using FT-IR spectroscopy, scanning electronmicroscopy (SEM) and thermogravimetric analysis (TGA). The catalytic activity of S-SO3H was investigated in the synthesis of 1,8-dioxo-octahydroxanthene derivatives. All of the reactions were fast and gave excellent yields. The catalyst was easily recovered and reused for 5 runs without significant loss of its catalytic activity.

Myo-inositol as an adjuvant to florfenicol against Aeromonas hydrophila infection in common carp Cyprinus carpio.

Florfenicol, a synthetic drug with chemical structure and spectrum of antibacterial activity similar to chloramphenicol, has been shown to be effective against a number of bacterial pathogens. However, there are increasing signs of florfenicol-resistant bacteria due to the misuse and overuse of florfenicol in aquaculture. In the present study, florfenicol had a higher bactericidal efficacy in the presence of myo-inositol, which may be due to the ability of myo-inositol to increase susceptibility of Aeromonas hydrophila to florfenicol. Furthermore, in two different infected models, co-administration of myo-inositol and florfenicol significantly reduced the bacterial load in the liver, kidney and spleen tissues of A. hydrophila-infected Cyprinus carpio, and greatly increased the survival rate of infected fish. Finally, it was also found that myo-inositol exhibited synergistic action with other antibiotic drugs including neomycin sulfate, ceftriaxone and enrofloxacin. The results obtained in this study suggest that myo-inositol as an efficient adjuvant to antibiotic drugs could be useful in increasing the antimicrobial activity of antibiotic drugs against A. hydrophila infection, and could also be useful to help decrease the occurrence of antibiotic overuse in aquaculture.

Pyruvate cycle increases aminoglycoside efficacy and provides respiratory energy in bacteria.

The emergence and ongoing spread of multidrug-resistant bacteria puts humans and other species at risk for potentially lethal infections. Thus, novel antibiotics or alternative approaches are needed to target drug-resistant bacteria, and metabolic modulation has been documented to improve antibiotic efficacy, but the relevant metabolic mechanisms require more studies. Here, we show that glutamate potentiates aminoglycoside antibiotics, resulting in improved elimination of antibiotic-resistant pathogens. When exploring the metabolic flux of glutamate, it was found that the enzymes that link the phosphoenolpyruvate (PEP)-pyruvate-AcCoA pathway to the TCA cycle were key players in this increased efficacy. Together, the PEP-pyruvate-AcCoA pathway and TCA cycle can be considered the pyruvate cycle (P cycle). Our results show that inhibition or gene depletion of the enzymes in the P cycle shut down the TCA cycle even in the presence of excess carbon sources, and that the P cycle operates routinely as a general mechanism for energy production and regulation in Escherichia coli and Edwardsiella tarda These findings address metabolic mechanisms of metabolite-induced potentiation and fundamental questions about bacterial biochemistry and energy metabolism.

C-Terminal Domain of Hemocyanin, a Major Antimicrobial Protein from Litopenaeus vannamei: Structural Homology with Immunoglobulins and Molecular Diversity.

Invertebrates rely heavily on immune-like molecules with highly diversified variability so as to counteract infections. However, the mechanisms and the relationship between this variability and functionalities are not well understood. Here, we showed that the C-terminal domain of hemocyanin (HMC) from shrimp Litopenaeus vannamei contained an evolutionary conserved domain with highly variable genetic sequence, which is structurally homologous to immunoglobulin (Ig). This domain is responsible for recognizing and binding to bacteria or red blood cells, initiating agglutination and hemolysis. Furthermore, when HMC is separated into three fractions using anti-human IgM, IgG, or IgA, the subpopulation, which reacted with anti-human IgM (HMC-M), showed the most significant antimicrobial activity. The high potency of HMC-M is a consequence of glycosylation, as it contains high abundance of α-d-mannose relative to α-d-glucose and N-acetyl-d-galactosamine. Thus, the removal of these glycans abolished the antimicrobial activity of HMC-M. Our results present a comprehensive investigation of the role of HMC in fighting against infections through genetic variability and epigenetic modification.

Synthesis of Carbazoles and Carbazole-Containing Heterocycles via Rhodium-Catalyzed Tandem Carbonylative Benzannulations.

Polycyclic aromatic compounds are important constituents of pharmaceuticals and other materials. We have developed a series of Rh-catalyzed tandem carbonylative benzannulations for the synthesis of tri-, tetra-, and pentacyclic heterocycles from different types of aryl propargylic alcohols. These tandem reactions provide efficient access to highly substituted carbazoles, furocarbazoles, pyrrolocarbazoles, thiophenocarbazoles, and indolocarbazoles. While tricyclic heterocycles could be derived from vinyl aryl propargylic alcohols, tetra- and pentacyclic heterocycles were synthesized from diaryl propargylic alcohols. The tandem carbonylative benzannulation is initiated by a π-acidic rhodium(I) catalyst-mediated nucleophilic addition to alkyne to generate a key metal-carbene intermediate, which is then trapped by carbon monoxide to form a ketene species for 6π electrocyclization. Overall, three bonds and two rings are formed in all of these tandem carbonylative benzannulation reactions.

Rhodium-Catalyzed Intramolecular [5+2] Cycloaddition of Inverted 3-Acyloxy-1,4-enyne and Alkyne: Experimental and Theoretical Studies.

By switching the position of the alkene and alkyne, a new type of 3-acyloxy-1,4-enyne (ACE) five-carbon building block was developed for Rh-catalyzed intramolecular [5+2] cycloaddition. An electron-withdrawing acyl group on the alkyne termini of the ACE was essential for a regioselective 1,2-acyloxy migration. This new method provided bicyclic [5.3.0]decatrienes that are different from previous methods because of the positions of the alkenes and the acyloxy group. Multiple mechanistic pathways become possible for this new [5+2] cycloaddition and they are investigated by computational studies.

Exogenous glycine and serine promote growth and antifungal activity of Penicillium citrinum W1 from the south-west Indian Ocean.

PcPAF is a novel antifungal protein identified by our recent study, which is produced by a fungal strain Penicillium citrinum W1 isolated from a south-west Indian Ocean sediment sample. The present study identified glycine as a potential metabolite which increased the fungal growth and promoted antifungal activity. Then, GC/MS based metabolomics was used to disclose the metabolic mechanism manipulated by glycine. With the aid of unsupervised hierarchical clustering analysis and supervised orthogonal partial least-squares-discriminant analysis, the intracellular metabolite profiles were distinguished among two glycine-treated groups and control. 43 and 47 significantly varied metabolites were detected in 2.5 mM or 5 mM glycine-treated groups and involved in seven and eight pathways, respectively. Furthermore, exogenous serine, which is converted from glycine, showed the same potential as glycine did. Our findings not only identify glycine and serine as nutrients which promoted P. citrinum W1 growth and increased antifungal activity, but also highlight the way to utilize metabolomics for an understanding of metabolic mechanism manipulated by an exogenous compound.

L-proline increases survival of tilapias infected by Streptococcus agalactiae in higher water temperature.

Streptococcosis causes massive tilapia kills, which results in heavy economic losses of tilapia farming industry. Out of the Streptococcosis, Streptococcus agalactiae is the major pathogen. The bacterium causes higher mortality of tilapias in higher than lower temperatures. However, effect of temperature on metabolic regulation which is related to the mortality is largely unknown. The present study showed 50% and 70% mortality of tilapias cultured in 25 °C and 30 °C, respectively, in comparison with no death in 20 °C following infection caused by S. agalactiae. Then, GC/MS based metabolomics was used to investigate a global metabolic response of tilapia liver to the two higher water temperatures compared to 20 °C. Thirty-six and forty-five varied abundance of metabolites were identified in livers of tilapias cultured at 25 °C and 30 °C, respectively. More decreasing abundance of amino acids and increasing abundance of carbohydrates were detected in 30 °C than 25 °C groups. On the other hand, out of the pathways enriched, the first five biggest impact pathways belong to amino acid metabolism. Decreasing abundance of l-proline was identified as a crucial biomarker for indexing higher water temperature and a potential modulator to reduce the high death. This was validated by engineering injection or oral addition of l-proline. Exogenous l-proline led to elevated amino acid metabolism, which contributes to the elevated survivals. Our findings provide a potential metabolic modulator for controlling the disease, and shed some light on host metabolic prevention to infectious diseases.

Gut CaVP is an innate immune protein against bacterial challenge in amphioxus Branchiostoma belcheri.

The importance of calcium-binding proteins in immune response of vertebrates is determined, but whether they have the role in invertebrates is largely unknown. In the present study, phylogenetic analysis indicated that calcium vector protein (CaVP), a protein unique to amphioxus, shared 68% similarity in amino acid sequence with human and mouse calmodulin (CaM). CaVP cDNA was cloned into a bacterial vector pET-32a, and its His-tagged fusion protein was produced in Eschherichia coli cells (BL21). The recombinant CaVP was purified by Ni-NTA column and SDS-PAGE, and then utilized for antibody preparing. The prepared antibodies could recognize amphioxus CaVP with high specificity. Further analysis by Western blotting showed that CaVP was detected in muscle and humoral fluid of normal animals and appeared in gut of bacterial immunized or challenged amphioxus. Interestingly, gut CaVP was significantly higher in a healthy sub-group than a wounded sub-group post bacterial challenge. This response was detected strongly in immunization and challenge by the same Gram-negative bacterium Vibro parahaemolyticus and weakly in immunization by V. parahaemolyticus and then challenge by Gram-negative Aeromonas hydrophila, whereas no any feedback was found in immunization by V. parahaemolyticus and challenge by Gram-positive Staphylococcus aureus. These findings indicate the importance of gut CaVP in response to bacterial challenge.