Exogenous Glucose Interferes with Antimicrobial-Mediated ROS Accumulation and Bacterial Death.

Glucose is widely used in the reconstitution of intravenous medications, which often include antimicrobials. How glucose affects antimicrobial activity has not been comprehensively studied. The present work reports that glucose added to bacteria growing in a rich medium suppresses the bactericidal but not the bacteriostatic activity of several antimicrobial classes, thereby revealing a phenomenon called glucose-mediated antimicrobial tolerance. Glucose, at concentrations corresponding to blood-sugar levels of humans, increased survival of Escherichia coli treated with quinolones, aminoglycosides, and cephalosporins with little effect on minimal inhibitory concentration. Glucose suppressed a ROS surge stimulated by ciprofloxacin. Genes involved in phosphorylated fructose metabolism contributed to glucose-mediated tolerance, since a pfkA deficiency, which blocks the formation of fructose-1,6-bisphosphate, eliminated protection by glucose. Disrupting the pentose phosphate pathway or the TCA cycle failed to alter glucose-mediated tolerance, consistent with an upstream involvement of phosphorylated fructose. Exogenous sodium pyruvate or sodium citrate reversed glucose-mediated antimicrobial tolerance. Both metabolites bypass the effects of fructose-1,6-bisphosphate, a compound known to scavenge hydroxyl radical and chelate iron, activities that suppress ROS accumulation. Treatment with these two compounds constitutes a novel way to mitigate the glucose-mediated antimicrobial tolerance that may exist during intravenous antimicrobial therapy, especially for diabetes patients.

Enhancement of ß-Lactam-Mediated Killing of Gram-Negative Bacteria by Lysine Hydrochloride.

Widespread bacterial resistance among Gram-negative bacteria is rapidly depleting our antimicrobial arsenal. Adjuvants that enhance the bactericidal activity of existing antibiotics provide a way to alleviate the resistance crisis, as new antimicrobials are becoming increasingly difficult to develop. The present work with Escherichia coli revealed that neutralized lysine (lysine hydrochloride) enhances the bactericidal activity of ß-lactams in addition to increasing bacteriostatic activity. When combined, lysine hydrochloride and ß-lactam increased expression of genes involved in the tricarboxylic acid (TCA) cycle and raised reactive oxygen species (ROS) levels; as expected, agents known to mitigate bactericidal effects of ROS reduced lethality from the combination treatment. Lysine hydrochloride had no enhancing effect on the lethal action of fluoroquinolones or aminoglycosides. Characterization of a tolerant mutant indicated involvement of the FtsH/HflkC membrane-embedded protease complex in lethality enhancement. The tolerant mutant, which carried a V86F substitution in FtsH, exhibited decreased lipopolysaccharide levels, reduced expression of TCA cycle genes, and reduced levels of ROS. Lethality enhancement by lysine hydrochloride was abolished by treating cultures with Ca2+ or Mg2+, cations known to stabilize the outer membrane. These data, plus damage observed by scanning electron microscopy, indicate that lysine stimulates ß-lactam lethality by disrupting the outer membrane. Lethality enhancement of ß-lactams by lysine hydrochloride was also observed with Acinetobacter baumannii and Pseudomonas aeruginosa, thereby suggesting that the phenomenon is common among Gram-negative bacteria. Arginine hydrochloride behaved in a similar way. Overall, the combination of lysine or arginine hydrochloride and ß-lactam offers a new way to increase ß-lactam lethality with Gram-negative pathogens. IMPORTANCE Antibiotic resistance among Gram-negative pathogens is a serious medical problem. The present work describes a new study in which a nontoxic nutrient increases the lethal action of clinically important ß-lactams. Elevated lethality is expected to reduce the emergence of resistant mutants. The effects were observed with significant pathogens (Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa), indicating widespread applicability. Examination of tolerant mutants and biochemical measurements revealed involvement of endogenous reactive oxygen species in response to outer membrane perturbation. These lysine hydrochloride-ß-lactam data support the hypothesis that lethal stressors can stimulate the accumulation of ROS. Genetic and biochemical work also revealed how an alteration in a membrane protease, FtsH, abolishes lysine stimulation of ß-lactam lethality. Overall, the work presents a method for antimicrobial enhancement that should be safe, easy to administer, and likely to apply to other nutrients, such as arginine.

Interplays of mutations in waaA, cmk, and ail contribute to phage resistance in Yersinia pestis.

Plague caused by Yersinia pestis remains a public health threat worldwide. Because multidrug-resistant Y. pestis strains have been found in both humans and animals, phage therapy has attracted increasing attention as an alternative strategy against plague. However, phage resistance is a potential drawback of phage therapies, and the mechanism of phage resistance in Y. pestis is yet to be investigated. In this study, we obtained a bacteriophage-resistant strain of Y. pestis (S56) by continuously challenging Y. pestis 614F with the bacteriophage Yep-phi. Genome analysis identified three mutations in strain S56: waaA* (9-bp in-frame deletion 249GTCATCGTG257), cmk* (10-bp frameshift deletion 15CCGGTGATAA24), and ail* (1-bp frameshift deletion A538). WaaA (3-deoxy-D-manno-octulosonic acid transferase) is a key enzyme in lipopolysaccharide biosynthesis. The waaA* mutation leads to decreased phage adsorption because of the failure to synthesize the lipopolysaccharide core. The mutation in cmk (encoding cytidine monophosphate kinase) increased phage resistance, independent of phage adsorption, and caused in vitro growth defects in Y. pestis. The mutation in ail inhibited phage adsorption while restoring the growth of the waaA null mutant and accelerating the growth of the cmk null mutant. Our results confirmed that mutations in the WaaA-Cmk-Ail cascade in Y. pestis contribute to resistance against bacteriophage. Our findings help in understanding the interactions between Y. pestis and its phages.

A broadly applicable, stress-mediated bacterial death pathway regulated by the phosphotransferase system (PTS) and the cAMP-Crp cascade.

Recent work indicates that killing of bacteria by diverse antimicrobial classes can involve reactive oxygen species (ROS), as if a common, self-destructive response to antibiotics occurs. However, the ROS-bacterial death theory has been challenged. To better understand stress-mediated bacterial death, we enriched spontaneous antideath mutants of Escherichia coli that survive treatment by diverse bactericidal agents that include antibiotics, disinfectants, and environmental stressors, without a priori consideration of ROS. The mutants retained bacteriostatic susceptibility, thereby ruling out resistance. Surprisingly, pan-tolerance arose from carbohydrate metabolism deficiencies in ptsI (phosphotransferase) and cyaA (adenyl cyclase); these genes displayed the activity of upstream regulators of a widely shared, stress-mediated death pathway. The antideath effect was reversed by genetic complementation, exogenous cAMP, or a Crp variant that bypasses cAMP binding for activation. Downstream events comprised a metabolic shift from the TCA cycle to glycolysis and to the pentose phosphate pathway, suppression of stress-mediated ATP surges, and reduced accumulation of ROS. These observations reveal how upstream signals from diverse stress-mediated lesions stimulate shared, late-stage, ROS-mediated events. Cultures of these stable, pan-tolerant mutants grew normally and were therefore distinct from tolerance derived from growth defects described previously. Pan-tolerance raises the potential for unrestricted disinfectant use to contribute to antibiotic tolerance and resistance. It also weakens host defenses, because three agents (hypochlorite, hydrogen peroxide, and low pH) affected by pan-tolerance are used by the immune system to fight infections. Understanding and manipulating the PtsI-CyaA-Crp­mediated death process can help better control pathogens and maintain beneficial microbiota during antimicrobial treatment.

Characterization of a novel class A carbapenemase PAD-1 from Paramesorhizobium desertii A-3-ET, a strain highly resistant to ß-lactam antibiotics.

Although clinical antibiotic-resistant bacteria have attracted tremendous attention in the microbiology community, the resistant bacteria that persist in natural environments have been overlooked for a longtime. We previously proposed a new species Paramesorhizobium desertii, isolated from the soil of the Taklimakan Desert in China that is highly resistant to most ß-lactam antibiotics. To identify potential ß-lactamase(s) in this bacteria, we first confirmed the carbapenemase activity in the freeze-thawed supernatant of a P. desertii A-3-ET culture using the modified Hodge assay. We then identified a novel chromosome-encoded carbapenemase (PAD-1) in strain A-3-ET, using a shotgun proteomic analysis of the supernatant and genomic information. The bioinformatics analysis indicated that PAD-1 is a class A carbapenemase. Subsequent enzyme kinetic assays with purified PAD-1 confirmed its carbapenemase activity, which is similar to that of clinically significant class A carbapenemases, including BKC-1 and KPC-2. Because the location in which A-3-ET was isolated is not affected by human activity, PAD-1 is unlikely to be associated with the selection pressures exerted by modern antibiotics. This study confirmed the diversity of antibiotic-resistant determinants in the environmental resistome.

A1S_2811, a CheA/Y-like hybrid two-component regulator from Acinetobacter baumannii ATCC17978, is involved in surface motility and biofilm formation in this bacterium.

Two-component systems in Acinetobacter baumannii are associated with its virulence, drug resistance, motility, biofilm formation, and other characteristics. In this study, we used RecAb , a genetic engineering method, to investigate the function of A1S_2811 in A. baumannii strain ATCC17978. A1S_2811, a hypothetical hybrid sensor histidine kinase/response regulator, has four histidine-containing phosphotransfer domains, a CheA-like regulatory domain, and a CheY-like receiver domain at its C terminus. Compared with the ATCC17978 strain, both surface motility and biofilm formation at the gas-liquid interface decreased significantly in the A1S_2811 knock-out strain. The number of pilus-like structures and the amount of extrapolymeric substances on the cell surface also decreased in the A1S_2811 null strain. Transcription of abaI, which encodes an N-acylhomoserine lactone synthase in A. baumannii , decreased significantly in the A1S_2811 null strain, and supplementation with synthetic N-(3-oxodecanoyl) homoserine-l-lactone rescued the surface motility and biofilm formation phenotype in the null mutant. We speculate that A1S_2811 regulates surface motility and biofilm formation, not by regulating type IV pili-associated genes expression, but by regulating the chaperone/usher pili-associated csuA/ABCDE operon and the AbaI-dependent quorum-sensing pathway-associated A1S_0112-0119 operon instead.