Aminoglycosides Antagonize Bacteriophage Proliferation, Attenuating Phage Suppression of Bacterial Growth, Biofilm Formation, and Antibiotic Resistance.

The common cooccurrence of antibiotics and phages in both natural and engineered environments underscores the need to understand their interactions and implications for bacterial control and antibiotic resistance propagation. Here, aminoglycoside antibiotics that inhibit protein synthesis (e.g., kanamycin and neomycin) impeded the replication of coliphage T3 and Bacillus phage BSP, reducing their infection efficiency and mitigating their hindrance of bacterial growth, biofilm formation, and tolerance to antibiotics. For example, treatment with phage T3 reduced subsequent biofilm formation by Escherichia coli liquid cultures to 53% ± 5% of that of the no-phage control, but a smaller reduction of biofilm formation (89% ± 10%) was observed for combined exposure to phage T3 and kanamycin. Despite sharing a similar mode of action with aminoglycosides (i.e., inhibiting protein synthesis) and antagonizing phage replication, albeit to a lesser degree, tetracyclines did not inhibit bacterial control by phages. Phage T3 combined with tetracycline showed higher suppression of biofilm formation than when combined with aminoglycosides (25% ± 6% of the no-phage control). The addition of phage T3 to E. coli suspensions with tetracycline also suppressed the development of tolerance to tetracycline. However, this suppression of antibiotic tolerance development disappeared when tetracycline was replaced with 3 mg/liter kanamycin, corroborating the greater antagonism with aminoglycosides. Overall, this study highlights this overlooked antagonistic effect on phage proliferation, which may attenuate phage suppression of bacterial growth, biofilm formation, antibiotic tolerance, and maintenance of antibiotic resistance genes. IMPORTANCE The coexistence of residual antibiotics and phages is common in many environments, which underscores the need to understand their interactive effects on bacteria and the implications for antibiotic resistance propagation. Here, aminoglycosides acting as bacterial protein synthesis inhibitors impeded the replication of various phages. This alleviated the suppressive effects of phages against bacterial growth and biofilm formation and diminished bacterial fitness costs that suppress the emergence of tolerance to antibiotics. We show that changes in bacteria caused by environmentally relevant concentrations of sublethal antibiotics can affect phage-host dynamics that are commonly overlooked in vitro but can result in unexpected environmental consequences.

A positive charge at position 33 of thioredoxin primarily affects its interaction with other proteins but not redox potential.

Oxidoreductases of the thioredoxin superfamily possess the C-X-X-C motif. The redox potentials vary over a wide range for these proteins. A crucial determinant of the redox potential has been attributed to the variation of the X-X dipeptide. Here, we substitute Lys for Gly at the first X of Escherichia coli thioredoxin to investigate how a positive charge would affect the redox potential. The substitution does not affect the protein's redox potential. The equilibrium constant obtained from pairwise reaction between the mutant and wild-type proteins equals 1.1, indicating that the replacement does not significantly affect the thiol-disulfide redox equilibrium. However, the catalytic efficiency of thioredoxin reductase on the G33K mutant decreases approximately 2.8 times compared to that of the wild type. The mutation mainly affects K(m), with little effect on k(cat). The mutation also inhibits thioredoxin's ability to reduce insulin disulfide by approximately one-half. Whether the mutant protein supports the growth of phages T3/7 and f1 was tested. The efficiency of plating (EOP) of T3/7 on the mutant strain decreases 5 times at 37 degrees C and 3 x 10(4) times at 42 degrees C relative to that of the wild-type strain, suggesting that interaction between phage gene 5 protein and thioredoxin is hindered. The mutation also reduces the EOP of phage f1 by 8-fold at 37 degrees C and 1.5-fold at 42 degrees C. The global structure of the mutant protein does not change when studied by CD and fluorescence spectra. Therefore, G33K does not significantly affect the overall structure or redox potential of thioredoxin, but primarily interferes with its interaction with other proteins. Together with the G33D mutation, the overall results show that a charged residue at the first X has a greater influence on the molecular interaction of the protein than the redox potential.

Gel electrophoretic analysis of bacteriophage assembly intermediates in bacteriophage plaques.

To increase the efficiency with which the phenotype of bacteriophage mutants is determined by gel electrophoresis, procedures are developed here for the preparation of the contents of bacteriophage plaques for gel electrophoresis. During the formation of plaques, the plaque-supporting upper layer gel is changed from the traditional agar gel to a gel made of a mixture of low-melt agaroses; the lower layer gel is eliminated. To extract particles from plaques, the plaque-supporting gel is disintegrated by both shaking and raising the temperature to 39-43 degrees C. During shaking, the gel is broken to domains that are 5-30 microns in diameter. After extraction, the contents of plaques are subjected to two electrophoretic analyses: (1) Nondenaturing agarose gel electrophoresis is performed after treatment with DNase. This procedure reveals both mature bacteriophage and immature capsids. (2) Nondenaturing agarose gel electrophoresis is performed after release of DNA from DNase-treated capsids. This latter procedure reveals both completely packaged (mature length) DNA and incompletely packaged (shorter than mature length) DNA. The amount of mature length DNA released per 2-3 mm plaque is 10-60 ng. In agreement with results previously obtained in liquid culture, most incompletely packaged DNA has the right, but not the left, mature T7 DNA end.