Within-species variability of antibiotic interactions in Gram-negative bacteria.

Treatments with antibiotic combinations are becoming increasingly important even though the supposed clinical benefits of combinations are, in many cases, unclear. Here, we systematically examined how several clinically used antibiotics interact and affect the antimicrobial efficacy against five especially problematic Gram-negative pathogens. A total of 232 bacterial isolates were tested against different pairwise antibiotic combinations spanning five classes, and the ability of all combinations in inhibiting growth was quantified. Descriptive statistics, principal component analysis (PCA), and Spearman's rank correlation matrix were used to determine the correlations between the different combinations on interaction outcome. Several important conclusions can be drawn from the 696 examined interactions. Firstly, within a species, the interactions are in general conserved but can be isolate-specific for a given antibiotic combination and can range from antagonistic to synergistic. Secondly, additive and antagonistic interactions are the most common observed across species and antibiotics, with 87.1% of isolate-antibiotic combinations being additive, 11.6% antagonistic, and only 0.3% showing synergy. These findings suggest that to achieve the highest precision and efficacy of combination therapy, not only isolate-specific interaction profiling ought to be routinely performed, in particular to avoid using drug combinations that show antagonistic interaction and an expected associated reduction in efficacy, but also discovering rare and potentially valuable synergistic interactions.IMPORTANCEAntibiotic combinations are often used to treat bacterial infections, which aim to increase treatment efficacy and reduce resistance evolution. Typically, it is assumed that one specific antibiotic combination has the same effect on different isolates of the same species, i.e., the interaction is conserved. Here, we tested this idea by examining how several clinically used antibiotics interact and affect the antimicrobial efficacy against several bacterial pathogens. Our results show that, even though within a species the interactions are often conserved, there are also isolate-specific differences for a given antibiotic combination that can range from antagonistic to synergistic. These findings suggest that isolate-specific interaction profiling ought to be performed in clinical microbiology routine to avoid using antagonistic drug combinations that might reduce treatment efficacy.

Novel regulatory mechanism of establishment genes of conjugative plasmids.

The principal route for dissemination of antibiotic resistance genes is conjugation by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugative elements contain genes that are important for their establishment in the new host, for instance by counteracting the host defense mechanisms acting against incoming foreign DNA. Little is known about these establishment genes and how they are regulated. Here, we deciphered the regulation mechanism of possible establishment genes of plasmid p576 from the Gram-positive bacterium Bacillus pumilus. Unlike the ssDNA promoters described for some conjugative plasmids, the four promoters of these p576 genes are repressed by a repressor protein, which we named Reg576. Reg576 also regulates its own expression. After transfer of the DNA, these genes are de-repressed for a period of time until sufficient Reg576 is synthesized to repress the promoters again. Complementary in vivo and in vitro analyses showed that different operator configurations in the promoter regions of these genes lead to different responses to Reg576. Each operator is bound with extreme cooperativity by two Reg576-dimers. The X-ray structure revealed that Reg576 has a Ribbon-Helix-Helix core and provided important insights into the high cooperativity of DNA recognition.

Influence of the Hfq and Crc global regulators on the control of iron homeostasis in Pseudomonas putida.

Metabolically versatile bacteria use catabolite repression control to select their preferred carbon sources, thus optimizing carbon metabolism. In pseudomonads, this occurs through the combined action of the proteins Hfq and Crc, which form stable tripartite complexes at target mRNAs, inhibiting their translation. The activity of Hfq/Crc is antagonised by small RNAs of the CrcZ family, the amounts of which vary according to carbon availability. The present work examines the role of Pseudomonas putida Hfq protein under conditions of low-level catabolite repression, in which Crc protein would have a minor role since it is sequestered by CrcZ/CrcY. The results suggest that, under these conditions, Hfq remains operative and plays an important role in iron homeostasis. In this scenario, Crc appears to participate indirectly by helping CrcZ/CrcY to control the amount of free Hfq in the cell. Iron homeostasis in pseudomonads relies on regulatory elements such as the Fur protein, the PrrF1-F2 sRNAs, and several extracytoplasmic sigma factors. Our results show that the absence of Hfq is paralleled by a reduction in PrrF1-F2 small RNAs. Hfq thus provides a regulatory link between iron and carbon metabolism, coordinating the iron supply to meet the needs of the enzymes operational under particular nutritional regimes.