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Microb Biotechnol. 2015 Jul;8(4):716-25. doi: 10.1111/1751-7915.12295. Epub 2015 Jun 8.

Efflux pump-deficient mutants as a platform to search for microbes that produce antibiotics.

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Department of Environmental Protection, Consejo Superior de Investigaciones Científicas, E-18008, Granada, Spain.
Bio-Iliberis R&D, Poligono Juncaril, calle Capileira nº 7, E-18121, Peligros, Granada, Spain.
Fundación MEDINA, Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía. Avda. del Conocimiento 3, Parque Tecnológico de Ciencias de la Salud, E-18100, Armilla, Granada, Spain.


Pseudomonas putida DOT-T1E-18 is a strain deficient in the major antibiotic efflux pump (TtgABC) that exhibits an overall increased susceptibility to a wide range of drugs when compared with the wild-type strain. We used this strain as a platform to search for microbes able to produce antibiotics that inhibit growth. A collection of 2400 isolates from soil, sediments and water was generated and a drop assay developed to identify, via growth inhibition halos, strains that prevent the growth of DOT-T1E-18 on solid Luria-Bertani plates. In this study, 35 different isolates that produced known and unknown antibiotics were identified. The most potent inhibitor of DOT-T1E-18 growth was an isolate named 250J that, through multi-locus sequence analysis, was identified as a Pseudomonas sp. strain. Culture supernatants of 250J contain four different xantholysins that prevent growth of Gram-positive bacteria, Gram-negative and fungi. Two of the xantholysins were produced in higher concentrations and purified. Xantholysin A was effective against Bacillus, Lysinibacillus and Rhodococcus strains, and the effect against these microbes was enhanced when used in combination with other antibiotics such as ampicillin, gentamicin and kanamycin. Xantholysin C was also efficient against Gram-positive bacteria and showed an interesting antimicrobial effect against Pseudomonas strains, and a synergistic inhibitory effect with ampicillin, chloramphenicol and gentamicin.

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