Jorge Olivares

Genetic Determinants Involved in the Susceptibility of Pseudomonas aeruginosa to β-Lactam Antibiotics

ABSTRACT The resistome of P. aeruginosa for three β-lactam antibiotics, namely, ceftazidime, imipenem, and meropenem, was deciphered by screening a comprehensive PA14 mutant library for mutants with increased or reduced susceptibility to these antimicrobials. Confirmation of the phenotypes of all selected mutants was performed by Etest. Of the total of 78 confirmed mutants, 41 demonstrated a reduced susceptibility phenotype and 37 a supersusceptibility (i.e., altered intrinsic resistance) phenotype, with 6 mutants demonstrating a mixed phenotype, depending on the antibiotic. Only three mutants demonstrated reduced (PA0908) or increased (glnK and ftsK) susceptibility to all three antibiotics. Overall, the mutant profiles of susceptibility suggested distinct mechanisms of action and resistance for the three antibiotics despite their similar structures. More detailed analysis indicated important roles for novel and known β-lactamase regulatory genes, for genes with likely involvement in barrier function, and for a range of regulators of alginate biosynthesis.

RND multidrug efflux pumps: what are they good for?

Multidrug efflux pumps are chromosomally encoded genetic elements capable of mediating resistance to toxic compounds in several life forms. In bacteria, these elements are involved in intrinsic and acquired resistance to antibiotics. Unlike other well-known horizontally acquired antibiotic resistance determinants, genes encoding for multidrug efflux pumps belong to the core of bacterial genomes and thus have evolved over millions of years. The selective pressure stemming from the use of antibiotics to treat bacterial infections is relatively recent in evolutionary terms. Therefore, it is unlikely that these elements have evolved in response to antibiotics. In the last years, several studies have identified numerous functions for efflux pumps that go beyond antibiotic extrusion. In this review we present some examples of these functions that range from bacterial interactions with plant or animal hosts, to the detoxification of metabolic intermediates or the maintenance of cellular homeostasis.

Characterization of the Polymyxin B Resistome of Pseudomonas aeruginosa

ABSTRACT Multidrug resistance in Pseudomonas aeruginosa is increasingly becoming a threat for human health. Indeed, some strains are resistant to almost all currently available antibiotics, leaving very limited choices for antimicrobial therapy. In many such cases, polymyxins are the only available option, although as their utilization increases so does the isolation of resistant strains. In this study, we screened a comprehensive PA14 mutant library to identify genes involved in changes of susceptibility to polymyxin B in P. aeruginosa. Surprisingly, our screening revealed that the polymyxin B resistome of this microorganism is fairly small. Thus, only one resistant mutant and 17 different susceptibility/intrinsic resistance determinants were identified. Among the susceptible mutants, a significant number carried transposon insertions in lipopolysaccharide (LPS)-related genes. LPS analysis revealed that four of these mutants (galU, lptC, wapR, and ssg) had an altered banding profile in SDS-polyacrylamide gels and Western blots, with three of them exhibiting LPS core truncation and lack of O-antigen decoration. Further characterization of these four mutants showed that their increased susceptibility to polymyxin B was partly due to increased basal outer membrane permeability. Additionally, these mutants also lacked the aminoarabinose-substituted lipid A species observed in the wild type upon growth in low magnesium. Overall, our results emphasize the importance of LPS integrity and lipid A modification in resistance to polymyxins in P. aeruginosa, highlighting the relevance of characterizing the genes that affect biosynthesis of cell surface structures in this pathogen to follow the evolution of peptide resistance in the clinic.

Overproduction of the multidrug efflux pump MexEF-OprN does not impair Pseudomonas aeruginosa fitness in competition tests, but produces specific changes in bacterial regulatory networks.

It is generally assumed that acquisition of antibiotic resistance leads to non-specific fitness costs. We have tested the alternative hypothesis that acquisition of antibiotic resistance may not always produce a general burden to the microorganisms, as measured in competition tests, but rather lead to specific changes in bacterial physiology. To this end we studied the effect of overproducing the multidrug efflux pump MexEF-OprN on Pseudomonas aeruginosa due to a constitutive activation of MexT, the transcriptional activator of the mexEF-oprN genes. We found that overexpression of MexEF-OprN does not cause a significant decrease in P.aeruginosa fitness in classical competition tests, indicating the absence of a large metabolic burden and that any possible negative effects might be observed only under specific conditions. Transcriptomic analyses revealed that overexpression of MexEF-OprN results in reduced expression of several quorum-sensing regulated genes. We traced back this phenotype to a delay in PQS production due to extrusion of kynurenine, a PQS precursor, through the efflux pump. Type VI secretion was also impaired. A Caenorhabditis elegans model demonstrated that overproduction of MexEF-OprN impairs virulence in P.aeruginosa. This effect was mainly due to the activity of the efflux pump, and not to MexT, despite the fact that the latter regulates Type III and Type VI secretion. Altogether, these data indicate that antibiotic resistance can produce modifications in the bacterial regulatory networks with relevant consequences for the bacterial behaviour in specific ecosystems, including the infected host.

The intrinsic resistome of bacterial pathogens

Intrinsically resistant bacteria have emerged as a relevant health problem in the last years. Those bacterial species, several of them with an environmental origin, present naturally low-level susceptibility to several drugs. It has been proposed that intrinsic resistance is mainly the consequence of the impermeability of cellular envelopes, the activity of multidrug efflux pumps or the lack of appropriate targets for a given family of drugs. However, recently published articles indicate that the characteristic phenotype of susceptibility to antibiotics of a given bacterial species depends on the concerted activity of several elements, what has been named as intrinsic resistome. These determinants comprise not just classical resistance genes. Other elements, several of them involved in basic bacterial metabolic processes, are of relevance for the intrinsic resistance of bacterial pathogens. In the present review we analyze recent publications on the intrinsic resistomes of Escherichia coli and Pseudomonas aeruginosa. We present as well information on the role that global regulators of bacterial metabolism, as Crc from P. aeruginosa, may have on modulating bacterial susceptibility to antibiotics. Finally, we discuss the possibility of searching inhibitors of the intrinsic resistome in the aim of improving the activity of drugs currently in use for clinical practice.

The intrinsic resistome of Pseudomonas aeruginosa to β-lactams.

Pseudomonas aeruginosa is a relevant opportunistic pathogen particularly problematic due to its low intrinsic susceptibility to antibiotics. Intrinsic resistance has been traditionally attributed to the low permeability of cellular envelopes together with the presence of chromosomally-encoded detoxification systems such as multidrug efflux pumps or antibiotic inactivating enzymes. However, some recently published articles indicate that several other elements can contribute to the phenotype of intrinsic resistance of bacterial pathogens. In a recently published article, we explored the chromosomally-encoded determinants that contribute to the phenotype of susceptibility of P. aeruginosa to ceftazidime, imipenem and carbapenem. Using a comprehensive library of transposon-tagged insertion mutants, we found 37 loci in the chromosome of P. aeruginosa that contributed to its intrinsic resistance, because mutants in these loci were more susceptible to antibiotics than their parental strain. 41 further loci could potentially be involved in the acquisition of resistance, because mutants in these loci were less susceptible than their wild-type counterpart. These results indicate that the intrinsic resistome of P. aeruginosa involves several elements, belonging to different functional families and cannot be considered as a specific mechanism of adaptation to the recent usage of antibiotics as therapeutic agents. In the current article, we summarize the findings of the paper and discuss their implications for understanding the evolution of antibiotic resistance and for defining novel targets for the search of new antimicrobials. Finally, the validity of recent theories on the mechanisms of action of antibiotics is discussed taken into consideration the results of our paper and other recently published works on the mechanisms of intrinsic resistance to antibiotics of P. aeruginosa.

Metabolic Compensation of Fitness Costs Associated with Overexpression of the Multidrug Efflux Pump MexEF-OprN in Pseudomonas aeruginosa

ABSTRACT The acquisition of antibiotic resistance has been associated with a possible nonspecific, metabolic burden that is reflected in decreased fitness among resistant bacteria. We have recently demonstrated that overexpression of the MexEF-OprN multidrug efflux pump does not produce a metabolic burden when measured by classical competitions tests but rather leads to a number of changes in the organisms physiology. One of these changes is the untimely activation of the nitrate respiratory chain under aerobic conditions. MexEF-OprN is a proton/substrate antiporter. Overexpression of this element should result in a constant influx of protons, which may lead to cytoplasmic acidification. Acidification was not observed in aerobiosis, a situation in which the MexEF-overproducing mutant increases oxygen consumption. This enhanced oxygen uptake serves to eliminate intracellular proton accumulation, preventing the cytoplasmic acidification that was observed exclusively under anaerobic conditions, a situation in which the fitness of the MexEF-OprN-overproducing mutant decreases. Finally, we determined that the early activation of the nitrate respiratory chain under aerobic conditions plays a role in preventing a deleterious effect associated with the overexpression of MexEF-OprN. Our results show that metabolic rewiring may assist in overcoming the potential fitness cost associated with the acquisition of antibiotic resistance. Furthermore, the capability to metabolically compensate for this effect is habitat dependent, as demonstrated by our results under anaerobic conditions. The development of drugs that prevent metabolic compensation of fitness costs may help to reduce the persistence and dissemination of antibiotic resistance.

Determination of minimal concentration of Piscirickettsia salmonis in water columns to establish a fallowing period in salmon farms

Abstract A highly sensitive real-time PCR procedure to detect and quantify the number of Pisciricketsia salmonis units in seawater samples from affected farm sites has been developed. The purpose was to determine a fallowing period that would allow safe restocking of the target farm with new fish. Bacterial load was determined in water samples by comparing the obtained amplification values against a standard curve generated by the amplification of known concentrations of the ITS-ribosomal component of P. salmonis DNA, cloned in a suitable vector. The standard curve was linear over the range of 10(1)-10(10) log units. Target samples were taken every 10 days over a 40-day period, at 5 m depth and at the surface. In a highly affected area of southern Chile, the number of bacterial units in farm water decreased to zero at day 50. Therefore, a fallowing period of 50 days post-removal of cages of affected fish appears to be appropriate before restocking. This procedure could be adapted to control disease problems because of other pathogens in fish farm waters.

Draft Genome Sequence of Antarctic Pseudomonas sp. Strain KG01 with Full Potential for Biotechnological Applications

ABSTRACT We report here the draft genome sequence of a free-living psychrotolerant, Pseudomonas sp. strain KG01, isolated from an Antarctic soil sample and displaying interesting antimicrobial and surfactant activities. The sequence is 6.3 Mb long and includes 5,648 predicted-coding sequences.

Antibiotics from Pseudomonas reptilivora II. Isolation, Purification, and Properties

Under well-established culture conditions, Pseudomonas reptilivora produced several antibiotics that have been purified by solvent extraction, chromatography in Sephadex G-25, electrophoresis, and paper chromatography in different solvent systems. Activity has been monitored at the different steps of isolation and purification by measurement of the inhibition of the growth of Staphylococcus aureus by the cylinder-plate method, as well as by bioautography of chromatograms and electropherograms. Three antibiotics have been isolated and named A, B1, and B2. The B1 and B2 activities were studied in greater detail than A. The B1 substance was crystallized, and its chemical properties were found to coincide with those of YC 73 or fluopsin C described by Egawa et al. and Itoh et al., respectively. Images