Fighting the Enemy: One Health Approach Against Microbial Resistance

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Draft whole-genome sequence of the antibiotic-producing soil isolate Pseudomonas sp. strain 250J

Bacteria of the genus Pseudomonas are becoming increasing well known for their ability to produce a wide range of antimicrobial compounds. In a largescale screening for antibiotic producers, we identified a soil isolate that uses 4-hydroxyphenylacetate as the sole carbon source, Pseudomonas sp. strain 250J, which produces cyclic lipodepsipeptides of the xantholysin family during the stationary phase of growth. The closest relatives of this strain are Pseudomonas mosselii, Pseudomonas soli and Pseudomonas entomophila. Sequencing of the 250J genome allowed us to find the genes relevant to antibiotic production, those which allow utilization of 4-hydroxyphenylacetate as a sole carbon source and a set of genes potentially involved in biocontrol.This work was supported by Fondo Social Europeo and Fondos FEDER from the European Union, through several projects Consolider-Ingenio CSD2007-00005 and BIO2010-17227

Genome Sequence of Serratia plymuthica A153, a Model Rhizobacterium for the Investigation of the Synthesis and Regulation of Haterumalides, Zeamine, and Andrimid.

The rhizobacterium Serratia plymuthica A153 is a Gram-negative bacterium belonging to the family Enterobacteriaceae Here, we present the genome sequence of this strain, which produces multiple bioactive secondary metabolites, including the halogenated macrolide oocydin A, the polyamino antibiotic zeamine, and the bacterial acetyl-CoA carboxylase inhibitor andrimid.Work in the Salmond laboratory was supported by the Biotechnology and Biological Sciences Research Council (BBSRC, United Kingdom). Miguel A. Matilla was supported by the EU Marie-Curie intra-European Fellowship For Career Development (FP7-PEOPLE-2011-IEF) grant 298003 and the Spanish Ministry of Economy and Competitiveness Postdoctoral Research Program, Juan de la Cierva (JCI-2012-11815). The Tino Krell laboratory is supported by FEDER funds and Fondo Social Europeo through grants from the Junta de Andalucía (grant CVI-7335) and the Spanish Ministry for Economy and Competitiveness (grants BIO2013- 42297 and RTC-2014-1777-3).This is the final version of the article. It first appeared from the American Society for Microbiology via http://dx.doi.org/10.1128/genomeA.00373-1

Iron Uptake Analysis in a Set of Clinical Isolates of Pseudomonas putida

Pseudomonas putida strains are frequent inhabitants of soil and aquatic niches and they are occasionally isolated from hospital environments. As the available iron sources in human tissues, edaphic, and aquatic niches are different, we have analyzed iron-uptake related genes in different P. putida strains that were isolated from all these environments. We found that these isolates can be grouped into different clades according to the genetics of siderophore biosynthesis and recycling. The pyoverdine locus of the six P. putida clinical isolates that have so far been completely sequenced, are not closely related; three strains (P. putida HB13667, HB3267, and NBRC14164T) are grouped in Clade I and the other three in Clade II, suggesting possible different origins and evolution. In one clinical strain, P. putida HB4184, the production of siderophores is induced under high osmolarity conditions. The pyoverdine locus in this strain is closely related to that of strain P. putida HB001 which was isolated from sandy shore soil of the Yellow Sea in Korean marine sand, suggesting their possible origin, and evolution. The acquisition of two unique TonB-dependent transporters for xenosiderophore acquisition, similar to those existing in the opportunistic pathogen P. aeruginosa PAO, is an interesting adaptation trait of the clinical strain P. putida H8234 that may confer adaptive advantages under low iron availability conditions.Work in our laboratories was supported by ERANET Pathogenomics program through the ADHERS project (Ref: BIO2008-04419-E) and Fondos FEDER from the European Union through project BIO2010-17227 of the Spanish Ministry of Economy and Competitivity. The work in Abengoa Research was funded by H2020 grant Empowerputida number 65703.Peer reviewedPeer Reviewe

Differential transcriptional response to antibiotics by Pseudomonas putida DOT-T1E

Multi-drug resistant bacteria are a major threat to humanity, especially because the current battery of known antibiotics is not sufficient to combat infections produced by these microbes. Therefore, the study of how current antibiotics act and how bacteria defend themselves against antibiotics is of critical importance. Pseudomonas putida DOT-T1E exhibits an impressive array of RND efflux pumps, which confer this microorganism high resistance to organic solvents and antibiotics that would kill most other microorganisms. We have chosen DOT-T1E as a model microbe to study the microbial responses to a wide battery of antibiotics (chloramphenicol, rifampicin, tetracycline, ciprofloxacin, ampicillin, kanamycin, spectinomycin and gentamicin). Ribonucleic acid sequencing (RNA)-seq analyses revealed that each antibiotic provokes a unique transcriptional response profile in DOT-T1E. While many of the genes identified were related to known antibiotic targets, others were unrelated or encoded hypothetical proteins. These results indicate that our knowledge of antibiotic resistance mechanisms is still partial. We also identified 138 new small RNAs (sRNAs) in DOT-T1E, dramatically adding to the 16 that have been previously described. Importantly, our results reveal that a correlation exists between the expression of messenger RNA and sRNA, indicating that some of these sRNAs are likely involved in fine tuning the expression of antibiotic resistance genes.Work of Juan LRamosin Denmark wassupported by an Otto Monsted Foundation visiting professorship. Work in Granada was founded by grants from the Spanish Ministry of Economics and Competitivity BIO2010-17227

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

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

Full Transcriptomic Response of Pseudomonas aeruginosa to an Inulin-Derived Fructooligosaccharide

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2020.00202/full#supplementary-materialPseudomonas aeruginosa is an ubiquitous gram-negative opportunistic human pathogen which is not considered part of the human commensal gut microbiota. However, depletion of the intestinal microbiota (Dysbiosis) following antibiotic treatment facilitates the colonization of the intestinal tract by Multidrug-Resistant P. aeruginosa. One possible strategy is based on the use of functional foods with prebiotic activity. The bifidogenic effect of the prebiotic inulin and its hydrolyzed form (fructooligosaccharide: FOS) is well established since they promote the growth of specific beneficial (probiotic) gut bacteria such as bifidobacteria. Previous studies of the opportunistic nosocomial pathogen Pseudomonas aeruginosa PAO1 have shown that inulin and to a greater extent FOS reduce growth and biofilm formation, which was found to be due to a decrease in motility and exotoxin secretion. However, the transcriptional basis for these phenotypic alterations remains unclear. To address this question we conducted RNAsequence analysis. Changes in the transcript level induced by inulin and FOS were similar, but a set of transcript levels were increased in response to inulin and reduced in the presence of FOS. In the presence of inulin or FOS, 260 and 217 transcript levels, respectively, were altered compared to the control to which no polysaccharide was added. Importantly, changes in transcript levels of 57 and 83 genes were found to be specific for either inulin or FOS, respectively, indicating that both compounds trigger different changes. Gene pathway analyses of differentially expressed genes (DEG) revealed a specific FOS-mediated reduction in transcript levels of genes that participate in several canonical pathways involved in metabolism and growth, motility, biofilm formation, b-lactamase resistance, and in the modulation of type III and VI secretion systems; results that have been partially verified by real time quantitative PCR measurements. Moreover, we have identified a genomic island formed by a cluster of 15 genes, encoding uncharacterized proteins, which were repressed in the presence of FOS. The analysis of isogenic mutants has shown that genes of this genomic island encode proteins involved in growth, biofilm formation and motility. These results indicate that FOS selectively modulates bacterial pathogenicity by interfering with different signaling pathways.This work was supported by grants from the Spanish Ministry for Economy and Competitiveness (AGL2017-85270-R). CS is funded by the program Juan de la Cierva-Formación (FJCI-2015-23810)

Specific Gene Loci of Clinical Pseudomonas putida Isolates

Pseudomonas putida are ubiquitous inhabitants of soils and clinical isolates of this species have been seldom described. Clinical isolates show significant variability in their ability to cause damage to hosts because some of them are able to modulate the host’s immune response. In the current study, comparisons between the genomes of different clinical and environmental strains of P. putida were done to identify genetic clusters shared by clinical isolates that are not present in environmental isolates. We show that in clinical strains specific genes are mostly present on transposons, and that this set of genes exhibit high identity with genes found in pathogens and opportunistic pathogens. The set of genes prevalent in P. putida clinical isolates, and absent in environmental isolates, are related with survival under oxidative stress conditions, resistance against biocides, amino acid metabolism and toxin/antitoxin (TA) systems. This set of functions have influence in colonization and survival within human tissues, since they avoid host immune response or enhance stress resistance. An in depth bioinformatic analysis was also carried out to identify genetic clusters that are exclusive to each of the clinical isolates and that correlate with phenotypical differences between them, a secretion system type III-like was found in one of these clinical strains, a determinant of pathogenicity in Gram-negative bacteria.Work in the authors’ laboratories was supported by ERANET Pathogenomics programme through the ADHERS project (Ref: BIO2008-04419-E) and Fondos FEDER from the European Union through project BIO2010-17227 of the Ministry of Economy and Competitivity of Spain. Bio-Iliberis R&D provided supportPeer reviewe

Characterization of an extremophile bacterial acid phosphatase derived from metagenomics analysis

Acid phosphatases are enzymes that play a crucial role in the hydrolysis of various organophosphorous molecules. A putative acid phosphatase called FS6 was identified using genetic profiles and sequences from different environments. FS6 showed high sequence similarity to type C acid phosphatases and retained more than 30% of consensus residues in its protein sequence. A histidine-tagged recombinant FS6 produced in Escherichia coli exhibited extremophile properties, functioning effectively in a broad pH range between 3.5 and 8.5. The enzyme demonstrated optimal activity at temperatures between 25 and 50°C, with a melting temperature of 51.6°C. Kinetic parameters were determined using various substrates, and the reaction catalysed by FS6 with physiological substrates was at least 100-fold more efficient than with p-nitrophenyl phosphate. Furthermore, FS6 was found to be a decamer in solution, unlike the dimeric forms of crystallized proteins in its family.Grants from the Ministry of Science through grants from the Plan Nacional 2021 (PID2021-123469OBI00) and Transición Ecológica (TED2021129632BI00) and European Commission projects EJP soils (EJPSOIL862695) and PREPSOIL (HE/CL5SOILM/0102