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      High pyocyanin production and non-motility of Pseudomonas aeruginosa isolates are correlated with septic shock or death in bacteremic patients

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          Abstract

          Studies of the outcome of Pseudomonas aeruginosa bacteremia ( Pab) have focused mainly on antibiotic appropriateness. However, P. aeruginosa possesses many virulence factors whose roles in outcomes have not been examined in humans, except for the type III secretion system (T3SS) toxins. The purpose of this study was to examine the role of virulence factors other than the T3SS toxins. Bacterial isolates were collected from 75 patients who suffered from Pa blood stream infections. Host factors such as neutropenia, immunosuppression, comorbidities, time to effective antibiotics, source of bacteremia, and presence of multidrug resistant (MDR) isolate were studied. The isolates were analyzed for the presence of toxin genes, proteolytic activity, swimming and twitching motility, and pyocyanin production. The data were analyzed to ascertain which virulence factors correlated with poor outcomes defined as septic shock or death (SS) within 7 days. Septic shock or death occurred in 25/75 patients. Univariate analysis identified age as a host factor that exerted a significant effect on these outcomes. Ineffective antibiotics administered during the first 24 hours of treatment or MDR P. aeruginosa did not influence the frequency of SS, nor did the presence of lasB, exoA, exoS exoU, plcH genes and proteolytic activity. However, 6/8 patients infected with non-motile isolates, developed SS, p = 0.014 and 5/6 isolates that produced large amounts of pyocyanin (>18ug/ml), were associated with SS, p = 0.014. Multivariate analysis indicated that the odds ratio (OR) for development of SS with a non-motile isolate was 6.8, with a 95% confidence interval (CI) (1.37, 51.5), p = 0.030 and with high pyocyanin producing isolates, an OR of 16.9, 95% CI = (2.27, 360), p = .017. This study evaluating the role of microbial factors that significantly effect outcomes following Pa bloodstream infection suggests that P. aeruginosa strains showing high pyocyanin production and the lack of motility independently increase the risk of SS.

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          Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications.

          Two anthranilate synthase gene pairs have been identified in Pseudomonas aeruginosa. They were cloned, sequenced, inactivated in vitro by insertion of an antibiotic resistance gene, and returned to P. aeruginosa, replacing the wild-type gene. One anthranilate synthase enzyme participates in tryptophan synthesis; its genes are designated trpE and trpG. The other anthranilate synthase enzyme, encoded by phnA and phnB, participates in the synthesis of pyocyanin, the characteristic phenazine pigment of the organism. trpE and trpG are independently transcribed; homologous genes have been cloned from Pseudomonas putida. The phenazine pathway genes phnA and phnB are cotranscribed. The cloned phnA phnB gene pair complements trpE and trpE(G) mutants of Escherichia coli. Homologous genes were not found in P. putida PPG1, a non-phenazine producer. Surprisingly, PhnA and PhnB are more closely related to E. coli TrpE and TrpG than to Pseudomonas TrpE and TrpG, whereas Pseudomonas TrpE and TrpG are more closely related to E. coli PabB and PabA than to E. coli TrpE and TrpG. We replaced the wild-type trpE on the P. aeruginosa chromosome with a mutant form having a considerable portion of its coding sequence deleted and replaced by a tetracycline resistance gene cassette. This resulted in tryptophan auxotrophy; however, spontaneous tryptophan-independent revertants appeared at a frequency of 10(-5) to 10(6). The anthranilate synthase of these revertants is not feedback inhibited by tryptophan, suggesting that it arises from PhnAB. phnA mutants retain a low level of pyocyanin production. Introduction of an inactivated trpE gene into a phnA mutant abolished residual pyocyanin production, suggesting that the trpE trpG gene products are capable of providing some anthranilate for pyocyanin synthesis.
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            Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa.

            Polyphosphate kinase (PPK), encoded by the ppk gene, is the principal enzyme in many bacteria for the synthesis of inorganic polyphosphate (poly P) from ATP. A knockout mutant in the ppk gene of Pseudomonas aeruginosa PAO1 is impaired in flagellar swimming motility on semisolid agar plates. The mutant is deficient in type IV pili-mediated twitching motility and in a "swarming motility" previously unobserved in P. aeruginosa. In swarming cultures, the polar monotrichous bacteria have differentiated into elongated and polar multitrichous cells that navigate the surface of solid media. All of the motility defects in the ppk mutant could be complemented by a plasmid harboring the ppk gene. Because bacterial motility is often crucial for their survival in a natural environment and for systemic infection inside a host, the dependence for motility on PPK reveals important roles for poly P in diverse processes such as biofilm formation, symbiosis, and virulence.
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              A four-tiered transcriptional regulatory circuit controls flagellar biogenesis in Pseudomonas aeruginosa.

              The single polar flagellum of Pseudomonas aeruginosa is an important virulence and colonization factor of this opportunistic pathogen. In this study, the annotation of the genes belonging to the fla regulon was updated and their organization was analysed in strains PAK and PAO1, representative type-a and type-b strains of P. aeruginosa respectively. The flagellar genes are clustered in three non-contiguous regions of the chromosome. A polymorphic locus flanked by flgJ and fleQ in Region I contains a glycosylation island in PAK. The expression and ordered assembly of the complex multicomponent flagellum is intricately regulated. Dedicated flagellar genes fleQ, fleS, fleR, fliA, flgM and fleN encode proteins that participate in the regulation of the flagellar transcriptional circuit. In addition, expression of the flagellum is coordinately regulated with other P. aeruginosa virulence factors by the alternative sigma factor sigma54, encoded by rpoN. In order to gain insight into the hierarchical regulation of flagellar genes, deletion mutations were constructed in fleQ, fleR, fliA and rpoN. The transcriptional impact of these mutations was examined by transcriptional profiling using a P. aeruginosa whole genome microarray. Analysis of the transcriptomes generated for each of these mutants indicates a four-tiered (Classes I-IV) hierarchy of transcriptional regulation. Class I genes are constitutively expressed and include the transcriptional regulator fleQ and the alternative sigma factor fliA (sigma28). Class II genes including fleSR, encoding a two-component regulatory system require FleQ and RpoN (sigma54) for their transcriptional activation. Class III genes are positively regulated by the activated response regulator FleR in concert with RpoN. The transcription of Class IV genes is dependent on the availability of free FliA following the export of the FliA specific antisigma factor FlgM through the basal body rod-hook structure (assembled from Class II and III gene products). Two previously uncharacterized genes, which are coordinately regulated with known flagellar genes have been identified by genome-wide analysis and their role in flagellar biogenesis was analysed.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: MethodologyRole: Supervision
                Role: Data curation
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS One
                plos
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                11 June 2021
                2021
                : 16
                : 6
                : e0253259
                Affiliations
                [1 ] Division of Infectious Diseases and Global Medicine, Department of Medicine, University of Florida, Gainesville, Florida, United States of America
                [2 ] Department of Pharmaceutical Outcomes & Policy, College of Pharmacy, University of Florida, Gainesville, Florida, United States of America
                COMSATS University Islamabad - Abbottabad Campus, PAKISTAN
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                https://orcid.org/0000-0002-3397-179X
                Article
                PONE-D-20-22229
                10.1371/journal.pone.0253259
                8195364
                34115807
                5b91baa3-a0a0-4dff-ad09-8631c41f494e

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 11 August 2020
                : 1 June 2021
                Page count
                Figures: 3, Tables: 5, Pages: 15
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: 1R01AI078770-01A1
                Award Recipient :
                This work was supported in part by grant from National Institutes of Health grant 1R01AI078770-01A1 to RR. This work was supported in part by University of Florida-Gatorade internal funding to AG. There was no additional external funding received for this study.
                Categories
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                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
                Pseudomonas Aeruginosa
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