Persister cells are able to survive in the presence of high concentrations of antibiotic, and re-grow once the antibiotic has been removed. Unlike conventional antibiotic resistance, the antibiotic tolerance of persister cells is due to phenotypic switching, and is non-inherited. There is growing evidence for a role of persisters in various persistent bacterial diseases. Burkholderia pseudomallei is a pathogen which causes melioidosis, which often persists in the host despite antibiotic treatment. As persister cells may contribute to persistent melioidosis, this study investigated persisters in B. thailandensis, as a model for B. pseudomallei. Treatment of B. thailandensis with ceftazidime, ciprofloxacin, imipenem or trimethoprim demonstrated persister cells which survived antibiotic treatment. Persister frequencies were increased in the absence of oxygen, and higher in stationary phase cultures compared with growing cultures. Drug concentration did not affect persister frequencies, and inherited antibiotic resistance was not detected. Different persister fractions were detected using treatment with multiple antibiotics, indicating heterogeneous susceptibility to antibiotics. In order to increase understanding of the molecular basis of B. thailandensis persister cells, a transposon mutagenesis-based sequencing approach was used on persister cultures. This indicated some issues with genome coverage and mutant diversity. Genes were identified from mutants present before and/or after ciprofloxacin treatment. In order to try to eradicate persister cells from a culture, two anti-persister strategies were tested. Itaconate appeared to stimulate growth of B. thailandensis, increasing susceptibility to the antibiotic ceftazidime. However, the overall effect of the combination was no greater than ceftazidime alone in the conditions tested. Metronidazole was effective against a persister culture under anaerobic conditions, suggesting it may be useful in treating anaerobic persisters. Treatment of B. pseudomallei infected mice with metronidazole and ceftazidime did not improve survival over ceftazidime treatment alone.

The historical notion of a microbial population has been of a clonal population of identical swimming planktonic cells in a laboratory flask. As the field has advanced, we have grown to appreciate the immense diversity in microbial behaviors, from their propensity to grow in dense surface-attached communities as a biofilm, to the consequences of social dilemmas between cells, to their ability to form spores able to survive nearly any environmental insult. However, the historically biased view of the clonal microbial population still persists – even when a rare phenotype is investigated, the focus simply shifts to that narrower focal population - and this bias can lead to some of the broader questions relating to the consequences of phenotypic diversity within populations to be overlooked. This work seeks to address this gap by investigating the evolutionary causes and consequences of phenotypic heterogeneity, with a focus on clinically relevant phenotypes. We first develop and experimentally validate a theoretical model describing the evolution of a microbial population faced with a trade-off between survival and fecundity phenotypes (e.g. biofilm and planktonic cells), which suggests that simultaneous investment in both types maximizes lineage fitness in heterogeneous environments. This model helps to inform the experimental studies in the following chapters. We find that biofilm-mediated phenotypic resistance to antibiotics is evolutionarily labile, and responsive to antibiotic dose and whether biofilm or planktonic cells are passaged. We also show that persistence in E. coli is age-independent, supporting the current hypothesis of stochastic metabolic fluctuations as the cause of this rare phenotype. Finally, we explore phenotypic variation across a library of natural isolates of P. aeruginosa, and find few organizing principles among key phenotypes related to virulence. Together these results suggest that phenotypic heterogeneity is a crucial component in the ecology and evolution of microbial populations, and directly affects pressing applied concerns such as the antibiotic resistance crisis.

Lyme disease is the most commonly reported vector-borne disease in the United States. The causative agent of Lyme disease, can alter gene expression to enable survival in a diverse set of conditions, including the tick midgut and the mammalian host. External environmental changes can trigger gene expression in B. burgdorferi, and the data demonstrate that B. burgdorferi can similarly alter gene expression as a stress-response when it is treated with the antibiotic doxycycine. After treatment with the minimum bactericidal concentration (MBC) of doxycycline, a subpopulation can alter its phenotype to survive antibiotic treatment, and to host adapt and successfully infect a mammalian host. Furthermore, our data demonstrate that if a population is treated with the MBC of doxycycline, a subpopulation may alter its phenotype to adopt a state of dormancy until the removal of the antibiotic, whereupon the subpopulation can regrow. We demonstrate that the chance of regrowth occurring increases as a population reaches stationary phase, and present a mathematical model for predicting the probability of a persister subpopulation within a larger population, and ascertain the quantity of a persister subpopulation. To determine which genes are expressed as stress-response genes, RNA Sequencing analysis, or RNASeq, was performed on treated, untreated, and treated and regrown B. burgdorferi samples. The results suggest several genes were significantly different in the treated group, compared to the untreated group, and in the untreated and regrown group compared to the untreated group, including a 50S ribosomal stress-response protein, coded from BB_0786. The appendices discuss the theory and methods that were used in RNA Sequencing (RNASeq) analysis, and provide an overview of the database that was created for the B. burgdorferi transcriptome. Additional studies may demonstrate further how persister subpopulations form, and which genes can trigger a persister state in B. burgdorferi. / acase@tulane.edu

Biofilms are formed in aquatic environments by the attachment of bacteria to submerged surfaces, to the air/liquid interface, and to each other. Although biofilms are associated with disease and biofouling, the robust nature of biofilms; for example, their ability to tolerate chemical and physical stresses, makes them attractive for beneficial biotechnology applications such as bioremediation and biofuels. Based on an understanding of diverse signals and regulatory networks during biofilm development, biofilms can be engineered for these applications by manipulating extracellular/intercellular signals and regulators. Here, we rewired the global regulator H-NS of Escherichia coli to control biofilm formation using random protein engineering. H-NS variant K57N was obtained that reduces biofilm formation 10-fold compared with wild-type H-NS (wild-type H-NS increases biofilm formation whereas H-NS K57N reduces it) via its interaction with the nucleoid-associated proteins Cnu and StpA. H-NS K57N leads to enhanced excision of the defective prophage Rac and results in cell lysis through the activation of a host killing toxin HokD. We also engineered another global regulator, Hha, which interacts with H-NS, to disperse biofilms. Hha variant Hha13D6 was obtained that causes nearly complete biofilm dispersal by increasing cell death by the activation of proteases. Bacterial quorum sensing (QS) systems are important components of a wide variety of engineered biological devices, since autoinducers are useful as input signals because they are small, diffuse freely in aqueous media, and are easily taken up by cells. To demonstrate that biofilms may be controlled for biotechnological applications such as biorefineries, we constructed a synthetic biofilm engineering circuit to manipulate biofilm formation. By using a population-driven QS switch based on the LasI/LasR system and biofilm dispersal proteins Hha13D6 and BdcAE50Q (disperses biofilms by titrating cyclic diguanylate), we displaced an existing biofilm and then removed the second biofilm. Persisters are a subpopulation of metabolically-dormant cells in biofilms that are resistant to antibiotics; hence, understanding persister cell formation is important for controlling bacterial infections. Here, we engineered toxin MqsR with greater toxicity and demonstrated that the more toxic MqsR increases persistence by decreasing the ability of the cell to respond to antibiotic stress through its RpoS-based regulation of acid resistance, multidrug resistance, and osmotic resistance systems.

The aim of this study was to identify and characterise type II toxin-antitoxin (TA) systems in Burkholderia pseudomallei, the causative agent of the human disease melioidosis. 8 putative TA systems were identified within the genome of B. pseudomallei K96243. 5 of these were located witihn genome islands. Of the candidate toxins, BPSL0175 (RelE1) or BPSS1060 (RelE2) caused growth to cease when expressed in Escherichia coli, whereas expression of BPSS0390 (HicA) or BPSS1584 (HipA) (in an E. coli ΔhipBA background) caused a reduction in the number of culturable bacteria. HicA also caused growth arrest in B. pseudomallei K96243 ΔhicAB. These toxin induced phenotypes were enhanced by an <3kDa extracellular factor that accumulated in the spent medium during growth. Expression of the cognate antitoxins could restore growth and culturability of cells. Expression of hicA in E. coli gave an increased number of persister cells in response to ciprofloxacin or ceftazidime. Site directed mutagenesis studies identified two key residues within the HicA toxin that were essential for both the reduced culturability and increased persistence phenotypes. Deletion of hicAB from B. pseudomallei K96243 did not affect persister cell or survival frequencies compared to the wild type following treatment with a variety of stress conditions. Deletion of the ΔhipBA locus from B. pseudomallei K96243 also had no affect on bacterial persistence or survival under the conditions tested.

No description available.

Four type II toxin-antitoxin (TA) systems were previously identified in Burkholderia pseudomallei K96243. Type II TA toxins are able to induce cell growth arrest or death by interfering with key processes within the organism. BPSS0390-0391 is one of the TA systems previously identified and has homology to hicBA system in Acinetobacter baumannii. B. pseudomallei HicA is able to cause a reduction in the number of culturable cells after expression in E. coli. This study aimed to characterise B. pseudomallei HicA in three ways: by inducing expression of HicA in bacterial species other than E. coli, by identifying amino acids in HicA involved in toxicity and neutralisation by the antitoxin HicB and by examining the interaction of HicA with other TA antitoxins identified within B. pseudomallei genome. A broad host range plasmid encoding BPSS0390 was transformed into a range of Gram negative bacteria including Yersinia pseudotuberculosis IP32953, Vibrio vulnificus E64MW, Salmonella enterica serovar Typhimurium SL1344 and Burkholderia thailandensis E264. Expression of BPSS0390 was toxic in all bacterial species tested, despite the presence of antitoxin BPSS0391 homologues in some species. Unregulated expression in E. coli resulted in the appearance of escape mutants encoding non-toxic variants of HicA. An alanine scanning mutagenesis study of HicA identified 20 mutants where toxicity was abolished despite high levels of expression, but identified no mutants that affected TA complex formation. Finally an existing co-expression assay was modified to examine interactions between HicA and other type II TA antitoxins in B. pseudomallei. The assay revealed no interaction between HicA and non-cognate antitoxins and clarified the role of IPTG as an inhibitor of PBAD promoter on the arabinose operon.

Acinetobacter baumanii fait aujourd’hui partie des bactéries les plus problématiques dans le monde. Responsable de nombreux pics épidémiques d’infections nosocomiales auxquelles sont associés de forts taux de mortalité, cette bactérie puise sa pathogénie dans de multiples caractéristiques qui lui permettent ainsi d’échapper au système immunitaire de l’hôte et à la plupart des traitements actuels. Capable d’adhérer à de multiples surfaces, A. baumanii persiste dans l’environnement hospitalier à travers un mode de vie communautaire au sein duquel ses capacités de survie sont exacerbées. Chez les espèces du genre Acinetobacter, le mode de vie communautaire peut prendre deux formes distinctes : le biofilm et la pellicule. Dans la première partie de cette thèse, nous avons cherché à discriminer ces deux modes de vie, chez la souche ATCC 17978, par une analyse protéomique à large échelle. Nous avons confirmé la présence de nombreux marqueurs communs aux deux communautés (transporteurs, systèmes de sécrétion, d’acquisition d’ions, adhésines et pili) et mis en exergue des systèmes spécifiquement reliés à la formation du biofilm (pilus Fim, T2SS, T1SS/pompe A1S_0535-38, LPS/LOS, motif capsulaire) et à celle de la pellicule (Gac). Grâce à l’étude de la souche A. baumannii SDF en mode biofilm, qui présente un génome plus compact, nous montrons que très peu de mécanismes moléculaires sont partagés par les deux souches étudiées. Ce résultat témoigne de la difficulté quant au développement d’un traitement dirigé contre les biofilms A. baumannii. Dans une deuxième partie, nous avons testé deux approches pour prévenir et éradiquer les biofilms à A. baumannii. La première a ciblé le Quorum Sensing (QS), système de communication essentielle à la coordination des cellules. Nous avons pu montrer que les acides gras mono-insaturés (acide palmitoléique et acide myristoléique), au même titre que la virstatine, limitait la formation de communautés à A. baumannii en inhibant l’expression du régulateur abaR nécessaire au QS. Dans une seconde stratégie, nous avons finalement évalué l’action antibactérienne et antibiofilm d’un nouveau composé d’origine naturelle : la squalamine. Dans cette étude, nous montrons pour la première fois qu’A. baumannii est capable d’entrer dans un état de dormance (persistant/VBNC) pour survivre à de fortes doses de ciprofloxacine, mais que la squalamine est capable d’éradiquer ces cellules persistantes grâce à des concentrations inférieures à la concentration hémolytique. / Today, Acinetobacter baumannii is one of the most problematic pathogens in the world. This bacterium is responsible for worldwide epidemic outbreaks associated with dramatic mortality rates. It possesses high capacities to evade the immune host system and to resist to numerous available antibacterial agents. A. baumannii is also able to persist into hospital environment due to high adhesion abilities which induce community development. This process is also associated to an enhanced survival rate. In Acinetobacter genus, community modes of lif can take two forms : biofilm and pellicle. In this study on the strain ATCC 17978, we tried to discriminate these two lifestyles by a large scale proteomic analysis. We have confirmed the presence of many common community markers (transporters, ion acquisition secretion systems, adhesins and pili) and highlighted systems specifically related to biofilm (pilus Fim, T2SS, T1SS / pump A1S_0535-38, LPS / LOS, capsular pattern) and pellicle communities. Furthermore the proteomic analysis of an avirulent A. baumannii strain, SDF, in biofilm allowed to highlight peculiar metabolic pathways, specific adhesion determinants but very few markers shared by ATCC 17978. This demonstrated the difficulty in developing a treatment directed against A. baumannii biofilm. Then, we tested different approaches to prevent and eradicate biofilms. The first one targeted the Quorum Sensing system (QS), an essential communication system for cell coordination. We have showed that monounsaturated fatty acids (palmitoleic acid and myristoleic acid), like virstatin prevent the community formation of A. baumannii by inhibiting the expression of the abaR regulator required for QS. In a second strategy, we have evaluated the antibacterial and antibiofilm activity of a new natural compound : the squalamine. We showed for the first time that if ciprofloxacin treatment was able to induce a dormancy population (persistent/VBNCs) in A. baumannii, squalamine was able to eradicate this population of dormant cells.