IL-1 is an essential pro-inflammatory factor in inflammation response. The effect of IL-1 is through binding to the IL-1 receptor that triggers the following signal transduction pathway. To study the role of IL-1 receptor-mediated signal pathway in inflammatory response, injecting P. aeruginosa into trachea of wild-type (WT) and type-1 IL-1 receptor knock-out (IL-1R1-/-) mice was used as the experimental model. Injecting bacterium into trachea of mice will induce pneumonia which increases accumulation of neutrophils, production of nitric oxide, expression of intercellular adhesion molecule-1 as well as many kinds of cytokines and causes the lung damage. The pneumonia-induced lung damage and inflammation at 24 hr after injecting P. aeruginosa into trachea were more severe in knock-out than in WT mice, as demonstrated by increases in extravasations of Evans blue dye (EBD), myeloperoxidase (MPO) activity, expression of iNOS, IL-1 beta and ICAM-1, and higher mortality of knock-out mice. The cause of the high mortality in knock-out mice was further investigated by culturing the lung and blood samples for bacterial counts. The bacterial counts of lung and blood of IL-1R1-/- mice were all higher than that of WT mice in 8 to 24 hr after injection of bacterium. Finally, chimeric mice (WT ¡÷ WT, IL1R1-/- ¡÷IL1R1-/-, WT ¡÷ IL1R1-/-, IL1R1-/- ¡÷ WT) were generated and used to determine the role of PMN cells of blood. Suggesting that increased amounts of bacteria in lung and blood is related to the higher mortality in knock-out mice and the type-1 IL-1 receptor is essential for mice to against pneumonia in this model.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen often infecting the lungs of individuals with the heritable genetic disease cystic fibrosis and the peritoneum of those undergoing continuous peritoneal dialysis. Often these infections are not caused by colonization with P. aeruginosa alone but instead by a consortium of pathogenic bacteria. Little is known about growth and persistence of P. aeruginosa in vivo, and less is known about the impact of coinfecting bacteria on P. aeruginosa pathogenesis and physiology. In this dissertation I used a rat dialysis membrane peritoneal model to evaluate the in vivo transcriptome of P. aeruginosa in monoculture and in coculture with Staphylococcus aureus. Monoculture results indicate that approximately 5% of all P. aeruginosa genes are differentially regulated during growth in vivo. Included in this analysis are genes important for iron acquisition and growth in lowoxygen environments. The presence of S. aureus caused decreased transcription of P. aeruginosa iron-regulated genes during in vivo coculture, indicating that the presence of S. aureus increases usable iron for P. aeruginosa in the environment. This lysis was shown to be dependent on antimicrobial quinolones produced by P. aeruginosa. I demonstrate that these quinolones are present in outer membrane vesicles (MVs). Not only were these quinolones present in MVs, but the quorum sensing molecule; 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal; PQS) was also packaged into MVs and was necessary for MV formation. These findings illustrate that a prokaryote possesses a signal trafficking system with features common to those used by higher organisms and outlines a novel mechanism for delivery of a signal critical for coordinating group behaviors in P. aeruginosa. Although MVs are involved in important processes besides signaling, the molecular mechanism is unknown. To provide insight into the molecular mechanism of MV formation, I examined the interaction of PQS with bacterial lipids. In this work, I demonstrated that PQS interacts strongly with the acyl chains and 4’-phosphate of bacterial lipopolysaccharide. The results of my studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non-signaling functions. Finally, I propose a model of PQSmediated MV formation where PQS interacts with specific outer membrane components to allow the necessary curvature for MV formation. / text

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is a primary constituent of chronic, polymicrobial infections in the lungs of individuals with cystic fibrosis (CF). A significant consequence of CF is production of thick mucus along epithelial surfaces. In the lungs, this mucus collects and serves as an excellent growth substrate for a range of bacteria including. CF lung fluids (sputum) also enhance the virulence of P. aeruginosa, as production of a signaling molecule critical for virulence, the Pseudomonas quinolone signal (PQS), is enhanced in the presence of phenylalanine and tyrosine in CF sputum. The goal of this dissertation is to better understand how phenylalanine and tyrosine affect PQS production and ultimately P. aeruginosa virulence. To address this, I use transcriptome profiling to determine that genes for phenylalanine and tyrosine catabolism, PQS biosynthesis, and a transcriptional regulator called PhhR are up-regulated in the presence of phenylalanine and tyrosine. I determine that PhhR regulates genes for aromatic amino acid catabolism but not genes for PQS biosynthesis. The PhhR regulon is further characterized by mapping of PhhR-regulated promoters with primer extension, and evidence for direct regulation is presented. To explain enhanced production of PQS in CF sputum, I favor a model in which flux of a shared metabolic precursor, chorismate, toward PQS biosynthesis is enhanced when phenylalanine and tyrosine are present. I investigate this model by examining the first step in PQS biosynthesis, conversion of chorismate to anthranilate by an anthranilate synthase (AS). P. aeruginosa possesses two AS enzymes encoded by the trpEG and phnAB genes, with the former generating anthranilate specifically for tryptophan biosynthesis while the latter generates anthranilate for PQS biosynthesis. I investigate the evolutionary origins of these two enzymes and generate unmarked deletion mutants to dissect their roles in tryptophan and PQS biosynthesis. The ability of PhnAB to compensate for loss of TrpEG at high cell densities is documented, and a model explaining anthranilate sequestering is developed. Knowledge gained from these studies will be useful in developing novel therapeutic strategies. / text

Pseudomonas aeruginosa is a ubiquitous bacterium that is commonly isolated from soil and water. Additionally, this bacterium can cause infections in individuals with compromised immune systems and in those with underlying debilitating conditions. Individuals with cystic fibrosis, burn wounds, AIDS and diabetes are more likely to being infected by P. aeruginosa than healthy individuals. In individuals with CF, there is a marked increase in the accumulation of lung mucus that serves as a source of nutrition for P. aeruginosa and other bacterial species resulting in chronic and often fatal infections. While CF lung infections are initially caused by more than one species of bacteria, over time P. aeruginosa emerges as the dominant species. P. aeruginosa also causes chronic infections in association with other bacteria in wounds. Microbes within these infections are engaged in complex interactions with each other. Often, these interactions are synergistic resulting in infections that are recalcitrant to antimicrobial therapy. While many studies have documented the occurrence of synergistic polymicrobial infections, little is known about the molecular mechanisms prevailing in these infections. Interestingly, production of virulence factors by P. aeruginosa has been shown to correlate with the presence of specific nutrients in their growth environment. Expanding on the idea of available nutrients affecting virulence, I demonstrate the ability of N-Acetylglucosamine (GlcNAc) and GlcNAc-containing polymers such as peptidoglycan to induce production of virulence factors in P. aeruginosa. Peptidoglycan shed by Gram-positive bacteria acts as a cue for P. aeruginosa in polymicrobial environments, to enhance production of virulence factors. In the context of a polymicrobial infection, this results in enhanced pathogenesis. Here, I provide insights into mechanisms influencing such interspecies interactions between the opportunistic pathogen Pseudomonas aeruginosa and S.aureus. / text

published_or_final_version / Microbiology / Master / Master of Science

published_or_final_version / Medicine / Master / Master of Research in Medicine

Bacterial communities engage in social activities, exhibiting behaviors such as communicating with small signaling molecules (quorum sensing [QS]) and building antibiotic-resistant biofilms. The opportunistic human pathogen Pseudomonas aeruginosa produces both freely diffusible QS molecules, as well as a QS molecule that is packaged or transported across cell membranes via the production of outer membrane vesicles. Despite the ubiquity of vesicle production in bacteria, the mechanism of outer membrane vesicle production has not been fully elucidated. In addition, most of our understanding of QS and biofilm formation arises from in vitro studies of bacterial communities containing large numbers of cells, often with greater than 10⁸ bacteria. However, many bacterial communities are comprised of small, densely packed aggregates of cells (≤10⁵ bacteria), and it is unclear how group behaviors and chemical interactions take place in densely packed, small populations. This dissertation has two main goals: i) to provide insights into the mechanism of bacterial membrane vesicle production, and ii) to understand how population size and the spatial distribution of cells affect cell-cell interactions and the nutritional microenvironment within a small (≤10⁵ bacteria) prokaryotic community. / text

Bacteria commonly use two-component sensors to sense and respond to their environment. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa has one of the largest sets of two-component sensors known in bacteria, which likely contributes to its ability to adapt to diverse environments, including the human host. Several of these sensors such as GacS have been shown to play a role in the regulation of virulence in this pathogen. However, the role of the majority of sensors remains unknown. In this thesis I show that the two-component sensor KinB is required for full P. aeruginosa virulence in the recently characterized model host Danio rerio. I found that KinB regulates several virulence-associated phenotypes in P. aeruginosa including pyocyanin and elastase production and motility. I show that KinB regulates these phenotypes through the global sigma factor AlgU, which plays a critical role in the repression of P. aeruginosa acute virulence factors and through its cognate response regulator, AlgB, albeit in a non-canonical manner. KinB’s primary role in the regulation of acute virulence is to act as a phosphatase to dephosphorylate AlgB.

Το γένος Pseudomonas ανήκει στην οικογένεια Pseudomonadaceae και περιλαμβάνει πολλά είδη. Το συχνότερο είδος ψευδομονάδας που προκαλεί νόσο στον άνθρωπο είναι η Pseudomonas aeruginosa. Η P.aeruginosa είναι ένα Gram αρνητικό, αυστηρά αερόβιο βακτηρίδιο, το οποίο αναπτύσσεται στους 37-42οC και καλλιεργείται σχετικά εύκολα σε κοινά θρεπτικά υλικά. Η P. aeruginosa έχει χαρακτηριστεί ως ένα από τα σημαντικότερα και σοβαρότερα ευκαιριακά παθογόνα βακτηρία, τα οποία είναι υπεύθυνα για νοσοκομειακές λοιμώξεις σε διάφορες ειδικές ομάδες ασθενών όπως ανοσοκατασταλμένα άτομα, άτομα με κυστική ίνωση (cystic fibrosis), ασθενείς με νεοπλασίες, κ.α. Οι λοιμώξεις που προκαλεί η P. aeruginosa χαρακτηρίζονται σοβαρές και επικίνδυνες αφενός μεν γιατί προκαλεί νόσο σε άτομα ήδη επιβεβαρυμένα από άλλες καταστάσεις, αφετέρου δε γιατί εμφανίζει ανθεκτικότητα στα περισσότερα αντιβιοτικά. Ο τρόπος με τον οποίο η P. aeruginosa ασκεί την παθογόνο δράση της είναι πολύπλοκος και δεν έχει διευκρινισθεί επακριβώς. Διάφοροι κυτταρικοί παράγοντες αλλά και εξωκυττάρια προϊόντα θεωρούνται υπεύθυνα για την έκφραση της παθογόνου δράσης του μικροοργανισμού. Οι πιο σημαντικοί κυτταρικοί παράγοντες είναι: ο λιποπολυσακχαρίτης, οι φίμπριες, οι πρωτεΐνες της εξωκυττάριας μεμβράνης, ο εξωκυττάριος πολυσακχαρίτης (Slime), καθώς επίσης και ο βλεννώδης πολυσακχαρίτης (αλγινικό οξύ) που σχετίζεται με τα βλεννώδη στελέχη. Επίσης διάφορα εξωκυττάρια προϊόντα-παράγοντες τα οποία θεωρούνται υπεύθυνα για τη λοιμογόνο δράση του βακτηρίου, είναι διάφορα ένζυμα και κυρίως το Εξωένζυμο-S και η Εξωτοξίνη-A. Στους μηχανισμούς άμυνας του ξενιστή έναντι της P. aeruginosa τα μακροφάγα που προσελκύονται στην εστία της λοίμωξης παίζουν το σπουδαιότερο ρόλο. Οι κυτταροκίνες που παράγονται από τα ενεργοποιημένα μακροφάγα καθορίζουν την έκταση της βλάβης και την έκβαση της λοίμωξης. Σε πειραματικά μοντέλα σήψης από P. aeruginosa η παραγωγή του παράγοντα νεκρώσεως όγκου (TNF-α) σε μικρά ποσά έχει συσχετισθεί με προστασία σε σοβαρές λοιμώξεις, ενώ η υπερπαραγωγή του οδηγεί σε σηπτικό σοκ. Η ρύθμιση της παραγωγής του TNF-α αντικατοπτρίζει τη ρύθμιση της ενεργοποίησης του μακροφάγου. Διαφορετικοί υποδοχείς επιφανείας όπως οι TLR’s, συμβάλουν στην αναγνώριση του μικροβίου και την επακόλουθη ενεργοποίηση του μακροφάγου. Τα ενδοκυττάρια γεγονότα που σχετίζονται με τη μετάδοση του σήματος ενεργοποίησης στα μακροφάγα δεν είναι απόλυτα αποσαφηνισμένα. Πρώιμα συμβάντα ενεργοποίησης των μακροφαγων που ρυθμίζουν την παραγωγή του TNF-α, είναι η ενεργοποίηση των MAP κινασών. Η οικογένεια των MAPK’s περιλαμβάνει 3 κινάσες: την p38, την Erk1,2 και την JNK, που έχουν ταυτοποιηθεί ως πιθανοί θεραπευτικοί στόχοι σε πειραματικά μοντέλα σήψης. Η διαδοχική φωσφορυλίωση υποστρωμάτων ενεργοποιεί διάφορους μεταγραφικούς παράγοντες, χαρακτηριστικότεροι των οποίων είναι ο NF-κΒ και ο AP-1, ο οποίοι συμμετέχουν στη μεταγραφή των γονιδίων των κυτταροκινών που εκφράζονται στη διάρκεια της σηπτικής καταπληξίας. Σημαντικότερη από αυτές είναι ο TNF-α. Η ρύθμιση της παραγωγής του TNF-α είναι αντιπροσωπευτικός στόχος μελέτης των μηχανισμών που διέπουν την ενεργοποίηση των μακροφάγων. Αυτή η ρύθμιση γίνεται τόσο στο επίπεδο μεταγραφής του γονιδίου όσο και στο μετά-μεταγραφικό και μεταφραστικό επίπεδο. Συνεργασία μεταξύ των πρωτεϊνών μεταγραφής και αυτών που δεσμεύονται στη 3-UTR περιοχή του mRNA του TNF-α συμβάλουν στην εκλεκτική ρύθμιση της παραγωγής του από τα μακροφάγα. Οι μηχανισμοί που ενέχονται στην ενεργοποίηση των μακροφάγων από την P. aeruginosa δεν έχουν πλήρως μελετηθεί. Ταυτοποίηση των συγκεκριμένων μορίων-στόχων στη μετάδοση του σήματος ενεργοποίησης στα μακροφάγα συμβάλει στην κατανόηση των μηχανισμών ελέγχου της σηπτικής καταπληξίας. Σκοπός της παρούσας διατριβής είναι η αποσαφήνιση της δράσης της P. aeruginosa στα διάφορα στάδια ενεργοποίησης των μακροφάγων όπως: α) το επίπεδο της φωσφορυλίωσης-ενεργοποίησης διαφόρων πρωτεϊνικών κινασών (π.χ. MAPK’s), β) την ενεργοποίηση-δράση μεταγραφικών παραγόντων με αντιπροσωπευτικότερους τον NF-κΒ και AP-1, και τέλος, γ) την διερεύνηση των υποδοχέων επιφανείας όπως οι Toll-like υποδοχείς στη μετάδοση του σήματος ενεργοποίησης, αλλά και σύνδεση αυτών με την ενεργοποίηση των MAPK’s. Σε επίπεδο παραγωγής TNF-α πρωτεΐνης και ενεργοποίησης μεταγραφικών παραγόντων, τα αποτελέσματά μας έδειξαν ότι: α) Το Slime-GLP είναι ισχυρότερος διεγέρτης από τον LPS P. aeruginosa για την παραγωγή TNF-α σε υπερκείμενα μονοκυτταρικών καλλιεργειών. β) Αυτή η διαφορετική ρύθμιση της παραγωγής πρωτεϊνικού TNF-α, αντικατοπτρίζεται και σε επίπεδο ενεργοποίησης μεταγραφικών παραγόντων. Συγκεκριμένα, το Slime-GLP μπορεί και επάγει περισσότερο την ενεργοποίηση του NF-κΒ από ότι ο LPS της P. aeruginosa και αυτή η ενεργοποίηση είναι σχεδόν ίση με αυτή που προκαλεί ολόκληρο το ζωντανό κύτταρο της P. aeruginosa. γ) Ο NF-κΒ που ενεργοποιείται με Slime-GLP αποτελείται από το ετεροδιμερές p50/p65. δ) Ο μεταγραφικός παράγοντας AP-1 ενεργοποιείται σχετικά πιο καθυστερημένα από ότι ο NF-κΒ, αλλά και σε αυτή την περίπτωση το Slime-GLP φαίνεται να είναι ισχυρότερος διεγέρτης από τον LPS P. aeruginosa. Σε επίπεδο ενεργοποίησης των MAP κινασών τα αποτελέσματά μας έδειξαν ότι η διαφορετική ρύθμιση της παραγωγής του TNF-α, σε επίπεδο πρωτεΐνης, μετά από διέγερση με LPS ή Slime-GLP αντικατοπτρίζεται και σε επίπεδο ενεργοποίησης των διαφορετικών MAP κινασών. Συγκεκριμένα το Slime-GLP ενεργοποιεί τις p38 και Εrk1,2 σε μεγαλύτερο βαθμό από ότι ο LPS της P. aeruginosa. Η ενεργοποίηση των p38 και Erk1,2 που αποτυπώνεται με αύξηση των επιπέδων φωσφορυλίωσής τους, αντιστοιχεί και με αύξηση της ενεργότητάς τους. Η χρήση ειδικών αναστολέων έναντι των p38 (SB203580) και Erk1,2 (PD98059) μπορεί και αναστέλλει σημαντικά την παραγωγή του πρωτεϊνικού TNF-α κατά 80-95% και 60-80% αντίστοιχα. Το Slime- GLP διαφέρει από τον ομόλογο LPS της P. aeruginosa μόνο στο βαθμό της ενεργοποίησης των MAP κινασών, δηλαδή μπορεί και επάγει ισχυρότερα τις MAP κινάσες από ότι ο ομόλογος LPS. Η συμμετοχή των Toll-like υποδοχέων στη μετάδοση του σήματος αυτής της διαφορετικής ενεργοποίησης των MAP κινασών από τους LPS και Slime-GLP της P. aeruginosa, μελετήθηκε με τη χρήση ειδικών μονοκλωνικών αντισωμάτων (anti-TLR2) και (anti-TLR4). Συγκεκριμένα, η ενεργοποίηση της p38, μετά από διέγερση με Slime-GLP, μειώνεται σημαντικά παρουσία του anti-TLR2, ενώ παρουσία του anti-TLR4 η μείωση της ενεργοποίησης της p38 δεν είναι τόσο προφανής. Αντίθετα, μείωση της ενεργοποίησης της p38, όταν αυτή επάγεται από τον LPS της P. aeruginosa, παρατηρούμε μόνο στην περίπτωση που αδρανοποιούμε τον TLR4. Τα αποτελέσματά μας υποδεικνύουν έναν μοριακό μηχανισμό με τον οποίο ο LPS και ο εξωκυττάριος πολυσακχαρίτης Slime-GLP της P. aeruginosa, χρησιμοποιούν διαφορετικούς υποδοχείς στην επιφάνεια των μακροφάγων για την διαφορετική ενεργοποίηση των MAP κινασών και τη ρύθμιση της παραγωγής του TNF-α στα ανθρώπινα μακροφάγα κύτταρα / Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that is associated with severe infections of immunocompromized or critically ill patients. P. aeruginosa infections both chronic and acute, are associated with high incidence of morbidity and mortality. Many gene products of the bacterium, involving lipolysaccharide (LPS), extracellular slime glycolipoprotein (Slime-GLP), exotoxin- A, and exoenzyme-S, contribute to the pathophysiology of P. aeruginosa infection, by stimulating different cell types of the immune system. Macrophages play a key role in execution of the innate and adaptive arms of the immune response to Pseudomonas infection. Activated macrophages exert their effects by producing several types of cytokines and/or other mediators. TNF-α an early proinflammatory cytokine produced by activated macrophages is probably the most important mediator of systemic toxicity. TNF-α production primarily by cells of monocytic lineage is a crucial event in the course of these infections. During in vivo infections with P. aeruginosa, both LPS and Slime-GLP produced by mucoid and non-mucoid strains are being released. Extracellular Slime-GLP causes systemic toxicity when administered in vivo. Signal transduction pathways and early activation intracellular events that mediate induction of TNF-α by human monocytes have been the subject of great interest by many investigators. A key component of many intracellular signaling pathways are the mitogen-activated protein (MAP) kinases. This superfamily includes the extracellular signal response kinases (ERK’s), c-jun N-terminal kinases and the p38 family of kinases. A wide range of bacterial compounds including LPS of Gram-negative bacteria, LTA and peptidoglycan of Gram-positive bacteria and Treponema components differentially activate the MAP kinase family members. Recently it has been shown that cell surface Toll-like receptor (TLR) proteins participate in the ability of the host to discriminate different LPS structural features, and/or other components of the bacterial cell. Although it is now clear that the TLR family of membrane proteins is linked to the activation of MAP kinase family members by different microbial components the subsequent pathways that lead to inflammatory mediator production are still being elucidated. The aim of our study was to delineate the molecular mechanisms leading to differential TNF-α induction by P. aeruginosa LPS and Slime-GLP, specifically theIdentification of specific molecular targets of P. aeruginosa Slime-GLP in human monocytes-macrophages may have important therapeutic implications for P. aeruginosa mediated systemic toxicity.

The main objective of the study was to understand the mode of interaction of protamine (Ptm), a cationic antibacterial peptide from fish milt on the Gram negative bacterial envelope. The present study was designed to resolve the question of Ptm translocation across the seemingly impermeable Gram negative cell envelope. The Gram negative pathogen Pseudomonas aeruginosa was studied as an example of a microorganism that is Ptm-sensitive but doesn’t lyse even at bactericidal concentrations. Acquired resistance to Ptm was induced in P. aeruginosa by continuous sub-culturing in nutrient rich media containing increasing concentrations of Ptm. Alterations in bacterial surface charge, LPS composition, cell morphology and Ptm localisation on acquiring resistance were also examined. Expression of outer membrane proteins significantly decreased as P. aeruginosa acquired resistance to Ptm. OprF, the major porin in P. aeruginosa was found to be stably expressed in control, revertant (Ptm-Rev) and resistant (Ptm-Res) groups. No change in expression of efflux proteins was observed as a result of induced Ptm resistance, indicating that efflux is not among the Ptm resistance mechanisms at least in P. aeruginosa. OprM, which is part of the major efflux system (MexAB-OprM) in P. aeruginosa, was found to be down-regulated in Ptm-resistant P. aeruginosa. Another outer membrane protein down-regulated in Ptm-resistant P. aeruginosa was found to be petidyl-prolyl cis trans isomerase (PPIase) which plays a major role in proper folding and maturation of channel proteins in the outer membrane. Among the sarcosinate soluble proteins, DNA dependent RNA polymerase ? and ?’ subunits were found to be down-regulated in Ptm-resistant group indicating lower transcription levels in them. Lipopolysaccharide (LPS) from the three groups of P. aeruginosa under study was isolated and separated by SDS-PAGE. LPS composition of Ptm-Res P. aeruginosa was found to be significantly different from that of the control and Ptm-Rev but was found to be similar with that of LPS from O-antigenic mutant (A+B-, which possessed only A band structures). Comparison of the zetapotential of control, Ptm-Rev and Ptm-Res P. aeruginosa, proved that electrostatic shielding was coincidental in acquired resistance to Ptm in P. aeruginosa. The MIC of the parent strain of P. aeruginosa (A+B+) and the O-antigenic mutants (A+B-, A-B+ and A-B-) were found to be the same which may be indicating that alterations in O-antigenic components alone cannot contribute to Ptm resistance. Effects of Ptm treatment on morphologies of E. coli, S. typhimurium and P. aeruginosa whole cells and spheroplasts were also studied using transmission immuno-electron microscopy. Condensation of cytoplasmic contents was observed when whole cells and spheroplasts were treated with Ptm. Also, Ptm-treated cells and spheroplasts were stained with colloidal gold-labelled antibodies against Ptm to determine distribution within the target cells. It was quite evident that Ptm internalised in whole cells and spheroplasts without lysis and was found to be concentrated in the cytoplasm. Morphological changes observed in Ptm-Rev P. aeruginosa when exposed to Ptm were comparable with that of the control. Condensation of cytoplasmic contents was not observed in Ptm-Res P. aeruginosa when challenged with Ptm. Most of the Ptm was localized at or near the outer membrane of Ptm-treated Ptm-Res P. aeruginosa, indicating decreased outer membrane permeability. Results obtained from these experiments confirm that the resistance to Ptm observed in P. aeruginosa is at the very least, coincidental with the pleiotropic mutations involving change in outer surface including change in LPS composition, loss of porins and or alterations of porin size in OprF.