Abdul N. Hamood

The role of quorum sensing in the in vivo virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide variety of infections. The cell-density-dependent signaling mechanisms known as quorum sensing play a role in several of these infections including corneal, lung and burn wound infections. In addition, the quorum-sensing systems contribute to the ability of P. aeruginosa to form biofilms on medically important devices. The quorum-sensing systems accomplish their effect by controlling the production of different virulence factors and by manipulating the host immune response.

Pseudomonas aeruginosa forms biofilms in acute infection independent of cell-to-cell signaling.

ABSTRACT Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 h of infection in thermally injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections as well. Using light, electron, and confocal scanning laser microscopy, P. aeruginosa biofilms were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates differentiation of biofilms in vitro, wild-type and QS-deficient P. aeruginosa strains formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independently of QS.

Isolation and characterization of a putative multidrug resistance pump from Vibrio cholerae

Multidrug‐resistant strains of Vibrio cholerae (the causative agent of the diarrhoeal disease cholera) have recently been described. In an attempt to identify a homologue of the Escherichia coli TolC in V. cholerae, we isolated a DNA fragment (pVC) that enabled an E. coli tolC mutant to grow in the presence of 0.05% deoxycholate (DOC). However, other TolC defects were not complemented. Nucleotide sequence analysis of this fragment revealed the presence of two open reading frames (ORF1 and ORF2) separated by 9 bp and encoding 42.4 and 55.8 kDa proteins respectively. The translational products of these two ORFs correlated closely with the molecular weights of the predicted proteins. The deduced amino acid sequences of ORF1 and ORF2 showed a high degree of similarity with conserved regions of the E. coli efflux pump proteins, EmrA and EmrB. The presence of pVC2 within the E. coli efflux pump mutants defective in either the emrAB or the acrAB genes provided the mutants with resistance against several antibiotics. A V. cholerae isogenic mutant defective in ORF2 was constructed by gene replacement. Characterization of this mutant has shown it to be more sensitive to CCCP, PMA, PCP, nalidixic acid and DOC than the parent strain. These results suggest that ORF1 and ORF2 constitute an operon encoding two components of a putative multidrug resistance pump in V. cholerae. In addition, the presence of both structural and functional similarities between VceAB and EmrAB suggests that VceAB is a homologue of EmrAB.

Pseudomonas aeruginosa Autoinducer Enters and Functions in Mammalian Cells

Quorum sensing (QS) is a cell density-dependent signaling mechanism used by many bacteria to control gene expression. Several recent reports indicate that the signaling molecules (autoinducers) that mediate QS in Pseudomonas aeruginosa may also modulate gene expression in host cells; however, the mechanisms are largely unknown. Here we show that two P. aeruginosa autoinducers, N-3-oxododecanoyl-homoserine lactone and N-butyryl-homoserine lactone, can both enter eukaryotic cells and activate artificial chimeric transcription factors based on their cognate transcriptional activators, LasR and RhlR, respectively. The autoinducers promoted nuclear localization of chimeric proteins containing the full LasR or RhlR coding region, and the LasR-based proteins were capable of activating transcription of a LasR-dependent luciferase gene. Responsiveness to autoinducer required the N-terminal autoinducer-binding domains of LasR and RhlR. Truncated proteins consisting of only the C-terminal helix-turn-helix DNA-binding domains of both proteins attached to a nuclear localization signal efficiently translocated to the nucleus in the absence of autoinducer, and truncated LasR-based proteins functioned as constitutively active transcription factors. Chimeric LasR proteins were only activated by their cognate autoinducer ligand and not by N-butyryl-L-homoserine lactone. These data provide evidence that autoinducer molecules from human pathogens can enter mammalian cells and suggest that autoinducers may influence gene expression in host cells by interacting with and activating as-yet-unidentified endogenous proteins.

Isolation and characterization of a Pseudomonas aeruginosa gene, ptxR, which positively regulates exotoxin A production.

Exotoxin A production in Pseudomonas aeruginosa is a complicated and highly regulated process that involves several genes. In this report, we describe the isolation of a new toxA regulatory gene (ptxR) which affects exotoxin A production in P. aeruginosa. In an iron‐deficient medium, the presence of a plasmid carrying ptxR in P. aeruginosa PAO1 resulted in a four‐ to fivefold increase in exotoxin A synthesis. No effect was observed on the levels of elastase, phospholipase C, exoenzyme S, and alkaline protease. Using subcloning and complementation experiments, ptxR was localized to a 2.1 kb KpnI–BglII fragment. Nucleotide sequence analysis revealed the presence of an open reading frame which encodes a 34.97 kDa protein (PtxR). The size of the predicted PtxR compares closely with the 34 kDa PtxR that was synthesized in Escherichia coli using the T7 expression system. The deduced amino acid sequence of PtxR is homologous to that of several members of the LysR family of transcriptional activators. The amino‐terminus region of PtxR contains a putative helix‐turn‐helix DNA‐binding motif. Specific ptxR‐deletion mutants in P. aeruginosa strains PAO1 and PA103 were constructed. In comparison with their parent strains, both mutants showed a significant reduction in the level of exotoxin A activity. However, upon extensive subculturing, the level of exotoxin A produced by the PAO1::ptxR mutant was similar to that of PAO1. Transcriptional studies, using both toxA–lacZ fusion and RNA analysis, confirmed that ptxR increases toxA and regA transcription. These results suggest that ptxR regulates (through regA) exotoxin A production at the transcriptional level.

Gallium Maltolate Treatment Eradicates Pseudomonas aeruginosa Infection in Thermally Injured Mice

ABSTRACT Gallium (Ga) is a semimetallic element that has demonstrated therapeutic and diagnostic-imaging potential in a number of disease settings, including cancer and infectious diseases. Galliums biological actions stem from its ionic radius being almost the same as that of ferric iron (Fe3+), whereby it can replace iron (Fe) in Fe3+-dependent biological systems, such as bacterial and mammalian Fe transporters and Fe3+-containing enzymes. Unlike Fe3+, ionic gallium (Ga3+) cannot be reduced, and when incorporated, it inactivates Fe3+-dependent reduction and oxidation processes that are necessary for bacterial and mammalian cell proliferation. Most pathogenic bacteria require Fe for growth and function, and the availability of Fe in the host or environment can greatly enhance virulence. We examined whether gallium maltolate (GaM), a novel formulation of Ga, had antibacterial activity in a thermally injured acute infection mouse model. Dose-response studies indicated that a GaM dose as low as 25 mg/kg of body weight delivered subcutaneously was sufficient to provide 100% survival in a lethal P. aeruginosa-infected thermally injured mouse model. Mice treated with 100 mg/kg GaM had undetectable levels of Pseudomonas aeruginosa in their wounds, livers, and spleens, while the wounds of untreated mice were colonized with over 108P. aeruginosa CFU/g of tissue and their livers and spleens were colonized with over 105P. aeruginosa CFU/g of tissue. GaM also significantly reduced the colonization of Staphylococcus aureus and Acinetobacter baumannii in the wounds of thermally injured mice. Furthermore, GaM was also therapeutically effective in preventing preestablished P. aeruginosa infections at the site of the injury from spreading systemically. Taken together, our data suggest that GaM is potentially a novel antibacterial agent for the prevention and treatment of wound infections following thermal injury.

Organoselenium Coating on Cellulose Inhibits the Formation of Biofilms by Pseudomonas aeruginosa and Staphylococcus aureus

ABSTRACT Among the most difficult bacterial infections encountered in treating patients are wound infections, which may occur in burn victims, patients with traumatic wounds, necrotic lesions in people with diabetes, and patients with surgical wounds. Within a wound, infecting bacteria frequently develop biofilms. Many current wound dressings are impregnated with antimicrobial agents, such as silver or antibiotics. Diffusion of the agent(s) from the dressing may damage or destroy nearby healthy tissue as well as compromise the effectiveness of the dressing. In contrast, the antimicrobial agent selenium can be covalently attached to the surfaces of a dressing, prolonging its effectiveness. We examined the effectiveness of an organoselenium coating on cellulose discs in inhibiting Pseudomonas aeruginosa and Staphylococcus aureus biofilm formation. Colony biofilm assays revealed that cellulose discs coated with organoselenium completely inhibited P. aeruginosa and S. aureus biofilm formation. Scanning electron microscopy of the cellulose discs confirmed these results. Additionally, the coating on the cellulose discs was stable and effective after a week of incubation in phosphate-buffered saline. These results demonstrate that 0.2% selenium in a coating on cellulose discs effectively inhibits bacterial attachment and biofilm formation and that, unlike other antimicrobial agents, longer periods of exposure to an aqueous environment do not compromise the effectiveness of the coating.

Contribution of the regulatory gene lasR to the pathogenesis of Pseudomonas aeruginosa infection of burned mice.

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that causes severe infections in patients with burns. The P aeruginosa regulatory gene, lasR, regulates the expression of several virulence factors. The specific lasR isogenic mutant, PAO-R1, is defective in the synthesis of the 2 elastases (LasB and LasA) and produces low levels of exotoxin A and alkaline proteases. In this study, we used a burned mouse model to examine the role of lasR in the pathogenesis of P aeruginosa infections. We have examined the following aspects of P aeruginosa infections: 1) lethality to the burned mouse, 2) the dissemination within the body of the burned mouse, and 3) the local spread within the burned skin. In comparison with its parent strain, PAO1, PAO-R1 was less lethal. In addition, the numbers of PAO-R1 microorganisms recovered from the livers and spleens of the burned mice were less than those of PAO1. Furthermore, at 8 hours postinfection, equivalent numbers of PAO1 and PAO-R1 were detected at the inoculation site of the burned skin. However, only PAO1 microorganisms were detected at other sites of the burned skin. These results suggest that the lasR gene contributes (directly and indirectly) to the dissemination of P aeruginosa within the body of burned mice and its horizontal spread within the burned skin.

An in vitro biofilm model to examine the effect of antibiotic ointments on biofilms produced by burn wound bacterial isolates

PURPOSE Topical treatment of burn wounds is essential as reduced blood supply in the burned tissues restricts the effect of systemic antibiotics. On the burn surface, microorganisms exist within a complex structure termed a biofilm, which enhances bacterial resistance to antimicrobial agents significantly. Since bacteria differ in their ability to develop biofilms, the susceptibility of these biofilms to topically applied antibiotics varies, making it essential to identify which topical antibiotics efficiently disrupt or prevent biofilms produced by these pathogens. Yet, a simple in vitro assay to compare the susceptibility of biofilms produced by burn wound isolates to different topical antibiotics has not been reported. METHODS Biofilms were developed by inoculating cellulose disks on agar plates with burn wound isolates and incubating for 24h. The biofilms were then covered for 24h with untreated gauze or gauze coated with antibiotic ointment and remaining microorganisms were quantified and visualized microscopically. RESULTS Mupirocin and triple antibiotic ointments significantly reduced biofilms produced by the Staphylococcus aureus and Pseudomonas aeruginosa burn wound isolates tested, as did gentamicin ointment, with the exception of one P. aeruginosa clinical isolate. CONCLUSIONS The described assay is a practical and reproducible approach to identify topical antibiotics most effective in eliminating biofilms produced by burn wound isolates.

Syndecan 1 Shedding Contributes to Pseudomonas aeruginosa Sepsis

ABSTRACT The innate immune system is comprised of many components that function coordinately to prevent bacterial sepsis. However, thermal injury suppresses many of these factors, and the opportunistic pathogen Pseudomonas aeruginosa takes advantage of this condition, making it one of the leading causes of morbidity and mortality in the setting of thermal injury. P. aeruginosa is extremely efficient at colonizing burn wounds, spreading systemically, and causing sepsis, which often results in a systemic inflammatory response, multiple-organ failure, and death. The pathogenicity of P. aeruginosa is due to the arsenal of virulence factors produced by the pathogen and the immunocompromised state of the host. Syndecan 1 is a major heparan sulfate proteoglycan present on many host cells involved in thermal injury. Syndecan 1 anchored to the cell surface can be cleaved in a process termed ectodomain shedding. Syndecan 1 shedding results in the release of intact, soluble proteoglycan ectodomains that have diverse roles in innate immunity. Here we show for the first time that thermal injury results in shedding of syndecan 1 from host tissue. Our data show that syndecan 1 null mice are significantly less susceptible to P. aeruginosa infection than their wild-type counterparts, as demonstrated by (i) significantly lower mortality; (ii) absence of systemic spread of P. aeruginosa; and (iii) significant reductions in some proinflammatory cytokines. These results suggest that shed syndecan 1 plays an important role in the pathogenesis of P. aeruginosa infection of thermal injury and that syndecan 1-neutralizing agents may be effective supplements to current P. aeruginosa treatments.