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1.  Presence or Absence of Lipopolysaccharide O Antigens Affects Type III Secretion by Pseudomonas aeruginosa▿  
Journal of Bacteriology  2007;189(6):2203-2209.
Pseudomonas aeruginosa is one of the major causative agents of mortality and morbidity in hospitalized patients due to a multiplicity of virulence factors associated with both chronic and acute infections. Acute P. aeruginosa infection is primarily mediated by planktonic bacteria expressing the type III secretion system (TTSS), a surface-attached needle-like complex that injects cytotoxins directly into eukaryotic cells, causing cellular damage. Lipopolysaccharide (LPS) is the principal surface-associated virulence factor of P. aeruginosa. This molecule is known to undergo structural modification (primarily alterations in the A- and B-band O antigen) in response to changes in the mode of life (e.g., from biofilm to planktonic). Given that LPS exhibits structural plasticity, we hypothesized that the presence of LPS lacking O antigen would facilitate eukaryotic intoxication and that a correlation between the LPS O-antigen serotype and TTSS-mediated cytotoxicity would exist. Therefore, strain PAO1 (A+ B+ O-antigen serotype) and isogenic mutants with specific O-antigen defects (A+ B−, A− B+, and A− B−) were examined for TTSS expression and cytotoxicity. A strong association existed in vitro between the absence of the large, structured B-band O antigen and increased cytotoxicity of these strains. In vivo, all three LPS mutant strains demonstrated significantly increased lung injury compared to PAO1. Clinical strains lacking the B-band O antigen also demonstrated increased TTSS secretion. These results suggest the existence of a cooperative association between LPS O-antigen structure and the TTSS in both laboratory and clinical isolates of P. aeruginosa.
PMCID: PMC1899375  PMID: 17209027
2.  Differences in biofilm formation and antimicrobial resistance of Pseudomonas aeruginosa isolated from airways of mechanically ventilated patients and cystic fibrosis patients 
Pseudomonas aeruginosa biofilms exhibit increased antimicrobial resistance compared with planktonic isolates and are implicated in the pathogenesis of both acute and chronic lung infections. Whilst antibiotic choices for both infections are based on planktonic antibiotic susceptibility results, differences in biofilm-forming ability between the two diseases have not previously been explored. The aim of this study was to compare differences in biofilm formation and antibiotic resistance of P. aeruginosa isolated from intubated patients and from patients with chronic pulmonary disease associated with cystic fibrosis (CF). The temporal evolution of antibiotic resistance in clonal P. aeruginosa strains isolated from CF patients during periods of chronic infection and acute pulmonary exacerbation was also evaluated. Biofilm formation and biofilm antibiotic susceptibilities were determined using a modified microtitre plate assay and were compared with antibiotic susceptibility results obtained using traditional planktonic culture. Clonality was confirmed using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Pseudomonas aeruginosa isolates collected from intubated patients produced substantially more biofilms compared with CF isolates. There was considerable heterogeneity in biofilm-forming ability among the CF isolates and this was unrelated to pulmonary status. Biofilm antibiotic resistance developed rapidly among clonal CF isolates over time, whilst traditional antibiotic resistance determined using planktonic cultures remained stable. There was a significant positive correlation between imipenem/cilastatin and ceftazidime resistance and biofilm-forming ability. The variability in biofilm-forming ability in P. aeruginosa and the rapid evolution of biofilm resistance may require consideration when choosing antibiotic therapy for newly intubated patients and CF patients.
PMCID: PMC3176759  PMID: 21382698
Pseudomonas aeruginosa; Bacterial biofilm; Antimicrobial resistance; Mechanical ventilation; Cystic fibrosis
3.  Secretion of Pseudomonas aeruginosa Type III Cytotoxins is Dependent on Pseudomonas Quinolone Signal Concentration 
Microbial pathogenesis  2010;49(4):196-203.
Pseudomonas aeruginosa is an opportunistic pathogen that can, like other bacterial species, exist in antimicrobial resistant sessile biofilms and as free-swimming, planktonic cells. Specific virulence factors are typically associated with each lifestyle and several two-component response regulators have been shown to reciprocally regulate transition between biofilm-associated chronic, and free-swimming acute infections. Quorum sensing (QS) signal molecules belonging to the las and rhl systems are known to regulate virulence gene expression by P. aeruginosa. However the impact of a recently described family of novel quorum sensing signals produced by the Pseudomonas Quinolone Signal (PQS) biosynthetic pathway, on the transition between these modes of infection is less clear. Using clonal isolates from a patient developing ventilator-associated pneumonia, we demonstrated that clinical observations were mirrored by an in vitro temporal shift in isolate phenotype from a non-secreting, to a Type III cytotoxin secreting (TTSS) phenotype and further, that this phenotypic change was PQS-dependent. While intracellular type III cytotoxin levels were unaffected by PQS concentration, cytotoxin secretion was dependent on this signal molecule. Elevated PQS concentrations were associated with inhibition of cytotoxin secretion coincident with expression of virulence factors such as elastase and pyoverdin. In contrast, low concentrations or the inability to biosynthesize PQS resulted in a reversal of this phenotype. These data suggest that expression of specific P. aeruginosa virulence factors appears to be reciprocally regulated and that an additional level of PQS-dependent posttranslational control, specifically governing type III cytotoxin secretion, exists in this species.
PMCID: PMC2935322  PMID: 20570614
Pseudomonas quinolone signal; Type III secretion; Pseudomonas aeruginosa; ventilator-associated pneumonia; endotrachael aspirate
4.  Novel Strategies to Combat Bacterial Virulence 
Current opinion in critical care  2008;14(5):593-599.
Purpose of review
Incidences of antimicrobial resistant infections have increased dramatically over the past several decades and are associated with adverse patient outcomes. Alternative approaches to combat infection are critical, and have led to the development of more specific drugs targeted at particular bacterial virulence systems or essential regulatory pathways. The purpose of this review is to highlight the recent developments in anti-bacterial therapy and the novel approaches toward increasing our therapeutic armory against bacterial infection.
Recent findings
Although classic antibiotic development is not occurring rapidly, alternative therapeutics that target specific bacterial virulence systems are progressing from the discovery stage through the FDA approval process. Here we review novel antibodies that target specific virulence systems as well as a variety of newly discovered small molecules that block bacterial attachment, communication systems (quorum sensing) or important regulatory processes associated with virulence gene expression.
The success of novel therapeutics could significantly change clinical practice. Furthermore, the complications of collateral damage due to antibiotic administration e.g. suprainfections or decreased host immunity due to loss of synergistic bacterial communities, may be minimized using therapeutics that specifically target pathogenic behavior.
PMCID: PMC2859961  PMID: 18787455
Bacterial virulence; antimicrobial therapy; Quorum sensing; type III secretion system; novel therapeutics
5.  Loss of Bacterial Diversity during Antibiotic Treatment of Intubated Patients Colonized with Pseudomonas aeruginosa▿  
Journal of Clinical Microbiology  2007;45(6):1954-1962.
Management of airway infections caused by Pseudomonas aeruginosa is a serious clinical challenge, but little is known about the microbial ecology of airway infections in intubated patients. We analyzed bacterial diversity in endotracheal aspirates obtained from intubated patients colonized by P. aeruginosa by using 16S rRNA clone libraries and microarrays (PhyloChip) to determine changes in bacterial community compositions during antibiotic treatment. Bacterial 16S rRNA genes were absent from aspirates obtained from patients briefly intubated for elective surgery but were detected by PCR in samples from all patients intubated for longer periods. Sequencing of 16S rRNA clone libraries demonstrated the presence of many orally, nasally, and gastrointestinally associated bacteria, including known pathogens, in the lungs of patients colonized with P. aeruginosa. PhyloChip analysis detected the same organisms and many additional bacterial groups present at low abundance that were not detected in clone libraries. For each patient, both culture-independent methods showed that bacterial diversity decreased following the administration of antibiotics, and communities became dominated by a pulmonary pathogen. P. aeruginosa became the dominant species in six of seven patients studied, despite treatment of five of these six with antibiotics to which it was sensitive in vitro. Our data demonstrate that the loss of bacterial diversity under antibiotic selection is highly associated with the development of pneumonia in ventilated patients colonized with P. aeruginosa. Interestingly, PhyloChip analysis demonstrated reciprocal changes in abundance between P. aeruginosa and the class Bacilli, suggesting that these groups may compete for a similar ecological niche and suggesting possible mechanisms through which the loss of microbial diversity may directly contribute to pathogen selection and persistence.
PMCID: PMC1933106  PMID: 17409203
6.  Escherichia coli Biofilms Formed under Low-Shear Modeled Microgravity in a Ground-Based System▿ †  
Applied and Environmental Microbiology  2006;72(12):7701-7710.
Bacterial biofilms cause chronic diseases that are difficult to control. Since biofilm formation in space is well documented and planktonic cells become more resistant and virulent under modeled microgravity, it is important to determine the effect of this gravity condition on biofilms. Inclusion of glass microcarrier beads of appropriate dimensions and density with medium and inoculum, in vessels specially designed to permit ground-based investigations into aspects of low-shear modeled microgravity (LSMMG), facilitated these studies. Mathematical modeling of microcarrier behavior based on experimental conditions demonstrated that they satisfied the criteria for LSMMG conditions. Experimental observations confirmed that the microcarrier trajectory in the LSMMG vessel concurred with the predicted model. At 24 h, the LSMMG Escherichia coli biofilms were thicker than their normal-gravity counterparts and exhibited increased resistance to the general stressors salt and ethanol and to two antibiotics (penicillin and chloramphenicol). Biofilms of a mutant of E. coli, deficient in σs, were impaired in developing LSMMG-conferred resistance to the general stressors but not to the antibiotics, indicating two separate pathways of LSMMG-conferred resistance.
PMCID: PMC1694224  PMID: 17028231
7.  Role and Regulation of σs in General Resistance Conferred by Low-Shear Simulated Microgravity in Escherichia coli 
Journal of Bacteriology  2004;186(24):8207-8212.
Life on Earth evolved in the presence of gravity, and thus it is of interest from the perspective of space exploration to determine if diminished gravity affects biological processes. Cultivation of Escherichia coli under low-shear simulated microgravity (SMG) conditions resulted in enhanced stress resistance in both exponential- and stationary-phase cells, making the latter superresistant. Given that microgravity of space and SMG also compromise human immune response, this phenomenon constitutes a potential threat to astronauts. As low-shear environments are encountered by pathogens on Earth as well, SMG-conferred resistance is also relevant to controlling infectious disease on this planet. The SMG effect resembles the general stress response on Earth, which makes bacteria resistant to multiple stresses; this response is σs dependent, irrespective of the growth phase. However, SMG-induced increased resistance was dependent on σs only in stationary phase, being independent of this sigma factor in exponential phase. σs concentration was some 30% lower in exponential-phase SMG cells than in normal gravity cells but was twofold higher in stationary-phase SMG cells. While SMG affected σs synthesis at all levels of control, the main reasons for the differential effect of this gravity condition on σs levels were that it rendered the sigma protein less stable in exponential phase and increased rpoS mRNA translational efficiency. Since σs regulatory processes are influenced by mRNA and protein-folding patterns, the data suggest that SMG may affect these configurations.
PMCID: PMC532419  PMID: 15576768
8.  Rearrangement of a Large Novel Pseudomonas aeruginosa Gene Island in Strains Isolated from a Patient Developing Ventilator-Associated Pneumonia 
Journal of Clinical Microbiology  2014;52(7):2430-2438.
Bacterial gene islands add to the genetic repertoire of opportunistic pathogens. Here, we perform comparative analyses of three Pseudomonas aeruginosa strains isolated sequentially over a 3-week period from a patient with ventilator-associated pneumonia (VAP) who received clindamycin and piperacillin-tazobactam as part of their treatment regime. While all three strains appeared to be clonal by standard pulsed-field gel electrophoresis, whole-genome sequencing revealed subtle alterations in the chromosomal organization of the last two strains; specifically, an inversion event within a novel 124-kb gene island (PAGI 12) composed of 137 open reading frames [ORFs]. Predicted ORFs in the island included metabolism and virulence genes. Overexpression of a gene island-borne putative β-lactamase gene was observed following piperacillin-tazobactam exposure and only in those strains that had undergone the inversion event, indicating altered gene regulation following genomic remodeling. Examination of a separate cohort of 76 patients with VAP for integration at this tRNAlys recombination site demonstrated that patients exhibiting evidence of integration at this site had significantly higher 28-day mortality. These findings provide evidence that P. aeruginosa can integrate, rapidly remodel, and express exogenous genes, which likely contributes to its fitness in a clinical setting.
PMCID: PMC4097748  PMID: 24789195
10.  Role of the rapA Gene in Controlling Antibiotic Resistance of Escherichia coli Biofilms▿ †  
Antimicrobial Agents and Chemotherapy  2007;51(10):3650-3658.
By using a high-throughput screening method, a mutant of a uropathogenic Escherichia coli strain affected in the rapA gene was isolated. The mutant formed normal-architecture biofilms but showed decreased penicillin G resistance, although the mutation did not affect planktonic cell resistance. Transcriptome analysis showed that 22 genes were down-regulated in the mutant biofilm. One of these genes was yhcQ, which encodes a putative multidrug resistance pump. Mutants with mutations in this gene also formed biofilms with decreased resistance, although the effect was less pronounced than that of the rapA mutation. Thus, an additional mechanism(s) controlled by a rapA-regulated gene(s) was involved in wild-type biofilm resistance. The search for this mechanism was guided by the fact that another down-regulated gene in rapA biofilms, yeeZ, is suspected to be involved in extra cell wall-related functions. A comparison of the biofilm matrix of the wild-type and rapA strains revealed decreased polysaccharide quantities and coverage in the mutant biofilms. Furthermore, the (fluorescent) functional penicillin G homologue Bocillin FL penetrated the mutant biofilms more readily. The results strongly suggest a dual mechanism for the wild-type biofilm penicillin G resistance, retarded penetration, and effective efflux. The results of studies with an E. coli K-12 strain pointed to the same conclusion. Since efflux and penetration can be general resistance mechanisms, tests were conducted with other antibiotics. The rapA biofilm was also more sensitive to norfloxacin, chloramphenicol, and gentamicin.
PMCID: PMC2043260  PMID: 17664315
11.  Effect of Chromate Stress on Escherichia coli K-12 
Journal of Bacteriology  2006;188(9):3371-3381.
The nature of the stress experienced by Escherichia coli K-12 exposed to chromate, and mechanisms that may enable cells to withstand this stress, were examined. Cells that had been preadapted by overnight growth in the presence of chromate were less stressed than nonadapted controls. Within 3 h of chromate exposure, the latter ceased growth and exhibited extreme filamentous morphology; by 5 h there was partial recovery with restoration of relatively normal cell morphology. In contrast, preadapted cells were less drastically affected in their morphology and growth. Cellular oxidative stress, as monitored by use of an H2O2-responsive fluorescent dye, was most severe in the nonadapted cells at 3 h postinoculation, lower in the partially recovered cells at 5 h postinoculation, and lower still in the preadapted cells. Chromate exposure depleted cellular levels of reduced glutathione and other free thiols to a greater extent in nonadapted than preadapted cells. In both cell types, the SOS response was activated, and levels of proteins such as SodB and CysK, which can counter oxidative stress, were increased. Some mutants missing antioxidant proteins (SodB, CysK, YieF, or KatE) were more sensitive to chromate. Thus, oxidative stress plays a major role in chromate toxicity in vivo, and cellular defense against this toxicity involves activation of antioxidant mechanisms. As bacterial chromate bioremediation is limited by the toxicity of chromate, minimizing oxidative stress during bacterial chromate reduction and bolstering the capacity of these organisms to deal with this stress will improve their effectiveness in chromate bioremediation.
PMCID: PMC1447458  PMID: 16621832
12.  Hilar tuberculosis in a patient with osteosarcoma 
Postgraduate Medical Journal  1981;57(670):530-531.
A case of hilar tuberculosis in a patient with osteosarcoma is described illustrating the importance of accurate diagnosis in this unusual association. This occurrence is discussed in the light of the use of potentially immunosuppressive treatments for osteosarcoma.
PMCID: PMC2426114  PMID: 6946409

Results 1-12 (12)