Gram-negative outer membrane vesicles: beyond the cell surface.

Considerable interest has recently mounted regarding the biological roles of Gram-negative outer membrane vesicles (MVs). The first discovery of MVs was made over four decades ago, and it is now clear that most Gram-negative bacteria produce MVs, with Pseudomonas aeruginosa and Escherichia coli as the most extensively studied. Much of our knowledge of the biological roles of MVs and mechanism of MV formation is due to T.J. Beveridge and colleagues. Beveridge pioneered the field of MV research not only by enhancing our understanding of MV function, but also through the application of a wide variety of physical, chemical, and genetic techniques to complement his elegant electron microscopy investigations. Here we review the contributions of Beveridge's group to our understanding of MV biology.

Dietary phytochemicals as quorum sensing inhibitors.

Quorum sensing (QS) is a cell density dependent expression of species in bacteria mediated by hormone like compounds called autoinducers (AI). Several processes responsible for successful establishment of bacterial infection are mediated by QS. Inhibition of QS is therefore being considered as a new target for antimicrobial chemotherapy. Dietary phytochemicals are secondary metabolites in plants known to have several health benefits including antimicrobial activity. However, their ability to inhibit QS has never been studied. Our objective was to investigate the effect of sub-lethal concentrations (SLC) of bioactive dietary phytochemical extracts from common dietary fruit, herb and spice extracts on modulating QS mediated by AI in model bioassay test systems. QS inhibition was measured in violacein pigment producing Chromobacterium violaceum O26 (CVO26) and CV 31532 system, mediated by AI known as acylated homeserine lactone (AHL). We also investigated the effect of the sub-lethal concentrations of the extracts on swarming motility of pathogens Escherichia coli (EC)O157:H7 and Pseudomonas aeruginosa (PA-01). Our results indicate that all extracts significantly inhibited quorum sensing. The mechanism of inhibition appeared to be combination of interfering with AHL activity and modulating the synthesis of AHL's. Our results also indicated that various phytochemical extracts which inhibited QS also inhibited swarming of pathogenic bacteria, known to be modulated by QS. The observation that phytochemicals from foods can inhibit QS related processes opens up an exciting new strategy for antimicrobial chemotherapy and lead to the discovery of new category of antibiotics which can overcome the issues related to antimicrobial resistance.

The effects of gabapentin and memantine in acquired and congenital nystagmus: a retrospective study.

BACKGROUND: Pharmacological treatment has been successful in some forms of acquired neurological nystagmus. However, drugs are not known to be effective in idiopathic infantile nystagmus or nystagmus associated with ocular diseases. METHODS: The authors retrospectively analysed Snellen visual acuity (VA), subjective visual function, and eye movement recordings of 23 patients with nystagmus (13 secondary to multiple sclerosis, three associated with other neurological diseases, two idiopathic infantile, and five with associated ocular diseases) treated with gabapentin or memantine. RESULTS: With gabapentin, 10 of 13 patients with nystagmus secondary to multiple sclerosis (MS) showed some improvement. Memantine improved the VA in all three patients with MS who did not improve on gabapentin. There was no change of nystagmus in other neurological disorders. Patients with congenital nystagmus showed reduction of nystagmus and their VA changes depended on the ocular pathology. CONCLUSION: Gabapentin and memantine may be effective in acquired nystagmus secondary to MS. To the authors' knowledge this is the first series of patients showing that gabapentin is effective in improving nystagmus in congenital nystagmus/nystagmus associated with ocular pathology. Memantine may be useful as an alternative drug in treating patients with nystagmus.

Identification and characterization of stripe rust resistance gene Yr34 in common wheat.

An uncharacterized source of seedling resistance to Puccinia striiformis f.sp. tritici was identified in an advanced wheat breeding line WAWHT2046. Genetic analysis based on a WAWHT2046/Carnamah-derived double haploid (DH) population demonstrated monogenic inheritance of seedling stripe rust resistance in WAWHT2046. The gene controlling stripe rust resistance in line WAWHT2046 was tentatively designated YrWA. The chromosome 5AL located awn inhibitor gene B1, possessed by WAWHT2046, also showed monogenic inheritance when the DH population was scored for the presence and absence of awns. Joint segregation analysis at the B1 and YrWA loci indicated genetic linkage between the two loci. A recombination value of 12.2 cM was computed using Mapmanager. This association located YrWA in the chromosome arm 5AL. Molecular mapping using microsatellite markers placed YrWA distal to B1. All molecular markers mapped proximal to the awn inhibitor locus B1. As no other stripe rust resistance gene is reported to be located in the chromosome arm 5AL, YrWA was permanently designated as Yr34. Yr34 produced an intermediate (23C) seedling infection type and expressed very low stripe rust response (10R-MR) on adult plants in the field, similar to the resistance gene Yr17. In addition to Yr34, this mapping population segregated for three genetically independent adult plant stripe rust resistance genes. The detection of DH lines with completely susceptible response, higher than that shown by the Yr34-lacking parent Carnamah, suggested that both parents contributed adult plant resistance. The use of WAWHT2046 as a parent in breeding programs would also contribute APR in addition to Yr34.

Bacteriophage T4 multiplication in a glucose-limited Escherichia coli biofilm.

An Escherichia coli K-12 biofilm was grown at a dilution rate of 0.028 h(-1) for 48 h in a glucose-limited chemostat coupled to a modified Robbins' device to determine its susceptibility to infection by bacteriophage T4. Bacteriophage T4 at a multiplicity of infection (MOI) of 10 caused a log reduction in biofilm density (expressed as colony forming units (CFU) per cm2) at 90 min postinfection. After 6 h, a net decrease and equilibrium in viral titer was seen. When biofilms were exposed to T4 phage at a MOI of 100, viral titer doubled after 90 min. After 6 h, viral titers (expressed as plaque forming units (PFU) per cm2) stabilized at levels approximately one order of magnitude higher than seen at a MOI of 10. Scanning confocal laser microscopy images also indicated disruption of biofilm morphology following T4 infection with the effects being more pronounced at a MOI of 100 than at a MOI of 10. These results imply that biofilms under carbon limitation can act as natural reservoirs for bacteriophage and that bacteriophage can have some influence on biofilm morphology.

Phenotype characterization of genetically defined microorganisms and growth of bacteriophage in biofilms.

Phenotypic characterization will be a pivotal aspect of future research in understanding the biofilm mode of growth. We hope that the concepts and techniques presented in this chapter will benefit other investigators in this field. Although initial studies will necessarily involve monocultures, eventually mixed culture work will have to be performed to understand biofilm growth in the natural environment. As the study of biofilm-phage interactions is new, there is considerable fundamental work that needs to be addressed. Here, we anticipate that some phage are better adapted to growth in biofilms, some are adept in growing in mixed culture biofilms, and others are better adapted to infecting planktonic organisms. Whereas biofilms are now widely accepted as a fundamental aspect of microbial growth in nature, the field of phage ecology is quite new and an exciting challenge for the future.

Bacterial biofilm formation under microgravity conditions.

Although biofilm formation is widely documented on Earth, it has not been demonstrated in the absence of gravity. To explore this possibility, Pseudomonas aeruginosa, suspended in sterile buffer, was flown in a commercial payload on space shuttle flight STS-95. During earth orbit, biofilm formation was induced by exposing the bacteria to sterile media through a 0.2-microm (pore size) polycarbonate membrane. Examination of these membranes by confocal microscopy revealed biofilms to be present and that these biofilms could persist in spite of vigorous agitation. These results represent the first report of biofilm formation under microgravity conditions.

Effects of community composition and growth rate on aquifer biofilm bacteria and their susceptibility to betadine disinfection.

Biofilm formation and function was studied in mixed culture using 20 bacterial strains isolated from a karst aquifer. When co-cultured in a glucose-limited chemostat, Vogesella indigofera and Pseudomonas putida were the dominant planktonic and biofilm organisms respectively. Biofilm formation and resistance to the iodine disinfectant betadine were then studied with monoculture and binary cultures of V. indigofera and P. putida and a 20-strain community. Biofilm population size [measured as colony-forming units (CFU) cm(-2)] increased with increasing species diversity. Significantly larger populations formed at dilution rates (DRs) of 0.0083 h(-1) than at 0.033 h(-1). P. putida populations were higher and V. indigofera lower in binary than in monoculture biofilms, suggesting that P. putida outcompeted V. indigofera. In binary biofilms, V. indigofera, a betadine-resistant organism, enhanced the survival of P. putida, a betadine-susceptible organism. In the 20-strain biofilms, this protective effect was not observed because of low concentrations of V. indigofera (< 1% of the total population), suggesting that resistant organisms contribute to overall biofilm disinfectant resistance. Growth at 0.033 h(-1) enhanced survival of V. indigofera biofilms against betadine. Although DR did influence survival of the other communities, its effects were neither consistent nor significant. All told, biofilm formation and betadine resistance are complex phenomena, influenced by community composition, growth rate and betadine concentration.

Proteus mirabilis viability after lithotripsy of struvite calculi.

PURPOSE: We tested the hypotheses that Proteus mirabilis viability of struvite calculi differs after exposure to different lithotripsy modalities and that the photothermal mechanism of holmium:YAG lithotripsy is antibacterial. MATERIALS AND METHODS: Human calculi of known struvite composition (greater than 90% magnesium ammonium phosphate hexohydrate) were incubated with P. mirabilis. Calculi were randomly distributed and fragmented with no lithotripsy (controls), or shock wave, intracorporeal ultrasonic, electrohydraulic, pneumatic, holmium:YAG or pulsed dye laser lithotripsy. After lithotripsy fragments were sonicated and specimens were serially plated for 48 hours at 38C. Bacterial counts and the rate of bacterial sterilization were compared. RESULTS: Median bacterial counts (colony-forming units per ml.) were 8 x 10(6) in controls and 3 x 10(6) in shock wave, 3 x 10(7) in ultrasonic, 4 x 10(5) in electrohydraulic, 8 x 10(6) in pneumatic, 5 x 10(4) in holmium:YAG and 1 x 10(6) in pulsed dye laser lithotripsy cases (p <0.001). The rate of bacterial sterilization was 50% for holmium:YAG lithotripsy treated stones versus 0% for each of the other cohorts (p <0.01). CONCLUSIONS: P. mirabilis viability varies among lithotrites. The photothermal mechanism of holmium:YAG lithotripsy is antibacterial.

Impact of rpoS deletion on Escherichia coli biofilms.

Slow growth has been hypothesized to be an essential aspect of bacterial physiology within biofilms. In order to test this hypothesis, we employed two strains of Escherichia coli, ZK126 (DeltalacZ rpoS(+)) and its isogenic DeltarpoS derivative, ZK1000. These strains were grown at two rates (0.033 and 0.0083 h(-1)) in a glucose-limited chemostat which was coupled either to a modified Robbins device containing plugs of silicone rubber urinary catheter material or to a glass flow cell. The presence or absence of rpoS did not significantly affect planktonic growth of E. coli. In contrast, biofilm cell density in the rpoS mutant strain (ZK1000), as measured by determining the number of CFU per square centimeter, was reduced by 50% (P < 0.05). Deletion of rpoS caused differences in biofilm cell arrangement, as seen by scanning confocal laser microscopy. In reporter gene experiments, similar levels of rpoS expression were seen in chemostat-grown planktonic and biofilm populations at a growth rate of 0.033 h(-1). Overall, these studies suggest that rpoS is important for biofilm physiology.

Influence of metal ions and temperature on the conformation of Escherichia coli K1 capsular polysaccharide.

Escherichia coli K1 secretes a homopolymer capsular polysaccharide (CPS) consisting of alpha 2, 8 linked N-acetylneuraminic acid (poly alpha 2,8NeuNAc). Typically poly alpha 2,8NeuNAc is arranged in low and high order alpha helices with carboxyl and hydroxyl groups extending from the helices. Several properties of CPS such as antigenicity and metal binding can be influenced by its structural conformation. We examined the influences of metal ions and temperature on the secondary structure of poly alpha 2,8NeuNAc. Conformation alteration was detected by ultraviolet (UV) spectroscopy and circular dichroism (CD). The majority of metal ions tested had no detectable influence on poly alpha 2,8NeuNAc structure. In contrast, Yb3+, Hg2+, and Cu2+ ions greatly altered the UV and CD spectra, which suggests that these ions had disrupted the alpha helical structure of poly alpha 2,8NeuNAc. These changes were influenced by the metal ion concentration. When poly alpha 2,8NeuNAc was incubated at temperatures ranging from 20-60 degrees C, alterations in its UV absorption spectra were also seen. The most significant change occurred between 35 and 40 degrees C. In summary, this study suggests that the higher order structure and function of bacterial CPS may be influenced by environmental factors.

The development of bacterial biofilms on indwelling urethral catheters.

The biofilm mode of growth has been implicated in the majority of human bacterial infections. In the urinary tract, notable biofilm-associated infections include prostatitis, chronic cystitis, struvite urolithiasis, and catheter-associated infections. Biofilms protect the causative organisms from host defences and antimicrobial therapy. Biofilm formation has traditionally been considered to result from adhesion and capsule formation by adherent microorganisms. Recent work has shown that a large number of genes are activated during this process, some of which have been associated with twitching motility, quorum sensing, and slow growth. In this paper, we review some of the recent work on biofilm biology and highlight its role in urinary tract infections, particularly those associated with urinary catheters.

Biofilms on indwelling urethral catheters produce quorum-sensing signal molecules in situ and in vitro.

Acylated homoserine lactones (AHLs) are chemical signals that mediate population density-dependent (quorum-sensing) gene expression in numerous gram-negative bacteria. In this study, gram-negative bacilli isolated from catheters were screened for AHL production by a cross-feeding assay utilizing an AHL-responsive Agrobacterium tumefaciens reporter strain. Positive reactions were obtained from 14 isolates of Pseudomonas aeruginosa; negative or weakly positive reactions were recorded for isolates of five other species. P. aeruginosa biofilms were then produced on catheters in a physical model of the bladder. Sections of colonized all-silicone catheters gave positive reactions for the quorum-sensing signal molecules as did sections that had been cleaned of biofilm and autoclaved. Control sections of unused catheters were negative in the tests. Sections from four of nine catheters that had been freshly removed from patients gave positive reactions for AHLs. Cleaned autoclaved sections of three of these catheters also gave strongly positive reactions for AHLs. These results demonstrate that AHLs are produced by biofilms as they develop on the catheters both in vitro in the model and in vivo in the patient's bladder. They represent the first demonstration of AHL production by biofilms in a clinical setting.

Evidence of autoinducer activity in naturally occurring biofilms.

N-Acyl homoserine lactone (AHL) molecules have been shown to act as mediators of population density-dependent (quorum-sensing) gene expression in numerous Gram-negative bacteria. Functions associated with AHL include light production in Vibrio fischeri, expression of virulence factors in Pseudomonas aeruginosa, and conjugation in Agrobacterium tumefaciens. In nature, bacteria often grow as surface-adherent biofilm communities. As biofilms typically contain high concentrations of cells, AHL activity and quorum-sensing gene expression have been proposed as essential components of biofilm physiology. However, proof of AHL production within biofilms has heretofore been lacking. In this study we have employed a cross-feeding assay, using A, tumefaciens A136 (traI::lacZ) as an AHL-responsive reporter strain, to show the presence of naturally occurring AHL production in aquatic biofilms growing on submerged stones. AHL was detected in living biofilms and biofilm extracts, but was not present in rocks lacking a biofilm. This represents the first report of AHL activity in naturally occurring biofilms.

Expression of a nonagglutinating fimbria by Proteus mirabilis.

We have clarified growth conditions and isolation strategies for the nonagglutinating fimbriae from Proteus mirabilis. Nonagglutinating fimbriae were expressed by all P. mirabilis strains we examined, and the major subunit proteins, which ranged from 23 to 29 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had highly conserved N-terminal sequences.

Unique ability of the Proteus mirabilis capsule to enhance mineral growth in infectious urinary calculi.

Struvite (MgNH4PO4.6H2O) calculi are a common complication of Proteus mirabilis urinary tract infections. Although urease is a major virulence factor in calculus formation, the polysaccharide capsule (CPS) of this organism also enhances struvite crystallization and growth in vitro (L. Clapham, R. J. C. McLean, J. C. Nickel, J. Downey, and J. W. Costerton, J. Crystal Growth 104:475-484, 1990). We obtained purified CPS, of known structure and varying anionic character, from P. mirabilis ATCC 49565 and several other organisms. Artificial urine was added to CPS, and the pH was elevated from 5.8 to 8.5 by the addition of urease or titration with 0.25 M NH4OH to induce struvite crystallization. Crystallization was measured by particle counting (Coulter counter), and the morphology (crystal habit) was examined by phase-contrast microscopy. In the presence of partially anionic P. mirabilis CPS, struvite formation occurred at a lower pH than in the absence of CPS or in the presence of other neutral, partially anionic, or anionic CPS. At pH 7.5 to 8.0, significantly more struvite crystals formed in the presence of P. mirabilis CPS than under other experimental conditions. With the exception of one polymer (curdlan) which did not bind Mg2+, enhancement of struvite formation by CPS polymers was inversely proportional to their Mg2+ binding ability. We speculate that the structure and partial anionic nature of P. mirabilis CPS enable it to enhance struvite formation by weakly concentrating Mg2+ ions during struvite crystal formation. This illustrates a new virulence aspect of bacterial CPS during infection.

Bacterial biofilms: influence on the pathogenesis, diagnosis and treatment of urinary tract infections.

The bacterial biofilm theory which describes bacterial populations in natural and pathogenic ecological systems in terms of a free-floating or 'planktonic' population of bacteria interacting with a more important matrix enclosed 'sessile' population of bacteria associated with or adherent to a surface, may help explain some of the problems linked to our understanding the nature of urinary tract infections. This paper reviews the role of bacterial biofilm formation in catheter-associated infection, prostatitis and struvite (infected stone) calculogenesis stressing, the importance of bacterial biofilms in the pathogenesis, persistence and hence the treatment of urinary tract infection.

Glycosaminoglycans and struvite calculi.

Glycosaminoglycans (GAGs) are suspended in urine and are present on tissue surfaces in the urinary tract. Consequently, they have the potential to influence any pathological disorder in this environment, including urinary tract infections by Proteus mirabilis and struvite (NH4MgPO4.6H2(0)) urolithiasis. Although GAGs, suspended in urine, may inhibit the formation of other types of calculus minerals, no such effect has been reported in struvite calculi. Nevertheless, GAGs are a major component of the organic matrix of all types of urinary calculi. In contrast, there is evidence that the GAG layer on the bladder surface may act as a defence mechanism against infection by inhibiting bacterial adhesion. More studies are needed to elucidate fully the role of GAGs in urinary infections and struvite urolithiasis.