Elucidating the genetic basis of crystalline biofilm formation in Proteus mirabilis.

Proteus mirabilis forms extensive crystalline biofilms on urethral catheters that occlude urine flow and frequently complicate the management of long-term-catheterized patients. Here, using random transposon mutagenesis in conjunction with in vitro models of the catheterized urinary tract, we elucidate the mechanisms underpinning the formation of crystalline biofilms by P. mirabilis. Mutants identified as defective in blockage of urethral catheters had disruptions in genes involved in nitrogen metabolism and efflux systems but were unaffected in general growth, survival in bladder model systems, or the ability to elevate urinary pH. Imaging of biofilms directly on catheter surfaces, along with quantification of levels of encrustation and biomass, confirmed that the mutants were attenuated specifically in the ability to form crystalline biofilms compared with that of the wild type. However, the biofilm-deficient phenotype of these mutants was not due to deficiencies in attachment to catheter biomaterials, and defects in later stages of biofilm development were indicated. For one blocking-deficient mutant, the disrupted gene (encoding a putative multidrug efflux pump) was also found to be associated with susceptibility to fosfomycin, and loss of this system or general inhibition of efflux pumps increased sensitivity to this antibiotic. Furthermore, homologues of this system were found to be widely distributed among other common pathogens of the catheterized urinary tract. Overall, our findings provide fundamental new insight into crystalline biofilm formation by P. mirabilis, including the link between biofilm formation and antibiotic resistance in this organism, and indicate a potential role for efflux pump inhibitors in the treatment or prevention of P. mirabilis crystalline biofilms.

A rapid, non-destructive method for the determination of Staphylococcus epidermidis adhesion to surfaces using quartz crystal resonant sensor technology.

AIMS: To investigate the use of quartz crystal resonant sensor (QCRS) technology to determine the adhesion of Staphylococcus epidermidis to fibronectin-coated surfaces. METHODS AND RESULTS: QCRS sensors (14 MHz) with 4 mm gold electrodes were coated with fibronectin and exposed for 15 min to suspensions of Staph. epidermidis ranging in concentration from 1 x 10(2) to 1 x 10(6) cfu ml(-1). Changes in resonant frequency were recorded and showed a linear relationship with the logarithm of cell concentration over the range tested. CONCLUSIONS: QCRS technology was shown to be a rapid, sensitive and non-destructive method for measuring the adhesion of bacteria to surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: This report demonstrates that QCRS technology has the potential to be used for a range of applications requiring measurement of bacteria on surfaces. In particular, it may be used for the real-time monitoring of bacterial biofilm formation.

Characterization of the stability of recombinant protein production in the GS-NS0 expression system.

The GS-NS0 system is an important mammalian expression system used largely within industry for the high-level expression of recombinant proteins for therapeutic use. It is essential that the productivity of this system remains stable throughout culture expansion for the successful long-term production of recombinant proteins. Here we present a study of the stability of recombinant protein production from unamplified GS-NS0 cell lines over extended period of continuous culture. The cell lines used in this study were generated by the transfection of NS0 cells with DNA encoding for a secreted recombinant protein and by two subsequent rounds of limiting dilution cloning prior to analysis of stability. The stability of recombinant protein production was assessed at intervals over a period of 134 days using repeated batch culture in shake flasks. Heterogeneous stability was identified. The productivity of some clones remained consistent throughout 134 days of continuous culture. Others exhibit rapid and progressive loss of productivity. Analysis of the causal relationships underlying stability indicates that the initial transfectant determines the susceptibility to loss or retention of productivity. Selection of production clones on the basis of growth and productivity alone will not predict stability during long-term culture. Our research indicates that stable high-producing clones can readily be obtained from use of the GS-NS0 system in the absence of amplification but there may be molecular features of the original transfectants that could serve as very important predictive indicators of the stability of recombinant protein production.

Advances in animal cell recombinant protein production: GS-NS0 expression system.

The production of recombinant proteins using mammalian cell expression systems is of growing importance within biotechnology, largely due to the ability of specific mammalian cells to carry out post-translational modifications of the correct fidelity. The Glutamine Synthetase-NS0 system is now one such industrially important expression system.Glutamine synthetase catalyses the formation ofglutamine from glutamate and ammonia. NS0 cellscontain extremely low levels of endogenous glutaminesynthetase activity, therefore exogenous glutaminesynthetase can be used efficiently as a selectablemarker to identify successful transfectants in theabsence of glutamine in the media. In addition, theinclusion of methionine sulphoximine, an inhibitor ofglutamine synthetase activity, enables furtherselection of those clones producing relatively highlevels of transfected glutamine synthetase and henceany heterologous gene which is coupled to it. Theglutamine synthetase system technology has been usedfor research and development purposes during thisdecade and its importance is clearly demonstrated nowthat two therapeutic products produced using thissystem have reached the market place.

Effect of milk proteins on adhesion of bacteria to stainless steel surfaces.

Stainless steel coupons were treated with skim milk and subsequently challenged with individual bacterial suspensions of Staphylococcus aureus, Pseudomonas fragi, Escherichia coli, Listeria monocytogenes, and Serratia marcescens. The numbers of attached bacteria were determined by direct epifluorescence microscopy and compared with the attachment levels on clean stainless steel with two different surface finishes. Skim milk was found to reduce adhesion of S. aureus, L. monocytogenes, and S. marcescens. P. fragi and E. coli attached in very small numbers to the clear surfaces, making the effect of any adsorbed protein layer difficult to assess. Individual milk proteins alpha-casein, beta-casein, kappa-casein, and alpha-lactalbumin were also found to reduce the adhesion of S. aureus and L. monocytogenes. The adhesion of bacteria to samples treated with milk dilutions up to 0.001% was investigated. X-ray photoelectron spectroscopy was used to determine the proportion of nitrogen in the adsorbed films. Attached bacterial numbers were inversely related to the relative atomic percentage of nitrogen on the surface. A comparison of two types of stainless steel surface, a 2B and a no. 8 mirror finish, indicated that the difference in these levels of surface roughness did not greatly affect bacterial attachment, and reduction in adhesion to a milk-treated surface was still observed. Cross-linking of adsorbed proteins partially reversed the inhibition of bacterial attachment, indicating that protein chain mobility and steric exclusion may be important in this phenomenon.

Immune studies in the depigmenting C57BL/Ler-vit/vit mice. An apparent isolated loss of contact hypersensitivity.

Vitiligo is a human disorder which destroys pigment cells in the skin, ears, eyes, and meningeal tissues and has often been associated with a variety of autoimmune disorders. The C57BL/Ler-vit/vit mouse is a mutant strain that exhibits a loss of epidermal pigment cells and a selective cell-mediated immune deficiency to epicutaneous-administered allergens. This observation is consistent with that observed in humans with vitiligo, who also exhibit loss of contact hypersensitivity (CHS), that appears to be associated with loss of pigment cells from the epidermis. Other cellular immune parameters such as delayed type hypersensitivity and antibody generation to both particulate and soluble Ag are normal or even hyperimmune in the vit/vit mice compared with congenic C57BL/6 controls. Cyclophosphamide treatment could reconstitute CHS responsiveness of the vit/vit mice to the allergen dinitrofluorobenzene. Further, this loss of CHS responsiveness to dinitrofluorobenzene could be restored with skin transplants from normal pigmented C57BL/6 mice to vit/vit mice. Normal C57BL/6 mice bearing white skin grafts from vit/vit mice did not contact sensitize. We suggest that this vit/vit mouse strain may serve as an excellent system to investigate various aspects of other contact hypersensitivity reactions as well as vitiligo.

The natural history of dysplastic nevi. A case history illustrating their evolution.

A 31-year-old man with familial dysplastic nevus syndrome presented with numerous histologically confirmed dysplastic nevi. Seven years before this, all of his nevi, numbering over 150, had been "prophylactically" removed. Despite the removal of all visible nevi, many new nevocellular nevi and atypical nevi appeared during the subsequent seven years. This case illustrates the natural history of dysplastic nevi and suggests that removal of all nevi to prevent melanoma may be futile.

Pemphigus vulgaris involving the esophagus. A case report and review of the literature.

A case of pemphigus vulgaris confined to the oral cavity, larynx, and hypopharynx for two years with subsequent involvement of the entire esophagus is presented. Histopathology and direct immunofluorescence of the esophagus confirmed the diagnosis.