De-repression of the smvA efflux system arises in clinical isolates of Proteus mirabilis and reduces susceptibility to chlorhexidine and other biocides.

Proteus mirabilis is a common pathogen of the catheterised urinary tract and often described as intrinsically resistant to the biocide chlorhexidine (CHD). Here we demonstrate that de-repression of the smvA efflux system has occurred in clinical isolates of P. mirabilis and reduces susceptibility to CHD and other cationic biocides. Compared to other isolates examined, P. mirabilis RS47 exhibited a significantly higher CHD MIC (≥512 µg/ml) and significantly greater expression of smvA. Comparison of the RS47 smvA and cognate smvR repressor with sequences from other isolates, indicated that RS47 encodes an inactivated smvR. Complementation of RS47 with a functional smvR from isolate RS50a (which exhibited the lowest smvA expression and lowest CHD MIC) reduced smvA expression by ∼59-fold, and markedly lowered the MIC of CHD and other cationic biocides. Although complementation of RS47 did not reduce MICs to concentrations observed in isolate RS50a, the significantly lower polymyxin B MIC of RS50a indicated that differences in LPS structure are also a factor in P. mirabilis CHD susceptibility. To determine if exposure to CHD can select for mutations in smvR, clinical isolates with the lowest CHD MICs were adapted to grow at increasing concentrations of CHD up to 512 µg/ml. Analysis of the smvR in adapted populations indicated that mutations predicted to inactivate smvR occurred following CHD exposure in some isolates. Collectively, our data show that smvA de-repression contributes to reduced biocide susceptibility in P. mirabilis, but differences in LPS structure between strains are also likely to be an important factor.

Bacterial biofilm formation on indwelling urethral catheters.

Urethral catheters are the most commonly deployed medical devices and used to manage a wide range of conditions in both hospital and community care settings. The use of long-term catheterization, where the catheter remains in place for a period >28 days remains common, and the care of these patients is often undermined by the acquisition of infections and formation of biofilms on catheter surfaces. Particular problems arise from colonization with urease-producing species such as Proteus mirabilis, which form unusual crystalline biofilms that encrust catheter surfaces and block urine flow. Encrustation and blockage often lead to a range of serious clinical complications and emergency hospital referrals in long-term catheterized patients. Here we review current understanding of bacterial biofilm formation on urethral catheters, with a focus on crystalline biofilm formation by P. mirabilis, as well as approaches that may be used to control biofilm formation on these devices. SIGNIFICANCE AND IMPACT OF THE STUDY: Urinary catheters are the most commonly used medical devices in many healthcare systems, but their use predisposes to infection and provide ideal conditions for bacterial biofilm formation. Patients managed by long-term urethral catheterization are particularly vulnerable to biofilm-related infections, with crystalline biofilm formation by urease producing species frequently leading to catheter blockage and other serious clinical complications. This review considers current knowledge regarding biofilm formation on urethral catheters, and possible strategies for their control.

Growth media and assay plate material can impact on the effectiveness of cationic biocides and antibiotics against different bacterial species.

The effectiveness of several cationic disinfectants as well as colistin and polymyxin B were assessed under different growth conditions against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa strains. These conditions included different media (MH1, MH2, TSB and LB) and plate material (polypropylene and polystyrene). Results showed that Minimum inhibitory and bactericidal concentrations (MIC/MBC) values of colistin and polymyxin B were significantly lower on polypropylene plates when compared to polystyrene plates regardless of media used. There were also differences in MIC/MBC values to certain biocides e.g. chlorhexidine and octenidine particularly for S. aureus and E. coli strains, with polypropylene again showing lower values. Other biocides appear to be mostly unaffected by plate type. Whether biocide efficacy was altered by media composition was organism dependent with S. aureus and E. coli more affected than P. aeruginosa. Lower MIC values were more commonly associated with MH2 media and higher MIC values with TSB media for both polypropylene and polystyrene plates, although there were exceptions. Results obtained for standard strains were, in general, indicative for other S. aureus, E. coli and P. aeruginosa strains tested. This study demonstrates the importance of media composition and plate material on biocide effectiveness and highlights the need for optimized disinfectant testing methods. SIGNIFICANCE AND IMPACT OF THE STUDY: There are an increasing number of reports of bacterial strains that are multi-drug resistant. The use of biocides as part of infection control is crucial in helping to combat the spread of these particular strains. Unlike for antibiotics, there are few standardized measuring techniques to understand if an isolate has become more resistant to biocides. This study demonstrates the importance of media composition and plate material on variation and reporting of susceptibility of several bacterial species to specific cationic biocides. It is a useful comparison study to highlight the need to standardize biocide susceptibility testing.

Real-time microfluidic recombinase polymerase amplification for the toxin B gene of Clostridium difficile on a SlipChip platform.

Clostridium difficile is one of the key bacterial pathogens that cause infectious diarrhoea both in the developed and developing world. Isothermal nucleic acid amplification methods are increasingly used for identification of toxinogenic infection by clinical labs. For this purpose, we developed a low-cost microfluidic platform based on the SlipChip concept and implemented real-time isothermal recombinase polymerase amplification (RPA). The on-chip RPA assay targets the Clostridium difficile toxin B gene (tcdB) coding for toxin B, one of the proteins responsible for bacterial toxicity. The device was fabricated in clear acrylic using rapid prototyping methods. It has six replicate 500 nL reaction wells as well as two sets of 500 nL control wells. The reaction can be monitored in real-time using exonuclease fluorescent probes with an initial sample volume of as little as 6.4 µL. We demonstrated a limit of detection of 1000 DNA copies, corresponding to 1 fg, at a time-to-result of <20 minutes. This miniaturised platform for pathogen detection has potential for use in resource-limited environments or at the point-of-care because of its ease of use and low cost, particularly if combined with preserved reagents.

Lipopolysaccharide structure modulates cationic biocide susceptibility and crystalline biofilm formation in Proteus mirabilis.

Chlorhexidine (CHD) is a cationic biocide used ubiquitously in healthcare settings. Proteus mirabilis, an important pathogen of the catheterized urinary tract, and isolates of this species are often described as "resistant" to CHD-containing products used for catheter infection control. To identify the mechanisms underlying reduced CHD susceptibility in P. mirabilis, we subjected the CHD tolerant clinical isolate RS47 to random transposon mutagenesis and screened for mutants with reduced CHD minimum inhibitory concentrations (MICs). One mutant recovered from these screens (designated RS47-2) exhibited ~ 8-fold reduction in CHD MIC. Complete genome sequencing of RS47-2 showed a single mini-Tn5 insert in the waaC gene involved in lipopolysaccharide (LPS) inner core biosynthesis. Phenotypic screening of RS47-2 revealed a significant increase in cell surface hydrophobicity and serum susceptibility compared to the wildtype, and confirmed defects in LPS production congruent with waaC inactivation. Disruption of waaC was also associated with increased susceptibility to a range of other cationic biocides but did not affect susceptibility to antibiotics tested. Complementation studies showed that repression of smvA efflux activity in RS47-2 further increased susceptibility to CHD and other cationic biocides, reducing CHD MICs to values comparable with the most CHD susceptible isolates characterized. The formation of crystalline biofilms and blockage of urethral catheters was also significantly attenuated in RS47-2. Taken together, these data show that aspects of LPS structure and upregulation of the smvA efflux system function in synergy to modulate susceptibility to CHD and other cationic biocides, and that LPS structure is also an important factor in P. mirabilis crystalline biofilm formation.

Neuroticism as a common dimension in the internalizing disorders.

BACKGROUND: Several theories have posited a common internalizing factor to help account for the relationship between mood and anxiety disorders. These disorders are often co-morbid and strongly covary. Other theories and data suggest that personality traits may account, at least in part, for co-morbidity between depression and anxiety. The present study examined the relationship between neuroticism and an internalizing dimension common to mood and anxiety disorders. METHOD: A sample of ethnically diverse adolescents (n=621) completed self-report and peer-report measures of neuroticism. Participants also completed the Structured Clinical Interview for DSM-IV (SCID). RESULTS: Structural equation modeling showed that a single internalizing factor was common to lifetime diagnosis of mood and anxiety disorders, and this internalizing factor was strongly correlated with neuroticism. Neuroticism had a stronger correlation with an internalizing factor (r=0.98) than with a substance use factor (r=0.29). Therefore, neuroticism showed both convergent and discriminant validity. CONCLUSIONS: These results provide further evidence that neuroticism is a necessary factor in structural theories of mood and anxiety disorders. In this study, the correlation between internalizing psychopathology and neuroticism approached 1.0, suggesting that neuroticism may be the core of internalizing psychopathology. Future studies are needed to examine this possibility in other populations, and to replicate our findings.

Implications for Creutzfeldt-Jakob disease (CJD) in dentistry: a review of current knowledge.

This review explores our current understanding of the risks of (variant) Creutzfeldt-Jakob disease transmission via dental practice, and whether they merit the rigorous enforcement of improved standards of instrument cleaning and decontamination. The recognition of prions as novel infectious agents in humans has caused significant concern among the public and medical professionals alike. Creutzfeldt-Jakob disease (CJD) in humans has been shown to be transmissible via several routes, including transplantation, contaminated medical products, and via neurosurgery. While the likelihood of transmission via dentistry is undoubtedly very low, this may be amplified considerably by unknown risk factors, such as disease prevalence (particularly in the UK), altered tissue distribution of vCJD, and the failure of decontamination processes to address the inactivation of prions adequately. Since current diagnostic techniques are unable to detect PrP(Sc) in human dental tissues, there is limited evidence for the presence of infectivity. Given these uncertainties, the control of risk by reinforced and improved decontamination practices seems the most appropriate response.

Pseudomonas aeruginosa adapts to octenidine in the laboratory and a simulated clinical setting, leading to increased tolerance to chlorhexidine and other biocides.

BACKGROUND: Octenidine is frequently used for infection prevention in neonatal and burn intensive care units, where Pseudomonas aeruginosa has caused nosocomial outbreaks. AIM: To investigate the efficacy and impact of using octenidine against P. aeruginosa. METHODS: Seven clinical isolates of P. aeruginosa were exposed to increasing concentrations of octenidine over several days. Fitness, minimum bactericidal concentrations after 1 min, 5 min and 24 h, and minimum inhibitory concentrations (MICs) of a variety of antimicrobials were measured for the parental and octenidine-adapted P. aeruginosa strains. Octenidine and chlorhexidine MICs of a population of P. aeruginosa isolated from a hospital drain trap, exposed to a diluted octenidine formulation four times daily for three months, were also tested. FINDINGS: Some planktonic cultures of P. aeruginosa survived >50% of the working concentration of an in-use octenidine formulation at the recommended exposure time. Seven strains of P. aeruginosa stably adapted following continuous exposure to increasing concentrations of octenidine. Adaptation increased tolerance to octenidine formulations and chlorhexidine up to 32-fold. In one strain, it also led to increased MICs of antipseudomonal drugs. Subsequent to continuous octenidine exposure of a multi-species community in a simulated clinical setting, up to eight-fold increased tolerance to octenidine and chlorhexidine of P. aeruginosa was also found, which was lost upon removal of octenidine. CONCLUSION: Incorrect use of octenidine formulations may lead to inadequate decontamination, and even increased tolerance of P. aeruginosa to octenidine, with resulting cross-resistance to other biocides.

Evaluation of Novel Process Indicators for Rapid Monitoring of Hydrogen Peroxide Decontamination Processes.

Geobacillus stearothermophilus spores on stainless steel discs are routinely used as biological indicators for the validation of hydrogen peroxide bio-decontamination processes. Given ongoing concerns about the reliability and response time of biological indicators, we explored the potential for an enzyme-based approach to decontamination process evaluation. Thermostable adenylate kinase enzyme was coated onto a solid support and exposed to hydrogen peroxide vapour, in parallel with standard commercial 6-log biological indicators, during a series of vapour-phase hydrogen peroxide cycles in a flexible film isolator. The exposed biological indicators were enumerated to define the degree of kill at different time intervals and the results compared to the thermostable adenylate kinase values, as determined by measuring adenosine triphosphate produced by residual active enzyme. Both biological indicators and the thermostable adenylate kinase indicators exhibited a biphasic inactivation profile during the process. There was significant variance between individual cycles, with some cycles showing complete inactivation of the biological indicators to the limit of detection of the assay, within 6 min, whereas biological indicators in some cycles were inactivated at a time greater than 12 min. The log-kill of the biological indicators at intermediate time points were plotted and compared to the fully quantifiable measurements derived from the thermostable adenylate kinase indicators at the same time points. The results demonstrated very similar inactivation profiles for the enzyme and for the biological indicators, thus it was possible to define a relationship between relative light units measurement and biological indicator kill. This indicates that it is possible to use thermostable adenylate kinase measurement as a direct measure of vapour-phase hydrogen peroxide bio-decontamination performance, expressed in terms of log reduction. Because thermostable adenylate kinase measurement can be achieved within a few minutes of vapour-phase hydrogen peroxide cycle completion, compared with a minimum of 7 days for the evaluation of biological indicator growth, this offers a potentially valuable tool for rapid vapour-phase hydrogen peroxide bio-decontamination cycle development and subsequent re-qualification.LAY ABSTRACT: Pharmaceutical product manufacture is performed in controlled cleanroom and closed chamber environments (isolators) to reduce the risk of contamination. These environments undergo regular decontamination to control microbial contamination levels, using a range of methods, one of which is to vaporize hydrogen peroxide (a chemical disinfectant) into a gas or an aerosol and disperse it throughout the environment, killing any microorganisms present. Biological indicators, which consist of a small steel coupon carrying a population of bacterial spores that are more resistant to hydrogen peroxide than are most microorganisms, are placed within the environment, and then tested for growth following treatment to ensure the process was effective. Confirmation of growth/no growth (and therefore hydrogen peroxide cycle efficacy) can take up to 7 days, which significantly increases time and cost of developing and confirming cycle efficacy. This study tests whether a new technology which uses a robust enzyme, thermostable adenylate kinase, could be used to predict biological indicator growth. The study shows this method can be used to confirm hydrogen peroxide cycle efficacy, by predicting whether the BI is killed at a specific time point or not and results are obtained in a few minutes rather than 7 days. This potentially offers significant time and cost benefits.

Surface decontamination of surgical instruments: an ongoing dilemma.

The issues of cross-infection and the survival of variant Creutzfeldt Jakob disease (vCJD) on surgical instruments have highlighted the importance of cleanliness of multiple-use surgical instruments. The aim of this study was to assess the levels of total protein contamination on a wide range of surgical instruments as an indication of the effectiveness of routine cleaning and disinfection in hospitals. Anonymized trays of wrapped and autoclaved instruments were supplied to two laboratories for analysis at the stage where they would normally be returned to operating theatres. Instruments were assessed for residual protein and total organic matter. Laboratory A showed that 17% (35/206) of instruments were above a threshold that equated to 200 microg. The worst examples, a McIvor gag, a Draffin rod (child) and a Yankaur sucker, had 1.028, 1.286 and 2.228 mg of extractable protein, respectively. The median (25th, 75th percentiles) amount of protein from instruments from different hospitals assessed in Laboratory B ranged from 8 (3, 30)mug (Hospital C) to 91 (35, 213) mug (Hospital D) (P=0.044). The residual matter washed from instruments varied from 0.62 (0.32, 0.81) mg (Hospital E) to 3.5 (3.5, 4.0) mg (Hospital A) (P=0.0001). In one case, 45 mg of residual organic matter was washed from an instrument (split stem). In conclusion, this study demonstrated that a proportion of instruments at the point of use show levels of protein that could pose a direct cross-infection risk via prion agents and other organic contamination that may reduce the effectiveness of cleaning/disinfection strategies targeted against either prions or traditional infectious agents.

Re-engineering the target specificity of Clostridial neurotoxins - a route to novel therapeutics.

The ability to chemically couple proteins to LH(N)-fragments of clostridial neurotoxins and create novel molecules with selectivity for cells other than the natural target cell of the native neurotoxin is well established. Such molecules are able to inhibit exocytosis in the target cell and have the potential to be therapeutically beneficial where secretion from a particular cell plays a causative role in a disease or medical condition. To date, these molecules have been produced by chemical coupling of the LH(N)-fragment and the targeting ligand. This is, however, not a suitable basis for producing pharmaceutical agents as the products are ill defined, difficult to control and heterogeneous. Also, the molecules described to date have targeted neuroendocrine cells that are susceptible to native neurotoxins, and therefore the benefit of creating a molecule with a novel targeting domain has been limited. In this paper, the production of a fully recombinant fusion protein from a recombinant gene encoding both the LH(N)-domain of a clostridial neurotoxin and a specific targeting domain is described, together with the ability of such recombinant fusion proteins to inhibit secretion from non-neuronal target cells. Specifically, a novel protein consisting of the LH(N)-domains of botulinum neurotoxin type C and epidermal growth factor (EGF) that is able to inhibit secretion of mucus from epithelial cells is reported. Such a molecule has the potential to prevent mucus hypersecretion in asthma and chronic obstructive pulmonary disease.

Establishment of a multi-species biofilm model to evaluate chlorhexidine efficacy.

BACKGROUND: Chronic infections, for example, diabetic foot ulcers, have a large impact in terms of patient morbidity and mortality. These wounds are characterized by complex polymicrobial communities of bacteria, which may include a number of difficult-to-eradicate multidrug-resistant pathogens. AIM: To establish a multi-species biofilm model to test the efficacy of chlorhexidine and chlorhexidine-containing formulas in eradication of polymicrobial biofilms. METHODS: A Centers for Disease Control and Prevention bioreactor was used to establish a multi-species biofilm incorporating Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis with equal numbers of each pathogen. This model was used to test the effectiveness of chlorhexidine at controlling the pre-formed biofilm. FINDINGS: Chlorhexidine digluconate (CHD) was added to the bioreactor at a range of concentrations. K. pneumoniae and P. aeruginosa survived within multi-species biofilms, up to and including 4% CHD, whereas S. aureus was reduced to below the level of detection at 1%. Wiping the biofilm-containing coupons from the bioreactor with chlorhexidine-containing medical wipes resulted in >3 to <4log10 reduction after 24h, for all species. When the coupons were embedded in a simulated wound bed, formed in an agar plate, CHD-containing medical dressings completely eliminated S. aureus (>8log10 reduction), but had minimal effect (<3log10) against the other species tested. CONCLUSION: The study demonstrates that the effectiveness of chlorhexidine may be limited in settings where it is required to act on multi-species biofilms. This may compromise the ability of chlorhexidine to control the infection and spread of these pathogens.

Varying activity of chlorhexidine-based disinfectants against Klebsiella pneumoniae clinical isolates and adapted strains.

BACKGROUND: Control of multi-drug-resistant (MDR) organisms relies increasingly on the use of biocides, including chlorhexidine, to limit the risk of infection. The concentration and formulation of chlorhexidine can vary hugely between products. AIM: To establish the activity of chlorhexidine and in-use chlorhexidine formulations against 14 clinical Klebsiella pneumoniae strains isolated before and since the use of chlorhexidine became routine, and strains that have adapted following sublethal chlorhexidine exposure. METHODS: Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of five chlorhexidine-containing formulations were measured at 5min, 15min, 30min and 24h for the panel of K. pneumoniae strains. FINDINGS: After 5min, MBCs of five formulations varied from 0.006 to >50% working concentration (WC) or from 78 to 2500µg/mL chlorhexidine. For one formulation, MBCs were >50% WC for five of the 14 strains, and for another formulation, four of the 14 strains could resist 25% WC. NCTC 13368 was consistently most tolerant to chlorhexidine, whereas the strains isolated before the use of chlorhexidine became routine were more sensitive. One pre-chlorhexidine era and five modern strains increased MICs up to 16-fold following exposure to sublethal concentrations of chlorhexidine. A hand disinfectant with MBCs of 0.39% WC for all six of the wild-type strains, had MBCs of 50% WC for the chlorhexidine-adapted strains. CONCLUSION: Not all chlorhexidine formulations kill MDR K. pneumoniae after the recommended exposure time. Activity, especially against chlorhexidine-adapted strains, depends on additional ingredients. Careful formulation of chlorhexidine products is therefore important to maintain and enhance the activity of chlorhexidine products, and avoid potential breakdown in infection control.

Randomized, double-blind, placebo-controlled dose-ranging study of tirofiban (MK-383) platelet IIb/IIIa blockade in high risk patients undergoing coronary angioplasty.

OBJECTIVES: The objectives of this double-blind, placebo-controlled, randomized dose-ranging study were 1) to examine the safety and tolerability of tirofiban (MK-383), a new nonpeptide platelet IIb/IIIa receptor antagonist, on a background of intravenous heparin and aspirin therapy; 2) to study the pharmacodynamics and pharmacokinetics of tirofiban; and 3) to evaluate the incidence of adverse cardiac outcomes (urgent repeat revascularization, myocardial infarction and death) with tirofiban versus placebo in a high risk subset of patients undergoing coronary angioplasty. BACKGROUND: Abrupt vessel closure complicates 4% to 8% of angioplasty procedures. Recent data have suggested that agents that antagonize the platelet glycoprotein IIb/IIIa receptor may reduce the incidence of adverse ischemic outcomes after coronary angioplasty. METHODS: Seventy-three patients received tirofiban in three sequential dose panels and 20 patients received placebo. Patients within each panel were randomized to receive either tirofiban or placebo in a 3:1 randomization design. Bolus doses of 5, 10 and 10 microg/kg and continuous infusion (16 to 24 h) doses of 0.05, 0.10 and 0.15 microg/kg per min were administered in panels I, II and III, respectively. Patients received concomitant heparin and aspirin for the angioplasty procedure. Data on patients receiving placebo (heparin and aspirin only) were pooled across panels for comparisons. The pharmacodynamic effect of tirofiban on ex vivo platelet aggregation to 5 micromol/liter adenosine diphosphate (ADP) and bleeding times were measured. Clinical outcomes were assessed in all patients, but the power to detect clinically meaningful differences (a one-third reduction in clinical events) between groups was limited (5%). RESULTS: Tirofiban was associated with a dose-dependent inhibition of ex vivo ADP-mediated platelet aggregation that was sustained during intravenous infusion and resolved rapidly after drug cessation. Adverse bleeding events, largely related to vascular access site hemorrhage, were slightly increased at the highest dose. Adverse clinical outcomes were infrequent in all patients and were not different among the small number of patients within each group. CONCLUSIONS: This study establishes a rational and generally well tolerated dosing regimen for administration of tirofiban as adjunctive therapy in high risk angioplasty patients. The impact of tirofiban on adverse clinical outcomes after angioplasty awaits definition by a larger clinical trial.

Analysis of the substrate recognition domain determinants of botulinum type B toxin using phage display.

The botulinum neurotoxin endopeptidases appear to recognise their intracellular protein substrates via two distinct sites: the cleavage site sequence and a 'recognition site' motif. In the present study phage display has been employed to generate a library of vesicle-associated membrane protein (VAMP2) variants in which the toxin recognition motif (part of the SNARE motif ELDDRADA) has been modified. VAMP (1-94) was displayed on the surface of M13 bacteriophage and this fragment was recognised and cleaved by botulinum neurotoxin type B (BoNT/B). A phage-displayed library was constructed in which six residues of the recognition domain (VAMP residues 63-68; wild-type sequence LDDRAD) were randomised, and a selection method established for identifying cleaved VAMP variants. Sequence analysis of 24 clones revealed that 5 contained two acidic residues although none corresponded to the native sequence. Cleavage was reduced compared to wild-type VAMP, and cleavage of mutants containing no acidic residues was also observed. The data are discussed in relation to the substrate recognition mechanism of BoNT/B.

Urinary eosinophils.

The sensitivity of the presence of urinary eosinophils for the diagnosis of acute interstitial nephritis is 60%. Of those with interstitial nephritis without urinary eosinophils, in most cases, few eosinophils are present in the renal interstitial inflammatory infiltrate. The specificity of urinary eosinophils for acute interstitial nephritis has not been determined but urinary eosinophils have been reported with eosinophilic cystitis, urinary tract infections, and various renal diseases. However, the incidence of eosinophiluria in any of these conditions has not been investigated.

Hyperammonemic encephalopathy due to essential amino acid hyperalimentation.

Hyperammonemic associated encephalopathy developed in an adult receiving essential amino acids. Evidence that her encephalopathy was related to her hyperammonemia included (1) elevated CSF glutamine and serum ammonia levels, (2) the absence of any other drug or metabolic cause of encephalopathy, and (3) resolution of her encephalopathy and abnormal ammonia levels with discontinuation of the hyperalimentation. The serum ammonia levels of patients receiving essential amino acid fluid should be monitored. If the levels remain elevated or if toxicity develops, consideration should be given to switching to an alternate fluid.

Study into the kinetic properties and surface attachment of a thermostable adenylate kinase.

A thermostable adenylate kinase (tAK) has been used as model protein contaminant on surfaces, so used because residual protein after high temperature wash steps can be detected at extremely low concentrations. This gives the potential for accurate, quantitative measurement of the effectiveness of different wash processes in removing protein contamination. Current methods utilise non-covalent (physisorbtion) of tAK to surfaces, but this can be relatively easily removed. In this study, the covalent binding of tAK to surfaces was studied to provide an alternative model for surface contamination. Kinetic analysis showed that the efficiency of the enzyme expressed as the catalytic rate over the Michaelis constant (kcat/KM) increased from 8.45±3.04 mM-1 s-1 in solution to 32.23±3.20 or 24.46±4.41 mM-1 s-1 when the enzyme was immobilised onto polypropylene or plasma activated polypropylene respectively. Maleic anhydride plasma activated polypropylene showed potential to provide a more robust challenge for washing processes as it retained significantly higher amounts of tAK enzyme than polypropylene in simple washing experiments. Inhibition of the coupled enzyme (luciferase/luciferin) system used for the detection of adenylate kinase activity, was observed for a secondary product of the reaction. This needs to be taken into consideration when using the assay to estimate cleaning efficacy.

Analysis of the C-terminal secretion signal of the Rhizobium leguminosarum nodulation protein NodO; a potential system for the secretion of heterologous proteins during nodule invasion.

We used deletions to analyze the domains required for secretion of the Rhizobium leguminosarum bv. viciae nodulation protein, NodO, by the sec-independent pathway. Deletion of the C-terminal 24 amino-acids (residues 261 to 284) reduced secretion by at least 95%. A monoclonal antibody that recognizes the C-terminal domain of NodO was used to identify four nested deletions that retained the C-terminal 24 residues of NodO but had lost up to 133 residues (amino acids 128 to 259); all four proteins were secreted into the growth medium with an efficiency between 50 and 90% of normal. A deleted derivative of NodO that retained residues 1 to 21 and 167 to 284 (and therefore lacked most of the N-terminal Ca(2+)-binding domain) was secreted at around 80% of normal efficiency. Taken together, these observations indicate that the C-terminal 24 amino acids are sufficient for NodO secretion although the region adjacent to this domain appears to affect secretion efficiency. A derivative of the Escherichia coli alkaline phosphatase (phoA) gene was cloned into two derivatives of nodO such that PhoA (lacking the N-terminal transit peptide) was in-frame at both ends, with the C terminus fused to either the last 24 or 50 amino acids of NodO. These fusion proteins were secreted at 40 and 80% of the wild-type level, respectively, and the larger of the two retained alkaline phosphatase activity. A hybrid protein, containing E. coli beta-glucuronidase (GUS) fused to the N terminus of NodO, was not secreted, and it reduced the levels of wild-type NodO secreted by R. leguminosarum bv. viciae. The nature of the NodO C-terminal secretion signal is discussed with regard to its use as a delivery system for heterologous proteins useful for investigating the Rhizobium-legume interaction.

Rhizobium leguminosarum NodT is related to a family of outer-membrane transport proteins that includes TolC, PrtF, CyaE and AprF.

Cells containing a protein fusion consisting of the Rhizobium leguminosarum bv. viciae nodulation protein, NodT, fused to PhoA, produced alkaline phosphatase activity, indicating that the N terminus of NodT could translocate PhoA across the inner membrane. Cellular fractionation suggested that the NodT::PhoA fusion is targetted to the outer membrane. NodT resembles a family of bacterial outer membrane proteins including TolC, PrtF, CyaE and AprF, which are involved in secretion. By analogy, NodT (together with the inner membrane putative transport proteins NodI and NodJ) is proposed to be involved in the secretion of nodulation factors.