A broadly applicable, stress-mediated bacterial death pathway regulated by the phosphotransferase system (PTS) and the cAMP-Crp cascade.

Recent work indicates that killing of bacteria by diverse antimicrobial classes can involve reactive oxygen species (ROS), as if a common, self-destructive response to antibiotics occurs. However, the ROS-bacterial death theory has been challenged. To better understand stress-mediated bacterial death, we enriched spontaneous antideath mutants of Escherichia coli that survive treatment by diverse bactericidal agents that include antibiotics, disinfectants, and environmental stressors, without a priori consideration of ROS. The mutants retained bacteriostatic susceptibility, thereby ruling out resistance. Surprisingly, pan-tolerance arose from carbohydrate metabolism deficiencies in ptsI (phosphotransferase) and cyaA (adenyl cyclase); these genes displayed the activity of upstream regulators of a widely shared, stress-mediated death pathway. The antideath effect was reversed by genetic complementation, exogenous cAMP, or a Crp variant that bypasses cAMP binding for activation. Downstream events comprised a metabolic shift from the TCA cycle to glycolysis and to the pentose phosphate pathway, suppression of stress-mediated ATP surges, and reduced accumulation of ROS. These observations reveal how upstream signals from diverse stress-mediated lesions stimulate shared, late-stage, ROS-mediated events. Cultures of these stable, pan-tolerant mutants grew normally and were therefore distinct from tolerance derived from growth defects described previously. Pan-tolerance raises the potential for unrestricted disinfectant use to contribute to antibiotic tolerance and resistance. It also weakens host defenses, because three agents (hypochlorite, hydrogen peroxide, and low pH) affected by pan-tolerance are used by the immune system to fight infections. Understanding and manipulating the PtsI-CyaA-Crp­mediated death process can help better control pathogens and maintain beneficial microbiota during antimicrobial treatment.

Post-stress bacterial cell death mediated by reactive oxygen species.

Antimicrobial efficacy, which is central to many aspects of medicine, is being rapidly eroded by bacterial resistance. Since new resistance can be induced by antimicrobial action, highly lethal agents that rapidly reduce bacterial burden during infection should help restrict the emergence of resistance. To improve lethal activity, recent work has focused on toxic reactive oxygen species (ROS) as part of the bactericidal activity of diverse antimicrobials. We report that when Escherichia coli was subjected to antimicrobial stress and the stressor was subsequently removed, both ROS accumulation and cell death continued to occur. Blocking ROS accumulation by exogenous mitigating agents slowed or inhibited poststressor death. Similar results were obtained with a temperature-sensitive mutational inhibition of DNA replication. Thus, bacteria exposed to lethal stressors may not die during treatment, as has long been thought; instead, death can occur after plating on drug-free agar due to poststress ROS-mediated toxicity. Examples are described in which (i) primary stress-mediated damage was insufficient to kill bacteria due to repair; (ii) ROS overcame repair (i.e., protection from anti-ROS agents was reduced by repair deficiencies); and (iii) killing was reduced by anti-oxidative stress genes acting before stress exposure. Enzymatic suppression of poststress ROS-mediated lethality by exogenous catalase supports a causal rather than a coincidental role for ROS in stress-mediated lethality, thereby countering challenges to ROS involvement in antimicrobial killing. We conclude that for a variety of stressors, lethal action derives, at least in part, from stimulation of a self-amplifying accumulation of ROS that overwhelms the repair of primary damage.

Reactive oxygen species play a dominant role in all pathways of rapid quinolone-mediated killing.

BACKGROUND: Quinolones have been thought to rapidly kill bacteria in two ways: (i) quinolone-topoisomerase-DNA lesions stimulate the accumulation of toxic reactive oxygen species (ROS); and (ii) the lesions directly cause lethal DNA breaks. Traditional killing assays may have underestimated the ROS contribution by overlooking the possibility that ROS continue to accumulate and kill cells on drug-free agar after quinolone removal. METHODS: Quinolone-induced, ROS-mediated killing of Escherichia coli was measured by plating post-treatment samples on agar with/without anti-ROS agents. RESULTS: When E. coli cultures were treated with ciprofloxacin or moxifloxacin in the presence of chloramphenicol (to accentuate DNA-break-mediated killing), lethal activity, revealed by plating on quinolone-free agar, was inhibited by supplementing agar with ROS-mitigating agents. Moreover, norfloxacin-mediated lethality, observed with cells suspended in saline, was blocked by inhibitors of ROS accumulation and exacerbated by a katG catalase deficiency that impairs peroxide detoxification. Unlike WT cells, the katG mutant was killed by nalidixic acid or norfloxacin with chloramphenicol present and by nalidixic or oxolinic acid with cells suspended in saline. ROS accumulated after quinolone removal with cultures either co-treated with chloramphenicol or suspended in saline. Deficiencies in recA or recB reduced the protective effects of ROS-mitigating agents, supporting the idea that repair of quinolone-mediated DNA lesions suppresses the direct lethal effects of such lesions. CONCLUSIONS: ROS are the dominant factor in all modes of quinolone-mediated lethality, as quinolone-mediated primary DNA lesions are insufficient to kill without triggering ROS accumulation. ROS-stimulating adjuvants may enhance the lethality of quinolones and perhaps other antimicrobials.

Suppression of Reactive Oxygen Species Accumulation Accounts for Paradoxical Bacterial Survival at High Quinolone Concentration.

When bacterial cells are exposed to increasing concentrations of quinolone-class antibacterials, survival drops, reaches a minimum, and then recovers, sometimes to 100%. Despite decades of study, events underlying this paradoxical high-concentration survival remain obscure. Since reactive oxygen species (ROS) have been implicated in antimicrobial lethality, conditions generating paradoxical survival were examined for diminished ROS accumulation. Escherichia coli cultures were treated with various concentrations of nalidixic acid, followed by measurements of survival, rate of protein synthesis, and ROS accumulation. The last measurement used a dye (carboxy-H2DCFDA) that fluoresces in the presence of ROS; fluorescence was assessed by microscopy (individual cells) and flow cytometry (batch cultures). High, nonlethal concentrations of nalidixic acid induced lower levels of ROS than moderate, lethal concentrations. Sublethal doses of exogenous hydrogen peroxide became lethal and eliminated the nalidixic acid-associated paradoxical survival. Thus, quinolone-mediated lesions needed for ROS-executed killing persist at high, nonlethal quinolone concentrations, thereby implicating ROS as a key factor in cell death. Chloramphenicol suppressed nalidixic acid-induced ROS accumulation and blocked lethality, further supporting a role for ROS in killing. Nalidixic acid also inhibited protein synthesis, with extensive inhibition at high concentrations correlating with lower ROS accumulation and paradoxical survival. A catalase deficiency, which elevated ROS levels, overcame the inhibitory effect of chloramphenicol on nalidixic acid-mediated killing, emphasizing the importance of ROS. The data collectively indicate that ROS play a dominant role in the lethal action of narrow-spectrum quinolone-class compounds; a drop in ROS levels accounted for the quinolone tolerance observed at very high concentrations.

Dimethyl Sulfoxide Protects Escherichia coli from Rapid Antimicrobial-Mediated Killing.

The contribution of reactive oxygen species (ROS) to antimicrobial lethality was examined by treating Escherichia coli with dimethyl sulfoxide (DMSO), an antioxidant solvent frequently used in antimicrobial studies. DMSO inhibited killing by ampicillin, kanamycin, and two quinolones and had little effect on MICs. DMSO-mediated protection correlated with decreased ROS accumulation and provided evidence for ROS-mediated programmed cell death. These data support the contribution of ROS to antimicrobial lethality and suggest caution when using DMSO-dissolved antimicrobials for short-time killing assays.

Moving forward with reactive oxygen species involvement in antimicrobial lethality.

Support for the contribution of reactive oxygen species (ROS) to antimicrobial lethality has been refined and strengthened. Killing by diverse antimicrobials is enhanced by defects in genes that protect against ROS, inhibited by compounds that block hydroxyl radical accumulation, and is associated with surges in intracellular ROS. Moreover, support has emerged for a genetic pathway that controls the level of ROS. Since some antimicrobials kill in the absence of ROS, ROS must add to, rather than replace, known killing mechanisms. New work has addressed many of the questions concerning the specificity of dyes used to detect intracellular ROS and the specificity of perturbations that influence ROS surges. However, complexities associated with killing under anaerobic conditions remain to be resolved. Distinctions among primary lesion formation, resistance, direct lesion-mediated killing and a self-destructive stress response are discussed to facilitate efforts to potentiate ROS-mediated bacterial killing and improve antimicrobial efficacy.

[Effect of triptolide on the expression of RANTES in the renal tissue of diabetic nephropathy rats].

OBJECTIVE: To investigate the effect of triptolide (TPL) on the renal tissue of diabetic rats and its possible mechanisms. METHODS: SD rats were randomly divided into the normal control group (as the normal group), the diabetic model group (the model group), the low dose TPL treatment group (the low dose TPL group, TPL 0.2 mg/kg by gastrogavage), the high dose TPL treatment group (the high dose TPL group, TPL 0.4 mg/kg by gastrogavage). Equal volume of normal saline was given to rats in the normal group and the model group. Five rats were randomly selected from each group at week 4, 8, and 12 of the experiment to detect body weight, kidney weight, 24 h urinary albumin (24 h UAL), plasma glucose (FBG), total cholesterol (TC), total triglyeride (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), white blood cell (WBC), and hemoglobin A1c (HbA1c). The mRNA and protein expression of regulated upon activation normal T-cell expressed and secreted (RANTES) in the renal tissue was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). The renal tissue was pathologically stained by HE, PAS, and Masson staining. The glomerular and renal tubular interstitial lesions were observed at each time point. The glomerular sclerosis index (GSI) was observed by PAS staining, and the renal interstitial filrosis index (RIFI) was calcutated. RESULTS: Compared with the same group at week 4, the expression of 24 h UAL, RANTES, GSI, and RIFI at week 12 significantly decreased in two TPL groups (P <0.01). Compared with the same group at week 8, the expression of 24 h UAL, RANTES, GSI, and RIFI at week 12 also significantly decreased in the two TPL groups (P <0. 05, P <0.01). Compared with the normal group, body weight and the kidney weight obviously decreased at week 4, 8, and 12 in the model group (P <0. 01); 24 h UAL, FBG, TG, TC, HbA1c, RANTES, GSI, and RIFI were obviously elevated (P <0.01). Compared with the model group, 24 h UAL, RANTES, GSI, and RIFI also decreased in the two TPL treatment groups (P <0.01). Compared with the low dose TPL group, they were attenuated in the high dose TPL group (P <0. 05, P <0. 01). CONCLUSION: TPL could not only inhibit the over-expression of RANTES, but also improve the glomerular sclerosis and renal interstitial fibrosis in the renal tissue of diabetic rats.

Phylogenetic analysis of bacterial community in the gut of American cockroach (Periplaneta americana).

OBJECTIVE: The present study was to fully evaluate the intestinal bacterial community of Periplaneta americana, an important model to study insects. METHODS: We investigated the bacterial community of P. americana gut by culture-independent methods, involving constructing the 16S rRNA gene library and microbial diversity analysis. RESULTS: The phylotypes were affiliated with Proteobacteria (66.4%), Bacteroidetes (17.8%), Firmicutes (14.5%), Fusobacteria (0.6%) and unclassified bacteria (0.6%). Phylogenetic analysis shows that 15% of the sequences clustered with that from a closely related omnivorous cockroach; and 59% clustered with that from more distantly related animals, including omnivorous, herbivorous, and carnivorous animals, which differ greatly in feeding habits. Moreover, 18% of the clones showed high sequence identity with potential pathogens closely related to human diseases, which also reinforces the concept of the cockroach as a carrier of pathogens. CONCLUSION: Due to their habits of feeding on a variety of foodstuffs, omnivorous cockroaches harbor a large and diverse microbial community in the gut. The host phylogeny and dietary habits might be critical for the intestinal bacterial community composition of cockroaches.

Side Chain-Modified Benzothiazinone Derivatives with Anti-Mycobacterial Activity.

Tuberculosis (TB) is a leading infectious disease with serious antibiotic resistance. The benzothiazinone (BTZ) scaffold PBTZ169 kills Mycobacterium tuberculosis (Mtb) through the inhibition of the essential cell wall enzyme decaprenylphosphoryl-ß-D-ribose 2'-oxidase (DprE1). PBTZ169 shows anti-TB potential in animal models and pilot clinical tests. Although highly potent, the BTZ type DprE1 inhibitors in general show extremely low aqueous solubility, which adversely affects the drug-like properties. To improve the compounds physicochemical properties, we generated a series of BTZ analogues. Several optimized compounds had MIC values against Mtb lower than 0.01 µM. The representative compound 37 displays improved solubility and bioavailability compared to the lead compound. Additionally, compound 37 shows Mtb-killing ability in an acute infection mouse model.

Single Escherichia coli bacteria detection using a chemiluminescence digital microwell array chip.

Rapid and sensitive Escherichia coli (E. coli) detection is important in determining environmental contamination, food contamination, as well as bacterial infection. Conventional methods based on bacterial culture suffer from long testing time (24 h), whereas novel nucleic acid-based and immunolabelling approaches are hindered by complicated operation, the need of complex and costly equipment, and the lack of differentiation of live and dead bacteria. Herein, we propose a chemiluminescence digital microwell array chip based on the hydrolysis of 6-Chloro-4-methylumbelliferyl-ß-D-glucuronide by the ß-D-glucuronidase in E. coli to achieve fast single bacterial fluorescence detection. Taking the advantage of the picoliter microwells, single bacteria are digitally encapsulated in these microwells, thus the accurate quantification of E. coli can be realized by counting the number of positive microwells. We also show that the chemiluminescence digital microwell array chip is not affected by the turbidity of the test samples as well as the temperature. Most importantly, our method can differentiate live and dead bacteria through bacterial proliferation and enzyme expression, which is confirmed by detecting E. coli after pH and chlorination treatment. By comparing with the standard method of plate counting, our method has comparable performance but significantly reduces the testing time from over 24 h-2 h and 4 h for qualitative and quantitative analysis, respectively. In addition, the microfluidic chip is portable and easy to operate without external pump, which is promising as a rapid and on-site platform for single E. coli analysis in water and food monitoring, as well as infection diagnosis.

A bacterial laccase from marine microbial metagenome exhibiting chloride tolerance and dye decolorization ability.

Laccases are blue multicopper oxidases with potential applications in environmental and industrial biotechnology. In this study, a new bacterial laccase gene of 1.32 kb was obtained from a marine microbial metagenome of the South China Sea by using a sequence screening strategy. The protein (named as Lac15) of 439 amino acids encoded by the gene contains three conserved Cu(2+)-binding domains, but shares less than 40% of sequence identities with all of the bacterial multicopper oxidases characterized. Lac15, recombinantly expressed in Escherichia coli, showed high activity towards syringaldazine at pH 6.5-9.0 with an optimum pH of 7.5 and with the highest activity occurring at 45 °C. Lac15 was stable at pH ranging from 5.5 to 9.0 and at temperatures from 15 to 45 °C. Distinguished from fungal laccases, the activity of Lac15 was enhanced twofold by chloride at concentrations lower than 700 mM, and kept the original level even at 1,000 mM chloride. Furthermore, Lac15 showed an ability to decolorize several industrial dyes of reactive azo class under alkalescent conditions. The properties of alkalescence-dependent activity, high chloride tolerance, and dye decolorization ability make the new laccase Lac15 an alternative for specific industrial applications.

A chimeric protein that functions as both an anthrax dual-target antitoxin and a trivalent vaccine.

Effective measures for the prophylaxis and treatment of anthrax are still required for counteracting the threat posed by inhalation anthrax. In this study, we first demonstrated that the chimeric protein LFn-PA, created by fusing the protective antigen (PA)-binding domain of lethal factor (LFn) to PA, retained the functions of the respective molecules. On the basis of this observation, we attempted to develop an antitoxin that targets the binding of lethal factor (LF) and/or edema factor (EF) to PA and the transportation of LF/EF. Therefore, we replaced PA in LFn-PA with a dominant-negative inhibitory PA (DPA), i.e., PA(F427D). In in vitro models of anthrax intoxication, the LFn-DPA chimera showed 3-fold and 2-fold higher potencies than DPA in protecting sensitive cells against anthrax lethal toxin (LeTx) and edema toxin (EdTx), respectively. In animal models, LFn-DPA exhibited strong potency in rescuing mice from lethal challenge with LeTx. We also evaluated the immunogenicity and immunoprotective efficacy of LFn-DPA as an anthrax vaccine candidate. In comparison with recombinant PA, LFn-DPA induced significantly higher levels of the anti-PA immune response. Moreover, LFn-DPA elicited an anti-LF antibody response that could cross-react with EF. Mice immunized with LFn-DPA tolerated a LeTx challenge that was 5 times its 50% lethal dose. Thus, LFn-DPA represents a highly effective trivalent vaccine candidate for both preexposure and postexposure vaccination. Overall, we have developed a novel and dually functional reagent for the prophylaxis and treatment of anthrax.

Structure of native laccase B from Trametes sp. AH28-2.

Fungal laccases are oxidoreductases that belong to the multinuclear copper-containing oxidases. They are able to oxidize a wide range of substrates, preferably phenolic compounds, which makes them suitable for employment in the bioremediation of soil and water as well as in other biotechnological applications. Here, the structural analysis of natural laccase B (LacB) from Trametes sp. AH28-2 is presented. This structure provides the opportunity to study the natural post-translational modifications of the enzyme. The overall fold shows a high homology to those of previously analyzed laccases with known three-dimensional structure. However, LacB contains a new structural element, a protruding loop near the substrate-binding site, compared with the previously reported laccase structures. This unique structural feature may be involved in modulation of the substrate recognition of LacB.

Soluble expression and purification of the anthrax protective antigen in E. coli and identification of a novel dominant-negative mutant N435C.

The anthrax toxin is an AB-type bacterium toxin composed of the protective antigen (PA) as the cell-binding B component, and the lethal factor (LF) and edema toxin (EF) as the catalytic A components. The PA component is a key factor in anthrax-related research and recombinant PA can be produced in general in Escherichia coli. However, such recombinant PA always forms inclusion bodies in the cytoplasm of E. coli, making difficult the procedure of its purification. In this study, we found that the solubility of recombinant PA was dramatically enhanced by fusion with glutathione S-transferase (GST) and an induction of its expression at 28 degrees C. The PA was purified to high homogeneity and a yield of 3 mg protein was obtained from 1 l culture by an affinity-chromatography approach. Moreover, we expressed and purified three PA mutants, I394C, A396C, and N435C, which were impaired in expression in previous study. Among them, a novel mutant N435C which conferred dominant-negative inhibitory activity on PA was identified. This new mutant may be useful in designing new antitoxin for anthrax prophylaxis and therapy.

Cloning and characterization of a beta-glucosidase from marine microbial metagenome with excellent glucose tolerance.

The demand for beta-glucosidases insensitive to product inhibition is increasing in modern biotechnology, for these enzymes would improve the process of saccharification of lignocellulosic materials. In this study, a beta-glucosidase gene which encodes a 442-amino-acid protein was isolated from a marine microbial metagenomic library by functional screening and named as bgl1A. The protein was identified to be a member of GH1 family, and was recombinantly expressed, purified and biochemically characterized. The recombinant beta-glucosidase, Bgl1A, exhibited high level of stability in the presence of various cations and high concentrations of NaCl. Interestingly, it was activated by glucose at concentrations lower than 400 mM. With glucose further increasing, the enzyme activity of Bgl1A was gradually inhibited, but remained 50% original value in even as high as 1,000 mM glucose. These findings indicate Bgl1A might be a potent candidate for industrial applications.

Cloning and characterization of a novel mannanase from Paenibacillus sp. BME-14.

A mannanase gene (man26B) was obtained from a sea bacterium, Paenibacillus sp. BME-14, through the constructed genomic library and inverse PCR. The gene of man26B had an open reading frame of 1,428 bp that encoded a peptide of 475- amino acid residues with a calculated molecular mass of 53 kDa. Man26B possessed two domains, a carbohydrate binding module (CBM) belonging to family 6 and a family 26 catalytic domain (CD) of glycosyl hydrolases, which showed the highest homology to Cel44C of P. polymyxa (60% identity). The optimum pH and temperature for enzymatic activity of Man26B were 4.5 and 60 degrees C, respectively. The activity of Man26B was not affected by Mg(2+) and Co(2+), but was inhibited by Hg(2+), Ca(2+), Cu(2+), Mn(2+), K(+), Na(+), and beta-mercaptoethanol, and slightly enhanced by Pb(2+) and Zn(2+). EDTA did not affect the activity of Man26B, which indicates that it does not require divalent ions to function. Man26B showed a high specific activity for LBG and konjac glucomannan, with K(m), V(max), and k(cat) values of 3.80 mg/ml, 91.70 micromol/min/mg protein, and 77.08/s, respectively, being observed when LBG was the substrate. Furthermore, deletion of the CBM6 domain increased the enzyme stability while enabling it to retain 80% and 60% of its initial activity after treatment at 80 degrees C and 90 degrees C for 30 min, respectively. This finding will be useful in industrial applications of Man26B, because of the harsh circumstances associated with such processes.

Gongronella sp induces overproduction of laccase in Panus rudis.

Laccase is usually produced via chemical induction and is also synthesized by hosts in interaction with the typical bio-control genus Trichoderma. In this study, we found that a newly isolated non-laccase-producing fungus, Gongronella sp. W5, could induce overproduction of laccase in Panus rudis. The enzyme activity, 148,200 U l(-1), was 25 times higher than the activity obtained from a chemical induction using copper/o -toluidine as inducers. A new laccase isozyme from the interaction of P. rudis and G. W5 was purified and characterized. A further test showed that some pH resistant metabolites secreted by G. W5 acted as signals to induce P. rudis laccase. Laccase is also highly expressed by Trametes sp. AH28-2 in interaction with Trichoderma sp. ZH1. However, no laccase activity was observed from the cross-over interactions of P. rudis -Trichoderma sp. ZH1 or Trametes sp. AH28-2-G. W5.

Rapid and dynamic detection of antimicrobial treatment response using spectral amplitude modulation in MZO nanostructure-modified quartz crystal microbalance.

We report a dynamic and rapid detection of the response of S. epidermidis to various antimicrobial treatments utilizing the real-time spectral amplitude modulations of the magnesium zinc oxide nanostructure-modified quartz crystal microbalance (MZOnano-QCM) biosensor. The sensor consists of a quartz crystal microbalance (QCM) with magnesium zinc oxide (MZO) nanostructures grown directly on the sensing electrode using metalorganic chemical vapor deposition (MOCVD). Combining the high sensitivity detection of bacteria provided by the MZO nanostructures with the QCM's dynamic acoustic spectrum makes a highly-sensitive dynamic biosensor well-suited for monitoring viscoelastic transitions during drug treatment compared to the QCM's conventional frequency shift signals. We demonstrated dynamically monitoring the response of S. epidermidis to various concentrations of the drug ciprofloxacin, and response to three different antimicrobials vancomycin, oxacillin, and ciprofloxacin, using spectral amplitude modulations of the MZOnano-QCM. Our results indicate that the amplitude modulations exhibit high sensitivity to S. epidermidis response to different drug treatments compared to the conventional frequency shift signals of the device, allowing for rapid determination (within 1.5 h) of the efficacy of the antimicrobial drug. The high sensitivity demonstrated by the spectral amplitude modulations is attributed to the direct relationship of these signals to the viscoelastic transitions of the bacterial cells on the device's sensing area while responding to drug treatment. This relationship is established by the Butterworth-Van-Dyke (BVD) model of the MZOnano-QCM. Standard microbiological protocols and assays were performed to determine the optimal drug dosages and the minimum inhibitory concentrations to serve as the benchmark for the sensor data.

Early stage detection of Staphylococcus epidermidis biofilm formation using MgZnO dual-gate TFT biosensor.

Early stage detection of biofilm formation is an important aspect of microbial research because once formed, biofilms show serious tolerance to antibiotics in contrast to the free-floating bacteria, which significantly increases the difficulty for clinical treatment of bacterial infections. The early stage detection technology is desired to improve the efficiency of medical treatments. In this work, we present a biosensor consisting of a magnesium zinc oxide (MZO) dual gate thin-film transistor (DGTFT) as the actuator and an MZO nanostructure (MZOnano) array coated conducting pad as the extended sensing gate for the early stage detection of Staphylococcus epidermidis (S. epidermidis) biofilm formation. S. epidermidis bacteria were cultured in vitro on the nanostructure modified sensing pad. Charge transfer occurs between microbial cells and the MZOnano during the initial bacterial adhesion stage. Such electrical signals, which represent the onset of biofilm formation, were dynamically detected by the DGTFT where the top gate electrode was connected to the extended MZOnano sensing pad and the bottom gate was used for biasing the device into the optimum characteristic region for high sensitivity and stable operation. The testing results show that a current change of ~80% is achieved after ~200 min of bacterial culturing. A crystal violet staining-based assay shows that tiny bacterial microcolonies just start to form at 200 min, and that it would take approximately 24 h to form matured biofilms. This technology enables medical professionals to act promptly on bacterial infection before biofilms get fully established.

Synergy between type 1 fimbriae expression and C3 opsonisation increases internalisation of E. coli by human tubular epithelial cells.

BACKGROUND: Bacterial infection of the urinary tract is a common clinical problem with E. coli being the most common urinary pathogen. Bacterial uptake into epithelial cells is increasingly recognised as an important feature of infection. Bacterial virulence factors, especially fimbrial adhesins, have been conclusively shown to promote host cell invasion. Our recent study reported that C3 opsonisation markedly increases the ability of E. coli strain J96 to internalise into human proximal tubular epithelial cells via CD46, a complement regulatory protein expressed on host cell membrane. In this study, we further assessed whether C3-dependent internalisation by human tubular epithelial cells is a general feature of uropathogenic E. coli and investigated features of the bacterial phenotype that may account for any heterogeneity. RESULTS: In 31 clinical isolates of E. coli tested, C3-dependent internalisation was evident in 10 isolates. Type 1 fimbriae mediated-binding is essential for C3-dependent internalisation as shown by phenotypic association, type 1 fimbrial blockade with soluble ligand (mannose) and by assessment of a type 1 fimbrial mutant. CONCLUSION: we propose that efficient internalisation of uropathogenic E. coli by the human urinary tract depends on co-operation between type 1 fimbriae-mediated adhesion and C3 receptor -ligand interaction.