Evaluation of the substrate specificity of human mast cell tryptase beta I and demonstration of its importance in bacterial infections of the lung

Human pulmonary mast cells (MCs) express tryptases alpha and beta I, and both granule serine proteases are exocytosed during inflammatory events. Recombinant forms of these tryptases were generated for the first time to evaluate their substrate specificities at the biochemical level and then to address their physiologic roles in pulmonary inflammation. Analysis of a tryptase-specific, phage display peptide library revealed that tryptase beta I prefers to cleave peptides with 1 or more Pro residues flanked by 2 positively charged residues. Although recombinant tryptase beta I was unable to activate cultured cells that express different types of protease-activated receptors, the numbers of neutrophils increased >100-fold when enzymatically active tryptase beta I was instilled into the lungs of mice. In contrast, the numbers of lymphocytes and eosinophils in the airspaces did not change significantly. More important, the tryptase beta I-treated mice exhibited normal airway responsiveness. Neutrophils did not extravasate into the lungs of tryptase alpha-treated mice. Thus, this is the first study to demonstrate that the two nearly identical human MC tryptases are functionally distinct in vivo. When MC-deficient W/W(v) mice were given enzymatically active tryptase beta I or its inactive zymogen before pulmonary infection with Klebsiella pneumoniae, tryptase beta I-treated W/W(v) mice had fewer viable bacteria in their lungs relative to zymogen-treated W/W(v) mice. Because neutrophils are required to combat bacterial infections, human tryptase beta I plays a critical role in the antibacterial host defenses of the lung by recruiting neutrophils in a manner that does not alter airway reactivity.     

Class B scavenger receptor types I and II and CD36 targeting improves sepsis survival and acute outcomes in mice

Class B scavenger receptors (SR-Bs), such as SR-BI/II or CD36, bind lipoproteins but also mediate bacterial recognition and phagocytosis. In evaluating whether blocking receptors can prevent intracellular bacterial proliferation, phagocyte cytotoxicity, and proinflammatory signaling in bacterial infection/sepsis, we found that SR-BI/II- or CD36-deficient phagocytes are characterized by a reduced intracellular bacterial survival and a lower cytokine response and were protected from bacterial cytotoxicity in the presence of antibiotics. Mice deficient in either SR-BI/II or CD36 are protected from antibiotic-treated cecal ligation and puncture (CLP)-induced sepsis, with greatly increased peritoneal granulocytic phagocyte survival (8-fold), a drastic diminution in peritoneal bacteria counts, and a 50-70% reduction in systemic inflammation (serum levels of IL-6, TNF-α, and IL-10) and organ damage relative to CLP in wild-type mice. The survival rate of CD36-deficient mice after CLP was 58% compared with 17% in control mice. When compensated for mineralocorticoid and glucocorticoid deficiency, SR-BI/II-deficient mice had nearly a 50% survival rate versus 5% in mineralo-/glucocorticoid-treated controls. Targeting SR-B receptors with L-37pA, a peptide that functions as an antagonist of SR-BI/II and CD36 receptors, also increased peritoneal granulocyte counts, as well as reduced peritoneal bacteria and bacterium-induced cytokine secretion. In the CLP mouse sepsis model, L-37pA improved survival from 6 to 27%, reduced multiple organ damage, and improved kidney function. These results demonstrate that the reduction of both SR-BI/II- and CD36-dependent bacterial invasion and inflammatory response in the presence of antibiotic treatment results in granulocyte survival and local bacterial containment, as well as reduces systemic inflammation and organ damage and improves animal survival during severe infections.     

Epinephrine intracerebroventricular stimulation modifies the LH effect on ovarian progesterone and androstenedione release

This study investigates the interaction between the effect of epinephrine intracerebroventricular (icv) injection and LH on the progesterone concentration in ovarian vein blood (Po) in vivo, and also, on the release of ovarian progesterone and androstenedione in vitro, in rats on dioestrus day 2. When 2 mg ovine LH were injected in vein (i.v.), Po increased reaching 120+/-12.2 and 151+/-17.5 ng ml(-1) at 22 and 25 min, respectively. Another group of rats was injected intracerebroventricular with 5 microgram epinephrine at time zero, and with 2 mg ovine LH i.v. 3 min later. This time Po decreased during the first 3 min, then increased, reaching 64+/-7.1 ng ml(-1) at 25 min, lower than the Po obtained 22 min after LH i.v. injection only (P<0.01). Moreover, rats were injected i.v. with 2 mg ovine LH at time zero, and 7 min later with epinephrine intracerebroventricular. Po increased during the first 7 min, decreased until the 13th minute and reached 70+/-8.9 ng ml(-1) at 25 min, lower than the Po obtained 25 min after LH i.v. injection only (P<0.01). In other experience, rats with one (either right or left) superior ovarian nerve transected (SON-t), were injected intracerebroventricular with epinephrine. Five minutes later, the ovaries were removed and incubated in vitro with LH. Both ovaries (right or left) in which the SON was intact at time of epinephrine i. c.v. injection, showed a reduction of progesterone and androstenedione released in vitro (P<0.05). These results suggest that, on dioestrus day 2, the central adrenergic stimulus competes with LH in the release of ovarian progesterone. Also, the neural input that arrives at the ovary through the SON would antagonize the ovarian progesterone and androstenedione response to LH.     

诃子水提物对泛耐药鲍曼不动杆菌的抑菌作用

摘要：目的 研究诃子水提物对泛耐药鲍曼不动杆菌（PDR-Ab）的抑菌作用。方法 采用二倍稀释法测定诃子水提物对临床分离的泛耐药鲍曼不动杆菌的最低抑菌浓度（MIC）和最低杀菌浓度（MBC）。小鼠腹腔注射上述PDR-Ab,建立急性肺炎模型。于造模前4天至造模后2天对小鼠连续灌胃给药,考察小鼠的死亡率、血液白细胞计数及肺组织病理变化。结果 体外实验表明诃子水提物能抑制PDR-Ab在液体培养基中的生长,MIC和MBC分别为0.75 mg/mL和12 mg/mL。体内实验表明,剂量为150 mg/kg诃子水提物灌胃给药能明显降低PDR-Ab感染性急性肺炎小鼠的死亡率,使血液白细胞计数较快恢复,肺组织病理改善明显。结论 诃子水提物对PDR-Ab在体内外均具有明显的抑菌作用,有望用于PDR-Ab感染性疾病的治疗。     

Antibacterial potential and synergistic interaction between natural polyphenolic extracts and synthetic antibiotic on clinical isolates

Emergence of antimicrobial resistance complicates treatment of infections by antibiotics. This has driven research on novel and combination antibacterial therapies. The present study evaluated synergistic antimicrobial activity of plant extracts and cefixime in resistant clinical isolates. Preliminary susceptibility profiling of antibiotics and antibacterial activity of extracts was done by disc diffusion and microbroth dilution assays. Checker-board, time-kill kinetics and protein content studies were performed to validate synergistic antibacterial activity. Results showed noteworthy quantities of gallic acid (0.24–19.7 µg/mg), quercetin (1.57–18.44 µg/mg) and cinnamic acid (0.02–5.93 µg/mg) in extracts of plants assessed by reverse-phase high performance liquid chromatography (RP-HPLC). Gram-positive (4/6) and Gram-negative (13/16) clinical isolates were intermediately susceptible or resistant to cefixime, which was used for synergistic studies. EA and M extracts of plants exhibited total synergy, partial synergy and indifferent characteristics whereas aqueous extracts did not show synergistic patterns. Time-kill kinetic studies showed that synergism was both time and concentration-dependent (2–8-fold decrease in concentration). Bacterial isolates treated with combinations at fractional inhibitory concentration index (FICI) showed significantly reduced bacterial growth, as well as protein content (5–62 %) as compared to extracts/cefixime alone treated isolates. This study acknowledges the selected crude extracts as adjuvants to antibiotics to treat resistant bacterial infections.     

Superoxide Dismutases and Anti-Oxidants Protected Mice from no-Reflow and Necrotic Damage Induced by Ischemia

A simple method in mice was established to screen anti-ischemic compounds. Thirteen times binding of rubber ring (1 × 1 mm, d = 42 mm) for 4.5 hrs, swelled the paws of 60% mice applied and 14 times binding swelled only of 5% mice. Critically reversible limit lay between these conditions. “All or none” rule dominated the paw swelling perhap due to different endogenous anti-oxidants' levels of individual mice. Determination of paw reversibility at 90 min of recirculation, was proved to be suitable. Swollen paws at this time returned normal and the paws with no-reflow dropped out by muscle necrosis after several days. Intravenous (i.v.) bovine Cu, Zn-SOD and bacterial Mn-SOD (3 - 10 × 10⁴ U/kg) or liposomal Cu, Zn-SOD (0.3 - 3 × 10⁴ U/kg) were protective (35-50%) by 14 times binding. Allopurinol (10-100 mg/kg) and D-mannitol (3-30mg/kg) was effective (25-55%). Catalase (i.v., up to 10⁵ U/kg) showed little protection, but local injection of 100 U/kg resulted in 50% protection. Glutathione (30 mg/kg) was suppressive only by local injection suggesting the importance of administration route. Desferal, heparin and nitric oxide synthesis inhibitor showed some protection, but indomethacin, mepyramine, ascorbate, vitamin E and dexamethasone were without effect. Excess dosing of all anti-oxidants tested, dramatically decreased their effects demanding caution for therapeutic trials.     

Superoxide Dismutases and Anti-Oxidants Protected Mice from no-Reflow and Necrotic Damage Induced by Ischemia

A simple method in mice was established to screen anti-ischemic compounds. Thirteen times binding of rubber ring (1 × 1 mm, d = 42 mm) for 4.5 hrs, swelled the paws of 60% mice applied and 14 times binding swelled only of 5% mice. Critically reversible limit lay between these conditions. “All or none” rule dominated the paw swelling perhap due to different endogenous anti-oxidants' levels of individual mice. Determination of paw reversibility at 90 min of recirculation, was proved to be suitable. Swollen paws at this time returned normal and the paws with no-reflow dropped out by muscle necrosis after several days. Intravenous (i.v.) bovine Cu, Zn-SOD and bacterial Mn-SOD (3 - 10 × 10⁴ U/kg) or liposomal Cu, Zn-SOD (0.3 - 3 × 10⁴ U/kg) were protective (35-50%) by 14 times binding. Allopurinol (10-100 mg/kg) and D-mannitol (3-30mg/kg) was effective (25-55%). Catalase (i.v., up to 10⁵ U/kg) showed little protection, but local injection of 100 U/kg resulted in 50% protection. Glutathione (30 mg/kg) was suppressive only by local injection suggesting the importance of administration route. Desferal, heparin and nitric oxide synthesis inhibitor showed some protection, but indomethacin, mepyramine, ascorbate, vitamin E and dexamethasone were without effect. Excess dosing of all anti-oxidants tested, dramatically decreased their effects demanding caution for therapeutic trials.     

Growth of legionella and other heterotrophic bacteria in a circulating cooling water system exposed to ultraviolet irradiation

The effect of ultraviolet irradiation on the growth and occurrence of legionella and other heterotrophic bacteria in a circulating cooling water system was studied. Water of the reservoir was circulated once in 28 h through a side-stream open channel u.v. radiator consisting of two lamps. Viable counts of legionellas and heterotrophic bacteria in water immediately after the u.v. treatment were 0—12 and 0·7—1·2% of those in the reservoir, respectively. U.v. irradiation increased the concentration of easily assimilable organic carbon. In the u.v. irradiated water samples incubated in the laboratory the viable counts of heterotrophic bacteria reached the counts in reservoir water within 5 d. The increase in viable counts was mainly due to reactivation of bacterialcells damaged by u.v. light, not because of bacterial multiplication. Despite u.v. irradiation the bacterial numbers in the reservoir water, including legionellas, did not decrease during the experimental period of 33 d. The main growth of bacteria in the reservoir occurred in biofilm and sediment, which were never exposed to u.v. irradiation.     

Factors that interact with the antibacterial action of thyme essential oil and its active constituents

The viable counts of Salmonella typhimurium on nutrient agar (NA) decreased upon the addition of either the essential oil of thyme or its constituent thymol, especially under anaerobic conditions. Antagonistic effects of thymol against Staphylococcus aureus were also greater under anaerobic conditions. In contrast to the phenolic constituents of the oil, thymol and carvacrol, the chemically related terpenes p -cymene and y -terpinene had no antagonistic effects against Salm. typhimurium.
The addition of Desferal to NA counteracted the antibacterial effects of both thyme oil and thymol. No support was obtained, however, for a possible role of iron in the oxygen-related antibacterial action of the thyme oil and thymol or for the observed effect of Desferal. In the presence of thymol, the viable counts of Salm. typhimurium obtained on a minimal medium (MM) were lower than those obtained on NA. Addition of bovine serum albumin (BSA) neutralized the antibacterial action of thymol. It is suggested that the effects of BSA or Desferal are due to their ability to bind phenolic compounds through their amino and hydroxylamine groups, respectively, thus preventing complexation reactions between the oil phenolic constituents and bacterial membrane proteins. This hypothesis is supported by the marked decrease in the viable counts of Salm. typhimurium caused by either thyme oil or thymol when the pH of the medium was changed from 6.5 to 5.5 or the concentration of Tween 80 in the medium was reduced.     

Use of immunoglobulin enriched bovine colostrum against oral challenge with enterohaemorrhagic Escherichia coli O157:H7 in mice

An immunoglobulin enriched bovine colostrum preparation, IMMULAC (New Zealand Dairy Group, Cambridge, New Zealand), contains antibodies against various bacterial antigens. In the present study, we investigated the protective effects of a commercial bovine colostrum preparation against infections with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 in a murine model. Balb/c mice were given drinking water containing streptomycin for 3 days before and following oral challenge with streptomycin-resistant EHEC O157:H7 strain (O157-SM(R)). In mice pretreated with streptomycin, EHEC O157:H7 maintained stable levels of bacterial colonization in the intestines for the 3-week experimental time period. Oral administration of colostrum resulted in rapid decrease in the bacteria numbers compared with administration of skim-milk. Colostrum showed no direct in vitro bactericidal properties against either EHEC O157:H7. When sections prepared from cecum walls of streptomycin-pretreated mice were incubated in vitro with EHEC O157:H7, the colostrum significantly prevented the attachment of the organisms to the sections when compared with skim-milk. These results indicate that oral administration of bovine colostrum effectively protects mice against food-borne infections by inhibiting bacterial attachment to the intestinal mucous membrane, colonization and growth in the intestinal tract.     

The Polysaccharide Capsule of Streptococcus pneumonia Partially Impedes MyD88-Mediated Immunity during Pneumonia in Mice

Toll-like receptors (TLR) and the downstream adaptor protein MyD88 are considered crucial for protective immunity during bacterial infections. Streptococcus (S.) pneumoniae is a human respiratory pathogen and a large majority of clinical pneumococcal isolates expresses an external polysaccharide capsule. We here sought to determine the role of pneumococcal capsule in MyD88-mediated antibacterial defense during S. pneumonia pneumonia. Wild type (WT) and Myd88^-/- mice were inoculated intranasally with serotype 2 S. pneumoniae D39 or with an isogenic capsule locus deletion mutant (D39∆cps), and analysed for bacterial outgrowth and inflammatory responses in the lung. As compared to WT mice, Myd88^-/- mice infected with D39 demonstrated a modestly impaired bacterial clearance accompanied by decreased inflammatory responses in the lung. Strikingly, while WT mice rapidly cleared D39∆cps, Myd88^-/- mice showed 10⁵-fold higher bacterial burdens in their lungs and dissemination to blood 24 hours after infection. These data suggest that the pneumococcal capsule impairs recognition of TLR ligands expressed by S. pneumoniae and thereby partially impedes MyD88-mediated antibacterial defense.     

The effects of prostacyclin (PGI2) and 6-keto-PGF1 alpha on bovine plasma progesterone and LH concentrations

Injections of 1 mg PGI2 directly into the bovine corpus luteum significantly increased peripheral plasma progesterone concentrations within 5 min. Concentrations were higher in the PGI2-treated heifers than in saline-injected controls between 5 and 150 min and at 3.5, 4, 5, and 7 h post-treatment. Levels tended to remain elevated through 14 h. Saline and 6-keto-PGF1 alpha were without effect on plasma progesterone levels. The luteotrophic effect of PGI2 was not due to alterations in circulating LH concentrations. An in vitro experiment assessed the effects of either PGI2 alone or in combination with LH on progesterone production by dispersed luteal cells. Progesterone accumulation over 2 h for control, 5 ng LH, 1 microgram PGI2, 10 micrograms PGI2, and 10 micrograms PGI2 plus 5 ng LH averaged 99 +/- 42, 353 +/- 70, 152 +/- 35, 252 +/- 45, and 287 +/- 66 ng/ml (n = 4), respectively. Thus PGI2 has luteotrophic effects on the bovine CL both in vivo and in vitro.     

Improved in vitro embryo development using in vivo matured oocytes from heifers superovulated with a controlled preovulatory LH surge

In bovine in vitro embryo production, the IVM step is rather successful with 80% of the oocytes reaching the MII stage. However, the extent to which the process limits the yield of viable embryos is still largely unknown. Therefore, we compared embryonic developmental capacity during IVC of IVF oocytes which had been matured in vitro with those matured in vivo. In vitro maturation was carried out for 22 h using oocytes (n = 417) obtained from 2- to 8-mm follicles of ovaries collected from a slaughterhouse in M199 with 10% fetal calf serum (FCS), 0.01 IU/mL LH, and 0.01 IU/mL FSH. In vivo matured oocytes (n = 219) were aspirated from preovulatory follicles in eCG/PG/anti-eCG-superovulated heifers 22 h after a fixed time GnRH-induced LH surge; endogenous release of the LH surge was suppressed by a Norgestomet ear implant. This system allowed for the synchronization of the in vitro and in vivo maturation processes and thus for simultaneous IVF of both groups of oocytes. The in vitro developmental potential of in vivo matured oocytes was twice as high (P < 0.01) as that of in vitro matured oocytes, with blastocyst formation and hatching rates 11 d after IVC of 49.3 +/- 6.1 (SEM; n = 10 heifers) vs 26.4 +/- 1.0% (n = 2 replicates), and 39.1 +/- 5.1% vs 20.6 +/- 1.4%, respectively. It is concluded that IVM is a major factor limiting in the in vitro production of viable embryos, although factors such as the lack of normal preovulatory development of IVM oocytes contributed to the observed differences.     

Effects of gonadotropins and steroids on the subsequent fertilizability of extrafollicular bovine oocytes cultured in vitro

Effects of gonadotropins (2 i.u./ml follicle stimulating hormone, FSH and 10 μg/ml luteinizing hormone, LH) and steroids (1 μg/ml oestradiol, E and progesterone, P) on the fertilizability of extrafollicular bovine oocytes cultured in vitro and transferred in either the rabbit oviduct (Experiment I) or glass test tubes (Experiment II) were investigated. Bovine oocytes collected from follicles of 2–5 mm in diameter were cultured in vitro for 27 h in a medium containing Ham's F-12, 20% (v/v) bovine fetal serum and antibiotics. The combination of the hormones added to the medium was as follows; (1) none (control), (2) E, (3) LH, (4) LH + E, (5) FSH + LH + E, and (6) FSH + LH + E + P. All oocytes were recovered 24 h after insemination and examined for the presence of the pronuclei and a sperm tail with the midpiece in the oocyte cytoplasm, and the extrusion of the second polar body.In Experiment I, 630 of 704 transferred oocytes (85.7%) were recovered from the rabbit oviduct. The maturation rates of these oocytes (overall 61.1%) were not significantly affected by gonadotropins and steroids. Of the 741 of 920 oocytes recovered from test tubes in Experiment II, the maturation rates of them (overall 64.2%) were significantly increased (P < 0.05) by addition of LH (72.9%) and FSH + LH + E + P (74.1%) as compared with controls (55.4%). Fertilization rates were increased (P < 0.05) by the addition of FSH + LH + E compared with the controls in both Experiments I (31.1% and 14.0%) and II (36.2% and 20.8%). Furthermore, the proportion of fertilized eggs in hormone treated groups was the highest in each experiment. The present study indicates that the addition of FSH, LH and E to a medium has improved the fertilizability of extrafollicular bovine oocytes cultured in vitro.     

The effects of the platinum anti-tumour agents on renal cell kinetics and the response to a second cytotoxic agent

Following 15 mg kg-1 cisplatin to mice, the labelling index (LI) in the kidney decreased from 0.4% to less than 0.01% at 1-3 d, increased to 1.9% at day 16 and returned to control levels by day 30. Cytotoxicity was assessed by counts of viable tubule cross-sections and recovery was incomplete up to 14 months after treatment. Cisplatin treatment impaired the regeneration response to a dose of 16 mg kg-1 uranyl nitrate (UN) given 14 d after cisplatin when assessed by an increase in LI and sub-capsular tubule count. However, there was recovery with greater time intervals. At nine months after cisplatin there was no difference in response to UN of controls and mice previously treated by cisplatin. Prior treatment with paraplatin or iproplatin at LD50 doses produced not only no histopathological changes but also no impairment of the subsequent responses to UN.     

Contribution of complement component C5 to the pathogenesis of experimental murine cryptococcosis

C5-deficient (C5-) mice succumb much sooner after intravenous inoculation with Cryptococcus neoformans than do C5-sufficient (C5+) mice. The C5- mice developed acute, fatal cryptococcal pneumonia, whereas the C5+ mice did not. The pneumonia was characterized by lung viable counts in C5- mice up to 1000-fold higher than in C5+, initial sequestration of twice as much 59Fe-labeled C. neoformans, and subsequent development of pulmonary edema. Chemotaxis of heterophils (PMNs) and mononuclear cells in response to C. neoformans was markedly greater in C5+ mice than in C5- animals. The effect of C5 on localization and growth of C. neoformans in the lung appeared to account for the disparate survival times of C5+ and C5+ mice after intravenous inoculation with C. neoformans.     

Methyl Gallate from Galla rhois Successfully Controls Clinical Isolates of Salmonella Infection in Both In Vitro and In Vivo Systems

Galla rhois is a commonly used traditional medicine for the treatment of pathogenic bacteria in Korea as well as in other parts of Asia. Methyl gallate (MG), a major component of Galla Rhois, exhibits strong antibacterial activity, but its mechanism of action against Salmonella spp. is unclear. In the present study, we investigated the antibacterial actions of MG against Salmonella. The antibacterial activity determined by broth dilution method indicated that the antibacterial activity of MG against Salmonella strains ranged from 3.9 to 125 µg/ml. In vitro bacterial viability test indicated that MG significantly decreased the viability of Salmonella over 40% when combined with ATPase inhibitors. The time-kill curves showed that a combined MG and ATPase inhibitors (DCCD and NaN3) treatment reduced the bacterial counts dramatically after 24 h. Oral administration of MG showed a strong anti-bacterial activity against WS-5 infected BALB/c mice. In contrast to the untreated Salmonella infected control animals, MG treated groups showed no clinical symptoms of the disease, such as lethargy and liver damage. It was observed that MG treatment significantly increased the survival of animals from Salmonella infection, while in untreated groups all animal succumbed to disease by the sixth day post infection. Thus, the present study demonstrates the therapeutic ability of MG against Salmonella infections.     

Antikinetoplastid activity of 3-aryl-5-thiocyanatomethyl-1,2,4-oxadiazoles

A series of 5-thiocyanatomethyl- and 5-alkyl-3-aryl-1,2,4-oxadiazoles were synthesized and evaluated for their activity against kinetoplastid parasites. Formation of the oxadiazole ring was accomplished through the reaction of benzamidoximes with acyl chlorides, while the thiocyanate group was inserted by reacting the appropriate 5-halomethyl oxadiazole with ammonium thiocyanate. The thiocyanate-containing compounds possessed low micromolar activity against Leishmania donovani and Trypanosoma brucei, while the 5-alkyl oxadiazoles were less active against these parasites. 3-(4-Chlorophenyl)-5-(thiocyanatomethyl)-1,2,4-oxadiazole (compound 4b) displayed modest selectivity for L. donovani axenic amastigote-like parasites over J774 macrophages, PC3 prostate cancer cells, and Vero cells (6.4-fold, 3.8-fold, and 9.1-fold, respectively), while 3-(3,4-dichlorophenyl)-5-(thiocyanatomethyl)-1,2,4-oxadiazole (compound 4 h) showed 30-fold selectivity against Vero cells but was not selective against PC3 cells. In a murine model of visceral leishmaniasis, compound 4b decreased liver parasitemia caused by L. donovani by 48% when given in five daily i.v. doses at 5mg/kg and by 61% when administered orally for 5 days at 50 mg/kg. These results indicate that aromatic thiocyanates hold promise for the treatment of leishmanial infections if the selectivity of these compounds can be improved.     

Biofilm culture of Pseudomonas aeruginosa expressing lux genes as a model to study susceptibility to antimicrobials

A simple in vitro model for culture of biofilm populations of self-bioluminescent Pseudomonas aeruginosa was used for real-time monitoring of the effects of ciprofloxacin. Biofilms of these organisms were established within Sorbarod filters, perfused with a chemically defined simple salts medium. The biofilm population was shown to achieve a pseudo-steady state which was reproducible and stable over several days. The viability of membrane-associated and eluted cells was assessed by spread plate viable counts and by monitoring bioluminescence as a measure of metabolic activity. Pseudo-steady state biofilms were exposed to 5x MIC ciprofloxacin (0.3 mg x l(-1)) in the perfusing medium for 1 h. Whilst both methods for viability assessment indicated an immediate reduction in viable cell numbers, the decline recorded with bioluminescence was greater. The use of bioluminescent bacteria proved to be a rapid and sensitive method for the measurement of real-time antibacterial effects on a bacterial biofilm.     

Protective effects of lactoferrin chimera and bovine lactoferrin in a mouse model of enterohaemorrhagic Escherichia coli O157:H7 infection

Mice orally infected with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 were used to evaluate the activity of bovine lactoferrin (bLF) and the synthetic peptide LFchimera. Groups of BALB/c mice inoculated intragastrically with EHEC O157:H7 showed chronic intestinal infection with the pathogen that persisted over 6 days and resulted in a high mortality rate (90%). LFchimera and kanamycin significantly decreased (40%) this mortality rate (P = 0.028). On the other hand, although mice administered with bLF showed an important reduction in mortality (50%), this was not statistically significant (P = 0.070). In infected and untreated mice, severe tubular necrosis, glomerular lesions, and moderate intratubular hyaline casts were found in the kidney. However, in the bLF and LFchimera groups we found a reduction in the damage and a substantial decrease in the bacterial concentration excreted in feces 48 h after infection. Furthermore, sepsis caused by EHEC was reduced by the treatments, evidenced by the fact that bacteria were not detected in the kidney or liver 72 h after infection. The results suggest the bLF and LFchimera could have potential as therapeutics in EHEC infections.