Safety and Efficacy of Topical Chitogel- Deferiprone-Gallium Protoporphyrin in Sheep Model.

Objectives: Increasing antimicrobial resistance has presented new challenges to the treatment of recalcitrant chronic rhinosinusitis fuelling a continuous search for novel antibiofilm agents. This study aimed to assess the safety and efficacy of Chitogel (Chitogel®, Wellington New Zealand) combined with novel antibiofilm agents Deferiprone and Gallium Protoporphyrin (CG-DG) as a topical treatment against S. aureus biofilms in vivo. Methods: To assess safety, 8 sheep were divided into two groups of 7 day treatments (n = 8 sinuses per treatment); (1) Chitogel (CG) with twice daily saline flush, and (2) CG-DG gel with twice daily saline flush. Tissue morphology was analyzed using histology and scanning electron microscopy (SEM). To assess efficacy we used a S. aureus sheep sinusitis model. Fifteen sheep were divided into three groups of 7 day treatments (n = 10 sinuses per treatment); (1) twice daily saline flush (NT), (2) Chitogel (CG) with twice daily saline flush, and (3) CG-DG gel with twice daily saline flush. Biofilm biomass across all groups was compared using LIVE/DEAD BacLight stain and confocal scanning laser microscopy. Results: Safety study showed no cilia denudation on scanning electron microscopy and no change in sinus mucosa histopathology when comparing CG-DG to CG treated sheep. COMSTAT2 assessment of biofilm biomass showed a significant reduction in CG-DG treated sheep compared to NT controls. Conclusion: Results indicate that CG-DG is safe and effective against S. aureus biofilms in a sheep sinusitis model and could represent a viable treatment option in the clinical setting.

Topical Colloidal Silver for the Treatment of Recalcitrant Chronic Rhinosinusitis.

Background: The management of recalcitrant chronic rhinosinusitis (CRS) is challenged by difficult-to-treat polymicrobial biofilms and multidrug resistant bacteria. This has led to the search for broad-spectrum non-antibiotic antimicrobial therapies. Colloidal silver (CS) has significant antibiofilm activity in vitro and in vivo against S. aureus, MRSA, and P. aeruginosa. However, due to the lack of scientific efficacy, it is only currently used as an alternative medicine. This is the first study looking at the safety and efficacy of CS in recalcitrant CRS. Methods: Patients were included when they had previously undergone endoscopic sinus surgery and presented with signs and symptoms of sinus infection with positive bacterial cultures. Twenty-two patients completed the study. Patients were allocated to 10-14 days of culture directed oral antibiotics with twice daily saline rinses (n = 11) or 10 days of twice daily 0.015 mg/mL CS rinses (n = 11). Safety observations included pre- and post-treatment serum silver levels, University of Pennsylvania Smell Identification Test (UPSIT) and adverse event (AE) reporting. Efficacy was assessed comparing microbiology results, Lund Kennedy Scores (LKS) and symptom scores using Visual Analog Scale (VAS) and Sino-Nasal Outcome Test (SNOT-22). Results: CS demonstrated good safety profile with no major adverse events, no changes in UPSIT and transient serum silver level changes in 4 patients. CS patients had 1/11 (9.09%) negative cultures, compared to 2/11 (18.18%) in the control group upon completion of the study. Whilst not statistically significant, both groups showed similar improvement in symptoms and endoscopic scores. Conclusion: This study concludes that twice daily CS (0.015 mg/mL) sinonasal rinses for 10 days is safe but not superior to culture-directed oral antibiotics. Further studies including more patients and looking at longer treatment or improving the tonicity of the solution for better tolerability should be explored.

Long-Term Safety of Topical Bacteriophage Application to the Frontal Sinus Region.

Background:Staphylococcus aureus biofilms contribute negatively to a number of chronic conditions, including chronic rhinosinusitis (CRS). With the inherent tolerance of biofilm-bound bacteria to antibiotics and the global problem of bacterial antibiotic resistance, the need to develop novel therapeutics is paramount. Phage therapy has previously shown promise in treating sinonasal S. aureus biofilms. Methods: This study investigates the long term (20 days) safety of topical sinonasal flushes with bacteriophage suspensions. The bacteriophage cocktail NOV012 against S. aureus selected for this work contains two highly characterized and different phages, P68 and K710. Host range was assessed against S. aureus strains isolated from CRS patients using agar spot tests. NOV012 was applied topically to the frontal sinus region of sheep, twice daily for 20 days. General sheep wellbeing, mucosal structural changes and inflammatory load were assessed to determine safety of NOV012 application. Results: NOV012 could lyse 52/61 (85%) of a panel of locally derived CRS clinical isolates. Application of NOV012 to the frontal sinuses of sheep for 20 days was found to be safe, with no observed inflammatory infiltration or tissue damage within the sinus mucosa. Conclusion: NOV012 cocktail appears safe to apply for extended periods to sheep sinuses and it could infect and lyse a wide range of S. aureus CRS clinical isolates. This indicates that phage therapy has strong potential as a treatment for chronic bacterial rhinosinusitis.

Subclinical ketosis in dairy cows: prevalence and risk factors in grazing production system.

The purpose of this study was to estimate the prevalence of Subclinical ketosis (SCK) between 4 and 19 days in milk (DIM) in a grazing production system and investigate the importance of potential risk factors for SCK. This cross-sectional study was conducted in dairy cows (n = 107), which had more of two parities. The concentration of ß-hydroxybutyric (BHB) in blood was quantified through a hand-held meter. Potential risk factors evaluated were calving interval (CI), milk yield in previous lactation, metritis, dystocia, calf sex (male), parity (≤3 vs. ≥4) and pre-partum body condition score (BCS ≤ 3.5 vs. ≥3.75). Prevalence of SCK was 10.3% (95% CI 4.7-15) between 4 and 19 DIM. Risk factors identified were the occurrence of both metritis and pre-partum BCS ≥ 3.75. Cows with metritis had 4.9 (95% CI 1.17-20.98) times more risk of developing SCK than cows without metritis. And the cows with pre-partum BCS ≥ 3.75 had 5.25 (95% CI 1.32-21.11) times more risk of developing SCK than cows with pre-partum BCS ≤ 3.5. Metritis could induce a lower feed intake and promote the development of SCK. High pre-partum BCS could induce a greater mobilization of body reserves altering liver function and aggravating post-partum NEB. The results are indicative of the expected prevalence of SCK in grazing production system. Factors associated could help to identify cattle at risk of SCK and improve the management of strategies to limit the effects.

In vivo effect on pig chromosomes of high dosage vaccine against classic swine fever.

Hog cholera virus (HCV) can induce chromosome abnormalities in diseased pigs as well as in those vaccinated with attenuated virus vaccine against classic swine fever. An experiment was made using animals from potency and safety control tests of commercial vaccines in Argentina. The different types of chromosomal alterations observed were chromatid and chromosome breaks, chromatid exchanges, polyploid, multiple aberrations cells, and chromosome pulverization. In this study the occurrence of chromosome alterations in pigs receiving either 1 or 10 vaccine doses was evaluated by means of blood sampling at different periods after vaccination. An essay comparing prolificity between treated and non-treated sows was also made. Significant differences in the amount of damaged chromosomes as well as differences in the type of predominant alterations between the two treatments were observed. Aberration frequencies increased from the 5-day postvaccination period reaching the highest value of 4.14% at the 10th, for the one-dose treatment; and highest value of 42.7% including 33.96% of cell with chromosome pulverization which was found in the 7th day interval when applying 10 doses. From then on, the proportion of affected cells dropped until the 20th day interval, which was the last recorded. The prolificity trial did not show any difference between treated and control sows, indicating that chromosome alteration might be limited to lymphocytes. It is concluded that HCV maintains its mutagenic potentiality in the attenuated vaccine, being able to induce chromosomal damage as it does in classic swine fever diseased animals.

[Use of the protoplast fusion and regeneration method for screening antibiotic producers among inactive strains of Streptomyces]. / Ispol'zovanie metoda sliianiia i regeneratsii protoplastov dlia poiska produtsentov antibiotikov sredi neaktivnykh shtammov streptomitsetov.

Intraspecies fusion of protoplasts of two strains of Streptomyces fradiae, i.e native protoplasts of an inactive strain INA 00708 and heat inactivated protoplasts of a neomycin-producing strain ATCC 10745, and regeneration of the protoplasts of the inactive strain INA 00708 resulted in formation of clones producing neomycin and clones synthesizing antibiotics of an unknown nature differing from neomycin. All the active clones were unstable and lost their antibiotic activity in subcultures. Regeneration of the protoplasts of 4 different inactive strains of Streptomyces sp. also resulted in formation of active clones which were unstable and lost their capacity for the antibiotic synthesis after the first subculture. The data in principal indicate to the possible use of protoplast fusion and regeneration in screening of cultures producing new antibiotics among inactive strains of streptomycetes. However, the efficiency of such procedures is low since the experiments are labor-consuming and the resulting active clones are genetically unstable.

[Protoplast fusion in Streptomyces fradiae strains producing neomycin and tylosin]. / Sliianie protoplastov shtammov Streptomyces fradiae, produtsentov neomitsina i tilozina.

Interspecies fusion of protoplasts of the Streptomyces fradiae strains producing neomycin (an aminoglycoside antibiotic) and tylosin (a macrolide antibiotic) was performed with a view to isolate strains producing novel antibiotics. Fusion of the protoplasts of the neomycin- and tylosin-producing strains labelled by the resistance to monomycin and lincomycin, respectively, caused no formation of stable strains producing antibiotics differing in chromatographic mobility from the antibiotics produced by the initial strains. In fusion of the protoplasts of the unlabelled strains, heat-inactivated protoplasts of the active line of one strain (donor) and native protoplasts of the inactive line of the other strain (recipient) were used. When the neomycin-producing culture was used as a recipient the fusion led to formation of strain 195-34 producing antibiotics of the benzo(a)anthraquinone group. One of these antibiotics, i.e. antibiotic 34-I, proved to be a novel biologically active substance. After regeneration of the protoplasts of the initial strains, no stable strains producing antibiotics differing from neomycin and tylosin were isolated.

[Comparative study of Streptomyces--producer of aminoglycoside antibiotics, monomycin and kanamycin, and the strain 344 obtained by fusion of their protoplasts by the method of restrictive total DNA fingerprinting]. / Sravnitel'noe izuchenie streptomitsetov--produtsentov monomitsina a kanamitsina i shtamma 344, poluchennogo pri zliianii ikh proto- plastov metodom restriktsionnykh "fingerprintov" total'noi DNK.

The method of total DNA restriction finger prints was applied to the study of Streptomyces monomycini INA 1465 producing monomycin, Streptomyces kanamyceticus INA K-13 producing kanamycin and strain 344 isolated after fusion of the protoplasts of strain 1465 and K-13, which produced albofungin and chloralbofungin, aminoglycoside antibiotics. For preparing the finger prints of the strains splitting by endonucleases BamHI, PstI, PvuII, and BgIII was used. The finger prints showed that strain 344 was related to the strain of S. monomycini and markedly differed from the strain of S. kanamyceticus. Strain 344 was likely to result from reconstruction (probably 20-kb deletion) of the genome of S. monomycini INA 1465 induced by the preparation and regeneration of its protoplasts. The reconstruction could affect the genome area with localization of the genes involved in monomycin biosynthesis and monomycin resistance genes.

[Albofungin formation by a strain isolated as a result of fusion of protoplasts of organisms producing aminoglycoside antibiotics]. / Obrazovanie al'bofungina shtammon, poluchennym v rezul'tate sliianiia protoplastov produtsentov aminoglikozidnykh antibiotikov.

Strain 344 synthesizing an antibiotic complex was isolated after fusion of the protoplasts of Streptomyces monomycini producing monomycin and Streptomyces kanamyceticus producing kanamycin. The major component of the complex was identified with albofungin and the minor one was suggested to be chloralbofungin. In the cultures of strain 344 variants forming monomycin were detected. After regeneration of the protoplasts of the parent strains there were isolated no stable clones synthesizing antibiotics differing from monomycin and kanamycin.

[Effect of protoplast formation and regeneration on the production of nebramycin complex in Streptomyces cremeus subsp. tobramycini]. / Vliianie obrazovaniia i regeneratsii protoplastov na produktsiiu nebramitsinovogo kompleksa u Streptomyces cremeus subsp. tobramycini.

Production of the nebramycin complex in Streptomyces cremeus subsp. tobramycini before and after the protoplast formation and regeneration was comparatively studied. The antibiotic production was estimated by the total activity and component composition of the nebramycin complex. It was found that formation and regeneration of the protoplast led to lowering of the activity and changing of the complex component composition. Strains mainly synthesizing each one of the three basic components of the nebramycin complex were isolated. The strains proved to be unstable by the antibiotic production property and after three subcultures lost the differences in the complex component composition.

Studies on the bioecological aspects of adult mosquitoes in the Prado Basin of southern California.

Studies on the bionomics of adult mosquitoes were carried out in the Prado Basin of southern California during 1985-86. The faunal composition of mosquitoes caught by species was (in descending order) Culex quinquefasciatus, Cx. tarsalis, Cx. erythrothorax, Cx. stigmatosoma (formerly Culex peus) followed by Anopheles freeborni, Culiseta particeps, Cs. inornata and Cs. incidens. The number of mosquitoes per trap night was the lowest during December through February, and the highest during August through October. Depending on both intrinsic and extrinsic factors, adult mosquitoes were active at dusk and dawn. In spatial distribution studies, both adult and larval collections showed that Cx. quinquefasciatus and Cx. stigmatosoma were associated with dairy lagoons and Cx. erythrothorax with duck ponds and a nearby wooded area. Culex tarsalis was found in greater number at all habitats. Anopheles freeborni and Culiseta spp. were found around the wooded area. In vertical distribution studies, more mosquitoes were captured at the highest (6 m) level than at lower (0.6 and 3 m) levels which was probably due to the large percentage of parous females present at this site.

Studies on potential biological control agents of immature mosquitoes in sewage wastewater in southern California.

Three biological control agents, a copepod, Mesocyclops leuckarti pilosa, and two fish, Cyprinodon macularius and Poecilia reticulata, were evaluated for their survival in secondary sewage effluent (SSE) and predation potential on mosquito larvae. Results showed that the survival of M. l. pilosa was not significantly affected in SSE or SSE diluted (50%) with water. In predation tests, the copepod consumed from 50 to 90% of the 1st-instar larvae of Culex quinquefasciatus in 24 to 72 hr and P. reticulata fed on almost all stages (egg to pupa) of the test mosquitoes. Survivorship of P. reticulata and C. macularius in SSE was not significantly affected by SSE under both greenhouse and sewage aquaculture conditions. Poecilia reticulata was distributed towards the influent end and C. macularius towards the effluent end of the aquaculture ponds, indicating the former species can tolerate higher levels of pollution which exists at the influent end of the pond. However, low water temperature and dissolved oxygen may be detrimental to these fish species in sewage aquacultural systems.

[Nucleotide specificity of sibiromycin interaction with DNA]. / Nukleotidnaia spetsifichnost' vzaimodeistviia sibiromitsina s DNK.

Sibiromycin binds selectively to poly(dG).poly (dC) and poly(dG--dC).poly (dG--dC) and does not interact with poly(dA--dT).poly(dA--DT), poly (dI).poly (dC) and poly(dI--dC).poly(dI-dC) as is evident from the changes in the UV spectrum of the antibiotic at 310 mn, differential CD spectrum and inhibition of the template activity of polynucleotides in the DNA-dependant RNA-polymerase system. Sibiromycin efficiently interacts with GC-rich DNA specifically methylated at N-7 of guanine and glucosilated DNA of T2 phage. Therefore, specific interaction of sibiromycin with the guanine sites may take place rather in the narrow groove than in the large groove of the double helix.

[Interaction of sibiromycin with soluble chromatin from the cells of murine NK/LI lymphadenosis]. / Vzaimodeistvie sibiromitsina s rastvorimym khromatinom iz kletok limfadenoza myshei NK/LI.

Interaction of sibiromycin with chromatin from NK/Li cells was studied. It was shown that the chromatin proteins had no significant effect on the amount of the antibiotic firmly bound with DNA. The difference observed in the kinetics of the sibiromycin interaction with chromatin and DNA from NK/Li cells was not induced by replacement of a part of the DNA bound proteins. Possibly the chromatin proteins hampered formation of DNA conformation necessary for sibiromycin binding.

[Density gradient ultracentrifugation method of studying sibiromycin interaction with linear and circular DNA]. / Issledovanie vzaimodeistviia sibiromitsina s lineinoi i kol'tsevoi DNK metodom ul'tratsentrifugirovaniia v gradientakh plotnosti.

Sibiromycin added to linear chromosomal E. coli DNA in vitro leads to the decrease of bouyant density in neutral CsCl density gradient. This decrease is a linear function of sibiromycin/DNA ratio and amounts to about 32 mg/ml at the ratio equal to 0.1. Binding sibiromycin does not change the degree of hydration of DNA as revealed by centrifugation in metrizamide density gradients. When added to the covalently closed or open circular DNA of PM-2 phage, sibiromycin decreased the bouyant density of these DNA species to a similiar extent. The antibiotic does not induce single-strand breaks in DNA in vitro as follows from the results of ethidium bromide-CsCl density gradient centrifugation of covalently closed PM-2 DNA.