Isolation and characterization of bacteria associated with silkworm gut under antibiotic-treated larval feeding / Isolamento e caracterização de bactérias associadas ao trato intestinal do bicho-da-seda sob alimentação larval tratada com antibióticos

Abstract The impact of antibiotics on growth, cocoon production was assessed in addition to isolation and characterization of bacteria associated with silkworm gut of infected larvae. Larval rearing was maintained at recommended conditions of temperature and humidity. Silkworm larvae showing abnormal symptoms were collected from the control group and dissected for gut collection. Bacteria were isolated from the gut content by spreading on agar plates and incubated at 37 °C for 48 hrs. Bacterial identification and phylogenetic analysis were carried out by 16S rRNA gene sequencing. The isolated bacteria were subjected to antimicrobial susceptibility test (disc diffusion methods) by using Penicillin (10 µg/mL), Tetracycline (30 µg/mL), Amoxicillin (25 µg/mL), Ampicillin (10 µg/mL), and Erythromycin (15 µg/mL). All isolated strains showed positive results for the catalase test. We isolated and identified bacterial strains (n = 06) from the gut of healthy and diseased silkworm larvae. Based on the 16S rRNA gene sequence, isolated bacteria showed close relation with Serratia, Bacillus, and Pseudomonas spp. Notably, 83.3% of strains were resistant to Penicillin, Tetracycline, Amoxicillin, Ampicillin, and Erythromycin but 16.6% showed antibiotic susceptibility to the above-mentioned commonly used antibiotics. Silkworm larvae fed on penicillin-treated leaves showed significant improvement in larval weight, larval length, and cocoon production. Significantly higher larval weight (6.88g), larval length (5.84cm), and cocoon weight (1.33g) were recorded for larvae fed on leaves treated with penicillin as compared to other antibiotics. Isolated bacterial strains showed close relation with Serratia spp., Bacillus spp. and Pseudomonas spp.

Resumo O impacto dos antibióticos no crescimento e na produção do casulo foi avaliado, além do isolamento e caracterização das bactérias associadas ao intestino de larvas infectadas do bicho-da-seda. A criação das larvas foi mantida nas condições recomendadas de temperatura e umidade. As larvas do bicho-da-seda com sintomas anormais foram coletadas do grupo controle e dissecadas para coleta do intestino. As bactérias foram isoladas do conteúdo intestinal por espalhamento em placas de ágar e incubadas a 37° C durante 48 horas. A identificação bacteriana e a análise filogenética foram realizadas pelo sequenciamento do gene 16S rRNA. As bactérias isoladas foram submetidas a teste de sensibilidade antimicrobiana (métodos de difusão em disco) com penicilina (10 µg / mL), tetraciclina (30 µg / mL), amoxicilina (25 µg / mL), ampicilina (10 µg / mL) e eritromicina (15 µg / mL). Todas as cepas isoladas apresentaram resultados positivos para o teste da catalase. Isolamos e identificamos cepas bacterianas (n = 06) do intestino de larvas de bicho-da-seda saudáveis e doentes. Com base na sequência do gene 16S rRNA, as bactérias isoladas mostraram estreita relação com Serratia, Bacillus e Pseudomonas spp. Notavelmente, 83,3% das cepas eram resistentes a penicilina, tetraciclina, amoxicilina, ampicilina e eritromicina, mas 16,6% mostraram suscetibilidade aos antibióticos comumente usados mencionados acima. As larvas do bicho-da-seda alimentadas com folhas tratadas com penicilina apresentaram melhora significativa no peso larval, comprimento larval e produção de casulo. Peso larval significativamente maior (6,88g), comprimento larval (5,84cm) e peso do casulo (1,33g) foram registrados para larvas alimentadas com folhas tratadas com penicilina, em comparação com outros antibióticos. Cepas bacterianas isoladas mostraram estreita relação com Serratia spp., Bacillus spp. e Pseudomonas spp.

Isolation and characterization of bacteria associated with silkworm gut under antibiotic-treated larval feeding

Abstract The impact of antibiotics on growth, cocoon production was assessed in addition to isolation and characterization of bacteria associated with silkworm gut of infected larvae. Larval rearing was maintained at recommended conditions of temperature and humidity. Silkworm larvae showing abnormal symptoms were collected from the control group and dissected for gut collection. Bacteria were isolated from the gut content by spreading on agar plates and incubated at 37 °C for 48 hrs. Bacterial identification and phylogenetic analysis were carried out by 16S rRNA gene sequencing. The isolated bacteria were subjected to antimicrobial susceptibility test (disc diffusion methods) by using Penicillin (10 µg/mL), Tetracycline (30 µg/mL), Amoxicillin (25 µg/mL), Ampicillin (10 µg/mL), and Erythromycin (15 µg/mL). All isolated strains showed positive results for the catalase test. We isolated and identified bacterial strains (n = 06) from the gut of healthy and diseased silkworm larvae. Based on the 16S rRNA gene sequence, isolated bacteria showed close relation with Serratia, Bacillus, and Pseudomonas spp. Notably, 83.3% of strains were resistant to Penicillin, Tetracycline, Amoxicillin, Ampicillin, and Erythromycin but 16.6% showed antibiotic susceptibility to the above-mentioned commonly used antibiotics. Silkworm larvae fed on penicillin-treated leaves showed significant improvement in larval weight, larval length, and cocoon production. Significantly higher larval weight (6.88g), larval length (5.84cm), and cocoon weight (1.33g) were recorded for larvae fed on leaves treated with penicillin as compared to other antibiotics. Isolated bacterial strains showed close relation with Serratia spp., Bacillus spp. and Pseudomonas spp.

Resumo O impacto dos antibióticos no crescimento e na produção do casulo foi avaliado, além do isolamento e caracterização das bactérias associadas ao intestino de larvas infectadas do bicho-da-seda. A criação das larvas foi mantida nas condições recomendadas de temperatura e umidade. As larvas do bicho-da-seda com sintomas anormais foram coletadas do grupo controle e dissecadas para coleta do intestino. As bactérias foram isoladas do conteúdo intestinal por espalhamento em placas de ágar e incubadas a 37° C durante 48 horas. A identificação bacteriana e a análise filogenética foram realizadas pelo sequenciamento do gene 16S rRNA. As bactérias isoladas foram submetidas a teste de sensibilidade antimicrobiana (métodos de difusão em disco) com penicilina (10 µg / mL), tetraciclina (30 µg / mL), amoxicilina (25 µg / mL), ampicilina (10 µg / mL) e eritromicina (15 µg / mL). Todas as cepas isoladas apresentaram resultados positivos para o teste da catalase. Isolamos e identificamos cepas bacterianas (n = 06) do intestino de larvas de bicho-da-seda saudáveis e doentes. Com base na sequência do gene 16S rRNA, as bactérias isoladas mostraram estreita relação com Serratia, Bacillus e Pseudomonas spp. Notavelmente, 83,3% das cepas eram resistentes a penicilina, tetraciclina, amoxicilina, ampicilina e eritromicina, mas 16,6% mostraram suscetibilidade aos antibióticos comumente usados mencionados acima. As larvas do bicho-da-seda alimentadas com folhas tratadas com penicilina apresentaram melhora significativa no peso larval, comprimento larval e produção de casulo. Peso larval significativamente maior (6,88g), comprimento larval (5,84cm) e peso do casulo (1,33g) foram registrados para larvas alimentadas com folhas tratadas com penicilina, em comparação com outros antibióticos. Cepas bacterianas isoladas mostraram estreita relação com Serratia spp., Bacillus spp. e Pseudomonas spp.

Isolation and characterization of bacteria associated with silkworm gut under antibiotic-treated larval feeding.

The impact of antibiotics on growth, cocoon production was assessed in addition to isolation and characterization of bacteria associated with silkworm gut of infected larvae. Larval rearing was maintained at recommended conditions of temperature and humidity. Silkworm larvae showing abnormal symptoms were collected from the control group and dissected for gut collection. Bacteria were isolated from the gut content by spreading on agar plates and incubated at 37 °C for 48 hrs. Bacterial identification and phylogenetic analysis were carried out by 16S rRNA gene sequencing. The isolated bacteria were subjected to antimicrobial susceptibility test (disc diffusion methods) by using Penicillin (10 µg/mL), Tetracycline (30 µg/mL), Amoxicillin (25 µg/mL), Ampicillin (10 µg/mL), and Erythromycin (15 µg/mL). All isolated strains showed positive results for the catalase test. We isolated and identified bacterial strains (n = 06) from the gut of healthy and diseased silkworm larvae. Based on the 16S rRNA gene sequence, isolated bacteria showed close relation with Serratia, Bacillus, and Pseudomonas spp. Notably, 83.3% of strains were resistant to Penicillin, Tetracycline, Amoxicillin, Ampicillin, and Erythromycin but 16.6% showed antibiotic susceptibility to the above-mentioned commonly used antibiotics. Silkworm larvae fed on penicillin-treated leaves showed significant improvement in larval weight, larval length, and cocoon production. Significantly higher larval weight (6.88g), larval length (5.84cm), and cocoon weight (1.33g) were recorded for larvae fed on leaves treated with penicillin as compared to other antibiotics. Isolated bacterial strains showed close relation with Serratia spp., Bacillus spp. and Pseudomonas spp.

Diagnostic value of clot examination for malignant cells in serous effusions.

OBJECTIVE: To assess the diagnostic value of clot examination for satisfactory processing and confirmation of malignancy in serous effusions in routine cytological evaluation and compare the results with those of conventional smear and cell block preparations. METHODOLOGY: Body cavity fluids (n = 600) received in our laboratory were processed according to a pre-designed protocol for the study as follows: Day1: on receipt of the specimen, smears were made and a cell block was prepared from the sediment. Day2: after overnight sample storage of the remaining specimen at 2-8 degrees C all fluids were examined for the presence of a clot at the bottom of the container. Fluids in which clot had formed were fixed in formalin. The clot was then placed on a lens paper, wrapped and processed routinely. Diagnostic yields were compared. RESULTS: In this study, we included 600 cases of serous fluids from pleural, pericardial and peritoneal effusions. In 73% (n = 437) of samples, clot formation was seen, while in 27%, (n = 163) no clot had formed. Routine smear and cell block preparations showed malignant cells in 9.6% (n = 42). However, with the addition of the clot preparation, the number of cases in which atypical/malignant cells were seen increased from 42 to 85 (19.4%), with a P < 0.001. Special stains and immunohistochemistry (IHC) were also performed on clot preparations in 10 difficult cases. CONCLUSION: Clot preparation from body cavity fluids on the second day can be used as an adjunct to smear and routine cell block preparation to improve the accuracy and yield of the cytological diagnosis and may also be of great help for special studies such as IHC staining.

Hypotensive constituents from the pods of Moringa oleifera.

Hypotensive activity of the ethanolic and aqueous extracts of Moringa oleifera whole pods and their parts, namely, coat, pulp, and seed was investigated. The activity of the ethanolic extract of both the pods and the seeds was equivalent at the dose of 30 mg/kg. The ethyl acetate phase of the ethanolic extract of pods was found to be the most potent fraction at the same dose. Its bioassay-directed fractionation led to the isolation of thiocarbamate and isothiocyanate glycosides which were also the hypotensive principles of the pods as observed in case of Moringa leaves. Two new compounds, O-[2'-hydroxy-3'-(2"-heptenyloxy)]-propyl undecanoate (1) and O-ethyl-4-[(alpha-L-rhamnosyloxy)-benzyl] carbamate (2) along with the known substances methyl p-hydroxybenzoate (3) and beta-sitosterol have also been isolated in the present studies. The latter two compounds and p-hydroxybenzaldehyde showed promising hypotensive activity. Structures of all these compounds have been deduced by spectroscopy and chemical reactions.

Enicostema littorale: a new source of swertiamarin.

Enicostema littorale a useful medicinal herb was subjected to phytochemical as well as pharmacological investigations. A secoiridoid glycoside, swertiamarin, was isolated and identified on the basis of UV, IR, Mass and NMR spectroscopic measurements. However, pharmacological studies on the blood pressure of rats and ileum of Guinea-pig could not provide significant results.

Traditional medicine Cassia absus L. (chaksu)-pharmacological evaluation.

A crude extract of Cassia absus L. produced a dose dependent decrease in systemic arterial blood pressure and heart rate in pentothal anaesthetized rats. Repeated injections of the same dose of the crude extract (CA) showed tachyphylaxis. The cardio-vascular actions were not modified by pretreatment with atropine, chloropheniramine, ranitidine or propranolol in vivo. The pressor response of norepinephnine was not altered, whereas the pressor response of carotid occlusion was less when this was performed before administration of CA. Moreover, nicotine induced an increase in arterial blood pressure which was completely abolished by the CA, suggesting that the decrease in blood pressure is unlikely to be mediated via cholinergic, histaminergic or ß-2-adrenergic receptor stimulation, or by α-1-adrenergic receptor blockade. Perhaps the crude extract has centrally acting/ganglion blocking action or anti-nicotinic action. In spontaneously beating guinea-pig atria, CA induced a concentration dependent decrease in force and rate of atrial contraction. Pretreatment of cardiac tissue with the crude extract blocked the pressor response of nicotine, without altering/modifying the response to isoprenaline. In the rabbit aorta, CA caused concentration-dependent relaxation of phenylephrine and K(+)-induced contractions. In the guinea-pig ileum, CA at a low concentrations significantly reduced the spasmogenic response of nicotine without modifying the effect of acetylcholine. At high concentrations, it non-specifically abolished all the spasmogenic responses to ACh, 5 HT and histamine. The neuromuscular activity of CA was found to be similar to that of d-tubocurarine in skeletal muscle preparations (Frog rectus abdominis). These results revealed that the CA of Cassia absus exerts centrally acting/ganglion blocking, anti-nicotinic, non-specific muscle relaxant and curare like activities.

Fully acetylated carbamate and hypotensive thiocarbamate glycosides from Moringa oleifera.

Six new and three synthetically known glycosides have been isolated from the leaves of Moringa oleifera, employing a bioassay-directed isolation method on the ethanolic extract. Most of these compounds, bearing thiocarbamate, carbamate or nitrile groups, are fully acetylated glycosides, which are very rare in nature. Elucidation of the structures was made using chemical and spectroscopic methods, including 2D NMR techniques. Thiocarbamates showed hypotensive activity.

Blood pressure lowering action of active principle from Trachyspermum ammi (L.) sprague.

Trachyspermum ammi (L.) Syn. Carum copticum (L.) Bth. (Apiaceae) is locally known as Ajowan. Bioassay-directed fractionation of Trachyspermum ammi has resulted in the isolation of thymol which is present in other plants as well. However, its action on blood pressure has not been studied so far. In anaesthetized rats, thymol (1-10mg/kg) produced dose-dependent fall in blood pressure and heart rate. These effects were not blocked by atropine (1 mg/kg) and thymol did not modify presser response of norepinepherine, which rules out the possibility of cholinergic stimulation or adrenergic blockade. In spontaneously beating atria, thymol caused decrease in force and rate of atrial contractions. These effects remained unaltered in the presence of atropine. In rabbit aorta, thymol caused relaxation of norepinepherine and potassium induced contractions in a concentration-dependent manner. These relaxant effects remained unchanged after the removal of endothelium. Moreover, atropine, propranolol, indomethacine and glibenclamide did not alter the vasorelaxation by thymol. These results suggest that Trachyspermum ammi contains a calcium channel blocker-like constituent (thymol) which may explain the hypotensive and bradycardiac effects observed in the in vivo studies.

Isolation and structure elucidation of new nitrile and mustard oil glycosides from Moringa oleifera and their effect on blood pressure.

Bioassay-guided analysis of an EtOH extract of Moringa oleifera leaves showing hypotensive activity led to the isolation of two nitrile glycosides, niazirin [1] and niazirinin [2], and three mustard oil glycosides, 4-[(4'-O-acetyl-alpha-L-rhamnosyloxy)benzyl]isothiocyanate [4], niaziminin A, and niaziminin B. Glycoside 2 is a new compound. Niaziminins A and B have previously been obtained from the left extract as a mixture, while compound 4 is new from this source. Structural determination was accomplished by means of spectroscopic methods including appropriate 2D nmr experiments and chemical reactions. This is the first report of the isolation of nitriles, an isothiocyanate, and thiocarbamates from the same plant species. Isothiocyanate 4 and the thiocarbamate glycosides niaziminin A and B showed hypotensive activity while nitrile glycosides 1 and 2 were found to be inactive in this regard.

Symphytoxide A, a triterpenoid saponin from the roots of Symphytum officinale.

A new triterpenoidal saponin of hederagenin named symphytoxide A has been isolated from the ethanolic extract of the roots of Symphytum officinale and characterized on the basis of chemical investigations and spectroscopic studies as 3-O-[beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->4)- alpha-L-arabinopyranosyl] hederagenin. The structure of this new saponin was established on the basis of 1D and 2D NMR experiments including heteroCOSY, COSY-45 degrees as well as HMBC measurements and other spectroscopic techniques. The saponin exhibited hypotensive activity in anesthetized rats.