Studying the Optimum Conditions of Hygromycin B Production and Detect their Toxicity

Hygromycin B was extracted with ethyl acetate, which separates organic phase from aqueous phase in the broth culture filtrate, only the aqueous phase showed significant antimicrobial activity by using agar well diffusion technique. At a concentration of 25mg/ml (as crude extract), this phase excreted its activity against the test microorganisms which include; one G(+) bacteria (Staphylococcus aureus), five G(–) bacteria (Pseudomonas aeruginosa , Proteus mirabilis, Escherichia coli , Klebsiella pneumoniae, Salmonella typhi) and one yeast (Saccharomyces cerevisiae). After detecting the aminoglycoside hygromycin B by the Thin Layer Chromatography (TLC) method to ensure presence of the antibiotic, same flow rate (Rf) value (0.357) as that of the standard hygromycin B was obtained. Results of the optimization conditions showed that the highest antimicrobial activity of hygromycin B was obtained at a medium pH of 8 and incubation temperature of 35°C for 10 days. When the toxicity of hygromycin B crude extract under such conditions was examined on mice liver, a mild effects were appeared.


Introduction
Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus that active against bacteria, fungi and higher eukaryotic cells by inhibiting polypeptide synthesis , It stabilizes the tRNA-ribosomal acceptor site, thereby inhibiting translocation (McGuire and Pettinger, 1953).
It has been reported to interfere with translocation1 and to cause mistranslation at the 70S ribosome (Gonzales, et al. 1978;Singh, 1979).
Hygromycin B was originally developed in the 1950s for use with animals and is still added into chicken feed as an anthelmintic or anti-worming agent (product name: Hygromix).. Resistance genes were discovered in the early 1980s (Burgett et al., 1983;Davies et al., 1983).Hygromycin B show in figure (1) has a chemical formula C 20 H 37 N 3 O 13 and molecular mass 275.53g/mol.In research it is used for the selection and maintenance of prokaryotic and eukaryotic cells that contain the hygromycin resistance gene.A more detailed examination of the mechanism of action has suggested that Hygromycin B also interferes with the translocation step in cell-free systems from both bacteria and yeasts (Cabanas et al., 1978).Most of the aminocyclitol antibiotics can be inactivated by at least one of three enzymatic mechanisms: (i) acetylation, (ii) adenylylation, or (iii) phosphorylation.Enzymes carrying out these reactions and the genes that code for them have been isolated from bacteria resistant to aminocyclitols (Davies and Smith, 1978.) and from Streptomyces species that produce these antibiotics (Leboul and Davies, 1982).Haas and Davies (1981) pointed out that the presence of these enzymes in bacteria usually makes the bacteria resistant to the appropriate antibiotic.Although resistance to Hm has been found in the Hm-producing organism Streptomyces hygroscopicus there has been no previous report of plasmid-encoded resistance to this antibiotic in enterobacteria.Moreover, none of the numerous aminoglycosidemodifying enzymes reported to date has been shown to use Hm as a substrate.
The aminocyclitol antibiotic hygromycin is produced by Streptomyces hygroscopicus and specifically blocks the translocation step on both 70s and 80s ribosomes (Cabanas et al., 1978;Gonzilez et al., 1978).The drug also induces misreading both in vivo and in vitro and therefore promotes phenotypic suppression (Singh., 1979) S. hygroscopicus contains a phosphotransferase (HPH) activity which phosphorylates hygromycin B, thus potentially providing the producing organism with autoimmunity against the toxic effects of the drug (Leboul & Davies, 1982).This interpretation appears to be valid since the gene (hyg) encoding the HPH activity has now been cloned in S. lividans and cells containing it became resistant to hygromycin B (Malpartida et al., 1983).Such an enzymic inactivation is one of several ways by which antibiotic-producing Streptomyces spp.become resistant to their own secondary metabolites.

Extraction of bioactive compounds:
Antibiotic production medium (500 ml )was inoculated with 1% of exponentially growing culture of the selected Streptomyces isolate and incubated at 35°C for 7 days.After that filtration was carried out through No.1 Whatman filter paper.The filtrate was centrifuged at5000 rpm for 15 min and antimicrobial activity was tested .
After pH was adjusted to 8 , the procedure proceed by extraction with ethyl acetate 1:1(v/v) in the rotary evaporator.The extract was separated into organic phase and aqueous phase; the organic phase was evaporated at room temperature for 4 days, while the aqueous phase evaporated in the oven at 40°C for 6 days.After the residual material was diluted with 5 ml of sterile distilled water, 1 ml was took from each phase and put in a beaker previously weighted then dry and weighted again to calculate the concentration of the active compounds in 1ml of D.W. each one was tested antimicrobial activity by using agar diffusion method.The extract of the phase that gave biological activity was used for further Hygromycin B detection .
-Detecting Hygromycin B by TLC technique: A small spot from each of the aqueous extract and the Hygromycin B standard solution (Sigma /USA) was applied to an aluminum foil coated thin-layer chromatoplates (20x20 cm) that placed in a separation chamber containing sodium acetate solution (eluent) as a mobile phase (taking in the consideration that the spots of the sample shouldn't touch surface of the eluent in the chamber).After the chamber was closed, it was left at 40°C .The solvent was then moved up via a capillary action and the sample was eluted, and the run was ended before the solvent reached the end of the plate.The spots was visualized by the ninhydrin spray reagent and heaing at 110 until the reddish spots appear and R f value was calculated.R f Value = Distance from Baseline travelled by Solute/ Distance from Baseline travelled by Solvent (Solvent Front) (Judit, 1972).

Optimization of antibiotic production conditions:
-Growing at different pH: Each of the four conical flasks, contained 25ml of hygromycin B production medium but with different pH values (4, 5, 6, 7, 8 and 9).They were prepared and inoculated with 1% of the exponentially growing culture of Streptomyces isolate, and incubated at 30˚C under for 7 days.By using the agar diffusion method, result was determined by exerting antimicrobial activity against Staph aureus, Protius mirabilis, and Saccharomyces cereviciae through measuring diameters (mm) of inhibition zones.

-Growing at different temperatures:
Each of the four conical flasks, contained 25ml of hygromycin B production medium with an optimum pH determined from the previous step prepared and inoculated with 1% exponentially growing culture of streptomyces isolates then incubated at different temperatures ( 25 , 30 , 35, 40 , 45˚C ) under aerobic condition for 7 days, By using the agar diffusion method, result was determined by exerting antimicrobial activity against Staph aureus, Protius mirabilis, and Saccharomyces cereviciae through measuring diameters (mm) of inhibition zones.

-Growing for different incubation periods:
In each of the four conical flasks, a volume of 25 ml of Hygromycin B production mediium with the optimum pH was inoculated with 1% of the active culture of Streptomyces isolate before incubation at the optimum temperature for different incubation periods (5 , 6 ,7 , 8,10 days ).By using agar diffusion method, results were read through measuring the diameters (mm) of inhibition zones formed the inhibitory activity of Hygromycin B against the three test microorganisms.

Detection of Hygromycin B toxicity:
The dose that prepared from the crude extract of Hygromycin B was in a concentration of (0.0002/gm body weight of mice, if we Know ,Practically, the recommended amount of Hygromycin B used as feed additive for chicken is 12g/ton and mean of chicken weight is 1500gm while the mean of mice weight is 15gm.The mice were divided into two groups; the first was daily treated by orally administration of 100µl of Hygromycin B for 14 day, while the second group was left without treatment as a control.A liver was taken and histologically analyzed

Production of Bioactive compounds:
Hygromycin B is an atypical aminoglycoside which is a large and diverse class of antibiotic has a unique structural and functional properties produced by Streptomyces isolates10cm/35pigment (Borovinskaya et al, 1996).
Clear filtrate which obtained after propagation of Streptomyces isolate 10cm/35pigment in cultural broth was tested for presence and activity of antimicrobial compounds against the seven test microorganisms.Separation of the filtrate by ethyl acetate resulted into two phases; organic and aqueous.
Results showed that when hygromycin B standard (100mg/ml) was used, no any antimicrobial activity was appeared in the organic phase at concentration of 3mg/ml, while in the aqueous phase, good antimicrobial activity was recorded at concentration of 25mg/ml of crude extract as illustrated in figure (2) and table (1).
Hygromycin B is soluble in water, at concentrations >50 mg/ml, methanol, buffer solution or in ethanol, but it is practically insoluble in less polar solvents (Merck Index,1996).In the study of Plozza et al. (2011), the aminoglycosides antibiotics were extracted from meat tissue or milk using an aqueous buffer.also Slolte (1999) stated that the aminoglycoside antibiotics (aminoglycosides) are hydrophilic molecules consisting of an aminated cyclitol associated with an amino sugar.Then, aminoglycosides are readily soluble in water.Thin layer chromatography (TLC) technique was used in detection of the aminoglycoside hygromycin B. The mobile phase carried the spot of the sample through a stable stationary phase, and each compound had a specific affinity for the mobile and stationary phases, therefore, migrates at a different speed.One spot of the sample mixture migrates at same speed of the hygromycin B standard and same Rf value that is constant under stable conditions of chromatography.It could be possible to say that the proposed antimicrobial agent is to be belonged to hygromycin B (Touchstone and Joseph, 1983).Medina MB and Unruh JJ.1996 also used thin chromatography method to separate and detect neomycin, gentamicin, spectinomycin, hygromycin B and streptomycin allowing multiresidue detection of these aminoglycosides.The respective RF values indicate the separation of these five compounds.This procedure provides a rapid and sensitive method for the semi-quantitative estimation of aminoglycosides.Hubicka et al.,(2009) stated that a TLC for identification and determination of amikacin, gentamicin, kanamycin, neomycin, netilmicin, and tobramycin (aminoglycosidic antibiotcs).

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Figure (3) shows the TLC plate of crude extract of hygromycin B in the aqueous .solution.It could be seen in this figure the movement of the extract and the standard where both of them reach the same distance from the bottom .

Figure (3) TLC analysis of crude extract hygromycin B produced by
Streptomyces isolate 10cm/35pigment.Rf ＝ A/B Rf for hygromycin B standard=2.5/7=0.357Rf for sample =2.5/7=0.357A= the distance from the starting point to the gravity center of the sample spot.B = the distance from the starting point to the front of the developing solvent.

Optimization of hygromycin B production conditions: -Optimization of pH:
To investigate the effects of the initial pH medium on hygromycin B production, the Streptomyces isolate was grown in hygromycin B production broth medium with different pH values (4 , 5 , 6 , 7 , 8 , 9) before incubated at 30˚C for 7 days .
Results in table (2) show that the isolate has recorded their highest activities when the pH of the medium was adjusted to 8. At this pH, Highest antimicrobial effect for hygromycin B was recorded with a (23) mm inhibition zone against the Gram negative bacteria Proteus mirabilis.Moreover, at this pH also, most efficient antimicrobial activity for hygromycin B against all three test organisms: Staph aureus (as Gram positive bacteria), Proteus mirabilis (as Gram negative bacteria) and Saccharomyces cerevisiae (as a yeast) was achieved when highest inhibition zone diameters (20, 23 and 18 mm, respectively) were recorded.Adversely, pH 4 led to the lowest antimicrobial activity for hygromycin B against all three test organisms with inhibition zones of (11, 11 and 9 mm, respectively).
Such results are closed to those obtained by Afifi et al., (2012)when they found that the optimum pH for hygromycin B production was 8 [16] and likely in agreement with result of Parthasarathi et al.,(2012) found that pH 7 showed maximum inhibition zone (26 mm) against Kleb.pneumoniae followed by Ps. aeruginosa (24 mm), S. aureus (20 mm), B. subtilis (19 mm) and E. coli (17 mm), in the substrate bombay rawa.Kavanag et al.,(1972) stated that , the assay of hygromycin B antimicrobial activity was more sensitive at pH 8.
The quantity of hygromycin B applied can be reduced by increasing the pH of the medium.At higher pH values, cells are more sensitive.The sensitivity of cells is pH dependent (i.e. the higher the pH of the culture medium the greater the sensitivity).Thus, the concentration of hygromycin B required for complete growth inhibition of given cells can be reduced by increasing the pH of the medium (Moazed, D. and Noller ,1987;Hemmi et al., 1992)  Optimization of growing temperature: The Streptomyces isolate was grown in the production medium after adjusting its pH to the optimum (8), then incubated the shaker incubator at different temperatures ( 25 , 30 , 35 , 40 , 45˚C) for 7 days.
Results in table (3) show that the Streptomyces isolate excreted its maximum biological activity when incubated at 35˚C, while less activity was detected at 45˚C.In this regard, Afifi et al. (2012) recorded 35˚C as the optimum temperature for hygromycin B produced from Streptomyces crystallinus AZ151.Parthasarathi et al.,(2012) used (25°C, 28°C, 37°C and 50°C) as growth temperatures and found that 28°C was the optimum one to give maximum antimicrobial activity by producing zones of inhibition of; (24 mm) against K. pneumoniae followed by P. aeruginosa (17 mm), E. coli (16 mm), S. aureus (14 mm), B. subtilis (12 mm) in the substrate bombay rawa.In another study, Oskay (2009) the researcher found the maximum zone of inhibition (18 mm) against S. aureus after incubation at 30°C temperature .

Optimization of incubation period:
Optimum production of hygromycin B was also determined after incubation at different incubation periods (5, 7, 10, 12 days).Results in table (4) show that the maximum antimicrobial activty was obtained after 10 days of incubation at an optimum pH 8 and a temperature 35°C .Afifi et al. (2012) recorded the maximum production of hygromucin B also after 10 days of incubation while in the study of Parthasarathi et al.,(2012) the maximum zone of inhibition (24 mm) against B. subtilis after 10 days of incubation.

Histological analysis of experimental mice:
Since liver is the foremost organ that receives all the materials absorbed by intestine through the portal vein, and it is the organ that must neutralize venoms, the toxic effect of most medications on liver manifest itself quicker than other organs (Vahidieyerisofla et al., 2014).
Oral addminstration with hygromycin B has been linked to mild effects which appeared after observing the microscopic slides of the liver tissue, of mice for both control and treated group .Microscopical examination of liver cross section slide of control mice showed normal structure, central vein,normal arrangement of hepatic cords, normal blood sinusoids and hepatocytes ,on the other hand the liver section of the treated mice with hygromycin B extract observed little hemorrhage with infiltration of PMN cell and degeneration of hepatocytes as show in figure (4).Khan et al., (2011) stated that the administration with gentamicin belong the class of aminoglycosides had been also linked to mild and asymptomatic elevations in serum alkaline phosphatase levels, but rarely affects aminotransferase levels or bilirubin, and changes resolve rapidly once gentamicin is stopped.Only isolated case reports of acute liver injury with jaundice have been associated with aminoglycoside therapy including gentamicin, most of which are not very convincing.Recovery typically occurs within 1 to 2 months and chronic injury has not been described.Aminoglycosides are not listed or mentioned in large case series of drug induced liver disease and acute liver failure; thus, hepatic injury due to gentamicin and other aminoglycosides is rare if it occurs at all.
Figure (4 ) A-Liver tissue of control showing normal structure, central vein (C.V.), normal arrangement of hepatic cords (H.C.), normal blood sinusoids (S) and hepatocytes., X 40x10.B and C-Liver tissue of Hygromycin B extract treated mice showing haemorrhage in the central vein, infiltration of PMN cell and degeneration of hepatocytes .at X 40x10 and X 10 respectively .

Table ( 3
) Antimicrobial activity of hygromycin B produced by Streptomyces isolate 10cm/35pigment grown in pH 8 production broth culture at different temperatures for 7 days.