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Isolation and identification of Bacterial flora from Catfish (Clarias gariepinus) with antimicrobial susceptibility and herbal sensitivity.

There is an increasing demand for fish and fish products around the world (1). Fish is a low fat food, a great source of protein, vitamins and minerals; so it is an important food source for human especially, freshwater fish as Catfish (Clarias gariepinus) in high economic and nutritional value (2,3).

Bacteria is the major group of water microbes are responsible for heavy mortalities and which invade the tissue fish host (4) and can contaminate the surface gills and intestinal tract of fresh shell frish and other sea foods (5).

Fish are susceptible to a wide variety of bacterial pathogens, the majority of bacterial infections are caused by Gram-negative (Aeromonas spp., Pseudomonas spp. and Vibrio spp.) which cause most diseases in tropical fish. Several workers have conducted investigations on these bacteria (6,7).

The genus Aeromonas comprises of several species of gram negative, rod shaped, motile and non-motile, oxidase and catalase positive (8). Owing to varied nature of these bacteria, they have been difficult to classify, making the taxonomy of the genus (9). There is therefore a need for a system for identification, RAPD fingerprinting is rapid, sensitive and making the detection and identification relatively easy (10) found that the motile Aeromonads, isolated from the intestines of farm raised fresh water fish, had been classified to species level on the morphological and physiological bases.

Uses of antibiotic to cure bacterial infection and prevent fish mortality in aquaculture is becoming limited as pathogen developed resistance to drugs (11), many studies showed that natural plants and their extracts have multi-anti-microbial properties (12). So treatments of bacterial disease with various herbs have been safely used such as cinnamon, clove, garlic, thyme, mint and vanilla (13-14) determined that association of antibiotics and plant showed synergistic antibacterial activity against antibiotic-resistant bacteria.

The aim of the present study was to isolate and identify the isolated from the affected catfish (Clarias fish) based on the biochemical properties and to screen their phenotypic and genotypic traits which might be helpful for identification of this species in the diagnostic laboratories and determination the sensitivity of bacterial isolates to antibiotic and some natural plant extract.

MATERIALS AND METHODS

Collection of fish

Total number of two hundred Clarias gariepinus were collected from different localities in Sharkia governorate fish were transferred alive to the laboratory and subjected to clinical, postmortem and bacteriological examinations. Forty of them were apparently healthy and chosen as a control; while hundred and sixty showing clinical abnormalities and lesions.

Isolation of bacteria

Isolation of bacteria was carried out aseptically from collected samples of the affected areas of skin, gills and the internal organs (kidney, liver, spleen and intestine) were cultivated on different media for the isolation of bacteria as tryptic soya broth at 25[degrees]C and at 37[degrees]C for (18-24)hours, then poured onto tryptic soya agar, blood agar, Rimler-Shoots agar, Thiosulfate Citrate Bilesalt Sucrose agar (TCBS), and incubate at the same time and temperature. Purification of the isolated bacterial strains was performed according to (15). Identification of bacterial isolates

All the bacterial isolates were preliminary differentiated according to their morphological characters, physiological and biochemical rests were performed to identify Aeromonas spp., Pseudomonas spp. and Gram-positive cocci according to (16,15) and so Vibrio spp. (17,18). The identification was performed by Gram staining, catalase activity, oxidase test, oxidation-fermentation (OF) test, methyl red test, Voges Proskaver (VP) test, lysine decarboxylase, ornithine decarboxylase, arginine dihydrolase, indole, urease and hydrogensulfide (H2S), production test, gelatin degradation, acid production and nitrate reduction (15).

RAPD-PCR Analysis DNA Extraction

The protocol developed by (19) with modifications was used for isolation of DNA from broth culture of bacteria at log phase. The modified protocol without the use of proteinase K has given good result, yielding quality DNA of approximately 20 [micro]g from a 2 ml bacterial culture.

Primers

A panel of 9 numbers of decamer random primers from M/S Operon Technologies, was used for PCR amplification of bacterial DNA template. Two out of 9 primers, viz., OPX-03, OPV-19 were selected for final screening as they only generated several reproducible amplicons that could be resolved as distinct bands by agarose gel electrophoresis.

PCR amplification and resolution of RAPD markers

PCR reactions has been optimized for important parameters such as annealing temperature, concentration of Mg[cl.sub.2], template DNA, Taq DNA polymerase, dNTP's and primers. The PCR reaction components consists of 200 mm dNTP, 7.5 pico moles of primer, 2 units of Taq DNA polymerase enzyme, assay buffer with working concentration of 1.5 mM Mg[cl.sub.2], 20-30 ng template DNA in an assay volume of 25 ml. These concentrations were determined by a series of preliminary standardizing experiments.

Thermal cycling was performed with Perkin-Elmer thermocycler (GeneAmp PCR System 2400). Each of the 35 PCR cycles standardized for this work consisted of denaturation of DNA at 92[degrees]C for one minute, primer annealing at 37[degrees]C for one minute and primer extension at 72[degrees]C for one minute. All PCR samples were subjected to an initial two minutes denaturation for 94[degrees]C and post amplification extension at 72[degrees]C for ten minutes. PCR products were stored at -20[degrees]C until electrophoresis was performed.

The PCR amplification products were resolved by carrying out electrophoresis using a 1.5% agarose gel, stained with ethidium bromide. The marker used was e DNA cut with HindIII/ EcoRI. The DNA bands were visualized and documented using a Vilber Lourmat Get documentation system.

Data analysis

The data of RAPD analysis were scored computer as (1) and (0) for the presence and absence of bands, respectively. Similarity coefficients among the studied Aeromonas isolates as implemented in the computer program SPSS version 10 (20).

Antibiotic Sensitivity test

Antibiotic susceptibility of isolates was determined using the disc diffusion technique (21,22). The antibiotics and the concentration ranges tested were: Ampicillin (AM 10 [micro]g), Clindamycin (DA 2 [micro]g), Ciprofloxacin (CIP 5 [micro]g), Erythromycin (E 15 [micro]g), Gentamycin (GN 10 [micro]g), Norofloxacin (NOR 10 [micro]g), Spectinomycin (SPT 100 [micro]g) and Cefuroxime (CRO 30 [micro]g).

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) method

Is the lowest concentration of an antimicrobial agent (MIC) that will inhibit the visible growth of a microorganism after over-night incubation [23]. A two-fold serial dilutions of the antibiotics were prepared in Muller-Hinton broth (MHB) and to determine (MBC), 100 [micro]l aliquot from the tube showing (MIC) was placed on (MBC) agar plate antibiotic free, after incubation and examine the plates at which 99.9% killing of bacteria concentration; which prevented visible growth.

Antimicrobial evaluation of medicinal plants

The disc diffusion method was used as described by (24). A total of six selected herb extracts was used in this study to examine their sensitivity to experimental isolates. Medicinal plants were collected from local market in Sharkia governorate. Cinnamon (Cinnamomum verum), Clove (Syzygium aromaticum), Thymus (Thymus vulgaris), Nutmeg (Myristica fragrans), Rosemary (Rosmarinus officinalis) and Hibiscus (Hibiscus sabdariffa) plants were finely grinded to powder, soaked in 100ml hot sterile water and allowed to stand for (72) hours (25), each plant material extracted in Soxhlet apparatus with 150 ml of 95% ethanol at 60[degrees]C for (4) hours (26). 15 [micro]l of the herb extract was added on top o the disc of 6mm diameters, the plate was incubated at 37[degrees]C for (24) hour, the antimicrobial activity was expressed as the mean of inhibition diameter (mm) produced by plant extracts.

Determination of MIC and MBC of ethanolic Clove extract against isolated bacteria

The micro dilution method was used to determine the MIC of Clove extract. A two-fold serial dilution of ethanolic extract of Clove were prepared in sterile Muller-Hinton broth. Each dilution was seeded with 100 [micro]l of standard bacterial inoculum, then inoculate the lowest concentration of extract; showing no visible growth (27).

RESULTS AND DISCUSSIONS

Clarias gariepinus is freshwater fish, a popular delicacy relished throughout tropical Africa. As we know, fishery products are of great importance for human nutrition worldwide and provide clear health benefits (3). Fish are susceptible to a wide variety of bacterial pathogens causes large mortality in the aquaculture.

Isolation of bacteria isolates

In the present study, bacteriological examination of 166 diseased Clarias gariepinus revealed the isolation of (289) different bacterial isolates in Table (1), isolate (1) was found to represent the most predominate isolates from clinically diseased Clarias gariepinus in an incidence of 46.7%, other bacteria were isolated in much lower numbers as 1.03% of isolate (8).

Identification of isolated bacteria

All the bacteria isolates were preliminary differentiated according to their morphological, physiological and biochemical properties. The results of this study, Aeromonas spp. were the most predominant bacterial species isolated from diseased fish in an incidence 83%, these results agreement with those recorded by (28,10,29,30).

In Table (2), the biochemical properties as the growth at 5[degrees]C and on 6% NaCl, acid production from sucrose, salicin and arabinose were used to differentiate between the four species of Aeromonas as described (31,32,33,15,22). all Aeromonas (group 1, 2, 3 and 4) isolates gave negative result with Inositol, Arginie dehydrolase and give positive result with Maltose and Mannitol. Group 1,3 and 4 gave negative result with Arabinose while group 2 gives positive result. Group 1,2 and 3 gave negative result with salicin, while group 4 gave positive result. Sucrose fermentation test gave positive result with group 1, 2 and 4 while group 3 gave negative result, that similar to those recorded by (30,33,15,29). so according to (17) the four groups are A. sobria, A. caviae, A. jandaei and A. veronii, respectively.

In Table (3), Vibrio spp. were gram negative motile, oxidase and catalase positive grown on TCBS gave yellow colonies and in oxidation fermentation test gave positive result, no growth appeared at 0% NaCl but positive at 2, 5, 7% NaCl and Indole, citrate utilization positive but arginine hydrolysis negative. They ferment carbohydrates with production of acid but no gas. Strain 1 had comma shape rods under oil immersion lens and growth 10% NaCl while Strain 2 was short thick bacilli, no growth at 10%NaCl. (34,35,36,37) as (17), strain 1 & 2 are most likely to be Vibrio alginolticus and Vibrio anguillarum, respectively.

The result in Table (4) showed that the biochemical properties for differentiation between Pseudomonas spp. were oxidation fermentation test, growth at 37 C, arginin dehydrolysis, production of acid from glucose, Arabinose, mannitol and sucrose. These results were in agreement with (38-39,40,41,33,15). Isolate 1 grows well onto blood agar, nutrient agar, Tryptic soya agar (TSA) media, on R-S medium produce green color, smooth colonies also Isolate 2 grows well onto nutrient, TSA agar and R-S media but producing large irregular spreading colonies. This exhibit that isolate 1 is Pseudomonas fluorescens and isolate 2 is Pseudomonas aerginosa.

Staphylococci spp. were gram positive, facultative anaerobes that grow by aerobic respiration or by fermentation Table (5). The bacteria were catalase--positive and oxidase negative, strain 1 formed a fairly large yellow colony on rich medium, hemolytic on blood agar and produce the coagulase enzyme. Strain 2 had a relatively small white colony, non hemolytic, lack coagulase enzyme, from the previous result strain 1 identified as S.aureus & strain 2 identified as S.epidermids. (33,42,43) which revealed that 73.5% of the examined fish were infected with various gram positive bacteria, namely Streptococcus spp., Staphylococcus spp. and Clostridium perfringens.

Table (6) revealed that the most common of bacterial isolates from organ and tissues of clinical diseased Clarias gariepinus were Aeromonas sobria (135 isolates) 40 of them from skin, 20 from liver and intestinal content, 25 from (kidney and spleen) while gills showed the lowest rate of isolation 5 isolates, also the highest rate of isolation was obtained from liver (67 isolates) followed by skin (62 isolates),intestinal contents (55), kidney (47 isolates) and spleen (46 isolates) and finally gills (12 isolates). Our results in agreement with those reported by (44,45) which demonstrated that the motile Aeromonas dominated the skin & gills (46) found that the highest rate of isolation was obtained from the intestine content (68 isolates) followed by the liver (43 isolates), kidney (16 isolates), while the lowest number of isolated bacteria were recovered from skin, spleen and gall bladder (12,11 and 9 isolates).

Fig (1a and b) illustrates the amplification products of four Aeromonas isolates. Nine 10-mer random primers were tested the present study to identify and discriminate among these isolates. But only primers (OPX-03 and OPV19) succeeded to get amplified and reproducible fragments. As shown in figure (1a and b) each land (PCR-DNA product of each isolate) appeared to be different. The molecular size of the PCR products generated by these primers are ranged from 2176 to 260 bp. Forty-seven polymorphic bands were generated by the two primers. A total of 15 polymorphic bands were scored as unique ones. The primer OPX-03 was found to be the more potent in generating 11 unique bands while the latter primer produced 7 unique bands.

The primer OPX-03 revealed clear variations in RAPD products between the studied bacterial isolates. Figure (1a) illustrates the DNA polymorphism obtained with this primer. A total of 28 reproducible bands were generated by the primer OPX-03, which not necessary generated by all isolates. All the generated bands were scored as polymorphic ones (100% polymorphism). Eight of these bands were identified as a unique bands (Table 7) the first and second unique bands were detected at about 980 and 1820 bp in A. caviae and A. jandaei, respectively. The third, forth, fifth and sixth unique bands were recognized at about 1033, 830, 750 and 570 bp in Aeromonas sobria. The remainder unique DNA bands were detected at about 570 and 820 bp in A. veronii (Table 8).

The generated RAPD profile by the primer OPV-19 shows a total of 19 bands, which not necessary identified in all isolates. No monomorphic bands (common bands) were detected by OPV-19 primer among the studied isolates (Figure 1b). The profile comprised 7 polymorphic unique bands and 12 non-unique ones. Three unique bands at apparent molecular size of 1800, 1700 and 615 bp were identified in the A.caviae. The two unique bands at about 680 and 780 bp were identified on A.jandaei and A.sobria, respectively. Also, the two bands at about 820 and 570 bp were observed in the A.veronii.

The average similarity index between Aeromonas isolates was calculated as Nei's original measures of genetic identity and genetic distance, considering all the amplicons resulting from all the primers. The coefficient of genetic identities ranged from 0.427 to 0.848, most of them were of high magnitude. This result confirmed by (47) used RAPD markers to analyze Avine E.coli isolates from the USA (predominately, Gorgia) It is concluded that RAPD provides a rapid and powerful tool to study the epidemiology of avain E. coli. As several authors have suggested (48,49,50,51) molecular typing has great discriminating power and could be used in epidemiological studies of Aeromonas species.

Fig (2) illustrated that, Aeromonas spp. high susceptible to Ciprofloxacin, Ceftriaxone. Moderate susceptible to Gentamycin, Norofloxacin but resistance to Ampicillin, Erythromycin, Spectomycin, this result agree with (52). While, Pseudomonas spp were found to be susceptible to Ceftriaxone and moderate susceptible to Ciprofloxacin but resistance to Ampicillin, Erythromycin, Spectomycin and Gentamycin these results nearly similar to the recorded by (53). Vibrio spp were highly sensitive to Ciprofloxacin, intermediated to Norofloxacin and Ceftriaxone and resistance to remaining antibiotic used. This result in agreement with those reported by (54,37). Staphylococcus spp. susceptible to Ciprofloxacin Ceftriaxone, Gentamycin, Norofloxacin, also resistance to Erythromycin, Ampicillin. Our result incorporate with those reported by (55).

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Ciprofloxacin against Aeromonas spp was recorded in Table (9) range from 0.39-1.5micro /ml, Pseudomonas spp. MIC and MBC was 25 micro/ml, Staphylococcus spp. gave (6.25 and 3.1 micro/ml) for Staph. aureus and Staph. epidermidis respectively, Vibrio spp. recorded 3.1 micro/ml (56,57), in an other hand (58) reported that MIC of Ciprofloxacin was (1 microgram/ml) for 90% of clinical isolates tested, on another hand (59) revealed that the Ciprofloxacin MICs 0.5 to 1.0 ug/ ml species for pathogenic bacteria such as Pseudomonas, Staphylococci and susceptible S.Pneumonia, in spite of this broad range in pathogen sensitivities.

Fig (3) showed that alcoholic clove (Syzygium aromaticum) extract has the highest inhibitory activity in comparison with other plant extracts, also ethanolic cinnamon (Cinnamomum verum) extract showed good inhibitory activity against bacterial isolates while alcoholic extracts of Nutmeg (Myristica fragrans) and Hibiscus (Hibiscus sabdariffa) have intermediated inhibitory activity against bacterial isolates, but alcoholic extracts of Thymus (Thymus vulgrais) and Rosemary (Rosmarinus officinalis) have low inhibitory activity, this accordance with (60).

Table (10) showed that minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ethanolic clove extract against isolated bacteria recorded 25 micro/L A. sobria, A. caviae and Staph.aureus resulted 3.1 micro/L while A.jandaei, A. veronii, Ps. aeruginosa and Staph.epidermids, Ps. Fluorescens gave 12.5 micro/L MIC and MBC. In the case of Vibrio spp. recorded 1.5 micro/L (61,62,63).

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Soheir S. Abd El-salam [1], F.M. Ghaly [1], Dina M. Baraka [1], Shahira H. Mahmoud [3] and Abeer A. El-makhzangy [3]

[1] Department of Botany, Faculty of Science, Benha University, Egypt

[2] Department of Botany, Faculty of Science, Zagazig University, Egypt.

[3] Animal Health Research Institute, Zagazig University, Egypt.

(Received: 12 May 2016; accepted: 22 June 2016)

* To whom all correspondence should be addressed.

Tel.: +2/01067140872; E-mail: Dr.Soheir_Saad@yahoo.com

Caption: Fig. 1. RAPD fingerprints of the four bacterial isolated using two random primers. Lanes 1-4 are Aeromonas sobria, A. caviae, A. jandae and A. veronii, respectively Lane M is the standard DNA marker

Caption: Fig. 2. Antibacterial sensitivity of bacteria isolated from naturally infected Clarias garpinus to different antibiotics

Caption: Fig 3. Antibacterial activity of different plant extracts against bacteria isolated from naturally infected Clarias garpinus
Table 1. Showed percentage (%) of bacterial
isolates from naturally diseased Clarias garpinus

Bacterial isolates   Total   Percentage %

Isolate (1)           135       46.7%
Isolate (2)           55        19.03%
Isolate (3)           25         8.6%
Isolate (4)           25         8.6%
Isolate (5)            6        2.07%
Isolate (6)            7         2.4%
Isolate (7)            8         2.7%
Isolate (8)            3        1.03%
Isolate (9)           13         4.5%
Isolate (10)          12        4.15%
Total                 289

Table 2. Morphological and biochemical characters of suspected
Aeromonas spp. Isolates from naturally infected Clarias garpinus

Biochemical tests                        Aeromonas spp.

                         Group 1     Group 2     Group 3     Group 4

Gram stain                 -ve         -ve         -ve         -ve
Shape                   Short rod   Short rod   Short rod   Short rod
Motility                    +           +           +           +
Cytochrome oxidase          +           +           +           +
O/F                         F           F           F           F
Growth at 5[degrees]C       +           -           -           -
Growth on 0% Nacl           +           +           +           +
Growth on 3.5% Nacl         +           +           +           +
Growth on 6% Nacl           +           -           -           -
Catalase test               +           +           +           +
[H.sub.2]S (TSI)            +           -           -           -
Indol                       +           +           +           +
Starch hydrolysis           +           +           -           -
Methyl red                  +           +           -           -
Vogus Proskauer             +           -           +           +
Citrate                     +           +           +           +
Gelatin Liquefaction        +           +           -           -
Hemolysis                   p           -           Y           -
Acid production
  from :
Arabinose                   -           +           -           -
Salicin                     -           -           -           +
Inositol                    -           -           -           -
Maltose                     +           +           +           +
Tween 80                    +           +           +           +
Glucose                     +           -           +           +
Ornithen decarboxylase      +           -           +           +
Lysine decarboxylase        +           -           +           +
Arginine dehydrolase        -           +           -           -

Table 3. Morphological and biochemical
character of suspected Vibrio spp. Isolated
from naturally infected Clarias garpinus

Biochemical tests                Vibrio spp.

                           Strain 1       Strain 2

Gram stain                    -ve           -ve
Shape                     Curved rod     Short rods
                         (comma) shape
Motility                       +             +
Cytochrome oxidase             +             +
O/F                            F             F
Catalase                       +             +
Growth on TCBS             + Yellow       + Yellow
                           colonies       colonies
Methyl red                     +             +
Vogus Proskauer                +             +
Arginine dehydrogenase                       -
Indole                         +             +
Citrate Utilization            +             +
Growth on 0% Nacl              -             -
Growth on 2% Nacl              +             +
Growth on 5% Nacl              +             +
Growth on 7% Nacl              +             +
Growth on 10% Nacl             +             -
Marg reaction              Evitage N     Evitage N

Table 4. Morphological and biochemical characters
of suspected Pseudomonas spp. Isolates
from naturally infected Clarias garpinus

Biochemical tests             Pseudomonas spp.

                          Isolate 1     Isolate 2

Gram stain                   -ve           -ve
Shape                       Long          Long
                         curved rods   curved rods
Motility                      +             +
Cytochrome oxidase            +             +
O/F                           o            o/-
Growth at 5[degrees]C         +             +
Growth on 0% Nacl             +             +
Arginine dehydrogenase        v             +
Catalase                      +             +
H2S (TSI)                     -             -
Indole                        -             -
Lysine decarboxylase          -             -
Gelatin degradation           +             v
Ornithen decarboxylase        -             -
Urease test                   v             v
Acid production from :
Glucose                       +             +
Sucrose                       -             -
Salicin                       -             -
Lactose                       v             -
Maltose                       +             +
Inositol                      +             -
Fructose                      -             -
Nitrate reduction             -             -
Esculin hydrolysis            -             -

Table 5. Morphological and biochemical character
of suspected Staphylococcus spp. Isolated from
naturally infected Clarias garpinus

Biochemical tests                Staphylococcus spp

                             Strain 1         Strain 2

Gram stain                      +ve             +ve
Shape                      Large yellow     Small white
                          colony on rich       colony
                               media
Growth temperature        15-45[degrees]C   37[degrees]C
Catalase                         +               +
Coagulase                        +               -
Cytochrom oxidase                -               -
D-mannitol fermentation          +               +
Hemolysis on blood agar      Hemolysis          Non
                              [beta]         hemolysis

Table 6. Distribution of bacterial isolates from organs and tissues
of naturally infected Clarias garpinus

Bacterial isolates    Liver   Gills   Kidney   Spleen   Intestinal
                                                         contents

Aeromonas sobria       20       5       25       25         20
Aeromonas caviae       10       5       11       9          15
Aeromonas jandaei       7       1       2        5          5
Aeromonas veronii       7       1       5        5          5
Pseudomonas             4      --       --       --         --
  fluorescens
Pseudomonas             2      --       --       --         2
  aeruginosa
Staphylococcus          5      --       --       --         3
  aureus
Staphylococcus          2      --       --       --         1
  epidermidis
Vibrio alginolitcus     5      --       2        1          2
Vibrio anguillarum      5      --       2        1          2
Total                  67      12       47       46         55

Bacterial isolates    Skin   Total

Aeromonas sobria       40     135
Aeromonas caviae       5      55
Aeromonas jandaei      5      25
Aeromonas veronii      2      25
Pseudomonas            2       6
  fluorescens
Pseudomonas            3       7
  aeruginosa
Staphylococcus         --      8
  aureus
Staphylococcus         --      3
  epidermidis
Vibrio alginolitcus    3      13
Vibrio anguillarum     2      12
Total                  62     289

Table 7. Code & sequence of the two DNA random primer
that revealed RAPD products in the
isolates as well as number & types of amplified DNA bands
generated by these primers

Primer        Sequences        Monomorphic     Unique      Polymorphic
code                              bands      polymorphic      bands
                                                bands

OPX-03   5'-TGG CGC AGT G-3'       --             8            20
OPV-19   5'-GGG TGT GCA A-3'       --             7            12

Primer   Total
code

OPX-03    28
OPV-19    19

Table 8. Molecular size in bp of the amplified
polymorphic (unique) DNA bands generated by 2
DNA random primers used to identify the four
Aeromonas isolates Monomorphic: the band
appeared in all isolates

Isolates         OPX-03           OPV-19

A.caviae          980          1800-1700-615
A.jandaei         1280              680
A.sobria    1033-830-750-570        780
A.veronii          --             820-570

Table 9. Minimum inhibitory concentration (MIC) & minimum
bactericidal concentration (MBC) for three antibiotics against
bacteria isolated from naturally infected Clarias garpinus

Isolated Bacteria             Ciprofloxacin     Cefuroxime (CRO)
                                 (CIP)

                              MIC       MBC       MIC       MBC
                           (0.39-100           (0.39-100
                           [micro]/L)          [micro]/L)

Aeromonas sobria              1.5       1.5        50       100
Aeromonas caviae              1.5       1.5        50       100
Aeromonas jandaei             0.39      0.39      1.5       1.5
Aeromonas veronii             1.5       1.5        50       100
Pseudomonas                    25        25       6.25      6.25
  fluorescens
Pseudomonas                    25        25       3.1       3.1
  aeruginosa
Staphylococcus                6.25      6.25      12.5      12.5
  aureus
Staphylococcusepidermids      3.1       3.1       6.25      6.25
Vibrio alginoliticus          3.1       3.1       6.25      6.25
Vibrio anguillarum            3.1       3.1       6.25      6.25

Isolated Bacteria            Gentamycin (CN)

                              MIC       MBC
                           (0.39-100
                           [micro]/L)

Aeromonas sobria               25        25
Aeromonas caviae              3.1       6.25
Aeromonas jandaei             6.25      6.25
Aeromonas veronii             6.25      12.5
Pseudomonas                    50        50
  fluorescens
Pseudomonas                    50        50
  aeruginosa
Staphylococcus                1.5       1.5
  aureus
Staphylococcusepidermids      1.5       1.5
Vibrio alginoliticus          12.5      12.5
Vibrio anguillarum            12.5      12.5

Table 10. Minimum inhibitory concentration (MIC) & Minimum
bactericidal concentration (MBC) of ethanolic clove extract against
bacteria isolated from naturally infected Clarias garpinus

Ethanolic clove                  Isolated bacteria
extract
                  1     2     3      4      5      6      7

MIC (mg/ml)       25   3.1   6.25   6.25   12.5   6.25   3.1
(0.19-100mg/ml)

MBC               25   3.1   6.25   6.25   12.5   6.25   3.1

Ethanolic clove    Isolated bacteria
extract
                   8      9    10

MIC (mg/ml)       6.25   1.5   1.5
(0.19-100mg/ml)

MBC               6.25   1.5   1.5
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Article Details
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Author:El-salam, Soheir S. Abd; Ghaly, F.M.; Baraka, Dina M.; Mahmoud, Shahira H.; El-makhzangy, Abeer A.
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Sep 1, 2016
Words:5761
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