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Original Article

Antimicrobial substance produced by Aeromonas KC954626and its effect on meat isolates

Periyasami Jagatheswaran a, Marimuthu Krishnaveni b,*aResearch Student, Department of Biochemistry, Periyar University, Salem 636 011, Tamil Nadu, IndiabAssistant Professor, Department of Biochemistry, Periyar University, Salem 636 011, Tamil Nadu, India

a r t i c l e i n f o

Article history:

Received 6 March 2013

Accepted 1 April 2013

Available online 23 May 2013

Keywords:

Aeromonas

Bacteriocin like antimicrobial sub-

stance

Food bacteria

Molecular weight

River soil

* Corresponding author. Tel.: þ91 9894829823E-mail address: krishnavenim2011@gmai

0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2013.05.001

a b s t r a c t

Aim: The urge to find a better antimicrobial substance made us to collect river soil sample

and Aeromonas was isolated to reach our goal, the crude antimicrobial substance produced

was optimized, its antimicrobial potency against microbes in meat sample was assessed.

Methods: Standard methods were adopted for screening, isolation, identification, agar well

diffusion but for optimizing antimicrobial substance production carbon and nitrogen

sources were replaced in the nutrient broth. SDS PAGE was done by kit method.

Results: The organism identified from river soil by 16S rDNA sequencing was Aeromonas sp.

Accession Number KC954626. The crude antimicrobial substance produced by Aeromonas

sp having molecular weight in the range of 14.3e98.4 kDa was active against Escherichia coli,

Pseudomonas, Staphylococcus present in the meat sample.

Conclusion: Form this present study,we can conclude that theAeromonas sp is able to produce

a substance, that have the ability to kill food borne gram positive as well gram negative

bacteria but at the same time it is not efficient in killing Klebsiella and Shigellawhich aremore

pathogenic. The bacteria present in the meat confirms undue human handling.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction isolated from river soil sample collected at Mohanur,

Aeromonas species are mesophilic, motile microorganism

present in aquatic and environmental habitats. It’s wide dis-

tribution depends on the seasonal changes, pollution level in

water. It is a Gram negative, short rod shaped, oxidase and

catalase positive, facultative anaerobes and non spore form-

ing. Antibiotics are organic molecules of microbes, at low

concentration, they are poisonous for the growth of other

microbes. In general, it acts against bacteria by attacking the

peptidoglycan cell wall. This study was designed towards the

search of antimicrobial compound from Aeromonas species

(mobile).l.com (M. Krishnaveni).2013, JPR Solutions; Publi

Namakkal District and its antimicrobial potency against bac-

teria isolated from meat samples.

2. Materials and methods

2.1. Isolation and identification of Aeromonas sp.

Wet soil samples collected in sterile bags were transported

immediately to the laboratory for analysis. One gram of

sample suspended in 9ml of sterile distilledwater was shaken

shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

Fig. 1 e Phylogenetic tree showing nearest sequences.

Plate II e Showing slime production by Staphylococcus sp.

j o u r n a l o f p h a rm a c y r e s e a r c h 6 ( 2 0 1 3 ) 5 3 8e5 4 2 539

well to homogenize the suspension. One millilitre of the su-

pernatant was diluted serially in tenfold 10�1e10�6. 0.1 ml

aliquot at 10�6 were dispensed in starch ampicillin agar1 for

24 h at 30 �C and observed for golden yellow colour colonies.

Standard biochemical tests were done and final confirmation

by 16S rDNA sequencing.

2.2. Screening of bacterial isolates in meat sample

One gram of meat sample collected from local market was

smashed in 2 ml phosphate buffered saline with mortar and

pestle, 0.1 ml was streaked directly on chromogenic,2

mannitol salt,3 SalmonellaeShigella agar4 plates prepared by

adopting standard procedures was incubated at 37 �C for 24 h

and pigmentation was observed. The identified isolates were

subjected to slime production on congo red plate as well for

beta lactamase on MullereHinton agar.3

2.3. Optimization, partial purification, antibacterialactivity of antimicrobial substance produced by Aeromonas

Optimization was carried out by maintaining the pH at 8.

Peptone in the nutrient broth was replaced with different

carbon sources such as sucrose, starch, glucose, fructose and

maltose. Similarly, beef extract with nitrogen sources like

ammonium chloride, ammonium nitrate, ammonium sul-

phate, potassium nitrate and sodium nitrate were added at a

final concentration of 1% (w/v) by keeping the remaining

same. The best carbon, nitrogen sources.

Plate I e Showing slime production by Pseudomonas,

Escherichia sp.

Antimicrobial substance and Aeromonas selected in the

optimization process was used for the bacteriocin like or

antimicrobial substance production, partial purification by

treating with solid ammonium sulphate at 40% saturation.

The contents were mixed for 2 h at 4 �C, centrifuged at

10,000 rpm for 20 min. The pellet obtained was dissolved in

500 ml phosphate buffered saline and 50 ml of this was used for

SDS PAGE,5 antimicrobial activity against identified meat

bacterial isolates by agar well diffusion method.6

Plate III e ShowingslimeproductionbyKlebsiella,Shigella sp.

Plate IV e Suseptibility of E. coli. to antimicrobial substance

produced by Aeromonas sp.Plate VI e Suseptibility of Staphylococcus sp. to

antimicrobial substance produced by Aeromonas sp.

j o u rn a l o f p h a rma c y r e s e a r c h 6 ( 2 0 1 3 ) 5 3 8e5 4 2540

3. Results and discussion

3.1. Isolation, identification of Aeromonas

River soil sample in selective media showed golden yellow

colour colonies, purified by repeated streaking in nutrient agar

was maintained for further analysis. Molecular identification

was carried out based on 16S r DNA sequence analysis. The

1.4 kb sequence obtained were aligned with sequences in the

GenBank database. A phylogenetic tree was constructed using

the neighbour joining method. Our sequence was found to be

very close to Aeromonas hydrophila and had 98% sequence

similarity with A. hydrophila strain WL-7 Genbank Accession

Number JQ034596 and GU227144. Phylogenetic analysis in

Plate V e Suseptibility of Pseudomonas sp. to antimicrobial

substance produced by Aeromonas sp.

Fig. 1 indicated that the bacterial isolate is Aeromonas sp. and

obtained Accession number KC954626.

3.2. Screening for meat bacterial isolates, its antibioticsusceptibility

The bacterial isolates in meat samples are Staphylococcus sp.,

Shigella sp., Escherichia coli, Klebsiella sp., and Pseudomonas sp.

Meat microbes when tested for biofilm production, mild pos-

itive result was observed with Escherichia Coli sp., moderate

with Staphylococcus sp. whereas Shigella sp. and Klebsiella sp.

was found to be strong positive. The results of biofilm assay is

shown in Plates IeIII.

Plate VII e Klebsiella sp. showing absence of zone

formation to antimicrobial substance produced by

Aeromonas sp.

Plate VIII e Shigella sp. showing absence of zone formation

to antimicrobial substance produced by Aeromonas sp.

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3.2.1. Optimization, partial purification, antibacterial activityof antimicrobial substance produced by AeromonasAmong the carbon sources selected, starch showed highest

antibacterial activity of 12 mm, 9 mm, 7 mm against Escher-

ichia coli, Pseudomonas sp, Staphylococcus sp. Whereas, the zone

of inhibition for sucrose was 10 mm, 6 mm, 4 mm. Glucose

and fructose showed similar results 9 mm, 7 & 8 mm, 5 mm

Fig. 2 e SDS PAGE of antimicrobial substance produced

from Aeromonas sp.

and 7 mm,8 mm,3 mm with maltose. Likewise, ammonium

nitrate showed 15 mm, 14 mm, 10 mm against Escherichia coli,

Pseudomonas and Staphylococcus. Ammonium chloride showed

14 mm, 11 mm, 5 mm. The zone of inhibition was lesser for

the other nitrogen sources. Best carbon, nitrogen sources

studied was used for the crude antimicrobial substance pro-

duction from Aeromonas sp. The antimicrobial activity in

terms of zone of inhibition measured are depicted in Plates

IVeVIII.

3.2.2. Molecular weight determinationMolecular weight of the partially purified antimicrobial sub-

stance was determined by SDS-PAGE,6 using kit, was ranged

from 14.3 to 98.4 kDa, shown in Fig. 2.

4. Conclusion

This study was carried out to synthesize bacteriocin like

antimicrobial substances from Aeromonas sp. The observed

result was positive against Escherichia coli, Staphylococcus sp. a

gram positive bacteria and Pseudomonas sp. a gram negative

bacteria. In our study, the Staphylococcus identified was

Staphylococcus epidermidis a non pathogenic strain as it showed

red colour pigmentation on mannitol salt agar screening.

Whereas negative result was observed with Klebsiella, Shigella

sp. a gram negative bacteria. Since, the produced bacteriocin

like antimicrobial substancewas ranged from 14.3 to 98.4 kDa,

this can be purified further to get a specific compound with

more antibacterial activity.

Conflicts of interest

All authors have none to declare.

Acknowledgement

The authors are thankful to Managing Director, Chrompark

Research Centre, D. Jagadeesh Kumar, Namakkal for

providing lab facilities and Dr. Sankara Pandian Selvaraj,

Helini Biomolecules, for helping us in doing bioinformatics

work.

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