Medical

Communication

Biosci. Biotech. Res. Comm. 10(4): 722-727 (2017)

Isolation and identi cation of pharmaceutically active

lipase producing

Bacillus

spp. from mangrove sediments

against Methicillin Resistant

Staphylococcus aureus

isolated from wound of patients

M.I. Farheena, A. Chithira and A. Mohankumar

Division of Microbial Technology, PG and Research Department of Zoology,

Chikkanna Govt. Arts College, Tirupur, Tamilnadu, India

ABSTRACT

Ninety two clinical wound samples were collected, among, 50 positive Staphylococcus aureus were isolated. These isolates were

characterized morphologically and biochemically. Twenty three antibiotics were used to determine susceptibility patterns by disc

diffusion method. The multiple antibiotic resistances (MAR) index were calculated according to the MAR index formula ranged

from 0.21 to 0.78. All the 50 isolates of MRSA were showed MAR index in between these ranges. Among these MAR index the

pathogenic isolates (100%) were resistant of Penicillin, while trimethoprim showed resistance (86%), cephoxitin (80%), kanamy-

cin (78%), vancomycin and cepfodoxamine (72%), moxalactam and quinupristin (70%), cotrimoxazole (66%), methicillin (64%),

novobiocin (62%) and erythromycin (56%) and there was no resistance found to chloramphenicol, and rifampicin. More than 65%

resistance MRSA isolates were selected for plasmid isolation. Natural products are boundless source for important novel com-

pounds having antagonistic activity against pathogenic organisms. Marine environment covers almost 70% of the earth surfaces.

Organisms present in these environments are extremely rich sources of bioactive compounds. The ocean remains as an unexploited

source of many drugs and pharmacologically active substances. Microbial enzymes have many advantages over the animal and

plant enzyme,  rstly; they are economical and can be produced on large scale within the limited space and time. Secondly, they

are capable of producing a wide variety of enzymes; they can grow a wide range of environmental condition. Enzymes have many

roles in the pharmaceutical and diagnostic industries. The bacteria Bacillus spp. was isolated from the mangrove sediment, for the

lipase production. These enzymes may inhibit the growth of MRSA. Recently with the advent of biotechnology, there has been a

growing interest and demand for enzymes with the novel properties.

KEY WORDS: PUS,

STAPHYLOCOCCUS AUREUS

, ANTIBIOTIC SUSCEPTIBILITY TESTING, ANTIMICROBIAL RESISTANCE PLASMID DNA, LIPASE

722

ARTICLE INFORMATION:

*Corresponding Author: farheena1980@gmail.com

Received 21

Oct, 2017

Accepted after revision 27

Dec, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2017: 4.31 Cosmos IF: 4.006

reserved.

Online Contents Available at:

http//www.bbrc.in/

DOI: 10.21786/bbrc/10.4/17

Farheena, Chithira and Mohankumar

INTRODUCTION

Wound infection is one of the major health problems

resulting from colonization of wounds by pathogenic

organisms Recurrently the predominant Staphylococcus

aureus is one of the most important opportunistic patho-

gen among Staphylococci belonging to Micrococaceae

family causing signi cant infections under appropriate

conditions (Prescott et al., 2002). About 20%–30% of

the general populations are carriers of S. aureus. The

anterior nasal cavity is the main site of S. aureus car-

riage. Among nasal S. aureus carriers, approximately

one-half also carry the organism on their skin. Recent

studies have established that S. aureus is often found at

nonnasal sites, particularly the pharynx and the gastro-

intestinal tract, with some carriers having colonization

con ned to these sites (Mertz et al., 2009; Acton et al.,

2009, Fagbomedo and Femi-Ola, 2017).

Staphylococcus aureus is one of the most versatile

nosocomial (i.e. acquired in hospital) and dangerous

human pathogen since publication of its role in sepsis

by Ogston in 1880 and 1882 (Lowy, 1998). At present,

Staphylococcal resistance to antibiotic has been associ-

ated with resistant plasmids (R-plasmid) that have the

ability to mediate the production of drug inactivated

enzymes such as -lactamase.The spread of resistance

to antimicrobial agents in S. aureus is largely due to the

acquisition of plasmids and or transposons (Lyon and

Skurray, 1987Adeleke and Odelola, 1997; Adeleke et al.,

2002 and other functions (King et al., 2006 and Diep

et al., 2008).

The un-hygienic hand swab showed presence of MRSA

and improvement in hand hygiene, coinciding with a

reduction of nosocomial infections and MRSA transmis-

sion (Pittet et al., 2000). Treatment options for patients

with serious invasive infections caused by methicillin-

resistant Staphylococcus aureus (MRSA) are limited

(Drew, 2007).Increasing resistance of MRSA in recent

years has had a signi cant impact on several aspects

of patient care and infection control. Antibiotic policies

need to be updated regularly, along with comprehen-

sive monitoring of antibiotic prescribing and antibiotic

consumption in healthcare settings. These facts clearly

highlight the need of a characterization of MRSA strains

at a regular basis at all levels. With the increasing inci-

dence of MDR, recourse to new antibiotics has become

necessary.Microbial enzymes have many advantages

over the animal and plant enzyme,  rstly; they are eco-

nomical and can be produced on large scale within the

limited space and time. Secondly, they are capable of

producing a wide variety of enzymes; they can grow a

wide range of environmental condition. Enzymes have

many roles in the pharmaceutical and diagnostic indus-

tries. Therefore the main objective of this study is to

isolate lipase enzyme from mangrove sediment bacteria

to treat against MRSA isolates.

MATERIAL AND METHODS

In the present study, 90 pus samples were collected from

various Hospitals in and around Tirupur District from

November 2012 to February 2013. The samples were

processed in the laboratory of PG and Research Depart-

ment of Zoology, CGAC. First step done was to isolate

the organisms from pus samples and then studied the

culture susceptibility of Staphylococcus aureus. All pus

samples were directly streaked on Mannitol Salt Agar

plates and incubated aerobically at 37ºC for 24 hours.

The isolates were identi ed with standard tests used to

identify S. aureus such as Gram stain, IMViC, Nitrate

reduction, Oxidase, catalase, slide and tube coagulase

tests. (Forbes et al., 2007).

Antibiotic sensitivity testing (AST) was done only for

con rmed S.aureus strains. Antibiotic sensitivity was

performed by Disc Diffusion Method (Bauer et al., 1966).

Overnight cultures of S.aureus in nutrient broth were

 ooded over the surface of Mueller Hinton agar plates.

The Mueller Hinton agar plates were allowed to dry

before applying antibiotic disc (Baddour et al., 2006).

Commercially available antibiotic discs were obtained

from (Himedia Labs, Mumbai, India) were gently and

 rmly placed on the agar plates, which were then left

at room temperature for 1 hour to allow diffusion of

the antibiotics into the agar medium. The plates were

then incubated at 37°C for 24 hours. If an antimicro-

bial activity was present on the plates, it was indicated

by an inhibition zone. The diameter of the inhibition

zones was measured in millimeter after 24 hours using a

standard chart and detected various pattern of antibiot-

ics as sensitive, intermediate and resistant to MRSA.

Multiple drug resistant strains of S. aureus which

showed 65% resistance to the antibiotics were selected

for plasmid extraction. Isolation of plasmids was per-

formed by Alkaline Lysis Method (Jegadeesh Babu and

Rajamanickam, 1998). The plasmids were observed in

0.7% Agarose gel electrophoresis.Extra cellular enzyme

such as lipase was detected from the marine bacteria

isolated from mangrove soil sediment. Using plate pre-

cipitation test with their speci c media screening of

lipolytic activity was done by using Rhodamine-B agar

plate (Kouker and Jaegar, 1987). The antibacterial activ-

ity of the lipase was performed by using well diffusion

method. So, hence in view to the signi cance of man-

grove ecosystem which provide a rich source of novel

lipase against MRSA isolates.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ISOLATION AND IDENTIFICATION OF PHARMACEUTICALLY ACTIVE LIPASE PRODUCING BACILLUS SPP. 723

Farheena, Chithira and Mohankumar

PLATE 1. Wound Sample

PLATE 2. Single colonies of S. aureus in MSA agar

plate

RESULTS AND DISCUSSION

Among the 92 clinical pus sample (Plate: 1) isolates of

S. aureus, 50 positive Staphylococcus aureus (Plate:

2) were identi ed as methicillin resistant Staphylococ-

cus aureus (MRSA) by disc diffusion method (Plate: 3).

Electrophoretic analysis of the plasmid DNA prepared

was carried out by agarose gel electrophoresis on 0.7%.

Five MRSA isolates (FA21, FA24, FA48, FA49 and FA50)

which was showed more than 65% resistance against

tested antibiotics were selected for plasmid isolation

(Plate: 4).Strain no FA49 harbored a single plasmid

DNA on basis of electrophoretic mobility on agarose gel.

Other four strains (FA21, FA42, FA48 and FA50) har-

bored a double plasmid DNA. The molecular size of the

plasmid DNA was calculated to be 1500bp and 1000bp

respectively. 100bp DNA ladder (Medox biotech, Chen-

nai) was used as marker DNA.

Marine bacterium Bacillus spp. was isolated from

marine sediment collected from Pitchavaram mangrove.

This bacterium was identi ed by biochemical characters.

This bacterium was identi ed by using ZoBell medi a and

lipase activity screening by Rhodamine B agar media

(Plate: 5) followed by the antimicrobial activity of lipase

enzyme was done by well diffusion assay (Plate: 6).

In Rhodamine B agar the isolates Bacillus spp. showed

halos around the bacterial colonies under ultra violet

irradiation. Kouker and Jaeger (1987) detected a plate

assay for microbial lipase in a medium containing tria-

cylglycerol with addition of  uorescent dye rhodamine

B. Substrate hydrolyses causes the formation of orange

 uorescence halos around bacterial colonies visible

upon UV irradiation. The isolated strains were screened

for lipase production.

Staphylococcus aureus is recognized as an important

bacterial pathogen contributing towards hospital infec-

tion, globally. Despite the use of potent antibiotic still

high mortality exist in case of Staphylococcus aureus

infection. In the present study, antibiotic susceptibility

pattern was assessed for Gram-positive cocci from pus

and a high resistance was recorded against antibiotics

tested. Despite the numerous studies found that advent

of antibiotics, it was though that warrant for the treat-

ment of the S. aureus related infection got issued but

due to the development of antibiotic resistant gene in

the plasmid of S. aureus could depend itself in a much

secured manner. So, these facts clearly highlight the

need of a characterization of MRSA strains at a regu-

lar basis at all levels. With the increasing incidence of

MDR, recourse to new antibiotics has become neces-

sary. In recent year the microbial enzymes have many

roles in the pharmaceutical and diagnostic industries.

Therefore the main objective of this study is to isolate

lipase enzyme from mangrove sediment bacteria to treat

against MRSA isolates.

Adame et al (2010) collected 250 samples from

healthy humans, cattle, sheep and goats for the isola-

tion of S. aureus and they reported that the antimi-

crobial susceptibility test showed highly susceptible

to Ciproxcin (91.1%), Nor oxacin (90.2%), Rifampicin

(73.2%), Streptomycin (72.3%), Erythromycin (71.4%),

Norbactin (64.3%), but the isolates showed resistant to

Ceftazimide (7.1%), Cefotaxime (14.3%), and Ampiclox

(31.3%). Totally 92 wound samples were collected from

infected human, among 50 S. aureus were isolated, Pen-

icillin showed 100% resistant against the isolates, Ami-

kacin and tetracycline showed 6% and 4% resistance

respectively in the present study.

Susmita Bhattacharya et al (2013) have isolated 280

MRSA strains from 714 Staphylococcus aureus of vari-

ous clinical samples. Among 280 MRSA, 21 strains were

724 ISOLATION AND IDENTIFICATION OF PHARMACEUTICALLY ACTIVE LIPASE PRODUCING BACILLUS SPP. BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ISOLATION AND IDENTIFICATION OF PHARMACEUTICALLY ACTIVE LIPASE PRODUCING BACILLUS SPP. 725

Farheena, Chithira and Mohankumar

PLATE 3. Antibiotic susceptibility test of S. aureus

found to be resistant to Vancomycin by disc diffusion

test. Similar method was used in the present inves-

tigation for antimicrobial susceptibility test. Among

50 MRSA strains, 36 strains were found to be resist-

ant to Vancomycin.High rates of resistance to Penicillin

among S.aureus have been observed since 1959, when

this frequency was recorded at 80% which have been

extended to amoxicillin and to ampicillin. Giarola et al

(2012) observed that the 90% resistance to Penicillin,

14% to rifampicin and 59% to azithromycin. Similarly

high rates of resistance was observed against Penicil-

lin (100%) but none of the isolates showed resistance

against rifampicin in this study.

All the isolates of S.aureus were multidrug resistant

and one isolate was pane resistant for all the tested drugs.

50 % resistance was observed in penicillin, methicillin,

polymyxin – B and Chloramphenicol 95.5%, 77.3%,

68.2% and 51.5% respectively (Alebachew et al 2009).

Similarly Penicillin and methicillin showed 100% and

64% resistance respectively, whereas none of the isolates

726 ISOLATION AND IDENTIFICATION OF PHARMACEUTICALLY ACTIVE LIPASE PRODUCING BACILLUS SPP. BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Farheena, Chithira and Mohankumar

PLATE 4. Plasmid Pro le of S. aureus

PLATE 5. Screening of Lipase enzyme from

Bacillus spp.

PLATE 6. Antimicrobial activity of lipase against S.

aureus

showed resistance against chloramphenicol. Totally 23

antibiotic drugs were tested, among 23 antibiotics, 12

antibiotics were showed were than 50% resistance.

Twenty four Staphylococcus aureus isolates were

tested invitro to determine their antibiotic susceptibil-

ity patterns by antibiotic disc diffusion method. All the

isolates showed multiple antibiotic resistances to the

antibiotics tested. All the isolates were 100% resistant

to Ampicillin, Amoxicillin Nalidixic acid (87.5%), Cef-

triaxone (75%), Streptomycin (54.16%), Erythromycin,

tetracycline, Kanamycin and Neomycin (25%), Oxacil-

lin (41.6%), Tobramycin and there was no resistance

found to Chloramphenicol, Vancomycin, Nor oxacin,

Cipro oxacin and Rifampicin (AI-Hamdani and Hamad,

2012). Totally 50 Staphylococcus aureus isolates were

tested invitro by similar method. Here also all the iso-

lates were showed multiple antibiotic resistance to the

drugs tested. But Chloramphenicol and Rifampicin were

showed 100% resistant.

Udo and Sarlchoo (2010) have characterized four

MRSA isolates , during plasmid analysis, four isolates

had showed three plasmid patterns, Isolate K6482 con-

tained two plasmids (28 and 26 Kb), K6531 and K6533

each contained three plasmids (28, 21 and41 Kb) and

K6552 contained two plasmids (41 and 41 Kb). In this

present study  ve strains which showed more than

65% resistance against all drug tested were taken for

plasmid isolation, Isolate FA49 contained one plasmid

(1.5Kb) and the isolates FA21, FA24, FA48 and FA50

each contained two plasmids (1.5kb and 1Kb).Herari et

al (2008) studied that the lipase production in an indig-

enous lipolytic Bacillus spp. in media containing tribu-

tyrin, tween 80 and rhodomine B- Olive oil. The statisti-

cal model was used to predict the optimum experimental

conditions for bacterial growth and lipase production.

Similarly in the present study Bacillus spp. was used

for lipase production, it was isolated from the sediment

of Pichavaram mangrove, lipase production was con-

 rmed by Rhodomine B-olive oil plate.Mohankumar and

Tamilselvi (2012) have isolated marine bacteria includes

Bacillus spp. Pseudomonas spp. Staphylococcus spp. and

Vibrio spp. for lipase production. Amount of lipase was

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ISOLATION AND IDENTIFICATION OF PHARMACEUTICALLY ACTIVE LIPASE PRODUCING BACILLUS SPP. 727

Farheena, Chithira and Mohankumar

estimated by plate assay method and titration. Similar

method was followed in the present study that the Bacil-

lus spp. was isolated from mangrove sediment samples

and the lipase production was estimated only by plate

assay method.

CONCLUSION

The present study has indicated that MRSA strains were

found to be develop resistance day by day to the currently

used antibiotics. This situation needs some novel thera-

peutic drugs from marine environment for an alternative

therapy for treatment of wound causing MRSA isolates.

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