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impetigo,
boils (furuncles),
cellulitis,
folliculitis,
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infections and is often the cause of postsurgical wound infections. Each year, some 500,000
patients in United States' hospitals contract a staphylococcal infection.
Role in Disease:
S. aureus is responsible for many infections but it may also occur as a commensal. The
presence of S. aureus does not always indicate infection. S. aureus can survive from hours to
weeks, or even months, on dry environmental surfaces, depending on strain.
S. aureus can infect tissues when the skin or mucosal barriers have been breached. This can
lead to many different types of infections including furuncles and carbuncles (a collection of
furuncles).
S. aureus infections can spread through contact with pus from an infected wound, skin-toskin contact with an infected person by producing hyaluronidase that destroys tissues, and
contact with objects such as towels, sheets, clothing, or athletic equipment used by an
infected person. Deeply penetrating S. aureus infections can be severe. Prosthetic joints put a
person at particular risk of septic arthritis, and staphylococcal endocarditis (infection of the
heart valves) andpneumonia. Strains of S. aureus can host phages, such as -PVL
(produces Panton-Valentine leukocidin), that increase virulence.
Antibiotic Resistance in Staphylococcus aureus:
In the 1940s, penicillin was introduced for the treatment of infection; as early as 1942, strains
of S. aureus resistant to penicillin had been detected in hospitals. Within 2 decades, 80% of
both hospital- and community-acquired S. aureus isolates was penicillin resistant. The
introduction of methicillin in 1961 was rapidly followed by reports of methicillin resistance
in S. aureus. Today, MRSA strains are found worldwide, and most are multidrug resistant.
Recently even the Vancomycin resistant Staphylococci were recovered from Hospitals, and
Cell phones.
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Flower details:
Campanula is one of several genera in the family Campanulaceae with the common
name bellflower. It takes both its common and its scientific name from its bellshaped flowers campanula is Latin for "little bell". The genus includes over 500 species and
several subspecies, distributed across the temperate and subtropical regions of the Northern
Hemisphere, with the highest diversity in the Mediterranean region east to the Caucasus. The
range also extends into mountains in tropical regions of Asia and Africa. The species
include annual, biennial and perennial plants, and vary in habit from dwarf arctic and alpine
species under 5 cm high, to large temperate grassland and woodland species growing to 2
meters (6 ft 7 in) tall.
The leaves are alternate and often vary in shape on a single plant, with larger, broader leaves
at the base of the stem and smaller, narrower leaves higher up; the leaf margin may be either
entire or serrated (sometimes both on the same plant). Many species contain white latex in the
leaves and stems.
The flowers are produced in panicles (sometimes solitary), and have a five-lobed corolla,
typically large (25 cm or more long), mostly blue to purple, sometimes white or pink. Below
the corolla, 5 leaf-like sepals form the calyx. Some species have a small additional leaf-like
growth termed an "appendage" between each sepal, and the presence or absence, relative size,
and attitude of the appendage is often used to distinguish between closely related species.
The fruit is a capsule containing numerous small seeds. Campanula species are used as food
plants by the larvae of some Lepidoptera species including Common Pug (recorded on
Harebell), Dot Moth, Ingrailed Clay (recorded on Harebell), Lime-speck Pug and Mouse
Moth.
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Datura is a genus of nine species of poisonous vespertine flowering plants belonging to the
family Solanaceae. They are known as angel's trumpets, sometimes sharing that name with
the closely related genus Brugmansia, and commonly as daturas. They are also sometimes
called moonflowers, one of several plant species to be so. Its precise and natural distribution
is uncertain, owing to its extensive cultivation and naturalization throughout the temperate
and tropical regions of the globe. Its distribution within the Americas and North Africa,
however, is most likely restricted to the United States and Mexico in North America,
and Tunisia in Africa, where the highest species diversity occurs.
All species of Datura are poisonous, especially their seeds and flowers.
Some South American plants formerly thought of as Datura are now treated as belonging to
the distinct genus Brugmansia (Brugmansia differs from Datura in that it is woody,
making shrubs or small trees, and it has pendulous flowers, rather than erect ones). Other
related genera include Hyoscyamus and Atropa.
REVIEW OF LITERATURE:
Staphylococci are Gram-positive bacteria, with diameters of 0.5 1.5 m and characterized
by individual cocci, which divide in more than one plane to form grape-like clusters. To date,
there are 32 species and eight sub-species in the genus Staphylococcus, many of which
preferentially colonies the human body (Kloos and Bannerman, 1994),
However Staphylococcus aureus and Staphylococcus epidermidis are the two most
characterized and studied strains. The staphylococci are non-motile, non-spore forming
facultative anaerobes that grow by aerobic respiration or by fermentation. Most species have
a relative complex nutritional requirement, however; in general they require an organic
source of nitrogen, supplied by 5 to 12 essential amino acids, e.g. arginine, valine, and B
vitamins, including thiamine and nicotinamide (Kloos and Schleifer, 1986; Wilkinson, 1997).
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Members of this genus are catalase-positive and oxidase-negative, distinguishing them from
the genus streptococci, which are catalase-negative, and have a different cell wall
composition to staphylococci (Wilkinson, 1997).
Staphylococci are tolerant to high concentrations of salt (Wilkinson, 1997) and show
resistance to heat (Kloos and Lambe 1991). Pathogenic staphylococci are commonly
identified by their ability to produce coagulase, and thus clot blood (Kloos and Musselwhite,
1975). This distinguishes the coagulase positive strains, S. aureus (a human pathogen), and S.
intermedius and S. hyicus (two animal pathogens), from the other staphylococcal species such
as S. epidermidis, that are coagulase-negative (CoNS).
Staphylococcus aureus is an opportunistic pathogen often carried asymptomatically on the
human body. Methicillin-resistant S. aureus (MRSA) strains have acquired a gene that makes
them resistant to all beta-lactam antibiotics. Infections caused by Staphylococcus aureus
pose serious threat in health care institutions. (Panlilio et al. 1992; and NNIS 2001, 2004). It
is one of the most widely spread and virulent nosocomial pathogen and is usually resistant to
multiple antibiotics making infections difficult to treat (Cooper et al. 2004). It appears to add
to the total burden of Staphylococcus infections in the hospitals, rather than replacing
sensitive S. aureus, and is associated with sharp risk in mortality attributable to
Staphylococcal infection (Crowcroft & Catchpole 2002).
Staphylococcus aureus strains continue to be a major problem in many healthcare institutions
especially with emergence of Methicillin resistant Staphylococcus aureus (MRSA) and now
account for more than 50% of S. aureus recovered from patients in intensive care units and
about 40% of S. aureus isolated from non intensive care unit (Boyce 2003).
Although, the clinical significance of methicillin resistance has been questioned in the past,
there is now widespread acknowledgement of the pathogenicity of MRSA. It has emerged as
a significant cause of both nosocomial and community-acquired infections. Recent report of
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strains of MRSA isolated from children in the community has led to speculation that the
epidemiology of S. aureus is changing (CDC 1999; and Boyce 1998).
Traditionally, MRSA infections have been acquired almost exclusively in hospitals, longterm care facilities or similar institutional settings (Thompson et al. 1982). Health-care
associated infection commonly caused by MRSA includes surgical site infections, bacteremia
and endocarditic, pneumonia, soft-tissue infections and urinary tract infections. However, the
emergence of community-associated MRSA (CA-MRSA) infections is of major concern to
both public health officials and clinicians.
The first report of CA-MRSA infection occurred among Australian aboriginals and Native
Americans in Canada in the early 1990s (Boyce 2003). The earliest reported cases of CAMRSA infection in the United States occurred in children with little or no recognized contact
with the hospitals or other health care institutions (Herold et al. 1998). Coagulase negative
Staphylococci (CNS) belong to the group of opportunistic pathogens since they are found as
normal flora of the skin and mucus membranes in different part of the body (Einsenstein and
Schaechter 1994). For this reason, CNS are often reported without further specification,
assuming that they are contaminating clinical samples but are not involved in the primary
infection.
However, there is mounting evidence that these bacteria may be responsible for primary
infections as a result of increased use of medical in dwelling plastic devices and
compromised or immune depressed patients (Jarvis andMartone1994; and Kloos and
Bannerman 1994). Methicillin resistance among CNS is particularly important due to cross
resistance to virtually al B-lactam agents and other antimicrobial classes. As a result,
therapeutic approaches are restricted to glycopetide and new antimicrobial agents as
Linezolid (Woods et al. 2002). Therefore, an accurate analysis of resistance between clinical
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and community strains may allow the provision of better antimicrobial therapy. Besides, the
importance for patient care the detection also has implications for the validity of antibiotic
resistance surveillance. Hence the purpose of this study is to isolate Staphylococcus species
from both clinical and community based samples and to determine the antibiogram of the
isolates against some selected commercial antibiotics.
Methicillin resistant S. aureus (MRSA) is a bacterium that has developed resistance to most
antibiotics such as methicillin, gentamycin, fucidic acid and clindamycin that are commonly
used for Staphylococcus infections, unfortunately, leading to failure of treatment (Shai et al.,
2004). The two major strains of MRSA are known to be hospital-acquired (HA) MRSA and
community-acquired (CA) MRSA. HA-MRSA includes cases in which the patient has had a
current or recent hospitalization receives dialysis, or resides in a long-term care facility.
During the period 1970 to 2010, strains of S. aureus resistant to multiple antibiotics including
methicillin were increasingly responsible for outbreaks of nosocomial infections in countries
around the world, for example, Saudi Arabia (Madani et al., 2001), Argentina (Reyes et al.,
2009), South Africa (Shittu et al., 2009), Italy (Soavi et al., 2010) and the United States
(Boyce, 1990). In many instances, these outbreaks were associated with individual wards,
neonatal, intensive care and burns units (Liu et al., 2011). Furthermore, increasing incidence
of CA-MRSA has been a growing public health concern (Mandell et al., 2005; Ma et al.,
2007) and has emerged as the predominant cause of skin infections in the USA (Stevens et
al., 2010).
Only few intervention studies have been published to explore alternative antimicrobial agents
to control and prevent diseases due to multidrug resistant S. aureus. Although other practices
have been explored such as bacterial interference therapy (Maibach and Aly, 1981) and phage
therapy (Jikia et al., 2005), medicinal plants have also been considered by some researchers
since they are frequently used in popular medicine as remedies for many infectious diseases
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(Geyid et al., 2005; Mohana et al., 2008; Rahim et al., 2010). The aim of the present study
was to determine the inhibitory effect of different plant extracts on the growth resistant
strains of S. aureus.
OBJECTIVES:
Collection of microbial samples from cell phones was carried out by swabbing the cell phone surfaces
on back and front by swabs immersed in sterile saline tubes (Duramz, et al., 2000). Same procedure
was followed for all the samples collected from different population groups .Samplings were
processed immediately after collection for isolation of bacterial pathogens (Kapdi, et al.2008). b.
B. ISOLATION:
The swabbed samples were serially diluted up to 10-9 dilution and plated by pour plate technique on
selective media like Nutrient agar media. The colonies were picked from these selective media by
noting down their colony characters and subjected for characterization studies by culturing them on
slants of respective selective media (Kolpin ,et al.,200 observed under microscope (Sepehri, et al,
2009).
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C. CHARACTERIZATION:
BIOCHEMICAL TESTS:
3% KOH solution was prepared and loop full of test culture was mixed with 3% KOH solution then
observed for the thread like appearance to identify Gram positive and Gram negative bacteria ( Aneja,
1996).
Starch hydrolysis: All the isolated organisms were inoculated on starch agar medium and
after incubation the clear zone was observed for the hydrolysis of starch (Siddiqui, et al,
1999).
Gelatin hydrolysis: Gelatin liquifaction was observed in all the test isolates when the
inoculated tubes were kept in at 4c to observe gelatin hydrolysis (Siddiqui, et al, 1999).
Sugar fermentation: All the test organisms were inoculated on 9ml of phenol red sucrose,
lactose; dextrose broth with 1ml broth was filled with Durhams tube. After incubation the
tubes were observed for acid and gas production (Arora, et al, 2009).
Catalase test: all the isolates were subjected for the catalase activity when the test organisms
were treated with 3% hydrogen peroxide by noticing the effervescence (Akinyemi, et al,
2009).
IMViC Test:
1) Indole production test: the test organism was inoculated on 1% tryptone broth after incubation
added with one ml of Kovacks reagent for indole production.
2) Methyl red test: Test organism was inoculated on methyl red broth. After incubation by adding
Five drops of methyl red indicator colour change was observed (Akinyemi, et al, 2009).
3) Voges-Proskaurs test: test organism were inoculated on V-P broth after incubation by adding
12 drops of VP-1 reagent and 3 drops of VP-2 reagent to observe the colour change from yellow to
ruby pink..(Akinyemi, et al, 2009).
simmon citrate agar medium after incubation to observe the colour change from green to blue.
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alpha, beta and gamma haemolysis was observed (Akinyemi, et al, 2009).
6) Mannitol fermentation: All the test organisms were inoculated on mannitol salt agar medium
after incubation observe the colour change from red to yellow was observed (Anderson, 2006).
Kirby-Bauer antibiotic sensitivity assay was followed for the selected isolates based on their
characterization studies. Different antibiotics like
Penicillin (at 10 micro gram concentration) (Hirematsuet al.,2008).Since all these were found to be
effective antimicrobial antibiotics (Fereen, et al.,2008).Broth cultures of test bacterial isolates were
swabbed on solidified nutrient agar media with sterile swabs (Anderson,2006). The antibiotic discs
were placed at equidistance on the agar surface and plates were incubated at 370 C for 24 hours, the
sensitivity or resistance was passed by measuring the zone of inhibition in millimetre (Boyce, 2008).
Fresh flowers, leaves of datura (dark lavender and light lavender), bell flower and fresh fruit of bell
flower were collected from the surrounding of Shivamogga, Holehonnur and Arasalu. The fresh
samples were washed with water and air dried. About 5 grams of samples were weighed and crushed
with the pestle and mortar respectively. The extract was mixed with 10ml of sterile water respectively
for sterile water extract. Well in agar method was followed to screen the antibacterial activity of
aqueous extracts.
SOLVENT EXTRACT PREPARATION:
Fresh flowers, leaves of datura (dark lavender and light lavender),bell flower and fresh fruit of bell
flower were collected from the surrounding of Shimogga, Holehonnur ,Arasalu. The fresh samples
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were washed with water and air dried. About five grams of samples were weighed and crushed with
the pestle and mortar respectively. The extract was mixed with 10ml of different solvents like
chloroform, petroleum ether, methanol, ethyl acetate respectively for solvent extract.
Sterilized Nutrient media was prepared and poured into sterile Petri plates.
The plates were labeled with the name of the bacterial culture Streptococcus aureus
which was swabbed on each plate and the type of compound was also labeled
respectively.
After solidification, swab was done on the surface of NA media with the selected
bacterial cultures.
After swabbing the well was digged using sterilized cork borer.
100-200L of the 10% solvent extract was pipette out using micropipette and added
into the well which was digged.
After incubation the zone of inhibition was observed in those plates, and was
measured by millimeter ruler. (Snehayadavet al, 2011).
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bellflower solvent extract was tested against S.aureus by WELL IN AGAR method. The
results indicated that the sterile water extract of datura leaf and other solvent extract show
better antimicrobial activity. In vitro antibacterial assay of flower extracts exhibited that the
solvent extracts contain bioactive compounds that can inhibit the growth of S.aureus.
When datura flower with dark lavender colour extract was mixed with sterile water was
transferred to a media which was swabbed with S.aureus organisms shown that zone of
inhibition of microorganism was found to be 26mm.When the Datura flower with light
lavender colour was tested to the above mentioned solvent, the zone of inhibition of
microorganism was found to be 30mm. The same light lavender colour flower was tested to
the solvents like ethyl acetate and methanol, the zone of inhibition of micro organism was
found to be 18mm and 26mm respectively. Where as in petroleum ether solvent, the flower
does not show any zone of inhibition of micro organism rather the microbial growth was very
abundantly that we could found any zone of inhibition. In our experiment, we can see that the
zone of inhibition was more in the solvent sterile water that is 30mm and zone of inhibition is
in ethyl acetate is 18mm with less growth . So the solvent extract, sterile water extract having
more antimicrobial potential and it can be used as drug (Table no2)
When Datura leaf extract treated with solvents was transferred to media we can see that the
zone of inhibition of micro organism is more in methanol 22mm, sterile water 18mm , ethyl
acetate 10mm and in chloroform and petroleum ether zone of inhibition is least (Table no3 ).
When bell flower fruit extract treated with solvents was transferred to media which was
swabbed with S.aureus shows that the zone of inhibition of micro organism in sterile water is
15mm and methanol is 22mm. Whereas zone of inhibition least in chloroform, petroleum
ether and ethyl acetate (Table no4 ).
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In this zone of inhibition of micro organism was more in sterile water 20mm , and chloroform
22mm where less growth of micro organism was found ( Table no 5).
Table no 1: Kirby Bauer Antibiotic Sensitivity Test:
Sl
Bacterial
Zone of inhibition in mm
No
isolates
Penicillin
Amphicillin
Methicilin
Vancomycin
01
S.aureus
17
02
S.aureus
18
03
S.aureus
18
04
S.aureus
<10
<10
22
05
S.aureus
17
06
S.aureus
16
Solvent Extract
Zone of inhibition in mm
control
Test
26
30
Ethyl acetate
18
Methanol
26
Petroleum ether
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Solvent Extract
Zone of inhibition in mm
control
Test
Sterile water
16
18
Ethyl acetate
10
10
Chloroform
Methanol
22
Petroleum ether
Staphylococcus aureus:
Sl No
Solvent Extract
Sterile water
Methanol
Zone of inhibition in mm
control
Test
11
15
22
Staphylococcus aureus:
Sl No
Solvent Extract
Zone of inhibition in mm
control
Test
Sterile water
20
Ethyl acetate
28
32
Chloroform
22
Methanol
14
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SUMMARY:
The present project work concentrated on isolation and characterisation of Staphylococcus aureus
isolates from cell phones. These isolates when tested for antibiotic sensitivity test by Kirby Bauer disc
diffusion method, few isolates exhibited resistance to the tested standard antibiotics like Penicillin,
Ampicillin, Methicillin and sensitive to Vancomycin.
Methicillin Resistant S.aureus isolates were subjected for further studies. Inhibitory effect of natural
products like flower extract of Datura and Bellflower were tested on these isolates by well in agar
method. 5% concentration of petroleum ether, ethyl acetate, chloroform and methanol extract were
capable of inhibiting MRSA to different extends. Among the solvent extract tested sterile water and
ethyl acetate were best in extracting the active fractions from flower and fruit extracts of Datura and
Bellflowers .
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CONCLUSION:
This study probably suggest the possibility of concurrent use of these plant extracts in
solvents like sterile water , ethyl acetate, methanol, chloroform in combination in
treating infection caused by S.aureus.
This study on antimicrobial potential of Datura and bellflower extract revealed that
the sterile water extract was more potent than other solvent extract.
Therefore our results revealed the importance of Datura and Bellflower of solvent
extracts when compared with antibiotics to control the S.aureus.
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REFERENCES
1. Abdul M.M, Sarker, A.A., Saiful, I.M. and Muniruddin, A. 2010. Cytotoxic and
Antimicrobial Activity of the Crude Extract of Abutilon Indicum.International
Journal of Pharmacognosy and Phytochemical Research. pp 2:1-4.
2. Akinyemi K. O., Atapu, A.D, Adetona, O. O and Coker, A. O. 2009. The potential
role of mobile phones in the spread of bacterial infections.Journal of Infect
DevCtries 3:628-632.
4. Amann, W. 1975. Acne vulgaris, Agruscastus(agnolyt). Jornal of
Medicine. 35:
1645-47.
5. Anas K, Jayasre P, Vijaykumar T, Kumar R (2008). In vitro antibacterial activity of
Psidium guajava Linn leaf extract on clinical isolates of multidrug resistance. Indian J.
Exp. Biol. 46:41-46.
6. Antrasen and AmlaBatra. Antimicrobial activity of different solvent extracts of
medicinal plants. 2012. International journal of current pharmaceutical
research.4 (2): pp.67-73.
7. Aswar, P.B., Khadabadi, S.S., B.S. Kuchekar, R.M. Rajurkar, S.S. Saboo and R.D.
Javarkar 2009. In-Vitro Evaluation of Anti-Bacterial and Vitexnigundo (Verbenaceae).
International journal of botony.12:13-46.
8. Bauer, A. W., Kirby, W. M. M., Sherris, J. C.
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NATURAL PRODUCTS
Bell flower
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Datura flower
Datura flower
Datura flower
Datura flower
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PHOTOS
Staphylococcus aureus from Cell phones:
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