World Journal of Pharmaceutical ReseaRch

Mullasseril World Journal of Pharmaceutical Research

Volume 3, Issue 3, 4155-4165. Research Article ISSN 2277 – 7105

COMPUTER ASSISTED DESIGNING (CADD), IN SILICO

PHARMACOLOGICAL AND IN VITRO STUDIES OF A SERIES OF
AZOMETHINE DERIVATIVES OF LEADING SEMI SYNTHETIC
AMINOPENICILLIN (AMOXICILLIN)

*Abhilash Mullasseril

Veliyanad Post, Ernakulam Dist, Kerala-682 313. India.

ABSTRACT
Article Received on
22 February 2014, The incorporation of computers and invention of much suitable
Revised on 13 March 2014, medicinal chemistry software attracted the interests of many scientists
Accepted on 31March 2014
to the field of drug designing and discovery. For an expert drug
designing scientist it is necessary to develop his interdisciplinary skills
*Correspondence for and knowledge to interpret the observations more scientifically to
Author achieve his goal of drug discovery. One of the majour research
Abhilash Mullasseril
problems facing recently by the scientists is the enhanced drug
Veliyanad Post, Ernakulam
Dist, Kerala, India.
resistances shown by common pathogens. It is not easy and economic
to develop a specific drug each time to overcome this challenging
problem. The easiest possible solution is the derivatization of the presently prescribing drugs
at their possible sites by strictly adhering to governing rules like Lipinski rules. In this
research paper application of Computer Assisted Drug Designing (CADD), In silico QSAR
studies, Synthesis and characterization, systematic antibacterial screening of a series of
azomethine derivatives of leadingly prescribing aminopenicillin (amoxicillin) etc. are
achieved in a fruitful manner to demonstrate the solution that can be adopted in an economic
manner by reducing the time and expenses for drug discovery processes.

Keywords: Computer Assisted Drug Designing (CADD), In silico QSAR Studies,

Amoxicillin, Docking, β-Lactams and β-Lactamases, Lipinski rules

1. INTRODUCTION
The field of drug research and drug designing is becoming more and more interdisciplinary in
nature by the introduction of computers. The intervention of computers reduced the workload
and increased the number of Drug designing scientists considerably. For an expert drug

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designing scientist it is necessary to develop his interdisciplinary skills and knowledge to

interpret the observations more scientifically to achieve his final target. One of the majour
research problems facing recently by the scientists is the enhanced drug resistances shown by
common pathogens. It is not easy and economic to develop a specific drug each time to
overcome this challenging problem. The easiest possible solution is the derivatization of the
presently prescribing drugs at their possible sites by strictly adhering to governing rules like
Lipinski rules. It is important to consider the modern definition of drug as; those are normally
low molecular weight chemicals that interact with macromolecular targets in the body to
produce a beneficial or harmful pharmacological effect. It is worthwhile to think on the idea
that “All molecules are not drugs but all drugs are molecules” because of this scientists are
behind the problem to correlate physical properties to the medicinal activity of the molecules
understudy. Thus the central objective of medicinal chemistry became how to explain the
relationship between chemical structure and molecular properties that will directly give the
idea of the usefulness of the molecule to humankind. Hence especially in the field of
medicinal and pharmaceutical chemistry research we have to give much importance for the
same so that the molecule understudy can lead to drug likeness. The previous century ended
with an explosion of activity in gene related studies and stem cell research. This century
emerges as the century of biomedical research. The bioavailability of the drugs at their site of
action can be enhanced or reduced by interaction with other groups or groups well
established. The extensive use of antibiotics in man and animals have caused a serious
problem called drug resistance and recently many bacteria developed this significantly [1]. For
example many of the β-Lactams were found deactivated by the specific β-Lactamases
produced by common pathogens those were previously were very susceptible to those
antibiotics. The classical methods for the invention of new specific drugs are very time
consuming and expensive at an unacceptable level. The present study aims at the application
of computer assisted drug designing and prediction of the QSAR endpoints using computer
software followed by synthesis, characterization of the designed drug derivatives of
amoxicillin and their systematic antibacterial screening in the wet laboratory. A leadingly
prescribing antibiotic Amoxicillin trihydrate, a semisynthetic aminopenicillin, which is an
effective antibacterial, was the drug that selected for the present study because the screening
for antibacterial activities is the easily achievable task in the wet laboratory [2]. By believing
one of the best possible methods to overcome the drug resistance might be the derivatisation
of the antibiotic at its easily possible functional group based on Lipinski rules that lead to an
[3]
acceptable range of structure activity relationships . The derivatives of the carboxyl group

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in penicillins except the metal salts proved that they are inferior in most cases to the parent
drug itself and hence the derivatives of amino group were focused in the present study [4]. The
reaction conditions were designed with the help of the developed concept called Distribution
of Active Electrons based on the theoretical studies conducted on many organic reactions and
their products.

2. MATERIALS AND METHODS

The antibiotic Amoxicillin trihydrate (Fluka, Germany) and all other materials (Sigma
Aldrich, Germany) were used without further purification. The bacteria used for biological
studies were isolated from nearby hospitals and were found β-Lactamase producing. The
culture media and biochemicals used were of HiMedia make. The software used in the
present study were run on a Windows Vista Business based Genuine Intel (R) CPU T2050
1.60GHz 512 MB of memory computer. The molecular modeling was achieved by free
[5]
softwares like MOPAC and ArgusLab4.0.1 and in silico pharmacological studies were
[6]
done according to the guidelines of EPA of United States . The methodology adopted can
be divided into the following three portions viz. Molecular Modelling and Drug Designing,
QSAR Studies, Synthesis and Antibacterial Screening.

2.1. Molecular Modelling and Drug Designing: The central aim of Computer-Assisted
Drug Design is to obtain a quantitative understanding of drug-receptor interactions at the
[7]
molecular level . For this purpose it is necessary to obtain detailed information about the
conformations of both the ligand and the receptor in their bound states. Various models like
AM1 (Austin Model1), MNDO (Modified Neglect of Diatomic Overlap), MNDO/3 and PM3
were used to calculate the theoretical characterization of the parent drug and its azomethine
derivatives. The MNDO, AM1 and PM3 methods are all based on the Neglect of Diatomic
[8, 9]
Differential Overlap (NDDO) approximate Hamiltonian . These methods have been
proved as very successful in treating a wide array of organic systems for conformation and
[10]
molecular properties with smallest overall errors with regard to experiment . Each type of
calculation has its advantages and applications for which it is particularly well suited. The
choice of the method is very important and for work on unknown systems the only alternative
[11]
is to use better and better levels of calculation . The structures of bacterial proteins were
downloaded from Protein Data Bank [Research Collaboratory for Structural Bioinformatics
(RCSB) (http://rcsb.org/pdb)]. The azomethine derivatives of amoxicillin were modeled to
attain a stable conformation and were designed strictly according to the Lipinski rules with

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[12]
great importance . These rules are based on strong physiochemical reasoning. Hydrogen
bonds increase solubility in water and must broken first in order to permit the drug to enter
into and through the lipid bilayer membrane and thus an increase in the number of possible
hydrogen bonds reduce partitioning from the aqueous phase into the lipid bilayer membrane
for permeation by passive diffusion. The more the molecular weight the more size of cavity
needed to make it soluble in water and as a result the solubility decreases. Increasing
molecular weight reduces the drug concentration at the surface of the intestinal epithelium
and net result is the reduced compound absorption. Poor absorption properties result in low
bioavailability or the need for dosing via an alternate route, either of which limits the
potential scope of the drug candidate. The computer assisted drug designing gives a clear idea
by calculating drug-like properties of the leads and the derivatives by predicting the
acceptance of them prior to their synthesis by using well formulated rules. The docking
studies were performed using ArgusLab4.0.1 which is a freeware developed by Mark
Thompson of Planaria software LLC to predict and compare the antibacterial activities of the
parent drug and its azomethine derivatives. In this process (Docking), the interaction energies
are generally calculated by computing the van der Waals and Coulombic energy contribution
between all atoms of the two interacting molecules. Docking results were ranked as poses and
they were recalculated further to check their consistency.

2.2. In Silico QSAR Studies: The molecular structures were modeled and the stablest
conformation of the drug and its derivatives were used to calculate the in silico QSAR
endpoints according to the guidelines accepted by EPA United States. The indicator species
include Daphnia magna, Fathead minnow (Pimephales promelas), Tetrahymena pyriformis
and Rattus norvegicus (Rat). The species D magna, F minnow and T pyriformis are the direct
measures of aquatic toxicity. The Rattus norvegicus is the mammal widely accepted for the
primary studies. The Daphnia act as an indicator species, particularly useful in that area
because of its short lifespan and reproductive capabilities. The QSAR end point LC50,
concentration of the test chemical in water in mg/L that causes 50% of Daphnia magna to die
after 48 hours is calculated. The FHM is widely used by the US Environmental Protection
Agency as an ecological test organism due to its wide distribution throughout North America,
availability, and relatively short generation time. Recent literature deals with the application
of in silico methodologies for calculating toxicity values are also mentioning the importance
of Fathead minnow. The QSAR end point LC50, the concentration of the test chemical in
water in mg/L that causes 50% of Fathead minnow to die after 96 hours is calculated. The

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Tetrahymena pyriformis, was used as a test animal for determining the lethal effect of certain
antimalarial drugs. The QSAR end point IGC50, the concentration of the test chemical in
water in mg/L that causes 50% growth inhibition to Tetrahymena pyriformis after 48 hours is
calculated. The Rattus norvegicus or common albino rats are commonly used as test animals.
The QSAR end point LD50, amount of chemical in mg/kg body weight that causes 50% of
rats to die after oral ingestion is calculated. Apart from these, other end points such as
Developmental toxicity (DTox), Bioaccumulation factor (BCF) and Mutagenicity were also
calculated. These have much importance in designing the industry process in an
environmentally benign manner for the production of the drug derivatives. These computed
end points were compared for their reliability by calculating for known drugs and chemicals.

2.3. Synthesis and Antibacterial Screening: The equimolar solutions of drug amoxicillin
trihydrate and corresponding aldehydes were refluxed (pH 7.5-8.0) for approximately 30-
40minutes and isolated by volume reduction and cooling. The isolated azomethine derivatives
were purified by common drug purification methods and kept in vacuum desiccators for
further use. The antibacterial studies were performed using stains of Staphylococcus aureus
[13]
and Escherichia coli that were β-Lactamase producning and resistant to β-Lactams . The
Staphylococci are non-motile, non-spore forming, occasionally capsulate gram-positive
cocci. The most important human pathogen in this class is Staphylococcus aureus and hence
selected for present study. Most clinical isolates of Staphylococcus aureus are resistant to
benzylpenicillin, due to the production of a β-Lactamase that destroys the activity of the drug
by opening it at the β-Lactam ring. The Escherichia coli a member of Enterobacteriaceae is
gram-negative non-spore-forming bacilli and is one of the most important common human
pathogen accounting for at least 80% of uncomplicated community acquired infections and
up to 50% stains may be resistant to penicillin due to acquisition of transmissible, plasmid-
determined TEM-type β-Lactamases [14]. The method adopted for antibacterial screening was
well diffusion method by dissolving sufficient equimolar quantity of the drug and derivatives
in DMSO as solvent. Micropipettes were used to fill the wells that were cut by sterilized
[15]
metallic instrument after the bacteria were inoculated to the Petri dish filled with broth .
The wet lab results were compared with that of computational studies.

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R5
HO 2
NH2 R4 CHO R 3
R
1
R
NH R3 R1 4
O R
HO
R2 N
S 5
pH (~8.0) R
O N
CH3
 NH
CH3 O
O
OH S
O N
CH3
Amoxicillin (D1) Derivative (D1S1-6) CH3
O
OH
Derivative Substituents

D1S1 R1= OH R2=R3=R4=R5=H

D1S2 R1= NO2 R2=R3=R4=R5=H

D1S3 R2= NO2 R1=R3=R4=R5=H

D1S4 R3= N(CH3)2 R1=R2=R4=R5=H

D1S5 R3=OH; R4=O(CH3 ) R1=R2= R5 =H

D1S6 R3= OH R1=R2=R4=R5=H

Scheme 1- Synthesis of Azomethine Derivatives (D1S1-6) of the Drug Amoxicillin (D1).

The structures of designed azomethine derivatives and the adopted scheme of synthesis of the
drug Amoxicillin are as shown in Scheme-1.

3. CHARACTERIZATION
The characterization of the synthesized drug derivatives were achieved by elemental analysis,
cryoscopy, measurements of conductance, absorption or UV-VIS spectroscopy, vibration or
IR spectroscopy and Nuclear Magnetic Resonance (NMR) spectroscopy. The results of
elemental analysis and cryoscopy were found in support to the hygroscopic nature of the
synthesized azomethine derivatives. UV-VIS spectroscopy results were found characteristic
to azomethine groups and β-Lactam ring. The conductance studies were in support to the
electron rich nature of the compounds as expected. The vibration spectra of compounds were
found characteristic of the β-Lactam rings. The colour varies from product to product. The
observed charring points were between 178oC to 190oC. The IR-Spectra were recorded
qualitatively by KBr Disc technique and the observed frequencies (cm-1) are plausibly
assigned (Table 1).

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Table 1: The observed prominent peaks and plausible functional groups assigned for
vibrational spectra.

Drug υOH υCO Lact υCO υCN NO2 As NO2 Sy δ CO

1
Derivative (cm- ) (cm-1) (cm-1) (cm-1) (cm-1) (cm-1) (cm-1)
D1S1 3528 1730 1626 1623 - - 753
(Broad) (Sharp)
D1S2 3410 1730 1607 1633 1443 1346 743
(Broad) (Sharp)
D1S3 3418(Broad) 1726(Sharp) 1649 1528 1440 1351 734
D1S4 3433(Broad) 1726 1659 1549 - - 813
(Sharp)
D1S5 3266 1775(Sharp) 1686 1586 - - 787
(Broad)
D1S6 3236(Broad) 1739(Sharp) 1673 1583 - - 788

The azomethine derivative Salicylideneamoxicillin (D1S1) is Orange in colour with an

observed conductance of 9.2 µS where as the 2-Nitrobenzaldiminamoxicillin (D1S2) is
Greenish Brown in colour with a conductance of 7.2 µS. Similarly 3-
Nitrobenzaldiminamoxicillin (D1S3) is found to be Yellow in colour with a conductance of
14.6 µS and the azomehtine derivative 4-(N,N Dimethyl)aminobenzaldiminamoxicillin
(D1S4) is Orange in colour with an observed conductance of 9.2 µS. The derivative
Vanilideneamoxicillin (D1S5) is Yellow in colour with a conductance of 18.9 µS and the
azomethine derivative 4-Hydroxybenzaldiminamoxicillin (D1S6) is Pale Orange in colour
with an observed conductance of 9.5µS.

Nuclear Magnetic Resonance (NMR) Studies: The NMR spectra recorded in DMSO-D6 as
solvent and were found much complicated to interpret completely but the shift of NH proton
attached to the β-Lactam was found to be more and more deshielded and shifts to higher δ–
values in all the azomethine derivatives than parent drug. The –NH2 protons were found
absent in the azomethine derivatives and the deshielded -N=CH protons in the derivative are
clearly observed in higher δ–values. All the spectra were showing characteristic peaks of
COOH protons around 9-10ppm and vanished in presence of few drops of D2O.

4. RESULTS AND DISCUSSION

The results can be classified into three (i) Drug Designing and Docking, (ii) The In silico
QSAR Studies, and (iii) Synthesis and In-vitro Studies.

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Drug Designing and Docking: The azomethine derivatives were designed and modeled
according to Lipinski rules. The results of the docking studies and parameters according
Lipinski rules are tabulated and given in the table (Table 2).

Table 2: The results obtained for Lipinski rules and docking studies of drug and its
derivatives.

Best Pose energy

Drug/
H-Bond H-Bond (Kcal mol-1)
Azomethine LogP
acceptors donors E.coli S.aureus
Derivative
(1LL9.pdb) (1BLH.pdb)
Amoxicillin 0.61+/
7 5 -8.40 -8.35
(D1) MW:365 -0.33
2.66+/
D1S1 MW:469 7 4 -8.65 -7.19
-0.43
2.79+/
D1S2 MW:498 8 3 -9.34 -7.72
-0.42
2.60+/
D1S3 MW:498 8 3 -9.26 -6.03
-0.42
2.86+/
D1S4 MW:496 6 3 -8.85 -8.68
-0.42
2.32+/
D1S5 MW:499 8 4 -8.83 -8.55
-0.44
2.45+/
D1S6 MW:469 7 4 -8.74 -7.37
-0.41

The docking studies were performed over the corresponding β-Lactamases viz. 1LL9.pdb and
1BLH.pdb that produced by E.coli and S.aureus respectively.

Amoxicillin series on E.coli: After analyzing the pose energies obtained from the docking
studies the activities were predicted. The pose energies showed that the derivative D1S2 is
the one that binds readily with the β-Lactamase 1LL9.pdb and the least binding one is D1 the
drug. Hence the most potent one is the drug amoxicillin trihydrate itself in the case of β-
Lactamase producing E.coli.
Amoxicillin series on S.aureus: The pose energies showed that the derivative D1S4 is the
one which easily binds with the β-Lactamase 1BLH.pdb and the least binding one is D1S3.
Hence the most potent one is D1S3, the azomethine drug derivative with 3-nitrobenzaldehyde
in the case of β-Lactamase producing S.aureus.

The In silico QSAR Studies: The possible toxicity end points were computed using several
different advanced Quantitative Structure Activity Relationship (QSAR) methodologies like,

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Hierarchical method (HM), FDA method, (FDA-M), Single model method (SMM), Group
contribution method (GCM), Nearest neighbour method (NNM), Consensus method (CM)
and Random method (RM) according the feasibility of the QSAR-method. The results for the
parent drug and azomethine derivatives are tabulated and discussed (Table 3).

Table 3: The computed QSAR endpoints for drug and its azomethine derivatives.

QSAR-Toxicity End points

DM FHM TP Oral rat Mutagenicity
Chemica BCF
LC50 LC50 IGC50 LD50 DTox value &
l Log
(mg/L) (mg/L) (mg/L) (mg/Kg) Result
/Drug
NonTo
D1 0.75 14.65 34.11 2638.56 0.30 0.12(-ve)
x
1.34 NonTo
D1S1 4.75 10.04 2803.12 3.65 0.23(-ve)
E-02 x
1.42 NonTo
D1S2 0.87 5.95 2911.22 4.10 0.28(-ve)
E-02 x
1.42 NonTo
D1S3 0.78 10.66 1995.90 4.10 0.31(-ve)
E-02 x
1.42 NonTo
D1S4 0.42 10.62 4035.31 4.10 0.18(-ve)
E-02 x
1.42 NonTo
D1S5 1.13 10.68 6278.85 4.10 0.20(-ve)
E-02 x
1.34 NonTo
D1S6 2.13 10.04 3709.82 3.65 0.29(-ve)
E-02 x

The FHM LC50 and TP IGC50 were calculated only using NNM and hence the results may not
be much reliable. DM LC50 was calculated using HM, FDA-M, SMM and NNM and hence is
much reliable. The aquatic toxicity of the drug derivatives are found more than the parent
drugs. The LD50 values were calculated using FDA-M and NNM and may be reliable. BCF
values calculated using NNM only. D.Tox values were calculated using HM, FDA-A, SMM
and NNM and are much reliable. Mutagenicities were calculated using HM, FDA-M and
NNM. All the drug derivatives were found developmental non toxic in nature. The
mutagenicity values were found much comparable to the parent drugs. The calculated Oral rat
LD50 (mg/Kg) values were found comparable and hence are promising as drugs. Systematic
laboratory studies have to be performed and are beyond the scope of this work. The
azomethine drug derivatives were found strictly obeying the Lipinski rules and the
corresponding QSAR endpoints calculated were found in support to the druggability of the
compounds.

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Synthesis and In vitro Studies: The syntheses and characterization of the azomethine
derivatives were performed. The observed vibrational frequencies and observations from
NMR studies were in accordance to the suggested derivative formation. A systematic
antibacterial studies were performed and zone of inhibitions were measured. The wet lab
studies showed the comparable result and found matching with the theoretically predicted
ones. In the case of E.coli the most potent one is the drug D1 and least active ones are the
D1S2 and D1S3. The potency of D1S1 is the best and more promising among the derivatives
as per the soft lab results. The activities of D1S5 and D1S4 were almost equal. The D1S3 and
D1S2 showed very less activity and were almost comparable. In the case of S.aureus also the
wet lab studies showed the result that is comparable and matching with the theoretically
predicted results. The wet lab studies also supporting the predicted results by showing most
potent as D1S3 and least active D1S4. The potencies of the three derivatives D1S3, D1S1 and
D1S6 are more than drug D1 and that of D1S2 is almost equal to the drug D1. The activities
of D1S5 and D1S4 are almost equal and much less than the parent drug D1.

5. CONCLUSION
The designed drugs by the derivatizing at the possible sites could considerably reduce the
time and expenses needed for drug discovery. Some of the azomethine derivatives of
amoxicillin were found promising from the studies. All the azomethine derivatives allow the
Lipinski rules and are promising as drugs and much QSAR studies have to be conducted for
the fully druggability of the derivatives which is beyond the scope of this research. It was
observed that the in silico QSAR studies can considerably reduce the use of animals and
organisms to an extent and even replace them with these green methods that are supportive
and complementary when applied intelligently. These studies were based on the novel
concept called Distribution of Active Electrons which was developed by closely observing
and designing many organic reactions. With that developed concept not only the designing of
the molecules but also the prediction of suitable reaction conditions to synthesize them are
also found possible.

REFERENCES
1. Wise R. The world wide threat of antimicrobial resistance. Current Science, 2008; 95(2):
181-87.
2. Amoxicillin trihydrate Official monographs USP

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3. Lipinski CA, Lombardo F, Dominy BW, Freeney PJ. Experimental and computational
approaches to estimate solubility and permeability in drug discovery and development
settings. Adv.Drug Deliv. Rev., 1997; 23: 3-25.
4. Imran M, Iqbal J, Latif S. Synthesis, characterization and in vitro screening of
Amoxicillin and its complexes with Ag(I), Cu(II),Zn(II) and Ni(II). J.Biol.Sci., 2006;
6(5): 946-949.
5. ArgusLab4.0.1, Mark Thompson of Planaria software LLC. www.arguslab.com
6. Environment Protection Agency, United States. www.epa.gov
7. Andersen K, Liljefors T, Gundertofte K, Perregaard J, Bogeso KP. Development of a
receptor-interaction model for serotonin 5-HT2 receptor antagonists. Prediction of
selectivity with respect to dopamine D2 receptors. J.Med.Chem., 1994; 37: 950-962.
8. Dewar MJS, Zoebisch EG, Healy EF, Stewart JJP. AM1: A general purpose quantum
mechanical molecular model. J.Am.Chem.Soc., 1985; 107: 3902-3909.
9. Michael JS, Dewar, Thiel W. The MNDO Method-Approximations and Parameters.
J.Am.Chem.Soc., 1977; 99: 4899-4907.
10. James JP, Stewart. Optimization of parameters for semiempirical methods.
J.Computational Chemistry, 1989; 10: 221-264.
11. Clark T. A handbook of computational chemistry. New York; John Wiley and Sons:
1985.
12. Larsen PK. Text book of drug design and discovery. New York; Taylor & Francis Inc.:
2002.
13. Baird B. Staphylococcus: Cluster-forming Gram-positive cocci. In: Collee JG, Marmion
BP, Fraser AG and Simmons A (Eds.). Mackie & McCartney Practical Medical
Microbiology 14th ed., New Delhi; Churchill Livingstone, Elsevier: 2006, pp.245-261.
14. Crichton PB. Enterobacteriaceae: Escherichia, Klebsiella, Proteus and other genera. In:
Collee JG, Marmion BP, Fraser AG and Simmons A (Eds.). Mackie & McCartney
Practical Medical Microbiology 14th ed., New Delhi; Churchill Livingstone, Elsevier:
2006, pp.361-384.
15. Cappuccino JG, Sherman N. Microbiology- A Laboratory manual. New Delhi: Dorling
Kindersley (India) Pvt. Ltd., Pearson Education: 2006.

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