CHEMICAL SCREENING AND ANTIBIOTIC

SUSCEPTIBILITY TEST

A PROJECT REPORT

Submitted by
Riti Kumari (22BBE10211)
Vaibhav Gandhi (22BBE10196)

Avantika Sharma (22BBE10209)

S.M. Osaid Rizvi (22BBE10228)
Jackline Steven Mwayeka (22BBE10244)

in partial fulfillment for the award of the degree of

Bachelors of Engineering
IN
BIOTECHNOLOGY

Chandigarh University
February - June 2023
 BONAFIDE CERTIFICATE

Certified that this project report “ CHEMICAL SCREENING AND

ANTIBIOTIC SUSCEPTIBILITY TEST ” is the bonafide work of “Riti
Kumari , Vaibhav Gandh , Avantika Sharma , Jackline Steven Mwayeka ,

S.M. Osaid Rizvi ” who carried out the project work under my/our

supervision.

Signature of the HoD : Signature of the Supervisor :

…………………….. ………………………….

Dr.Gyanendra Singh Goindi Dr.Swatantar Kumar

( HoD ) ( SUPERVISOR )

HEAD OF THE DEPARTMENT ACADEMIC COORDINATOR

( Bachelors Of Engineering ( Bachelors Of Engineering

In Biotechnology ) In Biotechnology )

Submitted for the project viva-voce examination held on :

INTERNAL EXAMINER EXTERNAL EXAMINER

 TABLE OF CONTENTS

List of Figures ..................................................................................................................... 05

CHAPTER 1. INTRODUCTION ......................................................................... 06

1.1. Identification of Client/ Need/ Relevant Contemporary issue ................................... 06

1.2. Identification of Problem ......................................................................................... 07

1.3. Identification of Tasks............................................................................................... 08

1.4. Timeline .................................................................................................................. 09

1.5. Organization of the Report ........................................................................................ 09

CHAPTER 2. LITERATURE REVIEW/BACKGROUND STUDY ............. 10

2.1. Timeline of the reported problem .............................................................................. 10

2.2. Existing solutions ..................................................................................................... 11

2.3. Bibliometric analysis .................................................................................................. 12

2.4. Review Summary ..................................................................................................... 13

2.5. Problem Definition..................................................................................................... 14

2.6. Goals/Objectives ....................................................................................................... 15

CHAPTER 3. DESIGN FLOW/PROCESS ........................................................ 16

3.1. Evaluation & Selection of Specifications/Features..................................................... 16

3.2. Design Constraints .................................................................................................... 17

3.3. Design Flow ............................................................................................................. 18

3.4. Design selection ....................................................................................................... 19

3.5. Implementation plan/methodology ............................................................................ 20

CHAPTER 4. RESULTS ANALYSIS AND VALIDATION .......................... 22
4.1. Implementation of solution ...................................................................................... 22

CHAPTER 5. CONCLUSION AND FUTURE WORK .................................. 25

5.1. Conclusion ................................................................................................................. 25

5.2. Future work ............................................................................................................... 25

REFERENCES .......................................................................................................... 26
 List of Figures

Figure 1.4 : Gantt Chart of Project Timeline

Figure 3.5.1 : MIC by agar di lution
Figure 3.5.2 : Antibiotic is diluted at various dilutions to test MIC
Figure 3.5.3 : Resistom/Resistance Profile
Figure 4.1 : Tools used
 CHAPTER 1.
INTRODUCTION

1.1. Identification of Relevant Contemporary Issue

MIC Assay (Minimum Inhibitory Concentration Assay) is a laboratory technique used to determine
the lowest concentration of an antimicrobial agent that inhibits the growth of a particular
microorganism. This assay is commonly used in microbiology and clinical settings to evaluate the
effectiveness of antibiotics or other antimicrobial agents.

Chemical screening is a process of identifying potential drug compounds or small molecules that
may have therapeutic effects on a particular disease or condition. This involves testing large
libraries of compounds using various assays and screening techniques to identify those that show
promise in treating the disease or condition of interest.

One of the relevant issues in chemical screening and antibiotic susceptibility tests is the increasing
incidence of multi-drug-resistant pathogens. The overuse and misuse of antibiotics in humans and
animals have led to the emergence of antimicrobial resistance.

This makes it more challenging to develop new drugs that are effective against these resistant
strains, and the need for effective screening methods becomes more critical.

Another issue is the lack of diversity in the screening libraries. Many screening libraries are biased
towards compounds that are easy to synthesize, and this may lead to the under-representation of
natural products or complex molecules that may have therapeutic potential. This makes it important
to diversify screening libraries to include more complex and diverse molecules.

Moreover, the accuracy of the assays used in chemical screening and antibiotic susceptibility tests
can also be a relevant issue. The assays should mimic the in vivo conditions as closely as possible
to improve the predictability of the results. Also, there is a need to validate the assays to ensure
they are reliable, consistent, and reproducible.

The cost and time required for chemical screening and antibiotic susceptibility tests can also be a
challenge. High-throughput screening assays are expensive and can requspecializedised equipment,
making it difficult for smaller research institutions to conduct these assays. Additionally, time can
be a limiting factor in identifying effective drugs as it takes several years to develop, test and approve
a new drug.

Finally, the need for the standardization of methods in chemical screening and antibiotic susceptibility
tests is an essential issue. Variability in assay conditions can impact assay results and make it
challenging to compare results between studies. Therefore, the development of standardised protocols
can improve the reproducibility and reliability of result
1.2. Identification of Problem

The problem statement for Chemical Screening and antibiotic susceptibility test may vary depending
on the specific research question or goal of the study. However, some common problem statements
for these techniques include:

• To identify new and effective antimicrobial agents for the treatment of infectious diseases
by screening a large library of compounds and determining their MIC values against target
microorganisms.

• To investigate the mechanism of action of known antimicrobial agents and determine their
efficacy against drug-resistant strains of microorganisms.

• To identify new drug compounds or small molecules that may have therapeutic effects on a
particular disease or condition by screening a large library of compounds and testing their
activity in various assays.

• To optimize the efficacy of known drugs or small molecules by modifying their chemical
structure and testing their activity in various assays, including MIC assays.

• To understand the molecular mechanisms underlying the activity of drugs or small

molecules identified through chemical screening, and to develop new drugs based on this
knowledge.

This project is going to investigate bacterial resistance towards the antibiotics and how to
resolve the condition by performing laboratory experiments and investigations in antibiotic
resistance in reference to Nutmeg seeds. Finally, we highlight the major challenges and potential
future efforts to improve clinical outcomes through rapid bacterial identification and development
of new antibiotics that would be non-resistive to antibiotics.
The emergence and spread of bacterial resistances to antibiotics is now leading to the antibiotic
crisis. This is driven by the antibiotic misuse and disabuse, rendered possible due to the absence of
fast and accurate technologies for antibiotic screening and resistant bacteria identification in
infectious diseases. Technologies that provide sensitive, quick and easy read-outs, conveying
information about the optimal treatment are required. A multitude of these tests is readily available
on the market, with many more being developed. However, current methods have their
shortcomings. An in-depth study overview and analysis of the field, with a deep focus on the
current market, will serve as a guideline for the future emerging technologies involving
susceptibility testing and antibiotic resistance evaluation. From such a dynamic and growing field,
the current review provides a critical discussion on how the susceptibility-testing field is
developing and where it is going.
1.3. Identification of Tasks

• Samples Collection: Collection of samples which are needed to perform the Lab
Experiments.

• Conduct Lab Experiment: Design and conduct lab experiments autonomously and

collaboratively by using the collected samples of the antibiotic and the naturally occurring
substance containing the antibiotic resistant properties (here Nutmeg)

• Interpretation: Document, interpret and present experiment results

• Literature Review: Incorporate the latest findings from the wider scientific community
into research methods and interpretation of findings

• Implement: Rely on state-of-the-art technology when conducting experiments

• Investigate: Conduct investigative procedures in relevant fields of expertise, including

pharmacology, environmental biology, or molecular biology.
 1.4. Timeline

Figure 1.4 (Timeline)

1.5. Organization of the Report

Chapter 1 Problem Identification: This chapter introduces the project and describes the
problem statement discussed earlier in the report.

Chapter 2 Literature Review: This chapter prevents review for various research papers
which help us to understand the problem in a better way. It also defines what has been done
to already solve the problem and what can be further done.

Chapter 3 Design Flow/ Process: This chapter presents the need and significance of the
proposed work based on literature review. Proposed objectives and methodology are
explained. This presents the relevance of the problem. It also represents logical and
schematic plan to resolve the research problem.
Chapter 4 Result Analysis and Validation: This chapter explains various
performance parameters used in implementation. Experimental results are shown in this
chapter. It explains the meaning of the results and why they matter.

Chapter 5 Conclusion and future scope: This chapter concludes the results and explain
the best method to perform this research to get the best results and define the future scope
of study that explains the extent to which the research area will be explored in the work.
 CHAPTER 2.
LITERATURE REVIEW

2 . 1 . Timeline of the reported problem

The problem of antibiotic resistance and the need for effective chemical screening and
antibiotic susceptibility tests has been recognized by the scientific community for several
decades. Here are a few examples of key events and documents that have contributed to our
understanding of this issue:

The discovery of penicillin in 1928 by Alexander Fleming was a major milestone in the history
of antibiotics. However, within a few years, reports of resistance to penicillin began to emerge,
highlighting the need for ongoing research into new antibiotics and methods for testing their
effectiveness.
In the 1960s and 1970s, the widespread use of antibiotics in agriculture and medicine led to the
emergence of antibiotic-resistant bacteria, including methicillin-resistant Staphylococcus
aureus (MRSA) and vancomycin-resistant enterococci (VRE). These outbreaks prompted
renewed efforts to develop new antibiotics and better testing methods.

In 1996, the World Health Organization (WHO) published a report on the global problem of
antibiotic resistance, highlighting the urgent need for action to address the issue. The report
called for increased surveillance and monitoring of antibiotic resistance, as well as improved
prescribing practices and infection control measures.

In 2000, the Centers for Disease Control and Prevention (CDC) in the United States launched
the National Antimicrobial Resistance Monitoring System (NARMS), which collects data on
antibiotic resistance in human, animal, and foodborne pathogens. This program has been
instrumental in identifying emerging resistance patterns and guiding public health policy.

In recent years, there have been numerous reports of antibiotic-resistant infections, including
the spread of carbapenem-resistant Enterobacteriaceae (CRE) and the emergence of Candida
auris, a multidrug-resistant fungus. These incidents highlight the ongoing need for effective
chemical screening and antibiotic susceptibility tests to guide treatment decisions and prevent
the spread of resistant infections.
Overall, the problem of antibiotic resistance and the need for effective screening and testing
methods has been recognized for many years, and ongoing efforts are needed to address this
global public health threat.

2.2. Existing solutions

Over the years, there have been several proposed solutions to improve chemical screening and
antibiotic susceptibility testing. Here are some examples:

Development of new antibiotics: Scientists have been working to develop new antibiotics to
treat resistant infections. This includes exploring new sources of antibiotics, such as soil
microbes and natural products, as well as developing synthetic compounds that can target
resistant bacteria.

Improved testing methods: There have been efforts to develop faster and more accurate testing
methods for antibiotic susceptibility. This includes the use of automated systems and
microfluidics, which can provide results in a matter of hours rather than days.

Antibiotic stewardship: Antibiotic stewardship programs aim to improve the use of antibiotics
in healthcare settings, by encouraging appropriate prescribing practices and reducing
unnecessary use of antibiotics. This can help to prevent the emergence of resistant bacteria.

Surveillance and monitoring: Improved surveillance and monitoring of antibiotic resistance

can help to identify emerging resistance patterns and guide public health policies. This includes
programs such as the National Antimicrobial Resistance Monitoring System in the United
States.

Alternative therapies: There is growing interest in alternative therapies for bacterial infections,
such as phage therapy and immunotherapy. These approaches use viruses or the immune
system to target bacteria, and may offer new options for treating resistant infections.

Overall, there is a need for a multifaceted approach to address the problem of antibiotic
resistance, including the development of new antibiotics, improved testing methods, and better
antibiotic stewardship practices
2.3.Bibliometric analysis

Chemical screening and antibiotic susceptibility testing are crucial tools in the diagnosis and
treatment of bacterial infections. Here is an analysis of their key features, effectiveness, and
drawbacks:

Key features:
Chemical screening tests involve exposing bacteria to various chemical compounds to
determine which ones are effective at inhibiting or killing the bacteria.

Antibiotic susceptibility tests involve exposing bacteria to different antibiotics to determine

which ones are effective at treating the infection.

Both types of tests rely on growing bacterial cultures in the laboratory, and results can take
several days to obtain.

Effectiveness:

Chemical screening and antibiotic susceptibility tests are generally effective at identifying
which compounds or antibiotics are effective against a particular strain of bacteria.

This information can help guide treatment decisions, ensuring that patients receive the most
appropriate therapy for their infection.
However, it is important to note that these tests are not foolproof, and resistance can still
develop over time.

Drawbacks:
One major drawback of these tests is that they require a bacterial culture, which can take several
days to obtain. This delay can lead to delayed treatment and potentially worse outcomes for
patients.
Additionally, these tests may not be effective at identifying all types of resistance mechanisms,
particularly those that are less well-understood or less common.

Another challenge is that some bacteria can be difficult to culture in the laboratory, which can
limit the effectiveness of these tests.

Finally, chemical screening and antibiotic susceptibility tests can be costly and time-
consuming, which can limit their accessibility in resource-limited settings.

Overall, chemical screening and antibiotic susceptibility testing are important tools in the fight
against antibiotic resistance, but there are some limitations to their effectiveness and
accessibility that need to be addressed. Researchers are working to develop faster, more
accurate testing methods that can provide more immediate results and better guide treatment
decisions.

2.4. Review Summary

A literature review of chemical screening and antibiotic susceptibility testing reveals that these
tests are essential tools for diagnosing and treating bacterial infections. However, there are
some limitations to their effectiveness and accessibility that need to be addressed. Here is a
summary of the key findings:

Chemical screening and antibiotic susceptibility testing are generally effective at identifying
which compounds or antibiotics are effective against a particular strain of bacteria, but results
can take several days to obtain.

Resistance can still develop over time, and some bacteria may be difficult to culture in the
laboratory.
Antibiotic stewardship programs can help to improve the use of antibiotics and prevent the
emergence of resistant bacteria.

Improved testing methods, such as automated systems and microfluidics, can provide faster
and more accurate results.

Alternative therapies, such as phage therapy and immunotherapy, may offer new options for
treating resistant infections.

In the context of a project, these findings suggest that chemical screening and antibiotic
susceptibility testing should be considered as part of a comprehensive approach to tackling
antibiotic resistance. Antibiotic stewardship programs and alternative therapies may also be
important components of such an approach. It is important to recognize the limitations of these
tests and to work towards developing faster and more accurate testing methods that can provide
more immediate results and better guide treatment decisions. Ultimately, a multifaceted
approach that addresses the underlying causes of antibiotic resistance and promotes the
responsible use of antibiotics is needed to effectively combat this global public health threat.

2.5. Problem Definition

The problem at hand is the increasing prevalence of antibiotic-resistant bacteria, which pose a
significant threat to public health. Chemical screening and antibiotic susceptibility testing are
important tools for diagnosing and treating bacterial infections, but there are limitations to their
effectiveness and accessibility. The challenge is to develop a comprehensive approach that
addresses the underlying causes of antibiotic resistance and promotes the responsible use of
antibiotics.

To address this problem, it is important to:

Develop new antibiotics: Scientists should continue to explore new sources of antibiotics and
develop synthetic compounds that can target resistant bacteria.

Improve testing methods: Efforts should be made to develop faster and more accurate testing
methods for antibiotic susceptibility, such as the use of automated systems and microfluidics.
Implement antibiotic stewardship programs: Healthcare providers should be encouraged to adopt
appropriate prescribing practices and reduce unnecessary use of antibiotics.

Monitor antibiotic resistance: Programs such as the National Antimicrobial Resistance Monitoring
System can help identify emerging resistance patterns and guide public health policies.

Explore alternative therapies: Alternative therapies such as phage therapy and immunotherapy
can be explored as potential options for treating resistant infections.

What not to be done:

Overuse of antibiotics: Antibiotics should not be used unnecessarily or inappropriately, as this can
lead to the development of antibiotic resistance.

Rely solely on chemical screening and antibiotic susceptibility testing: These tests are not
foolproof, and there may be other factors contributing to resistance that are not identified by these
tests.

In summary, the problem at hand is the emergence of antibiotic-resistant bacteria and the
challenge is to develop a multifaceted approach to address this issue. This includes the
development of new antibiotics, improved testing methods, implementation of antibiotic
stewardship programs, monitoring of resistance patterns, and exploration of alternative
therapies. It is important to avoid overuse of antibiotics and to recognize the limitations of
chemical screening and antibiotic susceptibility testing.

2.6. Goals/Objectives

 To determine the effectiveness of a chemical compound against a specific pathogen:

The objective is to identify the chemical compound that can inhibit the growth of a
pathogen in a specific environment. The goal is to find the most effective compound
that can be used as a treatment.
  To identify the minimum inhibitory concentration (MIC) of a chemical compound: The
objective is to determine the lowest concentration of a chemical compound that can
effectively inhibit the growth of a pathogen. The goal is to identify the most potent
concentration of the chemical that can be used as a treatment.

 To identify the mechanism of action of a chemical compound: The objective is to

understand how a chemical compound works against a pathogen. The goal is to identify
the mode of action of the chemical, which can be useful in developing new drugs.

 To identify the resistance of a pathogen to a chemical compound: The objective is to

determine if a pathogen is resistant to a particular chemical compound. The goal is to
identify the resistance mechanism, which can be useful in developing new drugs that
can overcome the resistance.

 To compare the efficacy of different chemical compounds against a pathogen: The

objective is to determine which chemical compound is most effective against a
pathogen in a specific environment. The goal is to identify the best treatment option for
a specific infection

Overall, the goals and objectives of chemical screening and susceptibility tests are to identify
effective treatments for infections, understand how treatments work, and develop new
treatments that can overcome resistance.
 CHAPTER 3.
DESIGN FLOW/PROCESS

3 . 1 . Evaluation & Selection of Specifications/ Features

Indications
Susceptibility testing for antimicrobials is necessary for patients who raise suspicion of
infection with specific pathogens based on disease manifestation and clinical
correlation. Antibacterial agents are then used to detect sensitivity or resistance from
bacteria. Although the purpose of this review is primarily towards the susceptibility testing for
bacterial pathogens, it is important to note that antifungal susceptibility tests also exist for
addressing fungal infection (e.g., Candida, Aspergillus spp.). Furthermore, antiviral
susceptibility tests are also available (e.g., influenza) via molecular technologies including
sequencing analysis such as Sanger and pyrosequencing methods.

Potential Diagnosis
A unique impact of AST on patient management is the identification of the specific diagnosis,
and additionally, targeting the particular etiologic agent causing the disease. No two patients
can be managed similarly, especially if they have the same signs and symptoms (disease
manifestation) but with different treatment regimens because the same causative organism can
have different resistance patterns. For example, two patients may present with an ordinary
strain of Staphylococcus aureus vs. methicillin-resistant Staphylococcus aureus (MRSA); and
another example would be patients with drug-susceptible (DS-TB) and drug-resistant
tuberculosis (DR-TB).

Normal and Critical Findings

For disk diffusion, measuring the zone of inhibition is done by using a dedicated
caliper. Correctly measure the diameter by the edges of the inhibition zone. For MIC panels,
reading each set of wells for an antibiotic drug is done. MIC determination is by either a clear
or slight whiteness on the well. Reporting the results of the inhibition zones and MIC
breakpoints is made using either the terms “susceptible” or “resistant” based on the set cut-off
range for zone diameter in the nearest whole millimeter and microgram per milliliter,
respectively. The Clinical Laboratory Standards Institute (CLSI) and the European Committee
on Antimicrobial Susceptibility Testing (EUCAST) developed expert-approved guidelines on
breakpoints for reporting results of these methods (e.g., CLSI M100-ED29:2019 Performance
Standards for Antimicrobial Susceptibility Testing, EUCAST Clinical breakpoints for
bacteria).

3.2.Design Constraints

When performing chemical screening and antibiotic susceptibility tests, several regulations,
economic, environmental, health, manufacturability, safety, professional, ethical, social,
political issues, and cost must be considered in the design. Here are some examples:

Regulations: The use of chemicals and antibiotics is regulated by various governmental

agencies. Regulations must be followed when testing chemicals and antibiotics, including
obtaining appropriate permits and licenses, and adhering to safety protocols and disposal
regulations.

Economic: The cost of chemical screening and antibiotic susceptibility tests can be significant,
and cost-effectiveness must be considered. The cost of manufacturing, testing, and distributing
the antibiotics must be taken into account, as well as the potential revenue from the sale of the
antibiotics.

Environmental: The disposal of chemicals and antibiotics can have a significant impact on
the environment. Proper disposal methods must be used to minimize the impact on the
environment and reduce the risk of contamination.

Health: The health of those performing the tests, as well as those who may come into contact
with the chemicals and antibiotics, must be protected. Safety protocols must be followed, and
protective equipment must be used.

Manufacturability: The ease of manufacturing the antibiotics must be considered when

designing the tests. The production process must be scalable, efficient, and cost-effective.
Safety: The safety of the chemicals and antibiotics must be considered. The toxicity and
potential side effects of the antibiotics must be evaluated, and appropriate warning labels must
be provided.

Professional: Professional standards must be maintained during the testing process. The tests
must be conducted according to accepted scientific methods, and results must be accurately
recorded and reported.

Ethical: The use of animals for testing purposes raises ethical concerns. Animal testing must
be done in accordance with ethical standards and with consideration for the welfare of the
animals.

Social & Political Issues: The use of antibiotics has social and political implications, including
the potential for overuse, the development of antibiotic resistance, and access to antibiotics in
developing countries.

Cost: The cost of developing and producing new antibiotics can be significant. Cost
considerations must be balanced against the potential benefits of the antibiotics in terms of
improved health outcomes and reduced healthcare costs.

3.3.Design Flow

 Specimen requirements for routine susceptibility testing using the disk diffusion
method and minimum inhibitory concentration (MIC) method are similar to the
guidelines for collecting samples for bacterial culture since a certain number of well-
isolated colonies (usually 3 to 5) grown from a culture is necessary to prepare a
suspension of inoculum. The usual specimens sent for culture and sensitivity tests are
blood, urine, cerebrospinal fluid, sputum, wound, stool, and other body fluids and
discharge.

 Special susceptibility tests via commercial systems may not always require bacterial
colonies from culture because they can detect resistance to certain antimicrobial drugs
by employing molecular techniques for detecting resistant genes. An example would
 be the Xpert MTB/Rif assay which determines sensitivity or resistance to rifampicin
directly from sputum specimens.

3.4.Design selection
Both disk diffusion and MIC methods employ the phenotypic identification of susceptibility,
and therefore, requires the following process:

Preparation of a standardized inoculum from a bacterial culture:

 Choosing well-isolated colonies

 Creating a bacterial suspension (inoculum)

 Standardizing the bacterial suspension using McFarland standards

Dilution of bacterial suspension (only for MIC method)

Inoculation of bacterial suspension to one of the following:

o A particular growth medium (e.g., Mueller Hinton Agar, MHA for disk
diffusion)

o A MIC panel

Addition of antimicrobial disks (only for disk diffusion)

Incubation of plates (disk diffusion) or panels (MIC)

Measuring the zone of inhibition or reading MIC panel

Interpretation of AST results

Disc diffusion tests involve the placement of paper discs containing a specific concentration of
an antibiotic on an agar plate inoculated with a bacterial culture. The plate is then incubated,
and the presence or absence of a zone of inhibition around the disc is measured to determine
the susceptibility of the bacteria to the antibiotic.

On the other hand, MIC tests involve diluting the antibiotic in a broth medium containing a
bacterial culture and determining the lowest concentration of the antibiotic that inhibits
bacterial growth.
The choice of test method often depends on the specific needs of the laboratory or clinical
setting, including the availability of resources, turnaround time, and the specific bacteria being
tested.

Here are some comparisons between the two methods:

 Accuracy: MIC tests are generally considered more accurate than disc diffusion tests
as they provide a quantitative measurement of antibiotic efficacy rather than a
qualitative assessment.
 Sensitivity: MIC tests are more sensitive than disc diffusion tests, as they can detect
smaller changes in bacterial susceptibility to antibiotics.
 Precision: Disc diffusion tests are generally considered more precise than MIC tests,
as they produce clear-cut zones of inhibition that are easy to interpret.
 Turnaround time: Disc diffusion tests are faster than MIC tests, as they can provide
results within 24 hours, while MIC tests may take 48 hours or more.

 Cost: Disc diffusion tests are generally less expensive than MIC tests, as they require
fewer reagents and materials.

3 . 5 . Implementation plan/ methodology

Here is a general implementation plan and methodology for performing chemical screening
and antibiotic susceptibility testing using the MIC (Minimum Inhibitory Concentration) test:

1. Sample Collection: The first step in implementing the test is to collect samples that
will be used for testing. These can include bacterial cultures from a patient's blood,
urine, sputum, or other bodily fluids.

2. Culture Preparation: The collected samples are then cultured on a suitable agar or
broth medium to grow the bacteria under optimal conditions. The growth of the
bacterial culture should be monitored to ensure it is healthy and pure.
3. Inoculation of Test Medium: The bacterial culture is then diluted and inoculated into
a test medium containing the antibiotic being tested. The test medium must be carefully
prepared to ensure consistency and accuracy of results.

4. Incubation: The inoculated test medium is then incubated for a specific period, usually
24-48 hours, at the appropriate temperature and under optimal growth conditions to
allow for bacterial growth and antibiotic interaction.

5. Observation and Recording of Results: After the incubation period, the test results
are observed and recorded. The MIC is determined as the lowest concentration of
antibiotic that inhibits bacterial growth. This is typically done visually, either through
the use of turbidity measurements or by observing the presence or absence of bacterial
growth.

6. Interpretation of Results: The results are then interpreted to determine the

susceptibility of the bacteria to the antibiotic. The susceptibility is determined based on
established guidelines and breakpoints that define the MIC values for different bacterial
species.

7. Reporting of Results: The results of the test are then reported to the appropriate parties,
such as physicians or public health officials, for further evaluation and decision-making
regarding treatment options.

Figure 3.5.1 (MIC by Agar Dilution)

 Figure 3.5.2 (Inhibitory Concentration)

Figure 3.5.3. The basic workflow of molecular-based techniques for antibiotic/antimicrobial susceptibility
testing. The routes from a clinical specimen to a final result are indicated by arrows (created with BioRender.com,
accessed on 27 February 2022. Reproduction of this figure requires permission from BioRender.com).
 CHAPTER 4.
RESULTS ANALYSIS AND VALIDATION

4 . 1. Implementation of solution

The results of a susceptibility test must be interpreted by the laboratory prior to communicating
a report to a patient's physician. Optimal interpretation of MICs requires knowledge of the
pharmacokinetics of the drug in humans, and information on the likely success of a particular
drug in eradicating bacteria at various body sites [. This is best accomplished by referring to an
expert source such as the CLSI, which publishes interpretive criteria for MICs of all relevant
antibiotics for most bacterial genera . Indeed, both MIC values and disk diffusion zone
diameters must be interpreted using a table of values that relate to proven clinical efficacy of
each antibiotic and for various bacterial species . The CLSI zone size and MIC interpretive
criteria are established by analysis of 3 kinds of data: (1.) microbiologic data, including a
comparison of MICs and zone sizes on a large number of bacterial strains, including those with
known mechanisms of resistance that have been defined either phenotypically or genotypically;
(2) pharmacokinetic and pharmacodynamic data; and (3) clinical studies results (including
comparisons of MIC and zone diameter with microbiological eradication and clinical efficacy)
obtained during studies prior to FDA approval and marketing of an antibiotic.

Figure 4.1 (Tools to be used)

Chemical screening and antibiotic susceptibility tests are important tools in microbiology and
clinical practice. Here is a brief overview of the testing, characterization, interpretation, and
data validation process:

 Testing:
Chemical screening tests are used to detect the presence or absence of certain chemical
compounds or elements in a sample. Antibiotic susceptibility tests are used to determine
the susceptibility of bacteria to specific antibiotics.

 Characterization:
Chemical screening tests can provide information about the identity of a chemical
compound or element present in a sample. Antibiotic susceptibility tests can provide
information about the effectiveness of different antibiotics against a particular bacterial
strain.

 Interpretation:
The interpretation of chemical screening tests will depend on the type of test and the
specific chemical being detected. Results can be compared to known standards or
reference materials to determine the identity and concentration of the chemical in
question. The interpretation of antibiotic susceptibility tests involves comparing the
growth of bacterial cultures in the presence of different antibiotics to determine the
minimum inhibitory concentration (MIC) or zone of inhibition (ZOI). The results can
be used to determine which antibiotics are most effective against the bacterial strain
being tested.

 Data Validation:
Data validation is the process of ensuring that the results of the tests are accurate and
reliable. This may involve running control samples to ensure that the testing process is
working correctly, verifying that the results are consistent with previous data, and
performing statistical analysis to confirm the significance of the results.
 Analysis: For chemical screening and antibiotic susceptibility testing, tools such as
spectrometers, chromatography systems, and microscopes can be used for sample
analysis. These tools allow for the identification and quantification of chemical
compounds and microorganisms present in the samples.

 Design Drawings/Schematics/Solid Models: Computer-aided design (CAD) software

can be used to create detailed drawings, schematics, and solid models of the equipment,
apparatus, or structures used during the tests. This helps to visualize and plan the testing
process, identify potential issues, and optimize the design for better performance.

 Report Preparation: Modern tools such as word processing software, data

visualization tools, and statistical analysis software can be used to create professional-
looking reports. These tools can be used to present data in a clear and concise manner,
making it easier to communicate the findings of the tests.

 Project Management: Project management tools such as Trello, Asana, and Microsoft
Project can be used to manage the workflow, assign tasks, and track progress during
the testing process. This helps to ensure that the project is completed on time and within
budget.

 Communication: Communication tools such as video conferencing software, instant

messaging apps, and email can be used to communicate with team members,
stakeholders, and clients. These tools help to keep everyone informed and on the same
page throughout the testing process.
 CHAPTER 5.
CONCLUSION AND FUTURE WORK

5.1.Conclusion
A “susceptible” result indicates that the patient's organism should respond to therapy with that
antibiotic using the dosage recommended normally for that type of infection and species [13,
20]. Conversely, an organism with a MIC or zone size interpreted as “resistant” should not be
inhibited by the concentrations of the antibiotic achieved with the dosages normally used with
that drug [13, 20]. An “intermediate” result indicates that a microorganism falls into a range of
susceptibility in which the MIC approaches or exceeds the level of antibiotic that can ordinarily
be achieved and for which clinical response is likely to be less than with a susceptible strain.
Exceptions can occur if the antibiotic is highly concentrated in a body fluid such as urine, or if
higher than normal dosages of the antibiotic can be safely administered (eg, some penicillins
and cephalosporins). At times, the “intermediate” result can also mean that certain variables in
the susceptibility test may not have been properly controlled, and that the values have fallen
into a “buffer zone” separating susceptible from resistant strains [13, 20]. Generally, reporting
of a category result of susceptible, intermediate, or resistant provides the clinician with the
information necessary to select appropriate therapy. Reporting of MICs could aid a physician
is selecting from among a group of similar drugs for therapy of infective endocarditis or
osteomyelitis, in which therapy is likely to be protracted.

5.2.Future work

 With drug resistance on the rise, improvements in clinical antibiotic susceptibility

testing and investment in widespread implementation are needed to usher in a new
generation of diagnostics that can inform on diverse types of drug resistance and
quickly predict drug susceptibility with high accuracy.

 Antibiotic resistant bacteria are a growing problem, and the spread of resistance means
that antibiotic susceptibility cannot easily be predicted based on bacterial species alone.
 Thus, it is increasingly important to specifically test for resistance to shorten treatment
times, ensure efficacy and reduce the likelihood of selecting for drug-resistant
pathogens.

 Current antibiotic susceptibility testing (AST) relies largely on time-consuming

culture-based methods. After pathogens are initially grown from patient samples, a pure
culture is challenged with different antibiotics and bacterial growth is assessed, either
on agar, by measuring zones of inhibition around a disk or strip impregnated with
antibiotic or by assessing turbidity in liquid media containing a drug. In both
approaches, the minimum inhibitory concentration (MIC) of a panel of antibiotics is
determined, and these values are compared against recommended resistance
breakpoints, which are derived from the pharmacokinetic and pharmacodynamic
profiles of the drugs for particular pathogens, as well as epidemiological data
comparing MICs to treatment efficacy.

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