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INTRODUCTION

Background of the Study

Hospitals these days supplement and enhance the viability of numerous

different pieces of the wellbeing framework, giving persistent accessibility of

administrations to intense and complex conditions. These facilities concentrate rare

assets inside well-arranged referral systems to react proficiently to populace wellbeing

needs. However, these facilities can also be a source for microbial growth. According

to Pati (2018), about 10,000 airborne organisms were found inside hospital

environments.

Emergency care plays a significant role in healthcare delivery. Emergency

Departments (ED) or commonly known as the Emergency Rooms are considered as

the most important area of a hospital since high degree of therapeutic thought being

passed on by emergency divisions starts from various factors: access to social

protection, client driven needs, an appreciation for the careful thought passed on by

emergency workplaces, and the limit of emergency workplaces to fill an essential gap

as for mind passed on to defenseless peoples. Emergency rooms are a major source of

medical care, especially for vulnerable populations (Marcozzi, Carr, Liferidge, Baehr,

& Browne 2017).

In a study by Allito, Juayno, & Kisteria (2019), which analyzed the presence of

microorganisms found air borne in emergency rooms and found pathogenic bacteria of

the genus, Staphylococcus and Bacillus which are commonly found in hospitals.
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According to the study of Rothman (2007), there is a risk of infection not only for the

patients but also for the staff, health care workers and hospital population.

Antibiotic resistance arises when certain bacteria exposed to the drug, survives

the attack and develop into a strain which then has the chance to occupy the space left

by the dead, non-resistant bacteria (Ventola 2015). The prevalent use of antibiotics in

farms and hospitals contributed greatly to the presence of resistant bacteria in that area

(Li, Liao, & Yao 2018). However, these bacteria is not only limited to the air-

conditioning units. The whole area may be at risk, including floors, walls, and even

hospital beds. In a study by Attaway, Fairey, Steed, Salgado, Michels & Schmidt

(2012), possibly pathogenic bacteria were recovered from hospital beds. Cleaning

agents such commercially available cleaning materials are commonly used as a

primary disinfectant. Most of these active agents demonstrate wide-spectrum of

antimicrobial activity, but only a little is understood about how these agents behave

relative to antibiotics (McDonell & Russell, 1999). Hence, the study seek to establish

what bacteria can be commonly found in hospital beds, the degree of antibiotic

resistance of the bacteria that will be isolated and determine if the cleaning agents

used is enough to disinfect the surface; furthermore, a comparison of effectiveness in

killing bacteria between the commonly used cleaning agents and antibiotics will be

considered.
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Statement of the Problem

This study aims to isolate and identify the degree of antibiotic resistance of

bacteria found in Emergency room beds in government hospitals. Specifically, it seeks

to answer the following questions:

1. What bacteria are found in hospital beds in the Emergency rooms?

2. What is the degree of antibiotic resistance of the bacteria?

3. Are the cleaning agents enough to be used as a disinfectant?

4. Are cleaning agents better at killing bacteria than antibiotics?

Objectives

Generally, this study aims to isolate and test the degree of antibiotic resistance

of the bacteria found in emergency room beds in government hospitals. Specifically, it

aims to:

1. isolate bacteria are found in hospital beds in the emergency rooms;

2. determine the degree of antibiotic resistance of the bacteria found; and

3. determine whether or not the cleaning agent must be used as a

disinfectant

4. establish how effective cleaning agents are relative to antibiotics

Significance of the Study

The study aims to determine what different kinds of bacteria are present in

emergency room beds in government hospitals and determine the degree of antibiotic
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resistance from those identified samples. In addition, this study could provide further

information on bacteria that inhabits emergency room beds and determine the best

way to sanitize the working environment in order to minimize or avoid the spreading

of some various diseases. Furthermore, this would also create awareness on the

presence of bacteria in a hospital environment and encourage more careful procedures

in handling patients and instruments.

Scope and Limitations of the Study

The study will only focus on the isolating and determining of the bacteria

found in the emergency room beds in a government hospital and determining the

degree of antibiotic resistance of the collected samples. The Collection of samples will

be limited to only one government hospital, specifically, in the emergency room of the

area.

Definition of Terms

Microorganisms are living things usually single - celled organisms that are

not visible to the naked eye and measure just a thousandth of a millimeter.

Antibiotic resistance refers to a microorganism survives an antibiotic and

adapts to the presence of antibiotics thus making it stronger.

.
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Procedural Framework

The figure below depicts the flowchart of procedures in gathering data.

ENTRY PROTOCOL

PREPARATION OF
MATERIALS

MICROBIAL ANALYSIS

STERILIZATION ISOLATION
SAMPLING PREPARATION OF CULTURE
MEDIA

PRESUMPTIVE
IDENTIFICATION

PREPARATION OF GRAM STAINING BIOCHEMICAL ANTIBIOTIC

SMEAR TESTS SUSCEPTIBLITY
TEST

DATA
PROCESSING

DECONTAMINATION

PHOTO
DOCUMENTATION

Figure 1. Flowchart of the data gathering.

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REVIEW OF RELATED LITERATURE

Since time immemorial, mankind has dealt with various microorganisms for

the duration of its existence here on earth. These microorganisms have affected the

way people lived for millennia but none as much as bacteria. There are a wide variety

of bacteria that exist in the world with some that do well for man whilst others do not.

Bacteria, along with other microorganisms, inhabit almost every surface on earth and,

with certain conditions, proliferate. Their presence in the human world is nothing new

but with the fact that these bacteria can also do harm means their presence in places

like restaurants, laboratories and hospitals needs to be regulated. In hospitals, in order

to avoid microbial contamination, aseptic procedures are followed in order to provide

maximum care to their patients (Eske, 2018). (add stuff about common bacteria found

in hospitals)

Cleaning Agents Used in Disinfection

Nosocomial Infection

The consequences when these aseptic procedures are disregarded are

sometimes fatal to the patient. The consequence often lead to nosocomial infections or

healthcare acquired infections. ‘Nosocomial’ or ‘healthcare associated infections’

occurs when a patient gets an infection which was not there at the time of admission

and can happen while giving health care for another disease and even after the patient

is discharged (Khan, Baig, & Mehboob, 2017). To avoid this, most equipment used
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are sterilized and/or decontaminated however places where the patients commonly

stay in such as the bed barely get considered in the aseptic procedures. The physical

surface that lies beneath the bed sheet is usually not included in these procedures.

Which could lead to a nosocomial infection originating from the surface underneath

the bed sheet.

One of the ways of preventing infections is through the administering of

antibiotics. An antibiotic is a kind of antimicrobial substance active against bacteria

and are utilized as a treatment for bacterial infection. They may either execute or

hinder the development of bacteria. Antibiotics are a marvel of modern medicine that

battle certain diseases and can spare lives when utilized appropriately. They either

prevent bacteria from repeating or decimate them (Nordqvist, 2019). Before bacteria

can duplicate and cause side effects, the insusceptible framework can regularly kill

them. However, antibiotics can have drastic effects if not used properly. Antibiotic

resistance is ascending to very concerning levels in all pieces of the world,

compromising the capacity to treat common infections. Pneumonia, tuberculosis,

blood harming and gonorrhea, and infections that affect animals, are getting to be

harder, and sometimes impossible, to treat as antibiotics become less viable. At the

point when bacteria in the body of a human or creature are to be exposed to

antibiotics, they change to oppose the impact of the medication. The more antibiotics

are utilized, the quicker resistance creates. (Serrano, 2019)

Microbes originating from establishments that commonly use antibiotics such

as farms and hospitals express antibiotic resistant genes (Li, et. al. 2019). If the
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antibiotics are misused in these establishments antibiotic resistant microbes come

about. Antibiotic resistance in microorganisms occurs when antibiotics are misused

and the misuse leads to strains of microorganism surviving the ordeal and mutating to

become stronger (Ventola, 2015). The air conditioning units found in hospital have a

high likelihood of having bacteria (Almagor, et. al. 2018) that are antibiotic resistant.

Having bacteria such as these are a threat to the hospital since, if these microbes are

airborne, could infect everyone susceptible and have even worse complications in

health. Such complications can be quantified through epidemiology.

Epidemiology

According to the Rose and Barker (1978), epidemiology is the quantification

of the occurrences of diseases and the reason behind it. Such that the information

gained is utilized for strategies to prevent illness or for the management of patients

which have that particular illness. This way of quantifying the information and raw

data gathered could lead to better strategies in preventing nosocomial infections and

managing the patients while in the care of the hospital.

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METHODOLOGY

Research Design

The study will be using the descriptive research design. Descriptive research

design is a scientific method which utilizes observation and description of the subject

without affecting the subject.

Entry Protocol

A letter will be given to the Chairperson of the Department of Biology,

College of Arts and Sciences, Central Mindanao University, Musuan, Bukidnon

asking permission to use the Microbiology Laboratory for the conduct of the study. A

letter will also be given addressed to the Natural Science Research Center to ask

permission for the use of other facilities needed for the conduct of the study such as

the equipment needed for sterilization of materials. Further, a request will also be

given to the medical directors of one government hospital in Maramag, Bukidnon

asking permission to collect samples from their respective emergency room beds.

Locale and Duration of the Study

Samples will be collected from one government hospital in Maramag,

Bukidnon specifically in the ward rooms of the hospitals. The name of the hospital is

to be kept confidential and anonymity is observed for ethical purposes. Possible cause

of contamination will be observed. Laboratory works will be conducted at the

Microbiology Laboratory of the College of Arts and Sciences at Central Mindanao

University, Musuan, Bukidnon.

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Microbial Analysis
a.) Sterilization

All the materials that will be utilized in the procedures will be sterilized

before the laboratory works. The materials will then be placed in Autoclave

cellophane and will be tied using rubber bands. They will be placed in the pressure

cooker and will decontaminate at 121°C for 15 minutes.

b.) Preparation of Culture Media

There are several types of culture media such as nutrient agar and nutrient

broth will be utilized in the study. The nutrient agar is solid at 37°C. It will be heated

at 100°C to melt the agar and then cooled.

c.) Sampling

Sampling will be done a week after the cleaning scheduled service clean-up of

the pre-identified air-conditioning units. The sample collection will be done once from

the selected hospitals. The dust will be collected using sterile cotton swabs. The

samples will be contained in a small airlock or zip lock bag and will be placed in a

refrigerator.

d.) Isolation

A well- isolated culture of bacterial colony will be separated using a sterile

inoculating loop and grown in an agar-based nutrient medium in a Petri-dish. These

colonies will serve as pure cultures.

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Streak-Plate Method

Pure cultures of bacteria or colonies can be isolated using the streak plate

method. It is a technique wherein a mixture of cells is strewn over the semi-solid, agar

based nutrient medium in a petri dish. The method should result in an uneven

distribution of cells with one end more condensed than the other. Incubation to

develop a colony will then be done after spread plating.

Presumptive Identification of Bacteria

1.) Preparation of smear

Creation of a “smear” of bacteria on a microscope slide will be done by

fixing, staining and drying without a coverslip and then examined using a

microscope. Sanitary techniques will be observed when taking samples of a culture

for making a smear. A culture on agar will be utilized as it is better than a liquid

culture. A thin and even layer will be made and used since this will enable its

shape and distribution of cell to be clearly observable and ensure that the staining

procedure is spread evenly.

2.) Gram Staining

A previously prepared smear will be stained with crystal violet for 30

seconds and washed immediately with running water for 5 seconds. The slide will

be covered with Gram’s Iodine Mordant for 1 minute and will be washed again

with water for 5 seconds. The smear will be decolorized using 95% for 20-40
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seconds. The slide will then be immediately washed for 10 seconds in order to

prevent the alcohol from losing color. After which, the said slide will be counter

stained with safranin for about 60 to 80 seconds and then washed again with water.

After which, the bacterial smear will be blotted dry. The Bacteria that keep the

violet or purple color are considered as Gram-positive while those that lose its

color are called Gram-negative, which are stained in a different color from pink to

red.

KOH String test

For the validation of the result of the Gram Staining, the KOH string will

be conducted. About 3% drop of KOH will be placed in a glass slide. It will then

be combined with a coil of 18-24 hours of culture utilizing the loop for 1 minute.

3.) Biochemical Tests

a.) Indole Test

Sulfide Indole Motility (SIM) agar (deep/based) medium will be used for

this experiment. The Agar (deep/based) will be inoculated with a loopful of

18-24 hours of culture of bacteria sample at 37°C. Ten (10) drops of Indole

reagent will then be added to the culture. Results will then be observed and

documented.

b.) Methyl Red Test

The Nutrient broth will be conducted. It will then be aseptically

inoculated which a loopful of bacteria sample incubated for 24 to 48 hours at

37°C. Ten (10) drops of methyl red reagent will be added.

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c.) Voges-Proskauer Test

0.5ml VP1 and 5 drops of VP2 will be added to the cultured bacteria.

The cultures will be left to stand for 15 seconds.

d.) Citrate Test

The preparation of the nutrient agar slant will be prepared for this test.

Incubation of the slant will be conducted for 24 hours at 37°C using stab-and-

streak inoculation.

4.) Antibiotic Susceptibility Test (CLSI Antimicrobial Susceptibility, 2006)

a.) Inoculation

By the use of the stock culture or pure isolates, the distilled water inside the

tube will undergo inoculation. It will be until the inoculated distilled water

inside the tube will reach the turbidity of the 0.5 McFarland standard. The

adjustment of suspension will follow using a sterile cotton swab and will be

dipped into the distilled water culture and will be pressed rotationally against

the inside of the tube to remove excess fluids. The cotton swab will be

swabbed in the surface of the previously prepared Mueller-Hinton Agar

(MHA) plate. The MHA plate will contain 15 ml of the medium. The cotton

swab will then be streaked two times more while rotating the plate at about 60°

after each streak.

b.) Application of Discs

Each battery of antimicrobial disc (amoxicillin, ampicillin,

streptomycin and chloramphenicol) will be dispensed to the previously

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streaked MHA plates by pressing down the discs using the whattman filter

paper. The discs should be evenly distributed and should not be less than 24

mm close from the center. Plates will be incubated for 8 to 16 hours. Water

will used as the control and the antibiotics will be used as the positive control.

c.) Reading and Interpretation of results

Incubation of the MHA plates will be analysed following 8 to 16 hours.

Quantifying the diameter of the zones of inhibition will be to the nearest whole

millimeter using a ruler at the back of the MHA plate by inverting it. The

measurement of the disc will also be taken. The measurements of the zones of

inhibition will be interpreted using the Zone Diameter Interpretative Standard

(CLSI Antibiotic Susceptibility).

d.) Computation of the zones of inhibition

After measuring the diameter of the zones of inhibition, it will then be

converted to the standard computation of the zones of inhibition with the

following formula:

𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 𝑜𝑓 𝑡ℎ𝑒 𝑧𝑜𝑛𝑒 𝑜𝑓 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 − 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 𝑜𝑓 𝑡ℎ𝑒 𝑑𝑖𝑠𝑘

𝑍𝑂𝐼 =
𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 𝑜𝑓 𝑡ℎ𝑒 𝑑𝑖𝑠𝑘

5.) Decontamination

The cultures that will be used in the procedures will be decontaminated

after the laboratory works. The culture plates of the colonies will be wrapped with

scratch papers and will be placed in an Autoclavable tied with rubber bands. They
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will be placed in the pressure cooker and will decontaminate at 121°C for 1 hour.

The glassware will then be washed.

6.) Statistical Analysis

The data will be summarized using frequency distribution and percentages.

A comparison of proportions of resistant organisms among the different antibiotics

and sources of samples will be established using the chi-square test.

7.) Documentation

Photographs of the sampling site, isolated colonies, laboratory works and

all the whole conduct of the study will be taken using a digital camera or android

phone’s camera.
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LITERATURE CITED

Allito, A. B., Juayno, J. S., & Kisteria, J. M. (2019). Microorganisms Isolated in Air
Conditioners in Emergency Rooms of Three Level -2 Hospitals in Valencia
City, Bukidnon.
Almagor, J., Temkin, E., Benenson, I., Fallach, N., & Carmeli, Y. on behalf of the
DRIVE-AB consortium (2018). The impact of antibiotic use on transmission of
resistant bacteria in hospitals: Insights from an agent-based model. Plos One,
13(5). doi: 10.1371/journal.pone.0197111
Eske, J. (2018, November 8). Aseptic technique: Purpose, benefits, and types.
Retrieved November 30, 2019, from
https://www.medicalnewstoday.com/articles/323615.php.
Khan, H. A., Baig, F. K., & Mehboob, R. (2017). Nosocomial infections:
Epidemiology, prevention, control and surveillance. Asian Pacific Journal of
Tropical Biomedicine, 7(5), 478–482. doi: 10.1016/j.apjtb.2017.01.019
Li, Y., Liao, H., & Yao, H. (2019). Prevalence of Antibiotic Resistance Genes in Air-
Conditioning Systems in Hospitals, Farms, and Residences. International
Journal of Environmental Research and Public Health, 16(5), 683.
doi:10.3390/ijerph16050683

McDonnell, Gerald & Russell, Denver A. (1999). Antiseptics and Disinfectants:

Activity, Actions and Resistance, 12(1), 147- 179.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC88911/
Marcozzi D, Carr B, Liferidge A, Baehr N, Browne B . (2017) Trends in the
Contribution of Emergency Departments to the Provision of Hospital-
Associated Health Care in the USA. 48(2):267-288. doi:
10.1177/0020731417734498
Nordqvist, C. (2019). What to Know About Antibiotics? Medical News Today.
Retrieved September 16, 2019 from
https://www.medicalnewstoday.com/articles/10278.php
Rose, G., & Barker, D. J. (1978). Epidemiology for the uninitiated. Bmj. doi:
10.1136/bmj.2.6147.1282

Rothman, Irvin, Moran, Sauer A, Bradshaw, Fry, Josephine, Ledyard, Hirshon.

(2007) Respiratory Hygiene in the Emergency Department, 33 (2), 119-134.
https://doi.org/10.1016/j.jen.2007.01.013

Serrano, R. (2019). Stop Overuse and Misuse of Antibiotics: Combat Resistance.

World Health Organization Western Pacific News. Retrieved September 16,
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2019 from https://www.who.int/westernpacific/news/detail/10-11-2017-stop-

overuse-and-misuse-of-antibiotics-combat-resistance

Ventola, C. L., (2015) The Antibiotic Crisis. P&T, 40(4), 277-283.