TMP UNIVERSAL JOURNAL OF RESEARCH AND REVIEW ARCHIVES

VOLUME 1 │ISSUE 2│YEAR 2022│OCT - DEC 2022

RECEIVED REVISED ACCEPTED
DATE DATE DATE
15/09/2022 20/10/2022 15/11/2022

Article Type: Research Article Available online: www.tmp.twistingmemoirs.com ISSN: N/A

CHARACTERIZATION OF MULTIDRUG RESISTANT

STAPHYLOCOCCUSAUREUS ISOLATED FROM VARIOUS
CLINICAL SAMPLES.
*1Farah B.Abd, 1Mohsen Hashem Risan, 1Dhafar N. Al-ugaili

*1Department of Biotechnology, College of Biotechnology, Al-Nahrain University, Baghdad, Iraq

ABSTRACT
Staphylococcus aureusis a major hospital and community pathogen that is attributed to a wide
variety of infections in humans and bio film production is one of the most important virulence
factors of S. aureus that contributes to its multiple drug resistance. Therefore, searching for a
valuable alternative to the used antibiotics is considered an important goal for study. For this
reason one hundred and fifty different clinical samples were collected from various clinical
sources and healthcare workers in Al-Imame in Al-Kadhimae in Medical City,Al-Numan
Teaching Hospital, Medical City/Teaching laboratories and Central Child Teaching Hospital
during the period from1/10/2020to 1/2/2021 in Baghdad City. Isolates were identified by
conventional methods (cultural, microscopic and biochemical tests) in addition to the
identification by the VITEK® 2Compact, and fifty isolates were recorded as Staphylococcus
aureus.

Keywords: Resistant, Staphylococcus, aureus, VITEK® 2, clinical samples.

CORRESPONDING AUTHOR

Name: Farah B.Abd

Affiliation: Department of Biotechnology, College of Biotechnology, Al-Nahrain University,

Baghdad, Iraq

Email: m_risan@yahoo.com

INTRODUCTION

The Staphylococci group is considered as one of the most important human pathogenic organisms
in the skin, nasal mucosa and oral mucosa; it is recognized as a group of opportunistic pathogens
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 Characterization of Multidrug Resistant Staphylococcusaureus isolated from various clinical samples.

that is responsible worldwide infections in hospitals. Staphylococci are positive for catalase,
which is (considered)a defining feature that distinguishes Staphylococci from Strep to coccus and
they were oxidase negative. Susceptibility to lysostaph in is considered to be another feature of
Staphylococci because it comprises many residues of glycine in the cross-bridge between
peptidogly can layers (Plata etal. 2009).

Staphylococcus aureus(S. aureus) is a Gram positive bacteria non-motile, catalase and coagulase
positive, non-spore-forming and as facultative anaerobic, cocci, blue-violet as single, pairs or
irregular as grape-like clusters, about (0.4-1.2μm) in diameter, during their growth have a yellow
colonies named(aureus; means golden)on nutrient rich media(Yan et al.,2016).Staphylococcus
aureus shows high degree of tolerance that enable them to grow under low humidity, high
pressure, and high salt concentration (15%NaCl); also, the bacteria can tolerate pH range (4.2-
9.3); It could grows at temperature between (15oC-45oC), and multiply to produce toxins that
lead to disease development(AlkhafajiandAlsaimary,2020).Staphylococcus aureus is
The main pathogenic bacterium responsible for nosocomial and public acquired infections, often
(25-50%) of the human are colonized with S. aureus and is one of the common human pathogenic
bacteria that cause diverse infections in both male and female (Suhailiet al.,2018). The bacteria
found to localize the mucous membranes, nose, mouth, upper respiratory tracts, intestinal, groin,
mammary glands, perinea area in males, hair, and genitourinary of human also its interpreted in
the production of abscesses, pus, fatal sepsisandsepsis.

MATERIALS AND METHODS

Sample collection of Staphylococcusaureus

A total of 150 samples were collected from patients (Skin swab, Nasal swab, Ear swab), health
care workers (Skin swab and Nasal swab),and operation theater (various places of operation
theater before and after sterilization), hospital words. Samples were collected in the period
between1/10/ 2020 and 2/1/2021, from several locations in Baghdad; Al-ImameinAl-Kadhimaein
Medical City, Al-Numan Teaching Hospital, Medical City/Teaching laboratories and Central
Child Teaching Hospital.

Isolation and identification of Staphylococcusaureus

The isolate was identified according to the standard laboratory test. S.aureus identified and
confirmed by the following: Specimenswere cultured in mannitol salt agar (MSA) and blood agar
base (BA), in cubatedat 37°C for 24 hours. All the primary screened isolates were then subjected
to various morphological and biochemical tests to ensure their identity. [1-10]

Morphological Examination
Staphylococcusaureus growth on Mannitol Salt Agar (MSA)
Staphylococcus can grow on mannitol salt agar medium with a highs alt concentration (7.5%)
NaCl, these media inhibits the growth of other than the staphylococci bacteria. Mannitol-
fermenting staphylococci changes color of MSA from the alka line(red)to the
acidic(yellow),while the rest of the Staphylococcus will grow without produce a color change of
the medium(Gilletet al.,2002).

Blood Hemolysis
Bacterial culture was streaked on blood agar(BA),and the nincubated for 24 h at 37°C. A Green
zone appeared around the S. aureuscolonies, denoting α- hemolysis; while a clear zone indicates
β- hemolysis. [11-20]

Microscopic Examinations
All bacterial isolates were subjected to Gram stain to check the irresponsive to the stain,

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 Characterization of Multidrug Resistant Staphylococcusaureus isolated from various clinical samples.

arrangement and the irshapes (the shape of the bacteria was observed as blue cocci, arranged in
grapes like irregular clusters). A small portion of the suspected colony of the positive culture was
placed and fixed on a clean microscopic glass slide. Gram staining technique was followed and all
slides were examined under oil emersion.

Bio chemical characteristics

Catalase test
From each clinical isolates a single colony was smeared on a slide and drops of (3%) H₂O₂ were
added. The appearance of bubbles indicated positive results.

Coagulase test
Two types of the coagulase tests were used to detect the presence of coagulaseenzymein S.aureus:

1. Coagulase slide test: Suspended S. aureus colony with a saline drop on a clean glass-slide,
mixed with a drop of human plasma, the result appears within ten seconds, as a coarse
coagulate that can be seen by the naked eye indicates a positive result.
2. Coagulase tube test:Anisolated colony is taken from the petri dish and is solved in 1 ml of
diluted plasma, the tube was incubated at 370C and achieve d after1-4h,positive results
will clot.

Oxidase Test
A few drops of the oxidase reagent (1%N, N, N, N-tetramethylep-phenylenediaminedihydro
chloride), were placed on a filter paper, then an isolated bacterial colony was added a paper by
wooden stick. The colony's color changes to dark purple within10-15 seconds when the result is
positive.

Bacterial Identification using VITEK2 System:

Bacterial isolates identification was carried out by VITEK2 system which is an automated
microbiology system employed growth-based technique. From clinical samples, a single colony of
bacterial culture was suspended in with 3ml of normal saline. The turbidity was checked to equal
(0.5) McFarland via turbidity meter for determining inoculums density of Gram positive bacterial
isolates. [21-24]

RESULT

Isolation and Identification of Staphylococcus aureus

One hundred and fifty different clinical samples that were collected from various clinical sources
and healthcare workers in Al-Imame in Al-Kadhimae in Medical City, Al-Numan Teaching
Hospital, Medical City/Teaching laboratories and Central Child Teaching Hospital. During the
period from 1/10/2020 to 1/2/2021 in Baghdad, Iraq. Fifty isolates (33%) were characterized as
S.aureus depending on the conventional cultural, biochemical and microscopic examination in
addition to a confirmatory test by the VITEK® 2Compact system. The rest of the clinical samples
which represent (67%) were found to be related to different genus of pathogenic bacteria. That the
number and percentage of isolates according to the sources in table (1)

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 Characterization of Multidrug Resistant Staphylococcusaureus isolated from various clinical samples.

Table:-1. Number of samples according to the sources of collection

Source of Samples No. of Samples No. Isolates Percentage

fromisolates (%)
Earswap 21 7 14

Hospitalwards 24 8 16

Noseswap 31 13 26

OperationRoom 20 4 8

HealthCareWorker 31 11 22

skinswap 23 7 14

Total 150 50 100

CULTURAL IDENTIFICATION

All the collected clinical specimens were streaked on MSA media, which is considered a selective
and differential medium containing high concentration of sodium chloride (7.5%) to inhibit the
growth of other than Staphylococci. The S.aureuson this medium appear edas form yellow
golden(Figure1)due to fermenting the mannitol salt changing the phenol red to golden, smooth,
raised, mucoid and glistening colonies while S. epidermidis tends to form colonies with pink
zones (Carroll et al., 2016). On the other hand; the same specimens were found to produce
hemolysis when cultured on blood agar media with smooth colony shape (Gillespie and Hawkey,
2006). Finally, the positively selected isolates were maintaining Ed for further steps in our work
using Brain Heart Infusion (BHI) broth and agar medium.

Figure:-1 Growing of Staphylococcus aureuscolonies on MSA (Mannitol salt agar) after 24

hours of incubationon37°C.

Microscopic Characterization
Microscopic examination showed Gram positive cocci that arranged inpaired or grape like
clusters but usually non- spore forming as mentioned by (Carroll etal.,2016).

Biochemical Characterizations
The basic biochemical tests of the all S. aureus isolates were showed a positive reaction for
catalase and coagulase tests which confirm the ability of the test ed isolates to synthesize the se
enzymes that act as a virulence factors enable the bacteria to develop infection. While negative

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 Characterization of Multidrug Resistant Staphylococcusaureus isolated from various clinical samples.

results were obtain for oxidase test which refer to the inability of the isolates to produce this
enzyme. All the tested bacterial isolates gave positive results for the catalase enzyme through the
formation of bubbles (figure 2).which refer to the release of O2from hydrogen peroxide H2O2.
Most isolates were able to yield the coagulase enzyme. This enzyme has an important role in S.
aureus pathogen city. As it enable the bacteria to form protective barriers of fibrin around
themselves, making them highly resistant to phagocytes is and some other anti microbial agents
(Aryal, 2018).

S. aureus secreted coagulase enzyme that converted the plasma to clot as shown in figure (2)by
effective prothromb into form thromb in which converts fibrinogen to fibrin. S. aureus can
produce fibrinolys in, which can lyseclots within4h.These strains would have misdiagnosed as
coagulase negative staphylococci if the tube coagulase test had been incubated overnight (Bello
and Qahtani, 2005).

Figure:-2 Detection of Staphylococcus aureus by Catalase and coagulase test.

Identifying with VITEK2 system.

Further confirmatory identification of the bacteria to the species level was carried out using the
vitek2 system that included a number of the biochemical test in addition to the antibiotic
susceptibility profile that specific for each bacterial species therefore it can give information to
the species level.

Detection of Hemolysin in Staphylococcus isolates

A total of 49 out from the 50 studied strains (90%) showed haemolysis on blood agar plates, S.
aureususually displays a golden yellow pigment as shown in the Figure(3). 42 of the 50 strains
(84%) had beta hemolytic phenol type, while five(10%)of the isolates showed alphahaemolytic
with dark and greenish color. Only three isolates (6%) were recorded as negative results as it
didn’t show any hemolysis, table (2).

This result came in accordance with AL-Khzarji (2020) who reported S.aureus haemolysis on
blood agarplate, 14 %(5/36)were alpha hemolysis and 86%(31/36)gave beta haemolysis. On the
other hand Almwafy (2020) showed that (30.95%)of S.aureus isolates were alpha haemolysis
while (30.95%) had the ability to make beta hemolysis and 38.09% posse the capacity to make
gammahemolysis.While,Jahanetal.(2015)reportedapositiveβ-hemolyicactivityforall the tested
isolates.

Table:-2. Haemolysis of Staphylococcusaureus isolates on blood agar

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 Characterization of Multidrug Resistant Staphylococcusaureus isolated from various clinical samples.

Hemolysis type Number(No.) Percentage (%)

Beta Hemolysis 42 84

Alpha Hemolysis 5 10

Gamma Hemolysis 3 6

Figure:-3 Hemolysis is by Staphylococcus aureus on blood agar medium

Hemolysins, that cause damage to the red blood cell membrane, is one of the main virulence
factors produce by S. aureus and play an important role in their pathogenesis as it participates in
the bio film (Al Lahamet al., 2015). And causes β-hemolytic (complete hemolytic); in addition a
number of S. aureus strains produceα-hemolysin (S.aureus in complete hemolytic
phenotype)(SIHP) strain have been related with nosocomial infection (Zhang et al.,
2016).In(2016)denReijer and h is colleagues noticed four toxins(alpha-toxin, gamma-Hemolysin
B and leukocidins D and E) in all bio films forming strains. The detection of S. aureus toxins,
notably alpha toxin, in bio films is well established roles in skin infections (Kobayashi et al.,
2015). Alpha-toxin causes pore-forming (cytolytic) including skin tissue, obstructs the innate and
adaptive immune responses, in addition, it is necessary for bio film growth on mucosal surfaces
(Anderson etal, 2012).

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