MINISTRY
OF EDUCATION AND TRAINING
MINISTRY
OF HEALTH
HANOI MEDICAL UNIVERSITY

LE KHANH TRAM
DETERMINATION OF CARRIER STATUS
AND TOXIN GENES OF Staphylococcus aureus
IN FOOD HANDLERS
Specialization: MEDICAL MICROBIOLOGY
Code: 62720115
PHD MEDICAL THESIS SUMMARY
HANOI-2013
THE WORKS WERE COMPLETED
AT THE HANOI MEDICAL UNIVERSITY

Supervisors:
1. Assoc. Professor Dinh Huu Dung, MD, PhD
2. Assoc. Professor Nguyen Vu Trung, MD, PhD

Referee 1: Assoc. Professor, PhD. Le Van Phung
Referee 2: Assoc. Professor, PhD. Nguyen Thi Khanh Tram
Referee 3: PhD. Tran Huy Thinh

Examination committee: Institutional Dissertation Council, Hanoi
Medical University
Examination date:
The thesis can be found at:
1. National library.
2. Central library of medicine information

1
INTRODUCTION

Staphylococcus aureus is a member of the bacterial flora in
humans. About 30% of healthy people carry S. aureus. These
bacteria can be found in many areas of human body such as on skin
of the necks, chest, abdominal, hands…and many natural cavities of
the body, especially the anterior nares. S. aureus can be persistent,
intermittent or non- carry on the human body. When there were skin
lesions, destroyed mucous membranes, the immunity defence of body
would decrease, giving the opportunities for the infection by S.
aureus. The presence and number of S. aureus in the body depends
on the immune status, sanitation, and many other factors of the hosts
as well as the bacteria.
S. aureus is one of the leading causes of food poisoning throughout
the world and it is still an unresolved problem. Food poisoning caused
by S. aureus accounted for about 25% of cases and 10% of patients
hospitalized for food poisoning treatment. There have been severe cases
of danger to life in Vietnam. S. aureus is the main cause of
contamination in food. Due to the hot and humid climate, the lack of
appropriate storage conditions, the presence of S. aureus with high
numbers in foods would potentially lead to the break out of food
poisoning, and consequently affect the economy. The individuals
working in the close contacts with food such as food processing, food
saling would be highly potential sources of contaminating S. aureus for
processing, manufacturing, packaging, transportation and storage of
food. In Vietnam there have been many studies on the pathogenicity and
antibiotic resistance of S. aureus but, no research on carrier status as
well as very low number of studies on toxin genes carried of these
pathogen.

1.1. MICROFLORA IN HUMAN
Normal microflora in human (normal microbial flora) is the
complex of microorganisms that live on the healthy human body.
Microflora on skin contains mainly Gram-positive bacteria, which
are usually Staphylococci (Staphylococcus epidermidis), anaerobic
bacteria (Propionibacterium acnes which cause acne). There is also
3
the Staphylococcus aureus, Streptococcus viridians, Bacillus like
diphtheria and spore bacteria in the air. It is accounted for about 10
10
bacteria on the skin. Normal microflora in narses are staphylococci
and streptococci, Corynebacterium, especially the S. aureus that have
the ability to infect and spread.
1.2. CHARACTERISTICS OF S. aureus
S. aureus is a Gram-positive cocci, with a diameter of 0.8-1 μm,
grouping into the form of grapes, no pilli, no spore and usually no
capsula. S. aureus can be easily cultivated, they grow at a
temperature of 10
0
C-45
0
C and salt concentrations as high as 10%,
appropriate in both anaerobic and aerobic conditions. The genome of
S. aureus is completely sequenced in size from 2820 to 2903 bp. G, C
contain is around 33% and from 2592 to 2748 bp sequence encodes
for proteins. In the genome, 75% of the sequences is highly
conserved between the strains of S. aureus. The set of all the
sequences of genomic region is called core genome.
1.3. CARIAGE PREVALENCE AND CARRIER STATUS OF S.
aureus

The coagulase gene of S. aureus has 6 basic segments. The
signal chain of the sequence starting at the N-end to the D1, D2
regions, central region, the repeated region (segments with 81 bp
nucleotid), and the C-end. The classification of the genotype of
coagulase gene in S. aureus based on the polymorphism of the DNA
fragments treated by the restriction enzyme AluI has a number of
advantages in the S. aureus strain identification such as simplicity,
5
fast, and accuracy.
1.5.2. Enterotoxin genes of S. aureus
Enterotoxin genes of S. aureus such as sea, seb, sec… encode
for more than 20 different types of enterotoxins. Most of the genes
coding for enterotoxins have been identified in the mobile genetic
factors such as plasmids, bacteriophages. Moreover, the pathogenic
genes can be scattered or concentrated into a group called enterotoxin
gene cluster. Thus, horizontal transmission of these genes between S.
aureus strains is common.
1.6. S. aureus INFECTIOUS DISEASES
Accoding to the statistic data of the 16 common disease-causing
bacteria in Vietnam, the rate of S. aureus constitutes from 18.5-
21.7%, ranking second only to E. coli. S. aureus can cause several
clinical diseases such as infections affecting skin and soft tissues,
scalded skin syndrome, infection of bones and joints, endocarditis,
bacteraemia, pneumonia, food poisoning and intestinal infections,
toxic shock syndrome and nosocomial infections.
1.7. LABORATORY ISOLATION AND IDENTIFICATION OF
S. aureus
1.7.1 Common techniques to identify S. aureus: direct microscopic
examination; isolation, cultivation, and identification slide;
aglutination, ELISA, identification by phages.

p (1-p)/(εp)
2

n: sample size for the research; Z: coefficient of reliability,
with α = 0.05 then Z
1-α/2
= 1.96
p: carriage prevalence of S. aureus in food handlers
according to previous studies (33%);
ε: accuracy relative to p, ε = 0.2, n = 195. Number of
eligible objects are studied was 212.
7
The objects were selected as random individuals.
- The isolated S. aureus strains are studied in coagulase and
entrerotoxin coding genes.
2.4. RESEARCH TECHNIQUES
2.4.1. Sampling:
Samples in the nose and hands of each participants will be
collected by using sterile swabs 4 times with one week intervals to
determine the carriage prevalence and carrier status of S. aureus.
2.4.2. Isolation and identification of S. aureus:
According to the microbiological procedures, the properties
will be used for the identifiction of S. aureus included:
+ Gram-positive cocci grouped into grapped-form clusters.
+ Enzyme coagulase.
+ Enzyme catalase
+ Fermentation of manitol
2.4.4. PCR for identification of toxin genes
2.4.4.1. DNA extraction (DNA QIAGEN QIAamp Minikit 50)
2.4.4.2.PCR for identification of coagulase gene polymorphism

SEC-2
gacataaaagctaggaattt
aaatcggattaacattatcc
Johnson et al.
(1991)
sed
SED-1
SED-2
ctagtttggtaatatctcct
taatgctatatcttataggg
Johnson et al.
(1991)
see
SEE-1
SEE-2
aggttttttcacaggtcatcc
cttttttttcttcggtcaatc
Merhotra et al.
(2000)
seg
SEG-1
SEG-2
aagtagacatttttggcgttcc
agaaccatcaaactcgtatagc
Omoe et al.
(2002)
sei
SEI-1
SEI-2
ggtgatattggtgtaggtaac

RESULTS
3.1. CARIAGE PREVALENCE AND CARRIER STATUS OF S.
aureus
3.1.1. Characteristics of study object
3.1.1.1. The distribution of subjects by gender
Table 3.1: Distribution of subjects by gender
Gender n %
Male 112 52.83
Female 100 47.17
Total 212 100.00
Among 212 employees participated in the study, the percentage
of male was 52.83% and female was 47.17%.
3.1.1.2. Distribution of subjects by age
Table 3.2: Distribution of research subjects by age
Age group n %
≤ 19 42 19.81
20-29 140 66.04
30-39 17 8.02
40-49 9 4.24
≥ 50 4 1.89
Total 212 100.00
The age group from 20 to 29 account for the highest percentage
10
(66.04%) among the total number of participates. The second was the
group ≤ 19 years, constituted in 19.81%. Finally the group ≥ 50 was
the lowest with 1.89%.
3.1.2. Carriage prevalence of S. aureus and relevant factors
3.1.2.1. Carriage prevalence of S. aureus
Chart 3.1: Carriage prevalence of S. aureus among the 212
individuals involving in the study

Table 3.14: Carriage prevalence of S. aureus in hands
Carrier status n %
Non carrier 193 91.04
Intermittent 19 8.96
Total 212 100.00
The carriage prevalence of S. aureus in hands was 8.96%, no
person carries these bacteria persistently in hands.
3.2. STUDIES ON TOXIN GENES OF S. auresus
101 strains of S. aureus including, 77 strains from nose and
24 strains from hands, were isolated and subjected to the study on
p<0,05
13
toxin genes.
3.2.1. Coagulase gene polymorphism of S. aureus strains
3.2.1.1. Distribution of genotype of coagulase gene of S. aureus
20 genotypes were identified based on the length of DNA
fragments obtained after digestion of the gene sequence by restriction
enzyme AluI. The most frequent was genotype 6 (53.47%) which
included 3 DNA fragments 465 bp, 250 bp and 165 bp. The second
was genotype 12 (6.93%) and the third was genotype 1 and genotype
2 (5.94%). The four genotypes have been accounted for nearly 75%
of the total number of strains.
Table 3.23: Distribution of genotype of coagulase gene
in 101 isolated S. aureus strains
Genotype
Number offragments
800 bp
550 bp
465 bp
250 bp

16 3 + + + 1 0.99
17 4 + + + + 1 0.99
18 4 + + + + 3 2.97
19 4 + + + + 1 0.99
20 4 + + + + 1 0.99
Total 101 100,00
3.2.1.3. Distribution S. aureus-harboring staffs according to the
number of the genotype of coagulase gene
Table 3.25: Distribution S. aureus-harboring staffs by the number
of the genotype of coagulase gene
Number of genotypes S. aureus-harboring staffs (%)
1 39 73.58
2 13 24.53
3 1 1.89
4 0 0.00
15
Total 53 100.00
S. aureus carriage prevalence with 1 genotype of the gene
encoding coagulase was the most common, 73.58%. The prevalence
with 2 genotypes constituted 24.53%. Only one S. aureus-harboring
staff showed 3 different genotypes, constituted 1.89%.
3.2.2. Study on enterotoxin genes of S. aureus
3.2.2.1. Overall carriage prevalence of enterotoxin genes of
S. aureus
Table 3.26: Overall carriage prevalence of enterotoxin genes of
S. aureus
Carrying gene n %
Carrying 64 63.37
Non carrying 37 36.63
Total 101 100.00

47.17%, respectively). This is different from other studies, which
were carried out with higher number of female than male, such as the
study of Gashaw on café serving staffs (63% and 37%), the study of
Oteri on 161 staffs of food handlers with 57.1% female and 42.9%
men. Concerning the age of participants in the present research, the
majority group was of people at the age 20 to 29 (accounting to
66.04%). This is the age of good health, and capability of active
labor. This result was similar to that in the study of Gashaw 2008.
4.1.2. Overall carriage prevalence of S. aureus and related factors
In this study, the percentage of people who carried S. aureus
counted for 25%, similar to the study of Bustan (26.6%) on 500 staffs
in restaurants in Kuwait, however was lower than Jonge's research in
the Netherlands in 2010 (33%) and research of Borges in 2010 in
Brazil (36%). It should be noted that here the carrier status of S.
aureus was determined as the analyses results of 4 times of sampling,
whereas the others studies based only on 1 time sampling. The
carriage prevalence of S. aureus found in this research was in the
range of normal limits (20-70%). The differences of prevalence may
be due to the study subject, races or geographical regions.
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4.1.3. Carriage prevalence and carrier status of S. aureus in the
nose and relevant factors
In this study, the carriage prevalence of S. aureus in the nose
was 21.7%, the intermittent carriage was 20.28% and the persistent
carriage was 1.42%. It has been reported by other authors that the
carriage prevalence of S. aureus in the nose ranged from 12-30%,
depending on geographical regions and races. Comparing with other
studies, the carriage prevalence of S. aureus in the nose found in this
research was similar to that (21.6%) reported by Humodi, who
carried out a study on employees in food processing and service

employees 79/212 (37.26%), whereas in the study of Humodi, staffs
in food processing were 152/259 (58.69%).
4.1.4. Carriage prevalence of S. aureus in hands and relevant
factors
The carriage prevalence of S. aureus in hands was low (8.96%)
and no persistent carrier was found in the study. This could be results
of the general hygiene conditions of employees such as regular hand-
washing, responsibility of staffs for food safety, good sanitation
conditions in the working environments. In case some employees
could wash their hands just before sampling, the results would not be
interfered since the sampling was performed 4 times with one week
intervals. Table 4.1 shows the carriage prevalence of S. aureus in
hands from different studies. Thus, the results obtained in our study
(8.96%) was comparable to those recently reported by Sospedra
(2012) in a study on 227 samples from hands of food handlers (8.4%)
in Spain. However, it was higher than reports of some other authors
such as Haluk (5.6%), Borges (2010) in Brazil (2.9%) and of Nguyen
Hung Long et al. (2009) in Vietnam (3.3%). In the same time, this
value was lower than that reported by Gashaw (16.5%), Soares
(16.5%) and Zaglol (17.5%). In the latter studies, the carriage
prevalence of S. aureus in hands was high because the study was
carried out on people selling food on the streets, who did not have
health care, had no training on food safety, and might not perform
hand hygiene as required. Nevertheless, the sampling procedure in
this study (4 times of sampling with one week intervals) could be
good explanation for the high accuracy of the results in comparison
20
to one time sampling procedure in other studies.
Table 4.1: Reports on the carriage prevalence of S. aureus
in hands of food handlers

genotype. Nevertheless, according to several authors, there should
always be one or several dominant genotypes, like in the study Kitai
(52.9%), Rodrigues, Su, Hinmabindu (15.5%).
Table 4.2: Polymorphism of coagulase gene in different studies
Author Year Country
Number of
strains of
S. aureus
Number of
genotypes
The present study 2012 Vietnam 101 20
Moradi Stefano 2008 Italy 25 16
Elizabete Rodrigues 2005 Brasil 64 49
Su. C 1999 The USA 425 40
Hinmabindu 2009 India 66 19
Swee-han Goh 1992 Canada 69 19
Schwarzkopf 1994 Germany 30 6
The polymorphism of the gene encoding coagulase has been
determines not only in S. aureus strains isolated from human but also
from animals and in different geographical regions. The
identification of coagulase genotypes of the S. aureus has confirmed
to be simple, accurate and very valuable in the classification of the
strains of S. aureus. Determining coagulase genotypes of S. aureus is
considered as the basis to assess the molecular epidemiology of S.
aureus. In our study, the most predominated genotype is 6 with 3
DNA fragments, accounting for 53.47% of the total number of S.
aureus strains isolated.
4.2.1.2. Distrbution of genotypes of the coagulase gene among
study objects