HANOI MEDICAL UNIVERSITY ---LE KHANH TRAM DETERMINATION OF CARRIER STATUS AND TOXIN GENES OF Staphylococcus aureus... Le Khanh Tram, Dinh Huu Dung, Nguyen Vu Trung2011, Determination of
Trang 1HANOI MEDICAL UNIVERSITY
-LE KHANH TRAM
DETERMINATION OF CARRIER STATUS
AND TOXIN GENES OF Staphylococcus aureus
Trang 2
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 TramReferee 3: PhD Tran Huy Thinh
Examination committee: Institutional Dissertation Council, HanoiMedical University
Examination date:
The thesis can be found at:
1 National library
2 Central library of medicine information
3 Library of Hanoi Medical University,
Trang 31 Le Khanh Tram, Dinh Huu Dung, Nguyen Vu Trung
(2011), Determination of carriage prevalence and carrier
status of Staphylococcus aureus, Journal of medical research, 77 (6), pp 29-33.
2 Le Khanh Tram, Dinh Huu Dung, Pham Thi Thuy Duong, Nguyen Vu Trung (2012), Distribution of
coagulase gene in genotypes of Staphylococcus aureus, Journal of medical research, 78 (1), pp 1-5
3 Le Khanh Tram, Dinh Huu Dung, Nguyen Vu Trung
(2012), Detection of enterotoxin genes in Staphylococcus aureus strains, Journal of medical research, 80 (3),
pp 11-16
4 Le Khanh Tram, Pham Thi Thuy Duong, Dinh Huu Dung, Nguyen Vu Trung (2010), Determination of carriage
prevalence and classification of Staphylococcus aureus based
in coagulase encoding gene of staffs working in restaurants in
Hanoi, at 16 th Scientific Conference for PhDs, Hanoi Medical University, Won the third prize.
5 Le Khanh Tram, Nguyen Vu Trung, Dinh Huu Dung
(2011), Detection of enterotoxin genes in Staphylococcus
Medical University, Won the third prize.
Trang 5Staphylococcus 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 skinlesions, 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 foodpoisoning, and consequently affect the economy The individualsworking 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 offood 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 thesepathogen
Thus, our study with the title: “Determination of carrier
status and toxin genes of Staphylococcus aureus in food handlers”
with the following two main purposes:
1 To determine the carriage prevalence and the carrier status
of Staphylococcus aureus in the nose and hands of staffs working in restaurants in Hanoi
2 To determine the distribution of coagulase gene polymorphism
Trang 6and enterotoxins genes of the isolated S aureus strains.
NEW CONTRIBUTIONS OF THE THESIS
1 This is the first study on the carrier status of S aureus in healthy
people in Viet Nam
2 Providing scientific information contributing to the carriage
prevalence of Staphylococcus aureus of food handlers working
in restaurants in Hanoi
3 This is the first study in Viet Nam on coagulase and enterotoxin
coding genes of S aureus isolated from noses and hands of food
handlers working in restaurants in Hanoi
4 Providing a better understanding in molecular epidemiology of
coagulase and enterotoxin coding genes of S aureus.
THE LAYOUT OF THE DISSERTATION THESIS
The thesis comprises of 117 pages Introduction 2 page; theconclusions 1 page; recommendations 1 page The main content isdivided into four chapters: Chapter 1: overview (35 pages); Chapter2: the object and methodology (20 pages); Chapter 3: study results(27 pages); Chapter 4: discussion (30 pages); There are 40 tables, 13figures and 7 charts, appendices and 131 references of those 28 were
in Vietnamese and 103 were in English
CHAPTER 1 OVERVIEW 1.1 MICROFLORA IN HUMAN
Normal microflora in human (normal microbial flora) is thecomplex 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
Trang 7the Staphylococcus aureus, Streptococcus viridians, Bacillus like
diphtheria and spore bacteria in the air It is accounted for about 1010
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,m,
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 100C-450C 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 encodesfor 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
S aureus is a member of the bacterial flora in humans About 20-30% of healthy people carry S aureus Long time ago, the
relationship between carriers of bacteria and food poisoning caused
by S aureus was mentioned and studied, particularly in those who
having direct contact with food during the processes of production,processing, transportation, distribution and storage of food Thesebacteria can be persistent, intermittent on the host body Also, insome individuals the existence of these bacteria has not been found
Carriage prevalence of S aureus among food handlers is about 40% according to several different studies The strains of S aureus
Trang 820-isolated from food handlers also possessed a high rate of antibioticresistance and contained the toxin genes such as the enterotoxin
genes se: sea, seb, sec, sed, see (80-90%).
1.4 THE PATHOGENIC FACTORS OF S aureus
There are many factors affecting to the establishment of
pathogenicity in S aureus These can be divided into three groups:
cell surface-associated, extra cellular proteins and others
1.4.1 Cell surface-associated protein: peptidoglycan, teichoic acid,
protein A, capsula, adhesins
1.4.2 Extra cellular proteins: enterotoxins, toxic shock syndrome
toxin, hemolyasin, exfoliatin
1.4.3 Other pathogenic factors: coagulase, alpha toxin, leukocidin,
staphylokinase enzymes, Panton-Valentine leukocidin,staphyloxanthin, some of others extracellular enzymes
1.5 TOXIN GENES OF S aureus
Pathogenic factors of S aureus are controlled by a gene or some
genes directly or indirectly through enzymes In this study we justfocused on the toxin genes of importance in these bacteria
1.5.1 Coagulase encoding gene 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, D2regions, central region, the repeated region (segments with 81 bpnucleotid), 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,
Trang 9fast, 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 genescoding for enterotoxins have been identified in the mobile geneticfactors such as plasmids, bacteriophages Moreover, the pathogenicgenes 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 21.7%, ranking second only to E coli S aureus can cause several
18.5-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
1.7.2 Molecular biology techniques: molecular biology techniques
have been applied more and more in the diagnosis and study of S aureus These methods are faster, less costly in some cases In
particular, these techniques give results with high sensitivity,specificity such as PCR, realtime PCR, nested PCR, RFLP, PFGE,
Trang 10MLST, gene sequencing
CHAPTER 2 OBJECTS, MATERIALS AND METHODS
2.1.2 Study objects for the distribution of toxin genes of S.
p: carriage prevalence of S aureus in food handlers
according to previous studies (33%);
εp): accuracy relative to p, εp) = 0.2, n = 195 Number ofeligible objects are studied was 212
Trang 11The 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 becollected 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
(according to the procedures described by Goh et al.)
4 sets primers:
COAG1:5’-ATACTCAACCGACGACACCG-3’ COAG2: 5’-CGAGACCAAGATTCAACAAG-3’ COAG3: 5’-AAAGAAAACCACTCACATCA-3’
COAG4: 5’-GATTTTGGATGAAGCGGATT-3’
The PCR products were treated with restriction enzymes AluI
Trang 122.4.4.3 Identification of enterotoxin coding genes (se)
Table 2.1: Primer sets for se genes
Gene Primer name Nucleotide sequence (5’-3’) Reference
sea SEA-1
SEA-2
tggaaacggttaaaacgaagaaccttcccatcaaaaaca
Johnson et al
(1991)
sed SED-1SED-2 ctagtttggtaatatctccttaatgctatatcttataggg Johnson et al (1991)see SEE-1SEE-2 aggttttttcacaggtcatcccttttttttcttcggtcaatc Merhotra et al.(2000)seg SEG-1
SEG-2
aagtagacatttttggcgttccagaaccatcaaactcgtatagc
Nashev et al
(2004)
2.6 CARRIER STATUS OF S aureus
Table 2.8: Evaluation standards to determine carrier status
of S aureusNumber of sampling with S aureus Carrier status
1 – 3/4 of the test results Intermittent
2.7 TIME PERIOD AND STUDY SITE:
The study was conducted from July 2009 to March 2012 at theMicrobiology Department, Hanoi Medical University and Clinical
Trang 13laboratories of National Hospital of Tropical Diseases in Hanoi,Vietnam.
2.8 ANALYTICAL METHOD AND DATA PROCESSING
+ The data are processed by using software SPSS 16
+ Test 2 is used to compare the difference between the two rates.Value of p<0.05 is considered the statistically significant differences
CHAPTER 3 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
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
Trang 14(66.04%) among the total number of participates The second was thegroup ≤ 19 years, constituted in 19.81% Finally the group ≥ 50 wasthe 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
3.1.3 Carriage prevalence and carrier status of S aureus in nose
and relevant factors
3.1.3.1 Carriage prevalence and carrier status of S aureus in nose
Trang 15Chart 3.2: Carriage prevalence and carrier status
Chart 3.3: Carriage prevalence and carrier status of S aureus in
nose by gender
Intermittent carriage of S aureus was higher in females than in
males (29% vs 12.5%), whereas persistent prevalence was higher inmales than females (1.79% vs 1%) This difference is statisticallysignificant with p < 0.05
3.1.3.4 Carriage prevalence and carrier status of S aureus in nose
by working sections
p<0,05
%
%
Trang 16Chart 3.4: Carriage prevalence and carrier status of S aureus in
nose by working sections
Intermittent carriage of S aureus in the nose was the highest in
group of waiters and waitresses (29.41%), the lowest was in thegroup of cooks (10.13%) Persistent carriage in these two groups was1.96% and 1.27%, respectively This difference is statisticallysignificant with p < 0.05
3.1.4 Carriage prevalence of S aureus in hands
3.1.4.1 Overall carriage prevalence of S aureus in hands
Table 3.14: Carriage prevalence of S aureus in hands
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