MOLECULAR CHARACTERIZATION OF NEISSERIA MENINGITIDIS STRAIN CIRCULATING IN THE NORTH OF VIETNAM

Meningococcal disease describes infections caused by the bacterium that effect to brain and medulla with the high death rate about 10%20%. There are four main species related to this disease: Streptococcus pneumoniae, Neisseria meningitidis, Listeria monocytogiens and Haemophilus influenzae. In which, Neisseria meningitidis causes the most in children. Yearly, in the world, the people die owing to meningococcal disease about 400.000 500.000 people. In Vietnam, the period from 2001 2010, an average of 650 recorded cases of the disease each year, mainly in the northern provinces. In 2012, there were 125 cases and in early 2013, sporadic cases found in several provinces and cities throughout the country, the risk of transmission in the community is still high. According to the Ministry of Health, in the first 6 months of 2015, the country recorded 24 cases of meningitis caused by Neisseria meningitidis, an increase of 16 cases over the same period last year. One of the reasons for the limited success of the strategy of vaccine and antibiotic therapy is the diversity of the serotypes serosubtype and rapid genetic transformation of Neisseria meningitidis through recombination making these bacteria resist the vaccine and antibiotic treatment. In Vietnam, the meningococcal disease precaution vaccine is currently imported from abroad.

Fistly, I would like to dedicate my sincere thanks to my supervisor, Associ Prof Dong Van Quyen Vice director of Institute Biotechnology and my co-supervisor Nguyen Huu Duc, Ph.D Head of Department of Animal of Faculty of Biotechnology

for their intellectual support though out this study Without their supervision, guardianand invaluable advice, I would not able to complete my study on time

My heartfelt gratitude is dedicated to personnel from the Department of MolecularMicrobiology of Institute of Biotechnology for supplying me the experimental bacteria.Especially, Ms Le Thu Trang who directly guide me in process I conduct thesis

My appreciation is extended to my friends for their wonderful advices, helps andsupports in these years

Last but not least my warmest gratitude to my parent Mr Pham Trong Truong andMdm Duong Thi Anh, my lovely old sister Pham Thuy Phuong and my little sisterPham Thi Bich My Their continuous support, care and love have built me to be abetter person Thanks for being with me always

Student

Pham Thi Ngoc

CONTENTS

CHAPTER I: INTRODUCTION 5

CHƯƠNG II: LITERATURE REVIEW 8

2.1 Meningococcal disease 8

2.1.1 History of discovery 8

2.2 Neisseria meningitidis research situation and meningococcal disease in the world and in Vietnam 10

2.2.1 Worldwide 10

2.2.2 In Vietnam 11

2.3 Biological Characteristics 11

2.3.1 The nomenclature and classification of Neisseria meningitidis 11

2.3.2 Culture medium 13

2.3.3 Resistance 14

2.4 Molecular characteristics 14

2.4.1 Target genes characteristics discovered Neisseria meningitidis 14

2.4.2 Target genes characteristics discovered serogroup of Neisseria meningitid 16 2.4.3 Known strains of Neisseria meningitidis 18

CHAPTER III: MATERIALS AND METHODS 20

3.1 Materials 20

3.2 Equipment and chemicals 20

3.3.1 Equipment 20

3.3.2 Chemicals 20

3.4 Methods 20

3.4.1 Total DNA extraction 20

3.4.2 Primer design 21

3.4.3 PCR amplification 21

3.4.4 DNA Electrophoresis 23 3.4.5 DNA sequencing 26 3.4.6 Phylogenetic tree analysis 27

TABLE OF CONTENTS

Table 2.1 Biology characteristic of Neisseria meningitidis 12

Table 2.2: Type of serogroup capsule and the target gene for genotype of 18Neisseria meningitidis 18

FIGURE OF CONTENTS

Figure 2.1: Global Distribution of Invasive Meningococcal Desease by Serogroup 9

Figure 2.2 : Gram stain of Neisseria meningitidis in cerebrospinal fluid (CSF) with

associated PMNs 13

Figure 2.5: Characteristics of genes coding for antigens Neisseria meningitides 16 Figure 2.6: Capsule gene map (CPS) of Neisseria meningitidis (37), ctrABCD

operon coding ATP-protein (frame grid) SynABC (gray), D / E / F / G (dots),

sacABCD (horizontal stripes), xcbABC (slash), encoding serogroup- specific enzymesfor synthesis capsule oatC (serogroup C) and oatWY (W135 and Y serogroups), combined with syn operons and encoding O-acetyltransferases LipA and liB coding protein Ctre and ctrF known as Lipa and LipB 18Figure 2.7: The principle of PCR 22Figure 2.8: The principle of electrophoresis 24

LIST OF ACRONYMS

CSF: cerebrospinal fluid

N.meningitidis: Neisseria meningitidis

PCR: Polymerase Chain Reaction

CHAPTER I: INTRODUCTION Meningococcal disease describes infections caused by the bacterium that effect tobrain and medulla with the high death rate about 10%-20% There are four main species

related to this disease: Streptococcus pneumoniae, Neisseria meningitidis, Listeria

monocytogiens and Haemophilus influenzae In which, Neisseria meningitidis causes the

most in children Yearly, in the world, the people die owing to meningococcal diseaseabout 400.000- 500.000 people In Vietnam, the period from 2001 - 2010, an average

of 650 recorded cases of the disease each year, mainly in the northern provinces In

2012, there were 125 cases and in early 2013, sporadic cases found in several provincesand cities throughout the country, the risk of transmission in the community is stillhigh According to the Ministry of Health, in the first 6 months of 2015, the country

recorded 24 cases of meningitis caused by Neisseria meningitidis, an increase of 16

cases over the same period last year One of the reasons for the limited success of thestrategy of vaccine and antibiotic therapy is the diversity of the serotypes / serosubtype

and rapid genetic transformation of Neisseria meningitidis through recombination

making these bacteria resist the vaccine and antibiotic treatment In Vietnam, themeningococcal disease precaution vaccine is currently imported from abroad.Therefore, the vaccine may not be well compatible with the antigenic capacity ofstrains circulating in the country resulting in a decrease of the vaccines effectiveness.Therefore, an urgent issue now is to study the distribution, molecular epidemiology and

characteristics, genetic variation and evolution of Neisseria meningitidis strains

circulating in Vietnam to have appropriate strategies to prevent human frommeningococcal disease effectively Based on this fact, we carried out this research:

''Molecular characterization of Neisseria meningitidis strain circulating in the north of

Vietnam’’ to define characteristics of these strains Results of the research will providemore information for further studies and especially to select suitable strains for vaccine

production to protect human from meningococcal disease caused by Neisseria

meningitidis meningococcus in Vietnam In this project, we will investigate, evaluate

epidemic meningitis situation due to Neisseria meningitidis in the northern of Viet

Nam; analyse the genetic characteristic, original evolution, genetic transformation in

nucleotide and amino acid level of Neisseria meningitidis strains circulating in the

northern of Vietnam The results of the study play important role in selecting strains toproduce suitable vaccine, oriented to the import and indigenous vaccine production and

meningitis epidemic precautionary strategy caused by Neisseria meningitidis in Viet

Nam

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CHAPTER II: LITERATURE REVIEW

2.1 Meningococcal disease

Meningococcal disease can refer to any illness that is caused by the type of

bacteria called Neisseria meningitidis Neisseria meningitidis, often referred to

as meningococcus, is a gram negative bacterium that can cause meningitis and otherforms ofmeningococcal disease such as meningococcemia, a life-threatening sepsis alsoknown as meningococcus These illnesses are often severe and include infections of thelining of the brain and spinal cord (meningitis) and bloodstream infections (bacteremia

or septicemia) Meningitis and meningococcemia are major causes of illness, death,and disability in both developed and under-developed countries There areapproximately 2,600 cases of bacterial meningitis per year in the United States, and onaverage 333,000 cases in developing countries The case fatality rate ranges between 10and 20 percent.[1] The incidence of endemic meningococcal disease during the last 13years ranges from 1 to 5 per 100,000 in developed countries, and from 10 to 25 per100,000 in developing countries During epidemics the incidence of meningococcaldisease approaches 100 per 100,000 The disease's pathogenesis is not fully understood.The pathogen colonises a large number of the general population harmlessly, but insome very small percentage of individuals it can invade the blood stream, and the entirebody but notably limbs and brain, causing serious illness Over the past few years,experts have made an intensive effort to understand specific aspects of meningococcalbiology and host interactions, however the development of improved treatments andeffective vaccines is expected to depend on novel efforts by workers in many differentfields

While meningococcal disease is not as contagious as the common cold (which is spreadthrough casual contact), it can be transmitted through saliva and occasionally throughclose, prolonged general contact with an infected person Meningococcal vaccines havesharply reduced the incidence of the disease in developed countries Meningococcusbacteria are spread through the exchange of respiratory and throat secretions like spit(e.g., by living in close quarters, kissing) Meningococcal disease can be treated with

antibiotics, but quick medical attention is extremely important Keeping up to date withrecommended vaccines is the best defense against meningococcal disease.

2.1.1 History of discovery

Meningococcal disease was described by Vieusseux in 1805 during an outbreakwith 33 deaths in the vicinity of Geneva, Switzerland [2] The Italian pathologistsMarchiafava and Celli first described intracellular oval micrococci in a sample of CSF.The Italian pathologists Marchiafava and Celli (1884) first described intracellular ovalmicrococci in a sample of CSF.[2]However, Anton Weichselbaum in 1887 firstidentified bacterium causing meningococcal disease in the CSF of six of eight patients

of bacterial meningitis and the bacterium was named Neisseria

Neisseria meningitidis causes a disease spectrum ranging from occult sepsis with

rapid recovery to fulminant disease Before the 1920s, meningococcal disease was fatal

in up to 70 percent of cases [3] Serum therapy with serum from immunized horses,introduced at the beginning of this century by Jochmann in Germany and Flexner in theUnited States, could reduce mortality from nearly 100% to 30%.[4], [5] The discovery

of sulfonamides and other antimicrobial agents led to a further decline in case fatalityrates Despite treatment with appropriate antimicrobial agents and optimal medicalcare, the overall case fatality rates have remained relatively stable over the past 20years, at 9 to 12%, with a rate of up to 40 % among patients with meningococcal sepsis.[6] Eleven percent to 19% of survivors of meningococcal disease have sequelae, such

as hearing loss, neurological disability, or loss of a limb.[7]

2.1.2 Distribution

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Figure 2.1: Global Distribution of Invasive Meningococcal Desease by Serogroup

2.2 Neisseria meningitidis research situation and meningococcal disease in the

world and in Vietnam.

2.2.1 Worldwide

Currently, in the world there are many studies from general to delve into the study of

genetic characteristics At least 8 complete genomes of Neisseria meningitidis strains

have been determined and bring in Genbank For example: the genome of strain MC58 (serogroup B), strain H44/76 and strain NMA510612 (serogroup A) Most recent is

complete Genome sequence of Neisseria meningitidis serogroup A strain NMA510612,

Isolated from a Patient with bacterial menigigtidis in China that published in 2014 Beside research, some developmental nations produced vaccine to prevent For instance,

In United States, a number of vaccines are available in the U.S to prevent

meningococcal disease Some of the vaccines cover serogroup B, while others cover A,

C, W, and Y.[8] A meningococcal polysaccharide vaccine (MPSV4) has been available since the 1970s and is the only meningococcal vaccine licensed for people older than 55 MPSV4 may be used in people 2–55 years old if the MCV4 vaccines are not available or contraindicated Two meningococcal conjugate vaccines (MCV4) are licensed for use in the U.S The first conjugate vaccine was licensed in 2005, the second in 2010 Conjugatevaccines are the preferred vaccine for people 2 through 55 years of age It is indicated in those with impaired immunity, such as nephrotic syndrome or splenectomy The Centers

for Disease Control and Prevention (CDC) publishes information about who should receive meningococcal vaccine.[9]

In June 2012, the U.S Food and Drug Administration (FDA) approved a combination vaccine against two types of meningococcal diseases and Hib disease for infants and children 6 weeks to 18 months old The vaccine, Menhibrix, was designed to prevent

disease caused by Neisseria meningitidis serogroups C and Y, and Haemophilus

influenzaetype b (Hib) It was the first meningococcal vaccine that could be given to

infants as young as six weeks old.[10]

In October 2014 the FDA approved the first vaccine effective against serogroup B, named Trumenba, for use in 10- to 25-year-old individuals.[11]

In 2010, the Meningitis Vaccine Project introduced a vaccine called MenAfriVac in the African meningitis belt It was made by generic drug maker Serum Institute of

India and cost 50 U.S cents per injection Beginning in Burkina Faso in 2010, it has been given to 215 million people across Benin, Cameroon, Chad, Ivory

Coast, Ethiopia, Ghana, Mali, Niger,Mauritania, Nigeria, Senegal, Sudan, Togo and Gambia.[12] The vaccination campaign has resulted in near-elimination of serogroup A

meningitis from the participating countries.[13]

2.2.2 In Vietnam

The literature on the epidemiological situation meningococcal meningitis in Vietnam isstill very limited According to some documents originally recorded epidemicmeningococcal meningitis owing to N meningitidis belong to serogroup C cause in thesouthern provinces of Vietnam from 1977 to 1979 The death rate from an estimated27.4% to 34.7% (Oberti, Hoi et al 1981) Seventy percent of cases occur in childrenaged 3 to 15 years old After the large epidemic of meningitis in 1977, four researcherstracked over time for meningitis was conducted from 1993 to 2005 (US, Diep et al.,1998; Tran, Le et al 1998; DD 2006 ; Nguyen, Tran et al 2007) In particular,meningococcal meningitis accounted for 0.5% of positive cases confirmed by culturemethod from blood samples and about 4 to 8.5% of cases of bacterial meningitis was thecause determined From 2000 to 2002, the estimated incidence of meningococcalmeningitis in Hanoi, Vietnam, is 21.8 cases / 100,000 children aged 7-11 months (95%

CI 5.0-94.4) and 2.6 cases / 100,000 children under 5 years of age (95% CI 0.8-8.5) (DD

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2006) However, no data on the disease-causing serogroups in this study Full study on

the most recent meningococcal meningitis and Neisseria meningitidis infection in

children in Vietnam was published by Kim et al (Kim, Kim et al 2012) according to a

survey on 700 samples collected children under age 5 in Hanoi, using PCR specificity,

detecting N meningitidis infection rate in this group was 14.2% According to our

understanding, so far no study has been conducted to assess the genetic variation or

genes regulated virulence of pathogenic serogroups of N meningitidis in Vietnam's

2.3 Biological Characteristics

2.3.1 The nomenclature and classification of Neisseria meningitidis

Nomenclature

Phylum: Proteobacteria Genus: Nesseria

Class: Beta Proteobacteria Species: N meningitides

Ordo: Neisseriales Serogroup: A, B, C, D, 29E, H,

I,L,W135,X,Y,Z

Genus Specific

species

Gram stain

Shape Capsule Arrange Mobile

Respira -tory

Grow Culture

Inside / outside cell

Tạo vỏ hoặc không

Phế cầu khuẩn

Không di động

Hiếu khí

martinBlood Agar orChocolate

Thayer-Gonococcus:inside cell

Neisseria meningitidis:

out cell

Table 2.1 Biology characteristic of Neisseria meningitidis

Meningococcal is Gram-negative cocci, size changes, can be found in the form thelonely or beans with flat sides facing each other and can be located inside or outside of a

neutrophil, hairless, no spores, most strains are capsule

Figure 2.2 : Gram stain of Neisseria meningitidis in cerebrospinal fluid (CSF) with

associated PMNs

Polysaccharide layer (PS) capsule: as antigens, that generated antibodiesprotection (except for group B) Based on the heterogeneity of the structure andproperties of PS antigen was found to be 13 serogroups of meningococcal bacteriaincluding: A, B, C, D, 29E, H, I, K, L , W135, X, Y, Z Mannosamine serogroup Acontains phosphate, while serogroups B, C, Y, W135 contains sialic acid - which plays

an important role in the survival and toxicity Capsular polysaccharide of serogroup Band C include acid homopolymer of N - acetyl - neuraminic associated with α – 2.8 and

α - 2.9 Small differences in the structure leads to different immne properties explicitly:while structural α - 2.9 is a strong antigen to the body and produce antibodies thatprotect the α - 2, 8 has antigenically very weak

The structure of the group antigen and subtypes in serum change in vivo quickly

on an individual person and in the community The loop (circuit) can be changed on theexposed surfaces of both porins, can change antigenically by adding or subtracting theamino-acid or by horizontal transfer of gene fragments representing parts

X antigens: antigens with Neisseria Gonorrhoeae, Streptococcus Pneumoniae This

antigen is used in a number of serological diagnostic techniques

2.3.2 Culture medium

Meningococcal bacteria are aerobic, developing in an environment with 5% bloodagar, Thayer-Martin agar environmental or chocolate, even Luria Bertani medium, at atemperature of 35℃-37℃, in 5-7% CO2 On blood agar, small colonies, round, opaque,

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