| BackgroundNeisseria meningitidis (N.m) is a Gram-negative human specific bacterium and despite effective antibiotics and partially effective vaccines, it remains one of the leading causes of bacterial meningitis worldwide, and can also cause sepsis, pneumonia, and other localized infections. Based on the immunogenicity and structure of the capsule polysaccharide, N.m can be classified into 12 serogroups, and the majority of meningococcal cases are caused by members of serogroups A, B, C, X, Y and W. The epidemiology of meningococcal disease varies substantially by geographic area. In Africa, most meningococcal disease is associated with serogroup A, but serogroups C, X and W also occur whereas in the Americas, serogroups C and B predominate, the latter being the most prevalent in Australia and Europe. Serogroups Y and W have also been associated with a substantial proportion of infections in various countries.In China, N. meningitidis serogroup A was responsible for most cases in the last century, and reported annual disease incidence reached up to 400 cases per 100,000 inhabitants., whilst B and C occurred only sporadically. Serogroup A meningococcal polysaccharide vaccine(MPV) was approved for use in 1980, and vaccination coverage of 93.6%. Then the incidence level showed a declining trend, and the incidence rate declined to<0.2 per 100,000 after 2000. However, during 2003 and 2005, a sudden increase in the number of cases with serogroup C strains occurred in Anhui province, and spread nationwide.Epidemiology of N.m contributes to understand bacterial spread and identify the hyper virulence clones. MLST(Multilocus sequence typing) indicated that sequence type (ST) 4821 complex, a new hyper virulent lineage, was the cause of outbreaks after 2003. MLST was considered as a golden standard for molecular typing, it cannot differentiate ST-4821 N.m. And N.m serogroup C ST-4821 spread nationwide as asymptomatic carriage in more than 22 provinces in China. Serogroup and ST results revealed that these strains were seemingly isolated from one clone. However, whole genome sequencing results tell us that there are no two identical strains. If diversity between N.m serogroup C ST-4821 isolates were tested by whole genome sequencing, it will cost a lot of time and labor, and is not suitable for all the strains.MLVA (multilocus variable-number tandem-repeat (VNTR) analysis) is a PCR-based technique that utilizes the variability in the number of short tandem repeat sequences that is used to create DNA fingerprints, and has proved to be highly discriminatory and provide useful information on phylogenetic relationships among several bacterial species. MLVA with various VNTR loci has also been applied for fine typing of meningococcus isolates with varying success in differentiating between and within some STs or ST complexes. However, these schemes were based on strains from Europe and America, and were not entirely suitable for N.m from China. Therefore, it is necessary to establish fit MLVA schemes for N.m from China and evaluate its practicability.Objectives1. Selecting VNTR loci for N.m from China, and establishing MLVA method.2. Establishing MLVA database and improving MLST database of N.m in China.3. Completing MLVA typing of some N.m isolates from China, and establishing different schemes according to the microbiologic and epidemiologic characteristics. Comparing MLVA results based on different schemes, and optimizing according to different aims.4. Analyzing phylogenetic characteristics and micro-evolution of N.m serogroup C including ST 4821 complex.5. Applying and evaluating MLVA in outbreaks caused by N.m, and establishing methods for rapidly identifing outbreak-associated isolates.Methods1. Strains selection. Quota sampling, proportion allocation in stratified random sampling and convenience sampling were used to select N.m strains based on the numbers, distribution and microbiologic characteristics.Quota sampling was used to select strains for identifying VNTR loci, and strains from the N.m database were stratified successively by serogroup, ST and PFGE types. And each strain with different serogroup, ST or PFGE type was selected.Proportion allocation in stratified random sampling was used to select strains for establishing MLVA database. Two hundred strains were selected first and strains were stratified successively by years, serogroup, ST and PFGE types. Convenience sampling were used when the above features are same, and the strains were selected when they were culturing or their DNA could be obtained.Quota sampling and proportion allocation in stratified random sampling were used according to objectives when adding strains.2. VNTR loci selection. VNTR loci were collected from references, and some of them were deleted first when their PCR results displayed ’multiple bands’, or’not in all the strains’, or’single copy’, or’diversity among flanking region’. The others were tested in N.m from China and were selected according to PCR products. Those with ’high diversity’,’existing in all tested strains’,’single band’ and’stable among flanking region’ were up to standard.3. MLVA. PCR of the VNTR loci was performed, and the forward primers targeting each VNTR locus were labeled at the 5’end with fluorescent dyes. Then the PCR products were tested on an automated DNA sequencer to know the ssessed sizing, which was used to calculate the number of repeat units in each locus. When the assessed PCR product showed 0.5 copy, DNA sequencing was used to identify the real data. The calculated numbers of repeats of the VNTR loci (alleles) are combined into a string which consists of integers and is referred to as the MLVA profile. Each unique MLVA profile was submitted to the bioinfotool internet to obtain the Nei’s and evalue the discriminatory power of each VNTR locus. The MLVA profile were importted to BioNumerics and can be used for comparison and clustering by UPGMA MLVA types and clonal culstering trees can also be obtained.4. MLST. From pubmlst internet, STs and ST complexes of N.m were obtained by MLST, which analyze seven housekeeping genes. Import the results into BioNumerics and analyze the distribution characteristics.5. Compare the discriminatory power of MLVA and MLST, and describe the similarity based on clutering results. Analyze the polygenetic and microevolutionay characteristics of N.m serogroup in China.Results1. Fifty-two strains were used to select VNTR loci. They were isolated from patients, contacts or healthy carriers among 22 provinces of China.Four hundred and thirty strains were used to establish MLVA database. Twenty-eight among them were isolated during 1977 to 1996, including 16 serogroup A and 12 serogroup B strains. Other 402 isolates were obtained during 2003 to 2014, including 49 isolates from an outbreak in 2010 and 43 from another outbreak in 2014. By entering MLVA results of 430 N.m isolates into Excel and BioNumerics, we established N.m MLVA database in China.2. A total of 56 VNTR loci were collected from three references. After initially selecting,19 loci were tested in 52 isolates. Then three loci were deleted for two of them were not found in all strains and one is not convenient due to sequencing. Finally,16 VNTR loci were used in our study to analyzeiN.m from China by MLVA.3. Four hundred and thirty N.m isolates were tested by MLVA with 16 VNTR loci, and 6880 records were obtained and imported into Excel to establish our MLVA database initially. MLST were carried on 122 N.m isolates without STs, and 854 records were imported into our MLST database.4. MLST and MLVA results among 215 N.m serogroup C isolates were clustered by BioNumerics. These strains were divided into 29 STs and many MLVA types due to different MLVA schemes.When 16 VNTR loci were used,215 isolates were divided into 201 MLVA types, including 182 ST-4821 complex strains into 176 types and 118 ST-4821 strains into 112 types. Six loci (VNTR1,2,4,5,18 and 19) with Nei’s above 0.5 were named high variable loci, and other ten were named stable loci. Among the six high variable loci, five displayed the same typing results to all the 16 ones. When the ten stable loci were used in MLVA,215 isolates were divided into 55 MLVA types, including 182 ST-4821 complex strains into 32 types and 118 ST-4821 into 11 types. Phylogenetic characteristics showed similar trends by MLST and MLVA results with ten stable VNTRloci.5. MLVA scheme with five stable VNTR loci (VNTR7,9,12,14 and 15) can be used to identify outbreak associated isolates. From clustering results that were generated by BioNumerics with this scheme, outbreak associated strains from the same outbreak clustered into one group and seperated from non-outbreak associated ones. However, some sporadic strains clustered into one group with outbreak associated isolates.Conclusion1. Combination of VNTR loci in MLVA should be different due to coutries, and the epidemiologic information should be considered.2. MLVA can be used to analyze diversity and phylogenetic pattern of N.m in China, and different schemes should be selected due to research aims. Five high variable loci could be used to analyze the diversities and compare differences among strains, while ten stable loci should be selected to find the phlogenetic characteristics and similarities.3. MLVA has higher resolution than MLST, and five high variable loci (VNTR1,2,4,18 and 19) can be used to analyze diversities of N.m serogroup C in China. Two hundred and fifteen N.m isolates were divided into 29 STs and 201 MLVA types.4. Diversity exists in N.m serogroup C in China, and the prevalent clone is ST-4821 complex. Micro-evolutionary characteristics of N.m ST-4821 complex serogroup C displayed the diversity of VNTR loci during the spread in last decade, but seven housekeeping genes of MLST and ten stable VNTR loci of MLVA shows similarity.5. MLVA with five stable VNTR loci can be used to rapidly screen outbreak associated strains in the same outbreak caused by N.m ST-4821 complex serogroup C.Innovation and Significance1. Developed MLVA method of N.m in China, and MLVA database was established initially. A great variety of strains in the database covered extensive years, areas and populations, and they were isolated during 1977 to 2014 from patients, contacts or healthy carriers among 23 prvinces. These strains iclude eight serogroups, 111 STs and 114 PFGE types.MLVA can be carried on N.m isolates or other samples including swabs from patients, therefore, pathogenic information will be obtained in spite of the lost of strains. MLVA type can be found from the database when results were submitted, and a new type will be assigned if the profile is different from every record. We can analyze the spread route by compring MLVA results and epidemiologic characteristics of the new isolate with those in the databae. If the isolate was assinged a new MLVA type, we can improved the database by importting the profile, and compare the loci among the same serogroup or ST complex. Based on the diversities of isolates belonging to special serogroup or ST complex, we can analyze their micorevolutionary characteristics comparing the differences of VNTR loci.2. Micorevolution of N.m serogroup C in China was first analyzed by MLVA, and results displayed that N.m ST-4821 serogroup C strains were not same for diverisities of VNTR loci. Also for the first time, we suggest that different MLVA schemes should be used in N.m studies due to research aims.N.m serogroup C ST-4821 has been isolated almost every year from patients or healthy carries. Although belonging to the same serogroup and ST, they are different in the copies of some VNTR loci, and this make the study of microevolution possible. It contributes to discover key hereditary change in N.m and provide bases to find the source of variations in pathogenicity or tolerance, and analyze the adaptations. Sixteen VNTR loci were tested in this study, but only some of them were used according to different aims. Bsed on these schemes, similarities and differences among N.m ST-4821 serogroup C strains were obtained, and this administer to the epidemiologic investigation. Then we could identify the advantageous heredity and disadvantageous variation of transmission, prevalence and pathogenicity among these strains.3. For the first time, MLVA was applied to outbreak investigation caused by N.m in China, and outbreak associated isolates were identified only by five stable VNTR loci.Clinical and epidemiologic doctors collected samples from patients and their contacts timely in outbreaks, however, not all of the bacterial culture were successful. Therefore, DNA of some samples including swabs and bloods could be extracted and used for MLVA by five VNTR loci to identify outbreak associated strains. In addition, we should pay close attention when many samples of the same new MLVA types happens, analyze prevalent trends according to epidemiologic information and take active measures for disease prevention and control. Priority should be give to those who carried outbreak associated N.m when health resources are limited. |
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