| Campylobacter jejuni is a major foodborne pathogen that cause gastroenteritis in the human world-wide, this pathogen can also cause Guillain-Barre syndrome and bacteremia when severe infection. With an increasing number of studies in recent years, it was found that diseases caused by C.jejuni and C.jejuni resistance are ingcreasing. In order to more effectively control drug-resistant C.jejuni and to prevent the related diseases, the resistant mechanisms of pathogenic bacteria must be studied. Some suspected clinical samples of C.jejuni have been collected in our laboratory, so we selected 6 clinical isolates from chickens for study, and a multi-drug resistant and pathogenic strain(No.1655) was screened out through a series rsistance and virulence phenotype tests, and then de novo genome sequencing technology and molecular biology methods was used to analyze the genomic features, resistance mechanisms and pathogenesis for screened strain, attempted to clarify the relationship between resistance and virulence. This topic explained the mechanism of clinical resistant and pathogenic C.jejuni to some extent, and provides favorable datas for biological repository, at the same time provides the theoretical guidance and technical support to prevent the resistant and pathogenic C.jejuni generation and dissemination.1. PCR identification and MIC determination for 6 suspected clinical C.jejunisAll organisms(No.1442,1447,1614,1622,1655,1685, RM1221 and ATCC33560, respectively) were stored in glycerol broth at -80℃, the Campylobacter strains were routinely cultured on improved Skirrow agar and incubated at 42℃ under microaerobic conditions (5%O2,10%CO2, and 85%N2) for 24-48 hours, observed colony morphology, single colonies were picked in MH broth and passaged twice. Extracted bacterial genomic DNA, according to literatures designed 16SrRNA and mapA primers, then PCR method was taken for identified the clinical strains with these two pairs of primers. Amplified fragment length with two pairs of primers were 857bp and 589bp respectively. After PCR amplification and sequencing analysis, ultimately determined the 6 suspected strains are C.jejunis.Agar dilution method was taken for drug susceptibility tests of 6 clinical isolates of C.jejunis, as ATCC33560 for quality control. Eight drugs of four kinds that more commonly used in the treatment of Campylobacteriosis are fluoroquinolones, macrolides, tetracyclines, and aminoglycosides. The results showed that 6 isolates were all resistant to the eight drugs. So drug resistance in clinical isolates is still very serious.2. In vivo toxicity tests and LDso determination of the clinical isolatesInfected mode chicks with 1mL bacteria by intraperitoneal injection and the pre-reagent volume was 1×109CFU/mL (obtained by counting),1442,1622 and 1655 group had suffered different degrees of death within 24h, and the most severe group was 1655 group. Therefore, initially screened this 3 strains to determine the LD50.According to the results, oral infected 2-day-old healthy chicks with 3.7×108CFU/mL, after two weeks the 1442 and 1622 groups only high concentrations appeared 1-2 deaths, so the LD50 can not be calculated, while the 1655 group had different mortality rates at different concentrations and the LD50 was 6.1×107CFU/mL. As the same concentration and dosage infected 2-day-old healthy chicks by intraperitoneal injection, although the 1442 and 1622 group occurred a different mortality at different concentrations,1655 group at the lowest concentration of 3.7×105CFU/mL also occurred 100% mortality. Combined with the results of oral infections, we determined 1655 strain has a very strong virulence. Then intraperitoneal infected 16-day-old healthy chicks with 3.1×108CFU/mL,1442 and 1622 grous did not appear death, but 1655 group still had a rather high mortality rate and the LD50 was 1.36×107CFU/mL. Thus, we initially concluded that 1655 was a pathogenic strains.3. Adhesion, invasion and intracellular survivability of C.jejunis to macrophage RAW264.7 cells.Determination of LD50 in order to inspected the in vivo toxicity, and it also need to verify the in vitro virulence, including detected adhesion and invasion of C.jejunis to macrophage RAW264.7 cells, as well as the intracellular survivability. Gentamicin protection method was used for cell experiments of 1442,1622 and 1655 strains, RM1221 as a reference strain.The results showed that:adhesion and invasion of 3 clinical isolates compared with RM1221 had a significant difference. For 3 clinical strains, adhesion and invasion between them there was no significant difference. Combined with intracellular viability experiments,1442 can survive to 48h, while 1622 and 1655 can survive to 72h. And will be apparent at each time point, the invasion number of 1655 strain still significantly higher than 1622, maybe it is the reason that 1655 strain exhibits more toxic than the other two strains. Therefore, the 1655 strain as the focus in subsequent trials and for whole genome sequencing and analyzed the resistance and toxicity-related mechanisms.4. Whole-genome sequencing-De novo sequencingBacterial genome extraction kit was used to extract the target genome DNA of target bacterial 1655, samples was sent to the company to conduct quality control, library construction and quality control, etc., and then sequenced the genome with machine, Solexa method was selected to sequencing. Our experiments required two-terminal sequencing, read length was 100nt and build a random library. Then conducted contig stitching, completed genome ring sketch and annotated and predicted genome when sequencing work was completed, bioinformatics was used to analyze the seqencing results of resistance and toxicity mechanisms of 1655 strain. According to the results, mechanisms that caused high levels resistance to 4 kinds drugs have been reported were all existed in 1655 strain, such as Thr-86-Ile mutation on the gyrA gene caused high-level resistance to fluoroquinolones, A2075G mutation on the 23SrRNA gene caused high-level resistance to macrolides, tetO exogenous genes obtained that mediates resistance to tetracycline and aminoglycoside, and the presence of aph-A and aadE gene resulted in resistance to aminoglycoside. Besides, there was a predicted protein with unknown function located on plasmids pCG8245 that is a resistance plasmid. Further studies of this plasmid-encoded protein with unknown function, maybe find some new resistance mechanisms. In addition, it also found some mutations and deletions have not been reported previously, but wether these mutations and deletions related to bacterial resistance need to be further verified. For the analysis of virulence mechanism showed that:1655 strain in addition to carrying some virulence factors that have an effect on pathogenicity, the phages and pathogenicity island that encode virulence genes and type IV secretion system homologous genes were not predicted, but the known causative factors may be used to explain its pathogenicity(such as virulence factors); However, comparative genomic analysis revealed a large number of SNP, as well as through the co-linear analysis with reference genome NCTC11168, RM1221 and 81-176 found there were serious deficiencies, rearrangements, and inversion in 1655 strain genome(including internal code and intergenic regions), this suggested that moderate genetic changes may enhance the virulence of 1655 strain. Besides, the variable gene region, phage, transposons and iron uptake systems on the chromosome and the plasmids pTet are all associated with toxicity may be involved in the virulence mechanisms. The results also showed, there were differences in gene contents between strains, it also provides a new perspective for subsequent analysis. Additionally, there were some predicted proteins with unknown function, some sequences with deletion and mutation, and biological components was differ from control strains, these genes work together may be have an impact on bacteria virulence and resistance. Given the complex environmental background of clinical isolates, the reasons that cause pathogenicity is relatively complex. So just to analyze the differences in contents and structure of the genome is not comprehensive enough, some mechanisms still can not be explained.To sum up, in this experiment, through a series phenotypic experiments we successfully screened out a multiple drug-resistant and pathogenic strain, de novo genome sequencing method and molecular biology techniques were taken for analyze the related mechanism. Clarified the reasons of multi-drug resistance and pathogenicity of clinical isolate, its complex pathogenic and resistance mechanisms were analyzed, and found some new possible mechanisms associated with resistance and virulence. Thus, the subject provides new foundation to study the multi-drug resistance of pathogenic C.jejuni; broaden the ideas to improve the resistance mechanisms; provides a new direction to study the pathogenesis of campylobacteriosis. In addition, for the analysis of gene structures and contents in this research, also provids favorable evidence to improve the C.jejuni genome information. |
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