Study On The Etiologic Genetic Diagnosis Of Antimicrobial-resistant Gram-negative Bacterial Infections

Gram-negative bacteria are one of the most important pathogens causing human infectious diseases. It composes the main part of the pathogens. The resistant rates of many gram-negative bacteria to commonly used antibacterial agents are now increasing. Bacteria which produce extended-spectrum β-lactamase are isolated frequently, and it is difficult to cure the infections. Thus, they are of particular concern. Infections caused by resistant pathogens are associated with significantly higher mortality rate than susceptible pathogens, and options for effective antimicrobial therapy are becoming increasingly limited. Furthermore, the absence of effective therapy will increase mortality . Accurate, early etiological diagnosis of bacterial infections lead to appropriate use of antibiotics. This in turn reduces side effects for the patient, decreases the cost and may prevent the spread of drug-resistance. Conventional microbiologic diagnostic method contains the culture of bacteria from clinical specimens and their subsequent antimicrobial susceptibility testing, thus, it need more time to provide a result (3~5day). With the advance in molecular biology and bacterial genomics, rapid, sensitive molecular methods were established as accepted methods for the detection of genes of resistant microbials. Based on the analysis of 23 S rRNA gene sequences and other genes sequence associated with antimicrobial resistance (SHV, CTX-M), we established a nucleic acid-based detection systems now offer rapid and sensitive methods to detect the presence of resistant microorganisms. It could provide identification and susceptibility data of pathogens rapidly, and may be helpful for the early etiological diagnosis and treatment of severe infections.Part 1 Study on molecular method to identify common pathogensThe rRNA (ribosomal RNA, rRNA) genes are essential for the survival of all bacteria and are highly conserved in bacteria. There are three genes, which make up of the rRNA functionality: the 5S, 16S, and 23S rRNA genes. Highly variable portions and conserved portion arrange discontinuous, the rRNA gene contain unique sequence for each bacterium in the variable portion, and provide useful information about therelationships between different bacteria, the information can be used to differentiate and identify bacteria. Furthermore, certain conserved regions of sequence are also found in all known bacteria, broad-range PCR primers may then be designed to recognize these conserved bacterial rRNA gene sequence and used to amplify variable regions. The 16S rRNA gene has been most commonly employed; however, more recently, attempt to employ 23 S rRNA gene to identify bacteria has become increasing. The objective of this part is to study the possibility of using 23S rRNA gene to identify pathogens. To analysis the sequence difference among the 23S rRNA genes from common bacteria, 23 S rRNA genes from 13 common bacteria were amplified with a broad-range PCR primer, and products were sequencing, data were analyzed with DNAStar software, including Staphylococcus aureus, coagulase- negative Staphylococcus, Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae, Escherichia coli, Klebsiella pneumoniae, Proteus spp., Enterobacter cloacae, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Burkholderia cepacia. The results show that there were a lot of diversity of 23S rRNA genes sequences of 13 pathogens. Based on the particular sequences, probes were designed and anchored on the nylon membrane; combination with digoxin labelled broad-range PCR primers, a novel PCR-reverse hybridization assay was established.This test permited identification of common bacteria by means of species-specific probes. The efficacy of PCR-reverse hybridization assay were evaluated by clinical isolates, and appear to be a specific and reproducable tool for detection or identification common bacteria. We can draw the conclusion that 23 S rRNA gene could be applied in bacteria detection. The methods we have established could identify 13 species, and it was specific and reproducable.Part 2: Study on the detection method of the antimicrobial-resistant gram-negative bacteria and antimicrobial-resistant genesIt is important to rapid identify antimicrobial resistant pathogens for diagnosis and treating severe infections. However, conventional methods are time-consuming, and could not satisfy the clinical needs for identification results. It is the main reason for the low cure rate and high mortality of severe infectious diseases. Thus, we need establish a rapid and sensitive detection method. There are a lot of species gram-negative bacteria which cause infectious diseases, and the mechanisms of antimicrobial agents resistance were complicated, so, it is a difficult problem. TheESBLs-producing pathogens threaten the health of human being most greatly. In China, ESBLs are mediated mainly by SHV and CTX-M gene. It leads to resistance to many commonly used antibiotics, it is hard to cure the infections due to this kind of bacteria and the mortality is high. Now, 20%~40% Escherichia coli and Klebsiella pneumoniae are found ESBLs-producing. It is very meaningful to detect this kind of resistant characteristic. We established PCR-reverse hybridization method to identify pathogens in the previous study, but it can only detect a few target genes concomitantly. In order to establish a new method to detect resistant genes and identify gram-negative bacteria rapidly, we conducted this study. Based on previous research, we studied the rapid genetic diagnostic method of bacterial identification and resistant genes using PCR-gene chip method. First, we sequenced and analyzed the 23 S rRNA genes of 26 species bacteria, identified the polymorphism points, designed probes, expanded the detection scope to 26 bacteria; at the same time, we analyzed the sequences of SHV, CTX-M ESBL genes, designed 3 pairs of primers(SHV, CTX-M-1 group, CTX-M-13 group), and constructed a multiplex PCR system. Combined with 23 S rRNA gene primers, we established multiplex PCR to amplify 23S rRNA gene and ESBL genes. Then, based on the probes used in the nylon membrane hybridization method, we designed more probes to detect bacteria and antimicrobial-resistant genes, all the probes were fixed to the DNA chip. After reverse hybridization of the multiplex PCR products to a piece of DNA chip, bacteria could be identified and its resistance could be determined. The detection system were evaluated with clinical isolates, drug-resistant standard strain, and appear to be a sensitive, specific, and reproducable tool for detection or identify common antimicrobial resistant bacteria.Part 3: Clinical study on the rapid etiological detection methodIt is very important to choose susceptible antibiotics to treat antimicrobial resistant bacterial infections as soon as possible, especially in the bloodstream infections, CNS infections. We can see good specificity and reproducibility of previous established genetic diagnostic method from standard strains and some clinical isolates. In order to know the clinical value of genetic method, and compare the consistent rate with traditional method, we conducted the study in different kind of infections to evaluate the practicability, at the same time, influence on the prognosis and medical cost. 173 blood samples, 339 cerebrospinal fluid, pleural fluid, ascitic fluid samples, 514 urine...