Genetic Diagnosis And Clinical, Molecular Epidemiology Of Nosocomial Infections By Multidrug-resistant Gram-negative Bacteria

Gram-negative bacteria is one of the most important pathogens which is account for about 60%-70% causing nosocomial infections. In recent years, gram-negative bacteria has a rising antibiotic resistance according to the global bacterial resistance surveillance data. Multidrug-resistant gram-negative pathogens, especially carbapenem-resistant bacteria causing high mortality are rapidly becoming an urgent and unmanageable clinical problem.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 (3～5days). With the advance in molecular biology, rapid, sensitive molecular methods were established as accepted methods for the detection of some pathogens and resistant genes. Based on the analysis of 23 S rRNA gene sequences and other genes sequence associated with antimicrobial resistance, we established a nucleic acid-based detection system which can be faster and more sensitive to detect the presence of resistant microorganisms. It could provide identification and susceptibility data of pathogens rapidly, and may be helpful to the early etiological diagnosis and treatment of severe infections.Prevention is more important compared to treatment. Most nosocomial infections can be controlled by cutting of the transmission. Epidemiology and clinical research of nosocomial infections by multidrug-resistant gram-negative may provide more evidence for controlling the growing bacterial resistance. A retrospective research of bloodstream infections and central nervous system infections by acinetobacter from 2006-2011 in HuaShan Hospital was analyzed in order to explore the relationship between bacterial sensitivity and prognosis. Resistance genes (carbapenemase, methylase, β-lacatamase) in the gram-negative pathogens separated from the blood and cerebrospinal fluid samples were be screening at the same time.Part One Study on genetic diagnosis to identify multidrug-resistant gram-negative bacteriaDNA microarray technology is developing rapidly in recent years because of owning a lot of advantages such as little testing time, higher sensitivity and less sample volume. It is widely used on the study of etiological diagnosis, resistance gene detection, gene mutation and analysis of gene expression profiles. However, it still has a lot of controversy on the application in clinical testing. 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. Broad-range PCR primers may be designed to recognize these conserved bacterial rRNA gene sequence and used to amplify variable regions. Until now there are many reports on etiological diagnosis by detecting 16S,16S-23S, and 23S rRNA genes while much fewer reports on resistance gene detection. Carbapenemase and methylase producing strains are difficulty to be cured which will be a huge threat to human health. Based on the study of etiological research of 23 S rRNA, we designed the primers and probes of carbapenemase resistance genes (KPC, NDM, VIM, IMP, OXA23,OXA51, OXA58) and methylase resistance genes (armA, rmtB). Multiple pairs of primers were integrated in only one PCR reaction and then all the probes were fixed on the same sheet of gene chip. We can know whether the strain produced carbapenemase or methylase after hybridization between PCR products and microarray at last. Experiments confirmed that DNA microarray technology could be applied in NDM, IMP, KPC, OXA23,OXA51, OXA58 carbapenemase and armA methylase resistant genes while it was not satisfactory in VIM carbapenemase and rmtB methylase resistant genes. The methods we have established could identify seven resistant genes and it was specific and reproducible.Part Two Clinical study on the rapid genetic diagnosis in multidrug-resistant gram-negative bacteriaBloodstream infection is one of the common clinical severe infectious diseases, with a high morbidity and mortality. The prognosis is closely related to early choice for the sensitive antibiotic therapy. In order to further assess the diagnostic value of DNA microarray detecting resistance genes in multidrug-resistant gram-negative bacteria, we collected 356 blood culture positive specimens from 2011.3-2012.2 in HuaShan Hospital to detect carbapenemase and methylase resistant genes. There were 119 gram-negative strains in all the specimens. The results showed that DNA microarray for detecting KPC, NDM-type carbapenemase and armA-type methylase resistance gene had 100% compliance rate compared with PCR method. The compliance rate in detecting OXA23, OXA51-type carbapenemase and rmtB-type methylase resistance gene were 90.0%,80% and 58.3%. Gene chip methods require for about 6～8 hours which can shorten 16～18 hours compared with K-B susceptibility testing. If using genetic method to detect pathogens and resistance genes simultaneously, we can get the result at least two days before the traditional method which may be helpful to reduce the inappropriate use antibiotics, decrease the unnecessary medical costs and improve the prognosis of patients.Part Three Clinical study and molecular epidemiology of nosocomial infections by Multidrug-resistant Gram-negative bacteriaEnterobacteriaceae and Acinetobacter are both important pathogens causing nosocomial infections in multidrug-resistant gram-negative bacteria. We collected the data of 74 bloodstream infections and 90 central nervous system infections by acinetobacter from 2006-2011 in HuaShan Hospital to summary the clinical characteristics and explore the relationship between bacterial sensitivity and prognosis. The results showed that nearly all bloodstream infections and central nervous system infections were nosocomial infections which mostly occurred in patients after surgery or with severe diseases. Acinetobacter had lower resistance rates to cefoperazone/sulbactam and minocycline in all the tested drugs. And the prognosis was closely related to acinetobacter sensitivity to antimicrobial drugs. We also collected 170 gram-negative strains separated from blood and cerebrospinal fluid culture from 2011.3-2012.2 in HuaShan Hospital to screen the resistance genes (carbapenemase, methylase, (3-lacatamase) by using PCR amplification. The results showed that it was about 77.6%(45/58) strains carrying carbapenemase resistant genes in carbapenem-nonsensitive strains. The mainly mechanism of carbapenem-resistant Klebsiella pneumoniae, Acinetobacter baumannii was due to producing KPC-type and OXA-type carbapenemase resistant genes. It was about 38.4%(33/86) strains carrying methylase resistance genes in aminoglycoside-nonsensitive strains. And these genes also mainly distributed in Klebsiella pneumoniae and Acinetobacter baumannii. By using PCR amplification there were 26 strains carrying SHV,24 strains carrying TEM and 69 strains carrying CTX-M-type [3-lactamase in all the specimens.75%(27/36) strains of Escherichia coli producing extended-spectrum β-lactamases which was the highest proportion of all the gram-negative pathogens. 23 strains of Acinetobacter baumannii producing OXA-type carbapenemase resistant genes could be divided into four subtypes by using ERIC-PCR while 17 stains of Klebsiella pneumoniae producing KPC-type carbapenemase resistant genes could be divided into two subtypes,4 strains of Loffi Acinetobacter producing NDM-type carbapenemase resistant genes could be divided into two subtypes. From these we can speculate that the strains may be homologous which causing nosocomial infections.