Study On The Mechanism Of The Porin-mediated Antibacterial Resistance In Clinical Shigella Spp.strains

Shigella spp., the pathogen of bacillary dysentery in humans, result in fever, abdominal pain and bloody diarrhea. It is an epidemic contagion throughout the world. Bacillary dysentery is a major cause of morbidity and mortality among diarrheal disease, especially in children. Dysentery caused by Shigella spp. has been pandemic throughout our country in history, and tend to be sporadic by now, which estimated more than millions infectious diarrhea cases each year.In developing countries, the bacillary dysentery is difficult to prevent due to the complex of the bacterial type, the easy variance, without the crossover immunity in Shigella. Since antimicrobial agents are widely employed to treat shigellosis, antibacterial resistance in shigella spp. has been rapidly emerged, frequency appeared and is multiple-antibiotic-resistance, which bring great hard to the clinical management. Therefore, it is desirable to study the mechanism of antibacterial resistance of shigella spp..The mechanisms of antibacterial resistance in Shigella spp. have been discovered at different aspects, but few study works on the mechanismof porin-mediated antibacterial resistance in Mar phenotype of shigella spp..Study on Escherichia coli. elucidated that many antibiotics such as β -lactamases, tetracycline, chloramphenicol and hydrophilic quinolone cross the outer membrane through porin channels OmpF (outer membrane protein F) and OmpC. A decrease in porin-mediated permeability may decrease drug accumulating concentration inside the cell and increase the level of resistance. Thus the bacterial bring about the resistance to multiple antibiotics. The chromosomal multiple-antibiotic-resistance (mar) locus ispopular accepted to be responsible for permeability decrease. Antibacterial resistance in Mar mutant is attributed to porin-deficient and active efflux. MarR or marO mutations may disrepress marA expressions, which activates micF expressions (post-transcriptionally regulates expression of outer membrane protein F), causing outer membrane protein F loss or deficient, resulting in multiple antibiotic resistance. It was found that mar locus is widespread among enteric bacteria. It has been proved that Shigella is phylogenetically indistinguishable from E. colLThese results mentioned above built a basis for the study on the mechanism of porin-mediated antibacterial resistance in Shigella spp. strains.This study was planned to amplify mar genes (marOR and marA) by polymerase chain reaction (PCR) and analyze all PCR products by single strand conformational polymorphism analysis (SSCP), analyze the outer membrane protein(Omp) of Mar strains and sequence the mar gene of outer membrane protein mutant. The aim of this study is to reveal whether porin-mediated antibacterial resistance and mar locus mediates multiple antibiotic resistance existed in Shigella spp.Methods1. Susceptibility test (Kirby-Bauer): We carried out antibacterial susceptibility tests of 41 strains of Shigella. Ciprofloxacin(CIP), Norfloxacin(NOR), Teracycline(TE), Chloramphenicol(C), ampicillin(Am), Cefalexin(CX) were used. The strains appearing resistance to 3 or more kinds of antibiotic were called as Mar, and those being susceptible to all of antibiotics were called as wild type(WT).2. PCR-SSCP of mar genes: marOR and marA genes were amplified by PCR. SSCP were applied to all PCR procucts.3. Prepare outer membrane protein: Outer membranes were prepared by treatment of membranes with 2% (w/v) sarkosyl for 60 min at 30°C followed by centrifugation at 100,000g for 30 min. The pellet, which contained the outer membrane proteins (OMPs), was washed twice with 0.5% (w/v) sarkosyl and stored at -70°C.4. Omps were extracted and analyzed on the 12%SDS-PAGE.5. DNA sequence analysis of a porin-deficient mutant. Results1. 41 clinical isolates were collected. 31 strains of Shigella flexneri. and 10 strains Shigella sonnei were identified.2. Identify the Mar strains and WT: 39 Mar strains and 2 WT were identified.3. This study amplified multiple-antibiotic-resistance locus marOR(647bp) and marA (425bp) by PCR in all strains, the lack of multiple-antibiotic-resistance locus were not found.4. PCR-SSCP analysis detected mar genes: none significant mutation was observed in both marA gene and marOR gene.5. Analysis of the outer membrane protein: The antibiotic resistance strains and standard susceptive strain were compared, 4 of 14 multiple antibiotic resistance strains had low level of the Mr43,000 outer membrane protein, overproduction of an outer membrane protein with an apparent molecular mass of 41 kDa and nomal level of the 3 8 kDa outer membrane protein.6. A mutation in marA gene of Shigella was first identified in this study by DNA sequence analysis, which located at 314 site of the PCR production(1734 site of mar gene on the Genbank), followed by 13 amino acids changes of MarA.Conclusion1. There are 28.5 percent of the multiple antibiotic resistance strains with Mr43,000 outer membrane decrease in the Shigella spp.. It reveals that the mechanism of porin-mediated antibacterial resistance is generally existed in multiple antibiotic resistance of Shigella spp..2. The decreased amounts of 43-kDa and nomal amounts of a 38-kDa outer membrane protein in the Mar phenotype of shigella spp. suggest that 43-kDa outer membrane protein plays an important role in the porin-mediated antibacterial resistance.3. The overproduction of an outer membrane protein with an apparent molecular mass of 41 kDa is first identified in the Mar phenotype of shigella spp.,which may be involved with multiple drug resistance.4. A mutation in marA gene of Shigella is first identified in this study by DNA sequence analysis, which may be associated with porin-mediated antibacterial resistance.