Study On The Molecular Epidemiology And Genotyping Of Extended Spectrum β-lactamases In Shigellae

Shigellae are one of the main pathogenic bacteria that cause gastrointestinal infection. They are pathogenic bacteria of shigellosis in mankind and primate, so Shigella is called as dysenteriae bacillary. Shigellae can produce endotoxin and ectotoxin that enduce Shigellae with invasiveness. Focal infections and an asymptomatic carrier state are main infective sources. Infection by Shigellae is peroral and usually transmitted by contaminated food and water. Infection induced by Shigellae can cause severe gastrointestinal tract reaction—bloody purulent stool and anhydration. A small portion of the Shigellae infected person can cause system complication such as eclampsia, uremic syndrome hemolytic, hyponatremia, hypoglycaemia , enterobrosis , and even death. The incidences of Shigellae infection are 164.7 million throughout the world every year. Among them about 1.1 million are fatal, while 69 % of the death are children under age 5 Shigellae cause severe public health problem. They are not only one of the main pathogenic bacteria for occasional diarrhea patients but also cause outbreak of food poisoning.It has been demonstrated that clinically isolated Shigellae are resistant to antimicrobial agents that have been widely employed to treat shigellosis. Some Shigellae are even resistant to multiple antibiotics. After that first line medicine that was used before were replaced by Extended Spectrum β-lactam antibiotics, Extended Spectrum β-lactam antibiotics are experiential medication for shigellosis. However, since SHV-11 extend-spectrum β-lactamase (ESBL) was reported in 1999 in India, ESBL-producing Shigellae were reported throughout the world. Furthermore, genes of ESBL are usually located in plasmid which are able to spread between the same species or even the different species, causing outbreak of drug resistant Shigellae. Therefore, resisntant to Extended Spectrum β-lactam antibiotics in Shigellae has brought great hurdle for the prevention and cure of shigellosis.In this study, 42 strains of Shigellae were collected from the Health Care Hospital for Maternal and Children of XiaoShan, ShaoXing People's Hospital, Centers for Disease Control of Hang Zhou from August 2006 to December 2006. Agar dilution for minimum inhibition concentration determination, pulse field gel electrophoresis (PFGE), and conjugation experiment were performed for these strains. Eleven clinical strains were detected to be ESBL positive by phenotype analysis (MIC, 3-D test and double disc confirmatory tests). PCR was performed for the 11 positive strains to detect ESBL genes using primers designed for TEM-1, SHV-1, CTX-M-1, CTX-M-2,CTX-M-8 and CTX-M-9. Sequencing was preformed for PCR products to confirm the genotypes of ESBL. The pIs of extend-spectrum β-lactamase were detected by isoelectric focusing (IEF). MIC was also determiend for the transconjugants to conform if they produced ESBL, while pIs of the expressed protein were detected by IEF.The MIC results showed that the clinical strains were highly sensitive to carbapenems and quinolones, sensitive rate are 100% and 95.2% respectively and secondary sensitive to Aztreonam, Extended Spectrum β-lactam antibiotics and antibiotics contained with enzyme inhibitor, but highly resistant to ampicillin and cidomycin, resistantrate are 90.5% and 80.5% respectively. Eleven clinical strains were detected to be ESBL positive and all of them are conjugated. The resistant spectrums of transconjugants were similar to their parent strains. The 11 transconjugants had isoelectric point around 9.0 or 7.9. The 11 transconjugants were positive in PCR analysis by using primers of CTX-M-1 or CTX-M-9. Nucleotide sequence analysis revealed that they are CTX-M-15 or CTX-M-14 ESBL, respectively.Among the 42 clinical strains, there are 11 S.flexneri isolated from Health Care Hospital for Maternal and Children of XiaoShan and 13 S.sonnei isolated from ShaoXing people's hospital. PFGE analysis for those 11 S.flexneri and 13 S.sonnei strains indicated that they were homogeneity, respectively.In summary, eleven ESBL detected from the 11 strains are the plasmid encoded CTX-M-15 or CTX-M-14. The same S. flexneri in Health Care Hospital for Maternal and Children of XiaoShan and S. sonnei in ShaoXing people's hospital, respectively, caused an epidemic outbreak.