| Enterobacter Sakazakii (E. Sakazakii) named after by Japanese microbiologist Riichi Sakazakii is a kind of Gram-negative Enterobacter which can produce a yellow pigment. It was first isolated in the case of baby sepsis happened in1929, and then attracted a world-wide attention. Now, E. sakazakii has been listed as the "serious harm to a specific group, or produce chronic sequelae" of microbes by ICMSF (International Commission for Microbiological Specifications for Foods), it’s equally dangerous as Clostridium botulinum (Clostridium botalinum)A、B type toxin and Listeria monocytogenes (Listeriamonocytogenes). Once infected, it would cause serious harm to the health of infants and young children, it can cause necrotizing enterocolitis, bacteremia, neonatal meningitis and some other diseases, even severe neurological sequelae, the mortality rate is as high as50%. With a wide range of infant food safety concern and attention, the Chongqing Municipal has been added the qualitative detection of E. sakazakii to the infant food safety risk monitoring project since2010, in order to ensure the safety of infant foods.In this paper, the E. sakazakii was detected in Chongqing commercially available baby food (from2012to2013) referred to the GB method of infant food. A table comprehensive analysis of phenotypic and genotypic characteristics of17E. sakazakii trains (including16infant food isolates and a standard strain) were carried on combined with API20E, automatic biochemical analyzer, susceptibility test (KB method),16S rRNA PCR technology and PFGE typing technique. The conclusions were as follows:(1) Seven E. sakazakii strains were detected from200samples sampled in2012, the detection rate was3.5%; nine E. sakazakii strains were detected from300samples sampled in2013, the detection rate was3.0%. Combined with the monitoring data of2010and2011, E. sakazakii detection rate of infant foods increased year by year, declined slightly in2013. The number of E. sakazakii detected from nutrition rice was the largest among various types of infant foods, followed by nutrition noodles, which had the highest detection rate.(2)17trains of E. sakazakii identified by API20E biochemical identification was divided into four kinds of biochemical spectrum:Type Ⅰ (3305373), ODC+VP+GEL+INO+; Type Ⅱ (3205373), ODC-VP+GEL+INO+; Type Ⅲ (3304373), ODC+VP-GEL+INO+; Type Ⅳ (3305173), ODC+VP+GEL+INO-. VITEK2showed that17trains of E. sakazakii all had more than95%certainty, the two biochemical analysis results were basically the same.(3) The tolerance temperature of ES.10was the strongest, up to70℃; followed by ES.15, the highest tolerable temperature was65℃. The temperature tolerance test results showed that in order to prevent the pollution of E. sakazakii in infant food had to heated at55℃for30min at least, or60℃5min more, or65℃lmin more, or70℃15s more, and heated at75℃can guarantee that all inactivated immediately.(4) The alkalinity tolerance of E. sakazakii was slightly stronger than acid. Standard strain ATCC29544had the strongest acid tolerance, still had some activity under the conditions of pH1.0, ES.09, ES.10and ES.16can be inactivated by pH2.0acid treatment; standard strain ATCC29544, ES.05and ES.06can survive after pH13.0alkali treatment, ES.07, ES.09, ES.16can be inactivated by pH12.0short-term effected.(5)17strains of E. sakazakii all appeared as resistance to antibiotic VA, PEN, OXA, and showed sensitive to antibiotic CXM and other15kinds of antibiotics. Resistance patterns were divided into five:Type I resistance spectrum of VA-PEN-OXA,1strain; Type Ⅱ (NA intermediate AMP sensitive) resistance spectrum of VA-PEN-OXA-KF,1strain; TypeⅢ (NA intermediate AMP intermediate) resistance spectrum of VA-PEN-OXA-KF,1strain; Type Ⅳ(NA sensitive AMP intermediate) resistance spectrum of VA-PEN-OXA-KF,3strains; Type Ⅴ(NA sensitive AMP sensitive) resistance spectrum of VA-PEN-OXA-KF,11strains.(6) With the comparison of BLAST search,16trains of E. sakazakii food isolates had respectively homology similarity with12standard strains, the similarity were up to99%, all the strains can be identified as E. sakazakii. The homology similar percentage of the isolates was between100%～96.01%, ES.07and ES.08had the highest similarity, was100%, ES.12and ES.16had the lowest similarity, was96.01%. Phylogenetic tree of mode isolates and food isolates were divided into four modes gene cluster, which ES.05alone into a branch; ES.02and ES.04was another branch; ES.12, ES.13, ES.14and mode strains can be classified as a strain gene cluster, the rest of the food isolates formed a gene cluster together.(7) PFGE with restriction endonuclease Xba Ⅰ gave13kinds of PFGE patterns. The number of digested bands was between14to19, the molecular weight range was about10～1200Kb, mostly concentrated in the30～700Kb.The results analyzed by BioNumerics software showed that the similarity among strains digested by restriction endonuclease Xba I was between28.6%-85.7%. ES.07had the highest similarity with ES.10; ES.01had the minimum similarity with ES.12. PFGE with restriction endonuclease Spe I gave13kinds of PFGE patterns too. The number of digested bands was between17to24, the molecular weight range was about10-1200Kb, mostly concentrated in the30-400Kb.The results analyzed by BioNumerics software showed that the similarity among strains digested by restriction endonuclease Spe I was between43.8%-87.2%. ES.04had the highest similarity with ES.08; ES.11had the minimum similarity with ES.12. |
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