Cronobacter Support
02-19-2009, 06:16 PM
Detection of E. sakazakii
Recently, FDA released a protocol for inclusion in the FDA's Bacteriological Analytical Manual (FDA/BAM) for quantitative enumeration of E. sakazakii in dehydrated powdered infant formula (cfsan.fda.gov/~comm/mmesakaz.html). In this method, a total of 333 g of product is assayed per sample with three replicate enrichments of 100, 10 and 1 g. Nine portions of Enterobacteriaceae Enrichment (EE) broth are pre-warmed at 45°C before addition to each of the nine subsamples. Samples are gently, but well mixed to sus pend solids. Each sample is incubated at 36°C overnight followed by addition of a 10-ml aliquot to 90 ml pre-sterilized EE broth that is also incubated at 36°C overnight. This second EE enrichment culture is streaked and directly plated onto two violet red bile glucose agar (VRBG) plates each. These plates are incubated at 36°C overnight, and up to five typical colonies are streaked onto Trypticase Soy Agar (TSA) and incubated at 25°C for 48-72 h. Only typical yellow-pigmented colonies on TSA are selected and confirmed by the API 20E biochemical identification system. The MPN per g of product is calculated using the table in the FDA/BAM, Appendix 2 (Most Probable Number from Serial Dilutions).
Media evaluated for the direct plating of food or clinical samples for isolating E. sakazakii include MacConkey agar, MacConkey-sorbitol agar, eosin methylene blue (EMB) agar, deoxycholate agar, tergitol 7 agar, xyloselysine-desoxycholate (XLD) agar, and violet red bile (VRB) agar. Recovery was less on XLD agar than on tergitol 7 agar. On MacConkey, EMB, or deoxycholate agars, E. sakazakii strains formed light to dark pink colonies with no precipitated bile around them. Unfortunately, a high percentage of false-negative results occur with direct plating methods. Addition of a preenrichment step to the direct plating approach (lauryl sulfate tryptose broth incubated for 48 h at 44°C and plated onto violet red bile agar for incubation at 44°C for 48 h) reduced the false-negative rate to 8% when combined with direct plating of environmental samples. Recovery of presumptive E. sakazakii colonies may be accomplished after transfer to a non-selective medium (trypticase soy agar) and formation of bright buttery yellow colonies on incubation at 25°C for 48 h. Diminis hed pigment production and lighter yellow colonies will be observed if incubated at 36°C or in the dark, or if multiple transfers of the organism have occurred. Final confirmation of E. sakazakii colonies can be done via several systems such as API 20 E™, API ZYM™, or Vitek™ assays.
Biochemical Reactions of E. sakazakii
Two major differences between E. sakazakii and the other Enterobacter species were observed in a study of 226 Enterobacter strains (of which 129 were E. sakazakii). a-Glucosidase activity was demonstrated in all E. sakazakii strains but in none of the other Enterobacter strains. Absence of the enzyme phosphoamidase was also unique to E. sakazakii isolates. E. sakazakii is also distinguished by its ability to ferment sucrose, raffinose, and a-methyl-D-glucoside, but not D-sorbitol, dulcitol, adonitol, or D-arabinol. Seventy to 90% of E. sakazakii strains would be classified as coliforms based on their ability to produce gas from lactose at 35°C. In addition, some could be identified as fecal coliforms based on their ability to produce gas in EC broth at 44.5°C within two days. We found that 2 of 4 environmental isolates of E. sakazakii produced gas in EC broth held at 44.5°C for 24 h.
The API ZYM™ system may be used to confirm E. sakazakii isolates. This system consists of a plastic strip and 20 cupules, with 19 cupules containing substrates and buffers, and 1 cupule serving as a negative control. The strip assays for alkaline phosphatase, butyrate esterase, caprylate esterase-lipase, myristate lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, acid phosphatase, phosphoamidase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, and a-fucosidase activities. After aerobic incubation in the dark at 36°C for 4 h, presence and degree of enzymatic activity is scored as color intensities 0 – 5 in accordance with a color comparison chart provided by the manufacturer. Distinguishing responses of E. sakazakii are the absence in practically all strains of myristate lipase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, b-glucuronidase, a-mannosidase, and a-fucosidase activities. E. sakazakii isolates also produce a strong caprylate esterase-lipase reaction and a moderate acid phosphatase reaction. Alteration in the enzyme profile occurs when cells are treated ultrasonically. In particular, positive responses previously observed by E. sakazakii for leucine arylamidase, b-galactosidase, b-glucosidase, a-glucosidase, acid phosphatase, and N-acetyl-b-glucosaminidase reactions become negative for ultrasound-treated cells.
Recently, FDA released a protocol for inclusion in the FDA's Bacteriological Analytical Manual (FDA/BAM) for quantitative enumeration of E. sakazakii in dehydrated powdered infant formula (cfsan.fda.gov/~comm/mmesakaz.html). In this method, a total of 333 g of product is assayed per sample with three replicate enrichments of 100, 10 and 1 g. Nine portions of Enterobacteriaceae Enrichment (EE) broth are pre-warmed at 45°C before addition to each of the nine subsamples. Samples are gently, but well mixed to sus pend solids. Each sample is incubated at 36°C overnight followed by addition of a 10-ml aliquot to 90 ml pre-sterilized EE broth that is also incubated at 36°C overnight. This second EE enrichment culture is streaked and directly plated onto two violet red bile glucose agar (VRBG) plates each. These plates are incubated at 36°C overnight, and up to five typical colonies are streaked onto Trypticase Soy Agar (TSA) and incubated at 25°C for 48-72 h. Only typical yellow-pigmented colonies on TSA are selected and confirmed by the API 20E biochemical identification system. The MPN per g of product is calculated using the table in the FDA/BAM, Appendix 2 (Most Probable Number from Serial Dilutions).
Media evaluated for the direct plating of food or clinical samples for isolating E. sakazakii include MacConkey agar, MacConkey-sorbitol agar, eosin methylene blue (EMB) agar, deoxycholate agar, tergitol 7 agar, xyloselysine-desoxycholate (XLD) agar, and violet red bile (VRB) agar. Recovery was less on XLD agar than on tergitol 7 agar. On MacConkey, EMB, or deoxycholate agars, E. sakazakii strains formed light to dark pink colonies with no precipitated bile around them. Unfortunately, a high percentage of false-negative results occur with direct plating methods. Addition of a preenrichment step to the direct plating approach (lauryl sulfate tryptose broth incubated for 48 h at 44°C and plated onto violet red bile agar for incubation at 44°C for 48 h) reduced the false-negative rate to 8% when combined with direct plating of environmental samples. Recovery of presumptive E. sakazakii colonies may be accomplished after transfer to a non-selective medium (trypticase soy agar) and formation of bright buttery yellow colonies on incubation at 25°C for 48 h. Diminis hed pigment production and lighter yellow colonies will be observed if incubated at 36°C or in the dark, or if multiple transfers of the organism have occurred. Final confirmation of E. sakazakii colonies can be done via several systems such as API 20 E™, API ZYM™, or Vitek™ assays.
Biochemical Reactions of E. sakazakii
Two major differences between E. sakazakii and the other Enterobacter species were observed in a study of 226 Enterobacter strains (of which 129 were E. sakazakii). a-Glucosidase activity was demonstrated in all E. sakazakii strains but in none of the other Enterobacter strains. Absence of the enzyme phosphoamidase was also unique to E. sakazakii isolates. E. sakazakii is also distinguished by its ability to ferment sucrose, raffinose, and a-methyl-D-glucoside, but not D-sorbitol, dulcitol, adonitol, or D-arabinol. Seventy to 90% of E. sakazakii strains would be classified as coliforms based on their ability to produce gas from lactose at 35°C. In addition, some could be identified as fecal coliforms based on their ability to produce gas in EC broth at 44.5°C within two days. We found that 2 of 4 environmental isolates of E. sakazakii produced gas in EC broth held at 44.5°C for 24 h.
The API ZYM™ system may be used to confirm E. sakazakii isolates. This system consists of a plastic strip and 20 cupules, with 19 cupules containing substrates and buffers, and 1 cupule serving as a negative control. The strip assays for alkaline phosphatase, butyrate esterase, caprylate esterase-lipase, myristate lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, acid phosphatase, phosphoamidase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, and a-fucosidase activities. After aerobic incubation in the dark at 36°C for 4 h, presence and degree of enzymatic activity is scored as color intensities 0 – 5 in accordance with a color comparison chart provided by the manufacturer. Distinguishing responses of E. sakazakii are the absence in practically all strains of myristate lipase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, b-glucuronidase, a-mannosidase, and a-fucosidase activities. E. sakazakii isolates also produce a strong caprylate esterase-lipase reaction and a moderate acid phosphatase reaction. Alteration in the enzyme profile occurs when cells are treated ultrasonically. In particular, positive responses previously observed by E. sakazakii for leucine arylamidase, b-galactosidase, b-glucosidase, a-glucosidase, acid phosphatase, and N-acetyl-b-glucosaminidase reactions become negative for ultrasound-treated cells.