Resumo
Cronobacter (Enterobacter sakazakii) é um novo gênero da família Enterobacteriaceae, reconhecido como responsável pelo elevado número de casos fatais em neonatos após consumo de fórmulas infantis. Os métodos convencionais de detecção desses micro-organismos são demorados e de sensibilidade inadequada. A ISO/TS 22964:2006 é a metodologia padronizada mais recentemente para detecção de Cronobacter. Este estudo teve como objetivo identificar como Cronobacter spp. os isolados brasileiros previamente classificados como E. sakazakii, comparar as características de 37 culturas de Cronobacter e não-Cronobacter e avaliar os kits miniaturizados e a ISO/TS 22964:2006. Foi também desenvolvido um protocolo convencional de PCR com alvo em dnaG. A maioria dos isolados brasileiros não foi confirmada como Cronobacter spp., e o enriquecimento seletivo da metodologia ISO foi inibitório para algumas cepas de Cronobacter. O kit ID 32E demonstrou desempenho mais confiável. O protocolo de PCR com alvo em gluA apresentou resultados consistentes com o ID 32E, e o protocolo com alvo em dnaG foi 100% sensível e específico. Portanto, os protocolos de PCR com alvo em gluA e dnaG podem ser usados para complementar a detecção e identificação de Cronobacter spp. após o emprego de métodos de isolamento e identificação convencionais.Referências
1. Iversen C, Mullane N, McCardell B, Tall BD, Lehner A, Fanning S et al. Cronobacter gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii and proposal of Cronobacter sakazakii gen. nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacterturicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov., Cronobacter genomospecies1, and of three subspecies, Cronobacter dublinensis subsp. dublinensis subsp. nov., Cronobacter dublinensis subsp. lausannensis subsp. nov. and Cronobacter dublinensis subsp. lactaridi subsp. nov. Int J Syst Evol Microbiol. 2008;58(6):1442-7.
2. Kuhnert P, Korczak BM, Stephan R, Joosten H, Iversen C. Phylogeny and prediction of genetic similarity of Cronobacterand related taxa by multilocus sequence analysis (MLSA). Int J Food Microbiol. 2009;136:152-8.
3. Food and Agricultural Organization / World Health Organization – FAO/WHO. Enterobacter sakazakii (Cronobacter spp.) in powdered follow-up formulae. Meeting Report. (Microbiological Risk Assessment Series, 15). Rome; 2008.
4. Lehner A, Stephan R. Microbiological, epidemiological and food safety aspects of Enterobacter sakazakii. J Food Prot. 2004;67:2850-7.
5. Biering G, Karlsson S, Clark NC, Jónsdóttir KE, Lúdvígsson P, Steingrímsson Ó. Three cases of neonatal meningitis caused by Enterobacter sakazakii in powdered milk. J Clin Microbiol. 1989;27:2054-6.
6. van Acker J, De Smet F, Muyldermans G, Bougatef A, Naessens, A, Lauwers, S. Outbreak of necrotizing enterocolitis associated with Enterobacter sakazakii in powdered milk formula. J Clin Microbiol. 2001;39:293-7.
7. Himelright I, Harris E, Lorch V, Anderson M. Enterobacter sakazakii infections associated with the use of powdered infant formula – Tenessee, 2001. MMWR. 2002;61:298-300.
8. Torres-Chavolla E, Ramirez-Cerda E, Gutiérrez-Rojo R. Isolation and identification of Enterobacter sakazakii in infant milk formulas. Foodborne Pathog Dis. 2007;4:164-8.
9. Simmons BP, Gelfand MS, Hass M, Metts L, Ferguson J. Enterobacter sakazakii infections in neonates associated with intrinsic contamination of a powdered infant formula. Infect Control Hosp Epidemiol. 1989;10:398–401.
10. Mullane N, Healy B, Meade J, Whyte P, Wall PG, Fanning S. Dissemination of Cronobacter (Enterobacter sakazakii) in a powdered milk protein manufacturing facility. Appl Environ Microbiol. 2008;74:5913-7.
11. Teramoto S, Tanabe Y, Okano E, Nagashima T, Kobayashi M, Etoh Y. A first fatal neonatal case of Enterobacter sakazakiiinfection in Japan. Pediatr Int. 2010;52(2):312-3.
12. Chap J, Jackson P, Siqueira R, Gaspar N, Quintas C, Park J et al. International survey of Cronobacter sakazakii and other Cronobacter spp. in follow up formulas and infant foods. Int J Food Microbiol. 2009;136:185-8.
13. Pagotto FJ, Farber, JM. Cronobacter spp. (Enterobacter sakazakii): Advice, policy and research in Canada. Int J Food Microbiol.2009;136:238-45.
14. Cawthorn D-M, Botha S, Witthuhn RC. Evaluation of different methods for the detection and identification of Enterobacter sakazakii isolated from South African infant formula milks and the processing environment. Int J Food Microbiol. 2008;127:129-38.
15. United States Food and Drug Administration – USFDA. Isolation and enumeration of E. sakazakii from dehydrated powdered infant formula. 2002. Disponível em: [http://www.cfsan.fda.gov/~comm/mmesakaz.html].
16. International Organization for Standardization / International Dairy Federation – ISO/IDF. Technical specification: milk and milk products – detection of Enterobacter sakazakii.ISO/TS 22964; IDF/RM 210;2006.
17. Iversen C, Forsythe SJ. Comparison of media for the isolation of Enterobacter sakazakii. Appl Environ Microbiol. 2007;73:48-52.
18. Iversen C, Lehner A, Mullane N, Marugg J, Fanning S, Stephan R et al. Identification of Cronobacter spp. (Enterobacter sakazakii). J Clin Microbiol. 2007;45:3814-6.
19. Fox EM, Jordan KN. Towards a one-step Enterobacter sakazakii enrichment. J Appl Microbiol. 2008;105:1091-7.
20. Giammanco GM, Aleo A, Guida I, Mammina C. Molecular epidemiological survey of Citrobacter freundii misidentified as Cronobacter spp. (Enterobacter sakazakii) and Enterobacter hormaechei isolated from powdered infant milk formula. Foodborne Pathog Dis. 2011;8(4):517-25.
21. Healy B, Cooney S, O’Brien S, Iversen C, Whyte P, Nally J et al. Cronobacter (Enterobacter sakazakii): an opportunistic foodborne pathogen. Foodborne Patholog Dis. 2010;7:339-50.
22. Seo KH, Brackett RE. Rapid, specific detection of Enterobacter sakazakii in infant formula using a real-time PCR assay. J Food Prot. 2005;68:59-63.
23. Lampel KA, Chen Y. Method for the isolation and detection of Enterobacter sakazakii (Cronobacter) from powdered infant formula. Int J Food Microbiol. 2009;136:179-84.
24. Hassan AA, Akineden Ö, Kress C, Estuningsih S, Schneider E, Usleber E. Characterization of the gene encoding the 16S rRNA of Enterobacter sakazakii and development of a species-specific PCR method. Int J Food Microbiol. 2007;116:214-20.
25. Lehner A, Nitzsche S, Breeuwer P, Diep B, Thelen K, Stephan R. Comparison of two chromogenic media and evaluation of two molecular based identification systems for Enterobacter sakazakii detection. BMC Microbiol. 2006;6-15.
26. Kothary MH, McCardell BA, Frazar CD, Deer D, Tall BD. Characterization of the zinc-containing metalloprotease encoded by zpx and development of a species-specific detection method for Enterobacter sakazakii. Appl Environ Microbiol. 2007;73:4142-51.
27. Farmer III JJ, Asbury MA, Hickman FW, Brenner DJ. The Enterobacteriaceae Study Group. Enterobacter sakazakii, new species of Enterobacteriaceae isolated from clinical specimens. Int J Systematic Bacteriol. 1980;30:569-84.
28. Iversen C, Lancashire L, Waddington M, Forsythe S, Ball G. Identification of Enterobacter sakazakii from closely related species: The use of Artificial Neural Networks in the analysis of biochemical and 16S rDNA data. BMC Microbiol. 2006;6-28.
29. Greenhalgh T. How to read a paper: papers that report diagnostic or screening tests. BMJ. 1997;315:540-3.
30. Restaino L, Frampton EW, Lionberg WC, Becker RJ. A chromogenic plating medium for the isolation and identification of Enterobacter sakazakii from foods, food ingredients and environmental sources. J Food Prot. 2006;69:315-22.
31. Ye Y, Wu Q, Yao L, Dong X, Wu K, Zhang J. A comparison of polymerase chain reaction and International Organization for Standardization Methods for determination of Enterobacter sakazakii contamination of infant formulas from Chinese mainland markets. Foodborne Pathog Dis. 2009;6:1229-34.
32. Drudy D, O’Rourke M, Murphy M, Mullane NR, O’Mahony R, Kelly L, et al. Characterization of a collection of Enterobacter sakazakii isolates from environmental and food sources. Int J Food Microbiol. 2006;110:127-34.
33. Mullane NR, Murray J, Drudy D, Prentice N, Whyte P, Wall PG et al. Detection of Enterobacter sakazakii in dried infant milk formula by cationic-magnetic-bead capture. Appl Environ Microbiol. 2006;72:6325-30.
34. O’Brien S, Healy B, Negredo C, Fanning S, Iversen C. Evaluation of a new one-step enrichment in conjunction with a chromogenic medium for the detection of Cronobacter spp. (Enterobacter sakazakii) in powdered infant formula. J Food Prot. 2009;72:1472-5.
35. Stephan R, Van Trappen S, Cleenwerck I, Vancanneyt M, De Vos P, Lehner A. Enterobacter turicensis sp. nov. and Enterobacter helveticus sp. nov., isolated from fruit powder. Int J Syst Evol Microbiol. 2007;57:820-6.
36. Guillaume-Gentil O, Sonard V, Kandhai MC, Marugg JD, Joosten H. A simple and rapid cultural method for detection of Enterobacter sakazakii in environmental samples. J Food Prot. 2005;68:64-9.
37. Iversen C, Druggan P, Forsythe SJ. A selective differential medium for Enterobacter sakazakii, a preliminary study. Int J Food Microbiol. 2004;96:133-9.
38. Iversen C, Druggan P, Forsythe SJ. A selective differential medium for Enterobacter sakazakii, a preliminary study. Int J Food Microbiol.2004;96:133-9.
39. El-Sharoud WM, O’Brien S, Negredo C, Iversen C, Fanning S, Healy B. Characterization of Cronobacter recovered from dried milk and related products. BMC Microbiol.2009;9:24.
40. Fanjat N, Leclerq A, Joosten H, Robichon D. Comparison of phenotyping methods ID 32E and Vitek 2 Compact GN with 16S rRNA gene sequencing for the identification of Enterobacter sakazakii. J Clin Microbiol. 2007;45:2048-50.
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Copyright (c) 2012 Márcia Barbosa Warnken, Marcelo Luiz Lima Brandão, Aline Encarnação Souza, Célia Maria Carvalho Pereira Araulo Romão, Ana Cristina Martins Almeida Nogueira, Maria Teresa Destro