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Fricker-Feer, Claudia (2012): Identification and further characterization of Enterobacteriaceae and Cronobacter spp. in a milk powder and infant processing plant. Dissertation, LMU München: Faculty of Veterinary Medicine



Due to technical reasons, milk powder and powdered infant formulae (PIF) are not sterile products. In order to achieve the requirements set by the Swiss and European regulations for microbiologic criteria extensive epidemiological studies are needed on each individual plant level. In this way contamination routes can be identified and appropriate measurements taken. A legally reglemented pasteurization process eliminates Enterobacteriaceae. Therefore recontamination must now be focussed on. PIF contaminated with Cronobacter spp. can lead to severe infections in neonates such a sepsis, meningitis or necrotizing enterocolitis. The reported prevalence of commercially available PIF appears to be gradually decreasing from estimates of 14% in 1988 to 2.0-2.5%, where it now seems to have become stabilized. In order to make a reasonable estimate concerning the prevalence of Cronobacter spp. on an individual plant level, 950 samples (raw material, finished products, environmental samples) were analysed. The high prevalence of 16% comes from the intentional sampling of critical raw material and environment samples. The PFGE analysis, however, did not reveal any correlation between raw material and environmental samples which would indicate a possible contamination via finished products. 470 PIF Enterobacteriaceae isolates were identified through biochemical tests as well as by rpoB sequencing. E. cloacae (35%), Pantoea spp. (11%) and K. pneumoniae (8%) were the most prevalent genus and species. In order to reveal possible contamination routes, a subtyping was conducted. The species E. cloacae, which can be found in the same niches as Cronobacter spp., could be used as a significant hygienic indicator organism. To complete the epidemiological picture, 363 milk based samples were analysed (raw milk, milk concentrate, milk powder). Raw milk contains Enterobacteriaceae but no Cronobacter spp. were detected. However, 12/172 samples of milk powder contained Cronobacter spp. due to recontamination (during the packaging process and/or further processing steps). In order to increase the sensitivity and specificity of today’s available analysis for the detection of Cronobacter spp. methodological improvements had to be undertaken. The currently used enrichment media (mLST, EE) contain components of too selective nature which can therefore lead to false negative results. The new “Cronobacter Screening Broth” (CSB) contains sucrose and 5-bromo-4-chloro-3-indolyl-α-D-Glucopyranoside which now leads to a sensitivity of 100% and a negative predicting value of 100% as well. The change in colour of the broth indicates a presumptively positive result whereby only these samples need to be streaked onto chromogenic agar. The visual intermediate result leads to a reduction in costs and working time. In order to increase specificity as well as the commercial pressure of fast product release, a PCR-based system where positive and negative results are clearly available in short time is recommended. Several real-time PCR based systems for detection of Cronobacter spp. have become commercially available. Two systems (one open platform (Biotecon Diagnostics, Potsdam, Germany) and one dedicated system (BioControl, Bellvue, USA)) generated neither false positive nor false negative results. Both systems were able to detect 9 target and 13 non-target strains. The dedicated system has the advantage of shorter hands-on and analysis time. In addition, contaminations due to handling faults are reduced. The existing rpoB based Cronobacter species PCR was upgraded for the recently described species C. condimenti which can now be detected with high reliability. Additional epidemiological data is needed in order to monitor the microbiological situation in industrial plants constantly as well as consequently. Based on information on individual plant level it is possible to implement adequate measurements such as HEPA filters, exact time for adding heat labile ingredients, personal and material flow, air management and cleaning (type, time). Scientific support is needed concerning adequate analytical methods, formation of biofilm, desiccation data, types of enrichment media, sample size as well as additional epidemiological data. Our recent study concerning genetic diversity showed that different Cronobacter isolates from one sample can contain different PFGE fingerprints. This observation suggests that analysis of one isolate per sample may not be sufficient for trace back studies. The analysis of at least five colony forming units per sample is suggested. This example shows that through a close collaboration between industrial companies and scientific institutes, knowledge can be actively turned into practice. – This helps prevent pre-term babies and newborns from falling ill to Cronobacter spp.