Detection, identification and live/dead differentiation of the emerging pathogen Enterobacter sakazakii from infant formula milk and the processing environment

[摘要] ENGLISH ABSTRACT: The World Health Organisation (WHO) estimates that at least 75% of infants receiveinfant formula milk (IFM) either entirely or in conjunction with breast milk during the firstfour months after birth. The presence of the emerging pathogen Enterobacter sakazakiiin IFM has been associated with rare but fatal cases of neonatal infections and deaths.There is thus a need for accurate methods for the rapid detection of E. sakazakii infoods. At present, the methods used to detect and identify this micro-organism areinadequate, controversial and contradictory. The aim of this study was to determine themost suitable method for E. sakazakii detection after evaluation of the currentlyavailable methods. A further aim was to optimise a polymerase chain reaction (PCR)method for the detection of only viable E. sakazakii cells utilising the DNA-intercalatingdyes ethidium monoazide (EMA) and propidium monoazide (PMA).The Food and Drug Administration (FDA) method for E. sakazakii detection wasutilised to select 50 isolates from IFM and 14 from the environment, regardless ofcolony appearance. These isolates were identified by sequencing a 1.5 kilobase (kb)fragment of the 16S ribosomal DNA (rDNA) and by using the National Centre forBiotechnological Information (NCBI) database to confirm the closet known relatives.Seven of the 50 (14%) IFM isolates and six of the 14 (43%) environmental isolates wereidentified as E. sakazakii. The methods that were evaluated for accuracy in detectingand identifying these E. sakazakii isolates included yellow pigment production ontryptone soy agar (TSA), chromogenic Druggan-Forsythe-Iversen (DFI) andEnterobacter sakazakii (ES) agars and PCR using six different species-specific primerpairs described in the literature.The suitability of the FDA method was lowered by the low sensitivity, specificityand accuracy (87%, 71% and 74%, respectively) of using yellow pigment production forE. sakazakii identification. DFI and ES agars were shown to be sensitive, specific andaccurate (100%, 98% and 98%, respectively) for the detection of E. sakazakii. Thespecificity of the PCR amplifications was found to vary between 8% and 92%, withEsakf and Esakr being the most accurate of the primer pairs evaluated.The current FDA method for E. sakazakii detection requires revision in the light ofthe availability of more sensitive, specific and accurate detection methods. Based onthe results obtained in this study, a new method is proposed for the detection ofE. sakazakii in food and environmental samples. This proposed method replaces theculturing steps on violet red bile glucose agar (VRBGA) and TSA with culturing on chromogenic DFI or ES agar. For identification and confirmation of presumptiveE. sakazakii isolates, the oxidase test, yellow pigment production and API biochemicalprofiling is replaced by DNA sequencing and/or species-specific PCR with the mostaccurate primer pair (Esakf and Esakr). The amendments to the current FDA methodwill reduce the time to detect E. sakazakii from approximately 7 days to 4 days andshould prove to be more sensitive, specific and accurate for E. sakazakii detection.In this study, a novel PCR-based method was developed which was shown to becapable of discriminating between viable and dead E. sakazakii cells. This wasachieved utilising the irreversible binding of bacterial DNA to photo-activated PMA orEMA in order to prevent PCR amplification from the dead cells. At concentrations of 50and 100 μg.ml-1, PMA completely inhibited PCR amplification from dead cells, whilecausing no significant inhibition of the PCR amplification from viable cells. EMA wasequally effective in preventing PCR amplification from dead cells, however, it alsoinhibited PCR amplification from viable cells. PMA-PCR in particular, will be useful forassessing the efficacy of processing techniques, as well as for monitoring theresistance, survival strategies and stress responses of E. sakazakii. This will be animportant step in the efforts to eliminate E. sakazakii from food and food productionenvironments.

[发布机构] Stellenbosch University