Detection of Klebsiella pneumoniae complex in carrots

Background Klebsiella pneumoniae (Kp) is an opportunistic pathogen now acknowledged as an urgent threat to human health because it is multi-drug resistant bacteria. The role of food as reservoir or carrier of Kp is under investigation, at the same time the optimization of the method to detect Kp in food is ongoing. The study was aimed to detect Kp in carrots using conventional method and to screen Real-time PCR performances, targeting intergenic sequence between zur and khe, based on INRA and Pasteur Institute method Methods A total of 60 samples of carrots were tested. The samples (25 g) were added to 225 ml of Buffered peptone water and incubated at 37 °C and at 44 °C for 24h. After incubation broths were subjected to Real-Time PCR, and one loop streaked on SCAI + inositol agar. Plates were then incubated at 37 °C and at 44 °C for 48 h. After incubation a maximum of 5 typical colonies were selected, subcultered on Nutritive agar and identified by MALDI-TOF MS. Results The RT PCR gave 29 positive samples, according to the presence of Kp detected at least in one of the two temperature conditions of broths incubation and 31 negative samples. Conventional culture gave, instead, 13 positive and 47 negative samples. Discordance was highlighted in 16 samples, all positives in RT-PCR and negatives with culture method. Conclusions Real-Time PCR gave not false negative results, then PCR decrease the time needed to perform the detection in negative samples. The discordance of results could be linked to lack of selectivity of the SCAI agar + inositol, not able to distinguish colonies of Klebsiella spp. Vs similar colonies not Klebsiella spp., the presence of DNA and not viable cells. The process of validation of the detection method is still ongoing, trying to improve selectivity using two selective media for plating. It's necessary to do new trials and to select more typical colonies to increase the probability to detect Kp colonies. Key messages Real-Time PCR is able to detect all positive samples, and to shorten the length of the analytical method. To improve sensibility of the analytical method more selective media need to be used.