Vicky Jasson

Characterization of Escherichia coli from raw poultry in Belgium and impact on the detection of Campylobacter jejuni using Bolton broth.

A comparative study examining Bolton broth and Preston broth for enrichment and reliable detection of Campylobacter jejuni (both healthy and freeze stressed cells) was performed. Tested as pure cultures, Bolton broth enabled faster resuscitation and growth of C. jejuni compared to Preston broth. When C. jejuni was co-incubated with extended-spectrum-beta-lactamase (ESBL) producing Escherichia coli isolated from Belgian poultry meat preparations, the latter dominated in the Bolton enrichment broth and crowded the mCCDA plates. This resulted in the inability to recover C. jejuni by ISO 10272-1:2006 standard method. Preston broth did not support the growth of the ESBL E. coli isolates, but showed longer detection time of C. jejuni compared to Bolton broth. The use of the same antibiotic (sodium cefoperazone) in Bolton broth and in mCCDA plates may explain the problems encountered for detection of C. jejuni, as high numbers of ESBL E. coli present after enrichment in Bolton broth, also caused overgrowth and masked the few C. jejuni colonies present on the mCCDA plates. The use of Campylobacter spp. specific real-time PCR circumvented these problems and enabled rapid detection of the pathogen after 24h enrichment in both Bolton and Preston broth, for both healthy and freeze stressed cells.

Screening of Fruit Products for Norovirus and the Difficulty of Interpreting Positive PCR Results

Despite recent norovirus (NoV) outbreaks related to consumption of fruit products, little is known regarding the NoV load on these foods. Therefore, 75 fruit products were screened for NoV presence by using an evaluated in-house NoV detection methodology consisting of a NoV extraction method and a reverse transcription quantitative PCR assay. Additionally, the fruit samples were screened for bacterial pathogens and bacterial hygiene indicators. Results of the NoV screening showed that 18 of 75 samples tested positive for GI and/or GII NoV despite a good bacteriological quality. The recovery of murine norovirus 1 virus particles acting as process control was successful in 31 of 75 samples with a mean recovery efficiency of 11.32% ± 6.08%. The level of detected NoV genomic copies ranged between 2.5 and 5.0 log per 10 g. NoV GI and/or GII were found in 4 of 10, 7 of 30, 6 of 20, and 1 of 15 of the tested raspberries, cherry tomatoes, strawberries, and fruit salad samples, respectively. However, confirmation of the positive quantitative PCR results by sequencing genotyping regions in the NoV genome was not possible. Due to the nature of the method used (reverse transcription quantitative PCR) for detection of genomic material, no differentiation was possible between infectious and noninfectious viral particles. No NoV outbreaks related to the tested fruit product types were reported during the screening period, which hampers a conclusion as to whether these unexpected high numbers of NoV-positive results should be perceived as a public health threat. These results, however, may indicate a prior NoV contamination of the tested food samples throughout the fresh produce chain.

Comparison of enrichment conditions for rapid detection of low numbers of sublethally injured Escherichia coli O157 in food.

A comparative study of lag phases and growth rates of healthy, stressed, and sublethally injured Escherichia coli O157 cells in 10 enrichment broths was performed. The evaluation of enrichment protocols was validated by different end point detection methods (two PCR and two combined capture-plate methods). Tryptic soy broth b [TSB (b)] provided the fastest growth (max = 1.00 1 0.06 h- ) but failed to recover oxidative-stressed E. coli O157. TSB (a), TSB-yeast extract medium, TSB supplemented with 8 mg/liter novobiocin plus 16 mg/liter vancomycin (TSB+), buffered peptone water (BPW), and BPW supplemented with 8 mg/liter vancomycin (BPW+V) enabled resuscitation of E. coli O157 cells independent from precultural conditions. Modified TSB plus 10 mg/liter novobiocin (mTSB+N), EC medium, EC reduced bile salts medium (ECred), TSB (b), and TSB supplemented with 8 mg/liter novobiocin plus 16 mg/liter vancomycin plus 2 mg/liter rifampin plus 1 mg/liter K-Telluriet plus 1.5 g/liter bile salts no. 3 (TSB++) all failed to recover E. coli O157 cells for at least one type of stress. The use of TSB (a), TSB+, BPW, and BPW+V was compared with that of mTSB+N (International Organization for Standardization reference broth) for reliable detection of low numbers of healthy, stressed, and sublethally injured E. coli O157 (approximately 10 CFU/10 g) from foods (sprouted seeds, fermented sausage, raw milk, and raw ground beef). When low numbers of healthy cells were inoculated, BPW, BPW+V, TSB, TSB+, and mTSB+N enabled growth until detectable numbers within 6 h of enrichment at 41.5 degrees C. Results showed that mTSB+N failed to recover to detectable numbers E. coli O157 cells sublethally injured by freeze and food stresses, in contrast to what was obtained with BPW and BPW+V. This study highlights that using mTSB+N for recovery of E. coli O157 from foods may yield false-negative results.

Kinetics of resuscitation and growth of L. monocytogenes as a tool to select appropriate enrichment conditions as a prior step to rapid detection methods

Rapid methods still rely on a prior (shortened) enrichment step before application. Quantitative information is a prerequisite for understanding the resuscitation kinetics of the growth during the enrichment step. In this study various basal and newly introduced selective enrichment broths were evaluated. First, growth parameters (lambda, mu(max)) of both healthy and sub-lethally injured cells were determined. Next, a selection of enrichment broths was compared for their capacity to support detection within 24h of low numbers of Listeria monocytogenes in artificially and naturally contaminated food samples. Detection was performed either by phage protein-based capture (Listeria Capture kit, Profos, Regensburg, Germany) combined with plating on chromogenic medium or by fluorescence in situ hybridization (FISH) using the VIT-Listeria kit (Vermicon, Munich, Germany). Kinetics of resuscitation and growth of L. monocytogenes in various enrichment broths showed that for detection of low numbers of sub-lethally injured L. monocytogenes cells at least an overnight enrichment was needed. A selective enrichment broth was needed to enable proliferation of L. monocytogenes within the indigenous bacterial flora present in foods. However, combination of an appropriate enrichment condition with advanced detection techniques may enable a 24h detection of L. monocytogenes.

Detection of low numbers of healthy and sub-lethally injured Salmonella enterica in chocolate

The capacity to detect low levels of healthy and sub-lethally injured Salmonella enterica cells in chocolate by two alternative rapid detection methods iQ-Check(TM)Salmonella II real-time PCR (Bio-Rad) and VIDAS® Easy SLM (BioMérieux) was assessed and compared with ISO 6579:2005. Chocolate, a low moisture food known to support the survival of Salmonella, was challenged as food matrix. Buffered peptone water (BPW) did not support the recovery of low levels of sub-lethally injured S. enterica independent of the detection method, while BPW supplemented with milk powder enabled detection by the three examined methods. However, inhibition of real-time PCR was observed since for one out of three repetitions of chocolate inoculated with a low number of sub-lethally injured S. enterica cells, no PCR signal was obtained. Therefore, attention should be paid to the enrichment step to avoid false negative results due to the presence of especially sub-lethally injured Salmonella cells in chocolate. An appropriate sample preparation (such as enrichment media and conditions for incubation) remains the key factor for reliable detection including sub-lethally injured cells and should be evaluated, if necessary optimized, for each detection assay.

PCR Applications in Food Microbiology

This article is a revision of the previous edition article by P.A. Bertram-Drogatz, F. Wilborn, P. Scheu, A. Pardigol, C. Koob, C. Gronewald, M. Fandke, A. Gasch, K. Berghof, volume 3, pp 1630–1641,