Extensive genetic variability linked to IS26 insertions in the fljB promoter region of atypical monophasic variants of Salmonella enterica serovar Typhimurium.

Fifty-nine monophasic Salmonella enterica serovar Typhimurium isolates, collected in Belgium during the period from 2008 to 2011, have been serotyped as 4,[5]:i:- and shown to harbor an fljB coding sequence. The genetic differences between these strains and phenotypically biphasic Salmonella Typhimurium were analyzed through PCR and DNA sequencing. Genetic alterations in the fljB promoter region affecting expression of the phase 2 flagellin were observed in 53 isolates. Other genetic events in the invertible region carrying the fljB promoter were observed in 2 isolates. For the remaining 4 isolates, no molecular differences with a reference biphasic Salmonella Typhimurium strain could be observed. Next-generation sequencing of one representative isolate affected in the fljB promoter region revealed a 26-kb IS26 composite transposon insertion along with a local genomic rearrangement. Several other IS26 element-mediated alterations of this genomic region were observed. This group of monophasic Salmonella Typhimurium isolates was genetically heterogeneous, as revealed by multilocus variable-number tandem-repeat analysis (MLVA), PCR, and sequencing. Pigs and pork represented a major source of such monophasic isolates in Belgium, as reported in other countries. Three out of 5 isolates of human origin presented genetic profiles identical to those of food isolates, demonstrating the pathogenic potential of the newly characterized variants and potential dissemination along the food chain. This study highlighted the key role played by IS26 insertions in the loss of phase 2 flagellin expression and the subsequent generation of multiple monophasic variant lineages from biphasic Salmonella Typhimurium ancestors.

Alternative microbial methods: An overview and selection criteria.

This study provides an overview and criteria for the selection of a method, other than the reference method, for microbial analysis of foods. In a first part an overview of the general characteristics of rapid methods available, both for enumeration and detection, is given with reference to relevant bibliography. Perspectives on future development and the potential of the rapid method for routine application in food diagnostics are discussed. As various alternative "rapid" methods in different formats are available on the market, it can be very difficult for a food business operator or for a control authority to select the most appropriate method which fits its purpose. Validation of a method by a third party, according to international accepted protocol based upon ISO 16140, may increase the confidence in the performance of a method. A list of at the moment validated methods for enumeration of both utility indicators (aerobic plate count) and hygiene indicators (Enterobacteriaceae, Escherichia coli, coagulase positive Staphylococcus) as well as for detection of the four major pathogens (Salmonella spp., Listeria monocytogenes, E. coli O157 and Campylobacter spp.) is included with reference to relevant websites to check for updates. In a second part of this study, selection criteria are introduced to underpin the choice of the appropriate method(s) for a defined application. The selection criteria link the definition of the context in which the user of the method functions - and thus the prospective use of the microbial test results - with the technical information on the method and its operational requirements and sustainability. The selection criteria can help the end user of the method to obtain a systematic insight into all relevant factors to be taken into account for selection of a method for microbial analysis.

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.

Establishment of procedures provoking sub-lethal injury of Listeria monocytogenes, Campylobacter jejuni and Escherichia coli O157 to serve method performance testing.

In this study procedures provoking sub-lethal injury for three different pathogens are described which may be used in determination of accuracy and robustness of methods, comparison studies and or validation of rapid detection methods. Three common food-borne pathogens were used, Listeria monocytogenes, Campylobacter jejuni and Escherichia coli O157. The pathogens were exposed to heat stress, cold stress, freeze stress, acid stress, oxidative stress and "food" stress. Sub-lethal injury was determined by plating in parallel on selective and non-selective media. The statistical significant differences in enumeration were established. The choice of stress to create sub-lethal injury to cells depended on the fact that the procedure must be easy to handle, repeatable and relevant for stress conditions in foods, but also on the micro-organism itself. Oxidative stress (1000 microM H(2)O(2)) was chosen to impose sub-lethal injury on L. monocytogenes and a specific "food" stress for E. coli O157. For C. jejuni a specific "food" stress as well as the oxidative stress (750 microM H(2)O(2)) were capable of creating a standardized procedure of provoking injury.

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.

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.

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.