Use of biochemical miniaturized galleries, rRNA based lateral flow assay and Real Time PCR for Cronobacter spp. confirmation.

Identification of Cronobacter represent a major challenge for laboratories testing powdered infant formula (PIF). In the present study, two biochemical galleries and three molecular methods have been applied to confirm 276 Cronobacter spp. and non-Cronobacter isolates from different sources. Using the latest database of API 20 E and ID 32 E biochemical miniaturized kits, 53% and 78% of the isolates were identified respectively. From the available results, total accuracy for Cronobacter detection was in 97% (API 20 E) and 99% (ID 32 E). The three molecular methods were based on rRNA based lateral flow, Real Time PCR combined with either a hybridization or hydrolysis probe. For all three methods total accuracy was more than 99%. A pilot trial using Next Generation Sequencing (NGS) correctly identified 58 out of 66 isolates (88%) in DNA mixtures. The results indicate that the commercially available approaches such as ID 32 E, rRNA based lateral flow and Real Time PCR are all suitable for Cronobacter identification at the genus level. The NGS method may become a suitable alternative in the future, provided that the sequence database is improved.

A Comparative Study of Nine SARS-CoV-2 IgG Lateral Flow Assays Using Both Post-Infection and Post-Vaccination Samples.

BACKGROUND: Since the SARS-CoV-2 pandemic, lateral flow assays (LFA) detecting specific antibodies have entered the market in abundance. Despite being CE-IVD-labeled, the antigenic compounds of the assays are often unknown, the performance characteristics provided by the manufacturer are often incomplete, and the samples used to obtain the data are not detailed. OBJECTIVE: To perform a comparative evaluation of nine lateral flow assays to detect IgG responses against SARS-CoV-2. For the evaluation, a carefully designed serum panel containing post-infection samples and post-vaccination (both mRNA vaccine and inactivated virus vaccine) samples was used. RESULTS: The sensitivity of the assays overall ranged from 9 to 90.3% and the specificity ranged from 94.2 to 100%. Spike protein-containing assays performed generally better than the assays with only nucleocapsid protein. The sensitivity of some assays was higher on post-infection samples, while other assays had a higher sensitivity to post-vaccination samples. CONCLUSION: A comparative approach in the verification of LFAs with an adequately designed serum panel enabled the identification of the antigens used in the assays. Sensitivities differed between post-infection and post-vaccination samples, depending on the assays used. This demonstrates that the verification of assays must be performed with samples representative of the intended use of the assay.

Detection of Listeria spp. and L. monocytogenes in pooled test portion samples of processed dairy products.

Listeria monocytogenes is a major foodborne pathogen. Testing multiple portions of the same final product is often required to verify the effectiveness of a food safety management system. Therefore, it will be advantageous to the laboratories to combine these test portions and process as one sample. However, combining samples for analysis, i.e., pooling, can be done only if there is no negative impact on the result. The objective of this study was to validate pooling of test portions for the detection of L. monocytogenes and Listeria spp. in dairy products as no scientific evidence currently exists to support this practice. Six representative matrices, namely, pudding, yogurt, brie cheese, 2% milk, ice cream and infant formula were spiked separately with stressed L. monocytogenes and Listeria spp. in 25 g and pooled test portions (375 g/250 g/125 g). Two methods, namely, ISO-11290-1:1996 Amd1:2004 and a validated alternative method Rapid'L.Mono were used for sample testing. Performance of a method in pooled test portions was considered to be satisfactory if the relative limit of detection (RLOD50; LOD50 [pooled test portion]/LOD50 [25 g test portion]) and limit of detection (LOD50) obtained was ≤2.5 and 1 CFU or MPN, respectively. Results obtained from L. monocytogenes and Listeria spp. trials were given equal weightage to decide on the impact of pooling. Acceptable RLOD50 and LOD50 values were consistently obtained in L. monocytogenes and Listeria spp. inoculation experiments when test portions were pooled up to 125 g for all matrices tested with both methods. While there was a slight delay for the primary enrichment of the pooled test portions to reach the desired incubation temperature when compared to the 25 g test portions, it did not negatively impact the outcome when samples were pooled up to 125 g. Background organisms were in general present at low concentrations and did not seem to adversely impact the recovery of the target organism in 125 g samples. Thus, pooling of test portions to up to 125 g for the detection of L. monocytogenes and Listeria spp. by two culture methods in processed dairy products has been validated.

Salmonella Serotyping; Comparison of the Traditional Method to a Microarray-Based Method and an in silico Platform Using Whole Genome Sequencing Data.

Salmonella is one of the most common causes of food-borne diseases worldwide. While Salmonella molecular subtyping by Whole Genome Sequencing (WGS) is increasingly used for outbreak and source tracking investigations, serotyping remains as a first-line characterization of Salmonella isolates. The traditional phenotypic method for serotyping is logistically challenging, as it requires the use of more than 150 specific antisera and well trained personnel to interpret the results. Consequently, it is not a routine method for the majority of laboratories. Several rapid molecular methods targeting O and H loci or surrogate genomic markers have been developed as alternative solutions. With the expansion of WGS, in silico Salmonella serotype prediction using WGS data is available. Here, we compared a microarray method using molecular markers, the Check and Trace Salmonella assay (CTS) and a WGS-based serotype prediction tool that targets molecular determinants of serotype (SeqSero) to the traditional phenotypic method using 100 strains representing 45 common and uncommon serotypes. Compared to the traditional method, the CTS assay correctly serotyped 97% of the strains, four strains gave a double serotype prediction. Among the inconclusive data, one strain was not predicted and two strains were incorrectly identified. SeqSero was evaluated with two versions (SeqSero 1 and the alpha test version of SeqSero 2). The correct antigenic formula was predicted by SeqSero 1 for 96 and 95% of strains using raw reads and assembly, respectively. However, 34 and 33% of these predictions included multiple serotypes by raw reads and assembly. With raw reads, one strain was not identified and three strains were discordant with phenotypic serotyping result. With assembly, three strains were not predicted and two strains were incorrectly predicted. While still under development, SeqSero 2 maintained the accuracy of antigenic formula prediction at 98% and reduced multiple serotype prediction rate to 13%. One strain had no prediction and one strain was incorrectly predicted. Our study indicates that the CTS assay is a good alternative for routine laboratories as it is an easy to use method with a short turn-around-time. SeqSero is a reliable replacement for phenotypic serotyping if WGS is routinely implemented.

A rapid and reliable alternative to ISO 21528-1:2004 for detection of Enterobacteriaceae.

Current legislation in Europe uses the Enterobacteriaceae as a parameter in process hygiene criteria for various food products and refers to the corresponding ISO standard (ISO 21528-1:2004) as mandatory analytical method for this purpose. The ISO procedure includes an enrichment step in EE ("Enterobacteriaceae Enrichment") broth, but it has been reported recently that some isolates of Enterobacteriaceae do not grow well or will even die off in this broth, which could lead to false negative results. To determine if this trait is common among the Enterobacteriaceae, a collection of 95 strains was screened for growth in EE broth. Inhibition was observed with 9 strains (7 Cronobacter sakazakii, 1 Cronobacter malonaticus and 1 Enterobacter amnigenus). Factors affecting cell death were found to be related mainly to the inclusion of bile salts and dyes in this medium. In a second step, an alternative method omitting the EE broth was evaluated using 326 samples, comprising 8 different food matrices and environmental samples from the corresponding manufacturing sites. Positive results were obtained for 235 samples using the ISO standard method and 232 samples using the alternative shortened method. No significant difference was found between the results for the two methods. It is proposed that the standard method for detection of Enterobacteriaceae is revised accordingly.

Lessons from the genomes of bifidobacteria.

The gut microbiota is a complex ecosystem composed of hundreds of different bacterial species that altogether play an important role in the physiology of their host. In the past few years the complete genome sequence of a number of bacterial strains isolated from the human gastrointestinal tract has been established including that of Bifidobacterium longum NCC2705 isolated from the feces of a healthy infant. Bifidobacteria are among the first species to colonise the human gastrointestinal tract and as such are believed to play an important role in gut homeostasis and normal development. The genome sequence of NCC2705 has revealed a number of features that suggest how this bacterium has adapted to its environment and that could help understanding how it interacts with its host. Here, we review general features of bifidobacteria and illustrate how genome-based approaches can help us better understand the biology of these organisms.

Validation of test portion pooling for Salmonella spp. detection in foods.

Pathogen monitoring programs play a crucial role in the verification of the effectiveness of implemented hygiene control measures. Sampling and testing procedures included in pathogen monitoring involve the analysis of multiple test portions where all samples must be negative for the presence of pathogens for a certain test portion size. Many food safety programs require increased testing due to the risks that a pathogen may be present. Analyzing more than one test portion could prove to be expensive and labor intensive. When more than one test portion for a specified food item is to be tested, the test portions could be combined to form a pooled test portion to reduce laboratory workload, costs of reagents and further confirmatory steps, but only when evidence is available that pooling does not affect on the number of false negative results for different matrices. This study has been performed to demonstrate the equivalence of test portion pooling for Salmonella detection with five different methods using cultural, ELISA and Real Time PCR technologies. Twenty-three (23) different food items including confectionary products, meal components, infant formula, pet food and powdered beverages were validated. Other complementary parameters like impact of minimum and maximum incubation time for pre-enrichment, temperature profile, pH and Salmonella concentration after the pre-enrichment and background flora have also been considered in the study. The results showed that pooling test portions up to 375g for Salmonella detection is valid for the methods that were tested. Relative level of detection (RLOD50) values for 22 of the food items tested were acceptable (i.e. lower than 2.5) when comparing the reference sample size (25g) against the alternative pooled sample size (375g), provided the enrichment broth was pre-warmed and maximum incubation time is respected.

Fully Closed Genome Sequences of Five Type Strains of the Genus Cronobacter and One Cronobacter sakazakii Strain.

Cronobacteris associated with infant infections and the consumption of reconstituted infant formula. Here we sequenced and closed six genomes ofC. condimenti(T),C. muytjensii(T),C. universalis(T),C. malonaticus(T),C. dublinensis(T), andC. sakazakiithat can be used as reference genomes in single nucleotide polymorphism (SNP)-based next-generation sequencing (NGS) analysis for source tracking investigations.