Multiplex Real-Time PCR for the Detection of Tetracycline, Ciprofloxacin, and Erythromycin Resistance Determinants from Human and Foodborne Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni and Campylobacter coli are the predominant thermophilic species responsible for foodborne gastroenteritis worldwide. Elevated resistance to certain antibiotics was observed due to antimicrobial therapy in farm animals and humans, while reduced antimicrobial usage partially reduced antibiotic resistance. Monitoring the antimicrobial resistance demonstrated a substantial fraction of multi-resistant isolates, indicating the necessity of reliable tools for their detection. In this study, resistance determinants in 129 German and 21 Vietnamese isolates were selected to establish a novel multiplex real-time PCR (qPCR), facilitating the simultaneous detection of four resistance determinants. These comprised tet(O) gene variants associated with tetracycline resistance, point mutations GyrA_T86I and GyrA_T86V associated with ciprofloxacin resistance, and the erm(B) gene together with the point mutation A2075G in the 23S rRNA gene, associated with erythromycin resistance. Moreover, the performance of the qPCR assay was evaluated by comparing the results of qPCR to phenotypic antimicrobial resistance profiles, obtained with standardized EUCAMP3 microdilution panel, which showed 100% similarity (inclusivity and exclusivity). Variation in measurement methods, including qPCR machines and master mixes showed robustness, essential for laboratories. The assay can be used for the rapid detection of resistance determinants, and is beneficial for monitoring the spread of antibiotic resistance in C. jejuni and C. coli.

GMO analysis results from official food control laboratories in Germany from 2017 to 2021.

In Germany, genetically modified organisms (GMO) analysis of food samples collected within the official food control is performed by the laboratories of the Federal States. The present report shows GMO analysis results from food samples of the years 2017 to 2021, including contaminations by unauthorized GMO, as well as genetically modified (GM) plant events authorized in the European Union. In addition to previous publications, evaluation of the aggregated food samples analysed for GMO components is shown. During this timeframe, 1077 (7.1%) out of 15,145 samples contained genetic modification. In 43 samples, DNA sequences of unauthorized GM plants were found. Additionally, for food derived from soybean, evaluations according to different product categories and the agronomic production (conventional and organic farming) are shown. Whereas in products from organic farming and in conventional soybeans labelled "without genetic engineering" GM soybeans were detected in 6.1% and 8.9%, of all tested samples, respectively, nearly 30% of all conventional soy samples yielded positive results below 0.1%. However, only in 0.7% of the overall analysed 5424 soybean samples GMO percentages of more than 0.1% were obtained. Generally, authorized GM plants were only found at low contamination levels. The labelling threshold of 0.9% for GM ingredients was exceeded only in 0.2% (maize) and 0.1% (soybean) samples, respectively. For monitoring purposes and risk evaluation, the data collection shall be continued.

[Assessment of available and currently applied typing methods including genome-based methods for zoonotic pathogens with a focus on Salmonella enterica]. / Bestandsaufnahme der verfügbaren und aktuell eingesetzten Typisierungsmethoden einschließlich genombasierter Verfahren von Zoonoseerregern am Beispiel von Salmonella enterica.

BACKGROUND: In recent years, whole genome sequencing (WGS) in combination with bioinformatic analyses has become state of the art in evaluating the pathogenicity/resistance potential and relatedness of bacteria. WGS analysis thus represents a central tool in the investigation of the resistance and virulence potential of pathogens, as well as their dissemination via outbreak clusters and transmission chains within the framework of molecular epidemiology. In order to gain an overview of the available genotypic and phenotypic methods used for pathogen typing of Salmonella and Shiga toxin-producing and enterohemorrhagic Escherichia coli (STEC/EHEC) in Germany at state and federal level, along with the availability of WGS-based typing and corresponding analytical methods, a survey of laboratories was conducted. METHODS: An electronic survey of laboratories working for public health protection and consumer health protection was conducted from February to June 2020. RESULTS AND CONCLUSION: The results of the survey showed that many of the participating laboratories provide a wide range of phenotypic and molecular methods. Molecular typing is most commonly used for species identification of Salmonella. In many cases, WGS-based methods have already been established at federal and state institutions or are in the process of being established. The Illumina sequencing technology is the most widely used technology. The survey confirms the importance of molecular biology and whole genome typing technologies for laboratories in the diagnosis of bacterial zoonotic pathogens.

Whole-Genome Sequence Comparisons of Listeria monocytogenes Isolated from Meat and Fish Reveal High Inter- and Intra-Sample Diversity.

Interpretation of whole-genome sequencing (WGS) data for foodborne outbreak investigations is complex, as the genetic diversity within processing plants and transmission events need to be considered. In this study, we analyzed 92 food-associated Listeria monocytogenes isolates by WGS-based methods. We aimed to examine the genetic diversity within meat and fish production chains and to assess the applicability of suggested thresholds for clustering of potentially related isolates. Therefore, meat-associated isolates originating from the same samples or processing plants as well as fish-associated isolates were analyzed as distinct sets. In silico serogrouping, multilocus sequence typing (MLST), core genome MLST (cgMLST), and pangenome analysis were combined with screenings for prophages and genetic traits. Isolates of the same subtypes (cgMLST types (CTs) or MLST sequence types (STs)) were additionally compared by SNP calling. This revealed the occurrence of more than one CT within all three investigated plants and within two samples. Analysis of the fish set resulted in predominant assignment of isolates from pangasius catfish and salmon to ST2 and ST121, respectively, potentially indicating persistence within the respective production chains. The approach not only allowed the detection of distinct subtypes but also the determination of differences between closely related isolates, which need to be considered when interpreting WGS data for surveillance.

MALDI mass spectrometry imaging: From constituents in fresh food to ingredients, contaminants and additives in processed food.

Mass Spectrometry imaging (MS imaging) provides spatial information for a wide range of compound classes in different sample matrices. We used MS imaging to investigate the distribution of components in fresh and processed food, including meat, dairy and bakery products. The MS imaging workflow was optimized to cater to the specific properties and challenges of the individual samples. We successfully detected highly nonpolar and polar constituents such as beta-carotene and anthocyanins, respectively. For the first time, the distributions of a contaminant and a food additive were visualized in processed food. We detected acrylamide in German gingerbread and investigated the penetration of the preservative natamycin into cheese. For this purpose, a new data analysis tool was developed to study the penetration of analytes from uneven surfaces. Our results show that MS imaging has great potential in food analysis to provide relevant information about components' distributions, particularly those underlying official regulations.

Detection and differentiation of murine leukemia virus (MLV) and murine stem cell virus (MSCV) and therefrom derived nucleic acids.

Murine leukemia virus (MLV) and murine stem cell virus (MSCV) and derived retroviral vectors are widely used to study retrovirus biology and as tools for gene delivery. The method described here represents a quantitative real time PCR (qPCR) with hydrolysis probe that can be applied within classical qPCR as well as in digital droplet PCR (ddPCR). The method targets a 60 bp long fragment located within the U5 region of the MLV/MSCV genome sequence. For the here described method a LOD95% of 25 copies per PCR reaction (DNA) and 80 copies per PCR reaction (RNA) was determined, and PCR efficiencies of 92.5 % and 98.5 %, respectively, were observed. This method enables the fast and simple titration of viral genomic RNA present in retroviral vector stocks for accurate and consistent transduction experiments. Furthermore, it enables the detection of proviral and transfer plasmid derived DNA sequences and can be modified to differentiate between retroviral RNA and DNA.

Enhanced Antiviral Function of Magnesium Chloride-Modified Heparin on a Broad Spectrum of Viruses.

Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.

Toward an Integrated Genome-Based Surveillance of Salmonella enterica in Germany.

Despite extensive monitoring programs and preventative measures, Salmonella spp. continue to cause tens of thousands human infections per year, as well as many regional and international food-borne outbreaks, that are of great importance for public health and cause significant socio-economic costs. In Germany, salmonellosis is the second most common cause of bacterial diarrhea in humans and is associated with high hospitalization rates. Whole-genome sequencing (WGS) combined with data analysis is a high throughput technology with an unprecedented discriminatory power, which is particularly well suited for targeted pathogen monitoring, rapid cluster detection and assignment of possible infection sources. However, an effective implementation of WGS methods for large-scale microbial pathogen detection and surveillance has been hampered by the lack of standardized methods, uniform quality criteria and strategies for data sharing, all of which are essential for a successful interpretation of sequencing data from different sources. To overcome these challenges, the national GenoSalmSurv project aims to establish a working model for an integrated genome-based surveillance system of Salmonella spp. in Germany, based on a decentralized data analysis. Backbone of the model is the harmonization of laboratory procedures and sequencing protocols, the implementation of open-source bioinformatics tools for data analysis at each institution and the establishment of routine practices for cross-sectoral data sharing for a uniform result interpretation. With this model, we present a working solution for cross-sector interpretation of sequencing data from different sources (such as human, veterinarian, food, feed and environmental) and outline how a decentralized data analysis can contribute to a uniform cluster detection and facilitate outbreak investigations.

Rapid point-of-care detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP).

BACKGROUND: Fast, reliable and easy to handle methods are required to facilitate urgently needed point-of-care testing (POCT) in the current coronavirus pandemic. Life-threatening severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread all over the world, infecting more than 33,500,000 people and killing over 1 million of them as of October 2020. Infected individuals without any symptoms might still transfer the virus to others underlining the extraordinary transmissibility of this new coronavirus. In order to identify early infections effectively, treat patients on time and control disease spreading, rapid, accurate and onsite testing methods are urgently required. RESULTS: Here we report the development of a loop-mediated isothermal amplification (LAMP) based method to detect SARS-CoV-2 genes ORF8 and N directly from pharyngeal swab samples. The established reverse transcription LAMP (RT-LAMP) assay detects SARS-CoV-2 directly from pharyngeal swab samples without previous time-consuming and laborious RNA extraction. The assay is sensitive and highly specific for SARS-CoV-2 detection, showing no cross reactivity when tested on 20 other respiratory pathogens. The assay is 12 times faster and 10 times cheaper than routine reverse transcription real-time polymerase chain reaction, depending on the assay used. CONCLUSION: The fast and easy to handle RT-LAMP assay amplifying specifically the genomic regions ORF8 and N of SARS-CoV-2 is ideally suited for POCT at e.g. railway stations, airports or hospitals. Given the current pandemic situation, rapid, cost efficient and onsite methods like the here presented RT-LAMP assay are urgently needed to contain the viral spread.

Comparison of Targeted (HPLC) and Nontargeted (GC-MS and NMR) Approaches for the Detection of Undeclared Addition of Protein Hydrolysates in Turkey Breast Muscle.

The adulteration of fresh turkey meat by the undeclared addition of protein hydrolysates is of interest for fraudsters due to the increase of the economic gain by substituting meat with low cost ingredients. The aim of this study was to compare the suitability of three different analytical techniques such as GC-MS and 1H-NMR with HPLC-UV/VIS as a targeted method, for the detection of with protein hydrolysates adulterated turkey meat. For this, turkey breast muscles were treated with different plant- (e.g., wheat) and animal-based (e.g., gelatin, casein) protein hydrolysates with different hydrolyzation degrees (15-53%: partial; 100%: total), which were produced by enzymatic and acidic hydrolysis. A water- and a nontreated sample (REF) served as controls. The data analyses revealed that the hydrolysate-treated samples had significantly higher levels of amino acids (e.g., leucine, phenylalanine, lysine) compared with REF observed with all three techniques concordantly. Furthermore, the nontargeted metabolic profiling (GC-MS and NMR) showed that sugars (glucose, maltose) and/or by-products (build and released during acidic hydrolyses, e.g., levulinic acid) could be used for the differentiation between control and hydrolysates (type, degrees). The combination of amino acid profiling and additional compounds gives stronger evidence for the detection and classification of adulteration in turkey breast meat.

Corrigendum to "Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179" [Biomol. Detect. Quantif. 17 (March) (2019) 100076].

[This corrects the article DOI: 10.1016/j.bdq.2018.12.001.].

Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179.

Genetically modified alfalfa is authorized for cultivation in several countries since 2005. On the other hand, cultivation in or export to the European Union is not allowed and thus neither certified reference material nor official event-specific detection methods are available. Therefore, based on patent sequence information, event-specific real-time PCR detection methods targeting the junction sequence of the alfalfa genome and the transgenic insert of the respective events J101, J163 and KK179 were developed. Newly developed plasmids were used as reference material for assay optimization and in-house validation. Plasmid standards were quantified using digital droplet PCR and LOD95%, PCR efficiency, robustness and specificity of the assays were determined using real-time PCR. A LOD95% of 10 copies per PCR reaction was observed and PCR efficiencies of 95-97 % were achieved. Different real-time PCR instruments and PCR conditions were applied to test for robustness of the assays using DNA at a concentration of 30 copies per µL for each gm alfalfa event. All replicates were positive independent of the instrument or the PCR condition. DNA from certified reference material of different genetically modified crops as well as reference materials of the three events was used to experimentally test for specificity. No unspecific amplification signal was observed for any of the assays. Validation results were in line with the "Minimum Performance Requirements for Analytical Methods of GMO Testing" of the European Network of GMO Laboratories. Furthermore, an inter-laboratory comparison study was conducted to show the transferability and applicability of the methods and to verify the assay performance parameters.

Molecular Tracing to Find Source of Protracted Invasive Listeriosis Outbreak, Southern Germany, 2012-2016.

We investigated 543 Listeria monocytogenes isolates from food having a temporal and spatial distribution compatible with that of the invasive listeriosis outbreak occurring 2012-2016 in southern Germany. Using forensic microbiology, we identified several products from 1 manufacturer contaminated with the outbreak genotype. Continuous molecular surveillance of food isolates could prevent such outbreaks.

Reverse Transcription Quantitative Polymerase Chain Reaction for Detection of and Differentiation Between RNA and DNA of HIV-1-Based Lentiviral Vectors.

The purpose of the described method is the detection of and differentiation between RNA and DNA of human immunodeficiency virus (HIV)-derived lentiviral vectors (LV) in cell culture supernatants and swab samples. For the analytical surveillance of genetic engineering, operations methods for the detection of the HIV-1-based LV generations are required. Furthermore, for research issues, it is important to prove the absence of LV particles for downgrading experimental settings in terms of the biosafety level. Here, a quantitative polymerase chain reaction method targeting the long terminal repeat U5 subunit and the start sequence of the packaging signal ψ is described. Numerous controls are included in order to monitor the technical procedure.

Strategies and methods for the detection and identification of viral vectors.

The production and application of viral vectors are frequently performed genetic engineering operations. HIV-1-based lentiviral vectors, AAV2-based, and adenoviral vectors are amongst the most abundant viral vectors utilized for gene delivery. They are generally classified into risk group 1 or 2 (according to EU directive 2000/54/EC on the protection of workers from risks related to exposure to biological agents at work).

Molecular characterization of an unauthorized genetically modified Bacillus subtilis production strain identified in a vitamin B2 feed additive.

Many food and feed additives result from fermentation of genetically modified (GM) microorganisms. For vitamin B2 (riboflavin), GM Bacillus subtilis production strains have been developed and are often used. The presence of neither the GM strain nor its recombinant DNA is allowed for fermentation products placed on the EU market as food or feed additive. A vitamin B2 product (80% feed grade) imported from China was analysed. Viable B. subtilis cells were identified and DNAs of two bacterial isolates (LHL and LGL) were subjected to three whole genome sequencing (WGS) runs with different devices (MiSeq, 454 or HiSeq system). WGS data revealed the integration of a chloramphenicol resistance gene, the deletion of the endogenous riboflavin (rib) operon and presence of four putative plasmids harbouring rib operons. Event- and construct-specific real-time PCR methods for detection of the GM strain and its putative plasmids in food and feed products have been developed.

[Food allergen regulations in the EU]. / Lebensmittelrechtliche Regelungen für Allergene in der EU.

With the implementation of EU regulation 1169/2011 in December 2014, labelling of allergenic ingredients has been extended to non-prepacked foods. The member states of the European Union were authorised to lay down national rules for the labelling of allergenic ingredients in non-prepacked foods. In Germany, this was accomplished through the introduction of the Vorläufige Lebensmittelinformations-Ergänzungsverordnung (VorlLMIEV). Regulation 1169/2011 also changed the way allergenic ingredients are to be labelled on prepacked foods.This article provides an overview over the current regulations regarding the labelling of food allergens on prepacked and non-prepacked foods in the EU and Germany.

Optimization of digital droplet polymerase chain reaction for quantification of genetically modified organisms.

Digital PCR in droplets (ddPCR) is an emerging method for more and more applications in DNA (and RNA) analysis. Special requirements when establishing ddPCR for analysis of genetically modified organisms (GMO) in a laboratory include the choice between validated official qPCR methods and the optimization of these assays for a ddPCR format. Differentiation between droplets with positive reaction and negative droplets, that is setting of an appropriate threshold, can be crucial for a correct measurement. This holds true in particular when independent transgene and plant-specific reference gene copy numbers have to be combined to determine the content of GM material in a sample. Droplets which show fluorescent units ranging between those of explicit positive and negative droplets are called 'rain'. Signals of such droplets can hinder analysis and the correct setting of a threshold. In this manuscript, a computer-based algorithm has been carefully designed to evaluate assay performance and facilitate objective criteria for assay optimization. Optimized assays in return minimize the impact of rain on ddPCR analysis. We developed an Excel based 'experience matrix' that reflects the assay parameters of GMO ddPCR tests performed in our laboratory. Parameters considered include singleplex/duplex ddPCR, assay volume, thermal cycler, probe manufacturer, oligonucleotide concentration, annealing/elongation temperature, and a droplet separation evaluation. We additionally propose an objective droplet separation value which is based on both absolute fluorescence signal distance of positive and negative droplet populations and the variation within these droplet populations. The proposed performance classification in the experience matrix can be used for a rating of different assays for the same GMO target, thus enabling employment of the best suited assay parameters. Main optimization parameters include annealing/extension temperature and oligonucleotide concentrations. The droplet separation value allows for easy and reproducible assay performance evaluation. The combination of separation value with the experience matrix simplifies the choice of adequate assay parameters for a given GMO event.