Nachweis von Shiga Toxin-bildenden Escherichia coli und thermophilen Campylobacter species bei Almkühen und in auf Almen produzierten Lebensmitteln

In Germany thermophilic Campylobacter spp. and Enterohemorrhagic Escherichia coli (EHEC) are - apart from Salmonella spp. and Yersinia spp. - the most frequent bacterial sources of human gastroenteritis. Analyzing the development in other European countries as well as in the USA and Canada, one can presume, that in near future thermophilic Campylobacter spp. will be at the top of the national statistics of food poisoning. The objective of this study was a possible STEC or thermophilic Campylobacter spp. contamination of natural food produced on alpine pastures, as in Bavarian Alps the food production on alpine pastures is still of great importance. In additon to this aspect of consumer´s health protection, it was on the other hand the intention to collect knowledge concerning a possible change of STEC and thermophilic Campylobacter spp. excretion between winter and summer time.Thermophile Campylobacter spp. und Enterohämorrhagische E. coli (EHEC) stehen in Deutschland, gemeinsam mit Salmonella spp. und Yersinia spp., an der Spitze der bakteriellen Enteritiserreger. Betrachtet man die Entwicklung in anderen europäischen Ländern sowie in den USA und Kanada, so kann man davon ausgehen, dass sich dieser Trend in den nächsten Jahren fortsetzen wird. Ziel dieser Arbeit war es deshalb zum einen zu untersuchen, ob und in welchem Ausmaß auf Almen produzierte, naturbelassene Lebensmittel mit STEC und thermophilen Campylobacter spp. kontaminiert sind, denn die Almwirtschaft spielt im bayerischen Alpenraum nach wie vor eine bedeutsame Rolle. Neben diesem Aspekt des gesundheitlichen Verbraucherschutzes sollten Daten darüber gewonnen werden, auf welche Weise sich die Ausscheidung beider Bakterien mit dem Kot durch die Älpung verändert

Genetic Characterization of Listeria from Food of Non-Animal Origin Products and from Producing and Processing Companies in Bavaria, Germany

Reported cases of listeriosis from food of non-animal origin (FNAO) are increasing. In order to assess the risk of exposure to Listeria monocytogenes from FNAO, the genetic characterization of the pathogen in FNAO products and in primary production and processing plants needs to be investigated. For this, 123 samples of fresh and frozen soft fruit and 407 samples of 39 plants in Bavaria, Germany that produce and process FNAO were investigated for Listeria contamination. As a result, 64 Listeria spp. isolates were detected using ISO 11290-1:2017. Environmental swabs and water and food samples were investigated. L. seeligeri (36/64, 56.25%) was the most frequently identified species, followed by L. monocytogenes (8/64, 12.50%), L. innocua (8/64, 12.50%), L. ivanovii (6/64, 9.38%), L. newyorkensis (5/64, 7.81%), and L. grayi (1/64, 1.56%). Those isolates were subsequently sequenced by whole-genome sequencing and subjected to pangenome analysis to retrieve data on the genotype, serotype, antimicrobial resistance (AMR), and virulence markers. Eight out of sixty-four Listeria spp. isolates were identified as L. monocytogenes. The serogroup analysis detected that 62.5% of the L. monocytogenes isolates belonged to serogroup IIa (1/2a and 3a) and 37.5% to serogroup IVb (4b, 4d, and 4e). Furthermore, the MLST (multilocus sequence typing) analysis of the eight detected L. monocytogenes isolates identified seven different sequence types (STs) and clonal complexes (CCs), i.e., ST1/CC1, ST2/CC2, ST6/CC6, ST7/CC7, ST21/CC21, ST504/CC475, and ST1413/CC739. The core genome MLST analysis also showed high allelic differences and suggests plant-specific isolates. Regarding the AMR, we detected phenotypic resistance against benzylpenicillin, fosfomycin, and moxifloxacin in all eight L. monocytogenes isolates. Moreover, virulence factors, such as prfA, hly, plcA, plcB, hpt, actA, inlA, inlB, and mpl, were identified in pathogenic and nonpathogenic Listeria species. The significance of L. monocytogenes in FNAO is growing and should receive increasing levels of attention

Innovative and Highly Sensitive Detection of Clostridium perfringens Enterotoxin Based on Receptor Interaction and Monoclonal Antibodies

Clostridium perfringens enterotoxin (CPE) regularly causes food poisoning and antibioticassociated diarrhea; therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different C. perfringens culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research.Peer Reviewe

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

Presence of Listeria at primary production and processing of food of non-animal origin (FNAO) in Bavaria, Germany

Several foodborne outbreaks associated with food of non-animal origin (FNAO) were reported within the last years. In recent years, Listeria monocytogenes has been associated with such outbreaks. For this reason, different producers of FNAO at the primary production and processing level in Bavaria, Germany, were inspected from July 2020 to June 2021. Environmental and food sampling as well as the sampling of irrigation and processing water was performed to investigate the prevalence of Listeria spp., including L. monocytogenes at facilities that produce ready-to-eat FNAO. Altogether, 39 producers of soft fruit, vegetables, ready-to-eat raw fruits, and vegetables/fresh cut were inspected. In addition to the on-spot inspections, 407 samples were taken in total, among them, 229 were swab samples from food contact material and the environment, 59 food samples (including soft fruit, vegetables and ready-to-eat vegetables), and 119 samples of irrigation and processing water. Samples were analyzed using methods according to ISO 11290-1:2017. Furthermore, the samples of irrigation and processing water were also quantitatively tested for the number of Escherichia coli (ISO 9308-2:2014-06), enterococci (ISO 7899-2:2000-11), and Pseudomonas aeruginosa (ISO 16266:2008-05). No contamination with E. coli, enterococci, and P. aeruginosa could be detected in most of the samples. Overall, in 12.53% of the samples, Listeria spp. were detected. L. monocytogenes was identified in 1.72% of the environmental and processing water samples, whereas L. monocytogenes was not detected in food samples.In addition to water sources and quality, this study demonstrates that irrigation regime, cultivation, hygienic handling, and maintenance protocols are highly important to reduce the potential contamination of ready-to-eat soft fruits and vegetables with Listeria spp

Diagnostic Real-Time PCR Assays for the Detection of Emetic Bacillus cereus Strains in Foods and Recent Food-Borne Outbreaks

Cereulide-producing Bacillus cereus can cause an emetic type of food-borne disease that mimics the symptoms provoked by Staphylococcus aureus. Based on the recently discovered genetic background for cereulide formation, a novel 5′ nuclease (TaqMan) real-time PCR assay was developed to provide a rapid and sensitive method for the specific detection of emetic B. cereus in food. The TaqMan assay includes an internal amplification control and primers and a probe designed to target a highly specific part of the cereulide synthetase genes. Additionally, a specific SYBR green I assay was developed and extended to create a duplex SYBR green I assay for the one-step identification and discrimination of the two emesis-causing food pathogens B. cereus and S. aureus. The inclusivity and exclusivity of the assay were assessed using a panel of 100 strains, including 23 emetic B. cereus and 14 S. aureus strains. Different methods for DNA isolation from artificially contaminated foods were evaluated, and established real-time assays were used to analyze two recent emetic food poisonings in southern Germany. One of the food-borne outbreaks included 17 children visiting a day care center who vomited after consuming a reheated rice dish, collapsed, and were hospitalized; the other case concerned a single food-poisoning incident occurring after consumption of cauliflower. Within 2 h, the etiological agent of these food poisonings was identified as emetic B. cereus by using the real-time PCR assay

Detection, differentiation, and identification of botulinum neurotoxin serotypes C, CD, D, and DC by highly specific immunoassays and mass spectrometry

Botulinum neurotoxin (BoNT) serotypes C and D and their mosaic variants CD and DC cause severe cases of botulism in animal husbandry and wildlife. Epidemiological data on the exact serotype or toxin variant causing outbreaks are rarely available, mainly because of their high sequence identity and the lack of fast and specific screening tools to detect and differentiate the four similar toxins. To fill this gap, we developed four highly specific sandwich enzyme-linked immunosorbent assays (ELISAs) able to detect and differentiate botulinum neurotoxins type BoNT/C, D, CD, and DC based on four distinct combinations of specific monoclonal antibodies targeting both conserved and divergent subdomains of the four toxins. Here, highly sensitive detection with detection limits between 2 and 24 pg mL−1 was achieved. The ELISAs were extensively validated and results were compared with data obtained by quantitative real-time PCR using a panel of Clostridium botulinum strains, real sample materials from veterinary botulism outbreaks, and non-BoNT-producing Clostridia. Additionally, in order to verify the results obtained by ELISA screening, the new monoclonal antibodies were used for BoNT enrichment and subsequent detection (i) on a functional level by endopeptidase mass spectrometry (Endopep-MS) assays and (ii) on a protein sequence level by LC-MS/MS spectrometry. Based on all technical information gathered in the validation study, the four differentiating ELISAs turned out to be highly reliable screening tools for the rapid analysis of veterinary botulism cases and should aid future field investigations of botulism outbreaks and the acquisition of epidemiological data

Multiplex PCR for Detection of Botulinum Neurotoxin-Producing Clostridia in Clinical, Food, and Environmental Samples▿

Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes

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

Detection, differentiation, and identification of botulinum neurotoxin serotypes C, CD, D, and DC by highly specific immunoassays and mass spectrometry

Botulinum neurotoxin (BoNT) serotypes C and D and their mosaic variants CD and DC cause severe cases of botulism in animal husbandry and wildlife. Epidemiological data on the exact serotype or toxin variant causing outbreaks are rarely available, mainly because of their high sequence identity and the lack of fast and specific screening tools to detect and differentiate the four similar toxins. To fill this gap, we developed four highly specific sandwich enzyme-linked immunosorbent assays (ELISAs) able to detect and differentiate botulinum neurotoxins type BoNT/C, D, CD, and DC based on four distinct combinations of specific monoclonal antibodies targeting both conserved and divergent subdomains of the four toxins. Here, highly sensitive detection with detection limits between 2 and 24 pg mL−1 was achieved. The ELISAs were extensively validated and results were compared with data obtained by quantitative real-time PCR using a panel of Clostridium botulinum strains, real sample materials from veterinary botulism outbreaks, and non-BoNT-producing Clostridia. Additionally, in order to verify the results obtained by ELISA screening, the new monoclonal antibodies were used for BoNT enrichment and subsequent detection (i) on a functional level by endopeptidase mass spectrometry (Endopep-MS) assays and (ii) on a protein sequence level by LC-MS/MS spectrometry. Based on all technical information gathered in the validation study, the four differentiating ELISAs turned out to be highly reliable screening tools for the rapid analysis of veterinary botulism cases and should aid future field investigations of botulism outbreaks and the acquisition of epidemiological data