Listeria monocytogenes can cause severe foodborne infections in humans and invasive diseases in different animal species, especially in small ruminants. Infection of sheep and goats can occur via contaminated feed or through the teat canal. Both infection pathways result in direct (e.g., raw milk from an infected udder or fresh cheese produced from such milk) or indirect exposure of consumers. The majority of dairy farmers produces a high-risk product, namely fresh cheese made from raw ewe's and goat's milk. This, and the fact that L. monocytogenes has an extraordinary viability, poses a significant challenge to on-farm dairies. Yet, surprisingly, almost no scientific studies have been conducted dealing with the hygiene and food safety aspects of directly marketed dairy products. L. monocytogenes prevalence studies on small ruminant on-farm dairies are especially limited. Therefore, it was our aim to focus on three main transmission scenarios of this important major foodborne pathogen: (i) the impact of caprine and ovine listerial mastitis; (ii) the significance of clinical listeriosis and outbreak scenarios; and (iii) the impact of farm management and feeding practices.

Humans and animals can become asymptomatic carriers of Listeria monocytogenes and introduce the pathogen into their environment with their feces. In turn, this environmental contamination can become the source of food- and feed-borne illnesses in humans and animals, with the food production chain representing a continuum between the farm environment and human populations that are susceptible to listeriosis. Here, we update a review from 2012 and summarize the current knowledge on the asymptomatic carrier statuses in humans and animals. The data on fecal shedding by species with an impact on the food chain are summarized, and the ways by which asymptomatic carriers contribute to the risk of listeriosis in humans and animals are reviewed.

Most Austrian dairies and cheese manufacturers participated in a Listeria monitoring program, which was established after the first reports of dairy product-associated listeriosis outbreaks more than thirty years ago. Within the Listeria monitoring program, up to 800 mL of product-associated liquids such as cheese smear or brine are processed in a semi-quantitative approach to increase epidemiological sensitivity. A sampling strategy within cheese production, which detects environmental contamination before it results in problematic food contamination, has benefits for food safety management. The liquid-based sampling strategy was implemented by both industrial cheese makers and small-scale dairies located in the mountainous region ofWestern Austria. This report considers more than 12,000 Listeria spp. examinations of liquid-based samples in the 2009 to 2018 timeframe. Overall, the occurrence of L. monocytogenes in smear liquid samples was 1.29% and 1.55% (n = 5043 and n = 7194 tested samples) for small and industrial cheese enterprises, respectively. The liquid-based sampling strategy for Listeria monitoring at the plant level appears to be superior to solid surface monitoring. Cheese smear liquids seem to have good utility as an index of the contamination of cheese up to that point in production. A modelling or validation process should be performed for the new semi-quantitative approach to estimate the true impact of the method in terms of reducing Listeria contamination at the cheese plant level.

Diagnostic strategies to detect foodborne pathogens such as L. monocytogenes in food processing environments are cost- and time-consuming necessities to ensure safe food. In this study we present in-house monitoring data of a relatively novel direct enrichment PCR (DEP) pooling approach for the detection of L. monocytogenes in the food industry. The DEP-pooling approach was initially developed and reported by our working group in 2017 and has since been implemented as the routine L. monocytogenes diagnostic method by an Austrian dairy producer in two of its internal diagnostic laboratories. The results of almost 50,000 individual routine samples demonstrate the overall applicability, reliability and cost-saving capability of the DEP-pooling approach. While the original DEP-pooling approach was designed and evaluated as a qualitative test, the quantitative PCR results of 168 positive samples show that a separation between samples that contained culturable or non-culturable L. monocytogenes is possible. In conclusion, the present study demonstrates the advantages of a straightforward combination of microbiological and molecular biological methods in a routine setup. It is not only cost- and time-efficient, but can also potentially identify the source of recurring contamination via non-culturable cells. These results should encourage researchers, biomedical companies and industrial customers alike to implement such new cost-efficient methods to ensure a safe food production environment.

Detection of pathogens is crucial in food production areas. While it is well established, swabbing as a state-of-the-art sampling method offers several drawbacks with respect to yield, standardization, overall handling, and long-term monitoring. This led us to develop and evaluate a method that is easier to use at a lower cost and that should be at least as sensitive. After evaluating sundry promising materials, we tested text-marking paper stickers for their suitability to take up and release Listeria monocytogenes with their nonsticky paper side over a 14-day time period using quantitative PCR. The recovery rate was similar to that in previous studies using conventional swabs, and we also confirmed the feasibility of pooling besides resilience to cleansing and disinfection. In a proof-of-concept experiment that sampled several locations, such as door handles, the occurrences of L. monocytogenes and Escherichia coli were determined. The results suggest that the presented sticker system might offer a promising cost-effective alternative sampling system with improved handling characteristics. IMPORTANCE As a ubiquitous bacterium, Listeria monocytogenes has a propensity to enter food production areas inadvertently via fomites such as door handles and switches. While the bacterium might not be in direct contact with the food products, knowing the microbial status of the surroundings is essential for risk assessment. Our investigation into a novel quantitative PCR (qPCR)-based sampling system with the highest sensitivity and ability to monitor over long periods of time, yet based on paper, proved to be cost-effective and reasonably convenient to handle.

Fatty acid-based ionic liquids (FAILs) are an emerging class of biodegradable ILs. Although long-chained FAILs in particular offer a promising alternative to problematic hydrophobic ILs in lubrication and extraction applications, recent studies have revealed the possibility of an anionic "side-chain length effect", which would adversely impact their eco-friendliness. This study therefore investigated the toxicity of FAILs with side-chain lengths between C1 and C18 and benign cations by applying a tripartite biological test system, including enzyme inhibition test, determination of virucidal activity against three model viruses, and bactericidal activity against 12 clinically relevant bacteria. Results do not confirm a general anionic "side-chain effect" with FAILs but rather demonstrate a diverse outcome depending on the biological test system employed. While Gram-negative bacteria and viruses were not affected by increasing FAIL anion alkyl chain length, medium- and long-chained FAILs exhibited pronounced effects at the enzyme level and on Gram-positive bacteria. While this study reinforces the necessity to establish a multilevel approach for the (eco)toxicological evaluation of FAILs, it also demonstrates a specific effectiveness of FAILs against enzymes and Gram-positive bacteria, which should be considered for possible future applications.

In recent years, a new potential measure against foodborne pathogenic bacteria was rediscoveredbacteriophages. However, despite all their advantages, in connection to their widespread application in the food industry, negative consequences such as an uncontrolled phage spread as well as a development of phage resistant bacteria can occur. These problems are mostly a result of long-term persistence of phages in the food production environment. As this topic has been neglected so far, this article reviews the current knowledge regarding the effectiveness of disinfectant strategies for phage inactivation and removal. For this purpose, the main commercial phage products, as well as their application fields are first discussed in terms of applicable inactivation strategies and legal regulations. Secondly, an overview of the effectiveness of disinfectants for bacteriophage inactivation in general and commercial phages in particular is given. Finally, this review outlines a possible strategy for users of commercial phage products in order to improve the effectiveness of phage inactivation and removal after application.

Due to a higher probability for violation of hygiene measures, reconstruction work is a substantial food safety challenge for food business operators (FBOs). Here, we monitored a Listeria monocytogenes contamination scenario during a timely enduring reconstruction period that aimed at an expansion of the main building of a leading meat processing facility. Reconstruction took place while food production was ongoing. We used a longitudinal sampling scheme targeting 40 floor water drains distributed over the food processing environment (FPE) over a five year period. The population structure of L. monocytogenes was determined by PCR-serogrouping, pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). While the first sampling deciphered a baseline of contamination (45%), intensified sanitation measures decreased L. monocytogenes prevalence before commencement of work (5%). The reconstruction activities increased the prevalence of L. monocytogenes in the FPE (20.5%) and changed the population structure to a higher proportion of disease-associated genotypes (61%). During the first sampling ST121 was prevalent throughout the FPE, even in the packaging area. After the second and third sampling, following increased application of hypochlorite during sanitation, ST121 was only present in the raw material preparation area. A resilient flora was detected during three sampling events (ST8, ST9 and ST37) which might have not been exposed to daily cleaning in the floor drains. After the accomplishment of reconstruction work, the L. monocytogenes population structure shifted to the condition initially found (45% and 20.5% during the first and sixth sampling event). This paper indicates that reconstruction phases are high risk episodes for food safety in FPEs. Special precautions must be taken to avoid cross-contamination of products since reconstruction is usually ongoing for extended periods of time.

Many bacteria enter the viable but non-culturable (VBNC) state to maximize resources and increase their tolerance to harmful conditions to cope with environmental stress, which has been described for a plethora of important human and foodborne pathogens. VBNC pathogens can potentially present a serious risk to human health as they are invisible to routine microbiological culture-based methods. Of high importance is the increased tolerance to antibiotics or disinfectant measures while in the VBNC state. The greatest remaining challenge for such investigations is the lack of an appropriate, cost-effective multi-species screening method due to experimental constraints. In this study, we investigated if de novo ATP production of cells in the VBNC state is a suitable indicator for overall cell viability that can be utilized to determine the minimum ATP inhibitory concentration (MAIC) of antibiotics and other antimicrobials. To validate this approach, heat-stress time-kill experiments were performed with both culturable and VBNC cells. We developed a comprehensive experimental setup and demonstrated the applicability of this VBNC-MIC assay for testing the tolerance of 12 strains of 4 important bacterial species (Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa, and Listeria monocytogenes) in the VBNC state to eight important antimicrobials including four different antibiotics. We confirmed that bacteria in the VBNC state were resistant to all tested antibiotics (ampicillin, imipenem, ciprofloxacin, and gentamicin) and additionally insensitive to disinfectants (benzalkonium chloride and trioctylmethylammonium chloride) and preservatives (bronopol and sodium azide). These data emphasize the need for further research regarding the characteristics of bacterial pathogens in the VBNC state and present the advantages and high-throughput capabilities of ATP determinations to investigate tolerance of VBNC pathogens to antimicrobials. The presented method should be helpful in order to identify appropriate countermeasures, treatments, or disinfectants when confronted with bacterial pathogens in the VBNC state.

Background: Bacteriophages are increasingly used as alternatives to conventional antibiotics, although very limited data is available in respect of consequences within the plant and for monitoring processes. Scope and approach: This commentary provides a critical view on the use of phages in food production processes and summarises possible disadvantages and problems which can arise. Further, prevention strategies and proposals for future handling and detection are discussed. Key findings and conclusions: Indiscriminate use of phages is not innocuous and can drastically reduce the potential of phages as an alternative class of anti-bacterial agent. It is important to avoid the development of phage-resistant bacteria, inhibition in standard microbiological monitoring, phage-spread and possibly the consumption of contaminated food. This could be expedited by a declaration of phage-treated food and the application of alternative detection methods for both, food-borne bacteria and applied phages. Additionally, use and development of new disinfectants effective against phages and awareness of the consequences of habitual use of phage products should ensure their future as alternatives to conventional antibiotics.

A cluster of 34 human cases of listeriosis was traced to consumption of contaminated quargel cheese, a sour milk specialty sold in Austria, Germany and Czech Republic. Here, we try to assess how many portions were consumed by the Austrian population at a certain contamination level (CL). In total, 1623 cheese lots were produced during the outbreak period resulting in > 3 million portions of cheese delivered to the market. From 650 sets of quality control data provided by the food business operator, we reconstructed the contamination scenario over time and identified 84 lots that were found to be positive. With regard to another sixteen lots, a CL was found ranging from one to 3,84 log(10) CFU L. monocytogenes/g, measured in product stored between one to 23 days after production. However the number of storage days at home before consumption is unknown. To resolve this issue, we modelled the theoretical CL of the product if consumed either 20, 30, 40 or 50 days post production. We found that 10 lots (approx. 27,350 portions) would have been contaminated at CLs higher than 3 log(10) CFU L. monocytogenes/g if all cheese had been consumed after 20 days of storage. This number shifts to 20 lots (approx. 54,700 portions) after 30 days of storage. If all cheese had been consumed at the end of shelf life (50 days of storage), theoretically 242,5 lots would have exceeded a CL of 6 log(10) CFU L. monocytogenes/g. We concluded that the extended shelf life given to the product was a driver of the outbreak scenario. It is stunning to note that so few cases were reported in spite of consumers' massive exposure to L monocytogenes. We hypothesized that a low pathogenicity of both quargel outbreak clones (QOC1 and QOC2) could have contributed to this discrepancy. Our hypothesis was falsified since both strains QOC1 and QOC2 are fully virulent in an oral infection mouse model, showing even higher pathogenicity than the reference strain EGDe.

16S rRNA amplicon sequencing is frequently used to investigate and compare microbiomes. This study focuses on the potential, applicability and limits of 16S rRNA amplicon sequencing for comparing the microbiomes of two dairy production plants, one of which experienced recurrent contamination with Listeria monocytogenes. During preceding in vitro studies, DNA extraction of Listeria monocytogenes and Salmonella enterica ser. Typhimurium with two extraction kits (PowerSoil and NucleoSpin) revealed differences between these exemplary bacteria and both extraction methods. When equivalent samples of the companies were processed independently with both methods, sequencing results demonstrated also bacterium specific variances. These variances do not reflect a direct correlation between Gram-positive or -negative bacteria and the performance of both kits. Despite these differences, the analysis with a non-metric multi-dimensional scaling plot showed grouping of respective samples independent of the DNA extraction method.Some operational taxonomic units (OTUs) occurred predominantly in one of the companies, but none of them generated a clear hypothesis why one of the plants had recurrent contamination with L. monocytogenes. This work provides insights into the microbiomes of the two dairy companies but advises cautions in over-interpreting sequencing results as selective DNA extraction and sampling only reflect components of prevalent bacterial communities.

Milk powder is a food for malnourished African children and for healthy infants of women with HIV/AIDS. High demand and low purchasing power has resulted in a huge informal, black market in Sub-Saharan Africa. Forty-three milk powder batches were analyzed for 43 chemical elements using ICP-MS One sample (2.3%) was contaminated at a lead concentration of 240 mu g/kg dry weight exceeding the European threshold (130 mu g/kg dry weight). Macroelement contents revealed a trend decreasing in concentration through skimmed, full cream products to infant formulae. Concentration ranges by dry weight differed in respect of uncertainty intervals of +/- 10%. Median Ca, K and P concentrations declined from 11.14 g/kg to 3.21 g/kg, 14.11 g/kg to 4.95 g/kg and 9.12 g/kg to 2.75 g/kg dry mass, respectively. Milk powder samples obtained from the Tanzanian black market were comparable in respect of nutritional and chemical content to international branded full cream products.

The quantitative real-time polymerase chain reaction (qPCR) is one of the most commonly molecular methods used today. It is central to numerous assays that have since been developed and described around its optimization. The Listeria monocytogenes prfA qPCR assay has been studied in great detail and due to its comprehensive knowledge, excellent performance (sensitivity of one single copy), and internal amplification control, it represents a suitable test platform for qPCR examinations. In this study, we compared ten different polymerases (or ready-to-use mastermixes) as possible (economic) alternatives to our gold standard Platinum Taq polymerase. We sought to determine the reproducibility of these assays under modified conditions, which are realistic because published assays are frequently used with substituted polymerases. Surprisingly, there was no amplification at all with some of the tested polymerases, even although the internal amplification control worked well. Since adaptation of the thermal profile and of MgCl2 concentration could restore amplification, simple replacement of the polymerase can destroy a well-established assay leading up to >106-fold less analytical sensitivity. Further, validation using Poisson and PCR-Stop analyses revealed limits to some assay-polymerase combinations and emphasize the importance of validation.

Unregulated international flow of foods poses a danger to human health, as it may be contaminated with pathogens. Recent studies have investigated neglected routes of pathogen transmission and reported the occurrence of Listeria monocytogenes in food illegally imported into the European Union (EU), either confiscated at four international airports or sold illegally on the Romanian black market. In this study we investigated the genotype diversity and the amino acid sequence variability of three main virulence factors of 57 L. monocytogenes isolates. These isolates were derived from 1474 food samples illegally imported into the EU and originated from 17 different countries. Multilocus sequence typing revealed 16 different sequence types (STs) indicating moderate genotype diversity. The most prevalent STs were ST2, ST9, and ST121. The pulsed-field gel electrophoresis (PFGE) analysis resulted in 34 unique pulsotypes. PFGE types assigned to the most prevalent STs (ST2, ST9, and ST121) were highly related in their genetic fingerprint. Internalin A (InlA) was present in 20 variants, including six truncated InlA variants, all harbored by isolates of ST9 and ST121. We detected eight ST-specific listeriolysin O (LLO) variants, and among them, one truncated form. The actin-assembly-inducing protein ActA was present in 15 different ST-specific variants, including four ActA variants with an internal truncation. In conclusion, this study shows that L. monocytogenes, isolated from illegally imported food, have moderate genotype diversity, but diverse virulence factors variants, mainly of InlA.

Effective monitoring of microbial pathogens is essential for a successful preventive food safety and hygiene strategy. However, as most monitoring strategies are growth-based, these tests fail to detect pathogenic bacteria that have entered the viable but non-culturable (VBNC) state. The present study reports the induction of the VBNC state in five human pathogens by commercially available household cleaners in combination with inorganic salts. We determined that non-ionic surfactants, a common ingredient in household cleaners, can induce the VBNC state, when combined with salts. A screening study with 630 surfactant/salt combinations indicates a correlation between the hydrophobicity of the surfactant and VBNC induction in L. monocytogenes, E. coli, S. enterica serovar Typhimurium, S. aureus and toxin-producing enteropathogenic E. coli. Cells that were exposed to combinations of surfactants and salts for 5 min and up to 1h lost their culturability on standard growth media while retaining their ATP production, fermentation of sugars and membrane integrity, which suggests intact and active metabolism. Screening also revealed major differences between Gram-negative and Gram-positive bacteria; the latter being more susceptible to VBNC induction. Combinations of such detergents and salts are found in many different environments and reflect realistic conditions in industrial and domestic surroundings. VBNC cells present in industrial environments, food-processing plants and even our daily routine represent a serious health risk due to possible resuscitation, unknown spreading, production of toxins and especially their invisibility to routine detection methods, which rely on culturability of cells and fail to detect VBNC pathogens.

Diagnostic strategies to detect foodborne pathogens such as L. monocytogenes in food processing environments are cost and time consuming necessities to ensure safe food products. While two-step pooling diagnostic strategies incorporating PCR have been successfully introduced in recent years, such strategies to date have not been employed for food and hygiene monitoring. The objective of this study was to develop and evaluate the applicability of a cost-effective PCR pooling approach called direct enrichment PCR (DEP)-pooling based on an existing L. monocytogenes monitoring setup. The proposed pooling strategy is based on a first enrichment step, subsequent PCR-pooling and re-evaluation of PCR-positive pools with the VIDAS DUO. Overall, more than 3000 individual routine samples from a European cheese production facility were tested with the proposed DEP-pooling and compared in parallel with existing L monocytogenes monitoring. In this approach equivalent results were obtained. Proposed DEP-pooling utilizes the advantages of both microbiological enrichment and PCR to identify negative samples faster in a cost efficient way with an overall cost reduction of 60% based on 1% positive samples. (C) 2017 Elsevier Ltd. All rights reserved.

Cleaning with solid CO2 (dry ice blasting) is an environmental friendly, fast and residue-free method applied in many industrial areas. In this study the disinfection potential of dry ice blasting and parameters influencing cleaning efficacy was investigated. The method removes bacterial cells to a similar extent from several surfaces and components of dairy production equipment, occasionally with a slight abrasive effect. Efficacy is affected by the quantity of dry ice and pressure applied but neither by the pellet size nor the initial quantity of bacterial cells on the surface. Since the bacteria removal rate is less than five log10 units, dry ice blasting cannot be recommended as a disinfection method, but it demonstrates efficient cleaning comparable to other conventional methods. In practice, dry ice blasting of food production equipment is recommended outside the production area because there is a high risk of recontamination due to spread of the bacteria.

The indigenous Maasai communities of Tanzania are traditional pastoralists who depend upon fresh cow's milk as a staple food. However, lack of clean water challenges conventional milk hygiene practices. To extend the life of milk in a hot environment, the internal surfaces of natural storage calabashes are smoke-treated. We conducted interviews in eight Maasai regions and 13 districts to establish the key indigenous plants preferred and their traditional manner of use. A semi-structured questionnaire was designed to determine: (i) plants used, (ii) parts used, (iii) preparation and utensil -smoking practices, (iv) therapeutic applications and health benefits and (v) other uses. Twenty plants were identified as most valuable, mostly hardwoods with strong aromas and astringent tastes. The most favoured plants were Zanthoxylum chalybeum (Knob wood; overall preference 26.6 %), Olea europaea subsp. africana (African wild olive; 11.9 %), Combretum molle (velvet bush willow; 11.4 %), Cordia ovalis/monoica (satin saucer berry; 9.5 %) and Cordia sinensis (grey-leaved saucer berry; 7.3 %). Pastoralists also use these plants medicinally and know-how is transmitted orally. However, the integrity of this competence is endangered by climate change and by inter-tribal territorial wars and land-grabs, which hamper community stability. Documentation of these indigenous, sustainable hygiene practices is warranted before traditional customs are abandoned.