Contamination of milk by enterococci and coliforms from bovine faeces

AIM: To determine the contribution of enterococci and coliforms from bovine faeces and teats to contamination of raw milk. Methods: Putative enterococci (n = 301) and coliforms (n = 365) were isolated from bovine faeces (n = 20), cows' teats (n = 20), the raw milk (n = 1) and the milking environment (n = 4) on one farm. The clonal relationships of each bacterial group were investigated using Pulsed-Field Gel Electrophoresis of genomic macrorestriction fragments. Representatives of the different clusters of enterococci were identified by molecular techniques including rep-PCR, SDS protein profiling, Fluorescent Amplified Fragment Length Polymorphism (FAFLP), phenylalanyl-tRNA synthase (pheS) sequence analysis and/or 16S rDNA gene sequencing. Coliforms were identified by API 20E strips. RESULTS: The majority of the bovine faecal enterococcal isolates were identified as a potential new species of Aerococcus (100 isolates); E. faecium (28 isolates), and Aerococcus viridans (28 isolates) were also found. All coliform isolates from the bovine faeces were identified as Escherichia coli. The coliforms present in the milk were Hafnia alvei, Serratia liquefaciens, Yersinia enterocolitica and Enterobacter amnigenus. No E. coli, Enterococcus or Aerococcus from the bovine faeces were found in the milk. A single clone of H. alvei was found in the water, the milking equipment and the milk, suggesting that the water was the source of the organism in the milk. No vancomycin-resistant aerococci or enterococci were found while most of the isolates tested showed the presence of at least one virulence gene. The milk-sock retained strains that adhered to particulate faecal material. Coliforms were present at approx. 2 orders of magnitude greater than enterococci in the bovine faeces. CONCLUSIONS: The results imply that bovine faeces are not an important source of contamination of raw milk with enterococci or coliforms. SIGNIFICANCE AND IMPACT OF THE STUDY: The results confirm those of two previous studies (Gelsomino et al. 2001, Int J Food Microbiol71, 177-188 and Kagkli et al. 2007, Int J Food Microbiol114, 243-251) on two other farms. The three studies show that contamination of milk by enterococci, lactobacilli and coliforms of bovine faecal origin is extremely low. The results also suggest that where raw milk is implicated in food infection, other factors in addition to faecal contamination must be involved.     

Detection of airborne genetically modified maize pollen by real-time PCR

The cultivation of genetically modified (GM) crops has raised numerous concerns in the European Union and other parts of the world about their environmental and economic impact. Especially outcrossing of genetically modified organisms (GMO) was from the beginning a critical issue as airborne pollen has been considered an important way of GMO dispersal. Here, we investigate the use of airborne pollen sampling combined with microscopic analysis and molecular PCR analysis as an approach to monitor GM maize cultivations in a specific area. Field trial experiments in the European Union and South America demonstrated the applicability of the approach under different climate conditions, in rural and semi-urban environment, even at very low levels of airborne pollen. The study documents in detail the sampling of GM pollen, sample DNA extraction and real-time PCR analysis. Our results suggest that this 'GM pollen monitoring by bioaerosol sampling and PCR screening' approach might represent an useful aid in the surveillance of GM-free areas, centres of origin and natural reserves.     

Differential Listeria monocytogenes Strain Survival and Growth in Katiki,a Traditional Greek Soft Cheese,at Different Storage Temperatures

Katiki Domokou is a traditional Greek cheese, which has received the Protected Designation of Origin recognition since 1994. Its microfloras have not been studied although its structure and composition may enable (or even favor) the survival and growth of several pathogens, including Listeria monocytogenes. The persistence of L. monocytogenes during storage at different temperatures has been the subject of many studies since temperature abuse of food products is often encountered. In the present study, five strains of L. monocytogenes were aseptically inoculated individually and as a cocktail in Katiki Domokou cheese, which was then stored at 5, 10, 15, and 20°C. Pulsed-field gel electrophoresis was used to monitor strain evolution or persistence during storage at different temperatures in the case of the cocktail inoculum. The results suggested that strain survival of L. monocytogenes was temperature dependent since different strains predominated at different temperatures. Such information is of great importance in risk assessment studies, which typically consider only the presence or absence of the pathogen.Listeria monocytogenes is a ubiquitous food-borne pathogen associated with outbreaks of listeriosis from consumption of various food commodities, especially dairy products, seafood, and meat (2, 26). The pathogen is of great health concern for the food industry because it is characterized by high mortality rates, amounting to 20 to 30% (14). Due to the severity of illness, especially for pregnant women, neonates, the elderly, and immunodeficient people, the level of the pathogen in food should remain low to ensure safe food products.The new regulation of the European Union (EU) for microbiological criteria for L. monocytogenes in foods has set maximum levels of 100 CFU g⁻¹ at the time of consumption for soft cheeses (8). In fact, the new EC 2073/2005 regulation in annex I lists the microbiological criteria for foodstuffs, which are classified into food safety criteria and process hygiene criteria. According to the new EU regulation, food safety criteria are those which “define the acceptability of a product or a batch of foodstuff applicable to products placed on the market” (8).Legislative amendments regarding the presence of L. monocytogenes in ready-to-eat (RTE) foods are of great importance. Indeed, for the first time RTE foods are legislatively distinguished according to the target population for which they are intended, i.e., whether they are intended for consumption (i) by infants, (ii) by people with special medical conditions (immunocompromised), or (iii) by other target human subpopulations. In the most recent amendment the RTE foods other than those intended for infants or for those with special medical needs are further subdivided into foods that are able to support the growth of L. monocytogenes and those that are not. Products with pH ≤ 5.0 and water activity of ≤0.94 and products with a shelf life of less than 5 days are automatically considered to belong to the category of RTE foods that are unable to support the growth of L. monocytogenes (8). The regulation also states that “other categories of products can also belong to this category, subject to scientific justification.” Last but not least, the food safety criteria for L. monocytogenes are adjusted according to the bacteria''s temporal stage in the food chain. Thus, for RTE foods that are able to support the growth of L. monocytogenes, the new regulation demands the absence of the pathogen (in 25 g) “before the food has left the immediate control of the food business operator, who has produced it” but allows up to 100 CFU g⁻¹ in “products placed on the market during their shelf life.” The 100-CFU g⁻¹ limit also applies throughout the shelf life of marketed RTE foods unable to support L. monocytogenes growth (8).The pH and the water activity of Katiki Domokou (Katiki), a spreadable RTE traditional Greek cheese, are within the limits mentioned in the regulation. This product, a white cheese with a creamy structure, was traditionally produced from goat milk or from a mixture of goat and sheep milk. It has been recognized as a Protected Designation of Origin product since 1994 (www.greekcheese.gr), and its consumption has readily increased in the last few years. The milk is initially pasteurized and cooled at 27 to 28°C. Coagulation is then conducted with or without the addition of rennet, and the mixture is left to stand at 20 to 22°C. The curd is pulped and placed in cloth sacks for draining, with high final moisture (ca. 75%) and low salt content (ca. 1%) and pH (4.3 to 4.5) while it is stored at 4 to 5°C.The quantitative estimation of kinetic parameters related to growth, survival, and death of L. monocytogenes has been described previously (2, 14, 20). The kinetic parameters of L. monocytogenes during storage at different temperatures have been the subject of many studies since temperature abuse of food products is often encountered (25, 28). However, strain characteristics or viability have not been taken into account (or have not been considered) as yet (20). This may explain the variability of findings in regard to different storage conditions (7, 17). Pulsed-field gel electrophoresis (PFGE) is a powerful subtyping tool, a gold standard for epidemiology, which provides repeatable results. It has the ability to generate profiles of a wide range of microorganisms and to discriminate strains with high fidelity (11, 19). PFGE has been used in several studies to type strains of epidemiological interest as well as to trace contaminants in the food chain (12, 13, 18).The purpose of the present study was to assess the survival of five strains of L. monocytogenes inoculated either individually or as a cocktail in Katiki cheese. The cheese was stored at 5, 10, 15, and 20°C over a period of 1 month. PFGE was used to monitor the strain(s) that might survive and/or grow at different temperatures in a complex ecosystem like Katiki. The strains used in the study to form the inoculum consisted of two type strains of serotype 4b and three isolates belonging to our laboratory collection that were isolated from soft cheese and the conveyor belt of RTE foods. The strains were chosen on the basis of their source of isolation since this could be crucial to the interpretation of the data. The population was monitored throughout storage with respect to its quantitative as well as its qualitative evolution.     

Application of the modular approach to an in-house validation study of real-time PCR methods for the detection and serogroup determination of verocytotoxigenic Escherichia coli

European Commission regulation 2073/2005 on the microbiological criteria for food requires that Escherichia coli is monitored as an indicator of hygienic conditions. Since verocytotoxigenic E. coli (VTEC) strains often cause food-borne infections by the consumption of raw food, the Biological Hazards (BIOHAZ) panel of the European Food Safety Authority (EFSA) recommended their monitoring in food as well. In particular, VTEC strains belonging to serogroups such as O26, O103, O111, O145, and O157 are known causative agents of several human outbreaks. Eight real-time PCR methods for the detection of E. coli toxin genes and their variants (stx(1), stx(2)), the intimin gene (eae), and five serogroup-specific genes have been proposed by the European Reference Laboratory for VTEC (EURL-VTEC) as a technical specification to the European Normalization Committee (CEN TC275/WG6). Here we applied a "modular approach" to the in-house validation of these PCR methods. The modular approach subdivides an analytical process into separate parts called "modules," which are independently validated based on method performance criteria for a limited set of critical parameters. For the VTEC real-time PCR module, the following parameters are being assessed: specificity, dynamic range, PCR efficiency, and limit of detection (LOD). This study describes the modular approach for the validation of PCR methods to be used in food microbiology, using single-target plasmids as positive controls and showing their applicability with food matrices.     

Towards a pathogenic Escherichia coli detection platform using multiplex SYBR®Green Real-time PCR methods and high resolution melting analysis

Escherichia coli is a group of bacteria which has raised a lot of safety concerns in recent years. Five major intestinal pathogenic groups have been recognized amongst which the verocytotoxin or shiga-toxin (stx1 and/or stx2) producing E. coli (VTEC or STEC respectively) have received a lot of attention recently. Indeed, due to the high number of outbreaks related to VTEC strains, the European Food Safety Authority (EFSA) has requested the monitoring of the "top-five" serogroups (O26, O103, O111, O145 and O157) most often encountered in food borne diseases and addressed the need for validated VTEC detection methods. Here we report the development of a set of intercalating dye Real-time PCR methods capable of rapidly detecting the presence of the toxin genes together with intimin (eae) in the case of VTEC, or aggregative protein (aggR), in the case of the O104:H4 strain responsible for the outbreak in Germany in 2011. All reactions were optimized to perform at the same annealing temperature permitting the multiplex application in order to minimize the need of material and to allow for high-throughput analysis. In addition, High Resolution Melting (HRM) analysis allowing the discrimination among strains possessing similar virulence traits was established. The development, application to food samples and the flexibility in use of the methods are thoroughly discussed. Together, these Real-time PCR methods facilitate the detection of VTEC in a new highly efficient way and could represent the basis for developing a simple pathogenic E. coli platform.     

Kluyveromyces lactis and Saccharomyces cerevisiae, two potent deacidifying and volatile-sulphur-aroma-producing microorganisms of the cheese ecosystem

Cheese flavour is the result of complex biochemical transformations attributed to bacteria and yeasts grown on the curd of smear-ripened cheeses. Volatile sulphur compounds (VSCs) are responsible for the characteristic aromatic notes of several cheeses. In the present study, we have assessed the ability of Kluyveromyces lactis, Kluyveromyces marxianus and Saccharomyces cerevisiae strains, which are frequently isolated from smear-ripened cheeses, to grow and deacidify a cheese medium and generate VSCs resulting from l-methionine degradation. The Kluyveromyces strains produced a wider variety and higher amounts of VSCs than the S. cerevisiae ones. We have shown that the pathway is likely to be proceeding differently in these two yeast genera. The VSCs are mainly generated through the degradation of 4-methylthio-oxobutyric acid in the Kluyveromyces strains, in contrast to the S. cerevisiae ones which have higher l-methionine demethiolating activity, resulting in a direct conversion of l-methionine to methanethiol. The deacidification activity which is of major importance in the early stages of cheese-ripening was also compared in S. cerevisiae and Kluyveromyces strains.     

Towards Plant Species Identification in Complex Samples: A Bioinformatics Pipeline for the Identification of Novel Nuclear Barcode Candidates

Monitoring of the food chain to fight fraud and protect consumer health relies on the availability of methods to correctly identify the species present in samples, for which DNA barcoding is a promising candidate. The nuclear genome is a rich potential source of barcode targets, but has been relatively unexploited until now. Here, we show the development and use of a bioinformatics pipeline that processes available genome sequences to automatically screen large numbers of input candidates, identifies novel nuclear barcode targets and designs associated primer pairs, according to a specific set of requirements. We applied this pipeline to identify novel barcodes for plant species, a kingdom for which the currently available solutions are known to be insufficient. We tested one of the identified primer pairs and show its capability to correctly identify the plant species in simple and complex samples, validating the output of our approach.     

L-methionine degradation pathway in Kluyveromyces lactis: identification and functional analysis of the genes encoding L-methionine aminotransferase

Kluyveromyces lactis is one of the cheese-ripening yeasts and is believed to contribute to the formation of volatile sulfur compounds (VSCs) through degradation of L-methionine. L-methionine aminotransferase is potentially involved in the pathway that results in the production of methanethiol, a common precursor of VSCs. Even though this pathway has been studied previously, the genes involved have never been studied. In this study, on the basis of sequence homology, all the putative aminotransferase-encoding genes from K. lactis were cloned in an overproducing vector, pCXJ10, and their effects on the production of VSCs were analyzed. Two genes, KlARO8.1 and KlARO8.2, were found to be responsible for L-methionine aminotransferase activity. Transformants carrying these genes cloned in the pCXJ10 vector produced threefold-larger amounts of VSCs than the transformant containing the plasmid without any insert or other related putative aminotransferases produced.