Jan De Block

Antimicrobial Resistance in the Food Chain: A Review

Antimicrobial resistant zoonotic pathogens present on food constitute a direct risk to public health. Antimicrobial resistance genes in commensal or pathogenic strains form an indirect risk to public health, as they increase the gene pool from which pathogenic bacteria can pick up resistance traits. Food can be contaminated with antimicrobial resistant bacteria and/or antimicrobial resistance genes in several ways. A first way is the presence of antibiotic resistant bacteria on food selected by the use of antibiotics during agricultural production. A second route is the possible presence of resistance genes in bacteria that are intentionally added during the processing of food (starter cultures, probiotics, bioconserving microorganisms and bacteriophages). A last way is through cross-contamination with antimicrobial resistant bacteria during food processing. Raw food products can be consumed without having undergone prior processing or preservation and therefore hold a substantial risk for transfer of antimicrobial resistance to humans, as the eventually present resistant bacteria are not killed. As a consequence, transfer of antimicrobial resistance genes between bacteria after ingestion by humans may occur. Under minimal processing or preservation treatment conditions, sublethally damaged or stressed cells can be maintained in the food, inducing antimicrobial resistance build-up and enhancing the risk of resistance transfer. Food processes that kill bacteria in food products, decrease the risk of transmission of antimicrobial resistance.

Heterogeneity of heat-resistant proteases from milk Pseudomonas species.

Pseudomonas fragi, Pseudomonas lundensis and members of the Pseudomonas fluorescens group may spoil Ultra High Temperature (UHT) treated milk and dairy products, due to the production of heat-stable proteases in the cold chain of raw milk. Since the aprX gene codes for a heat-resistant protease in P. fluorescens, the presence of this gene has also been investigated in other members of the genus. For this purpose an aprX-screening PCR test has been developed. Twenty-nine representatives of important milk Pseudomonas species and thirty-five reference strains were screened. In 42 out of 55 investigated Pseudomonas strains, the aprX gene was detected, which proves the potential of the aprX-PCR test as a screening tool for potentially proteolytic Pseudomonas strains in milk samples. An extensive study of the obtained aprX-sequences on the DNA and the amino acid level, however, revealed a large heterogeneity within the investigated milk isolates. Although this heterogeneity sets limitations to a general detection method for all proteolytic Pseudomonas strains in milk, it offers a great potential for the development of a multiplex PCR screening test targeting individual aprX-genes. Furthermore, our data illustrated the potential use of the aprX gene as a taxonomic marker, which may help in resolving the current taxonomic deadlock in the P. fluorescens group.

Furosine in consumption milk and milk powders

Abstract Furosine, a marker for the Maillard reaction, can be determined by a HPLC method. Concentrations of furosine in Belgian consumption milk samples were compared with contents of Italian and Spanish consumption milks reported in the literature. Further, the relation between acid-soluble β-lactoglobulin and furosine content in different milk samples prepared by a pilot UHT-installation was verified. As expected, low concentrations of furosine were found in samples with high levels of soluble β-lactoglobulin, but the relation was rather poor. In contrast, a good correlation was found between lactulose and furosine contents in UHT-milk and sterilized milk. Furosine determination on peroxidase-positive pasteurized milk can be used to demonstrate the addition of at least 6% reconstituted milk to raw milk. Further, the furosine content of skim milk powders produced under different process conditions was studied. It was demonstrated that only under extreme conditions (holding for more than 60 s at preheating temperatures >100 °C) was the furosine formation influenced by the process conditions. Apart from these extreme conditions, furosine formation was affected mainly by the drying process and storage; the preheating conditions seemed to have little or no effect. Finally, the formation of furosine in milk powders due to storage conditions was studied. Storage temperature seemed to be the most important parameter for the formation of furosine, followed by the relative humidity.

Seasonal influence on heat-resistant proteolytic capacity of Pseudomonas lundensis and Pseudomonas fragi, predominant milk spoilers isolated from Belgian raw milk samples.

Psychrotolerant bacteria and their heat-resistant proteases play a major role in the spoilage of UHT-processed dairy products. Summer and winter raw milk samples were screened for the presence of such bacteria. One hundred and three proteolytic psychrotolerant bacteria were isolated, characterized by API tests, rep-PCR fingerprint analysis and evaluated for heat-resistant protease production. Twenty-nine strains (representing 79% of the complete collection) were further identified by 16S rRNA gene sequencing, rpoB gene sequencing and DNA-DNA hybridizations. A seasonal inter- and intra-species influence on milk spoilage capacity (e.g. growth rate and/or protease production) was demonstrated. Moreover, this polyphasic approach led to the identification of Pseudomonas fragi and Pseudomonas lundensis (representing 53% of all isolates) as predominant producers of heat-resistant proteases in raw milk. The role of Pseudomonas fluorescens, historically reported as important milk spoiler, could not unequivocally be established. The use of more reliable identification techniques and further revision of the taxonomy of P. fluorescens will probably result in a different perspective on its role in the milk spoilage issue.

Capillary Electrophoresis of the Whey Protein Fraction of Milk Powders. A Possible Method for Monitoring Storage Conditions

A method is presented for the evaluation of the keeping quality of milk powders as a result of heating and storage conditions. Capillary electrophoresis of β-lactoglobulin reveals a native and a modified fraction of this protein. The modifications are a result of Maillard reaction, changing the apparent pI of the polypeptides and are due to heating and storage conditions. This change in pI is related to lysine damage and nutritional availability of this essential amino acid. Capillary electrophoresis allows a fast and easy determination of the ratio of native (unmodified) to total β-lactoglobulin for monitoring storage conditions of milk powders.

Evaluation of two methods for the determination of lactulose in milk

Abstract Two conventional methods for the determination of lactulose in milk were compared, namely the colorimetric-enzymatic method and the gas chromatographic method. Increasing lactulose concentrations were added to raw milk samples. For concentrations of lactulose higher than 50 mg L −1 both methods had a similar accuracy and precision. However, the enzymatic method was more sensitive and had a detection limit of about 10 mg L −1 . The detection limit for the gas chromatographic method was between 50 and 100 mg L −1 . In contrast to the gas chromatographic method, the enzymatic method can be used to discriminate between UHT-milk and high-pasteurized milk.

The Biodiversity of the Microbiota Producing Heat-Resistant Enzymes Responsible for Spoilage in Processed Bovine Milk and Dairy Products

Raw bovine milk is highly nutritious as well as pH-neutral, providing the ideal conditions for microbial growth. The microbiota of raw milk is diverse and originates from several sources of contamination including the external udder surface, milking equipment, air, water, feed, grass, feces, and soil. Many bacterial and fungal species can be found in raw milk. The autochthonous microbiota of raw milk immediately after milking generally comprises lactic acid bacteria such as Lactococcus, Lactobacillus, Streptococcus, and Leuconostoc species, which are technologically important for the dairy industry, although they do occasionally cause spoilage of dairy products. Differences in milking practices and storage conditions on each continent, country and region result in variable microbial population structures in raw milk. Raw milk is usually stored at cold temperatures, e.g., about 4°C before processing to reduce the growth of most bacteria. However, psychrotrophic bacteria can proliferate and contribute to spoilage of ultra-high temperature (UHT) treated and sterilized milk and other dairy products with a long shelf life due to their ability to produce extracellular heat resistant enzymes such as peptidases and lipases. Worldwide, species of Pseudomonas, with the ability to produce these spoilage enzymes, are the most common contaminants isolated from cold raw milk although other genera such as Serratia are also reported as important milk spoilers, while for others more research is needed on the heat resistance of the spoilage enzymes produced. The residual activity of extracellular enzymes after high heat treatment may lead to technological problems (off flavors, physico-chemical instability) during the shelf life of milk and dairy products. This review covers the contamination patterns of cold raw milk in several parts of the world, the growth potential of psychrotrophic bacteria, their ability to produce extracellular heat-resistant enzymes and the consequences for dairy products with a long shelf life. This problem is of increasing importance because of the large worldwide trade in fluid milk and milk powder.

Isolation and detection of Clostridium tyrobutyricum cells in semi-soft and hard cheeses using the polymerase chain reaction

Butyric acid fermentation in brine salted, semi-soft and hard cheeses (late blowing) can create considerable loss of product. Clostridium tyrobutyricum , a Gram-positive, sporeforming, anaerobic bacterium, has been identified as the causative agent (Klijn et al . 1995).

Identification and characterization of a heat-resistant protease from Serratia liquefaciens isolated from Brazilian cold raw milk.

The cold storage of raw milk before heat treatment in dairy industry promotes the growth of psychrotrophic microorganisms, which are known for their ability to produce heat-resistant proteolytic enzymes. Although Pseudomonas is described as the main causative genus for high proteolytic spoilage potential in dairy products, Serratia liquefaciens secretes proteases and may be found in raw milk samples as well. However, at the present there is no information about the proteolytic spoilage potential of S. liquefaciens in milk after heat-treatment. The main aim of this research was to assess the proteolytic spoilage potential of S. liquefaciens isolated from Brazilian raw milk and to characterize the involved protease. S. liquefaciens was shown to secrete one heat-resistant spoilage metalloprotease of, approximately, 52 kDa encoded by the ser2 gene. The heat-resistance of Ser2 was similar to the aprX encoded metalloprotease produced by Pseudomonas. Although the ser2 gene was detected in all S. liquefaciens isolates tested in this study, the proteolytic activity of the isolates in milk was highly heterogeneous. Since nucleotide and deduced amino acid sequences of ser2 of all tested isolates are identical, this heterogeneity may be attributed to differences in enzyme expression levels or post-translational modifications.

Cyclic Lipodepsipeptides Produced by Pseudomonas spp. Naturally Present in Raw Milk Induce Inhibitory Effects on Microbiological Inhibitor Assays for Antibiotic Residue Screening

Two Pseudomonas strains, identified as closely related to Pseudomonas tolaasii, were isolated from milk of a farm with frequent false-positive Delvotest results for screening putative antibiotic residues in raw milk executed as part of the regulatory quality programme. Growth at 5 to 7°C of these isolates in milk resulted in high lipolysis and the production of bacterial inhibitors. The two main bacterial inhibitors have a molecular weight of 1168.7 and 1140.7 Da respectively, are heat-tolerant and inhibit Geobacillus stearothermophilus var. calidolactis, the test strain of most of the commercially available microbiological inhibitor tests for screening of antibiotic residues in milk. Furthermore, these bacterial inhibitors show antimicrobial activity against Staphylococcus aureus, Bacillus cereus and B. subtilis and also interfere negatively with yoghurt production. Following their isolation and purification with RP-HPLC, the inhibitors were identified by NMR analysis as cyclic lipodepsipeptides of the viscosin group. Our findings bring to light a new challenge for quality control in the dairy industry. By prolonging the refrigerated storage of raw milk, the keeping quality of milk is influenced by growth and metabolic activities of psychrotrophic bacteria such as pseudomonads. Besides an increased risk of possible spoilage of long shelf-life milk, the production at low temperature of natural bacterial inhibitors may also result in false-positive results for antibiotic residue screening tests based on microbial inhibitor assays thus leading to undue production loss.