Nucleic Acid-Based Methods to Identify, Detect and Type Pathogenic Bacteria Occurring in Milk and Dairy Products

Culture‐Dependent and Culture‐Independent Nucleic‐Acid‐Based Methods Used in the Microbial Safety Assessment of Milk and Dairy Products

“…DNA‐based assays are widely used for pathogen detection and enumeration in milk and its derivatives. However, DNA is long‐term detectable in microbial cells regardless of their physiological conditions (McKillip and others ; Ercolini and others ; Wolffs and others ; Fusco and Quero ; Quero and others ). Herein, a brief description of several nucleic‐acid‐based methodologies for the detection of only viable pathogen cells is reported.…”

“…In any case, as such membrane‐damaged dead and injured/stressed cells will be indistinguishable by EMA/PMA PCR, an underestimation of the actual viable (even if unculturable) pathogenic population in a given food matrix may occur, thus enhancing the risk of adverse health effects besides economic losses. Indeed, under favorable environmental conditions, injured or stressed cells of a given pathogen can repair their damages and retrieve or even intensify some virulence features (Paiva de Sousa ; House and others ; Robinson and McKillip ; Dinu and Bach ; Fusco and others ; Fusco and Quero ). Due to the complex and variable composition and structure of milk and dairy products, and considering that foodborne pathogens may undergo inadequate or sublethal physical and chemical stress during the production/storage/distribution and consumption of foods, a population of viable, dead, and injured cells may accumulate (Ercolini and others ; Paiva de Sousa ; Wu ; Fusco and others ; Fusco and Quero ).…”

“…Indeed, under favorable environmental conditions, injured or stressed cells of a given pathogen can repair their damages and retrieve or even intensify some virulence features (Paiva de Sousa ; House and others ; Robinson and McKillip ; Dinu and Bach ; Fusco and others ; Fusco and Quero ). Due to the complex and variable composition and structure of milk and dairy products, and considering that foodborne pathogens may undergo inadequate or sublethal physical and chemical stress during the production/storage/distribution and consumption of foods, a population of viable, dead, and injured cells may accumulate (Ercolini and others ; Paiva de Sousa ; Wu ; Fusco and others ; Fusco and Quero ). Stressed or injured pathogens are still viable but, depending on the site and level of damage, might be highly sensitive to selective compounds usually utilized in standard liquid and solid detection and enumeration media.…”

“…Stressed or injured pathogens are still viable but, depending on the site and level of damage, might be highly sensitive to selective compounds usually utilized in standard liquid and solid detection and enumeration media. Thus, in such a “viable but nonculturable state” (VBNC) (Oliver ), they can be underestimated by the reference selective media (Ercolini and others ;Wu ; Fusco and others ; Fusco and Quero ; Quero and others ), which are needed to detect pathogens in foods with highly complex background microflora. In order to overcome such drawbacks, the solid‐ and liquid‐based enumeration methods may be modified to allow the greatest number of VBNC injured pathogens to restore, thus returning to conditions of perfect viability and cultivability (Fusco and others ; Fusco and Quero ).…”

“…Thus, in such a “viable but nonculturable state” (VBNC) (Oliver ), they can be underestimated by the reference selective media (Ercolini and others ;Wu ; Fusco and others ; Fusco and Quero ; Quero and others ), which are needed to detect pathogens in foods with highly complex background microflora. In order to overcome such drawbacks, the solid‐ and liquid‐based enumeration methods may be modified to allow the greatest number of VBNC injured pathogens to restore, thus returning to conditions of perfect viability and cultivability (Fusco and others ; Fusco and Quero ). The time‐ and labor‐consuming biochemical/serological confirmation required by the reference culture‐based methods may be replaced by a simplex or even a multiplex (real‐time) PCR (Fusco and others ; Quero and others ) that increases the speed of the whole detection assay.…”