4. Predictive microbiology models of L. monocytogenes

chemical composition of this EO since the concentration of phenolic compounds (i.e., thymol) was lower than the obtained in rosemary EO. With reference to [42], an antimicrobial effect of oregano, cinnamomum, rosmarinus, salvia and thymus EO against L. monocytogenes in meatballs stored at 4C was observed, while the extension of the antimicrobial effect varied according to the added EO and its concentration. A reduction of 1 and 2 log CFU/g, on average, was observed when concentrations of about 1 and 2% were added, respectively. It indicates that the antimicrobial effect of EO in foodstuffs is not enough to guarantee the safety of meat in case of high contamination. In addition, the negative impact on sensory acceptance

Regarding active packaging, the addiction of several substances with antimicrobial effect such as organic acids, chitosan or nisin among others has also been studied to improve meat safety against L. monocytogenes. With reference to [43], the authors observed that the use of chitosan diluted in acetic acid or lactic acid as coating in highly contaminated ready-to-eat roast beef (6.5 log/CFU) is useful to control L. monocytogenes. The use of sodium lactate and sodium diacetate in edible coating in combination with polysaccharide-based edible coatings have been studied by [44] in chilled and frozen roasted turkey. Although organic acids decreased the counts of L. monocytogenes, its combination with chitosan increased the antimicrobial effect. With reference to [45] who evaluated the decontaminating efficacy of lactic acid (2%), potassium sorbate (1%), sodium hypochlorite (200 ppm) and ethanol (10%) sprayed on the surface of meat previously inoculated with 100 <sup>μ</sup>L of a suspension of L. monocytogenes (1.5 104 CFU/g), the authors observed that samples treated with lactic acid showed significantly lower counts than the controls and other treatments. Lactic acid was shown to be promising in the control of L.

The use of Lactobacillus sakei to control L. monocytogenes in fresh beef was reported by [46]. Incorporation of lactic acid bacteria into sodium-caseinate films protected beef by lowering the growth of L. monocytogenes during storage under abusive temperatures. This strategy could be useful to guarantee the safety of fresh beef along the food chain in which temperature fluctu-

Bacteriophages harmless to human cells are considered natural biocontrol agents against foodborne pathogens [47]. Bacteriophages are bacterial viruses with host specificity and lysis

Phages used for biocontrol purposes should be virulent and feature abroad host range, that is, infect and kill as many target strains as possible [49, 50]. Virulent myoviruses closely related to P100 and A511 are the most popular and have been isolated from the sources in Europe, the US and New Zealand [51–53]. Commercially, "ListexTM P100" is available that was generally recognized as safe (GRAS) by FDA and USDA in 2007 for use in all food products. Several studies have showed its efficacy in foods such as ready-to-eat (RTE) meats and poultry [50, 51]. The phage A511, closely related to P100, also showed efficacy in various RTE foods [54]. According to [55], the direct immobilization of the viral particles in the cellulose membranes of the packaging materials can be used in alternative to the phage suspension as a possible

activities and can be used as preservatives or for pathogens rapid detection [48].

was also indicated by the authors.

30 Listeria Monocytogenes

monocytogenes presenting an early bactericidal effect.

ations may occur.

intervention strategy against Listeria.

Predictive microbiology models are used to infer about the evolution of microbial population considering the initial contamination and food environment, as the responses of microorganisms populations in a specific environment are reproducible [56]. Mathematical models may be generally categorized into three types: primary, secondary and tertiary models. The primary models are used to estimate the changes in the microbial population as a function of time, under a single set of conditions [57, 58]. The secondary models describe the microorganisms' responses to environmental conditions, according to one or more parameters of a primary model [59]. The tertiary models were defined by [60] as algorithms incorporated into software to integrate the effect of environmental variables on microbial responses and to provide predictions of the outcomes.

The increasing interest in the behavior of hazards such as L. monocytogenes promoted important advances in predictive microbiology, and it started to use the food matrix, instead of culture media [61]. Traditional strategies using fast-growing strains in optimal growth conditions usually overestimate the bacterial growth in a food product. This can lead to safe results but may also conduct to unnecessarily safety measures. A stochastic (or probabilistic) approach take into account the variability and uncertainty of various factors that affecting microbial behavior by using probability distributions of the input data. This provides safe enough predictions to avoid unacceptable health risks for consumers [62].

Predictive microbiology models are important tools to model bacterial growth in quantitative microbial risk assessments (MQRA) [63]. In this context, food business operators and authorities can use accessible predictive models, such as Pathogen Modeling Program [64], SymPrevius [65] and ComBase [66]. The incorporation of predictive microbiology models in MQRA must follow some guidelines [56]. The complexity of the predictive microbiology model elected in a MQRA depends on different factors, namely the needs of risk assessment, available model and data availability [63].

For an assessment of microorganisms' behavior in naturally contaminated foods, biological factors, food characteristics and storage conditions must be considered [67]. These authors emphasize the variability of L. monocytogenes growth in foods. According to [61], the role of microbial competition in models is now taken into consideration. Some studies were published regarding the survival of L. monocytogenes in fresh beef stored at two temperatures and different packaging systems as modified atmosphere packaging (MAP), using omnibus model based on the Weibull Equation [35]. Besides the increase of studies using predictive models, there are few data referred to the application of predictive models to composite foods containing raw and cooked ingredients [67].

According to [63], it is challenging for a risk assessor to choose an applicable predictive microbiology model in the abundant literature. This author suggests that the choice of a model should be done with the closed cooperation between microbiologists, mathematicians and risk managers.

#### 5. Conclusions

Besides the Regulation (EC) No 2073/2005, reviewed by the Regulation (EC) No 1441/2007 does not establish limits for L. monocytogenes in fresh meat, it is generally accepted a level of 100 cells on fresh meat, except for high-risk populations. Thus, the implementation of control procedures during processing and at retail level is important. These measures are closely dependent on intrinsic and extrinsic meat factors that could influence microbial growth, namely pH and storage temperature.

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Several strategies to improve meat safety and shelf-life have been studied in the latest years. From those, the use of alternative meat packaging systems has been strongly studied to obtain an attractive meat with a higher shelf-life. However, in some cases, these strategies associated to refrigerated storage can promote the survival and growth of some pathogenic microorganisms such as L. monocytogenes. However, some authors referred that independently of the refrigeration temperature, the presence of CO2 in the package atmosphere exerted a bactericidal effect on L. monocytogenes cells.

Food business operators and authorities can use predictive microbiology models as important tools to model bacterial survival or growth in quantitative microbial risk assessments. There are several mathematical models to predict the behavior of microorganisms in meat and meat products. However, predictive microbiological models must be carefully used and by whom who is expertise and has an understanding of their limitations and conditions of use.

#### Acknowledgements

The authors would like to thank CECAV-UTAD. This work was supported by the Portuguese Science and Technology Foundation (FCT) under the UID/CVT/00772/2013 and UID/CVT/ 00772/2016 projects.

### Conflict of interest

The authors declare no conflict of interests.

#### Author details

Cristina Saraiva\*, Juan García-Díez, Maria da Conceição Fontes and Alexandra Esteves

\*Address all correspondence to: crisarai@utad.pt

CECAV, Animal and Veterinary Research Centre, Department of Veterinary Sciences, School of Agrarian and Veterinary Sciences, University of Trás-os-Montes e Alto Douro, Vila Real, Portugal

#### References

5. Conclusions

32 Listeria Monocytogenes

namely pH and storage temperature.

cidal effect on L. monocytogenes cells.

Acknowledgements

00772/2016 projects.

Conflict of interest

Author details

Portugal

The authors declare no conflict of interests.

\*Address all correspondence to: crisarai@utad.pt

Besides the Regulation (EC) No 2073/2005, reviewed by the Regulation (EC) No 1441/2007 does not establish limits for L. monocytogenes in fresh meat, it is generally accepted a level of 100 cells on fresh meat, except for high-risk populations. Thus, the implementation of control procedures during processing and at retail level is important. These measures are closely dependent on intrinsic and extrinsic meat factors that could influence microbial growth,

Several strategies to improve meat safety and shelf-life have been studied in the latest years. From those, the use of alternative meat packaging systems has been strongly studied to obtain an attractive meat with a higher shelf-life. However, in some cases, these strategies associated to refrigerated storage can promote the survival and growth of some pathogenic microorganisms such as L. monocytogenes. However, some authors referred that independently of the refrigeration temperature, the presence of CO2 in the package atmosphere exerted a bacteri-

Food business operators and authorities can use predictive microbiology models as important tools to model bacterial survival or growth in quantitative microbial risk assessments. There are several mathematical models to predict the behavior of microorganisms in meat and meat products. However, predictive microbiological models must be carefully used and by whom

The authors would like to thank CECAV-UTAD. This work was supported by the Portuguese Science and Technology Foundation (FCT) under the UID/CVT/00772/2013 and UID/CVT/

Cristina Saraiva\*, Juan García-Díez, Maria da Conceição Fontes and Alexandra Esteves

CECAV, Animal and Veterinary Research Centre, Department of Veterinary Sciences, School of Agrarian and Veterinary Sciences, University of Trás-os-Montes e Alto Douro, Vila Real,

who is expertise and has an understanding of their limitations and conditions of use.


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**Section 3**

**Listeria monocytogenes in Medicine Research**


**Listeria monocytogenes in Medicine Research**

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38 Listeria Monocytogenes

**Chapter 4**

**Provisional chapter**

*Listeria monocytogenes* **in Medical Research**

*Listeria monocytogenes* **in Medical Research**

DOI: 10.5772/intechopen.74840

Bacteria are known to produce compounds of high value such as secondary metabolites used in biotechnological applications. It is therefore worthwhile to think how to exploit a pathogenic bacterium, e.g., *Listeria monocytogenes*, to be an effective source of bioactive compound used in particular in medicinal purposes. *Listeria monocytogenes* is considered as an acute contaminated bacterium in foods and could be a causal agent of food-borne diseases. This bacterium is the causal agent of listeriosis, a grave disease, caused by eating contaminated food. Although, *L. monocytogenes* is a pathogenic microorganism that threatens the progress of food industry, it would be also a reservoir of secondary metabolites such as antibiotics and other metabolites of economic importance when appropriate strain improvement will be addressed. This section would discuss in brief the negative and positive features of *L. monocytogenes* as either a pathogenic bacterium or an impor-

**Keywords:** productive strain, metabolic diseases, food-borne pathogens, valorization,

*Listeria monocytogenes* is a Gram-positive bacterium that is recognized as a facultative intracellular pathogen. An intracellular growth could therefore be observed among food and clinical

*L. monocytogenes* is a well-known food-borne pathogen, which has been found in many fresh and processed foods, and it is widely distributed in nature. In fact, this organism is able to survive in extreme environments including elevated osmolarity, cold, and acid shocks. Understanding the key stress adaptation is important for a better control of *L. monocytogenes*

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

tant microorganism in medical research.

biotechnological applications

http://dx.doi.org/10.5772/intechopen.74840

Nihed Ben Halima

Nihed Ben Halima

**Abstract**

**1. Introduction**

strains of such bacterium [1].

#### *Listeria monocytogenes* **in Medical Research** *Listeria monocytogenes* **in Medical Research**

DOI: 10.5772/intechopen.74840

#### Nihed Ben Halima Nihed Ben Halima

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74840

**Abstract**

Bacteria are known to produce compounds of high value such as secondary metabolites used in biotechnological applications. It is therefore worthwhile to think how to exploit a pathogenic bacterium, e.g., *Listeria monocytogenes*, to be an effective source of bioactive compound used in particular in medicinal purposes. *Listeria monocytogenes* is considered as an acute contaminated bacterium in foods and could be a causal agent of food-borne diseases. This bacterium is the causal agent of listeriosis, a grave disease, caused by eating contaminated food. Although, *L. monocytogenes* is a pathogenic microorganism that threatens the progress of food industry, it would be also a reservoir of secondary metabolites such as antibiotics and other metabolites of economic importance when appropriate strain improvement will be addressed. This section would discuss in brief the negative and positive features of *L. monocytogenes* as either a pathogenic bacterium or an important microorganism in medical research.

**Keywords:** productive strain, metabolic diseases, food-borne pathogens, valorization, biotechnological applications

#### **1. Introduction**

*Listeria monocytogenes* is a Gram-positive bacterium that is recognized as a facultative intracellular pathogen. An intracellular growth could therefore be observed among food and clinical strains of such bacterium [1].

*L. monocytogenes* is a well-known food-borne pathogen, which has been found in many fresh and processed foods, and it is widely distributed in nature. In fact, this organism is able to survive in extreme environments including elevated osmolarity, cold, and acid shocks. Understanding the key stress adaptation is important for a better control of *L. monocytogenes*

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

in food as well as in its resulted health diseases. Some authors have reported studies on the protein patterns expressed in response to salt shock in *L. monocytogenes* [2–4].

**3. Adaptation of** *L. monocytogenes* **to reduced temperature**

*genes* ATCC 19117 in raw minced beef meat under storage at 4°C [11].

and *Listeria monocytogenes* [14, 15].

*monocytogenes* [16].

*genes* in food [11, 18].

meat during refrigerated storage.

**4. Some traits highlighted in** *L. monocytogenes*

intracellular bacterial growth in Caco-2 cells.

butes [17].

cally packed cooked meat products and shelf-life failures of conserved foods.

Controlling *L. monocytogenes* in food has become a major preoccupation in the food industry and storage. For this end, many reports were investigated to determine the efficacy of natural drugs such as essential oils to inhibit the growth of such microorganism. In this regard, beef meat including plant essential oil such as that from lemon (*Citrus limon*) is an interesting target during refrigerated storage as contamination of beef meat by food spoilage and foodborne pathogens is considered one of the major problems to the progress of food industry. Indeed, the addition of such essential oil could substantially delay the growth of *L. monocyto-*

*Listeria monocytogenes* in Medical Research http://dx.doi.org/10.5772/intechopen.74840 43

*Listeria monocytogenes* is one of the most important psychrotrophic food pathogens, which is responsible for food-borne illnesses in particular listeriosis that has been known as one of the emerging zoonotic diseases nowadays [12, 13]. *L. monocytogenes* could be related to anaerobi-

Foods are exposed to contamination by several bacteria such as those reported as the causal agents of food-borne diseases, i.e., *Staphylococcus aureus*, *Salmonella*, *Escherichia coli* O157:H7,

The latter pathogen is the causal agent of listeriosis, a potential grave disease, and is often fatal in susceptible individuals, caused by eating contaminated food in particular with *Listeria* 

To prevent food contamination during the production, sale, and distribution and to extend the shelf-life time of raw and/or processed foods, additives should be used. However, the safety aspects of the synthetic additives could be paid attention, as these chemical preservatives are considered toxic and responsible for many carcinogenic and teratogenic attri-

Natural products such as plants and herbs and naturally derived compounds are regarded as new alternatives to prevent the proliferation of pathogens, e.g., strains of *Listeria monocyto-*

A particular interest has been focused on the potential application of plant essential oils such as those from lemon [11] and thyme [18] as safer additives for meat, in particular minced beef

Food and clinical strains of *Listeria monocytogenes* were used in the report of Kanki et al. [1] to study specific alleles in particular the activities of listeriolysin O (LLO) and phospholipases PlcA and PlcB that are known to promote rupture of the phagocytic vacuole, besides initial

*L. monocytogenes* could enter the food chain and lead to severe disseminated infection, as listeriosis, and this feature is very likely due to its ability to survive in both reduced temperature and high-salt conditions [5].

*L. monocytogenes* can be transmitted to humans through ingestion of contaminated food, particularly ready-to-eat meat, seafood, and dairy products [6].

*Listeria monocytogenes* is therefore an interesting microorganism to be studied mainly in food and medical research.

The present study focuses on reviewing and describing some important features of *Listeria monocytogenes* from different published reports.

### **2. Adaptation to salt stress and protein patterns of** *L. monocytogenes*

*Listeria monocytogenes* is able to tolerate salt stress. The mechanism of adaptation to increased salt concentration by this bacterium could be due to intracellular accumulation of compatible solutes. Indeed, the compatible solutes, such as carnitine and glycine betaine, protect the cell from deleterious effects of the external osmolarity and prevent water loss [7, 8].

Comparison of the salt-induced protein patterns of *L. monocytogenes* strain LO28 grown in a rich medium or in a chemically defined medium shows clear differences between these two media as reported by Duché et al. [2]. The later report revealed that the NaCl stress response of *L. monocytogenes* is a complex process. Indeed, there is synthesis of different proteins more or down-expressed in the presence of salt and either directly related or not to the salt stress response of *L. monocytogenes*. The protein pattern analysis revealed the synthesis of three proteins of the general metabolism (AckA, PdhD, and S6), which was modified after salt stress, but does not seem to be directly related to the salt stress response of *L. monocytogenes*. However, two proteins more expressed (GbuA and Ctc) in the presence of salt seem to be directly related with the response to salt stress of *L. monocytogenes*.

The report of Dussurget et al. [9] revealed the presence of an *L. monocytogenes*-specific putative gene encoding a bile salt hydrolase (BSH) and demonstrated that BSH is a novel PrfAregulated *L. monocytogenes* virulence factor involved in the intestinal and hepatic phases of listeriosis [9].

Protein patterns of *L. monocytogenes* were also analyzed by proteomic analysis in comparison with mode of growth either in biofilm or in planktonic mode [10]. The results showed a significant variation of the protein patterns of *L. monocytogenes* between the two growth conditions. The study indicated in particular that the biofilm development is probably controlled by specific regulation of protein expression involved at various levels of cellular physiology [10].
