**5. Microbial contaminants of prepared meals in catering establishments**

During the whole production chain, there is constant exposure of food to microbial contami‐ nation. Therefore, a strict quality and safety food control should be promoted with a view to minimize the incidence of food poisoning.

Undoubtedly, for catering establishments, the HACCP system assesses the condition under which the product was elaborated, determines the main risk factors of food contamination, and manages effective measures to reduce contamination by pathogenic and spoilage micro‐ organisms.

Microbial indicators are able to highlight deficiencies in the hygienic and sanitary food quality. Indeed, their presence at high levels leads to a reduction of shelf life and is probably related to the presence of pathogenic microorganisms.

According to the International Commission on Microbiological Specifications for Foods (ICMSF), microbial indicators do not offer a direct risk to human health. These groups are mainly aerobic mesophilic bacteria, lactic acid bacteria, total coliforms, fecal coliforms, enterococci, enterobacteriaceae, *Staphylococcus aureus* and *E. coli* [45]. In the next subsections, the most representative microbial indicators will be described. Besides, *Listeria monocyto‐ genes* is included due to its relevance and presence in a wide range of food commodities as well as for the current EU regulation (No. 1441/2007) [46] where it is included as safety criteria for ready to eat foods.

#### **5.1. Aerobic mesophilic microorganisms**

Microbial species belonging to this group are quite heterogeneous and include all bacteria, fungi, and yeasts growing at aerobic conditions. The presence of aerobic mesophilic microor‐ ganisms in fresh foods demonstrates the effectiveness of sanitary procedures during process‐ ing, handling, and storage before [47].

Ready to eat foods (apart from fermented foods, cheeses, and dairy products) with significant concentration levels of aerobic mesophilic microorganisms should not be considered suitable for human consumption, even if microbial species are not pathogenic.

Generally, contamination occurs because of the use of contaminated raw materials or ineffi‐ cient health treatments as well as inadequate conditions of storage time and temperature [48].

In general, high levels pose a greater risk of pathogen contamination. Several authors agree that the recommended concentrations for ready to eat foods should be less than 5.0 log cfu/g [49]. However, other guidelines for ready to eat foods such as those proposed by the Health Protection Agency (UK) [50] establish acceptable limits between 6 and 8 log cfu/g, depending on the food type.

#### **5.2. Lactic acid bacteria**

Lactic acid bacteria comprise a wide range of microorganisms with common morphological, metabolic, and physiological characteristics. Some of the most representative species are *Streptococcus* spp., *Pediococcus* spp., *Leuconostoc* spp., *Lactobacillus* spp., and *Lactococcus* spp. [51].

In the food industry, they have multiple uses as starter cultures in the manufacture of cheese, yogurt, and fermented meats. They are also recognized as natural antimicrobial agents against foodborne pathogens in biopreservation processes [52]. They represent the predominant group in fermented meat products reaching levels between 8 and 9 log cfu/g during the maturation processes. The most common species are *Lactobacillus sakei*, *Lactobacillus curvatus*, *Lactobacillus plantarum*, *Lactobacillus pentosus*, *Pediococcus acidilactici*, and *Pediococcus pentasaceus* [53].

Despite its protective function, they are able to produce end metabolites that lead to food spoilage and thus shortening its shelf life. Their final levels depend largely on the storage temperature and packaging methods [54]. Deterioration caused by the growth of lactic acid bacteria is shown by undesirable changes in smell, taste, color, and gas production. Some studies have found these changes in vacuum-packed meat products or modified atmosphere products [55].

#### **5.3. Enterobacteriaceae**

Enterobacteriaceae are considered as food quality indicators including *E. coli* being mainly related to fecal contamination. Generally, the presence of these microorganisms in foods is closely linked with the implementation of inadequate handling practices, inefficient cooking processes, cross-contamination, inadequate personal hygiene of food handlers, equipment and food-contact surfaces as well as inadequate holding time and temperature conditions [56].

Enterobacteriaceae species are Gram-negative bacteria, aerobic or facultative anaerobic, nonsporulated, mobile or immobile, and being able to ferment glucose and to reduce nitrate to nitrite. Some of the most representative species include *Salmonella enterica*, *Shigella* spp., *Yersinia* spp. (intestinal pathogens in humans), *Edwarsiella* spp., *Hafnia* spp., *Proteus* spp., *Morganella* spp., *Erwinia* spp., *E. coli*, *Enterobacter* spp., *Citrobacter* spp., *Serratia* spp., or *Klebsiella* spp. Most of them produce endotoxins and thermolabile and/or thermostable exotoxins. Some *E. coli* serotypes are producing verotoxins and shigatoxins, which are linked to a high rate of morbidity and mortality in humans [57].

Food commodities where Enterobacteriaceae can be found are processed meat products [58], nutritional formulas for infants [59], mixed salads, raw vegetables, and milk/dairy products, among others [60].

#### **5.4. Total and fecal coliforms**

Total and fecal coliforms are specific groups within the Enterobacteriaceae family, including species, such as *E. coli*, *Klebsiella* spp., *Enterobacter* spp., or *Citrobacter* spp. These are Gramnegative bacteria, aerobic or facultative anaerobes, non-sporulated, whose optimal growth temperature is around 35–40°C. These food quality indicators are taking part of the intestines of humans and warm-blooded animals and other organisms often located on the ground or plant.

The main difference between total and fecal coliforms is that the latter group ferments lactose at temperatures between 44 and 45°C. The group includes primarily *E. coli* (~90%) with certain *Klebsiella* and *Citrobacter* species. Coliforms are considered a reliable indicator of fecal contam‐ ination and are sometimes found in contaminated equipment and utensils, as well as in a wide variety of foods.

Contamination of ready to eat foods by coliforms is commonly attributed to environmental contamination, the use of inadequate hygiene practices, and/or insufficient control of the storage temperature. In the case of thermally treated food, the presence of coliforms is indicative of inadequate treatment or post-processing contamination as they are thermolabile microorganisms [47].

#### **5.5.** *Escherichia coli*

Enteropathogenic *E. coli* comprise different serotypes that can be present in contaminated foods. Most of them are able to produce Shiga-like toxins and/or other heat-labile or heat-stable toxins that can potentially cause diarrheagenic diseases in humans [61]. Besides, some serotypes of enterotoxigenic *E. coli* can also produce a cytotoxin to Vero cells (VTEC *E. coli*).

Normally, outbreaks caused by VTEC serotypes are of low prevalence (1.2 cases per 100,000 people in the EU) [39]; however, the high infectivity and severity of the disease increase the importance of performing novel research on this pathogen. It is reported that human outbreaks attributed to *E. coli* serotypes were mostly originated from catering services or restaurants [62].

Generally, *E. coli* can be present in animal origin foods (pork, beef, and poultry), water sources, or produce such as cabbage, lettuce, or spinach. They can enter the food chain through cross-contamination or recontamination phenomena [63] or through the irrigation with contaminated water, which may result in the internalization of certain *E. coli* sero‐ types in vegetables [64].

*E. coli* O157:H7 was the most studied serotype due to the severity of the illnesses caused and its low infective dose, around 100 cells [65]. However, other non-O157 serotypes have been associated to human infections through the ingestion of risk food products, such as fermented and minced meats or raw milk [66].

#### **5.6.** *Staphylococcus aureus*

*S. aureus* has been reported as a microbial indicator most likely associated to reduced water activity (*a*w) foods, such as ready to eat cooked or cured meats [67, 68]. The presence of *S. aureus* is often associated to contamination of raw material, such as poultry carcasses or raw chicken samples [69] or cross-contamination events occurring because of mishandling during processing and storage [63].

Food poisoning is attributed to the ingestion of foods that contain thermotolerant Staphylo‐ coccal Enterotoxins (SEs) in doses around 20–100 ng [70]. The staphylococcal enterotoxin A (SEA) is the one most frequently reported. A wide range of environmental factors, such as pH, *a*w, temperature, food type, and processing conditions, have been suggested to play an important role on SEs production. Generally, growth of *S. aureus* is necessary for SE produc‐ tion, although this phenomenon does not always accompany growth [67]. Indeed, some published studies consider hazardous *S. aureus* levels from 6 log cfu/g in contaminated foods for SE production [71].

#### **5.7.** *Listeria monocytogenes*

*L. monocytogenes* is a foodborne pathogen causing listeriosis, with high mortality rates between 20% and 30% [72]. It is mainly distributed in the field, soil, contaminated water sources, and decaying vegetation. It is also categorized as a psychrotrophic microorganism, being ubiqui‐ tous in food-processing environments. Consequently, *L. monocytogenes*is often found as a postcontamination pathogen in food products like sliced cooked meat products, smoked fish, cut vegetables, or ready-to-eat (RTE) products. Raw chicken, milk, and raw meat are frequently implicated in foodborne outbreaks [73]. The associated high mortality rates to pregnant women and their unborn child, neonates, elderly people, and immunocompromised people makes that its level in food products should remain low. The Commission Regulation No. 1441/2007 *on microbiological criteria for foodstuffs* states that, for *L. monocytogenes*, in the food category *RTE foods able to support the growth of L. monocytogenes other than those intended for infants and for special medical purposes, two different microbiological criteria are proposed: (i) L. monocytogenes levels should not exceed 100 cfu/g throughout the shelf life of the product and (ii) absence in 25 g of the product before the food has left the immediate control of the food business operator*, who has produced it. Their application depends on the ability of the food operator to demonstrate that the targeted food is able or not to support the growth of *L. monocytogenes* up to the end of the shelf life. Also in the United States, the limit of 100 cfu/g for *L. monocytogenes* that does not support growth of the microorganism in foods is being considered [74].
