**6. Risk factors associated to microbiological contamination and foodborne outbreaks**

Foods can become contaminated during growth and harvesting of raw materials, storage and transport to the industry, and processing into finished products. Recontamination can also occur during transport to retail outlets and before consumption at domestic homes and/or in catering establishments. Contamination vectors are mainly animals, surfaces, environment (air, water), and people in contact with foods (food handlers). Processing conditions, packag‐ ing materials, and equipment used can also be contamination sources. Survival of microor‐ ganisms on contaminated surfaces could lead to their multiplication at high levels, thus compromising food quality and safety [75].

Animals are important reservoirs of microorganisms, and slaughter of animals could introduce high concentration of microorganisms in food industries. Zoonotic pathogens are normally present on the skin and in the gastrointestinal and respiratory tracts. Pathogens carried on the hands are also a major source of contamination [76].

Airborne contamination represents a significant medium for the microbial transfer to food products. [77] Installation of proper air filters is recommended; otherwise, microorganisms can be present together with dust, debris, or insects.

Water sources are used in the food industry as an ingredient, a processing aid, and for cleaning. Therefore, not to increase both microbial and chemical contaminations, it is important to use decontaminated water (i.e., chlorinated and electrolyzed). Water used in hand-washing facilities can pose a potential risk because of the presence of condensations, leaking pipes, or aerosols. Microorganisms colonizing these surfaces can multiply rapidly if conditions are favorable. Thus, checking the microbiological quality of water is essential to guarantee food safety.

Food handlers can act as vectors for food contamination leading to the transmission of enteric and respiratory pathogens to food, e.g., through aerosol droplets from coughing near the processing line [78]. They can also favor cross-contamination through the skin if hand-washing is not properly done.

Finally, pests, such as birds, insects, and rodents, are potentially a major contamination problem. Therefore, care must be taken to avoid their entrance into food factories. Facilities should be designed in such a way they cannot live and breed. To do so, appropriate disinsec‐ tization fumigation methods must be achieved.

The above factors when combined together may increase the risk of food contamination. According to CDC data, 1527 foodborne disease outbreaks, resulting in 29,444 cases of illness, 1184 hospitalizations, and 23 deaths were reported within 2009–2010. [79] Among the 790 outbreaks with a laboratory-confirmed illness, norovirus was the most commonly reported infection, accounting for 42% of outbreaks, followed by *Salmonella*, with 30% of outbreaks. Outbreaks caused by some pathogens were particularly severe. For example, *Listeria* outbreaks resulted in the highest proportion of persons hospitalized (82%), followed by *Clostridium botulinum* (67%). Among the 23 deaths, 22 were linked to bacteria (9 *Listeria*, 5 *Salmonella*, *E. coli* O157, 3 *Clostridium perfringens*, and 1 *Shigella*), and 1 was linked to norovirus. Regarding European data [22], in 2013, *Campylobacter* continued to be the most commonly reported gastrointestinal bacterial pathogen in humans in the European Union (EU) and has been so since 2005 (214,779 cases) with an EU notification rate of 64.8 per 100,000 population. However, the high mortality rate associated to *L. monocytogenes* was confirmed since 191 deaths were reported in 2013, much higher than deaths associated to *Campylobacter* or *Salmonella* (59 or 56, respectively).

#### **6.1. Risk factors affecting microbial safety of foods in catering establishments**

#### *6.1.1. Hygienic food handlers` practices*

In production processes, storage, and distribution of prepared foods, the role of food handlers seems essential to ensure food safety, supported mainly on good hygienic practices and implementation of improved self-control measures. The food handler is defined as "anyone who by their work have direct contact with food during preparation, manufacture, processing, manufacturing, packaging, storage, transport, distribution, sale, supply and service."

Then existing laws applied to food handlers are cited as follows:


In a food catering environment, hygiene procedures may be improved as part of food poison‐ ing occurs as a result of the risk factors associated with food handling, related to poor hygiene, improper cooking procedures, cross-contamination, or improper storage of food [81].

Handlers sometimes act as vehicles for the spread of indicators or pathogens directly and indirectly through the hands to other food-contact surfaces and handlers. In the United States, the hygienic practices of food handlers are one of the five most important risk factors of food poisoning and about 89% of the outbreaks occur mainly by inadequate hygiene [78].

In certain circumstances, the hands may represent the most important vehicle of fecal and respiratory microorganisms [82]. It has been shown that microorganisms, such as *S. aureus*, *E. coli*, and *S. enterica.*, can survive on the hands if hygiene measures are not sufficiently appro‐ priate. Several studies describe outbreaks of food poisoning associated with catering estab‐ lishments [83]; these studies indicate that sanitary measures may be insufficient when dealing with consumer food safety.

Ayçiçek et al. [84] evaluated the sanitary measures of food handlers in a hospital central kitchen. They concluded that these measures were insufficient as significant counts of *S. aureus* and *E. coli* in both gloved hands and bare hands were presented when handling food. Specifically, *S. aureus* positive samples were obtained in 70% of the isolates. Other microor‐ ganisms, such as *Bacillus* spp. or *Staphylococcus* coagulase negative, were also isolated. Lues and Van Torden [85] attempted to relate the microbiological contamination found in the hands of food handlers and that presented in cloths and aprons. To do this, they visited several retail points in South Africa dedicated to the selling of RTE meat. In the study, it was found aerobic mesophilic counts (hands and aprons, respectively) in 98% and 8% of analyzed samples, total coliforms (40% and 26%), Enterobacteriaceae (44% and 16%), and *S. aureus* (88% and 40%). However, they did not find any significant correlation between the microbial counts, so potential cross-contamination could not be concluded. Besides, they considered that inade‐ quate hygiene could be a potential risk factor in the microbiological contamination of food, as 32% of the hands analyzed presented high counts for total coliforms. Fecal coliforms also prevailed in more than half of the samples from the hands (55.6%) of food handlers of several school canteens in Brazil [8]. The lack of the annual medical examination (51.9%), lack of regular training for handlers (74.1%), and poor hygiene practices (100%) could have an influence of these results, as stated by other authors [86]. Other microbial agents, such as norovirus and hepatitis A virus in humans, can survive in the hands of food handlers when they do not follow good hygienic practices [2]. In this case, contamination is often associated with asymptomatic carriers and direct hand contact with contaminated food. At the same time, contamination of food-contact surfaces is also promoted.

While hand-washing is a quick and simple method, it is also considered by many authors as the most convenient and effective way to reduce foodborne pathogens [82]. If done correctly, it prevents the risk of cross-contamination and the presence of high microbial loads in foods not submitted to intense inactivation treatments.

The principles for hand-washing are universal, though effective reduction of microorganisms depends on the following considerations [82]:


**•** Frequency and intensity of rubbing fingers and palms during rinsing.

In the light of the importance of maintaining adequate hygiene habits and attitudes of food handlers to ensure food safety, various methods have been used according to legal regulations for guidance handlers' hygiene. The Theory of Planned Behavior (TPB) has been advocated by many researchers to predict determinants of food handler's behavior [87].

However, some studies warn of possible deficiencies in hygiene by food handlers. For example, Green et al. [88] conducted a study to identify factors related to the hygienic practices of food handlers. To do this, they performed an observational study that evaluated the hand-washing and use of gloves of 321 food handlers. The results showed that washing hands and wearing gloves were most frequent in food preparation activities than when handling soiled equipment and direct contact with body parts. They also found that the use of gloves resulted in a decrease in the frequency of washes hands. In Clayton and Griffith [89], the habits of 115 handlers in 29 establishments during food preparation together with their corresponding hygiene measures were analyzed. The results indicated that only 9% of handlers washed their hands after touching their hair or face and 25% washed their hands after handling of contaminated equipment. Lubran et al. [90] examined the frequency of contact of the hands of food handlers with objects and food ready for consumption before the sale, washing hands and changing gloves during food preparation, as well as hygienic measures used for the equipment, utensils, and food-contact surfaces. The study was conducted in nine retail stores of RTE food and found a high percentage (60–80%) of handlers that did not wash their hands when handling food while maintaining contact with other surfaces. Likewise, the hygiene of the hands of food handlers in 15 retail shops was evaluated in another study [91]. Out of the 29 food handlers examined, only 48.3% used soap to wash their hands properly and rubbed hands including washing between fingers, fingertips, and wrists. They also found that most handlers who washed their hands in less than 10 s (41.4%) had slightly higher levels of contamination of aerobic mesophilic bacteria and *S. aureus* than those food handlers who took a longer time for hand-washing. To this end, some studies [82] highlight the importance of rubbing hands and increase in the frequency of hand-washing to have higher efficiency to remove microbial load.

On the other hand, hand-drying is also a very important in the hand-washing stage. Some authors consider most critical is the last washing stage and needs to be implemented correctly to ensure proper hygiene, thus reducing the risk of cross-contamination [92]. According to the study conducted by Michaels et al. [93], effective hand-drying may reduce microbial popula‐ tion up to 90%. As result of inappropriate drying practices, residual moisture of hands, drying hands with sheets of cloth, or inefficient air dryers that lengthen the drying time are encoun‐ tered [94]. It should be pointed out that rubbing hands during drying could promote skin contamination after washing. In an observational study by Clayton and Griffith [89], it was found that the effectiveness of hand-washing was affected by a high percentage of inappro‐ priate drying practices (61%).

#### *6.1.2. Availability of health resources*

Obviously, the availability of resources and the functioning of health facilities (sink, hot water, soap, etc.) in the catering establishments constitute also another factor that may adversely affect the personal hygiene practices of food handlers [94]. Another study aimed at evaluating the implementation of the HACCP system and knowledge of hygiene and food safety as well as food handlers' attitudes and practices in 20 establishments in Spain. [95] revealed that the difficulties encountered were improper maintenance of sinks and showers in 95% of the establishments visited, as well as the availability of hot water in bathrooms and changing rooms (50%). Likewise, another study evaluated 123 food retail outlets in the United States to investigate the knowledge and hygienic practices of food handlers, as well as the availability of resources for hand-washing [96]. The results indicated that the main constraints were linked to the absence of brushes to wash the nails (38%) and insufficient sinks (24%) concluding that only the fully equipped establishments (55%) had a properly trained supervisory staff.

#### *6.1.3. Gloves*

It is already demonstrated that hand-washing does not always guarantee complete removal of microorganisms. Thus, the use of gloves is necessary to prevent direct hand contact with food and food-contact surfaces as a measure of increasing food safety and minimizing risks of cross-contamination in the food industry. The most important issue is that sometimes the use of gloves can create a false sense of hygiene among handlers [97] and jeopardize the food safety.

It should be noted that the irregular change of gloves as well as their improper use could enhance cross-contamination. In the United States, this is attributed as one of the main reasons that favor the occurrence of food poisoning [98]. In another study [99], it was found that the use of the same pair of gloves for handling different foods increases the risk of transfer of pathogens. In this case, they found an increased risk of transfer of *L. monocytogenes* from contaminated raw chicken to cooked meat slices. In turn, if gloves are changed, this risk was minimized. Besides, not only the frequency of gloves changing but also their integrity is highly important to avoid cross-contamination. Some authors state that washing hands before and after the change of gloves is highly desirable to reduce this microbial contamination [81]. Even the process of changing gloves is fraught with hazards, because many glove materials cause excess moisture build-up, causing difficulty in disinfecting contamination from the nail region to spread all over the hand. However, gloves should be changed regularly because the risk of transmission of foodborne pathogens could be higher from dirty, unchanged gloves than from bare hands.

The pros and cons of using gloves are extensively explained [97]. However, it should be highlighted that a proper glove change must be done because their efficiency as physical barrier for microorganisms is limited over working time.

Besides this, physical properties of gloves (tensile strength, flexibility, resistance to puncture, and tears), material used (natural rubber gloves, vinyl, nitrile, polyurethane), and glove features (single use vs. multiuse, powder-free, allergenic reactions, etc.) should be primarily considered in the quality control system as they have a great impact on pathogens transmission from food handlers to prepared meals.

Gloves can be made of different types of material, according to their use and physical prop‐ erties. Polyethylene copolymer gloves could be the least expensive of all glove types. Density materials are quite variable, and they usually have a loose fit. They are built for using in shorttime periods, and some glove types contain antibacterial compounds. However, their use is quite limited in comparison to other gloves.

Vinyl gloves (PVC) can be used as an alternative to latex gloves being more resistant to heat damage. However, they are susceptible to being torn with snag on nail edges. They also have a limited use in food industries because of their short shelf life.

Nitrile gloves were also developed to replace latex gloves. They are less elastic but have a longer shelf life. They are more resistant to chemicals but sensitive to alcohol degradation. Although they are sensible to be torn, their garish color helps them to be distinguished within a food lot.

Natural rubber latex gloves are most commonly used because they are most cost-efficient and comfortable. They provide good tactile sensitivity and good dexterity. However, latex and chemical compounds added to the gloves can produce allergies and migration of particles to food, especially in the presence of bleach.

In summary, according to the intended use, convenience, and cost-effectiveness, glove material should be carefully chosen. However, it has been demonstrated that regardless of the gloves used, handling practices and gloves changing are critical steps that influence microbial transfer to foods.

#### *6.1.4. Cross-contamination*

As mentioned above, the cross-contamination is a major cause of food poisoning worldwide. Cross-contamination phenomena arise as a consequence of the application of inadequate hygiene practices, contact with contaminated equipment and utensils, by direct hand contact with foods prepared by the improper storage of food, bad processing food, by direct food contact with air or contaminated environments, and so on [94]. Recontamination routes and sources (e.g., raw materials, food contact surfaces, food handlers) were revised [100] demon‐ strating their relevance to foodborne disease outbreaks. These information sources should also be incorporated in Quantitative Microbiological Risk Assessments (QMRAs) to perform mitigation strategies and reduce foodborne disease [101].

Particularly, RTE foods are highly susceptible to be contaminated during handling. Some studies refer to the direct contact of the RTE food (e.g., meat) with food-contact surfaces and contaminated cutting utensils [58]. It is also emphasized its importance as a source of trans‐ mission of enteric pathogens, *L. monocytogenes*, or *S. aureus* [102].

#### *6.1.5. Food storage*

Temperature is the most important factor that governs microbial growth in food. Most microorganisms grow at temperatures between 5°C and 60°C (called danger zone), being the optimum growth temperature at 37°C. Thus, maintaining the cold chain and a correct heat treatment for hot foods are essential measures to maintain food safety, where food handlers play a key role throughout the production chain.

Table 4 presents the recommended temperature storage/transport and freezing of raw materials and finished products in catering establishments.

Maintaining food to inadequate time intervals/storage temperature often constitutes one of the most common risk factors for food poisoning. Previous studies indicate that in school canteens and other related catering establishments exposure to abuse refrigerated temperature for extended periods of time could lead to an increase of pathogens at hazardous levels for human health from preparation to the distribution thereof [103]. Other possible deficiencies are related to temperature control storage of raw and processed ready for consumption, lack of knowledge of food handlers about cooking and refrigeration temperatures suitable to prevent the growth and survival of microorganisms, inadequate cooling and warming food and preparing several hours before consumption [104], or joint cooling of raw and cooked foods [105].


**Table 4.** Recommended storage temperatures of raw ingredients and prepared meals

#### *6.1.6. Training of food handlers*

Training of food handlers has been considered an important measure as a part of the HACCP systems, given that it helps to prevent most foodborne diseases. Although knowledge alone is not enough to change practices, food handlers with adequate knowledge can change their practices easier if they are closely supervised and supported by their onsite managers. In addition, guidance and supervision by their managers during work improve attitudes and practices [106].

For some time ago, questionnaires or "checklists" have represented an effective tool to evaluate the level of knowledge and skills on food hygiene and safety of food handlers [98]. Neverthe‐ less, further studies detailing more sophisticated methods to obtain a greater quantity and reliability of information to improve the training of food handlers are needed.

Additionally, novel strategies leading to more effective training methods have been per‐ formed. For example, some studies proposed to strengthen the training of less experienced food handlers and validate the knowledge of those more experienced in a period not exceeding 10 years [107]. In this sense, a better knowledge on food safety by food handlers ensures better performance and motivation [108]. In this sense, it is highly important to food handlers in the HACCP systems companies to correct their attitudes and behavior at work. Also, a periodic training is found as an effective way to raise awareness of food handlers [9].

Training of food handlers in food hygiene is a mandatory requirement for the food industry. At EU level, requirements on food safety and hygiene procedures are stated in Regulation (EC) 852/2004 [10].

Specifically, in its Annex II (Chapter XII), the food business operators must ensure the following:


Recently, the European Federation of Food, Agriculture and Tourism (EFFAT) and the FERCO launched the project "Food hygiene training for all" [109]. The project is based on the devel‐ opment of a software tool for basic training of food handlers of the contract-catering sector across Europe. This tool is available online free of charge (www.contract-catering-guide.org/ food-hygiene-training-for-all) and aims at obtaining a better qualification of workers in catering establishments and also offers the opportunity for training staff in those companies that do not have sufficient resources to invest in training.

#### *6.1.7. Intervention strategies against microbial foodborne outbreaks*

The burden reduction of foodborne diseases is a major goal of societies. The strategies developed by countries to achieve this goal are numerous and very different depending on issues, such as political and socioeconomic status, actual or emerging pathogens, resources, trade (import/exports), temporal limitations, and inter-regional cooperation.

Woteki and Kineman [110] described different approaches to reducing foodborne illness and grouped them into four categories: (i) population surveillance and better outbreak detection, (ii) prevention-based regulatory approaches, (iii) information and education, and (iv) riskbased system.

Early identification of foodborne outbreaks and the implicated organism should be directed to controlling the outbreak, stopping exposure, and perhaps more importantly, preventing future outbreaks [111]. Also, a rapid and coordinated response is needed among state officials and federal agencies. Some authors [111, 112] pointed out that surveillance based on molecular analysis of foodborne pathogens involved in outbreaks and sporadic cases together with the creation of a platform to share this information would allow for anticipation of potential future episodes. In this sense, Fisher et al. [113] reported the creation in the EU of platforms where data and information on potential outbreaks of foodborne pathogens are available and can be disseminated rapidly to those who need to know; the Enter-net is a surveillance network database of bacterial enteric pathogens, while Salm-gene is a molecular typing network. In the United States, similar platforms are available [110], i.e., FoodNet, a system of disease surveil‐ lance that provides information on the incidence of foodborne illness, and Pulse-net, a common name for National Molecular Subtyping Network for Foodborne Disease Surveillance.

Regulatory agencies allow food business operators to set up performance standards in the industry through the well-known HAZARD plan. The HAZARD plan should be designed upon the analysis of the likely hazards in the food and the strategies put in place to eliminate them or to reduce them to acceptable levels [114]. This system has shown to have a very positive and crucial impact on food safety, thus on public health, as has been recognized by key organizations like the World Health Organization [115]. Special attention deserves establish‐ ments, which deliver meals to a large number of people, and even more, in those centers where there is an important proportion of consumers with a weak or impaired immune system like hospitals or nurseries. Unfortunately, in the past years it has been reported some cases where the HACCP plan was not fully implemented. This is the case of the study by Kokkinakis et al. [116], who reported that only two out of the seven major hospitals interviewed in Crete (Greece) had implemented the HACCP plan during the period of 2004–2009. These authors identified 14 crucial elements for HACCP implementation in hospitals. Shih and Wang [117], in their study on factors influencing HACCP implementation in 23 public hospitals in Taiwan, revealed that the most important concern perceived by managers was related to economic issues, i.e., "getting funds from the hospital" and "difficulty of allocation funds for facility improvement." In addition, it was shown that more support, HACCP training, and coordina‐ tion with other hospitals were necessary to avoid staff reluctance to implement the HACCP plan. Shih and Wang [117] also pointed out that kitchen design and flow charts of food production are the first two issues to consider before the HACCP implementation. The lack of financial support and poor HACCP training were also reported by Garayoa et al. [95] in their survey directed to staff from 20 contract catering companies throughout Spain.

In the food industry, emerging and existing technologies should be assessed in terms of food safety [118]. However, a new concept of food safety arisen in the early 2000s, with Regulation 178/2002, *laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety*. In this document, risk analysis was introduced as the pillar on which food law should be based on the aim to achieve the general objective of a high level of protection of human health and life. This risk-based approach would enable the setting of national and international targets for disease reduction as well as provide the basis for such reduction efforts [110, 119].

Education and information of consumers are highly relevant to prevent outbreaks, and above all, sporadic cases. The World Health Organization launched in 2001 an educational cam‐ paign called "Five keys to Safer Foods," where five brief and clear messages were given to food handlers: (i) keep clean, (2) separate raw and cooked, (3) cook thoroughly, (4) keep food at safe temperatures, and (5) use safe water and raw materials (WHO, 2006). Other campaigns like Fight bac™ [120] or Thermy™ [121] aimed at getting consumers informed about hygienic food handling practices the former, and the use of thermometers in the cooking of food products the latter. Other most recent campaigns like "The chicken challenge" clearly show short messag‐ es with the objective of cutting *Campylobacter* food poisoning in half by the end of 2015 [122].

The reduction of foodborne illness incidence is a challenge for governments, which should manage the different strategies to lower the risk posed by food hazards up to acceptable levels. Current knowledge and tools on risk assessment allow for science-based decision-making.
