Fish is considered a highly nutritious food that constitutes the human diet, produced through fishing and aquaculture activities, to be marketed and consumed around the world in different presentations and culinary preparations. Fish is also very susceptible to spoilage and contamination by microorganisms throughout the food chain, which may be part of the usual microbiota or incorporated into food derived from inadequate hygiene practices in the food industry. Fish has been associated worldwide with disease outbreaks derived from consumption, where various bacteria and/or metabolites (biogenic amines) are some of the main casual agents. Citrobacter sp. is considered a pathogen in fish, as well as in humans, derived from the consumption of contaminated food, generating infections or histamine poisoning as it is part of the generating microbiota.
1. Introduction
Fish is defined as any food that can be extracted from oceanic or continental waters (fresh or brackish) intended for human or animal feeding, being a generic term that includes fish, crustaceans, mollusks, algae, etc.
[1].
Fish is considered a traditional and popular food in different regions around the world, and for certain countries, it constitutes the main contribution of protein of animal origin
[2]. In addition, an increasing number of people choose fish as a healthy food option due not only to its source of protein of high biological value and digestibility, but also because of its content of polyunsaturated lipids, vitamins, and minerals
[1][2][3].
Fish and fish products are foods highly susceptible to contamination by various chemical hazards related to foodborne illnesses, including heavy metals, polychlorinated biphenyls, dioxins, insecticides, nitrosamines, antibiotics, hormones, biotoxins, biogenic amines (histamine), and biological hazards, such as parasites, viruses, and bacteria
[2][4][5][6]. They have also been related to numerous outbreaks of infections or poisoning in humans around the world, derived from consumption of microorganisms, in which bacteria are the most causative-related agents
[2][6][7][8].
Microorganisms can inhabit and survive in many natural environments, being transferred to food and contaminating it at different stages of the food chain through soil, air, water, insects, animals, and humans
[9]. Throughout the food chain, from capture or harvest to consumption, fish undergo various changes that alter their sensory, chemical, and microbiological features. During those various phases, a series of critical points, such as time, temperature, conditions, and hygiene practices in handling, among others, must be monitored because if they are not adequately controlled, they impact the sanitary quality of the product, transforming it into a high-risk food in terms of health intended for human consumption
[10][11]. In addition to this, the presence of hazards that put people’s health and perception at risk has implications for the acceptability of fish for consumption, as well as the contribution of this food to human nutrition and health
[12].
Citrobacter sp. has been related to agents of biological origin harmful to human and animal health. Therefore, the objective of this document is to provide a general informative perspective on the various contaminating hazards in fish, specifically those of biological origin such as Citrobacter sp., their impact on human and animal health. All of this in promotion of the safety of foods of aquatic origin destined for human consumption and the protection of public health.
2. Foodborne Diseases
Food safety refers to the guarantee that it will not cause harm or illness to the person who consumes it. This attribute is part of food’s basic characteristics, along with nutritional, sensory, and commercial features, which make up the total quality of food. There exists correspondence between the safety and health of consumers, and its obtention is fundamental and indisputable in terms of public health issues around the world
[11][13].
FDs constitute an important health problem worldwide due to their incidence, sequelae, mortality, new forms of transmission, vulnerable population groups, increased resistance of causative agents to antimicrobial compounds, loss of productivity, costs associated with health services, implementation, and monitoring of food safety policies, in addition to their effects on trade and tourism
[14][15][16][17]. It is estimated that every year, around 600 million people worldwide become sick, from which 420,000 die
[14][17].
Approximately 250 FD-causing agents have been reported, which may be chemicals released in nature (toxic inorganic compounds, antimicrobials, growth promoters, toxic food additives, lubricants, dyes, natural toxins, disinfectants, heavy metals, pesticides, and substances used in the cleaning industry), as well as those of physical origin (fragments of glass, metal, wood, or others) and biological origin (bacteria, parasites, viruses, fungi, and prions)
[15][16].
The most frequent FD, in cases and outbreaks, are those derived from biological contamination
[16][18].Generally, foods of animal or vegetable origin are rarely sterile, containing microbial associations whose composition depends on the organisms that reach it and how they multiply, survive, and interact in the food over time
[17]. The microorganisms responsible for diseases through food are numerous, with bacterial agents being the main culprits
[16][18], among which are adenovirus, rotavirus, norovirus, hepatitis A and E viruses,
Citrobacter freundii,
Salmonella spp.,
Vibrio sp.,
E. coli,
Clostridium sp.,
Cronobacter sp.,
L. monocytogenes,
Campylobacter sp.,
S. aureus,
Aeromonas sp.,
Y. enterocolitica,
B. cereus,
Shigella spp.,
Cryptosporidium parvum,
Entamoeba histolytica,
Anisakis sp.,
Giardia lamblia,
Trichinella sp., and
Toxoplasma sp., among others
[5][6][14][18][19][20][21].
3. Fish
Fish is a fundamental part of a healthy diet, being a source of proteins, lipids, vitamins, and minerals, contributing to nutritional needs through food by consumers
[1][22]. Fish for human consumption comes from capture fishing and aquaculture activities, estimating a worldwide production in both activities, in 2018, of around 178.5 million tons, allocating 156.4 million tons for human consumption with a per capita consumption of 20.5 kg
[23].
Fish and fish products are highly nutritious, but they are also susceptible to deterioration and contamination derived from autolysis, oxidation, and microbial activity due to high water activity, tissues’ neutral pH, and a high proportion of nutrients easily utilizable by microorganisms
[1][2][11], where the nutritional value and quality of the fish is related to factors such as species, age, their environment, food, capture conditions, handling, storage, transportation, and distribution
[11].
Fish and other aquatic organisms have a microbial population that is dependent on that present in the aquatic environment where they live
[24][25].Health risks can be caused by different pathogenic bacteria, which can be classified as follows: (1) autochthonous, which are those whose natural habitat is water (
C. botulinum,
Listeria spp.,
Vibrio spp.,
Plesiomonas shigelloides,
Edwardsiella tarda,
Pseudomonas spp., and
Aeromonas spp., among others) and are widely distributed in the aquatic environments of different places in the world, where temperature and salinity have a selective effect. They are also considered part of the natural microbiota of the fish
[2][4][26]; and (2) non-native, which includes bacteria present in water with fecal contamination and/or associated with inadequate hygiene practices and conditions during capture, transportation, processing, and handling phases, identifying various enterobacteria, such as
E. coli,
Shigella spp.,
Salmonella spp.,
Enterobacter cloacae,
Citrobacter sp.,
Serratia sp.,
P. vulgaris,
Listeria monocytogenes,
S. agalactiae,
Staphylococcus epidermidis, and
Staphylococcus aureus, among others
[2][4][25][27][28].
4. Citrobacter sp.
The
Citrobacter genus is found within the so-called coliform bacilli, along with the
Escherichia,
Klebsiella,
Enterobacter, and
Serratia genera, which include overt and opportunistic pathogens responsible for a wide range of infections
[29][30][31].
Citrobacter species are generally known as opportunistic pathogens in human and veterinary medicine, including aquaculture activities, due to the presence of immunosuppressive factors (stress and pollution, among others) that favor the appearance of diseases.
[32][33][34].
4.1. Citrobacter, Histamine, and Food Diseases by Fish Consumption
Fish, besides being a nutritious source of food, are susceptible to deterioration and endogenous or exogenous contamination by bacteria, particularly those that produce biogenic amines
[22]. Biogenic amines are biologically active low-molecular-weight, organic, non-protein nitrogenous compounds present in numerous foods, such as cheese, wine, meat, vegetables, and fish
[35][36].
Protein hydrolysis is carried out by the action of endoenzymes that release amino acids that, in turn, serve as a substrate for enzymes (amino acid decarboxylase) of microbial origin, resulting in the generation of biogenic amines
[32][36][37].Histamine-producing microorganisms are related to the natural microbiota of fish, but they can also come from contamination in post-capture stages, such as processing, conservation, storage, or distribution systems
[32][35][38][39]. Histamine-producing microorganisms in fish are varied, including species of the genera
Streptococcus spp.,
Clostridium spp.,
Tetragenococcus spp.,
Bacillus spp.,
Acinetobacter spp., and
Pseudomonas spp. Some enterobacteria considered indicators of food hygiene are
Citrobacter spp.,
Serratia spp.,
Hafnia spp.,
Vibrio spp.,
Escherichia spp.,
Edwarsiella spp.,
Klepsiella spp.,
Salmonella spp.,
Shigella spp.,
Photobacterium spp.,
Enterobacter spp.,
Plesiomonas spp.,
Proteus spp., and
Morganella, with the species
M. morganii considered the main producer
[35][38][40][41][42][43][44][45][46][47].
The consumption of foods with high levels of biogenic amines has been related to negative effects on the health of consumers, with gastrointestinal, neurological, and hemodynamic disorders with different symptoms, such as malaise, nausea, respiratory disorders, hot flashes, sweating, palpitations, migraines, headaches, head, itchy eyes, hyper and hypotension, stomach and intestinal problems, and pseudo-allergic reactions
[36][39].Among the different biogenic amines, histamine is mainly related to cases of poisoning from fish consumption due to its high levels
[36][48].
5. Isolation and Detection of Enterobacteria in Food Case: Citrobacter sp.
Commonly for the isolation and detection of microorganisms, such as enterobacteria in various samples, when the culture is viable, phenotypic identification is used, which includes the visible characteristics of bacteria, such as morphology, serological, biochemical, and metabolic properties using various simple, enriched media that are differential and/or selective
[49].
For the isolation and growth of
Citrobacter sp., different culture media can be used through culture, such as Luria–Bertani (LB) agar, tryptic soy agar (TSA), blood agar (BA), MacConkey agar, cystine–lactose–electrolyte-deficient (CLED) agar, Salmonella-Shigella (SS) agar, xylose lysine deoxycholate (XLD) agar, Eosin Methylene Blue (EMB) agar, and Violet Red Bile Glucose agar (VRBG), among others
[20][34][50][51][52][53]. The identification or confirmation of other enterobacteria, even at the species level, can be achieved by analysis of enzymatic activities, metabolic capacity, antigenic determinants, or susceptibility to bactericidal agents
[20][29][31][34][51][52][54].
Various generalized methods have been developed in liquid and/or solid media for the isolation and enumeration of enterobacteria in food, which are generally used as indicators of food contamination, inadequate hygiene practices during and after processing, and health risks from the consumption of contaminated food
[50], as well as the identification of where
Citrobacter species can be found and identified through the use of several differential and selective media. Among the existing methods are the ISO 21528-1:2017 method for detection and enumeration by the most probable number (NMP) technique, and ISO 21528-2:2017 for detection and enumeration by colony count. Jimenez et al.
[55] proposed a method for detection and enumeration by colony count which involves the dilution of the sample and subsequent inoculation in a selective culture medium and incubation for subsequent quantification of total coliforms and fecal coliforms, respectively, as well as subsequent identification of colonies through different biochemical tests. In addition, the methods proposed by Pascual and Calderón
[50] present the phases of pre-enrichment, enrichment, and sample dilution in different culture media for enterobacteria and subsequent growth and identification in differential culture medium and confirmation through various biochemical tests for enterobacteria.
Modern technologies for the detection and identification of
Citrobacter species in laboratory samples have been developed, which through molecular analysis of the subunit ribosomal RNA gene (16S rRNA) by polymerase chain reaction (PCR) in its different variants, have reported good utility and are even being used for epidemiological studies
[20][29][33][51][54]. Meanwhile, proteome analysis using matrix-assisted laser-desorption ionization and time-of-flight mass spectroscopy (MALDI-TOF MS) to identify strains in culture is also an alternative to traditional methods for greater sensitivity, specificity, and speed in obtaining results
[51][56][57][58].
6. Determination of Biogenic Amines (Histamine) in Fish and Fish Products
Histamine, once generated in food, cannot be eliminated by any heat treatment, so to guarantee the safety of fish and fish products, in relation to histamine content, various colorimetric, immunoenzymatical, and chromatographic methods have been developed and proposed, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), and thin-layer chromatography (TLC) of qualitative, semiquantitative, and quantitative natures
[46][59][60][61][62]. The choice of method to be used considers its efficiency, sensitivity, simplicity, speed, cost of materials, and used reagents
[63]. Among the different methods are fluorometric-AOAC 977.13, which is the most used procedure for fish samples. It is sensitive and reproducible, but it is also complex and consumes a lot of analysis time; this methodology presents extraction with methanol and passage through an ion exchange column, as well as derivatization and quantification with external standards
[37][64].
In the spectrophotometric method, the sample is subjected to processes of homogenization, centrifugation, and extraction with saline and butanol solutions, measuring the absorbance of extracts at 496 nm. The histamine concentration in the sample is calculated by means of a calibration curve using a histamine standard
[22]. Immunoenzymatic methods are useful for the detection of histamine in fish and are used for studies in case of surveillance of conditions and adequate hygienic practices for product preservation, suspected intoxication, and applications in HACCP systems; the method can be qualitative or semiquantitative
[61][65].
The basic principle of an immunoenzymatic study, such as the enzyme-linked immunosorbent assay (ELISA), is to use an enzyme to detect the binding of antigen to antibody. The enzyme converts a colorless (chromogenic) substrate to a colored product, indicating the presence of an antigen-binding antibody. Therefore, the ELISA can be used to detect the presence of antigens or antibodies in the sample, depending on its design
[66]. The commercial kits available to detect histamine in fresh fish products and meals can only discriminate products with less than 50 ppm, but for health regulation purposes, such as the European Union Regulation 2073/2005 that considers several detection levels (100, 200, and 400 mg/kg) of histamine content in fishery products, further confirmation of the positive sample is needed with higher-precision analytical techniques based on HPLC
[61].
In recent years, various methods based on high-performance liquid chromatography (HPLC) and/or gas chromatography (GC) have been developed for the determination of histamine in fish, which require chemical derivatization (dansyl chloride) or O-phthalaldehyde (OPA) to improve the detection of biogenic amine by UV–vis absorption or fluorescence, and where the derivatization step can be pre-column, column, or post-column, and quantification can be represented by a standard curve. These methods are fast, sensitive, and reproducible for the determination of one or more biogenic amines
[37][39][61][67][68][69].
7. Control and Prevention: Animal Health and Food Diseases by Consumption of Fish (Microbiology Contamination and Biogenic Amines)
The presence of various microorganisms, including enterobacteria, such as Citrobacter sp., in food production systems (aquaculture and fishing) is considered hazardous to animal health and a potential risk of contamination in the different phases of the food chain that can compromise food safety.
For the control and prevention of diseases in animals, related to aquaculture activities and due to various causative agents, including bacteria, it is recommended to implement good hygiene practices, including personal hygiene, cleaning and filling of ponds, the management of the quantity and quality of water (microbiological conditions, temperature, dissolved oxygen concentration, pH, turbidity), appropriate fish population density in ponds, removal and daily elimination of sick or dead fish, avoiding the use of chemical substances, favoring a nutritious diet adequate in quantity and quality for farmed fish, and the cleanliness of capture equipment and containers
[70][71].Meanwhile, in the post-capture or harvest stages, the implementation of different procedures, practices, and/or food-safety-management systems, such as good manufacturing practices, Sanitation Standard Operating Procedures (SSOPs), and the Hazard Analysis Critical Control Points (HACCP) System, are a fundamental part of reducing the presence of pathogens and achieving the safety of fish and fish products intended for human consumption
[71][72][73][74][75].