foods

Review
Antibiotic Use in Livestock and Residues in Food—A Public
Health Threat: A Review
Oana Mărgărita Ghimpet, eanu 1 , Elena Narcisa Pogurschi 2 , Dana Cătălina Popa 2 , Nela Dragomir 2 ,
Tomit, a Drăgotoiu 2 , Oana Diana Mihai 1 and Carmen Daniela Petcu 1, *

1 Faculty of Veterinary Medicine, University of Agronomic Sciences and Veterinary Medicine of Bucharest,
050097 Bucharest, Romania; ghimpe_marga@yahoo.com (O.M.G.); oprea_diana2008@yahoo.com (O.D.M.)
2 Faculty of Animal Productions Engineering and Management, University of Agronomic Sciences and
Veterinary Medicine of Bucharest, 011464 Bucharest, Romania; elena.pogurschi@usamv.ro (E.N.P.);
dana-catalina.popa@igpa.usamv.ro (D.C.P.); nela.dragomir@usamv.ro (N.D.);
tomita.dragotoiu@usamv.ro (T.D.)
* Correspondence: carmenpetcufmvb@gmail.com; Tel.: +40-723704482

Abstract: The usage of antibiotics has been, and remains, a topic of utmost importance; on the
one hand, for animal breeders, and on the other hand, for food safety. Although many countries
have established strict rules for using antibiotics in animal husbandry for the food industry, their
misuse and irregularities in compliance with withdrawal periods are still identified. In addition to
animal-origin foods that may cause antibiotic residue problems, more and more non-animal-origin
foods with this type of non-compliance are identified. In this context, we aim to summarize the
available information regarding the presence of antibiotic residues in food products, obtained in
various parts of the world, as well as the impact of consumption of food with antibiotic residues on
consumer health. We also aim to present the methods of analysis that are currently used to determine
antibiotic residues in food, as well as methods that are characterized by the speed of obtaining results
or by the possibility of identifying very small amounts of residues.

Citation: Ghimpet, eanu, O.M.; Keywords: antibiotic residues; food safety; antimicrobial resistance; public health
Pogurschi, E.N.; Popa, D.C.;
Dragomir, N.; Drăgotoiu, T.; Mihai,
O.D.; Petcu, C.D. Antibiotic Use in
Livestock and Residues in Food—A
1. Introduction
Public Health Threat: A Review.
Foods 2022, 11, 1430. https:// Antibiotics are produced by living organisms or in the laboratory and have the capacity
doi.org/10.3390/foods11101430 to kill or inhibit the growth of microorganisms [1]. Even if antibiotics represent a positive
aspect for both animals and humans, because of their great impact on their health status,
Received: 31 March 2022
their abusive use can lead to harmful consequences, and especially to the appearance of
Accepted: 12 May 2022
resistant germs [2–4].
Published: 16 May 2022
The practice of using antibiotics is still widespread for prophylactic and curative
Publisher’s Note: MDPI stays neutral purposes, and the problem of their remnants in animal products, groundwater, soil, and
with regard to jurisdictional claims in feed has caused (and is still causing) worldwide concern, generating vast costs to combat
published maps and institutional affil- antibiotic resistance [5]. In order to reduce this effect, a number of developed countries have
iations. enforced laws in order to decrease the excessive use of antibiotics as a means to prevent
diseases or as an acceleration tool in animals [6]. A number of monitoring programs regard-
ing the use of antibiotics in animal breeding have been implemented over the last 30 years,
starting with the Danish Integrated Antimicrobial Resistance Monitoring and Research
Copyright: © 2022 by the authors.
Program in 1995 [7]. Nowadays, a number of research projects have been developed in
Licensee MDPI, Basel, Switzerland.
this area; as an example, the Disseminating Innovative Solutions for Antibiotic Resistance
This article is an open access article
distributed under the terms and
Management project (DISARM), in which nine European countries are involved [8]. As a
conditions of the Creative Commons
counterbalance, in less developed countries in Asia or Africa, the use of antibiotics is still
Attribution (CC BY) license (https://
very high due to the great demand for animal products, and this leads to the uncontrolled
creativecommons.org/licenses/by/ use of these substances in the breeding systems [9].
4.0/).

Foods 2022, 11, 1430. https://doi.org/10.3390/foods11101430 https://www.mdpi.com/journal/foods

Foods 2022, 11, 1430 2 of 25

Most antibiotic residues can be found in a large range of food products, both ani-
mal and vegetal [10]. Although meat consumption is decreasing in developed countries,
especially in Europe, in less developed countries it is very high, so meat represents the
most important source of antibiotics. Primarily, the main source of antibiotics in food
is abusive use (overdosing, non-compliance with the withdrawal period) and the use of
antibiotic-contaminated water, or inappropriate disposal of animal dung.
The sources of antibiotics in humans are two-fold: first, prescriptions for people (most
commonly penicillins, macrolides, and fluoroquinolones), and, second, the substances used
in animal breeding (tetracycilies and sulfonamides). Antibiotics can cause serious effects
on human health that have led to the introduction of maximum residue limits (MRL) in
food safety legislation [11,12]. Long-term exposure is linked to impairment of the immune
system, digestive problems caused by the destruction of the intestinal flora, renal problems,
and even carcinogenic effects [13].
Yang et al. (2021) and Huang et al. (2020) mentioned that antibiotics have become
widespread in the environment due to their extensive and long-term use, influencing both
human health and the system, due to the emergence of antibiotic resistance [14,15]. Saeed
et al. (2020) addressed the contamination of food with antibiotic residues, mentioning that
it is a global problem, due to the improper use of antibiotics. The authors of the current
paper also mentioned methods of analysis to identify antibiotics in food [16].
This review focuses on three aspects: (1) antibiotic use in livestock; (2) antibiotic
residues in food and methods of detection; and (3) the impact of consumption of food with
antibiotic residues on consumer health.

2. Antibiotic Use in Livestock

Livestock farming plays a very important role in the agriculture of the European
Community. Achieving the best results largely depends on the use of safe and good quality
feed. Free movement of safe and good quality food and feed is a key aspect of the internal
market, and contributes significantly to the health and well-being of consumers [17].
In terms of animals of economic interest and poultry farms, various active substances
are used, including antibiotics, in order to maintain the health of the animals and to have a
better yield for breeding. Antibiotics can be administered via feed or by intramuscular injec-
tion [18,19]. Medicated feed is an oral route of veterinary medicinal product administration.
Medicated feed is a homogeneous mixture of feed and veterinary medicinal products [20].
Regulation (EU) 6/2019 establish rules for the authorization of use of veterinary medic-
inal products in feeding stuffs, including the manufacture, distribution, advertising, and
surveillance of such products [12]. Feed business operators, which handle manufacturing,
storing, transporting, or placing medicated feed and intermediate products on the market,
must be authorized by the competent authority, in accordance with the authorization sys-
tem, to ensure both the safety of the feed and the traceability of the products. The labeling
of medicated feed stuffs must comply with the general principles set out in Regulation
(EC) 767/2009, and are subject to specific labeling requirements, in order to provide users
with the information necessary for the correct administration of medicated feed [21]. Such
administration must be adequately described in the product information to ensure correct
administration and proper dosing of certain veterinary medicinal products, to be adminis-
tered orally to animals, in feed, or in drinking water, especially in the case of treating groups
of animals. The relevant legislation provides the establishment of additional instructions for
cleaning of the equipment used in the administration of respective medicinal products, in
order to avoid cross-contamination and to reduce resistance to antimicrobials [12]. A large
range of synthetic feed additives are included in this category of antibiotics and are used in
animal feeding to increase production efficiency and to control different diseases [8].
Recent studies have shown that a significant percentage of all manufacturers add an-
tibiotics to healthy animals’ feed to prevent, rather than cure, diseases [22–25]. According to
Erofeeva et al., livestock accounts for approximately 50% of all antibiotics produced in the
world. During the life of an animal, the use of antibiotics can significantly increase a gain in
Foods 2022, 11, 1430 3 of 25

weight by increasing the use of nutrients in the diet, which, in the end, makes it possible to
reduce feed costs and shorten the feeding period [25]. Feed manufacturers and authorities
establish procedures and instructions for the effective and safe use of authorized and
prescribed veterinary medicinal products, for oral administration, other than medicated
feeding stuff, such as mixing drinking water with veterinary medicinal products or manu-
ally mixing a veterinary medicinal product in feed, which are administered by farmers to
food-producing animals. These instructions take into account the scientific recommenda-
tions of the European Medicines Agency, established by Regulation (EC) No. 726/2004, on
measures to minimize over dosage or under dosage, unintended administration to animals
other than those targeted, the risk of cross-contamination, and the release of these products
into the environment [12,26]. Homogeneous dispersion of veterinary medicinal product in
feed is also essential for the manufacture of safe and effective medicated feed [20].
According to different researchers, reliable data regarding the quantity and patterns of
use, dose, and frequency, are not very accurate [27–29]. Disease prevention is more effective
than treating it. Drug treatment, with antimicrobials in particular, should in no way replace
good animal husbandry, biosecurity, and management practices [20]. Excessive weight
loss at the beginning of the lactation period leads to the appearance of stress, increasing
the susceptibility of animals to diseases, which is why medicated feed has been used for
animals for curative and preventive purposes [30]. The US Department of Agriculture
noted that approximately 88% of growing swine receive antibiotics in their feed for disease
prevention and growth promotion purposes, commonly tetracyclines or tylosin [22]. Some
of the most frequently used antibiotics in ruminants are ionophores, a distinctive class of
antibiotics that can influence intestinal flora to achieve increased energy and amino acid
availability and improved nutrient utilization [22]. Most beef calves in feed lots and some
dairy heifers receive this drug routinely in their feed. Ionophores have never been used
in humans or therapeutically in animals, because of their specific mode of action. The
impact that medicated food has on human health, and implicitly how antibiotic resistance
is installed after ingesting food with antibiotics residues, is a topic that we will detail
in a later section, but it is worth mentioning that while some bacteria are intrinsically
resistant to these drugs, there is currently no evidence to suggest that ionophore resistance
is transferable [22,28].
The presence of antibiotics in feed is not desirable due to the antibiotic resistance
that the bodies can later acquire. Therefore, the elimination of antibiotics from feed, but
improvements in the productivity of pigs and poultry, has been achieved through the use
of feed additives, such as omega 3, immunoglobulin, organic and inorganic acids, zinc
oxide, yeast derived β glucans, essential oils, prebiotics, probiotics, threonine, cysteine,
and herbs and spices [31,32]. On the other hand, the conclusion of another study was that
the elimination of prophylactic in-feed antibiotics leads to minor reductions in productive
performance and animal health [29].
Advertising for medicated feeding stuffs addressed persons who cannot properly
assess the risks associated with their use, and may lead to improper or excessive use of the
medicinal product, which may harm public or animal health or the environment [20].
Animal health management is mainly based on disease prevention, compliance with
hygiene conditions, and the correct application of disinfection actions. Preventive use of
synthetic chemical allopathic medicines is forbitten in order to obtain organic products. In
case antibiotics are compulsory, treatment should be carefully managed through minimal
use with a high efficiency. In such cases, in order to ensure the integrity of organic produc-
tion for consumers, an official withdrawal period after the use of such medicinal products
requires a longer period of time. In organic production, the use of antibiotics as growth or
production stimulants, coccidiostats, hormones, etc., is not permitted [33].

3. Antibiotic Residues in Food

Dozens of scientific papers have been published over the years, focusing on antibiotic
residues in food and feed. Antibiotics can be naturally produced by living organisms or
Foods 2022, 11, 1430 4 of 25

they can be synthetically produced in a laboratory. The main role of antibiotics is to inhibit
or kill microorganism growth [1,34]. Antibiotics can be used as feed additives in livestock
in order to maintain animal health; lately, however, there is an increasing attempt to stop
this phenomenon as antibiotic residues can be consumed by humans with food of animal
origin (meat, milk, eggs, fish, honey, etc.). It should be noted that, not only may food from
animal origin have antibiotic residues, but also plant foods, which can be contaminated by
soil and water [35,36]. From Table 1, it can be seen that antibiotic residues are found in all
foods intended for human consumption, whether they are of animal or non-animal origin.
It can be seen that most studies were performed to determine the presence of antibiotic
residues in milk, followed by meat, and then by honey. Another thing to note is that
research on antibiotic residues started in the 1980s, but the largest number of works in this
field appeared in the last 5 years.

Table 1. Bibliometric analysis according with “Web of Science” database.

Field of Research Number of Scientific Publications

Antibiotic residues in food 67
Antibiotic residues in feed 17
Antibiotic residues in animal origin food 12
Antibiotic residues in non-animal origin food 6
Antibiotic residues in meat 54
Antibiotic residues in meat products 4
Antibiotic residues in fish 12
Antibiotic residues in milk 292
Antibiotic residues in eggs 21
Antibiotic residues in honey 45

In order to ensure food safety for consumers, more and more studies have attempted
to find effective and rapid methods for the detection antibiotic residues in feed and
food [36]. Consumers are also increasingly interested in consuming quality food, and
are increasingly turning to organic products, which provide them with more safety relative
to conventional products.
Several studies have shown the presence of antibiotic residues in various types of
food (Table 2).

Table 2. Presence of varying concentrations of antibiotic residues in the different animal-derived

products in some developing countries. Reprinted from [37].

Antibiotic
Concentration Sample Country Literature
Residue
Chicken
2604.1 ± 703.7 µg/kg Muscle
Tanzania Kimeria et al. [38]
3434.4 ± 604.4 µg/kg Liver
3533.1 ± 803.6 µg/kg Kidney
Beef
Oxytetracycline
51.8 ± 90.53 µg/kg Muscle
Nigeria Olufemi and Agboola [39]
372.7 ± 366.8 µg/kg Kidney
1197.7 ± 718.9 µg/kg Liver
Cattle
15.92 to 108.34 µg/kg Muscle Ethiopia Bedada et al. [40]
9.02 to 112.53 µg/kg Kidney
Enrofloxacin 0.73 and 2.57 µg/kg
Chicken tissues
Chloramphenicol 1.34 and 13.9 µg/kg
Iran Tavakoli et al. [41]
Penicillin 0.87 and 1.3 µg/kg
Calves muscles
Oxytetracycline 3.5 and 4.61 µg/kg
Foods 2022, 11, 1430 5 of 25

Table 2. Cont.

Antibiotic
Concentration Sample Country Literature
Residue
30.81 ± 0.45 µg/kg Chicken
Quinolones Turkey Er et al. [42]
6.64 ± 1.11 µg/kg Beef
Chicken
124 to 5812 µg/kg Breast
Egypt Salama et al. [43]
107–6010 µg/kg Thigh
103 to 8148 µg/kg Livers
Chicken
Tetracyclines
150 ± 30 µg/g Liver Cameroon Guetiya-Wadoum et al. [44]
62.4 ± 15.3 µg/g Muscle
Beef
50 to 845µg/kg Kidney
Kenya Muriuki et al. [45]
50 to 573 µg/kg Liver
23–560 µg/kg Muscle
9.8 to 56.16 µg/mL Milk
Amoxicillin Bangladesh Chowdhury et al. [46]
10.46 to 48.8 µg/g Eggs
Sulfonamides 16.28 µg/kg
Raw milk China Zheng et al. [47]
Quinolones 23.25 µg/kg
Oxytetracycline 199.6 ± 46 ng/g
Beef Zambia Nchima et al. [48]
Sulphamethazine 86.5 ± 8.7 ng/g
15.22 ± 0.61 µg/L Fresh milk
Penicillin G 7.60 ± 0.60 µg/L Cheese (wara) Nigeria Olatoye et al. [49]
8.24 ± 0.50 µg/L Fermented milk (nono)
Chicken
Sulphonamides 0.08–0.193 µg/g Liver Malaysia Cheong et al. [50]
0.006–0.062 µg/g Breast
Tetracycline
Oxytetracycline
>0.1 µg/mL Raw milk India Kumari Anjana et al. [51]
Sulfadimidine
Sulfamethoxazole

3.1. Products of Non-Animal Origin

Although food products of animal origin are considered to be the major source of
antibiotic residues, studies in the literature have shown that even non-animal origin prod-
ucts represent an important problem regarding this type of contamination. The main
sources of these compounds in agriculture are irrigation water with antibiotics traces due to
inappropriate recycling processes or the use of manure as soil amendments, which leads to
spreading antibiotics through the food chain [52]. As a result of these agricultural practices,
antibiotics in soil can be taken up by plants, entering the food chain. Due to the fact that
plants are considered to be a minor source of antibiotic residues, studies have focused on
few compounds [29–31]. The most common vegetables that accumulate antibiotics are
considered to be cereals, such as wheat, rice, and oat, and coarse grains, such as maize and
barley. In this field, studies have focused on antibiotic detection in different matrices or
were conducted as experiments in a hydroponic environment [53–57].
In recent years, research has been conducted in order to study the relation between
intake of antibiotics in edible crops due to poorly management of wastewater used in irri-
gations and from manure [53,55,58–61]. Pan and Chu (2017) studied the influence of some
antibiotics (tetracycline, sulfamethazine, norfloxacin, erythromycin, and chloramphenicol)
on crops in relation to two types of contamination, irrigation with wastewater and soil
amendment with animal manure. Findings showed that the distribution of tetracycline,
norfloxacin, and chloramphenicol in crop tissues were as follows: fruit > leaf/shoot > root;
Foods 2022, 11, 1430 6 of 25

an opposite order was found for sulfamethazine (SMZ) and erythromycin (ERY), i.e., root >
leaf/shoot > fruit [58]. Research also revealed that the uptake of antibiotics in crops was
higher in the case of wastewater use and it was lower in the case of manure fertilization,
argued by the fact that crops are more likely to absorb residues during the continuous
process of irrigation. However, the levels of antibiotics ingested through the consumption
of edible crops under the different treatments were much lower than acceptable daily intake
(ADI) levels.
In Northern China, studies have shown that oxytetracycline, tetracycline, chlortetracy-
cline, sulfamethoxazole, sulfadoxine, sulfachloropyridazine, chloramphenicol, ofloxacin,
pefloxacin, and lincomycin were found in vegetables. In the same geographical area, rela-
tively high concentrations of norfloxacin, ciprofloxacin, and enrofloxacin were found in
vegetables, such as tomato, cucumber, pepper, spinach, eggplant, and crown daisy [62].
Other studies have shown that parts of vegetables, such as the roots of carrot and leaves
of lettuce, as well as cabbage and spinach, the stem of celery, and fruits of cucumber,
bell pepper, and tomato had 64% pharmaceutical residues, including antibiotics, due to
the wastewater used for irrigation [63]. Bassil et al. (2013) evaluated the uptake of gen-
tamicin and streptomycin in carrot (Daucus carota), lettuce (Lactuca sativa), and radish
(Rhaphanus sativus) due to the same type of fertilization. The conclusion of the study was
that three crops absorbed relatively higher amounts of gentamicin (small molecule) than
streptomycin (large molecule), and that the levels of antibiotics in plant tissues increased
when increasing the antibiotic concentration in manure [64]. The intake of antibiotics into
vegetables seedlings was also studied by Ahmed et al. (2015), who showed that cucumber
(Cucumis sativus), cherry tomato (Solanum lycopersicum), and lettuce had relatively high
levels of tetracyclines and sulfonamides in the non-edible parts, but lower concentrations
in fruit parts and were within acceptable daily intake levels [61].

3.2. Products of Animal Origin

The use of antibiotics that may lead to the accumulation of residues in meat, milk,
eggs, and honey should not be allowed in foods intended for human consumption. If the
use of antibiotics is necessary in the treatment or prevention of various animal diseases, a
withholding period must be respected until antibiotic residues are no longer detected [65].
The presence of antibiotic residues in meat from various species of economic interest is
considered a significant danger to public health.
The results of the study conducted by Al-Mashhadany in 2020 on sheep meat harvested
from supermarkets in Iraq showed that samples contained antibiotic residues at a level
higher than the maximum allowed limits. Cooling and freezing, as preservation methods,
slightly reduce antibiotic residues in meat. The same study showed that thermal processing
of lamb meat (cooking for about 45 min) leads to a transformation of antibiotic residues
into inactive residues against bacteria [66].
Another study conducted by Babapour. et al. in 2012 on meat samples collected
from Iran obtained similar results in terms of the incidence rate of antibiotic residues in
meat [67]. A higher incidence of antibiotic residue has been reported in Nigeria in beef sam-
ples [68]. In contrast, the lowest incidence rates were reported in sheep samples analyzed
in Spain [48]. The presence of fluoroquinolone residues (enrofloxacin and ciprofloxacin)
in some Indonesian chicken samples indicated that it were used by farmers in poultry
feed [69,70].
Milk with antibiotic residues significantly influences the technological process of ob-
taining dairy products, which includes the technology used for dairy yeasts. Antibiotics
can get into milk from treatments applied to sick animals or (less often and not recom-
mended) through the use of preservatives. Milk with antibiotic residues is considered a
rigged food on the market. By consuming unpasteurized milk, antibiotic-resistant bacte-
ria can be transmitted to consumers, especially in areas with a dense population and a
lower degree of development, where there is a risk of improper storage of milk and dairy
products [71,72]. Milk and dairy products are exposed to contamination by antibiotics and
Foods 2022, 11, 1430 7 of 25

other drug residues, but also to neutralizing and preserving substances [73–75]. Analysis
of antibiotic residues in dairy products (pasteurized drinking milk, yogurt, sour cream,
whipped milk, cheese) leads to the identification of the gentamicin/neomycin group, es-
pecially in sour cream. Macrolides sometimes appear in cheese, and tetracyclines in sour
cream and cheese. Here, we discuss about milk samples subject to confirmatory investi-
gations, used for human consumption. Moghadam et al. (2016) identified that 38.5% of
raw milk samples collected from the Iranian province of Khorasan Razavi had penicillin
residues, while Ghanavi et al. (2013) reported identification of residues of 11% antibiotics
of cow milk samples collected from different regions of Iran [76,77]. Studies conducted by
Vinu, (2021) showed that there is a direct correlation between the stage of lactation and
the presence of antibiotic residues in milk; 34.3% of positive samples came in the lactation
stage of 0–70 days, 20% between 70–140 days of lactation, and 45.7% between 140–305 days
of lactation [78]. Additionally, Knappstein et al. (2004) highlighted a direct correlation
between milk production and the presence of antibiotic residues (cefquinomas), their level
not being influenced by the frequency with which milking was performed [79].
In most countries, eggs are the main product generated from backyard poultry pro-
duction systems due to the fact that they can be quickly consumed or sold to meet essential
family needs. In a study by Cornejo et al. (2020), in Chile, the presence of antimicrobial
residues in eggs, such as tetracyclines, beta-lactams, aminoglycosides, and macrolides,
was analyzed. The survey showed that all samples were positive for at least one of the
four antimicrobials tested [80]. Another recent study from China concluded that careful
monitoring should be imposed on antibiotic residues in poultry eggs, after detecting 30%
positive egg samples for quinolones, tetracyclines, and sulphonamides [81].
The most common contaminations of honey can be explained by treatments in order
to control honeybee diseases and contaminants coming from procedures applied in agri-
culture [82]. The European Union has forbidden the use of antibiotics for bees, this aspect
is strictly enforced by recent legislation [83,84]. The most common and important antibi-
otics found in honey are beta-lactams (penicillin, ampicillin, cloxacillin, amoxicillin for
bacterial infections), amphenicols (thiamphenicol, florfenicol, chloramphenicol which are
carcinogenic antimicrobials), tetracyclines (oxytetracycline, chlortetracycline, tetracycline
for bacterial diseases), macrolides (erythromycin, tylosin, oleandomycin and spiramycin),
and aminoglycoside, fluoroquinolones (ciprofloxacin, enrofloxacin, norfloxacin—growth
enhancing) [85–87].

3.3. Methods of Analysis

Analytical techniques for determining antibiotics have gradually evolved with the
advent of increasingly advanced technology. If, 50 years ago, the usual technique was based
on inhibiting the development of known bacterial cultures, the so-called microtest (the
principle used in the antibiogram), today we are see the determination of antibiotics using
high performance liquid chromatography coupled with mass spectrometry (LC-MS/MS).
Analysis methods can be classified into screening analysis methods and confirmatory
analysis methods.
(a) Screening analysis methods list
Screening methods use equipment that is more readily available in terms of price
and mode of operation to identify a group of antibiotics or an antibiotic, with or without
quantification of that antibiotic. The general rule is that any result obtained by a screening
method must be confirmed using a confirmation method. The screening methods used
are the most varied, and are based on different principles, such as microbial inhibition,
enzyme immunoassay, stick format (lateral flow devices), radioimmunoassay (RIA), chemi-
luminescence immunoassay (CLIA), fluorescence immunoassay (FIA), and colloidal gold
immunoassay (CGIA). Screening methods for antibiotics have proven to be useful and fast
tools that provide results with a high accuracy and sensitivity, which can guarantee safe
food [17].
1. Microbial Inhibition Test (Microtest)
Foods 2022, 11, 1430 8 of 25

This test is based on the incubation of environmental plates with a suspension of

a known concentration of bacterial strains, which is added to the test sample. If the
test sample has an antibiotic, it will not allow the development of specific colonies, thus
opening a halo area around the sample to be analyzed [88]. This test is an expensive test
that involves specific endowments that are specific to a food microbiology laboratory, as
well as specialized personnel; another major disadvantage is the obtaining of results after
an average of 18 h of incubation, and not in 1–2 h as in other screening methods.
2. Delvotest
This is a classic test for determining antibiotics in milk and is based on the whole
principle of microbial inhibition. In the absence of an antibiotic, the bacterial suspension
develops and the opacity of the environment, or the change in color due to the appearance of
acid in the bacteria-growing activity, is noted. In the presence of an antibiotic, the bacterial
strain does not develop and there is an area of inhibition or a lack of environmental color
change [89]. This type of test is very sensitive to β-lactam antibiotics, but can also be used
for sulfonamides and other antimicrobials. This type of test requires incubation for several
hours before results can be visualized, so this test was modified by borrowing principles
from the enzyme immunoassay, thus forming antibody–antibiotic complexes that develop
a color reaction in the presence an enzyme. A low intensity usually means positive, while a
high intensity is considered negative. These tests are more expensive than conventional
tests with microbial inhibitors, but provide a result in minutes. The major disadvantage is
that they only detect substances that react immunologically with the receptor.
3. Enzyme-linked immunosorbent assay (ELISA)
This method is based on the classical antigen–antibody reaction in the presence of a
conjugate. The technique of obtaining the antibody is relatively simple and is based on
the body’s ability to generate antibodies to a particular antigen, usually using different
adjuvants that increase the body’s ability to produce antibodies [90]. This assay represents
the most common screening test for detecting of antibiotic residues, especially in food
samples [91], and the sensitivity of this method is sometimes superior to confirmatory meth-
ods [92]. Depending on the antibiotic detected, different immunoenzymatic techniques have
emerged. Thus, for the determination of fluoroquinolones, competitive ELISA methods
have been developed using the reaction between the antigen–conjugate (fluoroquinolone)–
antibody (bovine serum albumin) and the specific polyclonal antibody [93]. Addition-
ally, for the determination of chloramphenicol and tetracycline, the specific monoclonal
antibody is used in most cases [94,95]. ELISA can also assess multiple residues of an-
tibiotics in different foods, so a new colorimetric and dual-colorimetric ELISA test has
been developed for simultaneously determination of 13 fluoroquiunolone residues and
22 sulfonamides [96,97]. The sensitivity of the ELISA method has been improved by the
addition of a biotin-streptavidin compound that allows better catalysis of the substrate [98–
100]. Immunoenzymatic analysis techniques have been developed for the determination
of antibiotics in both animal products (milk, eggs, meat) and aquaculture products and
feed [101,102]. In conclusion, it can be said that the enzyme-linked immunosorbent assay is
a fast, sensitive, and easy to implement test [100], but that it also has some disadvantages,
including a fairly high percentage of false positive results due to cross-reactions and low
reproducibility [95].
4. Radioimmunotest (RIA)
This technique uses isotope-labeled, as well as unlabeled, antigens to react competi-
tively with antibodies. This technique is used to detect antibiotics in various products of
animal origin, as well as in various products in the aquatic environment [103].
5. Chemiluminescence Immunoassay (CLIA)
This test is widely used due to the fact that it is an easy, fast, sensitive, and selective
test [104]. CLIA is based on the combination of two systems, namely the immune response
and actual chemiluminescence analysis. Due to its high specificity and sensitivity, CLIA is
Foods 2022, 11, 1430 9 of 25

used in many fields, but it has limitations due to the compounds used such as acridinium
derivatives and the immediate emission of light is a disadvantage due to its measurement
problems [105].
6. Colloidal gold immunochromatographic assay (CGIA)
This new test uses colloidal gold as a tracer in an alkaline environment, which inter-
acts with negatively and positively charged groups and antibody protein molecules [106].
This technique has been developed for the rapid determination of chloramphenicol [107].
Another CGIA technique was developed for the simultaneous determination of quinolones,
tetracycline, and sulfonamide in milk, thus allowing the concomitant determination of
36 different antibiotics in less than 10 min [108]. A high-sensitivity CGIA test was devel-
oped for the determination of streptomycin in pig milk and urine, with a very low limit of
detection of 2.0 ng/mL for milk and 1.9 ng/mL in urine [99].
7. Fluorescence Polarization Assay (FPIA)
The principle of the test is competitive and is based on the binding of fluoroflora
to a specific antigen and highlights the fluorescent compound as a standard compound
needed to detect and identify an unknown antigen. If the antibiotic sought is not in the
sample, a tracer will be bound to the antibody and the signal will be high [109]. Various
FIA techniques have been developed for the simultaneous determination of several fluoro-
quinolones in food; these techniques are based on the use of monoclonal antibodies. Other
FPIA techniques have been developed for the simultaneous determination of cephalexin
and cefadroxil in milk samples, gentamicin in goat’s milk, as well as other antibiotic [99,110].
FIA is an easy-to-implement screening method that allows the simultaneous detection of
various antibiotics in a short period of time. As a disadvantage, this test requires a sample
preparation step to extract the antibiotic from the sample, as well as a filtering step to obtain
a colorless sample that does not affect the reading of the sample relative to the fluorescence
points [111].
8. Lateral flow immunoassay (LFIA)
Until a few years ago, this type of test had applications only in areas such as the diag-
nosis of various diseases, pregnancy, and identification of various toxins in the environment.
However, recently, there have been applications for the use of LFIA for the simultaneous
detection of beta-lactams, quinolones, sulfonamides, and tetracyclines in food [112]. The
advantages of using this test include its ease of use, increased shelf life—up to 2 years, and
use at room temperature. Like any very simple test, it has many disadvantages: many false
positive or false negative results, low reproducibility, etc. [113].
(b) Confirmatory analysis methods
Analysis techniques have evolved gradually and the need for more and more ad-
vanced methods has been a natural consequence. While screening methods, with the
exception of microtest, do not involve major costs or specialized personnel, confirmation
methods involve the use of expensive equipment (LCMSMS, GCMSMS) and highly quali-
fied personnel. Depending on the antibiotics of interest, the equipment and extraction steps
are different. A mass spectrometer (MS) is an equipment with an operating principle that
is the production of ions, their sorting according to the specific mass-to-load ratio (m/z),
and the analysis of the obtained signals. Each compound is characterized by a specific m/z
ratio, with data present in the literature, as well as in the software of the latest generation
of equipment, which comes equipped with data libraries that allow the identification of
compounds against reference values [114]. The use of MS is very common in the analysis
of antibiotic residues because it has a much higher specificity than screening methods and
allows the simultaneous determination of many classes of antibiotics. GCMS/MS has previ-
ously been used, but due to the fact that the processing of samples for gas chromatography
is more cumbersome and often requires derivatization steps for signal amplification, the
development of methods of analysis for the simultaneous determination of antibiotics of
several classes is more difficult [115].
Foods 2022, 11, 1430 10 of 25

1. Liquid chromatography coupled with mass spectrometry (LC/MS/MS)

Liquid chromatography coupled with mass spectrometry is a commonly used tech-
nique for determining antibiotic residues in food. This technique is used by both official
laboratories for routine analyses as well as by national reference laboratories and Euro-
pean reference laboratories. With liquid chromatography coupled with mass spectrometry,
determining seven classes of antibiotics was possible, totaling 30 antibiotics in less than
8 min. The preparation of a sample involves the weighing of 1 g of sample of a meat
obtained and is evaporated and then taken up again with 1 mL of ultrapure and used
for introduction into the LC/MS/MS. LC/MS/MS equipment which is optimized for the
identification and quantification of each compound of interest [114]. Another method of
determining antibiotics allows the determination of 46 antibiotics from different classes.
This method was developed for the determination of cows from cow’s milk, beef, sheep,
pigs, equines, and birds, as well as fish and shrimp [116]. As a general principle, a sample is
extracted with a mixture of solvents, purified by passing through an SPE column, and then
injected into the LC/MS/MS. For antibiotic residues, the method of analysis must meet the
performance criteria of European Commission Decision No. 2002/657 [117]. In general,
regardless of the laboratory in which antibiotic residues are determined by LC/MS/MS,
the extraction protocol is generally the same; namely, extraction of the sample with an
organic solvent, purification by passing through an SPE column, injection into LC/MS/MS,
and the criteria for performance must comply with European Commission.
2. Gas chromatography coupled with mass spectrometry (GC/MS/MS)
Using gas chromatography coupled with mass spectrometry, there are fewer applica-
tions because the derivatization stage is cumbersome and affects the long-term life of the
equipment, so applications are restricted to 1–2 classes of antibiotics that can be determined
simultaneously. The sample preparation protocol is generally the same as for LCMS/MS;
namely, sample extraction with organic solvent, purification by passing through an SPE col-
umn, specific sample derivatization, injection into GC/MS/MS, and performance criteria
must meet European Commission Decision No. 2002/657.
Official laboratories in Romania use methods that use LC/MS/MS equipment and
analyzed compounds are those provided by the Surveillance and Control Program in the
field of food safety; the developed methods are based on standardized methods or are
provided by European reference laboratories. Thus, in Romania a method for determining
14 classes of antibiotics totaling 83 antibiotics is used, and tissue samples for milk, eggs,
and honey are analyzed. Methods for LCMS/MS determination of chloramphenicol,
nitrofurans, nitroimidazole in food stuffs of animal origin are also developed.
The methods of analysis used to identify antibiotic residues have advantages and
disadvantages, as shown in Table 3.

Table 3. Advantages and disadvantages of different analysis methods.

Analytical Method Advantages Disadvantages

easy to operate mainly qualitative methods

Screening analysis methods any result obtained by a screening

low price method must be confirmed by a
confirmation method
expensive test that involves specific
specificity–if the test sample has an endowments specific to a food
antibiotic, it will not allow the microbiology laboratory as well as
(a) Microbial inhibition test (microtest) development of specific colonies, thus specialized personnel
opening a halo area around the sample to
be analyzed obtaining of results only after an average
of 18 h of incubation
Foods 2022, 11, 1430 11 of 25

Table 3. Cont.

Analytical Method Advantages Disadvantages

classic test for determining antibiotics
more expensive than conventional tests
in milk
(b) Delvotest
detects only substances that react
very sensitive to β-lactam antibiotics
immunologically with the receptor
sensitivity of this method is sometimes fairly high percentage of false positive
superior to confirmatory methods results due to cross-reactions
(c) Enzyme-linked immunosorbent
used for the multi-residue determination
assay (ELISA)
of antibiotics in different foods low reproducibility
fast, sensitive and easy to implement test
high selectivity high concentrations of other molecules
(d) Radioimmunotest (RIA) with antibody affinity could inactivate it
high sensitivity
measurement problems due to the
(e) Chemiluminescence compounds used, such as acridinium
easy, fast, sensitive and selective test
immunoassay (CLIA) derivatives and the immediate emission
of light
rapid determination of chloramphenicol
(f) Colloidal gold simultaneous determination of
high price
immunochromatographic assay (CGIA) quinolones, tetracycline and sulfonamide
in milk; 36 different antibiotics in less
than 10 min
requires a sample preparation step to
easy-to-implement screening method that extract the antibiotic from the sample
(g) Fluorescence polarization assay (FPIA) allows the simultaneous detection of a filtering step to obtain a colorless
various antibiotics in a short sample that does not affect the reading of
period of time the sample relative to the
fluorescence points
many false positive or false
ease of use
negative results,
(h) Lateral flow immunoassay (LFIA)
increased shelf life—up to 2 years at
low reproducibility
room temperature
higher specificity than screening methods use of expensive equipment
Confirmatory analysis methods allows the simultaneous determination of
super qualified personnel
many classes of antibiotics
Determination of 7 classes of antibiotics,
(a) Liquid chromatography coupled with 30 antibiotics in less than 8 min
high price
mass spectrometry (LC/MS/MS) method of analysis must meet the
performance criteria of European
Commission Decision No. 2002/657
applications are much lower because the
derivatization stage is cumbersome and
(b) Gas chromatography coupled with standardized methods or provided by affects the long-term life of the
mass spectrometry (GC/MS/MS) European reference laboratories equipment, so the applications are
restricted to 1–2 classes of antibiotics that
can be determined simultaneously

4. Impact of Food Consumption with Antibiotic Residues on Consumers’ Health

The concept of “One Health”, promoted by the World Health Organization and the
World Organization for Animal Health, emphasize the idea of an intimate relationship
between humans, animals, and the environment, all leading to a unique concept of health.
Foods 2022, 11, 1430 12 of 25

Thus, the entire chain must be considered in order to maintain equilibrium, carefully
using medicinal products at all three levels [20]. Today, there is major public awareness
about the consequences of prolonged and increased use of antibiotics in animal livestock
production [118]. Microorganisms have the ability to develop antibiotic-resistant genes,
resulting in increased survival, thus minimizing treatment options for microbial infections
and leading to increased mortality among humans [119–122]. Foods from animal origins
are considered key reservoirs of antibiotic residues, which occur as a result of the use of
antibiotics industrially, thus contributing to the induction of globally antibiotic resistance.
Antibiotic-resistant bacteria have been identified in animal-origin food products, in feeds,
as well as in humans. Globally, very large differences have been reported between geo-
graphical areas in terms of prevalence of animal-origin antibiotic-resistant bacteria and
antibiotic-resistance genes [123]. According to recent studies, antibiotics remain in animal-
origin food products, such as milk, meat, and eggs, even after heat treatment, and lead to the
development of gastrointestinal disorders and allergies in humans, or even the appearance
of antibiotics-resistant superbugs. Furthermore, along with antibiotic resistance, the ineffec-
tiveness of antibiotic therapy for human treatments is increasing [22]. There are a number
of studies that mention that the continuous and abusive use of antibiotics frequently causes
the development of antibiotic-resistant bacteria. Moreover, multidrug-resistant bacterial
infections can progressively increase mortality, thus posing a threat to public health [124].
The major problem frequently mentioned by researchers is that, over time, bacteria may
adapt and acquire resistance to active phenolic components similar to antibiotics; therefore,
their use must also be taken into account [8]. Kumar et al. (2020) reviewed this topic in
order to better understand the mechanisms of development and dissemination of antibiotic
resistance genes in nutritional, clinical, agricultural and environmental contexts [125–127].
In the same study, other dietary strategies were considered to replace medicated feed with
probiotics, essential oils, or antibodies, with a preventive role against bacterial infections.
A solution to antibiotic resistance needs efforts from several fields of activity, including
agriculture, but also veterinary medicine (microbiology, biochemistry, medical clinic, and
genetics), by replacing medicated feed with alternative therapies [124,128,129]. Animal
husbandry is a key component of the global economy, and is a major contributor to food
provision. In order for animals to gain weight, they receive medicated feed containing
antibiotics or antibiotics are introduced into drinking water. Antibiotics are introduced
into farm animal feed, even for preventive purposes. Allen mentioned (2014) that this
activity leads to massive accumulation of antibiotics in the environment, and subsequently
leads to the acquisition of antibiotic resistance by microorganisms [130]. Several stud-
ies have indicated that the spread of antibiotic-resistant microorganisms in humans is
mainly due to the consumption of animal-origin foods and beverages contaminated with
antibiotic residues, or through the consumption of water contaminated by environmental
pollution [25,124,131].
Therefore, in order to reduce antibiotic-resistant bacterial infections worldwide, mea-
sures regarding the use of antibiotics for non-therapeutic purposes, such as the use of
antibiotics in animals feed, have been taken, when the products are intended for human
consumption. Banning the use of avoparcin in animal feed in the European Union has
reduced the incidence of antibiotic resistance in animals, and thus its occurrence in hu-
mans [124,132]. Antibiotic resistance is a global problem that affects public health, with
socio-economic repercussions and it significantly influences the use of antibiotics in animal
feed of economic interest. The development of the WHO Global Action Plan and the FAO
Global Action Plan, in line with the One Health concept, is a requirement to prevent the
transmission of antibiotic resistance, from farm to fork [123].
Every year, 33,000 people die as a direct consequence of infections caused by antibiotic-
resistant bacteria, a number comparable to the passengers of more than 100 medium-sized
aircraft [133].
Many classes of antibiotics have been recognized, and their use should be limited. In
December 2019, the EMA classified antimicrobials into four categories, A to D (Figure 1).
 FoodsFoods
2022,2022, 11, x FOR PEER REVIEW
11, 1430 14 of 13
27of 25

Figure
Figure 1. Comparison listing 1. Comparison
of CIAs listing
by WHO (2019), of (2018),
OIE CIAs by WHO
and (2019), OIE
AMEG—EMA (2018),
(2019) for and AMEG—EMA
the major (2019) for the
classes of anti-
major classes of antibiotics adapted from [134].
biotics adapted from [134].

The
Theirrational
irrationaluse use of
of antibiotics inanimal
antibiotics in animalhusbandry
husbandry and
and subsequent
subsequent pollution
pollution of the
of the
environment
environment(wastewater,
(wastewater, animal-
animal- andandnon-animal-origin
non-animal-origin food
food products,
products, soilsoil in places
in places
FOR PEER REVIEW 15 of 27
wheremanure
where manurewas was applied),
applied), inevitably
inevitablyleads
leadstotothe
theformation
formationof of
antibiotic resistance.
antibiotic resistance.
Erofeevaetetal.al.(2021)
Erofeeva (2021)mentioned
mentionedthatthatthe
theproblem
problemisisthat
thatscientists
scientistshave
havenot
notdiscovered
discovered any
anygroup
new new group of antibiotics
of antibiotics sincesince
20042004
[25][25] (Figure
(Figure 2). 2).

3
15 1960-1970
27
1975-1979
1983-1987
1987-2004
55

Figure 2. Discovery of antibiotics adapted from [25].

Figure 2. Discovery of antibiotics adapted from [25].

In the European Union, the provisions of European Regulation 37/2010 concerning

maximum permitted limits by product categories and by food producing species are ap-
Foods 2022, 11, 1430 14 of 25

In the European Union, the provisions of European Regulation 37/2010 concerning

maximum permitted limits by product categories and by food producing species are
applicable (Table 4).

Table 4. Maximum residues limits of antibiotic in products of animal origin marketed in the European
Community [135].

Active Substance Animal Species Target Tissue MRL

Muscle 50 µg/kg
Fat 50 µg/kg
Amoxicillin All food-producing species Liver 50 µg/kg
Kidney 50 µg/kg
Milk 4 µg/kg
Muscle 50 µg/kg
Fat 50 µg/kg
Ampicilin All food-producing species Liver 50 µg/kg
Kidney 50 µg/kg
Milk 4 µg/kg
Muscle 50 µg/kg
Fat 100 µg/kg
Avilamycin Porcine, poultry, rabbit
Liver 300 µg/kg
Kidney 200 µg/kg
Bacitracin Bovine Milk 100µg/kg
Muscle 50 µg/kg
Fat 50 µg/kg
Benzylpenicillin All food-producing species Liver 50 µg/kg
Kidney 50 µg/kg
Milk 4 µg/kg
Cefacetrile Bovine Milk 125 µg/kg
Muscle 50 µg/kg
Fat 50 µg/kg
Cefapirin Bovine
Kidney 100 µg/kg
Milk 60 µg/kg
Cefazolin Bovine, ovine, caprine Milk 50 µg/kg
Muscle 100 µg/kg
Liver 300 µg/kg
Chlortetracycline All food-producing species Kidney 600 µg/kg
Milk 100 µg/kg
Eggs 200 µg/kg
Muscle 100 µg/kg
Fat 100 µg/kg
Clavulanic acid Bovine, porcine
Liver 200 µg/kg
Kidney 400 µg/kg
Muscle 300 µg/kg
Fat 300 µg/kg
Cloxacillin All food-producing species Liver 300 µg/kg
Kidney 300 µg/kg
Milk 30 µg/kg
Muscle 150 µg/kg
Fat 150 µg/kg
Liver 150 µg/kg
Colistin All food-producing species
Kidney 200 µg/kg
Milk 50 µg/kg
Eggs 300 µg/kg
Foods 2022, 11, 1430 15 of 25

Table 4. Cont.

Active Substance Animal Species Target Tissue MRL

Muscle 300 µg/kg
Fat 300 µg/kg
Cloxacillin All food-producing species Liver 300 µg/kg
Kidney 300 µg/kg
Milk 30 µg/kg
Muscle 300 µg/kg
Fat 300 µg/kg
Dicloxacillin All food-producing species Liver 300 µg/kg
Kidney 300 µg/kg
Milk 30 µg/kg
Muscle
Liver 100 µg/kg
Kidney 300 µg/kg
Not for use in animals from which milk is 600 µg/kg
produced for human consumption
Bovine
Doxycycline
Porcine, poultry Muscle
Skin and fat 100 µg/kg
Liver 300 µg/kg
Kidney 300 µg/kg
Not for use in animals from which eggs is 600 µg/kg
produced for human consumption
Muscle 100 µg/kg
Fat 100 µg/kg
Enrofloxacin Bovine, ovine Liver 300 µg/kg
Kidney 200 µg/kg
Milk 100 µg/kg
Muscle 100 µg/kg
Fat 100 µg/kg
Porcine, rabbit
Liver 200 µg/kg
Kidney 300 µg/kg
Muscle 100 µg/kg
Enrofloxacin Skin and fat 100 µg/kg
Poultry
Liver 200 µg/kg
Kidney 300 µg/kg
Muscle 100 µg/kg
Skin and fat 100 µg/kg
All other food-producing species
Liver 200 µg/kg
Kidney 200 µg/kg
Muscle 200 µg/kg
Fat 200 µg/kg
Liver 200 µg/kg
Erythromycin A All other food-producing species
Kidney 200 µg/kg
Milk 40 µg/kg
Eggs 150 µg/kg
Muscle 50 µg/kg
Fat 50 µg/kg
Gentamicin Bovine, porcine Liver 200 µg/kg
Kidney 750 µg/kg
Milk 100 µg/kg
Muscle 100 µg/kg
Fat 100 µg/kg
All food-producing species except
Kanamycin A Liver 600 µg/kg
fin fish
Kidney 2500 µg/kg
Milk 150 µg/kg
Foods 2022, 11, 1430 16 of 25

Table 4. Cont.

Active Substance Animal Species Target Tissue MRL

Muscle 100 µg/kg
Fat 50 µg/kg
Liver 500 µg/kg
Lincomycin All food-producing species
Kidney 1500 µg/kg
Milk 150 µg/kg
Eggs 50 µg/kg
Muscle 150 µg/kg
Fat 150 µg/kg
Marbofloxacin Bovine, porcine
Liver 50 µg/kg
Kidney 150 µg/kg
Muscle 500 µg/kg
Fat 500 µg/kg
Liver 500 µg/kg
Neomycin B All food-producing species
Kidney 5000 µg/kg
Milk 1500 µg/kg
Eggs 500 µg/kg
300 µg/kg
Muscle 300 µg/kg
Oxacillin All food-producing species Fat 300 µg/kg
Liver Kidney Milk 300 µg/kg
30 µg/kg
Muscle 100 µg/kg
Liver 300 µg/kg
Oxytetracycline All food-producing species Kidney 600 µg/kg
Milk 100 µg/kg
Eggs 200 µg/kg
Muscle 500 µg/kg
Fat 500 µg/kg
Streptomycin All ruminants, porcine, rabbit
Liver 500 µg/kg
Kidney 1000µg/kg
Muscle 100 µg/kg
Fat 100 µg/kg
Sulfonamides All food-producing species
Liver 100 µg/kg
Kidney 100 µg/kg
Muscle 100 µg/kg
Fat 100 µg/kg
Liver 100 µg/kg
Tylosin A All food-producing species
Kidney 100 µg/kg
Milk 50 µg/kg
Egg 200 µg/kg

Improper use of antibiotics in food-producing animals contributes to the appearance

of antibiotic resistance. WHO recommends that farmers and the food industry stop using
antibiotics routinely to promote growth and prevent disease in healthy animals. Recom-
mendations aim to help preserve the effectiveness of antibiotics that are important for
human medicine by reducing their unnecessary use in animals. WHO declares that, in
different countries, approximately 80% of total antibiotic consumption appears in the an-
imal sector, largely to promote the growth of healthy animals. Overuse and misuse of
antibiotics in animals and humans is contributing to the rising threat of antibiotic resistance.
Some types of bacteria that cause serious infections in humans have already developed
resistance. WHO strongly recommends a reduction in use of all classes of antibiotics for
food-producing animals, including a complete restriction of these antibiotics for growth pro-
motion and disease prevention without a diagnosis. “Scientific evidence demonstrates that
 Foods 2022, 11, 1430 17 of 25

overuse of antibiotics in animals can contribute to the emergence of antibiotic resistance”

says Dr. Kazuaki Miyagishima, Director of the Department of Food Safety and Zoonoses
at WHO. Many countries have already taken actions to reduce the use of antibiotics in
food-producing animals. For example, since 2006, the European Union has banned the
use of antibiotics for growth promotion. In addition, consumers promote the marketing
of meat raised without the routine use of antibiotics, with some major food chains adopt-
ing “antibiotic-free” policies for meat supplies. The general objective is to encourage the
prudent use of antibiotics in order to slow down antimicrobial resistance and maintain the
effectiveness of antibiotics for medicine [136]. About two-thirds of U.S. antibiotics that are
important to people are sold for use in food animal production. Yet, experts have long
warned of the public health threat from regularly exposing herds of thousands, even tens of
thousands, of animals to human-class antibiotics in their feed for prolonged periods of time.
The U.S. Food and Drug Administration (FDA) lists 89 medically important antibiotics
currently added to animal feeds. According to the www.nrdc.org website (accessed on
6 May 2022), since 2016, the FDA has flagged the lack of clear antibiotics use limits as a
significant problem needing a remedy, thus, at this moment in the USA, there are no clear
limits for the use of antibiotics. The same site mentions the deadline for establishing and
publishing these data as 2023 [137].
Numerous studies from different parts of the world mention that antibiotic residues
in feed stuffs are, at present, a large problem, and can lead to major associated health
Foods 2022, 11, x FOR PEER REVIEW
problems, including antibiotic resistance, toxicity, hypersensitivity reactions, teratogenicity,
and carcinogenicity (Figure 3) [1].

Antibiotic
resistance

Toxicity Hypersensitivity
reactions
Use of
antibiotics

Teratogenicity Carcinogenicity

Figure 3. Effects
Figure of antibiotics
3. Effects use on human
of antibiotics use onhealth.
human health.
Most antibiotics cause side effects in humans (Table 5) [134].
Mostnumber
A large antibiotics cause
of studies side
refer effects
to the in humans
management (Tableresidues,
of antibiotic 5) [134].antibiotic-
resistant bacteria (ARB), and antibiotic resistance genes (ARG) can be found in dairy manure
Table
and may5.contribute
Possible effects due toofantibiotics
to the spread antibiotic in human.(AR). More than 60 ARGs can
resistance
be found in milk manure (including β-lactam and tetracycline resistance genes), although
Group ofwith antibiotic use, residues, and ARBs have been inconsistent, possibly due to
correlations Main Effects Clinical Signs
Antimicrobials
sampling and analytical limitations. Antibiotic resistance genes often persist through these
systems, although optimal management and a higher operating temperature
Mild rash tomay facilitate
severe toxidermia a
their attenuation [138–140]. Elements related
Skin reactions to the mechanism of action and resistance
some of the skin reactionsin follow
humans are presented in Table 6 [141] (after Iwu et al., 2020).
human exposure to sulphonami
Hypersensitivity mentioned
Contact sensitization confirmed
averse reactions
topical medicinal products
Sulphonamides reactions
Hemolytic anemia, neutropenia
Blood dyscrasias
thrombocytopenia and pancyto
Sulfamethazine dose-dependen
Carcinogenicity
increase in follicular cells adeno
Foods 2022, 11, 1430 18 of 25

Table 5. Possible effects due to antibiotics in human.

Group of
Main Effects Clinical Signs
Antimicrobials
Mild rash to severe toxidermia are
Skin reactions some of the skin reactions following
human exposure to sulphonamide
Hypersensitivity mentioned
Contact sensitization confirmed for
averse reactions
Sulphonamides topical medicinal products
reactions
Hemolytic anemia, neutropenia,
Blood dyscrasias
thrombocytopenia and pancytopenia
Sulfamethazine dose-dependent
Carcinogenicity
increase in follicular cells adenomas
(thyroid)
of thyroid gland
Association with IgE-mediated
Hypersensitivity
allergic anaphylaxis 10% of the human population is believed to
reactions
be allergic
Human reaction based on penicilloyated (amoxicilloyated)
residues in milk and meat. Amoxicillin (AX), with or without
clavulanic acid, is the most common elicitor of allergy. Very low
Penicillins Anaphylaxis levels (6 µg/L) can cause this reaction; therefore, especially for
milk low MRLs (4 µg/kg) were established for the group of
penicillins by EMA and JECFA (Codex). USA—zero tolerance for
residues in milk
Sufficient evidence that consumption
Influence of
of beef or pork containing residues of
starter cultures in
penicillins exceeding MRLs causing
food processing
anaphylactic reactions
MRLs set based on the microbiological ADI. In the period of EMA
assessment, it was concluded that there is no induction of
resistant enterobacteria at the dose 2 mg per person per day—on
Possible the other hand, in an in vitro study to assess the impact of
influence of tetracycline on the human intestinal microbiome, there was
Tetracyclines
human intestine screened the variability of the presence of tet genes after exposure
microbiome of low concentrations 0.15, 1.5, 15 and 150 µg/mL of tetracycline,
after 24 h
and 40 days and variable to slight
increase of the tetracycline gene copies occurred.

Table 6. Mechanism of action and resistance mechanism of antibiotics in human. Adapted from [141].
(after Iwu et al., 2020).

Antibiotics Class Example (s) The Mechanism(s) of Action Resistance Mechanism(s)

Cephalosporins, Penicillins, Cleavage by β-lactamases, ESBLs,
Cell wall biosynthesis
β-lactams Cefotaxime, Monobactams, Carbapenemases, Cefotaximases, and
inhibition
Carbapenems altered Penicillin-binding proteins
Ribosomal mutations, enzymatic
Aminoglycosides Gentamicin, streptomycin Protein synthesis inhibition modification, 16S rRNA methylation,
and efflux pumps
Mutation of the 50S ribosomal subunit,
reduced membrane permeability, and
Phenicols Chloramphenicol Inhibition of protein synthesis
elaboration of chloramphenicol
acetyltransferase
Foods 2022, 11, 1430 19 of 25

Table 6. Cont.

Antibiotics Class Example (s) The Mechanism(s) of Action Resistance Mechanism(s)

Macrolides Erythromycin, azithromycin Alteration of protein synthesis Ribosomal methylation
Tetracyclines Minocycline, tigecycline Alteration of translation Mainly efflux
Rifamycins Rifampin Alteration of transcription Altered β-subunit of RNA polymerase
Alteration of cell
Glycopeptides Vancomycin, teicoplanin Altered cell walls, efflux
wall biosynthesis
Quinolones Ciprofloxacin Alteration of DNA synthesis Efflux, modification, target mutations
Alteration of cell Enzymatic cleavage,
Streptogramins Synercid, streptogramin B
wall biosynthesis modification, efflux
Alteration of formation of 70S Mutations in 23S rRNA genes followed
Oxazolidinones Linezolid
ribosomal complex by gene conversion
Depolarization of Modification of cell wall and
Lipopeptides Daptomycin
cell membrane cell membrane

Worldwide, and under the auspices of the World Health Organization (WHO), lec-
tures and interactive broadcasts are organized to emphasize the importance of judicious
administration of antibiotics in both human and veterinary use, or in agriculture. People
should be aware of the need for antibiotics to be used correctly and to reduce the abuse of
antibiotics. In addition, national programs aimed at the screening of antibiotic residues in
various types of food are being continuously updated.

5. Conclusions
The issue of the presence of antibiotic residues in food is intensely debated. Numerous
research studies have highlighted the irrational use of antibiotics and the risk of problems
with consumer antibiotic resistance, spread by foods with antibiotic residues.
The concentration and type of antibiotic found in the form of residues varies depending
on the geographical area and the type of food analyzed. Available studies present antibiotic
residues in all food groups: meat and meat products, milk and dairy products, eggs, honey,
and non-animal-origin products.
Although alarm signals are drawn regarding irrational antibiotic use, exceeding appli-
cable legal requirements are identified. While the European Union has clearly established
limits for antibiotic residues, in the United States, legislation does not include such values,
with a deadline set for 2023 to draw up these legislative requirements. Additionally, in recent
years, awareness of the irrational use of antibiotics programs have been launched, but still it is
necessary to develop these in order to increase the understanding of producers and consumers
regarding the use of antibiotics. These measures should be implemented worldwide.

Author Contributions: O.M.G., C.D.P., O.D.M. and D.C.P.: Conceptualization, writing—original

draft; E.N.P., N.D. and T.D.—writing, review and editing; E.N.P.: funding acquisition. All authors
have read and agreed to the published version of the manuscript.
Funding: This paper was funded by USAMV Bucharest Internal Research Projects Competition, 2021,
Contract No.2021-003/30.07.2021—Assessment of consumer exposure to antibiotics coming from
animal products in Romania.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Foods 2022, 11, 1430 20 of 25

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