Antibiotic Resistance in the Farming Environment

Abstract

Bacterial resistance to antibiotics is now an extremely important safety and health issue. Much of the research on this phenomenon focuses on its clinical aspects, while current findings confirm that it is only one of a number of potential sources of bacteria and genes responsible for drug resistance. There are many indications that one of the main contributors to this issue is currently agriculture and that this applies virtually worldwide. Increased awareness of issues of rational use of antibiotics in husbandry practice entails increased interest in phenomena related to the spread of antibiotic resistance in the environment, their specifics, and the scale of the existing threat. This work, based on current research, analyzed selected aspects of the use of antibiotics in animal production, the presence of antibiotic-resistant microorganisms in farm animals and in waste from agricultural production, in particular from animal breeding farms, the determinants of antibiotic resistance in farming practices and the potential threats related to emissions and spread of antibiotic resistance factors in the environment, including the possibility of transfer of resistant bacteria and resistance genes to humans.

1. Introduction

Antibiotic resistance in bacteria is currently one of the key challenges in ensuring health security. Recent years have seen both the phenomenon of increasing microbial resistance to commonly used antibiotics and the emergence of resistance to new-generation antibiotics, including those considered last resort pharmaceuticals [1,2]. This problem affects virtually every new group of antibiotics [3].

Agricultural ecosystems are an important area of the spread of antibiotic resistance [4]. Discharges from agricultural practices are now seen as one of the major sources of antibiotic pollution in the environment [5]. Up to 90% of antibiotics used in livestock production ultimately end up in various forms in the environment, resulting in intensified selection for resistant microorganisms [4,6,7]. The specificity of the antibiotics used is reflected in the profile of antibiotic resistance genes detected in the environment [8]. The development of molecular methods, including functional metagenomics and pyrosequencing, has allowed more efficient detection and identification of antibiotic resistance genes also in agricultural ecosystems [9].

The use of antibiotics in animal husbandry and livestock production has been practiced for a long time. As soon as the opportunity arose, breeders took advantage of measures that allowed them not only to avoid losses caused by massive disease in their animals or to intensify livestock production with a relatively small increase in costs but also to improve the welfare of their herds through widespread prevention. Nevertheless, in recent years, there has been an increase in public awareness regarding rational practices to limit the unjustified and excessive use of antibiotics, among other things, thanks to initiatives such as The Alliance for the Responsible Use of Medicines in Agriculture (RUMA) and the Food Industry Initiative on Antimicrobials (UK), which bring together various stakeholders related to agricultural production, food production, environmental issues and legislation in this area, aim to develop standards for the responsible use of antibiotics in animal husbandry, including good husbandry practices in this aspect [10,11]. In individual countries also at the level of the European Union, regulations are being introduced to reduce the use of antibiotics in various areas of farming activity [12]. However, there are still regions of the world where the use of antibiotics in animal production is poorly controlled, and awareness of the phenomenon of antibiotic resistance and the consequences of antibiotic overuse is still too low [13,14]. It should be pointed out that antibiotic resistance is becoming an increasingly dynamic phenomenon; therefore, it is necessary to constantly update the state of knowledge in this area.

This article presents the current state of research in the main aspects related to the issue of antibiotic resistance in the farming environment.

2. Antibiotics in Farming Practices

Antibiotics have been used in farming for decades not only for therapeutic purposes but also for prophylaxis and as growth promoters [10,15,16]. Today, thousands of tons of antibiotics are used every year for cattle, pig, and poultry farming [4,17,18] (

Table 1. Examples of antibiotics used in farming.

Type of Livestock	Antibiotics	References
Swine	Pleuromutilins, tetracyclines, macrolides	[15]
Swine	Penicillin, tetracyclines, cephalosporins, lincosamides, fluoroquinolones, amoxicillin, tulathromycin, tylosin, colistin	[19]
Poultry	Bacitracin, virginiamycin, salinomycin, tilmicosin	[20]
Cattle	β-lactams, aminoglycosides, quinolones, fluoroquinolones, macrolides, tetracyclines, sulfonamides, streptogramins, lincosamides	[20]
Cattle	Penicillins, tetracyclines and macrolides	[15]

).

According to the reports concerning European countries, penicillin, oxytetracycline, chlortetracycline, flavophospholipid, and bacitracin were among the most commonly used [1]. Numerous research results confirmed the fact that a wide range of antibiotics are detected in agricultural ecosystems on different continents, including sulfadiazine, sulfamethazine, sulfamethoxazole, erythromycin, tylosin, tilmicosin, lincomycin, chloramphenicol, florfenicol, chlortetracycline, doxycycline, oxytetracycline, tetracycline, ciprofloxacin, enrofloxacin, norfloxacin, monensin, and trimethoprim [21]. Due to the long history of the use of macrolide and tetracycline antibiotics in agriculture, it is the genes that determine resistance to these antibiotic groups that are detected most often [22,23,24].

Antibiotics are used both for therapeutic purposes for sick animals, as a metaphylactic treatment, when the entire herd is treated with an antibiotic (to both treat sick individuals and limit the spread of the disease), and for prophylactic purposes (e.g., to avoid transmission of an infection occurring in a particular area) [25,26] (Figure 1).

The amounts and types of antibiotics used depend on the stage of breeding and age of the animals [15], for example, they are commonly used during the suckling and post-weaning period [27]. In Belgium, more than 90% of the total antibiotics used in pig farming were used for prophylactic purposes [28]. Currently, the use of antibiotics as growth promoters is banned in many countries, and efforts are being made to limit their use in preventive treatments [13,25]. However, it should be noted that the ban on the use of antibiotics as growth promoters has not significantly affected the overall quantitative level of consumption of these pharmaceuticals [29].

The quantities of antibiotics used in animal production significantly exceed those used for human therapy [1,30,31]. For example, in the Netherlands, each year about a hundred tons of antibiotics are used for animal treatment [32]. Many of the antibiotics used in animal farming are structurally related to pharmaceuticals used in human medicine, which can contribute to the formation of cross-resistance [13].

It should be noted that antibiotics are also used in crop production. The use of antibiotics for crop protection involves up to 0.5% of all antibiotics used. Studies from the early 21st century report on the use of antibiotics on crops and trees in both European countries and the US, as well as Latin American countries and Israel. They address the use of antibiotics such as streptomycin, oxytetracycline, gentamicin, kasugamycin, and oxolinic acid, among others [21]. In recent years, there has been an increase in the use of streptomycin and oxytetracycline in the treatment of citrus plant diseases, which may result in an increase in the prevalence of antibiotic-resistant bacteria and drug resistance determinant genes in plant foods [16].

3. Farmed Animals as Reservoirs of Antibiotic Resistance

Intensive animal husbandry is seen as one factor in the spread of antibiotic resistance in the environment. Livestock is considered an important reservoir of antibiotic-resistant bacteria and genes responsible for drug resistance, while animal husbandry facilities are considered antibiotic resistance hotspots [33,34,35,36,37,38,39]. Livestock farms are also one of the sources of environmental emissions of bacteria showing increased tolerance to antibiotics [29]. It has been observed that the prevalence of multi-resistant Escherichia coli strains and resistance to extended-spectrum β- lactam antibiotics can be higher in animal isolates compared to strains isolated from humans [40].

The occurrence of antibiotic-resistant bacteria has been reported in various types of livestock [26]. The recent data concerning the resistance level and profile in different source animals are presented in

Table 2. Examples of antibiotic resistance of bacteria isolated from livestock.

Type of Livestock	Bacteria under Study	Resistance Level	Antibiotics	Detected Genes	References
Poultry	E. coli	35%	3rd generation cephalosporins		[16]
Poultry and swine	E. coli	29.5–54.7%	ampicillin, sulfonamide, tetracycline		[41]
Poultry	E. coli	57.6%	Ciprofloxacin		[41]
Poultry	E. coli	47.4%	Nalidixic acid		[41]
Poultry	E. coli	>10%	Cefotaxim		[41]
Poultry	E. coli	>50%	Sulfisoxazole, streptomycin		[41]
Poultry	E. coli	>40	Tetracycline, gentamicin		[41]
Poultry	Enterobacteriaceae	60–70%	Trimethoprim/sulfamethoxazol, tetracycline, chloramphenicol, amoxicillin/clavulanic acid	qnrS different tet genes	[42]
Poultry	Different isolates	>50%	Penicillin, ciprofloxacin, rifampicin, kanamycin, streptomycin, cefixime, erythromycin, ampicillin, Tetracyclin		[43]
Sheep	Staphylococcus aureus	23.5%	Methicillin, multi-resistant		[44]
Cattle	E. coli	24.5–30.6%	Ampicillin, streptomycin Sulfonamide, tetracycline		[41]
Cattle and goats	Staphylococcus aureus	17.6%	Methicillin, multi-resistant		[44]
Cattle	E. coli Salmonella sp.	Significant resistance	Azithromycin, tetracycline, erythromycin, oxytetracycline, ertapenem	tetA, tetB ereA	[45]
Cattle	Faecal bacteria	14.2–79.2%	Tetracycline		[46]
Cattle	E. coli			cfrB optrA	[47]
Swine	Enterobacteriaceae	80–90%	Tetracycline, trimethoprim/sulfamethoxazol		[42]
Swine	Campylobacter spp.	35.5–79.6	Nalidixic acid, erythromycin, tetracycline, azitromycine, Ciprofloxacin	tetO ermB	[48]
Swine	E. coli	70%	Tetracycline, streptomycin, florfenicol		[49]
Swine	Enterococcus faecalis	70–100%	Streptomycin, tetracycline		[50]
Swine	Enterococcus faecium	Up to 97%	Ampicillin, oxytetracycline, gentamicin, streptomycin		[50]

.

Zhu et al. [33] demonstrated the presence of 149 unique genes responsible for antibiotic resistance on three large-scale industrial pig farms in China using antibiotics in animal husbandry. Dohmen et al. [51] reported that one of the genes commonly found in livestock is blaCTX-M-1 encoding extended-spectrum β-lactamases (ESBL). The presence of tetracycline resistance genes on pig farms was found both in the animals themselves and in the soil or water at the site [19]. Lau et al. [52] identified as many as 34 new antibiotic resistance genes in soil samples contaminated with antibiotics used in animal production such as sulfamethazine, chlortetracycline, and tylosin.

The results of studies on the relationship between the use of antibiotics in animal husbandry and the prevalence of drug resistance are not entirely clear. On the one hand, the relationship between antibiotic use and the presence of resistant bacteria and antibiotic resistance determinant genes has undoubtedly been demonstrated [35]. The results of Zhu et al. [33] showed that the use of antibiotics in pig production can increase the frequency of drug resistance genes by up to 3–4 orders of magnitude. Monger et al. [26], on the other hand, cited findings also confirming the occurrence of antibiotic resistance in pig farms where antibiotics were not used, suggesting the natural origin of antibiotic resistance. AlSalerno et al. [38] found the surprising fact that antibiotic resistance genes: blaTEM, qnrS, sul2, and tetA were present in the broiler farm, where antibiotics were never used, and their relative abundance was comparable to that observed on typical industrial farms. Also, a study conducted by Faroq et al. [53] on poultry farms in Italy showed the presence of antibiotic resistance genes both at antibiotic-using and non-antibiotic broiler farms.

There is evidence that there may be a number of bacteria in the digestive tract of animals that exhibit natural antibiotic resistance, unrelated to the pressure caused by antibiotic use [54]. Colonization of livestock organisms by antibiotic-resistant bacteria may result not only from their selection in the presence of antibiotics used as feed additives but also from contact with drug-resistant microflora present in the environment, such as during grazing [18]. This could suggest that even abandoning the use of antibiotics in animal husbandry may not be enough to limit the spread of drug resistance in a farming environment. However, the relationship between the composition and structure of the intestinal microflora of cattle, pigs, and poultry and the drug resistance profile of the microorganisms can be observed [45,55].

There are some data suggesting that one of the determinants of the antibiotic resistance phenomenon in the farming environment is the breeding practices used (Figure 2).

Intensive animal husbandry involves the accumulation of a large number of individuals in a relatively small space, which increases the likelihood of the spread of infectious diseases, thus providing an argument for the use of antibiotics for prophylactic purposes. This is of particular importance, especially in large-scale poultry farming [1,35]. Meanwhile, a study conducted in Ecuador confirmed that the phenomenon of bacterial antibiotic resistance was more prevalent in poultry raised in an industrial system compared to domestic conditions (resistance to tetracycline 78% and 34%, respectively, to sulfisoxazole 69% and 20%, respectively, while to trimethoprim/sulfamethoxazole it was 63 and 17%, respectively) [56]. Also, in the case of pigs, herd size, intensity of contact between individuals, access to free space and enclosure, and feeding regime are indicated as factors determining the occurrence and spread of antibiotic resistance. Österberg et al. [57] compared the prevalence of antibiotic-resistant E. coli strains in the digestive tract of slaughtered pigs from organic and conventional farming in Denmark, Sweden, France, and Italy. The authors found that organic farming resulted in significantly lower antibiotic resistance to ampicillin, streptomycin, sulphonamides, trimethoprim, and tetracycline (in France and Italy also to chloramphenicol, ciprofloxacin, nalidixic acid, and gentamicin).

4. Husbandry Wastes as a Reservoir of Antibiotic Resistance and Its Carrier in the Environment

Some of the antibiotics used in animal husbandry end up in livestock waste. The level of metabolization of antibiotics in the body varies widely, ranging from 10 to 90% of the ingested dose, and most likely depends on the age and species of the animal [1]. Hence, the waste contains both the antibiotics themselves and the products of their metabolism. A French study found that solid waste from animal production contained 241 mg/kg d.w. of ciprofloxacin and about 12 mg/kg d.w. of doxycycline. For liquid waste, the concentrations of these antibiotics were 0.006 mg/L and 0.505 mg/L, respectively [5]. Comparing these values with the concentrations of these antibiotics suggested to cause resistance selection (0.064 µg/L for ciprofloxacin and 2 µg/L for doxycycline [58]), it can be concluded that these wastes pose a real threat in terms of pressure toward antibiotic resistance of the inhabiting microflora.

Bacteria isolated from livestock waste are characterized by resistance to numerous antibiotics. The addition of antibiotics to feed for prophylactic or therapeutic purposes causes not only an increase in drug-resistant intestinal microflora but also an increase in the number of drug-resistant bacteria in the manure of these animals [7]. Consequently, the agents responsible for the phenomenon of drug resistance are often detected in farming waste [25,59] (

Table 3. Antibiotic resistance in husbandry wastes.

Type of Wastes	Antibiotic Resistance	Resistance Genes	References
Poultry manure		ermB, sul2, tetA, sul1, and strB	[60]
Swine and cattle manure	Tetracyclines, sulfonamides, aminoglycoside, macrolide antibiotics, beta-lactam antibiotics and chloramphenicol	Tet and sul genes	[61,62]
Poultry litter	Amoxiclav, doxycycline, cefotaxime, levofloxacin, ciprofloxacin, amikacin, meropenem, linezolid, chloramphenicol, cefuroxime, ceftriaxone		[63]

).

Lima et al. [61] identified a number of factors determining the role of manure as a potential hotspot for the spread of antibiotic resistance traits through horizontal gene transfer: the abundance of nutrients, the presence of antibiotic residues that can act as a selection factor, the large number and diversity of microorganisms. The abundance of antibiotic resistance genes in manure can reach 10% relative to the number of 16S rRNA genes [62]. A number of pathogenic bacteria, including Klebsiella pneumoniae and bacteria from genera Campylobacter, Salmonella, Listeria, Coxiella, and Mycobacterium showing antibiotic resistance traits, have been isolated from manure of farm animals [64].

The use of manure sourced from antibiotic-treated animal farms as fertilizer is one of the factors promoting the development of drug resistance in the soil environment [62,63,65,66,67,68,69]. There are studies confirming the presence of tetracycline, sulfonamide, fluoroquinolone, and chloramphenicol resistance genes in manure-fertilized soils [15,19]. Ruuskanen et al. [70], in a study of antibiotic resistance conducted on Finnish cattle and pig farms, found that even when only small amounts of antibiotics were typically used in animal husbandry, soils amended with manure contained elevated amounts of resistance genes towards carbapenem (blaOXA-58), sulfonamide (sul1), and tetracycline (tetM). It was observed that drug-resistant microorganisms may persist in the environment for a relatively long time. The conditioning factors are climate, soil type, frequency of fertilization, and also the type of microflora inhabiting the soil [5].

A separate aspect is the potential for further spread of antibiotic resistance in the environment. An interesting phenomenon is an increase in the abundance of detected antibiotic resistance genes in the soil already some time after manure fertilization, which may indicate the transfer of these genes in the environment [15]. Antibiotics in manure may be an additional selection factor in soil bacteria for antibiotic resistance [59]. Kousar et al. [71] compared the frequency of antibiotic resistance in Pseudomonas aeruginosa strains isolated from the topsoil of a poultry farm where antibiotics were used to raise poultry and bacteria of this species from sites at least 500 m from the nearest poultry farm. They found indicators of antibiotic resistance were present in samples taken from both types of sites. What is interesting, florfenicol-resistant E. coli strains were isolated even from remote, geographically separated farms [46].

There is evidence that the increase in antibiotic resistance genes in the soil environment resulting from fertilization with manure is periodic. It was also observed that the concentration of resistance genes in the soil environment may decrease over time [66]. The results of a study conducted by Muurinen et al. [72] on Finnish dairy cattle and pig farms showed that despite some resistance genes being found in the manure, their abundance in manure-fertilized soils was gradually reduced. A very similar effect, a decrease in antibiotic-resistant bacteria over time, was observed for stored slurry by Baker et al. [73] However, it is worth noting that 163 genes determining antibiotic resistance were also detected in non-fertilized soil, while in manure-fertilized soil, depending on the type of livestock, there were 230–245 genes [74].

It should be noticed that a way to reduce the spread of drug resistance resulting from the use of natural fertilizers can be the use of the composting process. Manure composting promotes the elimination of antibiotic residues at levels as high as 50–99%, with higher temperatures and prolongation of the thermophilic phase having the greatest impact. Unfortunately, some antibiotics such as sulfamethazine, ofloxacin, and ciprofloxacin may not degrade under composting conditions, while their concentration in the final product remains high [21]. There is some evidence to confirm that the composting process can lead to the effective reduction of antibiotic-resistant microorganisms and resistance conditioning genes present in manure [5,75]. Liu et al. [76] pointed out the possibility of reducing the spread of antibiotic resistance genes by composting pig manure under conditions of appropriate temperature and pH. However, they found that the standard composting process did not provide effective control of antibiotic resistance. Even an increase in temperature in the thermophilic phase only resulted in a periodic decrease in the number of resistance-determining genes detected [77]. Moreover, research results are available indicating that during composting, there can be both a decrease, stabilization in the amount, and an increase in the antibiotic resistance in the composted material [67,70]. Wang et al. [67] found, a nine-fold increase in the abundance of antibiotic resistance genes (mainly sul1, sul2, tetQ, and tetX genes) in sheep manure aerobic heap composting. Wang et al. [78] observed that the frequency of antibiotic resistance genes increased 44-fold during heap composting, especially with regard to macrolide antibiotic resistance. On the other hand, during composting under thermophilic conditions, there was a 92% decrease in antibiotic resistance genes, with tetracycline resistance genes reaching as high as 97%. It was observed that the abundance of tetracycline resistance genes could both decrease and increase during the composting process, depending on microflora activity and environmental conditions (C/N, moisture, pH) [79]. A factor affecting the survival of antibiotic-resistant microorganisms in manure and compost and the persistence of antibiotic resistance genes in microbial communities may be the presence of heavy metals and antibiotic residues [80].

5. Determinants of Antibiotic Resistance in Farming

The phenomenon of drug resistance in the farming environment is determined by a number of factors related to both the characteristics of the organisms themselves and farming practices (Figure 3).

The increase in the frequency of antibiotic-resistant bacteria in the environment is the result of phenomena such as horizontal gene transfer, genetic mutations, and recombination [34]. The location of antibiotic resistance genes within mobile genetic elements (plasmids, transposons, integrons) facilitates their dissemination in microbial communities [61,62,81]. Typical mechanisms for spreading drug resistance via horizontal gene transfer include conjugation involving plasmids, transduction caused by bacteriophages, and natural transformation, allowing the bacterium to acquire DNA in extracellular form [82]. The interspecies spreading of genes determining antibiotic resistance includes also the transfer of drug resistance traits to pathogenic species [36,69]. For example, the transfer of drug resistance genes between strains of the Salmonella and Escherichia genera present in the gastrointestinal tract was observed [83]. In mixed microbial communities, β-lactam antibiotic-sensitive bacteria can survive due to their direct proximity to microorganisms that produce enzymes capable of hydrolyzing β-lactams [5]. There are also studies confirming that some avian pathogenic Escherichia coli strains have developed resistance to antibiotics such as ampicillin, amoxicillin, and tetracycline [84]. As reported by Skandalis et al. [16], resistance to cefepime, gentamicin, and chloramphenicol was detected in opportunistic pathogens Pseudomonas corrugata and Pectobacterium carotovorum.

It has also been confirmed that animals living in the wild in the immediate vicinity of livestock facilities show an increased presence of antibiotic-resistant microorganisms, suggesting the possibility of their transmission from livestock to the wild [85]. Potential vector species are indicated, among others, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), extended-spectrum beta-lactamase (ESBL), carbapenemase-producing Enterobacteriaceae, as well as multidrug-resistant Pseudomonas aeruginosa and Clostridium difficile. Importantly, vector strains do not need to be pathogens themselves; instead, it is sufficient that they have the ability to effectively colonize an organism [86].

One of the primary determinants of the development of antibiotic resistance in the farming environment is the fact that antibiotics are used in sub-therapeutic doses [34,87,88]. The presence of subinhibitory concentrations of antibiotics promotes the horizontal transfer of antibiotic resistance genes in the environment [81]. It was found that continuous exposure to low doses of pharmaceuticals creates a stronger selection pressure for antibiotic resistance than short-term use of higher doses [62].

Environmental stress is another factor that promotes the expression of antibiotic resistance genes and the development of multi-resistance [89]. Selective pressure toward antibiotic resistance can result not only from the presence of antibiotics themselves but also from other contaminants such as heavy metals or biocides [4,34,75]. Metal ions such as zinc or copper, used for growth promotion and high feed efficiency of cattle breeding, may contribute to the phenomenon of co-selection of antibiotic resistance genes in the case of bacteria inhabiting the digestive tract of animals [21]. Co-selection of antibiotic resistance and the resistance to other contaminants (heavy metals, pesticides, anti-fouling chemicals) is possible due to the close location of responsible genes in the same mobile genetic element [90]. It is also important to note the cross-resistance phenomenon between antibiotic-resistant microorganisms and those showing resistance to metals and biocides [69]. Interestingly, the occurrence of antibiotic resistance genes in the aquatic environment showed a stronger correlation with vanadium concentration than with antibiotic concentration [91].

One factor influencing the selection of antibiotic-resistant microorganisms may be their interactions with other substances used in agricultural and farming practices. It has been observed that a strain of E. coli subjected to simultaneous exposure to pesticides and ampicillin at sub-inhibitory concentrations also showed cross-resistance to such antibiotics as tetracycline, chloramphenicol, and ciprofloxacin [92]. Kurenbach et al. [93] observed that bacteria belonging to genera Escherichia and Salmonella exposed simultaneously to selected commercial herbicides and antibiotics of different classes showed a significant increase in antibiotic resistance. The presence of herbicides caused an increase in minimum inhibitory concentration (MIC) for some antibiotics, although the opposite effect was observed for other pharmaceuticals. However, there is no doubt that additional environmental factors related to agricultural practice can indirectly influence the formation of antibiotic resistance.

A source of long-term exposure in the agricultural environment to both antibiotics and antibiotic-resistant microorganisms may be the use of reclaimed wastewater for crop irrigation [94,95]. Irrigation of fields with reclaimed wastewater (treated sewage effluent) may be an important factor in the spread of antibiotic resistance genes in the environment [96]. Reclaimed water can contain numerous drug-resistant bacteria and drug resistance genes, including those to aminoglycosides, sulfonamides, tetracycline, β-lactam antibiotics, chloramphenicol, fluoroquinolones, macrolides, rifampicin, and trimethoprim [97]. Fahrenfeld et al. [98] in a study conducted under batch microcosm conditions showed the presence of sulfonamide resistance genes in soil subjected to repeated irrigation with reclaimed water. However, there are also studies that do not support the thesis that the use of irrigation with reclaimed water has an impact on the spread of antibiotic resistance agents [99] or suggest that the possible level of risk associated with this is not high [24].

6. Environmental Emissions and Human Health Risks

Farm workers are an occupational group that, due to the nature of their work, can be particularly exposed to antibiotic-resistant microflora [21]. Gao et al. [39] estimated that inhalation intake of antibiotic-resistant bacteria potentially pathogenic to humans for pig farm workers can be up to three times higher than for hospital employees.

An important role in the spread of antibiotic resistance in the farming environment is played by the air dust route, droplet dust route, direct skin contact, and to some extent the oral route [37,100]. Systematic contact with farm animals can contribute to the spread of drug-resistant microorganisms via direct transmission [25]. Van den Honert et al. [85] noted the fact of the evolution of antibiotic-resistant bacteria and their adaptation to new environments and organisms of new hosts. A strain of methicillin-resistant Staphylococcus aureus isolated in Finland was not only detected in livestock—pigs, cattle, and poultry—in subsequent years but the possibility of its transmission to humans was also found [101].

There are data supporting the possibility of similar and even the same resistant strains of E. coli and Enterococcus spp. in livestock, in samples collected on farms, and in farm workers [51,102,103,104]. Antibiotic-resistant ESBL-producing E. coli strains were found in more than 70% of healthy animal farm workers in Thailand and were also reported in 76.7% of pigs and over 40% of poultry broilers [105]. It was observed that the mcr-1 gene that determines resistance to colistin, one of the important antibiotics in human therapy was present in E. coli isolated from both pigs and farmers [106]. Pig farm workers have also been reported to have elevated antibiotic resistance genes in the nasopharyngeal microbiota [107]. Moreover, some similarities were found between the microbiome of the nasal or nasopharyngeal cavity of pig farmers and microorganisms present in the farm air [108].

Yang et al. [109] studied the occurrence of antibiotic resistance genes in poultry, in the air of poultry housing, and in samples from the nasopharynx of farm workers in China, and found some similarities in the distribution and structure of the environments studied in terms of antibiotic resistance genes. It confirms that the transmission of antibiotic-resistant bacteria from animals to humans can also occur indirectly—as a result of contamination with resistant microorganisms in the air around livestock facilities and in surface water and soil. Navajas-Benito et al. [110] tested for antibiotic resistance strains of E. coli isolated from the air and leachate from the grounds of and around a cattle farm in Spain. The authors found that of the isolated strains, nearly 22% showed resistance traits to antibiotics such as tetracycline, ampicillin, and trimethoprim/sulfamethoxazole, among others. Single strains were resistant to chloramphenicol and gentamicin–tobramycin. Among the antibiotic resistance genes identified were blaTEM-1, tet(A), tet(B), cmlA, flor, sul1, sul2, sul3. About 14% of the strains showed features of multi-resistance. Bhushan et al. [63] isolated antibiotic-resistant strains of E. coli and bacteria of the genera Staphylococcus and Salmonella from the samples related to poultry farming, including litter, exhaust air samples, and nearby surface waters and groundwater.

The role of air as a pathway for the spread of antibiotic-resistant agents in the agricultural work environment has been also pointed out by McEachran et al. [111], He et al. [112], Song et al. [80], and Gao et al. [39]. It was observed that the air microflora on pig farms was characterized by resistance to tetracyclines, lincosamides, and aminoglycosides, with the presence of antibiotic resistance genes detected in potentially pathogenic strains of the genera Clostridium, Streptococcus, and Aerococcus, among others [39]. The inhalation route is one route of exposure of farm workers to potentially pathogenic multi-resistant strains of the genus Staphylococcus [37]. It has been observed that not only direct contact but also the airborne route is responsible for the transmission of β-lactamase-encoding genes from animals to farm workers [51]. For example, the β-lactam resistance gene blaTEM, quinolone resistance gene qepA, and gene blaNDM-1 conferring resistance to carbapenems can be transported through the air [113]. Based on the air contamination assessment and atmospheric dispersion modeling, Bai et al. [37] found the possibility of spreading drug resistance genes from o area poultry and dairy farms by air over a distance of 10 km along the wind direction. Among the detected airborne bacteria containing antibiotic resistance genes, potentially pathogenic microorganisms were found, including those of the genera Staphylococcus, Sphingomonas, and Acinetobacter. It was further found that all isolated strains from poultry farms were characterized by multi-resistance (to more than three antibiotics), while 80% of them carried the methicillin resistance gene (mecA) [37].

It has been confirmed that one pathway for the spread of antibiotic-resistant bacteria may be farm dust [108]. Analysis of the microflora of pig and poultry farm dust for antibiotic resistance factors showed that farm dust had a greater diversity of genes responsible for antibiotic resistance than the intestinal microflora of farm animals [108]. This observation is important because it is the bacteria present in the dust that can easily spread through the air and travel even long distances from the farm site. When bioaerosol concentrations are high, the likelihood of transfer of antibiotic-resistant agents increases, including toward pathogenic microorganisms [39]. Studies have shown the possibility of spreading antibiotic resistance factors along with dispersed dust from large-scale beef cattle feed yards. It was found that the concentration of genes determining tetracycline resistance was significantly higher in dust samples collected downwind of feed yards compared to samples collected upwind [111]. The presence of antibiotic residues in farm dust accumulating in breeding facilities can be an important selection factor for bacteria present in the dust. Hamsher et al. [114] conducted a study of dust samples collected over a 20-year period from the same pig breeding facilities and found residues of antibiotics such as tetracycline, chloramphenicol, sulfamethazine, and tylosin in amounts as high as 12.5 mg/kg of the dust.

Leachates from waste lagoons and manure pits can be a source of contamination of surface and groundwater with resistant bacteria [30]. It has been found that close proximity to pig farms can result in elevated concentrations of both antibiotics and antibiotic resistance factors (bacteria, resistance genes) in groundwater [115]. AlSalah et al. [116] found widespread occurrence of sul1 and sul2 genes in sediments from rivers into which animal farming wastewater was discharged.

It is also worth mentioning an additional pathway for the spread of antibiotic-resistant microorganisms in the farming environment, which is various types of insects-houseflies, stable flies, and cockroaches, serving as vectors of microorganisms, primarily of fecal origin [18]. Studies conducted on cattle farms have shown that flies were vectors for multi-resistant E. coli O157:H7 and ESBL-producing E. coli [117,118].

The pathways for the spread of antibiotic resistance agents in the agricultural work environment, including their transfer to humans are summarized in Figure 4.

7. Antibiotic Resistance in Animal and Plant Products

When analyzing the scale of the risks associated with the spread of antibiotic resistance, their transmission through products of animal and plant origin cannot be ignored. Animal products may be one route for the transmission of antibiotic-resistant bacteria and antibiotic resistance genes to humans [16,35,119]. There are numerous data confirming the presence of antibiotic-resistant bacteria in meat, milk, and dairy products (

Table 4. Antibiotic resistance in animal products.

Type of Product	Resistant Microorganisms	Antibiotic Resistance Profile	References
Sheep milk	Staphylococci	Multi-resistant, tetM, ermB, ermC, and grlA genes	[120]
Pork meat	Enterococcus faecalis, E. faecium E. casseliflavus	80% of the strains resistant to sulfamethoxazole/trimethoprim, over 5% resistant to levofloxacin. A total of 40% of E. faecium strains resistant to quinupristin–dalfopristin. A total of 78% of the isolates were multi-resistant strains. Around 90% containing resistance genes towards tetracycline, aminoglycoside and macrolide antibiotics.	[121]
Poultry meat		A total of 88% of isolates were antibiotic resistant.	[122]
Chicken carcasses	E. coli, Salmonella sp.	Multi-resistant.
Ready-to-cook poultry products	Salmonella sp.	More than 80% strains resistant to at least 5 antibiotics.	[63]
Duck meat	Campylobacter sp.	Antibiotic resistant strains detected.	[123]
Poultry meat	E. coli	Resistance genes detected: tetA (tetracycline), ereA (erythromycin), aac-3-IV (gentamicin), cmlA and catA1 (chloramphenicol) aadA1 (streptomycin).	[124]
Chicken eggs		β-lactams, macrolides, tetracyclines and aminoglycosides.	[125]

).

Procedures used in the food animal industry may contribute to the spread of antibiotic resistance [18]. Friedman [105] cited an Austrian study showing that 20% of ground meat samples contained ESBL-producing bacteria. A study conducted by Amoako et al. [126] in Southern Africa showed that strains of Staphylococcus aureus isolated from samples taken at various stages of poultry meat production—from breeding through transportation, slaughter to sale of finished food products—were characterized by resistance to a number of antibiotics: more than 50% of the isolates were resistant to tetracycline, penicillin, and erythromycin, while at least 30% were resistant to clindamycin, doxycycline, ampicillin, moxifloxacin, amikacin, and trimethoprim/sulfamethoxazole. More than 39% were multi-resistant strains. Antibiotic-resistant bacteria can become a cause of meat contamination during slaughter [105] while slaughterhouses can be considered natural reservoirs of drug-resistant microorganisms and genes that determine resistance to pharmaceuticals [36]. Commensal bacteria found in livestock, carrying antibiotic resistance genes and transferred to food products of animal origin, pose a real health risk to consumers, especially given the possibility of transferring antibiotic resistance genes to potentially pathogenic microorganisms [3]. The results of studies conducted in China, India, and Ethiopia confirm that poultry and beef meat can be a factor in the transmission of drug-resistant microorganisms from livestock to humans [35]. There are known examples of transmission of antibiotic-resistant bacteria, including Staphylococcus aureus, to people working in the meat industry [119].

Similar to animal products, plant products are also not free from contamination by drug-resistant bacteria (

Table 5. Plant products with observed antibiotic resistance.

Type of Plant Product	References
Vegetables and fruits	[1,16,65]
Onion and garlic powders, ginger, goldenseal, mustard and rosemary	[105]
Tomatoes, salad vegetables, apples, blackberries and strawberries	[4]

). Consumption of vegetables in raw form can be a significant factor in exposure to antibiotic-resistant bacteria [127]. One obvious risk factor for contamination of crop products such as vegetables is the use of manure to fertilize fields [26,64], especially if it contains pathogenic microorganisms possessing genes that determine their resistance to commonly used antibiotics [128]. It was found that vegetables grown on manure-fertilized soil showed elevated levels of both antibiotics and antibiotic resistance conditioning genes, with the highest concentrations recorded in the root portion of the plants [129]. It has been observed that antibiotic-resistant bacteria can enter the aboveground parts of the plant through the root system [130]. In this context, soil should be considered one of the important reservoirs of drug resistance determinants in agricultural environments [85]. Interestingly, antibiotic resistance traits have also been found in microorganisms that are plant pathogens. Selection of streptomycin- and oxytetracycline-resistant strains was observed in the plant pathogen Erwinia amylovora [131]. In contrast, streptomycin resistance genes were detected in both E. amylovora and other plant pathogenic bacteria from the species Pseudomonas syringa and Xanthomonas campestris [132].

Drug resistance genes have been detected in soil in apple orchards, among others [133]. It is believed that one pathway for the spread of antibiotic resistance determinants in the environment may be contact between soil bacteria and plant-associated bacteria [97], while the specific occurrence of different antibiotic resistance genes in the soil environment may depend on soil type and crop characteristics [8]. The above clearly suggests that research on the spread of antibiotic resistance in the agricultural environment should be conducted comprehensively, taking into account both animal and plant production.

8. Summary and Conclusions

Resistance of microorganisms to antibiotics, especially new-generation pharmaceuticals, is now a real worldwide problem. Due to the non-decreasing importance of agriculture on a global scale and at the same time the fact of widespread use of antibiotics in farming practice, this area should be subject to special monitoring in terms of both the occurrence and the possibility of emission and spread of antibiotic-resistant microorganisms and genes determining drug resistance. We must be aware that the use of antibiotics in the context of large-scale animal production seems to be a difficult factor to eliminate at the moment, especially in the areas of veterinary therapy and prophylaxis. Therefore, it is becoming of utmost importance to take effective measures to reduce the threat of the spread and emission of antibiotic resistance agents from the farming environment and to develop technologies to effectively eliminate drug resistance agents from animal production waste. At the same time, it is extremely important to raise public awareness of drug resistance in the microflora of the agricultural environment as a consequence of the excessive use of antibiotics in farming practices.

Regardless of the scientific research conducted on antibiotic resistance, attention should be paid to the practical aspects of preventing and limiting the spread of this phenomenon in the farming environment. The simplest solution seems to be to provide livestock with such living conditions that prophylactic use of antibiotics is not necessary. It is also important to prevent external infections by applying biosecurity principles (e.g., avoiding the presence of people outside the staff, appropriate protective clothing, etc.), or vaccination, which will reduce the need for therapeutic use of antibiotics. It is also worth noting the possibility of using prebiotics and probiotics instead of antibiotic growth promoters. This should allow at least a partial reduction in the threat from farm animals, facilities, and products as sources of antibiotic resistance.

Based on the results of works published in recent years, it can be noted that they very often concern particular aspects: the occurrence of resistance to specific antibiotics in farm animals, the occurrence and behavior of the determinants of drug resistance in certain types of environments and living organisms, farming, and veterinary practice. Today, it should be emphasized that, more than ever before, research on the phenomenon of drug resistance in farming environments should be multifaceted and interdisciplinary, taking into account the various determinants of the occurrence of resistance to pharmaceuticals, the most commonly observed drug resistance profiles, the potential routes of spread (including animal and plant products), and the health risks to people employed in agriculture and living in areas used for agricultural purposes (Figure 5).

Due to the extremely dynamic nature of the phenomena taking place, the observations and results of quantitative and qualitative studies conducted a dozen years ago do not fully reflect the current situation and the associated health risks. Continued research in this area should focus on attempts to better understand the determinants of the process of spreading antibiotic resistance in the farming environment, also including the impact of seemingly unrelated factors, including broad environmental stress and environmental pollution.