Abstract
Antibiotics' action, once a ‘magic bullet’, is now hindered by widespread microbial resistance, creating a global antimicrobial resistance (AMR) crisis. A primary driver of AMR is the selective pressure from antimicrobial use. Between 2000 and 2015, antibiotic consumption increased by 65%, reaching 34.8 billion tons, 73% of which was used in animals. In the dairy cattle sector, antibiotics are crucial for treating diseases like mastitis, posing risks to humans, animals and potentially leading to environmental contamination. To address AMR, strategies like selective dry cow therapy, alternative treatments (nanoparticles, phages) and waste management innovations are emerging. However, most solutions are in development, emphasizing the urgent need for further research to tackle AMR in dairy farms.
Keywords: : alternative antimicrobial strategies, antimicrobial resistance, dairy cattle, One Health, resistance dissemination
Plain language summary
Antibiotics are becoming less effective at fighting infections because of antimicrobial resistance (AMR). This phenomenon is mainly caused by the abuse and misuse of antibiotics in both human and veterinary medicine. In the dairy cow industry, the use of antibiotics to treat diseases is a big concern. Ways to tackle this include the promotion of the responsible use of antibiotics, the development of alternative treatments and the discovery of better methods to deal with animal waste. However, much of these are still in development.
Plain language summary
Executive summary.
Antibiotics were once a ‘magic bullet’ against infectious diseases, improving life expectancy.
Widespread use has led to AMR dissemination, creating a global crisis.
Between 2000 and 2015, antibiotic consumption increased by 65%, reaching 34.8 billion tons.
The emergence and dissemination of antibiotic resistance pose a significant threat, emphasizing the urgent need for further research to combat AMR in dairy farms.
The influence of antibiotic usage in farm animals on human health: a One Health perspective
The One Health concept recognizes the interconnectedness of human, animal and environmental health.
Transmission of resistant bacteria, antibiotic residues and resistance genes among species is relevant.
Collaborative efforts are required from human doctors, veterinarians, researchers and the general population.
Antibiotic usage in cattle
Globally, 93,309 tons of antimicrobials were administered to food-producing animals in 2017.
The lowest quantity (42 mg/PCU) was used in cattle, with significant global variation.
In the USA, 41% of antimicrobials sold for cattle are medically relevant.
Dairy cattle diseases & AMR
Antimicrobial use in dairy farms, in both milk-producing animals and young stock, should be carefully evaluated.
Mastitis
Inflammation of the mammary gland, common in dairy cattle, causes a major financial burden.
Pathogens responsible for mastitis include S. aureus, NAS, Streptococcus spp. and E. coli.
Reproductive diseases
Metritis, especially puerperal metritis, is the second most common reason for antimicrobial treatment in dairy cows.
Trueperella pyogenes is a widespread opportunistic pathogen, associated with metritis and mastitis.
Hoof diseases
Lameness impacts animal welfare and is the third most common cause of culling.
Digital dermatitis is associated with bacteria from the Treponema genus.
Bovine Foot Rot is caused by Fusobacterium necrophorum.
Bovine respiratory disease
Mannheimia haemolytica, Histophilus somni, Pasteurella multocida and Mycoplasma bovis are major bacterial species.
Antimicrobials are the primary resort for controlling BRD.
Calf diarrhea
Is a multifactorial disease with various pathogens.
Salmonella enterica and E. coli are major calf diarrhea pathogens associated with antimicrobial use.
AMR spread sources
Resistance spread is associated with biological excreta like milk, urine and feces.
Waste milk
Includes unmarketable milk, repurposed for calf feeding to reduce costs.
Can be a reservoir for ARB and ARG.
European Commission and EFSA recognize the risk of AMR spread through WM.
There are inconsistencies in research on the direct link between WM feeding and AMR development.
Manure
Its misuse or overuse can lead to environmental contamination with antibiotic residues, ARB and ARG.
Antibiotic residues' effects depend on soil characteristics and climate.
Cattle manure is considered a reservoir for ARG with resistomes varying from herd to herd.
Strategies to fight antimicrobial resistance (AMR)
Monitoring & control
Effective global regulations and surveillance programs are imperative to oversee and control antibiotic use.
Selective antimicrobial treatment, guided by culture testing, can reduce unnecessary antibiotic use, particularly in dairy farming contexts.
Innovative alternatives
Nanoparticles, antimicrobial peptides like nisin and phage therapy offer promising avenues to reduce reliance on antibiotics.
Nisin applications show considerable potential for practical implementation on dairy farms.
Further research is essential to validate their safety and effectiveness.
Decontamination & remediation
Milk
Strategies such as utilizing β-lactamases for targeted antibiotic degradation and MIPs for selective residue removal are promising.
Manure
Implementing manure pretreatment methods, with a focus on biochar, holds substantial promise for the elimination of relevant bacteria.
Biochar's unique properties, including adsorption capacity, modification potential, selective ARG removal and synergy with other treatments, make it a compelling solution to reduce the risk of soil contamination with ARB and ARGs.
Before the beginning of the 20th century, several infectious diseases were responsible for high mortality rates, including pneumonia, typhoid fever, tuberculosis, typhus, syphilis and the plague, with humans presenting a life expectancy of around 47 years [1-3]. Fortunately, the world has evolved since then, with the increase in average life expectancy being mainly associated with the development of better hygiene practices and major scientific breakthroughs. The discovery of antibiotics was an important step toward the control of bacterial infections [2,4], however, in response to their use, microorganisms developed several resistance strategies, based on a panoply of mechanisms [5]. Shortly after penicillin's discovery in the 1940s, a penicillinase-producing Escherichia coli strain with the ability to inactivate penicillin was reported, and, only 2 years later, penicillin-resistant Staphylococcus aureus strains were isolated from hospitalized patients. The uprising of resistance continued throughout the years, in tandem with antibiotics use, with colistin resistance mediated by plasmids being reported in 2000, and resistance to ceftriaxone, a third-generation cephalosporin being reported in 2010 [6]. Furthermore, resistance to one of the most recent antibiotic combinations, ceftazidime-avibactam, was reported in 2015, only 1 year after its commercialization [7].
Antimicrobial resistance (AMR) can be defined as intrinsic, when bacteria traits, such as the cell wall of Gram-negative bacteria, render them resistant to antimicrobials. Alternatively, this resistance can derive from genetic mutations or the acquisition of new genetic material through horizontal transfer (transduction, conjugation and transformation), which is defined as acquired resistance [4,5,8,9]. As such, several antimicrobial resistance genes (ARG) can be present in the bacterial genome (reflecting genotypic resistance). When an antibiotic exerts its selective pressure on these genes, it can promote the activation of several mechanisms, including efflux pumps expression, cell wall recycling, porin reduction, target protein alteration and biofilm formation, which participate in bacterial resistance to several compounds [5]. In some cases, resistance ability may be reversible if antibiotic pressure disappears. This occurs due to alterations in membrane permeability, or in the activity of regulators involved in bacterial response to antimicrobials and related stressors. It has been shown that, after being exposed to an environment without antibiotics, drug resistance presented by some bacterial species can be reduced after 480 generations, albeit the rate of loss of resistance may vary [10].
Antibiotic-resistant bacteria pose a major global hazard to human and animal health. In the 2022 Global Research on Antimicrobial Resistance (GRAM) Global Burden Report, infections by resistant bacteria are estimated as being responsible for 4.95 million deaths that occurred in 2019 [11], surpassed only by cardiovascular diseases, responsible for 18.56 million deaths, and cancer, responsible for 10.08 million deaths. Of particular concern are the infections promoted by a group of pathogens classified by the World Health Organization (WHO) as of critical, high, and medium priority regarding antimicrobial resistance [12]. Enterococcus faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. (ESKAPE) are included in this group, due to their high level of resistance to most antibiotics and ability to disseminate resistance determinants between all One Health compartments [8]. As such, these pathogens pose a serious threat to both human and animal health, prompting the need for the development of new antimicrobial strategies.
Despite worldwide recommendations regarding the risk of antibiotic use and misuse, between 2000 and 2015 the global antimicrobial usage (AMU) rose 65%, from 21.1 billion to 34.8 billion tons. According to estimates, by 2030 the global consumption of antibiotics will increase by up to 200%, in comparison with the 42 billion daily doses administered in 2015 [13,14]. Of these, 73% are applied to animals to prevent and treat infections, but also to improve weight gain and productivity [15,16]. Despite antimicrobials use as growth promoters being forbidden in Europe [17], a study by Tiseo et al. [16] predicted an increase in the global consumption of antimicrobials used for animal health of 11.5% by 2030, from 93,309 tones in 2017 to 104,079 tones in 2030. Considering the safeguarding of animal health and the livelihood of billions of people who depend on animals for subsistence, the rise of antimicrobial usage and resistance in animals is a significant concern [18], especially since human infections involving drug-resistant bacteria can be prompted by animal-to-human transmission [19-23].
Dairy farming is mainly responsible for milk production, but male calves born on dairy farms and adult cows at the end of their productive life are also used for meat production. Due to the rapid growth of human populations, and consequently increase in the demand for safe food products of animal-origin, an increase in the use of antimicrobials in production animals can also be expected. This may lead to some concerns regarding the role of dairy farms in antimicrobial resistance dissemination [24].
This review employed a comprehensive methodology to gather and synthesize information on the intricate relationship between antibiotic usage, antimicrobial resistance (AMR), and the challenges faced by dairy farming. The literature consulted for this review was primarily obtained after an extensive search conducted on Scopus to identify relevant articles, research papers, and reviews. Additionally, legitimate scientific sources, such as reports from reputable organisations, were consulted to ensure the inclusion of diverse perspectives and up-to-date information on the subject. The search criteria were broad, encompassing expressions such as antibiotic consumption trends, global patterns of antimicrobial resistance, the impact of antibiotic use in cattle, resistance genes in cattle diseases and strategies to combat AMR in dairy farming. The main keywords used, alone or in combination, were AMR, dairy farms, resistance genes, alternative treatments, manure and milk. This search originated 532 articles which were narrowed down to the 167 referenced here.
This review aimed to explore the burden of AMR and antibiotic usage in animal production, particularly in dairy farms, as well as characterize the potential impact of the use of antibiotics on dairy farms on AMR development and point out the strategies in place or forthcoming to tackle this issue.
The influence of antibiotic usage in farm animals on human health: a One Health perspective
The potential connection between AMU and AMR is a noteworthy concern for both the scientific community and governmental authorities, particularly when evaluated through a One Health perspective. Furthermore, when trying to determine the exact link between antimicrobials usage and the development of resistance by different bacterial species, the number of factors to be studied and the limitations involved, make this issue even more complex. This complexity makes it difficult to consistently establish a clear connection between AMU and AMR.
For instance, Richardson et al. [25] conducted a study involving S. aureus isolates from samples collected in 50 different countries, aiming to explore the dissemination of AMR among various hosts. The ability of this bacterial pathogen to circulate between hosts is not new and can be traced back to the Neolithic period when animals were first domesticated. However, the intensification of livestock farming practices has created more opportunities for pathogens to spread between different hosts. According to Richardson et al. [25], cows may act as the primary animal reservoir for the emergence of S. aureus epidemic clones responsible for human infections. According to these authors, intensive farming directly influences the emergence of AMR in response to the selective pressure associated with the use of antibiotics [25].
Conversely, a collaborative report from multiple agencies, Joint Inter-agency Antimicrobial Consumption and Resistance Analysis (JIACRA) III [26], involving the European Centre for Disease Prevention and Control (ECDC), the European Food Safety Authority (EFSA) and the European Medicines Agency (EMA), did not yield conclusive findings regarding the relationship between AMU and AMR, nor regarding the transmission of resistant bacteria between humans and animals. For instance, this report states that an association between AMU and AMR can be observed in E. coli from both humans and animals regarding nearly all antimicrobial classes; on another end, for Campylobacter jejuni, the same association was only observed in the animal sector, but not in human settings; finally, regarding Salmonella, such association was not evident in neither in humans nor animals. In this report, differences between results were attributed to the typically larger datasets available for E. coli and C. jejuni when compared with Salmonella, and to various limiting factors, such as the inconsistency and variability of available data. The authors of the report concluded that further studies are required for a comprehensive understanding of the association between AMU and AMR and emphasized the importance of taking additional measures to reduce AMU, predicting that such measures could have a positive impact on the emergence of AMR [26].
While a consistent direct link between AMU, AMR and interspecies resistance transmission is not consistently reported, the majority of studies suggest that a comprehensive global strategy offers the greatest potential to effectively tackle AMR. The collective efforts to control and reduce the use of antimicrobials in both human and veterinary medicine could potentially mitigate overall AMR resistance, emphasizing the importance of the One Health approach [25-27].
Antibiotic usage in cattle
Worldwide data on AMU in dairy cattle is sparse and difficult to compile due to the lack of updated databases and the limited number of studies available. The latest study by Tiseo et al. [16] reports data from 2017 regarding 41 countries, stating that the total amount of antimicrobials administered to food-producing animals worldwide in that year was 93,309 tons. According to that study, which specifies antibiotic usage in different species, cattle was the animal production setting in which the lowest quantity of antimicrobials was used (42 mg/population correction unit – PCU), followed by poultry (68 mg/PCU), and swine production (193 mg/PCU) [16].
A study by Van Boeckel et al. [15] showed that there is a high variation between the number of antimicrobials used in different countries, ranging from 8 mg/PCU in Norway to 318 mg/PCU in China. As such, the countries with the higher consumption of antimicrobials in both relative (per PCU) and absolute terms should have a significant role in addressing AMR [15,16]. Nevertheless, several countries and regions, namely the EU and the USA, already monitor the sales of antimicrobials to be used in animals, especially those graded as of critical relevance for human health [28,29].
According to sales statistics reported by the US Food and Drug Administration (FDA), approximately 41% of the antimicrobials sold for administration to cattle correspond to medically relevant antimicrobials, as well as 42% of those used in pigs, 11% in turkeys and 3% in chickens [29]. Nevertheless, from 2016 to 2021, the sales of antibiotics for use in cattle decreased by 32% in the USA, as seen in Table 1. Table 2 displays the antimicrobial sales by class during this time period.
Table 1.
Total sales of antimicrobials for cattle administration in the USA in 2021 – The data presented in this table is adapted from the Summary Report on Antimicrobials Sold or Distributed for use in Food-producing Animals, FDA, 2021.
| Parameter | 2021 total sales (kg) in 2021 | Total sales (%) | Change between 2016 and 2021 (%) |
|---|---|---|---|
| Medically relevant | 2,460,766 | 41% | -32% |
| Non-medically relevant | 3,290,231 | 64% | 4% |
Data taken from [29].
Table 2.
Antimicrobial drugs sold in the USA from 2016 to 2021 for cattle use: domestic sales and distribution data reported by drug class estimated sales.
| Antimicrobial class | 2016 Estimated annual totals (kg) |
2017 Estimated annual totals (kg) |
2018 Estimated annual totals (kg) |
2019 Estimated annual totals (kg) |
2020 Estimated annual totals (kg) |
2021 Estimated annual totals (kg) |
Change 2016–2021 (%) | Change 2020–2021 (%) |
|---|---|---|---|---|---|---|---|---|
| Aminoglycosides | 161,646 | 124,675 | 133,842 | 139,445 | 174,132 | 177,173 | 10% | 2% |
| Amphenicols | † | † | † | † | 47,609 | 50,732 | ‡ | ‡ |
| Cephalosporins | 24,677 | 23,512 | 25,337 | 24,158 | 21,007 | 21,197 | -14% | 1% |
| Fluoroquinolones | † | † | † | 12,560 | 12,446 | 12,086 | ‡ | ‡ |
| Lincosamides | 118,916 | 128,642 | 104,527 | 114,398 | 128,562 | 158,036 | 33% | 23% |
| Macrolides | 194,811 | 274,479 | 274,837 | 286,438 | 247,581 | 303,371 | 56% | 23% |
| Penicillins | 99,935 | 96,936 | 96,591 | 78,887 | 82,008 | 66,347 | -34% | -19% |
| Sulfonamides | 234,955 | 196,902 | 187,603 | 197,486 | 161,220 | 136,147 | -42% | -16% |
| Tetracyclines | 2,840,519 | 1,560,542 | 1,732,416 | 1,741,883 | 1,703,391 | 1,693,680 | -40% | -1% |
Not enough data available – less than three sources.
Not enough data available – less than three sources – unable to determine the corresponding percentage.
The data presented in this table is adapted from the Summary Report on Antimicrobials Sold or Distributed for use in Food-producing Animals, FDA, 2021 [29].
In the EU, the latest report by the European Surveillance of Veterinary Antimicrobial Consumption (ESVAC) [28] presents data on the sales of antimicrobials up to 2021. The total sales in all 31 countries that submitted data for the report corresponded to 84.4 mg/PCU. Between 2020, in which the total sales corresponded to 88.8 mg/PCU, and 2021 a decrease of 4.9% in sales was observed. However, a significant disparity between sales in different countries was reported, ranging from 2.5–296.5 mg/PCU. The most predominant antibiotic class sold was penicillins, accounting for 31.2% of total sales (26.3 mg/PCU). This class, along with tetracyclines (21.8 mg/PCU, 25.8%), and sulphonamides (8.3 mg/PCU, 9.9%), accounted for 66.9% of all antimicrobial sales in 2021. In general, the sales of compounds from different classes varied greatly between countries. This was also true for the antibiotics from category B, established by the Antimicrobial Advice Ad Hoc Expert Group (AMEG). Sales for compounds from this group ranged from 0.01–0.5 mg/PCU for 3rd-generation cephalosporins, 0.01–14.8 mg/PCU for 4th-generation cephalosporins, 0.01–0.69 mg/PCU for fluoroquinolones and other quinolones and 0.01–12.7 mg/PCU, for polymyxins [28]. As seen in Table 3, which reports antimicrobials sold in 2021 for administration to cattle, France and Germany appear as the main consumers of these compounds.
Table 3.
Estimated PCU (in 1000 tones) of antimicrobials sold in 2021 for administration to cattle per EU country.
| Country | Cattle | Total |
|---|---|---|
| Austria | 420 | 945 |
| Belgium | 473 | 1770 |
| Bulgaria | 111 | 391 |
| Croatia | 96 | 331 |
| Cyprus | 29 | 152 |
| Czechia | 290 | 709 |
| Denmark | 378 | 2452 |
| Estonia | 58 | 114 |
| Finland | 203 | 492 |
| France | 2961 | 6758 |
| Germany | 2838 | 8071 |
| Greece | 74 | 1100 |
| Hungary | 191 | 846 |
| Iceland | 19 | 145 |
| Ireland | 1298 | 2196 |
| Italy | 1469 | 3813 |
| Latvia | 85 | 153 |
| Lithuania | 154 | 297 |
| Luxembourg | 42 | 54 |
| Malta | 4 | 15 |
| Netherlands | 1079 | 3092 |
| Norway | 215 | 2197 |
| Poland | 1538 | 4417 |
| Portugal | 212 | 1063 |
| Romania | 733 | 2943 |
| Slovakia | 83 | 230 |
| Slovenia | 100 | 184 |
| Spain | 1009 | 8245 |
| Sweden | 284 | 788 |
| Switzerland | 460 | 810 |
| United Kingdom | 1716 | 7054 |
| Total 31 countries | 18,623 | 61,825 |
| Percentage | 330% | 1100% |
This table was adapted from the report of Sales of veterinary antimicrobial agents in 31 European countries, European Medicines Agency, 2021 [28].
Several independent studies were also performed in developing countries aiming to quantify and understand antibiotic usage in food production in these regions [6,30-33]; however, the information available from farms and statistical data provided is insufficient to allow a comprehensive view of the reality of antimicrobials use in these countries. However, a substantial increase in antimicrobial use in livestock is anticipated, particularly in emerging nations such as Nigeria and Indonesia, where an increase in its use exceeding 200% is projected [30]. The overuse of antibiotics in developing countries can be associated with the increasing pressure to meet the population's demands for safe food in conjunction with the lack of access to veterinarians, easy accessibility to antibiotics, and a low knowledge of the risks associated with AMR [30-33].
Dairy cattle diseases & AMR
When reviewing antimicrobial use in dairy farms, it is of paramount importance to take into consideration not only milk-producing animals and their related illnesses (e.g., mastitis and metritis) but also those that affect young stock. Approximately 11.73% of the total number of antimicrobials used on dairy farms are administrated to calves, to treat frequent diseases such as bovine respiratory disease and diarrhea [34,35].
Mastitis
Mastitis, defined as the inflammation of the mammary gland generally resulting from microbial infection, is the most common disease affecting dairy cattle, being responsible for a major financial burden worldwide [36-41]. The microorganisms most commonly involved in bovine mastitis are S. aureus, as well as other non-aureus staphylococci (NAS), Streptococcus agalactiae, Streptococcus uberis, Streptococcus dysgalactiae and E. coli [37].
Staphylococcus
Regarding staphylococci, S. aureus is a common commensal microorganism with the ability to become an opportunistic pathogen, leading to superficial and invasive infections both in humans and animals, and being able to survive within the mammary gland. On the other hand, NAS are opportunistic invaders, with infections promoted by this group of staphylococci being generally associated with environmental contaminations [37].
Pathogens belonging to this genus express a variety of virulence factors, namely surface proteins responsible for bacteria's capacity to adhere to surfaces, form biofilms, and invade epithelial or immune cells [37].
S. aureus is intrinsically resistant to ciprofloxacin, daptomycin, gentamicin, linezolid, oxacillin and vancomycin. It has been documented that this bacterial species exhibits resistance and diminished susceptibility to quinolones and lincomycin, respectively, linked to the activation of efflux pumps. Additionally, NAS are intrinsically resistant to novobiocin [37].
Streptococcus
Streptococcus is a bacterial genus composed of Gram-positive and catalase-negative cocci, responsible for intramammary infections (IMI) resulting in both clinical and subclinical mastitis [42].
S. uberis became the most frequently reported mastitis pathogen, being mainly associated with the farm environment, with housed cows being at greater risk of developing an S. uberis infection than those at pasture. The same is observed for other environmental streptococci, such as S. dysgalactiae. Together, S. uberis and S. dysgalactiae are the most frequently reported cause of mastitis in Ireland, France, Sweden, and Finland. Formerly a prevalent pathogen, S. agalactiae, an obligate udder pathogen, has been less frequently described due to the continuing improvement of milking management practices, being more common in Portugal and Germany [37,42].
Typically, streptococci have low-level intrinsic resistance to quinolones due to the overexpression of ABC efflux pumps, and to aminoglycosides, due to being facultative anaerobes. These pathogens also present intrinsic resistance to fusidic acid [37].
Enterobacteriaceae: E. coli & Klebsiella pneumoniae
The Enterobacteriaceae family includes beneficial commensal microbiota, opportunistic pathogens and major pathogens. This family is composed by Gram-negative, non-spore-forming, facultative anaerobes that ferment glucose and other sugars and reduce nitrate to nitrite, being catalase positive and oxidase negative (except for Plesiomonas). Members of this family are often referred to as enteric bacteria since the main habitat of most of them is the lower GI tract of humans and animals [43]. From those, E. coli and K. pneumoniae are the species most often associated with mastitis and, therefore, the ones more explored in this review [37].
E. coli is a well-known opportunistic pathogen that can cause IMI in cattle, with a high clinical impact in animals with immunosuppression. Despite K. pneumoniae being a less common mastitis pathogen, it can cause severe clinical mastitis, inducing massive inflammation and necrosis of the mammary gland, and potentially death. Moreover, E. coli and K. pneumoniae are crucial in terms of public health and surveillance, as they can serve as a reservoir for antibiotic resistance determinants [37].
Due to the inability of macrolides, aminocoumarins, or glycopeptides to penetrate the outer membrane of Gram-negative bacteria, Enterobacteriaceae are considered intrinsically resistant to these antimicrobial classes [44]. Still, some exceptions have been reported, including the macrolide azithromycin, which has been found to be effective against E. coli [45].
Reproductive diseases
Due to the trauma of the birth canal, placental detachment, systemic inflammation, metabolic stress, immune suppression and shifts in the uterine microbiota that occur in the early postpartum period, dairy cows are susceptible of developing reproductive tract inflammatory diseases [46].
Metritis, particularly puerperal metritis, is the second most common reason for antimicrobial treatment of dairy cattle [47]. This inflammatory disease has a complex etiology, with several bacteria having already been associated with post-partum infections, such as E. coli, Trueperella pyogenes, Fusobacterium necrophorum and Prevotella melaninogenica. T. pyogenes is also responsible for severe cases of metritis, as well as for mastitis, therefore very relevant in terms of postpartum dairy cows' medicine. Considered one of the most frequent causes of antibiotic-resistant mastitis and metritis, this bacterial species will be our focus in this review [48].
Trueperella pyogenes
T. pyogenes is a Gram-positive, non-motile, β-hemolytic and widespread opportunistic pathogen, frequently present in the mucus layer of upper respiratory, urogenital and GI tracts of livestock. Additionally, this species can act as a primary pathogen following different traumas, being a causative agent of metritis, abortion, mastitis, infertility and pneumonia in dairy herds [48]. The resistance determinants previously reported in this bacterial species are summarized in Table 4.
Table 4.
Summary of resistance genes profiles reported for bacteria associated with diseases in dairy cattle.
| Disease | Bacterial species | Resistance gene | Ref. |
|---|---|---|---|
| Mastitis | Staphylococcus spp. | blaARL, blaZ, mecA, mecC, tet(K), tet(L), tet(38), tet(M), aphA3, aadE, ant(6)-Ia, str, dfrA1aadA1, aadA2, dfrA12-orfX2-aadA2, aadA, erm(A), erm(B), erm(C), erm(T), msr(A), msr(B), ere(A), mph(C), lnu(A), lnu(B) vga(A), vga(C), lsa(E), sal(A), dfr(A), dfr(D), dfr(G), dfr(K) parE and gyrB | [37] |
| Streptococcus spp. | bl2b, blaZ, tet(K), tet(L), tet(M), tet(O), tet(S), aphA-3, aad-6 erm(A), erm(B), mph(B), lnuA, lnuB, lnu(D), lnu(C), cat1, cat2, sul1, sul2 and sul3 | ||
| E. coli | blaTEM-, blaSHV-1, blaSHV-2, blaCTX-M-1, blaCTX-M-2, blaCTX-M-3, blaCTX-M-14, blaCTX-M-55, blaCTX-M-96, blaSHV-12, blaCMY-59, blaNDM-1, blaNDM-5, tet(A), tet(B), tet(C), tet(E), tet(G), rmtB, aac(6′)-Ib-cr, strA, strB, aph(3”)-I/II, aadA, aphA, gyrA, gyrB, sul1, sul2, dfrA1, dfrA5, dfrA7, dfrA12, dfrA15, dfrA16, dfrA17 and mcr-1 | ||
| K. pneumoniae | blaTEM-, blaSHV, blaSHV-2, blaSHV-11, blaSHV-27, blaSHV-28, blaSHV-52, blaSHV-61, blaSHV-83, blaSHV-98, blaSHV-108, blaSHV-148, blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, blaCTX-M-14, blaKPC, blaNDM-1, blaoxa-48, blaNDM-5, tet(A), tet(B), tet(D), aac(6′)-Ib-cr, strA, strB, aph(3")-I/II, oqxAB, sul1, sul2, dfrA1, dfrA5, dfrA7, dfrA12, dfrA15, dfrA16, dfrA17 and mgrB | ||
| Mastitis and Metritis | T. pyogenes | blaP1, tet(W), orfE, ermB, ermX, dfr2a, aadA2, aadA1 and aacC | [48] |
| BRD | M. haemolytica | floR, aph3-Ia, tet(H), strA, strB, erm42, ermF, sul2 and cat2 | [49] |
| P. multocida | ROB-1, floR, tet(H), aph3-Ia, erm42, strA, strB and sul2 | ||
| H. somni | floR, aph3-Ia, tet(H), strA, strB, MLSb, erm42, ermF, sul2 and dfrA14 | ||
| Calf diarrhea | E. coli | blaCTX-M, blaTEM, blaSHV, floR, tetB, tetA, tetD, parC, gyrA, qnrD, qnrS, strA-B, aadAI, and sul2 | [50] |
| Salmonella spp. | blaCMY-2, blaTEM-1, blaSHV-12, dfrA, aadA1, dfrA1-aadA1, aadA2, dfrA1-sat2-aadA1, qnrS and aac(6)-Ib-cr 15 | [51] |
Resistance to β-lactamic antibiotics: β-lactamase bla genes. Resistance to methicillin and oxacillin: mec genes; Resistance to Tetracyclines: tet genes; Resistance to Macrolides or Lincosamides or Streptogramins: erm, ere, Inu, lnu, lsa, MLSb, msr, mph, vga, and sal genes; Resistance to Aminoglycosides and Aminocyclitols: aph, aad, ant, str, dfrA1 and aadA1 genes; Resistance to trimethoprim: dfr genes; Resistance to Quinolones: gyr, par, qnr, and floR genes; Resistance to chloramphenicol: cat genes; Resistance to sulfonamides: sul genes, Resistance to colistin: mgr gene; Resistance to aminoglycoside: orf and str.
Hoof diseases
Lameness has major negative impacts on animal welfare, production and economy of dairy farms, being the third most common cause of culling or premature removal from the herd [52]. Some cases, including digital dermatitis, can be caused by infectious agents, such as bacteria from the Treponema genus, including T. denticola, T. maltophilum, T. medium, T. putidum, T. phagedenis and T. paraluiscuniculi. Additionally, Kot et al. [53] recently isolated Sphingomonas paucimobilis, Ochrobactrum intermedium I, Ochrobactrum intermedium II, Ochrobactrum gallinifaecis and Actinomyces odontolyticus for the first time from tissue, pus, blood and swab samples obtained from the limbs of cattle diagnosed with lameness.
Lameness can also be associated with Bovine Foot Rot (BFR), an infectious disease of the interdigital skin and subcutaneous tissues of beef and dairy cattle. A plethora of factors related with the host, agent, and the environment are linked to the development of BFR, being Fusobacterium necrophorum, Porphyromonas levii and Prevotella intermedia the pathogens most commonly associated with this disease [54].
Fusobacterium necrophorum
Fusobacterium necrophorum is an anaerobic Gram-negative bacterium involved in BFR pathogenesis. Early systemic antimicrobial therapy commonly leads to infection resolution, while delayed treatments may result in infection dissemination into deeper structures, such as bone, synovial structures or ligaments, and are responsible for a worse recovery prognosis [54]. Intrinsic AMR has not been described regarding this pathogen, however, the resistance genes already reported in isolates from this species and responsible for hoof diseases are summarized in Table 4.
Bovine respiratory disease
Bovine respiratory disease (BRD) can cover a range of respiratory illnesses, ranging from acute fatal respiratory disease to chronic respiratory disease, and consists of a multifactorial condition associated with several causative agents, including bacteria and viruses [52]. The main bacterial species responsible for BRD are Mannheimia haemolytica, Histophilus somni, Pasteurella multocida and Mycoplasma bovis.
A key hurdle in addressing this condition lies in the pathogens' capacity to concurrently infect cells within the respiratory tract [55].
Despite vaccination being available, it has shown inconsistent efficacy against M. haemolytica, P. multocida and H. somni infections, making antimicrobials the primary resort for controlling BRD [56]. However, a study from Klima et al. [57] performed with 68 isolates associated with BRD showed that 72% of M. haemolytica and 50% of P. multocida isolates evaluated were resistant to one or more of the antibiotics tested, which included ampicillin, penicillin, gentamicin, oxytetracycline, tilmicosin, tulathromycin, danofloxacin, enrofloxacin, spectinomycin, florfenicol, neomycin and chlortetracycline. Moreover, 30% of the M. haemolytica isolates and 12.5% of the P. multocida isolates tested were resistant to more than seven antimicrobial classes, including aminoglycosides, penicillins, fluoroquinolones, lincosamides, macrolides, pleuromutilins and tetracyclines [52].
From all agents responsible for BRD, in this review, we will focus on M. haemolytica, P. multocida and H. somni. The ARG associated with bacteria responsible for this disease are compiled in Table 4.
Mannheimia haemolytica
M. haemolytica is a Gram-negative species currently classified based on 12 capsular serotypes (A1, A2, A5, A6, A7, A8, A9, A12, A13, A14, A16 and A17), with A1 and A6 being most frequently associated with respiratory disease in cattle [56].
From 2008 to 2017, a decline in susceptibility of M. haemolytica isolates to tilmicosin, tulathromycin, florfenicol, fluoroquinolones and gentamicin was observed [49].
Pasteurella multocida
P. multocida, another Gram-negative bacterium, is currently classified into five capsular groups (A to E) and 16 somatic serotypes (1–16). In cattle, A:3 is the most common serotype isolated from animals displaying BRD [56].
Regarding AMR, P. multocida has shown low susceptibility to florfenicol, spectinomycin, tetracycline, tilmicosin and trimethoprim-sulfamethoxazole [51].
Histophilus somni
H. somni is also a Gram-negative bacterium that affects mainly cattle but also small ruminants, being weaned calves at a higher risk of infection. One of the main problems associated with H. somni infections is that the bacteria from this species have the capacity to colonize the lungs of the host and gain access to the bloodstream, causing systemic diseases including encephalitis, myocarditis and acute septicemia associated with sudden death. Treatment options for H. somni infections include the administration of large-spectrum antibiotics, such as florfenicol [56].
H. somni presents higher resistance to neomycin and sulfadimethoxine and reduced susceptibility to gamithromycin, clindamycin, tylosin, penicillin, spectinomycin and oxytetracycline. Additionally, like M. haemolytica, H. somni shows low susceptibility to tilmicosin, tulathromycin and gentamicin [49].
Calf diarrhea
Similarly to BRD, calf diarrhea is a multifactorial disease associated with several pathogens including viruses, bacteria, and protozoa, with studies reporting an incidence ranging between 5 and 23% in calves from dairy, beef and veal production systems [35,58,59].
Of the several agents implicated in calf diarrhea, the main ones are bovine rotavirus, bovine coronavirus, bovine viral diarrhea virus, Salmonella enterica, E. coli, Clostridium perfringens and Cryptosporidium parvum, along with newly emerging enteric pathogens such as bovine Torovirus and Caliciviruses (bovine Norovirus and Nebovirus) [60]. However, in this review, we will only focus on the most prevalent pathogens associated with calf diarrhea which control requires antimicrobial use: Salmonella enterica and E. coli [35]. Table 4 summarizes the ARG reported for bacterial species associated with this disease.
Salmonella spp.
Salmonella is a Gram-negative, facultative anaerobe and facultative intracellular bacteria. It is a well-known pathogen responsible for gastrointestinal diseases in a wide range of hosts, including humans and cattle. S. enterica serovar Typhimurium and S. enterica serovar Dublin are the most common causative agents of salmonellosis in cattle, with S. Typhimurium being mostly associated with acute diarrheal disease and S. Dublin with systemic disease [59].
In Salmonella, intrinsic antibiotic resistance is mainly associated with its outer membrane, the presence of efflux pumps and the expression of antibiotic-inactivating enzymes, associated with resistance to vancomycin and fluoroquinolones. Moreover, Salmonella‘s ability to form biofilms and persister cells play a critical role in their AMR capacity [61]. Persister cells are a special subpopulation of bacteria that can transiently tolerate antibiotics by presenting a slow or arrested growth, but that have the ability to resume growth after the removal of antibiotic stressors from the bacterial environment [60].
Escherichia coli
E. coli was already briefly described above regarding mastitis. Regarding calf diarrhea, the most common E. coli strains reported are enterotoxigenic strains (ETEC), namely E. coli K99, an ETEC strain that produces the K99 (F5) adhesion antigen, responsible for neonatal diarrhea [50].
Resistance genes
Table 4 summarizes available information regarding antimicrobial resistance genes (ARG) already reported in bacterial isolates responsible for the dairy cattle diseases mentioned above.
AMR spread sources
The possible relationship between antibiotic usage and AMR is a main concern of the scientific community and governmental authorities, especially from a One Health perspective. Despite no direct association between AMU, AMR and interspecies resistance transmission has been consistently reported, most studies support that a combined approach to tackle AMR is the most promising solution. Therefore, the combination of efforts to control and reduce the use of antimicrobials in human and veterinary medicine could potentially decrease the overall AMR resistance, which reinforces the importance of the One Health approach [25,27].
Resistance spread has also been associated with biological excreta such as milk, urine and feces. Thus, these sources can not only contribute to the dissemination of resistant bacteria but also of ARG and antibiotic residues [62].
Taking this into account, in the following topics we will focus on the different ways by which antimicrobial residues, resistant bacteria, and resistance genes can be disseminated between animals, the environment and humans, as well as on possible strategies to control this spread.
Waste milk
Waste milk (WM) comprises all milk produced that is not marketable, which includes colostral milk, milk from cows with clinical mastitis, milk collected during the withholding period after administration of veterinary drugs to the animals, milk with a high somatic cell count, and milk that exceeds the milk quota and cannot be marketed. However, to repurpose WM, farmers often feed it to calves, avoiding the high costs of milk replacers [63-65]. The importance of the different antimicrobials that may be present in WM differs from a public health perspective, and the risk associated with them depends on numerous factors, including the type of drug administered to the animals, dosage, timing of administration relative to milking and route of administration [66].
There are several studies available on bacterial isolates recovered from both raw milk and WM samples [67-72], portraying how milk can be a reservoir for AMR bacteria and ARG [32,66,72-80]. Aminoglycoside resistance genes ant(6)-Ia (conferring resistance to streptomycin), aac(6′)-Ie-aph(2′)-Ia (to gentamicin, tobramycin, amikacin), or aph(3′)-III (to kanamycin, neomycin, amikacin, gentamicin B, paromomycin) were found in NAS from bovine mastitic milk in Switzerland [67]. Additionally, the plasmid-borne gene blaCMY-2 was detected in E. coli isolates from bovine milk in Switzerland, Thailand, South Korea and Lebanon [69,70,81,82]. The blaNDM-1 gene has been detected in E. coli isolated from the milk of cattle with clinical or subclinical mastitis in India, whereas in Pakistan, cows' milk samples have been reported as containing K. pneumoniae carrying both the blaNDM-1 and the blaoxa-48 genes [83,84]. Also, the blaNDM-5 gene has been identified in E. coli and K. pneumoniae from milk samples from Algeria and China [68,85]. Last, the presence of rmtB in E. coli and K. pneumoniae isolates obtained from milk samples in China is one of the very few reports indicating the presence of this gene in bacteria from animal-derived products [68].
The presence of these AMR bacteria in WM indicates that this product can potentially be responsible for the dissemination of these strains to soil, water or calves through feeding. A study from 2013 by Aust et al. [64] on bacterial isolates from calves fed with bulk milk (BM) and raw or pasteurized WM showed that the proportion of resistant E. coli isolates, including isolates resistant to cefotaxime, nalidixic acid and sulfamethoxazole and trimethoprim, was significantly higher in calves fed with raw or pasteurized WM in comparison with calves fed with BM. These authors support that pasteurized WM from cows not treated with antimicrobials could be an acceptable feed option for young calves [64].
Maynou et al. [86] studied the antimicrobial resistance patterns of fecal E. coli and nasal Pasteurella multocida isolates from calves fed either with milk replacer (MR) or WM. These authors reported a higher number of E. coli isolates resistant to enrofloxacin, florfenicol and streptomycin, as well as multidrug-resistant E. coli phenotypes in the feces of calves fed with WM [86]. In further studies, these authors reported that feeding WM to animals increases the prevalence of pathogenic microbiota resistant to antimicrobials [86,87]. Additionally, they concluded that WM produced by cows treated with β-lactam antimicrobials contained drug residues in concentrations high enough for selecting resistant E. coli in the calf gut. Moreover, the presence of florfenicol-resistant E. coli in WM-fed calves to which this antibiotic was never administered, may suggest that the antimicrobial residues present in milk may exert selective pressure to the gut microbiota, leading to the development of bacterial resistance to other antimicrobials. However, the presence of high levels of both phenotypic and genotypic resistance to tetracycline and aminoglycosides in calves independently of feeding regimens impairs the establishment of a direct relation between WM feed and AMR transmission [87].
Following the inconsistent findings on the relation between WM feeding and AMR development, the European Commission (EC) requested EFSA to deliver a scientific opinion on the risk for the development of AMR due to feeding of calves with milk containing residues of antibiotics. In their report, they concluded that the risk of AMR and ARG spread through WM was a real threat [66].
Firth et al. [88] reviewed the subject, concluding that the available studies from 2016 to 2020 were mostly limited to E. coli, which is one of the most common bacteria in dairy farms, pointing to the need of addressing other bacterial species for a more comprehensive evaluation of WM feeding effects.
Another concern regarding feeding with WM is the duration of bacterial shedding. While WM appears to increase the excretion of AMR bacteria by dairy calves, Firth et al. [88] refer that such shedding is frequently temporary and transient, appearing not to pose a long-term threat. Moreover, authors state that, despite changes in the calves’ microbiome following WM feeding being commonly reported in the literature, there are no consistent results available on whether the effect that these changes have on calf health are indeed positive or negative [88].
Another review by Ma et al. [63] points out the positive advantages of WM feeding to the farm economy and to the growth and performance of dairy calves. However, authors also emphasize the risks related with feeding untreated WM to calves, mostly associated with direct bacterial transmission from cow to milk, and also with poor hygiene practices during milking, transportation and milk storage [63].
In general, the existing research on WM remains inconclusive regarding the existence of a direct link between WM use in calf feeding and AMR dissemination. This uncertainty is largely attributed to limited sample sizes and variations in testing methods performed in the different studies; as such, there is a crucial need for globally standardized research strategies for addressing this issue.
Manure
The use of manure as a soil amendment is a common agricultural practice worldwide, not only due to its richness in nutrients and organic matter but also because it is a cost-effective way of disposing of liquid manure (slurry), especially considering that a single dairy cow can produce an average of 54 kg of slurry per day [89,90]. Additionally, manure can enhance crop growth and development by actively cycling chemicals such as phosphorus and nitrogen. However, like other products, if it is misused or overused, it may have deleterious effects, including the accumulation of these compounds in the farm environment. Another possible side effect of manure application to soil is the increased risk of environmental contamination with antibiotic residues, antibiotic-resistant bacteria (ARB) and ARG [62,89]. However, several factors influence the potential risk associated with manure application to soil, such as time and conditions of manure storage, contaminants characteristics, application method, time period of UV exposure after soil application, and weather conditions during and after manure application [91].
For instance, the effect of antibiotic residues and their persistence in the farm environment depends on the soil's physicochemical characteristics, as well as on the climate. In sandy soils, the leaching of antibiotics is higher than in clay or silty soils. Additionally, the antibiotics themselves have different capabilities of penetrating the different soil layers, with sulfamethazine and erythromycin easily reaching the deeper layers and even groundwater, posing an increased risk of further spreading antibiotic residues [89].
Moreover, some antibiotics, such as tetracyclines and quinolones, are able to adhere to soil particles, thus accumulating in the soil, changing its natural microbiome and promoting ARG maintenance. For instance, the full biotransformation of oxytetracycline in cattle manure applied to soil takes up to 150 days; however, it is not clear if these antibiotics are still active after manure storage [89].
Cattle manure is considered a reservoir for ARG, with associated resistomes, which comprise all antibiotic-resistance genes found in a given environment, varying from herd to herd [62]. Moreover, Gram-negative bacteria are very prevalent in manure, which may contribute to increasing the flow of mobile genetic elements like ARG [92]. In dairy cow manure, ARG can coexist extracellularly on plasmids, as well as in transposable elements and bacteriophages. Recent research on ARG associated with E. coli has demonstrated herd-level resistome diversity, sometimes with variations in ARG encoding for resistance to the same antibiotic [93]. According to a sequencing effort of 160 antibiotic-resistant E. coli isolates and assessment of 28 fecal metagenomes, tetracycline resistance appears to be the most prevalent, corresponding to 61% of all detected ARG [94]. Similar results were found by Pereira et al. [95] who reported that the most frequent ARGs in cattle manure are tet, sul and erm. According to a recent study by Buta-Hubeny et al. [92], fresh bovine manure applied as fertilizer is colonized mostly by Actinobacteria (29%), Bacteroidetes (16%) and Proteobacteria (9%), presenting a predominance of genes associated with multidrug resistance, such as cfr (59%), followed by genes encoding for resistance to macrolides, lincosamides and streptogramins (9%), bacitracin (5%), fosmidomycin (5%), aminoglycosides (4%), vancomycin (4%), tetracyclines (4%) and sulfonamides (3%). Despite the high number of resistances found in bacteria from cattle manure, it is important to note that the concentrations of these ARG did not increase significantly after its use as soil fertilizer, nor lead to considerable changes in the structure of the soil resistome, in contrast with was observed for poultry manure, which was found to contribute for an increased diversity of ARGs in soil [92].
Besides antibiotics residues, ARB and ARG can also spread from soil to plants, with studies detecting sul2, ermF, blaPSE and blaOXA-2O genes in plants grown in dairy manure-amended soil [89]. Common soil bacteria can transfer ARG to plants through the root endophytes, being able to survive in these structures. In fact, Solomone et al. [96] reported the transmission of E. coli O157:H7 from manure-amended soil and water irrigation to lettuce, colonizing the vegetable through the root, after which spread to the edible parts.
In conclusion, several studies support that the application of manure containing ARB and ARGs increases the risk of ARB and ARG transmission to the environment. and therefore to humans [62,89,92,93,97-107]. However, due to the manure variability, several alternatives will be discussed later.
Strategies to fight back against AMR
To successfully tackle AMR, a combined approach is of paramount importance, focusing not only on the source of resistance but also on its dissemination routes through the different environments. Therefore, it should focus on three main pillars: controlling antibiotic usage; finding alternatives to conventional antimicrobials; and promoting the decontamination of residues and resistance genes.
Monitoring & control of AMU & AMR
Legislations & programs
In this review, we are going to mainly focus on the legislation and programs aiming to control antibiotic use in place, as well as on the procedures that can be applied in the field to improve the selection of antibiotics to be administered.
The World Health Organization (WHO) [108] paved the road for the fight against AMR with the publication, in 2015, of a Global Action Plan on Antimicrobial Resistance. Through the One Health project, the Food and Agriculture Organization (FAO) and the World Organization for Animal Health (WOAH) worked together with WHO to put into practice pertinent strategies to fight AMR worldwide. To address the global threat that AMR represents, this partnership promotes the development of efforts in the human and animal health settings, as well as in the environmental sector. Strengthening the knowledge and evidence basis through surveillance and research is one of the goals of this action plan [108].
With Directive 2003/99/EC and Decision 2013/652/EU, which has since been reinforced by the Decision (EU) 2020/1729, the EU established the requirement for AMR surveillance in zoonotic (Salmonella and Campylobacter) and indicator bacteria (E. coli) from healthy food-producing animals (cattle, poultry and pigs). Although a harmonized surveillance of veterinary clinical isolates is still not performed in the EU, several European countries, such as the Netherlands, Sweden, Denmark, Germany, France, United Kingdom and Portugal, are surveying AMR in veterinary clinical isolates as part of their AMR National Plans for some years now [37]. International harmonization methodologies for the investigation of veterinary AMR are of paramount importance since there are still major variations in the methods employed by different laboratories aiming at antimicrobial susceptibility testing (disk diffusion, minimal inhibitory concentration determination) and in the standards followed for interpretation (Clinical & Laboratory Standards Institute, CLSI; European Committee on Antimicrobial Susceptibility Testing, EUCAST; Le Comité de l'Antibiogramme de la Société Française de Microbiologie, CA-SFM; Animal Health and Veterinary Laboratories Agency, AHVLA), making the global comparison of results challenging [109].
Since 2017, the EU Joint Action on Antimicrobial Resistance and Healthcare-Associated Infections (EUJAMRAI) [109] published the best practices and policies for the proper implementation of national plans. The European Antimicrobial Resistance Surveillance network in Veterinary Medicine (EARS-Vet), which brings together professionals working in AMR surveillance in animals within the EU in an effort to establish best practices and standardize and harmonize antimicrobial susceptibility testing in the veterinary field, was born out of this project [109]. While a database of this magnitude can take years to implement, user-fed databases, such as resistancebank.org, offer complementary data with known limitations, primarily regarding the willingness of independent users to share their information, as well as the lack of results harmonization [18].
The EU Commission requested the European Medicines Agency (EMA) and the Antimicrobial Advice Ad Hoc Expert Group (AMEG) to provide scientific advice on the impact of antibiotics administration to animals on both public and animal health, and to propose steps to mitigate any potential danger to humans. In order to assist veterinarians in making treatment decisions, EMA has categorized antibiotics according to their risk of resistance development [110].
Moreover, Regulation (EU) 2019/6 on veterinary pharmaceutical goods and Regulation (EU) 2019/4 on medicated feed, established the need to restrict the use of antibiotics to prevent the emergence of new resistant strains. Through these new regulations and the reservation of specific antimicrobials solely for human use, this new legislation seeks to decrease the use of antibiotics in the field of animal health. This circumstance is expected to motivate veterinarians to ask for an antimicrobial susceptibility test before prescribing critical compounds, while also supporting antibiotic selection and maximizing therapeutic effectiveness [37].
However, in low-and-middle-income countries from Asia, Africa and South America, the absence of systematic surveillance systems and the lack of legislation limits the possibility of establishing global actions to control AMR. For instance, in 2016, China implemented a national pilot program to reduce unnecessary antimicrobial use, while in India the action plan for AMU was established in 2012, and is still being implemented [111-113]. However, in the new FAO action plan for 2021–2025, an incentive is predicted for developing countries to enforce the Implementation of AMU control strategies [113,114].
Selective antimicrobial treatment
Besides the current legislation that limits which antibiotics can be used, the identification of pathogens responsible for disease in dairy farm animals and the characterization of their antibiotic susceptibility profile would help reduce antibiotic use in these settings.
Regarding antimicrobial use in dairy farms, most literature points to dry cow therapy and clinical mastitis as the main reasons for antibiotic administration [115,116].
Blanket dry cow therapy (BDCT) consists of a treatment with a long-acting intramammary antibiotic infusion applied to all cows between lactation cycles, intending to treat existing infections and prevent new ones. However, BDCT implies administering antibiotics disregarding the animals' infection status or disease incidence risk during the dry period [115,116]. Due to the detection of genes conferring resistance to β-lactam antibiotics, third-generation cephalosporins and aminoglycosides in animals subjected to dry-off therapy, the European Commission prohibited the prophylactic use of antibiotics, including the routine antibiotic treatment of all quarters from cows at drying-off regardless of their infection status. However, in other parts of the world with no available regulation on this subject, these treatments are still in place, posing a risk for resistance dissemination and consequently a danger to animal and human health [117].
Selective dry cow therapy (SDCT) consists of only applying antibiotic treatment based on the risk of the animals developing an intramammary infection (IMI) during the dry period, as this is an important risk factor for mastitis in the early subsequent lactation. The selection of which cases to treat, instead of indiscriminately administering antibiotics, has largely reduced the use of antibiotics for mastitis treatment [117]. SDCT started being applied in Norden Europe since the 70s with favorable results. For instance, in Norway, the clinical cases of mastitis decreased 73% between 1994 and 2018 [118,119].
However, several studies found a concerning relation between SDCT and an increased risk of developing IMI in the subsequent lactation. Winder et al. [120] reviewed several of these reports, concluding that SDCT was associated with a higher risk of IMI at calving in comparison to BDCT (RR: 1.34, 95% CI: 1.13, 1.16). Still, this systematic review revealed a trend for the combination of teat sealants with SDCT not being associated with an increased risk of IMI. Moreover, this review emphasized the possibility that the variable criteria applied in the selection method may affect the association of SDCT with the risk of IMI [120]. McCubbin et al. [116] also reviewed the implications of SDCT for Dairy Farms, and their conclusion was aligned with the work by Winder et al. [120] regarding the possibility of complementing this treatment with teat sealants.
Nevertheless, a study by Rowe et al. [121] proposed that SDCT program failures may be attributed to insufficient diagnostic screening strategies to detect IMI, to the lack of a teat sealant to protect against new IMI during the dry period, or to both. To overcome the limitation of SDCT, this author suggested applying culture-guided (Cult-SDCT) and algorithm-guided SDCT (Alg-SDCT) programs to increase the selection sensitivity while using teat sealants. While Alg-SDCT allows to compare several criteria and protocols applied in the past, with statistical analysis results being summarized as low or high risk, Cult-SDCT is based on microbiological testing to detect and identify the pathogens responsible for the infection. Through a series of studies, Rowe et al. [121-123] concluded that both Cult-SDCT and Alg-SDCT could still contribute to substantially reduce AMU at dry-off, without negatively affecting IMI risk, or the milk yields.
Additionally, culture testing applied to the direct treatment of clinical mastitis could also contribute to reducing the AMU by identifying the causative agent. While samples processing by a laboratory can take more than 24 h to provide results, commercially available on-farm rapid tests can identify the causal agent quickly, contributing to the establishment of a targeted therapy. The first on-farm culture systems allowed the categorization of samples as presenting Gram-positive and Gram-negative microorganisms or no growth, within 24–32 h, with most of them allowing the identification of Gram-positive bacteria with a sensitivity ranging from 59 to 98%, and a specificity ranging from 48 to 97% [124,125]. More recent systems can presumptively differentiate between bacterial species and/or groups according to the color of the bacterial colonies obtained. According to a review by Tommasoni et al. [124], these chromogenic media can be highly sensible and specific. For instance, CHROMagar™ presents 100% sensibility and 99.8%, specificity for the identification of S. aureus in clinical mastitis milk samples, being also adequate for the identification of S. agalactiae, S. dysgalactiae, S. uberis, E. coli, Klebsiella spp. and Enterobacter spp. in subclinical mastitis. However, the observers' experience in interpreting results is crucial for the success of these systems and for the implementation of the right treatment protocol [124].
When comparing the application of culture testing of samples from animals with clinical mastitis subjected to BDCT, a review by Jong et al. concluded that selective treatment of non-severe clinical mastitis can be adopted to successfully reduce AMU without negatively impacting udder health [117].
Alternatives to antibiotics
Innovative antimicrobial approaches, including antimicrobial peptides, phage therapies and even nanoparticles, have been thoroughly investigated by the scientific community aiming to reduce antibiotic use, with new possible solutions and theories emerging every day. This review will focus on specific reports regarding the use of alternative antimicrobial strategies in dairy farms.
Nanoparticles
With the increasing development of nanotechnology, nanoparticles (NPs) have become a promising asset for several uses, including as targeted drug delivery systems, diagnostic systems, noninvasive imaging technologies and antimicrobial compounds. Their unique physicochemical properties, like resistance, durability, performance and flexibility, paired with action mechanisms completely different from the ones of traditional antibiotics, make them good candidates for substitute therapeutics [126]. Their use has been more explored regarding human medicine, still, there are already a few studies available directed towards animal health [53,126-131]. The applications and action mechanisms of several NPs with potential use in dairy farms are summarized in Table 5.
Table 5.
Summary of Nanoparticles with potential use in dairy farms, focusing on target microorganisms and action mechanisms.
| Nanoparticles | Target microorganisms | Action mechanism | Ref. |
|---|---|---|---|
| Silver (Ag) | P. aeruginosa, V. cholerae, K. pneumoniae, S. aureus, E. faecium, S. epidermidis, and T. pyogenes. | Adhesion to the bacterial cell surface causing membrane injury and affecting transport activity; Penetration of AgNPs inside the microbial cells, where interaction with cellular organelles and biomolecules damages the respective cellular machinery; Trigger an increase in reactive oxygen species inside the microbial cells which in turn cause cell damage; Modulation of cellular signal transduction pathway and finally cause cell death |
[126-128] |
| Copper oxide (CuO) | B. subtilis, S. aureus, E. coli, E. faecalis, E. cloace, S. agalactiae, C. albicans, K. pneumoniae, P. aeruginosa, Propionibacterium acnes, and Salmonella Typhi. | Reduce bacteria attachment to the cell wall; Disrupt the biochemical processes inside bacterial cells |
[126,129] |
| Gold (Au) | Methicillin-resistant S. aureus, S. paucimobilis, O. gallinifaecis, and A. odontolyticus. | Generate holes in the cell wall; Binds to the DNA and inhibits the transcription process |
[53,126] |
| Silver-Copper (AgCu) | S. aureus, S. epidermidis, E. coli, E. faecalis, E. cloacae, S. agalactiae, Candida albicans, S. paucimobilis, O. intermedium I, O. intermedium II, O. gallinifaecis and A. odontolyticus. | Same mechanism as AgNPs and CuONP separately with: • Better dissolution of Ag in the presence of Cu ions due to oxidation in the redox reaction • Production of more antibacterial Cu ions during the same redox reaction • Less favorable binding of Ag ions to medium proteins in the presence of Cu ions |
[53,126,130,131] |
For instance, Gurunathan et al. [128] tested the use of silver nanoparticles (AgNPs) as an alternative therapeutic for the treatment of uterine infections in dairy cattle, by synthesizing AgNPs using apigenin and testing their in vitro inhibitory potential toward Prevotella melaninogenica and T. pyogenes isolates obtained from uterine samples of cows. AgNPs are known for their antimicrobial potential toward drug-resistant bacteria. The silver ions (Ag+) discharged from AgNPs enhance bacterial membrane permeability and produce reactive oxygen species (ROS) that damage cell walls. Besides, the antibacterial activity of AgNPs is also linked to their penetration inside the bacterial cell, followed by the destruction of intracellular structures (ribosomes, mitochondria and vacuoles) and biomolecules (DNA, lipids and proteins) as well as by modulation of intracellular signals transduction pathways [127]. The AgNPs synthesized by Gurunathan et al. [128] exhibited significant antibacterial and anti-biofilm activity against the tested isolates in vitro. However, they still needed to be tested in vivo, since more recent in vitro studies reported that AgNPs present cytotoxic effects toward human cell lines, specifically those with sizes ≤10 nm. Besides, it has also been shown that AgNPs can cross the blood-brain barrier of mice, causing neurotoxicity, and neuronal death. Additionally, the accumulation of AgNPs in several organs of mice/rats was also observed, therefore raising concerns about the safety of the application of these NPs in mammals with therapeutic purposes [127].
Kalinska et al. [129] tested AgNPs, copper NPs (CuNPs), and combined silver and copper NPs (AgCuNPs) using both human and bovine cells, aiming to establish an alternative treatment for mastitis. Authors observed that these NPs, especially in lower concentrations, not only presented a positive antimicrobial effect against Enterococcus faecalis, E. coli, S. aureus, Enterobacter cloacae, S. agalactiae and Candida albicans but also appeared to be safe for human and bovine use since no toxic effects were observed neither toward a bovine mammary epithelial cell line (BME-UV1) or a human mammary epithelial cell line (HMEC) [129]. Furthermore, this research group is aiming to develop a disinfectant to be used in the milking routine of dairy cows. In their latest publication from 2023, they combined NPs with cosmetic substrates (collagen + elastin, glycerin, sorbitol, propylene glycol, d-panthenol, vitamin C, sodium lactate, urea and marigold flower extract) to protect the udder skin. They concluded that the combination of NPs and cosmetic substrates could be effective in preventing mastitis by S. aureus and E. coli, but also that, on their own, propyleneglycol and vitamin C can reduce bacteria presence by 35–50% [130].
Kot et al. [53] also investigated NPs potential as an alternative treatment for digital dermatitis, by studying the properties of AgNPs, CuNPs, gold NPs (AuNPs), platinum NPs (PtNPs), iron NPs (FeNPs) against pathogens isolated from cows suffering from hoof diseases: S. paucimobilis, O. intermedium I, O. intermedium II, O. gallinifaecis and A. odontolyticus. Similarly to the studies referred above, AgNPs, AuNPs and CuNPs exhibited the strongest antibacterial properties, only surpassed by the complex AgCuNPs, while PtNPs and (FeNPs) showed very weak antibacterial activity, even promoting bacterial growth in certain cases.
Based on these studies, it is evident that NPs, particularly AgCuNPs, seem to be a promising and viable alternative to antibiotics. Nevertheless, conducting additional toxicity studies using different cell lines and models is imperative to ensure its safety for a broader application.
Antimicrobial peptides: Nisin
Antimicrobial peptides (AMPs) are short proteins with 5–100 amino acids, produced by all living organisms, from prokaryotes to eukaryotes. These peptides play a vital role in innate immunity against a range of pathogens, including bacteria, both Gram-positive and Gram-negative, viruses, fungi and parasites, and even present anticancer activities [132,133]. They are very promising antimicrobials due to their broad-spectrum activity, higher efficiency against various bacterial strains and lesser tendency for microbial resistance development [132].
Their discovery is attributed to Rene Dubos, associated with the isolation of gramicidin from a soil Bacillus strain in 1939, which protected mice from pneumococcal infection [133]. According to the AMP Database's latest update, there are 3569 peptides currently classified as AMP, most of which are produced by animals, followed by bacteria, plants, fungi and archaea [134].
Of those AMP, one is particularly promising for use in dairy cattle. Nisin, a bacterial peptide with 34 amino acids produced by Lactococcus lactis, was the first bacteriocin approved by the WHO, FAO and FDA for use as a food additive to control microorganisms in several food products [132,134,135].
In 1992, Sears et al. [136] suggested nisin application as an effective compound against mastitis, including it as an active ingredient in a teat-dipping product to prevent mastitis.
In 2007, Cao et al. [135] evaluated the efficacy of a nisin-based formulation for intramammary infusion to be applied in the treatment of clinical mastitis. This study also evaluated the presence of pathogens and nisin residues in milk samples. The authors observed a similar cure rate promoted by nisin and by the control antibiotic treatment (gentamicin), including a bacteriological cure rate of 60.8 and 44.6% for nisin and gentamicin, respectively, and a clinical cure rate of 90.2% for nisin and 91.2% for gentamicin. Moreover, regarding the presence of pathogens in milk samples, more specifically S. agalactiae and S. aureus, nisin was more effective in their elimination than gentamicin (54.5 vs 33.3%). Finally, nisin was absent in milk samples after 12 h of intramammary infusion [135].
Moreover, Bennet et al. [137] reported that nisin can also be effectively applied as a teat-dipping agent to prevent intramammary infections and control mastitis, as its use allowed to control the proliferation of pathogens and reduce bacterial load on teat skin. The authors evaluated the efficacy of bacteriocin-based teat formulas, including bactofencin A, nisin and reuterin, applied alone or in combination. They observed that the combined application of nisin, bactofencin and reuterin produced a higher reduction of staphylococci, streptococci, and total bacteria counts, showing that bacteriocins could be considered a good alternative to be used as a teat disinfectant when compared with the iodine positive control [137].
Besides being effective in inhibiting S. aureus growth, as reported by Cao et al. [135], nisin also has the ability to impair biofilm formation by S. aureus and to reduce the density of established biofilms by methicillin-resistant S. pseudintermedius. Nisin also has a protective action against E. coli infection by enhancing host immunity, namely by downregulating the release of inflammatory factors, therefore exerting anti-inflammatory activity. However, little is known about the anti-inflammatory mechanisms of nisin as well as its in vivo cytotoxicity [133,135,136].
Huang et al. [138] conducted a study using nisin Z, a wild-type variant of nisin, aiming to understand its anti-inflammatory effect on mice and human cell lines. Results showed that nisin Z alleviates inflammation and reduces the release of inflammatory cytokines (IL-6, IL-1β), in both human cell lines and mastitis mice models. Moreover, nisin Z also decreased inflammation by promoting the blood-milk barrier in mastitis mice. Despite these promising results, further studies are required to support the in vivo use of this antimicrobial peptide in bovines [138].
Despite nisin potential, studies revealed that some Streptococcus strains may present a gene cluster encoding for a nisin resistance protein (NSR) and an ABC transporter, NsrFP, which confer resistance to nisin. NSR can degrade nisin by cleaving the peptide bond between MeLan28 in ring E and serine at position 29. After cleavage, the resulting molecule has significantly lower bactericidal efficacy and reduced affinity for cell membranes. However, this antimicrobial peptide can be modified through genetic engineering, aiming to produce molecules resistant to NRS action. For example, by replacing serine 29 and isoleucine 30 with proline and valine, respectively, a nisin derivative is obtained, Nisin PV, with enhanced resistance to proteolytic cleavage by NSR [139-142]. Besides having improved resistance to cleavage, a study by Pérez-Ibarreche et al. [140] comparing the efficacy of nisin A (wild-type) versus nisin PV regarding biofilms inhibition and eradication, also showed that the efficacy of nisin PV far exceeded that of nisin A.
Phage therapy
Bacteriophages are bacterial viruses that attach, invade, and multiply within their hosts, ultimately leading to bacterial lysis [143]. Due to their high specificity, phages are a promising alternative approach to substitute or complement the action of conventional antimicrobials. By acting exclusively in their host cells, they do not affect the host's normal microbiota, therefore preventing bacterial dysbiosis and subsequent infections [143,144]. Moreover, the high discovery rate of new phages and the possibility to combine different phages for a higher action spectrum and a reduced risk of resistance development, also support their use as antibiotic substitutes [145].
Mohamadiam et al. [143] tested two phages from the Podoviridae family, Staphylococcus phage M8 and Staphylococcus phage B4, obtained from dairy farm sewage, with specific lytic activity against S. aureus isolates. Despite Staphylococcus phage M8 showing better antimicrobial results, both phages kept their lytic activity in milk, reducing the S. aureus population in spiked milk by about 3 logs after 8 h of incubation, supporting their future potential application as biological control agents, alone or in combination [143].
However, it is important to refer that the use of phages as therapeutic alternatives has some limitations, such as stability, durability and the onset of immunological host responses, which must be addressed before its wide application in vivo [146].
Decontamination/remediation
Milk
There are limited solutions for the removal of antibiotic residues from milk, however, β-lactamases could be an option for the removal of penicillins from this type of substrate. In fact, a study by Li et al. [147] targeting penicillin G, penicillin V, and ampicillin residues present in milk showed that β-lactamases can effectively degrade penicillins; however, they cannot be used for its removal. Still, for future studies, the application of β-lactamases in conjugation with other targeting techniques may be a valid approach [147].
Moreover, studies focusing on other liquid substrates, such as contaminated water, could serve as a benchmark for assessing the effectiveness of methods aiming at the removal of antibiotics from milk. A study by Saitoh et al. [145] described the use of modified clay minerals, namely organoclay, as a promising alternative sorption method. Authors showed that hydrophobic organic pollutants and polar and ionizable compounds present in water, including penicillin G, nafcillin, cefazolin, cefotaxime and oxacillin, are incorporated into surfactant aggregates formed between the layers of clay minerals [145]. In this study, they tested the sorbent potential of didodecyldimethylammonium bromide (DDAB)-montmorill-onite (MT) organoclay, observing promising results in the removal of β-lactam antibiotics from water and also regarding its eco-friendly degradation [145]. Due to their successful application in water samples, these clay minerals could also be potentially tested in milk samples.
Recently, Hemmati et al. [148] reviewed the use of molecular imprinting polymers (MIPs) for the removal of antibiotics and other residues from milk samples. MIPs consist of synthetic materials containing specific recognition sites complementary to the target molecules. Due to their high selectivity, MIPs possess the advantage of allowing the extraction of specific chemical contaminants, without the interference caused by other constituents present in the milk matrix. Moreover, MIPs can be tailored to detect various chemical pollutants, both organic and inorganic, and to enhance their affinity toward particular chemical components [148].
The potential use of MIPs for the removal of milk residues allows the selective isolation of contaminants, having the advantage of being reusable, thereby mitigating the expenses and ecological consequences associated with the extraction procedure. These molecules have already been used in milk samples to isolate and purify milk proteins, such as casein and whey, and to detect milk allergens. Regarding the subtraction of antibiotic residues, including ampicillin, amoxicillin, oxacillin and penicillin G, they seem to present a higher selective ability of rebinding to ampicillin [148].
Despite MIP's efficiency and specificity, incomplete template removal and challenges in the scale-up production of MIPs have hampered their commercialization. Therefore, more studies are needed to improve the properties of MIPs as sorbents for commercial applications. Despite only being tested for the detection and removal of small molecules so far, the evaluation of their efficacy toward larger ones, such as bacterial toxins, would be an interesting approach for future studies [148].
MIPCs (molecularly imprinted polymer-coated stainless-steel sheets) are another promising alternative for fluoroquinolone detection in complex samples, such as milk. However, at present, the application of these compounds is still limited to the detection, and not to the removal, of fluoroquinolones from milk and other aqueous solutions [148].
Manure
There are an increasing number of available options for manure pretreatment, aiming to mitigate the threat of soil contamination with AMR bacteria and antibiotic residues originating from the application of untreated manure to the soil. Manure storing is the most common and cost-effective treatment applied prior to its application; However, there are several other treatment strategies that can be used for manure treatment, such as chemical treatments, liquid–solid separation and biological processes [91,149].
Chemical treatment through pH modifications can be a suitable alternative. For instance, manure acidification, a common technique in Northern Europe for minimizing ammonia emissions, exhibits a significant impact on mitigating antibiotic resistance as well as reducing E. coli survival. Nonetheless, the associated corrosion and potential toxicity to plants hinders its global use. On the other end, alkaline treatments with products like quicklime (CaO) or hydrated lime (Ca(OH)2) can effectively reduce pathogen concentration by disrupting pathogen cell membranes. However, resulting effluents would require neutralization prior to soil application [91,149].
Another common manure treatment is solid-liquid separation, that promotes manure separation into a liquid and a solid fraction. The specific characteristics of the resulting fractions depend on the type of separation equipment used. Initially, the liquid manure undergoes a separation process, yielding two fractions: a liquid portion with around 3–6% dry matter content, often used as fertilizer and a solid component. This separation process plays a crucial role in removing a substantial portion of tetracycline residues from the liquid fraction, as these antibiotics tend to bind to the solid fraction of the manure. Therefore, reducing the antibiotic load in the liquid fraction represents a critical initial step in manure treatment. In contrast, sulfonamides, owing to their limited absorption in the animal gut and/or reversible metabolization, are excreted in considerable quantities, persisting in the liquid manure, and subsequently entering the environment. The solid fraction can undergo further processing, including composting, drying, pelletization or incineration. Meanwhile, the liquid fraction can be utilized as a fertilizer, although this use may contribute to the dispersion of antibiotic residues, ARG, and zoonotic bacteria into the environment. Both fractions have potential applications in anaerobic digestion (AD) systems for biogas production, which represents one of the most prevalent manure treatment methods in Europe [91,150].
Biological processes constitute a vital aspect of manure treatment and encompass both aerobic and anaerobic techniques. Aerobic methods, exemplified by composting, play a pivotal role in stabilizing organic matter, mitigating odorous emissions and eliminating pathogens present in the manure. Composting, as a specific form of aerobic biological treatment, has been documented to effectively reduce concentrations of antibiotics and antibiotic-resistant pathogens within manure. In contrast, AD is another biological process that not only produces biogas as an energy resource, but also lowers the levels of antibiotic residues, antibiotic-resistant bacteria and antibiotic resistance genes in manure. Achieving effective pathogen inactivation during anaerobic digestion often requires the implementation of a thermophilic operational regime [91,150]. Studies showed that postdigestion composting of manure reduces the presence of ARGs by more than 80%, suggesting that different manure management practices have different efficiencies in removing antibiotics, ARB and ARG [150,151].
Composting and AD are considered the most cost-effective means of processing manure before its spread onto agricultural soils, with both being associated with the reduction of pathogens and also of manure mass and volume, making its handling and transport easier [152].
Despite the initial studies reporting that composting was more effective than anaerobic digestion in reducing antimicrobial residues, recent studies suggest that this removal is not complete. For instance, regarding cattle and dairy manure composting, a study showed that, while sulfamethazine and pirlimycin are almost fully removed by this tgreatment strategy, chlortetracycline removal can range between 71%–84%, tetracycline removal between 66 and 72%, and tylosin removal is almost null [152].
While conventional AD seems to be effective, especially in the removal of β-lactams, it shows poor results regarding sulfonamides, fluoroquinolones and macrolides subtraction, being reported to have highly consistent removal rates near zero [150,152]. Overall, despite both methods being able to partially reduce some antibiotic residues, neither is efficient in eliminating them completely.
Similarly, to what is observed regarding antibiotics' elimination, AD and composting have mixed results regarding the removal of ARB and ARG. Studies point to the better efficiency of composting, especially when associated with higher temperatures. However, its efficiency highly depends on the type of manure and composting duration, with short-term composting of cattle manure having the worst results [150].
A promising solution for eliminating ARB and ARG from cattle manure could be its supplementation with biochar. Biochar results from the combustion of agricultural waste, municipal sludge, or other biomass under oxygen-limited conditions. This compound is especially advantageous when considering that agricultural wastes, such as rice husk, corn straw, and wheat straw, are widely distributed and easily available, producing biochar with a high carbon content [153]. Recently, the interest in biochar has spiked due to its multitude of uses, including the improvement of soil quality [154], removal of emerging contaminants in water [155,156], and mitigation of greenhouse gas emissions [157]. Other advantages of this product include its low density, high stability and strong adsorption capacity [158]. Furthermore, biochar can be modified by acids, bases, and oxidants, in order to improve the physicochemical properties of its surface [159], and it has been demonstrated to decrease the bioavailability of heavy metals and antibiotics via adsorption [160].
According to Jang and Kan [161], the use of biochar can significantly remove ARGs (except for tetO and ermB) and genetic mobile elements such as intI1 from dairy manure. Furthermore, several studies suggest taking advantage of the porosity of biochar and combining it with other compounds to potentiate its ability to remove antibiotic residues and ARG [156,162,163]. For instance, Kang et al. [162] reviewed the efficacy of combining biochar with persulfate-based advanced oxidation process methods (persulfate-based-AOPs), concluding that biochar by itself was efficient in the removal of sulfamethoxazole, acetaminophen and cephalexin, but its combination with persulfate based-AOPs provided more stable results.
A recent study by Li et al. [163] tested an association of biochar with peroxydisulfate for manure composting. This study showed highly promising results, with biochar potentiating the action of peroxydisulfate, resulting in a decrease of most ARG. Additionally, the resulting environment was not favorable to the proliferation of several microorganisms, such as those belonging to the genera Thermopolyspora, Thermobifida and Saccharomonospora, contributing for decreasing the manure bacterial load and consequently ARG presence. The authors concluded that biochar-activated peroxydisulfate effectively reduced the risk of ARG transmission by optimizing the physicochemical characteristics of compost, reducing its moisture, adjusting the pH toward neutrality, and accelerating composting, which indirectly may lead to ARG decrease in manure [163].
Another study by Jauregi et al. [164] tested the amendment of soil with several compounds, including biochar, which was found to be one of the most promising compounds tested. In fact, the removal rate of sul1, sul2, tetA and intl1 genes during composting using 5% of biochar was significantly higher than the one observed in the control group.
These studies support the application of biochar as a promising way of altering the composition of the bacterial community present in cow's manure, and consequently of the associated ARG and resistome profile.
Conclusion
In conclusion, antimicrobial resistance (AMR) control in dairy farms has seen significant advancements in recent years.
The adoption of a One Health approach recognizes the interconnectedness of humans and food-producing animals in the spread of antibiotic-resistant bacteria and genes.
Three fundamental pillars for combating AMR have been elucidated:
-
▪
Monitoring and Control: global regulations, surveillance programs and selective antimicrobial treatment strategies are vital to reduce unnecessary antibiotic use;
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▪
Exploration of Alternative Antimicrobial Solutions: innovative alternatives like nisin and phage therapy offer promise in reducing reliance on traditional antibiotics, with nisin emerging as a particularly favorable option for dairy farms;
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▪
Decontamination and Remediation Strategies: approaches like β-lactamases and molecular imprinting polymers (MIPs) are being explored to target specific antibiotics and remove residues. Biochar incorporation into manure shows the most potential in reducing AMR prevalence, however, it should be noted that it still needs further research to reach its full potential.
Efforts to combat AMR should be based on a global and multisectoral approach, including public education on antibiotic risks, stewardship programs for farmers and veterinarians, collaborations for antibiotic alternatives, and the development of remediation systems to eliminate AMR in dairy farm environments and milk.
In summary, addressing AMR comprehensively requires a multifaceted strategy that spans healthcare, agriculture, research and environmental protection. By integrating these approaches, we can hope to slow the emergence and spread of antibiotic resistance, preserving these critical drugs for future generations.
Future perspective
From a forward-looking perspective for tackling AMR, strengthening global surveillance programs for AMR across both human and animal populations is indispensable. Increased data sharing and collaboration between countries and organizations can provide a comprehensive understanding of resistance patterns and identify emerging threats. Government and regulatory bodies must also persist in implementing and enforcing stringent regulations governing antibiotic use. Concurrently, the promotion of stewardship programs among farmers and veterinarians can foster responsible antibiotic usage and monitoring. Expanding public education campaigns on the responsible use of antibiotics and the associated risks linked to AMR is of paramount importance, since raising awareness among consumers can significantly influence the demand for dairy products sourced from responsible antibiotic use. Encouraging alignment of policies and regulations across sectors and countries also ensures a cohesive and coordinated approach to AMR mitigation, in both human and veterinary medicine.
Research should prioritize the development and validation of alternative antimicrobial therapies like peptides, nanoparticles and phage therapy. Advances in molecular diagnostics and precision medicine present opportunities to tailor antibiotic treatment regimens for individual animals – this personalized approach can minimize the indiscriminate use of broad-spectrum antibiotics, thereby reducing selective pressure for resistance. Encouraging collaboration among researchers from diverse fields, including microbiology, veterinary medicine, agriculture and environmental science, can also lead to innovative, holistic solutions to address AMR. Developing rapid and sensitive methods for monitoring antibiotic residues in dairy farms can further help reduce the risk of human exposure to antibiotics through the food supply. Fostering partnerships between the pharmaceutical industry, research institutions and governments is crucial to expedite the development and commercialization of viable antibiotic alternatives. Global collaboration is a driving force for innovation in this respect, and fostering international collaboration is imperative to comprehensively address AMR on a global scale. Promoting the One Health approach by supporting research and initiatives that recognize the interconnectedness of human, animal and environmental health within the context of AMR is of major relevance.
Funding Statement
This research was funded by CIISA – Centre for Interdisciplinary Research in Animal Health (CIISA), Faculty of Veterinary Medicine, University of Lisbon, Project UIDB/00276/2020, by Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS), LA/P/0059/2020–AL4AnimalS (funded by FCT) and by FCT Ph.D scholarship 2021.07175.BD.
Financial disclosure
This research was funded by CIISA – Centre for Interdisciplinary Research in Animal Health (CIISA), Faculty of Veterinary Medicine, University of Lisbon, Project UIDB/00276/2020, by Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS), LA/P/0059/2020–AL4AnimalS (funded by FCT) and by FCT Ph.D scholarship 2021.07175.BD. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
Competing interests disclosure
The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
Writing disclosure
No writing assistance was utilized in the production of this manuscript.
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