Opportunities and challenges in antimicrobial resistance policy including animal production systems and humans across stakeholders in Argentina: a context and qualitative analysis.

INTRODUCTION: Gaps in antimicrobial resistance (AMR) surveillance and control, including implementation of national action plans (NAPs), are evident internationally. Countries' capacity to translate political commitment into action is crucial to cope with AMR at the human-animal-environment interface. METHODS: We employed a two-stage process to understand opportunities and challenges related to AMR surveillance and control at the human-animal interface in Argentina. First, we compiled the central AMR policies locally and mapped vital stakeholders around the NAP and the national commission against bacterial resistance. Second, we conducted qualitative interviews using a semistructured questionnaire covering stakeholders' understanding and progress towards AMR and NAP. We employed a mixed deductive-inductive approach and used the constant comparative analysis method. We created categories and themes to cluster subthemes and determined crucial relationships among thematic groups. RESULTS: Crucial AMR policy developments have been made since 1969, including gradually banning colistin in food-producing animals. In 2023, a new government decree prioritised AMR following the 2015 NAP launch. Our qualitative analyses identified seven major themes for tackling AMR: (I) Cultural factors and sociopolitical country context hampering AMR progress, (II) Fragmented governance, (III) Antibiotic access and use, (IV) AMR knowledge and awareness throughout stakeholders, (V) AMR surveillance, (VI) NAP efforts and (VII) External drivers. We identified a fragmented structure of the food production chain, poor cross-coordination between stakeholders, limited surveillance and regulation among food-producing animals and geographical disparities over access, diagnosis and treatment. The country is moving to integrate animal and food production into its surveillance system, with most hospitals experienced in monitoring AMR through antimicrobial stewardship programmes. CONCLUSION: AMR accountability should involve underpinning collaboration at different NAP implementation levels and providing adequate resources to safeguard long-term sustainability. Incorporating a multisectoral context-specific approach relying on different One Health domains is crucial to strengthening local AMR surveillance.

National action plans on antimicrobial resistance in Latin America: an analysis via a governance framework.

In 2015, the World Health Assembly adopted a global action plan (GAP) on antimicrobial resistance (AMR). Member states were encouraged to develop their own national action plans (NAPs) in alignment with the GAP. To-date, in systematic assessments of NAPs, the Latin American specific context has not been previously analysed. Here we examined 11 Latin American NAPs published between 2015 and 2021 using content analysis. We focused on two approaches: (1) alignment between the strategic objectives and actions defined in the GAP, and those outlined in the NAPs via a content indicator; and (2) assessment of the NAPs via a governance framework covering 'policy design', 'implementation tools' and 'monitoring and evaluation' areas. We observed a high alignment with the strategic objectives of the GAP; however, the opposite was observed for the corresponding actions. Our results showed that the governance aspects contained within coordination and participation domains were addressed by every Latin American NAP, whereas monitoring and assessment areas, as well as incorporating the environment, would need more attention in subsequent NAPs. Given that AMR is a global health threat and collective efforts across regions are necessary to combat it, our findings can benefit member states by highlighting how to strengthen the AMR strategies in Latin America, while also supporting global policy formulation.

How did antibiotic growth promoters increase growth and feed efficiency in poultry?

It has been hypothesized that reducing the bioenergetic costs of gut inflammation as an explanation for the effect of antibiotic growth promoters (AGPs) on animal efficiency, framing some observations but not explaining the increase in growth rate or the prevention of infectious diseases. The host's ability to adapt to alterations in environmental conditions and to maintain health involves managing all physiological interactions that regulate homeostasis. Thus, metabolic pathways are vital in regulating physiological health as the energetic demands of the host guides most biological functions. Mitochondria are not only the metabolic heart of the cell because of their role in energy metabolism and oxidative phosphorylation, but also a central hub of signal transduction pathways that receive messages about the health and nutritional states of cells and tissues. In response, mitochondria direct cellular and tissue physiological alterations throughout the host. The endosymbiotic theory suggests that mitochondria evolved from prokaryotes, emphasizing the idea that these organelles can be affected by some antibiotics. Indeed, therapeutic levels of several antibiotics can be toxic to mitochondria, but subtherapeutic levels may improve mitochondrial function and defense mechanisms by inducing an adaptive response of the cell, resulting in mitokine production which coordinates an array of adaptive responses of the host to the stressor(s). This adaptive stress response is also observed in several bacteria species, suggesting that this protective mechanism has been preserved during evolution. Concordantly, gut microbiome modulation by subinhibitory concentration of AGPs could be the result of direct stimulation rather than inhibition of determined microbial species. In eukaryotes, these adaptive responses of the mitochondria to internal and external environmental conditions, can promote growth rate of the organism as an evolutionary strategy to overcome potential negative conditions. We hypothesize that direct and indirect subtherapeutic AGP regulation of mitochondria functional output can regulate homeostatic control mechanisms in a manner similar to those involved with disease tolerance.

Phenotype Alterations in the Cecal Ecosystem Involved in the Asymptomatic Intestinal Persistence of Paratyphoid Salmonella in Chickens.

The gastrointestinal ecosystem involves interactions between the host, gut microbiota, and external environment. To colonize the gut of poultry, Salmonella must surmount barriers levied by the intestine including mucosal innate immune responses and microbiota-mediated niche restrictions. Accordingly, comprehending Salmonella intestinal colonization in poultry requires an understanding of how the pathogen interacts with the intestinal ecosystem. In chickens, the paratyphoid Salmonella have evolved the capacity to survive the initial immune response and persist in the avian ceca for months without triggering clinical signs. The persistence of a Salmonella infection in the avian host involves both host defenses and tolerogenic defense strategies. The initial phase of the Salmonella-gut ecosystem interaction is characteristically an innate pro-inflammatory response that controls bacterial invasion. The second phase is initiated by an expansion of the T regulatory cell population in the cecum of Salmonella-infected chickens accompanied by well-defined shifts in the enteric neuro-immunometabolic pathways that changes the local phenotype from pro-inflammatory to an anti-inflammatory environment. Thus, paratyphoid Salmonella in chickens have evolved a unique survival strategy that minimizes the inflammatory response (disease resistance) during the initial infection and then induces an immunometabolic reprogramming in the cecum that alters the host defense to disease tolerance that provides an environment conducive to drive asymptomatic carriage of the bacterial pathogen.

Rats as sources of multidrug-resistant Enterobacteriaceae in animal production environments.

Rattus norvegicus and Rattus rattus are commensal pest rodents, considered reservoirs and vectors of zoonotic pathogens. In livestock farms, the wide use of antimicrobials and their release into the environment lead to high long-term residual concentrations, which may in turn lead to the occurrence of antimicrobial resistance (AMR). Farm environments serve as AMR sources, resulting in the transmission of antimicrobial-resistant bacteria and their AMR genes of livestock origin into wildlife. This study aimed to analyse the profile of enterobacteria carrying AMR determinants in rats captured in livestock farms to determine their potential vectors as for the spread of AMR. To this end, 56 rats (52 R. norvegicus and 4 R. rattus) were live-trapped on 11 farms (pig, dairy, poultry and mixed farms) located in central Argentina, from spring 2016 to autumn 2017. From 50 of the R. norvegicus individuals and three of the R. rattus individuals found in 10 of the farms, we isolated 53 Escherichia coli and five Salmonella strains. Susceptibility to antimicrobials, genotypic profiles, minimal inhibitory concentration of colistin and the presence of mcr-1 and genes encoding extended-spectrum ß-lactamase (ESBL) were determined. Of the 58 isolates not susceptible to different antimicrobial classes, 28 of the E. coli strains and two of the Salmonella strains were defined as multi-drug resistant (MDR). S. Westhampton and S. Newport recovered were not susceptible to ampicillin or all the cephems tested. One of the E. coli obtained showed resistance to colistin and harboured the mcr-1 gene, demonstrated by PCR and conjugation. In two ESBL-producing Salmonella isolated from rats, CTX-M-2 genes were responsible for the observed resistance to third-generation cephalosporins. The MDR E. coli isolates showed several different resistance patterns (23), although some of them were the same in different individuals and different farms, with six resistance patterns, evidencing the dispersion of strains. These findings suggest that rats play a role in the dissemination of AMR determinants between animal, humans and environmental reservoirs.

Use of adjuvant ISA VG 71 to produce neutralizing egg yolk antibodies against bothropic venom.

The use of egg yolk antibodies-IgY technology-represents an alternative to the production of mammalian immunoglobulins and has several advantages regarding animal welfare and lower costs of production. The use of adjuvants to achieve the hyperimmunization of laying hens plays a key role in the success of the production of high levels of the antibodies. In the present work, two different adjuvant systems (Freund's adjuvants and MontanideTM ISA 71 VG) were compared to produce IgY anti-Bothrops alternatus. For the first immunization, formalin-inactivated Salmonella was added to MontanideTM ISA 71 VG to emulate Freund's complete adjuvant which includes a mycobacteria antigen. After eight immunizations, IgY produced by using either adjuvant was able to neutralize the lethal activity of the venom in a mouse model, but differences were found regarding the recognition of components of the venom between the two adjuvants tested. Overall, MontanideTM adjuvant used in this work could be a good alternative choice to produce antibodies capable of neutralizing the lethality of complex antigens. This adjuvant is commercially available and used in the formulation of several poultry vaccines and could be used for the IgY technology instead of traditional immunomodulators such as Freund's adjuvants. Key points • IgY extracts recognized major components of the venom.• Avidity indexes of the IgY extracts increased after the successive immunizations.• IgY obtained by two adjuvant systems neutralized the lethal activity of the venom.

Effects of a blend of chestnut and quebracho tannins on gut health and performance of broiler chickens.

Antimicrobial restrictions prompted the search for cost and biologically effective alternatives to replace antimicrobial growth promoters (AGPs) in food-producing animals. In addition, the efficacy of this alternatives needs to be contrasted in field/commercial trials under different challenge conditions. However only a few studies describing the impact of tannins or others AGP-alternatives in commercial poultry production conditions are actually available. The aim of the present work is to study how the inclusion of a blend of chestnut and quebracho tannins can affect broiler productive performance and health under commercial conditions. Three experiments with different approaches were conducted: (1) a trial comparing the effects of both additives (tannins vs AGP) on different commercial farms at the same time; (2) the follow-up of one farm during an entire productive year; and (3) an experimental trial using a C. perfringens challenge model in broiler chickens. Although productive results from field trials were similar among treatments, evaluations of gut health indicators showed improvements in the tannins treated flocks. Frequency and severity of intestinal gross lesions were reduced in jejunum (42% vs 23%; p<0.05-1.37 vs. 0.73; p<0.01, respectively) and ileum (25% vs. 10%; p<0.0.5-1.05 vs. 0.58; p<0.01) in tannins treated birds. Results from 16S studies, show that cecal microbiota diversity was not differentially affected by AGPs or tannins, but changes in the relative abundance of certain taxa were described, including Lactobacillus and Bifidobacterium groups. Results from experimental C. perfringens necrotic enteritis showed that tannins treated birds had reduced incidence of gross lesions in jejunum (43.75 vs. 74.19%; p<0.01) and ileum (18.75% vs. 45.16%; p<0.05) compared with control. These results suggest that AGPs can be replaced by tannins feed additives, and contribute in the implementation of antimicrobial-free programs in broilers without affecting health or performance.

Geography as non-genetic modulation factor of chicken cecal microbiota.

The gastrointestinal tract of chickens harbors a highly diverse microbiota contributing not only to nutrition, but also to the physiological development of the gastrointestinal tract. Microbiota composition depends on many factors such as the portion of the intestine as well as the diet, age, genotype, or geographical origin of birds. The aim of the present study was to demonstrate the influence of the geographical location over the cecal microbiota from broilers. We used metabarcoding sequencing datasets of the 16S rRNA gene publicly available to compare the composition of the Argentine microbiota against the microbiota of broilers from another seven countries (Germany, Australia, Croatia, Slovenia, United States of America, Hungary, and Malaysia). Geographical location played a dominant role in shaping chicken gut microbiota (Adonis R2 = 0.6325, P = 0.001; Mantel statistic r = 0.1524, P = 4e-04) over any other evaluated factor. The geographical origin particularly affected the relative abundance of the families Bacteroidaceae, Lactobacillaceae, Lachnospiraceae, Ruminococcaceae, and Clostridiaceae. Because of the evident divergence of microbiota among countries we coined the term "local microbiota" as convergent feature that conflates non-genetic factors, in the perspective of human-environmental geography. Local microbiota should be taken into consideration as a native overall threshold value for further appraisals when testing the production performance and performing correlation analysis of gut microbiota modulation against different kind of diet and/or management approaches. In this regard, we described the Argentine poultry cecal microbiota by means of samples both from experimental trials and commercial farms. Likewise, we were able to identify a core microbiota composed of 65 operational taxonomic units assigned to seven phyla and 38 families, with the four most abundant taxa belonging to Bacteroides genus, Rikenellaceae family, Clostridiales order, and Ruminococcaceae family.

Natural Tannin Wood Extracts as a Potential Food Ingredient in the Food Industry.

Wood extracts are one of the most important natural sources of industrially obtained tannins. Their use in the food industry could be one of the biggest (most important) recent innovations in food science as a result of their multiple (many) possible applications. The use of tannin wood extracts (TWEs) as additives directly added in foods or in their packaging meets an ever-increasing consumer demand for innovative approaches to sustainability. The latest research is focusing on new ways to include them directly in food, to take advantage of their specific actions to prevent individual pathological conditions. The present review begins with the biology of TWEs and then explores their chemistry, specific sensorial properties, and current application in food production. Moreover, this review is intended to cover recent studies dealing with the potential use of TWEs as a starting point for novel food ingredients.

Microbiota, Gut Health and Chicken Productivity: What Is the Connection?

Gut microbiota and its relationship to animal health and productivity in commercial broiler chickens has been difficult to establish due to high variability between flocks, which derives from plenty of environmental, nutritional, and host factors that influence the load of commensal and pathogenic microbes surrounding birds during their growth cycle in the farms. Chicken gut microbiota plays a key role in the maintenance of intestinal health through its ability to modulate host physiological functions required to maintain intestinal homeostasis, mainly through competitive exclusion of detrimental microorganisms and pathogens, preventing colonization and therefore decreasing the expense of energy that birds normally invest in keeping the immune system active against these pathogens. Therefore, a "healthy" intestinal microbiota implies energy saving for the host which translates into an improvement in productive performance of the birds. This review compiles information about the main factors that shape the process of gut microbiota acquisition and maturation, their interactions with chicken immune homeostasis, and the outcome of these interactions on intestinal health and productivity.

IgY-based antivenom against Bothrops alternatus: Production and neutralization efficacy.

Antivenom for the treatment of bothropic snakebite is a priority for public health institutions from Latin America. An alternative to the conventional antivenom production is based on the use of egg yolk antibodies - IgY-technology - by immunizing laying hens. In this study, we produced, characterized and assessed the efficacy of IgY-based antivenoms against B. alternatus venom. Immunochemical studies (reactivity, avidity and antigen recognition pattern) as well as antivenom efficacy assays were performed. After the 3rd immunization, levels of specific IgY reached a maximum that was maintained throughout the observation period, while avidity indexes of the extracts increased after the successive immunizations. Furthermore, IgY against B. alternatus recognized protein complexes of the venom with high (>40 kDa), medium (20-40 kDa) and low (<20 kDa) molecular weights. IgY antivenoms obtained after 8 immunizations neutralized 35.65 µg of B. alternatus venom per mg of antivenom, while specific activities values ranged from 0.28 to 0.42. In conclusion, we produced and characterized IgY antivenoms capable of neutralizing the lethal activity of B. alternatus venom at a preclinical level. Thus, IgY-technology may allow the production of effective and affordable antivenoms fulfilling the urgent needs of many countries where conventional manufacture is unable to provide enough availability of antivenoms.

Characterization of Escherichia coli Carrying mcr-1-Plasmids Recovered From Food Animals From Argentina.

In this study, we found mcr-1.1 and mcr-1.5 genes carried by IncI2 plasmids in a subset of Escherichia coli isolates recovered from commercial broiler farms in Argentina. The comparative analysis of the sequences of these plasmids with those described in human clinical isolates suggests that this replicon-type is one of the main mcr-disseminator sources in Argentina.

Phytochemicals as antibiotic alternatives to promote growth and enhance host health.

There are heightened concerns globally on emerging drug-resistant superbugs and the lack of new antibiotics for treating human and animal diseases. For the agricultural industry, there is an urgent need to develop strategies to replace antibiotics for food-producing animals, especially poultry and livestock. The 2nd International Symposium on Alternatives to Antibiotics was held at the World Organization for Animal Health in Paris, France, December 12-15, 2016 to discuss recent scientific developments on strategic antibiotic-free management plans, to evaluate regional differences in policies regarding the reduction of antibiotics in animal agriculture and to develop antibiotic alternatives to combat the global increase in antibiotic resistance. More than 270 participants from academia, government research institutions, regulatory agencies, and private animal industries from >25 different countries came together to discuss recent research and promising novel technologies that could provide alternatives to antibiotics for use in animal health and production; assess challenges associated with their commercialization; and devise actionable strategies to facilitate the development of alternatives to antibiotic growth promoters (AGPs) without hampering animal production. The 3-day meeting consisted of four scientific sessions including vaccines, microbial products, phytochemicals, immune-related products, and innovative drugs, chemicals and enzymes, followed by the last session on regulation and funding. Each session was followed by an expert panel discussion that included industry representatives and session speakers. The session on phytochemicals included talks describing recent research achievements, with examples of successful agricultural use of various phytochemicals as antibiotic alternatives and their mode of action in major agricultural animals (poultry, swine and ruminants). Scientists from industry and academia and government research institutes shared their experience in developing and applying potential antibiotic-alternative phytochemicals commercially to reduce AGPs and to develop a sustainable animal production system in the absence of antibiotics.

Simultaneous Carriage of mcr-1 and Other Antimicrobial Resistance Determinants in Escherichia coli From Poultry.

The use of antimicrobial growth promoters (AGPs) in sub-therapeutic doses for long periods promotes the selection of resistant microorganisms and the subsequent risk of spreading this resistance to the human population and the environment. Global concern about antimicrobial resistance development and transference of resistance genes from animal to human has been rising. The goal of our research was to evaluate the susceptibility pattern to different classes of antimicrobials of colistin-resistant Escherichia coli from poultry production systems that use AGPs, and characterize the resistance determinants associated to transferable platforms. E. coli strains (n = 41) were obtained from fecal samples collected from typical Argentine commercial broiler farms and susceptibility for 23 antimicrobials, relevant for human or veterinary medicine, was determined. Isolates were tested by PCR for the presence of mcr-1, extended spectrum ß-lactamase encoding genes and plasmid-mediated quinolone resistance (PMQR) coding genes. Conjugation and susceptibility patterns of the transconjugant studies were performed. ERIC-PCR and REP-PCR analysis showed a high diversity of the isolates. Resistance to several antimicrobials was determined and all colistin-resistant isolates harbored the mcr-1 gene. CTX-M-2 cefotaximase was the main mechanism responsible for third generation cephalosporins resistance, and PMQR determinants were also identified. In addition, co-transference of the qnrB determinant on the mcr-1-positive transconjugants was corroborated, which suggests that these resistance genes are likely to be located in the same plasmid. In this work a wide range of antimicrobial resistance mechanisms were identified in E. coli strains isolated from the environment of healthy chickens highlighting the risk of antimicrobial abuse/misuse in animals under intensive production systems and its consequences for public health.

Overview of the role of Shiga toxins in porcine edema disease pathogenesis.

Shiga toxin-producing Escherichia coli (STEC) have been implicated as the cause of enterotoxemias, such as hemolytic uremic syndrome in humans and edema disease (ED) of pigs. Stx1 and Stx2 are the most common types found in association with illness, but only Stx2e is associated with disease in the animal host. Porcine edema disease is a serious affection which can lead to dead causing great losses of weaned piglets. Stx2e is the most frequent Stx variant found in porcine feces and is considered the key virulence factor involved in the pathogenesis of porcine edema disease. Stx2e binds with higher affinity to Gb4 receptor than to Gb3 which could be due to amino acid changes in B subunit. Moreover, this subtype also binds to Forssman glycosphingolipids conferring upon Stx2e a unique promiscuous recognition feature. Manifestations of edema disease are caused by systemic effects of Stx2e with no significant morphologic changes in enterocytes. Endothelial cell necrosis in the brain is an early event in the pathogenesis of ED caused by Stx2e-producing STEC strains. Further studies are needed to generate techniques and tools which allow to understand the circulation and ecology of STEC strains in pigs even in resistant animals for diagnostic and epidemiological purposes.

Tannins and Bacitracin Differentially Modulate Gut Microbiota of Broiler Chickens.

Antibiotic growth promoters have been used for decades in poultry farming as a tool to maintain bird health and improve growth performance. Global concern about the recurrent emergence and spreading of antimicrobial resistance is challenging the livestock producers to search for alternatives to feed added antibiotics. The use of phytogenic compounds appears as a feasible option due to their ability to emulate the bioactive properties of antibiotics. However, detailed description about the effects of in-feed antibiotics and alternative natural products on chicken intestinal microbiota is lacking. High-throughput sequencing of 16S rRNA gene was used to study composition of cecal microbiota in broiler chickens supplemented with either bacitracin or a blend of chestnut and quebracho tannins over a 30-day grow-out period. Both tannins and bacitracin had a significant impact on diversity of cecal microbiota. Bacitracin consistently decreased Bifidobacterium while other bacterial groups were affected only at certain times. Tannins-fed chickens showed a drastic decrease in genus Bacteroides while certain members of order Clostridiales mainly belonging to the families Ruminococcaceae and Lachnospiraceae were increased. Different members of these groups have been associated with an improvement of intestinal health and feed efficiency in poultry, suggesting that these bacteria could be associated with productive performance of birds.

Effects of Clostridium perfringens iota toxin in the small intestine of mice.

Iota toxin is a binary toxin solely produced by Clostridium perfringens type E strains, and is structurally related to CDT from C. difficile and CST from C. spiroforme. As type E causes hemorrhagic enteritis in cattle, it is usually assumed that associated diseases are mediated by iota toxin, although evidence in this regard has not been provided. In the present report, iota toxin intestinal effects were evaluated in vivo using a mouse model. Histological damage was observed in ileal loops treated with purified iota toxin after 4 h of incubation. Luminal iota toxin induced fluid accumulation in the small intestine in a dose dependent manner, as determined by the enteropooling and the intestinal loop assays. None of these changes were observed in the large intestine. These results suggest that C. perfringens iota toxin alters intestinal permeability, predominantly by inducing necrosis and degenerative changes in the mucosal epithelium of the small intestine, as well as changes in intestinal motility. The obtained results suggest a central role for iota toxin in the pathogenesis of C. perfringens type E hemorrhagic enteritis, and contribute to remark the importance of clostridial binary toxins in digestive diseases.

Clostridium perfringens epsilon toxin induces permanent neuronal degeneration and behavioral changes.

Clostridium perfringens epsilon toxin (ETX), the most potent toxin produced by this bacteria, plays a key role in the pathogenesis of enterotoxaemia in ruminants, causing brain edema and encephalomalacia. Studies of animals suffering from ETX intoxication describe severe neurological disorders that are thought to be the result of vasogenic brain edemas and indirect neuronal toxicity, killing oligodendrocytes but not astrocytes, microglia, or neurons in vitro. In this study, by means of intravenous and intracerebroventricular delivery of sub-lethal concentrations of ETX, the histological and ultrastructural changes of the brain were studied in rats and mice. Histological analysis showed degenerative changes in neurons from the cortex, hippocampus, striatum and hypothalamus. Ultrastructurally, necrotic neurons and apoptotic cells were observed in these same areas, among axons with accumulation of neurofilaments and demyelination as well as synaptic stripping. Lesions observed in the brain after sub-lethal exposure to ETX, result in permanent behavioral changes in animals surviving ETX exposure, as observed individually in several animals and assessed in the Inclined Plane Test and the Wire Hang Test. Pharmacological studies showed that dexamethasone and reserpine but not ketamine or riluzole were able to reduce the brain lesions and the lethality of ETX. Cytotoxicity was not observed upon neuronal primary cultures in vitro. Therefore, we hypothesize that ETX can affect the brain of animals independently of death, producing changes on neurons or glia as the result of complex interactions, independently of ETX-BBB interactions.