Special Issue "Antimicrobial Resistance and Antibiotic Alternatives in Livestock"

A special issue of Antibiotics (ISSN 2079-6382). This special issue belongs to the section "Antibiotics in Animal Health".

Deadline for manuscript submissions: closed (15 July 2022) | Viewed by 76903

Special Issue Editors

Dr. Laila Ben Said
E-Mail Website
Guest Editor
Institute of Nutraceuticals and Functional Foods (INAF), Laval University, Quebec, QC, Canada
Interests: antibiotics; antibio-resistance; alternatives to antibiotics; extended-spectrum beta-lactamases (ESBL); enterobacteria; pathogenic bacteria; bacteriocins; antimicrobial compounds; probiotics; food microbiology; food safety
Prof. Dr. Ismail Fliss
E-Mail Website
Guest Editor
Institute of Nutrition and Functional Foods, Universite Laval, Quebec, QC, Canada
Interests: lactic acid bacteculture; food microbiology; antimicrobial peptides; bacteriocins; antimicrobial activity; food and animal application
Prof. Dr. Carmen Torres
E-Mail Website
Guest Editor
Área de Bioquímica y Biología Molecular, Departamento de Agricultura y Alimentación, Universidad de La Rioja, Madre de Dios 51, 26006 Logroño, Spain
Interests: antimicrobial resistance; foodborne diseases; molecular microbiology

Special Issue Information

Dear Colleagues,

The discovery of antibiotics was one of the greatest scientific breakthroughs of the 20th century. However, the overuse of antibiotics in human medicine and in livestock production as therapeutic agents or growth promoters has led to the emergence of multidrug-resistant bacteria (MDR). Recently, the conditions for the use of antibiotics in food-producing animals have changed around the world and several countries have banned the use of antibiotics firstly as growth promoters and subsequently as prophylaxis agents. Food-producing animals are a major source of MDR transmission via the food chain. Several bacteria carried by animals can also cause disease in people. Consequently, consumers are now seeking alternatives and demonstrating an increasing preference for meat obtained without antibiotics. Over the years, several effective strategies have been intrdocued to maintain the health of food-producing animals. Among these alternatives, probiotics, prebiotics, bacteriocins and bacteriophages are promising. This Special Issue includes studies regarding the development of antibiotic resistance in livestock as well as those related to alternatives to antibiotics in food animal production and in the control of multidrug-resistant bacteria.

Dr. Laila Ben Said
Prof. Dr. Ismail Fliss
Prof. Dr. Carmen Torres
Guest Editors

Manuscript Submission Information

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Keywords

  • Antimicrobial resistance
  • Multidrug-resistant bacteria
  • Livestock
  • Food-producing animals
  • Alternatives
  • Bacterial infection
  • Bacteriocins
  • Probiotics
  • Prebiotics
  • Bacteriophages

Published Papers (11 papers)

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Research

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Article
Antimicrobial Susceptibility of Enterococcus Isolates from Cattle and Pigs in Portugal: Linezolid Resistance Genes optrA and poxtA
Antibiotics 2022, 11(5), 615; https://doi.org/10.3390/antibiotics11050615 - 03 May 2022
Cited by 1 | Viewed by 1536
Abstract
Enterococci are part of the commensal gut microbiota of mammals, with Enterococcus faecalis and Enterococcus faecium being the most clinically relevant species. This study assesses the prevalence and diversity of enterococcal species in cattle (n = 201) and pig (n = [...] Read more.
Enterococci are part of the commensal gut microbiota of mammals, with Enterococcus faecalis and Enterococcus faecium being the most clinically relevant species. This study assesses the prevalence and diversity of enterococcal species in cattle (n = 201) and pig (n = 249) cecal samples collected in 2017. Antimicrobial susceptibility profiles of E. faecium (n = 48) and E. faecalis (n = 84) were assessed by agar and microdilution methods. Resistance genes were screened through PCR and nine strains were analyzed by Whole Genome Sequencing. A wide range of enterococci species was found colonizing the intestines of pigs and cattle. Overall, the prevalence of resistance to critically important antibiotics was low (except for erythromycin), and no glycopeptide-resistant isolates were identified. Two daptomycin-resistant E. faecalis ST58 and ST93 were found. Linezolid-resistant strains of E. faecalis (n = 3) and E. faecium (n = 1) were detected. Moreover, oxazolidinone resistance determinants optrA (n = 8) and poxtA (n = 2) were found in E. faecalis (ST16, ST58, ST207, ST474, ST1178) and E. faecium (ST22, ST2138). Multiple variants of optrA were found in different genetic contexts, either in the chromosome or plasmids. We highlight the importance of animals as reservoirs of resistance genes to critically important antibiotics. Full article
(This article belongs to the Special Issue Antimicrobial Resistance and Antibiotic Alternatives in Livestock)
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Article
Antimicrobial Resistance in Escherichia coli from the Broiler Farm Environment, with Detection of SHV-12-Producing Isolates
Antibiotics 2022, 11(4), 444; https://doi.org/10.3390/antibiotics11040444 - 25 Mar 2022
Cited by 5 | Viewed by 1508
Abstract
Antimicrobial resistance is an important One Health challenge that encompasses the human, animal, and environmental fields. A total of 111 Escherichia coli isolates previously recovered from manure (n = 57) and indoor air (n = 54) samples from a broiler farm [...] Read more.
Antimicrobial resistance is an important One Health challenge that encompasses the human, animal, and environmental fields. A total of 111 Escherichia coli isolates previously recovered from manure (n = 57) and indoor air (n = 54) samples from a broiler farm were analyzed to determine their phenotypes and genotypes of antimicrobial resistance and integron characterization; in addition, plasmid replicon analysis and molecular typing were performed in extended-spectrum-beta-lactamase (ESBL) producer isolates. A multidrug-resistance phenotype was detected in 46.8% of the isolates, and the highest rates of resistance were found for ampicillin, trimethoprim–sulfamethoxazole, and tetracycline (>40%); moreover, 15 isolates (13.5%) showed susceptibility to all tested antibiotics. None of the isolates showed imipenem and/or cefoxitin resistance. Twenty-three of the one hundred and eleven E. coli isolates (20.7%) were ESBL producers and carried the blaSHV-12 gene; one of these isolates was recovered from the air, and the remaining 22 were from manure samples. Most of ESBL-positive isolates carried the cmlA (n = 23), tet(A) (n = 19), and aac(6′)-Ib-cr (n = 11) genes. The following genetic lineages were identified among the ESBL-producing isolates (sequence type-phylogroup-clonotype): ST770-E-CH116–552 (n = 12), ST117-B2-CH45–97 (n = 4), ST68-E-CH26–382/49 (n = 3), ST68-E-CH26–49 (n = 1), and ST10992-A/B1-CH11–23/41/580 (n = 4); the latter two were detected for the first time in the poultry sector. At least two plasmid replicon types were detected in the ESBL-producing E. coli isolates, with IncF, IncF1B, IncK, and IncHI1 being the most frequently found. The following antimicrobial resistance genes were identified among the non-ESBL-producing isolates (number of isolates): blaTEM (58), aac(6′)-Ib-cr (6), qnrS (2), aac(3)-II (2), cmlA (6), tet(A)/tet(B) (22), and sul1/2/3 (51). Four different gene-cassette arrays were detected in the variable region of class 1 (dfrA1-aadA1, dfrA12-aadA2, and dfrA12-orf-aadA2-cmlA) and class 2 integrons (sat2-aadA1-orfX). This work reveals the worrying presence of antimicrobial-resistant E. coli in the broiler farm environment, with ESBL-producing isolates of SHV-12 type being extensively disseminated. Full article
(This article belongs to the Special Issue Antimicrobial Resistance and Antibiotic Alternatives in Livestock)
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Article
Susceptibility to Nisin, Bactofencin, Pediocin and Reuterin of Multidrug Resistant Staphylococcus aureus, Streptococcus dysgalactiae and Streptococcus uberis Causing Bovine Mastitis
Antibiotics 2021, 10(11), 1418; https://doi.org/10.3390/antibiotics10111418 - 19 Nov 2021
Cited by 4 | Viewed by 1489
Abstract
Antibiotics are the most effective strategy to prevent and treat intramammary infections. However, their misuse has led to the dissemination of multidrug resistant bacteria (MDR) for both animals and humans. Efforts to develop new alternative strategies to control bacterial infections related to MDR [...] Read more.
Antibiotics are the most effective strategy to prevent and treat intramammary infections. However, their misuse has led to the dissemination of multidrug resistant bacteria (MDR) for both animals and humans. Efforts to develop new alternative strategies to control bacterial infections related to MDR are continuously on the rise. The objective of this study was to evaluate the antimicrobial activity of different bacteriocins and reuterin against MDR Staphylococcus and Streptococcus clinical isolates involved in bovine mastitis. A bacterial collection including S. aureus (n = 19), S. dysgalactiae (n = 17) and S. uberis (n = 19) was assembled for this study. Antibiotic resistance profiles were determined by the disk diffusion method. In addition, sensitivity to bacteriocins and reuterin was evaluated by determining minimum inhibitory concentrations (MIC). A total of 21 strains (37.5%) were MDR. MICs ranged from ≤1.0 μg/mL to ≥100 μg/mL for nisin and 2.0 to ≥250 μg/mL for bactofencin. Reuterin was active against all tested bacteria, and MICs vary between 70 and 560 μg/mL. Interestingly, 20 MDR strains were inhibited by bactofencin at a concentration of ≤250 μg/mL, while 14 were inhibited by nisin at an MIC of ≤100 μg/mL. Pediocin did not show an inhibitory effect. Full article
(This article belongs to the Special Issue Antimicrobial Resistance and Antibiotic Alternatives in Livestock)
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Article
Antibacterial Activity of Kaolin–Silver Nanomaterials: Alternative Approach to the Use of Antibiotics in Animal Production
Antibiotics 2021, 10(11), 1276; https://doi.org/10.3390/antibiotics10111276 - 20 Oct 2021
Cited by 3 | Viewed by 1309
Abstract
According to the search for alternatives to replace antibiotics in animal production suggested in the antimicrobial resistance action plans around the world, the objective of this work was to evaluate the bactericidal effect of kaolin–silver nanomaterial for its possible inclusion as an additive [...] Read more.
According to the search for alternatives to replace antibiotics in animal production suggested in the antimicrobial resistance action plans around the world, the objective of this work was to evaluate the bactericidal effect of kaolin–silver nanomaterial for its possible inclusion as an additive in animal feed. The antibacterial activity of the C3 (kaolin–silver nanomaterial) product was tested against a wide spectrum of Gram-negative and Gram-positive bacteria (including multidrug resistant strains) by performing antibiograms, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), as well as growth inhibition curves against seven strains causing infections in animals. The C3 product generated inhibition halos in all the tested strains, and a higher activity against Gram-negative bacteria was found, with MBC values ranged from 7.8 µg/mL (P. aeruginosa) to 15.6 µg/mL (E. coli and Salmonella). In contrast, it was necessary to increase the concentration to 31.3 µg/mL or 250 µg/mL to eliminate 99.9% of the initial population of S. aureus ATCC 6538 and E. faecium ATCC 19434, respectively. Conversely, the inhibition growth curves showed a faster bactericidal activity against Gram-negative bacteria (between 2 and 4 h), while it took at least 24 h to observe a reduction in cell viability of S. aureus ATCC 6538. In short, this study shows that the kaolin–silver nanomaterials developed in the framework of the INTERREG POCTEFA EFA183/16/OUTBIOTICS project exhibit antibacterial activity against a wide spectrum of bacteria. However, additional studies on animal safety and environmental impact are necessary to evaluate the effectiveness of the proposed alternative in the context of One Health. Full article
(This article belongs to the Special Issue Antimicrobial Resistance and Antibiotic Alternatives in Livestock)
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Article
Identification of Three Novel PmGRI1 Genomic Resistance Islands and One Multidrug Resistant Hybrid Structure of Tn7-like Transposon and PmGRI1 in Proteus mirabilis
Antibiotics 2021, 10(10), 1268; https://doi.org/10.3390/antibiotics10101268 - 18 Oct 2021
Cited by 4 | Viewed by 1258
Abstract
The widespread use of antibiotics in large-scale livestock production has led to serious antibiotic resistance. Proteus mirabilis is an important pathogenic bacterium on large-scale farms. Chromosomally localized mobilizable genetic elements (genomic islands) and mobile genetic elements (Tn7-like transposons) play an important [...] Read more.
The widespread use of antibiotics in large-scale livestock production has led to serious antibiotic resistance. Proteus mirabilis is an important pathogenic bacterium on large-scale farms. Chromosomally localized mobilizable genetic elements (genomic islands) and mobile genetic elements (Tn7-like transposons) play an important role in the acquisition and transmission of resistance genes by P. mirabilis. To study the prevalence and resistance characteristics of antibiotic-resistant genomic islands in P. mirabilis of animal origin in China, we performed whole genome sequencing of P. mirabilis isolated from large-scale pig and chicken farms. Three new variants of PmGRI1 (HN31, YN8, and YN9), and a hybrid structure (HN2p) formed by the multidrug-resistant Tn7-like-HN2p transposon and a genomic island PmGRI1-HN2p, were identified from P. mirabilis. All variants underwent homologous recombination mediated by insertion sequence IS26. A genomic rearrangement in the chromosome between the Tn7-like-HN2p transposon and PmGRI1-HN2p occurred in HN2p. The heterozygous structure contained various antimicrobial resistance genes, including three copies of fluoroquinolone resistance gene qnrA1 and 16S rRNA methylase gene rmtB, which are rarely found in P. mirabilis. Our results highlight the structural genetic diversity of genomic islands by characterizing the novel variants of PmGRI1 and enrich the research base of multidrug resistance genomic islands. Full article
(This article belongs to the Special Issue Antimicrobial Resistance and Antibiotic Alternatives in Livestock)
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Article
Butyrate, Forskolin, and Lactose Synergistically Enhance Disease Resistance by Inducing the Expression of the Genes Involved in Innate Host Defense and Barrier Function
Antibiotics 2021, 10(10), 1175; https://doi.org/10.3390/antibiotics10101175 - 27 Sep 2021
Cited by 7 | Viewed by 1439
Abstract
The rising concern of antimicrobial resistance highlights a need for effective alternatives to antibiotics for livestock production. Butyrate, forskolin, and lactose are three natural products known to induce the synthesis of host defense peptides (HDP), which are a critical component of innate immunity. [...] Read more.
The rising concern of antimicrobial resistance highlights a need for effective alternatives to antibiotics for livestock production. Butyrate, forskolin, and lactose are three natural products known to induce the synthesis of host defense peptides (HDP), which are a critical component of innate immunity. In this study, the synergy among butyrate, forskolin, and lactose in enhancing innate host defense, barrier function, and resistance to necrotic enteritis and coccidiosis was investigated. Our results indicated that the three compounds synergistically augmented the expressions of multiple HDP and barrier function genes in chicken HD11 macrophages. The compounds also showed an obvious synergy in promoting HDP gene expressions in chicken jejunal explants. Dietary supplementation of a combination of 1 g/kg sodium butyrate, 10 mg/kg forskolin-containing plant extract, and 10 g/kg lactose dramatically improved the survival of chickens from 39% to 94% (p < 0.001) in a co-infection model of necrotic enteritis. Furthermore, the three compounds largely reversed growth suppression, significantly alleviated intestinal lesions, and reduced colonization of Clostridium perfringens or Eimeria maxima in chickens with necrotic enteritis and coccidiosis (p < 0.01). Collectively, dietary supplementation of butyrate, forskolin, and lactose is a promising antibiotic alternative approach to disease control and prevention for poultry and possibly other livestock species. Full article
(This article belongs to the Special Issue Antimicrobial Resistance and Antibiotic Alternatives in Livestock)
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Article
Molecular Evaluation of Traditional Chicken Farm-Associated Bioaerosols for Methicillin-Resistant Staphylococcus aureus Shedding
Antibiotics 2021, 10(8), 917; https://doi.org/10.3390/antibiotics10080917 - 28 Jul 2021
Cited by 2 | Viewed by 1547
Abstract
The outbreak of airborne pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) through bioaerosol, and their molecular characterization around domestic poultry farming areas, was not completely understood. This imposes risk of a MRSA-associated health threat for the relevant livestock food production units. To address [...] Read more.
The outbreak of airborne pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) through bioaerosol, and their molecular characterization around domestic poultry farming areas, was not completely understood. This imposes risk of a MRSA-associated health threat for the relevant livestock food production units. To address this issue, the present study investigated the role of bioaerosol in transmitting MRSA strains in poultry house settings by combining molecular typing, phylogenetic classification, antibiotic susceptibility, and virulence gene distribution patterns. The present study highlights that all 18 bioaerosol and stool samples collected were MRSA positive, with a unique set of virulence factors. Out of 57 isolated MRSA isolates, 68.4% and 19.3% consisted of SCCmec I and IV elements, respectively, which are commonly linked with hospital-acquired and livestock-associated MRSA strains. It is worth noting that the exfoliative toxin eta and etb genes were carried by 100% and 70.2% of all isolates, respectively. Only 17.5% of strains showed the presence of enterotoxin entC. These MRSA isolates were resistant to chloramphenicol (C), ciprofloxacin (CIP), clindamycin (DA), erythromycin (E), and tetracycline (T), signifying their multi-drug resistance traits. A cluster of phylogenetic analysis described that 80.7% and 15.8% of total isolates belonged to Staphylococcus aureus protein A (spa) type t002 and t548. Whereas 3.5% were reflected as a new spa type. Additionally, as per the chi-squared test score value, these two spa types (t002 and t548) have a distribution correlation with HA-MRSA and LA-MRSA in all the samples (p < 0.005, chi-squared test; degree of freedom = 1). Ultimately, this study highlights the prevalence of MRSA colonization in the conventional poultry farm environment, showing the risk of bioaerosol transmission, which needs epidemiological attention and prevention strategies. Full article
(This article belongs to the Special Issue Antimicrobial Resistance and Antibiotic Alternatives in Livestock)
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Article
High Heritabilities for Antibiotic Usage Show Potential to Breed for Disease Resistance in Finishing Pigs
Antibiotics 2021, 10(7), 829; https://doi.org/10.3390/antibiotics10070829 - 08 Jul 2021
Cited by 1 | Viewed by 1302
Abstract
The use of antimicrobials in animal production is under public debate, mainly due to the risk of transfer of resistance to pathogenic bacteria in humans. Therefore, measures have been taken during the last few decades to reduce antibiotic usage in animals, for instance, [...] Read more.
The use of antimicrobials in animal production is under public debate, mainly due to the risk of transfer of resistance to pathogenic bacteria in humans. Therefore, measures have been taken during the last few decades to reduce antibiotic usage in animals, for instance, by national monitoring programmes and by improving animal health management. Although some initiatives exist in molecular genetic selection, quantitative genetic selection of animals towards decreased antibiotic usage is an underexplored area to reduce antibiotic usage. However, this strategy could yield cumulative effects. In this study, we derived new phenotypes from on-farm parenteral antibiotic records from commercially grown crossbred finishing pigs used in the progeny test of Piétrain terminal sires to investigate the heritability of antibiotics usage. Parenteral antibiotic records, production parameters and pedigree records of 2238 full-sib pens from two experimental farms in Belgium between 2014 and 2020 were analysed. Heritability estimates were moderate (18–44%) for phenotypes derived from all antibiotic treatments, and low (1–15%) for phenotypes derived from treatments against respiratory diseases only. Moreover, genetic correlations between these new phenotypes and mortality were low to moderate (0.08–0.60) and no strong adverse genetic correlations with production traits were found. The high heritabilities and favourable genetic correlations suggest these new phenotypes, derived from on-farm antibiotics records, to be promising for inclusion in future pig breeding programs to breed for a decrease in antibiotics usage. Full article
(This article belongs to the Special Issue Antimicrobial Resistance and Antibiotic Alternatives in Livestock)
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Article
Antibiotic Resistance and Virulence Gene Patterns Associated with Avian Pathogenic Escherichia coli (APEC) from Broiler Chickens in Qatar
Antibiotics 2021, 10(5), 564; https://doi.org/10.3390/antibiotics10050564 - 11 May 2021
Cited by 8 | Viewed by 2096
Abstract
Avian Pathogenic Escherichia coli (APEC) is the contributing agent behind the avian infectious disease colibacillosis, which causes substantial fatalities in poultry industries that has a significant impact on the economy and food safety. Several virulence genes have been shown to be concomitant with [...] Read more.
Avian Pathogenic Escherichia coli (APEC) is the contributing agent behind the avian infectious disease colibacillosis, which causes substantial fatalities in poultry industries that has a significant impact on the economy and food safety. Several virulence genes have been shown to be concomitant with the extraintestinal survival of APEC. This study investigates the antibiotic resistance patterns and APEC-associated virulence genes content in Escherichia coli isolated from non-healthy and healthy broiler chickens from a commercial poultry farm in Qatar. A total of 158 E. coli strains were isolated from 47 chickens from five different organs (air sac, cloacal, kidney, liver, and trachea). Based on genetic criteria, 65% were APEC strains containing five or more virulence genes, and 34% were non-pathogenic E. coli (NPEC) strains. The genes ompT, hlyF, iroN, tsh, vat, iss, cvi/cva, and iucD were significantly prevalent in all APEC strains. E. coli isolates showed 96% resistance to at least one of the 18 antibiotics, with high resistance to ampicillin, cephalothin, ciprofloxacin, tetracycline, and fosfomycin. Our findings indicate high antibiotic resistance prevalence in non-healthy and healthy chicken carcasses. Such resistant E. coli can spread to humans. Hence, special programs are required to monitor the use of antibiotics in chicken production in Qatar. Full article
(This article belongs to the Special Issue Antimicrobial Resistance and Antibiotic Alternatives in Livestock)
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Review

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Review
Insights in the Development and Uses of Alternatives to Antibiotic Growth Promoters in Poultry and Swine Production
Antibiotics 2022, 11(6), 766; https://doi.org/10.3390/antibiotics11060766 - 02 Jun 2022
Cited by 8 | Viewed by 58797
Abstract
The overuse and misuse of antibiotics has contributed to the rise and spread of multidrug-resistant bacteria. To address this global public health threat, many countries have restricted the use of antibiotics as growth promoters and promoted the development of alternatives to antibiotics in [...] Read more.
The overuse and misuse of antibiotics has contributed to the rise and spread of multidrug-resistant bacteria. To address this global public health threat, many countries have restricted the use of antibiotics as growth promoters and promoted the development of alternatives to antibiotics in human and veterinary medicine and animal farming. In food-animal production, acidifiers, bacteriophages, enzymes, phytochemicals, probiotics, prebiotics, and antimicrobial peptides have shown hallmarks as alternatives to antibiotics. This review reports the current state of these alternatives as growth-promoting factors for poultry and swine production and describes their mode of action. Recent findings on their usefulness and the factors that presently hinder their broader use in animal food production are identified by SWOT (strength, weakness, opportunity, and threat) analysis. The potential for resistance development as well as co- and cross-resistance with currently used antibiotics is also discussed. Using predetermined keywords, we searched specialized databases including Scopus, Web of Science, and Google Scholar. Antibiotic resistance cannot be stopped, but its spreading can certainly be hindered or delayed with the development of more alternatives with innovative modes of action and a wise and careful use of antimicrobials in a One Health approach. Full article
(This article belongs to the Special Issue Antimicrobial Resistance and Antibiotic Alternatives in Livestock)
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Other

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Case Report
Exudative Epidermitis in Combination with Staphylococcal Pyoderma in Suckling Piglets
Antibiotics 2021, 10(7), 840; https://doi.org/10.3390/antibiotics10070840 - 09 Jul 2021
Cited by 1 | Viewed by 2332
Abstract
A case of generalized exudative epidermitis (EE) is described, which occurred in a very small piglet producing farm in Austria. The antimicrobial treatment prescribed by the herd veterinarian did not improve the clinical problem. Therefore, the University Clinic for Swine intervened in the [...] Read more.
A case of generalized exudative epidermitis (EE) is described, which occurred in a very small piglet producing farm in Austria. The antimicrobial treatment prescribed by the herd veterinarian did not improve the clinical problem. Therefore, the University Clinic for Swine intervened in the case. Lab investigations were initiated in which Staphylococcus hyicus (SH) and Staphylococcus aureus (SA), both methicillin-resistant and susceptible strains, could be isolated from the skin of affected piglets. Poor hygiene and management practices were identified as predisposing factors on site. Adaptation of antimicrobial treatment according to results of the in vitro susceptibility testing and the implementation of proper hygiene measures resolved the clinical problem. Here, we describe a fatal coinfection of SH and SA in suckling piglets. Full article
(This article belongs to the Special Issue Antimicrobial Resistance and Antibiotic Alternatives in Livestock)
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