Alternativas y reducción del uso de antibióticos en el ganado porcino

  1. Pérez Fernández, Esther
Supervised by:
  1. César B. Gutiérrez Martín Director
  2. Sonia Martínez Martínez Director

Defence university: Universidad de León

Fecha de defensa: 01 December 2022

  1. Germán Naharro Carrasco Chair
  2. Ana Judith Martín de la Fuente Secretary
  3. Mónica Suárez Rodríguez Committee member

Type: Thesis


Traditionally, the misuse of antibiotics as growth promoters, as well as their prophylactic and metaphylactic abuse, has brought antibiotic resistant bacteria to a very critical position for human and animal health. Therefore, the relevant authorities at national and international level have designed various control and surveillance plans for this phenomenon, greatly reducing the use of antibiotics. Livestock farms, more specifically intensive pig farms, have started to make the transition to achieve a decrease in the use of antibiotics, and to ensure that these measures have as little impact as possible on the health of pigs and on production rates. For a proper understanding of antibiotic resistance, it is essential to study the resistance profile of the micro-organism. Therefore, isolation, identification and resistance profile of microorganisms is a key aspect as a strategy for control and appropriate treatment. On the other hand, biosecurity on farms is another aspect that should be considered, since the better the cleaning and disinfection, the more likely it is to cut the cycles of contagion between batches. In addition, a cleaner environment improves the overall health of the animals, preventing the spread of pathogenic micro-organisms. Finally, the use of feed additives as antimicrobials and microbiota modulators is becoming increasingly popular as alternative measures to the use of antibiotics. For all of the above reasons, this Doctoral Thesis has approached this problem from different perspectives. The identification of the bacterial species isolated from samples of respiratory origin was carried out by PCR and MALDI-TOF, obtaining 60 strains of Streptococcus suis, 48 of Pasteurella multocida, 18 of Actinobacillus pleuropneumoniae, and 5 of Glaesserella parasuis. The antibiotic resistance profile was also studied. Resistance of A. pleuropneumoniae and G. parasuis was studied by disc antibiograms. A. pleuropneumoniae was mostly resistant to trimethoprim-sulfamethoxazole and erythromycin, with 61.1 % of isolates resistant in both cases. For Glaesserella parasuis, resistance was mainly to tetracycline and ampicillin, with 60 % of the isolates. On the other hand, S. suis and P multocida were studied using the Sensititre methodTM. For P. multocida, resistance was obtained mainly for lincosamides (94.4 % of cases resistant to clinfamycin). For sulphamines and tiamulin, 55.5 % of isolates were resistant, and for all other antibiotics tested, at least 50 % of the isolates were found to be susceptible. The highest proportions of resistance among S. suis isolates were observed for tetracyclines with 95 % and 93,3 % for chlortetracycline and oxytetracycline, respectively. In addition, in the case of the two most prevalent bacterial species, the study of genes for both antibiotic resistance and virulence factors was carried out. Regarding virulence factors, S. suis presented 6.7 % of the isolates positive for the epf gene, 58.3 % positive for the mrp gene, 43.3 % for the sly gene, 76.7 % for the luxS gene and 53.3 % for the gapd gene. The most frequently found gene in these isolates was luxS, present in 76.6 % of the isolates. When studying P. multocida, we observed that all the isolates were positive for the oma87 gene (48 isolates). The hgbA, nanH, ompH, ptfA and sodA genes were positive in 95.8 %, followed by the tonB gene, with 83.3 % of positive cases. The least prevalent genes were toxA, with 27.1% of isolates, pfhA, with 10.4%, and tbpA, with only one positive isolate. Regarding the antibiotic resistance genes analysed in S. suis, of the four genes studied, for tetracyclines the tetO gene was present in 68.3 % of the bacteria, compared with tetM, which was present in only 1.7 %. For the study of macrolide resistance genes ermB was found in 46.7 % of the isolates, MefA/E was present in 8.3 % of the isolates. For tetracycline resistance, 12.5 % of isolates were positive for the tetA gene, and 39.6 % for tetB. For resistance to β-lactams, 27.1 % of isolates were positive for the bla geneROB1 compared to 8.3 % of isolates with blaTEM . Macrolide resistance was 16.7 % for the ermA gene, 41.7 % for ermC, 22.9 % for msrE and 0 % for mphE. In relation to the study of the action of disinfectants, in order to improve biosecurity, both in vitro and on-farm tests were carried out, as well as new disinfection methods, such as ozonisation, were tested. The capacity of the most common on-farm disinfectants on environmental and PRC bacteria was analysed and it was concluded that “polimorfo” and “limoseptic” disinfectants did not work as effectively as the others tested. In vitro tests were also carried out with ozone and ozonised water. There was a 100 % reduction in the number of colonies in the 100 litres of air at 1.5 ppm ozone: for bacteria in the air it was therefore very effective. For this ozone concentration and with ozonized water on objects with uneven surfaces, disinfection worked as well as conventional disinfectant on feeders, drinkers and walls, and better for heating plates, floors and toys. This finding was of great interest, as ozone is a disinfectant that does not leave residues, which is better for the environment. It also does not need rinsing, thus improving drying times, which speeds up the filling time with the next batch of pigs. Considering the use of food additives as an alternative to the use of antibiotics, in vitro studies were carried out to evaluate the antibacterial capacity of lauric acid in Gram-positive and Gram-negative bacteria, determining the minimum inhibitory concentration (MIC) for S. suis and P. multocida bacteria. These assays included diffusion-based methods (both plate and disc) to quantify the inhibition of bacteria against these compounds. A scanning electron microscopy study was also performed to observe the effect of lauric acid against S. suis. Plate diffusion tests could not be carried out due to the physicochemical characteristics of lauric acid with the culture medium, obtaining very heterogeneous plates. On the other hand, the disc diffusion methods could be carried out without any problem, obtaining very good results for lauric acid and monolaurin in the case of both bacteria studied and at intermediate concentrations. Concentrations from 2.4 to 1500 mg/ml of lauric acid or 5 % monolaurin were studied, obtaining inhibition for all concentrations of lauric acid in S. suis, and for P. multocida inhibition of bacterial growth was obtained from 40 mg/ml onwards. In addition, in the case of S. suis, a scanning electron microscopy study was carried out, in which a decrease in bacterial density and changes in morphology were observed when exposed to 900 mg/ml lauric acid. Finally, the effect of supplementing the diet with two different doses of lauric acid was evaluated in an on-farm experimental trial. This experiment was carried out in the transition stage, on 144 weaned piglets (25 days of age), which were distributed in three groups (two pens per group). All groups received feed without antibiotics and zinc oxide. The animals in the first group received pre-starter and starter feed without additives, the second group received feed supplemented with 2 kg lauric acid per tonne and the last group supplemented at 4 kg/T. The average daily gain (ADG) values during the 43 days of the experiment were very similar in all three cases, although somewhat higher in the group receiving the higher lauric acid dose. Feed intake was higher in the control group (28.64 kg/pig) compared to the groups receiving lauric acid (2 kg/T group: 27.17 kg/pig and 4 kg/T group: 27.48 kg/pig). Finally, the feed conversion ratio showed a slight improvement (more feed converted into productive kg) in the group receiving 4 kg/T (2.02) compared to the control group (2.29). The differences did not reach statistical significance. When studying the microbiota, the richness of bacterial species was higher in the case of the faecal microbiota, compared to the respiratory microbiota, which increased in diversity over time. The species diversity study was analysed using Simpson's diversity index, looking at both the bacterial species in the microbiota and their relative abundance. In the case of the faecal microbiota, this index decreased over time, so it can be affirmed that the bacterial species became more varied over time. Simpson's index of the nasal microbiota increased over time, so we can say that the diversity of species in this case settled and decreased over time. For the principal component analysis of the nasal microbiota analysis, we observed a segregation of nasal bacteria according to the age of the pigs, finding differences between species. Regarding faecal taxonomy, a segregation of faecal bacteria was observed, depending on the age of the pigs, between the final and initial stage, but no clear difference was observed between the intermediate and final stage. The most abundant bacterial species in the nasal microbiota were Clostridioides difficile, which appeared mostly during the first two samplings of the pigs, and Bergeyella zoohelcum, which appeared mostly in the first and last sampling. Bacteria occurring throughout the sampling, but especially after the 43 days of the trial, were S. suis and G. parasuis. Finally, several members of the genera Prevotella and Moraxella were identified throughout the trial. On the other hand, after analysis of the faecal microbiota, we obtained several members of the Prevotella genus, especially in the piglets that had been consuming the supplemented feed after the initial 14 days of the experiment. E. coli bacteria appeared in large numbers, especially at the beginning of the experiment, and could have been displaced by Prevotella. Ruminococcus was also found in high numbers, especially in piglets fed the highest dose of lauric acid. The genus Clostridium varied over time, as did the species Bacteroides fragilis.