Characterization of Antibiotic Resistance Gene Abundance and Microbiota Composition in Feces of Organic and Conventional Pigs from Four EU Countries

Abstract

One of the recent trends in animal production is the revival of interest in organic farming. The increased consumer interest in organic animal farming is mainly due to concerns about animal welfare and the use of antibiotics in conventional farming. On the other hand, providing animals with a more natural lifestyle implies their increased exposure to environmental sources of different microorganisms including pathogens. To address these concerns, we determined the abundance of antibiotic resistance and diversity within fecal microbiota in pigs kept under conventional and organic farming systems in Sweden, Denmark, France and Italy. The abundance of sul1, sul2, strA, tet(A), tet(B) and cat antibiotic resistance genes was determined in 468 samples by real-time PCR and the fecal microbiota diversity was characterized in 48 selected samples by pyrosequencing of V3/V4 regions of 16S rRNA. Contrary to our expectations, there were no extensive differences between the abundance of tested antibiotic resistance genes in microbiota originating from organic or conventionally housed pigs within individual countries. There were also no differences in the microbiota composition of organic and conventional pigs. The only significant difference was the difference in the abundance of antibiotic resistance genes in the samples from different countries. Fecal microbiota in the samples originating from southern European countries (Italy, France) exhibited significantly higher antibiotic resistance gene abundance than those from northern parts of Europe (Denmark, Sweden). Therefore, the geographical location of the herd influenced the antibiotic resistance in the fecal microbiota more than farm's status as organic or conventional.

Fig 1. Abundance of antibiotic resistance genes in fecal microbiota of conventional and organic pigs.

Antibiotic resistance gene abundance is presented as median, 25th and 75th percentiles (box) and the whiskers indicating the minimal and maximal values recorded. Black boxes, abundance in the microbiota from conventional pigs, green boxes, abundance in microbiota from organic pigs. As shown in the table embedded within the figure, country of sample origin (IT–Italy, FR–France, DNK–Denmark, SWE–Sweden) was a significant source of variation (ANOVA followed by Tukey HSD post-hoc test, P<0.01, asterisks indicate significant difference, NS–Non Significant). On the other hand, comparison of antibiotic resistance gene abundance in feces of pigs from organic and conventional farms within the same country reached statistical difference only for sul2, tet(B) and cat abundance in Italian samples (ANOVA followed by Tukey’s HSD post-hoc test P<0.01).

Fig 2. PCA clustering based on the antibiotic resistance gene abundance.

Each spot represents an individual sample tested in this study and clustered according to the abundance of 6 antibiotic resistance genes. Factor 1, accounting for nearly 58% of the variation negatively correlated with abundance of strA, sul1, sul2, tet(A) and tet(B) (correlation coefficients ranged from -0.84 to -0.76). Orange—samples from Italian farms, red—samples from French farms, blue—samples from Danish farms, green—samples from Swedish farms. Opened symbols–samples from pigs kept in conventional farms. Closed symbols–samples from pigs kept in organic farms.

Fig 3. Composition of fecal microbiota of conventional and organic pigs in four countries at family level.

Each color represents a particular bacterial family and the following numbers refer to the defined color codes. 1 –Veillonellaceae, 2—unclassified Clostridiales, 3—unclassified Bacteroidales, 4 –Streptococcaceae, 5 –Ruminococcaceae, 6 –Moraxellaceae, 7 –Rikenellaceae, 8 –Prevotellaceae, 9 –Porphyromonadaceae, 10 –Peptostreptococcaceae, 11 –Lachnospiraceae, 12 –Hydrogenophylaceae, 13 –Lactobacillaceae, 14 –Erysipelotrichaceae, 15—Clostridiaceae incertae sedis, 16 –Clostridiaceae, 17 –Acidaminococcaceae. IT–Italy, FR–France, DNK–Denmark, SWE–Sweden. Org–samples from organic farms, Con–samples from conventional farms. Sample 1–5 for each country and production system represent samples taken from 5 randomly selected pigs. The sixth samples (“P”) always represent pooled samples from particular country and production system.