Improvements of weaned pigs barn hygiene to reduce the spread of antimicrobial resistance

Abstract

The spread of antimicrobial resistance (AMR) in animal husbandry is usually attributed to the use of antibiotics and poor hygiene and biosecurity. We therefore conducted experimental trials to improve hygiene management in weaned pig houses and assessed the impact on the spread. For each of the two groups examined, the experimental group (EG) and the control group (CG), three replicate batches of piglets from the same pig breeder, kept in pre-cleaned flat decks, were analyzed. In the flat decks of the experimental groups, the hygiene conditions (cleaning, disinfection, dust removal and fly control) were improved, while regular hygiene measures were carried out in the control groups. The occurrence and spread of AMR were determined in Escherichia coli (E. coli; resistance indicator) using cultivation-dependent (CFU) and -independent (qPCR) methods as well as whole genome sequencing of isolates in samples of various origins, including feces, flies, feed, dust and swabs. Surprisingly, there were no significant differences (p > 0.05) in the prevalence of resistant E. coli between the flat decks managed with conventional techniques and those managed with improved techniques. Selective cultivation delivered ampicillin- and sulfonamide-resistant E. coli proportions of up to 100% and 1.2%, respectively. While 0.5% E. coli resistant to cefotaxime and no ciprofloxacin resistance were detected. There was a significant difference (p < 0.01) in the abundance of the bla_TEM-1 gene in fecal samples between EG and CG groups. The colonization of piglets with resistant pathogens before arrival, the movement of flies in the barn and the treatment of bacterial infections with antibiotics obscured the effects of hygiene improvement. Biocide tolerance tests showed no development of resistance to the farm regular disinfectant. Managing hygiene alone was insufficient for reducing antimicrobial resistances in piglet rearing. We conclude that the complex factors contributing to the presence and distribution of AMR in piglet barns underscore the necessity for a comprehensive management strategy.

Keywords: AMR; Escherichia coli; cultivation; disinfection; hygiene; pig; weaner barn.

Figure 1

Prevalence of ampicillin resistance in the indicated sample types. (A) Boxplots show the % proportion of AMR Escherichia coli compared to total E. coli (in CFU/g feces, flies, feed, or dust) in the experimental and control groups. Error bars are 95% confidence intervals, the bottom and top of the box are the 25th and 75th percentiles, and the line inside the box is the 50th percentile (median). The solid blue circles in the boxplots represent the means of ampicillin-resistant E. coli in each group in 2-week intervals. Each column represents the according sample type. “ns” at the top center of each column indicates no statistically significant difference between the two groups. (B) Line graphs show the average weekly prevalence of ampicillin-resistant E. coli over the four-week study period in feces, flies, feed, and dust samples.

Figure 2

Prevalence of Escherichia coli resistant to (A) sulfonamide and (B) cefotaxime compared to total E. coli in samples collected from piglet houses for hygiene improvement assessment.

Figure 3

Quantitative PCR results of total bacteria and resistance genes in fecal samples collected during a hygiene improvement experiment. The X-axis represents the sampling week (week: 0, 1, 2, 4) and the Y-axis represents the logarithm of gene copies per gram of feces. Boxplots show the distribution of gene copies above the limit of quantification (LoQ); gray area = below LoQ. The bottom and top of the boxes are the first and third quartiles, respectively. The black band within the box is the median and the ends of the whisker represent the maximum (largest gene copy number) and minimum (lowest gene copy number) values. The solid blue circles indicate the average number of gene copies. Each column represents the indicated gene name and ‘ns’ indicates statistically no significant difference, while ^*p < 0.05 and ^**p < 0.01 show statistically significant and strong significant differences, respectively (based on Wilcoxon rank sum test).

Figure 4

The relative abundance of resistance genes copy numbers normalized to the bacterial 16S rRNA gene. The X-axis shows the name of resistance gene, while Y-axis represents the proportion of resistance gene copy number to 16S rRNA gene copy number the in fecal samples. The samples were collected from flat decks where hygiene was managed using conventional techniques (red boxplot) and improved techniques (green boxplot). The boxes indicate the interquartile range of the data while the black line in the boxplot represents the median gene copy number for the samples tested. The top ‘ns’ means no significant difference, while ^**p < 0.01 means strong significant difference (based on Wilcoxon rank-sum test).

Figure 5

Heat map of the presence (light red) and absence (light yellow) of 41 different antimicrobial resistance genes and/or mutations in Escherichia coli strains isolated from 24 fecal and 6 swab samples collected weekly during the study period in experimental and control groups. At the top of the heat map are 13 classes of antibiotics to which the isolates were tested for resistance genes or mutations.

Figure 6

Results of the minimum inhibitory concentration determinations for the disinfectants Sorgene and Des Allround against Escherichia coli strains. (A) Comparison of susceptibility of CTX-susceptible E. coli isolated from control and experimental groups to both disinfectants; (B) Comparison of the MIC (%) of Sorgene and Des Allround against CTX-susceptible E. coli strains; (C) Comparison between CTX-susceptible and CTX-resistant to the biocidal effect of two disinfectants. ‘ns’ indicates no significant difference, while ^*p < 0.5 and ^***p < 0.001, means the difference is statistically significant and highly significant, respectively (based on Wilcoxon rank-sum test).