Impact of short-term storage on the quantity of extended-spectrum beta-lactamase-producing Escherichia coli in broiler litter under practical conditions

Abstract

Applying broiler litter containing extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) to arable land poses a potential risk for humans to get colonized by contact with contaminated soil or vegetables. Therefore, an inactivation of these bacteria before land application of litter is crucial. We performed 2 short-term litter storage trials (one in summer and winter, respectively), each covering a time span of 5 D to investigate the effectiveness of this method for inactivation of ESBL-producing E. coli in chicken litter. Surface and deep litter samples were taken from a stacked, ESBL-positive chicken litter heap in triplicates in close sampling intervals at the beginning and daily for the last 3 D of the experiments. Samples were analyzed quantitatively and qualitatively for ESBL-producing E. coli, total E. coli, and enterococci. Selected isolates were further characterized by whole-genome sequencing (WGS). In the depth of the heap ESBL-producing E. coli were detected quantitatively until 72 h and qualitatively until the end of the trial in winter. In summer detection was possible quantitatively up to 36 h and qualitatively until 72 h. For surface litter samples a qualitative detection of ESBL-producing E. coli was possible in all samples taken in both trials. In the deep samples a significant decrease in the bacterial counts of over 2 Log₁₀ was observed for total E. coli in the winter and for total E. coli and enterococci in the summer. Genetic differences of the isolates analyzed by WGS did not correlate with survival advantage. In conclusion, short-term storage of chicken litter stacked in heaps is a useful tool for the reduction of bacterial counts including ESBL-producing E. coli. However, incomplete inactivation was observed at the surface of the heap and at low ambient temperatures. Therefore, an extension of the storage period in winter as well as turning of the heap to provide aerobic composting conditions should be considered if working and storage capacities are available on the farms.

Keywords: E. coli; ESBL; antibiotic resistance; broiler litter; environment.

Figure 1

Development of mean sample temperatures and moistures in the winter trial (A) and summer trial (B).

Figure 2

Mean sample pH values in the winter trial (A) and summer trial (B) for each point in time.

Figure 3

Results of the quantitative and qualitative analysis of ESBL-producing E. coli in cfu/g of litter for the winter trial samples (A) and the summer trial samples (B). The geometric mean of 3 samples is shown for each point in time. The error bars indicate the upper and lower 95% confidence intervals. The graphs were shifted to improve the visibility of the error bars. ESBL, extended-spectrum beta-lactamase.

Figure 4

Results of the quantitative and qualitative analysis of total E. coli in cfu/g of litter, and the quantitative analysis of enterococci in cfu/g of litter for the winter trial samples (A) and the summer trial samples (B). The geometric mean of 3 samples is shown for each point in time. The error bars indicate the upper and lower 95% confidence intervals. The graphs were shifted to improve the visibility of the error bars.

Supplemental Figure 1

Phylogenetic NJ-Trees. Isolates are colored according to the season in which the experiment was performed (a), the sampling site in litter pile (b), the timepoint of sampling (c) or the resistance genes found by qPCR (d).

Supplemental Figure 2

Phylogenetic NJ-Trees. The isolates are colored according to their 7 gene MLST sequence type (a) or according to their Phylogroup determined via Enterobase (b)