Escherichia coli Probiotic Strain ED1a in Pigs Has a Limited Impact on the Gut Carriage of Extended-Spectrum-β-Lactamase-Producing E. coli

Abstract

Four trials were conducted to evaluate the impact of Escherichia coli probiotic strain ED1a administration to pigs on the gut carriage or survival in manure of extended-spectrum-β-lactamase-producing E. coli Groups of pigs were orally inoculated with strain E. coli M63 carrying the bla_CTX-M-1 gene (n = 84) or used as a control (n = 26). In the first two trials, 24 of 40 E. coli M63-inoculated pigs were given E. coli ED1a orally for 6 days starting 8 days after oral inoculation. In the third trial, 10 E. coli M63-inoculated pigs were given either E. coli ED1a or probiotic E. coli Nissle 1917 for 5 days. In the fourth trial, E. coli ED1a was given to a sow and its 12 piglets, and these 12 piglets plus 12 piglets that had not received E. coli ED1a were then inoculated with E. coli M63. Fecal shedding of cefotaxime-resistant Enterobacteriaceae (CTX-RE) was studied by culture, and bla_CTX-M-1 genes were quantified by PCR. The persistence of CTX-RE in manure samples from inoculated pigs or manure samples inoculated in vitro with E. coli M63 with or without probiotics was studied. The results showed that E. coli M63 and ED1a were good gut colonizers. The reduction in the level of fecal excretion of CTX-RE in E. coli ED1a-treated pigs compared to that in nontreated pigs was usually less than 1 log₁₀ CFU and was mainly observed during the probiotic administration period. The results obtained with E. coli Nissle 1917 did not differ significantly from those obtained with E. coli ED1a. CTX-RE survival did not differ significantly in manure samples with or without probiotic treatment. In conclusion, under our experimental conditions, E. coli ED1a and E. coli Nissle 1917 could not durably prevent CTX-RE colonization of the pig gut.

Keywords: antibiotic resistance; cephalosporin; cephalosporin resistance; microbiota; pig; probiotic.

FIG 1

Results of the first and second trials. Samples were cultured for ESCR Enterobacteriaceae, and qPCR for bla_CTX-M-1 and E. coli ED1a was performed on the different sampling days. (A to C) First trial; (D to F) second trial. (A and D) Titers of ESCR Enterobacteriaceae (log₁₀ number of CFU per gram, as indicated on the y axis); (B and E) titers of bla_CTX-M-1 (log₁₀ number of copies in 10 ng of DNA, as indicated on the y axis); (C and F) titers of E. coli ED1a (log₁₀ number of copies in 10 ng of DNA, as indicated on the y axis). For each date, asterisks indicate significant differences between groups (P < 0.05). For both trials, E. coli M63 was inoculated on day 0 (D0), and E. coli ED1a was given from day 8 (D8) to day 13 (D13). ND, not done. Samples from animals not inoculated with E. coli M63 were negative for ESCR Enterobacteriaceae and bla_CTX-M-1. Samples from animals not inoculated with E. coli ED1a were negative for E. coli ED1a.

FIG 2

Results of the third trial. Samples were cultured for ESCR Enterobacteriaceae, and qPCR for bla_CTX-M-1 and probiotics (E. coli ED1a and E. coli Nissle 1917) was performed on the different sampling days. (A) Titers of ESCR Enterobacteriaceae (log₁₀ number of CFU per gram, as indicated on the y axis); (B) titers of bla_CTX-M-1 (log₁₀ number of copies in 10 ng of DNA, as indicated on the y axis); (C) titers of probiotics E. coli ED1a and E. coli Nissle 1917 (log₁₀ number of copies in 10 ng of DNA, as indicated on the y axis). E. coli M63 was inoculated on day 0, and E. coli ED1a or E. coli Nissle 1917 was given from day 8 to day 12. Samples from animals not inoculated with E. coli M63 were negative for ESCR Enterobacteriaceae and bla_CTX-M-1. Samples from animals not inoculated with E. coli ED1a and E. coli Nissle 1917 were negative for E. coli ED1a and E. coli Nissle 1917, respectively.

FIG 3

Results of the fourth trial. Samples were cultured for ESCR Enterobacteriaceae, and qPCR for E. coli ED1a in fecal samples from piglets was performed on the different sampling days. (A) Titers of ESCR Enterobacteriaceae (log₁₀ number of CFU per gram, as indicated on the y axis); (B) titers of E. coli ED1a (log₁₀ number of copies in 10 ng of DNA, as indicated on the y axis). E. coli M63 was inoculated on day 0, and E. coli ED1a was given to the sow from before farrowing (days −42 to −28 before E. coli M63 inoculation) and to the piglets from birth to weaning day (days −27 to day −1 before E. coli M63 inoculation). Samples from animals not inoculated with E. coli M63 were negative for ESCR Enterobacteriaceae. Samples from animals not inoculated with E. coli ED1a were negative for E. coli ED1a.

FIG 4

Results of the first trial. A qPCR for bla_CTX-M and E. coli ED1a in manure samples collected on day 14 and stored at 5 ± 3°C was performed. (A) Titer of bla_CTX-M-1 (log₁₀ number of copies in 10 ng of DNA, as indicated on the y axis); (B) titer of E. coli ED1a (log₁₀ number of copies in 10 ng of DNA, as indicated on the y axis). E. coli M63 was inoculated on day 0, and E. coli ED1a was given from day 8 to day 13. Manure samples were collected on day 14 and analyzed after 0 (ST0), 7 (ST7), 56 (ST56), and 84 (ST84) days of storage at 5 ± 3°C. No isolates of the ESCR Enterobacteriaceae were cultured. Samples from animals not inoculated with E. coli M63 were negative for bla_CTX-M-1. Samples from animals not inoculated with E. coli ED1a were negative for E. coli ED1a.

FIG 5

Results of the second trial. Samples were cultured for ESCR Enterobacteriaceae, and a qPCR for the detection of E. coli ED1a in manure samples collected on day 13 and stored at room temperature was performed. (A) Titer of ESCR Enterobacteriaceae (log₁₀ number of CFU per gram, as indicated on the y axis); (B) titer of E. coli ED1a (log₁₀ number of copies in 10 ng of DNA, as indicated on the y axis). E. coli M63 was inoculated on day 0, and E. coli ED1a was given from day 8 to day 13. Manure samples were collected on day 13 and analyzed after 0 (ST0), 7 (ST7), 14 (ST14), 30 (ST30), or 60 (ST60) days of storage at room temperature. All samples from animals not inoculated with E. coli M63 were negative for ESCR Enterobacteriaceae, and all samples from animals not inoculated with E. coli ED1a were negative for E. coli ED1a. Most samples were negative for the bla_CTX-M gene.

FIG 6

Culture of ESCR Enterobacteriaceae and total E. coli (ECT) in in vitro-inoculated manure samples: segmentation of total E. coli and ESCR strains. (A) Titers of ESCR Enterobacteriaceae; (B) titers of all E. coli strains. Each leaf indicates the mean titer for the subgroup and the number of samples tested. The titers of ESCR Enterobacteriaceae were determined on days 2, 7, 21, 30, and 60. The titers of all E. coli strains were determined on days 2, 7, 15, 30, and 60. Temperat, temperature; RT, room temperature; EcN, E. coli Nissle 1917; ED, E. coli ED1a.