Prevalence, Characteristics and Clonal Distribution of Extended-Spectrum β-Lactamase- and AmpC β-Lactamase-Producing Escherichia coli Following the Swine Production Stages, and Potential Risks to Humans

Abstract

The worldwide spread of extended spectrum β-lactamase (ESBL)- and AmpC β-lactamase (AmpC)-producing Escherichia coli poses serious threats to public health. Swine farms have been regarded as important reservoirs of ESBL/AmpC-EC. This study aimed to determine the prevalence, ESBL/AmpC types, and clonal distribution of ESBL/AmpC-EC from swine farms and analyze the difference according to the swine production stages. In addition, we evaluated the potential risks of swine ESBL/AmpC-EC clones to humans. Individual fecal samples (n = 292) were collected from weaning, growing, finishing, and pregnant pigs in nine swine farms of South Korea between July 2017 and March 2020. In total, 161 ESBL/AmpC-EC isolates were identified (55.1%), with the highest prevalence detected in the weaning stage (86.3%). The dominant ESBL and AmpC types were CTX-M-55 (69.6%) and CMY-2 (4.3%), respectively. CTX-M found in all production stages, while CMY was only found in growing and finishing stages. In the conjugation assay, the high transferability of CTX-M gene (55.8%) was identified, while the transfer of CMY gene was not identified. The major clonal complexes (CCs) were CC101-B1 (26.8%), CC10-A (8.7%), and CC648-F (2.9%). There was similarity in clonal distribution between different swine production stages within swine farms, estimated using the k-means analysis, which suggested a clonal transmission between the different swine stages. Among swine ESBL/AmpC-EC sequence types (STs), seven STs (ST101, ST10, ST648, ST457, ST410, ST617, and ST744) were common with the human ESBL/AmpC-EC, which registered in National Center for Biotechnology Information database. The clonal population structure analysis based on the virulence factor (VF) presented that swine ESBL/AmpC-EC clones, especially ST101-B1, harbored a highly virulent profile. In conclusion, ESBL/AmpC-EC was distributed throughout the swine production stages, with the highest prevalence in the weaning stage. The CTX-M was present in all stages, while CMY was mostly found in growing-finishing stages. The swine ESBL/AmpC-EC was identified to harbor shared clone types with human ESBL/AmpC-EC and a virulent profile posing potential risk to humans. Considering the possibility of genetic and clonal distribution of ESBL/AmpC-EC among swine production stages, this study suggests the need for strategies considering the production system to control the prevalence of ESBL/AmpC-EC in swine farms.

Keywords: AmpC β-lactamase; Escherichia coli; clonal distribution; extended-spectrum β-lactamase; extra-intestinal pathogenic E. coli; multidrug resistance; swine production stages; virulence factor.

Figure 1

Prevalence of extended spectrum β-lactamase (ESBL)- or AmpC β-lactamase (AmpC)-producing E. coli (ESBL/AmpC-EC) according to different swine farms (A) and swine production stages (B).

Figure 2

ESBL/AmpC-EC showed significantly higher resistance rate to antibiotics compared to non-ESBL/AmpC-EC. Statistically significant (^*p < 0.05, GEE; ^†p < 0.05, Chi-square test).

Figure 3

ESBL/AmpC-EC carried higher number of extra-intestinal pathogenic E. coli associated virulence factors (ExPEC VFs) compared to non-ESBL/AmpC-EC. OR, odds ratio. Statistically significant (^*p < 0.05, GEEs; ^†p < 0.05, Chi-square test).

Figure 4

Prevalence and distribution of ESBL/AmpC types from swine farms. In the chord diagram, the size of segments on the top represent the number of ESBL/AmpC-EC isolates with a specific ESBL/AmpC types. Size of segments on the bottom represent the number of ESBL/AmpC-EC isolates detected in different farms (A) and production stages (B). Ribbons connecting the top and bottom segments represent the number of ESBL/AmpC-EC isolates with a specific ESBL/AmpC type found on the respective farms and production stages. The connected bar chart shows the composition of ESBL/AmpC types based on the number of ESBL/AmpC-EC isolates in different farms (A) or production stages (B). The chord diagram and bar chart were generated with R software (ver. 4.3.2).

Figure 5

Minimum spanning tree (MST) based on allele profiles of multi-locus sequence type (MLST): clonal distribution of ESBL/AmpC-EC between pig farms. The number shows the sequence type of each node, and the size of the node indicates the number of strains belonging to the sequence type (ST)-phylogenetic group. The gray shadow represents the clonal complex (CC). Branch line types represent differences in the number of alleles: bold solid line (1 allele), thin solid line (2–3 alleles), dashed line (4 alleles), and dotted line (above 5 alleles). CC, clonal complex; ST, sequence type; NT, non-typable ST (including two different non-typable STs).

Figure 6

k-means similarity clustering plot: similarity in the clonal distribution among swine production stages within farms. A total of 33 points were described and clustered into 9 (symbols and colors) using the k-means similarity clustering algorithm based on Euclidean distance. The k-means cluster plot was generated using the R software (ver. 4.3.2). Each point indicates the distribution of STs among swine production stages in each farm, consisting of 36 points based on combination of 9 farms (A to I) and 4 production stages (1, weaning piglets; 2, growing pigs; 3, finishing pigs; and 4, pregnant sows; e.g., A1 presents the clonal distribution of “Weaning piglets” of “Farm A”). Three points (H2, H4, and I2) were excluded as no ESBL/AmpC-EC strains were isolated from “Growing pigs” of “Farm H”, “Pregnant sows” of “Farm H,” and “Growing pigs” of “Farm I.”

Figure 7

Shared major STs of ESBL/AmpC-EC isolated from swine farms and humans. Venn diagram shows the STs shared between swine farm derived ESBL/AmpC-ECs from this study and ESBL/AmpC-ECs from human sources which registered in the NCBI Pathogen Isolation Database. The intersection area of the two circles represents to the seven shared STs (ST101, ST10, ST457, ST410, ST617, ST744, and ST648) of ESBL/AmpC-EC from swine farms and human sources. ST, sequence type.

Figure 8

A clonal population analysis of swine ESBL/AmpC-EC isolates using program structure. Each isolate was assigned to five populations based on their ExPEC VFs and E. coli phylogenetic group profile. The ExPEC VFs and E. coli phylogenetic group profile for each clonal population are presented in Table 2. Each isolate is represented by a vertical segment and aligned horizontally according to CCs and STs (x-axis). The proportion of population (Q value) for each isolate is shown as 100% stacked bar plots, with proportions of colored sections representing the probability of belonging to each population within each segment (y-axis). CC, clonal complex; ST, sequence type; STNT, ST non-typable. ^#a, CC205-B1; b, CC376-B1; c, CC12-B2; d, ST3285-B1; e, ST953-A; f, NT-B1; g, ST7203-A; h, STNT-A; I, ST767-B1; and j, ST1011-E.