Whole Genome Sequencing Differentiates Presumptive Extended Spectrum Beta-Lactamase Producing Escherichia coli along Segments of the One Health Continuum

Abstract

Antimicrobial resistance (AMR) has important implications for the continued use of antibiotics to control infectious diseases in both beef cattle and humans. AMR along the One Health continuum of the beef production system is largely unknown. Here, whole genomes of presumptive extended-spectrum β-lactamase E. coli (ESBL-EC) from cattle feces (n = 40), feedlot catch basins (n = 42), surrounding streams (n = 21), a beef processing plant (n = 4), municipal sewage (n = 30), and clinical patients (n = 25) are described. ESBL-EC were isolated from ceftriaxone selective plates and subcultured on ampicillin selective plates. Agreement of genotype-phenotype prediction of AMR ranged from 93.2% for ampicillin to 100% for neomycin, trimethoprim/sulfamethoxazole, and enrofloxacin resistance. Overall, β-lactam (100%; bla_EC, bla_TEM-1, bla_SHV, bla_OXA, bla_CTX-M-), tetracycline (90.1%; tet(A), tet(B)) and folate synthesis (sul2) antimicrobial resistance genes (ARGs) were most prevalent. The ARGs tet(C), tet(M), tet(32), bla_CTX-M-1, bla_CTX-M-14, bla_OXA-1, dfrA18, dfrA19, catB3, and catB4 were exclusive to human sources, while bla_TEM-150, bla_SHV-11-12, dfrA12, cmlA1, and cmlA5 were exclusive to beef cattle sources. Frequently encountered virulence factors across all sources included adhesion and type II and III secretion systems, while IncFIB(AP001918) and IncFII plasmids were also common. Specificity and prevalence of ARGs between cattle-sourced and human-sourced presumptive ESBL-EC likely reflect differences in antimicrobial use in cattle and humans. Comparative genomics revealed phylogenetically distinct clusters for isolates from human vs. cattle sources, implying that human infections caused by ESBL-EC in this region might not originate from beef production sources.

Keywords: One Health; antimicrobial resistance; comparative genomics; extended spectrum Beta-lactamase producing E. coli; whole genome sequencing.

Figure 1

Phenotypic antimicrobial resistance profiles of presumptive extended-spectrum β-lactamase (ESBL) E. coli obtained from different sources as described in materials and methods. OXYT—tetracycline; AMPI—ampicillin; CTIO—ceftiofur; STEP—streptomycin; SULF—sulfisoxazole; TMSZ—trimethoprim/sulfamethoxazole; FLOR—florfenicol; ENRO—enrofloxacin; CTZD—ceftazidime; AMCL—amoxicillin/clavulanic acid; NMYN—neomycin; MDR—multidrug resistance. MDR referred to resistance to two or more classes of antibiotics.

Figure 2

Heat map of resistance genes associated with various classes of antimicrobials as detected by the whole genome sequences of 162 presumptive ESBL-producing E. coli across a One Health continuum: Isolates originated from multiple segments of the One Health continuum including human clinical, municipal sewage, beef processing plant, cattle feces, catch basin, and surrounding streams. aac-(6′)-Ib-cr classified under aminoglycoside also encodes quinolone resistance. The overall prevalence of each determinant is displayed as a numeric percentage at the bottom of each determinant.

Figure 3

Phylogenetic tree generated on the basis of single-nucleotide polymorphisms (SNPs) of the core genes of 162 E. coli isolates obtained from multiple segments of the beef production system and human-associated isolates with reference genome E. coli str. K-12 substr. MG1655 (GenBank accession # GI: 545778205/U00096.3). Inner, middle ring, and outer rings are representative of isolate sources, phenotypic antimicrobial, and multidrug resistance, respectively.

Figure 4

Minimum spanning tree (MST) based on whole-genome multi-locus sequence typing (wgMLST) profiles of 162 presumptive ESBL-producing E. coli genomes along the One Health continuum from cattle feces, catch basins, surface streams, beef processing plants, municipal sewage, and humans: The MST includes a total of 9580 wgMLST loci and was constructed with BioNumerics (version 7.6.2). Each circle corresponds to a unique wgMLST profile and is colored based on sample origin. The size of the circle is proportional to the number of isolates sharing the same wgMLST profile, while the branch lengths correspond to the number of allele differences between isolates. Boxes highlight three major clusters of isolates of cattle and human origin.

Figure 5

Blast atlas (A—circular) showing sequences similarity and diversity among true ESBL and presumptive non-ESBL E. coli using E. coli MG1655 as a reference: Isolates shown in this analysis are all cattle-derived (cattle feces, catch basins, surface water, and processing plant) and do not include any human-derived presumptive ESBL-EC because WGS did not identify any non-ESBL-EC from humans or municipal sewage sources based on the presence of bla_SHV, bla_TEM, bla_OXA, or bla_CTX-M. Inner ring comprises GC skew, GC content, and sequence of reference E. coli MG1655, while middle and outer rings comprise ESBL and non-ESBL E. coli, respectively.

Figure 6

Percentage prevalence of specific sequence types (ST10 to ST7618 and unknown STs) identified in multiple segments of the One Health continuum using the E. coli Achtman scheme.

Figure 7

Prevalence of plasmid replicons per source of presumptive ESBL-producing E. coli from cattle feces, catch basins, surface streams, municipal sewage, humans, and beef processing along a One Health continuum.