Genomic Analysis of Third Generation Cephalosporin Resistant Escherichia coli from Dairy Cow Manure

Abstract

The production of extended-spectrum β-lactamases (ESBLs) conferring resistance to new derivatives of β-lactams is a major public health threat if present in pathogenic Gram-negative bacteria. The objective of this study was to characterize ceftiofur (TIO)- or cefotaxime (FOX)-resistant Escherichia coli isolated from dairy cow manure. Twenty-four manure samples were collected from four farms and incubated under anaerobic conditions for 20 weeks at 4 °C or at 25 °C. A total of 37 TIO- or FOX-resistant E. coli were isolated from two of the four farms to determine their susceptibility to 14 antibiotics. Among the 37 resistant E. coli, 10 different serotypes were identified, with O8:H1 being the predominant serotype (n = 17). Five isolates belonged to each of serotypes O9:NM and O153:H42, respectively. All 37 cephalosporin resistant isolates were multi-resistant with the most prevalent resistance spectrum being amoxicillin-clavulanic acid-ampicillin-cefoxitin-ceftiofur-ceftriaxone-chloramphenicol-streptomycin-sulfisoxazole-tetracycline-trimethoprim-sulfamethoxazole. The genomes of 18 selected isolates were then sequenced and compared to 14 selected human pathogenic E. coli reference genomes obtained from public repositories using different bioinformatics approaches. As expected, all 18 sequenced isolates carried at least one β-lactamase bla gene: TEM-1, TEM-81, CTX-M115, CTX-M15, OXA-1, or CMY-2. Several other antibiotic resistance genes (ARGs) and virulence determinants were detected in the sequenced isolates and all of them harbored antimicrobial resistance plasmids belonging to classic Inc groups. Our results confirm the presence of diverse ESBL producing E. coli isolates in dairy cow manure stored for a short period of time. Such manure might constitute a reservoir of resistance and virulence genes for other bacteria that share the same environment.

Keywords: E. coli; cefotaxime; ceftiofur; dairy cow; manure; whole genome sequencing.

Figure 1

The temperature manipulations of manure samples. The graph shows isolation of ceftiofur or cefotaxime resistant Gram-negative bacteria from different manure samples incubated under anaerobic conditions for over 20 weeks at two temperature regimes (4 °C or at 25 °C). The gradual increase or decrease in temperature is shown in blue and red lines.

Figure 2

Antimicrobial Susceptibility test (AST). The AST results of the 18 E. coli selected isolates against 14 different antibiotics are presented as the percentage of isolates that were resistant (red), or susceptible (green) to the antibiotics listed on the y-axis.

Figure 3

Heat map showing the presence (green color) and absence (red color) of (a) 38 adherence factors; (b) 24 iron uptake proteins; (c) 22 toxins; and, (d) secretions factors, across 18 TIO or FOX resistant from day cow manure and 14 pathogenic reference E. coli genomes. The bottom labels indicate the gene names. The tree on the left show a hierarchical clustering of the labelled strains based on the genes profile.

Figure 3

Heat map showing the presence (green color) and absence (red color) of (a) 38 adherence factors; (b) 24 iron uptake proteins; (c) 22 toxins; and, (d) secretions factors, across 18 TIO or FOX resistant from day cow manure and 14 pathogenic reference E. coli genomes. The bottom labels indicate the gene names. The tree on the left show a hierarchical clustering of the labelled strains based on the genes profile.

Figure 3

Heat map showing the presence (green color) and absence (red color) of (a) 38 adherence factors; (b) 24 iron uptake proteins; (c) 22 toxins; and, (d) secretions factors, across 18 TIO or FOX resistant from day cow manure and 14 pathogenic reference E. coli genomes. The bottom labels indicate the gene names. The tree on the left show a hierarchical clustering of the labelled strains based on the genes profile.

Figure 3

Heat map showing the presence (green color) and absence (red color) of (a) 38 adherence factors; (b) 24 iron uptake proteins; (c) 22 toxins; and, (d) secretions factors, across 18 TIO or FOX resistant from day cow manure and 14 pathogenic reference E. coli genomes. The bottom labels indicate the gene names. The tree on the left show a hierarchical clustering of the labelled strains based on the genes profile.

Figure 4

The heat map showing the presence (green color) and absence (red color) of antibiotic resistance genes (ARGs) across 18 TIO or FOX resistant and 14 pathogenic reference E. coli genomes. The bottom labels indicate the gene names. The tree on the left shows a hierarchical clustering of the labelled strains based on the genes profile.

Figure 5

Nucleotide sequence alignments of bla_CMY-2 containing plasmids identified in three sequenced E. coli isolates in this study to the previously characterized 101,461 bp plasmid pCVM29199-101 (GenBank accession # NC_011077) of Salmonella Kentucky isolated from poultry. The asterisk above the alignment indicates the position of class C β-lactamase bla_CMY-2 present in all of the plasmids. The proteins absent in the plasmids are listed above the alignment. ParA and SugE are a chromosome partitioning and a quaternary ammonium compound-resistance protein, respectively. The E. coli isolates containing the plasmids are indicated on the right.

Figure 6

Schematic representation of the various gene cassette arrangements found in class 1 integrons detected in this study. The arrows display the open reading frames of the different genes. All aadA genes are presented as white arrows and dfrA genes as black arrows. The grey boxes indicate the 3′ and 5′ CSs regions typical of class 1 integrons harboring additional resistance determinants. On the right are the strains Ids containing the specific integron types. The gene cassettes are not drawn to scale.

Figure 7

Genome-wide phylogenetic tree produced in PhyML using the “Generalized Time Reversible” model of nucleotide substitution showing the relationship among 18 E. coli isolates (of 10 different serotypes) recovered from dairy cow manure and their comparison with 14 reference genomes. The red and blue box show related strains. The scale is number of substitutions per site.

Figure 8

Comparative genomic analysis of (a) E. coli O157:H7 (strain EDL933) with E. coli O8:H1 (ID 6297) and (b) E. coli 0104:H4 (strain 2011C-3493) with E. coli O25:H11 (ID 5951). The innermost ring shows the reference genomes coverage. The innermost second and third rings show GC content and GC Skew. The outer ring (pink) shows the sequence homology with the reference genomes and the outermost ring highlights the SP sites (prophage regions) annotated in black. The circular plot was generated using BLAST Ring Image Generator (BRIG) software (http://brig.sourceforge.net/).

Figure 8

Comparative genomic analysis of (a) E. coli O157:H7 (strain EDL933) with E. coli O8:H1 (ID 6297) and (b) E. coli 0104:H4 (strain 2011C-3493) with E. coli O25:H11 (ID 5951). The innermost ring shows the reference genomes coverage. The innermost second and third rings show GC content and GC Skew. The outer ring (pink) shows the sequence homology with the reference genomes and the outermost ring highlights the SP sites (prophage regions) annotated in black. The circular plot was generated using BLAST Ring Image Generator (BRIG) software (http://brig.sourceforge.net/).