Integrative Conjugative Element ICE Hs 1 Encodes for Antimicrobial Resistance and Metal Tolerance in Histophilus somni

Abstract

The objectives of this study were to determine antimicrobial resistance and metal tolerance, and identify associated genes and mobile genetic elements in clinical strains of Histophilus somni isolated from feedlot cattle in Alberta during years 2012-2016 (contemporary isolates, n = 63) and years 1980-1990 (historical isolates, n = 31). Comparison of antimicrobial resistance (AMR) showed a significant increase in resistance among contemporary isolates compared to historical isolates (P < 0.001). Tolerance to copper (Cu) and zinc (Zn) concentrations above 1 mM was observed in 68 and 52% of the contemporary isolates, respectively. The tet(H) gene associated with oxytetracycline resistance and multicopper oxidase (mco) and cation efflux (czcD) genes associated with Cu and Zn tolerance were identified. An integrative conjugative element; ICEHs1, was identified in whole genome sequences of strains resistant to oxytetracycline, which had Cu and Zn minimum inhibitory concentrations (MIC) >1 mM. The length of ICEHs1 was 64,932 bp and it contained 83 genes, including tetracycline resistance gene tetH, a multidrug efflux pump gene ebrB, and metal tolerance genes mco, czcD, and acr3. Comparative genomics of ICEs revealed that ICEHs1 shares high homology with previously described ICEs of Histophilus somni, Pasteurella multocida, and Mannheimia haemolytica. The ICEHs1 is an active element capable of intra- and inter-genus transfer as demonstrated by successful transfer to H. somni and P. multocida recipients. All isolates carrying ICEHs1 were resistant to tetracycline, a commonly used antibiotic in feedlots, and had Cu and Zn MIC higher than 1 mM. Since Cu and Zn are routinely used in feedlots, there is the possibility of co-selection of AMR in H. somni due to selection pressure created by Cu and Zn. Based on results of in-vitro conjugation experiments, ICEHs1 mediated transmission of antimicrobial and metal resistance genes is possible between BRD pathogens in the respiratory tract, potentially undermining treatment options available for histophilosis and BRD.

Keywords: Histophilus somni; antimicrobial resistance; copper; feedlot cattle; integrative conjugative element; metal tolerance; zinc.

Figure 1

Percentage of antimicrobial resistant contemporary (2012–2016), (n = 63) and historical (1980's), (n = 31) clinical isolates of Histohilus somni from feedlot cattle in Alberta. Fisher's exact test was used for statistical comparison of antimicrobial resistance between contemporary and historical isolates.

Figure 2

Distribution of minimum inhibitory concentration (mM) of copper (left) and zinc (right) for 63 contemporary (2012–2016) and 31 historical (1980's) isolates of Histophilus somni. The MIC value for the isolates tested was in triplicate in three replicate experiments (Experiments 1 and 2). The median value of copper and zinc in contemporary and historical isolates was consistent in all three experiments, indicating the reproducibility of the metal tolerance assay. A Mann-Whitney U test was used for comparing median MIC between historical and contemporary isolates and a significant difference was detected (P < 0.001).

Figure 3

Schematic structure of ICEHs1 identified in this study compared to previously described ICEHso2336. The 83 open reading frames (ORFs) present in the ICEHs1 are represented by arrows with the arrowhead representing direction of transcription. The ORFs are color coded according to their biological function. The area between the ICEs shaded in dark gray indicates 100% and light gray indicates 67% shared sequence similarity based on the BLASTn. Function of genes present in ICE region have been inferred from BLAST hits and annotation with PROKKA.

Figure 4

Schematic comparison of ICEHs1 identified in this study with ICEPmu1 and ICEMh1. Open reading frames (ORFs) present in each ICE are represented by arrows with the arrowhead representing direction of transcription. The ORFs are color coded according to their biological function. The areas between the ICEs shaded in dark gray indicates 100% and light gray indicates 67% shared sequence similarity based on the BLASTn. Function of genes present in ICE region have been inferred from BLAST hits and annotation with PROKKA. These three ICEs from three common BRD pathogens (P. multocida, H. somni, and M. haemolytica) share 90% nucleotide identity. ICEPmu1 and ICEMh1 carry 12 and 5 antimicrobial resistance genes, respectively. However, ICEHs1 carries only tet(H) and a multidrug efflux pump gene (ebrB), along with 3 different metal tolerance genes, among which only two were present in ICEMh1 and ICEPmu1.