Antimicrobial Resistance Profile of Zoonotic Clinically Relevant WHO Priority Pathogens

Abstract

The World Health Organization announced critically important bacterial and fungal pathogens displaying alarming levels of antimicrobial resistance, which currently represent difficult-to-treat cases of morbidity. Within this grouping, the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are causative of significant morbidity and mortality. Studies described herein demonstrate the presence of critically important fungal and ESKAPE bacterial species in companion animals which are zoonotic in nature. The relationship between the environment, animals, and human infectious disease has long been recognized as part of One Health. This research investigates the resistance patterns of isolated zoonotic pathogens using recognized in vitro methodologies, namely disk diffusion, minimum inhibitory concentration testing, and genetic screening. Antibiotic susceptibility testing and gene analysis demonstrated an association between multi-drug resistance and extended beta spectrum lactamase production in critical-priority bacteria. Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa exhibit great levels of multi-drug resistance. Fungal isolates demonstrated high levels of resistance, with Amphotericin B proving the most effective antifungal agent investigated. The level of antimicrobial resistance present in clinically relevant bacterial and fungal pathogens isolated from animal cases of morbidity in this study is alarming. In conclusion, this study shows that animals can act as a reservoir facilitating the transmission of antibiotic-resistant pathogens and genes zoonotically.

Keywords: ESKAPE; critical; fungal; mortality; resistance; zoonosis.

Figure 1

Schematic diagram detailing (1) how samples of infection were collected and received, (2) how samples were cultured, (3) how colonies were isolated, and (4) how isolates were identified based on growth on selective agars and PCR.

Figure 2

Growth of ESBL-producing (a) E. coli (pink-reddish-mauve colonies indicating a positive result) and (b) K. pneumonia (green-blue colonies indicating a positive result) isolates post 24 h incubation at 37 °C on ‘Chromatic ESBL’ chromogenic media for determination of ESBLs in Enterobacterale species. Reading of results for chromogenic agar is followed as per manufacture guidelines as provided on the manufacturer’s website. See link CHROMagar™ ESBL-Chromagar (https://www.chromagar.com/en/product/chromagar-esbl/, accessed on 7 November 2024).

Figure 3

Double Disk Synergy test for phenotypic analysis of ESBL activity in Gram-negative Enterobacterale isolates using cefpodoxime (CPD) and cefpodoxime + clavulanic acid (CD) disks. ESBL production was judged if the zone diameter ratios for the antibiotic comparative to antibiotic plus inhibitor was ≥5 mm. Imaged (a) E. coli (testing positive for ESBL production), (b) K. pneumoniae (testing positive for ESBL production), and (c) A. asburiae (testing negative for ESBL production). Reading of results for Double Disk Synergy testing is followed as per manufacturer guidelines.

Figure 4

Phenotypic confirmation of AmpC production in Enterobacterale isolates (a) K. pneumoniae and (b) E. coli using MIC combination test strips containing cefotetan (CTT)/cefotetan + cloxacillin (CXT). AmpC production was assumed if the MIC ratios of antibiotic comparative to antibiotic plus inhibitor was ≥8.

Figure 5

Agarose gel electrophoresis of PCR products of (a) ESBL and (b) AmpC-producing isolates. Image 4 (a) Lane 1: 1 kb DNA ladder; Lane 2: quality control strain carrying bla_TEM gene (positive control); Lane 3: negative control; Lane 4: bla_TEM positive (A. baumannii); Lane 5: bla_TEM positive (K. pneumoniae); Lane 6: bla_TEM positive (E. coli). Image 4 (b) Lane 1: negative control; Lane 2: quality control strain carrying bla_AmpC gene (positive control); Lane 3: bla_AmpC positive (E. coli); Lane 4: bla_AmpC positive (P. aeruginosa); Lane 5: 1 kb DNA ladder.