Investigation of antimicrobial resistance and its relation with mobile genetic elements among Australian equine Klebsiella isolates
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2026
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Melbourne Authors
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Minerva Elements
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PhD thesis
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This item is embargoed and will be available on 2028-10-21.
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© 2026 Amin Kawarizadeh
Abstract
Antimicrobial resistance (AMR) is a global crisis that can threaten human and animal health. AMR can complicate the treatment and management of bacterial infections, leading to increased economic burden in veterinary settings. In addition, close contact between animals and humans may facilitate the transmission of resistant and potentially virulent bacterial strains across the animal-human interface. This study focuses on Klebsiella pneumoniae which is a Gram-negative bacterium with a great potential to acquire and spread antimicrobial resistance genes (ARGs). In this study the frequency of AMR among presumptive K. pneumoniae isolates in reproductive and non-reproductive samples from horses located in New South Wales, Australia between 2020 and 2022 was studied. Additionally, the genetic diversity and the potential role of mobile genetic elements (MGEs) in distribution and development of ARGs among these isolates were studied. The genome and AMR profile of a Raoultella terrigena isolate, which was initially misidentified as K. pneumoniae using conventional laboratory methods was thoroughly investigated. This isolate exhibited extensively drug-resistant (XDR) profile.
Clinical isolates were initially identified as presumptive K. pneumoniae in a private diagnostic laboratory based on the combination of colony morphology, biochemical and motility tests. Following transfer to the University of Melbourne, antimicrobial susceptibility testing was performed using broth microdilution method with the SensititreTM system, and the results were interpreted according to the guidelines of the Clinical & Laboratory Standards Institute (CLSI). All isolates were further characterized to assess their hypermucoviscosity phenotype, and their virulence was evaluated using a silk moth larvae infection model. Subsequently, all isolates underwent short-read sequencing using Illumina NovaSeq 6000 platform. The resulting sequencing data were used for taxonomic classification of the presumptive K. pneumoniae isolates. In addition, these sequencing data were used to identify ARGs and virulence-associated genes, and to perform capsule locus typing of the isolates. Furthermore, whole genome sequencing data were utilized for phylogenetic analysis to investigate the genetic relatedness between the isolates in this study and previously characterized strains available in public databases. Finally, a subset of multidrug-resistant (MDR) reproductive isolates was selected for long-read sequencing using Oxford Nanopore technology. These isolates were chosen based on their sequence type, ARGs profile, and plasmid content. Hybrid genome assemblies were generated by combining short- and long-read data, and the resulting assemblies were used to examine the association between ARGs and MGEs.
In this study, a total of 25,513 samples, including 23,772 reproductive and 1,741 non-reproductive samples were tested by the laboratory in NSW. From these, 120 isolates were recovered from reproductive samples, among them 117 presumptive K. pneumoniae isolates were available for further analysis. Furthermore, 24 isolates were recovered from non-reproductive isolates, among them 13 isolates were available for further analysis. Genomic analysis revealed that 14 of the reproductive isolates and one non-reproductive isolate were species other than K. pneumoniae. Antimicrobial susceptibility testing revealed that 55% of reproductive isolates and 70% of non-reproductive isolates were classified as MDR. Additionally, resistance to critically important antimicrobial, as defined by the World Health Organization, was observed, with rates among reproductive and non-reproductive isolates of 38% and 30% for ceftazidime, 52% and 50% for ceftiofur and 47% and 60% for enrofloxacin, respectively.
Among reproductive isolates from mares, over breeding season (August-December), a strong positive relationship between months and the isolation rate of MDR isolates (R2 = 0.960, p = 0.003) was observed.
Hypermucoviscosity phenotype was observed in 14% and 20% of reproductive and non-reproductive isolates, respectively. Among clinically relevant virulence loci, the most prevalent loci in reproductive and non-reproductive isolates were yersiniabactin (74% and 70%, respectively), followed by salmochelin (16% and 20%, respectively). Additionally, among reproductive isolates, a small subset of isolates carried colibactin (9%) and aerobactin (2%). No significant trend in mean virulence score over the month of breading season (R2 = 0.459, p = 0.209) was observed.
Genomic analysis revealed a high level of genetic diversity among the recovered isolates, particularly among reproductive isolates, in which 31 sequence types and 23 capsule locus types were identified. Among them, ST323 (29%) and ST145 (10%) were the most prevalent sequence types, while KL30 (42%) and KL22 (9%) were the most identified capsule locus types. Phylogenetic analysis of reproductive isolates revealed that 52 isolates belonging to eight sequence types in this study were closely related to equine isolates reported in horses from Australia and France in publicly available databases. Additionally, phylogenetic analysis of isolates belonging to ST323 demonstrated a high level of similarity to other equine isolates from Australia, as well as to a human-derived isolate reported in Australia. Among non-reproductive isolates, ST39 (30%) and KL149 (30%) were the most frequently identified sequence type and capsule locus type. Phylogenetic analysis demonstrated a substantial genetic divergence between isolates belonging to ST39 in this study and human-derived isolates of this sequence type from Australia.
Additionally, all isolates that were initially misidentified as K. pneumoniae were susceptible to all tested antimicrobials, except for one isolate recovered from a uterine lavage of a mare. Following genomic analysis, this isolate was identified as R. terrigena and exhibited an XDR profile. Phylogenetic analysis of this isolate, together with other isolates of R. terrigena available in publicly accessible databases, revealed two distinct clades, with equine isolates clustering together with other animal-derived isolates.
Among reproductive MDR K. pneumoniae isolates, the genes conferring resistance to critically important antimicrobials were blaCTX-M-15 (68%), blaCTX-M-14 (16%) and blaCMY-2 (12%), associated with resistance to 3rd generation cephalosporins, as well as qnrB1(66%) and qnrS1(20%) associated with fluoroquinolone resistance. Among non-reproductive isolates blaCTX-M-15 (30%) and blaCTX-M-14 (20%), were linked to 3rd generation cephalosporin resistance, and qnrS1(30%) and qnrB1(30%) to fluoroquinolone resistance. In R. terrigena isolate, blaCTX-M-14 associated with 3rd generation cephalosporin resistance, was identified. However, despite the presence of qnrB6, no phenotypic resistance to fluoroquinolones was observed.
Analysis of the association between ARGs and MGEs demonstrated that, among reproductive MDR K. pneumoniae isolates, most acquired resistance determinants are in association with transposons, integrons and insertion sequences and were carried on IncC and hybrid and non-hybrid variants of IncF plasmids. Furthermore, multiple chromosomal regions linked to acquired resistance genes were identified. Among these, a chromosomal copy of blaCTX-M-14 associated with IS5075, IS903 and ISEcp1 elements was identified, not previously reported in K. pneumoniae. In the R. terrigena isolate, all acquired resistance genes (n=14), including blaCTX-M-14 and qnrB6 were identified within three mobile genetic element regions carried on an IncQ1/IncU plasmid.
This study represents the largest combined genomic and phenotypic investigation of reproductive K. pneumoniae isolates from horses worldwide, as well as the largest investigation of non-reproductive K. pneumoniae isolates from horses in Australia. The findings of this study indicated a high level of AMR, virulence and genetic diversity among K. pneumoniae isolates from horses. The identified resistance genes were frequently associated with MGEs, suggesting their potential role in the dissemination of resistance determinants. Furthermore, the close genetic relatedness between some isolates from this study and human-derived isolates suggests a potential risk for interspecies transmission of this bacterium. In addition, the presence of similar lineages of K. pneumoniae among horses in Australia and France may reflect their wider dissemination. These findings underscore the importance of continuous monitoring and antimicrobial stewardship programs in veterinary settings.
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Keywords
Klebsiella pneumoniae, Raoultella terrigena, Horse, Antimicrobial Resistance Genes, Whole Genome Sequencing, Phylogenetic Analysis, Veterinary Microbiology