Journal Articles
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Item 20 years later: unravelling the genomic success of New Zealand’s home-grown AK3 community-associated methicillin-resistant Staphylococcus aureus(Microbiology Society, 2025-07-25) White RT; Bakker S; Bloomfield M; Burton M; Elvy J; Eustace A; French NP; Grant J; Greening SS; Grinberg A; Harland C; Hutton S; Karkaba A; Martin J; Matthews B; Miller H; Straub C; Tarring C; Taylor WT; Ussher J; Velasco C; Voss EM; Dyet KMethicillin-resistant Staphylococcus aureus (MRSA) represents a significant public health challenge. In New Zealand, the community-associated MRSA sequence type (ST)5, carrying the staphylococcal cassette chromosome mec (SCCmec) type IV genetic element (which confers methicillin resistance), has been predominant since its detection in 2005. Known informally as the AK3 strain, it also exhibits resistance to fusidic acid. Here, we investigated the genomic evolution of the AK3 strain by analysing 397 genomes, comprising 361 MRSA and 36 closely related methicillin-susceptible S. aureus (MSSA) genomes, including 285 recently sequenced isolates from New Zealand spanning 2020 (n=30), 2021 (n=77), 2022 (n=88), 2023 (n=73) and 2024 (n=17). Phylogenetic analysis revealed that the AK3 strain evolved through stepwise acquisition of mobile genetic elements, with an MSSA ancestor likely introduced to New Zealand in the late 1970s. The lineage first acquired a SaPITokyo12571-like pathogenicity island, which contains the staphylococcal enterotoxin C bovine variant (sec-bov) and an enterotoxin-like protein (sel), between 1984 and 1991. This was followed by the integration of SCCmec type IV and adjacent fusidic acid resistance operon between 1997 and 2000. This timing coincides with increased community fusidic acid use in New Zealand. The AK3 strain then diversified into three major clades, spreading throughout New Zealand and Australia, with sporadic detection in European countries and Samoa. Our findings demonstrate how the sequential acquisition of mobile genetic elements, combined with antibiotic selection pressure, likely contributed to the successful emergence of AK3 and its spread in the South Pacific region.Item Mutations in the riboflavin biosynthesis pathway confer resistance to furazolidone and abolish the synergistic interaction between furazolidone and vancomycin in Escherichia coli.(Microbiology Society, England, 2025-02-11) Wykes H; Le VVH; Rakonjac JThe combined application of furazolidone and vancomycin has previously been shown to be synergistic against Gram-negative pathogens, with great therapeutic promise. However, the emergence and mechanism of resistance to this antibiotic combination have not been characterized. To fill this gap, we here selected Escherichia coli progeny for growth on the furazolidone-vancomycin combination at the concentration where the parent was sensitive. We show that selected clones were associated with increased resistance to neither, only one drug, or both furazolidone and vancomycin, but in all cases were associated with a decrease in the growth inhibition synergy. Using whole-genome sequencing, we identified various gene mutations in the resistant mutants. We further investigated the mechanism behind the most frequently arising mutations, those in the riboflavin biosynthesis genes ribB and ribE, that represent novel mutations causing furazolidone resistance and diminished vancomycin-furazolidone synergy. It was found that these ribB/ribE mutations act predominantly by decreasing the activity of the NfsA and NfsB nitroreductases. The emergence of the ribB/ribE mutations imposes a significant fitness cost on bacterial growth. Surprisingly, supplementing the medium with riboflavin, which compensates for the affected riboflavin biosynthesis pathway, could restore the normal growth of the ribB/ribE mutants while having no effects on the furazolidone resistance phenotype. Searching the ribB/ribE mutations in the public sequencing database detects the presence of the furazolidone-resistance-conferring ribE mutations (TKAG131-134 deletion or duplication) in clinical isolates from different countries. Hypotheses explaining why these ribE mutations were found in clinical isolates despite having poor fitness were further discussed.Item In vitro synergy of 5-nitrofurans, vancomycin and sodium deoxycholate against Gram-negative pathogens(Microbiology Society, 2021-01-15) Olivera C; Le VVH; Davenport C; Rakonjac JIntroduction. There is an urgent need for effective therapies against bacterial infections, especially those caused by antibiotic-resistant Gram-negative pathogens. Hypothesis. Synergistic combinations of existing antimicrobials show promise due to their enhanced efficacies and reduced dosages which can mitigate adverse effects, and therefore can be used as potential antibacterial therapy. Aim. In this study, we sought to characterize the in vitro interaction of 5-nitrofurans, vancomycin and sodium deoxycholate (NVD) against pathogenic bacteria. Methodology. The synergy of the NVD combination was investigated in terms of growth inhibition and bacterial killing using checkerboard and time-kill assays, respectively. Results. Using a three-dimensional checkerboard assay, we showed that 5-nitrofurans, sodium deoxycholate and vancomycin interact synergistically in the growth inhibition of 15 out of 20 Gram-negative strains tested, including clinically significant pathogens such as carbapenemase-producing Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii, and interact indifferently against the Gram-positive strains tested. The time-kill assay further confirmed that the triple combination was bactericidal in a synergistic manner. Conclusion. This study demonstrates the synergistic effect of 5-nitrofurans, sodium deoxycholate and vancomycin against Gram-negative pathogens and highlights the potential of the combination as a treatment for Gram-negative and Gram-positive infections.Item Correlated transcriptional responses provide insights into the synergy mechanisms of the furazolidone, vancomycin and sodium deoxycholate triple combination in Escherichia coli(American Society for Microbiology, 2021-10-27) Olivera C; Cox MP; Rowlands GJ; Rakonjac J; Bradford PAEffective therapeutic options are urgently needed to tackle antibiotic resistance. Furazolidone (FZ), vancomycin (VAN), and sodium deoxycholate (DOC) show promise as their combination can synergistically inhibit the growth of, and kill, multidrug-resistant Gram-negative bacteria that are classified as critical priority by the World Health Organization. Here, we investigated the mechanisms of action and synergy of this drug combination using a transcriptomics approach in the model bacterium Escherichia coli. We show that FZ and DOC elicit highly similar gene perturbations indicative of iron starvation, decreased respiration and metabolism, and translational stress. In contrast, VAN induced envelope stress responses, in agreement with its known role in peptidoglycan synthesis inhibition. FZ induces the SOS response consistent with its DNA-damaging effects, but we demonstrate that using FZ in combination with the other two compounds enables lower dosages and largely mitigates its mutagenic effects. Based on the gene expression changes identified, we propose a synergy mechanism where the combined effects of FZ, VAN, and DOC amplify damage to Gram-negative bacteria while simultaneously suppressing antibiotic resistance mechanisms. IMPORTANCE Synergistic antibiotic combinations are a promising alternative strategy for developing effective therapies for multidrug-resistant bacterial infections. The synergistic combination of the existing antibiotics nitrofurans and vancomycin with sodium deoxycholate shows promise in inhibiting and killing multidrug-resistant Gram-negative bacteria. We examined the mechanism of action and synergy of these three antibacterials and proposed a mechanistic basis for their synergy. Our results highlight much-needed mechanistic information necessary to advance this combination as a potential therapy.Item Co-selection of Heavy Metal and Antibiotic Resistance in Soil Bacteria from Agricultural Soils in New Zealand(MDPI (Basel, Switzerland), 2022-02-04) Heydari A; Kim ND; Horswell J; Gielen G; Siggins A; Taylor M; Bromhead C; Palmer BRAccumulation of trace elements (including heavy metals) in soil from usage of superphos-phate fertilisers induces resistance of soil bacteria to trace elements of environmental concern (TE-oEC) and may co‐select for resistance to antibiotics (Ab). This study aimed to investigate selection of co‐resistance of soil bacteria to Cd, Zn and Hg, and Ab in soils with varied management histories. Genetic diversity of these bacteria and horizontal transfer of Cd resistance genes (cadA and czcA) were also investigated. Soils with either pastoral and arable management histories and either high levels of Cd and Zn, or indigenous bush with background levels of these TEoEC from the Waikato region, New Zealand were sampled. Plate culturing with a range of TEoEC and Ab concentrations, Pollution Induced Community Tolerance (PICT) assay, antibiotic sensitivity, terminal restriction fragment length polymorphism (TRFLP) and horizontal gene transfer (HGT) analyses were em-ployed to investigate co‐selection of TEoEC and Ab resistance. Higher levels of bacterial resistance to TEoEC and Ab correlated with higher levels of TEoEC in soil. Bacterial community structures were altered in soils with high TEoEC levels. Cd resistance genes were transferred from donor bacterial isolates, to recipients and the transconjugants also had resistance to Zn and/or Hg and a range of Ab.Item Co-Selection of Bacterial Metal and Antibiotic Resistance in Soil Laboratory Microcosms.(18/04/2023) Heydari A; Kim ND; Biggs PJ; Horswell J; Gielen GJHP; Siggins A; Taylor MD; Bromhead C; Palmer BRAccumulation of heavy metals (HMs) in agricultural soil following the application of superphosphate fertilisers seems to induce resistance of soil bacteria to HMs and appears to co-select for resistance to antibiotics (Ab). This study aimed to investigate the selection of co-resistance of soil bacteria to HMs and Ab in uncontaminated soil incubated for 6 weeks at 25 °C in laboratory microcosms spiked with ranges of concentrations of cadmium (Cd), zinc (Zn) and mercury (Hg). Co-selection of HM and Ab resistance was assessed using plate culture on media with a range of HM and Ab concentrations, and pollution-induced community tolerance (PICT) assays. Bacterial diversity was profiled via terminal restriction fragment length polymorphism (TRFLP) assay and 16S rDNA sequencing of genomic DNA isolated from selected microcosms. Based on sequence data, the microbial communities exposed to HMs were found to differ significantly compared to control microcosms with no added HM across a range of taxonomic levels.Item Correlated Transcriptional Responses Provide Insights into the Synergy Mechanisms of the Furazolidone, Vancomycin, and Sodium Deoxycholate Triple Combination in Escherichia coli.(27/10/2021) Olivera C; Cox MP; Rowlands GJ; Rakonjac JEffective therapeutic options are urgently needed to tackle antibiotic resistance. Furazolidone (FZ), vancomycin (VAN), and sodium deoxycholate (DOC) show promise as their combination can synergistically inhibit the growth of, and kill, multidrug-resistant Gram-negative bacteria that are classified as critical priority by the World Health Organization. Here, we investigated the mechanisms of action and synergy of this drug combination using a transcriptomics approach in the model bacterium Escherichia coli. We show that FZ and DOC elicit highly similar gene perturbations indicative of iron starvation, decreased respiration and metabolism, and translational stress. In contrast, VAN induced envelope stress responses, in agreement with its known role in peptidoglycan synthesis inhibition. FZ induces the SOS response consistent with its DNA-damaging effects, but we demonstrate that using FZ in combination with the other two compounds enables lower dosages and largely mitigates its mutagenic effects. Based on the gene expression changes identified, we propose a synergy mechanism where the combined effects of FZ, VAN, and DOC amplify damage to Gram-negative bacteria while simultaneously suppressing antibiotic resistance mechanisms. IMPORTANCE Synergistic antibiotic combinations are a promising alternative strategy for developing effective therapies for multidrug-resistant bacterial infections. The synergistic combination of the existing antibiotics nitrofurans and vancomycin with sodium deoxycholate shows promise in inhibiting and killing multidrug-resistant Gram-negative bacteria. We examined the mechanism of action and synergy of these three antibacterials and proposed a mechanistic basis for their synergy. Our results highlight much-needed mechanistic information necessary to advance this combination as a potential therapy.Item Carriage of Extended-Spectrum-Beta-Lactamase- and AmpC Beta-Lactamase-Producing Escherichia coli Strains from Humans and Pets in the Same Households.(American Society for Microbiology, 24/11/2020) Toombs-Ruane LJ; Benschop J; French NP; Biggs PJ; Midwinter AC; Marshall JC; Chan M; Drinković D; Fayaz A; Baker MG; Douwes J; Roberts MG; Burgess SAExtended-spectrum-beta-lactamase (ESBL)- or AmpC beta-lactamase (ACBL)-producing Escherichia coli bacteria are the most common cause of community-acquired multidrug-resistant urinary tract infections (UTIs) in New Zealand. The carriage of antimicrobial-resistant bacteria has been found in both people and pets from the same household; thus, the home environment may be a place where antimicrobial-resistant bacteria are shared between humans and pets. In this study, we sought to determine whether members (pets and people) of the households of human index cases with a UTI caused by an ESBL- or ACBL-producing E. coli strain also carried an ESBL- or ACBL-producing Enterobacteriaceae strain and, if so, whether it was a clonal match to the index case clinical strain. Index cases with a community-acquired UTI were recruited based on antimicrobial susceptibility testing of urine isolates. Fecal samples were collected from 18 non-index case people and 36 pets across 27 households. Eleven of the 27 households screened had non-index case household members (8/18 people and 5/36 animals) positive for ESBL- and/or ACBL-producing E. coli strains. Whole-genome sequence analysis of 125 E. coli isolates (including the clinical urine isolates) from these 11 households showed that within seven households, the same strain of ESBL-/ACBL-producing E. coli was cultured from both the index case and another person (5/11 households) or pet dog (2/11 households). These results suggest that transmission within the household may contribute to the community spread of ESBL- or ACBL-producing E. coliIMPORTANCEEnterobacteriaceae that produce extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases (ACBLs) are important pathogens and can cause community-acquired illnesses, such as urinary tract infections (UTIs). Fecal carriage of these resistant bacteria by companion animals may pose a risk for transmission to humans. Our work evaluated the sharing of ESBL- and ACBL-producing E. coli isolates between humans and companion animals. We found that in some households, dogs carried the same strain of ESBL-producing E. coli as the household member with a UTI. This suggests that transmission events between humans and animals (or vice versa) are likely occurring within the home environment and, therefore, the community as a whole. This is significant from a health perspective, when considering measures to minimize community transmission, and highlights that in order to manage community spread, we need to consider interventions at the household level.