Publications
21 results found
Di Pilato V, Codda G, Niccolai C, et al., 2024, Functional features of KPC-109, a novel 270-loop KPC-3 mutant mediating resistance to avibactam-based β-lactamase inhibitor combinations and cefiderocol., Int J Antimicrob Agents, Vol: 63
OBJECTIVES: To investigate a ceftazidime/avibactam (CZA)-resistant Klebsiella pneumoniae (NE368), isolated from a patient exposed to CZA, expressing a novel K. pneumoniae carbapenemase (KPC)-3 variant (KPC-109). METHODS: Antimicrobial susceptibility testing was performed by reference broth microdilution. Whole-genome sequencing (WGS) analysis of NE368 was performed combining a short- and long-reads approach (Illumina and Oxford Nanopore Technologies). Functional characterization of KPC-109 was performed to investigate the impact of KPC-109 production on the β-lactam resistance phenotype of various Escherichia coli and Klebsiella pneumoniae strains, including derivatives of K. pneumoniae with OmpK35 and OmpK36 porin alterations. Horizontal transfer of the KPC-109-encoding plasmid was investigated by conjugation and transformation experiments. RESULTS: K. pneumoniae NE368 was isolated from a patient after repeated CZA exposure, and showed resistance to CZA, fluoroquinolones, piperacillin/tazobactam, expanded-spectrum cephalosporins, amikacin, carbapenems and cefiderocol. WGS revealed the presence of a large chimeric plasmid of original structure (pKPN-NE368), encoding a novel 270-loop mutated KPC-3 variant (KPC-109; ins_270_KYNKDD). KPC-109 production mediated resistance/decreased susceptibility to avibactam-based combinations (with ceftazidime, cefepime and aztreonam) and cefiderocol, with a trade-off on carbapenem resistance. However, in the presence of porin alterations commonly encountered in high-risk clonal lineages of K. pneumoniae, KPC-109 was also able to confer clinical-level resistance to carbapenems. Resistance of NE368 to cefiderocol was likely contributed by KPC-109 production acting in concert with a mutated EnvZ sensor kinase. The KPC-109-encoding plasmid did not appear to be conjugative. CONCLUSIONS: These findings expand current knowledge about the diversity of emerging KPC enzyme variants with 270-loop alterations that can be encountered in the
Frankel G, David S, Low WW, et al., 2023, Plasmids pick a bacterial partner before committing to conjugation, NUCLEIC ACIDS RESEARCH, Vol: 51, Pages: 8925-8933, ISSN: 0305-1048
Wong J, David S, Sanchez Garrido J, et al., 2022, Recurrent emergence of Klebsiella pneumoniae carbapenem resistance mediated by an inhibitory ompK36 mRNA secondary structure, Proceedings of the National Academy of Sciences of USA, Vol: 119, Pages: 1-12, ISSN: 0027-8424
Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In Klebsiella pneumoniae (KP), modifications in the porin OmpK36 are implicated in increasing resistance to carbapenems. Analysis of large KP genome collections, encompassing major healthcare-associated clones, revealed the recurrent emergence of a synonymous cytosine to thymine transition at position 25 (25c>t) in ompK36. We show that the 25c>t transition increases carbapenem resistance through depletion of OmpK36 from the outer membrane. The mutation attenuates KP in a murine pneumonia model, which accounts for its limited clonal expansion observed by phylogenetic analysis. However, in the context of carbapenem treatment, the 25c>t transition tips the balance towards treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c>t transition mediates an intramolecular mRNA interaction between a uracil encoded by 25t and the first adenine within the Shine-Dalgarno sequence. This specific interaction leads to the formation of an RNA stem structure, which obscures the ribosomal binding site thus disrupting translation. While mutations reducing OmpK36 expression via transcriptional silencing are known, we uniquely demonstrate the repeated selection of a synonymous ompK36 mutation mediating translational suppression in response to antibiotic pressure.
David S, Wong JLC, Sanchez-Garrido J, et al., 2022, Widespread emergence of OmpK36 loop 3 insertions among multidrug-resistant clones of Klebsiella pneumoniae., PLoS Pathogens, Vol: 18, Pages: 1-23, ISSN: 1553-7366
Mutations in outer membrane porins act in synergy with carbapenemase enzymes to increase carbapenem resistance in the important nosocomial pathogen, Klebsiella pneumoniae (KP). A key example is a di-amino acid insertion, Glycine-Aspartate (GD), in the extracellular loop 3 (L3) region of OmpK36 which constricts the pore and restricts entry of carbapenems into the bacterial cell. Here we combined genomic and experimental approaches to characterise the diversity, spread and impact of different L3 insertion types in OmpK36. We identified L3 insertions in 3588 (24.1%) of 14,888 KP genomes with an intact ompK36 gene from a global collection. GD insertions were most common, with a high concentration in the ST258/512 clone that has spread widely in Europe and the Americas. Aspartate (D) and Threonine-Aspartate (TD) insertions were prevalent in genomes from Asia, due in part to acquisitions by KP sequence types ST16 and ST231 and subsequent clonal expansions. By solving the crystal structures of novel OmpK36 variants, we found that the TD insertion causes a pore constriction of 41%, significantly greater than that achieved by GD (10%) or D (8%), resulting in the highest levels of resistance to selected antibiotics. We show that in the absence of antibiotics KP mutants harbouring these L3 insertions exhibit both an in vitro and in vivo competitive disadvantage relative to the isogenic parental strain expressing wild type OmpK36. We propose that this explains the reversion of GD and TD insertions observed at low frequency among KP genomes. Finally, we demonstrate that strains expressing L3 insertions remain susceptible to drugs targeting carbapenemase-producing KP, including novel beta lactam-beta lactamase inhibitor combinations. This study provides a contemporary global view of OmpK36-mediated resistance mechanisms in KP, integrating surveillance and experimental data to guide treatment and drug development strategies.
Low WW, Wong J, Beltran L, et al., 2022, Mating pair stabilization mediates bacterial conjugation species specificity, Nature Microbiology, Vol: 7, Pages: 1016-1027, ISSN: 2058-5276
Bacterial conjugation mediates contact-dependent transfer of DNA from donor to recipient bacteria, thus facilitating thespread of virulence and resistance plasmids. Here we describe how variants of the plasmid-encoded donor outer membrane(OM) protein TraN cooperate with distinct OM receptors in recipients to mediate mating pair stabilization and efficient DNAtransfer. We show that TraN from the plasmids pKpQIL (Klebsiella pneumoniae), R100-1 (Shigella flexneri) and pSLT (SalmonellaTyphimurium), and the prototypical F plasmid (Escherichia coli) interact with OmpK36, OmpW and OmpA, respectively.Cryo-EM analysis revealed that TraN pKpQIL interacts with OmpK36 through the insertion of a β-hairpin in the tip of TraN intoa monomer of the OmpK36 trimer. Combining bioinformatic analysis with AlphaFold structural predictions, we identified afourth TraN structural variant that mediates mating pair stabilization by binding OmpF. Accordingly, we devised a classifica-tion scheme for TraN homologues on the basis of structural similarity and their associated receptors: TraNα (OmpW), TraNβ(OmpK36), TraNγ (OmpA), TraNδ (OmpF). These TraN-OM receptor pairings have real-world implications as they reflect thedistribution of resistance plasmids within clinical Enterobacteriaceae isolates, demonstrating the importance of mating pairstabilization in mediating conjugation species specificity. These findings will allow us to predict the distribution of emergingresistance plasmids in high-risk bacterial pathogens.
Stephens J, Wong J, Broomhead R, et al., 2021, Raised serum amylase in patients with COVID-19 may not be associated with pancreatitis, British Journal of Surgery, Vol: 108, Pages: 152-153, ISSN: 0007-1323
Zheng W, Pena A, Low WW, et al., 2020, Cryoelectron-Microscopic Structure of the pKpQIL Conjugative Pili from Carbapenem-Resistant <i>Klebsiella pneumoniae</i>, STRUCTURE, Vol: 28, Pages: 1321-+, ISSN: 0969-2126
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Wong JLC, Romano M, Kerry LE, et al., 2019, OmpK36-mediated Carbapenem resistance attenuates ST258 <i>Klebsiella pneumoniae</i> in vivo, NATURE COMMUNICATIONS, Vol: 10, ISSN: 2041-1723
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- Citations: 59
Wong JLC, Mason A, Gordon A, et al., 2018, Are large randomized controlled trials in severe sepsis and septic shock statistically disadvantaged by repeated inadvertent underestimates of required sample size, BMJ Open, Vol: 8, ISSN: 2044-6055
Objectives: We sought to understand why randomized controlled trials in septic shock have failed to demonstrate effectiveness in the face of improving overall outcomes for patients and seemingly promising results of early phase trials of interventions. Design: We performed a retrospective analysis of large critical care trials of severe sepsis and septic shock. Data were collected from the primary trial manuscripts, pre-published statistical plans or by direct communication with corresponding authors. Setting: Critical care randomized control trials in severe sepsis and septic shock. Participants: 14619 patients randomized in 13 trials published between 2005 to 2015, enrolling greater than 500 patients and powered to a primary outcome of mortality. Intervention: Multiple interventions including the evaluation of treatment strategies and novel therapeutics. Primary and secondary outcome measures: Our primary outcome measure was the difference between the anticipated and actual control arm mortality. Secondary analysis examined the actual effect size and the anticipated effect size employed in sample size calculation. Results: In this post-hoc analysis of 13 trials with 14 619 patients randomised, we highlight a global tendency to overestimate control arm mortality in estimating sample size (absolute difference 9.8%, 95% confidence interval, -14.7% to -5%, p<0.001). When we compared anticipated and actual effect size of a treatment there was also a substantial overestimation in proposed values (absolute difference 7.4%, 95% confidence interval -9.0% to -5.8%, p<0.0001). Conclusions: An interpretation of our results is that trials are consistently underpowered in the planning phase by employing erroneous variables to calculate a satisfactory sample size. Our analysis cannot establish if, given a larger sample size, a trial would have had a positive result. It is disappointing so many promising phase II res
Mullineaux-Sanders C, Colins JW, Ruano-Gallego D, et al., 2017, Citrobacter rodentium relies on commensals for colonization of the colonic mucosa, Cell Reports, Vol: 21, Pages: 3381-3389, ISSN: 2211-1247
We investigated the role of commensals at the peak of infection with the colonic mouse pathogen Citrobacter rodentium. Bioluminescent and kanamycin (Kan)-resistant C. rodentium persisted avirulently in the cecal lumen of mice continuously treated with Kan. A single Kan treatment was sufficient to displace C. rodentium from the colonic mucosa, a phenomenon not observed following treatment with vancomycin (Van) or metronidazole (Met). Kan, Van, and Met induce distinct dysbiosis, suggesting C. rodentium relies on specific commensals for colonic colonization. Expression of the master virulence regulator ler is induced in germ-free mice, yet C. rodentium is only seen in the cecal lumen. Moreover, in conventional mice, a single Kan treatment was sufficient to displace C. rodentium constitutively expressing Ler from the colonic mucosa. These results show that expression of virulence genes is not sufficient for colonization of the colonic mucosa and that commensals are essential for a physiological infection course.
Grailey KE, Singleton JM, Simon JB, et al., 2015, Peaks and Troughs: Variations in the Availability of Therapeutic Drug Monitoring in Critical Care Units across London
Singleton J, Grailey KE, Simon JB, et al., 2015, Variability in aminoglycoside dosing in Intensive Care Units across London – should our methods be standardized?
Cunningham ME, Javaid A, Waters J, et al., 2015, Development and validation of a “capture‐fusion” model to study drug sensitivity of patient‐derived hepatitis C, Hepatology, Vol: 61, Pages: 1192-1204, ISSN: 0270-9139
<jats:p>Emerging therapies for chronic hepatitis C viral (HCV) infection involve inhibition of viral enzymes with drug combinations. Natural, or treatment‐induced, enzyme polymorphisms reduce efficacy. We developed a phenotyping assay to aid drug selection based on viral transfer from monocytes to hepatocytes. We studied HCV in monocytes from infected patients and developed a model in which patient‐derived HCV is “captured” by the cell line THP‐1 and replication assessed after fusion to hepatoma cells. We found that monocytes from HCV‐infected patients harbor virus that replicates when cells are fused to hepatocytes. THP‐1 cells incubated with infected sera capture HCV, which replicates when fused to hepatocytes. Inhibitable replication of all HCV genotypes was achieved (42 of 52 isolates). We measured sensitivity of telaprevir (TVR) and alisporivir (AVR) in different genotypes, and showed differences in 50% inhibitory concentration (IC<jats:sub>50</jats:sub>) correlating with clinical response (TVR IC<jats:sub>50</jats:sub> for genotype (G)1 was 0.042 ± 0.003 vs. 0.117 ± 0.015 μM for G3, whereas AVR IC<jats:sub>50</jats:sub> for G1 was 0.139 ± 0.013 vs. 0.044 ± 0.007 μM for G3). We tested TVR‐resistant viral isolates and identified changes in IC<jats:sub>50</jats:sub>. One patient with a poor clinical response to TVR and wild‐type viral sequence showed reduced TVR sensitivity in our assay. We studied samples from a 2‐week TVR monotherapy study in which 5 of 8 patients with G3 HCV did not respond whereas 3 of 8 patients did. The “capture‐fusion” assay correctly identified responders. <jats:italic toggle="yes">Conclusion</jats:italic>: The capture‐fusion model represents a promising new technique that may help identify appropriate treatment strategies for patients with chronic HCV infection. (H<jats:sc>epatology</jats:sc> 2
Wong JLC, Harb HF, Bamford KB, et al., 2014, Time post out-of-hospital cardiac arrest determines the prevalence of different pathogenic organisms from respiratory secretions., ESCIM LIVES 2014
Wong JLC, Bamford KB, Harb HF, et al., 2014, Gram-negative resistance is highly prevalent in initial out-of-hospital cardiac arrest respiratory isolates., ESICM LIVES 2014
Wong JLC, Harb HF, Bamford KB, et al., 2014, A retrospective analysis of respiratory isolates post out-of-hospital cardiac arrest (OOHCA) to establish choices in empirical antibiotic cover., ISICEM 2014
Lahiri R, Wong JLC, Bhattacharya S, et al., 2013, Enhanced monocyte function and surface TLR expression predict post-operative systemic inflammatory response syndrome in patients immediately following major hepatic and pancreatic resection., British Society for Gastroenterology
Lahiri R, Wong JLC, Waters J, et al., 2013, Increased expression of TLR4 and TLR5 following complex hepato-pancreatico-biliary (HPB) surgery predicts development of post-operative systemic inflammatory response syndrome (SIRS)., Society of Academic and Research Surgery (SARS) January Conference
Wong JLC, Brett SJ, 2013, The assessment and risk stratification of psychological morbidity in critical care survivors, CRITICAL CARE, Vol: 17, ISSN: 1466-609X
Mckie AB, Vaughan S, Zanini E, et al., 2012, The OPCML Tumor Suppressor Functions as a Cell Surface Repressor–Adaptor, Negatively Regulating Receptor Tyrosine Kinases in Epithelial Ovarian Cancer, Cancer Discovery
McKie AB, Vaughan S, Okon I, et al., 2011, The OPCML tumor suppressor negatively regulates a specific repertoire of 5 receptor tyrosine kinases via a novel proteasomal mechanism, and its recombinant derivative is a potent in-vivo anticancer protein therapy, CANCER RESEARCH, Vol: 71, ISSN: 0008-5472
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