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Marina Harper

Publications and source records attributed to Marina Harper.

7 recordsLinked to original sources
PubMed2026

Dual β-lactam therapy against high-risk Pseudomonas aeruginosa isolates: a dynamic in-vitro infection model study integrating population genomics with quantitative systems pharmacology modelling and simulations.

BACKGROUND: Pseudomonas aeruginosa has an extraordinary capacity for resistance emergence during treatment, even with newer antipseudomonals. There is a gap in understanding how resistance mechanisms affect the time-course of bacterial response to these newer agents. Traditional approaches for predicting pathogen response to an antibiotic do not apply to combination therapy. We aimed to develop a modelling framework to predict treatment response based on resistome information, using isolates of the worldwide-disseminated high-risk clone sequence type (ST) 235 and β-lactam antibiotics as the example. METHODS: In this hollow-fibre in-vitro infection study, we used three extensively drug-resistant ST235 clinical isolates from the national collection of the Clinical Microbiology Department of the Hospital Son Espases (Palma de Mallorca, Spain) that were hospital-acquired, were isolated following routine microbiological procedures from different patients between 2017 and 2022, were susceptible to ceftolozane-tazobactam, and had different levels of meropenem resistance. The selected isolates (ST235-05, ST235-09, and ST235-10) showed classical β-lactam resistance mechanisms pre-treatment. The isolates were investigated in 240-h dynamic hollow-fibre in-vitro infection models (HFIMs). The studies exposed the isolates to pharmacokinetic profiles of ceftolozane-tazobactam (simulating 1 g of ceftolozane and 0·5 g of tazobactam as a 3-h infusion every 8 h) and meropenem (simulating 6 g per day continuous infusion) as observed in hospitalised patients, as monotherapy and in combination. Treatment response was assessed through the quantification of the time-courses of viable total and resistant bacteria. Whole-genome sequencing identified the mechanisms of emerging resistance. A quantitative systems pharmacology (QSP) approach was used to model total and resistant bacterial counts and corresponding pharmacokinetic data from the HFIM. Monte Carlo simulations were used to predict treatment responses in 1000 virtual infected patients treated with ceftolozane-tazobactam and meropenem as monotherapies or in combination over 10 days. FINDINGS: In the HFIMs, each antibiotic alone amplified resistance by approximately 48 h for all isolates; that is, monotherapies resulted in a higher concentration of resistant bacteria compared with the control treatment at the respective time, except ceftolozane-tazobactam against ST235-10. Combination of ceftolozane-tazobactam and meropenem was synergistic (bacterial counts ≥2 log10 colony forming units [CFU] per mL lower than the best performing monotherapy and initial inoculum) against all isolates and suppressed resistance. Against ST235-10, ceftolozane-tazobactam monotherapy reduced counts to less than 1 log10 CFU per mL from 192 h onwards, whereas the combination reached less than 1 log10 CFU per mL by 24 h. Across strains, population genomics confirmed monotherapy failures were associated with emerging resistance mechanisms (ceftolozane-tazobactam: ampC Ω-loop mutations; meropenem: ftsl mutation). The developed QSP model incorporated baseline resistance mechanisms and those emerging in resistant mutant subpopulations. The model explained and predicted the monotherapy failures involving amplification of these subpopulations, and synergistic killing and resistance suppression by the combination. Simulations using the model predicted bacterial regrowth above the initial inoculum for more than 90% of patients after 0 to approximately 3 days for meropenem monotherapy across all strains and for ceftolozane-tazobactam monotherapy against ST235-05 and ST235-09. For ceftolozane-tazobactam monotherapy against ST235-10, regrowth was predicted for approximately 30% of patients. In contrast, the simulations predicted sustained bacterial killing of at least 2 log10 CFU per mL compared with the initial inoculum by the combination for more than 89% of patients across all strains. INTERPRETATION: To our knowledge, this model is the first to characterise and predict the time-course of responses of clinical isolates to antibiotics only by the resistance mechanisms present and their complex interplay, representing a step towards pathogen-specific, personalised medicine. FUNDING: Australian National Health and Medical Research Council.

Pseudomonas aeruginosa↗
PubMed2006

Pasteurella multocida pathogenesis: 125 years after Pasteur.

Pasteurella multocida was first shown to be the causative agent of fowl cholera by Louis Pasteur in 1881. Since then, this Gram-negative bacterium has been identified as the causative agent of many other economically important diseases in a wide range of hosts. The mechanisms by which these bacteria can invade the mucosa, evade innate immunity and cause systemic disease are slowly being elucidated. Key virulence factors identified to date include capsule and lipopolysaccharide. The capsule is clearly involved in bacterial avoidance of phagocytosis and resistance to complement, while complete lipopolysaccharide is critical for bacterial survival in the host. A number of other virulence factors have been identified by both directed and random mutagenesis, including Pasteurella multocida toxin (PMT), putative surface adhesins and iron acquisition proteins. However, it is likely that many key virulence factors are yet to be identified, including those required for initial attachment and invasion of host cells and for persistence in a relatively nutrient poor and hostile environment.

Animals↗
PubMed2005

Structural analysis of the lipopolysaccharide of Pasteurella multocida strain VP161: identification of both Kdo-P and Kdo-Kdo species in the lipopolysaccharide.

The structure of the lipopolysaccharide from the Pasteurella multocida strain VP161 was elucidated. The lipopolysaccharide was subjected to a variety of degradative procedures. The structures of the purified products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structures for the lipopolysaccharides were determined on the basis of the combined data from these experiments. [structure: see text]. Based on the NMR data, all sugars were found in pyranose ring forms, and Kdo is 2-keto-3-deoxy-octulosonic acid, L-alpha-D-Hep is L-glycero-D-manno-heptose, PPEtn is pyrophosphoethanolamine and PCho is phosphocholine. Intriguingly, when the O- and fully deacylated LPS was examined, it was evident that there was variability in the arrangement of the Kdo region of the molecule. Glycoforms were found with a Kdo-P moiety, as well as glycoforms elaborating a Kdo-Kdo group. Furthermore the Glc II residue was not attached to Hep I when two Kdo residues were present, but it was attached when the Kdo-P arrangement was elaborated, suggesting a biosynthetic incompatibility due to either steric hindrance or an inappropriate acceptor conformation. This variation in the Kdo region of the LPS was also observed in several other Pasteurella multocida strains investigated including the genome strain Pm70.

Carbohydrate Sequence↗
PubMed2004

Genomic-scale analysis of Pasteurella multocida gene expression during growth within liver tissue of chickens with fowl cholera.

We have recently reported the gene expression profile of Pasteurella multocida during growth in the blood of chickens with fowl cholera. Here we report the gene expression profile of P. multocida during growth in the livers of similarly infected chickens. We compared expression profiles of bacteria harvested from the livers of infected chickens with late-stage fowl cholera with those of bacteria grown in rich medium. Independent analysis of bacterial expression profiles from three individual chickens indicated that 93 P. multocida genes were always differentially expressed during growth in liver tissue. Of these 93 genes, 49 were upregulated and 44 downregulated in the host. Many of the upregulated genes were involved in energy production and conversion (9/49) and carbohydrate transport and metabolism (8/49), and a number of these have been shown to be induced under anaerobic conditions in other species. The downregulated genes were generally of unknown or poorly characterised functions (14/44). Comparison of the differentially regulated gene sets identified for growth in liver with those identified previously for growth in blood allowed the identification of a core set of 13 upregulated and 16 downregulated genes that were differentially regulated in at least five of the six infections studied.

Anaerobiosis↗
PubMed2004

A heptosyltransferase mutant of Pasteurella multocida produces a truncated lipopolysaccharide structure and is attenuated in virulence.

Pasteurella multocida is the causative agent of fowl cholera in birds. In a previous study using signature-tagged mutagenesis, we identified a mutant, AL251, which was attenuated for virulence in mice and in the natural chicken host. Sequence analysis indicated that AL251 had an insertional inactivation of the gene waaQ(PM), encoding a putative heptosyl transferase, required for the addition of heptose to lipopolysaccharide (LPS) (M. Harper, J. D. Boyce, I. W. Wilkie, and B. Adler, Infect. Immun. 71:5440-5446, 2003). In the present study, using mass spectrometry and nuclear magnetic resonance, we have confirmed the identity of the enzyme encoded by waaQ(PM) as a heptosyl transferase III and demonstrated that the predominant LPS glycoforms isolated from this mutant are severely truncated. Complementation experiments demonstrated that providing a functional waaQ(PM) gene in trans can restore both the LPS to its full length and growth in mice to wild-type levels. Furthermore, we have shown that mutant AL251 is unable to cause fowl cholera in chickens and that the attenuation observed is not due to increased serum sensitivity.

Animals↗
PubMed2003

Signature-tagged mutagenesis of Pasteurella multocida identifies mutants displaying differential virulence characteristics in mice and chickens.

Pasteurella multocida is the causative agent of fowl cholera in birds. Signature-tagged mutagenesis (STM) was used to identify potential virulence factors in a mouse septicemia disease model and a chicken fowl cholera model. A library of P. multocida mutants was constructed with a modified Tn916 and screened for attenuation in both animal models. Mutants identified by the STM screening were confirmed as attenuated by competitive growth assays in both chickens and mice. Of the 15 mutants identified in the chicken model, only 5 were also attenuated in mice, showing for the first time the presence of host-specific virulence factors and indicating the importance of screening for attenuation in the natural host.

Animals↗
PubMed2002

Genomic scale analysis of Pasteurella multocida gene expression during growth within the natural chicken host.

Little is known about the genomic-scale transcriptional responses of bacteria during natural infections. We used whole-genome microarray analysis to assess the transcriptional state of the gram-negative pathogen Pasteurella multocida, the causative agent of fowl cholera, during infection in the natural chicken host. We compared the expression profiles of bacteria harvested from the blood of septicemic chickens experiencing late-stage fowl cholera with those from bacteria grown in rich medium. Independent analysis of bacterial expression profiles from the infection of three individual chickens indicated that 40 genes were differentially expressed in all three individuals, 126 were differentially expressed in two of the three individuals, and another 372 were differentially expressed in one individual. Real-time reverse transcription-PCR assays were used to confirm the expression ratios for a number of genes. Of the 40 genes differentially expressed in all three individuals, 17 were up-regulated and 23 were down-regulated in the host compared with those grown in rich medium. The majority (10 of 17) of the up-regulated genes were involved in amino acid transport and metabolism and energy production and conversion, clearly indicating how P. multocida alters its biosynthetic and energy production pathways to cope with the host environment. In contrast, the majority (15 of 23) of down-regulated genes were of unknown or poorly characterized functions. There were clear differences in gene expression between the bacteria isolated from each of the three chickens, a finding consistent with individual host variation being an important factor in determining pathogen gene expression. Interestingly, bacteria from only two of the three infected animals had a gene expression profile highly similar to that observed during growth under iron-limiting conditions, suggesting that severe iron starvation may not always occur during P. multocida infection.

Animals↗