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Engineered live bacteria suppress Pseudomonas aeruginosa lung infection in mouse and dissolve biofilm in endotracheal tubes from patients

ABSTRACT: Engineered live bacteria could provide a new modality for treating lung infections , a major cause of mortality worldwide. Here, we engineered a genome-reduced human lung bacterium, Mycoplasma pneumoniae, to treat ventilator-associated pneumonia (VAP), a disease with high hospital mortality when associated with Pseudomonas aeruginosa biofilms. After validating the biosafety of an attenuated M. pneumoniae chassis in mice, we introduced four transgenes in the chromosome by transposition, to implement bactericidal and biofilm degradation activities. We show that this engineered strain has high efficacy against an acute P. aeruginosa lung infection in a mouse model. In addition, we demonstrate that the engineered strain can dissolve biofilms formed in endotracheal tubes of VAP patients and can be combined with antibiotics targeting the peptidoglycan layer to increase efficacy against gram-positive and gram-negative bacteria. We expect that our

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mycoplasma Pneumoniae (strain Atcc 29342 / M129)

SUBMITTER: Rocco Mazzolini

LAB HEAD: Maria Lluch-Senar

PROVIDER: PXD037233 | Pride | 2023-10-24

REPOSITORIES: Pride

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Engineered live bacteria suppress Pseudomonas aeruginosa infection in mouse lung and dissolve endotracheal-tube biofilms.

Mazzolini Rocco R Rodríguez-Arce Irene I Fernández-Barat Laia L Piñero-Lambea Carlos C Garrido Victoria V Rebollada-Merino Agustín A Motos Anna A Torres Antoni A Grilló Maria Jesús MJ Serrano Luis L Lluch-Senar Maria M

Nature biotechnology 20230119 8

Engineered live bacteria could provide a new modality for treating lung infections, a major cause of mortality worldwide. In the present study, we engineered a genome-reduced human lung bacterium, Mycoplasma pneumoniae, to treat ventilator-associated pneumonia, a disease with high hospital mortality when associated with Pseudomonas aeruginosa biofilms. After validating the biosafety of an attenuated M. pneumoniae chassis in mice, we introduced four transgenes into the chromosome by transposition ...[more]

PMID: 36658340

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Project description:Diagnosis of acute infection in the critically ill remains a challenge. Changes in physiologic parameters and existing molecular diagnostics are not specific and microbiologic confirmation of infection can take days. As cellular first responders, preclinical studies indicate that circulating leukocytes are activated in response to bacterial infection, manifesting infection-specific transcriptional programs. We hypothesized that circulating leukocyte transcriptional profiles can be used to diagnose infection and monitor response to therapy in the critically ill. Experiment Overall Design: A translational research approach was employed. Fifteen mice underwent intratracheal (i.t.) injections of live P. aeruginosa, P. aeruginosa endotoxin, live S. pneumoniae, or normal saline. At 24 hours after injury, GeneChip microarray analysis of circulating buffy coat RNA identified 219 genes that distinguished between the pulmonary insults and differences in 7-day mortality. Similarly, buffy coat microarray expression profiles were generated from 20 mechanically ventilated patients every two days for up to three weeks; 11 of these patients developed pneumonia. Principal components analysis of the expression levels of the human orthologs for the mouse genes resulted in gene expression trajectories that describe the cohort of 11 patients as they develop, are treated for, and convalesce from ventilator-associated pneumonia (VAP). When the VAP microarray profiles were analyzed independently, 85 genes were identified with consistent changes in abundance during the seven days bracketing the diagnosis of VAP. The estimated prediction accuracy in this small patient cohort was 91%. The abundance of select transcripts was validated using real-time PCR. Principal components analysis of the average microarray-measured abundances of these genes defined a general trajectory (riboleukogram) for the onset of acute infection in this patient population. Individual riboleukograms demonstrated that the onset and course of infection are specific for each individual, but as patients recover from infection the riboleukograms converge. The gene ontologies of the co-expression networks indicate that leukocyte chemokines and granule maturation are especially informative for pneumonia diagnostics.

Project description:Multispecies biofilms are the predominant form of bacterial growth in natural and human-associated environments. Although the pathways involved in monospecies biofilm have been well characterized, less is known about the metabolic pathways and emergent traits of a multispecies biofilm community. Here, we performed a transcriptome survey of the developmental stages of a 3-species biofilm community and combined it with quantitative imaging and growth experiments. We report the remodelling of central metabolism of two of the three species in this community. Specifically, we observed an increase in the expression of genes associated with glycolysis and pentose phosphate pathways in K. pneumoniae. Similarly, a decrease in the expression of the same pathways in P. protegens was observed along with an increase in expression of glyoxalate cycle genes when grown as a mixed species biofilm, suggesting reorganisation of metabolic pathways and metabolite sharing for the community biofilms. To test the possibility of cross-feeding for the community, planktonic growth experiments revealed that both the Pseudomonads grew well in TCA cycle intermediates, while K. pneumoniae grew poorly when given those carbon sources. Despite this poor growth in mono-culture, K. pneumoniae was still the dominant species in mixed species biofilms cultivated in TCA intermediates as the sole source of carbon. The biofilm growth data, combined with the transcriptomics data, suggests there is reorganisation of metabolism for the community members and may allow for cross-feeding that allows K. pneumoniae to dominate the community. We also demonstrated that sdsA1 of P. aeruginosa was induced upon exposure to the surfactant SDS and that this gene was essential in protecting mono and mixed species biofilms from surfactant stress. This also suggests that the community members can share defence mechanisms. Overall, this study describes a comprehensive transcriptomics level investigation of shared resources, metabolites and stress defence that may underpin the emergent properties of mixed species biofilm communities.

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Dataset Information

Engineered live bacteria suppress Pseudomonas aeruginosa lung infection in mouse and dissolve biofilm in endotracheal tubes from patients

Publications

Engineered live bacteria suppress Pseudomonas aeruginosa infection in mouse lung and dissolve endotracheal-tube biofilms.

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