Pseudomonas aeruginosa modulates Inquilinus limosus tolerance to acute antibiotherapy under cystic fibrosis variable oxygen conditions

Cystic Fibrosis (CF) airways disease involves a complex polymicrobial infection whereby different bacterial species can interact and influence each other. To gain insights into the role that Pseudomonas aeruginosa and Inquilinus limosus interactionsmay play during CF infection, the reciprocal effect during biofilm formation, as well as ciprofloxacin activity against mixed biofilms under in vitro atmospheres with different oxygen availabilities were evaluated. The kinetics of biofilm formation showed that P. aeruginosa negatively affected I. limosus growth, under both aerobic and anaerobic environments. On the other hand, under aerobic conditions, I. limosus led to a decrease in biofilm production by P. aeruginosa, although biofilm-cells viability of remains unaltered.Given the differences measured by the crystal violet [biofilm biomass, consisting of both extracellular polymeric substance (EPS) and cells] and the viable count (biofilm viability) assays, these results may indicate that in mixed biofilms the presence of I. limosus, under aerobic conditions, leads to a reduction in P. aeruginosa EPS.Interestingly, P. aeruginosa might be responsible for the protection of I. limosus against ciprofloxacin activity. The analysis of the viable count dynamics revealed that I. limosus is less susceptible to ciprofloxacin when co-cultured in mixed biofilms with P. aeruginosa. Taken together, the results suggest a reciprocal interference between different bacterial species in CF lung. Alterations of bacterial behaviour due to interspecies interactions may be important for disease progression in CF infection

Impact of variable oxygen environments on resistance to acute antibiotherapy by cystic fibrosis related bacteria

The existence of steep oxygen gradients within the cystic fibrosis (CF) airways mucus is well known, with zones ranging from aerobic to completely anaerobic. Those environments, of heterogeneous availabilities of oxygen, contribute for the proliferation of a phylogenetically diverse ecosystem. This study aimed to inspect whether CF-related bacteria - Staphylococcus aureus and Pseudomonas aeruginosa and other emerging species Acinetobacter baumannii, Dolosigranulum pigrum, Inquilinus limosus, Klebsiella pneumoniae and Stenotrophomonas maltophilia are able to develop in vitro biofilms and be tolerant towards ciprofloxacin, an in-use antibiotic in acute CF infections. Single biofilms were formed in vitro, under aerobic and anaerobic environments, and further evaluated in terms of biomass and CFU counting. The antibiotic resistance profiles were analysed by constructing time-kill-curves.All species were able to growth under environments with distinct oxygen availability, demonstrating a great biofilm-forming ability highlighted by higher amount of biofilm mass, particularly under aerobic atmospheres.Biofilm time-kill curves showed augmented antibiotic tolerance of the bacteria, which was independent of the oxygen availability, except for D. pigrum where total eradication of biofilm-cells was noticed. Data highlighted that CF-related bacteria could persist under atmospheres with restricted oxygen availability, and form biofilms resilient to ciprofloxacin. Therefore, a more detailed knowledge about the effect of CF environments on the ability of the bacteria to proliferate and resist to antibiotics might be crucial for the success of CF infection treatment

Cystic fibrosis bacteria under variable oxygen tensions: biofilm formation ability and resilience to acute antibiotherapy

Distinct availabilities of oxygen, nutrients and antibiotics in the cystic fibrosis (CF) airways have contributed to the colonization of a large polymicrobial community, which may have further repercussion in the development of chronic biofilms and in antibiotherapy. This study aimed to inspect whether CF-related bacteria - Staphylococcus aureus (Sa) and two less common species Acinetobacter baumanni (Ab) and Klebsiella pneumoniae (Kp), are able to develop in vitro biofilms and resist to ciprofloxacin under aerobic/hypoxic conditions. Thus, biofilm-cells were estimated by CFU counting and time-kill-curves determined by absorbance (planktonic) and CFU (biofilm). All species showed alike results for biofilm growth, with higher bacteria (~109 CFU/cm2) adhering under aerobiosis than for hypoxic atmospheres (~108 CFU/cm2) in 24 h. Regarding the susceptibility profiles, Sa was the most sensitive species (MIC/MBC: 0.5 mg/L), with Kp keeping the most resistant profile against ciprofloxacin (MIC: 16 mg/L; MBC: >512 mg/L). Planktonic and biofilm time-kill curves were equivalent for both atmospheres. Interestingly, ciprofloxacin affected notably Sa biofilms under both conditions (adhesion rates declining 4log from 1/4MIC to 4MIC), but Ab and Kp biofilms were not disturbed even by the presence of abnormal ciprofloxacin concentrations, preserving initial adhesion rates from 106 - 107 cells/cm2/h, respectively. Data highlighted that CF unusual species could persist under hypoxia and form biofilms resilient to ciprofloxacin currently applied in acute infections, eventually progressing for severe biofilms hard to eradicate with powerful antibiotherapy

Revealing the dynamics of polymicrobial infections: update on the qPCR as a promising tool for the quantification of bacterial jungles

Microbiotec'17 - Congress of Microbiology and Biotechnology 2017P. aeruginosa and S. aureus are important opportunistic human pathogens in many polymicrobial infections. Interactions between them change the infection dynamics, resulting in increased potential for disease development. Determining the relative bacterial abundance through culture-dependent approaches is hindered by the presence of cells in a viable but poorly-cultivable state, or underestimated by the presence of cell-aggregates. The monitoring of bacterial pathogens by the use of culture-independent tools has led to fresh insights into the complex relationships between host and microbes, but often key experimental controls are lacking. This study aimed at examining changes in microbial composition in P. aeruginosa (PA) and S. aureus (SA) communities by quantitative PCR (qPCR). Total RNA was extracted and normalized against the amount of an exogenous RNA control-molecule. The Cq value for each gene of interest was transformed into relative quantities taking the differences between the target and the calibrator exogenous mRNA.info:eu-repo/semantics/publishedVersio

Interactions of Pseudomonas aeruginosa and Staphylococcus aureus in biofilm-related infections: insights through network reconstruction and creation of a new online database

Introduction: Despite important advances in biofilm research, these consortia remain a critical concern for many biomedical applications. Their naturally occurring polymicrobial nature is characterised by the development of complex communities, where pathogen interactions can promote disease progression and severity. Intra- and inter-species communication within these consortia is majorly regulated by quorum-sensing, affecting the expression of virulence factors and biofilm formation, making it a promising target for new anti-infective strategies. P. aeruginosa and S. aureus are two major pathogens that co-occur in many biofilm-related infections and whose competitive interaction is highly related to infection resilience. Hypothesis and aims: Information on P. aeruginosa-S. aureus interactions is currently scattered in the ever-growing scientific literature, making it difficult for researchers to grasp critical information. Therefore, this study aimed at systematically collecting and analysing experimental information presented in the biomedical literature on the molecular basis of P. aeruginosa-S. aureus interactions, identifying promising therapeutic targets, and making this data available to the research community. Methodology: Full-text papers were optimally retrieved from PubMed and classified by their relevance. Interaction data was methodically annotated, reconstructed as networks to identify promising therapeutic targets, and integrated with specialized databases to identify promising antimicrobials. A new online database was created to deposit the gathered interaction data in searchable format. Results: Network analysis revealed key entities regulating P. aeruginosa-S. aureus interactions, for instance the PqsABCDE/PqsR quorum-sensing system, which affects S. aureus growth and biofilm formation. By identifying the most reported P. aeruginosa virulence factors affecting S. aureus, e.g. HQNO and siderophores, a list of experimentally validated agents affecting those factors, ranging from synthetic drugs to natural plant extracts, was constructed. Conclusion: The complex experimental data on P. aeruginosa-S. aureus interactions was for the first time thoroughly retrieved, systematized, and made publically available in the new Inter-Species CrossTalk Database (www.ceb.uminho.pt/ISCTD).Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit; European Regional Development Fund under the scope of Norte 2020 - Programa Operacional Regional do Norte for the BioTecNorte operation (NORTE - 01 - 0145 - FEDER - 000004 ) ; COMPETE 2020 and FCT for the project POCI - 01 - 0145 - FEDER - 029841 . The authors also thank FCT for the PhD Grant of Andreia Patricia Magalhães [grant number SFRH/BD/ 132165 / 2017 ] and ESCMID for the Young Scientist Members Attendance Grantinfo:eu-repo/semantics/publishedVersio

S. aureus modulates P. aeruginosa small-colony variants formation

EUROBIOFILMS 2017 - 5th European Congress on Microbial BiofilmsSome studies reported that, in polymicrobial consortia, some P. a eruginosa exoproducts suppresses the growth of S.aureus and provokes the emergence of SCV, associated with antimicrobial resistance, altered metabolism and reduced immunogenicity. However, the role of S.aureus in P.aerugionsa behavior has still not been deciphered. This work aimed to deeply investigate the phenotypic changes undergone by P. aeruginosa and S.aureus in a co-infection scenario. The cells recovered from 24 -h-old single and dual-species biofilms were inspected regarding their colony morphology differentiation, antibiotic susceptibility profiles and expression of virulence factors. The population analysis profiles showed the existence of two P.aeruginosa SCV resulting only from the dual-species biofilms. These SCV exhibited impressive ability to form biofilm, impaired swimming, twitching and swarming abilities, in comparison with the wild -type morphotype. Their morphologies remained unchangeable over 10 passages onto solid media, which may mean these phenotypic alterations were not adaptive. Yet, the two SCV were susceptible to the action of several classes of antibiotics. In conclusion, the presence of S.aureus in biofilms seemed to modulate some phenotypic alterations in P.aeruginosa that may be clinically relevant as two SCV were detected. As t he role of S. aureus over P. aeruginosa stills not full clear, some tests are being run to more comprehensively know how these P. aeruginosa SCV modulate the dual -species community response to in - use antimicrobials.Portuguese Foundation for Science and Technology (FCT), through the strategic funding of UID/BIO/04469/2013 and COMPETE 2020 (POCI- 01-0145-FEDER-006684), and FCT and the European Community fund FEDER, through COMPETE and BioTecNorte operation (NORTE-01-0145-FEDER -000004) funded by the European Regional Development Fund under the scope of Norte2020. Grants of SPL (SFRH/BPD/95616/2013) and APM (UMINHO/BD/25/2016)info:eu-repo/semantics/publishedVersio

The fate of adhering bacteria on antimicrobial surfaces: transcriptomic analysis of resistance associated genes and macrophage-mediated phagocytosis

Book of Abstracts of CEB Annual Meeting 2017info:eu-repo/semantics/publishedVersio

Pseudomonas aeruginosa-Candida albicans polymicrobial biofilms in ventilator-associated infections (VAP): evaluating the post-antimicrobial effect of amphotericin B/polymyxin B combined activity

Introduction: Mixed bacterial-fungal colonization of the endotracheal tubes is now evident, with microbial interplay withstanding common antimicrobial therapy and paying for persistent and severe VAP infections. While alternative therapeutic strategies effectively targeting inter-kingdom biofilms are required, the role of each microorganism need to be appraised to deliver effective treatments. Hypothesis and aims: We earlier reported the combination therapy involving polymyxin B (PMB) and amphotericin B (AMB) as holding an attractive therapeutic option to treat dual-species biofilms. This study aimed to determine the post-antimicrobial phenomenon of PMB/AMB combined action in P. aeruginosa (PA) +Candida albicans (CA) biofilms, and to ascertain the events underlying biofilm growth restoration. Methodology: Post-antimicrobial effect of PMB combined with AMB was assessed in 24-h dual-species biofilms. Cell culturability and viability were evaluated by CFU and Live/Dead staining, respectively. The gene expression profile was assessed by qPCR. Results: Results showed that PA+CA biofilms lost their culturability straightaway being exposed to PMB/AMB combined solution. However, 24h was enough to both species recover their growth onto agar medium, with microbial counts approximating those observed for pre-treated biofilms. Following the subsequent treatment cycle, CFU estimation was only slightly disturbed. L/D results revealed that PA and CA populations displayed a compromised status at the end of the first PMB/AMB treatment cycle. Finishing the 24-h-regrowth cycle, most biofilm-encased species exhibited viability, which endured after the second treatment period. Transcriptional analysis of dual-species biofilms exposed to PMB/AMB combined action showed a high expression level in all PA resistance-encoded genes anrB, galU, mexA and algD and in ERG3 and ALS2 CA genes. Conclusion: Our finsings showed that PA+CA biofilms were able to escape to the combined action of PMB/AMB, and both species had a preeminent role while retaining adaptive resistance mechanisms that likely contributed for their recovery and adaptation on the ensuing treatments.info:eu-repo/semantics/publishedVersio

Unveiling co-infection in cystic fibrosis airways: transcriptomic analysis of Pseudomonas aeruginosa and Staphylococcus aureus dual-species biofilms

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene.2022.883199/ full#supplementary-materialCystic fibrosis (CF) is a common heritable genetic disorder caused by a defect in the cystic fibrosis conductance regulator gene, resulting in several complications in the human body (Kreda et al., 2012). So far, the pathological changes in the lungs are best studied due to the high mortality rates linked to poor lung function and the recurrent development of severe biofilm-related infections (Flume et al., 2009; Ciofu et al., 2015). Staphylococcus aureus and Pseudomonas aeruginosa are the most prevalent pathogens that colonize structurally abnormal airways such as those diagnosed with CF and other chronic obstructive lung diseases (Lyczak et al., 2002; Hubert et al., 2013). Although these bacteria seem to succeed with one another, CF patients acquire coinciding P. aeruginosa and S. aureus pulmonary infections, being co-infection usually associated with decreased lung function and increased frequency of pulmonary exacerbations (Limoli et al., 2016). Furthermore, P. aeruginosa and S. aureus pathogens adopt a biofilm mode of growth, which contributes to high tolerance to antibiotic treatment (Schobert and Jahn, 2010) and the recalcitrant nature of these chronic co-infections (Burmølle et al., 2006; Lopes et al., 2012), leading to significant patient morbidity and mortality (Cox et al., 2010). Interactions between P. aeruginosa and S. aureus have been widely studied, and it is commonly admitted that P. aeruginosa outcompetes S. aureus, perhaps outcompeting S. aureus for limited nutrients (Mashburn et al., 2005) or producing antistaphylococcal compounds (DeLeon et al., 2014; Fugère et al., 2014), having S. aureus a minimal contribution to the overall course of the CF-associated biofilm infections (Bragonzi et al., 2012; Filkins et al., 2015). However, P. aeruginosa and S. aureus have been identified in the same lobe of CF lungs (Hogan et al., 2016; Wakeman et al., 2016) and are frequently diagnosed (Limoli et al., 2016; Zolin et al., 2019) as co-infecting species in CF patients. Moreover, P. aeruginosa strains isolated from early infection outcompete S. aureus, while strains isolated from chronic infection are less aggressive and can be co-cultivated with S. aureus (Frydenlund Michelsen et al., 2016; Limoli et al., 2017), suggesting that these pathogens can interact in vivo. In a previous study, we showed that S. aureus can grow and coexist with P. aeruginosa under dualspecies biofilm conditions (Magalhães et al., 2021). Following up on these findings, and acknowledging that the molecular mechanisms behind these interactions are largely unknown, the purpose of the present study was, therefore, to identify the major transcriptomic features of P. aeruginosaS. aureus dual-species biofilms, using high-throughput RNA-sequencing (RNA-seq). Herein, we described the full transcriptome of P. aeruginosa and S. aureus single- and dual-species biofilms and used a data analysis approach based on direct and functional gene interactions, namely gene set enrichment. These results will be invaluable for future functional studies involving P. aeruginosaS. aureus interactions.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte. The authors also acknowledge the support, through the Programa Operacional Competitividade e Internacionalização (COMPETE 2020) and by national funds, through FCT, of the PhD Grant of APM (SFRH/BD/132165/2017).info:eu-repo/semantics/publishedVersio