Proteus mirabilis biofilm expansion microscopy yields over 4-fold magnification for super-resolution of biofilm structure and subcellular DNA organization.

Bacterial biofilms form when bacteria attach to surfaces and generate an extracellular matrix that embeds and stabilizes a growing community. Detailed visualization and quantitative analysis of biofilm architecture by optical microscopy are limited by the law of diffraction. Expansion Microscopy (ExM) is a novel Super-Resolution technique where specimens are physically enlarged by a factor of ∼4, prior to observation by conventional fluorescence microscopy. ExM requires homogenization of rigid constituents of biological components by enzymatic digestion. We developed an ExM approach capable of expanding 48-h old Proteus mirabilis biofilms 4.3-fold (termed PmbExM), close to the theoretic maximum expansion factor without gross shape distortions. Our protocol, based on lytic and glycoside-hydrolase enzymatic treatments, degrades rigid components in bacteria and extracellular matrix. Our results prove PmbExM to be a versatile and easy-to-use Super-Resolution approach for enabling studies of P. mirabilis biofilm architecture, assembly, and even intracellular features, such as DNA organization.

In house-development of a rapid immunochromatographic test for the detection of Escherichia coli in urine samples.

Urinary tract infections (UTIs) are a common health concern. Urine culture is the "gold standard" for UTI diagnosis but takes 48h. Rapid methods like dipstick tests are used as point-of-care tests. However, their sensitivity and specificity are variable. In this work, a rapid immunochromatographic test (IT) for detecting Escherichia coli in urine was developed, and its performance was evaluated in urine samples from patients with suspected UTI. The "universal lateral flow assay kit" was employed using an E. coli capture antibody. One hundred and five (105) urine samples were analyzed using the IT, dipstick test, and urine culture. The sensitivity of the IT was 74.5%, specificity 88.9%, positive predictive value (PPV) 86.3%, and negative predictive value (NPV) 78.7%. The combination of the IT with the dipstick test increases sensitivity to 94.1%, specificity to 66.7%, PPV to 72.7%, and NPV to 92.3%. Using the IT for detecting E. coli in urine could be a valuable technique for UTI screening, showing better specificity and diagnostic precision but lower sensitivity than the dipstick test. Based on these results, we propose that the combined use of both screening techniques would allow a rapid and more precise diagnosis of UTI, rationalizing the indication for empirical antibiotics.

Role of Proteus mirabilis flagella in biofilm formation / Papel de los flagelos de Proteus mirabilis en la formación de biofilms

Abstract Proteus mirabilis (P. mirabilis) is a common etiological agent of urinary tract infec-tions, particularly those associated with catheterization. P. mirabilis efficiently forms biofilms on different surfaces and shows a multicellular behavior called 'swarming', mediated by flagella. To date, the role of flagella in P. mirabilis biofilm formation has been under debate. In this study, we assessed the role of P. mirabilis flagella in biofilm formation using an isogenic allelic replacement mutant unable to express flagellin. Different approaches were used, such as the evaluation of cell surface hydrophobicity, bacterial motility and migration across catheter sections, measurements of biofilm biomass and biofilm dynamics by immunofluorescence and confocal microscopy in static and flow models. Our findings indicate that P. mirabilis flagella play a role in biofilm formation, although their lack does not completely avoid biofilm genera-tion. Our data suggest that impairment of flagellar function can contribute to biofilm prevention in the context of strategies focused on particular bacterial targets.

Resumen Proteus mirabilis (P mirabilis) es un agente etiológico común de infecciones del tracto urinario, en particular de aquellas asociadas con cateterización. P. mirabilis forma biofilms eficientemente en diferentes superficies y muestra un comportamiento multicelular llamado swarming, mediado por flagelos. Hasta el momento, el papel de los flagelos en la formación de biofilms de P. mirabilis ha estado en discusión. En este estudio, se evaluó el papel de los flagelos de P. mirabilis en la formación de biofilms, utilizando una mutante isogénica generada por reemplazo alélico, incapaz de expresar flagelina. Se utilizaron diferentes enfoques, como la evaluación de la hidrofobicidad de la superficie celular, de la movilidad y la migración bacteriana sobre secciones de catéteres y medidas de biomasa y de la dinámica del biofilm mediante inmunofluorescencia y microscopia confocal, tanto en modelos estáticos como de flujo. Nuestros hallazgos indican que los flagelos de P. mirabilis desempeñan un papel en la formación de biofilms, aunque su falta no suprime por completo su generación. Asimismo, evidencian que la interferencia de la función flagelar puede contribuir a evitar la formación de biofilms en el contexto de estrategias centradas en blancos bacterianos particulares.

Gold-, silver- and magnesium-doped zinc oxide nanoparticles prevents the formation of and eradicates bacterial biofilms.

Aim: This work aimed to synthesize magnesium-doped zinc oxide, silver and gold nanoparticles (Nps) and to evaluate their potential to prevent and eradicate Escherichia coli, Proteus mirabilis, Staphylococcus aureus, Acinetobacter baumannii and Pseudomonas aeruginosa biofilms. Materials & methods: The Nps were synthesized by precipitation and metallic reduction techniques. Physicochemical and biological characterization of Nps was performed. Results: All the Nps tested were able to inhibit the formation of E. coli, P. mirabilis, S. aureus and A. baumannii biofilms. The effects on the eradication of preformed biofilms were variable, although all the Nps tested were able to eradicate A. baumannii biofilms. Conclusion: The observed effects make the Nps suitable for coating surfaces and/or antibiotic carriers with medical interest.

Role of Proteus mirabilis flagella in biofilm formation.

Proteus mirabilis(P. mirabilis) is a common etiological agent of urinary tract infections, particularly those associated with catheterization. P. mirabilis efficiently forms biofilms on different surfaces and shows a multicellular behavior called 'swarming', mediated by flagella. To date, the role of flagella in P. mirabilis biofilm formation has been under debate. In this study, we assessed the role of P. mirabilis flagella in biofilm formation using an isogenic allelic replacement mutant unable to express flagellin. Different approaches were used, such as the evaluation of cell surface hydrophobicity, bacterial motility and migration across catheter sections, measurements of biofilm biomass and biofilm dynamics by immunofluorescence and confocal microscopy in static and flow models. Our findings indicate that P. mirabilis flagella play a role in biofilm formation, although their lack does not completely avoid biofilm generation. Our data suggest that impairment of flagellar function can contribute to biofilm prevention in the context of strategies focused on particular bacterial targets.

Salmonella Typhimurium Triggers Extracellular Traps Release in Murine Macrophages.

Salmonella comprises two species and more than 2500 serovars with marked differences in host specificity, and is responsible for a wide spectrum of diseases, ranging from localized gastroenteritis to severe life-threatening invasive disease. The initiation of the host inflammatory response, triggered by many Pathogen-Associated Molecular Patterns (PAMPs) that Salmonella possesses, recruits innate immune cells in order to restrain the infection at the local site. Neutrophils are known for killing bacteria through oxidative burst, amid other mechanisms. Amongst those mechanisms for controlling bacteria, the release of Extracellular Traps (ETs) represents a newly described pathway of programmed cell death known as ETosis. Particularly, Neutrophil Extracellular Traps (NETs) were first described in 2004 and since then, a number of reports have demonstrated their role as a novel defense mechanism against different pathogens. This released net-like material is composed of cellular DNA decorated with histones and cellular proteins. These structures have shown ability to trap, neutralize and kill different kinds of microorganisms, ranging from viruses and bacteria to fungi and parasites. Salmonella was one of the first microorganisms that were reported to be killed by NETs and several studies have confirmed the observation and deepened into its variants. Nevertheless, much less is known about their counterparts in other immune cells, e.g. Macrophage Extracellular Traps (METs) and Salmonella-induced MET release has never been reported so far. In this work, we observed the production of METs induced by Salmonella enterica serovar Typhimurium and recorded their effect on bacteria, showing for the first time that macrophages can also release extracellular DNA traps upon encounter with Salmonella Typhimurium. Additionally we show that METs effectively immobilize and reduce Salmonella survival in a few minutes, suggesting METs as a novel immune-mediated defense mechanism against Salmonella infection. Of note, this phenomenon was confirmed in primary macrophages, since MET release was also observed in bone marrow-derived macrophages infected with Salmonella. The evidence of this peculiar mechanism provides new incipient insights into macrophages´ role against Salmonella infection and can help to design new strategies for the clinical control of this transcendental pathogen.

Selection of Effective Antibiotics for Uropathogenic Escherichia coli Intracellular Bacteria Reduction.

Urinary tract infections (UTI) are one of the most frequent bacterial infections in humans, being Uropathogenic Escherichia coli (UPEC), the most common etiological agent. The ability of UPEC to invade urothelial cells and to form intracellular bacterial communities (IBC) has been described. Therefore, UPEC can persist in the urinary tract producing recurrent infections, resisting antibiotic activity. The objective of the present work was to analyze the ability of a collection of UPEC clinical isolates to invade bladder epithelial cells in vitro and the activity of different classes of antibiotics on intracellular bacteria. We selected 23 UPEC clinical isolates that had been previously detected intracellularly in desquamated bladder epithelial cells from patients' urine. A cellular invasion assay using the T24 bladder cell line was used. Intracellular bacteria was confirmed by laser confocal microscopy. All the strains were able to invade the cells with different percentages of intracellular bacterial survival (0.7 to 18%). However, no significant relationship was found between the percentage of in vitro infection and the presence of IBC in desquamated urine cells. In vitro, intracellular bacteria were confirmed in four representative strains by confocal laser microscopy. Ceftriaxone, ciprofloxacin and, azithromycin in vitro activity on intracellular bacteria were evaluated. Amikacin was used as a negative control. All the antibiotics tested, except amikacin, significantly decreased the number of intracellular bacteria. Ciprofloxacin was the antibiotic that induced the highest decrease percentage. Conclusions: All UPEC clinical isolates could invade bladder epithelial cells in vitro. Ceftriaxone, ciprofloxacin, and azithromycin can reduce the percentage of intracellular bacteria in vitro. In vivo studies are needed to confirm the utility of these antibiotics for intracellular bacteria reduction in UTI.

Visualizing the invisible: class excursions to ignite children's enthusiasm for microbes.

We have recently argued that, because microbes have pervasive - often vital - influences on our lives, and that therefore their roles must be taken into account in many of the decisions we face, society must become microbiology-literate, through the introduction of relevant microbiology topics in school curricula (Timmis et al. 2019. Environ Microbiol 21: 1513-1528). The current coronavirus pandemic is a stark example of why microbiology literacy is such a crucial enabler of informed policy decisions, particularly those involving preparedness of public-health systems for disease outbreaks and pandemics. However, a significant barrier to attaining widespread appreciation of microbial contributions to our well-being and that of the planet is the fact that microbes are seldom visible: most people are only peripherally aware of them, except when they fall ill with an infection. And it is disease, rather than all of the positive activities mediated by microbes, that colours public perception of 'germs' and endows them with their poor image. It is imperative to render microbes visible, to give them life and form for children (and adults), and to counter prevalent misconceptions, through exposure to imagination-capturing images of microbes and examples of their beneficial outputs, accompanied by a balanced narrative. This will engender automatic mental associations between everyday information inputs, as well as visual, olfactory and tactile experiences, on the one hand, and the responsible microbes/microbial communities, on the other hand. Such associations, in turn, will promote awareness of microbes and of the many positive and vital consequences of their actions, and facilitate and encourage incorporation of such consequences into relevant decision-making processes. While teaching microbiology topics in primary and secondary school is key to this objective, a strategic programme to expose children directly and personally to natural and managed microbial processes, and the results of their actions, through carefully planned class excursions to local venues, can be instrumental in bringing microbes to life for children and, collaterally, their families. In order to encourage the embedding of microbiology-centric class excursions in current curricula, we suggest and illustrate here some possibilities relating to the topics of food (a favourite pre-occupation of most children), agriculture (together with horticulture and aquaculture), health and medicine, the environment and biotechnology. And, although not all of the microbially relevant infrastructure will be within reach of schools, there is usually access to a market, local food store, wastewater treatment plant, farm, surface water body, etc., all of which can provide opportunities to explore microbiology in action. If children sometimes consider the present to be mundane, even boring, they are usually excited with both the past and the future so, where possible, visits to local museums (the past) and research institutions advancing knowledge frontiers (the future) are strongly recommended, as is a tapping into the natural enthusiasm of local researchers to leverage the educational value of excursions and virtual excursions. Children are also fascinated by the unknown, so, paradoxically, the invisibility of microbes makes them especially fascinating objects for visualization and exploration. In outlining some of the options for microbiology excursions, providing suggestions for discussion topics and considering their educational value, we strive to extend the vistas of current class excursions and to: (i) inspire teachers and school managers to incorporate more microbiology excursions into curricula; (ii) encourage microbiologists to support school excursions and generally get involved in bringing microbes to life for children; (iii) urge leaders of organizations (biopharma, food industries, universities, etc.) to give school outreach activities a more prominent place in their mission portfolios, and (iv) convey to policymakers the benefits of providing schools with funds, materials and flexibility for educational endeavours beyond the classroom.

Characterization of the Different Stages of Biofilm Formation and Antibiotic Susceptibility in a Clinical Acinetobacter baumannii Strain.

Acinetobacter baumannii is a relevant opportunistic pathogen, and one of the main microorganisms responsible for outbreaks in nosocomial infections worldwide. Its pathogenicity is mainly due to its resistance to multiple antibiotics and to its ability to form biofilms on abiotic surfaces. The objective of this study was to characterize the biofilm formation cycle of A. baumannii isolated from a patient in a hospital and compare its antibiotic resistance with the planktonic cells. To study biofilm formation, the classical microtiter assay was used, with crystal violet staining and optical density reading to classify the type of biofilm. Also, the effect of gentamicin and colistin on bacterial biofilm was studied with an extra step of antibiotic addition. For the characterization of the different biofilm formation stages, the strain was grown on a coverslip, and the stain was made with a mixture of fluorophores markers to visualize the biofilm with a confocal laser microscope. It was possible to differentiate the A. baumannii biofilm formation stages. Through these observations, it was possible to estimate the time elapsed between each stage. As the strain was susceptible to colistin and gentamicin, both antibiotics were evaluated after the biofilm was formed. Neither antibiotics showed an effect on the eradication of A. baumannii biofilm.

Microbiology can be comic.

For microbiologists, the importance of microorganisms in our daily lives and their impact on our well-being is evident. However, microbiology literacy in our society is far from being enough for individuals to make informed choices and to demand actions based on that information. The vaccine hesitation movement and the alarming increase in antimicrobial resistance due to overuse and misuse of antibiotics are just two examples of how much work is needed to make our society literate in topics related to microbiology. Considering the challenges of communicating a discipline surrounded by misconceptions, which studies the role of living organisms that cannot be seen in plain sight, we need to explore different strategies to effectively contribute to microbiology literacy in our society. Here, we will comment on the use of comics for such a task.

Purification of Native Flagellin.

Flagella are effective organelles of locomotion and one of several virulence factors in Proteus mirabilis. To study their properties and role in virulence, we describe a protocol to extract and purify the native flagellin of P. mirabilis. Purified flagellin can be visualized by SDS-PAGE or immunoblot and is suitable for downstream applications such as immunization.

Adherence of Proteus mirabilis to Uroepithelial Cells.

Bacterial adherence to eukaryotic cells is mediated by different adhesins that can act at different stages in bacteria-host interaction. Abundant evidence has suggested that adherence is critical for infection by bacterial pathogens. Proteus mirabilis is an opportunistic pathogen which frequently infects the human urinary tract, particularly in patients with indwelling urinary catheters. Sequencing of the genome of this pathogen has revealed the existence of a remarkable amount of complete fimbrial operons. In this chapter, we describe in vitro adherence assays of P. mirabilis to uroepithelial cells, which can provide relevant results to assess virulence of uropathogenic strains.

Magnesium-doped zinc oxide nanoparticles alter biofilm formation of Proteus mirabilis.

Aim:Proteus mirabilis biofilms colonize medical devices, and their role in microbial pathogenesis is well established. Magnesium-doped zinc oxide nanoparticles (ZnO:MgO NPs) have potential antimicrobial properties; thus, we aimed at evaluating the antibiofilm activity of ZnO:MgO NPs against P. mirabilis biofilm. Materials & methods: After synthesis and characterization of ZnO:MgO NPs and their addition to a polymer film, we evaluated the stages of P. mirabilis biofilm development over glass coverslip covered by different concentrations of ZnO:MgO NPs. Results: Low concentrations of ZnO:MgO NPs affect the development of P. mirabilis biofilm. Descriptors showed reduced values in bacterial number, bacterial volume and extracellular material. Conclusion: Our results highlight this new application of ZnO:MgO NPs as a potential antibiofilm strategy in medical devices.

Fosfomycin tromethamine activity on biofilm and intracellular bacterial communities produced by uropathogenic Escherichia coli isolated from patients with urinary tract infection.

Fosfomycin tromethamine (FT), an old antibiotic revived as a new strategy to overcome antibiotic resistance, is an excellent option for the treatment of lower urinary tract infection (UTI). During UTI, Escherichia coli produces biofilms and could invade the bladder epithelial cells, developing intracellular bacterial communities (IBC). The present work aimed to evaluate the activity of FT on biofilms and IBC from clinical isolates of E. coli. A total of 38 E. coli clinical UTI isolates previously characterized as biofilm and IBC producers were studied. FT susceptibility was evaluated and its activity on 48 h biofilm was determined by microtiter plate-based biofilm assay comparing three different antibiotic concentrations. Two UPEC strains were selected to evaluate FT activity on IBC in vitro using T24 bladder cells. The survival percentage of intracellular bacteria after 24 h exposure to FT was calculated and compared to the percentage of intracellular bacteria without antibiotic. All the strains were susceptible to FT. FT produced a significant reduction of biofilms at the three concentrations tested, compared to the control. However, no statistically effect on IBC was observed after 24 h of fosfomycin exposure in cell culture. FT is a good option for bacterial biofilm reduction within UTI. However, it does not affect IBC.

Herbaspirillum seropedicae Differentially Expressed Genes in Response to Iron Availability.

Herbaspirillum seropedicae Z67 is a nitrogen-fixing endophyte that colonizes many important crops. Like in almost all organisms, vital cellular processes of this endophyte are iron dependent. In order to efficiently acquire iron to fulfill its requirements, this bacterium produces the siderophores serobactins. However, the presence in its genome of many others iron acquisition genes suggests that serobactins are not the only strategy used by H. seropedicae to overcome metal deficiency. The aim of this work was to identify genes and proteins differentially expressed by cells growing in low iron conditions in order to describe H. seropedicae response to iron limitation stress. For this purpose, and by using a transcriptomic approach, we searched and identified a set of genes up-regulated when iron was scarce. One of them, Hsero_2337, codes for a TonB-dependent transporter/transducer present in the serobactins biosynthesis genomic locus, with an unknown function. Another TonB-dependent receptor, the one encoded by Hsero_1277, and an inner membrane ferrous iron permease, coded by Hsero_2720, were also detected. By using a proteomic approach focused in membrane proteins, we identified the specific receptor for iron-serobactin internalization SbtR and two non-characterized TonB-dependent receptors (coded by genes Hsero_1277 and Hsero_3255). We constructed mutants on some of the identified genes and characterized them by in vitro growth, biofilm formation, and interaction with rice plants. Characterization of mutants in gene Hsero_2337 showed that the TonB-dependent receptor coded by this gene has a regulatory role in the biosynthesis of serobactins, probably by interacting with the alternative sigma factor PfrI, coded by gene Hsero_2338. Plant colonization of the mutant strains was not affected, since the mutant strain normally colonize the root and aerial part of rice plants. These results suggest that the strategies used by H. seropedicae to acquire iron inside plants are far more diverse than the ones characterized in this work. In vivo expression studies or colonization competition experiments between the different mutant strains could help us in future works to determine the relative importance of the different iron acquisition systems in the interaction of H. seropedicae with rice plants.

Development of a High-Throughput ex-Vivo Burn Wound Model Using Porcine Skin, and Its Application to Evaluate New Approaches to Control Wound Infection.

Biofilm formation in wounds is considered a major barrier to successful treatment, and has been associated with the transition of wounds to a chronic non-healing state. Here, we present a novel laboratory model of wound biofilm formation using ex-vivo porcine skin and a custom burn wound array device. The model supports high-throughput studies of biofilm formation and is compatible with a range of established methods for monitoring bacterial growth, biofilm formation, and gene expression. We demonstrate the use of this model by evaluating the potential for bacteriophage to control biofilm formation by Staphylococcus aureus, and for population density dependant expression of S. aureus virulence factors (regulated by the Accessory Gene Regulator, agr) to signal clinically relevant wound infection. Enumeration of colony forming units and metabolic activity using the XTT assay, confirmed growth of bacteria in wounds and showed a significant reduction in viable cells after phage treatment. Confocal laser scanning microscopy confirmed the growth of biofilms in wounds, and showed phage treatment could significantly reduce the formation of these communities. Evaluation of agr activity by qRT-PCR showed an increase in activity during growth in wound models for most strains. Activation of a prototype infection-responsive dressing designed to provide a visual signal of wound infection, was related to increased agr activity. In all assays, excellent reproducibility was observed between replicates using this model.

A multilevel trait-based approach to the ecological performance of Microcystis aeruginosa complex from headwaters to the ocean.

The Microcystis aeruginosa complex (MAC) clusters cosmopolitan and conspicuous harmful bloom-forming cyanobacteria able to produce cyanotoxins. It is hypothesized that low temperatures and brackish salinities are the main barriers to MAC proliferation. Here, patterns at multiple levels of organization irrespective of taxonomic identity (i.e. a trait-based approach) were analyzed. MAC responses from the intracellular (e.g. respiratory activity) to the ecosystem level (e.g. blooms) were evaluated in wide environmental gradients. Experimental results on buoyancy and respiratory activity in response to increased salinity (0-35) and a literature review of maximum growth rates under different temperatures and salinities were combined with field sampling from headwaters (800km upstream) to the marine end of the Rio de la Plata estuary (Uruguay-South America). Salinity and temperature were the major variables affecting MAC responses. Experimentally, freshwater MAC cells remained active for 24h in brackish waters (salinity=15) while colonies increased their flotation velocity. At the population level, maximum growth rate decreased with salinity and presented a unimodal exponential response with temperature, showing an optimum at 27.5°C and a rapid decrease thereafter. At the community and ecosystem levels, MAC occurred from fresh to marine waters (salinity 30) with a sustained relative increase of large mucilaginous colonies biovolume with respect to individual cells. Similarly, total biomass and, specific and morphological richness decreased with salinity while blooms were only detected in freshwater both at high (33°C) and low (11°C) temperatures. In brackish waters, large mucilaginous colonies presented advantages under osmotic restrictive conditions. These traits values have also been associated with higher toxicity potential. This suggest salinity or low temperatures would not represent effective barriers for the survival and transport of potentially toxic MAC under likely near future scenarios of increasing human impacts (i.e. eutrophication, dam construction and climate change).

Fluoxetine and thioridazine inhibit efflux and attenuate crystalline biofilm formation by Proteus mirabilis.

Proteus mirabilis forms extensive crystalline biofilms on indwelling urethral catheters that block urine flow and lead to serious clinical complications. The Bcr/CflA efflux system has previously been identified as important for development of P. mirabilis crystalline biofilms, highlighting the potential for efflux pump inhibitors (EPIs) to control catheter blockage. Here we evaluate the potential for drugs already used in human medicine (fluoxetine and thioridazine) to act as EPIs in P. mirabilis, and control crystalline biofilm formation. Both fluoxetine and thioridazine inhibited efflux in P. mirabilis, and molecular modelling predicted both drugs interact strongly with the biofilm-associated Bcr/CflA efflux system. Both EPIs were also found to significantly reduce the rate of P. mirabilis crystalline biofilm formation on catheters, and increase the time taken for catheters to block. Swimming and swarming motilies in P. mirabilis were also significantly reduced by both EPIs. The impact of these drugs on catheter biofilm formation by other uropathogens (Escherichia coli, Pseudomonas aeruginosa) was also explored, and thioridazine was shown to also inhibit biofilm formation in these species. Therefore, repurposing of existing drugs with EPI activity could be a promising approach to control catheter blockage, or biofilm formation on other medical devices.