Open Access
Experimental polymorphism survey in intergenic regions of the icaADBCR locus in Staphylococcus aureus isolates from periprosthetic joint infections
(MDPI, 2022) Morales Laverde, Liliana Andrea; Echeverz Sarasúa, Maite; Trobos, Margarita; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; Ciencias de la Salud; Osasun Zientziak
Staphylococcus aureus is a leading cause of prosthetic joint infections (PJI) characterized by bacterial biofilm formation and recalcitrance to immune-mediated clearance and antibiotics. The molecular events behind PJI infection are yet to be unraveled. In this sense, identification of polymorphisms in bacterial genomes may help to establish associations between sequence variants and the ability of S. aureus to cause PJI. Here, we report an experimental nucleotide-level survey specifically aimed at the intergenic regions (IGRs) of the icaADBCR locus, which is responsible for the synthesis of the biofilm exopolysaccharide PIA/PNAG, in a collection of strains sampled from PJI and wounds. IGRs of the icaADBCR locus were highly conserved and no PJI-specific SNPs were found. Moreover, polymorphisms in these IGRs did not significantly affect transcription of the icaADBC operon under in vitro laboratory conditions. In contrast, an SNP within the icaR coding region, resulting in a V176E change in the transcriptional repressor IcaR, led to a significant increase in icaADBC operon transcription and PIA/PNAG production and a reduction in S. aureus virulence in a Galleria mellonella infection model. In conclusion, SNPs in icaADBCR IGRs of S. aureus isolates from PJI are not associated with icaADBC expression, PIA/PNAG production and adaptation to PJI.
Open Access
Genomics of Staphylococcus aureus and Staphylococcus epidermidis from periprosthetic joint infections and correlation to clinical outcome
(American Society for Microbiology, 2022) Trobos, Margarita; Firdaus, Rininta; Malchau, Karin Svensson; Tillander, Jonatan; Arnellos, Dimitrios; Rolfson, Ola; Thomsen, Peter; Lasa Uzcudun, Íñigo; Ciencias de la Salud; Osasun Zientziak
The approach of sequencing or genotyping to characterize the pathogenic potential of staphylococci from orthopedic device-related infection (ODRI) has been applied in recent studies. These studies described the genomic carriage of virulence in clinical strains and compared it with those in commensal strains. Only a few studies have directly correlated genomic profiles to patient outcome and phenotypic virulence properties in periprosthetic joint infections (PJIs). We investigated the association between genomic variations and virulence-associated phenotypes (biofilm-forming ability and antimicrobial resistance) in 111 staphylococcal strains isolated from patients with PJI and the infection outcome (resolved/unresolved). The presence of a strong biofilm phenotype in Staphylococcus aureus and an antibiotic-resistant phenotype in Staphylococcus epidermidis were both associated with treatment failure of PJI. In S. epidermidis, multidrug resistance (MDR) and resistance to rifampicin were associated with unresolved infection. Sequence type 45 (ST45) and ST2 were particularly enriched in S. aureus and S. epidermidis, respectively. S. epidermidis ST2 caused the majority of relapses and was associated with MDR and strong biofilm production, whereas ST215 correlated with MDR and non/weak biofilm production. S. aureus agr II correlated with resolved infection, while S. epidermidis agr I was associated with strong biofilm production and agr III with non/weak production. Collectively, our results highlight the importance of careful genomic and phenotypic characterization to anticipate the probability of the strain causing treatment failure in PJI. Due to the high rate of resistant S. epidermidis strains identified, this study provides evidence that the current recommended treatment of rifampicin and a fluoroquinolone should not be administered without knowledge of the resistance pattern.
Open Access
Regulation of gene expression by non-phosphorylated response regulators
(Institut d'Estudis Catalans, 2021) Gómez Arrebola, Carmen; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; Ciencias de la Salud; Osasun Zientziak
Two-component systems (TCSs) are a prominent sensory system in bacteria. A prototypical TCS comprises a membrane-bound sensor histidine kinase (HK) responsible for sensing the signal and a cytoplasmic response regulator (RR) that controls target gene expression. Signal binding activates a phosphotransfer cascade from the HK to the RR. As a result, the phosphorylated RR undergoes a conformational change that leads to activation of the response. Growing experimental evidence indicates that unphosphorylated RRs may also have regulatory functions, and thus, the classical view that the RR is only active when it is phosphorylated needs to be revisited. In this review, we highlight the most recent findings showing that RRs in the non-phosphorylated state control critical bacterial processes that range from secretion of factors to the host, antibiotic resistance, iron transport, stress response, and cell-wall metabolism to biofilm development.
Open Access
Bacteriophages avoid autoimmunity from cognate immune systems as an intrinsic part of their life cycles
(Nature Research, 2024) Rostøl, Jakob T.; Quiles Puchalt, Nuria; Iturbe Sanz, Pablo; Lasa Uzcudun, Íñigo; Penadés, José R.; Ciencias de la Salud; Osasun Zientziak
Dormant prophages protect lysogenic cells by expressing diverse immune systems, which must avoid targeting their cognate prophages upon activation. Here we report that multiple Staphylococcus aureus prophages encode Tha (tail-activated, HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain-containing anti-phage system), a defence system activated by structural tail proteins of incoming phages. We demonstrate the function of two Tha systems, Tha-1 and Tha-2, activated by distinct tail proteins. Interestingly, Tha systems can also block reproduction of the induced tha-positive prophages. To prevent autoimmunity after prophage induction, these systems are inhibited by the product of a small overlapping antisense gene previously believed to encode an excisionase. This genetic organization, conserved in S. aureus prophages, allows Tha systems to protect prophages and their bacterial hosts against phage predation and to be turned of during prophage induction, balancing immunity and autoimmunity. Our results show that the fne regulation of these processes is essential for the correct development of prophages’ life cycle.
Open Access
Evaluation of the use of sonication combined with enzymatic treatment for biofilm removal in the microbiological diagnosis of prosthetic joint infection
(American Society for Microbiology, 2024) Henríquez, Lucía; Martín Contero, María del Carmen; Echeverz Sarasúa, Maite; Lasa Uzcudun, Íñigo; Ezpeleta Baquedano, María Carmen; Portillo, Eugenia; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua
Sonicating explanted prosthetic implants to physically remove biofilms is a recognized method for improving the microbiological diagnosis of prosthetic joint infection (PJI); however, chemical and enzymatic treatments have been investigated as alternative biofilm removal methods. We compared the biofilm dislodging efficacy of sonication followed by the addition of enzyme cocktails with different activity spectra in the diagnosis of PJI with that of the sonication of fluid cultures alone. Consecutive patients who underwent prosthesis explantation due to infection at our institution were prospectively enrolled for 1 year. The diagnostic procedure included the collection of five intraoperative tissue cultures, sonication of the removed devices, and conventional culture of the sonication fluid. The resulting sonication fluid was also treated with an enzyme cocktail consisting of homemade dispersin B (0.04 ¿g/mL) and proteinase K (Sigma; 100 ¿g/mL) for 45 minutes at 37°C. The resulting sonication (S) and sonication with subsequent enzymatic treatment (SE) fluids were plated for aerobic and anaerobic culture broth for 7 days (aerobic) or 14 days (anaerobic). Identification was performed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (Bruker). We included 107 patients from whom a prosthetic implant had been removed, among which PJI was diagnosed in 36 (34%). The sensitivity of S alone was significantly greater than that of SE alone (82% vs 71%; P < 0.05). Four patients with PJI were positive after sonication alone but negative after sonication plus enzymatic treatment. The four microorganisms missed after the addition of the enzyme cocktail were Staphylococcus aureus, two coagulase-negative Staphylococci, and Cutibacterium acnes. In conclusion, sonication alone was more sensitive than sonication followed by enzymatic treatment. The combination of these two methods had no synergistic effect; in contrast, the results suggest that the combination of both dislodgment methods affects the viability of gram-positive microorganisms.
Open Access
Biofilm properties in relation to treatment outcome in patients with first-time periprosthetic hip or knee joint infection
(Elsevier, 2021) Malchau, Karin Svensson; Tillander, Jonatan; Zaborowska, Magdalena; Hoffman, Maria; Lasa Uzcudun, Íñigo; Thomsen, Peter; Malchau, Henrik; Rolfson, Ola; Trobos, Margarita; Ciencias de la Salud; Osasun Zientziak
Background: periprosthetic joint infections (PJI) are challenging complications following arthroplasty. Staphylococci are a frequent cause of PJI and known biofilm producers. Biofilm formation decreases antimicrobial susceptibility, thereby challenging favourable treatment outcomes. The aims of this study were to characterize the biofilm abilities and antimicrobial susceptibilities of staphylococci causing first-time PJI and correlate them to clinical outcome (infection resolution and recurrence). Methods: reoperations for PJI of the hip or knee between 1st January 2012 to 30th June 2015 performed at the Sahlgrenska University Hospital were identified in a local database. Medical records were reviewed and clinical parameters recorded for patients whose intraoperative bacterial isolates had been stored at the clinical laboratory. Staphylococcal strains isolated from reoperations due to first-time PJI were characterised by their ability to form biofilms using the microtiter plate test. Antimicrobial susceptibility of the strains was determined by minimum inhibitory concentration (MIC) when grown planktonically, and by minimum biofilm eradication concentration (MBEC) when grown as biofilms. MBEC determination was conducted using the Calgary biofilm device (CBD) and a custom-made antimicrobial susceptibility plate containing eight clinically relevant antimicrobial agents. Results: the study group included 49 patients (70 bacterial strains) from first-time PJI, whereof 24 (49%) patients had recurrent infection. Strong biofilm production was significantly associated with recurrent infection. Patients infected with strong biofilm producers had a five-fold increased risk for recurrent infection. Strains grown as biofilms were over 8000 times more resistant to antimicrobial agents compared to planktonic cultures. Biofilms were more susceptible to rifampicin compared to other antimicrobials in the assay. Increased biofilm susceptibility (MBEC > MIC) was observed for the majority of the bacterial strains and antimicrobial agents. Conclusions: Strong biofilm production was significantly associated with increased antimicrobial resistance and PJI recurrence. This underscores the importance of determining biofilm production and susceptibility as part of routine diagnostics in PJI. Strong staphylococcal biofilm production may have implications on therapeutic choices and suggest more extensive surgery. Furthermore, despite the increased biofilm resistance to rifampicin, results from this study support its use in staphylococcal PJI. The Translational Potential of this Article: Like for many biomaterial-associated infections, staphylococci are a common cause of PJI. Their ability to adhere to surfaces and produce biofilms on medical devices is proposed to play a role. However, clinical studies where biofilm properties are directly linked to patient outcome are scarce. This study demonstrates that the majority of staphylococci isolated from first-time PJI were biofilm producers with increased antimicrobial resistance. Patients suffering an infection caused by a staphylococcal strain with strong biofilm production ability had a five-fold greater risk of recurrent infection. This novel finding suggests the importance of evaluating biofilm production as a diagnostic procedure for the guidance of treatment decisions in PJI.
Open Access
Systematic reconstruction of the complete two-component sensorial network in staphylococcus aureus
(American Society for Microbiology, 2020) Rapún Araiz, Beatriz; Haag, Andreas F.; Gil Puig, Carmen; Dorado Morales, Pedro; Lasa Uzcudun, Íñigo; Ciencias de la Salud; Osasun Zientziak
In bacteria, adaptation to changes in the environment is mainly controlled through two-component signal transduction systems (TCSs). Most bacteria contain dozens of TCSs, each of them responsible for sensing a different range of signals and controlling the expression of a repertoire of target genes (regulon). Over the years, identification of the regulon controlled by each individual TCS in different bacteria has been a recurrent question. However, limitations associated with the classical approaches used have left our knowledge far from complete. In this report, using a pioneering approach in which a strain devoid of the complete nonessential TCS network was systematically complemented with the constitutively active form of each response regulator, we have reconstituted the regulon of each TCS of S. aureus in the absence of interference between members of the family. Transcriptome sequencing (RNA-Seq) and proteomics allowed us to determine the size, complexity, and insulation of each regulon and to identify the genes regulated exclusively by one or many TCSs. This gain-of-function strategy provides the first description of the complete TCS regulon in a living cell, which we expect will be useful to understand the pathobiology of this important pathogen. IMPORTANCE Bacteria are able to sense environmental conditions and respond accordingly. Their sensorial system relies on pairs of sensory and regulatory proteins, known as two-component systems (TCSs). The majority of bacteria contain dozens of TCSs, each of them responsible for sensing and responding to a different range of signals. Traditionally, the function of each TCS has been determined by analyzing the changes in gene expression caused by the absence of individual TCSs. Here, we used a bacterial strain deprived of the complete TC sensorial system to introduce, one by one, the active form of every TCS. This gain-of-function strategy allowed us to identify the changes in gene expression conferred by each TCS without interference of other members of the family.
Open Access
Elevated c-di-GMP levels promote biofilm formation and biodesulfurization capacity of Rhodococcus erythropolis
(Society for Applied Microbiology, 2020) Dorado Morales, Pedro; Martínez, Igor; Rivero Buceta, Virginia; Díaz, Eduardo; Bähre, Heike; Lasa Uzcudun, Íñigo; Solano Goñi, Cristina; Ciencias de la Salud; Osasun Zientziak
Bacterial biofilms provide high cell density and a superior adaptation and protection from stress conditions compared to planktonic cultures, making them a very promising approach for bioremediation. Several Rhodococcus strains can desulfurize dibenzothiophene (DBT), a major sulphur pollutant in fuels, reducing air pollution from fuel combustion. Despite multiple efforts to increase Rhodococcus biodesulfurization activity, there is still an urgent need to develop better biocatalysts. Here, we implemented a new approach that consisted in promoting Rhodococcus erythropolis biofilm formation through the heterologous expression of a diguanylate cyclase that led to the synthesis of the biofilm trigger molecule cyclic di-GMP (c-di-GMP). R. erythropolis biofilm cells displayed a significantly increased DBT desulfurization activity when compared to their planktonic counterparts. The improved biocatalyst formed a biofilm both under batch and continuous flow conditions which turns it into a promising candidate for the development of an efficient bioreactor for the removal of sulphur heterocycles present in fossil fuels.
Open Access
Antibiofilm activity of flavonoids on staphylococcal biofilms through targeting BAP amyloids
(Nature Research, 2020) Matilla Cuenca, Leticia; Gil Puig, Carmen; Cuesta Ferre, Sergio; Rapún Araiz, Beatriz; Mira, Alex; Lasa Uzcudun, Íñigo; Valle Turrillas, Jaione; Ziemité, Miglé; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua, PI011 KILL-BACT
The opportunistic pathogen Staphylococcus aureus is responsible for causing infections related to indwelling medical devices, where this pathogen is able to attach and form biofilms. The intrinsic properties given by the self-produced extracellular biofilm matrix confer high resistance to antibiotics, triggering infections difficult to treat. Therefore, novel antibiofilm strategies targeting matrix components are urgently needed. The biofilm associated protein, Bap, expressed by staphylococcal species adopts functional amyloid-like structures as scaffolds of the biofilm matrix. In this work we have focused on identifying agents targeting Bap-related amyloid-like aggregates as a strategy to combat S. aureus biofilm-related infections. We identified that the flavonoids, quercetin, myricetin and scutellarein specifically inhibited Bap-mediated biofilm formation of S. aureus and other staphylococcal species. By using in vitro aggregation assays and the cell-based methodology for generation of amyloid aggregates based on the Curli-Dependent Amyloid Generator system (C-DAG), we demonstrated that these polyphenols prevented the assembly of Bap-related amyloid-like structures. Finally, using an in vivo catheter infection model, we showed that quercetin and myricetin significantly reduced catheter colonization by S. aureus. These results support the use of polyphenols as anti-amyloids molecules that can be used to treat biofilm-related infections.
Open Access
Bacterial biofilm functionalization through Bap amyloid engineering
(Springer Nature, 2022) Matilla Cuenca, Leticia; Taglialegna, Agustina; Gil Puig, Carmen; Toledo Arana, Alejandro; Lasa Uzcudun, Íñigo; Valle Turrillas, Jaione; Ciencias de la Salud; Osasun Zientziak
Biofilm engineering has emerged as a controllable way to fabricate living structures with programmable functionalities. The amyloidogenic proteins comprising the biofilms can be engineered to create self-assembling extracellular functionalized surfaces. In this regard, facultative amyloids, which play a dual role in biofilm formation by acting as adhesins in their native conformation and as matrix scaffolds when they polymerize into amyloid-like fibrillar structures, are interesting candidates. Here, we report the use of the facultative amyloid-like Bap protein of Staphylococcus aureus as a tool to decorate the extracellular biofilm matrix or the bacterial cell surface with a battery of functional domains or proteins. We demonstrate that the localization of the functional tags can be change by simply modulating the pH of the medium. Using Bap features, we build a tool for trapping and covalent immobilizing molecules at bacterial cell surface or at the biofilm matrix based on the SpyTag/SpyCatcher system. Finally, we show that the cell wall of several Gram-positive bacteria could be functionalized through the external addition of the recombinant engineered Bap-amyloid domain. Overall, this work shows a simple and modulable system for biofilm functionalization based on the facultative protein Bap. © 2022, The Author(s).