Multisubstituted pyrimidines effectively inhibit bacterial growth and biofilm formation of Staphylococcus aureus

Biofilms are multicellular communities of microorganisms that generally attach to surfaces in a self-produced matrix. Unlike planktonic cells, biofilms can withstand conventional antibiotics, causing significant challenges in the healthcare system. Currently, new chemical entities are urgently needed to develop novel anti-biofilm agents. In this study, we designed and synthesized a set of 2,4,5,6-tetrasubstituted pyrimidines and assessed their antibacterial activity against planktonic cells and biofilms formed by Staphylococcus aureus. Compounds 9e, 10d, and 10e displayed potent activity for inhibiting the onset of biofilm formation as well as for killing pre-formed biofilms of S. aureus ATCC 25923 and Newman strains, with half-maximal inhibitory concentration (IC50) values ranging from 11.6 to 62.0 mu M. These pyrimidines, at 100 mu M, not only decreased the number of viable bacteria within the pre-formed biofilm by 2-3 log(10) but also reduced the amount of total biomass by 30-50%. Furthermore, these compounds were effective against planktonic cells with minimum inhibitory concentration (MIC) values lower than 60 mu M for both staphylococcal strains. Compound 10d inhibited the growth of S. aureus ATCC 25923 in a concentration-dependent manner and displayed a bactericidal anti-staphylococcal activity. Taken together, our study highlights the value of multisubstituted pyrimidines to develop novel anti-biofilm agents.Peer reviewe

Repurposing the Sphingosine-1-Phosphate Receptor Modulator Etrasimod as an Antibacterial Agent Against Gram-Positive Bacteria

New classes of antibiotics are urgently needed in the fight against multidrug-resistant bacteria. Drug repurposing has emerged as an alternative approach to accelerate antimicrobial research and development. In this study, we screened a library of sphingosine-1-phosphate receptor (S1PR) modulators against Staphylococcus aureus and identified five active compounds. Among them, etrasimod (APD334), an investigational drug for the treatment of ulcerative colitis, displayed the best inhibitory activity against S. aureus when growing as free-floating planktonic cells and within biofilms. In follow-up studies, etrasimod showed bactericidal activity and drastic reduction of viable bacteria within 1 h of exposure. It also displayed a potent activity against other Gram-positive bacteria, including penicillin- and methicillin-resistant S. aureus strains, S. epidermidis, and Enterococcus faecalis, with a minimum inhibitory concentration (MIC) ranging from 5 to 10 mu M (2.3-4.6 mu g/mL). However, no inhibition of viability was observed against Gram-negative bacteria Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa, showing that etrasimod preferably acts against Gram-positive bacteria. On the other hand, etrasimod was shown to inhibit quorum sensing (QS) signaling in Chromobacterium violaceum, suggesting that it may block the biofilm formation by targeting QS in certain Gram-negative bacteria. Furthermore, etrasimod displayed a synergistic effect with gentamicin against S. aureus, thus showing potential to be used in antibiotic combination therapy. Finally, no in vitro toxicity toward mammalian cells was observed. In conclusion, our study reports for the first time the potential of etrasimod as a repurposed antibacterial compound against Gram-positive bacteria.Peer reviewe

Food-Grade Bacteria Combat Pathogens by Blocking AHL-Mediated Quorum Sensing and Biofilm Formation

Disrupting bacterial quorum sensing (QS) signaling is a promising strategy to combat pathogenic biofilms without the development of antibiotic resistance. Here, we report that food-associated bacteria can interfere with the biofilm formation of a Gram-negative pathogenic bacterium by targeting its AHL (acyl-homoserine lactone) QS system. This was demonstrated by screening metabolic end-products of different lactobacilli and propionibacteria using Gram-negative and biofilm-forming Chromobacterium violaceum as the QS reporter and our anti-QS microscale screening platform with necessary modifications. The method was optimized in terms of the inoculation technique and the concentrations of D-glucose and L-tryptophan, two key factors controlling the synthesis of violacein, a purple pigment indicating the activation of the QS system in C. violaceum. These improvements resulted in ca. 16-times higher violacein yields and enabled revealing anti-QS effects of Lactobacillus acidophilus, Lentilactobacillus kefiri, Lacticaseibacillus rhamnosus and Propionibacterium freudenreichii, including new cheese-associated strains. Our findings also suggest that acetate and propionate excreted by these species are the main factors that interrupt the QS-mediated signaling and subsequent biofilm growth without affecting the cell viability of the C. violaceum reporter. Thus, the present study reports a revised anti-QS screening method to accurately define new bacteria with an ability to combat pathogens in a safe and sustainable way

Food-Grade Bacteria Combat Pathogens by Blocking AHL-Mediated Quorum Sensing and Biofilm Formation

Disrupting bacterial quorum sensing (QS) signaling is a promising strategy to combat pathogenic biofilms without the development of antibiotic resistance. Here, we report that food-associated bacteria can interfere with the biofilm formation of a Gram-negative pathogenic bacterium by targeting its AHL (acyl-homoserine lactone) QS system. This was demonstrated by screening metabolic end-products of different lactobacilli and propionibacteria using Gram-negative and biofilm-forming Chromobacterium violaceum as the QS reporter and our anti-QS microscale screening platform with necessary modifications. The method was optimized in terms of the inoculation technique and the concentrations of D-glucose and L-tryptophan, two key factors controlling the synthesis of violacein, a purple pigment indicating the activation of the QS system in C. violaceum. These improvements resulted in ca. 16-times higher violacein yields and enabled revealing anti-QS effects of Lactobacillus acidophilus, Lentilactobacillus kefiri, Lacticaseibacillus rhamnosus and Propionibacterium freudenreichii, including new cheese-associated strains. Our findings also suggest that acetate and propionate excreted by these species are the main factors that interrupt the QS-mediated signaling and subsequent biofilm growth without affecting the cell viability of the C. violaceum reporter. Thus, the present study reports a revised anti-QS screening method to accurately define new bacteria with an ability to combat pathogens in a safe and sustainable way

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Comprehensive Exploration of Bacterial Biofilm Responses to Chemical, Physicochemical and Biological Factors

Bacteria are highly versatile and able to adapt to numerous environmental conditions to ensure their survival. The formation of bacterial biofilms, a form of bacterial life, notably contribute to such protection via diverse mechanisms. In the healthcare system, most of the microbial infections are biofilm-related, including chronic opportunistic and medical devices bacterial infections, Staphylococcus aureus and Pseudomonas aeruginosa being among the most commonly isolated pathogens. Biofilms have an increased tolerance to both the host immune system and exogenous antimicrobials. Consequently, the biofilm lifestyle is linked with the alarming increasing rate of antimicrobial resistance worldwide. The production of the matrix serves as a shield to biofilm cells. The biofilm matrix is composed of macromolecules i.e., polysaccharides, proteins and eDNA and it has a dynamic complexity. Because of the detrimental impact of biofilms in the clinic, further explorations on factors able to alter the biofilm formation may shed some light on improving biofilm control strategies. Therefore, the main aim of this thesis project was to examine the responses of S. aureus biofilm formation to modified surfaces and newly synthesized chemical agents as well as when it was grown in co-culture with P. aeruginosa. In the first study, the ability of S. aureus to form biofilms when grown on a set of nanostructured polymer-coated surfaces was characterized by measuring cell viability, polysaccharide levels and surface-associated proteins in the biofilm matrix. The effect of two different biofilm formation assays on the S. aureus biofilm formation was also assessed. Certain surface chemistry and topography properties led to different S. aureus biofilm responses, depending on the growth assay used. The proteomics analysis showed that there were surface-associated proteins, which were differentially expressed depending upon the used biofilm formation assay and the modified surface where these biofilms were grown. Also, the expression of a subset of virulence-related proteins was shown to correlate with certain underexplored parameters of surface roughness. In the second study, a set of pyrimidine derivatives was evaluated for its S. aureus biofilm inhibition activity, followed by the chemical optimization and functional characterization of the most active compounds (9e, 10d and 10e). S. aureus biofilm was shown to be inhibited by these compounds, including established biofilms that were affected via reduction of the cell viability, total biomass and polysaccharide abundance. Additionally, structure-activity relationship study was carried out. In the third study, after establishing optimal conditions for growing S. aureus and P. aeruginosa biofilms in co-culture, a proteomics approach was used for analyzing the interactions via proteome profiles between mono- and co-cultured biofilms. Several phenotypic assays were performed and thus they confirmed the findings from the proteomics analysis. Furthermore, additional bacterial responses were studied by using the dual-species biofilms model to evaluate a subset of nanostructured polymer-coated surfaces and the most effective pyrimidine 10d. Overall, this thesis aims to provide comprehensive insights on the responses of bacterial biofilms when grown on modified surfaces with different physicochemical properties and exposed to chemical agents, which could ultimately contribute towards the development of enhanced biofilm control strategies.Bakteerit ovat monimuotoisia eliöitä, jotka kykenevät sopeutumaan erilaisiin elinolosuhteisiin varmistaakseen selviytymisensä. Biofilmi on bakteerien kasvutapa, joka edistää tätä sopeutumista suojaamalla bakteereja ympäristötekijöiltä useiden eri mekanismien välityksellä. Biofilmit liittyvät suurimpaan osaan terveydenhuollossa hoidetuista mikrobi-infektiosta, kuten kroonisiin opportunisti-infektioihin sekä lääkinnällisiin laitteisiin liittyviin infektioihin. Staphylococcus aureus- ja Pseudomonas aeruginosa -bakteerilajit lukeutuvat näistä tavallisimmin eristettyihin taudinaiheuttajiin. Biofilmeillä on kohonnut kyky sietää sekä isäntäorganismin immuunipuolustusta että mikrobilääkkeitä, ja kasvutapa on liitetty mikrobilääkeresistenssin hälyttävän nopeaan maailmanlaajuiseen lisääntymiseen. Matriksi on puolestaan rakenne, jonka biofilmi tuottaa solujen suojaksi. Se koostuu makromolekyyleistä, kuten polysakkarideista, proteiineista ja solunulkoisesta DNA:sta, ja rakenne on dynaamisesti kompleksinen. Hoidollisesti haasteellisten biofilmien muodostumiseen vaikuttavien tekijöiden tutkimusta tarvitaan torjuntakeinojen kehittämiseksi, ja tämän työn päätavoitteena onkin tutkia, miten muunnellut pinnat, uudet synteettiset yhdisteet sekä yhteisviljely P. aeruginosa -bakteerin kanssa vaikuttavat S. aureus -biofilmin muodostumiseen. Ensimmäisessä osatyössä S. aureus -bakteerin kykyä muodostaa biofilmejä erilaisilla nanorakenteisilla polymeeripinnoitteilla tutkittiin mittaamalla solujen elinkelpoisuutta sekä polysakkaridimääriä ja pintaproteiinien esiintymistä biofilmimatriksissa. Samalla tutkittiin kahden erilaisen viljelymenetelmän vaikutusta biofilmimuodostukseen. Tietyt pintakemia- ja topografiaominaisuudet saivat aikaan biofilmeissä erilaisia vasteita riippuen käytetystä kasvatusmenetelmästä. Proteomiikka-analyysillä tunnistettiin pintaproteiineja, joiden määrä vaihteli viljelytavan ja pinnoitteen mukaan, ja taudinaiheuttamiskykyyn vaikuttavien proteiinien osajoukon ilmenemisen havaittiin korreloivan eräiden vielä heikosti tunnettujen pintakarkeusparametrien kanssa. Toisessa osatyössä tutkittiin pyrimidiinijohdannaisten kykyä estää S. aureus -biofilmin kasvua, ja aktiivisimmat yhdisteet (9e, 10d ja 10e) valittiin kemiallisen rakenteen optimointiin sekä toiminnalliseen kartoitukseen. Näiden yhdisteiden todettiin paitsi estävän biofilmimuodostusta myös vaikuttavan jo muodostuneisiin biofilmeihin vähentämällä solujen elinkelpoisuutta, biofilmin kokonaisbiomassaa ja polysakkaridin määrää. Lisäksi tutkittiin näiden yhdisteiden kemiallisen rakenteen ja biologisen aktiivisuuden välisiä yhteyksiä. Kolmannessa osatyössä optimoitiin S. aureus ja P. aeruginosa -yhteisviljelmän kasvatusolosuhteet ja verrattiin puhdas- ja yhteisviljelminä kasvatettujen biofilmien proteomiprofiileja. Proteomiikka-analyysin löydökset vahvistettiin fenotyyppisillä määrityksillä. Biofilmiyhteisviljelmämallia käytettiin myös nanorakenteisten polymeeripinnoitteiden sekä tehokkaimmaksi osoittautuneen pyrimidiinijohdannaisen 10d jatkoarviointiin. Tämä väitöskirja pyrkii tarjoamaan kattavan näkemyksen vasteista, joita bakteeribiofilmeissä saadaan aikaan kasvattamalla niitä fysikaalis-kemiallisilta ominaisuuksiltaan eroavilla, muunnelluilla pintamateriaaleilla sekä koeyhdisteille altistettuna. Nämä voivat edistää tehokkaampien biofilmin torjuntakeinojen kehittämistä

Modulation of virulence factors of Staphylococcus aureus by nanostructured surfaces

Investigating and understanding the response of microbes to various surfaces requires a versatile parametrisation of the surface, and multiple assays that captures the complexity of the biofilm structures. Here, Staphylococcus aureus biofilm viability, polysaccharide poly-N-acetylglucosamine, and proteins on the cell surface were analysed with agar plate- and well plate-based biofilm formation assays. Biofilms were grown on a set of nanostructured polymeric surfaces, which were thoroughly characterised for their surface chemistry and topography. Surface hydrophobicity, summit density as well as peak and valley structure were found to influence the microbial viability and exopolysaccharide abundance level in the agar plate assay. In the well plate assay, surface chemical parameters had a lesser influence on the viability, but roughness caused by valley structures increased the viability and decreased the exopolysaccharide expression. Surface proteins relating to pathogenicity were affected by the biofilm formation assay. The abundance profile of these proteins correlated clearly with several roughness parameters, especially fine structure parameters in the agar plate assay and lateral roughness in the well plate assay. These results highlight the necessity of describing the surfaces with a versatile set of different roughness parameters to completely understand what specific features of a surface drive a certain bacterial response.Peer reviewe

Surfaceome and Exoproteome Dynamics in Dual-Species Pseudomonas aeruginosa and Staphylococcus aureus Biofilms

Bacterial biofilms are an important underlying cause for chronic infections. By switching into the biofilm state, bacteria can evade host defenses and withstand antibiotic chemotherapy. Despite the fact that biofilms at clinical and environmental settings are mostly composed of multiple microbial species, biofilm research has largely been focused on single-species biofilms. In this study, we investigated the interaction between two clinically relevant bacterial pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) by label-free quantitative proteomics focusing on proteins associated with the bacterial cell surfaces (surfaceome) and proteins exported/released to the extracellular space (exoproteome). The changes observed in the surfaceome and exoproteome of P. aeruginosa pointed toward higher motility and lower pigment production when co-cultured with S. aureus. In S. aureus, lower abundances of proteins related to cell wall biosynthesis and cell division, suggesting increased persistence, were observed in the dual-species biofilm. Complementary phenotypic analyses confirmed the higher motility and the lower pigment production in P. aeruginosa when co-cultured with S. aureus. Higher antimicrobial tolerance associated with the co-culture setting was additionally observed in both species. To the best of our knowledge, this study is among the first systematic explorations providing insights into the dynamics of both the surfaceome and exoproteome of S. aureus and P. aeruginosa dual-species biofilms.Peer reviewe