Relationship between biofilm formation and antibiotic resistance in commensal isolates of Staphylococcus epidermidis

Staphylococcus epidermidis is a common bacterial coloniser of the normal human microflora and usually have a benign relationship with the host. For several years, S. epidermidis was regarded as a harmless commensal microorganism. However, this bacterium is now recognised as an opportunistic pathogen, representing a leading cause of healthcare-associated infections. The major recognised determinants in the pathogenesis of S. epidermidis infections are its ability to form thick and multilayered biofilms along with high resistance to several classes of antibiotics. Biofilms are defined as structured communities of microorganisms embedded in a self-produced matrix of extracellular polymeric. It is well established that bacteria exhibiting a biofilm phenotype are more recalcitrant to antibiotic therapy. Hence, these two pathogenic features stated above appear to be intimately related. The present study aimed to evaluate the pathogenic potential of commensal S. epidermidis isolates through the assessment of their biofilm formation ability and antibiotic susceptibility profiles, as well as to analyse the relationship between biofilm formation and antibiotic resistance. To achieve that, thirty-one S. epidermidis isolates from Portuguese healthy volunteers (obtained from September 2012 to April 2013) were tested for biofilm formation ability, carriage of biofilm-associated genes (icaA, aap and bhp) and antibiotic susceptibility to six antibiotics (clindamycin, erythromycin, gentamicin, penicillin, rifampicin and vancomycin). The study of biofilm formation revealed that 20 (65%) isolates were able to produce biofilm at different levels, while 11 (35%) did not form biofilm. Moreover, 12 (39%) isolates were positive for icaA, 18 (58%) for aap, and 6 (19%) for bhp. With regards to the results of antibiotic susceptibility assays, the highest rates of resistance were detected for penicillin (n = 16, 52%), followed by erythromycin (n = 15, 48%) and gentamicin (n = 13, 42%), while the lowest rate was exhibited for ciprofloxacin (n = 2, 6%). All isolates were susceptible to rifampicin and vancomycin. By comparing the data on biofilm formation and antibiotic susceptibility assays, we found a significant higher frequency of antibiotic resistance in biofilm-formers than in non-biofilm formers (p = 0.02). Additionally, we also found a significant higher proportion of multidrug-resistant isolates among biofilm formers comparing with non-biofilm formers (p = 0.03), demonstrating a clear trend of isolates with biofilm formation ability to be resistant to two or more antibiotics simultaneously. In order to elucidate the nature of the relationship between biofilm formation and antibiotic resistance, we also compared the data on detection of biofilm-associated genes with the data on antibiotic susceptibility assays. In general, we also observed a tendency of isolates that carry the icaA and/or aap genes to be resistant to two or more antibiotics simultaneously. Interestingly, and when analysing the frequency of resistance for each of the antibiotics tested, the association between the presence of icaA gene and antibiotic resistance was particularly evident for gentamicin, with a p-value much close to the significance level (p = 0.07). Conversely, and with regards to aap gene, the same association was not observed. Overall, our findings provide evidence that commensal S. epidermidis strains are well equipped with biofilm formation determinants as well as are resistant to different groups of antibiotics. Moreover, our results support the existence of an association between biofilm formation and antibiotic resistance. Nevertheless, and taking into account other previously published results, our findings lead us to challenge the hypothesis that this association is common throughout all antibiotic classes. We rather hypothesise that this association might be restricted to some classes of antibiotics, especially aminoglycosides, and that it may be primarily associated with icaADBC operon and not with the biofilm phenotype per se

Efficacy studies of a S. epidermidis bacteriophage against stationary and biofilm cells

Staphylococcus epidermidis has recently become known as a common cause of nosocomial infections, predominantly in patients with indwelling medical devices. Although, S. epidermidis infections only rarely develop into life-threatening diseases, they are very frequent and difficult to treat due to the ability of this bacterium to adhere to the surfaces of indwelling medical devices and form biofilms. A biofilm is a three‐dimensional microbial structure consisting of a multicellular community composed of cells embedded in a matrix composed, at least partially, of material synthesized by the sessile cells in the community. When S. epidermidis cells are in a biofilm they are more resistant to antibiotics and to the immune system. The importance of biofilms in the pathogenesis of the S. epidermidis infections is becoming more understandable, consequently several studies are needed, in order to develop effective methods for biofilm control. The use of (bacterio)phages to eradicate biofilms has been considered a potentially valuable approach. Phages are virus that infect bacteria and are the most abundant organisms on Earth. They are generally very efficient antibacterial agents and possess many advantages over antibiotics. Our aim is to search for virulent phages with broad host range for S. epidermidis biofilm therapy. Using wastewater treatment plants raw effluents we were able to isolate 5 phages. Their activity against 40 clinical S. epidermidis isolates with different genetic profiles was screened and was found to be different ranging from 46% to 95% of positive results. Further morphologic and genetic characterization of these isolated phages is now being performed. Preliminary results show that, one of the phages (phiIBB-Se1), using a MOI of 1 is able to cause a 6 Log CFU/ml reduction of the cell titre in <2h for some of the clinical strains at exponential phase and in <4h for stationary phase cells. This phage has also the capacity of reducing by up to 2 Log CFU/ml 24h biofilm cells. These are promising results, since phage phiIBB-Se1 presents a broad host range and ability to control S. epidermidis under different metabolic states. Ongoing studies are being performed with 4 other phages, with the purpose of developing a phage cocktail to be used against S. epidermidis biofilm infections

Isolation and optimization of the production of Staphylococcus epidermidis bacteriophage from environmental samples

Staphylococcus epidermidis is now among the most important nosocomial pathogenic agents owing its virulence to the adhesion and biofilm-forming abilities on medical surfaces, such as catheters. Biofilm control by antibiotics is often innefective and new strategies of biofilm control are being sought. One promising strategy is the use of bacteria-specific virus, known as bacteriophages, to control infections by pathogenic bacteria. Bacteriophages, also know as phages, have been suggested to be one of the most abundant biological agents on the planet. Phages are currently suggested as possible alternatives to antibiotics for the treatment of bacterial diseases in humans to minimize the pathogen loads in medical devices . The goal of this study was to isolate new phages with potential bactericidal activity against S. epidermidis clinical isolates. Bacteriophages were isolated from an effluent from Waste Water Treatment Plants or from Hospital efluents, using a set of 40 bacterial strains as background. Five phages were isolated but when determining the phage titer the achieved concentration was around 10E5 pfu/ml and this titer was reduced 1 fold in two week‘s time. In order to increase the concentration of bacteriophages, since the obtained concentration was not sufficient to use in biofilm assays, severall optimization steps were performed, using previous described isolation protocols, namelly: using different concentrations of CaCl2, using different concentrations of top agar, using different buffers, and using different phage filtration systems. For the optimization protocols we selected the bacteriophage with the higher titters and found that an optimized protocol was achieved by using Tris Buffer, Top Agar at 0.4%, and purification with CsCl2 gradient (q = 1.3, 1.5, and 1.7) with ultracentrifugation at 100,000g for 1 h at 4⁰C. This phage titter was determined to be around 10E8-10E9 pfu/ml. The optimized produced phage was then characterized by determining the lytic spectrum. The phage was able to lyse 13 strains, and of these strains 10 had the biofilms genes present. Finally, the 10 selected strains were tested for biofilm formation, using the microtiter assay, and it was confirmed that they formed biofilms in TSB supplemented with 1% glucose. For future work; we need to determine if we have lytic or temperate phages with DNA sequence analyses and to test the Phage against to the Biofilm formation of relevant bacterial strains

Can bacteriofages be effective in controlling harmful biofilms?

(Bacterio)phages are viruses that specifically infect bacteria, causing cell lysis and therefore can be considered a valuable strategy for bacterial control. Recent studies have demonstrated the potential of using phages to control bacterial biofilms. Phages are able to penetrate the extracellular matrnc and can cause up to 90% of biofilm mass reduction even in old biofilms. However phage action can be impaired by components of the biofilm matrix, the slow growth of biofilm bacteria and the fast emergence of phage resistant phenotypes. We have conducted several studies of phage biofilm interaction and based on our experimental data, we have hypothesized that the general mechanisms of a virulent phage-biofilm infection, in a very simplistic model, can occur in four stages: 1) Transport of the phage particles through the biofilm matrix (by diffusion or convection mechanisms); 2) Settlement and/or attachment of phages onto bacterial cells embedded in the biofilm matrix, followed by adsorption and phage replicatiOn inside host cells; 3) Release of phage progeny to planktonic and biofilm phases, through host cell lysis and infection of neighbourhood biofilm cells resulting in biofilm biomomass reduct1on; 4) Detachment of biofilm portions and phages into the planktomc phase. Nevertheless, the interaction between phage and biofilms is a rather complex process. Theoretically, a biofilm should be rapidly infected because cells are more close to each other and this fact can enhance phage replication, when compared to the less accessible bacteria of planktonic cultures. On the other hand, the structure and compositiOn of the biofilm as well the physiology of the biofilm cells may impose some limitations to biofilm infection. Indeed, phage·biofilm interaction is greatly inFluenced by the biofilm age, biofilm structure, biofilm mode of growth and most importantly the host and phage characteristics. This work is a summary of all phage/biofilm interaction studies conducted by our team involving different phage types and host species

Correction to: The first Paenibacillus larvae bacteriophage endolysin (PlyPl23) with high potential to control American foulbrood

The Data Availability statement for this paper is incorrect. The correct statement is: All relevant data are within the paper and its Supporting Information files, except the aminoacidic sequence of the endolysin, which is available in RefSeq, accession number YP_008320357.1

Phage therapy as an alternative or complementary strategy to prevent and control biofilm-related infections

The complex heterogeneous structure of biofilms confers to bacteria an important survival strategy. Biofilms are frequently involved in many chronic infections in consequence of their low susceptibility to antibiotics as well as resistance to host defences. The increasing need of novel and effective treatments to target these complex structures has led to a growing interest on bacteriophages (phages) as a strategy for biofilm control and prevention. Phages can be used alone, as a cocktail to broaden the spectra of activity, or in combination with other antimicrobials to improve their efficacy. Here, we summarize the studies involving the use of phages for the treatment or prevention of bacterial biofilms, highlighting the biofilm features that can be tackled with phages or combined therapy approaches.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the project PTDC/BBB-BSS/6471/2014, the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684). This work was also supported by BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Efficacy studies of phage phiIBB-Sep1 against S. epidermidis cells under different metabolic states

Staphylococcus epidermidis has recently become known as a usual cause of nosocomial infections, predominantly in patients with indwelling medical devices. Although, S. epidermidis infections only rarely develop into life-threatening diseases, they are very frequent and difficult to treat due to the ability of this bacterium to adhere to the surfaces of indwelling medical devices and form biofilms. When S. epidermidis cells are in a biofilm they are more resistant to antibiotics and to the immune system. The importance of biofilms in the pathogenesis of the S. epidermidis infections is becoming more understandable, consequently several studies are needed, in order to develop effective methods for biofilm control. The use of bacteriophages (phages) to eradicate biofilms can be seen as a potentially valuable approach. Phages are virus that infect bacteria and are the most abundant organisms on Earth. They are generally very efficient antibacterial agents and possess many advantages over antibiotics. Our aim is to search for virulent phages with broad host range for S. epidermidis biofilm therapy. Using wastewater treatment plants raw effluents we were able to isolate 5 phages. Their lytic activity was screened against 40 clinical S. epidermidis isolates with different genetic profiles and it was found to be different ranging from 46% to 95% of positive results. Further morphologic and genetic characterization of these isolated phages is now being performed. Efficacy studies results show that phage phiIBB-Sep1 is able to cause a 6 Log CFU/ml reduction of the cell titre in <2h for some of the clinical strains at exponential phase and in <4h for stationary phase cells (using a MOI of 1). This phage has also the capacity of reducing by up to 2 Log CFU/ml 24h biofilm cells and in some strains it was observed 50% cell reduction on biofilms. Besides CFU counting, all the cell counts were confirmed by flow cytometry assays. Additionally, flow cytometry allow the observation that this phage kill cells under different metabolic states from the biofilm. Work developed with non biofilm forming strains showed that possibly PNAG might be the cell receptor of the phage. The high amounts of PNAG on biofilms, might the lower activity of this phage on biofilms. These are promising results, since phage phiIBB-Se1 presents a broad host range and ability to control S. epidermidis under different metabolic states. Ongoing studies are being performed with 4 other phages, with the purpose of developing a phage cocktail to be used against S. epidermidis biofilm infections

Environmentally-friendly technology for rapid identification and quantification of emerging pollutants from wastewater using infrared spectroscopy

Supplementary material related to this article can be found, in theonline version, at doi:https://doi.org/10.1016/j.etap.2020.103458.The monitoring of emerging pollutants in wastewaters is nowadays an issue of special concern, with the classical quantification methods being time and reagent consuming. In this sense, a FTIR transmission spectroscopy based chemometric methodology was developed for the determination of eight of these pollutants. A total of 456 samples were, therefore, obtained, from an activated sludge wastewater treatment process spiked with the studied pollutants, and analysed in the range of 200cm1 to 14,000cm1. Then, a k-nearest neighbour (kNN) analysis aiming at identifying each sample pollutant was employed. Next, partial least squares (PLS) and ordinary least squares (OLS) modelling approaches were employed in order to obtain suitable prediction models. This procedure resulted in good prediction abilities regarding the estimation of atrazine, desloratadine, paracetamol, -estradiol, ibuprofen, carbamazepine, sulfamethoxazole and ethynylestradiol concentrations in wastewaters. These promising results suggest this technology as a fast, eco-friendly and reagent free alternative methodology for the quantification of emerging pollutants in wastewaters.The authors thank the Portuguese Foundation for Science andTechnology (FCT) under the scope of the strategic funding of UIDB/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fundunder the scope of Norte2020 - Programa Operacional Regional doNorte.info:eu-repo/semantics/publishedVersio

Effect of design and operating variables on biofouling

Biofouling, like other types of fouling, may cause serious problems in heat transfer equipment. In general, the formation of deposits is dependent on several physical and chemical variables, but in what respects bioufouling other conditions have to be considered. These are related with the growth and reproduction of microorganisms in the fluid and also in the films. In this work reference is made specially to the effects on bioufouling of: fluid velocity; surface characteristics; surface and fluid temperature; pH of the fluid; nutrient concentration; presence of inorganic suspended particlesinfo:eu-repo/semantics/publishedVersio

Genomic characterization of the Staphylococcus epidermidis-specific bacteriophage SEP1 and evaluation of its lytic activity against bacterial under different metabolic states

Staphylococcus epidermidis is one of the most frequent causative agents of nosocomial infections, predominantly in patients with indwelling medical devices. This microrganism may form biofilms which are microbial structures very tolerant to the host immune defenses and to antibiotherapy. Therefore, studies are needed in order to develop effective methods for biofilm control. Currently, bacteriophages (phages) are seen as an important tool to combat pathogenic organisms. These bacteria-specific viruses are generally very efficient antibacterial agents and possess many advantages over antibiotics. The present study concerns the search for virulent phages with broad host range for S. epidermidis biofilm therapy. Using wastewater treatment plant raw effluents, a novel phage was isolated and characterized. This virus was named phiIBB-SEP1 and TEM micrographs suggested that it belonged to the Twortlikevirus genus. Phage phiIBB-SEP1 is able to infect 41 S. epidermidis clinical isolates used in this study, and contrarily to other polyvalent viruses of the Twortlikevirus genus, phiIBB-SEP1 is highly specific for S. epidermidis strains. The genome of this phage was fully sequenced and presents the typical structure of a member of the Twortlikevirus. However, when compared to other staphylococcal members of this genus, it showed DNA sequence identities no greater than 58.2%, suggesting that phiIBB-SEP1 is a new species within this subfamily. Efficacy studies results showed that phage phiIBB-SEP1 is able to cause a 6 Log CFU per ml reduction of the cell titre in less than 2h for some of the clinical strains in exponential phase; and, in less than 4h for stationary phase cells (using a multiplicity of infection of 1). This phage has also the capacity of reducing, by up to 2 Log CFU per ml, 24h scraped biofilm cells, and in some strains it was observed 50% cell reduction. Besides CFU counting, this cell reduction was confirmed by flow cytometry counting. Additionally, live/death flow cytometry staining allowed the observation that this phage kills biofilms bacteria in different metabolic states. These are promising results, since phage phiIBB-SEP1 presents a broad host strain range and the ability to control S. epidermidis bacteria in different metabolic states. Keywords: bacteriophage, Twortlikevirus, S. epidermidis, staphylococci, biofilm