The N-terminal domain of enterococcal surface protein, Esp, is sufficient for Esp-mediated biofilm enhancement in Enterococcus faecalis

Enterococci have emerged as one of the leading causes of nosocomial bloodstream, surgical site, and urinary tract infections. More recently, enterococci have been associated with biofilms, which are bacterial communities attached to a surface and encased in an extracellular polymeric matrix. The enterococcal cell surface-associated protein, Esp, enhances biofilm formation by Enterococcus faecalis in a glucose-dependent manner. Mature Esp consists of a nonrepeat N-terminal domain and a central region made up of two types of tandem repeats followed by a C-terminal membrane-spanning and anchor domain. This study was undertaken to localize the specific domain(s) of Esp that plays a role in Esp-mediated biofilm enhancement. To achieve this objective, we constructed in-frame deletion mutants expressing truncated forms of Esp in an isogenic background. By comparing strains expressing the mutant forms of Esp to those expressing wild-type Esp, we found that the strain expressing Esp lacking the N-terminal domain formed biofilms that were quantitatively less in biovolume than the strain expressing wild-type Esp. Furthermore, an E. faecalis strain expressing only the N-terminal domain of Esp fused to a heterologous protein anchor formed biofilms that were quantitatively similar to those formed by a strain expressing full-length Esp. This suggested that the minimal region contributing to Esp-mediated biofilm enhancement in E. faecalis was confined to the nonrepeat N-terminal domain. Expression of full-length E. faecalis Esp in heterologous host systems of esp-deficient Lactococcus lactis and Enterococcus faecium did not enhance biofilm formation as was observed for E. faecalis. These results suggest that Esp may require interaction with an additional E. faecalis-specific factor(s) to result in biofilm enhancement.     

Identification of fitness determinants in Enterococcus faecalis by differential proteomics

Enterococcus (E.) faecalis is found as commensal in healthy humans, in a variety of fermented foods. It can serve as probiotic but also as pathogen causing endocarditis, bacteremia and urinary tract infections. We have employed a proteomic study with E. faecalis strain OG1RF under different growth conditions and in contact to mouse intestinal cells to identify novel latent and adaptive fitness determinants. These relate to changes in catabolic pathways (BudA), protein biosynthesis (AsnS), cellular surface biosynthesis (RmlA) and regulatory mechanisms (OmpR). This knowledge can be used to derive novel evidence-based targets, which can be used to further elucidate gene expression changes enhancing pathogenicity or fitness in a commensal strain and possibly delineate this species into groups of higher and lower risk for applications in a food or a medical context versus improved treatment strategies of the so far hard to cure diseases.     

Characterization of Mucosal Candida albicans Biofilms

C. albicans triggers recurrent infections of the alimentary tract mucosa that result from biofilm growth. Although the ability of C. albicans to form a biofilm on abiotic surfaces has been well documented in recent years, no information exists on biofilms that form directly on mucosal surfaces. The objectives of this study were to characterize the structure and composition of Candida biofilms forming on the oral mucosa. We found that oral Candida biofilms consist of yeast, hyphae, and commensal bacteria, with keratin dispersed in the intercellular spaces. Neutrophils migrate through the oral mucosa and form nests within the biofilm mass. The cell wall polysaccharide β-glucan is exposed during mucosal biofilm growth and is more uniformly present on the surface of biofilm organisms invading the oral mucosa. We conclude that C. albicans forms complex mucosal biofilms consisting of both commensal bacterial flora and host components. These discoveries are important since they can prompt a shift of focus for current research in investigating the role of Candida-bacterial interactions in the pathogenesis of mucosal infections as well as the role of β-glucan mediated signaling in the host response.     

Antimicrobial activity of tigecycline alone or in combination with rifampin against Staphylococcus epidermidis in biofilm

Staphylococcus epidermidis is a commensal inhabitant of the healthy human skin, but in the recent years, it has been recognized as a nosocomial pathogen especially in immunocompromised patients. The pathogenesis of S. epidermidis is thought to be based on its capacity to form biofilms on the surface of medical devices, where bacterial cells may persist, protected from host defence and antimicrobial agents. Rifampin has been shown to be one of the most active antimicrobial agents in the eradication of the staphylococcal biofilm. However, this antibiotic should not be used in monotherapy. Therefore, one of the objectives of our research was to study the efficacy of the tigecycline/rifampin combination against methicillin-resistant S. epidermidis embedded in biofilms. Of the 80 clinically significant S. epidermidis isolates, 75 strains possess the ability to form a biofilm. These bacteria formed the biofilm via ica-dependent mechanisms. However, other biofilm-associated genes, including aap (encoding accumulation-associated protein) and bhp (coding cell wall-associated protein), were present in 85 and 29 % of isolates, respectively. The biofilm structures of S. epidermidis strains were also analyzed in confocal laser scanning microscopy (CLSM) and the obtained image demonstrated differences in their architecture. In vitro studies showed that the MIC value for tigecycline against S. epidermidis growing in the biofilm ranged from 0.125 to 2 μg/mL. Tigecycline in combination with rifampin demonstrated higher activity against bacteria embedded in biofilms than tigecycline alone.     

In Vitro Biofilm Activity of Non-Candida albicans Candida Species

Candidosis has been attributed to C. albicans; however, infections caused by non-Candida albicans Candida (NCAC) species are increasingly being recognised. The ability of Candida to grow as a biofilm is an important feature that promotes both infection and persistence in the host. The biofilms’ activity is significant since high activity might be associated with enhanced expression of putative virulence factors, whilst in contrast low activity has previously been suggested as a mechanism for resistance of biofilm cells to antimicrobials. The aim of this study was to determine the metabolic activity of in vitro biofilms formed by different clinical isolates of NCAC species. The in situ total metabolic activity of C. parapsilosis, C. tropicalis and C. glabrata biofilms was determined using 2,3-(2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay, and the number of cultivable cells was also established by CFU (colony forming unit) counts. The biofilm structure was assessed by scanning electron microscopy (SEM). Results showed that total biofilm metabolic activity was species and strain dependent. C. glabrata exhibited the lowest biofilm metabolic activity despite having the highest number of biofilm cultivable cells. Similarly, the metabolic activity of resuspended C. glabrata biofilm and planktonic cells was lower than that of the other species. This study demonstrates the existence of intrinsic activity differences amongst NCAC species, which could have important implications in terms of species relative virulence. Furthermore, the absence of an obvious correlation, between cultivable cells number and total biofilm activity, raises the question about which parameter is the most appropriate for the in vitro assessment of biofilms and their potential clinical significance.     

Chemical composition of Enterococcus faecalis in biofilm cells initiated from different physiologic states

Enterococcus faecalis is a ubiquitous bacterium of the gut that is observed in persistent periradicular infections. Its pathogenicity is associated with biofilm formation and the ability to survive under nutrient-poor (starvation) conditions. However, characteristics of chemical composition of biofilm cells developed by starved E. faecalis cells remain poorly understood. In this study, E. faecalis cells in exponential, stationary, and starvation phases were prepared and separately cultured to form biofilms. Confocal laser scanning microscopy was performed to verify biofilm formation. Raman microscopy was used to investigate the chemical composition of cells within the biofilms. Compared to cells in exponential or stationary phase, starved cells developed biofilms with fewer culturable cells (P?E. faecalis.     

Characterization and comparison of biofilm development by pathogenic and commensal isolates of Histophilus somni

Histophilus somni (Haemophilus somnus) is an obligate inhabitant of the mucosal surfaces of bovines and sheep and an opportunistic pathogen responsible for respiratory disease, meningoencephalitis, myocarditis, arthritis, and other systemic infections. The identification of an exopolysaccharide produced by H. somni prompted us to evaluate whether the bacterium was capable of forming a biofilm. After growth in polyvinyl chloride wells a biofilm was formed by all strains examined, although most isolates from systemic sites produced more biofilm than commensal isolates from the prepuce. Biofilms of pneumonia isolate strain 2336 and commensal isolate strain 129Pt were grown in flow cells, followed by analysis by confocal laser scanning microscopy and scanning electron microscopy. Both strains formed biofilms that went through stages of attachment, growth, maturation, and detachment. However, strain 2336 produced a mature biofilm that consisted of thick, homogenous mound-shaped microcolonies encased in an amorphous extracellular matrix with profound water channels. In contrast, strain 129Pt formed a biofilm of cell clusters that were tower-shaped or distinct filamentous structures intertwined with each other by strands of extracellular matrix. The biofilm of strain 2336 had a mass and thickness that was 5- to 10-fold greater than that of strain 129Pt and covered 75 to 82% of the surface area, whereas the biofilm of strain 129Pt covered 35 to 40% of the surface area. Since H. somni is an obligate inhabitant of the bovine and ovine host, the formation of a biofilm may be crucial to its persistence in vivo, and our in vitro evidence suggests that formation of a more robust biofilm may provide a selective advantage for strains that cause systemic disease.     

<Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> glucose uptake in biofilm versus planktonic cells

The aim of this work was to compare the glucose uptake of biofilms formed by four different Staphylococcus epidermidis strains as well as to compare between sessile and planktonic cells of the same strain. Biofilm cells showed a lower level of glucose uptake compared to planktonic cells. Moreover, glucose uptake by cells in the sessile form was strongly influenced by biofilm composition. Therefore, this work helps to confirm the phenotypic variability of S. epidermidis strains and the different behaviour patterns between sessile and free cells.     

Enhanced Biofilm Formation and Increased Resistance to Antimicrobial Agents and Bacterial Invasion Are Caused by Synergistic Interactions in Multispecies Biofilms

Most biofilms in their natural environments are likely to consist of consortia of species that influence each other in synergistic and antagonistic manners. However, few reports specifically address interactions within multispecies biofilms. In this study, 17 epiphytic bacterial strains, isolated from the surface of the marine alga Ulva australis, were screened for synergistic interactions within biofilms when present together in different combinations. Four isolates, Microbacterium phyllosphaerae, Shewanella japonica, Dokdonia donghaensis, and Acinetobacter lwoffii, were found to interact synergistically in biofilms formed in 96-well microtiter plates: biofilm biomass was observed to increase by >167% in biofilms formed by the four strains compared to biofilms composed of single strains. When exposed to the antibacterial agent hydrogen peroxide or tetracycline, the relative activity (exposed versus nonexposed biofilms) of the four-species biofilm was markedly higher than that in any of the single-species biofilms. Moreover, in biofilms established on glass surfaces in flow cells and subjected to invasion by the antibacterial protein-producing Pseudoalteromonas tunicata, the four-species biofilms resisted invasion to a greater extent than did the biofilms formed by the single species. Replacement of each strain by its cell-free culture supernatant suggested that synergy was dependent both on species-specific physical interactions between cells and on extracellular secreted factors or less specific interactions. In summary, our data strongly indicate that synergistic effects promote biofilm biomass and resistance of the biofilm to antimicrobial agents and bacterial invasion in multispecies biofilms.     

Prevalence of Virulence Genes and Antibiotic Resistance Pattern in Enterococcus Faecalis Isolated from Urinary Tract Infection in Shahrekord,Iran

Background:This study aims to specify the antimicrobial resistance pattern and virulence genes of Enterococcus faecalis isolated from urinary tract infections in Shahrekord, Iran. Methods:Urine samples of 1000 people suspected of having urinary tract infections referred to Shahrekord medical diagnostic laboratories were examined. Biofilm assays were performed by microtiter plate test through reading the OD490. Polymerase Chain Reaction (PCR) was applied to study the virulence factors.Results:Enterococcus faecalis was detected in 60 samples. After performing microbiological tests, all samples were positive in the molecular analysis. Strong, moderate and weak biofilm reactions reported 66.67%, 25%, and 8.33% respectively. The most resistance reported to cotrimoxazole, vancomycin and amikacin and the lowest resistance to nitrofurantoin (8.33%) was reported. Statistical analysis with Fisher''s exact test showed a statistically significant relationship between biofilm production and resistance to cotrimoxazole, vancomycin and cefotaxime. Prevalence of efe A, ace, gel E, esp, cyl M, agg, cyl A and cyl B in strong biofilm formation isolates was reported 100%, 87.5%, 82%, 62.5%, 55%, 37.5% 25% and 22.5% respectively. There was a significant relationship between the frequency of efa A and strong biofilm reaction.Conclusion:The presence of E. faecalis strains resistant to co-trimoxazole and vancomycin and present of some virulence factors is alarming the researchers. Since antibiotic resistance genes are probably transmitted among enterococci, and Staphylococci, controlling infections made by enterococci as well as the appropriate administration of antibiotics could treat the nosocomial infections effectively.Key Words: Antibiotic Resistance, Enterococcus faecalis, Urinary Tract Infection, Virulence genes     

Distinct SagA from Hospital-Associated Clade A1 Enterococcus faecium Strains Contributes to Biofilm Formation

Enterococcus faecium is an important nosocomial pathogen causing biofilm-mediated infections. Elucidation of E. faecium biofilm pathogenesis is pivotal for the development of new strategies to treat these infections. In several bacteria, extracellular DNA (eDNA) and proteins act as matrix components contributing to biofilm development. In this study, we investigated biofilm formation capacity and the roles of eDNA and secreted proteins for 83 E. faecium strains with different phylogenetic origins that clustered in clade A1 and clade B. Although there was no significant difference in biofilm formation between E. faecium strains from these two clades, the addition of DNase I or proteinase K to biofilms demonstrated that eDNA is essential for biofilm formation in most E. faecium strains, whereas proteolysis impacted primarily biofilms of E. faecium clade A1 strains. Secreted antigen A (SagA) was the most abundant protein in biofilms from E. faecium clade A1 and B strains, although its localization differed between the two groups. sagA was present in all sequenced E. faecium strains, with a consistent difference in the repeat region between the clades, which correlated with the susceptibility of biofilms to proteinase K. This indicates an association between the SagA variable repeat profile and the localization and contribution of SagA in E. faecium biofilms.     

A Widely Used In Vitro Biofilm Assay Has Questionable Clinical Significance for Enterococcal Endocarditis

Biofilm formation may play an important role in the pathogenesis of infections caused by Enterococcus faecalis, including endocarditis. Most biofilm studies use a polystyrene dish assay to quantify biofilm biomass. However, recent studies of E. faecalis strains in tissue and animal models suggest that polystyrene dish results need to be interpreted with caution. We evaluated 158 clinical E. faecalis isolates using a polystyrene dish assay and found variation in biofilm formation, with many isolates forming little biofilm even when different types of media were used. However, all tested clinical isolates were able to form biofilms on porcine heart valve explants. Dextrose-enhanced biofilm formation in the polystyrene dish assay was found in 6/12 (50%) of clinical isolates tested and may explain some, but not all of the differences between the polystyrene dish assay and the heart valve assay. These findings suggest that in studies assessing the clinical relevance of enterococcal biofilm-forming ability, ex vivo biofilm formation on a relevant tissue surface may be warranted to validate results of in vitro assays.     

Effect of glucose on Listeria monocytogenes biofilm formation,and assessment of the biofilm’s sanitation tolerance

Listeria monocytogenes is an important cause of human foodborne infections and its ability to form biofilms is a serious concern to the food industry. To reveal the effect of glucose conditions on biofilm formation of L. monocytogenes, 20 strains were investigated under three glucose conditions (0.1, 1.0, and 2.0% w v^–1) by quantifying the number of cells in the biofilm and observing the biofilm structure after incubation for 24, 72, and 168 h. In addition, the biofilms were examined for their sensitivity to sodium hypochlorite. It was found that high concentrations of glucose reduced the number of viable cells in the biofilms and increased extracellular polymeric substance production. Moreover, biofilms formed at a glucose concentration of 1.0 or 2.0% were more resistant to sodium hypochlorite than those formed at a glucose concentration of 0.1%. This knowledge can be used to help design the most appropriate sanitation strategy.     

Candida albicans and Non-C. albicans Candida Species: Comparison of Biofilm Production and Metabolic Activity in Biofilms, and Putative Virulence Properties of Isolates from Hospital Environments and Infections

Candida albicans and, more recently, non-C. albicans Candida spp. are considered the most frequent fungi in hospitals. This study analyzed Candida spp. isolates and compared the frequency of different species, that is, C. albicans and non-C. albicans Candida spp., and the origins of isolates, that is, from hospital environments or infections. Yeast virulence factors were evaluated based on biofilm production and metabolic activity. Hemolysin production and the antifungal susceptibility profiles of isolates were also evaluated. Candida spp. were highly prevalent in samples collected from hospital environments, which may provide a reservoir for continuous infections with these yeasts. There were no differences in the biofilm productivity levels and metabolic activities of the environmental and clinical isolates, although the metabolic activities of non-C. albicans Candida spp. biofilms were greater than those of the C. albicans biofilms (p < 0.05). Clinical samples had higher hemolysin production (p < 0.05) and lower susceptibility to fluconazole (p < 0.05). Non-C. albicans Candida spp. predominated in samples collected from hospital environments and infections (p < 0.05). These species had a lower susceptibility to fluconazole and amphotericin B, and their biofilms had higher metabolic activities than those produced by C. albicans, which may explain the increased incidence of fungal infections with these yeasts during recent years.     

Urinary catheter indwelling clinical pathogen biofilm formation,exopolysaccharide characterization and their growth influencing parameters

Self-reproducing microbial biofilm community mainly involved in the contamination of indwelling medical devices including catheters play a vital role in nosocomial infections. The catheter-associated urinary tract infection (CA-UTI) causative Staphylococcus aureus, Enterobacter faecalis, and Pseudomonas aeruginosa were selectively isolated, their phenotypic as well as genotypic biofilm formation, production and monomeric sugar composition of EPS as well as sugar, salt, pH and temperature influence on their in vitro biofilm formation were determined. From 50 culture positive urinary catheters S. aureus (24%), P. aeruginosa (18%), E. faecalis (14%) and others (44%) were isolated. The performed assays revealed their varying biofilm forming ability. The isolated S. aureus ica, E. faecalis esp, and P. aeruginosa cup A gene sequencing and phylogenetic analysis showed their close branching and genetic relationship. The analyzed sugar, salt, pH, and temperature showed that the degree of CA-UTI isolates biofilm formation is an environmentally sensitive process. EPS monosaccharide HPLC analysis showed the presence of neutral sugars (ng/μl) as follows: glucose (P. aeruginosa: 44.275; E. faecalis: 4.23), lactose (P. aeruginosa: 7.29), mannitol (P. aeruginosa: 2.53; S. aureus: 2.62; E. faecalis: 2.054) and maltose (E. faecalis: 7.0042) revealing species-specific presence and variation. This study may have potential clinical relevance for the easy diagnosis and management of CA-UTI.     

Enterococcus faecalis inhibits Klebsiella pneumoniae growth in polymicrobial biofilms in a glucose-enriched medium

Abstract

Catheter-related urinary tract infections are one of the most common biofilm-associated diseases. Within biofilms, bacteria cooperate, compete, or have neutral interactions. This study aimed to investigate the interactions in polymicrobial biofilms of Klebsiella pneumoniae and Enterococcus faecalis, two of the most common uropathogens. Although K. pneumoniae was the most adherent strain, it could not maintain dominance in the polymicrobial biofilm due to the lactic acid produced by E. faecalis in a glucose-enriched medium. This result was supported by the use of E. faecalis V583 ldh-1/ldh-2 double mutant (non-producer of lactic acid), which did not inhibit the growth of K. pneumoniae. Lyophilized cell-free supernatants obtained from E. faecalis biofilms also showed antimicrobial/anti-biofilm activity against K. pneumoniae. Conversely, there were no significant differences in planktonic polymicrobial cultures. In summary, E. faecalis modifies the pH by lactic acid production in polymicrobial biofilms, which impairs the growth of K. pneumoniae.     

Staphylococcus epidermidis in Orthopedic Device Infections: The Role of Bacterial Internalization in Human Osteoblasts and Biofilm Formation

Background

Staphylococcus epidermidis orthopedic device infections are caused by direct inoculation of commensal flora during surgery and remain rare, although S. epidermidis carriage is likely universal. We wondered whether S. epidermidis orthopedic device infection strains might constitute a sub-population of commensal isolates with specific virulence ability. Biofilm formation and invasion of osteoblasts by S. aureus contribute to bone and joint infection recurrence by protecting bacteria from the host-immune system and most antibiotics. We aimed to determine whether S. epidermidis orthopedic device infection isolates could be distinguished from commensal strains by their ability to invade osteoblasts and form biofilms.

Materials and Methods

Orthopedic device infection S. epidermidis strains (n = 15) were compared to nasal carriage isolates (n = 22). Osteoblast invasion was evaluated in an ex vivo infection model using MG63 osteoblastic cells co-cultured for 2 hours with bacteria. Adhesion of S. epidermidis to osteoblasts was explored by a flow cytometric approach, and internalized bacteria were quantified by plating cell lysates after selective killing of extra-cellular bacteria with gentamicin. Early and mature biofilm formations were evaluated by a crystal violet microtitration plate assay and the Biofilm Ring Test method.

Results

No difference was observed between commensal and infective strains in their ability to invade osteoblasts (internalization rate 308+/−631 and 347+/−431 CFU/well, respectively). This low internalization rate correlated with a low ability to adhere to osteoblasts. No difference was observed for biofilm formation between the two groups.

Conclusion

Osteoblast invasion and biofilm formation levels failed to distinguish S. epidermidis orthopedic device infection strains from commensal isolates. This study provides the first assessment of the interaction between S. epidermidis strains isolated from orthopedic device infections and osteoblasts, and suggests that bone cell invasion is not a major pathophysiological mechanism in S. epidermidis orthopedic device infections, contrary to what is observed for S. aureus.     

Effects of Subinhibitory Concentrations of Ceftaroline on Methicillin-Resistant Staphylococcus aureus (MRSA) Biofilms

Ceftaroline (CPT) is a novel cephalosporin with in vitro activity against Staphylococcus aureus. Ceftaroline exhibits a level of binding affinity for PBPs in S. aureus including PBP2a of methicillin-resistant S. aureus (MRSA). The aims of this study were to investigate the morphological, physiological and molecular responses of MRSA clinical strains and MRSA biofilms to sub-MICs (1/4 and 1/16 MIC) of ceftaroline by using transmission, scanning and confocal microscopy. We have also used quantitative Real-Time PCR to study the effect of sub-MICs of ceftaroline on the expression of the staphylococcal icaA, agrA, sarA and sasF genes in MRSA biofilms. In one set of experiments, ceftaroline was able to inhibit biofilm formation in all strains tested at MIC, however, a strain dependent behavior in presence of sub-MICs of ceftaroline was shown. In a second set of experiments, destruction of preformed biofilms by addition of ceftaroline was evaluated. Ceftaroline was able to inhibit biofilm formation at MIC in all strains tested but not at the sub-MICs. Destruction of preformed biofilms was strain dependent because the biofilm formed by a matrix-producing strain was resistant to a challenge with ceftaroline at MIC, whereas in other strains the biofilm was sensitive. At sub-MICs, the impact of ceftaroline on expression of virulence genes was strain-dependent at 1/4 MIC and no correlation between ceftaroline-enhanced biofilm formation and gene regulation was established at 1/16 MIC. Our findings suggest that sub-MICs of ceftaroline enhance bacterial attachment and biofilm formation by some, but not all, MRSA strains and, therefore, stress the importance of maintaining effective bactericidal concentrations of ceftaroline to fight biofilm-MRSA related infections.