Bacteriophage-encoded depolymerases: their diversity and biotechnological applications

Bacteriophages (phages), natural enemies of bacteria, can encode enzymes able to degrade polymeric substances. These substances can be found in the bacterial cell surface, such as polysaccharides, or are produced by bacteria when they are living in biofilm communities, the most common bacterial lifestyle. Consequently, phages with depolymerase activity have a facilitated access to the host receptors, by degrading the capsular polysaccharides, and are believed to have a better performance against bacterial biofilms, since the degradation of extracellular polymeric substances by depolymerases might facilitate the access of phages to the cells within different biofilm layers. Since the diversity of phage depolymerases is not yet fully explored, this is the first review gathering information about all the depolymerases encoded by fully sequenced phages. Overall, in this study, 160 putative depolymerases, including sialidases, levanases, xylosidases, dextranases, hyaluronidases, peptidases as well as pectate/pectin lyases, were found in 143 phages (43 Myoviridae, 47 Siphoviridae, 37 Podoviridae, and 16 unclassified) infecting 24 genera of bacteria. We further provide information about the main applications of phage depolymerases, which can comprise areas as diverse as medical, chemical, or food-processing industry.

Bacterial vaginosis biofilms: challenges to current therapies and emerging solutions

Bacterial vaginosis (BV) is the most common genital tract infection in women during their reproductive years and it has been associated with serious health complications, such as preterm delivery and acquisition or transmission of several sexually transmitted agents. BV is characterized by a reduction of beneficial lactobacilli and a significant increase in number of anaerobic bacteria, including Gardnerella vaginalis, Atopobium vaginae, Mobiluncus spp., Bacteroides spp. and Prevotella spp.. Being polymicrobial in nature, BV etiology remains unclear. However, it is certain that BV involves the presence of a thick vaginal multi-species biofilm, where G. vaginalis is the predominant species. Similar to what happens in many other biofilm-related infections, standard antibiotics, like metronidazole, are unable to fully eradicate the vaginal biofilm, which can explain the high recurrence rates of BV. Furthermore, antibiotic therapy can also cause a negative impact on the healthy vaginal microflora. These issues sparked the interest in developing alternative therapeutic strategies. This review provides a quick synopsis of the currently approved and available antibiotics for BV treatment while presenting an overview of novel strategies that are being explored for the treatment of this disorder, with special focus on natural compounds that are able to overcome biofilm-associated antibiotic resistance.

Influence of oxygen conditions on bacterial interactions within biofilms related with cystic fibrosis

Dissertação de mestrado em Bioengineering

The influence of biofilm formation by Gardnerella vaginalis and other anaerobes on bacterial vaginosis

Bacterial vaginosis (BV) is the worldwide leading vaginal disorder in women of reproductive age. BV is characterized by the replacement of beneficial lactobacilli and the augmentation of anaerobic bacteria. Gardnerella vaginalis is a predominant bacterial species, however, BV is also associated with other numerous anaerobes, such as Atopobium vaginae, Mobiluncus mulieris, Prevotella bivia, Fusobacterium nucleatum and Peptoniphilus sp.. Currently, the role of G. vaginalis in the etiology of BV remains a matter of controversy. It is however known that, in BV patients, a biofilm is usually formed on the vaginal epithelium and G. vaginalis is typically the predominant species. So, the current paradigm is that the establishment of a biofilm plays a key role in the pathogenesis of BV. This review provides background on the influence of biofilm formation by G. vaginalis and other anaerobes in the polymicrobial etiology of BV, through its initial adhesion until biofilm formation and discusses the commensal and synergic interactions established between them to understand the phenotypic shift of G. vaginalis' biofilms into BV establishment.

Novel strategies to combat gram-negative bacterial pathogens using bacteriophage proteins

Dissertação de mestrado em Bioengenharia

Thermo-compression molding of chitosan with a deep eutectic mixture for biofilms development

Supplementary information available at: http://www.rsc.org/suppdata/c5/gc/c5gc02231b/c5gc02231b1.pdf

Bacterial cellulose as a novel substrate for retinal pigment epithelium cells transplantation in age-related macular degeneration

Tese de Doutoramento em Engenharia Química e Biológica.

Poly-N-acetyl glucosamine expression in Staphylococcus epidermidis biofilm cells affects the immune response and promotes tissue pathology in infected hosts

Staphylococcus epidermidis is a biofilm - forming bacterium and a leading etiological agent of nosocomial infections. The ability to establish biofilms on indwelling medical devices is a key virulence factor for this bacterium. Still, the influence of poly - N - acetyl glucosamine (PNAG), the major component of the extracellular biofilm matrix, in the host immune response has been scarcely studied. Here, t h is influence was assessed in mice challenged i.p. with PNAG - p roducing (WT) and isogenic - mutant lacking PNAG (M10) bacteria grown in biofilm - inducing conditions. Faster bacterial clearance was observed in the mice infected with WT bacteria than in M10 - infected counterparts , which w as accompanied by earlier neutrophil recruitment and higher IL - 6 production. Interestingly, in the WT - infected mice, but not in those infected with M10 , elevated serum IL - 10 was detected . To further study the effe ct of PNAG in the immune response, mice were primed with WT or M10 biofilm bacteria and subsequently infected with WT biofilm - released cells. WT - primed mice presented a higher frequency of splenic IFN - γ + and IL - 17 + CD4 + T cells, and more severe liver patho logy than M10 - primed counterparts. Nevertheless, T reg cells obtained from the WT - primed mice presented a higher suppressive function than those obtained from M10 - primed mice. This effect was abrogated when IL - 10 - deficient mice were similarly primed and infected indicating that PNAG promotes the differentiati on of highly suppressive T reg cells by a mechanism dependent on IL - 10. Altogether, these results provide evidence help ing explain ing the coexistence of inflammation and bacterial persistence often observed in biofilm - originated S. epidermidis infections

Influence of glucose concentration on the structure and quantity of biofilms formed by Candida parapsilosis

Candida parapsilosis is nowadays an emerging opportunistic pathogen and its increasing incidence is part related to the capacity to produce biofilm. In addition, one of the most important C. parapsilosis pathogenic risk factors includes the organisms\textquoteright selective growth capabilities in hyper alimentation solutions. Thus, in this study, we investigated the role of glucose in C. parapsilosis biofilm modulation, by studying biofilm formation, matrix composition and structure. Moreover, the expression of biofilm-related genes (BCR1, FKS1 and OLE1) were analyzed in the presence of different glucose percentages. The results demonstrated the importance of glucose in the modulation of C. parapsilosis biofilm. The concentration of glucose had direct implications on the C. parapsilosis transition of yeast cells to pseudohyphae. Additionally, it was demonstrated that biofilm related genes BCR1, FKS1 and OLE1 are involved in biofilm modulation by glucose. The mechanism by which glucose enhances biofilm formation is not fully understood, however with this study we were able to demonstrate that C. parapsilosis respond to stress conditions caused by elevated levels of glucose by up-regulating genes related to biofilm formation (BCR1, FKS1 and OLE1).

Evidence for inter- and intra-species biofilm formation variability among a small group of coagulase-negative staphylococci

Coagulase-negative staphylococci (CoNS) are common bacterial colonisers of the human skin. They are often involved in nosocomial infections due to biofilm formation in indwelling medical devices. While biofilm formation has been extensively studied in Staphylococcus epidermidis, little is known regarding other CoNS species. Here, biofilms from six different CoNS species were characterised in terms of biofilm composition and architecture. Interestingly, the ability to form a thick biofilm was not associated with any particular species, and high variability on biofilm accumulation was found within the same species. Cell viability assays also revealed different proportions of live and dead cells within biofilms formed by different species, although this parameter was particularly similar at the intra-species level. On the other hand, biofilm disruption assays demonstrated important inter- and intra-species differences regarding extracellular matrix composition. Lastly, confocal laser scanning microscopy (CLSM) experiments confirmed this variability, highlighting important differences and common features of CoNS biofilms. We hypothesised that the biofilm formation heterogeneity observed was rather associated with biofilm matrix composition than with cells themselves. Additionally, our results indicate that polysaccharides, DNA and proteins are fundamental pieces in the process of CoNS biofilm formation.

Engineering modular viral scaffolds for targeted bacterial population editing

Bacteria are central to human health and disease, but existing tools to edit microbial consortia are limited. For example, broad-spectrum antibiotics are unable to precisely manipulate bacterial communities. Bacteriophages can provide highly specific targeting of bacteria, but assembling well-defined phage cocktails solely with natural phages can be a time-, labor- and cost-intensive process. Here, we present a synthetic biology strategy to modulate phage host ranges by engineering phage genomes in Saccharomyces cerevisiae. We used this technology to redirect Escherichia coli phage scaffolds to target pathogenic Yersinia and Klebsiella bacteria, and conversely, Klebsiella phage scaffolds to target E. coli by modular swapping of phage tail components. The synthetic phages achieved efficient killing of their new target bacteria and were used to selectively remove bacteria from multi-species bacterial communities with cocktails based on common viral scaffolds. We envision this approach accelerating phage biology studies and enabling new technologies for bacterial population editing.

Filamentous fungi in free water and biofilms from Brazilian drinking water systems

In some regions of Brazil, especially where the water is scarce, drinking water is stored in water storage tanks. This practice gives the consumer the guarantee of available water. The water storage conditions such as the exposure to hot weather when the tanks are on rooftops allow the development of microorganisms and microbial biofilms which can deteriorate the water quality and increase the risk to human health [1,2]. This study describes the filamentous fungi (FF) detected in free water and biofilms in drinking water storage tanks in Recife - Pernambuco, Brazil. Five sampling times in triplicate were performed at two distinct points. Colony-forming units (CFU) of FF fungi were determined with 0.45 µm filtration membranes using peptone glucose rose Bengal agar (PGRBA). From the 30 samples analysed a total of 1136 CFU were obtained. The water biofilms were collected from samplers consisting of polyethylene coupons, previously installed in the reservoirs. These coupons were transferred to PGRBA plates and incubated using with the same conditions described for free FF. For the in situ detection of FF in biofilms the Calcofluor White staining technique was used. This procedure demonstrated FF forming biofilms on the surfaces of the coupons. Brazilian legislation does not define limits for FF in drinking water. However considering the potential risk of fungal contamination, the data obtained in this study will contribute to developing future quantitative and qualitative parameters for the presence of fungi in drinking water distribution systems in Brazil. [1] HageskaL, G, Lima, N, Skaar, I. The study of fungi in drinking water. Mycological Research, 113, 2009, 165-172. [2] Skaar I, Hageskal G. Fungi in Drinking Water. In.: Paterson RRM, Lima N. (Eds.) Molecular Biology of Food and Water Borne Mycotoxigenic and Mycotic Fungi. CRC Press, Taylor & Francis Group, Boca Raton, 2015, 597-606.

A novel small-caliber bacterial cellulose vascular prosthesis: production, characterization, and preliminary in vivo testing

Vascular grafts are used to bypass damaged or diseased blood vessels. Bacterial cellulose (BC) has been studied for use as an off-the-shelf graft. Herein, we present a novel, cost-effective, method for the production of small caliber BC grafts with minimal processing or requirements. The morphology of the graft wall produced a tensile strength above that of native vessels, performing similarly to the current commercial alternatives. As a result of the production method, the luminal surface of the graft presents similar topography to that of native vessels. We have also studied the in vivo behavior of these BC graft in order to further demonstrate their viability. In these preliminary studies, 1 month patency was achieved, with the presence of neo-vessels and endothelial cells on the luminal surface of the graft.

Strategies to combat infections of Acinetobacter baumannii biofilms

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)