Effect of temperature, pH and plasmids on in vitro biofilm formation in Escherichia coli

Acid resistance (AR) in Escherichia coli is an important trait that protects this microorganism from the deleterious effect of low-pH environments. Reports on biofilm formation in E. coli K12 showed that the genes participating in AR were differentially expressed. Herein, we investigated the relationship between AR genes, in particular those coding for specific transcriptional regulators, and their biofilm-forming ability at the phenotypic level. The latter was measured in 96-well plates by staining the bacteria attached to the well, following 24-hour growth under static conditions, with crystal violet. The growth conditions were as follows: Luria Bertani (LB) medium at neutral and acidic pH, at 37°C or 25°C. We observed that the three major transcriptional regulators of the AR genes (gadX, gadE, gadW) only marginally affected biofilm formation in E. coli. However, a striking and novel finding was the different ability of all the tested E. coli strains to form a biofilm depending on the temperature and pH of the medium: LB, pH 7.4, strongly supported biofilm formation at 25°C, with biofilm being hardly detectable at 37°C. On the contrary, LB, pH 5.5, best supported biofilm formation at 37°C. Moreover, we observed that when E. coli carried a plasmid, the presence of the plasmid itself affected the ability to develop a biofilm, typically by increasing its formation. This phenomenon varies from plasmid to plasmid, depends on growth conditions, and, to the best of our knowledge, remains largely uninvestigated

The yhiM gene codes for an inner membrane protein involved in GABA export in Escherichia coli

In order to survive the exposure to acid pH, Escherichia coli activates molecular circuits leading from acid tolerance to extreme acid resistance (AR). The activation of the different circuits involves several global and specific regulators affecting the expression of membrane, periplasmic and cytosolic proteins acting at different levels to dampen the harmful consequences of the uncontrolled entry of protons intracellularly. Many genes coding for the structural components of the AR circuits (protecting from pH ≤ 2.5) and their specific transcriptional regulators cluster in a genomic region named AFI (acid fitness island) and respond in the same way to global regulators (such as RpoS and H-NS) as well as to anaerobiosis, alkaline, cold and respiratory stresses, in addition to the acid stress. Notably some genes coding for structural components of AR, though similarly regulated, are non-AFI localised. Amongst these the gadBC operon, coding for the major structural components of the glutamate-based AR system, and the ybaS gene, coding for a glutaminase required for the glutamine-based AR system. The yhiM gene, a non-AFI gene, appears to belong to this group. We mapped the transcription start of the 1.1 kb monocistronic yhiM transcript: it is an adenine residue located 22 nt upstream a GTG start codon. By real-time PCR we show that GadE and GadX equally affect the expression of yhiM under oxidative growth conditions. While YhiM is partially involved in the RpoS-dependent AR, we failed to detect a significant involvement in the glutamate- or glutamine-dependent AR at pH ≤ 2.5. However, when grown in EG at pH 5.0, the yhiM mutant displays impaired GABA export, whereas when YhiM is overexpressed, an increases of GABA export in EG medium in the pH range 2.5–5.5 is observed. Our data suggest that YhiM is a GABA transporter with a physiological role more relevant at mildly acidic pH, but not a key component of AR at pH < 2.5

New perspectives for transcatheter aortic valve implantation: the "coup d'essai" of bicuspid aortic valves

Outline of the thesis The thesis is divided in four parts. Part I: Aortic valve disease and innovation in percutaneous treatment by transcatheter aortic valve implantation. In this first section of the thesis we present some research projects published in the field of transcatheter aortic valve replacement in general population, describing the progress of this percutaneous technique during the past years until nowadays practice. In this section there are some considerations about TAVI in particular scenarios and procedural settings requiring dedicated technical adjustments. Part II: TAVI in a complex anatomical setting: Bicuspid Aortic Valve management and percutaneous treatment. In this second section we collected research papers focused on the challenging anatomy of bicuspid aortic valves (BAV) undergoing TAVI treatment for aortic stenosis (AS). In contemporary TAVI practice BAV anatomy still represents a challenge for percutaneous treatment. This section is the real focus of the three years of PhD research, since the complexity of the topic and the time required for data collection and analysis. Moreover, this part represents the result of an International collaboration and knowledge sharing among renowned centres for the percutaneous treatment of aortic disease. Part III: Imaging for TAVI in Bicuspid Aortic Valve. This section includes research papers investigating the role of imaging, in particular of multi-sliced computed tomography (MSCT), in BAV patients undergoing TAVI procedure, considering the lack of standardized protocols for sizing and device choice in this complex aortic valve anatomy. Part IV: Discussion and Conclusions. The last part of the thesis is a discussion of the presented topics with some conclusions

Biochemical and spectroscopic properties of Brucella microti glutamate decarboxylase, a key component of the glutamate-dependent acid resistance system

In orally acquired bacteria, the ability to counteract extreme acid stress (pH < 2.5) ensures survival during transit through the animal host stomach. In several neutralophilic bacteria, the glutamate-dependent acid resistance system (GDAR) is the most efficient molecular system in conferring protection from acid stress. In Escherichia coli its structural components are either of the two glutamate decarboxylase isoforms (GadA, GadB) and the antiporter, GadC, which imports glutamate and exports γ-aminobutyrate, the decarboxylation product. The system works by consuming protons intracellularly, as part of the decarboxylation reaction, and exporting positive charges via the antiporter. Herein, biochemical and spectroscopic properties of GadB from Brucella microti (BmGadB), a Brucella species which possesses GDAR, are described. B. microti belongs to a group of lately described and atypical brucellae that possess functional gadB and gadC genes, unlike the most well-known "classical" Brucella species, which include important human pathogens. BmGadB is hexameric at acidic pH. The pH-dependent spectroscopic properties and activity profile, combined with in silico sequence comparison with E. coli GadB (EcGadB), suggest that BmGadB has the necessary structural requirements for the binding of activating chloride ions at acidic pH and for the closure of its active site at neutral pH. On the contrary, cellular localization analysis, corroborated by sequence inspection, suggests that BmGadB does not undergo membrane recruitment at acidic pH, which was observed in EcGadB. The comparison of GadB from evolutionary distant microorganisms suggests that for this enzyme to be functional in GDAR some structural features must be preserved

The glutaminase-dependent acid resistance system. Qualitative and quantitative assays and analysis of its distribution in enteric bacteria

Neutralophilic bacteria have developed several strategies to overcome the deleterious effects of acid stress. In particular, the amino acid-dependent systems are widespread, with their activities overlapping, covering a rather large pH range, from 6 to <2. Recent reports showed that an acid resistance (AR) system relying on the amino acid glutamine (AR2_Q), the most readily available amino acid in the free form, is operative in Escherichia coli, Lactobacillus reuteri and some Brucella species. This system requires a glutaminase active at acidic pH and the antiporter GadC to import L-glutamine and export either glutamate (the glutamine deamination product) or GABA. The latter occurs when the deamination of glutamine to glutamate, via acid-glutaminase (YbaS/GlsA), is coupled to the decarboxylation of glutamate to GABA, via glutamate decarboxylase (GadB), a structural component of the glutamate-dependent AR (AR2) system, together with GadC. Taking into account that AR2_Q could be widespread in bacteria and that until now assays based on ammonium ion detection were typically employed, this work was undertaken with the aim to develop assays that allow a straightforward identification of the acid-glutaminase activity in permeabilised bacterial cells (qualitative assay) as well as a sensitive method (quantitative assay) to monitor in the pH range 2.5-4.0 the transport of the relevant amino acids in vivo. The qualitative assay is colorimetric, rapid and reliable and provides several additional information, such as co-occurrence of AR2 and AR2_Q in the same bacterial species and assessment of the growth conditions that support maximal expression of glutaminase at acidic pH. The quantitative assay is HPLC-based and allows to concomitantly measure the uptake of glutamine and the export of glutamate and/or GABA via GadC in vivo and depending on the external pH. Finally, an extensive bioinformatic genome analysis shows that the gene encoding the glutaminase involved in AR2_Q is often nearby or in operon arrangement with the genes coding for GadC and GadB. Overall, our results indicate that AR2_Q is likely to be of prominent importance in the AR of enteric bacteria and that it modulates the enzymatic as well as antiport activities depending on the imposed acidic stress

Bone morphogenetic proteins and growth factors: Emerging role in regenerative orthopaedic surgery

Bone morphogenetic proteins (BMPs) were discovered by Urist and colleagues in 1965 and later defined as multifunctional cytokines involved in osteoinduction. BMPs are members of the transforming growth factor-β superfamily, with the exception of the BMP-1. Presently, at least 20 BMPs have been identified and studied, but only BMP 2, 4 and 7 have been able in vitro to stimulate the entire process of stem cell differentiation into osteoblastic mature cells. In preclinical and clinical studies, BMPs have demonstrated potential in osteoinduction and have been approved for clinical use in treating open fractures of the long bones and nonunions and in vertebral arthrodesis. Additional clinical uses of these molecules are under investigation and the possibility of using gene therapy in selected pathologies seems the most appealing

Biomimetic implant surface functionalization with liquid L-PRF products: in vitro study

Abstract Objective: Platelet-rich fibrin (PRF) clots and membranes are autologous blood concentrates widely used in oral surgical procedures; less is known, however, about the liquid formulations of such products. The aim of this in vitro study is to assess the behavior of different implant surfaces when in contact with two liquid leucocyte- and platelet-rich fibrin (L-PRF) products. Methods: Six commercial pure titanium discs, of 9.5 mm diameter and 1.5 mm thickness, were used. Three of these samples had a micro/nano-rough surface; three were machined. Three different protocols were tested. Protocols involved the immersion of the samples in (1) a platelets, lymphocytes, and fibrinogen liquid concentrate (PLyF) for 10 minutes, (2) an exudate obtained from L-PRF clots rich in fibronectin and vitronectin for 5 minutes, and (3) the fibronectin/vitronectin exudate for 2 minutes followed by immersion in the PLyF concentrate for further 8 minutes. After these treatments, the samples were fixed and observed using a scanning electron microscope (SEM). Results: Under microscopic observation, (1) the samples treated with the PLyF concentrate revealed a dense fibrin network in direct contact with the implant surface and a significant number of formed elements of blood; (2) in the samples treated with the fibronectin/vitronectin exudates, only a small number of white and red blood cells were detectable; and (3) in samples exposed to the combined treatment, there was an apparent increase in the thickness of the fibrin layer. When compared to the machined surface, the micro/nano-rough samples showed an overall increased retention of fibrin, leading to a thicker coating. Conclusions: Liquid L-PRF products promote the formation of a dense fibrin clot on micro/nano-rough implant surfaces in vitro. The adjunctive treatment of surfaces with the fibronectin/vitronectin exudate could provide support to contact of the fibrin with the surface, though it is not essential for the clot formation. Further studies are necessary to better elucidate the properties and benefits of liquid L-PRF products