Comparison of titres of IgM and IgG antibodies against staphylococcal slime polysaccharide antigens (SSPA) (ELISA units [EU]) in sera from patients with an ongoing staphylococcal late-onset infection of synthetic vascular graft (LO-SVGI) and in controls.
Abstract
Biofilm infections represent a new medical challenge that drives towards the discovery of new diagnostics and new drugs specifically designed for this purpose. All living organisms offer a huge source of compounds which represent the biochemical substrate of the biological competition on the Earth and can be used to this aim. We describe an innovative diagnostic tool to early diagnose medical device infections sustained by Staphylococci; then we list new compounds that modulate bacterial phenotype and reduce virulence without affecting bacterial viability so as to avoid the emergence of genetic resistances. These compounds are all derived from natural sources: prokaryotes, plants, and human body. From prokaryotes we studied new compounds extracted from different environmental bacterial species, including Antarctic species growing in extreme environments. We describe also the anti-biofilm properties of extracts obtained from plants well known since centuries in folk medicine. The humoral immune response is the source of the last anti-biofilm compound: transferrin (Tf), a protein derived from human plasma involved in inflammation and natural immunity. All these compounds can be used as scaffolds for the design of new drugs active on the sessile form of pathogens prevalent in human biofilm infections.
Keywords
- human
- diseases
- infection
- therapy
- diagnosis
1. Introduction
Since 1929, the discovery of penicillin radically changed the history of human infection diseases, opening the path to the discovery of a wide array of new antibacterial compounds. After a few decades, the appearance of genotypic resistances undermined the dream of definitive human victory on infectious diseases. In the 1970s, some unexplainable cases of drug resistance sustained by strains prone to conventional antibiotics were attributed to biofilm. At the beginning, sessile phenotype has been related only to medical devices infections, but as long as knowledge on biofilm behavior increased, the majority of chronic human infections have been attributed to sessile phenotype. It has been demonstrated that sessile bacteria resist to antibiotics due to a variety of causes including, a reduced penetration of drugs in the deep layers of biofilm, a favored diffusion of resistance genes, a drift of bacterial metabolism towards anaerobiosis causing a reduced cellular division, and a dramatic reduction of susceptibility to drugs. Efforts have been devoted to restore sensitivity of sessile bacteria to antibiotics and to identify new compounds to treat biofilm. In this chapter, we describe our efforts in this field. Since the early 1990s, our lab dedicated many efforts to identify new diagnostics and new therapeutical strategies to counteract biofilm infections in humans. We shall first describe a diagnostic tool based on enzyme-linked immunosorbent assay (ELISA) technique to early identify Staphylococcal colonization of medical devices, then we shall list an array of different compounds active on bacterial biofilms.
2. An ELISA assays for early diagnosis of biofilm infections
Specimen culturing is the gold standard for diagnosing bacterial infections, but growing bacteria from a biofilm is not reliable. Other testing modalities, such as polymerase chain reaction Polymerase chain reaction (PCR) and serology assays are nonspecific for biofilm infections and include the risk of contamination during sampling. In recent years, many attempts have been performed to create new for early, noninvasive diagnosis. A main feature of infections on implanted medical devices is the absence or paucity of local signs and general symptoms of infection/inflammation. The first signs are due to dysfunction of the device itself (loosening of orthopedic prostheses, cardiac valve regurgitation) and device-related damage to the surrounding tissues. Prompt detection of biofilm infection in the initial asymptomatic phases can allow earlier medical/surgical treatment. Highly virulent organisms (e.g.,
Group | ||||||
---|---|---|---|---|---|---|
A (n = 38) | B (n = 10) | C (n = 16) | D (n = 17) | E (n = 58) | B+C+D+E (n = 101) | |
IgM titre (mean, SD) (EU) | 0.69 (0.37) | 0.28 (0.07) | 0.26 (0.06) | 0.18 (0.05) | 0.17 (0.06) | 0.19 (0.08) |
IgG titre (mean, SD) (EU) | 1.13 (0.36) | 0.86 (0.46) | 0.82 (0.57) | 0.46 (0.27) | 0.55 (0.26) | 0.60 (0.37) |
Positive tests for an IgM titre (number, %) (EU) |
||||||
≥0.35 | 38 (100%) | 2 (20%) | 0 | 0 | 0 | 2 (2%) |
≥0.40 | 37 (97%) | 0 | 0 | 0 | 0 | 0 |
The highest titers of IgG antibody to SSPA were noted in individuals with ongoing staphylococcal LO-SVGIs (group A). However, high titers were also seen in the control groups, which precluded the use of IgG titers for diagnostic purposes. In contrast, titers of IgM antibodies against SSPA were higher in the group A patients. There was virtually no overlap between the titers of these patients and those of controls. IgM antibody of 0.4 ELISA units (EU) or more indicated ongoing staphylococcal LO-SVGIs with detection rates of 97% and no false positives. When a cutoff of 0.35 ELISA units was used, the detection rate increased to 100%, but the false-positive rate also rose to 2%. The substantial difference observed between patients with ongoing versus previous staphylococcal LO-SVGIs (group A vs. group C) suggested that levels of IgM antibody against SSPA decrease rapidly after successful graft substitution. Recurrence of graft infection was associated with the return of elevated IgM antibody titers. We concluded that SSPA ELISA positivity can be used as a marker of active staphylococcal graft infections. Anti-SSPA ELISA was also tested for the diagnosis of orthopedic joint prosthetic infections (DOJP-Is). To this aim, we compared the titers of IgM antibodies against SSPA in the sera of 90 subjects [2]. Studied population included 29 subjects with ongoing staphylococcal DOJP-Is (group A), 34 subjects with orthopedic joint prostheses implanted at least 1 year previously without infection (group B), and 27 subjects not previously operated for orthopedic implants, attending the hospital for noninfectious diseases (group C). All subjects in group A underwent surgical removal of the infected prosthesis, and staphylococcal infection had been microbiologically confirmed by intraoperative cultures. For orthopedic applications, we adopted a cutoff value of 0.35 EU. The main results, summarized in Table 2 , show that high anti-SSPA IgM levels may provide for noninvasive detection of the immune response elicited by biofilm colonization on artificial orthopedic implants. We did not evaluate IgG titers, because they were not associated with current infection both in the previous study on vascular grafts and in a preliminary analysis on five cases and eight controls. According to these results, we can affirm that anti-SSPA ELISA assay is effective in detecting antibodies in DOJP-Is caused by different staphylococcal species.
Group (no. of samples) | Mean (SD) IgM titre (EU) | % (no.) of positive tests for an IgM titre: | |
---|---|---|---|
≥0.35 EU | ≥0.40 EU | ||
Prosthesis infection |
0.72 (0.55) | 89.7 (26) | 69.0 (20) |
Prosthesis, no infection |
0.21 (0.09) |
8.8 (3) | 5.9 (2) |
No Prosthesis, no infection (27) | 0.20 (0.05) |
0 (0) | 0 (0) |
All controls (61) | 0.21 (0.07) |
4.9 (3) | 3.3 (2) |
Periodic testing for IgM antibodies against SSPA could prove useful in the follow-up of patients with implanted vascular and orthopedic devices. The SSPA ELISA displays a strong advantage over other available methods used to diagnose biofilm-related infections, in fact it is versatile because it can detect antibodies in biofilm infections caused by different staphylococcal species. The higher diagnostic value of the IgM titers depends on the choice of the antigen: a mixture of purified polysaccharide antigens extracted from the biofilm matrix. Polysaccharide antigens are known to elicit a thymus-independent humoral response based exclusively on IgM production. This response is maintained as long as the antigenic stimulus is present, and there is no shift to IgG production. IgMs elicited by antigens of this type are synthesized by a particular subpopulation of B lymphocytes (B1): blood IgM + IgD + CD27 + cells that correspond to splenic marginal zone B cells. Because of the absence of thymic involvement and IgG production, these responses are regarded as expressions of innate immunity. The peculiar behavior of the immune response to polysaccharide antigens represents a diagnostic advantage since it can be used at any time to evaluate the possibility of device infection, even during the post-replacement follow-up. The interest in the development of alternative anti-infective approaches for the prevention and treatment of staphylococcal infections has increased in recent years [3–5]. But our group has been working on the search of new compounds active as anti-biofilm drugs since the early 1990s [6].
2.1. New compounds from prokaryotes for the therapy of biofilm infections.
Our first attempt, for the search of new anti-biofilm drugs, was based on the clinical observation that the administration of proteolytic enzymes can enhance therapeutic outcomes in the treatment of contact lens and endo-ocular prosthetic devices infections. The hypothesis was that proteolytic treatment could damage the proteic structure of biofilm matrix reverting bacteria to a condition of susceptibility to antibiotics. In our first study [6] four different proteases were tested on 10 bacterial strains (5
Serratiopeptidase is a metalloprotease (containing zinc) cloned from
We found no relationship between bacterial gene profile and proteases activity. Cell surface protein samples from treated and untreated cultures of all staphylococcal strains were simultaneously analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The ability of each protease to interfere with
A successful strategy to hinder bacterial infection should not affect processes essential for bacterial survival in order to avoid the rapid appearance of escape mutants. A smarter approach should target the main virulence factors of
Biofilm formation | ||
---|---|---|
Control | SPEP-treated | |
6538P | 1.64 ± 0.15 | 0.086 ± 0.015 |
25923 | 1.68 ± 0.23 | 0.63 ± 0.12 |
12598 | 0.35 ± 0.05 | 0.21 ± 0.03 |
BAA1556 | 2.15 ± 0.08 | 0.47 ± 0.04 |
Two steps lead to biofilm formation are adherence to a surface by bacterial cells and progressive growth of cell clusters in multilayers. The study of the factors that gather cells into a biofilm has evidenced the existence of strains producing either polysaccharide intercellular adhesion/poly-N-acetylglucosamine (PIA/PNAG) or a protein-dependent biofilm, even if in staphylococci the best-known biofilm mechanism depends on the production of PIA/PNAG, an extracellular polysaccharide adhesin [13, 14]. It has been shown that, besides their best-known role in the eukaryotic invasion process, fibronectin-binding proteins (FnBPs) play a relevant role in biofilm-associated foreign-body infections. Some proteins are multifunctional factors involved in metabolic pathways, in adhesion to extracellular matrix and invasion of host cells, such serine-aspartate repeat-containing protein D (SdrD), Elongation factor-Tu (EF-Tu), Elongation factor-G (EF-G), Atl, SsA2, and second immunoglobulin-binding protein (Sbi). In order to understand the molecular mechanism of SPEP action on
Experiments in BioFlux system, performed on MSSA 6538P and MRSA BAA1556, showed that SPEP clearly impairs biofilm formation as already seen in static system. The SPEP action on the proteome of tested strains was assessed on surface proteins extracted according to the method of Tabouret [17]. Extracted proteins were separated on SDS-PAGE. Many specific protein bands detected in the untreated
Untreated | SPEP-treated | |||
---|---|---|---|---|
Adhesion a | Invasion b | Adhesion a | Invasion b | |
6538P | 2.75 ± 0.45 | 0.7 × 10–2 ± 0.1 × 10–2 | 2.80 ± 1.80 | 0.3 × 10–4 ± 0.0 × 10–4 |
BAA1556 | 4.94 ± 0.97 | 2.5 ± 0.79 | 2.82 ± 1.46 | 0.7 × 10–3 ± 0.1 × 10–3 |
It is not yet clear if serratiopeptidase action is mediated by its proteolytic function or to the activation of specific signal transduction pathways that regulate expression of proteins.
In the last years, our group started working also on compounds obtained from marine bacteria. Marine bacteria from Antarctica represent an untapped reservoir of biodiversity and produce several compounds which may be of potential biotechnological interest; culture supernatants derived from most of them have been shown to exhibit anti-biofilm activity against both gram-positive and gram-negative bacteria, including
The efficacy of
2.2. New compounds from Eukaryotes for the therapy of biofilm infections.
2.2.1. Compounds from plants.
Considering that plants have already yielded compounds with inhibiting activities against gram-positive bacteria [45], and that the use of medicinal and herbal remedies to treat infectious diseases is common in many countries [46], we have also attempted the discovery of new leads from plants. We explored several plant extracts searching for specific antibacterial activity from fractionated pools [47]. We considered extracts from
MW | Compound |
|
|
||||||
---|---|---|---|---|---|---|---|---|---|
M.I.C. [μM] a |
M.B.C. [μM] a |
EC50 (μM) b |
slope | M.I.C. [μM] a |
M.B.C. [μM] a |
EC50 (μM) |
slope | ||
383.8 |
|
16.3; 32.6 | >260 | 15.2 ± 2.3 | 2.4 ± 0.7 | 16.3; 32.6 | >260 | 8.6 ± 0.4 | 3.1 ± 0.5 |
367.8 |
|
34.0; 68.0 | 34.0; 68.0 | 24.5 ± 3.6 | 2.3 ± 0.7 | 8.5; 17.0 | 34.0; 68.0 | 4.4 ± 1.3 | 1.0 ± 0.2 |
226.2 |
|
55.3; 110.6 | 55.3; 110.6 | 8.2 ± 1.2 | 1.5 ± 0.3 | 55.3; 110.6 | 55.3; 110.6 | 23.5 ± 0.6 | 4.9 ± 0.6 |
592.6 |
|
>168.7 | >168.7 | 6.9 ± 2.4 | 0.8 ± 0.2 | >168.7 | >168.7 | 7.6 ± 2.7 | 1.0 ± 0.3 |
According to our proteomics data, we observed that SA, CH, and proAc down-regulate proteins involved in
2.2.2. Compounds from human body.
Studies performed on various fish species and in swine which led to the identification of innate immuno factors important to be selected for resistance to gram-negative infections, have pointed the attention on Tf as a candidate gene for disease resistance [51, 52]. Therefore, Tf is considered as a relevant safeguard for human body, facing infections sustained by bacteria. Tf is a glycoprotein; its molecular structure shows the presence of two lobes, each binding one iron III ion. Apo-Tf is the denomination of its iron-depleted form; holo-Tf is the iron-loaded form; both forms are collectively named Tfs. Tf has been shown to exert both a bacteriostatic and bactericidal effect in vitro on a variety of microbial pathogens [53, 54]. The antimicrobial activity of transferrin is conventionally related to the iron-depleted form but some studies demonstrated that the mechanism of its antibacterial activity could not be referred only to iron deprivation [55, 56].
We investigated the effect of human apo-Tf and holo-Tf on biofilm formation by
3. Conclusions
More than four billion years ago, bacteria appeared on the earth and rapidly evolved in different species which spread and colonized nearly all ecological niches. Since that time prokaryotes and then eukaryotes (plants and animals, unicellular and multicellular) struggle to maintain a perpetual condition of equilibrium between cohabitants. This equilibrium is obtained by the selection of organisms that can coexist and by the elimination of those who exert a negative influence on others. From the point of view of bacteria, this means that a bacterial cell has to defend its position in a niche; in fact in a prokaryotic community, all cells communicate by means of small molecules in order to allow only the presence of commensals, while multicellular organisms distinguish and select saprophytes from pathogens in order to allow the residency of the first ones and to counteract or even kill the last ones. In this struggle for survival, all living organisms including prokaryotes and all eukaryotes (plants and animals) produce compounds that counteract undesired bacteria by killing them or by modulating their virulence. Molecules that modulate bacterial virulence can be considered as a huge source of molecules to be studied as lead compounds for the development of new antibacterial drugs. We gave some examples derived from our experience in this field, by the description of compounds obtained from prokaryotes, from plants and from humans that can interfere with bacterial phenotype in order to reduce virulence. They represent thus promising scaffolds to use for further development of antibacterial drugs, which may overcome the insurgence of resistance.
Acknowledgments
We gratefully acknowledge Marco Tilotta and Andrea Cellini for skilful technical and scientific assistance.
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