Systematic Reconstruction of the Complete Two-Component Sensorial Network in Staphylococcus aureus.

In bacteria, adaptation to changes in the environment is mainly controlled through two-component signal transduction systems (TCSs). Most bacteria contain dozens of TCSs, each of them responsible for sensing a different range of signals and controlling the expression of a repertoire of target genes (regulon). Over the years, identification of the regulon controlled by each individual TCS in different bacteria has been a recurrent question. However, limitations associated with the classical approaches used have left our knowledge far from complete. In this report, using a pioneering approach in which a strain devoid of the complete nonessential TCS network was systematically complemented with the constitutively active form of each response regulator, we have reconstituted the regulon of each TCS of S. aureus in the absence of interference between members of the family. Transcriptome sequencing (RNA-Seq) and proteomics allowed us to determine the size, complexity, and insulation of each regulon and to identify the genes regulated exclusively by one or many TCSs. This gain-of-function strategy provides the first description of the complete TCS regulon in a living cell, which we expect will be useful to understand the pathobiology of this important pathogen.IMPORTANCE Bacteria are able to sense environmental conditions and respond accordingly. Their sensorial system relies on pairs of sensory and regulatory proteins, known as two-component systems (TCSs). The majority of bacteria contain dozens of TCSs, each of them responsible for sensing and responding to a different range of signals. Traditionally, the function of each TCS has been determined by analyzing the changes in gene expression caused by the absence of individual TCSs. Here, we used a bacterial strain deprived of the complete TC sensorial system to introduce, one by one, the active form of every TCS. This gain-of-function strategy allowed us to identify the changes in gene expression conferred by each TCS without interference of other members of the family.

Design of a suspended germanium micro-antenna for efficient fiber-chip coupling in the long-wavelength mid-infrared range.

Recent developments of photonic integrated circuits for the mid-infrared band has opened up a new field of attractive applications for group IV photonics. Grating couplers, formed as diffractive structures on the chip surface, are key components for input and output coupling in integrated photonic platforms. While near-infrared optical fibers exhibit large mode field diameters compared to the wavelength, in the long-wave regime commercially available single-mode optical fibers have mode field diameters of the order of the operating wavelength. Consequently, an efficient fiber-chip surface coupler designed for the long-wave infrared range must radiate the power propagating in the waveguide with a higher radiation strength than a conventional grating coupler in the near-infrared range. In this article, we leverage the short electrical length required for long-wave infrared couplers to design a broadband all-dielectric micro-antenna for a suspended germanium platform at 7.67 µm. The design methodology is inspired by fundamental grating coupler equations, which remain valid even when the micro-antenna has only two or three diffractive elements. A simulated coupling efficiency of ~ 40% is achieved with a 1-dB bandwidth broader than 430 nm, which is almost twice the typical fractional bandwidth of a conventional grating coupler. In addition, the proposed design is markedly tolerant to fiber tilt misalignments of ±10°. This all-dielectric micro-antenna design paves the way for efficient fiber-chip coupling in long-wavelength mid-infrared integrated platforms.

Silicon-on-insulator free-carrier injection modulators for the mid-infrared.

Experimental demonstrations of silicon-on-insulator waveguide-based free-carrier effect modulators operating at 3.8 µm are presented. PIN diodes are used to inject carriers into the waveguides, and are configured to (a) use free-carrier electroabsorption to create a variable optical attenuator with 34 dB modulation depth and (b) use free-carrier electrorefraction with the PIN diodes acting as phase shifters in a Mach-Zehnder interferometer, achieving a VπLπ of 0.052 V·mm and a DC modulation depth of 22 dB. Modulation is demonstrated at data rates up to 125 Mbit/s.

Suspended low-loss germanium waveguides for the longwave infrared.

Germanium is a material of high interest for mid-infrared (MIR) integrated photonics due to its complementary metal-oxide-semiconductor (CMOS) compatibility and its wide transparency window covering the 2-15 µm spectral region exceeding the 4 and 8 µm limit of the silicon-on-insulator platform and Si material, respectively. In this Letter, we report suspended germanium waveguides operating at a wavelength of 7.67 µm with a propagation loss of 2.6±0.3 dB/cm. To the best of our knowledge, this is the first demonstration of low-loss suspended germanium waveguides at such a long wavelength. Suspension of the waveguide is achieved by defining holes alongside the core providing access to the buried oxide layer and the underlying Si layer so that they can be wet etched using hydrofluoric acid and tetramethylammonium hydroxide, respectively. Our MIR waveguides create a new path toward long wavelength sensing in the fingerprint region.

Suspended silicon waveguides for long-wave infrared wavelengths.

In this Letter, we report suspended silicon waveguides operating at a wavelength of 7.67 µm with a propagation loss of 3.1±0.3 dB/cm. To our knowledge, this is the first demonstration of low-loss silicon waveguides at such a long wavelength, with loss comparable to other platforms that use more exotic materials. The suspended Si waveguide core is supported by a sub-wavelength grating that provides lateral optical confinement while also allowing access to the buried oxide layer so that it can be wet etched using hydrofluoric acid. We also demonstrate low-loss waveguide bends and s-bends.

Suspended silicon mid-infrared waveguide devices with subwavelength grating metamaterial cladding.

We present several fundamental photonic building blocks based on suspended silicon waveguides supported by a lateral cladding comprising subwavelength grating metamaterial. We discuss the design, fabrication, and characterization of waveguide bends, multimode interference devices and Mach-Zehnder interferometers for the 3715 - 3800 nm wavelength range, demonstrated for the first time in this platform. The waveguide propagation loss of 0.82 dB/cm is reported, some of the lowest loss yet achieved in silicon waveguides for this wavelength range. These results establish a direct path to ultimately extending the operational wavelength range of silicon wire waveguides to the entire transparency window of silicon.

III-V-on-silicon integrated micro - spectrometer for the 3 µm wavelength range.

A compact (1.2 mm2) fully integrated mid-IR spectrometer operating in the 3 µm wavelength range is presented. To our knowledge this is the longest wavelength integrated spectrometer operating in the important wavelength window for spectroscopy of organic compounds. The spectrometer is based on a silicon-on-insulator arrayed waveguide grating filter. An array of InAs0.91Sb0.09 p-i-n photodiodes is heterogeneously integrated on the spectrometers output grating couplers using adhesive bonding. The spectrometer insertion loss is less than 3 dB and the waveguide-referred responsivity of the integrated photodiodes at room temperature is 0.3 A/W.

Angled multimode interferometer for bidirectional wavelength division (de)multiplexing.

We have demonstrated a bidirectional wavelength division (de)multiplexer (WDM) on the silicon-on-insulator platform using two 4-channel angled multimode interferometers (AMMIs) sharing the same multimode interference waveguide. An excellent match of the peak transmission wavelength of each channel between the two AMMIs was achieved. The input and output access waveguides were arranged in a configuration such that the propagation of light of one AMMI in the multimode interference waveguide suffered minimal perturbation by the input and output waveguides of the other AMMI. This type of device is ideal for the WDM system for datacom or telecom applications, e.g. an integrated optical transceiver, where the transmission wavelengths are required to match with the receiving wavelengths. The device also benefits from simple fabrication (as only a single lithography and etching step is required), improved convenience for the transceiver layout design, a reduction in tuning power and circuitry and efficient use of layout space. A low insertion loss of 3-4 dB, and low crosstalk of -15 to -20 dB, was achieved.

Suspended SOI waveguide with sub-wavelength grating cladding for mid-infrared.

We present a new type of mid-infrared silicon-on-insulator (SOI) waveguide. The waveguide comprises a sub-wavelength lattice of holes acting as lateral cladding while at the same time allowing for the bottom oxide (BOX) removal by etching. The waveguide loss is determined at the wavelength of 3.8 µm for structures before and after being underetched using both vapor phase and liquid hydrofluoric acid (HF). A propagation loss of 3.4 dB/cm was measured for a design with a 300 nm grating period and 150 nm holes after partial removal (560 nm) of BOX by vapor phase HF etching. We also demonstrate an alternative design with 550 nm period and 450 nm holes, which allows a faster and complete removal of the BOX by liquid phase HF etching, yielding the waveguide propagation loss of 3.6 dB/cm.

Mid-infrared wavelength division (de)multiplexer using an interleaved angled multimode interferometer on the silicon-on-insulator platform.

A low-cost and high-performance wavelength division (de)multiplexing structure in the mid-IR wavelength range is demonstrated on the silicon-on-insulator platform using an interleaved angled multimode interferometer (AMMI). As compared to a single AMMI, the channel count was doubled and the channel spacing halved with negligible extra insertion loss and crosstalk and with only a slight increase in device footprint. The device requires only single lithography and etching steps for fabrication. Potential is also shown for achieving improved performance with further optimized design.

Protection from Staphylococcus aureus mastitis associated with poly-N-acetyl beta-1,6 glucosamine specific antibody production using biofilm-embedded bacteria.

Staphylococcus aureus vaccines based on bacterins surrounded by slime, surface polysaccharides coupled to protein carriers and polysaccharides embedded in liposomes administered together with non-biofilm bacterins confer protection against mastitis. However, it remains unknown whether protective antibodies are directed to slime-associated known exopolysaccharides and could be produced in the absence of bacterin immunizations. Here, a sheep mastitis vaccination study was carried out using bacterins, crude bacterial extracts or a purified exopolysaccharide from biofilm bacteria delivered in different vehicles. This polysaccharide reacted specifically with antibodies to poly-N-acetyl-beta-1,6-glucosamine (PNAG) and not with antibodies to other capsular antigens or bacterial components. Following intra-mammary challenge with biofilm-producing bacteria, antibody production against the polysaccharide, milk bacterial counts and mastitis lesions were determined. Bacterins from strong biofilm-producing bacteria triggered the highest production of antibodies to PNAG and conferred the highest protection against infection and mastitis, compared with weak biofilm-producing bacteria and non-cellular inocula. Thus, bacterins from strong biofilm bacteria, rather than purified polysaccharide, are proposed as a cost-efficient vaccination against S. aureus ruminant mastitis.

Biotechnological war against biofilms. Could phages mean the end of device-related infections?

Microorganisms universally attach to surfaces, resulting in biofilm formation. These biofilms entail a serious problem in daily clinical practice because of the great prevalence of implantable device-related infections. Differences in antibiotic activity against planktonic and sessile bacteria may relate to clinical failures in the treatment of biofilm-related infections (BRI). Bacteriophages have several characteristics that make them potentially attractive therapeutic agents in some selected clinical settings, like for example BRI. They are highly specific and very effective in lysing targeted bacteria, moreover, they appear to be safe for humans. Many studies have shown the potential of phages for the treatment of infectious diseases in plants and animals, including infections with highly drug-resistant bacteria. The therapeutic use of bacteriophages, possibly in combination with antibiotics, may be a valuable approach in BRI. However, many important questions still remain that must be addressed before phages can be endorsed for therapeutic use in humans.

Staphylococcal infections in rabbit does on two industrial farms.

The main reasons for culling adult rabbit does on two Spanish rabbit farms were investigated for a year. The most important conditions were mastitis (33.3 per cent), followed by subcutaneous abscesses (9.9 per cent) and pyometra (8.7 per cent). Staphylococcus aureus infections were the most severe problem, the organism being isolated from 69.2 per cent of infected animals. Pasteurella species were more prevalent in cases of pyometra and pneumonia. Two strains of S aureus were identified by using polymorphism of the coagulase gene as the criterion. One of these strains was responsible for the majority of the staphylococcal infections and was isolated from several pathological processes.

Genotypic characterization of Staphylococcus aureus strains isolated from rabbit lesions.

Since staphylococcal infections are the main pathological problem in rabbit does, the objective of this study was to characterize epidemiologically Staphylococcus aureus isolates from different lesion types in rabbits. Using 3 genetic markers (coagulase, staphylococcal protein A and clumping factor B genes), 22 different genotypes were identified among 301 isolates recovered from 259 rabbit does with 10 different kinds of chronic purulent lesions. These infected rabbits were obtained from 30 herds located in the Valencia province on the Spanish Mediterranean coast. The most frequent genotype was designated A1/II1/delta (coa/spa/clfB combination genotype) and represented 70.76% of the isolates. Although most genotypes were previously identified in other countries, novel types were also documented. No specificity between genotypes and nature of the pathologic process could be identified. After genetic comparison between strains from different origins, the results may suggest that rabbit, bovine and human S. aureus isolates are not clonally related, suggesting that specific host-dependent pathogenic factors may have evolved independently in these species. These differences indicate that a rational and effective strategy to control infections caused by rabbit-specific isolates may be advantageous.

Biofilm related infections: is there a place for conservative treatment of port-related bloodstream infections?

Vascular catheters are the most frequently used indwelling medical devices and have become necessary tools for patients with chronic or critical illness. Surgically or percutaneously placed venous access ports are used to facilitate long-term intravenous therapy. The widespread use of these devices has resulted in a dramatic increase in catheter-related infections. It implies considerable morbidity, occasional mortality, and an increase in medical costs derived from its diagnosis, treatment, and mainly, prolongation of the patient's in-hospital stay. Treatment of such infections is often difficult due to the presence of biofilms on the port inner surface; inside the biofilms, bacteria are less vulnerable to antimicrobial agents. Current diagnostic strategies are suboptimal, and most successful treatment options require removal of the infected device followed by a course of antimicrobial therapy. There are limited data concerning the efficacy of antibiotic treatment of port-related bloodstream infections without catheter removal.

[Bacterial biofilms and infection]. / Biofilms bacterianos e infección.

In developed countries we tend to think of heart disease and the numerous forms of cancer as the main causes of mortality, but on a global scale infectious diseases come close, or may even be ahead: 14.9 million deaths in 2002 compared to cardiovascular diseases (16.9 million deaths) and cancer (7.1 million deaths) (WHO report 2004). The infectious agents responsible for human mortality have evolved as medical techniques and hygienic measures have changed. Modern-day acute infectious diseases caused by specialized bacterial pathogens such as diphtheria, tetanus, cholera, plague, which represented the main causes of death at the beginning of XX century, have been effectively controlled with antibiotics and vaccines. In their place, more than half of the infectious diseases that affect mildly immunocompromised patients involve bacterial species that are commensal with the human body; these can produce chronic infections, are resistant to antimicrobial agents and there is no effective vaccine against them. Examples of these infections are the otitis media, native valve endocarditis, chronic urinary infections, bacterial prostatitis, osteomyelitis and all the infections related to medical devices. Direct analysis of the surface of medical devices or of tissues that have been foci of chronic infections shows the presence of large numbers of bacteria surrounded by an exopolysaccharide matrix, which has been named the "biofilm". Inside the biofilm, bacteria grow protected from the action of the antibodies, phagocytic cells and antimicrobial treatments. In this article, we describe the role of bacterial biofilms in human persistent infections.

The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation.

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.

Bap, a Staphylococcus aureus surface protein involved in biofilm formation.

Identification of new genes involved in biofilm formation is needed to understand the molecular basis of strain variation and the pathogenic mechanisms implicated in chronic staphylococcal infections. A biofilm-producing Staphylococcus aureus isolate was used to generate biofilm-negative transposon (Tn917) insertion mutants. Two mutants were found with a significant decrease in attachment to inert surfaces (early adherence), intercellular adhesion, and biofilm formation. The transposon was inserted at the same locus in both mutants. This locus (bap [for biofilm associated protein]) encodes a novel cell wall associated protein of 2,276 amino acids (Bap), which shows global organizational similarities to surface proteins of gram-negative (Pseudomonas aeruginosa and Salmonella enterica serovar Typhi) and gram-positive (Enteroccocus faecalis) microorganisms. Bap's core region represents 52% of the protein and consists of 13 successive nearly identical repeats, each containing 86 amino acids. bap was present in a small fraction of bovine mastitis isolates (5% of the 350 S. aureus isolates tested), but it was absent from the 75 clinical human S. aureus isolates analyzed. All staphylococcal isolates harboring bap were highly adherent and strong biofilm producers. In a mouse infection model bap was involved in pathogenesis, causing a persistent infection.

Phosphorylation of the Goodpasture antigen by type A protein kinases.

Collagen IV is the major component of basement membranes. The human alpha 3 chain of collagen IV contains an antigenic domain called the Goodpasture antigen that is the target for the circulating immunopathogenic antibodies present in patients with Goodpasture syndrome. Characteristically, the gene region encoding the Goodpasture antigen generates multiple alternative products that retain the antigen amino-terminal region with a five-residue motif (KRGDS). The serine therein appears to be the major in vitro cAMP-dependent protein kinase phosphorylation site in the isolated antigen and can be phosphorylated in vitro by two protein kinases of approximately 50 and 41 kDa associated with human kidney plasma membrane, suggesting that it can also be phosphorylated in vivo. Consistent with this, the Goodpasture antigen is isolated from human kidney in phosphorylated and non-phosphorylated forms and only the non-phosphorylated form is susceptible to phosphorylation in vitro. Since this motif is exclusive to the human alpha 3(IV) chain and includes the RGD cell adhesion motif, its phosphorylation might play a role in pathogenesis and influence cell attachment to basement membrane.

Characterization and expression of multiple alternatively spliced transcripts of the Goodpasture antigen gene region. Goodpasture antibodies recognize recombinant proteins representing the autoantigen and one of its alternative forms.

Collagen IV, the major component of basement membranes, is composed of six distinct alpha chains (alpha 1-alpha 6). Atypically among the collagen IV genes, the exons encoding the carboxyl-terminal region of the human alpha 3(IV) chain undergo alternative splicing. This region has been designated as the Goodpasture antigen because of its reactivity in the kidney and lung with the pathogenic autoantibodies causing Goodpasture syndrome. The data presented in this report demonstrate that, in human kidney, the gene region encompassing the Goodpasture antigen generates at least six alternatively spliced transcripts predicting five distinct proteins that differ in their carboxyl-terminus and retain, except in one case, the exon that harbors the characteristic amino-terminus of the antigen. Goodpasture antibodies specifically recognize recombinant proteins representing the antigen and the alternative form that retains the amino-half of the antigen, suggesting that this moiety could be involved in the in vivo binding of the pathogenic antibodies. Furthermore, the sera of control individuals contain autoantibodies against the antigen that can be differentiated from those causing the syndrome based on their specific reactivities, suggesting that the binding of the pathogenic autoantibodies to a specific determinant likely trigger a distinct and unique cascade of events causing the disease.