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.

Noncontiguous operon is a genetic organization for coordinating bacterial gene expression.

Bacterial genes are typically grouped into operons defined as clusters of adjacent genes encoding for proteins that fill related roles and are transcribed into a single polycistronic mRNA molecule. This simple organization provides an efficient mechanism to coordinate the expression of neighboring genes and is at the basis of gene regulation in bacteria. Here, we report the existence of a higher level of organization in operon structure that we named noncontiguous operon and consists in an operon containing a gene(s) that is transcribed in the opposite direction to the rest of the operon. This transcriptional architecture is exemplified by the genes menE-menC-MW1733-ytkD-MW1731 involved in menaquinone synthesis in the major human pathogen Staphylococcus aureus We show that menE-menC-ytkD-MW1731 genes are transcribed as a single transcription unit, whereas the MW1733 gene, located between menC and ytkD, is transcribed in the opposite direction. This genomic organization generates overlapping transcripts whose expression is mutually regulated by transcriptional interference and RNase III processing at the overlapping region. In light of our results, the canonical view of operon structure should be revisited by including this operon arrangement in which cotranscription and overlapping transcription are combined to coordinate functionally related gene expression.

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.

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.

Intrinsic defects explain altered proliferative responses of T lymphocytes and HVS-derived T-cell lines in gastric adenocarcinoma.

We have taken advantage of a recently described technique of transformation and immortalization of T lymphocytes using the lymphotropic Herpesvirus saimiri, to achieve long-lasting T-cell lines from gastric cancer patients and healthy volunteers. Blood samples were drawn and T lymphocytes were transformed. Once sustained growth was observed, lines were subjected to phenotypic and functional analyses, and the results compared with freshly isolated peripheral blood mononuclear cells. Cytofluorometric analysis revealed that CD3 and CD45 were found at lower proportion in primary cells from patients than from control individuals (54% vs 75%, p<0.001, 90% vs 96%, p<0.05, respectively), and in HVS-derived T-cell lines (90% vs 98%, p<0.05, 97% vs 100%, p<0.05, respectively). Proliferative analyses showed that primary isolated cells were unable to respond adequately to CD3-, CD2-, and PHA-mediated stimulation, as compared to controls. Similarly, T-cell lines from patients proliferated to a lesser extent when CD3- and CD2-mediated stimuli were considered, especially when simultaneous stimulation via CD3 and CD2 molecules was carried out (47,824 counts per minute [cpm] vs 121,478 cpm, p<0.05). Altogether these results show that the defects reported in T cells from patients with cancer are not exclusively due to tumour-derived factors, since the alterations persist in long-lasting, HVS-transformed, T-cell lines, suggesting that this model seems a suitable one to disclose them.

Meat traceability using DNA markers: application to the beef industry.

Consumer concerns about beef demands instruments to assure its traceability. A methodology using DNA markers is proposed for beef identification focussing on a Spanish beef certification, Ternera de Navarra (Beef of Navarra). To validate this methodology the number of markers used and the implications of population structure in individual identification were evaluated. In order to get practical implementation, the sampling levels required, depending on the number of markers and amount of possible fraud, is also discussed. Using at least eight very informative markers the origin of retailed meat is always found independent of genetic population structure. The total control of fraud would be very expensive using large-scale application of DNA analyses and a strategy based on anonymous sampling is proposed.

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.

Adenosine diphosphate sugar pyrophosphatase prevents glycogen biosynthesis in Escherichia coli.

An adenosine diphosphate sugar pyrophosphatase (ASPPase, EC ) has been characterized by using Escherichia coli. This enzyme, whose activities in the cell are inversely correlated with the intracellular glycogen content and the glucose concentration in the culture medium, hydrolyzes ADP-glucose, the precursor molecule of glycogen biosynthesis. ASPPase was purified to apparent homogeneity (over 3,000-fold), and sequence analyses revealed that it is a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated as "nudix" hydrolases. Insertional mutagenesis experiments leading to the inactivation of the ASPPase encoding gene, aspP, produced cells with marginally low enzymatic activities and higher glycogen content than wild-type bacteria. aspP was cloned into an expression vector and introduced into E. coli. Transformed cells were shown to contain a dramatically reduced amount of glycogen, as compared with the untransformed bacteria. No pleiotropic changes in the bacterial growth occurred in both the aspP-overexpressing and aspP-deficient strains. The overall results pinpoint the reaction catalyzed by ASPPase as a potential step of regulating glycogen biosynthesis in E. coli.

Clinical characteristics of nonneoplastic cavernomatous transformation of the portal vein at a Gastroenterology Service in Spain.

OBJECTIVE: To identify predisposing factors, clinical characteristics and effective treatment in patients with nonneoplastic cavernomatous transformation of the portal vein in our gastroenterology service. METHODS: We retrospectively reviewed the clinical records of 2,201 patients diagnosed as having portal hypertension (2,165 with cirrhosis and 36 with noncirrhotic portal vein hypertension) during the period from 1977 to 1998. The diagnosis of cavernomatous transformation was confirmed with angiographic or Doppler echographic studies, or both. RESULTS: Thirteen patients (6 males, 7 females, age range 8 to 69 years) with cavernomatous transformation were found. Predisposing factors were omphalitis (1), echinococcal cyst (1), major abdominal surgery (3), liver cirrhosis (3), Sjögren syndrome (1), and no apparent cause (4). Eleven of the 13 patients had upper digestive tract bleeding from varices, 9 had splenomegaly, and 2 had cirrhotic decompensation. Splenectomy was done in 3 patients on admission, and in 5 patient shunts were used (splenorenal in 4, mesenteroatrial in 1) because of repeated bleeding. CONCLUSIONS: Of the patients with noncirrhotic portal hypertension, 27.7% had nontumoral cavernomatous transformation of the portal vein. Previous abdominal surgery was the most frequent predisposing factor; the 2 cases of echinococcal liver disease and Sjögren disease were exceptional. Age younger than 30 years, bleeding esophageal varices and splenomegaly were the most frequent clinical features. Portosystemic shunt was the only effective treatment alternative in these patients.

[Gastric carcinoma associated with other primary malignant neoplasms. A retrospective study of 25 cases]. / Carcinoma gástrico asociado a otras neoplasias malignas primarias. Estudio retrospectivo de 25 casos.

BACKGROUND: The risk of developing a second neoplasm in a person with gastric carcinoma (GC) is higher than among general population. OBJECTIVE: To analyze the clinical findings in patients with GC associated with other primary malignant neoplasms. PATIENTS AND METHODS: A total of 25 patients with GC associated with extragastric tumours were retrospectively studied. The following characteristics were studied: age, sex, location and staging, free interval, therapy, and survival. Survival of 13 patients with GC diagnosed as primary tumour was compared with that observed in a control group of 62 patients with GC alone. RESULTS: Twenty-five out of 792 (3.1%) patients with GC had other primary malignant neoplasms (seven synchronous and 18 metachronous). GC was associated with respiratory tumours in 7 cases. Sixty percent of patients with GC who had a second neoplasm had it diagnosed within the first year after gastric tumour was diagnosed (8 out of 13). Survival at 18 months was similar, both in the GC group with a second tumour as in the control group. CONCLUSIONS: The development of a second neoplasm among patients with GC usually occurs within the first year after diagnosis. Most commonly, the second neoplasm seats in the respiratory tract.

[Bleeding of the upper digestive tract due to gastric metastasis of squamous lung carcinoma]. / Hemorragia digestiva alta por metástasis gástrica de un carcinoma escamoso de pulmón.

Bronchogenic lung carcinoma, melanoma and breast cancer are the neoplasms which have most frequently been reported to metastasize to the stomach. These lesions are usually located on the fundus and on the upper part of the gastric body. They are usually asymptomatic with the diagnosis being made at necropsy. We present a patient who developed gastrointestinal bleeding as the first symptom of squamous lung cancer secondary to a gastric metastasis.

Detection and characterization of cerein 7, a new bacteriocin produced by Bacillus cereus with a broad spectrum of activity.

A bacteriocin-producing strain of Bacillus cereus was identified and isolated from a soil sample. The bacteriocin could be purified by a two-step procedure: ammonium sulfate precipitation of culture supernatants followed by a butanol extraction step, the antibiotic was recovered from the organic phase. The peptidic nature of the bacteriocin was proven by its sensitivity to proteolytic enzymes; its molecular mass, determined by mass spectrometry, was 3940 Da; and its amino-terminal sequence (GWGDVL) is unique in the databases. The compound was active against most Gram-positive but not Gram-negative bacteria. This is to our knowledge the first bacteriocin with these characteristics reported to be produced by B. cereus.

A comparative study of the actin-based motilities of the pathogenic bacteria Listeria monocytogenes, Shigella flexneri and Rickettsia conorii.

Listeria monocytogenes, Shigella flexneri, and Rickettsia conorii are three bacterial pathogens that are able to polymerize actin into 'comet tail' structures and move within the cytosol of infected cells. The actin-based motilities of L. monocytogenes and S. flexneri are known to require the bacterial proteins ActA and IcsA, respectively, and several mammalian cytoskeleton proteins including the Arp2/3 complex and VASP (vasodilator-stimulated phosphoprotein) for L. monocytogenes and vinculin and N-WASP (the neural Wiskott-Aldrich syndrome protein) for S. flexneri. In contrast, little is known about the motility of R. conorii. In the present study, we have analysed the actin-based motility of this bacterium in comparison to that of L. monocytogenes and S. flexneri. Rickettsia moved at least three times more slowly than Listeria and Shigella in both infected cells and Xenopus laevis egg extracts. Decoration of actin with the S1 subfragment of myosin in infected cells showed that the comet tails of Rickettsia have a structure strikingly different from those of L. monocytogenes or S. flexneri. In Listeria and Shigella tails, actin filaments form a branching network while Rickettsia tails display longer and not cross-linked actin filaments. Immunofluorescence studies revealed that the two host proteins, VASP and (&agr;)-actinin colocalized with actin in the tails of Rickettsia but neither the Arp2/3 complex which we detected in the Shigella actin tails, nor N-WASP, were detected in Rickettsia actin tails. Taken together, these results suggest that R. conorii may use a different mechanism of actin polymerization.