Substrate translocation involves specific lysine residues of the central channel of the conjugative coupling protein TrwB.

Conjugative transfer of plasmid R388 requires the coupling protein TrwB for protein and DNA transport, but their molecular role in transport has not been deciphered. We investigated the role of residues protruding into the central channel of the TrwB hexamer by a mutational analysis. Mutations affecting lysine residues K275, K398, and K421, and residue S441, all facing the internal channel, affected transport of both DNA and the relaxase protein in vivo. The ATPase activity of the purified soluble variants was affected significantly in the presence of accessory protein TrwA or DNA, correlating with their behaviour in vivo. Alteration of residues located at the cytoplasmic or the inner membrane interface resulted in lower activity in vivo and in vitro, while variants affecting residues in the central region of the channel showed increased DNA and protein transfer efficiency and higher ATPase activity, especially in the absence of TrwA. In fact, these variants could catalyze DNA transfer in the absence of TrwA under conditions in which the wild-type system was transfer deficient. Our results suggest that protein and DNA molecules have the same molecular requirements for translocation by Type IV secretion systems, with residues at both ends of the TrwB channel controlling the opening-closing mechanism, while residues embedded in the channel would set the pace for substrate translocation (both protein and DNA) in concert with TrwA.

A novel chimeric phage lysin with high in vitro and in vivo bactericidal activity against Streptococcus pneumoniae.

OBJECTIVES: Streptococcus pneumoniae is becoming increasingly antibiotic resistant worldwide and new antimicrobials are urgently needed. Our aim was new chimeric phage endolysins, or lysins, with improved bactericidal activity by swapping the structural components of two pneumococcal phage lysozymes: Cpl-1 (the best lysin tested to date) and Cpl-7S. METHODS: The bactericidal effects of four new chimeric lysins were checked against several bacteria. The purified enzymes were added at different concentrations to resuspended bacteria and viable cells were measured after 1 h. Killing capacity of the most active lysin, Cpl-711, was tested in a mouse bacteraemia model, following mouse survival after injecting different amounts (25-500 µg) of enzyme. The capacity of Cpl-711 to reduce pneumococcal biofilm formation was also studied. RESULTS: The chimera Cpl-711 substantially improved the killing activity of the parental phage lysozymes, Cpl-1 and Cpl-7S, against pneumococcal bacteria, including multiresistant strains. Specifically, 5 µg/mL Cpl-711 killed ≥7.5 log of pneumococcal R6 strain. Cpl-711 also reduced pneumococcal biofilm formation and killed 4 log of the bacterial population at 1 µg/mL. Mice challenged intraperitoneally with D39_IU pneumococcal strain were protected by treatment with a single intraperitoneal injection of Cpl-711 1 h later, resulting in about 50% greater protection than with Cpl-1. CONCLUSIONS: Domain swapping among phage lysins allows the construction of new chimeric enzymes with high bactericidal activity and a different substrate range. Cpl-711, the most powerful endolysin against pneumococci, offers a promising therapeutic perspective for the treatment of multiresistant pneumococcal infections.

Improving the lethal effect of cpl-7, a pneumococcal phage lysozyme with broad bactericidal activity, by inverting the net charge of its cell wall-binding module.

Phage endolysins are murein hydrolases that break the bacterial cell wall to provoke lysis and release of phage progeny. Recently, these enzymes have also been recognized as powerful and specific antibacterial agents when added exogenously. In the pneumococcal system, most cell wall associated murein hydrolases reported so far depend on choline for activity, and Cpl-7 lysozyme constitutes a remarkable exception. Here, we report the improvement of the killing activity of the Cpl-7 endolysin by inversion of the sign of the charge of the cell wall-binding module (from -14.93 to +3.0 at neutral pH). The engineered variant, Cpl-7S, has 15 amino acid substitutions and an improved lytic activity against Streptococcus pneumoniae (including multiresistant strains), Streptococcus pyogenes, and other pathogens. Moreover, we have demonstrated that a single 25-µg dose of Cpl-7S significantly increased the survival rate of zebrafish embryos infected with S. pneumoniae or S. pyogenes, confirming the killing effect of Cpl-7S in vivo. Interestingly, Cpl-7S, in combination with 0.01% carvacrol (an essential oil), was also found to efficiently kill Gram-negative bacteria such as Escherichia coli and Pseudomonas putida, an effect not described previously. Our findings provide a strategy to improve the lytic activity of phage endolysins based on facilitating their pass through the negatively charged bacterial envelope, and thereby their interaction with the cell wall target, by modulating the net charge of the cell wall-binding modules.

Microarray studies reveal a 'differential response' to moderate or severe heat shock of the HrcA- and HspR-dependent systems in Corynebacterium glutamicum.

This article has been retracted at the request of Microbiology because identical bands for the 16S rRNA probe controls in the Northern blots were reported to correspond to experiments using different strains and experimental conditions in articles published in this journal and in Journal of Bacteriology over a period of 5 years.

Structural independence of conjugative coupling protein TrwB from its Type IV secretion machinery.

The stability of components of multiprotein complexes often relies on the presence of the functional complex. To assess structural dependence among the components of the R388 Type IV secretion system (T4SS), the steady-state level of several Trw proteins was determined in the absence of other Trw components. While several Trw proteins were affected by the lack of others, we found that the coupling protein TrwB is not affected by the absence of other T4SS components, nor did its absence alter significantly the levels of integral components of the complex, underscoring the independent role of the coupling protein on the T4SS architecture. The cytoplasmic ATPases TrwK (VirB4) and TrwD (VirB11) were affected by the absence of several core complex components, while the pilus component TrwJ (VirB5) required the presence of all other Trw proteins (except for TrwB) to be detectable. Overall, the results delineate a possible assembly pathway for the T4SS of R388. We have also tested structural complementation of TrwD (VirB11) and TrwJ (VirB5) by their homologues in the highly related Trw system of Bartonella tribocorum (Bt). The results reveal a correlation with the functional complementation data previously reported.

Role for Rhizobium rhizogenes K84 cell envelope polysaccharides in surface interactions.

Rhizobium rhizogenes strain K84 is a commercial biocontrol agent used worldwide to control crown gall disease. The organism binds tightly to polypropylene substrate and efficiently colonizes root surfaces as complex, multilayered biofilms. A genetic screen identified two mutants in which these surface interactions were affected. One of these mutants failed to attach and form biofilms on the abiotic surface although, interestingly, it exhibited normal biofilm formation on the biological root tip surface. This mutant is disrupted in a wcbD ortholog gene, which is part of a large locus predicted to encode functions for the biosynthesis and export of a group II capsular polysaccharide (CPS). Expression of a functional copy of wcbD in the mutant background restored the ability of the bacteria to attach and form normal biofilms on the abiotic surface. The second identified mutant attached and formed visibly denser biofilms on both abiotic and root tip surfaces. This mutant is disrupted in the rkpK gene, which is predicted to encode a UDP-glucose 6-dehydrogenase required for O-antigen lipopolysaccharide (LPS) and K-antigen capsular polysaccharide (KPS) biosynthesis in rhizobia. The rkpK mutant from strain K84 was deficient in O-antigen synthesis and exclusively produced rough LPS. We also show that strain K84 does not synthesize the KPS typical of some other rhizobia strains. In addition, we identified a putative type II CPS, distinct from KPS, that mediates cell-surface interactions, and we show that O antigen of strain K84 is necessary for normal cell-cell interactions in the biofilms.

[Opinions and experiences of healthcare professionals in the nutritional management of oncology patients: the ONA study]. / Opiniones y experiencias de profesionales sanitarios en el manejo nutricional del paciente oncológico: estudio ONA.

Introduction: Objectives: an inadequate approach to prevent malnutrition in cancer patients may worsen their quality of life and reduce their response to treatment. This study aims to describe the nutritional management of cancer patients in clinical practice, as well as the opinions of the healthcare professionals (HCPs) involved. Methods: this was an observational, descriptive, cross-sectional study addressed to HCPs in the Spanish healthcare setting. The online questionnaire was designed based on a literature review, one focus group of patients (n = 6), and the experience of the multidisciplinary scientific committee (n = 5), and was distributed by the scientific societies endorsing the study. Results: a total of 461 HCPs answered the survey. Most of them considered that a nutrition expert (95.0 %) is essential for the nutritional management of patients. However, 22.8 % of HCPs did not have access to this expert, and only 49.0 % had received training. Nutritional screening or patient referral for screening was performed by 58.4 % of HCPs. Of the total of HCPs, 86.6 % stated that nutritional information is provided to patients and considered them moderately satisfied with the information received. In malnourished patients or in those at risk of malnutrition, a complete nutritional assessment was performed by HCPs (73.1 %). Most HCPs (87.4 %) reported prescribing or recommending nutritional support if needed, and assessing adherence (86.8 %). Conclusions: despite malnutrition being a common problem in cancer patients, almost half of professionals do not perform any nutritional screening. In addition, patient information and assessment of nutritional adherence appear to be suboptimal.

Introducción: Objetivos: un abordaje inadecuado de la desnutrición en el paciente con cáncer puede conducir a un empeoramiento de su calidad de vida y una respuesta deficiente al tratamiento. El estudio ONA (Oncología, Nutrición y Adherencia) tiene como objetivo describir el manejo nutricional del paciente con cáncer en la práctica clínica, así como las opiniones de los profesionales sanitarios involucrados en el mismo. Métodos: estudio observacional, descriptivo y transversal dirigido a profesionales sanitarios españoles. El cuestionario online fue diseñado a partir de una revisión bibliográfica, un grupo focal de pacientes (n = 6) y un comité científico multidisciplinar (n = 5), y distribuido por las sociedades científicas que avalan el estudio. Resultados: de los 461 profesionales sanitarios participantes, el 95,0 % consideraron fundamental la figura del profesional sanitario con formación específica en nutrición, pero el 22,8 % no tenían acceso a ella y solo el 49,0 % habían recibido formación. El 58,4 % afirmaron realizar el cribado nutricional o derivar al paciente para este fin. El 86,6 % de los participantes indicaron que se informa al paciente sobre aspectos nutricionales y consideraron que este estaba moderadamente satisfecho con la información recibida. En caso de detectarse desnutrición o riesgo de desnutrición, los profesionales afirmaron realizar una evaluación nutricional completa (73,1 %) y, de necesitarse soporte nutricional, este se prescribiría/recomendaría (87,4 %), evaluándose la adherencia al mismo (86,8 %). Conclusiones: a pesar de que la desnutrición es un problema común en el paciente con cáncer, casi la mitad de los profesionales no realizan un cribado nutricional. Además, el proceso de información y evaluación de la adherencia nutricional es subóptimo.

Transfer of R388 derivatives by a pathogenesis-associated type IV secretion system into both bacteria and human cells.

Bacterial type IV secretion systems (T4SSs) are involved in processes such as bacterial conjugation and protein translocation to animal cells. In this work, we have switched the substrates of T4SSs involved in pathogenicity for DNA transfer. Plasmids containing part of the conjugative machinery of plasmid R388 were transferred by the T4SS of human facultative intracellular pathogen Bartonella henselae to both recipient bacteria and human vascular endothelial cells. About 2% of the human cells expressed a green fluorescent protein (GFP) gene from the plasmid. Plasmids of different sizes were transferred with similar efficiencies. B. henselae codes for two T4SSs: VirB/VirD4 and Trw. A ΔvirB mutant strain was transfer deficient, while a ΔtrwE mutant was only slightly impaired in DNA transfer. DNA transfer was in all cases dependent on protein TrwC of R388, the conjugative relaxase, implying that it occurs by a conjugation-like mechanism. A DNA helicase-deficient mutant of TrwC could not promote DNA transfer. In the absence of TrwB, the coupling protein of R388, DNA transfer efficiency dropped 1 log. The same low efficiency was obtained with a TrwB point mutation in the region involved in interaction with the T4SS. TrwB interacted with VirB10 in a bacterial two-hybrid assay, suggesting that it may act as the recruiter of the R388 substrate for the VirB/VirD4 T4SS. A TrwB ATPase mutant behaved as dominant negative, dropping DNA transfer efficiency to almost null levels. B. henselae bacteria recovered from infected human cells could transfer the mobilizable plasmid into recipient Escherichia coli under certain conditions, underscoring the versatility of T4SSs.

Management of prediabetes from the perspective of Spanish physicians and community pharmacists: Detecta2 study.

INTRODUCTION: Prediabetes is a high-risk state for diabetes. The study aims to describe routine clinical practice and the views of physicians and pharmacists on prediabetes management. MATERIALS AND METHODS: An observational, descriptive, cross-sectional study was conducted using a structured questionnaire. RESULTS: A total of 410 physicians and 393 pharmacists completed the questionnaire. Self-adherence to clinical practice guidelines (CPGs) was reported by 51.5% and 23.2% of physicians and pharmacists, respectively. Less than 60% of participants defined prediabetes according to main CPG. Regarding the use of screening strategies to detect prediabetes (physicians: 96%; pharmacists: 42.1%), reports indicate the opportunistic strategy is widely employed (≥75%) whereas systematic screening is unusual (<20%). Changes in lifestyle were deemed essential by almost all participants (≥95%), but in clinical practice only 58.3% of healthcare centers and 28.0% of pharmacies were found to provide awareness-raising/instruction. The role of pharmacists in the prevention of prediabetes/diabetes was judged useful by most participants. CONCLUSIONS: Use of CPG, systematic prediabetes screening strategies, and specific strategies for patient education are scarce. The support of community pharmacists in prediabetes management was well valued. Therefore, it is crucial that the lines of action followed by both physicians and pharmacists align with each other and with the CPG.

Management of prediabetes from the perspective of Spanish physicians and community pharmacists: Detecta2 study.

INTRODUCTION: Prediabetes is a high-risk state for diabetes. The study aims to describe routine clinical practice and the views of physicians and pharmacists on prediabetes management. MATERIALS AND METHODS: An observational, descriptive, cross-sectional study was conducted using a structured questionnaire. RESULTS: A total of 410 physicians and 393 pharmacists completed the questionnaire. Self-adherence to clinical practice guidelines (CPGs) was reported by 51.5% and 23.2% of physicians and pharmacists, respectively. Less than 60% of participants defined prediabetes according to main CPG. Regarding the use of screening strategies to detect prediabetes (physicians: 96%; pharmacists: 42.1%), reports indicate the opportunistic strategy is widely employed (≥75%) whereas systematic screening is unusual (<20%). Changes in lifestyle were deemed essential by almost all participants (≥95%), but in clinical practice only 58.3% of healthcare centers and 28.0% of pharmacies were found to provide awareness-raising/instruction. The role of pharmacists in the prevention of prediabetes/diabetes was judged useful by most participants. CONCLUSIONS: Use of CPG, systematic prediabetes screening strategies, and specific strategies for patient education are scarce. The support of community pharmacists in prediabetes management was well valued. Therefore, it is crucial that the lines of action followed by both physicians and pharmacists align with each other and with the CPG.

Cost, microbiological, and nutritional properties of pureed food production in nursing homes. The ABADIA Study.

INTRODUCTION: Introduction: although nutritional differences between different types of texture-modified diet (TMD) have been evaluated, the resources and costs associated with their preparation have been less studied. Objective: to describe the nutritional, microbiological properties and costs of: 1) in-home produced pureed food (hTMD); 2) concentrated nutrient-dense commercial food products, hand-blended (cTMD); 3) food prepared using the MixxPro® automatic food mixer (cTMD-Mix). Methods: an observational, prospective study carried out in three geriatric nursing-homes. Patients ≥ 65 years, receiving TMD, with a stable clinical condition, estimated survival/expected internment > 1 month, and sufficient cognitive capacity were included. The following data were recorded: 1) patient socio-demographic and clinical variables; 2) TMD compliance and symptoms related to dysphagia during the meal; 3) patient appetite; and 4) kitchen information and resources used to prepare a TMD. Results: sixty-two residents were included (65.0 % women, 88.3 years (SD: 9.3); 43.5 % malnourished, 79.0 % with good appetite). The proportion of food eaten/median kcal served/portion/mean kcal consumed were: hTMD: 95.5 % (SD: 10.7)/92.4 kcal (IQR: 75.6-128.1)/88.2 kcal (IQR: 72.2-122.3); cTMD: 89.2 % (SD: 15.9)/323.4 kcal (IQR: 284.2-454.1)/288.5 kcal (IQR: 253.5-325.1); and cTMD-Mix: 80.3 % (SD: 21.4)/358.0 kcal (IQR: 344.0-372.1)/287.5 kcal (IQR: 276.5-298.8). No microorganisms were detected. The average time spent in preparing each portion and its costs were: hTMD: 11.2 min (SD: 3.89)/€2.33 (SD: 0.63); cTMD: 1.7 min (SD: 0.28)/€2.01 (SD: 0.39); and cTMD-Mix: 1.6 min (SD: 0.00)/€2.00 (SD: 0.33). Conclusions: in patients with dysphagia and/or chewing difficulties, concentrated nutrient-dense food products, particularly those produced using the MixxPro® automatic food mixer, ensure a high caloric intake and allow quick and safe food preparation.

INTRODUCCIÓN: Introducción: aunque existe evidencia acerca de las diferencias nutricionales entre los distintos tipos de dieta de textura modificada (DTM), los recursos y los costos asociados a su preparación se han estudiado menos. Objetivo: describir las propiedades nutricionales, las microbiológicas y los costes de: 1) una dieta triturada de manera artesanal (hDTM); 2) una dieta preparada con alimentación básica adaptada (ABA) (cDTM); y 3) una ABA preparada con el mezclador automático de alimentos MixxPro® (cDTM-Mix). Métodos: estudio observacional prospectivo realizado en tres residencias. Se incluyeron pacientes ≥ de 65 años que recibían DTM, con estado clínico estable, con supervivencia/internamiento estimado > 1 mes y capacidad cognitiva suficiente. Se registraron: 1) las variables sociodemográficas y clínicas del paciente; 2) el cumplimiento y los síntomas relacionados con la disfagia durante la comida; 3) el apetito del paciente, y 4) la información de la cocina y los recursos utilizados para preparar la DTM. Resultados: se incluyeron 62 residentes (65,0 % mujeres, 88,3 años (SD: 9,3), 43,5 % desnutridos, 79,0 % con buen apetito). La proporción de alimentos consumidos/mediana de kcal servidas/porción/media de kcal media consumidas fueron: hDTM 95,5 % (SD: 10,7)/92,4 kcal (IQR: 75,6-128,1)/88,2 kcal (IQR: 72,2-122,3); cDTM: 89.2 % (SD: 15.9)/323,4 kcal (IQR: 284.2-454.1)/288,5 kcal (IQR: 253.5-325.1), y cDTM-Mix: 80,3 % (SD: 21.4)/358,0 kcal (IQR: 344.0-372.1)/287,5 kcal (IQR: 276.5-298.8). No se detectaron microorganismos. El tiempo medio empleado en la preparación y el coste por porción fueron: hDTM: 11,2 min (SD: 3,89)/2,33 € (SD: 0,63); cDTM: 1,7 min (SD: 0,28)/2,01 € (SD: 0,39), y cDTM-Mix: 1,6 min (SD: 0,00)/2,00 € (SD: 0,33). Conclusiones: en los pacientes con disfagia y/o dificultades para masticar, los productos de ABA comerciales, en particular los que se producen con el mezclador automático de alimentos MixxPro®, aseguran una elevada ingesta calórica y permiten una preparación rápida y segura.

Functional dissection of the conjugative coupling protein TrwB.

The conjugative coupling protein TrwB is responsible for connecting the relaxosome to the type IV secretion system during conjugative DNA transfer of plasmid R388. It is directly involved in transport of the relaxase TrwC, and it displays an ATPase activity probably involved in DNA pumping. We designed a conjugation assay in which the frequency of DNA transfer is directly proportional to the amount of TrwB. A collection of point mutants was constructed in the TrwB cytoplasmic domain on the basis of the crystal structure of TrwB Delta N70, targeting the nucleotide triphosphate (NTP)-binding region, the cytoplasmic surface, or the internal channel in the hexamer. An additional set of transfer-deficient mutants was obtained by random mutagenesis. Most mutants were impaired in both DNA and protein transport. We found that the integrity of the nucleotide binding domain is absolutely required for TrwB function, which is also involved in monomer-monomer interactions. Polar residues surrounding the entrance and inside the internal channel were important for TrwB function and may be involved in interactions with the relaxosomal components. Finally, the N-terminal transmembrane domain of TrwB was subjected to random mutagenesis followed by a two-hybrid screen for mutants showing enhanced protein-protein interactions with the related TrwE protein of Bartonella tribocorum. Several point mutants were obtained with mutations in the transmembranal helices: specifically, one proline from each protein may be the key residue involved in the interaction of the coupling protein with the type IV secretion apparatus.

Physicians' and Pharmacists' Clinical Considerations for Elderly Patients with Type 2 Diabetes Mellitus: The IMPLICA2 Study.

BACKGROUND: Management of elderly patients with type 2 diabetes mellitus (T2DM) is complex due to their age-related conditions. Several clinical guidelines provide specific recommendations for management of these patients but little is known about their implementation in clinical practice. OBJECTIVE: To describe physician and community pharmacist perceptions and routine clinical practice in the management of elderly T2DM patients. METHODS: Cross-sectional study. RESULTS: A total of 993 physicians and 999 community pharmacists completed the questionnaire. More physicians than pharmacists agreed on the need to establish more flexible HbA1c targets for elderly (79.4% vs. 30.6%; p < 0.001) and frail (92.6% vs. 31.4%; p < 0.001) patients than for the general diabetic population. HbA1c targets < 7.5% for elderly patients and < 8.5% for frail patients (as recommended by the principle guidelines) were set by 38.9% and 28.7% of physicians, respectively. Furthermore, 62.8% of physicians stated they follow guideline recommendations but, based on their prescription decisions for hypothetical patients, less than 50% were aligned with them. In addition, 73.1% of physicians monitor treatment adherence, mainly by using dispensing control (59.1%). Specific nutritional approaches for elderly patients are provided by 62.9% of physicians and 56.0% of pharmacists, whilst 57.4% and 21.7%, respectively, deliver specific physical exercise programs. CONCLUSIONS: Low adherence to guideline recommendations (i.e. setting more stringent HbA1c targets or delaying treatment intensification) may lead to suboptimal glycaemic control in elderly patients. The standardization of processes, extensive monitoring of patient treatment adherence and providing advice regarding specific personal lifestyle habits may improve the management of elderly T2DM patients.

Validation of a loop-mediated isothermal amplification assay for rapid diagnosis of pertussis infection in nasopharyngeal samples.

OBJECTIVE: To develop and validate a novel loop-mediated amplification (LAMP) assay for rapid diagnosis (<1 hour) of whooping cough in nasopharyngeal samples versus the gold standard: real-time PCR. METHODS: The study included all nasopharyngeal samples (n = 213) collected from children with clinical suspicion of pertussis admitted to Children's University Hospital Sant Joan de Déu (Barcelona, Spain) during July-December 2014. Fresh samples were routinely analyzed by real-time PCR and stored for retrospective LAMP analysis, following an easy 30 minute DNA extraction step by Chelex-100. RESULTS: Performance results of the LAMP assay were: linearity, 10(5)-10(1) CFU/ml; Limit of Detection, 2 CFU/ml; precision (mean CV), 7.38%; diagnostic sensitivity, 96.55%; diagnostic specificity, 99.46%; time to detection, 12-30 minutes. CONCLUSION: The new test was shown to be 2.5-fold faster than real-time PCR while maintaining similar levels of analytical and clinical performance. Therefore it could become a useful diagnostic tool for molecular point-of-care testing.

Differences in Bordetella pertussis DNA load according to clinical and epidemiological characteristics of patients with whooping cough.

OBJECTIVE: To identify associations between nasopharyngeal Bordetella pertussis DNA load and clinical and epidemiological characteristics and evaluate DNA load prognostic value in pertussis severity. METHODS: Prospective observational multi-centre study including nasopharyngeal samples positive to pertussis DNA by real-time PCR collected from children and adult patients in more than 200 health centres of Catalonia (Spain) during 2012-2013. RESULTS: B. pertussis load was inversely correlated with age (rho = -0.32, p < 0.001), time to diagnosis (rho = -0.33, p < 0.001) and number of symptoms (rho = 0.13, p = 0.002). Median bacterial load was significantly higher in inpatients versus outpatients (4.91 vs. 2.55 log10 CFU/mL, p < 0.001), patients with complications versus those without (6.05 vs. 2.82 log10 CFU/mL, p < 0.001), disease incidence in summer and autumn versus spring and winter (3.50 vs. 2.21 log10 CFU/mL, p = 0.002), and unvaccinated-partially vaccinated patients versus vaccinated (4.20 vs. 2.76 log10 CFU/mL, p = 0.004). A logistic regression model including bacterial load and other candidate prognostic factors showed good prediction for hospital care (AUC = 0.94) although only age and unvaccinated status were found to be prognostic factors. CONCLUSIONS: We observed strong positive associations of nasopharyngeal bacterial load with severity outcomes of hospitalisation and occurrence of complications. Bacterial load and other independent variables contributed to an accurate prognostic model for hospitalisation.

Recurrent invasive pneumococcal disease in children: underlying clinical conditions, and immunological and microbiological characteristics.

PURPOSE: Clinical, immunological and microbiological characteristics of recurrent invasive pneumococcal disease (IPD) in children were evaluated, differentiating relapse from reinfection, in order to identify specific risk factors for both conditions. METHODS: All patients <18 years-old with recurrent IPD admitted to a tertiary-care pediatric center from January 2004 to December 2011 were evaluated. An episode of IPD was defined as the presence of clinical findings of infection together with isolation and/or pneumococcal DNA detection by Real-Time PCR in any sterile body fluid. Recurrent IPD was defined as 2 or more episodes in the same individual at least 1 month apart. Among recurrent IPD, we differentiated relapse (same pneumococcal isolate) from reinfection. RESULTS: 593 patients were diagnosed with IPD and 10 patients died. Among survivors, 23 episodes of recurrent IPD were identified in 10 patients (1.7%). Meningitis was the most frequent form of recurrent IPD (10 episodes/4 children) followed by recurrent empyema (8 episodes/4 children). Three patients with recurrent empyema caused by the same pneumococcal clone ST306 were considered relapses and showed high bacterial load in their first episode. In contrast, all other episodes of recurrent IPD were considered reinfections. Overall, the rate of relapse of IPD was 0.5% and the rate of reinfection 1.2%. Five out of 7 patients with reinfection had an underlying risk factor: cerebrospinal fluid leak (n = 3), chemotherapy treatment (n = 1) and a homozygous mutation in MyD88 gene (n = 1). No predisposing risk factors were found in the remainder. CONCLUSIONS: recurrent IPD in children is a rare condition associated with an identifiable risk factor in case of reinfection in almost 80% of cases. In contrast, recurrent IPD with pleuropneumonia is usually a relapse of infection.

Microarray studies reveal a 'differential response' to moderate or severe heat shock of the HrcA- and HspR-dependent systems in Corynebacterium glutamicum.

Genome-wide transcription profile analysis of the heat-shocked wild-type strain under moderate (40 degrees C) and severe heat stress (50 degrees C) revealed that a large number of genes are differentially expressed after heat shock. Of these, 358 genes were upregulated and 420 were downregulated in response to moderate heat shock (40 degrees C) in Corynebacterium glutamicum. Our results confirmed the HrcA/controlling inverted repeat of chaperone expression (CIRCE)-dependent and HspR/HspR-associated inverted repeat (HAIR)-dependent upregulation of chaperones following heat shock. Other genes, including clusters of orthologous groups (COG) related to macromolecule biosynthesis and several transcriptional regulators (COG class K), were upregulated, explaining the large number of genes affected by heat shock. Mutants having deletions in the hrcA or hspR regulators were constructed, which allowed the complete identification of the genes controlled by those systems. The up- or downregulation of several genes observed in the microarray experiments was validated by Northern blot analyses and quantitative (real-time) reverse-transcription PCR. These analyses showed a heat-shock intensity-dependent response ('differential response') in the HspR/HAIR system, in contrast to the non-differential response shown by the HrcA/CIRCE-regulated genes.

Functional interactions between type IV secretion systems involved in DNA transfer and virulence.

This paper reports an analysis of the functional interactions between type IV secretion systems (T4SS) that are part of the conjugative machinery for horizontal DNA transfer (cT4SS), and T4SS involved in bacterial pathogenicity (pT4SS). The authors' previous work showed that a conjugative coupling protein (T4CP) interacts with the VirB10-type component of the T4SS in order to recruit the protein-DNA complex to the transporter for conjugative DNA transfer. This study now shows by two-hybrid analysis that conjugative T4CPs also interact with the VirB10 element of the pT4SS of Agrobacterium tumefaciens (At), Bartonella tribocorum (Bt) and Brucella suis (Bs). Moreover, the VirB10 component of a cT4SS (protein TrwE of plasmid R388) could be partially substituted by that of a pT4SS (protein TrwE of Bt) for conjugation. This result opens the way for the construction of hybrid T4SS that deliver DNA into animal cells. Interestingly, in the presence of part of the Bs T4SS the R388 T4SS protein levels were decreased and R388 conjugation was strongly inhibited. Complementation assays between the Trw systems of R388 and Bt showed that only individual components from the so-called 'core complex' could be exchanged, supporting the concept that this core is the common scaffold for the transport apparatus while the other 'peripheral components' are largely system-specific.