Burkholderia cepacia complex isolates survive intracellularly without replication within acidic vacuoles of Acanthamoeba polyphaga

We have previously demonstrated that isolates of the Burkholderia cepacia complex can survive intracellularly in murine macrophages and in free-living Acanthamoeba. In this work, we show that the clinical isolates B. vietnamiensis strain CEP040 and B. cenocepacia H111 survived but did not replicate within vacuoles of A. polyphaga. B. cepacia-containing vacuoles accumulated the fluid phase marker Lysosensor Blue and displayed strong blue fluorescence, indicating that they had low pH. In contrast, the majority of intracellular bacteria within amoebae treated with the V-ATPse inhibitor bafilomycin A1 localized in vacuoles that did not fluoresce with Lysosensor Blue. Experiments using bacteria fluorescently labelled with chloromethylfluorescein diacetate demonstrated that intracellular bacteria remained viable for at least 24 h. In contrast, Escherichia coli did not survive within amoebae after 2 h post infection. Furthermore, intracellular B. vietnamiensis CEP040 retained green fluorescent protein within the bacterial cytoplasm, while this protein rapidly escaped from the cytosol of phagocytized heat-killed bacteria into the vacuolar lumen. Transmission electron microscopy analysis confirmed that intracellular Burkholderia cells were structurally intact. In addition, both Legionella pneumophila- and B. vietnamiensis-containing vacuoles did not accumulate cationized ferritin, a compound that localizes within the lysosome. Thus, our observations support the notion that B. cepacia complex isolates can use amoebae as a reservoir in the environment by surviving without intracellular replication within an acidic vacuole that is distinct from the lysosomal compartment.

β-Lactam Monotherapy vs β-Lactam-Macrolide Combination Treatment in Moderately Severe Community-Acquired Pneumonia: A Randomized Noninferiority Trial.

IMPORTANCE: The clinical benefit of adding a macrolide to a β-lactam for empirical treatment of moderately severe community-acquired pneumonia remains controversial. OBJECTIVE: To test noninferiority of a β-lactam alone compared with a β-lactam and macrolide combination in moderately severe community-acquired pneumonia. DESIGN, SETTING, AND PARTICIPANTS: Open-label, multicenter, noninferiority, randomized trial conducted from January 13, 2009, through January 31, 2013, in 580 immunocompetent adult patients hospitalized in 6 acute care hospitals in Switzerland for moderately severe community-acquired pneumonia. Follow-up extended to 90 days. Outcome assessors were masked to treatment allocation. INTERVENTIONS: Patients were treated with a β-lactam and a macrolide (combination arm) or with a β-lactam alone (monotherapy arm). Legionella pneumophila infection was systematically searched and treated by addition of a macrolide to the monotherapy arm. MAIN OUTCOMES AND MEASURES: Proportion of patients not reaching clinical stability (heart rate <100/min, systolic blood pressure >90 mm Hg, temperature <38.0°C, respiratory rate <24/min, and oxygen saturation >90% on room air) at day 7. RESULTS: After 7 days of treatment, 120 of 291 patients (41.2%) in the monotherapy arm vs 97 of 289 (33.6%) in the combination arm had not reached clinical stability (7.6% difference, P = .07). The upper limit of the 1-sided 90% CI was 13.0%, exceeding the predefined noninferiority boundary of 8%. Patients infected with atypical pathogens (hazard ratio [HR], 0.33; 95% CI, 0.13-0.85) or with Pneumonia Severity Index (PSI) category IV pneumonia (HR, 0.81; 95% CI, 0.59-1.10) were less likely to reach clinical stability with monotherapy, whereas patients not infected with atypical pathogens (HR, 0.99; 95% CI, 0.80-1.22) or with PSI category I to III pneumonia (HR, 1.06; 95% CI, 0.82-1.36) had equivalent outcomes in the 2 arms. There were more 30-day readmissions in the monotherapy arm (7.9% vs 3.1%, P = .01). Mortality, intensive care unit admission, complications, length of stay, and recurrence of pneumonia within 90 days did not differ between the 2 arms. CONCLUSIONS AND RELEVANCE: We did not find noninferiority of β-lactam monotherapy in patients hospitalized for moderately severe community-acquired pneumonia. Patients infected with atypical pathogens or with PSI category IV pneumonia had delayed clinical stability with monotherapy. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00818610.

β-Lactam Monotherapy vs β-Lactam-Macrolide Combination Treatment in Moderately Severe Community-Acquired Pneumonia: A Randomized Noninferiority Trial.

IMPORTANCE: The clinical benefit of adding a macrolide to a β-lactam for empirical treatment of moderately severe community-acquired pneumonia remains controversial. OBJECTIVE: To test noninferiority of a β-lactam alone compared with a β-lactam and macrolide combination in moderately severe community-acquired pneumonia. DESIGN, SETTING, AND PARTICIPANTS: Open-label, multicenter, noninferiority, randomized trial conducted from January 13, 2009, through January 31, 2013, in 580 immunocompetent adult patients hospitalized in 6 acute care hospitals in Switzerland for moderately severe community-acquired pneumonia. Follow-up extended to 90 days. Outcome assessors were masked to treatment allocation. INTERVENTIONS: Patients were treated with a β-lactam and a macrolide (combination arm) or with a β-lactam alone (monotherapy arm). Legionella pneumophila infection was systematically searched and treated by addition of a macrolide to the monotherapy arm. MAIN OUTCOMES AND MEASURES: Proportion of patients not reaching clinical stability (heart rate <100/min, systolic blood pressure >90 mm Hg, temperature <38.0°C, respiratory rate <24/min, and oxygen saturation >90% on room air) at day 7. RESULTS: After 7 days of treatment, 120 of 291 patients (41.2%) in the monotherapy arm vs 97 of 289 (33.6%) in the combination arm had not reached clinical stability (7.6% difference, P = .07). The upper limit of the 1-sided 90% CI was 13.0%, exceeding the predefined noninferiority boundary of 8%. Patients infected with atypical pathogens (hazard ratio [HR], 0.33; 95% CI, 0.13-0.85) or with Pneumonia Severity Index (PSI) category IV pneumonia (HR, 0.81; 95% CI, 0.59-1.10) were less likely to reach clinical stability with monotherapy, whereas patients not infected with atypical pathogens (HR, 0.99; 95% CI, 0.80-1.22) or with PSI category I to III pneumonia (HR, 1.06; 95% CI, 0.82-1.36) had equivalent outcomes in the 2 arms. There were more 30-day readmissions in the monotherapy arm (7.9% vs 3.1%, P = .01). Mortality, intensive care unit admission, complications, length of stay, and recurrence of pneumonia within 90 days did not differ between the 2 arms. CONCLUSIONS AND RELEVANCE: We did not find noninferiority of β-lactam monotherapy in patients hospitalized for moderately severe community-acquired pneumonia. Patients infected with atypical pathogens or with PSI category IV pneumonia had delayed clinical stability with monotherapy. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00818610.

A Novel Pathway for Inducible Nitric-oxide Synthase Activation through Inflammasomes

Innate immune recognition of flagellin is shared by transmembrane TLR5 and cytosolic Nlrc4 (NOD-like receptor family CARD (caspase activation recruitment domain) domain containing 4)/Naip5 (neuronal apoptosis inhibitory protein 5). TLR5 activates inflammatory genes through MYD88 pathway, whereas Nlrc4 and Naip5 assemble multiprotein complexes called inflammasomes, culminating in caspase-1 activation, IL-1 beta/IL-18 secretion, and pyroptosis. Although both TLR5 and Naip5/Nlrc4 pathways cooperate to clear infections, little is known about the relative anti-pathogen effector mechanisms operating through each of them. Here we show that the cytosolic flagellin (FLA-BSDot) was able to activate iNOS, an enzyme previously associated with TLR5 pathway. Using Nlrc4- or Naip5-deficient macrophages, we found that both receptors are involved in iNOS activation by FLA-BSDot. Moreover, distinct from extracellular flagellin (FLA-BS), iNOS activation by intracellular flagellin is completely abrogated in the absence of caspase-1. Interestingly, IL-1 beta and IL-18 do not seem to be important for FLA-BSDot-mediated iNOS production. Together, our data defined an additional anti-pathogen effector mechanism operated through Naip5 and Nlrc4 inflammasomes and illustrated a novel signaling transduction pathway that activates iNOS.

Pneumonia adquirida na comunidade em adultos hospitalizados : estudo da etiologia, epidemiologia e fatores prognósticos

OBJETIVO: Determinar a etiologia, epidemiologia e os fatores prognósticos de pneumonia adquirida na comunidade (PAC) em adultos imunocompetentes hospitalizados. MÉTODOS: Durante um período de 3 anos, foram estudados, prospectivamente, 110 pacientes consecutivos com diagnóstico de PAC. RESULTADOS: Sessenta e seis (60%) pacientes eram homens, a idade média foi de 54 anos, 42 (38,2%) eram maiores de 65 anos, 81 (73,6%) apresentavam comorbidades, 70 (63%) pertenciam às classes IV e V de Fine e 24 (21,8%) pacientes foram admitidos em unidade de terapia intensiva (UTI). Um agente etiológico foi identificado em 60 (54,5%) casos, incluindo Streptococcus pneumoniae em 23 (20,9%) casos, Staphylococcus aureus em 14 (12,7%), Pseudomonas aeruginosa em 7 (6,4%), Haemophilus influenzae em 5 (4,6%) e Legionella pneumophila em 5 (4,6%) casos, como os patógenos mais freqüentemente isolados. O uso de antimicrobianos previamente à admissão hospitalar ocorreu em 33,6% dos casos e foi significativamente associado com etiologia desconhecida. Houve 15 (13,6%) óbitos e três variáveis foram estatisticamente associadas ao desfecho: idade > 65 anos, índice de Fine V e IV e internação em UTI. Alterações no tratamento empírico inicial foram realizadas em 43 (39%) casos devido à obtenção do diagnóstico etiológico. CONCLUSÕES: Em nosso estudo, S. pneumoniae foi o principal agente etiológico de PAC, seguido de S. aureus e P. aeruginosa, que apresentaram freqüência elevada em indivíduos com pneumonia grave e/ou fatores de risco conhecidos. A determinação do agente etiológico serviu para otimizar o tratamento proposto pelos consensos e estimar a prevalência local dos patógenos.

Strain variability in the DNA immigration control region (ICR) of Xylella fastidiosa

The genome of the bacterium Xylella fastidiosa contains four ORFs (XF2721, XF2725, XF2739 and XF0295) related to the restriction modification type I system, ordinarily named R-M. This system belongs to the DNA immigration control region (ICR). Each CIRF is related to different operon structures, which are homologues among themselves and with subunit Hsd R from the endonuclease coding genes. In addition, these ORFs are highly homologous to genes in Pseudomonas aeruginosa, Methylococcus capsulatus str. Bath, Legionella pneumophila, Helicobacter pylori, Xanthomonas oryzae pv. Oryzae and Silicibacter pomeroyi, as well as to genes from X. fastidiosa strains that infect grapevine, almond and oleander plants. This study was carried out on R-M ORFs from forty-three X. fastidiosa strains isolated from citrus, coffee, grapevine, periwinkle, almond and plum trees, in order to assess the genetic diversity of these loci through PCR-RFLP. PCR-RFLP analysis of the four ORFs related to the R-M system from these strains enabled the detection of haplotypes for these loci. When the haplotypes were defined, wide genetic diversity and a large range of similar strains originating from different hosts were observed. This analysis also provided information indicating differences in population genetic structures, which led to detection of different levels of gene transfer among the groups of strains. (c) 2005 Elsevier SAS. All rights reserved.

Structural and Functional Insights from the Metagenome of an Acidic Hot Spring Microbial Planktonic Community in the Colombian Andes

A taxonomic and annotated functional description of microbial life was deduced from 53 Mb of metagenomic sequence retrieved from a planktonic fraction of the Neotropical high Andean (3,973 meters above sea level) acidic hot spring El Coquito (EC). A classification of unassembled metagenomic reads using different databases showed a high proportion of Gammaproteobacteria and Alphaproteobacteria (in total read affiliation), and through taxonomic affiliation of 16S rRNA gene fragments we observed the presence of Proteobacteria, micro-algae chloroplast and Firmicutes. Reads mapped against the genomes Acidiphilium cryptum JF-5, Legionella pneumophila str. Corby and Acidithiobacillus caldus revealed the presence of transposase-like sequences, potentially involved in horizontal gene transfer. Functional annotation and hierarchical comparison with different datasets obtained by pyrosequencing in different ecosystems showed that the microbial community also contained extensive DNA repair systems, possibly to cope with ultraviolet radiation at such high altitudes. Analysis of genes involved in the nitrogen cycle indicated the presence of dissimilatory nitrate reduction to N2 (narGHI, nirS, norBCDQ and nosZ), associated with Proteobacteria-like sequences. Genes involved in the sulfur cycle (cysDN, cysNC and aprA) indicated adenylsulfate and sulfite production that were affiliated to several bacterial species. In summary, metagenomic sequence data provided insight regarding the structure and possible functions of this hot spring microbial community, describing some groups potentially involved in the nitrogen and sulfur cycling in this environment. Citation: Jimenez DJ, Andreote FD, Chaves D, Montana JS, Osorio-Forero C, et al. (2012) Structural and Functional Insights from the Metagenome of an Acidic Hot Spring Microbial Planktonic Community in the Colombian Andes. PLoS ONE 7(12): e52069. doi:10.1371/journal.pone.0052069

[Community-acquired pneumonia--pathogens and assessment]

Pneumonia is one of the most important infectious diseases, both in terms of incidence as well as potential severity. Streptococcus pneumoniae remains the most prevalent etiologic agent, accounting for about two-thirds of bacteremic cases. Diagnostic procedures include chest radiography, blood culture, Gram staining and culture of expectorated sputum, urine antigen assays for Legionella pneumophila and pneumococci, and asservation of an initial serum sample for comparative serologic investigations. Molecular biology techniques continue to gain importance for the diagnosis of Chlamydia pneumoniae, Mycoplasma pneumoniae, Legionellae and viral respiratory infections, however, their availability at present is mainly restricted to research and reference laboratories.

Water quality in dental chair units. A random sample in the canton of St. Gallen

This study aimed to identify the microbial contamination of water from dental chair units (DCUs) using the prevalence of Pseudomonas aeruginosa, Legionella species and heterotrophic bacteria as a marker of pollution in water in the area of St. Gallen, Switzerland. Water (250 ml) from 76 DCUs was collected twice (early on a morning before using all the instruments and after using the DCUs for at least two hours) either from the high-speed handpiece tube, the 3 in 1 syringe or the micromotor for water quality testing. An increased bacterial count (>300 CFU/ml) was found in 46 (61%) samples taken before use of the DCU, but only in 29 (38%) samples taken two hours after use. Pseudomonas aeruginosa was found in both water samples in 6/76 (8%) of the DCUs. Legionella were found in both samples in 15 (20%) of the DCUs tested. Legionella anisa was identified in seven samples and Legionella pneumophila was found in eight. DCUs which were less than five years old were contaminated less often than older units (25% und 77%, p<0.001). This difference remained significant (0=0.0004) when adjusted for manufacturer and sampling location in a multivariable logistic regression. A large proportion of the DCUs tested did not comply with the Swiss drinking water standards nor with the recommendations of the American Centers for Disease Control and Prevention (CDC).

Studying the fate of non-volatile organic compounds in a commercial plasma air purifier

Degradation of non-volatile organic compounds-environmental toxins (methyltriclosane and phenanthrene), bovine serum albumin, as well as bioparticles (Legionella pneumophila, Bacillus subtilis, and Bacillus anthracis)-in a commercially available plasma air purifier based on a cold plasma was studied in detail, focusing on its efficiency and on the resulting degradation products. This system is capable of handling air flow velocities of up to 3.0m s(-1) (3200Lmin(-1)), much higher than other plasma-based reactors described in the literature, which generally are limited to air flow rates below 10Lmin(-1). Mass balance studies consistently indicated a reduction in concentration of the compounds/particles after passage through the plasma air purifier, 31% for phenanthrene, 17% for methyltriclosane, and 80% for bovine serum albumin. L. pneumophila did not survive passage through the plasma air purifier, and cell counts of aerosolized spores of B. subtilis and B. anthracis were reduced by 26- and 15-fold, depending on whether it was run at 10Hz or 50Hz, respectively. However rather than chemical degradation, deposition on the inner surfaces of the plasma air purifier occured. Our interpretation is that putative "degradation" efficiencies were largely due to electrostatic precipitation rather than to decomposition into smaller molecules.

TREM-1 deficiency can attenuate disease severity without affecting pathogen clearance

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune reactions. While TREM-1-amplified responses likely aid an improved detection and elimination of pathogens, excessive production of cytokines and oxygen radicals can also severely harm the host. Studies addressing the pathogenic role of TREM-1 during endotoxin-induced shock or microbial sepsis have so far mostly relied on the administration of TREM-1 fusion proteins or peptides representing part of the extracellular domain of TREM-1. However, binding of these agents to the yet unidentified TREM-1 ligand could also impact signaling through alternative receptors. More importantly, controversial results have been obtained regarding the requirement of TREM-1 for microbial control. To unambiguously investigate the role of TREM-1 in homeostasis and disease, we have generated mice deficient in Trem1. Trem1(-/-) mice are viable, fertile and show no altered hematopoietic compartment. In CD4(+) T cell- and dextran sodium sulfate-induced models of colitis, Trem1(-/-) mice displayed significantly attenuated disease that was associated with reduced inflammatory infiltrates and diminished expression of pro-inflammatory cytokines. Trem1(-/-) mice also exhibited reduced neutrophilic infiltration and decreased lesion size upon infection with Leishmania major. Furthermore, reduced morbidity was observed for influenza virus-infected Trem1(-/-) mice. Importantly, while immune-associated pathologies were significantly reduced, Trem1(-/-) mice were equally capable of controlling infections with L. major, influenza virus, but also Legionella pneumophila as Trem1(+/+) controls. Our results not only demonstrate an unanticipated pathogenic impact of TREM-1 during a viral and parasitic infection, but also indicate that therapeutic blocking of TREM-1 in distinct inflammatory disorders holds considerable promise by blunting excessive inflammation while preserving the capacity for microbial control.

Aspectos a considerar en una actualización de la normativa nacional en materia de legionelosis

Objetivo: En España, el vigente Real Decreto 865/2003 establece una serie de medidas destinadas a la prevención de la legionelosis que inciden especialmente en el mantenimiento de las instalaciones de riesgo relacionadas con la proliferación y transmisión de Legionella. Este trabajo pretende revisar determinados aspectos estructurales, de mantenimiento, operacionales y documentales que, desde el punto de vista del Control Oficial, podrían ser incluidos en una futura nueva norma que pudiera ser más efectiva, si cabe, en la prevención y control de la legionelosis en las instalaciones de riesgo. Materiales y Métodos: Se ha utilizado la normativa nacional vigente en materia de la legionelosis, documentación técnica de apoyo, otras normativas de aplicación en países de la Unión Europea, así como servicios en línea de información científica (ISI Web of Knowledge) y bases de datos de bibliografía médica (MEDLINE PubMed). Resultados y Discusión: Tras más de una década en vigor, es necesario que ciertos aspectos técnicos de las instalaciones de riesgo en la transmisión de la legionelosis sean revisados y adecuados al conocimiento actual y a la experiencia adquirida. Entre ellos se encuentran la clasificación de riesgo de las instalaciones, aspectos técnicos y operacionales de los sistemas de agua fría de consumo humano y caliente sanitaria, de los equipos de enfriamiento evaporativos, de los sistemas de agua climatizada y de los procedimientos de toma de muestras. Por último, se discute sobre la inclusión de técnicas analíticas basadas en la Biología Molecular (qPCR) como método oficial en la determinación de Legionella. Conclusiones: Una posible nueva normativa sobre prevención y control de la legionelosis debe contemplar una clasificación de las instalaciones en función del riesgo, que recoja las de reciente aparición que han sido asociadas a brotes. También deberá contar con anexos sobre las medidas preventivas para cada una de ellas, que desarrollen protocolos de toma de muestras específicos y modelos para la evaluación de riesgos adecuados que tengan en cuenta establecimientos especialmente sensibles, debería estudiarse la inclusión de las técnicas analíticas moleculares como métodos oficiales o complementarios a los oficiales. Esta actualización permitirá una actuación más rápida y eficaz ante la identificación de casos y la asociación de los mismos a las instalaciones de riesgo implicadas.

Acute Psychosis as Major Clinical Presentation of Legionnaires’ Disease

We report a case of a 61-year-old woman who presented with acute psychosis as a major manifestation of Legionnaires’ disease in the absence of other neuropsychiatric symptoms. Clinical history revealed dry cough and nausea. Observation showed fever and auscultation crackles in the lower lobe of the right lung. Laboratory testing demonstrated elevated C-reactive protein and lung chest radiograph showed patchy peribronchial and right lower lobe consolidation. Soon after admission, she started producing purulent sputum. Epidemiological data suggested Legionella pneumophila as possible cause of the clinical picture that was confirmed by urinary antigen detection and polymerase chain reaction of the sputum. She was treated with levofloxacin 750 mg/day for 10 days with complete remission of pulmonary and psychiatric symptoms. She has not had further psychotic symptoms.