Legionellosis is mostly caused by Legionella pneumophila and is defined by a severe respiratory illness with a case fatality rate ranging from 5 to 80%. In vitro and in vivo, interactions of L. pneumophila with lung epithelial cells are mediated by the sulfated glycosaminoglycans (GAGs) of the host extracellular matrix. In this study, we have identified several Legionella heparin binding proteins. We have shown that one of these proteins, designated Lcl, is a polymorphic adhesin of L. pneumophila that is produced during legionellosis. Homologues of Lcl are ubiquitous in L. pneumophila serogroups but are undetected in other Legionella species. Recombinant Lcl binds to GAGs, and a ⌬lpg2644 mutant demonstrated reduced binding to GAGs and human lung epithelial cells. Importantly, we showed that the ⌬lpg2644 strain is dramatically impaired in biofilm formation. These data delineate the role of Lcl in the GAG binding properties of L. pneumophila and provide molecular evidence regarding its role in L. pneumophila adherence and biofilm formation.
BackgroundPertussis continues to challenge medical professionals; recently described increases in incidence may be due to age-cohort effects, vaccine effectiveness, or changes in testing patterns. Toronto, Canada has recently experienced increases in pertussis incidence, and provides an ideal jurisdiction for evaluating pertussis epidemiology due to centralized testing. We evaluated pertussis trends in Toronto using all available specimen data, which allowed us to control for changing testing patterns and practices.MethodsData included all pertussis culture and PCR test records for Greater Toronto from 1993 to 2007. We estimated incidence trends using Poisson regression models; complex relationships between disease incidence and test submission were explored with vector autoregressive models.ResultsFrom 1993 to 2007, 26988 specimens were submitted for testing; 2545 (9.4%) were positive. Pertussis incidence was 2 per 100,000 from 1993 to 2004 and increased to 10 per 100,000 from 2005-2007, with a concomitant 6-fold surge in test specimen submissions after the introduction of a new, more sensitive PCR assay. The relative change in incidence was less marked after adjustment for testing volumes. Bidirectional feedbacks between test positivity and test submissions were identified.ConclusionsToronto's recent surge in pertussis reflects a true increase in local disease activity; the apparent size of the outbreak has likely been magnified by increasing use of pertussis testing by clinicians, and by improved test sensitivity since 2005. These findings may be applicable to changes in pertussis epidemiology that have been noted elsewhere in North America.
Bordetella holmesii is a human pathogen found mainly in immunocompromised patients. A specific real-time PCR assay was developed and successfully used to identify specimens from which B. holmesii was misidentified as Bordetella pertussis and to establish the prevalence of B. holmesii in Ontario patients with pertussis-like symptoms.The Gram-negative bacterium Bordetella holmesii was first described in 1995. It is known to cause disease (e.g., septicemia) in patients with serious underlying medical conditions (2,5,7,12,14) and has also been detected in nasopharyngeal specimens from patients with a pertussis-like illness (6, 15). Two insertion sequences, IS481 and IS1001, are commonly used PCR targets for the diagnosis of Bordetella pertussis and Bordetella parapertussis, respectively (4, 11). Since both targets are also present in B. holmesii, disease due to this pathogen may be mistakenly attributed to other Bordetella species (8, 9). The aim of this study was to develop a duplex real-time PCR assay able to detect and discriminate between B. pertussis and B. holmesii. Additionally, 1,775 specimens positive for IS481 and/or IS1001 collected during 2007 and 2008 were retrospectively tested with the new assay to determine the prevalence of B. holmesii in Ontario, Canada.To date, the B. holmesii genome has not been fully sequenced, and thus, it is challenging to identify novel targets for specific PCR identification of this organism. The housekeeping gene recA has been used in previous studies for detection of B. holmesii (1, 13) and was selected as a PCR target for this study. Specific detection of B. holmesii was accomplished by designing a real-time PCR assay targeting a 50-bp segment of the recA gene (GenBank accession no. AF399664) that is polymorphic between B. holmesii, B. pertussis, B. parapertussis, Bordetella bronchiseptica, Bordetella hinzii, and Bordetella avium (Fig. 1).To confirm the conservation of B. holmesii recA (BHrecA) among clinical isolates, the full gene from 8 B. holmesii strains cultured from patients was amplified and sequenced. Sequencing revealed that BHrecA was 100% conserved in the 8 isolates tested.Analytical sensitivity of the assay was determined using a dilution series (10-fold dilutions, 5.5 ng to 5.5 ϫ 10 Ϫ10 ng) of BHrecA cloned into pCR2.1 (Invitrogen, Carlsbad, CA). Each dilution was tested in triplicate, and the experiment was performed 3 separate times to ensure accuracy of the results. The assay found a 100% probability that detection of 55 ag of cloned material (equal to 2 copies of recA) was possible at a threshold cycle (C T ) of 38.3 Ϯ 1.0. Furthermore, specificity of the real-time PCR assay was verified by testing purified genomic DNA from 32 different bacterial, fungal, and viral pathogens, including B. pertussis (n ϭ 54), B. parapertussis (n ϭ 32), B. bronchiseptica (n ϭ 1), B. hinzii (n ϭ 1), Acinetobacter sp., To develop our novel duplex real-time PCR assay for the simultaneous detection of B. pertussis and B. holmesii, the BHrecA primers and probe were combined wit...
Legionella species are increasingly recognized as a cause of both healthcare- and community-acquired pneumonia (so-called "Legionnaire's disease"). These pathogens are ubiquitous in the environment, but environmental factors in the occurrence of sporadic legionellosis remain poorly understood. We analyzed all legionellosis cases identified in the Greater Toronto Area of Ontario from 1978 to 2006, and evaluated seasonal and environmental patterns in legionellosis case occurrence by using both negative binomial models and case-crossover analysis. A total of 837 cases were reported during the study period. After adjusting for seasonal effects, changes in the local watershed, rather than weather, were the strongest contributors to legionellosis risk. A 3.6-fold increase (95% confidence interval (CI), 2.4-5.3) in odds of disease was identified with decreasing watershed levels approximately 4 weeks before case-occurrence. We also found a 33% increase (95% CI, 8-64%) in odds of disease with decreasing lake temperature during the same period and a 34% increase (95% CI, 14-57%) with increasing humidity 5 weeks before case-occurrence. We conclude that local watershed ecology influences the risk of legionellosis, notwithstanding the availability of advanced water treatment capacity in Toronto. Enhancement of risk might occur through direct contamination of water sources or via introduction of micronutrients or commensal organisms into residential and hospital water supplies. These observations suggest testable hypotheses for future empiric studies.
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