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.

Identifying geographic distribution can improve surveillance and clinical testing procedures.

Summary Legionella, the aetiological agent responsible for Legionellosis, is an opportunistic pathogen that infects humans upon the inhalation of contaminated aerosolized water droplets. Legionella is pleomorphic and its different morphotypes exhibit varying degrees of virulence. While the filamentous forms of Legionella pneumophila (Lp) have been reported in patient samples since the first description of legionellosis, their role in disease has not been studied. Our results show that both E‐cadherin and β1 integrin receptors mediate filamentous Lp (FLp) attachment to lung epithelial cells (LECs). The activation of these receptors induces the formation of actin enriched membrane surface structures that we designated ‘hooks’ and ‘membrane wraps’. These structures entrap the filaments on the cell surface leading to their gradual internalization through a zipper mechanism of phagocytosis dependent on actomyosin activity. The supply of E‐cadherin receptors from the recycling pathway and β1 integrins released from focal adhesion turnover are required to sustain this process. Intracellular FLp inhabits a vacuolar compartment where filaments differentiate into short rods and replicate to produce infective progeny. Here we are reporting a first description of the invasion mechanism used by FLp to invade LECs. Therefore, filamentous morphotype of Lp can induce its own uptake by LECs and has the potential ability to cause disease.

Antimicrobial drug resistance rates for Mycoplasma pneumoniae was determined in clinical specimens and isolates obtained during 2011–2012 in Ontario, Canada. Of 91 M. pneumoniae drug-resistant specimens, 11 (12.1%) carried nucleotide mutations associated with macrolide resistance in the 23S rRNA gene. None of the M. pneumoniae specimens were resistant to fluoroquinolones or tetracyclines.

Pertussis, or whooping cough, is a contagious respiratory disease caused by Bordetella pertussis. Despite the availability of a vaccine, pertussis remains common. Currently, there is no FDA-cleared diagnostic test for Bordetella pertussis, and laboratories have resorted to the use of "home-brewed" tests. A number of targets have been used for real-time PCR assays, yet these targets are often polymorphic and have homology to other Bordetella species. At the Ontario Public Health Laboratories, the multiple-copy target IS481 is used for the diagnosis of B. pertussis. Use of this target leads to the interpretative dilemma where high cycle threshold (C T ) values (i.e., Ͼ35 cycles) may represent concentrations of DNA target of Ͻ1 genome per assay reaction. Cummings et al. (2) suggested BP3385, a gene of unknown function, as a possible diagnostic target because it is well conserved within B. pertussis and has not been observed in any other Bordetella species. The goal of this study was to evaluate the use of this single-copy PCR target as a means of confirming IS481 PCRs with C T values of Ͼ35.Bordetella pertussis ATCC 9797 was used as a control for culture, DNA extraction, and PCR steps. Cultures were maintained on charcoal agar (Oxoid, Cambridge, United Kingdom) (10% [vol/vol] horse blood, Bordetella selective supplement, 20 mg/liter cephalexin) at 37°C in a humidified 5% CO 2 incubator for up to 10 days. For real-time PCR optimization, positive control, and initial limit of detection studies, the entire gene sequence of BP3385 (GenBank accession no. EU727200) was cloned and confirmed by Sanger sequence analysis using standard M13 primers. Nasopharyngeal specimens from patients with suspected pertussis were obtained using Dacron-tipped swabs and transported in 500 l of phosphate-buffered saline. Patient swabs were processed for culture and then subjected to rapid boiling extraction (100°C for 20 min) for real-time PCR amplification using the targets IS481 and IS1001, as described by Kösters et al. (6).Real-time PCR targeting BP3385 used the primers BP3385_ fwd (5Ј-GGTTTCTTCAGGCCCTAAATGG-3Ј) and BP3385_ rev (5Ј-TCGTCCTCGACGTGTGTGTAG-3Ј) and the TaqMan MGB probe 5Ј-6-carboxyfluorescein-CTCTACCAACGCGCTC T-BNFQ-3Ј. An internal control (pAcGFP-1; Clontech, Mountain View, CA) was detected using the primers GFP_fwd (5Ј-A AGCTGACCCTGAAGTTCATCTG-3Ј) and GFP_rev (5Ј-AA GTCGTGCTGCTTCATGTGA-3Ј) and the TaqMan MGB probe 5Ј-VIC-CCTGAGCTACGGCGTG-BNFQ-3Ј. PCR mixtures contained 1ϫ TaqMan gene expression master mix (Applied Biosystems), a 0.4 M concentration of each primer, a 0.2 M concentration of each probe, 5 fg pAcGFP-1, and 5 l of template. Real-time PCR was performed using an Applied Biosystems 7900HT instrument (1 cycle of 50°C for 2 min, 1 cycle of 95°C for 15 min, and 45 cycles of 94°C for 15 s and 60°C for 1 min). The specificity of the real-time PCR assay was verified with purified genomic DNAs from 32 different viral, fungal, and bacterial pathogens, including B. parapertussis, B. bronchiseptica, B. holmesii, and B. hin...