Pneumophila DNA Research Articles

Structural basis for recognition of transcriptional terminator structures by ProQ/FinO domain RNA chaperones

The ProQ/FinO family of RNA binding proteins mediate sRNA-directed gene regulation throughout gram-negative bacteria. Here, we investigate the structural basis for RNA recognition by ProQ/FinO proteins, through the crystal structure of the ProQ/FinO domain of the Legionella pneumophila DNA uptake regulator, RocC, bound to the transcriptional terminator of its primary partner, the sRNA RocR. The structure reveals specific recognition of the 3’ nucleotide of the terminator by a conserved pocket involving a β-turn-α-helix motif, while the hairpin portion of the terminator is recognized by a conserved α-helical N-cap motif. Structure-guided mutagenesis reveals key RNA contact residues that are critical for RocC/RocR to repress the uptake of environmental DNA in L. pneumophila. Structural analysis and RNA binding studies reveal that other ProQ/FinO domains also recognize related transcriptional terminators with different specificities for the length of the 3’ ssRNA tail.

Evaluation of an Environmental Transport Medium for Legionella pneumophila Recovery

The collection and storage of water-related matrices such as biofilm from collection to processing are critical for the detection of Legionella pneumophila by cultural and molecular tests. SRK™ is a liquid medium that acts both as an antimicrobial neutralizing agent and a transport medium for bacterial culture enumeration and is useful to maintain the stability of the sample from collection to analysis. The aims of this study were to evaluate Legionella pneumophila viability and bacterial nucleic acids’ stability in SRK™ medium over time at different storage conditions. Artificial bacterial inoculates with an approximate concentration of 104, 103 and 102 CFU/mL were made using Legionella pneumophila certified reference material suspended in SRK™ medium. Bacteria recovery was analyzed by cultural and molecular methods at time 0, 24 and 48 h at room temperature and at 0, 24, 48 and 72 h at 2–8 °C, respectively. SRK™ medium supported Legionella pneumophila culture viability with CFU counts within the expected range. The recovery after 72 h at 2–8 °C was 83–100% and 75–95% after 48 h at room temperature. Real-time PCR appropriately detected Legionella pneumophila DNA at each temperature condition, dilution and time point. Results demonstrated a good performance of SRK™ medium for the reliable recovery of environmental Legionella.

Confirming the Presence of Legionella pneumophila in Your Water System: A Review of Current Legionella Testing Methods.

Legionnaires’ disease has been recognized since 1976 and Legionella pneumophila still accounts for more than 95% of cases. Approaches in countries, including France, suggest that focusing risk reduction specifically on L. pneumophila is an effective strategy, as detecting L. pneumophila has advantages over targeting multiple species of Legionella. In terms of assays, the historically accepted plate culture method takes 10 days for confirmed Legionella spp. results, has variabilities which affect trending and comparisons, requires highly trained personnel to identify colonies on a plate in specialist laboratories, and does not recover viable-but-non-culturable bacteria. PCR is sensitive, specific, provides results in less than 24 h, and determines the presence/absence of Legionella spp. and/or L. pneumophila DNA. Whilst specialist personnel and laboratories are generally required, there are now on-site PCR options, but there is no agreement on comparing genome units to colony forming units and action limits. Immunomagnetic separation assays are culture-independent, detect multiple Legionella species, and results are available in 24 h, with automated processing options. Field-use lateral flow devices provide presence/absence determination of L. pneumophila serogroup 1 where sufficient cells are present, but testing potable waters is problematic. Liquid culture most probable number (MPN) assays provide confirmed L. pneumophila results in 7 days that are equivalent to or exceed plate culture, are robust and reproducible, and can be performed in a variety of laboratory settings. MPN isolates can be obtained for epidemiological investigations. This accessible, non-technical review will be of particular interest to building owners, operators, risk managers, and water safety groups and will enable them to make informed decisions to reduce the risk of L. pneumophila.

Legionella pneumophila occurrence in drinking water supplied by private wells.

Unregulated private wells are understudied potential sources of community-acquired Legionnaires' disease. Here we conducted a comprehensive survey of 44 homes supplied by private wells in Wake County, North Carolina, quantifying Legionella spp. DNA, Legionella pneumophila DNA, and total bacterial 16S rRNA genes via real-time polymerase chain reaction in hot and cold drinking water samples, along with culturable L. pneumophila via IDEXX Legiolert in cold drinking water samples. Legionella spp. DNA, L. pneumophila DNA and culturable L. pneumophila were detected in 100, 65·5 and 15·9% of the 44 homes, respectively, and culturable levels were comparable to some municipal surveys applying the same methods. Total coliforms and Escherichia coli were monitored as representative faecal indicators and were found in 20·4 and 0·0% of homes. Within certain sample types, Legionella spp. and L. pneumophila gene copy numbers were positively associated with total bacteria (i.e. total 16S rRNA genes) and water softener use, but were not associated with faecal indicator bacteria, inorganic water parameters or other well characteristics. These findings confirm that occurrence of Legionella and L. pneumophila is highly variable in private wells. SIGNIFICANCE AND IMPACT OF THE STUDY: Legionella is the leading identified cause of waterborne disease outbreaks associated with US municipal water systems. While Legionella is known to occur naturally in groundwater, prior efforts to characterize its occurrence in unregulated private wells are limited to sampling at the wellhead and not in the home plumbing where Legionella can thrive. This work documents much higher levels of Legionella in home plumbing versus water directly from private wells and examines factors associated with higher Legionella occurrence.

The 2015 New York City Legionnaires' Disease Outbreak: A Case Study on a History-Making Outbreak.

The 2015 New York City Legionnaires' Disease Outbreak: A Case Study on a History-Making Outbreak.

Prevalence of Pulmonary Infections Caused by Atypical Pathogens in non-HIV Immunocompromised Patients.

Although atypical bacteria are important causes of lower airway infections, data on their role in immunocompromised patients are scarce. The aim of the study was to evaluate the prevalence of atypical pulmonary infections in patients with various types of immunosuppression, and to analyze clinical characteristics of these infections. Eighty non-HIV immunocompromised patients with different underlying diseases and clinical and radiological signs of pulmonary infection were enrolled. Due to incomplete data on eight patients, 72 patients were eligible for final analysis (median age 58 years). All patients underwent fiberoptic bronchoscopy and bronchoalveolar lavage. Bronchoalveolar lavage fluid (BALF) fluid samples were sent for direct microscopy, cultures, and fungal antigen detection, when appropriate. Commercial qualitative amplification assay (PNEUMOTRIS oligomix Alert Kit®), based on nested PCR method, was used to detect specific DNA sequences of L. pneumophila, C. pneumoniae, and M. pneumoniae in BALF. There were 61 (84.7 %) patients with hematologic diseases, 3 (4.2 %) after solid organ transplantation, and 8 (11.1 %) with miscellaneous diseases affecting immune status. Specific sequences of M. pneumoniae, C. pneumoniae, and L. pneumophila DNA were found in 7 (9.7 %), 2 (2.8 %), and 0 patients, respectively. In 8 of these patients co-infections with different microorganisms were demonstrated. Co-infection with A. baumanii and P. aeruginosa was diagnosed in three patients who died. We conclude that atypical lower airway infections are uncommon in immunocompromised patients. The majority of these infections are co-infections rather than single pathogen infections.

P182: Detection of legionella pneumophila DNA from environment of hospitals

The risk of infection and epidemiological surveillance and control of legionellosis depends on the challenges of detecting the Legionella pneumophila DNA in the hospital environment.

Method development for genomic Legionella pneumophila DNA quantification by inductively coupled plasma mass spectrometry

The development of a method for the quantification of Legionella pneumophila genomic deoxyribonucleic acid is considered. The method is based on the quantification by inductively coupled plasma mass spectrometry (ICP–MS) of the mass fraction of phosphorus, stoichiometrically presented in the DNA molecules. Through the DNA sequencing data, it was possible to convert the ICP–MS analysis results into DNA genome units. L. pneumophila DNA samples were analyzed using ICP–sector field MS and ICP–quadrupole MS with a collision/reaction cell. Spectrophotometric measurements of the absorbance at 260nm and real-time PCR techniques were used to independently confirm the ICP–MS results. The comparison of the methods showed that the ICP–MS method provides better accuracy with respect to currently applied analytical techniques such as UV spectrophotometry, fluorescent dye methods, and real-time PCR. Moreover, with the use of calibration standards whose values are traceable to the International System of Units and the possibility of evaluating the contribution to the overall uncertainty of each step of the measurement procedure, the method enables long-term comparability of the measurement results. These advantages make the ICP–MS method suitable for nucleic acid investigation, from nucleotides to genomic DNA, as well as for the certification of the reference materials containing nucleic acids.

Application of Legionella pneumophila-specific quantitative real-time PCR combined with direct amplification and sequence-based typing in the diagnosis and epidemiological investigation of Legionnaires’ disease

The detection of Legionella pneumophila DNA in clinical specimens using quantitative real-time polymerase chain reaction (qPCR) combined with direct sequence-based typing (SBT) offers rapid confirmation and timely intervention in the investigation of cases of Legionnaires' disease (LD). We assessed the utility of a specific L. pneumophila qPCR assay targeting the macrophage infectivity potentiator (mip) gene and internal process control with three clinical specimen types from confirmed LD cases. The assay was completely specific for L. pneumophila, as demonstrated by positive results for 39/39 strains from all subspecies and 16 serogroups. No cross-reaction was observed with any of the 54 Legionella non-pneumophila (0/69 strains) or 21 non-Legionella (0/58 strains). All L. pneumophila culture-positive respiratory samples (81/81) were qPCR-positive. Of 80 culture-negative samples tested, 47 (58.8%) were qPCR-positive and none were inhibitory. PCR was significantly more sensitive than culture for samples taken ≤ 2 days of hospitalisation (94.7% vs. 79.6%), with the difference being even more marked for samples taken between 3 and 14 days (79.3% vs. 47.8%). Overall, the sensitivity of the qPCR was ∼30% greater than that of culture and direct typing on culture-negative PCR-positive samples resulted in full 7-allele profiles from 23/46, 5 to 6 alleles from 8/46 and ≥ 1 allele from 43/46 strains.

Detection of microorganisms in exhaled breath condensate during acute exacerbations of COPD

One hallmark of COPD is colonization and infection of the lung. Acute exacerbations of COPD (AECOPD) are acute deteriorations of the chronic disease and are associated with a change of the pulmonary microbial balance. The collection of exhaled breath condensate (EBC) can be used to non-invasively determine markers of lung disease. The aim of the present study was to compare the results of assays based on the detection of microbial nucleic acids from EBC and from spontaneous sputum in patients with AECOPD. EBC and sputa of 29 adults with AECOPD were obtained. Isolated DNA or RNA were used as starting material for the PCR assays to detect Staphylococcus aureus, Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia pneumoniae, influenza viruses (AH 1, AH 3) and respiratory syncytial virus. Bacterial or viral nucleic acids were identified in 14 EBC and 21 sputa from 29 patients. Results from EBC did not correlate well with those from sputum. Viral and S. pneumoniae nucleic acids were detected only in sputum, whereas L. pneumophila DNA was only found in EBC. In three EBC and 10 sputa nucleic acids of more than one microorganism was detected. Bacterial nucleic acids can be identified in EBC of COPD patients with exacerbations. The results obtained from EBC and sputum did not correlate well.

Legionella pneumophila monoclonal antibody subgroups and DNA sequence types isolated in Canada between 1981 and 2009: Laboratory Component of National Surveillance

Legionella pneumophila (Lp) is a significant cause of nosocomial, community-acquired, and travel-associated pneumonia in industrialized regions. Legionellosis has been a nationally notifiable disease in Canada since 1986, with an average of 75 cases reported annually; however, only the most severe, and often fatal, cases are reported or investigated. Here, epidemiological relationships, types, and distribution of Lp referrals to the Canadian national reference center were studied. Lp strains from different years, sources, and geographic locations were subtyped using a sequence-based typing (SBT) scheme and by the 'Joly' and/or 'Dresden' monoclonal antibody panels. Included were 128 epidemiologically unrelated clinical and 86 unrelated environmental strains. Sixty-four (index of diversity [IOD] = 0.964) and 45 (IOD = 0.888) sequence types (STs) were observed among clinical and environmental sources, respectively. Serogroup (sg) 1 was represented by 60.2% (77/128) and 52.3% (45/86) of clinical and environmental strains, respectively, and 63.6% (49/77) and 15.6% (7/45) of those were mAb2-positive, respectively. Serogroup 1, ST1 accounted for 14.1% (18/128) and 30.2% (26/86) of unrelated clinical and environmental isolates, respectively. This database will serve as a basis for Canadian epidemiological surveillance efforts and is linked to global surveillance initiatives curated by the European Working Group for Legionella Infections (EWGLI) network.

Legionella pneumophila DNA in serum samples during Legionnaires’ disease in relation to C-reactive protein levels

Legionella pneumophila DNA can be detected in serum from patients with Legionnaires' disease (LD). We explored this observation studying the kinetics of L. pneumophila DNA in serum samples in relation to C-reactive protein (CRP). Eleven hospitalized patients with LD were studied. Diagnosis was made by Legionella urinary antigen test in 8 patients and seroconversion in 3 patients. A macrophage infectivity potentiator (MIP) real-time PCR was performed on 31 serum samples, including 20 follow-up serum samples. Serum samples obtained on the day of admission were MIP PCR-positive in 7 (64%) and MIP PCR-negative in 4 (36%) patients. Three (75%) of the 4 patients with a MIP PCR-negative serum sample on the day of admission became positive during follow-up. Overall, L. pneumophila DNA was detected in serum samples from 10 of the 11 patients (91%). CRP levels in the 7 patients with a positive MIP PCR serum sample on day of admission (499 +/- 144 mg/l; median +/- SD) were significantly higher than those in the 4 patients with a negative MIP PCR serum sample on the day of admission (244 +/- 97 mg/l). No difference in the severity of the disease on the day of admission was found between these patients. The presence of L. pneumophila DNA in serum is a common phenomenon in hospitalized patients with LD, although in some cases it is not yet present on the day of admission. L. pneumophila DNA in serum on the day of admission correlates with high CRP levels, but not with the severity of the disease.

Sensitivity of Legionella pneumophila DNA detection in serum samples in relation to disease severity

Sensitivity of Legionella pneumophila DNA detection in serum samples in relation to disease severity

Legionnaires' disease among residents of a long-term care facility: The sentinel event in a community outbreak

A long-term care facility (LTCF) reported an outbreak of Legionnaires' disease (LD) in September 2004. We conducted case finding through enhanced surveillance, medical record review (n = 131), and community surveys (n = 258). We cultured water samples from the LTCF and assayed their outdoor air-intake filters for Legionella DNA. We also investigated a cooling tower, the only nearby outdoor aerosol source. Among 7 confirmed cases, 2 LTCF residents never exited, and 2 community residents never entered the LTCF during the incubation period. Among 63 water and biofilm samples collected from throughout the LTCF, we found no evidence of Legionella colonization, either in the potable water or air-handling systems. Conversely, we isolated a common outbreak-causing strain of Legionella pneumophila serogroup 1 from an industrial cooling tower located 0.4 km from the LTCF and recovered L pneumophila DNA from the LTCF's outdoor air-intake filters, suggesting that aerosolized Legionella from the cooling tower most likely entered the LTCF through the air-intake system or, possibly, through open windows. Residents of LTCFs can acquire LD from community sources. A cluster of LD cases among LTCF residents does not necessarily indicate transmission from within the LTCF.

P1462 Sensitivity of Legionella pneumophila DNA detection in serum samples in relation to disease severity

P1462 Sensitivity of Legionella pneumophila DNA detection in serum samples in relation to disease severity

Evaluation of real-time PCR for the early detection of Legionella pneumophila DNA in serum samples

Legionella pneumonia can be difficult to diagnose. Existing laboratory tests all have shortcomings, especially in the ability to diagnose Legionnaires' disease (LD) at an early stage of the disease in a specimen that is readily obtainable. The aim of this study was to assess the performance of PCR as a rapid diagnostic method and to compare the results of different PCR assays of serum samples from patients with LD. Samples included 151 serum samples from 68 patients with proven LD and 60 serum samples from 36 patients with respiratory tract infections other than Legionella. PCR assays were based on the 5S rRNA gene, 16S rRNA gene and the mip gene. The samples from patients with infections caused by pathogens other than Legionella all tested negative in PCR. Among the patients with proven LD 54.4 % (37/68) tested positive in 5S rRNA PCR, 52.9 % (36/68) in mip gene PCR and 30.9 % (21/68) in 16S rRNA PCR in the first available serum sample. The association between threshold cycle value in 5S PCR positive serum samples (n=49) and C-reactive protein value was determined, and showed a strong negative correlation (Pearson correlation coefficient r=-0.63, P<0.0001). In addition to existing tests for the diagnosis of LD, detection of Legionella DNA in serum could be a useful tool for early diagnosis of LD caused by any Legionella species and serogroup, and has the potential to provide a diagnosis in a time frame that could affect initial infection management.

Detection and quantification of Legionella pneumophila DNA in serum: case reports and review of the literature

Legionella pneumonia can be difficult to diagnose. Existing laboratory tests all have shortcomings, especially the ability to diagnose all Legionella spp. at an early stage. Detection of Legionella DNA in serum can be a valuable tool for the diagnosis of Legionnaires' disease (LD). This report describes two patients with LD diagnosed by PCR using serum samples. In addition, quantification of L. pneumophila DNA using real-time PCR during the course of illness was carried out. The results obtained mirrored both the clinical condition and C-reactive protein values during the course of the illness. Quantification of Legionella DNA in serum using real-time PCR could be a valuable tool to monitor the effects of antimicrobial therapy in patients with LD.

Co-expression and Immunity of &amp;lt;italic&amp;gt;Legionella pneumophila mip&amp;lt;/italic&amp;gt; Gene and Immunoadjuvant &amp;lt;italic&amp;gt;ctxB&amp;lt;/italic&amp;gt; Gene

The mip gene of Legionella pneumophila and the ctxB gene of Vibrio cholerae were amplified by PCR respectively. The amplified cDNA was ligated to the pcDNA3.1(+) vector. The recombinant plasmids pcDNA3.1-mip and pcDNA3.1-ctxB were identified by restriction analysis and PCR, and further confirmed by sequencing analysis. NIH3T3 cells were transfected with pcDNA3.1-mip and pcDNA3.1-ctxB according to the Lipofection method. Transient and stable products of the co-expression of the mip gene and ctxB gene were detected by immunofluorescence and Western blotting. The results showed that NIH3T3 cells were successfully transfected, and that the transiently and stably co-expressed products can be detected in the transfected cells. To detect the humoral and cellular immune response in immunized mice induced by the co-mmunization of the mip and ctxB genes, female BALB/c mice were immunized intramuscularly with pcDNA3.1-mip and pcDNA3.1-ctxB. The results showed that the specific antibody titer and the cytotoxic T-lymphocyte response for pcDNA3.1-mip immunization and co-immunization were increased compared with that of pcDNA3.1(+) immunization. Furthermore, the specific antibody titer and cytotoxic T-lymphocyte response for co-immunization were increased compared with that of pcDNA3.1-mip immunization. Statistical analysis using one-way analysis of variance (ANOVA) showed that there was a significant difference between the groups (P<0.01). The results indicated that the ctxB gene enhanced the humoral and cellular immune response to the mip gene immunization. These findings provide experimental evidence to support the development of the L. pneumophila DNA vaccine.

Detection of Legionella pneumophila DNA in urine and serum samples from patients with pneumonia

albicans strains adhere better to epithelial cells of the oral cavity or the esophagus, but it is accepted that esophageal candidiasis represents a form of invasive candidiasis which requires higher dosages of antifungal drugs compared to OPC [11]. In this patient, replacement of a fluconazole-resistant C. albicans strain by a genetically different fluconazole-sensitive strain was observed; this finally caused further recurrences of OPC, which responded well to intermittent therapy with itraconazole solution. However, it is unclear whether the disappearance of the resistant genotype is due to the temporary increase of the CD4 cell count or because of therapy with itraconazole despite highlevel resistance to azoles in vitro. It may be concluded that serial DNA typing of C. albicans strains, together with antifungal susceptibility testing, may help us to monitor the susceptibility of clinical isolates to azole therapy.

Legionella pneumophila DNA coding for major outer membrane protein; serogroup-1 Philadelphia strain 29 kDa recombinant major outer membrane protein production; DNA sequence; application in diagnosis and recombinant vaccine : Wolf H Germany 4419 294; 7 December 1995

Legionella pneumophila DNA coding for major outer membrane protein; serogroup-1 Philadelphia strain 29 kDa recombinant major outer membrane protein production; DNA sequence; application in diagnosis and recombinant vaccine : Wolf H Germany 4419 294; 7 December 1995