Characterization of a small cryptic plasmid pK50-2 isolated from Lactobacillus reuteri K50.

The complete nucleotide sequence of a cryptic plasmid, pK50-2, from Lactobacillus reuteri K50 had been determined. It consisted of an 1866 bp circular molecule with a G+C content of 35%, from which two putative open reading frames (orfs) could be predicted. Based on sequence similarity, the orf1 was not homologous to any known protein, while the N-terminus of the orf2 shared 56% and 64% identities with RepB proteins of plasmid pAR141 and an unnamed plasmid in L. reuteri strain PA-16, members of the rolling-circle replication (RCR) pMV158 family, respectively. Downstream of orf2, a sequence containing two conserved regions (i.e., bind and nick), possibly involved in the binding and nicking of Rep protein, similar to the dso (double strand origin) of RCR-pMV158 family was also identified. Furthermore, a sequence capable of forming the characteristic secondary structure of ssoT (single-strand origin type T) was subsequently determined upstream of the orf2 gene. Thus, the three elements essential for a RCR plasmid (i.e., dso, sso, and rep gene) were all deducible in the pK50-2. Noteworthy was that a conserved alpha helix-turn-alpha helix (HTH) motif, thus far only seen in theta-type plasmids, was for the first time identified in Rep protein of RCR plasmid, pK50-2. To estimate the pK50-2 could be an expression vector to deliver exogenous antigens, a shuttle vector pK50-S containing both pK50-2 and pUE80 (-) was used to analyze the segregational stability and copy-number, which were shown that pK50-S in L. reuteri DSM 20016 were estimated to be 98%, 77%, and 75% after 36, 72, and 100 generations and about 50 copies per chromosome equivalent by real-time PCR.

Comparison of genospecies and antimicrobial resistance profiles of isolates in the Acinetobacter calcoaceticus-Acinetobacter baumannii complex from various clinical specimens.

This study was conducted to compare the prevalences of antimicrobial resistance profiles of clinical isolates in the Acinetobacter calcoaceticus-Acinetobacter baumannii complex from sterile and nonsterile sites and to further study the relationship of antimicrobial resistance profiles and genospecies by amplified rRNA gene restriction analysis (ARDRA). A total of 1,381 isolates were tested with 12 different antibiotics to show their antimicrobial susceptibility profiles. A total of 205 clinical isolates were further analyzed by ARDRA of the intergenic spacer (ITS) region of the 16S-23S rRNA gene. It was found that the overall percentage of isolates from nonsterile sites (urine, sputum, pus, or catheter tip) that were resistant to the 12 antibiotics tested was significantly higher than that of isolates from sterile sites (cerebrospinal fluid [CSF], ascites fluid, and bloodstream) (46% versus 22%; P < 0.05). After ARDRA, it was found that 97% of the 62 isolates resistant to all antibiotics tested were the A. baumannii genospecies, which was identified in only 31% of the isolates susceptible to all antibiotics tested. More genospecies diversity was identified in the isolates susceptible to all antibiotics tested, including genospecies of 13TU (34%), genotype 3 (29%), and A. calcoaceticus (5%). Furthermore, as 91% (10/11) of the isolates from CSF were susceptible to all antibiotics tested, the A. calcoaceticus-A. baumannii complex isolates with multidrug resistance could be less invasive than the more susceptible isolates. This study also indicated current emergence of carbapenem-, fluoroquinolone-, aminoglycoside-, and cephalosporin-resistant A. calcoaceticus-A. baumannii complex isolates in Taiwan.

NagZ-dependent and NagZ-independent mechanisms for ß-lactamase expression in Stenotrophomonas maltophilia.

ß-N-Acetylglucosaminidase (NagZ), encoded by the nagZ gene, is a critical enzyme for basal-level ampC derepression (ampC expression in the absence of ß-lactam challenge) in ampD and dacB mutants of Pseudomonas aeruginosa. Three mutants with a phenotype of basal-level L1 and L2 ß-lactamase derepression in Stenotrophomonas maltophilia have been reported, including KJΔDI (ampD(I) mutant), KJΔmrcA (mrcA mutant), and KJΔDIΔmrcA (ampD(I) and mrcA double mutant). In this study, nagZ of S. maltophilia was characterized, and its roles in basal-level ß-lactamase derepression, induced ß-lactamase activities, and ß-lactam resistance of KJΔDI, KJΔmrcA, and KJΔDIΔmrcA were evaluated. Expression of the nagZ gene was constitutive and not regulated by AmpR, AmpD(I), AmpN, AmpG, PBP1a, and NagZ. Introduction of ΔnagZ into KJΔDI nearly abolished basal-level derepressed ß-lactamase activity; conversely, introduction of ΔnagZ into KJΔmrcA did not affect it. At least two activator ligands (ALs) are thus considered responsible for ß-lactamase expression in the S. maltophilia system, specifically, the NagZ-dependent (AL1) and NagZ-independent (AL2) ligands responsible for the basal-level derepressed ß-lactamase activities of KJΔDI and KJΔmrcA, respectively. The contributions of AL1 and AL2 to the induced ß-lactamase activities may vary with the types of ß-lactams. nagZ inactivation did not affect aztreonam-, cefoxitin-, and carbenicillin-induced ß-lactamase activities, but it attenuated cefuroxime- and piperacillin-induced ß-lactamase activities. Introduction of ΔnagZ into KJ, KJΔDI, KJΔmrcA, and KJΔDIΔmrcA did not significantly change the MICs of the ß-lactams tested except that the MICs of cefuroxime and piperacillin moderately decreased in strains KJΔZ and KJΔDIΔZ (nagZ mutants).

Characterization of tetracycline resistance lactobacilli isolated from swine intestines at western area of Taiwan.

To investigate the frequency of tetracycline resistance (Tet-R) lactobacilli in pig intestines, a total of 256 pig colons were analyzed and found to contain typical colonies of Tet-R lactic acid bacteria in every sample, ranging from 3.2 × 10(3) to 6.6 × 10(5) CFU/cm(2). From these samples, a total of 159 isolates of Tet-R lactobacilli were obtained and identified as belonging to 11 species, including Lactobacillus reuteri, Lactobacillus amylovorus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus ruminis, Lactobacillus kefiri, Lactobacillus fermentum, Lactobacillus sakei, Lactobacillus coryniformis, Lactobacillus parabuchneri and Lactobacillus letivazi. Based on the EFSA (2008) breakpoints, all isolates, after MIC analysis, were qualified as Tet-R, from which the significant high Tet-R MIC(50) and MIC(90) values indicated an ecological distribution of Tet-R lactobacilli mostly with high resistance potency in pig colons. PCR-detection identified 5 tet genes in these isolates, the most predominant one being tet (W), followed by tet (M), (L), (K), and (Q). Their detection rates were 82.0%, 22.5%, 14.4%, 8.1% and 0.9%, respectively. Noteworthily, isolates of the same species carrying identical tet gene(s) usually had a wide different MIC values. Furthermore, strain-subtyping of these isolates by REP-PCR demonstrated a notable genotypic biodiversity % (average = 62%).

Inactivation of mrcA gene derepresses the basal-level expression of L1 and L2 ß-lactamases in Stenotrophomonas maltophilia.

OBJECTIVES: To characterize the relationship between inactivation of the mrcA gene and ß-lactamase expression and ß-lactams resistance in Stenotrophomonas maltophilia KJ and to investigate the involvement of ampR, ampN-ampG, ampD(I) and creBC in this. METHODS: The mrcA deletion mutant KJΔmrcA was constructed to investigate the role of this putative penicillin-binding protein 1a (PBP1a) in ß-lactamase expression and ß-lactam resistance. The ΔampR, ΔampNG, ΔampDI and ΔcreBC alleles were introduced into KJΔmrcA, and KJΔDIΔBC and KJΔDIΔmrcAΔBC were also constructed for comparison. All the mutants and their corresponding parent strains were assayed for ß-lactamase activities and MICs of ß-lactams. RESULTS: Inactivation of mrcA caused basal L1/L2 ß-lactamase production to increase by ∼100-fold, but made little difference to cefuroxime-induced ß-lactamase activity and the MICs of ß-lactams. The ΔmrcA-derived basal ß-lactamase hyperproduction was ampR and ampN-ampG dependent. Simultaneous inactivation of ampD(I) and mrcA did not augment ß-lactamase production over and above that seen in an ampD(I) mutant alone. Furthermore, we could find no evidence for a role of the creBC two-component regulatory system in ß-lactamase hyperproduction in a ΔampD(I) or ΔmrcA background. CONCLUSIONS: Inactivation of mrcA, predicted to encode PBP1a, causes basal L1/L2 ß-lactamase hyperproduction in S. maltophilia.

Establishment of an arabinose-inducible system in Stenotrophomonas maltophilia.

A pBBad22T-derived conditioned arabinose (Ara)-inducible expression system was evaluated in Stenotrophomonas maltophilia (an opportunistic pathogen and has gained increasing attention as a cause of healthcare-associated infection). S. maltophilia cannot grow well when Ara is the sole available carbon source. The induction kinetic study, optimal inducer concentration determination, and depletion experiment were performed by using a xylE gene fusion construct, pBxylE, to monitor the expression of pBBad22T in S. maltophilia. For induction survey, the expression of catechol 2,3-dioxygenase (C23O), encoded by xylE gene, continuously increases during an 8-h induced course and can be modulated by different inducer concentrations. The applied induction condition of pBBad22T in S. maltophilia is the inducer concentration ranging from 0.1% to 0.5% for an induction time of 4 h. For repression evaluation, the C23O expression is rapidly turned off within 30 min after the removal of Ara. Accordingly, the established Ara-inducible system can provide a convenient tool for the study of S. maltophilia.

SmQnrR, a DeoR-type transcriptional regulator, negatively regulates the expression of Smqnr and SmtcrA in Stenotrophomonas maltophilia.

OBJECTIVES: To characterize the role of SmqnrR in the expression of Smqnr and SmtcrA, and the role of SmtcrA in drug resistance in Stenotrophomonas maltophilia. METHODS: SmqnrR, a DeoR-type regulator gene, is situated between a quinolone resistance gene (Smqnr) and a putative major facilitator superfamily transmembrane transporter gene (SmtcrA). To assess the regulatory role of SmQnrR in the expression of Smqnr and SmtcrA, the transcripts of Smqnr and SmtcrA genes were determined in the wild-type KJ and the SmqnrR isogenic mutant KJΔQnrR. An SmqnrR polar mutant, KJΔQnrRΩ, was constructed to investigate the possibility that SmqnrR and SmtcrA form an operon. The contribution of Smqnr and SmtcrA genes to the intrinsic and acquired resistance of S. maltophilia was evaluated using susceptibility testing. RESULTS: SmQnrR acted as a repressor for the expression of Smqnr and SmtcrA genes. SmqnrR and SmtcrA genes formed an operon, which was negatively autoregulated by SmQnrR. Smqnr and SmtcrA contributed only slightly to intrinsic resistance in S. maltophilia. Nevertheless, overexpression of Smqnr and SmtcrA by inactivating SmqnrR conferred a slight increase in quinolone MICs and a more marked increase in tetracycline MIC. CONCLUSIONS: The SmQnrR protein is a transcriptional repressor for the contiguous Smqnr and SmtcrA genes, and SmQnrR is a negative regulator of SmqnrR-SmtcrA operon expression. Inactivation of SmqnrR contributes to an acquired increase in quinolone and tetracycline MICs for S. maltophilia.

AmpN-AmpG operon is essential for expression of L1 and L2 beta-lactamases in Stenotrophomonas maltophilia.

AmpG is an inner membrane permease which transports products of murein sacculus degradation from the periplasm into the cytosol in Gram-negative bacteria. This process is linked to induction of the chromosomal ampC beta-lactamase gene in some members of the Enterobacteriaceae and in Pseudomonas aeruginosa. In this study, the ampG homologue of Stenotrophomonas maltophilia KJ was analyzed. The ampG homologue and its upstream ampN gene form an operon and are cotranscribed under the control of the promoter P(ampN). Expression from P(ampN) was found to be independent of beta-lactam exposure and ampN and ampG products. A DeltaampN allele exerted a polar effect on the expression of ampG and resulted in a phenotype of null beta-lactamase inducibility. Complementation assays elucidated that an intact ampN-ampG operon is essential for beta-lactamase induction. Consistent with ampG of Escherichia coli, the ampN-ampG operon of S. maltophilia did not exhibit a gene dosage effect on beta-lactamase expression. The AmpG permease of E. coli could complement the beta-lactamase inducibility of ampN or ampG mutants of S. maltophilia, indicating that both species have the same precursor of activator ligand(s) for beta-lactamase induction.

Multiple screening of urolithic organic acids with copper nanoparticle-plated electrode: potential assessment of urolithic risks.

There is yet to be a reliable prediction of urolithiasis. To facilitate early diagnosis, a simple and rapid high performance liquid chromatography method with electrochemical detection using disposable copper-nanoparticle-plated electrodes (Cu(n)-SPE) was developed for multiple detection of creatinine and 4 urolithic organic acids. A total of 206 normal and urolithic human and canine urines and urolith samples were collected for direct analysis of creatinine, cystine, uric acid, oxalic acid, and citric acid without sample cleanup and derivatization processes. Urinary organic acids were separated in 11 min and were devoid of ascorbic acid interference. The detection limits (S/N>3) were at the nanomolar level with linear dynamic ranges spanning 2-3 orders of magnitude. Recoveries in urine ranged from 99.5% for creatinine to 86.5% for citric acid. The analytical variations (RSD) were less than 6.2% in phosphate buffer and 7.7% in urine. Important differences in organic acid levels/profiles between animal species and among normal and urolithic urines/urolith were unveiled and corresponded well (70-90%) with the urolithic risk in a retrospective assessment. The simplicity and reproducibility of this method using disposable Cu(n)-SPE has made routine urine analysis possible and can be of great clinical and diagnostic potential in the screening of urolithiasis and abnormal states related to excess secretion of organic acids and amino acids in humans and animals.

Identification of legionella species by use of an oligonucleotide array.

The genus Legionella contains a diverse group of motile, asaccharolytic, nutritionally fastidious gram-negative rods. Legionella pneumophila is the most important human pathogen, followed by L. micdadei, L. longbeachae, L. dumoffii, and other rare species. Accurate identification of Legionella spp. other than L. pneumophila is difficult because of biochemical inertness and phenotypic identity of different species. The feasibility of using an oligonucleotide array for identification of 18 species of Legionella was evaluated in this study. The method consisted of PCR amplification of the macrophage infectivity potentiator mip gene, followed by hybridization of the digoxigenin-labeled PCR products to a panel of 30 oligonucleotide probes (16- to 24-mers) immobilized on a nylon membrane. A collection of 144 target strains (strains we aimed to identify) and 50 nontarget strains (44 species) were analyzed by the array. Both test sensitivity (144/144 strains) and specificity (50/50 strains) of the array were 100%. The whole procedure for identification of Legionella species by the array can be finished within a working day, starting from isolated colonies. It was concluded that species identification of clinically relevant Legionella spp. by the array method is very reliable and can be used as an accurate alternative to conventional or other molecular methods for identification of Legionella spp.

Pseudomonas aeruginosa exotoxin A-induced hepatotoxicity: an animal model in rats.

Pseudomonas aeruginosa exotoxin A (PEA) has been generally used to induce liver injury in mice for experimental study. No PEA-induced hepatotoxicity study has ever been conducted in rats, although rats are the most common rodents used in toxicologic bioassay and pharmacological evaluation. The present study was conducted in male Wistar rats that were injected (i.v.) with PEA at doses of 0, 10, 20, 30 or 40 microg/kg body weight and evaluated at 12, 24, 36, 48 and 60 hr post-exposure (HPE). Rats exposed to PEA at 40 microg/kg died before 36 HPE, and the mortality was dose and time dependent. Liver injury was noted as increases in serum enzymes, along with alterations of liver histology in the 40 microg/kg group at 12 HPE. TUNEL-positive staining indicative of hepatocyte apoptosis was observed in the 20 microg/kg group at 12 HPE. Significant levels of DNA fragmentation ladder were observed in the 30 microg/kg group starting at 24 HPE. Serum levels of TNF-alpha was increased in the 30 and 40 microg/kg groups at 48 and 24 HPE, respectively. Other cytokines, such as IL-2, IL-6, and IL-10 were also increased at various doses and times. Furthermore, the elevated serum hepatic index levels decreased significantly by dexamethasone pretreatment. In contrast, these markers were exacerbated by co-administration of a non-toxic dose LPS. In overall evaluation, the PEA-induced liver injury can be used as a model for study of hyperimmune-mediated hepatotoxicity.

Co-exposure of lipopolysaccharide and Pseudomonas aeruginosa exotoxin A-induced multiple organ injury in rats.

Pseudomonas aeruginosa Exotoxin A (PEA) induces hepatotoxicity in experimental animals. Lipopolysaccharide (LPS) interacts synergistically with xenotoxics to induce severe organ injury. We examined the combination of non-injurious doses of LPS and sub-hepatotoxic PEA in the induction of multiple organ injury (MOI). Rats treated with 20 or 40 microg/kg LPS plus 10 microg/kg PEA developed severe liver, kidney, and lung injury; elevation of TNF-alpha, IFN-gamma, and IL-2; and high mortality. Depletion of Kupffer cells or T-cells by pretreatment with Gadolinium Chloride or FK506, respectively, attenuated MOI. Thus LPS + PEA acted synergistically on Kupffer and T-cells to induce proinflammatory cytokines contributing to MOI.

Mice protected by oral immunization with Lactobacillus reuteri secreting fusion protein of Escherichia coli enterotoxin subunit protein.

A green fluorescent protein (gfp) gene was ligated to the Lactobacillus reuteri-specific nisin-inducible expression-secretion vector pNIES, generating a pNIES-GFP vector capable of secreting the cloned gene as a GFP-fusion protein with fluorescent activity. To develop this system as a live vehicle carrying the heat-stable enterotoxin (ST) and heat-labile enterotoxin B (LT(B)) of the enterotoxigenic Escherichia coli (ETEC), a recombinant 5'-ST-LT(B)-3' DNA fragment was cloned into pNIES-GFP. The resulting L. reuteri/pNIES-GFP:STLT(B) system was found to possess the capability of adhering to the mice gut, secreting GFP:STLT(B) product at 0.14 and 0.026 pgcell(-1) under induced and noninduced conditions, respectively. Further analysis of the GFP:STLT(B) product confirmed its ganglioside-binding ability, LT(B) antigenicity and relative freedom from the ST-associated toxicity, making it suitable for use as an oral vaccine in mice. Oral inoculation of the L. reuteri/pNIES-GFP:STLT(B) culture in mice elicited significant (P<0.01) serum IgG and mucosal IgA antibodies against the STLT(B) antigen. These immunized mice were subsequently challenged with ETEC and showed full protection against the fluid influx response in the gut. This is the first report of using L. reuteri as a vaccine carrier to induce complete immunologic protection against ETEC.

Prevalence of melioidosis in the Er-Ren River Basin, Taiwan: implications for transmission.

An increase in melioidosis cases compared to other areas in Taiwan was observed in the Er-Ren River Basin, southwestern Taiwan, from November 2001 to August 2006. The objective of this study was to determine the association between the level of exposure to Burkholderia pseudomallei and the incidence rate of melioidosis and to survey the transmission modes of B. pseudomallei in the Er-Ren River Basin. The serosurveillance of melioidosis gave seropositivity rates of 36.6%, 21.6%, and 10.9%, respectively, for residents in regions A, B, and C within the Er-Ren Basin area. Culture and PCR-based detection of B. pseudomallei from soil demonstrated that the geographical distribution of this bacterium was confined to a particular site in region B. The distribution of seropositive titers was significantly associated with the incidence rate of melioidosis (120, 68, or 36 incidence cases per 100,000 population in region A, B, or C in 2005), whereas it did not correlate with the geographical distribution of B. pseudomallei within the soil. A survey of transmission modes showed that residents with seropositivity were linked to factors such as having confronted flooding and having walked barefoot on soil, which are potential risk factors associated with exposure to B. pseudomallei. Our findings indicated that the Er-Ren River Basin in Taiwan has the potential to become a high-prevalence area for melioidosis. This is the first report that documents a high prevalence of melioidosis in an area north of latitude 20 degrees N.

A legionellosis case due to contaminated spa water and confirmed by genomic identification in Taiwan.

Tracing the source of a legionellosis (LG) case revealed that the Legionella pneumophila (LP) strain isolated from patient's sputum shared the same serogroup (SG) and PFGE-type with 4 LP strains obtained from a spa center. With a high LP-contamination rate (81.2%, 13/16) in all of its 16 basins, this spa center was also found to have a multi-genotypic distribution among its 13 LP isolates, which can be categorized into 5 PFGE-types. Despite such a serious contamination in the spa center, which usually had ca. 100 visitors per day, this male patient, bearing LG-risk factors of long-term heavy smoking and alcoholism, was the only case identifiable after an active investigation. To explore the possible reason for this sporadic infection, all 5 PFGE-types of LP isolated were assayed for their presence of two important virulent genes (lvh and rtx A) and were identified as either less-virulent (lvh (+) , rtx A(+)) or non-virulent (lvh (-), rtx A (-)) types. The strong virulent type (lvh (+), rtx A (+)) usually seen in clinical strains elsewhere was not found here. Moreover, the LG-causative type in this infection was the only one to be classified as the less-virulent type, with the presence of lvh gene indicating its relatively more virulent potential than other 4 PFGE-types. Accordingly, mutual interaction between LP's virulent potential and patient's health-status was suggested to be the force directing the opportunistic infection of this sporadic case. This is the first spa-associated infection caused by SG 2 of LP.

Green fluorescent protein is a reliable reporter for screening signal peptides functional in Lactobacillus reuteri.

A signal peptide (SP)-probe vector pNICE-gfpSP, which employed a green fluorescent protein (Gfp) as the SP-selection marker, was constructed for use in Lactobacillus reuteri. This chimerical plasmid allowed cloning and screening DNA fragments with the SP function by direct visualization of the expressed fluorescence activity around cells. Assay of fluorescent intensity in their culture supernatant with spectrofluorometry further enabled quantifying the secretion efficiency of the identified SP fragment.

Construction and characterization of nisin-controlled expression vectors for use in Lactobacillus reuteri.

The Nisin-controlled gene expression (NICE) system, which was discovered in Lactococcus lactis, was adapted to Lactobacillus reuteri by ligating nisA promoter (PnisA) and nisRK DNA fragments into the Escherichia coli-Lb. reuteri shuttle vector pSTE32. This chimerical plasmid (pNICE) was capable of expressing the heterologous amylase gene (amyL) under nisin induction. Optimization of induction factors for this Lb. reuteri/pNICE system, including nisin concentration (viz. 50 ng/ml), growth phase of culture at which nisin be added (viz. at the early exponential phase), and the best time for analyzing the gene product after inoculation (viz. at the 3rd h), allowed the amylase product to be expressed in high amounts, constituting up to about 18% of the total intracellular protein. Furthermore, the signal peptide (SP) of amyL gene (SPamyL) from Bacillus licheniformis was ligated to the downstream of PnisA in pNICE, upgrading this vector to a NICE-secretion (NIES) level, which was then designated pNIES (Sec+, secretion positive). Characterization of pNIES using an amyL-SPDelta gene (amyL gene lacking its SP) as a reporter revealed the 3rd h after induction as the secretion peak of this system, at which the secretion efficiency and the amount of alpha-amylase being secreted into the culture supernatant were estimated to reach 77.6% and 27.75 mg/l. Expression and secretion of AmyL products by pNIES in Lb. reuteri was also confirmed by SDS-PAGE and immunoblotting analysis.

Legionella pneumophila infection in the Taiwan area.

The aim of this study was to explore the epidemiological distribution of legionellosis among pneumonia patients in Taiwan. From January 2001 to December 2003, specimens (i.e., sputum, urine, and serum) from a total of 5097 patients with pneumonia or pneumonia-like disease registered at hospitals in the Taiwan area were analyzed for possible Legionella infection. Following the guideline issued by the Centers for Disease Control and Prevention (CDC) in the United States, a total of 237 pneumonia patients were diagnosed with legionellosis, with an incidence rate among pneumonia patients in this area of 4.7% (237/5097). The paired-serum antibody test was found to be the most effective detection method, followed by urine-antigen detection and the sputum culture method. Analysis of distribution showed that: (1) male and female occurrence rates were 70.9% (168/237) and 29.1% (69/237), respectively; (2) occurrence rates in different age groups, i.e., those aged between 61 and 80 years, those aged between 41 and 60, and those aged between 21 and 40 were 50.2% (119/237), 26.2% (62/237), and 12.2% (29/237), respectively; (3) autumn was the peak season for infection, followed by winter, summer, and spring, sequentially. This is the first study in Taiwan to have followed the three-method guideline issued by the CDC and it is the second report in Taiwan involving the investigation of a large series of pneumonia patients for legionellosis detection.