Distribution of Sequence-Based Types of Legionella pneumophila Serogroup 1 Strains Isolated from Cooling Towers,Hot Springs,and Potable Water Systems in China

Legionella pneumophila serogroup 1 causes Legionnaires'' disease. Water systems contaminated with Legionella are the implicated sources of Legionnaires'' disease. This study analyzed L. pneumophila serogroup 1 strains in China using sequence-based typing. Strains were isolated from cooling towers (n = 96), hot springs (n = 42), and potable water systems (n = 26). Isolates from cooling towers, hot springs, and potable water systems were divided into 25 sequence types (STs; index of discrimination [IOD], 0.711), 19 STs (IOD, 0.934), and 3 STs (IOD, 0.151), respectively. The genetic variation among the potable water isolates was lower than that among cooling tower and hot spring isolates. ST1 was the predominant type, accounting for 49.4% of analyzed strains (n = 81), followed by ST154. With the exception of two strains, all potable water isolates (92.3%) belonged to ST1. In contrast, 53.1% (51/96) and only 14.3% (6/42) of cooling tower and hot spring, respectively, isolates belonged to ST1. There were differences in the distributions of clone groups among the water sources. The comparisons among L. pneumophila strains isolated in China, Japan, and South Korea revealed that similar clones (ST1 complex and ST154 complex) exist in these countries. In conclusion, in China, STs had several unique allelic profiles, and ST1 was the most prevalent sequence type of environmental L. pneumophila serogroup 1 isolates, similar to its prevalence in Japan and South Korea.     

Geographical and Temporal Structures of Legionella pneumophila Sequence Types in Comunitat Valenciana (Spain), 1998 to 2013

Legionella pneumophila is an accidental human pathogen associated with aerosol formation in water-related sources. High recombination rates make Legionella populations genetically diverse, and nearly 2,000 different sequence types (STs) have been described to date for this environmental pathogen. The spatial distribution of STs is extremely heterogeneous, with some variants being present worldwide and others being detected at only a local scale. Similarly, some STs have been associated with disease outbreaks, such as ST578 or ST23. Spain is among the European countries with the highest incidences of reported legionellosis cases, and specifically, Comunitat Valenciana (CV) is the second most affected area in the country. In this work, we aimed at studying the overall diversity of Legionella pneumophila populations found in the period from 1998 to 2013 in 79 localities encompassing 23 regions within CV. To do so, we performed sequence-based typing (SBT) on 1,088 L. pneumophila strains detected in the area from both environmental and clinical sources. A comparison with the genetic structuring detected in a global data set that included 20 European and 7 non-European countries was performed. Our results reveal a level of diversity in CV that can be considered representative of the diversity found in other countries worldwide.     

High Prevalence,Genetic Diversity and Intracellular Growth Ability of Legionella in Hot Spring Environments

Background

Legionella is the causative agent of Legionnaires'' disease, and hot springs are a major source of outbreaks of this disease. It is important from a public health perspective to survey hot spring environments for the presence of Legionella.

Methods

Prospective surveillance of the extent of Legionella pollution was conducted at three hot spring recreational areas in Beijing, China in 2011. Pulsed-field gel electrophoresis (PFGE) and sequence-based typing (SBT) were used to describe the genetic polymorphism of isolates. The intracellular growth ability of the isolates was determined by interacting with J774 cells and plating the dilutions onto BCYE agar plates.

Results

Overall, 51.9% of spring water samples showed Legionella-positive, and their concentrations ranged from 1 CFU/liter to 2,218 CFU/liter. The positive rates of Legionella were significantly associated with a free chlorine concentration of ≥0.2 mg/L, urea concentration of ≥0.05 mg/L, total microbial counts of ≥400 CFU/ml and total coliform of ≥3 MPN/L (p<0.01). The Legionella concentrations were significantly associated with sample temperature, pH, total microbial counts and total coliform (p<0.01). Legionella pneumophila was the most frequently isolated species (98.9%), and the isolated serogroups included serogroups 3 (25.3%), 6 (23.4%), 5 (19.2%), 1 (18.5%), 2 (10.2%), 8 (0.4%), 10 (0.8%), 9 (1.9%) and 12 (0.4%). Two hundred and twenty-eight isolates were analyzed by PFGE and 62 different patterns were obtained. Fifty-seven L. pneumophila isolates were selected for SBT analysis and divided into 35 different sequence types with 5 main clonal groups. All the 57 isolates had high intracellular growth ability.

Conclusions

Our results demonstrated high prevalence and genetic polymorphism of Legionella in springs in Beijing, China, and the SBT and intracellular growth assay results suggested that the Legionella isolates of hot spring environments were pathogenic. Improved control and prevention strategies are urgently needed.     

Distribution of Legionella Species from Environmental Water Sources of Public Facilities and Genetic Diversity of L. pneumophila Serogroup 1 in South Korea

A total of 560 Legionella species were isolated from environmental water sources from public facilities from June to September 2008 throughout South Korea. The distribution of Legionella isolates was investigated according to geographical region, facility type, and sample type. The genetic diversity of 104 isolates of Legionella pneumophila serogroup 1 (sg 1) was analyzed by sequence-based typing (SBT). L. pneumophila was distributed broadly throughout Korea, accounting for 85.0% of the isolates, and L. pneumophila sg 1 predominated in all of the public facilities except for the springs. Legionella anisa and Legionella bozemanii predominated among non-L. pneumophila species (48.1% and 21.0%, respectively). The second most dominant strain differed depending on the facility type: L. anisa was the second most dominant strain in the buildings (10.8%), L. pneumophila sg 5 in public baths (21.6%), L. pneumophila sg 6 in factories (12.0%), and L. pneumophila sg 7 in hospitals (13.1%). In the SBT analysis, 104 L. pneumophila sg 1 isolates were differentiated into 26 sequence types (STs) and categorized into 3 clonal groups (CGs) and 10 singleton STs via the eBURST V3 program. ST1, a potential founder of major CG1, was commonly distributed (48.1%). The dominant ST in hot water was ST-K1 (7, 12, 17, 3, 35, 11, 11), which was designated in this study (36.1%). The second most dominant strain differed depending on the type of facility from which the samples were obtained. The unique allelic profile of ST-K1, obtained from hot water, was not found in the European Working Group for Legionella Infections (EWGLI) SBT database.Legionella species, ubiquitous Gram-negative bacteria, are found in a variety of artificial water systems, natural freshwaters, and soils. Currently, the Legionella genus includes 52 species and more than 70 different serogroups, and more than 20 species have been proven to be causative agents of Legionnaires'' disease (LD). The species Legionella pneumophila accounts for approximately 90% of confirmed cases of legionellosis, and L. pneumophila serogroup 1 (sg 1) has been recognized as the most important agent in this regard, as that specific strain was initially implicated as the pathogen causative of LD in 1977 (15; http://www.bacterio.cict.fr/l/legionellaceae.html). The other non-L. pneumophila sg 1 strains, sg 2 to 15, accounted for 7.4% of cases, and Legionella longbeachae (3.9%) and Legionella bozemanii (2.4%) have also been associated with the pathogen of LD. In particular, L. longbeachae has been recognized as accounting for 30.4% of community-acquired Legionella isolates in Australia and New Zealand (53).The most common transmission mechanism of legionellosis is the inhalation of aerosols from the water systems of artificial facilities, including large buildings, hotels, hospitals, public baths, spas, or decorative fountains contaminated by Legionella species (1). Therefore, hot water and water from cooling towers have been perceived as sources of infection in cases of community-acquired, nosocomially acquired, or travel-associated LD (15, 26, 31, 37, 38, 39, 41, 43). Thus, it is important from a public health perspective to continually survey environmental water systems for the presence of Legionella species (2, 34, 35). In particular, hot-water systems used as public baths, such as springs, spas, or tubs, have become a popular means of recreation in a lot of countries, including South Korea. The contamination of hot-water systems has gradually become recognized as an important risk factor all over the world (4, 12, 18, 23, 42, 50), as sources of legionellosis have been detected increasingly since 1982 (52) and many cases of nosocomially acquired (32, 51) and community-acquired (6, 7, 48) LD have been detected in Legionella-contaminated hot-water systems or hot springs.In South Korea, several cases of nosocomial infection and community-acquired pneumonia have occasionally been reported (9, 45) since the first recognized outbreak in South Korea in 1984, which was associated with Legionella gormanii (27). Since 2006, the Korean National Infectious Disease Surveillance (NIDS) program (http://dis.cdc.go.kr/) has reported an average of 20 cases of LD per year (29). In South Korea, surveys of Legionella acquired from environmental water in public facilities such as hot springs and public baths has been gradually enhanced since 2007. An annual training program for the detection of Legionella species from environmental water systems and clinical specimens is currently conducted for the personnel of 16 Provincial Institute of Health and Environment locations (PIHEs) throughout South Korea. Recently, the rate of detection of environmental Legionella bacteria has been gradually increasing (8.1% in 2006, 9.4% in 2007, and 10.3% in 2008).The principal objectives of this study were to assess the current distribution of Legionella species from environmental water sources from public facilities such as buildings, hotels, public baths, springs, hospitals, or factories throughout South Korea. Additionally, the molecular typing of L. pneumophila sg 1 isolates was conducted using sequence-based typing (SBT) to assess the genetic diversity among the isolates.     

Development of a DNA Microarray Method for Detection and Identification of All 15 Distinct O-Antigen Forms of Legionella pneumophila

Legionella is ubiquitous in many environments. At least 50 species and 70 serogroups of the Gram-negative bacterium have been identified. Of the 50 species, 20 are pathogenic, and Legionella pneumophila is responsible for the great majority (approximately 90%) of the Legionnaires'' disease cases that occur. Furthermore, of the 15 L. pneumophila serogroups identified, O1 alone causes more than 84% of the Legionnaires'' disease cases that occur worldwide. Rapid and reliable assays for the detection and identification of L. pneumophila in water, environmental, and clinical samples are in great demand. L. pneumophila bacteria are traditionally identified by their O antigens by immunological methods. We have recently developed an O serogroup-specific DNA microarray for the detection of all 15 distinct O-antigen forms of L. pneumophila, including serogroups O1 to O15. A total of 35 strains were used to verify the specificity of the microarray, including 15 L. pneumophila O-antigen standard reference strains and seven L. pneumophila clinical isolates as target strains, seven reference strains of other non-pneumophila Legionella species as closely related strains, and six non-Legionella bacterial species as nonrelated strains. The detection sensitivity was 1 ng of genomic DNA or 0.4 CFU/ml in water samples with filter enrichment and plate culturing. This study demonstrated that the microarray allows specific, sensitive, and reproducible detection of L. pneumophila serogroups. To the best of our knowledge, this is the first report of a microarray serotyping method for all 15 distinct O-antigen forms of L. pneumophila.     

Presence and Persistence of Legionella spp. in Groundwater

Groundwater samples (111) from six different boreholes located in two geographical areas were examined for the presence of legionellae over a 7-year period. The number of Legionella isolates detected was generally low. The colonization of the aquifers was not uniform, and the persistence of Legionella was independent of the hydraulic pumps and the plumbing system present in the borehole. A total of 374 isolates identified by fatty acid methyl ester analysis belonged to Legionella pneumophila, L. oakridgensis, L. sainthelensi, and L. londiniensis. In area 1, L. oakridgensis constituted the major population detected, exhibiting only one random amplified polymorphic DNA (RAPD)-PCR profile. L. sainthelensi strains were less frequently isolated and also displayed a single RAPD profile, while L. pneumophila was only sporadically detected. In contrast, L. pneumophila comprised the vast majority of the isolates in area 2 and exhibited six distinct RAPD patterns, indicating the presence of different genetic groups; three L. londiniensis RAPD types were also detected. Two of the L. pneumophila and one of the L. londiniensis RAPD types were persistent in this environment for at least 12 years. The genetic structure of L. pneumophila groundwater populations, inferred from rpoB and dotA gene sequences, was peculiar, since the majority of the isolates were allied in a discrete group different from the lineages containing most of the type and reference strains of the three subspecies of L. pneumophila. Furthermore, gene exchange events related to the dotA allele could be envisioned.     

Validation of IRS PCR,a molecular typing method,for the study of the diversity and population dynamics of Legionella in industrial cooling circuits

Legionella bacteria are ubiquitous in aquatic environments. Members of the species Legionella pneumophila are responsible for more than 98% of cases of Legionnaires' disease in France. Our objective was to validate a molecular typing method called infrequent restriction site PCR (IRS PCR), applied to the study of the ecology of Legionella and to compare this method with reference typing methods, pulsed‐field gel electrophoresis (PFGE) and sequence‐based Typing (SBT). PFGE and SBT are considered as gold methods for the epidemiological typing of Leg. pneumophila strains. However, these methods are not suitable to an ecological monitoring of Legionella in natural environments where a large number of strains has to be typed. Validation of IRS PCR method was performed by the identification of 45 Leg. pneumophila isolates from cooling circuits of thermal power plants by IRS PCR, PFGE and SBT. The parameters of each method were measured and compared to evaluate the effectiveness of IRS PCR. The results of this study showed that IRS PCR has a discriminating power similar or better than that of the reference methods and thus that, by its speed and low cost represents an appropriate tool for the study of the ecology of Legionella in cooling circuits.     

Distribution of monoclonal antibody subgroups and sequence-based types among Legionella pneumophila serogroup 1 isolates derived from cooling tower water, bathwater, and soil in Japan

Legionella pneumophila serogroup (SG) 1 is the most frequent cause of legionellosis. This study analyzed environmental isolates of L. pneumophila SG 1 in Japan using monoclonal antibody (MAb) typing and sequence-based typing (SBT). Samples were analyzed from bathwater (BW; n = 50), cooling tower water (CT; n = 50), and soil (SO; n = 35). The distribution of MAb types varied by source, with the most prevalent types being Bellingham (42%), Oxford (72%), and OLDA (51%) in BW, CT, and SO, respectively. The ratios of MAb 3/1 positive isolates were 26, 2, and 14% from BW, CT, and SO, respectively. The environmental isolates from BW, CT, and SO were divided into 34 sequence types (STs; index of discrimination [IOD] = 0.973), 8 STs (IOD = 0.448), and 11 STs (IOD = 0.879), respectively. Genetic variation among CT isolates was smaller than seen in BW and SO. ST1 accounted for 74% of the CT isolates. The only common STs between (i) BW and CT, (ii) BW and SO, and (iii) CT and SO were ST1, ST129, and ST48, respectively, suggesting that each environment constitutes an independent habitat.     

Listeria monocytogenes Associated with New Zealand Seafood Production and Clinical Cases: Unique Sequence Types,Truncated InlA,and Attenuated Invasiveness

Listeriosis is caused by the food-borne pathogen Listeria monocytogenes, which can be found in seafood and processing plants. To evaluate the risk to human health associated with seafood production in New Zealand, multi-virulence-locus sequence typing (MVLST) was used to define the sequence types (STs) of 31 L. monocytogenes isolates collected from seafood-processing plants, 15 from processed foods, and 6 from human listeriosis cases. The STs of these isolates were then compared with those from a collection of seafood isolates and epidemic strains from overseas. A total of 17 STs from New Zealand clustered into two lineages: seafood-related isolates in lineages I and II and all human isolates in lineage II. None of the New Zealand STs matched previously described STs from other countries. Isolates (belonging to ST01-N and ST03-N) from mussels and their processing environments, however, were identical to those of sporadic listeriosis cases in New Zealand. ST03-N isolates (16 from mussel-processing environments, 2 from humans, and 1 from a mussel) contained an inlA premature stop codon (PMSC) mutation. Therefore, the levels of invasiveness of 22 isolates from ST03-N and the three other common STs were compared using human intestinal epithelial Caco-2 cell lines. STs carrying inlA PMSCs, including ST03-N isolates associated with clinical cases, had a low invasion phenotype. The close relatedness of some clinical and environmental strains, as revealed by identical MVLST profiles, suggests that local and persistent environmental strains in seafood-processing environments pose a potential health risk. Furthermore, a PMSC in inlA does not appear to give L. monocytogenes a noninvasive profile.     

Evaluation of the Duopath Legionella Lateral Flow Assay for Identification of Legionella pneumophila and Legionella Species Culture Isolates

Duopath Legionella (Merck KGaA, Darmstadt, Germany) is a new immunochromatographic assay for the simultaneous identification of cultured L. pneumophila and Legionella species other than L. pneumophila. In tests of 89 L. pneumophila strains and 87 Legionella strains other than L. pneumophila representing 41 different species, Duopath and a widely used latex agglutination assay detected L. pneumophila with 100% and 98% accuracy, respectively, whereas the percentages differed significantly for other Legionella spp. (93% versus 37% [P < 0.001]). Since many countries’ regulations require the identification of Legionella spp. in water and environmental samples, the use of Duopath Legionella to comply with those regulations could contribute to significantly fewer false-negative results.     

Specific Detection of Legionella pneumophila: Construction of a New 16S rRNA-Targeted Oligonucleotide Probe

Based on comparative sequence analysis, we have designed an oligonucleotide probe complementary to a region of 16S rRNA of Legionella pneumophila which allows the differentiation of L. pneumophila from other Legionella species without cultivation. The specificity of the new probe, LEGPNE1, was tested by in situ hybridization to a total of four serogroups of six strains of L. pneumophila, five different Legionella spp. and three nonlegionella species as reference strains. Furthermore, L. pneumophila cells could be easily distinguished from Legionella micdadei and Pseudomonas aeruginosa cells by using in situ hybridization with probes LEGPNE1, LEG705, and EUB338 after infection of the protozoan Acanthamoeba castellanii.     

Molecular Typing of Pathogenic Leptospira Serogroup Icterohaemorrhagiae Strains Circulating in China during the Past 50 Years

Background

Leptospirosis is one of the most important neglected tropical infectious diseases worldwide. Icterohaemorrhagiae has been throughout recent history, and still is, the predominant serogroup of this pathogen in China. However, very little in detail is known about the serovars or genotypes of this serogroup.

Methodology/Principal Findings

In this study, 120 epidemic strains from five geographically diverse regions in China collected over a 50 year period (1958~2008), and 8 international reference strains characterized by 16S rRNA sequencing and MLST analysis. 115, 11 and 2 strains were identified as L. interrogans, L. borgpetersenii, and L. kirschneri, respectively. 17 different STs were identified including 69 ST1 strains, 18 ST17, 18 ST128, 9 ST143 and 2 ST209. The remaining 12 strains belonged to 12 different STs. eBURST analysis demonstrated that, among the clonal complexes isolated (CCs), CC1 accounted for 73.3% (88/120) strains representing three STs: ST1, ST128 and ST98. ST1 was the most likely ancestral strain of this CC, followed by singleton CC17 (17/120) and CC143 (11/120). Further analysis of adding 116 serogroup Icterohaemorrhagiae strains in the MLST database and studies previously described using global eBURST analysis and MST dendrogram revealed relatively similar ST clustering patterns with five main CCs and 8 singletons among these 244 strains. CC17 was found to be the most prevalent clone of pathogenic Leptospira circulating worldwide. This is the first time, to our knowledge, that ST1 and ST17 strains were distributed among 4 distinct serovars, indicating a highly complicated relationship between serovars and STs.

Conclusions/Significance

Our studies demonstrated a high level of genetic diversity in the serogroup Icterohaemorrhagiae strains. Distinct from ST17 or ST37 circulating elsewhere, ST1 included in CC1, has over the past 50 years or so, proven to be the most prevalent ST of pathogenic leptospires isolated in China. Moreover, the complicated relationship between STs and serovars indicates an urgent need to develop an improved scheme for Leptospira serotyping.     

Targeting Species-Specific Low-Affinity 16S rRNA Binding Sites by Using Peptide Nucleic Acids for Detection of Legionellae in Biofilms

Using fluorescence in situ hybridization to detect bacterial groups has several inherent limitations. DNA probes are generally used, targeting sites on the 16S rRNA. However, much of the 16S rRNA is highly conserved, with variable regions often located in inaccessible areas where secondary structures can restrict probe access. Here, we describe the use of peptide nucleic acid (PNA) probes as a superior alternative to DNA probes, especially when used for environmental samples. A complex bacterial genus (Legionella) was studied, and two probes were designed, one to detect all species and one targeted to Legionella pneumophila. These probes were developed from existing sequences and are targeted to low-binding-affinity sites on the 16S rRNA. In total, 47 strains of Legionella were tested. In all cases, the Legionella spp. PNA probe labeled cells strongly but did not bind to any non-Legionella species. Likewise, the specific L. pneumophila PNA probe labeled only strains of L. pneumophila. By contrast, the equivalent DNA probes performed poorly. To assess the applicability of this method for use on environmental samples, drinking-water biofilms were spiked with a known concentration of L. pneumophila bacteria. Quantifications of the L. pneumophila bacteria were compared using PNA hybridization and standard culture methods. The culture method quantified only 10% of the number of L. pneumophila bacteria found by PNA hybridization. This illustrates the value of this method for use on complex environmental samples, especially where cells may be in a viable but noncultivable state.     

Molecular Determination of Infection Source of a Sporadic Legionella Pneumonia Case Associated with a Hot Spring Bath

To determine the infection source of a sporadic Legionella pneumonia case associated with a hot spring bath, we used five molecular methods, including repetitive element polymerase chain reaction (rep-PCR), arbitrarily primed PCR (AP-PCR), ribotyping, restriction endonuclease analysis (REA), and macrorestriction endonuclease analysis (MREA) by pulsed-field gel electrophoresis. L. pneumophila serogroup (SG) 3 strain EY 3702, isolated from an intratracheal specimen of a 71-year-old Japanese female who developed pneumonia after nearly drowning in a hot spring spa bath, produced rep-PCR and AP-PCR fingerprints identical to those of L. pneumophila SG 3 strains EY 3768 and EY 3769 isolated from the bath water. Four epidemiologically unrelated L. pneumophila SG 3 strains showed different rep-PCR or AP-PCR fingerprints from those of the three EY strains (EY 3702, 3768, and 3769). The three EY strains were also genotypically indistinguishable by ribotyping with EcoRI and PstI, by REA with EcoBI or HindIII, and by MREA with NotI. Based on these results, we identified the bath water of the hot spring spa as the source of infection of this patient, even though the viable number of the organisms in the bath water was low (3 CFU/100 ml) when determined 27 days after her nearly drowning.     

Detection of Legionella pneumophila serogroup 1 antigen in respiratory samples using an immunochromatographic membrane test

The immunochromatographic membrane test (ICT) efficacy of Legionella antigen detection (Binax Now Legionella®) was evaluated using respiratory samples, including bronchial washings (44 cases) and sputum (128 cases), from suspected Legionella pneumonia patients. The ICT results using respiratory samples agreed well with isolation of L. pneumophila SG1 and ICT using urines.     

A New Oligonucleotide Microarray for Detection of Pathogenic and Non-Pathogenic Legionella spp.

Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS) of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii) and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp.     

Comparative Genomics Reveal That Host-Innate Immune Responses Influence the Clinical Prevalence of Legionella pneumophila Serogroups

Legionella pneumophila is the primary etiologic agent of legionellosis, a potentially fatal respiratory illness. Amongst the sixteen described L. pneumophila serogroups, a majority of the clinical infections diagnosed using standard methods are serogroup 1 (Sg1). This high clinical prevalence of Sg1 is hypothesized to be linked to environmental specific advantages and/or to increased virulence of strains belonging to Sg1. The genetic determinants for this prevalence remain unknown primarily due to the limited genomic information available for non-Sg1 clinical strains. Through a systematic attempt to culture Legionella from patient respiratory samples, we have previously reported that 34% of all culture confirmed legionellosis cases in Ontario (n = 351) are caused by non-Sg1 Legionella. Phylogenetic analysis combining multiple-locus variable number tandem repeat analysis and sequence based typing profiles of all non-Sg1 identified that L. pneumophila clinical strains (n = 73) belonging to the two most prevalent molecular types were Sg6. We conducted whole genome sequencing of two strains representative of these sequence types and one distant neighbour. Comparative genomics of the three L. pneumophila Sg6 genomes reported here with published L. pneumophila serogroup 1 genomes identified genetic differences in the O-antigen biosynthetic cluster. Comparative optical mapping analysis between Sg6 and Sg1 further corroborated this finding. We confirmed an altered O-antigen profile of Sg6, and tested its possible effects on growth and replication in in vitro biological models and experimental murine infections. Our data indicates that while clinical Sg1 might not be better suited than Sg6 in colonizing environmental niches, increased bloodstream dissemination through resistance to the alternative pathway of complement mediated killing in the human host may explain its higher prevalence.     

LAMP-based method for a rapid identification of Legionella spp. and Legionella pneumophila

Legionella pneumophila is accounted for more than 80% of Legionella infection. However it is difficult to discriminate between the L. pneumophila and non-L. pneumophila species rapidly. In order to detect the Legionella spp. and distinguish L. pneumophila from Legionella spp., a real-time loop-mediated isothermal amplification (LAMP) platform that targets a specific sequence of the 16S rRNA gene was developed. LS-LAMP amplifies the fragment of the 16S rRNA gene to detect all species of Legionella genus. A specific sequence appears at the 16S rRNA gene of L. pneumophila, while non-L. pneumophila strains have a variable sequence in this site, which can be recognized by the primer of LP-LAMP. In the present study, 61 reference strains were used for the method verification. We found that the specificity was 100% for both LS-LAMP and LP-LAMP, and the sensitivity of LAMP assay for L. pneumophila detection was between 52 and 5.2 copies per reaction. In the environmental water samples detection, a total of 107 water samples were identified by the method. The culture and serological test were used as reference methods. The specificity of LS-LAMP and LP-LAMP for the samples detection were 91.59% (98/107) and 93.33% (56/60), respectively. The sensitivity of LS-LAMP and LP-LAMP were 100% (51/51) and 100% (18/18). The results suggest that real-time LAMP, as a new assay, provides a specific and sensitive method for rapid detection and differentiation of Legionella spp. and L. pneumophila and should be utilized to test environmental water samples for increased rates of detection.     

The Legionella pneumophila Collagen-Like Protein Mediates Sedimentation,Autoaggregation, and Pathogen-Phagocyte Interactions

Although only partially understood, multicellular behavior is relatively common in bacterial pathogens. Bacterial aggregates can resist various host defenses and colonize their environment more efficiently than planktonic cells. For the waterborne pathogen Legionella pneumophila, little is known about the roles of autoaggregation or the parameters which allow cell-cell interactions to occur. Here, we determined the endogenous and exogenous factors sufficient to allow autoaggregation to take place in L. pneumophila. We show that isolates from Legionella species which do not produce the Legionella collagen-like protein (Lcl) are deficient in autoaggregation. Targeted deletion of the Lcl-encoding gene (lpg2644) and the addition of Lcl ligands impair the autoaggregation of L. pneumophila. In addition, Lcl-induced autoaggregation requires divalent cations. Escherichia coli producing surface-exposed Lcl is able to autoaggregate and shows increased biofilm production. We also demonstrate that L. pneumophila infection of Acanthamoeba castellanii and Hartmanella vermiformis is potentiated under conditions which promote Lcl dependent autoaggregation. Overall, this study shows that L. pneumophila is capable of autoaggregating in a process that is mediated by Lcl in a divalent-cation-dependent manner. It also reveals that Lcl potentiates the ability of L. pneumophila to come in contact, attach, and infect amoebae.     

Genetic Variability of Vancomycin-Resistant Enterococcus faecium and Enterococcus faecalis Isolates from Humans,Chickens, and Pigs in Malaysia

Vancomycin-resistant enterococci (VRE) have been reported to be present in humans, chickens, and pigs in Malaysia. In the present study, representative samples of VRE isolated from these populations were examined for similarities and differences by using the multilocus sequence typing (MLST) method. Housekeeping genes of Enterococcus faecium (n = 14) and Enterococcus faecalis (n = 11) isolates were sequenced and analyzed using the MLST databases eBURST and goeBURST. We found five sequence types (STs) of E. faecium and six STs of E. faecalis existing in Malaysia. Enterococcus faecium isolates belonging to ST203, ST17, ST55, ST79, and ST29 were identified, and E. faecium ST203 was the most common among humans. The MLST profiles of E. faecium from humans in this study were similar to the globally reported nosocomial-related strain lineage belonging to clonal complex 17 (CC17). Isolates from chickens and pigs have few similarities to those from humans, except for one isolate from a chicken, which was identified as ST203. E. faecalis isolates were more diverse and were identified as ST4, ST6, ST87, ST108, ST274, and ST244, which were grouped as specific to the three hosts. E. faecalis, belonging to the high-risk CC2 and CC87, were detected among isolates from humans. In conclusion, even though one isolate from a chicken was found clonal to that of humans, the MLST analysis of E. faecium and E. faecalis supports the findings of others who suggest VRE to be predominantly host specific and that clinically important strains are found mainly among humans. The infrequent detection of a human VRE clone in a chicken may in fact suggest a reverse transmission of VRE from humans to animals.