Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6759-y
ENVIRONMENTAL BIOTECHNOLOGY
Improved PCR assay for the species-specific identification and quantitation of Legionella pneumophila in water Min Seok Cho 1,2 & Tae-Young Ahn 2 & Kiseong Joh 3 & Eui Seok Lee 4 & Dong Suk Park 1
Received: 25 March 2015 / Revised: 2 June 2015 / Accepted: 5 June 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Legionellosis outbreak is a major global health care problem. However, current Legionella risk assessments may be compromised by uncertainties in Legionella detection methods, infectious dose, and strain infectivity. These limitations may place public health at significant risk, leading to significant monetary losses in health care. However, there are still unmet needs for its rapid identification and monitoring of legionellae in water systems. Therefore, in the present study, a primer set was designed based on a LysR-type transcriptional regulator (LTTR) family protein gene of Legionella pneumophila subsp. pneumophila str. Philadelphia 1 because it was found that this gene is structurally diverse among species through BLAST searches. The specificity of the primer set was evaluated using genomic DNA from 6 strains of L. pneumophila, 5 type strains of other related Legionella species, and other 29 reference pathogenic bacteria. The primer set used in the PCR assay amplified a 2 6 4 - b p p r o d u c t f o r o n l y t a rg e t e d si x s t r a i n s o f L. pneumophila. The assay was also able to detect at least 1.39×103 copies/μl of cloned amplified target DNA using purified DNA or 7.4×100 colony-forming unit per reaction
* Dong Suk Park [email protected] 1
National Academy of Agricultural Science, Rural Development Administration, Jeonju 560-500, Republic of Korea
2
Department of Microbiology, Dankook University, Cheonan 330-714, Republic of Korea
3
Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Wangsan, Yongin, Gyeonggi 449-791, Republic of Korea
4
Department of Oral and Maxillofacial Surgery, Korea University Guro Hospital, Seoul 152-703, Republic of Korea
when using calibrated cell suspension. In addition, the sensitivity and specificity of this assay were confirmed by successful detection of Legionella pneumophila in environmental water samples. Keywords Legionella pneumophila . LysR transcriptional regulator . Detection . Real-time PCR
Introduction Legionellae are facultative intracellular Gram-negative bacteria that may cause Legionnaires’ disease (legionellosis) as well as Pontiac fever, which is a mild flu-like lung infection (Costa et al. 2005). In particular, Legionella pneumophila is the most common pathogenic species, accounting for more than 90 % of legionellosis cases although the genus Legionella comprises 59 species/subspecies, and at least 24 species have been found to cause human pneumonia (DSMZ. 2013; Gomez-Valero et al. 2009; Rizzardi et al. 2015; WHO. 2007). Therefore, the risk assessment for L. pneumophila is especially significant for public health officials and managers responsible for maintenance of water supply systems and cooling towers within industrial or public buildings. However, there are limited data regarding human dose response for L. pneumophila, and the concentration of Legionella required to result in an outbreak is unknown (Whiley et al. 2014). In general, it has been known that the clinical symptoms of Legionnaires disease are difficult to distinguish from those of other forms of pneumonia. Thus, a rapid and accurate method for the detection of Legionella at the species level is important to identify nosocomial infections or community outbreaks and to locate the source of infection. In addition, the development of more rapid and sensitive methods for the detection and
Appl Microbiol Biotechnol
quantification of Legionella cells without cultivation is a matter of increasing importance for water monitoring (DelgadoViscogliosi et al. 2005). Currently, diagnosis of L. pneumophila infection is based on isolation of the pathogen, followed by biochemical or serological tests (Ditommaso et al. 2010; Whiley et al. 2011). Such tests require at least 10 days before final confirmation is obtained. The sensitivity of the enzyme-linked immunosorbent assay (around 106 CFU/ml) is adequate only for detection in symptomatic samples. In some cases, cross-reactions with L. pneumophila and other bacteria are also observed (Füchslin et al. 2010; Keserue et al. 2012). In general, molecular assays based on 5S rRNA, 16S rRNA, the 23S–5S ribosomal RNA intergenic spacer regions (ISRs), RNA polymerase β-subunit (rpoB), macrophage infectivity potentiator (mip), defective organelle trafficking (dotA), and gyrase subunit B (gyrB) genes are frequently used for the detection of L. pneumophila strains, but there have been critical defects in the diagnosis and identification of all isolates of L. pneumophila (Ko et al. 2003; Merault et al. 2011; Nazarian et al. 2008; Stolhaug and Bergh 2006; Wilson et al. 2003; Yáñez et al. 2005; Zhou et al. 2011). In particular, it was determined that the allelic diversity of dotA is predominantly found in L. pneumophila isolates from natural environments, suggesting that niche-specific selection pressures have been operating on this gene (Costa et al. 2010). Actually, the high level of dotA allelic diversity may reflect fitness variation in the persistence of those strains in distinct environmental niches and/or tropism to various protozoan hosts (Costa et al. 2010). Consequently, detection specificity, which depends on both the uniqueness of the sequence to the pathogen of interest, as well as the specific binding of the primers or probes to the target, is significant to the efficacy of any PCR detection method (Lang et al. 2010). In recent, the enormous and increasing number of available bacterial whole-genome sequences in databases, together with employed bioinformatics tools, offers potential for more reliable, fast, and cost-effective methods for bacterial identification in a wide range of samples in coming years (Albuquerque et al. 2009; Segerman et al. 2011). Thus, we have investigated the LysR-type transcriptional regulator (LTTR) family genes to develop a specific primer set for identifying L. pneumophila. The nucleotide sequences of the genes were assessed for specificity and variety among Legionella spp. through BLASTn searches. We designed species-specific primers based on the LysR-type transcriptional regulator (LTTR) family gene of str. Philadelphia 1 and found that the primer set showed high specificity for detecting the targeted pathogen in water samples. In the present study, we have established a reliable and efficient procedure for quantitative detection of L. pneumophila in water samples by SYBR Green Direct polymerase chain reaction (PCR). Serial dilutions of
L. pneumophila were used to determine the detection limits of the single assay, and DNA extraction and real-time PCR were performed within 2 h. The specificity of the assay was also tested against other strains of L. pneumophila and related bacterial species, and environmental samples. These results revealed that this real-time PCR-based method can be particularly useful for analysis of environmental water samples in which the densities of L. pneumophila are very low and provides a platform capable of rapid screening of samples from drinking water, wells, lakes, rivers, cooling towers, and recreational swimming pools for trace levels of L. pneumophila.
Materials and methods Bacterial strains, culture media, and growth conditions All bacterial strains were obtained from the American Type Culture Collection (ATCC) in the USA, the Belgian Coordinated Collections of Micro Organisms (BCCM/LMG) in Belgium, the Korean Agricultural Culture Collection (KACC) and the Korean Culture Center of Microorganisms (KCCM) and the National Culture Collection for Pathogens (NCCP) in the Republic of Korea, and the National Collection of Type Cultures (NCTC) in the UK. All bacterial strains used in this study are listed in Table 2. The suitable culture media and optimal incubation conditions were in accordance with the Handbook of Microbiological Media (Atlas. 2004). L. pneumophila strains were grown on buffered charcoal yeast extract agar (activated charcoal, 2.0 %; yeast extract, 10 %; agar, 1.3 %) at 35 °C in 2.5 % CO2 for 1 day; E. coli, Shigella strains, Salmonella strains, and other bacterial strains were grown on nutrient agar (peptone, 0.5 %; NaCl, 0.5 %; yeast extract, 0.2 %; beef extract, 0.1 %; agar, 1.5 %) at 37 °C for 1 day. DNA preparation The genomic DNA of all microorganisms was prepared using a DNeasy Tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. To measure the quantity and purity of genomic DNA, NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) was used. The genomic DNA was eluted in a final volume of 25 ng/μl at TE buffer and stored at −20 °C until use. Genomic analysis and primer design The genomes of Legionella pneumophila and other Legionella species were compared using the bioinformatics sequence analysis technology. All L. pneumophila and other bacterial genomes used in this study were downloaded from the NCBI bacterial genome database (ftp://ftp.ncbi.nlm.nih.
Appl Microbiol Biotechnol
gov/genomes/bacteria/). Candidate genes were selected by the modified method of the Chen and Lang (Chen et al. 2010; Lang et al. 2010). To specifically detect L. pneumophila, the LysR-type transcriptional regulator (LTTR) family gene of L. pneumophila subsp. pneumophila str. Philadelphia 1 was selected. A specific primer set based on the LysR-type transcriptional regulator (LTTR) family gene of L. pneumophila subsp. pneumophila str. Philadelphia 1 was designed. The primer set was designed using PrimerSelect of Lasergene (version 7.2.1; DNASTAR Inc., USA). The nucleotide sequences of primer set were assessed for specificity through NCBI-BLAST searches (http://blast.ncbi.nlm.nih.gov). The primer set, LP264F (5′-ACAGCTTGAAGAGGAGTTAG-3′) and LP264R (5′-ACAAGCTCTACTTCAATGCC-3′), for PCR reaction was synthesized by Bioneer Corporation (Daejeon, Korea), respectively.
Real-time PCR reactions The real-time PCR assay was performed in the CFX96 realtime PCR system (Bio-Rad Laboratories, Inc., USA). The mixture consisted of 10-μl iQTM SYBR® Green Supermix (Bio-Rad laboratories, Inc., USA), 5 pM each of LP264F/R primers, and 5 ng purified DNA from each sample in total volume of 20 μl with distilled water. The cycling program was adjusted at 95 °C for 30 s, 45 cycles of 95 °C for 5 s, and 47 °C for 30 s. The data collection was performed during each annealing step. Following amplification, during melting curve analysis, the temperature increases to obtain productspecific melting temperature from 65 to 95 °C with an increment of 0.5 °C. Determination of cycle threshold (Ct) and data analysis were set automatically by the CFX ManagerTM Software system (Version 1.6, Bio-Rad laboratories, Inc., USA).
Conventional PCR The PCR amplifications were performed with the above primers (0.2-μM final concentration) and GoTaq® Flexi DNA polymerase (1× buffer, 4.0 mM MgCl2, 0.2 mM of each dNTP, GoTaq® DNA polymerase 1.25 U final concentration; Promega, Madison, Wisconsin, USA) in a final volume of 50 μl according to the manufacturer’s instructions, and 25 ng genomic DNA from a given bacterial strain. Amplifications were done in a PTC-225 thermocycler (MJ Research, Watertown, MA, USA) with the following cycling conditions: initial denaturation of 5 min at 95 °C; then 35 cycles of 1 min at 95 °C, 30 s at 47 °C, and 1 min at 72 °C; and final extension of 7 min at 72 °C. After PCR reaction, each amplified PCR product (10 μl) was loaded onto a 1.5 % (w/v) agarose gel and subjected to electrophoresis for 1 h at 100 V in 1× TAE buffer, stained with ethidium bromide (EtBr), and visualized on an UV transilluminator and imaged using a VersaDoc 1000 gel imaging system (Bio-Rad laboratories, Inc., USA). Construction of target plasmid DNA The amplification product (264 bp) was then cloned into pGEM-T Easy vector (Promega, Madison, Wisconsin, USA) and amplified with a combination of genespecific and vector-targeted primers using PCR with GoTaq® DNA po lymera se (Prome ga, Madiso n, Wisconsin, USA). The recombinant plasmid DNA was transformed into the E. coli DH5α and purified by QIAprep Spin Miniprep Kit (QIAGEN Inc., Valencia, CA). The concentration and quality (ratio A260/A280) of the extracted DNA were determined with a NanoDrop ND-1000 spectrophotometer prior to calculating the concentration.
LOQ and LOD of real-time PCR assay for L. pneumophila The limit of quantification (LOQ) and limit of detection (LOD) of the real-time PCR assay were determined using 10-fold dilutes of cloned-plasmid DNA, genomic DNA, and bacterial cell suspension of L. pneumophila KCCM 41777 in a 20-μl reaction mixture containing 10-μl iQ™ SYBR® Green Supermix (Bio-Rad laboratories, Inc.) and 5 pmol each of LP264F/R primers. This LOQ and LOD were reproducible with 10-fold serial dilution and real-time PCR testing being done in triplicate (Table 3). The copy number of the cloned plasmid DNA was calculated with the following equation:(Whelan et al. 2003) 23 Copies=μl ¼ 6:022 10 ðcopy=molÞ amount ðgÞ = ½length ðbpÞ 660 ðg=mol=bpÞ The efficiency (E) was calculated using the equation E = 10− − 1, where the slope referred to the log-linear portion of the standard curve (Bustin et al. 2009). 1/slope
Environmental water samples for SYBR Green qPCR A total of 144 samples were collected at 12 sites of four major rivers in South Korea from 2010 to 2012: Han River (37° 35′ 25″ N, 127° 09′ 52″ E), (37° 32′ 57″ N, 127° 06′ 37″ E), (38° 31′ 30″ N, 127° 17′ 13″ E), Geum River (36° 22′ 56.7″ N, 127° 24′ 37.7″ E), (36° 27′ 28.9″ N, 127° 23′ 59.4″ E), (36° 28′ 9.6″ N, 127° 28′ 4.8″ E), Nakdong River (36° 8′ 34.5″ N, 128° 22′ 34.7″ E), (36° 6′ 29.8″ N, 128° 24′ 5.1″ E), (36° 8′ 0.3″ N, 128° 27′ 1.6″ E), and Soyang River (38° 02′ 26″ N,
Appl Microbiol Biotechnol
128° 09′ 11″ E), (38° 03′ 01″ N, 128° 09′ 56″ E), (38° 04′ 39″ N, 128° 11′ 17″ E) (Table 4). Environmental water sampling conditions were in accordance with Handbooks for Water-Resources Investigations (U.S. Geological Survey USGS 2003). All collected samples were stored at 4 °C upon arrival at the laboratory and analyzed within 24 h after collection. To survey for the presence of L. pneumophila in the water samples, 500 ml of each water sample was filtered through 0.45-μm pore size membrane filter (Pall Corporation, New York, USA) and using a vacuum pump. The genomic DNA was extracted from water concentrates using a Fast DNA spin kit for soil (MP Biomedicals, OH, USA). The genomic DNA concentrations of the water samples were measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc). All genomic DNA (5 ng) was used in the real-time PCR assays as described above.
Results Genomic analysis and in silico specificity test of designed primer set In recent, a large number of available bacterial sequences in databases and bioinformatics tools could result in more reliable, fast, and cost-effective methods for bacterial identification in a broad range of samples (Albuquerque et al. 2009). Also, there is an unmet need for specific markers for early detection and identification of L. pneumophila. To identify candidate gene specific to L. pneumophila, we compared microbial genomes by bioinformatics tools. In particular, we found overlap gene region across analysis between putative candidate genes for the same traits in the genomes for L. pneumophila 12 strains (str. Philadelphia 1, str. Paris, str. Lens, str. Corby, str. 2300/99 Alcoy, str. ATCC 43290, str. Hextuple_2q, str. Hextuple_3a, str. HL06041035, str. L o r r a i n e , s t r. L P E 5 0 9 , a n d s t r. T h u n d e r B a y ) , L. longbeachae str. NSW150, and L. oakridgensis str. ATCC 33761 (Table 1). Among them, a species-specific primer set was designed based on the sequences of the LysR-type transcriptional regulator (LTTR) family protein gene of L. pneumophila subsp. pneumophila str. Philadelphia 1 (GenBank accession No. AAU26322 region: 247787..248671). The specificity of the primer sequences was tested in silico by searching for similar sequences in bacterial genomes using the BLAST and e-PCR analysis (http:// www.ncbi.nlm.nih.gov/). The BLASTn searches showed similarity to the transcriptional regulator, LysR family sequences [identity = 78 %, score = 679 bits (752), and expected=3e-23] from Tatlockia micdadei genome assembly LMI, chromosome: I and putative transcriptional regulator,
LysR family sequences [identity = 74 %, score = 545 bits (604), and expected = 3e-151] from L. longbeachae NSW150. The BLASTx results with the predicted protein sequence revealed the closest similarity to the LysR family transcriptional regulator sequence [identity=89 %, score= 159 bits (402), and expected=1e-45] from L. longbeachae NSW150. Conventional PCR and real-time PCR The species-specific molecular marker, the transcriptional regulator LysR family gene sequence, was amplified by using the LP264F/R primers (Fig. 1). The specificity validation test on 11 strains of represented 6 Legionella species and 29 nonLegionella strains revealed that 6 strains of L. pneumophila gave a consistent positive result regardless of the existence of other species of the pathogen. As expected, a 264-bp DNA fragment was amplified with conventional PCR (Fig. 1), and the amplification plot and a unique dissociation melting peak at 81.50 °C were observed with the real-time PCR assay (Table 2). LOD and LOQ sensitivity test by real-time PCR We used a real-time PCR analysis of L. pneumophila subsp. pneumophila str. Philadephia 1 (KCCM 41777) to generate a standard curve by plotting the mean Ct (n=3) versus the logarithmic concentration of the cloned plasmid DNA, the genomic DNA, and density of the bacterial cell suspension (range, 1.39 ×109 to 1.39 ×103 copies/μl, 5 × 100 to 5 × 10−6 ng/μl, and 7.4×106 to 7.4×100 colony-forming unit (CFU) per reaction, respectively) (Fig. 2a and Table 3). The LOQ assay showed a good linear response and a high correlation coefficient (cloned plasmid DNA, R2 =1.000; genomic DNA, R2 = 0.999; bacterial cell suspension, R2 = 0.996). A standard curve analysis of the linear part of the slope resulted in a coefficient of −3.627, which generated a PCR efficiency of 88.7 % and y-intercept value=35.014 (Fig. 2b). The analysis of the melting temperature and melting peaks of L. pneumophila KCCM 41777 produced from a real-time PCR reaction was shown a reproducible melting temperature of 81.50 °C and specific peaks (Fig. 2c). The standard curve results of the genomic DNA and the bacterial cell suspension showed that there was a linear correlation between the Ct (n=3) values and the concentrations of input the genomic DNA (R2 =0.999, slope=−3.350) and the bacterial cell suspension (R2 =0.996, slope=−3.531). They also revealed that the detection limit of the real-time PCR was 5 fg/μl (fg per μl reaction mix) and 7.4×100 CFU/ml (CFU per ml reaction mix) of L. pneumophila KCCM 41777, respectively. The real-time PCR assay of L. pneumophila KCCM 41777 presented excellent quantification characteristics and accurate detection.
Appl Microbiol Biotechnol Table 1
In silico specificity test of the target gene by BLASTn searches
Organism/Name
Results dotAa
mipb
gyrBc
This studyd
Legionella pneumophila subsp. pneumophila str. Philadelphia 1 Legionella pneumophila str. Paris Legionella pneumophila str. Lens Legionella pneumophila str. Corby Legionella pneumophila 2300/99 Alcoy Legionella pneumophila subsp. pneumophila ATCC 43290 Legionella pneumophila subsp. pneumophila str. Hextuple_2q Legionella pneumophila subsp. pneumophila str. Hextuple_3a Legionella pneumophila subsp. pneumophila str. HL06041035 Legionella pneumophila subsp. pneumophila str. Lorraine Legionella pneumophila subsp. pneumophila LPE509 Legionella pneumophila subsp. pneumophila str. Thunder Bay Legionella pneumophila str. G2762 Legionella pneumophila str. MDC1252 (serogroup 2)
N.D + + N.D + N.D N.D N.D + + N.D N.D -
+e + + + + + + + + + + + -
+ + + + + + + + + + + + -
+ + + + + + + + + + + + -
Legionella pneumophila str. MDC1223 (serogroup 2) Legionella sp. CD-2 Legionella sp. CD-3 Legionella sp. CD-4 Legionella worsleiensis Legionella fairfieldensis Tatlockia micdadei isolate Pavia 16 Legionella longbeachae NSW150 Legionella oakridgensis ATCC 33761
-
+ + + + + + -
-
-
a
dotA (GenBank accession No. AY720956, Yáñez et al. 2005)
b
mip (GenBank accession No. 52628139, Nazarian et al. 2008)
c
gyrB (GenBank accession No. JF718229, Zhou et al. 2011)
d
LysR-type transcriptional regulator (LTTR) family protein gene (GenBank accession No. AAU26322, This study)
e
+, detected; -, not detected; N.D not determined
Specific detection of Legionella pneumophila in environmental water samples A total of 144 environmental water samples were examined by real-time PCR for the presence of L. pneumophila.
Fig. 1 Specific PCR amplification of a gene fragment of Legionella pneumophila using the LP264F/R primer set. Lane M, size marker (1 kb DNA plus ladder; Gibco BRL); lanes 1-40re described in Table 2; lane 41 distilled water
Location of the sampled environmental water used and the results detected in environmental water samples from major four rivers of Korea are described in Table 4. The minimum detection range of L. pneumophila in environmental water samples was 7.4×103 to 7.4×104 CFU/ml.
Appl Microbiol Biotechnol Table 2
Bacterial strains used in the PCR specificity test
No.
Bacterial strains
Source
This studya
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Legionella pneumophila subsp. pneumophila (serogroup 1) Legionella pneumophila subsp. pneumophila (serogroup 2) Legionella pneumophila subsp. pneumophila (serogroup 2) Legionella pneumophila subsp. pneumophila (serogroup 8) Legionella pneumophila subsp. fraseri (serogroup 5) Legionella pneumophila (serogroup 7) Legionella jordanis Legionella oakridgensis Legionella maceachernii Legionella erythra Legionella rubrilucens Shigella sonnei Shigella flexneri Shigella boydii Shigella dysenteriae Escherichia coli O1:K1:H7 Escherichia coli O157:H7 (Non-toxigenic)
KCCM 41777T ATCC 33154 ATCC 33155 KCCM 41784 KCCM 41782 KCCM 41783 NCCP 11013T NCCP 11031T NCCP 11137T NCCP 11138T NCCP 11139T KCCM 40949T KCCM 40948T KCCM 41649T NCTC 9952 LMG 2092T LMG 21756
+b + + + + + -
18 19 20 21 22 23 24 25 26 27 28 29 30
Salmonella enterica subsp. enterica serovar Typhimurium Salmonella enterica subsp. enterica serovar Typhimurium Salmonella enterica subsp. enterica serovar Vichow Salmonella enterica subsp. enterica serovar Paratyphi A Salmonella enterica subsp. enterica serovar Paratyphi B Salmonella enterica subsp. enterica serovar Typhi Salmonella enterica subsp. enterica serovar Choleraesuis Salmonella enterica subsp. enterica serovar Enteritidis Salmonella enterica subsp. arizonae Salmonella enterica subsp. diarizonae Salmonella enterica subsp. houtenae Salmonella enterica subsp. indica Salmonella bongori
ATCC 43971T ATCC 13311 ATCC 51955 ATCC 9150 ATCC 8759 ATCC 33459 ATCC 7001 ATCC 13076 ATCC 13314 ATCC 43973 ATCC 43974 ATCC 43976 ATCC 43975T
-
31 32 33 34 35 36
Campylobacter jejuni subsp. jejuni Campylobacter coli Helicobacter pylori Enterobacter aerogenes Citrobacter freundii Enterobacter aerogenes
LMG 8841 LMG 6440T LMG 19449T KACC 13732T KACC 11404T KACC 13732T
-
37 38 39 40
Serratia marcescens Serratia odorifera Straphylococcus capitis subsp. capitis Stenotrophomonas maltophilia
ATCC 990 KACC 12324T KACC 13242T KACC 10244
-
T
ATCC American Type Culture Collection, United States, KCCM Korean Culture Center of Microorganisms, Republic of Korea, LMG The Belgian Coordinated Collections of Microorganisms (BCCMTM ), Belgium, NCCP National Culture Collection for Pathogens, Republic of Korea, NCTC National Collection of Type Cultures, United Kingdom, KACC Korean Agricultural Culture Collection, Republic of Korea T
type strain
a
Experiments to assess the specificity of real-time PCR assays
b
+, detected; -, not detected
Appl Microbiol Biotechnol
Discussion
Fig. 2 Specificity, melting peak, and standard curve of the LP264F/R primer set with SYBR Green qPCR. a Fluorescence intensity as a function of the concentration of the template. For each assay, a series of 10fold dilutions of cloned DNA (range, 1.39×109 to 1.39×103 copies/μl) was used as the template for PCR (1-7, sample dilutions; 8, no-template control). b Standard curve derived from the amplification plot. c Melting peak analysis (1-7, sample dilutions; 8, no template control). The negative first derivative of the relative fluorescence units (–d(RFU)/dT) is plotted as a function of the temperature. The melting temperature is 81.50 °C. The high peak indicates the amplified product; the low peak is the notemplate control
In current, over 90 % of legionellosis cases are caused by infection of L. pneumophila (Marston et al. 1997; Muder and Yu 2002; Yu et al. 2002). Until now, many studies have been performed to develop sensitive and specific technologies to improve the bacterial pathogen detection and ensure successful control measures (Bielaszewska et al. 2011; Müller et al. 2007). However, despite recent advances in molecular and immunological methods, the official and internationally accepted detection method for Legionella spp. in water samples (ISO 11371) is still based on cultivation. This method has major disadvantages such as a long assay time of 10 days and the detection of cultivable cells only, including extremely slow growth rate strains (Bedrina et al. 2013). Therefore, direct detection of L. pneumophila DNA in water samples is a challenging alternative, and much effort has been put into developing PCR method and, more recently, real-time PCR assays. Previous different molecular targets for diagnosis of L. pneumophila have been employed such as mip gene, 5S ribosomal DNA, 16S ribosomal DNA, the 23S-5S spacer, dotA, and gyrB gene. However, it has been reported that the 5S and 16S rRNA genes are such wellconserved regions that it is difficult to differentially detect L. pneumophila and other Legionella species on a real-time basis without subsequent tests, or that there is a risk of amplification of the DNAs of other than Legionella species (Herpers et al. 2003; Ko et al. 2003; Merault et al. 2011; Nazarian et al. 2008; Stolhaug and Bergh 2006; Wilson et al. 2003). In addition, through in silico screen with BLAST engines, it was also confirmed that mip gene is highly conserved within Legionella, whereas dotA is extremely variable between L. pneumophila strains, with an average level of nucleotide diversity four times greater than that of mip (Bumbaugh et al. 2002; Costa et al. 2010) (Table 1). Accordingly, these molecular probes may place public health at significant risk, leading to significant monetary losses in health care. Consequently, as seen in previous studies, development of specific diagnostic tools is even now facing the difficulty in identifying unique and distinguishing features among the target microorganisms. In recent, as a consequence of the rapid development of technology in the area of high-throughput sequencing, the availability of complete Legionella genome sequences presents a great opportunity for improving the existing molecular diagnostic tools by identifying new targets for more sensitive and specific detection. Despite the existence of genomic information for
Appl Microbiol Biotechnol Table 3 Mean Ct end-point fluorescence of 10-fold serial dilutions of Legionella pneumophila cloned-plasmid DNA, genomic DNA and a cell suspension determined with a real-time PCR assay Cloned DNA
Genomic DNA
Cell suspension
Plasmid copies/μl 1.39×109 1.39×108 1.39×107
Ct±SD (n = 3) 13.18±0.09 16.93±0.28 20.55±0.10
Weight/μl reaction mix 5 ng 500 pg 50 pg
Ct±SD (n = 3) 15.51±0.21 18.37±0.15 21.61±0.10
CFU/ml reaction mix 7.4×106 7.4×105 7.4×104
Ct±SD (n = 3) 15.91±0.11 19.32±0.13 22.89±0.09
1.39×106 1.39×105 1.39×104 1.39×103
24.19±0.27 27.75±0.21 31.26±0.06 35.08±0.22
5 pg 500 fg 50 fg 5 fg
25.24±0.15 28.60±0.29 32.21±0.10 35.22±0.18
7.4×103 7.4×102 7.4×101 7.4×100
26.38±0.01 29.90±0.38 33.48±0.15 37.13±1.26
the most abundant type of transcriptional regulator in prokaryote. The conservation of LTTRs within the genomes of extremely diverse bacteria means that they have evolved a regulatory role over genes with similarly diverse functions, whose products can be involved in metabolism, cell division, quorum sensing, virulence, motility, nitrogen fixation, oxidative stress responses, toxin production, attachment, and secretion (Henikoff et al. 1988; Maddocks and Oyston. 2008). In conclusion, this newly developed real-time PCR assay is detecting L. pneumophila with high specificity, sensitivity, and without any false-positive signals from other Legionella species from pure cultures, or DNA mixtures extracted from natural fresh water. This real-time PCR assay might be a useful method for the detection and quantitation of L. pneumophila in water for quality management purposes.
Legionella spp. for many years, the majority of molecular diagnostic tools still rely on 16S rRNA gene sequences. Therefore, in the present study, a computational genomics pipeline was used to compare sequenced genomes of Legionella spp. and to rapidly identify unique regions for development of highly specific diagnostic markers (Lang et al. 2010). We selected the species-specific sequence region and developed a primer set for the species-specific detection and quantitation of L. pneumophila. The species-specific primers were obtained from a LysR-type transcriptional regulator (LTTR) family protein gene of L. pneumophila subsp. pneumophila str. Philadelphia 1 (GenBank accession No. AAU26322 region: 247787..248671). It has been reported that the LysR family of transcriptional regulators represents
Table 4
SYBR Green quantitative PCR assay for specific detection of Legionella pneumophila at four major rivers in South Korea
Location of water samples
Coordinates 2010
Han River
Geum River
Nakdong River
Soyang River
a
2012
Latitude
Longitude
Ia
IIb
IIIc
IVd
I
II
III
IV
I
II
III
IV
37° 35′ 25″ N 37° 32′ 57″ N 37° 31′ 30″ N 36° 22′ 56.7″ N 36° 27′ 28.9″ N 36° 28′ 9.6″ N 36° 8′ 34.5″ N 36° 6′ 29.8″ N 36° 8′ 0.3″ N 38° 02′ 26″ N
127° 09′ 52″ E 127° 06′ 37″ E 127° 17′ 13″ E 127° 24′ 37.7″ E 127° 23′ 59.4″ E 127° 28′ 4.8″ E 128° 22′ 34.7″ E 128° 24′ 5.1″ E 128° 27′ 1.6″ E 128° 09′ 11″ E
-
-
-
+ -
+ + + -
-
+ + +
+
+ -
+ + + -
+ + +
+ -
38° 03′ 01″ N 38° 04′ 39″ N
128° 09′ 56″ E
-
-
+
-
-
-
-
-
-
-
+
-
128° 11′ 17″ E
+
-
-
-
-
-
-
-
-
-
-
-
I, April; a II, June; c III, August; d IV, October
+, detected; -, not detected
2011
Appl Microbiol Biotechnol Acknowledgments This study was funded by the National Academy of Agricultural Science, Rural Development Administration, Republic of Korea (Research Program for Agricultural Science and Technology Development; Project No. PJ010831). Conflict of interest The authors declare that they have no competing interests. Ethics statement This article does not contain any studies with human participants or animals performed by any of the authors.
References Albuquerque P, Mendes MV, Santos CL, Moradas-Ferreira P, Tavares F (2009) DNA signature-based approaches for bacterial detection and identification. Sci Total Environ 407:3641–3651 Atlas RM (2004) Handbook of microbiological media, 3rd edition. CRC Press Bedrina B, Macian S, Solis I, Fernandez-Lafuente R, Baldrich E, Rodriguez G (2013) Fast immunosensing technique to detect Legionella pneumophila in different natural and anthropogenic environments: comparative and collaborative trials. BMC Microbiol 13:88. doi:10.1186/1471-2180-13-88 Bielaszewska M, Mellmann A, Zhang W, Köck R, Fruth A, Bauwens A, Peters G, Karch H (2011) Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study. Lancet Infect Dis 11: 671–676 Bumbaugh AC, McGraw EA, Page KL, Selander RK, Whittam TS (2002) Sequence polymorphism of dotA and mip alleles mediating invasion and intracellular replication of Legionella pneumophila. Curr Microbiol 44(5):314–322 Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesomele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622 Chen J, Zhang L, Paoli GC, Shi C, Tu SI, Shi X (2010) A real-time PCR method for the detection ofSalmonella entericafrom food using a target sequence identified by comparative genomic analysis. Int J Food Microbiol 137:168–174 Costa J, Tiago I, Costa MS, Verissimo A (2005) Presence and persistence of Legionella spp. in groundwater.Appl. Environ Microbiol 71(2): 663–671 Costa J, Tiago I, Da Costa MS, Veríssimo A (2010) Molecular evolution of Legionella pneumophila dotA gene, the contribution of natural environmental strains. Environ Microbiol 12(10):2711–2729 Delgado-Viscogliosi P, Simonart T, Parent V, Marchand G, Dobbelaere M, Pierlot E, Pierzo V, Menard-Szczebara F, Gaudard-Ferveur E, Delabre K, Delattre JM (2005) Rapid method for enumeration of viable Legionella pneumophila and other Legionella spp. in water. Appl Environ Microbiol 71:4086–4096 Ditommaso S, Giacomuzzi M, Gentile M, Zotti CM (2010) Evaluation of the usefulness of a new direct immunofluorescence assay (ScanVITLegionella) for monitoring hospital water systems contaminated with Legionella spp. Lett Appl Microbiol 50:341–346 DSMZ (2013) Prokaryotic Nomenclature Up-to-Date - Legionella. [WWW document]. URL http://www.dsmz.de/microorganisms/ pnu/bacterial_nomenclature_info_mm.php?genus= Legionella&show_genus_info=1 Füchslin HP, Kötzsch S, Keserue HA, Egli T (2010) Rapid and quantitative detection of Legionella pneumophila applying immunomagnetic separation and flow cytometry. Cytometry A 77(3):264–274
Gomez-Valero L, Rusniok C, Buchrieser C (2009) Legionella pneumophila: population genetics, phylogeny and genomics. Infect Genet Evol 9:727–739 Henikoff S, Haughn GW, Calvo JM, Wallace JC (1988) A large family of bacterial activator proteins. Proc Natl Acad Sci U S A 85:6602– 6606 Herpers BL, de Jongh BM, van der Zwaluw K, van Hannen EJ (2003) Real-time PCR assay targets the 23S-5S spacer for direct detection and differentiation of Legionella spp. and Legionella pneumophila. J Clin Microbiol 41(10):4815–4816 Keserue HA, Baumgartner A, Felleisen R, Egli T (2012) Rapid detection of total and viable Legionella pneumophila in tap water by immunomagnetic separation, double fluorescent staining and flow cytometry. Microb Biotechnol 5(6):753–763 Ko KS, Hong SK, Lee HK, Park MY, Kook YH (2003) Molecular evolution of the dotA gene in Legionella pneumophila. J Bacteriol 185: 6269–6277 Lang JM, Hamilton JP, Diaz MGQ, Van Sluys MA, Burgos MRG, Vera Cruz CM, Buell CR, Tisserat NA, Leach JE (2010) Genomics-based diagnostic marker development for Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola. Plant Dis 94:311–319 Maddocks SE, Oyston PCF (2008) Structure and function of the LysRtype transcriptional regulator (LTTR) family proteins. Microbiology 154:3609–3623 Marston BJ, Plouffe JF, File TM Jr, Hackman BA, Salstrom SJ, Lipman HB, Kolczak MS, Breiman RF (1997) Incidence of communityacquired pneumonia requiring hospitalization. Results of a population-based active surveillance study in Ohio. The community-based pneumonia incidence study group. Arch Intern Med 157(15):1709–1718 Merault N, Rusniok C, Jarraud S, Gomez-Valero L, Cazalet C, Marin M, Brachet E, Aegerter P, Gaillard JL, Etienne J, Herrmann JL, DELPH-I Study Group, Lawrence C, Buchrieser C (2011) Specific real-time PCR for simultaneous detection and identification of Legionella pneumophila serogroup 1 in water and clinical samples. Appl Environ Microbiol 77(5):1708–1717 Muder RR, Yu VL (2002) Infection due to Legionella species other than L. pneumophila. Clin Infect Dis 35(8):990–998 Müller D, Greune L, Heusipp G, Karch H, Fruth A, Tschäpe H, Schmidt MA (2007) Identification of unconventional intestinal pathogenic Escherichia coli isolates expressing intermediate virulence factor profiles by using a novel single-step multiplex PCR. Appl Environ Microbiol 73:3380–3390 Nazarian EJ, Bopp DJ, Saylors A, Limberger RJ, Musser KA (2008) Design and implementation of a protocol for the detection of Legionella in clinical and environmental samples. Diagn Microbiol Infect Dis 62(2):125–132 Rizzardi K, Winiecka-Krusnell J, Ramliden M, Alm E, Andersson S, Byfors S (2015) Legionella norrlandica sp. nov., isolated from the biopurification systems of wood processing plants. Int J Syst Evol Microbiol 65(Pt 2):598–603 Segerman B, De Medici D, Ehling Schulz M, Fach P, Fenicia L, Fricker M, Wielinga P, Van Rotterdam B, Knutsson R (2011) Bioinformatic tools for using whole genome sequencing as a rapid high resolution diagnostic typing tool when tracing bioterror organisms in the food and feed chain. Int J Food Microbiol 145(Suppl 1):S167–176 Stolhaug A, Bergh K (2006) Identification and differentiation of Legionella pneumophila and Legionella spp. with real-time PCR targeting the 16S rRNA gene and species identification by mip sequencing. Appl Environ Microbiol 72:6394–6398 U.S. Geological Survey (USGS) (2003) National Field Manual for the Collection of Water-Quality Data, Book 9. Handbooks for WaterResources Investigations, USGS, Washington, DC Whelan JA, Russel NB, Whelan MA (2003) A method for the absolute quantification of cDNA using real time PCR. J Immunol Meth 278: 261–269
Appl Microbiol Biotechnol Whiley H, Taylor M, Bentham RH (2011) Detection of Legionella species in potting mixes using fluorescent in situ hybridisation (FISH). J Microbiol Methods 86:304–309 Whiley H, Keegan A, Fallowfield H, Ross K (2014) Uncertainties associated with assessing the public health risk from Legionella. Front Microbiol 24(5):501 WHO (2007) Guide to Ship Sanitation, 2nd edn. WHO, Geneva Wilson DA, Yen-Lieberman B, Reischl U, Gordon SM, Procop GW (2003) Detection of Legionella pneumophila by real-time PCR for the mip gene. J Clin Microbiol 41:3327–3330 Yáñez MA, Carrasco-Serrano C, Barberá VM, Catalán V (2005) Quantitative detection of Legionella pneumophila in water samples
by immunomagnetic purification and real-time PCR amplification of the dotA gene. Appl Environ Microbiol 71(7):3433–3441 Yu VL, Plouffe JF, Pastoris MC, Stout JE, Schousboe M, Widmer A, Summersgill J, File T, Heath CM, Paterson DL, Chereshsky A (2002) Distribution of Legionella species and serogroups isolated by culture in patients with sporadic community-acquired legionellosis: an international collaborative survey. J Infect Dis 186(1):127–128 Zhou G, Cao B, Dou Y, Liu Y, Feng L, Wang L (2011) PCR methods for the rapid detection and identification of four pathogenic Legionella spp. and two Legionella pneumophila subspecies based on the gene amplification of gyrB. Appl Microbiol Biotechnol 91(3):777–787
ENVIRONMENTAL BIOTECHNOLOGY
Improved PCR assay for the species-specific identification and quantitation of Legionella pneumophila in water Min Seok Cho 1,2 & Tae-Young Ahn 2 & Kiseong Joh 3 & Eui Seok Lee 4 & Dong Suk Park 1
Received: 25 March 2015 / Revised: 2 June 2015 / Accepted: 5 June 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Legionellosis outbreak is a major global health care problem. However, current Legionella risk assessments may be compromised by uncertainties in Legionella detection methods, infectious dose, and strain infectivity. These limitations may place public health at significant risk, leading to significant monetary losses in health care. However, there are still unmet needs for its rapid identification and monitoring of legionellae in water systems. Therefore, in the present study, a primer set was designed based on a LysR-type transcriptional regulator (LTTR) family protein gene of Legionella pneumophila subsp. pneumophila str. Philadelphia 1 because it was found that this gene is structurally diverse among species through BLAST searches. The specificity of the primer set was evaluated using genomic DNA from 6 strains of L. pneumophila, 5 type strains of other related Legionella species, and other 29 reference pathogenic bacteria. The primer set used in the PCR assay amplified a 2 6 4 - b p p r o d u c t f o r o n l y t a rg e t e d si x s t r a i n s o f L. pneumophila. The assay was also able to detect at least 1.39×103 copies/μl of cloned amplified target DNA using purified DNA or 7.4×100 colony-forming unit per reaction
* Dong Suk Park [email protected] 1
National Academy of Agricultural Science, Rural Development Administration, Jeonju 560-500, Republic of Korea
2
Department of Microbiology, Dankook University, Cheonan 330-714, Republic of Korea
3
Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Wangsan, Yongin, Gyeonggi 449-791, Republic of Korea
4
Department of Oral and Maxillofacial Surgery, Korea University Guro Hospital, Seoul 152-703, Republic of Korea
when using calibrated cell suspension. In addition, the sensitivity and specificity of this assay were confirmed by successful detection of Legionella pneumophila in environmental water samples. Keywords Legionella pneumophila . LysR transcriptional regulator . Detection . Real-time PCR
Introduction Legionellae are facultative intracellular Gram-negative bacteria that may cause Legionnaires’ disease (legionellosis) as well as Pontiac fever, which is a mild flu-like lung infection (Costa et al. 2005). In particular, Legionella pneumophila is the most common pathogenic species, accounting for more than 90 % of legionellosis cases although the genus Legionella comprises 59 species/subspecies, and at least 24 species have been found to cause human pneumonia (DSMZ. 2013; Gomez-Valero et al. 2009; Rizzardi et al. 2015; WHO. 2007). Therefore, the risk assessment for L. pneumophila is especially significant for public health officials and managers responsible for maintenance of water supply systems and cooling towers within industrial or public buildings. However, there are limited data regarding human dose response for L. pneumophila, and the concentration of Legionella required to result in an outbreak is unknown (Whiley et al. 2014). In general, it has been known that the clinical symptoms of Legionnaires disease are difficult to distinguish from those of other forms of pneumonia. Thus, a rapid and accurate method for the detection of Legionella at the species level is important to identify nosocomial infections or community outbreaks and to locate the source of infection. In addition, the development of more rapid and sensitive methods for the detection and
Appl Microbiol Biotechnol
quantification of Legionella cells without cultivation is a matter of increasing importance for water monitoring (DelgadoViscogliosi et al. 2005). Currently, diagnosis of L. pneumophila infection is based on isolation of the pathogen, followed by biochemical or serological tests (Ditommaso et al. 2010; Whiley et al. 2011). Such tests require at least 10 days before final confirmation is obtained. The sensitivity of the enzyme-linked immunosorbent assay (around 106 CFU/ml) is adequate only for detection in symptomatic samples. In some cases, cross-reactions with L. pneumophila and other bacteria are also observed (Füchslin et al. 2010; Keserue et al. 2012). In general, molecular assays based on 5S rRNA, 16S rRNA, the 23S–5S ribosomal RNA intergenic spacer regions (ISRs), RNA polymerase β-subunit (rpoB), macrophage infectivity potentiator (mip), defective organelle trafficking (dotA), and gyrase subunit B (gyrB) genes are frequently used for the detection of L. pneumophila strains, but there have been critical defects in the diagnosis and identification of all isolates of L. pneumophila (Ko et al. 2003; Merault et al. 2011; Nazarian et al. 2008; Stolhaug and Bergh 2006; Wilson et al. 2003; Yáñez et al. 2005; Zhou et al. 2011). In particular, it was determined that the allelic diversity of dotA is predominantly found in L. pneumophila isolates from natural environments, suggesting that niche-specific selection pressures have been operating on this gene (Costa et al. 2010). Actually, the high level of dotA allelic diversity may reflect fitness variation in the persistence of those strains in distinct environmental niches and/or tropism to various protozoan hosts (Costa et al. 2010). Consequently, detection specificity, which depends on both the uniqueness of the sequence to the pathogen of interest, as well as the specific binding of the primers or probes to the target, is significant to the efficacy of any PCR detection method (Lang et al. 2010). In recent, the enormous and increasing number of available bacterial whole-genome sequences in databases, together with employed bioinformatics tools, offers potential for more reliable, fast, and cost-effective methods for bacterial identification in a wide range of samples in coming years (Albuquerque et al. 2009; Segerman et al. 2011). Thus, we have investigated the LysR-type transcriptional regulator (LTTR) family genes to develop a specific primer set for identifying L. pneumophila. The nucleotide sequences of the genes were assessed for specificity and variety among Legionella spp. through BLASTn searches. We designed species-specific primers based on the LysR-type transcriptional regulator (LTTR) family gene of str. Philadelphia 1 and found that the primer set showed high specificity for detecting the targeted pathogen in water samples. In the present study, we have established a reliable and efficient procedure for quantitative detection of L. pneumophila in water samples by SYBR Green Direct polymerase chain reaction (PCR). Serial dilutions of
L. pneumophila were used to determine the detection limits of the single assay, and DNA extraction and real-time PCR were performed within 2 h. The specificity of the assay was also tested against other strains of L. pneumophila and related bacterial species, and environmental samples. These results revealed that this real-time PCR-based method can be particularly useful for analysis of environmental water samples in which the densities of L. pneumophila are very low and provides a platform capable of rapid screening of samples from drinking water, wells, lakes, rivers, cooling towers, and recreational swimming pools for trace levels of L. pneumophila.
Materials and methods Bacterial strains, culture media, and growth conditions All bacterial strains were obtained from the American Type Culture Collection (ATCC) in the USA, the Belgian Coordinated Collections of Micro Organisms (BCCM/LMG) in Belgium, the Korean Agricultural Culture Collection (KACC) and the Korean Culture Center of Microorganisms (KCCM) and the National Culture Collection for Pathogens (NCCP) in the Republic of Korea, and the National Collection of Type Cultures (NCTC) in the UK. All bacterial strains used in this study are listed in Table 2. The suitable culture media and optimal incubation conditions were in accordance with the Handbook of Microbiological Media (Atlas. 2004). L. pneumophila strains were grown on buffered charcoal yeast extract agar (activated charcoal, 2.0 %; yeast extract, 10 %; agar, 1.3 %) at 35 °C in 2.5 % CO2 for 1 day; E. coli, Shigella strains, Salmonella strains, and other bacterial strains were grown on nutrient agar (peptone, 0.5 %; NaCl, 0.5 %; yeast extract, 0.2 %; beef extract, 0.1 %; agar, 1.5 %) at 37 °C for 1 day. DNA preparation The genomic DNA of all microorganisms was prepared using a DNeasy Tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. To measure the quantity and purity of genomic DNA, NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) was used. The genomic DNA was eluted in a final volume of 25 ng/μl at TE buffer and stored at −20 °C until use. Genomic analysis and primer design The genomes of Legionella pneumophila and other Legionella species were compared using the bioinformatics sequence analysis technology. All L. pneumophila and other bacterial genomes used in this study were downloaded from the NCBI bacterial genome database (ftp://ftp.ncbi.nlm.nih.
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gov/genomes/bacteria/). Candidate genes were selected by the modified method of the Chen and Lang (Chen et al. 2010; Lang et al. 2010). To specifically detect L. pneumophila, the LysR-type transcriptional regulator (LTTR) family gene of L. pneumophila subsp. pneumophila str. Philadelphia 1 was selected. A specific primer set based on the LysR-type transcriptional regulator (LTTR) family gene of L. pneumophila subsp. pneumophila str. Philadelphia 1 was designed. The primer set was designed using PrimerSelect of Lasergene (version 7.2.1; DNASTAR Inc., USA). The nucleotide sequences of primer set were assessed for specificity through NCBI-BLAST searches (http://blast.ncbi.nlm.nih.gov). The primer set, LP264F (5′-ACAGCTTGAAGAGGAGTTAG-3′) and LP264R (5′-ACAAGCTCTACTTCAATGCC-3′), for PCR reaction was synthesized by Bioneer Corporation (Daejeon, Korea), respectively.
Real-time PCR reactions The real-time PCR assay was performed in the CFX96 realtime PCR system (Bio-Rad Laboratories, Inc., USA). The mixture consisted of 10-μl iQTM SYBR® Green Supermix (Bio-Rad laboratories, Inc., USA), 5 pM each of LP264F/R primers, and 5 ng purified DNA from each sample in total volume of 20 μl with distilled water. The cycling program was adjusted at 95 °C for 30 s, 45 cycles of 95 °C for 5 s, and 47 °C for 30 s. The data collection was performed during each annealing step. Following amplification, during melting curve analysis, the temperature increases to obtain productspecific melting temperature from 65 to 95 °C with an increment of 0.5 °C. Determination of cycle threshold (Ct) and data analysis were set automatically by the CFX ManagerTM Software system (Version 1.6, Bio-Rad laboratories, Inc., USA).
Conventional PCR The PCR amplifications were performed with the above primers (0.2-μM final concentration) and GoTaq® Flexi DNA polymerase (1× buffer, 4.0 mM MgCl2, 0.2 mM of each dNTP, GoTaq® DNA polymerase 1.25 U final concentration; Promega, Madison, Wisconsin, USA) in a final volume of 50 μl according to the manufacturer’s instructions, and 25 ng genomic DNA from a given bacterial strain. Amplifications were done in a PTC-225 thermocycler (MJ Research, Watertown, MA, USA) with the following cycling conditions: initial denaturation of 5 min at 95 °C; then 35 cycles of 1 min at 95 °C, 30 s at 47 °C, and 1 min at 72 °C; and final extension of 7 min at 72 °C. After PCR reaction, each amplified PCR product (10 μl) was loaded onto a 1.5 % (w/v) agarose gel and subjected to electrophoresis for 1 h at 100 V in 1× TAE buffer, stained with ethidium bromide (EtBr), and visualized on an UV transilluminator and imaged using a VersaDoc 1000 gel imaging system (Bio-Rad laboratories, Inc., USA). Construction of target plasmid DNA The amplification product (264 bp) was then cloned into pGEM-T Easy vector (Promega, Madison, Wisconsin, USA) and amplified with a combination of genespecific and vector-targeted primers using PCR with GoTaq® DNA po lymera se (Prome ga, Madiso n, Wisconsin, USA). The recombinant plasmid DNA was transformed into the E. coli DH5α and purified by QIAprep Spin Miniprep Kit (QIAGEN Inc., Valencia, CA). The concentration and quality (ratio A260/A280) of the extracted DNA were determined with a NanoDrop ND-1000 spectrophotometer prior to calculating the concentration.
LOQ and LOD of real-time PCR assay for L. pneumophila The limit of quantification (LOQ) and limit of detection (LOD) of the real-time PCR assay were determined using 10-fold dilutes of cloned-plasmid DNA, genomic DNA, and bacterial cell suspension of L. pneumophila KCCM 41777 in a 20-μl reaction mixture containing 10-μl iQ™ SYBR® Green Supermix (Bio-Rad laboratories, Inc.) and 5 pmol each of LP264F/R primers. This LOQ and LOD were reproducible with 10-fold serial dilution and real-time PCR testing being done in triplicate (Table 3). The copy number of the cloned plasmid DNA was calculated with the following equation:(Whelan et al. 2003) 23 Copies=μl ¼ 6:022 10 ðcopy=molÞ amount ðgÞ = ½length ðbpÞ 660 ðg=mol=bpÞ The efficiency (E) was calculated using the equation E = 10− − 1, where the slope referred to the log-linear portion of the standard curve (Bustin et al. 2009). 1/slope
Environmental water samples for SYBR Green qPCR A total of 144 samples were collected at 12 sites of four major rivers in South Korea from 2010 to 2012: Han River (37° 35′ 25″ N, 127° 09′ 52″ E), (37° 32′ 57″ N, 127° 06′ 37″ E), (38° 31′ 30″ N, 127° 17′ 13″ E), Geum River (36° 22′ 56.7″ N, 127° 24′ 37.7″ E), (36° 27′ 28.9″ N, 127° 23′ 59.4″ E), (36° 28′ 9.6″ N, 127° 28′ 4.8″ E), Nakdong River (36° 8′ 34.5″ N, 128° 22′ 34.7″ E), (36° 6′ 29.8″ N, 128° 24′ 5.1″ E), (36° 8′ 0.3″ N, 128° 27′ 1.6″ E), and Soyang River (38° 02′ 26″ N,
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128° 09′ 11″ E), (38° 03′ 01″ N, 128° 09′ 56″ E), (38° 04′ 39″ N, 128° 11′ 17″ E) (Table 4). Environmental water sampling conditions were in accordance with Handbooks for Water-Resources Investigations (U.S. Geological Survey USGS 2003). All collected samples were stored at 4 °C upon arrival at the laboratory and analyzed within 24 h after collection. To survey for the presence of L. pneumophila in the water samples, 500 ml of each water sample was filtered through 0.45-μm pore size membrane filter (Pall Corporation, New York, USA) and using a vacuum pump. The genomic DNA was extracted from water concentrates using a Fast DNA spin kit for soil (MP Biomedicals, OH, USA). The genomic DNA concentrations of the water samples were measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc). All genomic DNA (5 ng) was used in the real-time PCR assays as described above.
Results Genomic analysis and in silico specificity test of designed primer set In recent, a large number of available bacterial sequences in databases and bioinformatics tools could result in more reliable, fast, and cost-effective methods for bacterial identification in a broad range of samples (Albuquerque et al. 2009). Also, there is an unmet need for specific markers for early detection and identification of L. pneumophila. To identify candidate gene specific to L. pneumophila, we compared microbial genomes by bioinformatics tools. In particular, we found overlap gene region across analysis between putative candidate genes for the same traits in the genomes for L. pneumophila 12 strains (str. Philadelphia 1, str. Paris, str. Lens, str. Corby, str. 2300/99 Alcoy, str. ATCC 43290, str. Hextuple_2q, str. Hextuple_3a, str. HL06041035, str. L o r r a i n e , s t r. L P E 5 0 9 , a n d s t r. T h u n d e r B a y ) , L. longbeachae str. NSW150, and L. oakridgensis str. ATCC 33761 (Table 1). Among them, a species-specific primer set was designed based on the sequences of the LysR-type transcriptional regulator (LTTR) family protein gene of L. pneumophila subsp. pneumophila str. Philadelphia 1 (GenBank accession No. AAU26322 region: 247787..248671). The specificity of the primer sequences was tested in silico by searching for similar sequences in bacterial genomes using the BLAST and e-PCR analysis (http:// www.ncbi.nlm.nih.gov/). The BLASTn searches showed similarity to the transcriptional regulator, LysR family sequences [identity = 78 %, score = 679 bits (752), and expected=3e-23] from Tatlockia micdadei genome assembly LMI, chromosome: I and putative transcriptional regulator,
LysR family sequences [identity = 74 %, score = 545 bits (604), and expected = 3e-151] from L. longbeachae NSW150. The BLASTx results with the predicted protein sequence revealed the closest similarity to the LysR family transcriptional regulator sequence [identity=89 %, score= 159 bits (402), and expected=1e-45] from L. longbeachae NSW150. Conventional PCR and real-time PCR The species-specific molecular marker, the transcriptional regulator LysR family gene sequence, was amplified by using the LP264F/R primers (Fig. 1). The specificity validation test on 11 strains of represented 6 Legionella species and 29 nonLegionella strains revealed that 6 strains of L. pneumophila gave a consistent positive result regardless of the existence of other species of the pathogen. As expected, a 264-bp DNA fragment was amplified with conventional PCR (Fig. 1), and the amplification plot and a unique dissociation melting peak at 81.50 °C were observed with the real-time PCR assay (Table 2). LOD and LOQ sensitivity test by real-time PCR We used a real-time PCR analysis of L. pneumophila subsp. pneumophila str. Philadephia 1 (KCCM 41777) to generate a standard curve by plotting the mean Ct (n=3) versus the logarithmic concentration of the cloned plasmid DNA, the genomic DNA, and density of the bacterial cell suspension (range, 1.39 ×109 to 1.39 ×103 copies/μl, 5 × 100 to 5 × 10−6 ng/μl, and 7.4×106 to 7.4×100 colony-forming unit (CFU) per reaction, respectively) (Fig. 2a and Table 3). The LOQ assay showed a good linear response and a high correlation coefficient (cloned plasmid DNA, R2 =1.000; genomic DNA, R2 = 0.999; bacterial cell suspension, R2 = 0.996). A standard curve analysis of the linear part of the slope resulted in a coefficient of −3.627, which generated a PCR efficiency of 88.7 % and y-intercept value=35.014 (Fig. 2b). The analysis of the melting temperature and melting peaks of L. pneumophila KCCM 41777 produced from a real-time PCR reaction was shown a reproducible melting temperature of 81.50 °C and specific peaks (Fig. 2c). The standard curve results of the genomic DNA and the bacterial cell suspension showed that there was a linear correlation between the Ct (n=3) values and the concentrations of input the genomic DNA (R2 =0.999, slope=−3.350) and the bacterial cell suspension (R2 =0.996, slope=−3.531). They also revealed that the detection limit of the real-time PCR was 5 fg/μl (fg per μl reaction mix) and 7.4×100 CFU/ml (CFU per ml reaction mix) of L. pneumophila KCCM 41777, respectively. The real-time PCR assay of L. pneumophila KCCM 41777 presented excellent quantification characteristics and accurate detection.
Appl Microbiol Biotechnol Table 1
In silico specificity test of the target gene by BLASTn searches
Organism/Name
Results dotAa
mipb
gyrBc
This studyd
Legionella pneumophila subsp. pneumophila str. Philadelphia 1 Legionella pneumophila str. Paris Legionella pneumophila str. Lens Legionella pneumophila str. Corby Legionella pneumophila 2300/99 Alcoy Legionella pneumophila subsp. pneumophila ATCC 43290 Legionella pneumophila subsp. pneumophila str. Hextuple_2q Legionella pneumophila subsp. pneumophila str. Hextuple_3a Legionella pneumophila subsp. pneumophila str. HL06041035 Legionella pneumophila subsp. pneumophila str. Lorraine Legionella pneumophila subsp. pneumophila LPE509 Legionella pneumophila subsp. pneumophila str. Thunder Bay Legionella pneumophila str. G2762 Legionella pneumophila str. MDC1252 (serogroup 2)
N.D + + N.D + N.D N.D N.D + + N.D N.D -
+e + + + + + + + + + + + -
+ + + + + + + + + + + + -
+ + + + + + + + + + + + -
Legionella pneumophila str. MDC1223 (serogroup 2) Legionella sp. CD-2 Legionella sp. CD-3 Legionella sp. CD-4 Legionella worsleiensis Legionella fairfieldensis Tatlockia micdadei isolate Pavia 16 Legionella longbeachae NSW150 Legionella oakridgensis ATCC 33761
-
+ + + + + + -
-
-
a
dotA (GenBank accession No. AY720956, Yáñez et al. 2005)
b
mip (GenBank accession No. 52628139, Nazarian et al. 2008)
c
gyrB (GenBank accession No. JF718229, Zhou et al. 2011)
d
LysR-type transcriptional regulator (LTTR) family protein gene (GenBank accession No. AAU26322, This study)
e
+, detected; -, not detected; N.D not determined
Specific detection of Legionella pneumophila in environmental water samples A total of 144 environmental water samples were examined by real-time PCR for the presence of L. pneumophila.
Fig. 1 Specific PCR amplification of a gene fragment of Legionella pneumophila using the LP264F/R primer set. Lane M, size marker (1 kb DNA plus ladder; Gibco BRL); lanes 1-40re described in Table 2; lane 41 distilled water
Location of the sampled environmental water used and the results detected in environmental water samples from major four rivers of Korea are described in Table 4. The minimum detection range of L. pneumophila in environmental water samples was 7.4×103 to 7.4×104 CFU/ml.
Appl Microbiol Biotechnol Table 2
Bacterial strains used in the PCR specificity test
No.
Bacterial strains
Source
This studya
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Legionella pneumophila subsp. pneumophila (serogroup 1) Legionella pneumophila subsp. pneumophila (serogroup 2) Legionella pneumophila subsp. pneumophila (serogroup 2) Legionella pneumophila subsp. pneumophila (serogroup 8) Legionella pneumophila subsp. fraseri (serogroup 5) Legionella pneumophila (serogroup 7) Legionella jordanis Legionella oakridgensis Legionella maceachernii Legionella erythra Legionella rubrilucens Shigella sonnei Shigella flexneri Shigella boydii Shigella dysenteriae Escherichia coli O1:K1:H7 Escherichia coli O157:H7 (Non-toxigenic)
KCCM 41777T ATCC 33154 ATCC 33155 KCCM 41784 KCCM 41782 KCCM 41783 NCCP 11013T NCCP 11031T NCCP 11137T NCCP 11138T NCCP 11139T KCCM 40949T KCCM 40948T KCCM 41649T NCTC 9952 LMG 2092T LMG 21756
+b + + + + + -
18 19 20 21 22 23 24 25 26 27 28 29 30
Salmonella enterica subsp. enterica serovar Typhimurium Salmonella enterica subsp. enterica serovar Typhimurium Salmonella enterica subsp. enterica serovar Vichow Salmonella enterica subsp. enterica serovar Paratyphi A Salmonella enterica subsp. enterica serovar Paratyphi B Salmonella enterica subsp. enterica serovar Typhi Salmonella enterica subsp. enterica serovar Choleraesuis Salmonella enterica subsp. enterica serovar Enteritidis Salmonella enterica subsp. arizonae Salmonella enterica subsp. diarizonae Salmonella enterica subsp. houtenae Salmonella enterica subsp. indica Salmonella bongori
ATCC 43971T ATCC 13311 ATCC 51955 ATCC 9150 ATCC 8759 ATCC 33459 ATCC 7001 ATCC 13076 ATCC 13314 ATCC 43973 ATCC 43974 ATCC 43976 ATCC 43975T
-
31 32 33 34 35 36
Campylobacter jejuni subsp. jejuni Campylobacter coli Helicobacter pylori Enterobacter aerogenes Citrobacter freundii Enterobacter aerogenes
LMG 8841 LMG 6440T LMG 19449T KACC 13732T KACC 11404T KACC 13732T
-
37 38 39 40
Serratia marcescens Serratia odorifera Straphylococcus capitis subsp. capitis Stenotrophomonas maltophilia
ATCC 990 KACC 12324T KACC 13242T KACC 10244
-
T
ATCC American Type Culture Collection, United States, KCCM Korean Culture Center of Microorganisms, Republic of Korea, LMG The Belgian Coordinated Collections of Microorganisms (BCCMTM ), Belgium, NCCP National Culture Collection for Pathogens, Republic of Korea, NCTC National Collection of Type Cultures, United Kingdom, KACC Korean Agricultural Culture Collection, Republic of Korea T
type strain
a
Experiments to assess the specificity of real-time PCR assays
b
+, detected; -, not detected
Appl Microbiol Biotechnol
Discussion
Fig. 2 Specificity, melting peak, and standard curve of the LP264F/R primer set with SYBR Green qPCR. a Fluorescence intensity as a function of the concentration of the template. For each assay, a series of 10fold dilutions of cloned DNA (range, 1.39×109 to 1.39×103 copies/μl) was used as the template for PCR (1-7, sample dilutions; 8, no-template control). b Standard curve derived from the amplification plot. c Melting peak analysis (1-7, sample dilutions; 8, no template control). The negative first derivative of the relative fluorescence units (–d(RFU)/dT) is plotted as a function of the temperature. The melting temperature is 81.50 °C. The high peak indicates the amplified product; the low peak is the notemplate control
In current, over 90 % of legionellosis cases are caused by infection of L. pneumophila (Marston et al. 1997; Muder and Yu 2002; Yu et al. 2002). Until now, many studies have been performed to develop sensitive and specific technologies to improve the bacterial pathogen detection and ensure successful control measures (Bielaszewska et al. 2011; Müller et al. 2007). However, despite recent advances in molecular and immunological methods, the official and internationally accepted detection method for Legionella spp. in water samples (ISO 11371) is still based on cultivation. This method has major disadvantages such as a long assay time of 10 days and the detection of cultivable cells only, including extremely slow growth rate strains (Bedrina et al. 2013). Therefore, direct detection of L. pneumophila DNA in water samples is a challenging alternative, and much effort has been put into developing PCR method and, more recently, real-time PCR assays. Previous different molecular targets for diagnosis of L. pneumophila have been employed such as mip gene, 5S ribosomal DNA, 16S ribosomal DNA, the 23S-5S spacer, dotA, and gyrB gene. However, it has been reported that the 5S and 16S rRNA genes are such wellconserved regions that it is difficult to differentially detect L. pneumophila and other Legionella species on a real-time basis without subsequent tests, or that there is a risk of amplification of the DNAs of other than Legionella species (Herpers et al. 2003; Ko et al. 2003; Merault et al. 2011; Nazarian et al. 2008; Stolhaug and Bergh 2006; Wilson et al. 2003). In addition, through in silico screen with BLAST engines, it was also confirmed that mip gene is highly conserved within Legionella, whereas dotA is extremely variable between L. pneumophila strains, with an average level of nucleotide diversity four times greater than that of mip (Bumbaugh et al. 2002; Costa et al. 2010) (Table 1). Accordingly, these molecular probes may place public health at significant risk, leading to significant monetary losses in health care. Consequently, as seen in previous studies, development of specific diagnostic tools is even now facing the difficulty in identifying unique and distinguishing features among the target microorganisms. In recent, as a consequence of the rapid development of technology in the area of high-throughput sequencing, the availability of complete Legionella genome sequences presents a great opportunity for improving the existing molecular diagnostic tools by identifying new targets for more sensitive and specific detection. Despite the existence of genomic information for
Appl Microbiol Biotechnol Table 3 Mean Ct end-point fluorescence of 10-fold serial dilutions of Legionella pneumophila cloned-plasmid DNA, genomic DNA and a cell suspension determined with a real-time PCR assay Cloned DNA
Genomic DNA
Cell suspension
Plasmid copies/μl 1.39×109 1.39×108 1.39×107
Ct±SD (n = 3) 13.18±0.09 16.93±0.28 20.55±0.10
Weight/μl reaction mix 5 ng 500 pg 50 pg
Ct±SD (n = 3) 15.51±0.21 18.37±0.15 21.61±0.10
CFU/ml reaction mix 7.4×106 7.4×105 7.4×104
Ct±SD (n = 3) 15.91±0.11 19.32±0.13 22.89±0.09
1.39×106 1.39×105 1.39×104 1.39×103
24.19±0.27 27.75±0.21 31.26±0.06 35.08±0.22
5 pg 500 fg 50 fg 5 fg
25.24±0.15 28.60±0.29 32.21±0.10 35.22±0.18
7.4×103 7.4×102 7.4×101 7.4×100
26.38±0.01 29.90±0.38 33.48±0.15 37.13±1.26
the most abundant type of transcriptional regulator in prokaryote. The conservation of LTTRs within the genomes of extremely diverse bacteria means that they have evolved a regulatory role over genes with similarly diverse functions, whose products can be involved in metabolism, cell division, quorum sensing, virulence, motility, nitrogen fixation, oxidative stress responses, toxin production, attachment, and secretion (Henikoff et al. 1988; Maddocks and Oyston. 2008). In conclusion, this newly developed real-time PCR assay is detecting L. pneumophila with high specificity, sensitivity, and without any false-positive signals from other Legionella species from pure cultures, or DNA mixtures extracted from natural fresh water. This real-time PCR assay might be a useful method for the detection and quantitation of L. pneumophila in water for quality management purposes.
Legionella spp. for many years, the majority of molecular diagnostic tools still rely on 16S rRNA gene sequences. Therefore, in the present study, a computational genomics pipeline was used to compare sequenced genomes of Legionella spp. and to rapidly identify unique regions for development of highly specific diagnostic markers (Lang et al. 2010). We selected the species-specific sequence region and developed a primer set for the species-specific detection and quantitation of L. pneumophila. The species-specific primers were obtained from a LysR-type transcriptional regulator (LTTR) family protein gene of L. pneumophila subsp. pneumophila str. Philadelphia 1 (GenBank accession No. AAU26322 region: 247787..248671). It has been reported that the LysR family of transcriptional regulators represents
Table 4
SYBR Green quantitative PCR assay for specific detection of Legionella pneumophila at four major rivers in South Korea
Location of water samples
Coordinates 2010
Han River
Geum River
Nakdong River
Soyang River
a
2012
Latitude
Longitude
Ia
IIb
IIIc
IVd
I
II
III
IV
I
II
III
IV
37° 35′ 25″ N 37° 32′ 57″ N 37° 31′ 30″ N 36° 22′ 56.7″ N 36° 27′ 28.9″ N 36° 28′ 9.6″ N 36° 8′ 34.5″ N 36° 6′ 29.8″ N 36° 8′ 0.3″ N 38° 02′ 26″ N
127° 09′ 52″ E 127° 06′ 37″ E 127° 17′ 13″ E 127° 24′ 37.7″ E 127° 23′ 59.4″ E 127° 28′ 4.8″ E 128° 22′ 34.7″ E 128° 24′ 5.1″ E 128° 27′ 1.6″ E 128° 09′ 11″ E
-
-
-
+ -
+ + + -
-
+ + +
+
+ -
+ + + -
+ + +
+ -
38° 03′ 01″ N 38° 04′ 39″ N
128° 09′ 56″ E
-
-
+
-
-
-
-
-
-
-
+
-
128° 11′ 17″ E
+
-
-
-
-
-
-
-
-
-
-
-
I, April; a II, June; c III, August; d IV, October
+, detected; -, not detected
2011
Appl Microbiol Biotechnol Acknowledgments This study was funded by the National Academy of Agricultural Science, Rural Development Administration, Republic of Korea (Research Program for Agricultural Science and Technology Development; Project No. PJ010831). Conflict of interest The authors declare that they have no competing interests. Ethics statement This article does not contain any studies with human participants or animals performed by any of the authors.
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