Streszczenie

Wprowadzenie: Przez ostatnie pięć lat coraz więcej uwagi w Polsce zaczęto poświęcać problemowi infekcji wy­woływanych przez bakterie Legionella sp. Ma to związek m.in. z pojawieniem się nowych re­gulacji prawnych, dotyczących jakości wody przeznaczonej do spożycia przez ludzi. Było to in­spiracją do opracowania testu umożliwiającego oznaczenie wielu genów wirulencji Legionella pneumophila w celu lepszego zrozumienia ich dystrybucji w szczepach środowiskowych i klinicz­nych. Metoda może być nieocenioną pomocą w skuteczniejszym przewidywaniu ryzyka wywoła­nia choroby przez dany szczep i stanowić nowe narzędzie podczas dochodzeń epidemiologicznych. <br>Materiał/Metody: Mikromacierz bazuje na systemie Array Tubes, zawiera trzy kontrole pozytywne i jedną nega­tywną. Geny docelowe kodują elementy strukturalne T4SS, białka efektorowe oraz geny wiru­lencji niezwiązane z systemem sekrecji. Sondy zaprojektowano używając programu OlogoWiz, mikromacierz analizowano z użyciem programu IconoClust. W celu izolacji szczepów klinicz­nych i środowiskowych zgromadzono próbki BAL i pobrano próbki wody z różnych, niezależ­nych instalacji ciepłej wody w budynkach użyteczności publicznej.
Wyniki: Zaprojektowano i wyprodukowano mikromacierz do identyfikacji 66 genów L. pneumophila bio­rących udział w patogenezie. Czułość mikromacierzy pozwala na analizę DNA wyizolowanego z pojedynczej kolonii L. pneumophila. Analizowano 7 szczepów środowiskowych, z których dwa różniły się wzorem hybrydyzacji od szczepu referencyjnego.
Dyskusja: Prezentowana metoda nie jest czasochłonna i kosztowna. Analiza szczepów środowiskowych wy­kazała możliwe różnice w obecności genów kodujących białka efektorowe. Dalsze analizy mogą pozwolić na identyfikację genów zwiększających ryzyko wywołania choroby oraz na ocenę zja­dliwości szczepów.

Słowa kluczowe:mikromacierz DNA • czynnik wirulencji • białko efektorowe • Legionella pneumophila • legioneloza

Summary

Introduction: For the last five years, Legionella sp. infections and legionnaire’s disease in Poland have been re­ceiving a lot of attention, because of the new regulations concerning microbiological quality of drinking water. This was the inspiration to search for and develop a new assay to identify many virulence genes of Legionella pneumophila to better understand their distribution in environmen­tal and clinical strains. The method might be an invaluable help in infection risk assessment and in epidemiological investigations. <br>Material/Methods: The microarray is based on Array Tube technology. It contains 3 positive and 1 negative control. Target genes encode structural elements of T4SS, effector proteins and factors not related to T4SS. Probes were designed using OligoWiz software and data analyzed using IconoClust software. To isolate environmental and clinical strains, BAL samples and samples of hot water from different and independent hot water distribution systems of public utility buildings were collected. r>Results: We have developed a miniaturized DNA microarray for identification of 66 virulence genes of L. pneumophila. The assay is specific to L. pneumophila sg 1 with sensitivity sufficient to perform the assay using DNA isolated from a single L. pneumophila colony. Seven environmental strains were analyzed. Two exhibited a hybridization pattern distinct from the reference strain. >Discussion: The method is time- and cost-effective. Initial studies have shown that genes encoding effector proteins may vary among environmental strains. Further studies might help to identify set of ge­nes increasing the risk of clinical disease and to determine the pathogenic potential of environ­mental strains.

Key words:DNA microarray • virulence factor • effector protein • Legionella pneumophila • legionellosis

Abbreviations:

AFLP – amplified fragment length polymorphism; BAL – broncho-alveolar lavage fluid; BLAST – basic local alignment search tool; EWGLI – European working group for Legionella infections; LCV – Legionella containing vacuole; MLVA – multiple-locus variable number of tandem repeats analysis; NSI – normalized signal intensity; PCR – polymerase chain reaction; PFGE – pulsed-field gel electrophoresis; SBT – sequence-based typing; T4SS – type IV secretion system.

Introduction

Legionella spp. are causative agents of legionnaire’s dise­ase. For most infections Legionella pneumophila sg 1 is re­sponsible [55]. Water is the major reservoir for Legionellae. Legionellae have been detected in natural waters (e.g. gro­und water, surface water, hot spring water) and manmade aquatic environments (e.g. hot and cold water system, co­oling towers and evaporative condensers, respiratory the­rapy equipment). Conditions in manmade aquatic environ­ments that are favorable for the amplification of Legionella growth include temperatures of approximately 25-42oC, sta­gnation, scale and sediments, biofilms and the presence of protozoa. Legionellae survive in aquatic environments as in­tracellular parasites of free-living protozoa [20]. The main transmission route of Legionella is inhalation of droplets of water- air aerosol containing bacteria. Occasionally, inhaled by humans it multiplies in alveolar macrophages [26]. After uptake to the host cell via phagocytosis, L. pneumophila starts to export effector proteins through T4SS [9]. More than 200 potential effectors have been identified using bio­informatic methods [8]. Many various assays have been de­veloped to confirm the translocation of putative effectors. The methods apply cya-fusion assay, immunofluorescen­ce microscopy, inter-bacterial transfer, SidC-based translo­cation assay, and fusion to β-lactamase. The methods have also been used to identify effectors not selected previously by bioinformatics. Discovered proteins affect vesicle traf­ficking, regulate host GTPases, inhibit apoptosis, mimic host ubiquitination pathways and induce a stress response [5,10,12,14,19,29,30,33,39,40,49].

After infection the disease may develop up to 20 days. This means that identification of the source of infection is difficult, especially if the disease developed after travel. To establish the link between environmental and clinical strains, genetic studies are required. Routinely PFGE, considered as a gold standard, SBT, AFLP and MLVA are used. These techniqu­es take advantage of identification of DNA polymorphism after restriction enzymes digestion (PFGE, AFLP), analysis of multiple locus variable number tandem repeats (MLVA) and sequencing of selected genes (SBT) [21,22,41,51,60]. The methods have different ability to discriminate strains ba­sed on the presence in the genome of DNA polymorphisms unspecific for virulence, but do not allow particular virulen­ce genes to be identified. The technique which allow this are microarrays. The method has been used for the rapid identi­fication of antimicrobial resistance genes in Gram-negative bacteria [6], genotyping of enterohemorrhagic Escherichia coli (EHEC) [24] and Staphylococcus ureus [32], detec­tion of herpesvirus and adenovirus co-infections [38] and for haemagglutinin subtyping and pathotyping of avian in­fluenza viruses [23]. Simultaneous identification of many virulence genes is essential, because effector proteins of L. pneumophila display a high level of redundancy. Deletion of a single gene often does not cause a noticeable effect on phenotype [17]. Identification of many virulence genes in environmental strains will give a more reliable view on the virulence potential of a given strain and will help to better understand their distribution. In future it may help to pre­dict with higher accuracy risk of infection.

Risk assessment of infection is usually made on the basis of the number of Legionella sp. in tested water. Routinely, for quantitative analysis, membrane filtration and cultiva­tion on solid microbiological media is performed. The me­thod in detail is described in PN – EN ISO 11731-2: 2008 [45]. In Polish law a regulation that relates directly to this issue is the decree of Ordinance of the Minister of Health of March 29, 2007 on the requirements related to the qu­ality of water intended for human consumption [47,48]. European guidelines, formed by EWGLI in 2005, contain many instructions about risk assessment and management in order to minimize risk of infection [27]. Attachment No. 7 to the Polish decree defines the risk based on the number of Legionella sp. determined according to PN – EN ISO 11731-2: 2008. The risk gradation is consistent with the EWGLI guidelines, but the regulation says nothing about risk assessment and management performed individually for each installation. The assessment of risk of infection based on the number of Legionella sp. is very difficult, be­cause infection dose is still not determined and not all in­dividuals exposed will develop the disease.

To date, five genomes of L. pneumophila have been sequ­enced. Pan-genome analysis reveals that only 67% of iden­tified genes are common for all strains, while 33% are stra­in-specific [15]. Such high genetic diversity might be very useful for pathotyping of a strain.

This was the inspiration for our study, in which we present a low-density oligonucleotide DNA microarray dedicated to identification of 66 virulence genes of Legionella pneumophila sg 1. Genes of interest are involved in translocation of effector proteins into the eukaryotic cell (dot/icm genes encode struc­tural elements of T4SS, and proteins with function similar to molecular chaperones), growth on solid microbiological media, intracellular growth, inhibition of apoptosis, matura­tion of LCV, iron acquisition, and cytochrome c synthesis. Function of many target genes encoding effector proteins is not determined. Target genes encoding effector proteins were selected on the basis of studies where their translocation to a eukaryotic cell was proven. Target genes are listed in Table 1. All probes and primers were designed in this study.

Table 1. Genes of interest

Materials and Methods

Microarray design and manufacture

The designed microarray is based on the Array Tube sys­tem (Alere Technologies GmbH). On the surface 9 mm2 of glass slide probes are immobilized via amino-linker C7. Each slide has its own hybridization chamber. In the first step of the detection procedure, products of linear PCR, la­beled with biotin, hybridize to specific probes. During the next step, streptavidin conjugated with horseradish peroxi­dase binds to the biotin. The enzyme transforms the solu­ble and colorless substrate into an insoluble black product. In the last step each microarray is analyzed in an array re­ader and clarity of all spots is calculated.

All probes were designed using OligoWiz 2.0 softwa­re. Legionella pneumophila subsp. pneumophila str. Philadelphia 1 genome was used as a reference sequence. Probes were designed in two stages. In the first stage, a set of probes specific to each target gene was designed (up to 15 probes for each gene). Probe sequences were selected to be specific for the target gene of the reference strain and to have specified GC contents, lengths, and Tm in order to yield high hybridization efficiency. In the second stage the BLAST algorithm was used to select one probe speci­fic to the consensus region of the target gene.

Primers were designed using Primer-BLAST. Primers were selected to be specific and to have similar GC contents, lengths, and Tm in order to have similar template binding efficiency. Probes and primers are specific to a consensus region of target genes. The primer binding site is situated up to 100 bp upstream of the probe-binding site (Figure 1). Probes and primers are listed in Table 2.

Figure 1. Principle of the method. Location of hybridization sites of probes and primers on a DNA template. Arrow indicates the direction of elongation.

Table 2. Names and sequences of all designed probes immobilized on the microarray and primers used in linear multiplex PCR

Each target gene has one specific probe and each spot is duplicated. Spot distance: 180 µm, spot diameter: 150 µm (Figure 2). The microarray has one negative control (NC) – a probe and primer complementary to human papillo­ma virus E7 gene – and 3 positive controls – probes and primers complementary to the gene encoding 16S rRNA (lpg0302), mip gene (lpg0791) encoding macrophage in­fectivity potentiator, and pgi gene (lpg0759) encoding glu­cose-6-phosphate isomerase.

Figure 2. Layout of oligonucleotide probes on the microarray. Each spot is duplicated. Violet and blue color correspond to two areas of the microarray. In each area one duplication of each spot is placed. Rectangles coded marker (red color) correspond to spots with immobilized biotin (positive controls for Poly-HRP-streptavidin). Each microarray has 5 marker spots. Black rectangles correspond to reference points of the microarrays. Green rectangles corresponds to positive control spots. Yellow rectangles corresponds to negative control spots.

Bacterial strains

Legionella pneumophila was isolated from water samples collected from the hot water systems according to PN – EN ISO 11731-2: 2008. Serogroup 1 was identified using an L. pneumophila latex kit (BIORAD) according to the manu­facturer’s instructions. (Five strains were obtained from the Department of Communal Hygiene, National Institute of Public Health – National Institute of Hygiene, Warsaw. Two strains were isolated in this study.) Legionella pneumophila sg 1 Philadelphia 1 (ATCC 31152) was used as a reference strain. In order to isolate clinical strains of L. pneumophila, BAL samples were collected from 31 patients with chronic cough, hemoptysis, chest pain, diarrhea, respiratory disor­ders, abdominal pain, confusion. Samples were cultivated on BCYE-a with cysteine and confirmed on BCYE-α without cysteine and BMPA plates. Not a single strain was isolated. To confirm the negative result of the cultivation, DNA from BAL samples was isolated and used as a template for real time PCR reaction to detect and quantify Legionella sp. (BioRad). In all tested samples Legionella sp. DNA was not detected.

DNA isolation and preparation

One colony of each strain of L. pneumophila was resuspen­ded in lysis buffer from DNeasy Blood & Tissue Kit and the DNA was isolated according to the manufacturer’s instruc­tions. BAL samples (5 ml) were centrifuged (10 min., 12000 rpm), the pellet was resuspended in lysis buffer from DNeasy Blood & Tissue Kit and the DNA was isolated according to the manufacturer’s instructions. DNA concentration was measured using a spectrophotometer. DNA isolated from bacterial strains was subsequently diluted in double-distil­led water to the expected concentration. DNA isolated from BAL samples was concentrated to the expected concentra­tion using BlueMATRIX PCR/DNA Clean-up Purification Kit (Eurex) according to the manufacturer’s instructions.

Labeling and PCR

Each elongation reaction contained 1 µl dNTP mix (Fermentas), 1 µl Terminator DNA polymerase buffer 10× (New England Biolabs), 0.1 µl Terminator DNA po­lymerase (New England Biolabs), 0.35 µl Biotin-16-dUTP (Roche), 1 µl primer mix (Genomed), and 1 µg genomic DNA (up to 6.55 µl). The PCR conditions were as follows: 3 min at 96°C, followed by 40 cycles of 20 s at 51°C, 40 s at 72°C and 60 s at 96°C.

Hybridization and detection

Each Array Tube was in the first step washed with 500 µl of double-distilled water and incubated at 55°C using a ther­momixing device for 5 min at 550 rpm. In the next step the water was discarded and 500 µl of hybridization buffer was added for 5 min at 55°C and 550 rpm. The hybridization buffer was removed from the hybridization chamber and 100 µl of a hybridization mix (90 µl of hybridization buffer and 10 µl of PCR product) was added for 60 min at 55°C and 550 rpm. In the next step the Array Tube was washed once with 500 µl of 2 × SSC with Triton X-100 (10 × SSC is 1.5 M NaCl, 0.15 M sodium citrate, pH 7.0) for 5 min at 30°C and 550 rpm, once with 500 µl of 2 × SSC for 5 min at 30°C at 550 rpm and finally with 500 µl of 0.2 SSC for 5 min at 20°C and 550 rpm. The washing buffer was discar­ded and 100 µl of blocking solution was added (blocking solution is 6 × SSPE containing 0.005% Triton X-100 and 2% milk powder; 6 × SSPE is 60 mM sodium phospha­te, 1.08 M NaCl, 6 ml EDTA, pH 7.4) for 15 min at 30°C and 550 rpm. Blocking solution was removed and horsera­dish peroxidase-streptavidin conjugate solution was added (Poly-HRP-streptavidin was diluted 1: 3000 in 6 × SSPE with Triton X-100) for 15 min at 30°C and 550 rpm. To re­move excess HRP, the Array Tubes were washed using wa­shing buffers as described above. In the last step, 100 µl of peroxidase substrate was added (Seranum Grün, Seranum diagnostica GmbH) and the staining reaction was perfor­med at ambient temperature without shaking for 10 min. Array Tubes were placed in an Array Tube reader (Alere Technologies GmbH). Data were acquired and analyzed using IconoClust 3.2 software (Alere Technologies GmbH).

Data analysis

Analysis was performed using IconoClust 3.2 software. Raw data were normalized and the Normalized Spot Intensity (NSI) was calculated. NSI=1-(M/BG) where M represents the average intensity of a spot and the BG represents the intensity of the local background. NSI value >0.1 of a gi­ven spot was considered to be “positive”.

Results

Assay validation

In the first step of the validation, quality control of the designed and manufactured microarrays was performed. To exclude contamination of probes with biotin, the full hybridization procedure was performed using double-di­stilled water in place of biotinylated PCR product. NSI of spots corresponding to the legL1 gene was higher in com­parison with background and other spots on the microar­ray. In further analysis these spots were not considered be­cause of possible contamination with biotin. All data were normalized to minimize the effect of heterogeneous back­ground signal emission (Figure 3D).

Figure 3. Images of the microarray after hybridization procedure. Each arrow points to an air-bladder. (A) E. coli DNA used as a PCR template. (B) L. pneumophila DNA used as a PCR template. (C) DNA isolated from BAL sample used as a PCR template. (D) 3D visualization of background signal of the microarray before data normalization. Distilled water was used instead of PCR product and the full hybridization procedure was performed.

Specificity of the microarray was tested using geno­mic DNA isolated from Legionella pneumophila subsp. pneumophila str. Philadelphia 1, Escherichia coli (O157: H7), Pseudomonas aeruginosa, Haemophilus influenzae and DNA isolated from BAL samples. The PCR product used for hybridization, where DNA of Escherichia coli (O157: H7), Pseudomonas aeruginosa and Haemophilus influenzae was the template, hybridized only to the probes corresponding to 16S rRNA (Figure 3A). The PCR product from BAL samples gave no positive signal in any spot area (Figure 3C). The PCR product from the L. pneumophila re­ference strain hybridized to all probes corresponding to tar­get genes. The spot corresponding to the icmL probe cha­racterized low value of the NSI in all tested microarrays. The shape of the spot indicates that probes were incorrectly spotted during the manufacturing process. Hybridization to probes corresponding to negative control was not de­tected (Figure 3B).

Assay sensitivity

To determine the minimal DNA concentration for the ana­lysis, a series of dilutions of reference DNA was made: 250 ng/µl, 150 ng/µl, 100 ng/µl and 50 ng/µl. The detec­tion limit was 975 ng DNA/reaction. It is the equivalent of 2.66×10⁸ DNA copies, based on a genome size of 3.4×10⁶ nucleotide residues corresponding to a 3.6×10^-15 g/1 genome copy. In experiments with DNA dilutions near to the detec­tion limit, heterogeneous signals from different spots were observed. To overcome this effect, 1.2 µg of DNA should be used in routine experiments.

Tested strains

In the last stage of the study we tested DNA isolated from 7 environmental strains. Analysis of DNA isolated from five strains showed a positive signal in all spots correspon­ding to target genes. DNA from two tested strains, named LPE06 and LPE07, gave a spot signal pattern different to the reference strain and the other environmental strains (Figure 4). Exact analysis of spot signal level indicated that both strains give a positive signal of spots corresponding to dot/icm genes. Strain LPE06 gives no signal of spots corresponding to legC8, legLC4, legU2, sidG, SidH and wipB genes. Strain LPE07 gives no signal of spots corre­sponding to legA12, legC4, legC8, legL7, legLC4, legU1, legU2, lepB, sidG, sidH, vipD and wipB genes.

Figure 4. Images of the microarray after hybridization procedure. (A) DNA of LPE06 strain was used as a PCR template. (B) DNA of LPE07 strain was used as a PCR template.

Discussion

Routinely, legionellosis risk assessment is made on the basis of the number of Legionella sp. detected in a wa­ter sample using microbiological methods. Results of the analysis determine actions which should be put into prac­tice to reduce the level of contamination. These actions are often expensive and cause difficulties in usage of the in­stallation. If dead ends are present, effective chemical or thermal disinfection is very difficult to perform. The me­thod also has limitations. Identification of L. pneumophila in water from cooling towers has shown that the number of bacteria may vary in a short period of time. Single wa­ter testing may not give reliable information about the le­vel of contamination of installation [7]. Other studies have shown that genotypes of L. pneumophila in cooling towers do not change while the number of bacteria vary [46]. For this reason, the bacteria number count should not be the only method for the assessment of risk of infection. The microbiological method should be supplemented by ana­lysis of virulence potential of a strain, which is determi­ned by specific virulence genes.

The presented microarray allowed identification of two stra­ins with a hybridization pattern different to the reference stra­in. Strain LPE07 does not give a positive signal of spots cor­responding to genes encoding 6 effector proteins and strain LPE07 to 12 effector proteins. Previous studies with micro­arrays have shown that genes sidG and sidH are not highly conserved. Gene sidG was present in 52% of tested strains while sidH was present in 72%. Genes encoding structural elements of T4SS were present in all tested strains except the gene icmX, which was present in 65% of tested strains [11]. These data are compatible with the results of our stu­dy, because all spots corresponding to dot/icm genes give a positive signal. Strains LPE06 and LPE07 give no positive signal for three and seven eukaryotic-like genes respectively. Leg genes have been discovered recently and their function in many cases is still not known. Further analysis of clinical and environmental strains isolated from different installa­tions made of different materials with parallel identification of protozoa will help to better understand their distribution and functions in pathogenesis. Highly conserved virulen­ce genes are important for survival of Legionella. Strains with deletions in these genes may have reduced ability to infect protozoa and therefore to survive in water with bio­cides and to infect humans. Knowledge about the virulence potential of a strain identified in an installation of interest with information about the number of bacteria will help to choose the most effective intervention.

Sensitivity of the array allows one to analyze DNA isola­ted only from one colony and is similar to sensitivity le­vels reached in other studies with this technology [2,4,37]. Furthermore, there are several advantages of this technolo­gy. In one experiment detection of up to 100 genes in du­plicate is possible in only 8 hours. It is less costly and ti­me-consuming than 200 PCR reactions. The Array Tube system allows one to perform the assay with standard la­boratory equipment, because each Array Tube has the Eppendorf tube format. Moreover, PCR is prone to conta­mination, which is almost impossible with the Array Tube system, and a free PCR product environment is not rigoro­usly required. It is an open system and changes in microar­ray layout are easy to introduce. In future the method mi­ght be a supplementation of the microbiological method in routine diagnostics of environmental samples.

Studies performed at the National Institute of Public Health – National Institute of Hygiene showed that the bacteria were present in most tested samples collected from hot wa­ter distribution systems in inpatient healthcare facilities [34]. Moreover, no method of disinfection applied once guarantees permanent elimination of the pathogen. Only constant monitoring will give valid information about the risk of infection. The microbiological method has limi­tations and because of that the microarray was designed. The technique has been used in many fields of environ­mental and medical diagnostics and it is also a promising method for identification and graduation of risk of infec­tion of Legionella pneumophila.

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The authors have no potential conflicts of interest to declare.

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