Molecular Depiction Of lepa , lida , ralf , rtxa and lvhb Virulence factors Of Legionella Pneumophila Isolated from Respiratory Tract Infections

Background: Among all bacterial species in the genus Legionella, Legionella pneumophila is responsible for 90% of Legionella infections in humans. Putative virulence genes are the main factors in pathogenesis of L. pneumophila. The aim of this study was to determine the incidence of L. pneumophila in the broncho alveolar lavages of patients hospitalized due to respiratory tract infections as well as study the distribution of lepA, lidA, ralF, rtxA and lvhB virulence factors in bacterial strains.


Background
Respiratory Tract Infections (RTIs) are the most common, and possibly most severe clinically infectious diseases.There were 3,084 deaths attributable to RTIs and pneumonia in recent years in developed countries like Australia [1].RTIs accounted for a total of 43,953 hospital admissions with an average length of stay of 6.3 days [1].RTIs are usually caused by viruses, however the roles of bacteria are also significant.One of the most commonly considered pathogens in the cases of RTIs and pneumonia is Legionella species (Legionella spp.).Although several species of the genus Legionella were subsequently identified in the cases of RTIs, Legionella pneumophila (L.pneumophila) is the most frequent cause of human legionellosis or Legionnaires Disease (LD) and also a comparatively common cause of community-acquired and nosocomial pneumonia in adults [2].In children, L pneumophila is also a significant, although quite scarce, cause of pneumonia.It has been estimated that 18,000 patients are admitted annually in the United States due to the LD which is quite significant [3].
Most outbreaks and sporadic cases of RTIs caused by L. pneumophila have been attributed to presence of certain virulence factors.The products of genes are involved in the initial attachment to host cells and early stages of intracellular infection like the pore formation protein rtxA [7].Another group of genes are those required for bacterial survival and intracellular replication like a number of effectors including ralF, lidA and lepA [8].Legionella vir homolog (lvh) is a type IV secretion system involved in conjugation [8,9].Another important virulence factor is legAS4.LegAS4 contained an active SET domain that methylated H3.Previous bioinformatics efforts have identified LegAS4 as a putative secreted effector owing to the presence of a eukaryotic specific ankyrin repeat domain [7][8][9].The amino terminus of LegAS4 also contained a SET domain and tandem nuclear localization signals (NLS).LegAS4 was efficiently translocated from L. pneumophila into host macrophages.
The epidemiology and prevalence of L. pneumophila and its virulence factors in BAL samples are essentially unknown in Iran.From a clinical, microbiological and epidemiological perspective, it is important to know the exact prevalence of L. pneumophila among clinical samples taken from patients suffering from RTIs.It is also important to know

Conclusion:
Results indicate that sex and age of patients and climate conditions may constitute risk factors for incidence of L. pneumophila.Due to the high prevalence of L. pneumophila, wide-ranging amendments should be done in the principles of clinical care in some Iranian hospitals.

Bacterial isolation
Prior to culture, BAL samples were centrifuged for 15 min at 2,500 rpm, and the top 7.5 ml of the resulting suspension was removed.The remaining cell concentrate was mixed and used for culture.Aliquots of 100 µL of prepared samples were spread on duplicate plates of aBCYE selective medium Agar (Difco Laboratories, Detroit, Mich., USA) and to plates containing L-cysteine (0.44mg mL -1 ), ferric pyrophosphate (0.250 mg mL -1 ), glycine (3.0 gL -1 ), vancomycin (0.0025 mg mL -1 ) and polymyxin B (0.006 mgmL -1 ), which are named αBCYE-GVP selective agar medium.Plates were incubated at 37ºC in a humidified atmosphere without CO2 during 5 days.Colonies with the typical ground glass appearance of Legionella were sub cultured on two nonselective media, sheep-blood agar and αBCYE agar without L-cysteine.Colonies that grew on αBCYE-GVP but not on non-selective media were consi-dered putative Legionella strains, and were Gram stained and subcultured on a selective medium.The identification of putative Legionella strains as L. pneumophila was carried out using Legionella specific latex reagents (Oxoid, Hampshire, England) and direct immunofluorescence assay with poly clonal rabbit sera (m-Tech Alpharetta, Ga., USA).

Gel electrophoresis
The PCR amplification products (10 μl) were subjected to electrophoresis in a 1% agarose gel in 1X TBE buffer at 80 V for 30 min, stained with ethidium bromide, and images were obtained in a UVIdoc gel documentation system (UK).The PCR products were identified by 100 bp DNA size marker (Fermentas, Germany).A DNA of L. pneumophila ATCC 33152 was used as positive control and DNA of a laboratory isolate strain of E. coli as negative control.

Sequencing
In order to confirm the PCR results, the sequencing method was used.For this reason, PCR products of some positive samples were purified with High pure PCR product purification kit (Roche Applied Science, Germany) according to manufacturer's recommendations.Single DNA strands were sequenced with ABI 3730 XL device and Sanger sequencing method (Macrogen, Korea).Result of the sequence of each gene was aligned with the gene sequences recorded in the GenBank database located at NCBI.

Statistical analysis
The data were analyzed using SPSS (Statistical Package for the Social Sciences) software and P values were calculated using Chi-square and Fisher's exact tests to identify statistically significant relationships for the distribution of L. pneumophila and putative genes between various studied groups of patients.A P value < 0.05 was considered statistically significant.

Ethical issues
The present study was authorized by the ethical committee of the education health care centers of the Shahrekord city, Iran, and the Biotechnology Research Center of the Islamic Azad University of Shahrekord Branch, Iran.All patients or their parents signed the written informed consent.

Results and discussion
Table 1 shows the distribution of L. pneumophila in patients suffering from respiratory infections.Of 150 BAL samples studied, 18 samples (12%) were positive for L. pneumophila.In the other hand, 13 out of 90 male BAL samples (14.4%) and 5 out of 60 female BAL samples (8.3%) were positive for L. pneumophila.There were significant differences (P =0.046) in the incidence of L. pneumophila between male and female patients.We found that the over 50 years old patients had the highest incidence of L. pneumophila (20%), while the less than 15 years old children had the lowest incidence (4.1%).There also were significant differences (P =0.017)  (12) in the incidence of L. pneumophila between young and old patients.Total incidence of L. pneumophila based on the clinical signs of patients is shown in table 3. We found that all of the positive patients had fever, while 7.6% of male patients had nausea.The most commonly recorder clinical signs in patients suffered from respiratory infections caused by L. pneumophila were cough (77.7%), dyspnea (77.7%), chest pain (66.6%) and headache (44.4%).There were no female patient with nausea and myalgia.We found a statistically significant association between the incidence of fever and abdominal pain (P =0.009), cough and nausea (P =0.012), chest pain and diarrhea (P =0.035) and dyspnea and myalgia (P =0.019) in patients infected with L. pneumophila.
Results of the gel electrophoresis for identification of putative virulence factors in the L. pneumophila isolates are shown in figure 1-4.Table 4 shows the distribution of putative virulence factors in patients suffering from L. pneumophila respiratory infections.The most commonly detected virulence factors among L. pneumophila isolates were lidA (50%), followed by ralF (27.7%).Totally, males had a higher incidence of virulence factors than females but there were no statistically significant differences between these two groups.There were significant differences between the incidence of lidA and lepA (P =0.028), lidA and ralF (P =0.042), lidA and rtxA (P =0.021) and lidA and lvhB (P =0.033) virulence factors.
The incidence of L. pneumophila and its putative virulence factors in the BAL samples of patients suffering from RTIs has been investigated in the present study.Results showed that 14.4% of male and 8.3% of female patients were infected with L. pneumophila.One possible explanation for the higher prevalence of L. pneumophila in male than female patients is that men usually have more contact with the external contaminated environment.They work outside the house but women usually stay at home and are not in close contact with outside.In fact, most of the Iranian women prefer to work at home.In a study conducted by Ngeow et al. (2005) [10] 1800 patients were analyzed for presence of respiratory pathogens.Totally, 1756 patients with a diagnosis of CAP were enrolled in their investigation, comprising 1263 adults and 493 children.Total prevalence of respiratory patho-      gens in the male and female patients of adults and children groups were 537 and 389 and 245 and 203, respectively which showed a higher incidence of respiratory pathogens in male patients.Similar results have been reported by Nagalingam et al. (2005) [11] and Amemura-Maekawa et al., (2010) [12].These differences observed between the two sexes are consistent with animal and human studies [13].Smoking and alcoholism are commonly acknowledged to be predisposing factors for legionellosis [14].Smoking and alcoholism are more prevalent in male than female in Iran.Our results showed that 10 out of 13 male (76.92%) and 3 out of 5 female (60%) patients had the history of smoking in their life.Total incidence of L. pneumophila in the BAL samples of our study were 12% (18/150).Climatic variables such as heat, thunderstorms and rain, together with variable barometric pressure may have affected the patients' autonomic nervous systems and causes to high incidence of L. pneumophila in our study.These variables might affect immunity, thus making people more susceptible to infections.Alternatively, the higher prevalence of L. pneumophila may be related to cold climate of the study region.The samples of our study were also collected from November to March which were the cold months of the year in Iran.Environmental factors such as high humidity and increased rainfall also increase the risk for legionellosis [15].Herrera-Lara et al. (2013) [16] suggested that the highest incidence of community acquired pneumonia was in the cold seasons of the year.Another explanation for the high incidence of L. pneumophila in our study is that some health guidelines may have been ignored in Iranian hospitals.
Total prevalence of L. pneumophila in the clinical samples of our study were 12% which was higher than the results of Ghotaslou et al. (2013) (Iran, 2.85%) [17] and Bozzoni et al. (1995) (Switzerland, 5.1%) [18], while was lower than the results of (2000) (India, 13%) [20], and Azara et al. (2006) (Italy, 26%) [21].Lim et al. (2001) [22] reported that of 309 patients hospitalized due to Community Acquired Pneumonia (CAP) 135 (50.60%) were men and the prevalence of L. pneumophila was 3%.A total of 202 patients hospitalized with a diagnosis of CAP were enrolled in a Korean study and 3 samples (2.4%) were positive for L. pneumophila which also was lower than our results [23].Total incidence of L. pneumophila in Southeast Asia was 0.28 cases per 100,000 population (0.1%) [24], while its incidence among European countries were 1.18% (100,000 cases in 2008) [25].The above investigations highlight large differences in the prevalence of L. pneumophila.This could be related to differences in the type of sample (BAL, water of hospital, stool, blood, urine, and other clinical samples) tested, number of samples, method of sampling, season of sampling, experimental methodology, geographical area, and climate differences in the areas where the samples were collected, which would have differed between each study.
Our results also showed that patients older than 50 years had the highest incidence of L. pneumophila (20%), while patients younger than 15 years old had the lowest incidence (4.1%).Nagalingam et al. (2005) [11] reported that hospitals, gender and ethnicity did not significantly (P>0.05;chisquared) affect the seroprevalence of L pneumophila.However, Sopena et al. (2007) [26] showed that elderly patients with CAP caused by L. pneumophila had a higher frequency of underlying comorbidities and presented less frequently with fever and classical non respiratory symptoms and laboratory abnormalities of Legionnaires' disease than younger patients.
To date, there were no exclusive investigation on the molecular detection of virulence factors among clinical isolates of L. pneumophila in Iran.The results of our study showed that the distribution of lepA, lidA, ralF, rtxA and lvhB virulence genes were 11.1% (2/18), 50% (9/18), 27.7% (5/18), 5.5% (1/18) and 16.6% (3/18) with the higher prevalence in male patients with the exception of ralF and lvhB gene.Huang et al. (2006) [28] showed that the incidence of lvh and rtxA genes in patients with clinical manifestations of L. pneumophila were 57.6% and 64.4%, respectively.RtxA positive strains of L. pneumophila have been shown to have a high capability to enter to monocytes and epithelial cells, and increased cytotoxicity and intracellular duplication [7].An isolate with the rtxA gene would have improved abilities for attachment, intracellular growth and cytotoxicity.Consequently, isolates bearing either lvh or rtxA, or both, seem to be more virulent.Therefore, they could be considered as indicators of the infection potential of an isolate.Presence of lepA, lidA, ralF, rtxA and lvhB virulence genes is essential for the existence and growth of L. pneumophila in macrophages, for avoidance of phagosome acifidication and lysosome fusion and is necessary for instruction of apoptosis in human macrophages [29,30].

Conclusions
In conclusion, we identified a large number of positive samples (12%) in L. pneumophila isolated from BAL samples of patients with RTIs in Iranian health centers.Marked sexual and age-dependent variation in the distribution of L. pneumophila were also found.Higher levels of health care and diagnostics should be performed on elder male patients with clinical signs of fever, cough, dyspnea, chest pain, headache and diarrhea who are referred to hospitalization in cold months of the year.In fact, sex, season and certain clinical signs are risk factors for legionellosis caused by L. pneumophila.In an overall view, bacterial strains of our study harbored several virulence factors i.e. lepA, lidA, ralF, rtxA and lvhB.Therefore, virulent L. pneumophila should be diagnosed rapidly and carefully.This is the first molecular identification of virulence factors in L. pneumophila strains of RTIs in Iran.Our results represents higher amounts of lidA (50%) and ralF (27.77%) virulence factors in bacterial isolates.More complementary studies in larger groups of L. pneumophila strains are necessary to confirm our finding.

Figure 1 :
Figure 1: Results of the gel electrophoresis for identification of ralF and lepA genes of the L. pneumophila strains.M: 100 bp DNA ladder (Fermentas, Germany), Line 1: Positive control, Line 2: Positive sample for ralF gene (230 bp), Line 3: Positive control and Line 4: Positive sample for lepA gene (354 bp).

Figure 3 :
Figure 3: Results of the gel electrophoresis for identification of lidA gene of L. pneumophila in BAL samples, M:100 bp DNA ladder (Fermentas, Germany), Lane 1: Positive control, 2: Positive samples for lidA gene (270 bp band) and Line 3: Negative control.

Figure 4 :
Figure 4: Results of the gel electrophoresis for identification of rtxA gene of L. pneumophila in BAL samples, M:100 bp DNA ladder (Fermentas, Germany), Lane 1: Positive control, Line Positive samples for rtxA gene (265 bp band) and Line 3: Negative control.

Table 1
lists the primers used for detection of lepA, lidA, ralF, rtxA and lvhB virulence factors of L. pneumophila and program and the condition for each

Table 1 .
IThe oligonucleotide primers and the PCR programs used for amplification of lepA, lidA, ralF, rtxA and lvhB virulence factors of L. pneumophila isolated from BAL samples.

Table 2 .
Total distribution of L. pneumophila in the BAL samples of male and female patients with respiratory infections.

Table 3 .
Total Distribution of L. pneumophila with respect to the various clinical symptoms.

Table 4 .
Distribution of L. pneumophila in the BAL samples of hospitalized male and female patients with and without history of smoking.