Detection of Pseudomonas fluorescens from broth , water and infected tissues by loop-mediated isothermal amplification ( LAMP ) method

Loop mediated isothermal amplification is rapid, highly sensitive and specifically developed method for detection of bacterial infections. AprX gene for alkaline metalloprotease of Pseudomonas fluorescens was used to design four primers and loop mediated isothermal amplification (LAMP) conditions were standardized for amplification of DNA. LAMP primers successfully amplified P. fluorescens from DNA and bacterial cells taken directly from broth, water and infected tissues with high specificity and sensitivity (10 pg) under isothermal condition at 61°C.


INTRODUCTION
Pseudomonas fluorescens is a member of the fluorescent pseudomonad group and (unlike Pseudomonas aeruginosa) has generally been regarded to be of low virulence and an infrequent cause of human infection (Hsueh et al., 1998).But later on, six strains grown in transfusion blood having characteristic features of P. fluorescens had caused severe to fatal reactions in the recipients (Pittman, 1953).Sutter (1968) also isolated P. fluorescens repeatedly from the blood of a patient with an abdominal abscess following bowel resection.P. fluorescens has been reported to cause occasional cases of transfusion-associated septicemia in blood recipients, including fatal reactions (Scott et al., 1988), and catheterrelated bacteremia in patients with cancer (Hsueh et al., 1998).P. fluorescens is an aerobic, rod shaped, Gram-negative bacteria that grows best at temperature range between 25 and 30°C.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Eissa et al. (2010) isolated different strains of Pseudomonas species namely Pseudomonas putida, P. aeruginosa, P. fluorescens and P. anguilliseptica from Oreochromis niloticus in Qaroun and Wadi-El-Rayan Lakes, Egypt.They reported that infected fishes showed irregular hemorrhages on body surface, especially at the ventral part of abdomen, eyes cloudiness, scales detachment and congested gills were observed.Internally, there were sanguineous fluids in the abdominal cavity of some fish.Also, these results are in agreement with those of EL-Hady and Samy (2011) who isolated P. putida, P. aeruginosa, P. fluorescens and P. anguilliseptica from cultured O. niloticus and Cyprinus carpio from different fish farms at different localities in Egypt.This data clearly indicates that infection is responsible for huge losses to aquaculture.
Pseudomonas spp.causes septicaemia in aquatic animals (Roberts, 1978) and a number of aquatic animals including fish, frogs and soft-shelled turtles are reported to be susceptible to Pseudomonas spp. with moderate to high losses (Somsiri and Soontornvit, 2002).Pseudomonas spp.bacteria are considered as opportunistic indoor pathogens as their infection initiates an inflammatory response (Hirvonen et al., 2005;Huttunen et al., 2003).Bacteria invades the host tissue and cause infection and bacteremia in immunocompromised hosts (HIV/AIDS, cystic fibrosis, bronchiectasis, severe chronic obstructive pulmonary disease, burns, malignancy or diabetes mellitus) (Feldman et al., 1998;Liu and Mercer, 1963).Identification of P. fluorescens is very tedious as it is not a frequent cause of human infections; prefers versatile environments (like water, soil, foods, etc.) and grow poorly at the standard hospital microbiology incubation temperature of approximately 36°C (Weyant et al., 1996;CDC, 2005).
Detection of P. fluorescens by standard plate count method, polymerase chain reaction (PCR) and RT-PCR is time consuming and also such methods require the use of special equipments.On the other hand, loop mediated isothermal amplification (LAMP) (Notomi et al., 2000;Nagamine et al., 2002) is simple, easy, rapid and costeffective method which have been widely used for the diagnosis of bacteria (Gahlawat et al., 2009;Chen at al., 2011;Han et al., 2011;Saharan et al., 2014a), viruses (Wang et al., 2011;Zhao et al., 2011;Saharan et al., 2014b) and parasites (Ikadai et al., 2004;Iseki et al., 2007;Lu et al., 2011).Seeking the benefits of LAMP method, the present study is planned to determine application of LAMP assay for rapid detection of P. fluorescens from DNA and bacterial cells taken directly from broth, water and infected tissues.

Bacterial strains
Aeromonas hydrophila (MTCC 646), Lactobacillus acidophilus(MTCC 447), P. flourescens (MTCC 7200), Staphylococcus aureus (MTCC 87) and Bacillus cereus (MTCC 6728) were purchased from Microbial Type Culture Collection (MTCC) and Gene Bank, Institute of Microbial Technology Chandigarh (India) in the lyophilized form.The lyophilized cultures were revived in nutrient broth and cultured on nutrient agar plates.After 24 h of incubation at 28±2°C, the bacteria were resuspended in nutrient broth for the isolation of genomic DNA.

Test sample
Clarius batracus (10-12 cm in length) were collected from local pond and kept in aquarium of 50 L capacity in 1:1 ratio of tap water: pond water.Fish were challenged by adding P. fluorescens broth in aquarium water (approximately 1:100 ratio).Mucus, kidney and water samples were collected after 24 h and stored at -20°C.Further genomic DNA was isolated from these samples.

Genomic DNA extraction from bacterial broth, pond water and infected tissue
Genomic DNA was isolated from five bacterial strains (P.fluorescens, A. hydrophila, L. acidophilus, S. aureus and B. cereus) grown in nutrient broth overnight, pond water, mucus and kidney tissue (isolated from infected fish crushed in liquid nitrogen) using Qiagen DNA extraction kit (Gentra Puregene protocol, 2010) according to the manufacturer's instructions.

Design of oligonucleotide primers for LAMP method
Pseudomonas sp.strains high in proteolytic activity in milk are responsible for bacterial deterioration of dairy products.Protease digestion of milk can lead to clotting and gelation of milk casein.So, a rapid test for detection of Pseudomonas strains in milk is required because conventional plate-counting procedure to detect psychrotrophic contamination in milk products is time-consuming and not useful to prevent food degradation.The aprX gene encoding an alkaline metalloprotease is considered the responsible agent for milk spoilage.LAMP primers targeting for this sequence can accelerate the detection process.Hence, DNA sequence of P. fluorescens Apr X gene for alkaline metalloprotease, was retrieved (partial sequence) from NCBI (http://www.ncbi:nlm.nhi.gov/) and used for primer designing.The specific LAMP DNA oligonucleotide primers were designed by using Eiken Genome site via free online software, that is, Primer-Explorer IV software program (http://loopamp.eiken.co.jp/e/lamp/).
Four sets of primers (described in detail below) based on the following six distinct regions of the target gene: the F3c, F2c and F1c regions at the 3' side and the B1, B2 and B3 regions at the 5' side were designed (Table 1) and got synthesized from Sigma Aldrich.

Visualization of LAMP product
The amplified products were analyzed in 2.0% agarose gel in Tris Borate EDTA (1x) buffer stained with ethidium bromide and photographed using the Bio Red gel documentation and analysis system (XR).

Specificity test
The ability of LAMP assay developed for detection of P. fluorescens specifically was evaluated by using different bacterial DNA viz.P. fluorescens, L. acidophilus, A. hydrophila, S. aureus and B. cereus.The specificity of the LAMP was determined on the basis of DNA amplification.

Sensitivity test
DNA of P. fluorescens was serially diluted up to 10 -10 dilutions with nuclease free water and lower detection limit was tested.

Detection of P. fluorescens in broth, pond water, infected tissue and mucus samples
Bacterial broth, pond water, mucus and infected crushed kidney tissue samples of fish were used to check the potential of LAMP method to amplify nucleic acid directly from live cells of P. fluorescens.Finally, LAMP assay was performed with these test samples under optimized conditions.

RESULTS AND DISCUSSION
LAMP method successfully detected P. fluorescens DNA as ladder like pattern on gel after isothermal amplification by Bst polymerase.

Optimization of LAMP reaction conditions for P. fluorescens
P. fluorescens DNA was tested under different conditions of temperature and time, that is, 60 to 65°C for 1 h followed by heat inactivation step at 80°C for 5 to 10 min in order to determine best amplification reaction conditions.Results show that optimum temperature and time for amplification of P. fluorescens DNA was found to be 61°C for 60 min and 80°C for 5 to 7 min (Figure 1 (Su et al., 2014).

Sensitivity limit of LAMP
The detection limit of LAMP assay was tested with serially diluted DNA samples of P. fluorescens.Samples were tested up to 10 -10 dilution and last detection limit of LAMP was found to be approximately 10 pg according to dilution (Figure not shown).Similar sensitivity was reported by Pan et al. (2011) in Brucella species.
In sensitivity evaluations, Hui et al. ( 2013) successfully detected a serial dilution of extracted Bordetella bronchiseptica DNA with a detection limit of 9 copies, which was 10 times more sensitive than that of PCR.He also considered that there is no need for the complex instrumentation making this LAMP assays a promising alternative for the diagnosis of B. bronchiseptica in rural areas and developing countries where there is lack of complex laboratory services.
Direct detection of P. fluorescens from bacterial broth, pond water, tissue and mucus LAMP assay was also found an to be an effective tool for the detection of P. fluorescens bacteria directly from field samples, that is, pond water and infected kidney tissue of fish (Figure 2).Although there are very few reports related to direct detection of bacteria from broth, water as well as from infected tissue by using LAMP method (Iwamoto et al., 2003;Savan et al., 2004;Poon et al., 2006;Kubota et al., 2008;Gahlawat et al., 2009) but still, this method may prove useful for direct detection of bacteria from the sites of infection and thus can be helpful in field testing.However, this method can detect P. florescens DNA from kidney tissue easily (Figure 2).So, LAMP can directly detect the bacteria from pond water and tissue samples and thus, it can be used as valuable tool for easy and robust on-the-spot detection of this bacterium in clinical laboratories and field conditions.

Figure 1 .
Figure1.Agarose gel illustrating the amplification of the designed primers to the P. fluorescens DNA.The reaction was carried out at 61°C using the 4 primer set for 1 h followed by heat inactivation step at 80°C for 5 to 7 min.Lanes: 1, negative control; 2, empty; 3, P. fluorescens DNA showing good amplification (ladder like band pattern).
) and no amplification was observed in negative control.While at other temperatures, very poor (smear like band pattern at 63°C) or no amplification was observed(Gahlawat et  al., 2009).Fernandez-Soto et al. (2014)  detected Schistosoma mansoni by using Loopamp DNA amplification Kit tested with the S. mansoni primer set and found that LAMP reaction successfully take place at temperatures of 61, 63 and 65°C within the temperature range (60-65°C).Similarly,Uemura et al. (2008) detected Pneumocystis pneumonia by using LAMP assay at 61°C temperature.Specificity of LAMP assay for P. fluorescensDNA samples of P. fluorescens, L. acidophilus, A. hydrophila, S. aureus and B. cereus were tested for

Figure 2 .
Figure 2. Agarose gel illustrating the direct detection of bacteria from collected field samples infected with P. fluorescens by LAMP method.The reaction was carried out at 61°C using the 4 primer set for 1 h.Lanes: 1, DNA ladder (100 bp); 2, negative control; 3, positive control (P.fluorescens DNA); 4, kidney tissue; 5: DNA isolated from infected tissue; 6, pond water sample; 7, DNA isolated from infected pond water; 8, DNA isolated from test water infected with mixture of pathogenic bacteria.

Table 1 .
Details of primers used for LAMP assay.