Bertani (LB) medium and on Lowenstein Jensen. (LJ) medium. The obtained cultures were Gram's stained. Following DNA extraction by the standard CTAB ... decontamination using modified Petroff's method (Tripathi et al., 2014). All the ...
NO. 905/A
Identification of Bacteria in Patients with Bronchiectasis and Suspected Lung Cancer; A Preliminary Study A. Ekanayake1, D. Magana-Arachchi1*, D. Madegedara2 1National Institute of Fundamental Studies, Hantana Road, Kandy, Sri Lanka 2Respiratory Disease Treatment Unit, Teaching Hospital, Kandy, Sri Lanka
ABSTRACT Respiratory diseases are a group of diseases which affects and burden humans throughout the world. A diseased lung may present altered diversity of commensal bacteria as well as pathogenic bacteria depending on the disease type and severity. According to scientific research carried, lung cancer and bronchiectasis are two diseases where microbial dysbiosis is observed. As a tropical country, Sri Lanka accounts for many lung cancers and bronchiectasis patients and it is timely to address the effect of bacterial microbiome. The aim of the study was to identify culturable bacterial microbiome of respective patients. The ethical clearance for the study was obtained by Teaching Hospital, Kandy. Study population consisted; Lung cancer suspects (according to symptoms) (n=20), bronchiectasis (n=20) patients. Oropharyngeal (OP) swabs and bronchoalveolar lavage (BAL) samples were collected from all patients as per the representation of upper and lower respiratory tract, respectively. Samples were collected and processed for culturing on Luria Bertani (LB) medium and on Lowenstein Jensen (LJ) medium. The obtained cultures were Gram’s stained. Following DNA extraction by the standard CTAB (N-Cetyl-N, N, N-trimethyl ammonium bromide) method, 16S rDNA gene amplification was carried out. The amplified DNA was sequenced to identify the bacterial species present by standard nucleotide BLAST. No cultures were obtained on LJ medium and LB medium yielded 60 isolates. Gram’s staining resulted, Gram positive rods (n=5), Gram negative rods (n=41), Gram positive cocci (n=3), Gram negative cocci (n=11). Bacterial sequences belonging to six families; Enterobacteriaceae,Bacillaceae, Enterococcaceae, Neisseriaceae, Pseudomonadaceae and Paenibacillacea were identified. Further analysis to genus level revealed seven genera as Enterobacter, Klebsiella, Bacillus, Enterococcus, Nesisseria, Pseudomonas and Paenibacillus. Therefore, the organisms belong to the two phyla, Preoteobacteria and Firmicutes. The commonest were genera Enterobacter and Pseudomonas in both Lung cancer (27.3% each) and bronchiectasis (28.5% each). An important observation made was that OP swabs failed to produce cultures compared to BAL. The results further confirm that chronically diseased airways tend to habitat many bacteria belonging to phylum Proteobacteria. A limited but supportive impression can be gained from identifying culturable bacteria, yet molecular methods might provide a specific insight.
RESULTS
INTRODUCTION Human microbiota comprises of commensal, symbiotic and pathogenic microbes. The importance of studying the microbiota in human body is to understand how microbial communities affect and interfere the functionality. Throughout the world, chronic respiratory diseases alone has accounted for 7% of all deaths (4.2 million) in year 2011 (Bloom et al., 2011). Lung cancer and bronchiectasis are chronic respiratory diseases. Culture dependent and culture independent methods are being used for identification of lung microbiota. n this study, bacteria in lower respiratory tract (LRT) and upper respiratory tract (URT) of lung cancer suspected patients and bronchiectasis patients were studied using culture based method.
DISCUSSION
Following incubation, 60 cultures were yielded. Many samples which were culture negative were OP swabs. The sixty cultures consisted of Gram positive rods (n=5), Gram negative rods (n=41), Gram positive cocci (n=3) and Gram negative cocci (n=11). Nine bacterial species belonging to of two phyla were identified following 16S rDNA sequence analysis. Sequences were submitted to NCBI GenBank nucleotide database (Table. 1).
Objective To identify culturable bacteria in lung cancer suspected patients and in bronchiectasis patients.
Samples were cultured on Luria- Bertani culture medium for 48- 72 hours and on Lowenstein- Jensen slants up to 8 weeks to investigate mycobacterial species following decontamination using modified Petroff’s method (Tripathi et al., 2014). All the cultured plates and slants were incubated at 37 °C. Cultures obtained were Gram’s stained. Bacterial genomic DNA was extracted by CTAB/NaCl method (Somerville et al., 2005) and amplified 16S rDNA was sequenced.
CONTACT
Bacterial culturing
DNA amplification (PCR) Genomic DNA isolation
Figure 1. Isolated bacteria and Gram’s stain observations
A
The results observed in the current study support the existing data as these species have identified in patients with lung cancer and bronchiectasis previously. According to the study, Proteobacteria and Firmicutes were the dominant families in the culturable bacterial population. It can be concluded that culture based methods may provide general results for the understanding, yet not suffice for a more vivid perception. Advanced molecular techniques are in need in order for a clear insight of relationship between bacterial microbiota with each disease individually.
B
REFERENCES Figure 2. Bacterial population composition. A) composition of bacteria in phylum level. B) Composition of bacteria in species level
Organism
NCBI GenBank Accession Number
Pseudomonas stutzeri
MF498510.1
Klebsiella pneumoniae
MF498509.1
Paenibacillus glucanolyticus
MF498508.1
Pseudomonas aeruginosa
MF498503.1
Enterobacter cloacae
MF498504.1
Neisseria sp.
MF498500.1
Enterococcus hirae
MF498499.1
Enterococcus faecalis
MF498498.1
Bacillus kochii
MF498496.1
16S Sanger sequencing Agarose gel electrophoresis
The bacterial families identified (Figure 2.A) are commonly seen in lung microbiome, but Paenibacillaceae is rarely being observed. Although bacteria belonging to Enterococcaceae were observed in lung cancer and bronchiectasis lungs, Enterococcus hirae is considered as a rare clinical isolate (Anghinah et al., 2013). According to Kim et al. (2005), Enterococcus faecalis has shown the ability to promote cancer in animal models.
CONCLUSIONS
METHODOLOGY Ethical clearance was obtained from Teaching Hospital, Kandy for the study. Patients attending to the Teaching hospital, Kandy with confirmed bronchiectasis (n=20) and lung cancer suspected patients (n=20), were selected by clinical presentation. Specimens representing URT; oropharyngeal (OP) swabs, and LRT specimens; Bronchoalveolar lavage (BAL) were collected.
Only 75% samples were able to produce a culture. Many OP swabs were culture negative. This can be due to intake of antibiotics or anesthetic agents administered during and prior to bronchoscopy, as swabs were obtained after bronchoscopy.
Table 1. Identified organisms with respective NCBI GenBank accession numbers
1. Anghinah R., Watanabe R., Simabukuro M., Guariglia C., Pinto L. and Gonçalves D. (2013) Case Reports in Neurological Medicine, 2013E, 1-3. 2. Bloom D.E., Ca¬ero E.T., Jané-Llopis E., Abrahams-Gessel S., Bloom L.R., Fathima S., Feigl A.B., Gaziano T., Mowa¬ M., Pandya A., Prettner K., Rosenberg L., Seligman B., Stein A.Z., and Weinstein C. (2011) Geneva: World Economic Forum. 3. Kim S., Tonkonogy S., Albright C., Tsang J., Balish E., Braun J., Huycke M. and Sartor R. (2005). Gastroenterology, 128, 891-906. 4. Somerville, W., Thibert L and Behr M A (2005) Journal of Clinical Microbiology, 43, 2996-2997 5. Tripathi K., Tripathi P., Nema S., Shrivastava A., Dwiwedi K., and Dhanvijay A. (2014) International Journal of Recent Trends in Science And Technology, 10, 461-464.
