Bacterial and fungal microflora in surgically removed lung cancer samples

Background Clinical and experimental data suggest an association between the presence of bacterial and/or fungal infection and the development of different types of cancer, independently of chemotherapy-induced leukopenia. This has also been postulated for the development of lung cancer, however the prevalence and the exact species of the bacteria and fungi implicated, have not yet been described. Aim To determine the presence of bacterial and fungal microflora in surgically extracted samples of patients with lung cancer. Materials and methods In this single-center prospective, observational study, tissue samples were surgically extracted from 32 consecutive patients with lung cancer, and reverse-transcription polymerase chain reaction (RT-PCR) was used to identify the presence of bacteria and fungi strains. Results The analysis of the electrophoresis data pointed out diversity between the samples and the strains that were identified. Mycoplasma strains were identified in all samples. Strains that appeared more often were Staphylococcus epidermidis, Streptococcus mitis and Bacillus strains, followed in descending frequency by Chlamydia, Candida, Listeria, and Haemophilus influenza. In individual patients Legionella pneumophila and Candida tropicalis were detected. Conclusions A diversity of pathogens could be identified in surgically extracted tissue samples of patients with lung cancer, with mycoplasma strains being present in all samples. These results point to an etiologic role for chronic infection in lung carcinogenesis. Confirmation of these observations and additional studies are needed to further characterize the etiologic role of inflammation in lung carcinogenesis.


Introduction
Lung cancer is the most common cancer worldwide, with 1.35 million incident cases annually, and consists one of the leading causes of mortality worldwide [1]. In addition to cigarette smoking, the major lung cancer risk factor [1], recent studies underscore an etiologic role for chronic pulmonary infection in lung carcinogenesis, acting either independently or as a cofactor to tobacco smoke in increasing lung cancer risk [2][3][4][5]. Experimental and clinical data correlate cancer development with the presence of certain pathogens, independently of chemotherapy-induced leucopenia [6][7][8].
Importantly, previous retrospective and prospective studies have relied on serologic characterization of chronic bacterial and fungal infections [14]. To the best of our knowledge, the prevalence of bacterial and/or fungal infection in surgically extracted samples of patients with lung cancer has not been previously reported. The aim of the present study, therefore, was to determine the presence of bacterial and fungal microflora in surgically removed tissue samples of patients with lung cancer, by using PCR methods and special primers.

Materials and methods
In this single-center prospective, observational study, tissue samples were surgically removed from 32 consecutive patients with lung cancer. The samples were maintained in RPMI culture medium (Sigma, R0883, Germany). The tissue was dissociated and 2 ml Trypsin -0,25% EDTA (Invitrogen, 25200-072, California) was added in order to detach the cells. The trypsin has been inactivated by using FBS (Invitrogen, 10106-169, California) and cells were centrifuged at 1,200 rpm for 10 min. Then cells were incubated in 25 cm 2 flasks (Orange Scientific, 5520200, Belgium) at 37°C, in a 5% CO2 atmosphere, until well developed. RNA was extracted using TRIZOL (Invitrogen, 15596-026, California) and was used as a template to generate cDNA using the First strand cDNA synthesis kit (Fermentas, K1612, Canada). The First strand cDNA was used as a template for the Gradient-PCR reaction, which was performed using GoTaq Flexi polymerase (Promega, M8305, USA). Primers have been designed with Gene Expression 1.1 software. The PCR conditions were set as follows: initial denaturation at 95°C for 10 min to activate the polymerase, 35 cycles of denaturation at 94°C for 45 sec, followed by annealing at 52-58°C for 45 sec and an extension step at 72°C for 60 sec. A final extension step was performed at 72°C for 10 min. The PCR products were then separated on 1.5% agarose gel (Merck, 1012360500, USA) stained with Gel-Green (Gentaur, 41005, Belgium), and finally observed under UV-light. A 100-bp ladder (Promega, G2101, USA) was used as marker.
This study was in compliance with the Helsinki Declaration. All participants gave informed consent and the study was approved by the institutional board review. Table 1 shows the primer pairs that were used in PCR to identify the specific pathogen strains. Table 2 presents the frequency of different species and strains in the samples that were examined. The analysis of the electrophoresis data pointed out diversity between the samples and the strains that were identified in them. Mycoplasma strains
These data strongly support the idea that lung cancer is a biofilm associated chronic infection. Biofilms are microorganism populations organized in a form of colonies using self-produced extracellular matrix that works as infrastructure material. The vast majority of the micro-"colonists" establish biofilms on any inert or diseased biological surface. They adhere to each other, divide, cooperate, and, progressively, their bio-mass grows, matures and finally disperses. It resembles malignant behavior (tumors composed by cancer cells and by stroma cells-monocytes, lymphocytes, microvessels, can metastasize). Therefore, many researchers imply that lung malignancies are communities of diverse pathogens resistant to antibiotics.
One of the major limitations in most of the previous studies was the use of serologic characterization to identify chronic bacterial or fungal infections [14]. This has resulted in conflicting results and great variability in relative risk estimations among seropositive individuals [14,15,[26][27][28][29]. This wide variability could also reflect the retrospective nature of most of the studies, the small sample sizes, or inadequate adjustment for confounding factors [14]. New techniques, such as PCR-RFLPs, Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and microcolony methods allow examination and analysis of microbial communities [30,31]. Analyzing the constituents of microbial biofilms responsible for lung disease may help us discover novel strategies to control malignancies.
To the best of our knowledge, the prevalence of bacterial and/or fungal infection in surgically extracted samples of patients with lung cancer has not been previously reported. Therefore, the main purpose of the present study was to determine the presence of bacterial and fungal microflora in surgically removed tissue samples of patients with lung cancer, by using PCR methods and special primers. In this study, specific primers were designed in order to amplify as many different strains of microorganisms. Pairs of primers that were designed were capable of amplifying Treponema, Neisseria, Legionella, Borrelia, Listeria, Helicobacter, Staphylococcys, Haemophilus, Bacillus, Leptospira, Streptococcus, Mycoplasma, Candida and Brachyspira species. It is worth noting that Mycoplasma species were observed in all samples. Staphylococcus epidermidis and Streptococcus mitis were almost seen in one quarter of patients. Neither Treponema strains nor Leptospira, Helicobacter, and Staphylococcus aureus strains were observed in this study.

Conclusion
A diversity of pathogens could be identified in surgically extracted tissue samples of patients with lung cancer, with mycoplasma strains being present in all samples. These results point to an etiologic role for chronic infection in lung carcinogenesis. Confirmation of these  observations and additional studies are needed to further characterize the etiologic role of inflammation in lung carcinogenesis, thus making it possible to apply new therapeutic modalities.