Chemotherapy Could Induce Antibiotic Resistance in E. Faecalis in Patients with Colorectal Cancer

| Background: Colorectal cancer is one of the most common cancers in Iran. There are many effective methods of treatment of it. As a conventional treatment, chemotherapy has become a part of treatment scheme for patients with colorectal cancer. Enterococci are intestinal commensals. They are opportunistic pathogens which cause millions of human and animal infections annually. The aim of this study was to investigate the side effects of chemotherapy of sufferers from colon cancer on the antibiotic resistance of microflora. Methods: In this study, participants were divided into three groups: Group A: 300 colorectal cancer patients before the start of the cancer chemotherapy, group B: 300 healthy people living with patients at least for recent 12 months and group C includes 300 patients with colorectal cancer after six weeks chemotherapy. RNA was extracted from the stool of all the participants of the study. Following the RNA extraction from stool samples, cDNA libraries were constructed. Eight virulent genes (vanA, vanB, gelE, esp, asa1, aggA, efaA and enlA) of E. faecalis were evaluated by real-time qPCR. results: The results were showed the expression level of the virulent genes in the group of the patients after chemotherapy was significantly higher than the two groups of B and C (P<0.05). Although the expression of these genes in the group of patients before chemotherapy was higher than that of the control group, this increase was not significant (P>0.05). conclusions: It seems that chemotherapy could change the balance of mRNA expression of microflora such as antibiotic resistance genes. These could be responsible for infections arisen after ending the chemotherapy of cancer.

cancers in the country (Khosravi Shadmani et al., 2017, Mohagheghi et al., 2009. Several chemotherapy regimens are used in the treatment of this disease, the common basis of which is the 5-FU (5-fluorouracil) (Tang et al., 2017, Fang et al., 2016.Chemotherapy aims at shrinking primary tumors, slowing the tumor growth, and killing cancer cells that may have metastasized to other parts of the body from the original (WELCH, 1959). An injectable 5-FU drug has been used as an effective drug in the first line of colorectal cancer metastasis treatment. 5-FU has a finite therapeutic dose range and its usage indicate remarkable individual difference (Baker et al., 2002, Gamelin andBoisdron-Celle, 1999).
One of the most pivotal side effects of every cancer treatment method in human is dysregulation of microbial flora functions (Tayebe Talebzade, 2017, Soha Sadeghi, 2018. That is related to decrease in the number and diversity of microbiota and susceptibility of under treatment's patients to infections such as gastrointestinal mucositis had reported in patients and rat models (Fijlstra et al., 2015, Tayebe Talebzade, 2017, Nicolatou-Galitis et al., 2006, Soha Sadeghi, 2018. Post treatment infections in cancer patients is a significant problem; and most reported infection in these patients which is commonly induced by E.Coli and Enterococcus faecalis (Seo and Lee, 2013, Liss et al., 2011, Hakim et al., 2018, Soha Sadeghi, 2018. Several studies have been conducted on the association between microbial flora isolated from feces and colorectal cancer. In some researches were reported arate of the microbial flora (such as Streptococcus bovis, Enterococcus faecalis, bacterioidophagylis, and Clostridium) isolated from stool of people with colorectal cancer had much more than healthy people (Michaux et al., 2014, Verneuil et al., 2005. an array of research had been done on the mechanism and function of these bacteria. Generally, these bacteria produce their metabolites in the cycle of proliferation, which may stimulate and proliferate colon mucous membrane cells and, in the long run, maybe lead to cancer (Montalto et al., 2004, Trenkner et al., 1987. The Gram-positive Commensal Enterococcus faecalis is a pivotal member of the human and other animals intestinal core microbiota. Also, they are isolated from environmental sources (Qin et al., 2010, Ocvirk et al., 2015. The ability to acquire several virulence genes (such as the antibiotic resistance) and the emerging importance in nosocomial infections (such as gastroenteritis, intestinal infections and endocarditis) clearly indicate its role as an opportunistic pathogen (Comerlato et al., 2013, Arias and Murray, 2012, Pinholt et al., 2014, Sievert et al., 2013. Several regulatory proteins with known functions were identified as pathogenicity factors in clinical and commensal Enterococcus isolates. Attendance and increase in expression levels of the virulence genes are considerably associated with infection, biofilm construction and antibiotic resistance in Enterococcus isolates (Semedo et al., 2003, Tayebe Talebzade, 2017. Some environmental and biological changes may be responsible for altering mRNA level of virulence genes of microbial flora (Lenz et al., 2010).
The purpose of this research was to scrutinize the side effects of the chemotherapy on the microflora of body as well as investigation of the mechanism of the antibiotic resistance of bacteria in cases with colon cancer undergoing chemotherapy.

SubjecT SelecTionS and STool Sampling
The Patient group includes 300 patients (168 males and 132 female) with early diagnosed adenocarcinoma of the colon which were candidates for chemotherapy with different protocols based on 5-FU or capecitabine that have been investigated for six period of cancer chemotherapy. The criteria for entering the study included age range of 40 to 60 years, hematologic status, and function of renal and hepatic were at normal levels. Patients should not have other cancer history and any chemotherapy. 300 healthy subjects selected from individuals which were lived with patients at least for recent 12 months due to microbial flora is strongly related to the life style of individuals. Also, age range, BMI and socioeconomic situation of the normal group were matched with patients group. The subjects with current and history of the severe medical condition including any infection or allergy were omitted from the study. All subjects were explained on the purpose of the study and next, written informed consent has been provided. The demographic characteristics of the subjects participating in the study are presented in Table 1.

gene expreSSion STudy by QuanTiTaTive real-Time pcr
Total RNA was directly extracted from fecal specimens using Viral RNA Mini Kit (catalog number: 52906, QIA-GEN®, USA). The quantity of extracted RNA examined by Denovix Nano drop device (Model Ds-11). The cDNA was synthesized according to the protocol of Transcription first strand cDNA synthesis kit (Revert Aid Premium First Strand cDNA Synthesis Kit #K1652, Thermo Scientific, Latvia). 16srRNA was selected as a housekeeping gene, then Specific primers for all genes was designed by "primer3" software. Quantitative RT-PCR in atriplicate method performed by using Rotor-Gene 6000 (Corbett Research, Mortlake, NSW, Australia), and 2X RealQ PCR master mix with Green DNA I dye (Ampliqon, Denmark) according to manufacturer's protocol.

STaTiSTical analySiS
Descriptive data are expressed as mean ± SD (range) and

Journal of Infection and Molecular Biology
December 2018 | Volume 6 | Issue 2 | Page 24 a= capable of normal activity, few symptoms or signs of disease; b= normal activity with some difficulty, some symptoms or signs; c= caring for self, not capable of normal activity or work; d= requiring some help, can take care of most personal requirements level of statistical significance was set at P < 0.05.Gene expression changes between studied groups were examined by paired t test and independent sample t test. SPSS (version 22) was used for statistical assessments.

rESultS
Demographic, clinical and pathological data of patients are presented at table1.The patients were in stage II, III and IV.

gene expreSSion reSulTS
The results of the expression level of the virulent genes in the group of the patients after chemotherapy was significantly higher than the two groups of healthy subjects and Patients before chemotherapy (P<0.05). However, the expression of studied genes in the group of patients before chemotherapy was higher than that of the control group, this increase was not significant (P>0.05) ( Table 2, 3 and  4).
From 300 treated patients, 128 patients were shown symptoms of infection between 3 and 5 chemotherapy periods. 101 patients showed Vancomycin (Van) antibiotic resistance. Therefore 27 patients were treated with vancomycin and 101 patients treated by ciprofloxacin (Cipro).

DIScuSSIon
Regarding the growth of the use of chemotherapy drugs in the treatment of various cancers in hospitals in Iran, the number of recipients of immunosuppressive medications is dramatically increasing daily. Unfortunately, this group of patients is continuously at risk of various types of infections (Hoos, 2012, Tayebe Talebzade, 2017, Soha Sadeghi, 2018.

Journal of Infection and Molecular Biology
December 2018 | Volume 6 | Issue 2 | Page 25 Results of the present study have been showing chemotherapy could change virulence gene expression of microbial flora. Moreover, it could be responsible for significant over expression of antibiotic resistance genes (vanA and vanB) in the group of the patients after treatment compared to normal subjects and patients before chemotherapy (P<0.05).This outcome has confirmed some result of the previous study in the side effect of the cancer therapy ways on germs and viruses. Tayebe  Nevertheles, our result was shown increased negligible expressionin patients group in comparison with related normal individuals which mean cancer ostensibly would not change gene expression pattern of virulence genes in microflora (Pvalue>0.05). This result was inconsistent with some results of previous studies. Some of the previous studies have indicated the rate of the prevalence of the microbial flora (such as Streptococcusbovis, Enterococcus faecalis, bacterioidophagylis, and Clostridium) isolated from the stool of people with colorectal cancer had much more than healthy people (Michaux et al., 2014, Verneuil et al., 2005 Also, several researchers claimed microbial flora could lead to colorectal cancer in their host's body by some their mechanism (Montalto et al., 2004, Trenkner et al., 1987. The difference between these results can be due to the difference in the studied method, the statistical society and the select of the community studied. Striking over-expression of all eight virulence genes in E. faecalis from treated patients' flora was detected. It seems six-period chemotherapy triggered the of the gene expression alterations of E. faecalis in patients which may increase the ability of microflora of body to infection. 101 participants with colorectal cancer after chemotherapy were observed vancomycin antibiotic resistance. It may be proofed the causing of the over expression of the vanA and vanB genes in the group of the patients after chemotherapy in compared with patients before chemotherapy. A pivotal question could be asked is that how cancer chemotherapy could affect microflora. The normal microflora acts as a barrier against overgrowth of opportunistic microorganisms as well as against colonization of potentially pathogenic microorganisms. Control of growth of opportunistic microorganisms is termed colonization resistance. Administration of antimicrobial agents, therapeutically such as chemotherapy, causes disturbances in the ecological balance between the host and the normal microflora (Sullivan et al., 2001). It is known that chemotherapy reduced or altered white blood cells production. That may be responsible for the development of bacteremia caused by Gram-positive and Gram-negative bacteria. A well-balanced microflora prevents the establishment of resistant microbial strains. An antimicrobial agents cause the risk of emergence and spread of resistant strains between patients due to they do not disturb colonization resistance. It is possible to presume that reduced number of the white blood cells could set up a natural selection in microflora population like what antibiotics effect do (Nowak et al., 2002).Therefore, if preventive measures are not done, an infection will be caused.
The most primary of this study is the investigation of side effects of chemotherapy on antibiotic resistance genesand pathogenic genes of microbial flora in CRC cancer patients. Therefore, level of the expression of virulence genes and VanA and VanB as two antibiotic resistances which is essential genes in E. faecalis were investigated. In order to reduce error in this study, patients were scrutinized before and after chemotherapy as well as healthy individuals were selected from familiar member of patients. Our result was shown chemotherapy might cause temporary antibiotic resistance and change balance microflora. Therefore, it is possible that measuring and evaluating viral and antibiotic-resistant genes of the body's natural microflora can be useful for patients under chemotherapy as well as it may contribute to prognosticate the onset of infection and select the appropriate antibiotic to treat the patient.