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790 Increase in Bacteroides/Firmicutes Ratio After Early-Life Repeated Administration of Cefixime In ORIGINAL ARTICLE Bali Medical Journal (Bali Med J) 2017, Volume 6, Number 3: 368-371 P-ISSN.2089-1180, E-ISSN.2302-2914 Increase in bacteroides/firmicutes ratio after ORIGINAL ARTICLE early-life repeated administration of Cefixime in Rat CrossMark Doi: http://dx.doi.org/10.15562/bmj.v6i3.790 Komang Januartha Putra Pinatih,1* Ketut Suastika,2 I Dewa Made Sukrama,1 I Nengah Sujaya3 Published by DiscoverSys Volume No.: 6 ABSTRACT The antibiotic administration could potentially to affects metabolism group. Both groups were transplanted orally with feces from 7-months and physiology by altering the gut microbiota composition and old infant boy prior to antibiotic administration. Cefixime group was Issue: 3 diversity. As early life is a critical period for metabolic development, given cefixime orally twice a day for five days in 3 consecutive periods microbiota disruption during this window period could have a with one-week interval. Microbiome analysis from caecal content was significant implication to the health status. In humans, early-life conducted to determine the changes in gut microbiota composition. microbiota disruption has been proven to be associated with increased This study found an increase in relative abundance of Bacteroides First page No.: 368 risk of overweight later in childhood. As the use of cefixime is rising and decrease in relative abundance of Firmicutes. An increase in in pediatric clinical infections, it is important to determine the Bacteroidetes/Firmicutes ratio by 32% was also observed. It was changes in gut microbiota composition after early-life exposure of concluded that early-life-repeated-administration of cefixime in rat P-ISSN.2089-1180 this antibiotic. In this study, six-week old male Wistar rats were used has a significant increase in Bacteroidetes/Firmicutes ratio. as an animal model, divided equally into a control group and cefixime E-ISSN.2302-2914 Keywords: Cefixime, Dysbiosis, Bacteroidetes/Firmicutes Ratio Cite This Article: Pinatih, K.J.P., Suastika, K., Sukrama, I.D.M., Sujaya, I.N. 2017. Increase in bacteroides/firmicutes ratio after early-life repeated administration of Cefixime in Rat. Bali Medical Journal 6(3): 368-371. DOI:10.15562/bmj.v6i3.790 1Department of Clinical INTRODUCTION Microbiology, Faculty of Medicine, Udayana University, Bali - Antibiotics are widely used in various clinical becomes the frontier in the various field of research. Indonesia infections and saved the millions life of people However, study about gut microbiota in Asia or 2 Department of Internal Medicine, worldwide every year. Despite their beneficial developing countries is still scarce while popu- Faculty of Medicine, Udayana University, Bali - Indonesia properties to combat infections, the use of antibi- lations in these regions have increased the risk of 3School of Public Health, Faculty of otic is one of the common and significant causes of chronic diseases related to changes in gut microbi- Medicine, Udayana University, Bali major alterations in normal gut microbiota.1 This ota composition due to change in diet or antibiotics - Indonesia event could potentially result in dysbiosis between overused. host and microbes which could be described as a Early life is a critical period for metabolic state in which the microbial community in the gut development. Thus, microbiota disruption during produces harm, through qualitative and quantita- this window period could lead to changes in body tive changes in the intestinal microbiota, bacterial composition.11 In humans, early-life microbiota metabolic activity and the local distribution of disruption is associated with increased risk of bacteria.2 Broad-spectrum antibiotics can signifi- overweight status later in childhood.12-15 Since cantly causing rapid drops in bacterial diversity. antibiotic is widely used in childhood, especially Short-term disturbances in the human gut microbi- in Indonesia, it can be postulated that antibiotics ota have been observed after antibiotics treatment, could potentially affect host metabolism and phys- and incomplete recovery of the microbiota to its iology by altering the composition of gut micro *Corresponding author: Komang initial composition has been found.3-6 biota. Januartha Putra Pinatih, Department Evidence in the last decade has shown that gut Recently, there is an increasing trend of the of Clinical Microbiology, Faculty of Medicine, Udayana University, JL. PB microbiota had a significant impact on host physi- usage of cefixime in a clinical setting, especially Sudirman, Denpasar, Bali, Indonesia ology.7,8 It significantly associated with some of the in children. Cefixime is a beta-lactam antibiotic 80232, Phone: +62 361 222510, most notorious chronic diseases such as obesity, with broad-spectrum activity and it usually used [email protected] type II diabetes mellitus, inflammatory bowel to treat respiratory tract and gastrointestinal infec- disease, and colorectal cancer.7,9 It even implicated tion, two of the most common diseases in children. Received: 2017-08-03 in the effectiveness of cancer chemotherapy as it Unfortunately, there is no report regarding the Accepted: 2017-08-25 modulates host immune system.10 Because of its effect of this antibiotic to gut microbial diver- Published: 2017-09-1 wide clinical implications, gut microbiota quickly sity. Therefore, this study was aimed to reveal the 368 Open access: www.balimedicaljournal.org and ojs.unud.ac.id/index.php/bmj ORIGINAL ARTICLE change in the gut microbial diversity after repeated reads were clustered into Operational Taxonomic exposure of cefixime in early-life. Units (OTUs) based on 97% of sequence iden- tity using USEARCH v.5.2.236 algorithm within RESEARCH DESIGN AND METHODS QIIME v.1.8.0 software. After OTU picking, representative sequence set was extracted from the Animal model clustered OTUs. Both extracted reads 1 and reads 2 Six 6-weeks old male Wistar rats were used as an were separately aligned onto the Greengenes v.13.8 animal model. The rats randomly divided into database to obtain the taxonomy annotation. The two groups (3 rats per group), i.e., control group abundance of each microbial taxon is estimated (receiving no antibiotic treatment) and cefixime from the absolute abundance values of the OTUs group. All rats were singly housed under controlled which further normalized by the expected number conditions (12:12 light-dark cycle, 22-24°C and of copies of 16S rRNA genes present in the genome 50% relative humidity) with free access to water and of each microbial species. standard rodent chow. Prior to antibiotic exposure, all rats were transplanted orally with feces from Statistical Analysis 7-months old infant boy once a day for five days. The data were analyzed using a statistical computer program. Shapiro-Wilk test was used to deter- Antibiotic Administration mine normality of data. Homogeneity of data was One week after last fecal transplant, antibiotic analyzed using Levene’s test. Normal data presented treatment was started. Cefixime 8 mg/100 grams as mean ± SD, and median (minimum-maximum) of body weight were given orally by gastric gavage, was used for data that were not distributed normally. twice a day for five days. Antibiotics was given in Statistical significance between the two groups was 3 consecutive periods with a one-week interval. determined using Independent t-test, with p value Two weeks after last antibiotic administration, rats of < 0.05 was considered significant. were sacrificed and caecal content was collected for microbiome analysis. RESULTS Microbiome Analysis At the end of the study, the rats were sacrificed and Microbiome analysis was conducted by using Next the gut microbiome data were analyzed using the Generation Sequencing on Illumina Nextseq plat- caecal content. Before proceeding to bivariate anal- form. The 16S rRNA genes were amplified at V3-V4 ysis, all data obtained were first tested for normality hypervariable regions and the resulted amplicon dan homogeneity. Shapiro-Wilk’s test showed that library was sequenced in paired-end (PE) mode all data were normally distributed. Meanwhile, with length 150 bases. The surviving paired-end Levene’s test showed homogeneity in all data except for Firmicutes relative proportion (Table 1). Microbiome analysis revealed that community of Table 1 Normality and variance analysis of the research data gut microbiota in control group was dominated by Normality test/ phyla Firmicutes (66,22% ± 3,52) and Bacteroides Kolmogorov Smirnov Levene Test (14,78% ± 0,78), with a Bacteroides/Firmicutes ratio Statistic p-value F P- value 0,22 ± 0,02. Cefixime exposure significantly increase Firmicutes 0,264 0.200 9,234 0,038 the relative proportion of Bacteroides to 17,40% ± 0,40 (p=0,011) and Bacteroides/Firmicutes ratio Bacterioidetes 0,232 0.200 2,483 0,190 to 0,29 ± 0,01 (p=0,005). A decrease in the relative Bacteroidetes/Firmicutes 0,252 0.200 0,617 0,476 proportion of Firmicutes (58,69% ± 0,60) was also Ratio observed but not statistically significant (p = 0,062). All results were presented in Table 2. Table 2 Relative Abundance of Firmicutes, Bacteroidetes, and Bacteroidetes/Firmicutes Ratio DISCUSSION Group Besides genetics, age and dietary habits, antibi- Control Cefixime otic treatment is a major factor contributing to (n=3) (n=3) alteration in gut microbiota composition. Because Firmicutes 66,22% ± 3,52 58,69% ± 0,60 of non-specific nature of most antibiotics, their Bacteroidetes 14,78% ± 0,78 17,40% ± 0,40 * administration is not only affecting the targeted Bacteroidetes/Firmicutes Ratio 0,22 ± 0,02 0,29 ± 0,01 * pathogen but also the normal microbe of the gut3. Data is presented in Mean and Standard Deviation The potency of an antimicrobial agent to influence * p<0,05 the gut microbiota is related to its spectrum of Published by DiscoverSys | Bali Med J 2017; 6(3): 368-371 | doi: 10.15562/bmj.v6i3.790 369 ORIGINAL ARTICLE activity, pharmacokinetics, and dosage, modes of obesity and type II DM in younger age.
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