IDENTIFICATION OF INTESTINAL MICROBES IN CHILDREN WITH DIARRHEA ANDNON-DIARRHEA USING POLYMERASE CHAIN REACTION / ELECTROSPRAY IONIZATION-MASS SPECTROMETRY (PCR / ESI-MS) Teguh Sarry Hartono1, Dewi Murniati2, Andi Yasmon3, Lucky H Moehario3 1 Infectious Disease HospitalProf Dr Sulianti Saroso, Microbiology Resident–Departement of Microbiology, Faculty of Medicine, University of Indonesia. 2Infectious Disease Hospital Prof Dr Sulianti Saroso, 3Department of Microbiology, Faculty of Medicine, University of Indonesia. Abstract :Microbiota present in human intestinal are diverse, and imbalance in composition of intestinal flora may cause diarrhea.This study aimed to obtain a profile of intestinal bacteria in children with and without diarrhea and assess their presence with incidence of diarrhea. An analitical descriptive with cross sectional design study was carried out. A stool specimen was collected from each children of 2-12 years old with and without diarrhea who lived in North Jakarta. DNA extraction was performed prior to detection of microbes using Polymerase Chain Ceaction/Electrospray Ionization-Mass Spectrometry. Eighty stool specimens consisted of 33 and 47 specimens from children with and without diarrhea were included in the study. Thirty single and 6 multiple matches were detected in 30 specimens of the diarrhea group; 28 single and 8 multiple matches were found in 34 specimens of the non-diarrhea.Escherechiacoli and Klebsiella pneumonia were predominant in both groups. Firmicutes, Proteobacteria and Bacteroidetes were deteced in the diarrhea group, while Actinobacteria, Proteobacteria and Verrucomicrobia were in the non-diarrhea. The relationship of incidence of diarrhea and the present of enteropathogens in the stool was not significant, however, there was a strong correlation of the risk of suffering diarrhea due to the presence of enteropathogens (OR = 0.724 with 95%, CI: 0.237-2.215). In conclusion, most bacteria detected in both groups were similar, nonetheless, Actinobacteria was present only in the non-diarrhea. The chance to have diarrhea was higher when enteropathogen was detected in the stool. Keywords:Gut microbiota, diarrhea, PCR/ESI-MS Correspondence : were more prevalent4.Infection will affect dr. Teguh Sarry Hartono, Sp.MK on interaction among microbiota and Infectious Disease Hospital Prof Dr Sulianti trigger inflammation responses, and Saroso, Jalan Baru Sunter Permai Raya, consequently influence the composition of Jakarta 14350 intestinal microbiota5. Lupp C et al, 2007 Email : [email protected] reported that intestinal mucosal inflammatory process due to infection INTRODUCTION altered the residentobligate anaerobic Gastrointestinaltract (GIT) is themost bacteria and triggered excessive growth of heavily colonized organ; the colon alone commensal Enterobacteriaceae to 5 contains over 70% of all the microbes in become pathogenic . the human body1,2.The intestinal A variety of approacheswhich include 6 microbiota composed of more than 1000 full-length 16S rRNA sequencing , species3. Almost all of these species denaturing gradient gel electrophoresis by 7 (98%) belonged to only four bacterial Zoetendal et al , and fluorescent insitu 8 phyla i.e. Firmicutes (64%), Bacteroidetes hybridization by Franks et al had been (23%), Proteobacteria (8%), and used in the study of intestinal microbial Actinobacteria (3%)4.Other species such communities. Further, Polymerase Chain as members of Verrumicrobia, Reaction coupled with Electrospray Fusobacteria and Cyanobacteria exist in Ionization/Mass Spectrometry (PCR/ESI- small amount3. Microbes in small intestine MS), a technology based on PCR were enriched with the Bacilliclass of technique, and dispersion technology of Firmicutes and Actinobacteria, while in electro ionization mass spectrometry, has colon, Bacteroidetes and the been utilized for a complete identification 9,10 Lachnospiraceae, family of Firmicutes of a largenumber of microorganisms . It The Indonesian Journal of Infectious Disease 1 has a capability to identy multiple PCR-ESI/MS assay organism present in a sample up to Prior to PCR-ESI/MS, inhibitor in the species level simultaneously without prior samples were analized by conventional cultivication9,11,12. PCR. PCR-ESI/MS was performed by In this study we employed the using PLEX-ID Broad Bacteria panel in PCR/ESI-MS to identify intestinal accordance with manufacture’s instruction. microbes from stools of children aged 2-12 The data treshold is 0.85 confidence, in years with and without diarrhea in Jakarta, that less than 0.85 were reported and analyse their relationship to incidence uninterpratable14. of diarrhea. Such information will be valuablefor the development of Statistical analysis management of diarrhea in the context of Statistical analysis was performed by intestinal microbial balance. using SPSS ver 17 software. The relationship between entero pathogens and diarrhea cases was done using Chi- MATERIALS AND METHODS square test with a P-value of < 0.05 was considered significant. The risk Clinical specimens relationship (Odds ratio) between those The research was an analitical two was also measured. descriptive study with cross sectional design. Stool samples were collected from RESULT September 2012 to December 2012. Diarrhea samples were collected from out 1. Subjects profiles patients in Infectious Diseases Hospital Overall 80 subjects was obtained in (IDH) Prof Dr Sulianti Saroso and primary accordance with the inclusion criteria, health centers highlighted the close consisted of 33 children who had diarrhea relation area of study population in North (diarrhea subjects) and 47 children who Jakarta, Indonesia. Non diarrhea samples did not have diarrhea (non-diarrheal were collected from healthy children in subjects). Diarrhea subjects consisted of toddler classes and kindergartens in the 24 boys and 9 girls with an age range of 2 same area as mentioned above. The years to 9 years and the median age of 3 0 samples were stored at 4 C for not more years. While the non-diarrheal subjects than 24 hours and immediately consisted of 27 boys and 20 girls with an transported to laboratory using special age range of 2 years 3 months to 11 years 0 coolbox. All samples were stored at -70 C with a median age of 4 years and 4 until further process. Inclusion criteria for months. diarrhea sample were children aged 2-12 year which had an episode of acute 2. PLEX-ID result 13 diarrhea according to WHO criteria and PLEX-ID psitive detection means that has not received antibiotic.For non- the processor PLEX-ID reads one or more diarrhea sample,were children aged 2-12 microorganisms with a Q score > 0.85 in years, without episodes of diarrhea.This accordance with the spectrometer study was approved by Ethical Committee database PLEX-ID. If more than one of Faculty of Medicine, University of bacteria detected in one sample with a Indonesia. different Q score, means that there is more than one bacteria that has the same DNA extraction opportunities as the detected bacteria. Total DNA was extracted from 180-220 PLEX-ID detection would give three mg solid stool or 200 µl liquid stool. The kindsof result,as showedin Figure 1. extraction was performed by using Of 33 samples in diarrhea QIAamp® DNA Stool Mini Kit according to groupshowed the presence of 30 single the manufacturer's instructions with 200 µl matchbacteria and 6 multiple matches of the elution. The elution was stored at - bacteria, while the 3 other samples 0 70 C until processed. showed no detectable bacteria. In the non- diarrhea group (47 samples) showed 28 2 The Indonesian Journal of Infectious Disease single bacteria, 8 multiple matches and all cases with enteropathogens, 48.2% 13 samples showed no detectable (27/56) had diarrhea. While in the cases bacteria. with non-enteropathogens, which had diarrhea was 56.5% (9/16). Using Chi- 3. Detection of bacteria in samples square test gave P= 0.571. It means there Table 2 shows Escherechia coli is no significant relationship between dominated the diarrhea group, followed by enteropathogens with the incidence of Klebsiella pneumonia, both from the diarrhea.Odds ratio is used to see the phylum Proteobacteria. Another species strength of the relationship between the were Campylobacter jejuni, Acidovorax incidentof diarrhea by enteropathogens avenae and Aquaspirillum gracile, all from (OR 0,724 [95% CI: 0,237-2,215]). This Proteobacteria, while Staphylococcus means that the chance of having diarrhea epidermidis, Clostridium perfringens, caused by enteropathogens was 0.724 Streptococcus vestibularfrom Firmicutes fold than by non-enteropathogen. and Prevotella albensisfrom Bacteroidetes. DISCUSSION The non-diarrhea group also dominated by Echerechia coliand Klebsiella This study was conducted in four health pneumonia. Another Protobacteria centers and one hospital in North Jakarta. detected was Escherichia fergusonii. The health centers were selected on the Additionally it also detected the presence basis of the distance between the sites, of Actinobacteria (Bifidobacterium longum) which ranges from 4 km to IDH, to and Verrucomicrobia (Akkermansia facilitate sample collection. In addition, it muciniphila). was assumed homogenization of social, The diversity ofbacteria detected in the economic, environmental and climate of diarrhea group (12 of 30 samples) was the subjects. Specimens obtained were more than in non-diarrheal group (5 of placed in a cooler box with temperatures 0 28).Figure 2 shows Firmicutes and around 8 C before transfer to a