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Home , Escherichia fergusonii, Shigella, Shigella boydii, Shigella dysenteriae, Shigella flexneri

IDENTIFICATION OF INTESTINAL MICROBES IN CHILDREN WITH 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 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 were predominant in both groups. , and were deteced in the diarrhea group, while , Proteobacteria and 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 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 and exist in Ionization/Mass Spectrometry (PCR/ESI- small amount3. Microbes in 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 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 .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 , 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 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 collection Bacteroidetes were only detected in point in IDH on the day or the next day. diarrhea group, while Actinobacteria and From the published literature, stool 0 Verrumicrobia were only detected in non- specimens can be stored at 2-8 C prior to 15 diarrhea group. Proteobacteria was processing . detected in both groups by the number of Of the 33 diarrhea subjects, there were samples in non diarrhea more than in 24 boys and 9 girls with an age range of 2 diarrhea group (25 vs. 24). years to 9 years. This is in accordance The results of multiple matches were with the Basic Health Survey Indonesia in dominated by clusters of / 2007 that claimed the prevalence of flexneri / in each diarrhea in boys was higher than girls, as sample group, 4 samples (D02, D13, D14 well as the prevalence by age group ie and D33) in the diarrhea group, and 4 age of 24-48 monthshad a higher samples (S10, S17, S23 and S38) in the prevalence than the age of 48 months 16 non diarrhea group. Sample D11, D33 and upwards . S11 were also detected single match The use of PLEX - ID of the faecal bacteriai. Streptococcus sp on D11, samples directly, as far as we know just Aquaspirillum gracile species on sample recently conducted in this study. In D33, and Vibrio sp. on sample S1 (Table previous studies on blood culture, the 3). suitability of the results obtained at the In this study, enteropathogens were species level by standard methodswas 9 determined using the assumption that the 86.75 % ( n = 234 ) , while from clinical organisms identified using PLEX-ID were isolates was 74% ( n = 156 ) identified organisms known as enteropathogens. In appropriately, with only 9 % were 17 single match, they were Campylobacter incorrectly identified .In this study, due to jejuni, Clostridium perfringens and E. coli; not using the comparison method, the in multiple matches, they were Shigella results of which can be spp and Vibrio spp. Table 4 shows that of expressed,wasobtained 80 % ( 64 of 80 ) The Indonesian Journal of Infectious Disease 3 detected the presence of bacteria, with 14 The non diarrhea group, was not of 64 samples detection results were the detected bacteria, such as result of multiple matches ( Table 3 ). The Campylobacter sp and Shigella sp. While detection results can not be distinguished in the diarrhea group, no detection of further thusreported as a cluster ( M. Rost, probiotic bacteria that play a role in personal communication, April 12, 2013 maintaining immunological conditions of )18. Of the 14 multiple matches samples, children.There may be an imbalance in the dominated by a cluster of Escherichia coli microbiota of the gastrointestinal tract of andShigella sp. This happens due to E. children with diarrhea than non diarrhe, coli and Shigella is a bacterial species with although it is more confirmed when the close ties phylogenic17. number of samples involved in this study Escherechia coli is the dominant is greater. bacteria in the gastrointestinal tract[19].It Detection of E.coli and K.pneumonia in is showed in both groups of the study that both groups of samples as most bacteria most of bacteria detectedwereE. coli. detected, and the similarity in the results Detection of Campylobacter jejuni and of multiple matches, implies similarities of , which are the intestinal microbiota in the subjects enteropathogens, in the diarrhea group, studied. Likely, this is due to the similarity distinguish diversity detection results with of subject demographics, which causes non-diarrhea group. Despite the similar environmental, habits and eating patterns study with different detection methods have similarities. In a study conducted by conducted by Bodhidatta (2010) in Yatsunenko et al (2012) stated that Thailand found that Campylobacter and differences in cultural tradition also affect Shigellawere also found in non-diarrheal food, exposure to pets and livestock, and samples20. manyother factors that could influence Moreover, it also detected the presence how and from where a gut of the Aquaspirillum gracilewhich have a microbiota/microbiome isacquired25. new name as Hylemonella gracilis. This The dominant phyla in diarrhea and bacterium iscommonly found in aquatic non-diarrhea group was Proteobacteria. ecosystems21. Other bacteria detected in were only detected in the diarrhea group wasAcidovorax diarrhea group were Firmicutes and avenae. This bacterium is a Gram- Bacteroidetes whereas that was only negative rod bacteria, not pigmented and found in non-diarrheal group were do not ferment lactose . Usually found in Actinobacteria and Verrumicrobia. The soil and water, as well known as plant composition of those bacterial phyla are pathogens. There are case reports stating intestinal microbial composition3,4.In this that the bacteria associated with the onset study, analysis of the intestinal microbial of catheter- related sepsis22.Although very composition were obtained from faecal rare, the role of environmental bacteria samples only describe the intestinal which are not usually found associated microbial composition in general and do with diarrhea should be evaluated and not describe the composition of intestinal studied in the future. microbes on the anatomical location. That All bacteria that were detected in the is because most of the results obtained in group of non-diarrhea is normal intestinal each sample only one bacterium and to be flora.Akkermansia muciniphila colonizes in able to know the different microbial the intestinal mucosal lining and amounted composition at each anatomical location, to 3-5 % of the entire community of samples should be obtained from the intestinal bacteria23.Bifidobacterium anatomical locations, such as the research species was intestinal commensal bacteria conducted by Frank et al4, Monstein et associated with the synthesize of al[26] and Morteau et al27. compounds that affect a human. B.longum Few studies noted that E. coli, express serine protease inhibitor which Campylobacter jejuni, Shigella spp and plays a role in the function of Vibrio spp as a cause of diarrhea in immunomodulator24. children15,20,28, while Clostridium perfringens is known to cause food 4 The Indonesian Journal of Infectious Disease poisoning causes gastroenteritis29,30. REFERENCES Investigation of pathogenic strains of E. coli, was not included in the study, thus 1. Sekirov I, Russell SL, Antunes LC, the analysis it is to be assumed that allE. Finlay BB. Gut microbiota in health coli detected as a pathogen. In the study and disease. Physiol Rev the relationship between enteropathogens 2010;90:859-904 and diarrhea incidence is not significant (P 2. Ley RE, Peterson DA, Gordon JI. = 0.571). There are two possibilities that Ecological and evolutionary forces support this statistical analysis: (1) all E. shaping microbial diversity in the coli detected (diarrhea = 18 and non- human intestine. 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Escherichia coli and Klebsiella gastrointestinal tract are uniformly pneumoniae were two most detected distributed along the colon and differ bacteria in both groups, suggest the gut from the community recovered from microbiota was influenced by feces. Appl Environ Microbiol environmenta, habits and eating patterns. 2002;68:3401-7. One of the well known probiotic bacteria 8. Franks AH, Harmsen HJ, Raangs GC, i.e. Bifidobacterium longum were found Jansen GJ, Schut F, Welling GW. only in non diarrhea group. Likely the Variations of bacterial populations in chance of children with enteropathogen measured by fluorescent detected in the stool would have diarrhea in situ hybridization with group- 0.724 fold more than children with no specific 16S rRNA-targeted enteropathogen detected. oligonucleotide probes. Appl Environ Microbiol 1998;64:3336-45

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9. Kaleta EJ, Clark AE, Johnson DR, 18. Rost M. PLEX-ID enquiries from Gamage DC, Wysocki VH, Cherkaoui Indonesia. In: Meulila F, ed.; 2013. A, et al. Use of PCR coupled with 19. Bannister B, Gillespie S, Jones J. electrospray ionization mass Infection Microbiology and spectrometry for rapid identification of bacterial and yeast bloodstream Management 3rd ed. Massachusetts: pathogens from blood culture bottles. Blackwell Publishing; 2006. J ClinMicrobiol 2011;49:345-53. 20. Bannister B, Gillespie S, Jones J. 10. Ecker DJ, Sampath R, Blyn LB, Infection Microbiology and Eshoo MW, Ivy C, Ecker JA, et al. Management 3rd ed. Massachusetts: Rapid identification and strain-typing Blackwell Publishing; 2006. of respiratory pathogens for epidemic 21. Pawlowski D, Raslawsky A, Siebert surveillance. Proc Natl AcadSci U S A G, Metzger D, Koudelka G, Karalus R. 2005;102:8012-7. Identification of Hylemonella gracilis 11. Brinkman CL, Vergidis P, Uhl JR, Pritt as an Antagonist of BS, Cockerill FR, Steckelberg JM, et Persistence J BioterrBiodef S3:004 al. PCR-electrospray ionization mass 2011:doi:10.4172/2157-526. spectrometry for direct detection of 22. Malkan AD, Strollo W, Scholand SJ, pathogens and antimicrobial Dudrick SJ. Implanted-port-catheter- resistance from heart valves in related sepsis caused by Acidovorax patients with infective endocarditis. J avenae and methicillin-sensitive Clin Microbiol 2013;51:2040-6. Staphylococcus aureus. J 12. Jacob D, Sauer U, Housley R, ClinMicrobiol 2009;47:3358-61. Washington C, Sannes-Lowery K, 23. Everard A, Belzer C, Geurts L, Ecker DJ, et al. Rapid and high- Ouwerkerk JP, Druart C, Bindels LB, throughput detection of highly et al. Cross-talk between by Ibis PLEX-ID Akkermansia muciniphila and technology. PLoS One 2012;7:e39928 intestinal epithelium controls diet- 13. WHO. Diarrhoeal disease. In: World induced obesity. Proc Natl AcadSci U Health Organization Fact Sheet 2009. S A 2013;110:9066-71. 14. Inc AM. PLEX-ID System Customer 24. Buffie CG, Pamer EG. Microbiota- Training Guide. In; 2011. mediated colonization resistance 15. Oyofo B, Subekti D, Tjaniadi P, against intestinal pathogens. Nature Machpud N, Komalarini S, Setiawan reviews Immunology 2013;13:790- B. Enteropathogen associated with 801. acute diarrhea in community and 25. Yatsunenko T, Rey FE, Manary MJ, hospital patients in Jakarta, Trehan I, Dominguez-Bello MG, Indonesia. FEMS Immun and Med Contreras M, et al. Human gut Microbiol 2002:139-46. microbiome viewed across age and 16. Kementerian Kesehatan RI. Situasi geography. Nature 2012;486:222-7. diare di Indonesia. Buletin Jendela 26. Monstein HJ, Tiveljung A, Kraft CH, Data &Informasi Kesehatan; 2011. Borch K, Jonasson J. Profiling of 17. Sampath R, Mulholland N, Blyn LB, bacterial flora in gastric biopsies from Massire C, Whitehouse CA, patients with - Waybright N, et al. Comprehensive associated gastritis and histologically biothreat cluster identification by normal control individuals by PCR/electrospray-ionization mass temperature gradient gel spectrometry. PLoS One electrophoresis and 16S rDNA 2012;7:e36528 6 The Indonesian Journal of Infectious Disease

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Figure

Figure 1. PLEX-ID result sheet which shows the detection of bacteria, with each having Q-score and the different levels (arrow).a.Shows the detection of a single bacterium, b.Shows the detection of the bacteria, and bacterial detection with the results of multiple matches and c. Shows no detectable.

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Figure 2. Phyla comparison chart based on the number of species bacteria detected in a sample group of diarrhea and non-diarrhea.

Tables

Table 1. Types of PLEX-ID detection results Detection of bacteria species No detectable Single match* Multiple matches bacteria detection

Diarrhea (n =33) 30 6 3 Non diarrhea (n =47) 28 8 13 Total (n=80) 58 14 16 *Single match means detection of bacteria in sample with definitive species name, it can be a multiple (more than one bacteria detected) or together with multiple matches detection.

Table 2. Bacteri detected in samples group diarrhea and non-diarrhea Bacteria in Number Family Phylum Bacteria in non Number Family Phylum diarrhea of diarrhea of samples samples samples samples Escherechia 18 Enterobacteriaceae Proteobacteria Escherechia 21 Enterobacteriaceae Proteobacteria coli coli Klabsiella 2 Enterobacteriaceae Proteobacteria Klabsiella 3 Enterobacteriaceae Proteobacteria pneumonia pneumonia Prevotella 1 Bacteroidaceae Bacteroidetes Bifidobacterium 2 Bifidobacteriaceae Actinobacteria albensis longum Staphylococ 1 Straphylococcaceae Firmicutes Eschenchia 1 Enterobacteriaceae Proteobacteria cus fergusonii epidermidis Eubacterium 1 Eubacteriaceae Firmicutes Akkermansia 1 Verrucomicrobiaceae Verrucomicrobia rectale muciniphila Clostnium 1 Clostriadiaceae Firmicutes perfringens Streptococcu 1 Streptococcaceae Firmicutes s vestibularis Streptococcu 1 Streptococcaceae Firmicutes s sp Acidovorax 1 Comamonadaceae Proteobacteria avenae Aquapinillum 1 Comamonadaceae Proteobacteria gracile Campylobact 1 Campylobacteraceae Proteobacteria er jejuni Shigella 1 Enterobacteriaceae Proteobacteria boydii

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Table 3. The results of multiple matches Diarrhea Bacteria Non Bacteria Sample code diarrhea Sample code D02 E.coli//S.sonnei S10 E.coli/Shigella flexneri/S.sonnei D11 E.coli/ S11 Vibrio proteolyticus/Vibrio sp. D13 E.coli/Shigella flexneri/S.sonnei S14 E.coli/Escherichia coli O157:not H7/Shigella sonnei D14 E.coli/Shigella flexneri/S.sonnei S17 E.coli/Shigella dysenteriae D15 E.coli/Escherichia coli O157:not S23 E.coli/Shigella flexneri/S.sonnei H7/Shigella boydii/Shigella sonnei D33 E.coli/Shigella flexneri/S.sonnei S26 E.coli/Shigella flexneri/S.sonnei S38 E.coli/Shigella flexneri/S.sonnei S46 E.coli/Escherichia coli O157:not H7/Shigella sonnei

Table 4. Cross tabulation between entropathogen and diarrhea incidence Enteropathogen Total + - diarrhea + 27 9 36 - 29 7 36 Total 56 16 72

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