Molecular Studies of Fecal Anaerobic Commensal Bacteria inAcute Diarrhea in Children
�Ramadass Balamurugan, �Harish P. Janardhan, �Sarah George, yM. Venkata Raghava,yJayaprakash Muliyil, and �Balakrishnan S. Ramakrishna
Journal of Pediatric Gastroenterology and Nutrition46:514–519 # 2008 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition andNorth American Society for Pediatric Gastroenterology, Hepatology, and Nutrition
as during administrarhea (3). Several bacresidents of the gut o
Received April 22, 200Address correspondenc
Department of GastrointeVellore 632004, India (e-
Supported by an internWellcome Trust, UK; by aCouncil of Medical ReseDepartment of Science &
The authors report no
of Gastrointestinal Sciences, and {Department of Community Health, Christian Medic
Vellore, Tamil Nadu, India
ABSTRACT
Background and Objective: The commensal bacterial floraof the colon may undergo changes during diarrhea, owing tocolonization of the intestine by pathogens and to rapidintestinal transit. This study used molecular methods todetermine changes in the composition of selected com-mensal anaerobic bacteria during and after acute diarrhea inchildren.Materials and Methods: Fecal samples were obtained from46 children with acute diarrhea in a rural community duringan episode of acute diarrhea, immediately after recoveryfrom diarrhea, and 3 months after recovery. DNA wasextracted and quantitative polymerase chain reaction usingSYBR green and genus- and species-specific primers targeting16S rDNA were undertaken to quantitate the following groupsof bacteria: Bifidobacterium spp., Bifidobacterium longumgroup, Bacteroides-Prevotella group, Bacteroides fragilis,
Lippincott Williams & Wilkins.Un
tion of antibiotics, may lead to diar-terial species, derived from normalr from natural foods such as yogurt,
7; accepted September 23, 2007.e and reprint requests to B.S. Ramakrishna,stinal Sciences, Christian Medical College,mail: [email protected]).ational trilateral collaborative grant from theSenior Research Fellowship from the Indian
arch (R.B.); and by a FIST grant from theTechnology, New Delhi, India.
conflicts of interest.
514
Results: Bacteria belonging to the Bacteroides-Prevotella-Porphyromonas group, E rectale, L acidophilus, and Fprauznitzii groups were low during acute diarrhea comparedwith their levels after recovery from diarrhea. The pattern wassimilar in rotavirus diarrhea and nonrotavirus diarrhea.Administration of amylase-resistant maize starch as adjuvanttherapy was associated with lower levels of F prauznitzii at thetime of recovery but did not lead to other changes in the floralpattern.Conclusions: Specific classes of fecal bacteria are lower duringepisodes of acute diarrhea in children than during periods ofnormal gastrointestinal health, suggesting specific alterations inthe flora during diarrhea. JPGN 46:514–519, 2008. Key Words:Anaerobes—Bacteroides—Bifidobacteria—Children—Commensal bacteria—Diarrhea—Lactobacilli—Quantitativepolymerase chain reaction. # 2008 by European Society for
Pediatric Gastroenterology, Hepatology, a and Eubacterium rectale, relative to amplification of universal
nd Nutrition and NorthAmerican Society for Pediatric Gastroenterology, Hepatology,
and Nutrition bacterial domain 16S rDNA.
The human gastrointestinal tract plays host to a largenumber of bacteria, consisting of more than 400 species,their total number exceeding the number of cells in thehuman body. The large majority of these bacteria areanaerobic. These bacteria play an important role in humanhealth by producing nutrients, preventing colonization ofthe gut by potential pathogens, and affecting immuneresponses (1,2). Alterations in the bacterial flora, such
are used as probiotics to hasten recovery from diarrhea.Bacteria of the Lactobacillus genus and Bifidobacterium
genus, in particular, can prevent infective diarrhea inexperimental animals and in children (4–6).
Acute diarrhea is an important cause of childhoodmortality and morbidity in developing countries (7).During diarrhea the intestinal milieu is altered, andovergrowth of the intestine by pathogens is likely tochange the normal bacterial flora in the gut. Acutediarrhea may sometimes eventuate in persistent diarrheaand malnutrition (8), usually attributed to changes in theepithelial cells lining the intestine (9); it is conceivablethat persistence of diarrhea can actually be due tochanges in the normal bacterial flora of the gut. Previousstudies that examined the gastrointestinal flora in acutediarrhea using conventional culture techniques indicatedthat the fecal anaerobic flora are significantly reduced
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in number in acute diarrhea (10–12), leading to arelative predominance of aerobic bacteria. In onestudy, Bacteroides, Bifidobacterium, Lactobacillus, and
Eubacterium were found to be lower during diarrhea.Anaerobic bacteria are difficult to cultivate, in particularbifidobacteria, for which several media are used for cul-ture. Recent techniques to examine the anaerobic bacteriause molecular approaches that target 16S rDNA (13).
The management of acute gastroenteritis is based onadequate hydration using glucose-based oral rehydrationsolution (ORS) and early refeeding (14). In addition tothese measures, administration of amylase-resistant starchto children with diarrhea hastened recovery (15). It hasbeen suggested that this occurs through fermentation of thestarch to short-chain fatty acids by colonic bacteria. Pro-duction of short-chain fatty acids, such as butyrate, fromunabsorbed carbohydrate is one of the significant contri-butions of the colonic flora to human health. Inasmuchas amylase-resistant starch reaches the colon and is avail-able as a substrate for bacterial nutrition and growth, it ispossible that it may also have a prebiotic effect andselectively stimulate the growth of beneficial commensalbacteria.
The present study used real-time polymerase chainreaction (PCR) targeting the 16S rDNA to quantitativelycompare selected fecal commensal bacteria in childrenwith acute gastroenteritis: during, immediately after, and4 months after an episode of acute diarrhea. Given thatbifidobacteria and lactobacilli have probiotic effects andare known to affect the course of diarrhea (16), we specifi-cally targeted these bacterial groups, whereas Bacteroideswere included for comparison, being another major fecalanaerobegroup that may sometimes also have a deleteriouseffect on intestinal health. We also examined the effect ofshort-term administration of amylase-resistant high-amy-lose maize starch (HAMS) during diarrhea on the selectedfecal bacteria; to do this, the 2 major human fecal butyrate-producing bacteria, Eubacterium rectale and Faecalibac-terium prauznitzii (17), were additionally targeted.
MATERIALS AND METHODS
Study Participants and Study Design
The study was undertaken in a block of 20 villages coveredby the Community Health and Development Department of theChristian Medical College, Vellore. A trial of ORS acceptabilityis ongoing in these villages. In each village, primary health careis administered by a community health worker who resides inthe village. Children with acute diarrhea receive their firstassessment and care from this health worker, who dispensesORS to children with no dehydration or mild dehydration,whereas the parents of a child with severe dehydration areadvised to take the child to the nearest health center. In 10 of thevillages, health workers were provided with regular ORSpackets, whereas in the other 10 villages, health workers wereprovided ORS with adjuvant HAMS, which has a high content
of amylase-resistant starch, in separate sachets to be added tothe ORS at the time of reconstitution. Each village also had atrained volunteer who was responsible for detection and report-
ing of diarrhea cases in children to the base team. The healthvolunteers were informed about the study and were given stoolsampling kits to be provided to parents of the children undersurvey. When a child had acute diarrhea and the parent waswilling to comply with the study, the health volunteer immedi-ately informed the base team via telephone, and providedinstructions and a kit to the parent to collect a stool sampleat the earliest. Simultaneously, a person was dispatched fromthe base hospital to the appropriate village to collect the stoolsample, which was brought back immediately to the laboratory.Children 3 months to 5 years of age affected with acute diarrhea(defined as >3 episodes of watery stools in 24 hours) and withmild dehydration were recruited for this study. A second groupof student volunteer health informants in each village identi-fied the cases and reported them to the investigating team bytelephone. The health informants were provided with stoolcollection kits that included a screw-capped plastic tube, tissuenapkins, and a wooden spatula. Three samples of feces werecollected from each affected child: the first sample duringthe diarrheal episode, the second being the first formed stoolafter cessation of diarrhea, and the third at 3 months after theindex episode of diarrhea. All of the samples were collected bythe team member within 2 hours of being passed and weretransported immediately to the laboratory in a cold box andstored at �808C to be processed in batches. A medical researchofficer from the investigating team visited the village andinterviewed the parents of each child to obtain demographicinformation including age, sex, mode of delivery, and age atweaning. The study was approved by the Research Committeeof the Christian Medical College, Vellore.
DNA Extraction and PCR
DNAwas extracted from 200 to 250 mg (wet weight) of fecesusing the QIAamp DNA stool mini kit (QIAgen, Germany) (18),eluted in a final volume of 200 mL and stored at �208C.
Oligonucleotide primers were targeted at the 16srRNA gene(rDNA) sequences of the bacterial species or groups shown inTable 1. Primers were also used to amplify a conserved 16SrDNA sequence present in all bacteria (universal primer set,recognizing domain bacteria) (19), the amplification of whichserved as the denominator against which the amplifications ofthe other bacteria were compared. All of the primer sequenceswere derived from the previously published studies mentionedin Table 1 (20–22) with the exception of the primer sets forE rectale and F prauznitzii. The latter were designed usingPRIMER3 software, using sequences retrieved from the ribo-somal database project II (RDP-II), and screened using theBLAST program of the National Center for BiotechnologyInformation. The PCR primers against Bacteroides-Prevo-tella-Porphyromonas have been shown to amplify target bac-teria from the genera mentioned, whereas the Bifidobacteriumgenus–specific primers amplified total Bifidobacteriumspecies. The Bifidobacterium longum primers detected a smallgroup of bifidobacteria, including B infantis, B longum,B pseudolongum, and B suis. Similarly, the L acidophilusprimers amplified rDNA from a closely related group of
NSAL BACTERIA IN ACUTE DIARRHEA 515
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lactobacilli, including L acidophilus, L amylovorus, L amylo-lyticus, L crispatus, L gasseri, and L johnsonii. Eubacteriumrectale and Faecalibacterium prauznitzii primer sets were
J Pediatr Gastroenterol Nutr, Vol. 46, No. 5, May 2008
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TABLE 1. Primers used in study
Target organism Primer Sequence (50-30) Product size, bp Reference
Universal Forward TCCTACGGGAGGCAGCAGT 466 Nadkarni et al, 2002 (19)Reverse GGACTACCAGGGTATCTAATCCTGTT
species specific. The primers were synthesized by Genosys,SiGMA (Bangalore, India).
A gradient PCR was performed initially to standardize thePCR conditions. PCR amplification was performed with initialdenaturation at 958C for 10 minutes, followed by 40 cycles ofdenaturation at 958C for 30 seconds, annealing at 618C for30 seconds, and extension at 728C for 30 seconds. PCR productswere analyzed on agarose gel electrophoresis for the specificband of the amplified product. After standardization, theseconditions were then used to perform quantitative PCR (qPCR)to quantify bacterial levels from fecal samples. Quantificationof bacterial DNA was performed using the Chromo4 real timePCR system (Biorad, USA), using SYBR Green master mix(Eurogentec, Belgium). All of the PCR reactions were per-formed in duplicate in a volume of 20 mL, using 96-well fullskirt clear PCR microplate and PCR strip caps (AXYGENScientific, USA). Melting curve analysis was performed from408 to 958C with a plate read step after every 18C, and held at aparticular temperature for 10 seconds to check the specificity ofthe product formed. The Opticon 3.1 software (Biorad) plots therate of change of the relative fluorescence units (RFU) withreference to time (T) (-d (RFU)/dT) on the y axis versus thetemperature on the x axis, with the curve peaking at the meltingtemperature (Tm), and melting curve analysis was alwaysdone to check the specificity of the amplification. Quantifi-cation was based on the fluorescence intensity obtained fromthe intercalated SYBR Green dye. The cycle number at whichthe signal was first detected—the threshold cycle (Ct)—corre-lated with the original concentration of DNA template. DNAcopy was not expressed as absolute number but was expressedby the relative cycle threshold at which DNA for each target wasdetected relative to the cycle threshold at which universalbacterial DNA was detected after amplification. This relativequantification is done automatically by the Opticon 3.1 softwareand expressed as relative fold difference compared withthe reference (universal) amplicon.
The specificity of the primers was checked by performingPCR using standard strains of representative bacteria from eachbacterial group, and on the results of in silico PCR. Strains used
to check specificity of the PCR included Bifidobacterium ado-lescentis CIP64.59, Bifidobacterium angulatum CIP104167, Bifi-dobacterium bifidum CIP56.7, Bifidobacterium breve CIP64.69,
J Pediatr Gastroenterol Nutr, Vol. 46, No. 5, May 2008
Bifidobacterium infantis/longum CIP64.67, Bifidobacterium lac-tis CIP105265, Bifidobacterium longum CIP64.62, B. longumCIP64.63, Bacteroides uniformis ATCC8492, Clostridium per-fringens ATCC13124, Lactobacillus acidophilus ATCC4356,Bacteroides fragilis NCTC 9343, B. sporogenes, Clostridiumperfringens NCTC 8346, Enterococcus faecalis ATCC 51299,Eubacterium rectale DUN-128, and Faecalibacterium prauznit-zii. The Bifidobacterium-type strains were grown at 378C underanaerobic conditions in M20 broth or Bifidobacterium broth(HiMedia). The other organisms were cultured on enriched bloodagar. Bacterial colonies were removed, washed with phosphate-buffered saline, and resuspended in phosphate-buffered saline byadjusting against MacFarland tubes. Resuspended bacteria wereserially diluted (log dilutions from 107 to 100) and added tocontrol fecal samples. Fecal samples were also tested for rotavirus(the single most common cause of acute gastroenteritis in this agegroup) antigen using a commercially available enzyme-linkedimmunosorbent assay.
Statistical Analysis
Statistical analysis was carried out using GraphPad Prismversion 4.0 (www.graphpad.com). Relative fold differences ofthe selected bacteria were expressed as median values (inter-quartile range). Significance of differences between groups wastested using the Kruskal-Wallis analysis of variance with theDunn multiple comparison test for post-hoc analysis of differ-ences between individual groups. Two-tailed P< 0.05 wereconsidered statistically significant.
RESULTS
A total of 46 children (30 male, 16 female) were enrolledinto the study. They ranged in age from 3 months to 5 years,with a median age of 12 months. All but 3 of the childrenhad been weaned and were a median of 6 months afterweaning. Of these children, 13 had positive results
CTG GCT TCCCT TCA GT 203AAA CAC AT
authorized reproduction of this article is prohibited.
for rotavirus by enzyme-linked immunosorbent assay.The median socioeconomic status (23) was IV (rangeIII–V), indicating a relatively homogeneous low to
there were no significant differences between diarrhealperiods and healthy periods in the relative levels ofBifidobacterium species. To date, quantitative studies on
FIG. 1. Fecal Bifidobacterium group (Bifido) and Bifidobacterium FIG. 2. Fecal Bacteroides-Prevotella-Porphyromonas group (BP)
FIG. 3. Fecal Eubacterium rectale (E rectale), Faecalibacteriumprauznitzii (Fp) and Lactobacillus acidophilus (La) organismsduring diarrhea (1), immediately after (2), and 3 months later�
MOLECULAR STUDIES OF ANAEROBIC COMMENSAL BACTERIA IN ACUTE DIARRHEA 517
low-to-middle socioeconomic group. Of the total 46 chil-dren in the study, 23 received standard therapy with ORSand early refeeding, and the other 23 received ORStogether with early refeeding and supplemental HAMSon the first day of diarrhea.
As shown in Figure 1, the relative levels of Bifidobac-terium species and B longum group were not significantlydifferent between children during diarrhea, immediatelyafter diarrhea, or during a period without diarrhea.By contrast, relative levels of Bacteroides-Prevotella-Porphyromonas species were significantly lower duringand immediately after acute diarrhea than during theperiod without diarrhea (Fig. 2). The relative levels ofBacteroides fragilis were not significantly different duringdiarrhea, immediately after diarrhea, or during a period ofnormal health without diarrhea.
Levels of Eubacterium rectale were significantly lowerduring and immediately after diarrhea than during adiarrhea-free period of normal health (Fig. 3). Faecali-bacterium prauznitzii were also significantly less abundantduring or immediately after diarrhea than during normalhealth. The Lactobacillus acidophilus group showed aninteresting phenomenon, increasing immediately uponcessation of diarrhea with subsequent reduction in numberduring normal health. There was no major difference inbacterial predominance between the children positivefor rotavirus and those who were negative for rotavirus(data not shown). Table 2 shows the characteristics ofchildren receiving HAMS supplements on Day 1 com-pared with those receiving ORS alone. There was no majordifference in fecal bacteria between the children receivingHAMS and those receiving only conventional therapy,
longum (B longum) organisms during diarrhea (1), immediatelyafter (2), and 3 months later (3). Bars represent mean (SEM).None of the differences was statistically significant.
with the exception of Faecalibacterium prauznitzii, whichwas significantly lower in the HAMS group at the time ofrecovery from diarrhea (Table 3).
DISCUSSION
The present study indicates that there are subtle changesin the composition of the fecal anaerobic bacterial floraduring acute diarrhea in children. Inparticular, the numbersof Bacteroides-Prevotella group, the predominant fecalanaerobic bacteria, were lower during acute diarrhea.Lower levels ofEubacterium rectale andFaecalibacteriumprauznitzii were also noted during diarrhea. By contrast,
and B. fragilis (B. fragilis) organisms during diarrhea (1), immedi-ately after (2), and 3 months later (3). Bars represent mean (SEM).�P<0.01 when comparing healthy period to period during and
after diarrhea.
nauthorized reproduction of this article is prohibited.
(3). Bars represent mean (SEM). P<0.01 when comparinghealthy period to period during and after diarrhea. @P<0.01for diarrhea compared to nondiarrheal period. #P<0.01 for diar-rhea compared to nondiarrheal period.
J Pediatr Gastroenterol Nutr, Vol. 46, No. 5, May 2008
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TABLE 2. Characteristics of children in study
HAMS Control P
n 23 23Age, mo 15.7 (2.6) 15.9 (2.3) NSSex 12 M, 11 F 18 M, 5 F NSDiarrhea duration, days 4.7 (0.7) 5.3 (0.6) NSAntibiotic use 2 2 NSMonths since weaning 5.5 (0.4) 6.0 (0.6) NSDelivery by cesarean section 2 3 NS
TA
BL
E3.
Co
mp
ari
son
of
feca
lb
act
eria
inch
ild
ren
rece
ivin
gei
ther
HA
MS
OR
So
rst
an
da
rdth
era
py
Du
rin
gd
iarr
hea
Aft
erd
iarr
hea
No
rmal
hea
lth
HA
MS
Con
tro
lP
HA
MS
Co
ntr
ol
PH
AM
SC
on
trol
P
teri
um
spp
.0
.12
51
(0.0
30
6,
0.2
19
1)
0.1
44
3(0
.02
01
,0
.411
3)
NS
0.2
06
2(0
.15
86
,0
.32
06
)0
.207
5(0
.00
56
,0
.665
4)
NS
0.1
87
3(0
.06
68
,0
.33
60
)0
.03
28
5(0
.01
18
,0
.307
6)
NS
teri
um
0.0
02
35
(7.2
9E
-05
,0
.070
3)
0.0
26
83
(7.5
2E
-05
,0
.11
15
)N
S0
.006
63
1(0
.00
04
5,
0.0
16
0)
0.0
09
8(0
.00
03
9,
0.1
02
5)
NS
0.0
01
6(0
.00
04
7,
0.0
26
2)
0.0
01
59
(0.0
003
8,
0.0
07
7)
NS
es-
ella
0.0
78
62
(0.0
10
4,
0.1
52
1)
0.0
14
29
(0.0
02
4,
0.1
60
6)
NS
0.0
31
7(0
.00
71
,0
.06
78
)0
.016
64
(0.0
108
,0
.041
0)
NS
0.1
25
7(0
.04
00
,0
.52
48
)0
.07
25
7(0
.02
12
,0
.340
8)
NS
esfr
ag
ilis
9.6
2E
-05
(5.6
6E
-06
,0
.011
0)
0.0
00
21
4(8
.06
E-0
6,
0.0
00
27
)N
S9
.12E
-05
(3.8
3E
-06
,0
.001
6)
0.0
00
16
2(6
.20
E-0
6,
0.0
01
3)
NS
0.0
00
31
(5.0
1E
-05
,0
.002
6)
0.0
01
13
4(0
.00
01
4,
0.0
15
43
)N
S
ium
rect
ale
5.3
2E
-05
(1.6
7E
-05
,0
.000
2)
1.7
0E
-05
(9.5
5E
-07
,0
.00
01
7)
NS
3.7
3E
-06
(2.1
2E
-07
,3
.73E
-05
)5
.54E
-06
(8.0
8E
-08
,0
.000
76
)N
S0
.000
40
(2.6
3E
-05
,0
.007
4)
0.0
00
11
(4.6
8E
-05
,0
.001
8)
NS
cter
ium
itzi
i4
.52
1E
-06
(1.3
4E
-08
,2
.40E
-05
)1
.27
E-0
6(2
.46
E-0
9,
1.2
9E
-05
)N
S5
.491
E-0
7(2
.11
E-0
7,
3.3
4E
-05
)2
.45E
-05
(7.4
2E
-07
,0
.000
13
)0
.035
0.0
00
18
(4.8
0E
-06
,0
.002
0)
3.9
6E
-05
(1.6
2E
-05
,0
.000
11
)N
S
illu
sh
ilus
1.3
5E
-06
(6.9
6E
-09
,6
.24E
-06
)1
.60
8E
-07
(6.1
0E
-01
1,
2.9
5E
-06
)N
S4
.753
E-0
5(3
.39
E-0
5,
0.0
00
99
)0
.000
12
(1.2
2E
-05
,0
.000
32
)N
S2
.26E
-06
(6.4
2E
-08
,3
.53E
-05
)4
.57
E-0
6(1
.25
E-0
8,
2.4
3E
-05
)N
S
n(i
nte
rqu
arti
lera
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fre
lati
ve
dif
fere
nce
val
ues
of
the
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ance
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wh
ich
the
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imal
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dso
fort
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dic
ativ
eo
flo
wab
un
dan
ceb
acte
rial
gro
ups
or
spec
ies.
518 BALAMURUGAN ET AL.
the human gut microbiota in acute diarrhea have been doneexclusively through culture-based methods. These haveimportant limitations, primarily related to the fastidiousnature of the gut microbiota. The advent of real-time PCRmethods targeting 16S rDNA prompted this reexaminationof specified fecal anaerobic commensal bacteria in acutediarrhea.
Limited information is available on the commensalbacterial flora of the gut in acute diarrhea. An early studyfrom this institution (10) noted that there was a markedshift in fecal bacteria from predominance of anaerobes(which normally outnumber aerobes by a factor of�10) toa predominance of aerobes during acute diarrhea in chil-dren. This was attributed to an overgrowth of the gut bypathogens and to an alteration in the redox environment ofthe colon. A study from Kenya confirmed the markedreduction in anaerobes and found that there were alsolower short-chain fatty acid concentrations in the fecesduring acute diarrhea (11). The same group reported thatBacteroides, Bifidobacterium, Lactobacillus, and Eubac-terium were fewer during acute diarrhea than after recov-ery (12). In the present study, there was no apparentdisturbance of Bifidobacterium species, in contrast tothe culture-based study from Kenya. Bifidobacteria aredifficult to grow, and B adolescentis in particular mayrequire special media for its culture.
The significantly lower levels of the Bacteroides-Prevotella-Porphyromonas group and of Eubacteriumrectale and of the Faecalibacterium prauznitzii speciesduring acute diarrhea may be important in the pathophy-siology of diarrhea. Each component of the anaerobicbacterial flora contributes to fermentation in the gut.Metabolic interactions between different groups of bac-teria are important in the entire process of carbohydratefermentation. Studies using gnotobiotic animals showthat the capacity of Bacteroides to use carbohydrate isenhanced by cooperation with Bifidobacterium and Lac-tobacillus (24). Bacteroides populations have beenshown to be susceptible to changes in pH (being reducedin numbers at lower colonic pH) and peptide supply (25).
Socioeconomic status IV IV NS
Values mean (SEM). Socioeconomic status was determined by use ofthe modified Kuppuswamy scoring system (23).
Another observation in the present study was thatLactobacillus was increased in the feces when the chil-dren were recovering from diarrhea. Levels were low B
ifid
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J Pediatr Gastroenterol Nutr, Vol. 46, No. 5, May 2008
Cop
MME
during the phase of diarrhea, increased significantly justafter recovery, but then again declined to extremely lowlevels. The reason for this phenomenon was not inves-tigated in this study, but it could be related to dietarychanges and perhaps an increase in milk or yogurt intakeduring diarrhea. The bacterial changes were similar in thesubgroup of children positive for rotavirus, and it is likelythat changes were secondary to alterations in colonicmilieu rather to any specific effect of the pathogen.
There were no differences between children whoreceived HAMS and those who did not, except for areduction in Faecalibacterium prauznitzii in the formergroup. Again, there is no obvious explanation for this, butthe fact that it occurred immediately after recoverysuggests that it may be related to the adjuvant HAMS,which was the only ongoing intervention at that time thatwas different between the 2 groups. HAMS has been notedto shorten diarrhea, and its effect is attributed to colonicfermentation to short-chain fatty acids. The lack of effectof HAMS on fecal bacterial composition suggests that itdid not have an obvious prebiotic effect. However, childrenin the present community-based study received smallamounts of starch, compared with dehydrated infants inthe previous hospital-based study (15), who receivedgreater quantities of the starch. This may be one reasonfor the lack of a discernible prebiotic effect. This iscorroborated by another study that failed to find anyalteration in counts of Eubacterium, Clostridium, or Rumi-nococcus after the intake of resistant starch in healthyhuman volunteers (26). It is, of course, possible thatHAMS does not have a prebiotic effect at all but may stillserve as a substrate for short-chain fatty acid production.
In summary, alterations in the fecal anaerobic commen-sal bacterial populations are noted during acute diarrheain children. Further studies are needed to examine theimpact of these alterations on disease course and compli-cations in acute diarrhea.
Acknowledgement: The authors thank Prof GagandeepKang for the rotavirus tests.
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J Pediatr Gastroenterol Nutr, Vol. 46, No. 5, May 2008
