Microbial composition and fecal fermentation end products from colicky infants - a probiotic supplementation pilot

ORIGINAL ARTICLE

Microbial composition and fecal fermentation end products fromcolicky infants � a probiotic supplementation pilot

SILJA MENTULA1, TUULA TUURE2, RAITA KOSKENALA3, RIITTA KORPELA2,4 &

EIJA KONONEN1

1Anaerobe Reference Laboratory, Department of Bacterial and Inflammatory Diseases, National Public Health Institute

(KTL), 2Research and Development Centre Valio Ltd, 3Department of Applied Chemistry and Microbiology, University of

Helsinki and 4Institute of Biomedicine, Pharmacology, University of Helsinki, Helsinki, Finland

AbstractObjectives: The etiology of infantile colic has remained unknown. The present study aimed to identify possible differences inthe fecal microbial composition and metabolic end products between colicky and non-colicky infants and to examinewhether an orally administered probiotic supplementation has any effect on the microbiota and relief in colicky symptoms.Subjects and methods: The study population consisted of nine colicky and nine non-colicky infants. Five colicky infantsreceived probiotic supplementation (Lactobacillus rhamnosus GG, Lactobacillus rhamnosus LC705, Bifidobacterium breveBbi99, and Propionibacterium freudenreichii ssp. shermanii JS) and four colicky infants received placebo for 2 weeks. Fecalmicrobiota of all infants was studied by culture, cellular fatty acid (CFA) analysis, and gas and short chain fatty acid (SCFA)production in 48 h fermentation. Microbial parameters and crying profiles were compared between the cases and controls atbaseline and between the probiotic and placebo supplementation after 2 weeks. Results: Although SCFA and gas productionand bacterial total counts were similar, the prevalence of indole-producing coliforms was significantly higher in colickyinfants compared with controls (89% vs 33%), while many aerobic genera present in controls were not detected in colickyinfants. CFA composition reflected the differences, since �2% of the CFAs were present exclusively in the colicky group.After probiotic supplementation, the total counts of anaerobic bacteria, especially lactobacilli and bifidobacteria, increasedsignificantly. Fermentation parameters were not extensively affected; however, acetic and lactic acid production tended toincrease and hydrogen production tended to decrease. No significant difference was observed in crying patterns between theprobiotic and placebo group. Conclusion: The composition of the intestinal microbiota differs between colicky and non-colicky infants. Although no apparent relief in colicky symptoms was achieved, the probiotic supplementation seems toincrease the bacterial diversity and strengthen the succession towards a balanced commensal gut microbiota.

Key words: infantile colic, fecal bacteria, probiotics, colonization, SCFA

Introduction

Infantile colic is a common problem; the prevalence

rate varies between 9% and 17%, depending on the

criteria and methodology used (1). The etiology of

colic has remained unknown despite the fact that a

wide range of behavioral, gastrointestinal, and func-

tional factors havebeen connected with it (2). The role

of the composition and activities of the intestinal

microbiota has not been fully determined yet. No sin-

glebacterial by-product,pathogen,or specieshasbeen

found to be the immediate cause of infantile colic.

The commensal microbiota of neonates develops

gradually; in general, the very first colonizers are

facultative enterobacteria and enterococci, followed

by bifidobacteria and other anaerobic species (3). A

number of factors, such as the mode of delivery,

microbes in the immediate environment, and form of

feeding, influence the composition of the early

intestinal microbiota (4,5). Birth by cesarean section

delays infants’ colonization by lactobacilli, bifido-

bacteria, and the B. fragilis group organisms early in

life (4,5).

Correspondence: Silja Mentula, Anaerobe Reference Laboratory, National Public Health Institute (KTL), Mannerheimintie 166, 00300 Helsinki, Finland.

Tel: �358 9 4744 8161. Fax: �358 9 4744 8238. E-mail: [email protected]

Microbial Ecology in Health and Disease. 2008; 20: 37�47

(Received 23 February 2007; accepted 18 December 2007)

ISSN 0891-060X print/ISSN 1651-2235 online # 2008 Taylor & Francis

DOI: 10.1080/08910600801933846

In addition to intrinsic factors, the intestinal

microbiota can be modified with bacterial therapy.

Probiotic strains of Lactobacillus, Bifidobacterium,

and Propionibacterium are known to provide several

beneficial effects on the host (6,7). Long-term

consumption of infant formula containing Bifidobac-

terium lactis and Streptococcus thermophilus was well

tolerated and reduced colic and irritability as well as

antibiotic usage in infants aged 3�24 months (8). A

significantly greater reduction in colicky cry was

reported with Lactobacillus reuteri compared with

simethicone (9). A newborn baby is, at least in

theory, an ideal object for bacterial therapy because

his/her indigenous microbiota is still immature and,

therefore, may be easily manipulated (10).

Fermentation capacity measured as production of

gas and short chain fatty acids (SCFAs) has been

used to evaluate the microecology of the gut and

functionality of the microbiota (11,12). These para-

meters and cellular fatty acid (CFA) composition

(13) also reflect the composition of the microbiota.

Bacterial metabolites are used as energy source by

the host and other bacteria (6). On the other hand,

when accumulated some end products of especially

protein breakdown and dissimilatory amino acid

metabolism (indoles, amines, phenols) are bioactive

and may become harmful (11). If the basis of colic

lies in the disturbance of the intestinal microbiota,

probiotic supplementation might be a proper means

to balance the microbial composition, metabolites,

and activities in the gut.

The aims of the present study were to determine

whether the composition of the fecal microbiota,

studied by culture and culture-independent CFA

analysis, or microbial metabolites (SCFAs, gases)

differ between colicky and non-colicky infants aged

1�6 weeks and whether an orally administered

probiotic supplementation (Lactobacillus rhamnosus

GG, L. rhamnosus LC705, Bifidobacterium breve

Bbi99, and Propionibacterium freudenreichii ssp. sher-

manii JS) has any effect on the monitored symptoms

or microbial parameters. In this pilot study a wide

variety of laborious methodology was used for a

relatively small but carefully chosen group of

infants.

Subjects and methods

Participants and study design

Infants in the Helsinki area were recruited in

maternity clinics, maternity hospitals, and child

health care centers. Their mothers gave a written

informed consent, and the City of Helsinki Ethical

Committee of Health Care approved the protocol.

The mothers were interviewed and instructed on

keeping a daily diary of sleeping, eating, and crying

habits of the baby, including the type (mild, moder-

ate, severe) and duration of crying. The inclusion

criteria for infants were: age 3�6 weeks and colicky

symptoms as described by Wessel et al. (14), i.e. 3 h

of crying per day for 3 days per week for 3 weeks.

The exclusion criteria were: congenital malforma-

tions or abnormalities, bowel diseases, prematurity

(B38 gestational weeks or birth weight B2500 g),

antimicrobial therapy during 2 preceding weeks,

flatulence associated with feeding or consumption

of other probiotic-containing products. Altogether

18 breast-fed infants were recruited in the case group

and 9 infants in the control group. However, after

the first study week (week 0) nine infants were

excluded from the case group because the amount of

crying did not strictly fulfill the inclusion criteria.

Demographic characteristics of the 18 infants (9

cases and 9 controls) who fulfilled the inclusion

criteria are shown in Table I.

Capsules each containing a mixture of probiotic

bacteria (L. rhamnosus GG, ATCC 53103 (LGG),

5�109 cfu; L. rhamnosus LC705, 5�109 cfu; B.

breve Bbi99, 2�108 cfu; and P. freudenreichii ssp.

shermanii JS, 2�109 cfu), and microcrystalline

cellulose as a filling agent (Valio Ltd, Helsinki,

Finland) were given to five of nine cases, whereas

the remaining four infants received placebo capsules

(microcrystalline cellulose) once a day for 2 weeks in

a randomized, double-blind manner. The contents

of the capsules were suspended in water or breast

milk by the mothers.

Table I. Demographic characteristics of the infants at enrolment.

Parameter Colicky Control

Gender

Males 3 3

Females 6 6

Age (weeks)

Mean 3 4

Range 2�5 1�6

Mode of delivery

Vaginal 8 6

Cesarean 1 3

Birth weight (kg)

Mean 4.2 4.2

Range 3.3�5.0 3.7�4.6

Type of feeding

Breast-fed 9 8

Formula-fed 0 1*

Colicky cry (h/week)

Mean 16 �$Range 9�28 �$

*Fed with both breast milk and formula.

$Sporadic, infrequent.

38 S. Mentula et al.

Microbiology

Fecal samples were collected at baseline and after 2

weeks of the probiotic or placebo supplementation.

The samples were transported anaerobically in

containers sealed in gas-impermeable plastic

pouches (AnaerocultA, Merck, Darmstadt, Ger-

many) within 4 h of defecation and processed

immediately. After homogenization the pH of the

feces was measured (Benchtop 420 pH Meter, Orion

Inc., Beverly, ME, USA), and the homogenate was

serially diluted (10�1�10�7) in prereduced PY

(peptone-yeast extract) broth (pH 7.0). Aliquots of

10 ml or 100 ml of the homogenates or appropriate

dilutions were plated onto several non-selective and

selective agar media and incubated at 368C as

follows: blood (5% sheep blood) agar for total

aerobes in 5% CO2 atmosphere for 2�4 days; cystine

lactose electrolyte-deficient (CLED) agar for coli-

forms, bile esculin (BE) agar for enterococci, and

Sabouraud agar with chloramphenicol for yeasts in

ambient air for 2�4 days; brucella agar supplemented

with hemin and vitamin K1 for total anaerobes,

bacteroides bile esculin (BBE) agar for bile-resistant

Bacteroides spp., tomato juice-based agar supplemen-

ted with hemin and maltose for bifidobacteria,

deMann Rogosa Sharpe (MRS) agar for lactoba-

cilli, cycloserine cefoxitin fructose egg yolk (CCFA)

agar for Clostridium difficile, and neomycin egg-

yolk (NEYA) agar for clostridia in anaerobic jars

filled with gas mixture (90% N2, 5% CO2, 5% H2)

for 5�7 days.

The total counts and main groups of anaerobic

and aerobic bacteria and yeasts were enumerated

from applicable plates (detection limit 102 cfu/g wet

weight). Different colony morphotypes were isolated

under a stereomicroscope and identified using es-

tablished methods, including aerotolerance testing,

Gram stain, various biochemical tests, metabolic end

product analysis by gas-liquid chromatography, and

enzyme profiling (15,16). Coliforms were further

identified with Api 20E and lactobacilli with Api

50CHL (bioMerieux, Marcy l’Etoile, France). The

presence of C. difficile toxins A and B in feces was

determined with commercial kits (Premier Toxins

A&B, Meridian Bioscience Inc., Cincinnati, OH,

USA, and C. difficile toxin A test, Oxoid, Hampshire,

England) according to the manufacturers’ instruc-

tions. The species and clonal identity of LGG

isolates were confirmed by arbitrarily primed PCR

(17). Other probiotics were identified by conven-

tional methods including metabolic end product

analysis.

Cellular fatty acids (CFAs)

For the analysis, 100 mg of feces was suspended in

5 ml PY (peptone-yeast extract) broth, allowed to

sediment at 48C for 2 h, remixed, and allowed to

sediment for 15 min. Supernatant was removed and

centrifuged for 15 min (1000 g). Fatty acids were

extracted from the pellets as described in the

operating manual of the Microbial Identification

System (MIS) software package by MIDI (Microbial

ID, Inc., Newark, DE, USA). The procedure

includes saponification (cell lysis), methylation (for-

mation of methyl esters to increase volatility for

GC), extraction, and wash. CFA analysis was

performed with an HP 5890 series II gas chromato-

graph equipped with an HP U2 cross-linked 5%

phenyl methyl silicone fused capillary column with a

flame ionization detector (Hewlett-Packard, Palo

Alto, CA, USA). Hydrogen was used as carrier gas.

Injection temperature was 2508C, that of detector

3008C, and a cycle of 1708C, 2608C, and 3108C for

the oven.

Fermentation

Fermentations were performed in triplicate in 20 ml

glass head-space vials containing 10 ml of either PY

broth or PY with 1% glucose (PYG) inoculated in an

anaerobic chamber with fresh fecal homogenate

(final concentration 0.2% w/v) and sealed with

rubber stoppers and aluminium seals. The vials

were incubated at 368C in a shaker (100 rpm/min)

and after 48 h transferred to 48C until analyzed

within few hours.

Percentage of carbon dioxide (CO2), hydrogen

(H2), methane (CH4), and air of the bottles head-

space gas volume were measured at 0 h and 48 h by

HP gas chromatograph model 5890 equipped with

Porapak N column and molecular sieve (Hewlett-

Packard). The amount of oxygen (air) was moni-

tored to control the atmospheric conditions of

fermentation. Before the measurement, the syringe,

valve, and needle were flushed with helium to

remove air. Helium was used as the carrier gas (total

flow 20 ml/min). The temperature of the injector

was 1508C, that of oven 458C, and detector 2008C.

The baseline (0 h) values were subtracted from

the 48 h values. As only traces or no methane-

thiol (CH3SH) or hydrogen sulfide (H2S) were

expected from breast-fed infants (18) they were not

monitored.

Both volatile and non-volatile metabolic fatty

acids were analyzed. Before analysis, the fermenta-

tion suspension was acidified with sulfuric acid and

centrifuged (Labofuge GL, Heraeus, Hannover,

Microbial parameters in infantile colic 39

Germany) for 30 min (5000 g). SCFAs were

extracted with methyl tert-butyl ether (16 Jousi-

mies-Somer) and analyzed with an HP Agilent Plus

GC 6890 equipped with an HP Innowax column

(Hewlett-Packard). The carrier gas was hydrogen

(constant flow 15 ml/min). Injection and FID-

detector temperatures were 2508C and oven tem-

perature was 2008C (initial 2 min 1008C).

The gas production of selected coliform isolates

from baseline samples was analyzed using pure

cultures by adding 200 ml bacterial suspension (1:10

dilution of Mcfarland 4 in NaCl) similarly in head-

space vials containing 10 ml PY broth with 5% lactose

(PYL). Only the total volume (ml) of gas produced

after 5 h incubation at 368C in a shaker (100 rpm/

min) was measured using a gas-tight syringe and

allowing the piston to be driven up by the pressure

generated in the vial during fermentation.

Statistics

Statistical analyses were performed using SPSS14

(SPSS, Chicago, IL, USA) and Epi Info 6 Statcalc

(CDC, Atlanta, GA, USA) software for Windows.

Related samples t test and single table test were used

to compare the concentrations and prevalence be-

tween the groups, and paired samples t test for

comparison before and after the probiotic or placebo

supplementation. A p value B0.05 was considered

statistically significant.

Results

Microbial findings in colicky and non-colicky infants at

baseline

The number of different isolates per sample was

similar between the colicky and control infants (9.3

vs 9.7) and the fecal pH values were also similar (5.5

vs 5.3). Table II presents the prevalence and

concentration of microbial taxa detected in the cases

and controls. The predominant anaerobic findings

were bifidobacteria followed by the Bacteroides fragilis

group organisms and lactobacilli, and predominant

aerobes were indole-positive coliforms, coagulase-

negative staphylococci, and enterococci. The pre-

valence of indole-positive coliforms (Escherichia coli

and Klebsiella oxytoca) was significantly higher in the

colicky infants than in the controls: 89% (8/9) vs

33% (3/9), p�0.016. The total load measured as the

proportion of indole-positive coliforms of the total

count varied but was slightly higher in colicky than

control infants: 8.7% (SD 16.9) vs 2.3% (SD 6.2)

(NS). When grouped together, gram-negative anae-

robes covered a slightly higher proportion of the

microbiota in the colicky group than in controls:

Table II. Microbial findings (log cfu/g wet weight) in nine colicky and nine control infants at baseline.

Control Colicky

Parameter n Mean Range n Mean Range

Total anaerobic growth 9 10.2 8.5�10.7 9 10.3 8.8�10.7

Total lactobacilli 6 9.0 5.0�9.5 7 9.5 3.4�10.3

Non-LGG lactobacilli 6 8.9 5.0�9.5 7 9.5 3.4�10.3

L. rhamnosus GG 1 8.2 0.0�9.2 0

Bifidobacteria 8 10.0 8.3�10.7 8 9.9 8.6�10.5

Clostridium spp. 2 7.8 8.5 4 7.5 6.5�8.3

Gram-positive rods$ 4 8.8 6.0�9.6 2 8.8 8.0�9.8

Bacteroides fragilis group 6 9.3 5.3�10.0 7 9.6 5.0�10.3

Gram-negative rods% 1 3.0 0.0�4.0 2 7.3 3.0�8.3

Gram-positive cocci 3 8.6 8.3�9.5 1 7.0 0.0.�8.0

Gram-negative cocci 1 8.1 0.0�9.0 3 9.0 8.0�10.0

Total aerobic growth 9 9.1 8.0�9.4 9 9.5 7.0�10.1

Coliform, indole-positive 3 9.0 8.0�9.4 8* 9.3 4.0�10.0

Coliform, indole-negative 2 7.9 2.0�8.9 1 7.5 0.0�6.6

Pseudomonas spp. 3 2.6 2.7�3.5 4 5.6 2.2�6.5

Staphylococcus aureus 5 6.5 2.6�7.3 4 5.9 1.3�6.8

Coagulase-negative staphylococci 7 8.0 6.7�8.5 9 8.2 5.3�8.7

Enterococci 5 7.5 7.0�8.0 2 8.1 8.7�8.8

Streptococci 2 7.2 7.8�8.0 0

Micrococcus spp. 1 5.0 0.0�6.0 0

Corynebacteria 3 8.0 4.0�8.9 0

Bacillus spp. 3 8.5 4.0�9.3 0

Yeasts 2 2.3 2.0�3.2 0

*pB0.05.

$Includes Actinomyces spp., Eubacterium-like organisms, and Propionibacterium spp.

%Includes Bilophila spp., Fusobacterium spp., and Prevotella spp.


19.4% (SD 13.3) vs 10.9% (SD 15.7) of the total

bacterial count (NS). In contrast, the colicky infants

lacked several aerobic genera; Streptococcus, Micro-

coccus, Corynebacterium, Bacillus spp. and yeasts were

cultivable, although in low prevalence, from the

controls only. The ratio anaerobes:aerobes varied

drastically between infants in both groups (range

0.7�2000) with similar median values, 12.2 in the

colicky and 14.3 in the control group. The ratio

lactic acid bacteria:total bacterial count, with lactic

acid bacteria including lactobacilli, bifidobacteria,

streptococci, and enterococci, varied less and was

similar in the colicky and in the control group (0.41

vs 0.53, NS). Also the species distribution of

lactobacilli was similar between the groups with

Lactobacillus paracasei ssp. paracasei and L. rhamnosus

dominating in both groups. Only two infants were

colonized with more than one Lactobacillus species,

although several infants harbored two subtypes of

the same species with clearly different colony

morphologies. One control infant carried a strain

identical to LGG (log10 9.2 cfu/g feces). All fecal

samples were negative for C. difficile toxins A and B

and no C. difficile was isolated at baseline. None

of the four infants delivered by cesarean section

carried E. coli, and the B. fragilis group organisms

and bifidobacteria were absent from three infants

and lactobacilli from two infants.

Microbe-derived fecal cellular fatty acids (CFAs)

A total of 45 CFAs were identified and their

percentage of the total fatty acid content was

calculated (percentage of the total peak area in

chromatogram). The four most abundant fatty acids

� 16:0 (palmitic), 18:0 (stearic), 14:0 (myristic), and

18:1 cis 9 � comprised 75% (each 4�34%) of the

total CFA content. Altogether 44 CFAs (mean 20

acids per infant) were detected among colicky

infants and 34 CFAs (mean 21) among control

infants. Acids comprising 0.5% or more are pre-

sented in Figure 1. Of the individual fatty acids,

significant differences between colicky and control

infants were found in the prevalence of saturated

17:0 and hydroxy 18:0 12OH, both being more

common among controls. Grouped together, unsa-

turated and branched fatty acids were slightly more

prevalent in colicky infants than controls: unsatu-

rated 20.3% (SD 4.6) vs 12.5% (SD 3.1) of the total

CFAs, p�0.049 and branched 7.1% (SD 1.4) vs

2.2% (SD 0.4), p�0.048, respectively, while satu-

rated fatty acids and dimethyl acetals were equally

common. The 11 fatty acids that were present only

among colicky infants were dispersed among 6

infants and included cyclic, straight, branched,

saturated and unsaturated acids, aldehydes, and

dimethyl acetals, with varying carbon chain length.

Their proportions of the total fatty acids remained

low (mean 0.19%, range 0.01�0.70) as well as the

number of infants carrying them (mean 1 infant,

range 1�3). When grouped together they comprised

2.3% of the total CFAs.

Short chain fatty acids (SCFAs) and gas as metabolic

fermentation end products

A total of nine SCFAs were identified and their

concentrations measured. The individual SCFA

concentration varied considerably (between 14.5

and 928.6 mmol/l) and the number of different fatty

acids (variety) between three and seven. Variation

between the three parallel (triplicate) samples was

subtle. The profile of the fermentation products

differed as expected between carbohydrate-rich

PYG and peptone-rich PY media (lacking fermen-

table carbohydrate) (Figure 2). Despite individual

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Figure 1. Proportions of bacterial cellular fatty acids (CFAs) of total fatty acids (mean area percentage and SD) and number of infants

carrying them among nine colicky and nine control infants at baseline. Only CFAs comprising at least 0.5% of the total CFAs are presented.

*Difference between groups was significant (pB0.05). Dma, dimethyl acetal.


variation the overall distribution and mean concen-

trations of SCFAs were similar in colicky and control

infants in both fermentation media.

Variation in the fecal gas production between the

study subjects was also substantial; however, no

significant differences between the groups were

found. CO2 had systematically the highest percen-

tages (percentage of the bottle’s head-space gas

volume) (see Table IV). In PY the relation CO2:H2

was slightly smaller in the colicky (3.3) than in the

control group (5.0); however, no significant differ-

ence between the groups was seen in the proportion

of CO2 (11.6% vs 10.0%, NS), while there was a

trend of a higher proportion of H2 in the colicky

group compared with the control group (3.5% vs

1.9%, p�0.051). In PYG fermentation no trend was

seen. The highest levels of H2 were measured among

infants harboring coliforms, especially Klebsiella and/

or Clostridium perfringens. Infants harboring lactoba-

cilli produced slightly less H2 in PYG (2.6% vs

8.8%, NS). No methane was detected.

Gas production by coliform isolates

Variation in volumes of the total gas production

between the isolates after 5 h in pure culture was

dependent on the species, being either zero (Enter-

obacter spp., Serratia spp.) or ranging between 7.1

and 12.0 ml (E. coli, K. oxytoca, K. terrigena). There

was no difference in gas volumes between the iso-

lates from the colic and control group or between

the most prevalent coliforms (E. coli, K. oxytoca, and

K. terrigena). Altogether 15 coliform strains originat-

ing from baseline samples were analyzed.

Microbial findings in colicky infants after the probiotic or

placebo supplementation

After the probiotic supplementation, the total counts

of anaerobic bacteria increased significantly com-

pared with the baseline, whereas after the placebo

supplementation the total counts of both anaerobic

and aerobic bacteria were similar to those at baseline

(Table III). This was due to a considerable increase

of bifidobacteria and LGG. The number of different

isolates per sample increased slightly with both

supplementations but tended to be higher after the

probiotic than after the placebo supplementation

(seven vs six anaerobic findings, and five vs four

aerobic findings per sample, respectively). The

infants randomized for the placebo supplementation

happened to have higher numbers of coliforms (the

most numerous aerobic finding) at baseline than did

the infants randomized for the probiotic supplemen-

tation. The relative proportion of coliforms of the

total microbial concentration in infants receiving

probiotics was 2.4% (range 0�5.2%) at baseline

and 1.2% (range 0.05�3.3%) after the probiotic

supplementation and in infants receiving placebo it

was 18% (range 0�51%) at baseline and 13% (range

0.3�25%) post supplementation.

After the probiotic supplementation, LGG ap-

peared in all five infants in high concentrations

(mean log10 8.6 cfu/g), and the counts of bifido-

bacteria increased significantly (Table III). Both

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Colicky gas

Figure 2. Fecal volatile and nonvolatile short chain fatty acids (SCFAs) (mean mmol/l and SD) and hydrogen and carbon dioxide gases

(percentage of total gas volume, SD) produced at baseline in 48 h fermentation in peptone-yeast extract broth (PY) and peptone-yeast

extract-glucose broth (PYG) by nine colicky and nine control infants. Also minor amounts (1�5 mmol/l) of isovaleric, isobutyric,

isocapronic, and phenylacetic acids were produced from PY and pronionic acid from PYG (not shown). No capronic or valeric acids were

detected. *Difference between the groups is in the limit of being significant, p�0.051.


lactobacilli and bifidobacteria counts exceeded the

corresponding counts in the placebo group. L.

rhamnosus LC705 was isolated in one (log10 8.0

cfu/g) but Propionibacterium JS in none of the infants.

In addition, the prevalence of enterococci increased

significantly. In general, the prevalence and mean

concentration of anaerobic bacteria increased, with

the exception of gram-negative cocci. Aerobic bac-

teria either increased or decreased, depending on the

species and infant. The proportion of lactic acid

bacteria of the total bacterial count increased after

probiotic supplementation from 47% to 65%.

After the placebo supplementation, the counts of

lactobacilli and bifidobacteria were similar to those

at baseline (Table III); similarly the proportion of

lactic acid bacteria of the total bacterial count was

unaffected by placebo supplementation (34% before

and 36% after). The only statistically significant

change in the placebo group was the decrease in the

prevalence and mean concentration of coagulase-

negative staphylococci. A strain identical to LGG

was isolated from one infant (log10 4.2 cfu/g) after

the placebo supplementation.

All fecal samples were negative for C. difficile

toxins A and B. C. difficile was found (log10 3 cfu/

g) in one infant as a new finding after the probiotic

supplementation. In pure culture, the strain pro-

duced toxin A but the carrier infant had no

symptoms other than colicky cry.

After the probiotic or placebo supplementation,

there was no difference in fecal pH values (5.7 vs

5.3) between the groups. No side effects related to

the supplementation were recorded.

SCFAs and gas production after supplementation

Probiotic supplementation increased the production

of acetic acid from PYG markedly in four of five

infants and the production of lactic acid in three of

five infants (Table IV). Acetic acid production from

PY increased markedly in two infants in the placebo

group. Otherwise SCFA concentrations remained

fairly similar to the baseline.

In both supplementation groups the amounts of

CO2 were unaffected and it remained the major

fermentation gas. After probiotic supplementation

fecal H2 production from PY decreased in four of

five infants and remained the same in one infant,

while in the placebo group and in PYG no trends

were seen (Table IV). In PY the mean proportion of

H2 was 0.6% after the probiotic and 3.4% after the

placebo supplementation (percentage of the bottle’s

head-space gas volume), and there was a 10-fold

difference in the relation CO2:H2 (20.7 vs 2.9,

respectively) between the supplementations.

Cellular fatty acids (CFAs) after supplementation

Altogether, 33 fatty acids were detected post supple-

mentation, of which 30 were detected after the

probiotic and 28 after the placebo supplementation.

Probiotic supplementation had no systematic effect

on the major fatty acids. Some minor acids were

replaced individually with both supplementations.

Crying patterns

The crying patterns of the colicky infants during the

week preceding the baseline sample collection and

those during the second week of the probiotic or

placebo supplementation were compared. At base-

line, the total crying time turned out to be longer in

the placebo group (Table V). The mean duration of

total crying time per week decreased markedly after

both supplementations, as did the colicky cry,

including moderate and severe crying. No statistical

differences in the crying patterns between the

probiotic and placebo group were found. The

mode or amount of crying could not be linked to

bacterial findings.

Table III. Microbial key findings (log cfu/g wet weight) in colicky infants before and after supplementation with the probiotic (five infants)

or placebo (four infants).

Probiotic supplementation Placebo supplementation

Before After Before After

Bacteria n Mean Range n Mean Range n Mean Range n Mean Range

Total anaerobes 5 10.3 8.8�10.6 5 10.7* 10.2�10.8 4 10.4 9.9�10.7 4 10.5 9.8�10.9

Bifidobacteria 5 9.9 8.6�10.5 5 10.4* 10.0�10.5 3 9.8 9.8�10.0 3 10.2 8.8�10.7

L. rhamnosus GG 0 0 5 8.6* 7.5�8.9 0 0 1 3.6 0.0�4.2

Non-LGG lactobacilli 4 9.0 3.4�9.7 3 9.6 2.0�10.3 4 9.7 8.6�10.3 3 8.4 3.1�9.0

Total lactobacilli 4 9.0 3.4�9.7 5 9.6 7.5�10.3 4 9.7 8.6�10.3 4 8.4 3.1�9.0

Enterococci 0 0 4 7.9* 5.0�8.5 2 8.5 8.7�8.8 3 8.2 7.9�8.5

Total aerobes 5 9.5 7.7�10.0 5 9.7 8.5�10.2 4 9.5 7.0�10.1 4 9.3 8.3�9.7

*pB0.05.


Table IV. Fecal volatile and nonvolatile short chain fatty acids (SCFA mmol/l) and hydrogen and carbon dioxide gases (% of gas volume) produced in 48 h fermentation in peptone-yeast extract

broth (PY) and peptone-yeast extract-glucose broth (PYG) before and after probiotic (five infants) or placebo (four infants) supplementation.

Probiotic supplementation Placebo supplementation

Before After Before After

Products n Mean Range n Mean Range n Mean Range n Mean Range

PY

Acetic 5 119 33�232 5 97 15�231 4 63 11�127 4 160 102�201

Propionic 5 35 22�66 5 30 8.0�74 4 18 3.6�47 4 31 19�52

Isobutyric 1 0.3 0�1.4 1 0.7 0�3.4 1 1.5 0�6.1 2 0.4 0�1.6

Butyric 4 13 0�33 5 13 0.8�32 3 7.7 0�21 4 24 0�42

Isovaleric 2 0.7 0�2.4 2 1.4 0�7.0 2 1.2 0�3.5 1 1.3 0�5.2

Valeric 0 0 0 0 1 B0.1 0�0.01 0 0

Isocapronic 0 0 0 0 1 1.5 0�5.9 1 1.5 0�5.9

Lactic 1 0.9 0�4.4 0 0 0 0 0 0

Succinic 2 3.3 0�8.9 0 0 2 3.3 0�6.9 2 4.5 0�7.9

Phenylacetic 0 0 1 0.8 0�3.9 1 0.5 0�1.8 1 0.5 0�1.8

H2 5 3.5 0.4�5.6 3 0.6 0�4.5 4 4.3 2.1�6.2 4 3.4 3.4�4.6

CO2 5 11 10�12 5 11 9.1�13 4 12 9.0�14 4 9.7 8.0�14

PYG

Acetic 5 148 6.6�462 5 436 53�962 4 74 17�206 4 80 19�181

Butyric 1 1.6 0�8.1 1 3.6 0�18 0 0 1 23 0�90

Lactic 5 159 89�247 5 196 185�211 4 159 73�278 4 157 99�233

Succinic 4 1.4 0�3.2 5 4.8 1.0�15 4 2.5 0.8�4.8 4 8.6 0.1�3.3

H2 5 1.5 0.2�5.0 5 1.8 0.2�4.2 4 5.6 2.8�9.3 4 4.6 1.2�7.5

CO2 5 5.6 2.5�8.6 5 5.3 1.1�11 4 10.9 6.8�15 4 9.5 6.3�13

Note that statistics were not performed.

44

S.

Men

tula

etal.

Discussion

In the present study wide-ranging methods were

chosen to gain an extensive description of the gut

microbiota as a functional organ, as well as to give an

insight to the associations between the microbiota,

microbial by-products, and colicky symptoms. In-

deed, colicky infants had a significantly higher

prevalence of indole-producing coliforms in their

feces than did their controls. In contrast, various

aerobic genera were absent from colicky infants.

Minor differences were recorded also among fecal

cellular fatty acids, as over 2% of the CFAs in the

colicky group were absent from the control group.

CFA composition shifts and reflects all bacteria,

including those that are nonviable and uncultivable.

In addition to the bacterial composition, the patterns

of bacterial fermentation products are affected by

growth conditions. Still, no consistent difference in

the major fermentation end products was detected.

Moore et al. (19) speculated that the immaturity of

the gut might affect motility, absorption, fermenta-

tion conditions, and handling of colonic gas or other

bioactive compounds. Our findings suggest that colic

is not connected to bacterial growth or fermentation

conditions but more likely to factors influencing

microbial colonization patterns and bacterial com-

position and/or factors affecting the host reactions to

bacteria and their metabolites.

We found no previous reports on elevated levels

of coliforms in colicky infants; however, coliform-

derived indole, having physiologic effects in mam-

mals, may have relevance. Higher urinary levels of

5-OH indole acetic acid, a serotonin metabolite,

have been reported in colicky than control infants

(20). Fecal indole levels were not monitored in the

present study. We tested the total gas production

capacity of the coliform isolates but found no

difference between isolates from the colicky and

control groups.

Previously, Lehtonen et al. (21) reported C.

difficile more frequently in stool samples from colicky

infants than their age-matched controls and, in

addition, differences in saturated 15:0 and branched

17:0 iso and 17:0 anteiso fatty acids between infants

with severe colic and controls. We found C. difficile in

one colicky infant and differences between the

groups in hydroxy acid 18:0 12OH and branched

acids grouped together, but unlike Lehtonen et al.

(21), we found branched CFAs more often in the

colicky group. Palmitic and stearic fatty acids, which

are also the major CFAs in most microbial cell walls,

predominated. Hydroxy acids typical of gram-nega-

tive bacteria were more prevalent in the control

group, contradicting the proposal that gram-negative

bacteria made up a slightly higher proportion of

the total bacterial count in the colicky group. The

amount of dimethyl acetals and aldehydes common

in anaerobic bacteria remained relatively low. This

observation agrees with the culture results showing

only mild dominance of anaerobes. Generally anae-

robes are 100�1000-fold more numerous than aero-

bes, while in these infants the relation was 1:13. Our

culture results agree with Savino et al. (22) in

reporting higher yield of gram-negative anaerobes

in colicky than control infants but disagree with the

reported decreased lactobacilli. Later Savino et al.

reported an altered Lactobacillus species composition

in colicky infants but this time no difference in

lactobacillar counts (23). Although no difference

was seen among Lactobacillus species or counts in the

present study, the proportion of lactic acid bacteria

in the total bacterial count tended to be lower in the

colicky group. The colonization patterns of lactoba-

cilli in our study are similar to those reported in

healthy infants using PCR-DGGE, with some in-

fants already carrying several lactobacilli at 2 months

(24,25).

In our study the ranking order of fecal SCFAs

produced was fairly uniform but the individual

concentrations differed significantly. All infants pro-

duced detectable amounts of acetic and propionic

acid and often also butyric acid. Samples yielding

high butyric acid concentration yielded butyrate-

producing genera such as Clostridium or Porphyro-

monas. Fecal fermentation in carbohydrate-poor PY

media, chosen to resemble the substrate-poor distal

colon, yielded SCFAs in a molar ratio 73:18:8 for

acetate:propionate:butyrate. Corresponding colonic

values for adults are approximately 60:20:18 (26).

Breath hydrogen production has been reported

to be significantly more frequent by colicky than

Table V. Mean crying times (SD) (h/week) preceding the baseline (week 0) and at the second week of the probiotic or placebo

supplementation (week 2).

Week 0 Week 2 Difference

Type of crying Probiotic Placebo Probiotic Placebo Probiotic Placebo

Moderate/severe 16.1 (2.8) 16.2 (7.3) 9.6 (6.5) 6.8 (3.7) �6.5 (40%) �9.4 (58%)

Mild 11.6 (6.6) 22.0 (10.9) 13.7 (5.3) 23.9 (16.3) 2.1 (19%) 1.9 (9%)

Total 27.7 (7.9) 38.3 (13.9) 23.4 (4.8) 30.7 (19.2) �4.3 (16%) �7.6 (20%)


non-colicky infants (19). In contrast, Belson et al.

(27) failed to show any association between hydro-

gen concentration and colic but instead an associa-

tion between low levels of methane at 6 months of

age and colic history. We detected no significant

difference in hydrogen production and no methane

in either the colic or control group at B2 months of

age. This may be reasonable, as hydrogen produc-

tion begins immediately after intestinal colonization,

while methane production is individual and more

age-related (27). Intestinal gases are produced but

simultaneously also consumed by numerous colonic

bacteria (18). Like fecal culture, in vitro fermenta-

tion may be a reflection rather than an exact

description of the microbiota and its activities in

the upper gut. However, due to practical problems in

direct examination, fermentation offers a feasible

approach to studying the intestinal microbiota.

Probiotic supplementation aims to balance the gut

microbiota and its function and to ease symptoms

originating from the gut. Probiotics are generally

well tolerated, as was also the case in the present

study, where none of the infants had diarrhea or

other side effects during the period of supplementa-

tion. Supplemented lactobacilli and bifidobacteria

effectively colonized the infant’s gut, being isolated

in high concentrations from all infants in the

probiotic group. B. breve is known to colonize infants

effectively (28). The ubiquitous colonization of

LGG demonstrated in the present study was higher

than previously reported: 67% in healthy newborns

(10), 90% in premature infants receiving antibiotics

(29), 47% in infants with low birth-weight (30), and

83% in healthy newborns delivered vaginally or by

cesarean section whose mothers were colonized with

LGG during pregnancy (31). Restrictions in the

methodology and sample material probably led to

lower recovery rate of Propionibacterium JS and

Lactobacillus LC705 than expected, since fecal bac-

terial composition differs markedly from that of the

upper gut where many probiotics are aimed to act.

In general, no major changes or effects on coloniza-

tion or growth patterns of other bacteria have been

reported after probiotic supplementation (29�31). In

our study, there was a tendency towards the dom-

inance of anaerobes and vast species diversity in the

probiotic group. Probiotics influenced the fermenta-

tion patterns by increasing acetic and lactic acid

production in glucose-rich PYG media and by

decreasing the release of hydrogen in glucose-poor

PY, even though the effect was mild. Changes in

individual infants may also reflect the natural succes-

sion progress of the immature microbiota as seen in

the placebo group. Hydrogen release in PYG tended

to be lower at baseline in infants harboring lactoba-

cilli. In theory, proliferation of anaerobic lactic acid

bacteria accompanied by increased acid production

may strengthen the development of colonization

resistance upheld by commensal microbiota.

In the present study, the counts of total anaerobes

tended to increase slightly in infants receiving the

placebo supplementation, but there were no clear

trends at the species level. Despite the strict exclu-

sion criteria, two infants not receiving probiotics

harbored an isolate similar to LGG (one in the

control group at baseline and one in the placebo

group after supplementation). LGG, which was

originally isolated from the human microbiota, may

have been residing in the microbiota of their

mothers, since various LGG-containing products

are on the market and widely used in Finland. These

two cases may be explained by salivary transmission

between the mother and her child.

The overall changes in crying patterns between the

probiotic and placebo group were comparable, and

the infants thrived well with both supplementations.

Colicky cry decreased dramatically in both groups,

as colic normally does by the age of 4 months. In the

present study, mean total crying time and colicky cry

corresponded well with those presented in previous

studies (9,32). At baseline, the total crying time

happened to be longer among the infants receiving

placebo; thus, a more marked reduction was ex-

pected and recorded in that group. Consistently, the

total crying time remained longer in the placebo

group. As to colicky cry, however, the groups did not

differ markedly from each other at any time of

measurement.

Conclusions

A shift towards less lactic acid bacteria and more

indole-producing coliforms differentiated colicky

infants from non-colicky infants. To thoroughly

assess the observed differences in coliform popula-

tions, indole production, CFA profiles and fermen-

tation parameters, larger study groups, and selected

molecular technology are required. Succession to-

wards a balanced commensal microbiota, i.e. in-

creased counts of anaerobic bacteria, bifidobacteria,

and lactobacilli, and towards lactic acid metabolism

and improved gas utilization, may be achieved by

probiotic supplementation.

Acknowledgements

The authors thank Maija Saxelin PhD for cons-

tructive comments on the manuscript, and Soile

Tynkkynen PhD for her help on identification of

LGG isolates. The late Professor Hannele Jousimies-

Somer is acknowledged for her contributions to the

study.


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