Enumeration of Lactobacilli in the Fecal Flora of
Infant Using Two Different Modified de-Man Rogosa Sharpe Media
under Aerobic and Anaerobic Incubation
Regarding the importance of the presence of intestinal
lactobacilli and their population in infants, four different treatments
were evaluated for Lactobacillus isolation efficiency via reduction
in the growth of other groups of bacteria capable of growing on de-Man
Rogosa Sharpe (MRS) medium from fecal samples of 11 Iranian infants. MRS-Vancomycin
(1 mg L-1) was used as a base medium and application of lactic
acid and aerobic incubation of inoculated plates were performed as selective
factors. Each fecal sample was cultivated as duplicate on to the base
medium with or without lactic acid to reduce the pH to 5.4 ±0.2.
Half of the plates were incubated aerobically and the rest of them incubated
under 10% CO2 concentration. Total count and Lactobacillus
count of all samples were recorded according to the age differences of
infants. The counts of false positive colonies were recorded with respect
to their cell morphology and gram reaction in all treatments. Anaerobic
incubation of lactic acid modified MRS-Vancomycin gave the most Lactobacillus
percentage coverage, about 93% among the Lactobacillus positive
samples. Using this treatment, the median Lactobacillus count yielded
8.29 log10 cfu g-1 in the younger and 5.70 log10
cfu g-1 in the elder group. It could be concluded that lactic
acid might be a proper pH reducing agent when enumeration of lactobacilli
from fecal samples is of interest.
to cite this article:
M. Mirlohi, S. Soleimanian-Zad, M. Sheikh-Zeiondin and Hossein Fazeli, 2008. Enumeration of Lactobacilli in the Fecal Flora of
Infant Using Two Different Modified de-Man Rogosa Sharpe Media
under Aerobic and Anaerobic Incubation. Pakistan Journal of Biological Sciences, 11: 876-881.
Lactobacilli are a heterogeneous group of microaerophilic,
lactic acid producing, bacteria. They are usually present in the intestinal
microbiota of healthy adults, although in substantially lower population
counts than several other bacteria, through to represent 0-2% of gut microbiota
(Bjorksten et al., 2001; Mikelsaar et al., 2004; Ahrne et
al., 2005). During the past twenty years researches on isolation,
identification and prevalence of Lactobacillus flora of the gastrointestinal
tract have received special interest because of their health promoting
effects. The gut flora as such is essential for mucosal immune stimulation,
amplification of immune-competent cells and competitive exclusion of pathogens;
these actions appear correlated in infants (Ahrne et al., 2005;
Westerbeek et al., 2006). In newborn infants and small children,
the extent to which lactobacilli colonize the intestine and their colonization
rate is controversial (Ahrne et al., 2005) From the physiological
point of view, the Lactobacillus flora change by age (Mitsuoka,
1992;Mikelsaar et al., 2004) Some studies claim that the Lactobacillus
colonization pattern was completely different before and after 6 months
of age (Ahrne et al., 2005) and from the ecological point of view,
the result of some studies claim lower-level presence of lactobacilli
in infants and children in industrial countries rather than in developing
countries (Bjorksten et al., 2001; Ahrne et al., 2005).
Variation in methodologies and different geographical areas may account
for these differences (Ahrne et al., 2005). Although Lactobacillus
selective (LBS) medium or Rogosa medium (Rogosa et al., 1951) provides
specific condition for Lactobacillus growth via pH reduction by
acetic acid and sodium acetate, enumeration of lactobacilli from the stool
samples using this medium is not very easy because of the interference
of bifidobacteria and occasional streptococci (Sharpe, 1986; Kandler and
Weiss, 1986; Ahrne et al., 2005) with the growth of these bacteria.
It has been shown that the prevalence of these microorganisms is more
usual than lactobacilli in the fecal microflora of infants (Mitsuoka,
1992;Mikelsaar et al., 2004). Several studies have claimed that
bifidobacteria were dominant over lactobacilli especially in breast fed
infants before 6 month of age (Mountzouris et al., 2002; Ahrne
et al., 2005). Bifidobacteria are strictly anaerobes (Scardovi,
1986; Mitsuoka, 1992) and aerobic incubation of Lactobacillus selective
media improved specificity of the medium when selective isolation of lactobacilli
from fecal samples was of interest (Vael et al., 2005). Lactobacillus
anaerobic MRS agar with Vancomycin and Bromocresol (LAMVAB) medium was
also designed for isolation and enumeration of lactobacilli from fecal
samples based on low pH and Vancomycin resistance of lactobacilli which
is unusual for any Gram-positive bacteria (Hartemink et al., 1997).
The results of recent studies have shown that these media still are not
efficient enough in enumeration of lactobacilli from fecal samples; Rogosa
agar was more likely to support growth of non-Lactobacillus species
(Hartemink and Rombouts, 1999; Jackson et al., 2002; Beasley, 2004;
Vael et al., 2005) and LAMVAB agar could not support the growth
of some strains of Lactobacillus acidophilus group and gave lower
Lactobacillus counts than unspecific MRS (Jackson et al.,
2002; Leuschner et al., 2003; Vael et al., 2005). It is
known that acetic acid used in above mentioned selective media at a given
pH has more growth inhibitive effect on microorganisms than lactic acid
(Halm et al., 2004), in addition, bifidobacteria produce acetic
acid and lactic acid in the molar ratio of 3:2 during fermentation (Scardovi,
1986). So they may be more resistant to acetic acid than lactobacilli
in acetic acid modified medium. On the other hand, lactic acid is the
main metabolite of lactobacilli (Siegumfeldt et al., 2000; Pieterse
et al., 2005) and their tolerance to lactic acid may provide their
selective dominance in the isolation medium as it happens in the natural
This study aimed to assess the application of lactic acid
as the pH reducing component of the isolation media and aerobic incubation
to inhibit the growth of complete anaerobes such as bifidobacteria and
other inhabitants of the intestine which are capable of growth in MRS
and therefore it was desirable to validate the efficacy of the methods
by identification and enumeration of the false positive colonies, Lactobacillus
count and Lactobacillus percentage coverage of each treatment.
Because there was not any previous data of the Lactobacillus count
in the fecal flora of Iranian infants, categorization of subjects before
and after 6 month was performed to reduce the variation in the Lactobacillus
count in the data analyzing step.
MATERIALS AND METHODS
Preparation of samples: The feces samples of eleven healthy infants
(a) 3-6 month, n = 5 and (b) 12-21 month, n = 6 were used for the experiments.
The experiments were carried out during March-May 2007. Fresh feces was
collected from the infants by their parents and placed in tight plastic
boxes at home, 8-12 h before running the experiment. The samples were
kept refrigerated up until received by the laboratory, where they were
processed as soon as possible.
Experimental design and inoculation of culture media: Isolation
of lactobacilli was performed using four different treatments. In all
treatments MRS (Scharlou, Spain)-Vancomycin (1 mg L-1) (VMRS)
agar was used as the base culture medium. The pH of half of the media
was reduced to 5.5±0.2 by 90% lactic acid (Merck, Germany) and it was
called LVMRS. For each sample, a pair of plates containing the same medium
was prepared. After inoculation, one of the plates was kept aerobically
and the other one was incubated under 10% CO2 anaerobic environment,
obtained with the CO2 generating Gas pack A system (Merck,
Darmstadt, Germany). Half a gram of each sample was placed in a sterilized
flask, mixed with 5 mL of sterilized normal saline and centrifuged at
low speed (100 rpm) for 1 min. One milliliter of the upper phase was collected
and serially diluted to 10-8 dilution. One hundred micro liters
of the diluted samples (10-3 to 10-8) was inoculated
on the MRS-Vancomycin or MRS-Vancomycin-lactic acid agar and all the plates
were then incubated at 37°C for 72 h.
Bacterial counting: Plates containing 25-250 colonies were selected
and representative colonies of each morphotype (differing in size, shape,
color or texture from other colonies) were enumerated. Cell morphology
and Gram reaction were examined using phase contrast microscopy. Cell
morphotypes were categorized in four groups; Gram- positive cocci or oval
shape bacteria, Gram-positive branched rods, Gram-positive rods, Gram
negative rods and yeast-like morphotypes. Each cell morphotype was enumerated.
Gram-positive, rod isolates were sub-cultured to purity (1-4 per sample)
on MRS and tested further for catalase reaction by 3% H2O2
and spore formation. All rod, aerotolorant, Gram-positive, catalase-negative,
non-spore-former, non-motile isolates were regarded as lactobacilli. Colony
and cell morphology were recorded by a single observer. The number of
Lactobacillus and total colonies (log10 cfu g-1)
developed on the media of each particular treatment were calculated for
each stool sample.
Lactobacillus percentage coverage: The proportion percentage of
the number of Lactobacillus count to the total colony count for
each Lactobacillus-positive sample in each treatment was recorded
as a representative data of the Lactobacillus percentage coverage
of that treatment.
Statistical methods: Data analysis was carried out with Minitab
15 software. One way ANOVA was used to study significant differences between
means, at a = 0.05 level. Tukey`s test was used to perform multiple comparisons
of the means.
Lactobacilli isolated from 6 out of 11 total tested fecal
samples using at least one of the four tested treatments. The range and
median viable counts (log10 cfu g-1) of different
morphotypes containing Gram-positive cocci (enterococci, staphylococci
and streptococci) and oval morphotypes, Gram-positive rods, Gram-positive-branched
rods, Gram-negative rods and yeast-like cell morphotypes which regarded
as false positive colonies in Lactobacillus isolation media of
6 Lactobacillus positive fecal samples, using four different treatments
are shown in Table 1.
Vancomycin, at the concentration used for this study, could
not induce an inhibitive effect on the growth of other groups of bacteria
on MRS especially, Gram-positive cocci observed on all tested samples
at the high population range of 5.37-9.3 log10 cfu g-1,
Evaluation of different cell
morphotypes from 6 Lactobacillus positive fecal samples
*: Minimum-maximum range count
(log cfu g-1 of the feces), **: Median count (log
cfu g-1 of the feces)
VMRS in spite of aerobic or anaerobic incubation. Gram-
positive branched rods also occurred on anaerobic incubated plates containing
this medium at a population range of 6-8.45 log10 cfu g-1.
They were also observed a substantially lower number on a few aerobic
incubated plates containing this medium. These two cell morphotypes were
also the most frequent morphotypes in Lactobacillus negative samples.
Gram negative rods and yeast like morphotypes were detected in VMRS although
they did not seem to be main disturbing microorganisms because of their
rare incidence in tested agars. Addition of lactic acid to VMRS reduced
the viable counts of Gram-positive cocci and Gram-positive branched rods.
None of the Gram-positive branched rods were found on aerobic incubated
plates containing this media anymore and no Gram-negative rod cell morphotype
grew under both aerobic and anaerobic incubated condition. The counts
of yeast-like microorganisms seemed unaffected by addition of lactic acid,
although they were mostly detected from fecal samples belonging to the
elder subjects and developed under aerobic environments. Gram positive
rods primarily regarded as lactobacilli grew in VMRS at the population
range of 0-8.9 log10 cfu g-1 under anaerobic and
0-8.47 log10 cfu g-1 under aerobic incubation. The
minimum count of this morphotype was increased when lactic acid was added
to the media but only anaerobic incubation of LVMRS gave about 1.7 log10
cfu g-1 median counts of this group more than that in VMRS
under the same incubation condition.
The results of total and Lactobacillus counts of
11 fecal samples enumerated on examined agars using different treatments
in 3-6 months infants (group a) and in 12-21 months infants (group b)
have been shown in Table 2. Comparison of the total and
Lactobacillus counts between two age groups, resulted significant
differences; using four treatments total and Lactobacillus count
of samples from younger group was more than that in the elder group. In
both age groups the total anaerobes which grew in each medium out numbered
Counts (log10 cfu
g-1) of total and Lactobacillus colonies and
Lactobacillus percentage coverage obtained by different
treatments in two age groups of infant
Ta: Total count and La: Lactobacillus
count in the younger g roup (3-6 months), Tb: Total count and
Lb: Lactobacillus count in the elder group (12-21 months).
*: Mean counts (log10 cfu g-1) are expressed
as mean±standard error of means, Means in the same column followed
by different uppercase letter(s) are significantly different
(p<0.05), **: Median counts (lo10g cfu g-1),
***: Lactobacillus percentage coverage of VMRS and LVMRS
under aerobic and anaerobic incubation from Lactobacillus
aerobes capable of growth in that medium by about 1-1.4
log10 cfu g-1. However, only aerobic incubation
of LVMRS reduced the mean total colony count significantly in the younger
(p = 0.01) and in the elder (p = 0.036) groups. Three fecal samples in
each age group harbored lactobacilli. None of the treatments carried out,
revealed a significant difference in the Lactobacillus count but
the Lactobacillus count obtained in anaerobic incubation of LVMRS
in both age groups was relatively higher than in other treatments, giving
the median Lactobacillus count of 8.29 log10 cfu g-1
in the younger and 5.7 log10 cfu g-1 in the elder
group in addition the most Lactobacillus percentage coverage about
93% was obtained by this treatment in Lactobacillus positive samples.
The result of this study revealed that the reduction of
the pH of the medium is a main selective factor for isolation and enumeration
of lactobacilli from fecal samples. lactic acid modified VMRS used in
this study could effectively differentiate Lactobacillus colonies
and other microorganisms capable of growth on VMRS medium therefore, lactic
acid which is the predominant metabolite biosynthesized during the growth
and fermentation of lactobacilli, could be a proper pH-reducing ingredient
for Lactobacillus isolation from environments such as fecal samples.
Acidified MRS media, by acetic acid or acetate introduced earlier for
this purpose (Rogosa et al., 1951; Sabine and Vaselekos, 1965;
Kandler and Weiss, 1986; Sharpe, 1986) but recent studies, based on molecular
methods, have claimed that these media still do not have sufficient efficiency
for enumeration of Lactobacillus species from human fecal samples
(Jackson et al., 2002; Ahrne et al., 2005). In another study,
plating the canine fecal samples on the LBS amended with acetic acid yielded
no colonies (Beasley, 2004). As the results showed that false positive
cocci and branched rods also could grow in the lactic acid modified VMRS
(Table 1), comparing the Lactobacillus recovery
of this medium, containing higher concentration of lactic acid with acetic
acid modified MRS could be helpful.
The significant higher counts of total and Lactobacillus
colonies in the younger infants in all treatments showed that paying attention
to the age group of infants could reduced the variation in the Lactobacillus
count of their fecal flora however high variation in Lactobacillus
count was obtained in each age group because of the little size of the
study. Former study showed that the intestinal Lactobacillus colonization
pattern is different before and after 6 month of age, between these two
phases, the lactobacilli reach their lowest prevalence and population
(Ahrne et al., 2005).
Regarding incubation condition, aerobic incubation could
not be a selective factor for the purpose of the study. When VMRS is used
as isolation media, false positive branched rods were detected in a few
aerobic plates. These plates were mostly cultivated from fecal samples
belonging to the younger subjects. As these morphotypes were regarded
as Bifidobacterium species, their growth under aerobic environments
may because of their high population in these fecal samples. When the
lactic acid modified VMRS was used, aerobic incubation both reduced the
total colony count and false positive colonies of Gram-positive cocci.
It also reduced the median Lactobacillus count of Lactobacillus
positive samples in both age groups of infants. This is in agreement with
previous report which claimed aerobic incubation of MRS improved its specificity
and reduced its sensitivity for Lactobacillus recovery among different
strains isolated from fecal samples (Vael et al., 2005).
Modification of MRS with 1 mg L-1 Vancomycin,
could not be a selective medium for Lactobacillus recovery from
fecal samples. This medium introduced earlier for enumeration of some
probiotic Lactobacillus species in mixed cultures containing some
other lactic acid bacteria and bifidobacteria species under anaerobic
environment (Thamaraj and Shah, 2003). in the preliminary examination
of this study, performed on a few fecal samples, this medium gave about
1 log cfu g-1 lower total counts than MRS under anaerobic environments
(data was not shown) therefore, higher concentration of Vancomycin was
not tested in this experiment. Concentration of Vancomycin in LAVAB medium
is 20 times greater than that in this study (Hartemink et al.,
1997). LAVAB was shown to inhibit the growth of some Lactobacillus
species (Jackson et al., 2002). High concentration of Vancomycin
(10-20 mg L-1) also used for selective enumeration of L.
caei group from probiotic cheese (Phillips et al., 2006) and
fecal samples (Marzotto et al., 2006).
Based on former documentations, the lactoflora of infants
develop during the first year of life (Mitsuoka, 1992) and the intestinal
bacterial structure of infant aged 12-24 months is in a transitional state
combining neonate and adult-like features (Marzotto et al., 2006).
When a child starts to eat solid food, the fecal flora of children closely
resembles that of adults where the counts of lactobacilli are usually
less than 107 cfu g-1 of the feces (Mitsuoka, 1992)
In this study, the types of nutrition was not the focus because tested
infants in the younger group were both breast-fed and formula-fed and
most of the elder infants still received breast milk in addition to bottle
milk and solid foods. In this study, most of examined morphotypes seemed
to be enterococci, streptococci and bifidobacteria in both age groups
with higher counts in the younger group. Yeast-like cell morphotypes developed
on aerobic methodologies only from the samples belonged to the elder group.
This may be because of variation in fermented foods introduced in to the
elder children`s diet.
As the type of nutrition is a principal factor for establishment
of Lactobacillus flora, further studies aimed at enumeration of
lactobacilli in Iranian infants concerning the impact of food in addition
to age group with larger populations, will be of interest to provide more
knowledge of colonization phase and pattern of these important parts of
our intestinal microbiota.
Authors would like to appreciate Dr. Moayednia, Head
of Aria diagnostic laboratory and his assistants for their collaboration.
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