up to 10% of newborn infants require admission to the neonatal intensive care unit ( nicu ) for ongoing medical care [ 13 ] .
antibiotic administration is known to perturb the composition of the intestinal microbiota , resulting in suppression of anaerobic bacteria ( with the exception of clostridia , which remain at detectable levels ) and increased numbers of potentially pathogenic bacteria such as klebsiella , enterobacter , citrobacter , and pseudomonas [ 4 , 5 ] . since the pioneering microbiota of infancy probably create perpetual habitats for themselves , defining the life - long composition of the gut microbiota and consequently contributing to host health and well - being [ 6 , 7 ]
have several reported health - promoting effects , including inhibition of growth of harmful bacteria , stimulation of the immune system , alleviation of constipation , and prevention of intestinal infections [ 813 ] .
species commonly associated with humans include bifidobacterium infantis , b. longum , b. bifidum , b. breve , b. catenulatum , b. pseudocatenulatum , b. angulatum , b. gallicum , and b.   adolescensis , of which the first four are prevalent in infants .  
suppression of the bifidobacteria population as occurred following antibiotic exposure in children [ 5 , 15 , 16 ] may have negative effects on host well - being and can be associated with higher susceptibility to enteropathogenic bacterial infection . however , the impact , if any , of parenteral antibiotics prescribed shortly following birth on the developing bifidobacterial population has not been addressed .
the aim of this study was therefore to determine the impact of intravenous antibiotic treatment in the neonatal period on the evolution of bifidobacterial colonization over time .
faecal material was sampled , using swabs , from 18 infants , nine infants previously treated with antibiotics and nine healthy controls ( table 1 ) .
approval for the study protocol was obtained from the clinical research ethics committee of the cork teaching hospitals , cork , ireland .
inclusion criteria were defined as full or near term infants requiring parenteral antibiotic administration within the first 48 hours of life .  
control subjects were eligible if they were full or near term infants who were otherwise well and did not require admission to the nicu .  
infants placed on oral antibiotics , those kept nil by mouth or requiring surgery , infants with congenital anomalies , or those born before term were excluded from the study .
the indications for antibiotic treatment were determined clinically , based on symptoms and/or signs of suspected sepsis , and independent of the study investigators .
stool samples were taken by the same clinician at four and eight weeks after birth and stored at 20c pending analysis .
bacterial dna was extracted from swabs using a qiaamp dna stool minikit ( qiagen , hilden , germany ) by following the manufacturer 's instructions ( lysis temperature , 95c ) . to investigate the bifidobacterial population in the samples ,   pcr was performed as a nested approach .
the first pcr applied primers im26-f ( 5- gattctggctcaggatgaacg -3 ) and im3-r ( 5- cgggtgcticcccactttcatg -3 ) described by kaufmann et al .   amplified   a 1,417  bp fragment of the bifidobacterial 16s rrna gene .
pcr volumes of 50  l contained 5  l of 10 pcr buffer , 5  l of mgcl2 ( 2.5  mm ) , 8  l of dntps ( 0,2  mm each ) , 1  l of each primer ( 5  pmol ) , 0.5  l of taq polymerase ( 5  u/l ) , 28.5  l of sterile milli - q water , and 1  l of dna solution .
the following pcr program was used : initial denaturation at   94c for 5  min ; 3 cycles of denaturation at 94c for 45  s , annealing at   57c for 2  minutes , and extension at   72c for 1  min ; 30 cycles of denaturation at   94c for 20  s , annealing at   57c for 1  min , and extension at   72c for 1  min ; and final extension at   72c for 5  min followed by cooling to   4c .
the presence of pcr products was verified on a 1.5% ( w / v ) agarose gel . in order to eliminate the remaining oligonucleotides and original template dna , purification of the amplicons
was performed by use of the qiaquick pcr purification kit ( qiagen ) according to the manufacturer 's instructions .
a second pcr was performed using the amplicons of the first pcr as template dna .
the primer set used ( f357-gc and 518r ) ( 5gc - clamp- gcctacggaggcagcag -3 and 5- attaccgcggctgctgg -3 , resp . )
the forward primer contained a gc clamp ( 5- cgcccgccgcgcgcggcgggcggggcgggggcacggggg -3 ) to facilitate separation of the amplicons in a dgge gel .
pcr volumes of 50  l contained 5  l of 10 pcr buffer , 2  l of mgcl2 ( 1  mm ) , 8  l of dntps ( 0.2  mm each ) , 2  l of each primer ( 5  m ) , 0.5  l of taq polymerase ( 5 u/l ) , 29.5  l of sterile milli - q water , and 1  l of 10-fold diluted dna solution ( obtained from the first pcr ) .
the following pcr program was used : initial denaturation at   94c for 5  min , 30 cycles of denaturation at   94c for 20  s , annealing at   58c for 45  s , and extension at   72c for 1  min , and final extension at   72c for 7  min , following by cooling to   4c .
pcr products were analyzed on dgge gels based on the protocol by temmerman et al . .
a dcode universal mutation detection system ( bio - rad laboratories , hercules , ca , usa ) was used , utilizing 16  cm by 16  cm by 1  mm gels .
eight percent polyacrylamide gels were prepared and run with 1 tae buffer diluted from 50 tae buffer ( 2  m tris base , 1  m glacial acetic acid , and 50  mm edta ) .
the denaturing gradient was formed with two 8% acrylamide ( acrylamide - bis / acrylamide 40% ) stock solutions ( severn biotech ltd , worcestershire , uk ) .
a 100% denaturing solution contained 40% ( v / v ) formamide and 7.0  m urea .
the gels were poured from the top by using a gradient maker ( cbs scientific linear gradient maker , bio - rad laboratories ) and a pump ( bio - rad laboratories ) and gradients of 50% to 70% were used for the separation of the generated amplicons . before polymerization of the denaturing gel ( 24  ml gradient volume ) , a 6  ml stacking gel without denaturing chemicals was added , and the appropriate comb was subsequently inserted .
pcr amplicons were separated by electrophoresis at a constant voltage of 60  v in a 0.5  tae buffer at a constant temperature of   60c for 16  h. gels were stained in ethidium bromide for 30  min , allowing digital capturing of the dgge band profiles under uv light . a mixture of five different species of bifidobacteria which are the most commonly isolated species of bifidobacteria from the infant gastrointestinal tract ( git ) [ 14 , 20 , 21 ] was used to create a reference ladder in order to enable a visual comparison of the bands .
dna extraction from the reference strains , bifidobacterium breve , b. bifidum , b. infantis , b. adolescentis , and b. longum , was performed according to the method described by hoffman and winston .
nine subjects were recruited to both control and antibiotic - treated groups in this study .
while the gender distribution and feeding history was similar in both groups , the antibiotic treated infants were more likely to be born by caesarean section ( 5/9 infants ) and all control infants were born by spontaneous vaginal delivery .
ampicillin and gentamicin were the only antibiotics used and the median duration of therapy was 2 days ( range 29 days ) .   in order to investigate the population of bifidobacteria in the samples ,
amplicons obtained using these primers served as template dna for the v3 primer combination v3r and v3f , during a second pcr step .
the mobility of the pcr products obtained by these primers in dgge was compared with the pcr pattern of bifidobacteria reference strains .
because of the high g+c content of bifidobacteria , the conventional 35% to 70% denaturing gradient was replaced with a 50% to 70% denaturing gradient . at four weeks of age , species of bifidobacteria were detected in six out of nine infants treated with antibiotics ( subjects a 
r ) . the samples from the antibiotic treated infants exhibited a lower number of bands in the dgge gel ( figure 1(a ) ) compared with controls ( figure 1(b ) ) at four weeks of age , representing a less diverse population of bifidobacteria at this age .
five of six infants in the antibiotic - treated group showed a single dominant band , which corresponded to b. longum ( figure 1(a ) ) .
all five infants had been treated with intravenous ampicillin / gentamicin for two days and there were both breast fed ( subject f , g ) and formula fed ( subject d , h , i ) infants within this group .
the sample lacking a band in the position of b. longum was obtained from an infant that had received antibiotics for five days ( subject e ) . according to the band pattern of this subject , some bifidobacterium species were able to colonize this infant despite antibiotic treatment for five days ; that is , the lower band appears to be b. bifidum , while the upper band is not comparable to any of the species within the reference ladder .
all samples from the formula fed controls ( k , l , m , q , r ) exhibited a band in the same position as b. longum at four weeks of age , except for one infant ( subject j ) ( figure 1(b ) ) .
in contrast , samples from the breast fed infants in the control group ( n , o , p ) showed bands in the positions of b. infantis and b. bifidum .
these two species were only found in one of the formula fed infants . while all vaginally born infants in the antibiotic - treated group harboured bifidobacteria by four weeks of age , two out of five infants born by caesarean section harboured bifidobacteria ( b. bifidum and b. longum ) at this time point . at eight weeks of age , eight out of nine antibiotic - treated infants and all controls had detectable levels of bifidobacteria in their stools .
interestingly , the only sample in which bifidobacteria was not detected was from an infant that had received antibiotics for nine days ( subject a ) ( the other infants were treated with antibiotics for 25 days ) .
between four and eight weeks , an increase in bifidobacteria diversity in both groups was observed as evidenced by the increased number of bands ( figure 2 ) .
nonetheless , while both groups were similar at eight weeks , samples from antibiotic - treated infants continued to display a less diverse population of bifidobacteria when compared with controls ( figure 2 ) .
the samples obtained from the breast - fed infants in the control group had in general a more diverse population of bifidobacteria compared with formula - fed infants ( 7 - 8 bands compared with 25 bands , resp . ) . however , no differences between the samples from the breast - fed and formula - fed infants in the antibiotic - treated groups were observed .
moreover , the band with highest intensity at four weeks , identified as b. longum , was replaced by another dominant strain , identified as b. breve , after eight weeks .
four of the five infants in the antibiotic - treated group born by caesarean section harboured bifidobacteria by week eight . as found at week four ,
all vaginally born infants harboured bifidobacteria at eight weeks of age , with a higher diversity observed compared with four weeks of age .  
overall , b. longum appeared to be the most commonly detected bifidobacterium species in the infants at eight weeks of life ( present in 15 out of 17 infants harbouring bifidobacteria ) ( figure 2 ) .

 bifidobacterium and lactobacillus species are considered among the most important beneficial bacteria in the human git and their presence in large numbers in the microbiota is regarded as beneficial [ 23 , 24 ] .
suppression of these beneficial components could facilitate growth of potentially pathogenic bacteria such as klebsiella and clostridium and have consequences for the well - being of the host .
this study demonstrates that even brief parenteral antibiotic treatment alters the pattern of bifidobacterial evolution over time .
infants treated with antibiotics showed a reduction in both colonisation and diversity of bifidobacteria compared with controls . while control and antibiotic - treated groups were similar by eight weeks of age , a number of control subjects continued to display a more diverse population of bifidobacteria .
reported that the colonisation of bifidobacteria in infants is normally well established by one week of age .
it is tempting to speculate that the observed difference in detectable levels of bifidobacteria between the antibiotic - treated infants and controls in our study reflects the early antibiotic exposure .
previously described a significant difference in the microbiota of a single infant receiving antibiotics compared to four nonantibiotic treated infants , using similar methodology that is , pcr - dgge and bifidobacterium - specific primers as used in our study .
were absent in the antibiotic - treated infant at all time points assessed , from five days to five months of age .
however , in that study , the antibiotic - treated subject received prolonged antibiotic therapy with both parenteral and oral agents ( thirteen days of parenteral coamoxiclav followed by oral cotrimoxazole ) .
in contrast , the majority of participants in our study had relatively brief periods of intravenous antibiotic administration and yet differences were still observed compared with controls . 

b. longum was the dominant species in both the antibiotic - treated infants and in the control group .
this finding is consistent with the study by grnlund et al .   which found b. longum to be the most common bifidobacterium species present in infants .
this result correlates with a recent finding by mtt et al . , demonstrating that b. longum has a higher minimum inhibitory concentration ( mic ) for ampicillin compared with other bifidobacterium species .
thus , it appears that the antibiotic treatment itself may have selected for this species .
one infant showed a decrease in numbers of species from three species at four weeks of age to one species at eight weeks of age .
the species that showed the highest intensity in the dgge gel after four weeks was replaced by another dominant species after eight weeks .
these results are in agreement with a study by favier et al .   in which the initially dominant bifidobacteria in two infants were replaced following the emergence of new species within the study time period .   
although less than half of the infants in this study were breast - fed , our study confirms previous reports that breast - feeding is positively associated with a more diverse bifidobacterial population compared with formula - fed infants [ 16 , 28 , 29 ] .
bifidobacterium species are considered among the most important beneficial bacteria in the human git [ 18 , 19 , 24 ] .
a higher density of beneficial bacteria in infants may confer a degree of protection against childhood diseases .
for example , bifidobacterial colonisation has been associated with a lower risk of atopy [ 21 , 30 , 31 ] .   in a prospective study ,
children with lower numbers of fecal bifidobacteria and increased numbers of clostridia from the first weeks of life were more likely to become sensitized to common allergens .
in addition , it has been reported that differences in the gut microbiota during infancy may precede the onset of obesity . in this study by kalliomki et al . , the numbers of bifidobacterium spp . were higher during infancy in children who remained at normal weight than those in children who became overweight .
factors such as breast - feeding , vaginal delivery , and the absence of antibiotic exposure would promote microbial diversity with higher numbers of bifidobacteria .      in conclusion , the present study demonstrates that short - term parenteral antibiotic treatment of neonates causes a disturbance in the expected colonization pattern of bifidobacteria in the first months of life .
whether a delay in bifidobacterial colonisation could adversely influence health status , either in infancy or later life remains unknown .
however , adequately powered prospective studies that document changes in the microbiota and incorporate long - term health status outcomes are needed to further our understanding of the sequelae , if any , of early neonatal antibiotic administration . until such data are available , supplementation with bifidobacteria in this patient group can not be justified .
 we investigated the impact of parenteral antibiotic treatment in the early neonatal period on the evolution of bifidobacteria in the newborn . 
 nine babies treated with intravenous ampicillin / gentamicin in the first week of life and nine controls ( no antibiotic treatment ) were studied . 
 denaturing gradient gel electrophoresis was used to investigate the composition of bifidobacterium in stool samples taken at four and eight weeks . 
 bifidobacteria were detected in all control infants at both four and eight weeks , while only six of nine antibiotic - treated infants had detectable bifidobacteria at four weeks and eight of nine at eight   weeks . 
 moreover , stool samples of controls showed greater diversity of bifidobacterium spp . compared with antibiotic - treated infants . in conclusion , short - term parenteral antibiotic treatment of neonates causes a disturbance in the expected colonization pattern of bifidobacteria in the first months of life . 
 further studies are required to probiotic determine if supplementation is necessary in this patient group . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion