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Early Human Development 90 (2014) 579–585
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Early Human Development
j ourna l homepage: www.e lsev ie r .com/ locate /ear lhumdev
Gut microbiota in preterm infants with gross blood in stools:A prospective, controlled study
Mohamed Ben Said a,b, Stephane Hays a,c, Deplhine Maucort-Boulch d,e,f, Abdallah Oulmaati a,Stefanyia Hantova g, Claire-Marie Loys a, Estelle Jumas-Bilak g,h, Jean-Charles Picaud a,c,f,⁎a Department of Neonatology, University Hospital Croix Rousse, Hospices Civils de Lyon, F-69004 Lyon, Franceb Faculty of Medicine of Tunis, University of Tunis El-Manar, 1007 Bab Saâdoun, Tunis, Tunisiac Rhone-Alpes Human Nutrition Research Center, F-69310 Pierre Bénite, Franced Department of Biostatistics, Hospices Civils de Lyon, F-69003 Lyon, Francee CNRS, Laboratoire Biostatistique Santé, UMR 5558, F-69310 Pierre Bénite, Francef Lyon-Sud Charles Merieux Medical School, Claude Bernard University Lyon 1, F-69310 Pierre Bénite, Franceg University Montpellier 1, Laboratoire de Bacteriologie-Virologie, EA 3755 UM1, Faculte de Pharmacie, F-34093 Montpellier, Franceh Department of Hospital Hygiene, CHU de Montpellier, F-34000 Montpellier, France
Abbreviations: NEC, necrotizing enterocolitis; OTUsTTGE, temporal temperature gel electrophoresis.⁎ Corresponding author at: Service de Néonatologie, H
Grande rue de la Croix Rousse, 69004 Lyon, France. Tel.: +E-mail address: [email protected] (J.-C.
http://dx.doi.org/10.1016/j.earlhumdev.2014.07.0040378-3782/© 2014 Elsevier Ireland Ltd. All rights reserved
a b s t r a c t
a r t i c l e i n f oArticle history:
Received 21 March 2014Received in revised form 6 July 2014Accepted 8 July 2014Keywords:PrematurityGut microbiotaStaphylococcusInfectionRectal bleedingHuman milk
Objective:Gross blood in stools is a peculiar entity in preterm infants, but little is known about its etiology. As gutmicrobiota can be distorted in preterm infants, we aimed to evaluate the gut microbiota in infants with grossblood in stools.Study design: In a prospective, controlled, single-center study, we enrolled all infants born before 34 weeksof gestational age presenting gross blood in stools that was either completely isolated or associated with mildclinical symptoms or radiological signs. Each case was paired with two controls who were hospitalized in thesame unit and were matched for gestational age and birth weight. The diversity of the gut microbiota was ana-lyzed using 16S rRNA gene PCR and temporal temperature gel electrophoresis. We calculated a diversity scorecorresponding to the number of operational taxonomic units present in the microbiota.Results: Thirty-three preterm infants with gross blood in stools were matched with 57 controls. Clinicalcharacteristics were similar in cases and controls. There was no statistically significant difference in the diversity
score between the two groups, but microbiota composition differed. The proportion of infants with Escherichiacoli was significantly higher in cases than in controls (p = 0.045) and the opposite pattern occurred forStaphylococcus sp. (p = 0.047).Conclusion: Dysbiosis could be a risk factor for gross blood in stools in preterm infants. Additional, larger studiesare needed to confirm the implications of the presence of different genotypes of E. coli and to evaluate preventiveactions such as the prophylactic use of probiotics and/or prebiotics.© 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
In premature infants gross blood in stools may be related to severediseases (necrotizing enterocolitis (NEC), colitis in Hirschsprung'sdisease, infectious colitis, or hemorrhagic disease of the newborn) orto milder diseases (allergic colitis, anal fissure, or swallowing bloodsyndrome) [1,2]. Apart from these conditions, gross blood in stools is apeculiar entity either completely isolated or is associated with mildclinical symptoms or radiological signs (gastric residuals, vomiting,
, operational taxonomic units;
ôpital de la Croix Rousse, 10333 472 001 550.Picaud).
.
mild abdominal distension, and mild radiological intestinal dilation).Isolated rectal bleeding has been related to ecchymotic colitis [3].
Occurrence of gross blood in stools has a significant impact on neo-natal care, as the management of this disorder often requires a fastingperiod to start or to extend parenteral nutrition, which increases therisk of catheter-related sepsis [4]. However, published data on grossblood in stools in preterm infants are scarce.
While the predisposing factors of severe NEC (≥stage II) are well-known, including colonization by potentially pathogenic bacteria orgut dysbiosis [5–8], it is not the case for gross blood in stools. Maayan-Metzger et al. reported that a feeding regimen that did not includebreast milk was the only variable that predicted isolated rectal bleedingand emphasized the benign nature of this bleeding [9]. Luoto et al. didnot identify any risk factors in a small number of very-low-birth weightinfants fed human milk supplemented with probiotics [10]. Luoto et al.
580 M.B. Said et al. / Early Human Development 90 (2014) 579–585
did not detect significant changes in the composition of the gut micro-flora in preterm infants with gross blood in stools, but they focusedtheir investigation on a few main bacterial genera and species [10].
In the present investigation, we evaluated the relationship betweenthe composition of the intestinal microbiota and gross blood in stools inpreterm infants.
2. Methods
2.1. Study design and population
We performed a prospective, controlled, single-center study. Allpreterm infants hospitalized between January and October 2011 inour tertiary care unit at the University Hospital Croix Rousse, Lyon,France were eligible for study enrolment if they met the inclusioncriteria of birth gestational age ≤34 weeks, absence of congenital mal-formation, and gross blood in stools. The nurses in charge of daily carefor cases and controls collected systematically few times per day infor-mation about digestive tolerance as routinely recommended in ourunit: gastric residuals, vomiting, abdominal distension, stool consisten-cy and gross blood in stools. Diagnosis was confirmed by physicians byclinical observation. Gross blood in stoolswas either completely isolatedor associated with mild clinical symptoms or radiological signs (gastricresiduals, vomiting, mild abdominal distension, and mild radiologicalintestinal dilation). We did not include infants who developed NEC≥stage II according to the modified Bell classification [11] or exhibitedspontaneous intestinal perforation. The study protocol was approvedby the ethics committee of Lyon (Comité de protection des personnesSud Est IV Lyon).
Each case was matched with two controls. Controls were the firsttwo preterm infants hospitalized in the same unit during the same peri-od, and whose birth weight and gestational age were similar (±100 gand±1 week, respectively) to those of the cases. These infantswere se-lected by researchnurses amonghospitalized infants fulfilling the abovecriteria, independently from clinicians who took care of these infants.Infants included as controls weremonitored as routinely recommendedin our unit: daily monitoring of gastric residuals, vomiting, abdominaldistension, stool consistency and blood in stools.When infants includedas controls later presented rectal bleeding they were excluded from thecontrol group and not replaced by another one, then included in thegroup of cases and paired with two controls.
2.2. Routine care protocol
The feeding regimen of the very-low-birth weight infants was notchanged during the study period. Cases and controls were fed accordingto the same NICU protocol. Enteral feeding was started at day 1 or 2 andcomplementary parenteral feeding was administered until the enteralintake reached 100 mL/kg/day. All infants were fed pasteurized humanmilk (their own mother's milk or donor milk) according to Frenchregulations [12] until their bodyweightwas ~1500 g. Then, if themotherhad no milk, feeding with the same preterm formula (PreMilumel,Milumel, Torce, France)was commenced. Humanmilkwas supplement-ed with a cow's milk protein basedmulticomponent fortifier (Eoprotine,Milumel, Torce France). None of the infants included in our studyreceived probiotics. Full enteral feeding was 160 mL/kg/day and wasachieved by increasing the feeding by 10–20 mL/kg/day depending onthe feeding tolerance of the infant. According to the NICU protocol enter-al feeding was started as continuous feeding followed by bolus feedingwhen full enteral feeding was reached and digestive tolerance was satis-factory. After full enteral feeding was achieved, a thickener was usedwhen infants presented signs of gastro-esophageal reflux (carob-basedwhen the infant was fed human milk and starch-based when the infantwas fed preterm formula). If the signs of gastro-esophageal refluxpersisted, we administered domperidone with or without a protonpump inhibitor (omeprazole or esomeprazole) depending on the
severity of the signs. Infants who presented clinical and ultrasoundsigns of persistent ductus arteriosus were treated with ibuprofen(10, 5, and 5 mg/kg/day at days 1, 2, and 3, respectively).
2.3. Management of gross blood in stools
When gross blood in stools occurred, we systematically assessedblood count cells, serum C-reactive protein levels, serum procalcitoninlevels, and blood culture (aerobic and anaerobic). Stool samples werecollected for bacteriological culture and for the identification of rotavi-rus and adenovirus. Abdominal x-rays were also performed. Then, in-fants received no enteral feeding for a few days, depending on theclinical and radiographic data collected at the time of rectal bleedingand in the following days. For each case with gross blood in stools,stool samples were collected from the two matched control infants.
2.4. Data collection
We collected data on the pregnancies, the deliveries (antenatal ste-roids, mode of delivery, maternal diseases), the infants' characteristicsat birth (gestational age, birth weight, gender, Apgar score), growth re-striction (body weight less than −2SD for gestational age) [13] treat-ments during hospitalization before gross blood in stools (ventilation,antibiotics, ibuprofen treatment, postnatal steroids, anti-reflux treat-ment, breast milk or formula), treatments at the time of gross blood instools (body weight, clinical examination, x-ray results, C-reactiveprotein levels, procalcitonin levels, complete blood count, blood cul-ture), and information about the infant after gross blood in stools(fasting period, recurrence of rectal bleeding).
2.5. Analysis of gut microbiota
The qualitative composition of the gut microbiota was assessed viaextraction of bacterial DNA from stool samples, PCR, temporal tempera-ture gradient electrophoresis (TTGE), and identification of amplified se-quences. Stool samples collected from cases and controls were stored at−80 °C until analysis. Approximately 50mg of each homogenized stoolspecimenwas placed in 1mL of sterile DNA-freewater in a 1.5-mL tube.The suspension was centrifuged for 10 min at 10,000 ×g. DNA wasextracted from the pellet using the MasterPure Gram Positive DNAPurification Kit (Epicentre, Madison, WI, USA) according to thesupplier's instructions and optimized by Roudière et al. [14].
The V2–V3 region (233 bp) of the 16S rRNAgenewas amplifiedwithprimers HDA1 (with a GC-clamp) and HDA2 [14]. The reaction mixture(50 μL) contained 200 μM of deoxynucleoside triphosphate mix,10 pmol of each primer, 2.5 U of TaqDNA polymerase in the appropriatebuffer (FastStart High Fidelity PCR system, Roche, Basel, Switzerland),and 1 μL of template DNA. The amplification program, which was car-ried out with a Mastercycler apparatus (Eppendorf, Le Pecq, France),was 95 °C for 2 min, 30 cycles of 95 °C for 1 min, 62 °C for 30 s, and72 °C for 1 min, with a final extension of 72 °C for 7 min. PCRs werechecked by electrophoresis migration on a 1.5% agarose gel stainedwith ethidium bromide (500 μg/mL) and visualized on an ultraviolettransillumination system.
The DCode universal mutation detection system (Bio-Rad Laborato-ries, Marne La Coquette, France) was used for TTGE. Five microliters ofthe PCR product was added to 5 μL of loading buffer. The gels were pre-pared with 8% (wt/vol) bisacrylamide (37.5:1) and 7 M urea andwere run with 1X Tris–acetate–EDTA buffer. Denaturating electrophore-sis was performed at 46 V for 16 h with a temperature gradient of63–70 °C (0.4 °C/h). Gels were stained with 10 mg/mL ethidium bro-mide and photographed with an ultraviolet transillumination system.
Identification of the TTGE bands was performed as described previ-ously, via comparison of migration distances to the “gut microbiota di-versity ladder” and sequencing on an ABI 3730XL sequencer (Takeley,United Kingdom) [14,15]. The resulting DNA sequenceswere compared
Table 1Clinical characteristics of 33 cases before gross blood in stools and 57 controls beforeinclusion.
Casesn = 33
Controlsn = 57
p-Value
PregnancyHypertension 07 (21) 9 (16) 0.517Gestational diabetes 0 (0) 2 (3) 0.530Transfusion syndrome 2 (6) 3 (5) 1.000Cesarean section 28 (85) 46 (81) 0.620Intrapartum antibiotics 11 (33) 27 (47) 0.194
At birthGestational age in weeks 30 [25, 34] 30 [23, 34] 0.694Birth weight in g 1200 [635, 2160] 1240 [570, 2280] 0.714Birth weight ≤ 2 SDs 1 (3) 2 (3) 1.000Male gender 11 (33) 28 (49) 0.145
Drugs before rectal bleedingDomperidone 3 (9) 0 (0) 0.046Proton pump inhibitor 1 (3) 1 (2) 1.000Ibuprofen 6 (18) 9 (16) 0.769Postnatal steroids 2 (6) 1 (2) 0.552Postnatal antibiotics 18 (54) 30 (53) 1.000Duration of postnatal antibiotics 2 [2, 10] 2 [2, 3] 0.489
Respiratory support before rectal bleedingAssisted ventilation, in hours 0 [0, 1479] 0 [0, 1512] 0.530CPAP, in hours 218 [0, 1600] 190 [0, 1655] 0.933Oxygen therapy, in hours 3 [0, 1291] 20 [0, 1500] 0.443
Values are given as number (percentage) or median [minimum, maximum].SD, standard deviation; CPAP, continuous positive airway pressure.
581M.B. Said et al. / Early Human Development 90 (2014) 579–585
with Genbank (http://www.ncbi.nlm.nih.gov/) and with the RibosomalDatabase Project II (http://rdp.cme.msu.edu/) using the Basic LocalAlignment Search Tool (BLAST) and Seqmatch, respectively. Each se-quence was affiliated with an operational taxonomic unit (OTU) thatmay be defined first at the species level, and otherwise at the genuslevel on the basis of the percent of sequence identity with the closestrelative in the sequence databases. Each OTU was further classifiedinto groups of taxonomic or clinical relevance (genus or family). The di-versity score corresponded to the number of different OTUs in a sampleas previously validated [14,15].
Complementary TTGE experiments were performed to obtain spe-cies level affiliation, for enterobacteria and staphylococci. Tuf-PCR-TTGE [16] and rpoB-PCR-TTGE [17] were performed as described inorder to determine the species in the genus Staphylococcus and in thefamily Enterobacteriaceae, respectively.
2.6. Statistical analysis
Main variables were described with numbers (percentage) for cate-gorical variables and medians [minimum, maximum] for continuousvariables.We used the chi-squared test or Fisher's exact test to comparequalitative variables and the Wilcoxon test to compare quantitativevariables. p-Values less than 0.05 were considered significant. Weused SPSS version 16.0 (SPSS Inc., Chicago, IL, USA) to perform thestatistical analyses.
3. Results
Of the 270 preterm infants with gestational age less than 34 weeksthat were hospitalized in our neonatal intensive care unit betweenJanuary and October 2011, 33 (12.2%) presented gross blood in stools.We matched 57 controls to these 33 cases (Fig. 1). Approximately twoout three cases had isolated rectal bleeding (n = 22). Nine infantsinitially selected as controls presented a gross blood in stools.
270 preterm infants
≤34 wks
Not included for :
. Spontaneous intestinal
perforation (n=1)
. NEC grade 2 (n=1)
Grossblood in stools
(n=9)
Gross blood in stools (n=33)
No rectal bleeding(n=244)
Rectal bleeding(n=26)
Gross blood in stools (n=24)
Controls(n=48)
Controls
n=18
Controls(n=57)
Fig. 1. Flow chart of study design.
The clinical characteristics of the two groups before and at inclusionwere comparable, with the exceptions of treatment with domperidoneand use of thickener. There was no significant difference in antibioticexposure between the two groups (Tables 1 and 2).
The proportion of singletons was similar in both groups (57% ofcases vs. 47% of controls, p = 0.351). Most infants received antenatalsteroids (91% of cases vs. 96% of controls, p = 0.352).
The bacterial diversity scores observed in the overall study popula-tion were low, ranging from 1 to 10, but the scores varied between pa-tients. Therewasno significant difference in the diversity score betweencases and controls (3 [1, 9] vs. 3 [1, 10] OTUs, respectively; p = 0.654).
A total of 39 OTUs were detected in the populations studied. TheseOTUs were distributed through four phyla, with a predominance ofOTUs in Firmicutes and Proteobacteria (Fig. 2), and 10 major genera orfamilies, with a predominance of OTUs in Staphylococcus, Enterobacteri-aceae and Clostridium (Fig. 3). The distributions of phyla (Fig. 2) andmost-represented genera or families (Fig. 3) did not significantly differbetween cases and controls. However, enterobacteria occurred morefrequently in cases but the difference was not statistically significant(Fig. 3). The proportion of infants harboring Escherichia coli was
Table 2Clinical characteristics of preterm infants at the time of gross blood in stools (cases) and atinclusion (controls).
Casesn = 33
Controlsn = 57
p-Value
Postmenstrual age, in weeks 33 [27, 46] 33 [29, 38] 0.427Body weight, in g 1752
[880, 3454]1670[940, 2900]
0.110
Postnatal age, in days 21.5 [3, 102] 17 [4, 75] 0.147Postnatal growth restriction 3 (9.4) 1 (1.8) 0.131Enteral feeding at time of rectal bleeding
Human milka 18 (54) 31 (54) 0.988Preterm formula 11 (33) 16 (28) 0.600Human milka + preterm formula 4 (12) 10 (17) 0.494
Thickener 10 (30) 6 (10) 0.018
Values are presented as number (percentage) or median [minimum, maximum].a Humanmilk fortifiedwith amulticomponent fortifier (Eoprotine,Milumel, Torce France:
4 g/dL).
Fig. 2. Distribution of phyla in the gut microbiota of 90 preterm infants (33 cases and 57 controls). No significant differences were detected between the two groups.
582 M.B. Said et al. / Early Human Development 90 (2014) 579–585
significantly higher in cases than in controls (27.3% of cases vs. 8.8% ofcontrols, p = 0.045) (Fig. 4a); the opposite pattern occurred for theStaphylococcus sp. (12% of cases vs. 32% of controls, p = 0.047)(Fig. 4b). Staphylococcus sp. corresponded to a species level OTU thatcould not be affiliated with certainty to a taxonomic species. Noother significant differences were identified at the genus or specieslevels, particularly concerning colonization with bacteria of the genusClostridium. A systematic search for evidence of rotavirus and adenovi-rus in infants with gross blood in stools revealed the presence of bothviruses in one child and the presence of rotavirus in another infant.
We analyzed isolated rectal bleeding separately and we found re-sults close to what is observed in the whole population (significantlyhigher proportion of infants with E. coli in the cases).
4. Discussion
In our population of preterm infants born before a gestational age of34 weeks, we observed modifications in gut microbiota in infants withgross blood in stools which was recently reported with NEC [18,19].
Fig. 3.Distribution of themost-represented bacterial groupsdefined to the genus or to the familysignificant between-group differences.
We observed a high incidence of rectal bleeding which is in-linewith the study from Luoto et al. in a similar population of preterminfants [10].
The number of subjects in each group was not equal because wewere not able to pair each case with two controls infants. Nine infantsinitially included as controls later presented gross blood in stools. How-ever, the matching of cases and controls was successful, as there wereno significant differences in the characteristics of the included subjects.
Differences in the use of anti-reflux medication and thickenerbetween the two groups indicated that feeding tolerance was lower ininfants presenting gross blood in stools. The potential for thickenedfoods to increase the risk of NEC has been suggested but not confirmed[20,21]. We were not able to carry out multivariate analyses due to thesmall number of events (only three infants received domperidone andthickening) and the small number of subjects in each group.We cannotexclude that thickening could modify intestinal microbiota but there isno data from the literature that supports that hypothesis.
One strength of our study is that our controls were selected from theset of infants hospitalized in the same unit at the same time as the cases,which is crucial when considering the influence of environment on gut
level in the gutmicrobiota of 90preterm infants (33 cases and 57 controls). Therewere no
p = 0.045
p = 0.047
a
b
Fig. 4. Enterobacterial (a) and staphylococcal (b) operational taxonomic units (OTUs) in stool samples from 90 preterm infants (33 cases and 57 controls). A significant difference indistribution was detected for E. coli (p = 0.045) and Staphylococcus sp. (p = 0.047).
583M.B. Said et al. / Early Human Development 90 (2014) 579–585
microbiota in these infants [22]. The diversity of the gut microbiota waslow, as suggested previously [15], and the composition of themicrobio-ta was roughly similar between cases and controls; OTUs were distrib-uted through four bacterial phyla and 10 major genera or families,with the absence of an OTU specific to one patient group. However,the relative frequencies of E. coli and Staphylococcus sp. varied signifi-cantly between cases and controls, suggesting that gut dysbiosis(more E. coli and less Staphylococcus) could be another cause for grossblood in stools. Dysbiosis has been suggested to explain the occurrenceof NEC [8,18] and late-onset sepsis [23]. Mai et al. suggested that a dis-tortion in the normal microbiota composition, and not an enrichmentof pathogens, could be associated with late-onset sepsis in preterm in-fants [23]. Jenke et al. suggested that expansion of E. coli in ELBW infantsis associated with NEC [24]. In preterm infants, staphylococci are pre-dominant during the first month of life [15]. In our study, stool samplewas collected from cases and controls at approximately three weeks ofage. Therefore, the reduction of the presence of Staphylococcus togetherwith an increase in the presence of E. coli in cases represents a substan-tial distortion of the gut microbiota in these subjects.
The significant increase in the presence of E. coli in infants with grossblood in stools is of particular interest. This species consists of severalgenotypes (pathovars) that display particular pathogen behaviors.Classical enteropathogenic pathovars, genotypes that are consideredto be strict pathogens, are rarely involved in the infections of infants
hospitalized in neonatal intensive care units. However, some genotypesseem to display virulence that differs from the classical acute gastroen-teritis associated with most enteropathogenic pathovars. For instance,the enterohemorrhagic strain O157:H7 has been associated with acase of NEC [25], and strains with particular virulence determinantsfor adhesion and invasion seem to be involved in bacteremia [26] andin inflammatory bowel diseases in infants [27]. A recently describedpathovar, diffusely adherent E. coli, not only harbors genes associatedwith virulence, but it also contributes to the normal microbiota presentin asymptomatic patients [28]. Consequently, this pathovarmay exhibitan opportunistic behavior linked to dysbiosis, with the augmentation ofits representation in the microbiota leading to symptoms.
The microbiological approach used in this study allowed the identi-fication of bacteria to the species level, but not to the genotype level.Consequently, we cannot associate the clones of E. coli detected inpatients with a particular pathovar. Additional investigations will berequired in order to describe the E. coli genotypic diversity in the gutmicrobiota of premature neonates.
In preterm infants humanmilk prophylactically supplemented withLactobacillus rhamnosus GG, Luoto et al. found no alterations in gutmicroecology associated with gross blood in stool samples from a verysmall number of infants (25 cases in Luoto et al. vs. 89 in the present in-vestigation) [10]. In contrast to our results, Luoto et al. were unable toidentify differences between cases and controls, probably because
584 M.B. Said et al. / Early Human Development 90 (2014) 579–585
they focused a priori on a limited number of species. It is possible thatprophylactic supplementation with a probiotic contributed in theirstudy to successful gut colonization with bifidobacteria and lactobacilli,but further investigation will be necessary before concluding that aparticular gut microbiota composition is typical of healthy breastfedinfants, as claimed by Luoto et al. [10].
A limitation of our study was the limited number of subjects. There-fore we were unable to correlate, in the cases, the clinical and/or radio-graphic characteristics with micro-organisms. We were neither able toinvestigate whether clinical pattern of infants with E. coli was worsethan the others. Further prospective studies in larger population shouldbe performed to answer these questions. Another limitation of ourstudy was that the diagnosis was performed by sole clinical observation,which could be associated to a degree of subjectivity. However, the diag-nosis was assessed by experienced physicians. We did not confirm thediagnosis as biological testing (e.g. lab analysis or bedside test such asHemoccult®) is not considered reliable in pediatric patients and thereis no published validation in neonates [29]. Another limitation couldhave been related to the bacteriological method we used (TTGE). Micro-biota descriptions based onfingerprintmethods allowed the detection ofonly majority sub-populations forming the community. Recent studiescomparing fingerprinting and high-throughput sequencing showedthat fingerprint methods gave a valuable survey for the detection ofmajor differences between groups of patients [30]. High-throughput se-quencing should be a second step to study deeply the communities inorder to detect dysbiosis affecting minority bacterial populations. Asother sequencing methods focused on a small variable part of the 16SrRNA gene, communities TTGE fingerprints lack of resolution to identifyto the species level with accuracy, particularly in some particular taxa.Here in, we confirmed and complete the identification by the use ofalternative markers tuf and rpoB recognized to have higher resolutionfor species level identification in staphylococci and enterobacteria.
The very high incidence of cesarean section in our study populationcould represent a limitation to the generalizability of our results. Thereare still debates about the influence of the mode of delivery on gut mi-crobiota. In term neonates, cesarean deliver may hamper the develop-ment of digestive microbiota [31,32]. In preterm infants, there are stilldiscrepancies about the influence of the delivery mode on gut microbi-ota. Jacquot et al. reported a faster development of gutmicrobiota after acesarean delivery [15] while other authors did not find any associationbetween the mode of delivery and the number of bacterial species,in small number of subjects [33,34]. Finally, Penders et al. observeda significant impact on colonization rates, which were lower forbifidobacteria and higher for Clostridium difficile and counts of E. coli ininfants born after C-section [35].
In conclusion, our study suggests that dysbiosis could be a risk factorfor gross blood in stools. Additional, larger studies are needed to confirmthe implications of the presence of various genotypes of E. coli and toevaluate preventive actions such as the use of probiotics and/orprebiotics.
Conflicts of interest statement
The authors have no conflicts of interest to declare. Authors have noactual or potential conflict of interest including any financial, personalor other relationships with other people or organizations within thatcould inappropriately influence their work.
All authors made substantial contribution to the design of the study,acquisition of data, analysis and interpretation of data, drafting or revis-ing the article and approved the final version of the manuscript.
Acknowledgments
The authors thank Brigitte Guy, Blandine Pastor-Diez, MarionMasclef, and Bernadette Reygrobellet for their assistance in collectingstools, and Paul Kretchmer for editing of the manuscript.
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