What Is Known
- Preterm birth is associated with an aberrant intestinal colonization pattern.
- Preterm infants are routinely tube fed for several days until they are ready for sucking.
- The inner portion of nasogastric enteral feeding tubes has been shown to be colonized by neonatal intensive care unit–associated microorganisms.
What Is New
- A thick bacterial biofilm is formed inside the external feeding tube and connectors within 24 hours contributing to the bacterial composition of the milk that passes through it.
- Sharing of enterobacterial, staphylococcal, and enterococcal strains between milk samples collected after they pass through the feeding system and infant feces was observed.
The colonization of the infant gastrointestinal tract is an essential process that has important short- and long-term consequences for human health (1). Many factors affect the acquisition, composition, and evolution of the infant gut microbiota, including gestational age, mode of delivery, diet, or medical treatments (2). Preterm infants are known to have an abnormal intestinal colonization pattern during the first weeks of life (3,4), which may lead to increased susceptibility to disease (5–7). Compared with infants born at term, the intestinal microbiota of preterm infants exhibits a significantly reduced bacterial diversity and an abundance of microorganisms usually related to hospital environments (8–10).
Breast-feeding is the natural and most recommended way of supporting the growth and development of healthy term infants (11,12). When, for any reason, breast-feeding is not possible and own mother's milk (OMM) is not available, donor human milk (DM) becomes the next best alternative (11,13). Preterm neonates frequently receive a mixed diet regimen, including alternating OMM, DM, and/or preterm infant formula, depending on their health status, internal hospital management, and availability of human milk during their stay at the neonatal intensive care unit (NICU).
Preterm infants are routinely tube fed until they are physiologically ready for the coordination of sucking, swallowing, and breathing, which often occurs at 33 to 36 weeks of postmenstrual age (14). Therefore, any type of feed must be administered through a feeding device. The inner portion of nasogastric enteral feeding tubes has been shown to be colonized by NICU-associated microorganisms (15–17). As a consequence, any nutritional source passing through the tubes may take along bacteria and have a strong effect on the infant intestinal colonization.
In this context, the objective of this work was to evaluate whether the external feeding tube, which is connected with the nasogastric enteral feeding tube through a connecting device, may also act as a site that sustains growth of nosocomial bacteria and thereby affecting the early gut colonization of preterm neonates.
Subjects and Sampling
Thirty-one preterm infants were recruited among those born at the Hospital Universitario 12 de Octubre of Madrid (Spain) from October 2009 to June 2010. The protocol of this prospective study was approved by the local ethics committee (09/157) and written informed parental consent was obtained for each preterm before inclusion. To be eligible for enrolment, preterm infants had to be born at a gestational age of 32 weeks or less or with a birth weight of 1200 g or less. Neonates with any malformation or those experiencing any genetic metabolic disorder were excluded from the study.
All of the infants were fed with human milk (OMM and/or DM) and, occasionally, with preterm formula; however, there was a high individual variability in the feeding pattern. That issue had made impossible to distinguish whether the microbiota composition differed depending on the type of milk fed.
Mother's milk was extracted using an electric pump and stored either refrigerated (5°C) for maximum of 24 hours or frozen (−18 °C) up to 6 months. DM is normally pasteurized (62.5°C, 30 minutes) and stored frozen (−18°C) after collection up to 3 months. The commercial sterilized formula milk used in the hospital is already prepared in individual doses. All milks were warmed during 10–15 minutes at 37°C–40°C before administration. Syringe barrels used as reservoirs were connected through an external feeding tube (EFT) to the infant's nasogastric EFT (NEFT) (Fig. 1). Feeding tubes were routinely replaced and discarded every 24 hours, which means that different feed types could pass through the same tube during that period.
First spontaneously released meconium and weekly fecal samples were collected by the medical staff of the Department of Neonatology of the Hospital from the diapers of the infants during their stay at the NICU. All the samples were stored at −20°C until analysis. Routinely nonused diapers were placed inside the incubators to be used as controls.
Details regarding culture analysis, bacterial identification, and genetic relatedness among selected bacteria from mother and infants with random amplification polymorphism DNA and pulse field gel electrophoresis, and statistical analysis of microbial composition and clinical data are presented in the supplementary methods document (http://links.lww.com/MPG/A597).
Furthermore, 6 different nasogastric enteral feeding tubes, connectors, and external feeding tubes were selected from 6 neonates taking into account numerous conditions that could be involved in microbial growth, such as temperature (cot or incubator), last type of feeding that passed through the tube, time that the tube was placed into the neonate, or infusion rate (gavage or pump). Bacteria present on the internal surface of these devices were analyzed by scanning electron microscopy (SEM) as explained in the supplementary methods document (http://links.lww.com/MPG/A597).
Characteristics of the Infants
Of the 31 infants recruited, 5 dropped out from the trial: 2 infants died before the meconium release and the parents of 3 infants revoked the consent. Twenty-six infants were included in the study. Their main clinical characteristics are described in Table 1. All of them, except 2, received antibacterial prophylaxis at least for the first 3 days of life. Infants were fed with their OMM, DM, and/or formula by nasogastric enteral feeding tube for, at least, 17 days after delivery.
Microbiological Characterization of OMM, DM, and Preterm Formula After Their Pass Through the External Feeding Tubes
The 135 feeding (OMM: 85; DM: 35; and infant formula: 15) samples analyzed in the present study using culture-based methods were the last fraction obtained after their passage through the external feeding tube, immediately before entering the nasogastric tube at the connector level (Fig. 1). The same bacterial profile could be observed in the 3 different feeding types (supplementary Table 1, http://links.lww.com/MPG/A598). Staphylococcus was the genus most frequently isolated from OMM samples (93%) in contrast to DM and formula feeding, in which it was present at 37% and 11%, respectively. Enterococcus was the Gram-positive genus most frequently found in DM and formula samples (49% and 27%, respectively) but it could also be isolated from a high percentage (61%) of OMM samples. In addition, some Gram-negative bacteria, such as Escherichia coli, Klebsiella spp, or Serratia spp, were also isolated from the 3 feeding types (supplementary Table 1, http://links.lww.com/MPG/A598). Mean bacterial counts of Enterococcus (P = –0.004), Staphylococcus (P < 0.001), and Serratia (P = 0.05) were significantly higher in OMM samples (5.03, 4.82, and 5.58 log10 CFU/mL, respectively) than in the other 2 feeding types (supplementary Table 1, http://links.lww.com/MPG/A598).
At the species level, Staphylococcus epidermidis was the dominant species in OMM samples, whereas Enterococcus faecalis was the most abundant in those of DM and infant formula (supplementary Fig. 1, http://links.lww.com/MPG/A599). Serratia marcescens accounted for >23% of the total bacteria isolated from infant formula samples. Three Lactobacillus species (L fermentum, L gasseri, and L salivarius) and 2 Bifidobacterium species (B longum and B breve) were detected in the present study. Among them, L salivarius, B longum, and B breve were only detected in OMM samples.
The diversity and evenness of the microbial communities of the different feed samples after their passage through the external portion of the tubes were determined using the Shannon diversity index. The results obtained showed that the diversity present in OMM (1.15 ± 0.09) samples was higher than that observed in those of DM and infant formula (0.59 ± 0.11 and 0.41 ± 0.06, respectively).
Culture Analysis of the Meconium and Fecal Samples
The time required for spontaneous release of the first meconium varied between the first minutes after birth to 6 days of age. A total of 17 meconium and 128 weekly fecal samples were collected during hospitalization of the infants; on average, 6.4 samples per infant were analyzed. Globally, inoculation of suitable dilutions of all the samples led to bacterial growth on the culture media tested, with the exception of 5 meconium samples. The dominant classes in all the samples analyzed were Bacilli and Gammaproteobacteria.
Meconium samples were characterized by an absence of Gram-negative bacteria, such as E coli, Klebsiella spp, or Serratia spp (P < 0.001), a higher frequency of Streptococcus spp (P = 0.089) and a lower frequency of Staphylococcus spp (P = 0.047) when compared to the fecal samples (supplementary Table 2, http://links.lww.com/MPG/A600).
Mean counts of all bacterial groups were higher in feces than in meconium samples but, at the genus level the differences were statistically significant for Klebsiella (P = 0.012), Serratia (P = 0.034), and other Gram-negative bacteria (P = 0.049) (supplementary Table 2, http://links.lww.com/MPG/A600).
In relation to the succession of the different microbial groups in the fecal samples throughout the infants’ hospitalization, the prevalence of the genus Enterococcus remained high in all the samples, whereas that of Staphylococcus decreased after the first month of life (supplementary Table 2, http://links.lww.com/MPG/A600). Although the number of tested samples decreased as the number of months increased, the prevalence of Klebsiella was significantly lower (P = 0.003) after the third month of life (data not shown).
The number of species detected in the meconium and fecal samples ranged from 1 to 4 and 2 to 9, respectively, increasing with participants’ growth and including up to 5 classes and 12 genera (supplementary Fig. 2, http://links.lww.com/MPG/A601). Despite the interindividual variability observed in the composition of the cultivable fecal microbiota of the infants, E faecalis and S epidermidis were isolated most frequently, being present in 100% and 96% of samples, respectively. Other abundant species belonging to the Class Bacilli were E faecium (77%), L fermentum (42%), and S aureus (38%). In addition, L gasseri and L salivarius were isolated from 8% of the samples. With respect to the Class Gammaproteobacteria, the predominant species were Klebsiella pneumoniae (88%), E coli (81%), S marcescens (81%), Enterobacter cancerogenus (46%), K oxytoca (35%), and E fergusonii (31%).
Finally, 6 different genera of the phylum Actinobacteria (Rothia, Micrococcus, Microbacterium, Dermabacter, Corynebacterium, and Bifidobacterium) were detected. Two species of Bifidobacterium, B breve and B longum, could be isolated from a total of 4 infants.
The diversity of the microbial communities, assessed using the Shannon-Weaver index, was lower in meconium samples (0.42) than in the fecal ones (1.19–1.35), whereas the highest microbial diversity was reached after the third month of life (1.35).
Potential associations between demographic and clinical parameters and isolation of the different genera were assessed using the Fisher exact test (supplementary Fig. 3, http://links.lww.com/MPG/A602). The isolation of the genus Serratia seemed to be strongly influenced by demographic or clinical variables related to prematurity, whereas the presence of E coli was higher in fecal samples from infants with a lower degree of prematurity (supplementary Fig. 3, http://links.lww.com/MPG/A602).
Genotyping of Milk and Fecal Isolates
The isolates belonging to the most frequently detected species in all the samples and sampling points (E faecalis, E faecium, S epidermidis, S aureus, K pneumoniae) were genotyped by random amplification polymorphism DNA and pulse field gel electrophoresis.
Sharing of 34 bacterial strains between milk samples collected after their passage through the feeding system and infant feces was observed at different time points in 23 of the 26 cases (Fig. 2). In most of the cases (26 times), the genotypes were detected simultaneously in milk samples collected after their passage through the feeding system and in infant feces. In addition to this, in 15 occasions the strain was observed first in the milk and at a later time point in the feces, although the opposite occurred in 16 occasions and only 1 time was found 1 strain only in fecal samples. The number of bacterial genotypes shared in each case varied between 1 and 6. The highest strain diversity was detected in the S epidermidis and E faecalis species in which 17 and 9 different genotypes were distinguished, respectively, followed by K pneumoniae with 8 different genotypes. Examples of the genotypes diversity observed in S epidermidis and E faecalis species are shown in supplementary Figure 4, (http://links.lww.com/MPG/A603). Among the S aureus isolates 4 different genotypes were found and only 1 among the E faecium isolates (Fig. 2).
Some genotypes of E faecalis, K pneumonia, or S epidermidis were found in different cases suggesting that these strains may have an environmental origin (Fig. 2). In fact, one of the genotypes of E faecalis (orange circle in Fig. 2) was found in 9 different cases and a different one (blue circle in Fig. 2) in 4 cases. Additionally, one genotype of K. pneumonia (pink cross in Fig. 2) was detected in 3 cases as well as one genotype of S. epidermidis (maroon diamond in Fig. 2).
SEM Analysis of the Nasogastric Enteral Feeding Tubes
The selected parts of 6 external feeding tubes with their respective connectors and nasogastric enteral feeding tubes were analyzed by SEM. The time that the tubes were placed into a preterm infant was the factor that exerted the highest influence on bacterial growth. Thick bacterial biofilms were observed inside the external feeding tube (Fig. 3A–C) and connectors (Fig. 3D–F) that were used for 24 hours and they seemed to be particularly complex in nasogastric enteral feeding tubes that were used for >48 hours (Fig. 3G–J). In contrast, only milk residues (but no bacteria) could be observed in the inner surfaces of those nasogastric enteral feeding tubes that were placed for <12 hours (Fig. 3K and L).
In the present study, the succession of the bacterial species in meconium and feces of preterm infants during their stay in the NICU was assessed by culture-based methods. In addition, bacterial diversity was also studied in preterm feed (OMM, DM, preterm formula) after their passage through the external part of the feeding tubes.
Few studies have considered the role of neonatal nasogastric enteral feeding tubes as a site of bacterial colonization and as a source of bacteria for preterms, and the influence of the feeding regimen on the pattern of colonization of such devices. Such studies have, however, revealed the consistent presence of staphylococci (S epidermidis, S aureus), enterococci (E faecalis, E faecium), and Enterobacteriaceae (K pneumoniae, S marcescens, E cancerogenus, E cloacae, E coli), including clones harboring antibiotic resistance genes, from the inner wall of most enteral feeding tubes analyzed (17–19). All these studies investigated nasogastric enteral feeding tubes (the part of the enteral feeding system that is introduced into the neonate's digestive tract), whereas our work was focused, for the first time, on the external feeding tube, which are not in direct physical contact with the preterm (Fig. 1). The same or closely related microorganisms could also, however, be detected in our study of OMM, DM, and formula samples, after their passage through the external feeding tube. Therefore, the practice of prolonged placement of nasogastric enteral feeding tubes, external feeding tube, or connectors in neonates may need to be reconsidered.
SEM analysis of the internal surfaces of some sets of external feeding tube, connectors, and nasogastric enteral feeding tubes, revealed that complex microbial biofilms were formed when such devices were placed for at least more than 12 hours. As previously described, nasogastric enteral feeding tubes are kept at 37°C and can be in place for more than 48 hours, a perfect environment for opportunistic pathogens arising from the host gastrointestinal tract (17). As biofilms age, bacteria will break off in clumps which, subsequently, will inoculate any fresh feed in the tube lumen leading to further bacterial multiplication and to the reinoculation of the preterm gut with hospital-related microbiota. On the contrary, nasogastric enteral feeding tubes will contaminate external feeding tubes at the connector level, becoming an additional source and reservoir of the host's own gastrointestinal bacteria. Therefore, it is quite logical that the fecal microbiota of preterm infants is usually dominated by cultivable bacteria that are prevalent in antibiotic-rich NICUs environments (3,4,10).
In the present study, OMMs were the samples showing the highest bacterial concentrations after their passage through the tubes, which may be explained by the natural presence of bacteria in human milk as previously shown (20). OMM is provided either immediately after collection or after being refrigerated, which preserves bacterial diversity of this biological fluid (21). In contrast, single-use infant formula is sterilized, whereas DM is pasteurized, a treatment that kills all vegetative bacterial cells whereas allowing Bacillus spp spores to survive (22). Therefore, temperature and hygiene control must be extreme when dealing with postpasteurized donor's milk to avoid contaminations or spores’ germination.
The major difference between OMM and the other 2 feed types was the isolation frequency and concentration of staphylococci (mainly S epidermidis), which were significantly higher in OMM. Staphylococci constitute, at least quantitatively, the main bacterial group in human milk (23–25). Previously, culture-based methods revealed that S epidermidis is the most prevalent species both in human milk and in feces of breast-fed infants, whereas being absent in those of formula-fed infants (26). In fact, this species can be considered as a differential trait of the fecal microbiota of breast-fed infants in contrast to enterococci which are widespread in infant feces, independent of the type of feeding (26). Studies carried 20 years ago already described that staphylococci were common in feces of breast-fed infants (27–30). More recently, it has been shown that coagulase-negative staphylococci colonized 100% of breast-fed Western infants from day 3 onward (31), both in the case of infants born by vaginal delivery and a C-section. Interestingly, staphylococci seem to be the bacteria with the highest ability to use human milk oligosaccharides (32).
Bifidobacterium and Lactobacillus are often described as dominant genera in breast-fed infants and to be present in human milk (33). In our study, they could be isolated from a low percentage of samples and, in the case of bifidobacteria, only after the passage of OMM samples through the external feeding tube. Such bacteria can also be isolated or detected, albeit at a lower rate, in nonheated human milk (34); however, their prevalence may be underrated using either culture-dependent or culture-independent techniques because their isolation is often more difficult than that of other bacteria while DNA isolation methods and currently used primers often lead to the preferential amplification of DNA sequences belonging to other bacterial groups (35,36). It was shown that the detection of lactobacilli or bifidobacterial DNA in the milk samples is significantly lower in those women who receive antibiotherapy during pregnancy, delivery, or lactation (33). It has been long known that antibiotics are responsible for dysbiosis processes in the human microbiota, leading to antibiotic-associated diarrhea and gastroenteritis, urogenital, and oral infections. In this context, one should consider that a high proportion of women having a preterm delivery receive antibiotherapy.
E coli, Klebsiella spp, and other enterobacteria, which comprise nonpathogenic and potentially pathogenic bacteria, can be occasionally detected in breast milk of healthy women (37,38). Molecular studies have shown that they are among the first colonizers of the infant gut coexisting with Gram-positive bacteria (39). Cronobacter sakazakii, which is a major concern in NICUs, was also isolated from the internal surface of nasogastric enteral feeding tubes in previous studies (17–19). This species could, however, not be detected in our work.
Despite differences in bacterial diversity and concentrations, a common bacterial pattern was observed in the 3 types of feed after their passage through the EFTs. As previously mentioned, this may reflect the enteric contamination from the NEFTs at the connector level but also the retention of bacteria present in nonheated milk because the neonate may have received different types of feed through the same EFT (supplementary Table 1, http://links.lww.com/MPG/A598). Therefore, our results strongly suggest that the practice of prolonged placement of NEFTs, EFTs or connectors in neonates may need to be reconsidered.
In addition to the bacterial inoculum, human milk also provides a plethora of other biologically active compounds, including antibacterial agents, such as maternal antibodies, lactoferrin, and lysozyme. This complex composition may explain why even though the initial bacterial inoculum is higher it results in a lower numbers of Enterobacteriaceae in the inner portions of nasogastric enteral feeding tubes in comparison with other feeding regimens (17). This effect can contribute to health beneficial effects that are associated with the use of breast milk for preterm at NICU settings.
Preterm neonates are usually characterized by an abnormal pattern of gut colonization in comparison to that of healthy full-term ones (34,40–42). This is important for infant's health because such a pattern is considered a risk factor in developing gastrointestinal infections and necrotizing enterocolitis (43). Analysis of the meconium and fecal samples led to results similar to those obtained with a similar (although smaller) cohort of preterm infants born in the same hospital (44). Globally, the analysis of the meconium and fecal samples revealed a low species diversity and high interindividual variability, as previously described (3,9,40). Because meconium and fecal samples were frozen at −20°C after their collection the viability and recovery of some bacteria could be affected. At the genus level, Staphylococcus, Streptococcus, Enterococcus, and Lactobacillus predominated in meconium samples, whereas members of the family Enterobacteriaceae, such as Escherichia spp, Klebsiella spp, or Serratia spp, rapidly became dominant in feces, which has been repeatedly reported (4,44,45). The presence of Serratia spp was strongly associated with several hospital-related parameters, which confirms the results obtained by Moles et al (44).
Fear of sepsis and other infections often leads to an early and widespread use of broad-spectrum antibiotics at the NICUs that, in turn, increases the risk of colonization with those bacteria highly prevalent in hospital settings, including resistant bacterial strains. The high influence of the environment explains the tendency to uniformity in the bacterial communities of preterm infants during their stay at the NICU (10). Actually, the same bacterial genotypes of different genera have been found in the feeds after their passage through the external feeding tube and the fecal samples of different infants but it has not been proven that colonization of the feeding tube precedes intestinal colonization. Hospital-associated bacterial species that integrate the thick biofilms formed in the feeding tubes may influence the fecal microbial composition of preterm neonates. Some bacterial strains have, however, been observed first in fecal samples and a later time point in milk, which could mean that there may exist another source of bacteria for the baby. In addition, the low bacterial concentration in milk may hinder the detection of specific strains that are easier to isolate from fecal samples in which the bacterial concentration is at least 3 log units higher (46,47).
Future work should address novel strategies to minimize the effect of the NICU environment on the early colonization of the preterm gut.
The authors acknowledge all the families that participated in the present study. The authors also thank Ana Vicente from the Centro Nacional de Microscopía Electrónica (Madrid) for her helpful assistance with SEM technique, and are also grateful to Hans Heilig and Dr Rocio Martin for the revision of the manuscript.
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