Journal of Developmental Origins of Health and Disease, page 1 of 10. © Cambridge University Press and the International Society for Developmental Origins of Health and Disease 2015 doi:10.1017/S2040174415001506
ORIGINAL ARTICLE
High frequencies of antibiotic resistance genes in infants’ meconium and early fecal samples M. J. Gosalbes1,2, Y. Vallès1, N. Jiménez-Hernández1, C. Balle3, P. Riva1, S. Miravet-Verde1, L. E. de Vries3,4, S. Llop2,5, Y. Agersø6, S. J. Sørensen3, F. Ballester2,5 and M. P. Francino1,2,7* 1
Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana (FISABIO), Unitat Mixta d’Investigació en Genòmica i Salut, FISABIO-Salut Pública/Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Valencia, Spain 2 CIBER en Epidemiología y Salud Pública (CIBEResp), Madrid, Spain 3 Department of Biology, Section of Microbiology, University of Copenhagen, Copenhagen, Denmark 4 Department of Technology, Metropolitan University College,Copenhagen, Denmark 5 FISABIO-UJI-University of Valencia Epidemiology and Environmental Health Unit of Research, Valencia, Spain 6 National Food Institute, Technical University of Denmark, Lyngby, Denmark 7 School of Natural Sciences, University of California Merced, Merced, CA, USA
The gastrointestinal tract (GIT) microbiota has been identified as an important reservoir of antibiotic resistance genes (ARGs) that can be horizontally transferred to pathogenic species. Maternal GIT microbes can be transmitted to the offspring, and recent work indicates that such transfer starts before birth. We have used culture-independent genetic screenings to explore whether ARGs are already present in the meconium accumulated in the GIT during fetal life and in feces of 1-week-old infants. We have analyzed resistance to β-lactam antibiotics (BLr) and tetracycline (Tcr), screening for a variety of genes conferring each. To evaluate whether ARGs could have been inherited by maternal transmission, we have screened perinatal fecal samples of the 1-week-old babies’ mothers, as well as a mother–infant series including meconium, fecal samples collected through the infant’s 1st year, maternal fecal samples and colostrum. Our results reveal a high prevalence of BLr and Tcr in both meconium and early fecal samples, implying that the GIT resistance reservoir starts to accumulate even before birth. We show that ARGs present in the mother may reach the meconium and colostrum and establish in the infant GIT, but also that some ARGs were likely acquired from other sources. Alarmingly, we identified in both meconium and 1-week-olds’ samples a particularly elevated prevalence of mecA (>45%), six-fold higher than that detected in the mothers. The mecA gene confers BLr to methicillin-resistant Staphylococcus aureus, and although its detection does not imply the presence of this pathogen, it does implicate the young infant’s GIT as a noteworthy reservoir of this gene. Received 30 April 2015; Revised 23 July 2015; Accepted 18 August 2015 Key words: antibiotic resistance, gastrointestinal microbiota, meconium, mecA, tetracycline
Introduction Not even 100 years after the discovery of the first antibiotic, we are faced with a worldwide public health concern as antibiotic resistant strains are rapidly spreading as a consequence of antibiotic misuse and abuse in human and animal disease control and treatment as well as a growing connectivity between populations. Endowed with the most diverse microbiome of the body, the human gastrointestinal tract (GIT) has been shown to be a significant reservoir of antibiotic resistance.1–7 Furthermore, not only the adult GIT constitutes such a reservoir, but the infant GIT also harbors a variety of antibiotic resistances as early as the 1st month of life.8–12 By analyzing stool samples taken 1 month after childbirth, de Vries et al.10 were able to show that specific tetracycline resistance genes with identical sequences were recovered from both mother and infant, suggesting vertical inheritance of such genes. Traditionally, because the womb was *Address for correspondence: M. P. Francino, Unitat Mixta d’Investigació en Genòmica i Salut, FISABIO-Salut Pública, Ave. Catalunya 21, Valencia 46020, Spain. (Email
[email protected])
considered sterile, such vertical transmission could only be conceived as an after-birth event due to the exposure of the infant to the external and proximal environment. However, today, in the era of metagenomics, this dogma has been laid to rest. On the mother’s side, there are important physiological changes occurring in the GIT during pregnancy and in particular during the third trimester, in which the mother shows symptoms similar to those presented by individuals with metabolic disorders, obesity and diabetes.13–15 These conditions entail an inflammatory state of the intestinal epithelium that could enhance translocation events of bacteria to the mesenteric lymphatic nodes (MLN) and blood. In fact, Berg16 suggests that in healthy immunocompetent individuals a low level of translocation of bacteria to the MLN is the norm rather than the exception, and that mechanisms such as intestinal bacterial overgrowth, increased mucosal barrier permeability and/or host immune deficiencies would allow for such events to increase in frequency. Both of the latter mechanisms are exacerbated during pregnancy, as a low-grade inflammation of the intestinal epithelium takes place involving a reduction of epithelium integrity17 and immune responsiveness is altered in order to allow for the development of the fetus and placenta.
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These physiological changes presumably could foster translocation events, facilitating transport of maternal GIT bacteria to the fetal GIT.18–20 In fact, in mice enhanced bacterial translocation from the gut has been shown to take place both during late pregnancy21,22 and at the early onset of type 2 diabetes,23 and, in the latter case, the dendritic cells of the immune system have been implicated in mediating the increased translocation level. On the fetus side, the dendritic cells, essential for their immune function of antigen sensing and presenting, play key roles in the establishment of a tolerogenic environment, providing a delicate immunological balance throughout fetal development. This is possible in part thanks to the fact that fetal T-cell differentiation is biased towards producing regulatory (Treg) cells upon stimulation. The establishment of such a robust tolerogenic environment is essential particularly in the fetal GIT, permitting the initiation of commensal microbe colonization.24 In accordance with the notion of maternal transmission of bacteria to the fetus, a substantial number of both culturedependent and 16S-rRNA-based analyses have demonstrated the presence of bacteria in amniotic fluid,25–32 fetal membranes,27,33 umbilical cord,34 placenta35–37 and meconium.19,38–45 Although it is difficult to completely rule out the possibility of external bacterial contamination of intrauterine human samples, experimental work in mice has confirmed that an efflux of bacteria from the mother’s gut to that of the fetus does exist, as genetically labeled bacteria orally inoculated to pregnant mice are recovered from the meconium of offspring obtained by C-section.38 The bacteria that reach the fetal GIT could have a strong influence on the process of microbial succession in this niche as well as on the trajectories of immune, metabolic and somatic development.18–20 Therefore, it is important to investigate the composition of the microbiota present in meconium, and the type of genes that it carries. 16S rRNA analyses show that meconium microbial communities in term neonates are predominantly composed by enterobacteria42 and/or lactic acid bacteria (LAB).19 Remarkably, this composition coincides with the reported levels of translocation efficiency from the GIT to the MLN for different bacteria,16 which are highest for the Gram-negative, facultatively anaerobic enterobacteria, intermediate for the Gram-positive, oxygen-tolerant LAB, and lowest for obligately anaerobic bacteria, such as Bacteroides, which is scarcely represented in meconium in spite of its high abundance in the adult GIT. Moreover, Cabrera-Rubio et al.46 have identified in colostrum, the first ‘milk’ secreted by the human breast, a bacterial microbiota composed mainly of the same LAB genera often detected in meconium, suggesting that these bacteria may travel through the maternal circulation and reach both the fetus and the mammary gland. In addition, we have detected similarities in composition between the microbiota in meconium and that present in the infant during the 1st weeks of life, while showing that some of the bacterial strains found in meconium remained in the infant GIT up to 7 months of age.19 In the present work, we have used a culture-independent approach to explore whether the bacteria present in meconium and in the fecal microbiota of 1-week-old babies carry detectable
frequencies of antibiotic resistance genes (ARG). For comparison, and to assess the possibility of maternal transmission, we have also investigated the ARGs present in the perinatal fecal microbiota of the mothers of the 1-week-old babies. Furthermore, for one of the mother–infant pairs (MIPs), we have evaluated the presence of ARGs in an entire series of fecal samples spanning the infant’s 1st year of life (including meconium), as well as in colostrum and in maternal fecal samples collected during the perinatal period and 1 year after delivery. Our screenings have targeted resistances to β-lactam antibiotics (BLr) and tetracycline (Tcr), which are known to be widespread in the GIT microbiota. Specifically, we have screened for the presence of different types of genes encoding β-lactamases, which convey resistance by breaking up the β-lactam ring in the antibiotic,47 including the extendedspectrum β-lactamase genes blaCTX-M and blaKPC, the broadspectrum β-lactamase gene blaTEM, and the metallo-β-lactamase genes blaNDM and blaVIM. We have also screened for mecA, which encodes the penicillin-binding protein 2a that provides resistance to methicillin and a wide array of β-lactam antibiotics.48 Finally, we have screened for 11 tet genes encoding proteins that confer Tcr by several mechanisms,49 including active efflux [tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(K), tet(L)], ribosomal protection [tet(O), tet(Q)] and monooxigenase inactivation [tet(X)]. Method Study subjects This study employs meconium samples and metadata from 20 children enrolled in the Valencia birth cohort of project INMA (Infancia y Medio Ambiente – Childhood and the Environment, http://www.proyectoinma.org).50 Briefly, pregnant women from a well-defined geographic area in Valencia and attending the first prenatal visit at La Fe Hospital during November 2003 to June 2005 were recruited before week 13 of gestation and followed up until delivery. Their children were enrolled at birth and have been followed up until 9 years of age. The mothers of the 20 children were all born in Spain, had a non-vegetarian diet and, in most cases (18/20), had normal body mass indexes (18.5–24.9) before pregnancy. All had healthy pregnancies with no complications (no fever, urine infection, gestational diabetes, high blood pressure or amniotic fluid losses). Six of the mothers underwent a C-section and likely received amoxicillin during delivery, as this is the standard practice in Spanish hospitals, although this was not specified in the Valencia INMA cohort clinical metadata. Two of these mothers also received some antibiotic treatment before the 12th week of pregnancy and one of them received antibiotic treatment after the 12th but before the 32nd week. In addition, one of the 14 mothers who delivered vaginally also received antibiotics before the 12th week (Table 1). The 20 infants were born at term (>37 weeks of gestation), none of them had a low birth weight (
