At the moment of birth, the human intestine is sterile. During the first days of life, the infant gut begins to acquire the first bacterial organisms, and by 12 months, the baby acquires a gut flora that approximates the flora of the adult intestine. Hall and O'Toole (1) reported a new anaerobe in the stools of 3 of 6 healthy infants. Because the organism was difficult to isolate, they christened it Bacillus difficilis(1). To their surprise, this harmless commensal organism was extremely toxigenic. When broth culture extracts of B difficilis were injected into guinea pigs, the animals quickly developed convulsions, coma, and death. Here rose a paradox: an organism that caused severe toxicity in test animals was completely harmless to infants. This bacterium was subsequently renamed Clostridium difficile, and is now recognized as the major cause of antibiotic-associated diarrhea and colitis in adults. However, it remains a harmless commensal in neonates and infants (2,3). We review here the acquisition and asymptomatic carriage of C difficile in infants and the implications of the carrier state for C difficile–associated disease in adults.
C DIFFICILE COLONIZATION IN NEONATES AND INFANTS
To determine the colonization rate of C difficile in healthy infants, we searched the PubMed database for studies that surveyed infants younger than 2 years of age with no overt evidence of gastrointestinal (GI) illness. When stratified for age at sampling, a definite pattern emerged (Fig. 1). Among healthy infants younger than 1 month of age, C difficile was recovered from an average of 37% of individuals. Between 1 and 6 months of age, colonization decreased to an average rate of 30%. This trend continued, with recovery dropping to 14% between 6 and 12 months, and 10% >1 year of age. The asymptomatic carriage rate falls after the first year of life, approximating the 0% to 3% carriage rate in adults thereafter (4). The early peak of colonization within the first month of life and its subsequent decline are suggestive of ecological succession, a term used to describe the process in which mammals are initially born with sterile GI tracts, then gradually acquire certain flora and reject others, until a balance of predominant flora is permanently established (5). For example, other Clostridium species isolated in the first week of life, such as Clostridium butyricum and Clostridium sartagoformum, are rarely found in adults (6).
FACTORS AFFECTING RECOVERY RATE OF C DIFFICILE
The wide variation of reported colonization rates in Figure 1 may reflect in part differences in bacteriological techniques, intermittent carriage, and other host or environmental factors.
Techniques to Assay for C Difficile
In most studies C difficile was identified by plating stool specimens on semiselective media under anaerobic conditions, which can yield variable results because of choice of media or freezing of specimens. Real-time polymerase chain reaction (PCR) techniques can amplify conserved 16s rDNA bacterial sequences that allow for identification at the genus and species levels. However, 2 recent reports based on real-time PCR identified C difficile in healthy infants 1 month of age at rates of 44% and 18.2%, respectively (7,8), consistent with the range described for culture techniques.
C difficile carriage in some neonates and infants may be transient (2), whereas others may be colonized by different strains over time, with stool positive for toxigenic and nontoxigenic strains intermittently (3). When typing techniques were used to differentiate strains, some infants had varying serotypes over time (9). In contrast, 1 investigator reported stable colonization in 90% of infants prospectively followed in 1 year (10). We conclude that studies reporting on a single stool sample underestimate overall colonization rates.
Host and Environmental Effects
Cesarean section may influence carriage of C difficile as the infant is delivered under sterile conditions. However, studies in infants born by vaginal, cesarean, and instrumental delivery had similar yields of C difficile in stool, suggesting that method of delivery has little impact (11,12) (Table 1). Furthermore, premature rupture of membranes (12,13), sex (11,12), and prior administration of antibiotics to the mother or the neonate had no effect on carriage rates (2,9,14). Longer duration of hospital stay appears to increase the prevalence of the carrier state (13), possibly through increased exposure to C difficile. However, neonates requiring longer hospital stays are often premature or have comorbid conditions, factors that may independently increase susceptibility to colonization.
Environmental conditions appear to have a strong influence on C difficile recovery rates in infancy: for example, recovery rates from neonates in intensive care units were higher (54.9%) than those from neonates in regular nurseries (11%). Infants in day care nurseries were more likely than neonates in the hospital environment to be colonized with C difficile, suggesting that colonization may depend on frequent interactions between infant, staff, and environment (15). In 1 study, neonates hospitalized in November and December had higher colonization rates (50%) compared with colonization in other months (1%–30%) (14). Colonization rates appear to be similar in infants from developing countries compared with developed countries (15–17).
ACQUISITION OF C DIFFICILE BY NEONATES AND INFANTS
Maternal Transmission of C difficile
C difficile is seldom isolated from the maternal genital tract with reported rates of vaginal culture near 0 (2,3,15,18–20). However, 1 study reported that 71% of the women attending venereal disease clinics had vaginal carriage of C difficile(21). This finding has been subsequently disputed because the criteria used for identification of C difficile were not sufficiently precise (22). As noted above, vaginal delivery has not been shown to predispose to higher rates of C difficile carriage in the neonate (30%) when compared with cesarean deliveries (37.5%), suggesting that vaginal delivery is not a risk factor for acquisition (2). Furthermore, when colonization rates are examined in the first week of life, rates of colonization were near 0% in the first 2 days of life, rising only at the end of the week or weeks later (2,3,12,23,24). Breast-feeding appears to decrease subsequent colonization in the neonate (7,8,10). Furthermore, because rectal swabs from mothers are rarely positive for C difficile (0%–1%) (3,15,19), direct maternal–infant transmission seems unlikely.
Environmental Acquisition of C difficile
Nosocomial acquisition of C difficile occurs frequently among hospitalized adults, with staff and patient rooms often being positive with the same strains cultured from symptomatic patients (4,11). Similarly, when the daily incidence of C difficile was monitored across 3 neonatal hospital wards, the rate of symptomatic carriage was highest in the ward with the highest density of environmental contamination with C difficile(3). C difficile was also frequently recovered from the hands of hospital personnel, from shared equipment such as baby baths and oximeters, and from hospital floors (3,19,25). The time course of acquisition of C difficile in infancy is consistent with environmental transmission because most neonates are not colonized until several days after birth, as environmental exposure in the nursery increases. Moreover, in support of environmental acquisition is the finding that the serotype of strains found in neonates most often matches that recovered from the local environment (9).
DISEASE RESISTANCE TO C DIFFICILE TOXINS IN INFANTS
Following colonization of the lower intestinal tract in adults, C difficile causes colitis through the effects of its 2 protein toxins, designated A and B, which bind receptors on the luminal-facing plasma membrane of colonocytes. After entering the cytoplasm, toxins A and B disrupt the cytoskeleton by inactivating ρ family proteins, triggering colonocyte apoptosis and an acute inflammatory reaction resulting in diarrhea and colitis (26).
The infant gut appears to be completely resistant to the effects of toxin A and B, and clinical infection is rarely reported in infants. After 12 to 24 months of life, the C difficile carrier state disappears, as the child acquires the adult barrier flora. At this time, children become sensitive to the toxins and may develop pseudomembranous colitis similar to adults. Potential mechanisms for disease resistance in neonates include relatively low numbers of the pathogen in the infant gut, preferential colonization of the infant by nontoxigenic or less pathogenic C difficile strains, absence of toxin receptors or downstream signaling pathways in the immature gut mucosa, and protective factors in breast milk and neonatal gut flora.
Degree of Colonization and Toxigenicity
Reduced colony counts of C difficile may explain disease resistance among colonized infants, but evidence suggests similar bacterial counts of C difficile in symptomatic adults and healthy infants. Adult patients with pseudomembranous colitis harbor C difficile ranging from 105 to 109 bacteria per gram of wet feces (27), whereas healthy infants have counts as high as 108.1 bacteria per gram of feces without any overt symptoms, suggesting that low colonization density is not a factor (10,28).
Toxin titers in stool were also found to be similar for healthy neonates and adults with C difficile–associated diarrhea (2,11). Nine studies examined the rate at which toxin-producing strains were recovered from healthy infants. Pooling the data from these studies, 13% of 928 healthy infants carried toxigenic C difficile strains versus 17% carrying nontoxigenic strains (Fig. 2). Thus, infants remain healthy despite the presence of toxigenic strains of C difficile. This observation parallels the important observation that between birth and 24 months, a progressive increase occurs in the number of infants with serum IgG antibodies against toxins A and B. More than 50% of infants test positive for antitoxin antibodies at 1 year of age (29). Moreover, the high levels of serum antitoxins after infancy indicate that infants must be colonized by toxigenic strains of C difficile (Fig. 3) without developing acute colitis.
Absence of Mediators of Toxicity
How may colonized infants remain free of disease in the presence of C difficile toxins in the colonic lumen? Infants may lack the cellular machinery necessary for cellular internalization of the toxin, such as toxin receptors, or elements of the pathogenesis cascade identified in animal studies (26). In 1 experimental model, newborn rabbit ileum was demonstrated to lack intestinal toxin A receptors that are considered necessary for toxin-mediated mucosal damage (30). A similar situation occurs in newborn rabbits that are resistant to Shiga toxin. This is attributed to age-related development of the glycolipid Shiga toxin receptor CB3, which is low or absent in neonatal rabbits and only reaches adult levels after weaning (31).
Protective Factors in Breast Milk and Defenses Provided by Neonatal Commensal Flora
Breast-fed infants are reported to have lower rates of C difficile colonization compared with formula-fed infants (Fig. 4). Clostridium species, in general, were recovered more frequently in formula-fed infants (49%–66%) than in breast-fed infants (6%–20%), suggesting that a factor in human breast milk may inhibit the growth of Clostridium(32). The prevalence of specific colonization by C difficile was nearly 2-fold higher in formula-fed infants (30%) compared with breast-fed infants (14%) (8). Other studies confirm this association between formula feeding and higher rates of C difficile recovery from healthy infants (7,14,33). When breast-fed or formula-fed infants were prospectively studied, these differences in colonization rate in early infancy seemed to vanish at 12 months (14,34), suggesting that breast-feeding may decrease colonization by C difficile in early infancy, with a “catch-up” phenomenon after weaning.
The mechanisms by which breast-feeding prevents C difficile colonization are unclear. Breast-fed infants have a reduced mean fecal pH of 5.29 compared with pH 6.48 in bottle-fed infants, possibly related to the reduced buffering capacity of breast milk versus formula (7). Increased acidity of GI contents may inhibit C difficile growth, perhaps by facilitating sporulation and reducing toxin-producing vegetative forms (35). Breast-feeding may also play a role in interrupting pathogenesis and preventing the action of toxin. Proteins in the aqueous phases of human milk inhibited toxin A binding to hamster colonic receptors by up to 90% (36). Secretory IgA from breast milk had significantly more neutralizing activity against toxin A than other immunoglobulin subtypes. Secretory IgA may exert protection because the molecule contains a domain similar to the intestinal receptor for toxin A. Oligosaccharides in breast milk may also bind toxin A in the gut lumen, thus limiting its attachment to colonocytes (37).
Breast-feeding may also enhance the development of protective organisms that inhibit C difficile colonization. The predominant gut flora in bottle-fed babies consisted of Bacteroides, Clostridium, and Enterobacter, all of which were recovered in higher numbers in breast-fed infants compared with bottle-fed infants (38). Babies who were breast-fed often had decreased fecal pH, which would favor growth of Bifidobacterium while suppressing growth of Bacteroides and Clostridium(38). Bifidobacterium and other anaerobic flora may compete with C difficile for luminal nutrients such as essential amino acids while producing factors that adversely affect survival of C difficile, such as lactic acid, hydrogen peroxide, volatile fatty acids, and bacteriocins (39–41). Increased frequency and carriage of Lactobacillus were inversely related to C difficile colonization (28). A prospective study of 6 healthy babies revealed that C difficile colonization diminished at 1 year of age as the levels of Bacteroides and Eubacterium increased. Both Bifidobacterium and Lactobacillus were able to inhibit the growth of specific C difficile strains (40,42).
To summarize, the infant large bowel is frequently colonized by toxigenic strains of C difficile with colony counts as high as those seen in adults with pseudomembranous colitis. The resistance of infants to disease is likely related to the absence of adequate machinery to bind and process toxin, protective components in breast milk, and competition by other commensal flora (Table 2). The eventual eradication of C difficile from the flora after 12 months of age likely occurs as nutritional and physiological changes in the gut favor the succession of competing flora, which suppresses the growth of C difficile.
C DIFFICILE COLITIS IN LATER INFANCY
Despite frequent carriage by infants, C difficile–associated diarrheal illness before 12 months is rare. In a US survey of 20,642 C difficile–associated deaths from 1999 to 2004, only 17 occurred in the first year of life (43). However, a survey of C difficile infection in a children's hospital in the United States from 2004 to 2006 revealed that 26% of 4895 cases were children younger than 1 year of age (44). Infantile diarrhea is generally related to viral pathogens such as rotavirus and norovirus (45,46). Infants with diarrhea who have C difficile in their stool are clinically indistinguishable from those without the pathogen, suggesting no pathogenic role. Treating infants with diarrhea with antibiotics directed against C difficile usually does not alter infantile diarrhea even if C difficile is present. No correlation seems to exist between diarrhea in infants and C difficile colonization rates, supporting the idea that this organism is rarely pathogenic for infants (9,17,18,47).
Necrotizing enterocolitis, a common GI emergency in neonates, has an unclear etiology, although some have surmised that an altered gut flora may trigger the cascade of hyperactive inflammation. C difficile and its toxin, however, are not recovered at increased frequencies or at higher rates from patients with necrotizing enterocolitis (48–50). A causal relation between C difficile and sudden infant death syndrome has also been discounted, given the high C difficile carriage rate in normal infants and epidemiological observations about sudden infant death syndrome that cannot be explained by carriage of this pathogen (51). The increased worldwide prevalence of atopic disease has prompted speculation that gut microbiota may trigger aberrant immune responses. Healthy infants colonized with C difficile in the first month of life were at higher risk for the development of eczema and atopic dermatitis during the first 2 years of life (52,53). Administration of probiotics during the period of infancy may protect against development of atopic diseases; however, additional work needs to be done to identify relations between C difficile carriage in healthy infants and atopy in later life.
HUMORAL IMMUNITY TO TOXINS IN PATHOGENESIS OF COLITIS
Perhaps the most beneficial outcome of infantile carriage of C difficile is related to its critical influence on C difficile disease expression in adults. As noted above (Fig. 3), serum antitoxins are present in approximately 60%–70% of older children and adults (29). Binding of C difficile toxins by IgG prevents toxin-receptor binding and subsequent diarrhea and inflammation in animal models (54). It is also known that vaccination of laboratory animals against C difficile toxin A is strongly protective against subsequent challenge (55,56). Several recent studies support the potential importance of the infantile carrier state in hospitalized adults exposed to C difficile. Kyne et al (57) reported protection by preexisting toxin A antibodies on C difficile diarrhea and colitis in hospitalized patients receiving antibiotics. Patients with higher levels of antitoxin A at the time of admission were more likely to be asymptomatic carriers of C difficile, whereas patients with low or absent antitoxin A were significantly more likely to develop diarrhea and other manifestations of acute colitis. In a follow-up report from the same cohort, acutely infected patients who developed an IgM- and IgG-positive primary immune response to toxin A eventually experienced complete and lasting recovery, whereas those who failed to respond immunologically were much more likely to experience recurrent disease (58). These data strongly support the view that during the infantile carrier state C difficile serum IgG antitoxins develop, which protect older adults exposed to infection from this pathogen. Vaccination is a promising strategy for the eventual control of C difficile infection, with early studies showing a robust humoral IgG response in healthy adults (59,60).
An unsolved mystery in neonatal biology relates to the curious ability of healthy newborns and infants to tolerate large numbers of the toxigenic pathogen C difficile, the cause of pseudomembranous colitis, a growing scourge in adults exposed to antibiotics (61). Babies who harbor even huge amounts of potent toxins A and B remain entirely healthy, yet the toxins are recognized and processed by the gut immune system, resulting in lifelong immunity. This unique symbiotic relation raises a number of interesting questions regarding the complex interactions between the components of the colonic microbiome and the human host. Solving this puzzle will likely provide interesting insights into the role of the colonic flora in health and disease.
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