Stress Induced Alterations in Intestinal Microflora

The number and types of bacteria that reside as part of the indigenous microflora are thought to be relatively stable, but environmental and physiological challenges have been shown to disrupt this stability. For example, early studies by Schaedler and Dubos (1962) demonstrated that rehousing mice into new cages significantly decreased lactobacilli levels (Schaedler and Dubos 1962). And, chronic sleep deprivation in rats was shown to induce a significant overgrowth of microflora in the ileum and cecum (Everson and Toth 2000), with more recent studies indicating that intrinsic factors such as age and gender can also affect the composition of the microflora of laboratory animals (Ge et al. 2006). Fewer studies have focused on environmental affects on the microflora of humans, but an early study in cosmonauts demonstrated that the intestinal microflora were significantly affected during space flight (Lizko 1987), with others suggesting that some of the effects could be due to the stress of confinement (Holdeman et al. 1976). To further study the potential impact of psychological stress on the stability of the intestinal microflora, we assessed the microflora of young rhesus monkeys that were being separated from their mothers for husbandry purposes (Bailey and Coe 1999).

In captive colonies, rhesus monkeys are routinely separated from their mothers at approximately 6 months of age. At this age, the monkeys are no longer nursing and are eating solid foods. Yet, they still show a strong physiological and emotional reaction to separation from their mothers. This transition from living with the mother to living with other peer monkeys is associated with an increased incidence of diseases, including GI diseases. While much of this can be explained by exposure to new contagion or the actions of the nervous system on the immune system, we hypothesized that the stress response during maternal separation could significantly affect microflora levels in the infants, and thus reduce the barrier effects of the intestinal microflora.

Culture-based enumeration of shed microflora revealed significant alterations in bacterial levels the week following maternal separation compared to levels when the infants were still residing with their mothers. This was evident for Gramnegative and total aerobic and facultatively anaerobic microflora, but only reached statistical significance when a single genus of bacteria was enumerated. The number of aerobically grown lactobacilli was significantly altered after maternal separation (Fig. 11.1). In most cases, the alterations followed a standard profile of increased levels immediately after separation, followed by significantly lower levels 3 days after separation and a return to baseline by the end of the week. Interestingly, the magnitude of the reduction in microflora 3 days after maternal separation could be predicted by the infants' behavior on day 2 post-separation. Three stress-indicative behaviors, cooing, barking, and lip smacking, were associated with microflora levels; in general, those animals that had the highest number of stress-indicative behaviors shed the fewest lactobacilli and total aerobic and facultatively anaerobic bacteria on day 3 post-separation (Fig. 11.2) (Bailey and Coe 1999).

Fig. 11.1 Aerobically grown lactobacilli were enumerated from coprocultures before and for 1 week following maternal separation. Results are mean (S.E.) number of colony forming units (CFU) per gram of fecal matter (wet weight). *p < 0.05 versus preseparation values. Reproduced from Developmental Psychobiology, 1999 with permission from Wiley

Days Post-separation

Fig. 11.1 Aerobically grown lactobacilli were enumerated from coprocultures before and for 1 week following maternal separation. Results are mean (S.E.) number of colony forming units (CFU) per gram of fecal matter (wet weight). *p < 0.05 versus preseparation values. Reproduced from Developmental Psychobiology, 1999 with permission from Wiley

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Log (10) Stress-Indicative Behaviors r=-0.81, p<.0001

Log (10) Stress-Indicative Behaviors

Fig. 11.2 Log transformed stress-indicative behaviors were significantly associated with log(10) CFU/g of intestinal microflora. (a) Total aerobic and facultatively anaerobic microflora. (b) Aerobically grown lactobacilli. Reproduced from Developmental Psychobiology, 1999 with permission from Wiley

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Lactic acid bacteria, such as members of the genus Lactobacillus, are thought to be important contributors to microflora-mediated colonization exclusion. Thus, stressor-induced reductions in lactobacilli would be hypothesized to be associated with enhanced susceptibility to enteric infection. In this experiment, none of the monkeys were intentionally infected, but many nonhuman primate colonies have endemic levels of enteric pathogens, notably Shigella flexneri and Campylobacter jejuni. And, 45% (i.e., 9/20) of the infant monkeys became colonized with either S. flexneri or C. jejuni during the week following maternal separation. On the first day that pathogen colonization was observed there was a weak, marginally significant (p = 0.07) inverse association between the number of lactobacilli and pathogens shed from the intestines (Bailey and Coe 1999). These data are consistent with the idea that lactobacilli are important in colonization resistance against enteric pathogens, but further studies are needed to conclude that stressor-induced alterations in microflora result in increased susceptibility to enteric infection.

Stressor-induced reductions in lactobacilli have also been found in college students during stressful periods (Knowles et al. 2008). In this study, lactobacilli levels were determined during a low stress period (i.e., the first week of the semester) and a high stress period (i.e., final exam week). The exam period was associated with significantly higher levels of perceived daily stress and weekly stress, as well as an increase in GI upset. Moreover, when compared to the low stress period, levels of lactic acid bacteria, primarily lactobacilli, shed in the stool were significantly lower for up to 5 days following examination, with differences in bacterial levels reaching one half log unit in magnitude (e.g., baseline values of 6 x 107 CFU/ml vs. 1 x 107 on day 5 post-examination). It should be noted, however, that significant differences in diet did occur across the two time periods; most notable were significant reductions in vegetable consumption and a significant increase in coffee consumption (Knowles et al. 2008). But, given that stressor exposure alters lactobacilli levels in laboratory animals fed a standardized diet, it is likely that stress-associated changes in human microflora reflect an impact of the stressor as well as potential effects of diet.

Healthy adults are somewhat resistant to the impact of stressors on various physiological systems. For example, stressor-induced alterations in the immune response tend to return to baseline upon termination of the stress response. However, the stress response can have a more prolonged effect on immunity in the very old and the very young (Coe and Lubach 2003). And, stressor exposure in the very young, or even during the prenatal period, is thought to set the infant on a significantly different developmental trajectory, resulting in larger stressor induced effects later in life (Coe and Lubach 2003). One of the most consistent findings in regards to exposure to prenatal stressors is that fetal growth and birth weight are reduced after women experience stressful situations during pregnancy (Field et al. 1985; Lederman et al. 1981; Lederman 1986). Rhesus monkeys have been used extensively to investigate the influence of prenatal stress on infant development. And, it has been shown that prenatal stress affects nueromotor development (Schneider and Coe 1993), emotional reactivity to stressors (Clarke and Schneider 1993), brain monoamine levels (Schneider et al. 1998), cell density in the brain (Coe et al. 2002, 2003), and immune reactivity (Coe et al. 1996, 1999, 2007). Our studies focused on the impact of gestational stress on the intestinal microflora across the four phases of microflora development.

Bacteria colonize the GI tract of newborns in a sequential pattern that is tightly related to developmental milestones in the infant (Cooperstock and Zed 1983). The first phase of colonization begins at birth when bacteria from the mother's reproductive tract colonize the otherwise sterile newborn. These bacteria do not predominate for long and are quickly overcome by maternal aerobic intestinal microflora, which are thought to persist in the intestines for the first few days of life (Tannock et al. 1990). These aerobic species, such as E. coli and Streptococcus spp. consume molecular oxygen as they grow and begin to reduce the oxidation-reduction potential in the intestines creating a more favorable environment for the growth of anaerobic species (Meynell 1963). As a result, high levels of Enterobacteriaceae are evident 1 day after birth, but anaerobes, such as bifidobacteria, predominate by 6 days of age and throughout the period of exclusive breast feeding (Sakata et al. 1985).

Members of the genus Bifidobacterium thrive in breastfed infants and are the predominant bacteria in the intestines due to growth factors found in human milk that bifidobacteria readily use for energy, such as lactose. As bifidobacteria grow, they produce pronounced levels of lactic and acetic acids that can not be buffered by human milk, thus inhibiting the growth of acid sensitive microbes. Breast milk also contains large amounts of immune factors, such as secretory immunoglobu-lins, lactoferrin, lysozymes, and even leukocytes that can inhibit colonization of certain bacteria (Balmer and Wharton 1991; Wharton et al. 1994a, b). The combination of immune factors and acidic fermentation products gives bifidobacteria a tremendous ecological advantage over other species (Heine et al. 1992; Beerens et al. 1980).

The initiation of weaning from breast milk is associated with a resurgence of aerobic and facultatively anaerobic species, such as E. coli, Streptococci, and Clostridia spp., that are naturally found in newly ingested foods. The concentrations of these newly arrived bacteria fluctuate greatly during this period, but as the diet becomes more consistent, microbial populations in the intestines also stabilize and will remain quite stable throughout the lifespan. This stability is important for maintaining intestinal homeostasis (O'Hara and Shanahan 2006), and if disrupted could contribute to the development of GI infections or cancers (O'Hara and Shanahan 2006).

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