Intrinsic host factors that influence the composition of the Lactobacillus G

microbiota.

Lactobacilli that occupy the oral cavity must adapt to certain environmental stresses that intestinal Lactobacillus species are not usually subjected to, such as oxygen exposure, sharp fluctuations in local pH and temperature during meal-times and recurring exposures to anti-microbials as part of routine oral hygiene practices.

To colonise the intestines, a bacterium must first withstand challenges to its structural integrity presented by digestive enzymes and acid in the mouth and the stomach. The bacteria that enter the intestines may therefore be sublethally damaged. Mechanical and chemical forces, such as the peristaltic contractions of the intestines and the secretion of anti-microbial substances by the host and the microbiota alike, may further hinder the ability of a bacterium to colonise this niche.

1.1.3.1.1 Influence of pH.

The pH in the mouth is in the range of 6.75 - 7.25 (Marsh, 2003), and the lactobacilli are a subdominant component of the oral microbiota (Dewhirst et al., 2010). Dental plaque is a multi-species biofilm that forms on teeth. However, the species composition of dental plaque may be influenced by the prevailing conditions (Marsh, 2003), such as such as pH, oxygen and nutrient availability. This is summarised in the “ecological plaque hypothesis” which is founded on the assumption that microbial homeostasis is established in the oral cavity and that any sustained alteration to the prevailing conditions could favour a cariogenic oral environment. Changes to the oral environment would select for species that are more tolerant of the new conditions (Marsh, 1994). A major ecological pressure such as an enrichment of fermentable sugars in the host diet, may favour an acidic oral

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environment and a dental plaque that is dominated by aciduric and acidogenic

Lactobacillus and Streptococcus species (Marsh, 1994).

Gastric acid (pH = 1 - 2) is a major colonisation barrier that restricts outgrowth of microorganisms in both the upper and lower GI tracts. Lactobacilli were more numerous in the faecal microbiotas of persons with hypochlorhydria, (a condition defined by reduced hydrochloric acid secretion in the stomach), when compared to asymptomatic individuals without chronic atrophic gastritis (CAG) (Kanno et al., 2009).

Importantly, Lactobacillus strains intended for use as probiotics or as drug and vaccine delivery agents, must be able to withstand gastric transit (FAO/WHO, 2001). During colonisation of the murine stomach, L. reuteri induces expression of acid- tolerance genes, including glutamate decarboxylase and urease (Wilson, 2011; Wilson

et al., 2012). Presumably, the aciduric Lactobacillus species that colonise the human

stomach have a similar complement of acid-tolerance genes.

The pH of the intestinal lumen is influenced by gastric acid, pancreatic secretions, microbial fermentations and host diet. The small intestine and colon have mildly acidic pHs (pH ~ 5 - 7) which increase distally (Bown et al., 1974; Ridlon et

al., 2006). The unequal availability of fermentable carbohydrate throughout the colon

is probably partly responsible for the proximal to distal pH gradient (Macfarlane et

al., 1992). Specifically, carbohydrate and protein catabolism by enteric microbes

tends to reduce the luminal pH by the production of acidic metabolic by-products such as short-chain fatty acids (SCFAs), phenols, indoles, thiols, hydrogen sulphide, carbon dioxide and hydrogen gas (Macfarlane et al., 1992; Macfarlane & Macfarlane, 2012). Protein fermentation additionally yields molecules which have basic properties, such as ammonia (Macfarlane et al., 1992). A combination of pH levels

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and peptide availability influence the composition of the intestinal microbiota and also the intestinal SCFA profile produced (Walker et al., 2005). Experiments performed in a continuous flow fermentor showed that more butyrate was present at a maintained fermentor pH of 5.5 than at pH 6.5, under conditions of both high and low peptide availability. This coincided with expansion of the butyrogenic bacterial population at the lower pH. Propionate formation was greatest under high peptide (0.6 %) conditions at pH 6.5 (Walker et al., 2005). Thus, it may be inferred that in the colon, the local pH, peptide availability and utilisation of bacterial-derived metabolites cumulatively influence the composition of the microbiota.

1.1.3.1.2 Influence of digestive enzymes and bile.

The duodenum receives enzymatic secretions from the pancreas and biliary secretions from the gall bladder as part of the normal digestive process. Bile acids have a dual purpose; they are important for the emulsification of dietary lipids, but also as anti-microbials (Begley et al., 2005). Thus, bile acids have a significant influence on the composition of the intestinal microbiota and also play a role in enteroprotection by stimulating the host’s anti-microbial defences (Inagaki et al., 2006).

When bile acids are synthesised from cholesterol in the liver they are usually conjugated to either taurine or glycine, which increases their solubility (Hofmann, 1994). These conjugated bile acids are released into the duodenum from the gall bladder following a meal to assist the digestion and absorption of dietary fats and fat soluble vitamins. Bile acids are modified by the actions of the resident microbiota in the intestines, yielding unconjugated and secondary bile acids (Hofmann, 1994; Ridlon et al., 2006). Bile acids and their derivatives have significant anti-microbial properties (Begley et al., 2005). Membrane damage caused by the affinity of the

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inherently hydrophobic bile acids for the phospholipid bilayers of bacteria is fundamental to the bactericidal effect of bile (Kurdi et al., 2006). Bile acids are efficiently reabsorbed in the ileum, so bile concentrations are greatest in the proximal small intestine and decrease distally (Hofmann, 1994).

The Lactobacillus bile-resistance phenotype is conferred by a number of genes and proteins which respond to different aspects of bile challenge (Hamon et al., 2011; Hamon et al., 2012). Those with a role in modifying the cell surface or in maintaining the structural integrity of the cell wall and membrane were found to contribute to the bile tolerance phenotype of L. casei and L. plantarum strains (Hamon et al., 2011; Hamon et al., 2012). In L. delbrueckii subsp. lactis and L. casei, some central aspects of cellular function such as translation and carbohydrate metabolism were altered in response to bile (Burns et al., 2010; Hamon et al., 2012). Bile induced L. plantarum strains to produce glutathione reductases, presumably to cope with bile-induced oxidative damage (Hamon et al., 2011). In L. casei and L.

delbrueckii subsp. lactis, proteins with a role in cell protection, such as the molecular

chaperone-protease ClpL, contributed to the early stages of the bile-resistance phenotype (Burns et al., 2010; Hamon et al., 2012). L. plantarum expressed an Opu ABC transporter to overcome the osmotic stresses imposed on the bacterium by bile

(Hamon et al., 2011), and expression of an F0F1-ATPase (AtpH) may have

contributed to the resistance phenotype by maintaining intracellular pH (Hamon et al., 2011). Bile salt hydrolases are enzymes with a role in the detoxification of bile acids (Begley et al., 2006). While lactobacilli that are intended for use as probiotics are routinely screened for the presence of bile salt hydrolases (BSH) (FAO/WHO, 2001), these enzymes may not play a central role in the bile-resistance phenotype of

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lactobacilli (Begley et al., 2005; Fang et al., 2009; Hamon et al., 2011; Hamon et al., 2012; Moser & Savage, 2001).

1.1.3.1.3 Influence of oxygen availability.

The concentration of oxygen in the gut decreases distally (He et al., 1999), and oxygen tension is particularly low in the rectal lumen (Lind Due et al., 2003). The intestine is essentially an anaerobic environment (Hartman et al., 2009) and any swallowed oxygen that arrives to the intestine is rapidly consumed by facultative anaerobes. Lactobacilli can be detected in the microbiota of vaginally delivered neonates 48 hours after birth (Karlsson et al., 2011) and are continually represented as part of the intestinal microbiota of infants through weaning and during the first 18 months of life (Ahrne et al., 2005). This suggests that the enteric Lactobacillus community is tolerant of aerobic and anaerobic conditions.

1.1.3.1.4 Influence of mucus and cationic anti-microbial compounds.

Mucus is a viscous fluid that lines the GI tract and provides a physical barrier between the luminal contents and the underlying epithelium and also acts as a non- specific immune defence against invading bacteria. The mucus lining that covers the GI epithelium consists of a thick outer layer which is in direct contact with the intestinal microbiota, and a thinner inner layer that is effectively devoid of microbes (McGuckin et al., 2011; Swidsinski et al., 2007). The outer mucus layer is thickest at sites of potential injury, such as in the stomach and colon (Atuma et al., 2001). However, the outer mucus layer is constantly sloughed off by the movement of undigested food boluses and faecal material through the GI tract, and also by the digestive activities of the ~ 1 % of the resident microbiota that can use mucus components as a substrate (Derrien et al., 2004; Hoskins et al., 1985; Ruas-Madiedo

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Lactobacilli do not usually degrade mucins (Ruseler-van Embden et al., 1995; Turpin et al., 2012; Zhou et al., 2001). Rather, lactobacilli such as L. rhamnosus (Kankainen et al., 2009) and L. fermentum (Macias-Rodriguez et al., 2009) have evolved mechanisms to adhere to components of colonic mucus. This may represent a colonisation factor that helps these bacteria to persist in the intestines.

Cationic anti-microbial compounds (CAMPs) are peptides (Peschel & Sahl, 2006) found in mucus that influence the composition of the intestinal microbiota. The lactobacilli may either stimulate CAMP secretion or they may be sensitive to CAMP molecules. For example, Lactobacillus rhamnosus GG is sensitive to human beta- defensin-2, but not to beta-defensin-1 (De Keersmaecker et al., 2006a). A number of

Lactobacillus species stimulate beta-defensin-2 secretion in vitro (Schlee et al., 2008)

and are presumably resistant to the anti-microbial effects of these peptides. L. reuteri 100-23 is also resistant to human beta-defensins 1 - 4 (Walter et al., 2007). L.

delbrueckii subsp lactis CIDCA 133 but not L. delbrueckii subsp. bulgaricus CIDCA

331 was resistant to human defensins (Hugo et al., 2010).

Thus, the fate of the microorganisms that reach the duodenum alive is dependant on their ability to further withstand the many barriers to colonisation imposed by the host. However, the resident microbiota may also manipulate the microbiota locally, which facilitates the colonisation or adherence of some species at the expense of others.