La inducción de los principios

The power-time curves of pure cultures of C. difficile, LA-5^® and BB-12^® in BHIct are shown in Figures 4.4, 4.5 and 4.6 respectively. The power time curves were characteristic for the different species. Both LA-5^® and BB-12^® showed exponential growth in this medium without significant time lag unlike C. difficile which metabolised energetically prior ca. 7 h of dormancy in the microcalorimeter. The long lag phase that occurred for C. difficile could be associated with its slow rate of growth (Carroll, 2013).

It could as well be associated with the period required for the spores to adapt, sense the suitable environment for germination and subsequently germinated and commenced vegetative growth (Stringer et al., 2011). The former events may have been associated with metabolic activity (Stringer et al., 2011) but possibly with related heat outputs, which was below the detection limit of the microcalorimeter.

Figure 4.4. Power-time curves of 3 repeats of C. difficile in BHIct

0 10 20 30 40

0 100 200 300 400 500 600 700 800 900 1000 1100 1200

P (W)

TIME (h)

C.diff BHIct repeat 2 repeat 3

Figure 4.5. Power-time curves of 3 repeats of L. acidophilus, LA-5^® in BHIct

Figure 4.6. Power-time curves of 3 repeats of B. lactis, BB-12^® in BHIct

The total heat outputs associated with the metabolism of C. difficile was 9.93 ± 0.38 J and was significantly higher (P< 0.05) than that of LA-5^® and BB-12^® in BHIct which were 6.05 ± 0.40 and 6.93 ± 0.36 J, respectively. The higher heat output associated with the metabolism of C. difficile relative to the probiotic species could be due to the

together might have been a higher energy yielding process relative to sole vegetative growth. A previous study by Kalakoutskiai and Pozharitska, (1968) has shown that the emergence of germ tubes of spores of Actinomyces streptomycini in the microcalorimeter was associated with great heat output which was noted to be in the upper limit of heat generated by growing cultures of E. coli. The authors also demonstrated that the swelling prior to exponential growth was not associated with significant heat production (Kalakoutskiai and Pozharitska, 1968). Also, it is likely the medium might have favoured the growth C. difficile relative to the probiotic species, which could have been inhibited by the bile salt content of the medium (Begley et al., 2005). Exclusion of bile salt from the medium however did not change the power-time curves of the probiotic species.

A post TAM analysis of the culture showed a significantly less turbid culture of LA-5^® relative to the other species and previous cultures of it in CMMg. It was also noted that the probiotic species did not produce as many acidic metabolites in BHIct as they had previously done in CMMg (Chapter 3). Nonetheless, they significantly reduced (P<0.05) the pH of the medium to 5.13 ± 0.04 and 4.92 ± 0.01 respectively for LA-5^® and BB-12^® whilst C. difficile also reduced the pH of the medium to 5.88 ± 0.05 (P<0.05) from an initial value of 6.77 ± 0.01.

The power-time curves of mixed cultures of C. difficile with LA-5^® and C. difficile with BB-12^® are shown in Figures 4.7 and 4.8. The power-time curves of the mixed cultures lacked the characteristic curve of C. difficile and showed only the growths of the probiotics.

In the previous section, it was demonstrated that C. difficile could germinate and grow in the environment created, BHIct, pH 6.8. This medium could simulate the environment of the small intestine (due to the bile salt) (Kalantzi et al., 2006) or the colon (Paredes-Sabja et al., 2008) due to the potassium and phosphate content of BHI (Paredes-(Paredes-Sabja et al., 2008). Also, the culture of C. difficile that was inoculated into BHIct consisted of mainly spores of C. difficile, which could simulate the oral transmission of the infection (Gerding et al., 2008a, Burns et al., 2010, Burke and Lamont, 2014). The results suggest that the consequent vegetative growth and production of toxins associated after germination may be prevented in the intestine by the probiotics since C. difficile germination may not have occurred in the presence of the probiotics.

Figure 4.7. Comparison of the power-time curves of pure culture of L. acidophilus, LA-5^® and C.

difficile, Cdiff and their mixed culture (LA-5+Cdiff) in BHIct

Figure 4.8. Comparison of the power-time curves of pure culture of B. lactis, BB-12^® and C. difficile, Cdiff and their mixed culture (BB-12+Cdiff) in BHIct

As indicated earlier, spores of the organism are pivotal for transmission of the infection;

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production and clinical manifestations of the infection. In the previous section, bile salt was demonstrated to play an important role in the germination of the organism but in the presence of the probiotic species, germination may not have progressed. These probiotic species could potentially play a major role in the prevention or treatment of the infection in susceptible individuals since no toxins would be consequently produced to cause CDAD.

The mechanism of inhibition of C. difficile by the probiotic species was likely due to the probiotic species competitively utilizing the nutrient sources in the media before C.

difficile could be adequately capable of metabolic activity. Furthermore, the probiotics produced acids, or likely other non-acidic metabolites during their growth, which might have caused C. difficile to remain in spore form. The pH of the medium after growth of the mixed cultures of LA-5^® and C. difficile and BB-12^® and C. difficile were significantly reduced (P<0.05) to 5.24 ± 0.19 and 4.92 ± 0.03 respectively showing that they indeed still produced acidic metabolites in mixed culture which could have inhibited the growth of C. difficile.

In the previous experiments of mixed culture of the probiotic species with P. aeruginosa, S. aureus and E. coli in Chapter 3, it was observed that growth of the probiotics occurred after or concurrently with the growth of the other faster growing species. To determine whether C. difficile could germinate and grow in the presence of other species, C.

difficile was also co-cultured with P. aeruginosa. The power-time curve showed the presence of both species (Figure 4.9), which implied that the inhibition of C. difficile by the probiotics might have been specific.

Enumerations of vegetative cells of C. difficile post TAM experiments on selective medium, Clostridium difficile selective agar supplemented with Clostridium difficile selective supplement (cycloserine 250 mg/L, cefoxitin 8 mg/L) and 0.7% v/v defibrinated horse blood “CDSAsb” gave no recoveries of C. difficile on plates for the mixed cultures with probiotics (Table 4.2).

Figure 4.9. Power-time curve of mixed culture of P. aeruginosa (first peak) and C. difficile in BHIct

Table 4.2. A table comparing the mean heat output and post TAM profile of the pure culture and mixed culture of probiotics and C. difficile. n=3 for numerical values

Culture in BHIct Heat output (J) pH post TAM Cell count post TAM (log CFU/mL)

C. difficile 9.93 ± 0.38 5.88 ± 0.05 6.00± 0.60

B. lactis BB-12^® 6.93 ± 0.36 4.92 ± 0.01 6.47± 0.07

L. acidophilus LA-5^® 6.05 ± 0.40 5.13 ± 0.04 5.19 ± 0.19

C. difficile and B. lactis BB-12^® 6.63 ± 0.35 4.92 ± 0.03 Cdiff BB-12^®

0 6.50 ±

0.03 2.1x10⁵ C. difficile and L. acidophilus LA-5^® 5.79 ± 0.52 5.24 ± 0.19 Cdiff LA-5^®

0 5.04 ±

0.09

0 10 20 30 40

0 100 200 300 400 500 600 700 800 900 1000 1100 1200

P (W)

TIME (h)

Pa with Cdiff