Evaluation of propanediol and cobalamin metabolism in the intestinal colonization and systemic invasion of Salmonella Enteritidis in laying hens

Annually approximately 80.3 million cases of human foodborne diseases worldwide are caused by Salmonella, specially Salmonella Enteritidis which is one of the most prevalent serotypes isolated from human and non-human sources. Products containing raw eggs or even crosscontamination of vegetables by contaminated chicken meat during manipulation are the main sources of S. Enteritidis to consumers (Salmonella..., 2018). In poultry, S. Enteritidis has been one of the most important serotypes responsible for avian paratyphoid. In young birds it is able to colonize the gut and establish systemic infection. On the other hand, infections provoked in mature birds are generally not severe, but birds become carriers and capable to vertically or horizontally transmit the microorganism for long periods (Gast et al., 2017).

Annually approximately 80.3 million cases of human foodborne diseases worldwide are caused by Salmonella, specially Salmonella Enteritidis which is one of the most prevalent serotypes isolated from human and non-human sources. Products containing raw eggs or even crosscontamination of vegetables by contaminated chicken meat during manipulation are the main sources of S. Enteritidis to consumers (Salmonella…, 2018). In poultry, S. Enteritidis has been one of the most important serotypes responsible for avian paratyphoid. In young birds it is able to colonize the gut and establish systemic infection. On the other hand, infections provoked in mature birds are generally not severe, but birds become carriers and capable to vertically or horizontally transmit the microorganism for long periods (Gast et al., 2017).
Salmonella enterica uses 1.2 propanediol (1.2-Pd) as carbon and energy source throughout a vitamin B12-dependent route. 1.2-Pd is a product of the fermentation of two common pentoses, rhamnose and fucose. Within the gastrointestinal tract it can be found naturally as a metabolized product of herbal origin and also as a terminal sugar of gut mucins (Muraoka and Zhang, 2010). B12-dependent anaerobic catabolism of small molecules in the intestine may play a central role on Salmonellae physiology, enhancing gut colonization under conditions which normally induce starvation (Bobik et al., 1999).
Previous reports indicated that 1.2-Pd metabolism is important for S. Typhimurium during infection in both humans and murine (Klumpp and Fuchs, 2007). Based on these data, we propose to investigate herein the importance of cobS, cbiA, pduC, pduD and pduE genes to S. Enteritidis gut colonization and systemic infection in white and brown laying-hen varieties.
In this study, Salmonella Enteritidis strain P125109 (SE) provided the genetic background to construct the mutant strains and was used as a positive control since its capability to extensively colonize the chicken gut and cause systemic infection is known (Thomson et al., 2008). The knockout of pduC, pduD, pduE, cobS and cbiA genes were done by standard Lambda Red methodology (Datsenko and Wanner, 2000). Constructions of single and triple mutant strains were carried out using the plasmids pKD46, pKD3, pKD4 and pCP20 and transduction using the bacteriophage ØP22. All strains and plasmids used are shown in Table 1  The presence of SE in feces was evaluated twice a week over a month by cloacal swabs. The swabs were placed within tubes containing 2mL of Selenite Broth (OXOID, UK) with 0.04 % novobiocin (Sigma Aldrich, USA) (SN) and, after incubation, were streaked with Brilliant Green agar supplemented with the same antibiotics as described above, according to the respective bacterial strain. Tubes and plates were incubated at 37°C for 24h. Data on fecal shedding were compared by Chi-square Test and Statistical differences amongst viable bacteria numbers recovered from caeca contents, livers and spleens were determined using two-way ANOVA followed by Tukey's Test. Both analyses were performed using GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla, California, USA).
To our knowledge, the operons cob/cbi and pdu are located side by side in the Salmonella enterica genome. During anoxic growth condition they are induced by 1.2-Pd and are positively regulated by the PocR and the global regulators cAMP and ArcA/ArcB (Bobik et al., 1992). It is noteworthy that the anaerobic growth of Salmonella Typhimurium on 1.2-Pd depends on both the electron acceptor tetrathionate and the endogenously synthesized B12. The degradation of 1.2-Pd is mediated by B12dependent diol dehydratase (PduCDE) within a polyhedral organelle (Bobik et al., 1999) which, in turn, prevents a consequent toxicity and/or carbon loss. Thus, the intermediate product, propionaldehyde, can be converted into propionate by PduP, PduL and PduW or 1propanol by PduQ (Bobik et al., 1999). On the other hand, cobalamin is synthesized again only under anaerobiosis and the ability to synthesize and import B12 requires more than 35 genes (Richter-Dahlfors et al., 1994).
In the present study, the onset of clinical signs was at three dpi when chickens of all groups had diarrhea with traces of blood and persisted until the 12 th dpi. Moreover, mortality was not observed in any group. Mutant strains were shed amidst feces in higher number by laying hens of brown variety in comparison to WT (P<0.05). However, both strains were excreted at the same amount by white laying hens (P>0.05) (Figure 1).
At three days post-infection, gross lesions such as hepato-splenomegaly with congestion in both organs were observed. No significant difference between the recovery of WT and mutant strains in organs or in caeca content was found for chicken varieties (P> 0.05). Means of bacterial counts in liver, spleen and caeca contents are presented in Figure 2.  Despite data from previous work have showed that the use of 1.2-Pd leads to advantages in the pathogenic potential of Salmonella and Listeria (Klumpp and Fuchs, 2007), we demonstrated herein that its usage is not essential for SE pathogenicity in chickens, both in the presence and absence of cobalamin synthetic pathway. No differences were found among the counts of mutant and wild-type strains recovered from liver, spleen and caeca content of both laying hens varieties. A higher number of positive cloacal swabs were recovered from brown egg layers inoculated with mutant strains in comparison to the wild type. Paiva et al. (2011) also previously found that mutations in cobS and cbiA genes did not affect neither the ability of SE mutant strain to colonize the gut nor to provoke systemic infection in challenged chickens. Steeb et al. (2013)  infection. The results revealed that this bacterium was able to use at least 31 different nutrients present in host tissues by expressing versatile catabolic pathways to simultaneously exploit multiple nutrients. Possibly SE is capable of to use alternative substrates such as ethanolamine, glycogen, D-mannitol and D-sorbitol available in the chicken intestine (Kim et al., 2013). It has been demonstrated that S. Typhimurium can use ethanolamine by inducing the mouse gut to produce a respiratory electron acceptor (tetrathionate) which supports anaerobic growth (Thiennimitr et al., 2011). On the other hand, the chicken-restricted S. Gallinarum does not efficiently colonize the gut as a result of mutations affecting several metabolism genes, including cbi and pdu operons (Thomson et al., 2008).
In the present study, the lack of cobS, cbiA and pdu CDE genes in SE did not alter its ability to colonize the intestine or to cause systemic infection in both laying hens varieties. It is probably that other energy, carbon and nitrogen sources compensate the lack of 1.2-Pd usage during the pathogen infectious process in chickens. Thus, further studies are being carried out to confirm the importance of tetrathionate and ethanolamine metabolism for S. Enteritidis and other serovars in poultry.