Curr Microbiol (2010) 61:357–360 DOI 10.1007/s00284-010-9619-0
Isolation of Streptococcus thoraltensis from Rabbit Faeces Stina Borø • Christine A. McCartney Tim J. Snelling • Hilary J. Worgan • Neil R. McEwan
•
Received: 11 January 2010 / Accepted: 19 February 2010 / Published online: 9 March 2010 Ó Springer Science+Business Media, LLC 2010
Abstract Bacteria were isolated from rabbit faeces using equine caecal fluid as a growth medium. Two new isolates of the genus Streptococcus are described in terms of their biochemical properties. One of these has a 16S rRNA gene with 97.7%, and the other 98.5%, identity to Streptococcus thoraltensis. While S. thoraltensis has been described in the intestinal tract of pigs, it is generally considered to inhabit the porcine genital tract. The biochemical properties of these bacteria indicate that both new isolates showed an ability to digest xylose, an adaptation beneficial for survival in a niche where much of the nutrient supply is of plant origin. Moreover, having bacteria able to digest xylose in the digestive tract should be beneficial to the rabbit, allowing more effective utilisation and digestion of food. This work provides one of the few examples of an analysis of the physiological properties of a bacterium found in the hindgut of the rabbit. By building up a number of such studies, the mechanisms of bacterial digestion in the rabbit will become better understood.
Introduction As with other herbivores, the rabbit does not have the enzymes necessary for complete digestion of all of the plant material which it ingests. Instead, these herbivores rely on a symbiotic relationship between bacteria of the digestive tract allowing more efficient digestion of food to take place. Initial investigations into the bacterial composition of the digestive tract of the rabbit have demonstrated S. Borø C. A. McCartney T. J. Snelling H. J. Worgan N. R. McEwan (&) Aberystwyth University, IBERS, Aberystwyth, UK e-mail:
[email protected]
that, although they perform a role similar to that of the rumen microbes, the species carrying out these tasks in the two different environments are remarkably different. In the first study of the genetic diversity of the rabbit caecum, of the forty-six 16S rRNA sequences studied only a single sequence had more than 97% identity to anything already present in the EBI database, and 24 of these sequences clustered as a discrete, previously unidentified clade [1]. More recently, a further similar investigation [11] carried out on a larger scale corroborated these initial observations; that the rabbit has a bacterial population unlike that described previously for the digestive tract of other herbivores such as ruminants. This difference in population composition may be a reflection of the caecotrophic, as opposed to ruminant, digestive strategy of the rabbit. However, it is assumed that the microbes of the rabbit caecum, as with those of the rumen, are responsible for the digestion of plant material, thereby making nutrients available to the host animal, which would otherwise be lost and excreted. In order that the microbial diversity of the rabbit hindgut may be more clearly understood, it is essential that experimental work addresses the issue of characterising bacteria which have been isolated from this environment.
Materials and Methods Isolation of Bacteria Fresh rabbit faecal material was collected for bacterial isolation. Hard faeces, as opposed to caecotrophs were used for this isolation as these are known to contain viable bacteria, and have been shown to be those ingested by young animals following excretion by their mother [17].
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Although the caecotrophs may be richer in live bacteria, in the rabbit these are generally ingested directly from the anus by the rabbit excreting them and so are not generally available for ingestion by other animals. Thus, the use of hard faeces was thought to be justifiable as a source of initial inoculum in pups. In total 0.69 g of faeces was collected from two animals, pooled together and diluted with 0.85% (w/v) NaCl. Samples were diluted serially from 10-1 to 10-8. Two forms of M8 media [12] were produced; based on clarified rumen fluid or equine caecal fluid and used in 1.5% agar plates to generate isolation medium. Diluted solutions were streaked out on plates and incubated in an anaerobic growth chamber at 39°C. Discrete, individual colonies were picked and re-streaked to ensure purity of starter colony. Morphology, Growth and Biochemical Properties Bacterial morphology was assessed as part of a Gramstaining assay. Tolerance of aerobic conditions was tested by growing tubes in the presence of oxygen. Substrate utilisation was assessed using API-20A strips for anaerobic microbes ((BioMe´rieux, Marcy-l’Etoile, France). Although the manufacturers recommend assays be carried out overnight, the slow-growing properties of both of these isolates required incubations of 48 h to permit detection. DNA Analysis and Phylogenetics DNA was extracted by using a QIAmpÒ DNA mini stool kit (QIAGEN Ltd, West Sussex, England) following the manufacturer’s instructions with the exception of increasing the initial incubation at 95°C to 10 min. This extended heating step was adopted to increase the yield of DNA due to the isolates being identified as Gram-positive bacteria, which as a group have been previously shown to be difficult to lyse. PCR was used to amplify the 16S rRNA gene from both isolates using a bacterial-specific primer pair, 8F (50 -AGA GTT TGA TCC TGG CTG AG-30 ) and 1389R (50 -AGG GGG GGT GTG TAG AAG-30 ) [9]. Amplification was performed using a BIORAD MyCyclerTM thermal cycler with the following program: an initial 4 min denaturation at 94°C followed by 25 cycles of 1 min denaturation at 94°C, 1 min annealing at 55°C and 1 min extension at 72°C. A final cycle of 1 min at 94°C, 1 min at 55°C and elongation for 5 min at 72°C completed the PCR. The following reaction cocktail was used: 0.05 U/ll Taq DNA polymerase (Promega); 19 reaction buffer as supplied by manufacturer; 1.5 mM MgCl2; 0.25 lM of each primer; and 0.2 mM of each of the dNTPs with 2 ll of template. All reactions were carried out in a final volume of 50 ll.
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Amplicons were ligated into pCRÒ–TOPOÒ vector (Invitrogen) and transformed into competent E. coli TOP10 cells (Invitrogen). Recombinant clones were selected and checked for the presence of an insert of the appropriate size before being sequenced in both directions using a Beckman CEQ8000 sequencer. Following contig assembly of the complete 16S rRNA for both of these sequences were checked for similarity to existing related sequences in the EBI database using BLAST [2]. The sequences of the 20 best ‘hits’ in BLAST for both sequences were downloaded and used for construction of a phylogenetic tree. A sequence from Enterococcus faecalis (accession number FN377815) was used as an outlier in the tree construction. Sequences were aligned using ClustalW [16] and the *.phy file was used as an input file for PHYLIP [6]. An initial NJ tree was constructed using DNAdist, followed by Neighbor. The tree was then validated by bootstrapping with 1000 iterations by Seqboot, DNAdist, Neighbor and Consense. Output trees were viewed using TreeView [13].
Results Morphology and Growth Characteristics After incubation for 24 h there was minimal growth on only the most concentrated plates. Plates were then allowed a further 24 h growth and by this time there were enough discrete colonies on the more dilute plates to permit discrete colonies to be picked for enrichment. In total, 16 of the colonies which grew on the M8 supplemented with equine caecal fluid were picked for analysis, two of which (S2 and S6) are reported here. Following Gram-staining, both colonies S2 and S6 were identified as being Grampositive organisms with cocci chains, with individual cells within the range of around 1.0–1.5 lm in diameter. Both organisms had been isolated under anaerobic conditions, and attempts to grow them aerobically failed to produce any growth, suggesting that they may be obligate anaerobes, although growth was achieved under microaerophilic conditions (5% CO2). 16S rRNA Analysis and Phylogeny BLAST results identified the organism with best identity to both of these isolates was from Streptococcus thoraltensis (accession number Y09007), an organism which had been isolated from the genital tract of sows [5], although the authors report that this organism can also be found in the intestinal tract of the pig. Isolate S6 showed the higher identity (98.5%) of the two new isolates, with Isolate S2 also showing high identity levels (97.7%). Comparisons
S. Borø et al.: Isolation of Streptococcus thoraltensis from Rabbit Faeces
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Table 1 API strip properties of the two new isolates, with comparison to the properties reported for the only other previous S. thoraltensis isolate: positive reaction (?); no reaction detected (-); most isolates showed a positive reaction (?/-) or not reported (NR)
Fig. 1 Neighbor-Joining tree showing the relationship between the two isolates presented in this study, together with the other S. thoraltensis isolate and other Streptococcus isolates identified by the BLAST searches. All bootstrap values in excess of 70% are shown
between the two new isolates confirmed the similarity of these organisms, with 97.8% identity to each other. Phylogenetic tree construction supported the grouping of S. thoraltensis and these two new isolates as a single cluster (Fig. 1), as evidenced by them cluster together as a group of three sequences, with a bootstrap value of 100%. Biochemical Properties The use of API 20A strips verified that the presence of many of the metabolic properties described in the work of Ref. [5] are similar in both of the new isolates and the original S. thoraltensis isolate (Table 1). One particular point to note is that both of these new isolates are able to utilise xylose, whereas a number of the earlier S. thoraltensis isolates were unable to use this as a substrate.
Discussion The fact that two of the 16 colonies selected for further study turned out to be S. thoraltensis, despite this organism not having been detected in random 16S rRNA studies suggests that there may be some degree of unintentional selectivity for this organism in the equine caecal fluid— even though it has not been reported in the equine hindgut.
Reactions/enzymes Isolate tested using the API 20A number 2 test strips (FN377818)
Isolate Streptococcus number 6 thoraltensis (FN377819) (Y09007)
Indole formation
-
-
NR
UREase
-
?
-
Acidification of glucose ?
-
?
Acidification of mannitol ?
?
?
Acidification of lactose
?
?
?
Acidification of saccharose
?
?
?
Acidification of maltose ?
?
?
Acidification of salicin
?
?
?
Acidification of D-xylose ?
?
?/-
Acidification of arabinose
?/-
-
-
Hydrolysis of gelatin
-
-
NR
Hydrolysis of esculin
?
?
?
Acidification of glycerol Acidification of ? cellobiose
?
?
Acidification of mannose ?
?
?
Acidification of melezitose
?
-
?/-
Acidification of raffinose -
-
?/-
Acidification of sorbitol -
-
?/-
Acidification of rhamnose
-
-
-
Acidification of trehalose
-
?
?
Catalase activity
-
-
-
Data for Streptococcus thoraltensis (Y09007) are from the work of Devriese et al. [5]
Another interesting observation from this work is the presence of positive acidification reactions for both D-xylose and D-cellobiose in both of the current isolates. In the previous S. thoraltensis isolates acidification of D-cellobiose took place in all isolates, but acidification of D-xylose was not found universally. This suggests that the new isolates can contribute to the breakdown of large molecules that are indigestible once they reach the hindgut. There are possible explanations for this difference, as D-xylose is a subunit of the hemicellulose xylan, which is a major component of straw [7, 8]. Straw and hay were provided for the rabbits, and constituted a large portion of the diet, suggesting that there was an ample supply of xylan in the gastrointestinal tract of the rabbit, although these are of course not the only sources of xylose in the diet. Conversely, farmed pigs are not normally fed large amounts of
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high fibre feeds such as hay and straw, although these may constitute a larger amount of the diet in wild pigs. Nevertheless, it is interesting to note that although the pig’s diet is unlikely to be devoid of xylan, it can be assumed that its gastrointestinal tract will contain less xylan than that of the rabbit. Both of these new isolates were isolated on, and then subsequently grown on medium derived from equine caecal fluid. Attempts to maintain them on rumen fluid were also successful, but this did not appear to improve growth rates. Hence their slow growth rates may be a factor of selection in less than optimal conditions, but this was done primarily on the basis of problems associated with obtaining enough rabbit caecal fluid for large-scale in vitro culturing experiments. Nevertheless, it is also interesting to note that despite these organisms being present in the hindgut of the rabbit, they are also capable of growing in medium derived from the caecum of the horse. This point coupled with the original description of the organism in the digestive tract and reproductive tract of pigs suggests an organism which can survive in more than one niche. Streptococci are amongst the first organisms to infect the digestive tract in the newborn animal [14]. The level of these species can be critical to the host animal, as has been demonstrated in the linkage of laminitis and streptococci in the horse [10]. It has also been shown for a wide range of animals that the stomach and small intestine can support an extensive microflora including large numbers of streptococci [15]. However, this is not the pattern seen in rabbits, where streptococci were only present in the caecum and faeces [15]. Streptococci are also described as being facultatively anaerobes [4], with the previous isolate of this species being classified as capnophilic [5]. Neither of the two new isolates was able to grow in an aerobic environment, but growth under microaerophilic conditions was achieved, suggesting that they are indeed facultatively anaerobic. The preference for capnophilic conditions is easily understood, since these organisms were isolated from an environment where microbes can break down nutrients into gases including CO2 [3]. In conclusion, this work reports the isolation of two bacterial strains which appear to be members of the S. thoraltensis species, and that they both show metabolic properties in keeping with those which might be expected from an organism in the hindgut of the rabbit. Moreover, due to their ability to digest D-xylose and D-cellobiose, it is likely that these organisms are able to the breakdown of plant material, and in so doing be beneficial to the nutrition of the rabbit.
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