Isolation and identification of candidate probiotics
A total of 307 isolates were cultured from 21 rectal swabs, 25 gastric fluid specimens, 2 oral swabs and 3 milk specimens obtained from 38 dolphins, using MRS and Brucella media. Each isolate corresponded to a distinct morphotype obtained per plate with the sample and culture conditions employed.
The majority of isolates were obtained using MRS (150 isolates, 48·9%) and Brucella (118 isolates, 38·4%) media; some isolates were cultured using sheep blood (27 isolates, 8·8%) and chocolate (12 isolates, 3·9%) agar. One hundred and fifty-three (49·8%) isolates were recovered from rectal swabs, 97 isolates (31·6%) were obtained from gastric fluid, 53 isolates (17·3%) were derived from milk, and four isolates (1·3%) were cultured from nursing calf oral swabs.
Two hundred and fifty isolates (81·4%) were identified by partial 16S rDNA sequencing; 58 (18·9%) isolates remained unidentified. The most prevalent genera cultured from dolphin samples under the conditions employed were Staphylococcus (77 isolates, 25·1%) and Escherichia/Shigella (61 isolates, 19·9%), not surprisingly as both are able to grow in MRS media. Staphylococci were isolated from all specimen types and were predominant in gastric fluid and milk, whereas Escherichia/Shigella were mainly isolated from rectal swabs. Other genera recovered included Achromobacter, Actinobacillus, Alcanivorax, Bacillus, Citrobacter, Clostridium, Corynebacterium, Desulfovibrio, Edwardsiella, Enterococcus, Eubacterium, Lactobacillus, Macrococcus, Ochromobactrum, Peptostreptococcus, Photobacterium, Pigmentiphaga, Pseudomonas, Rhodococcus, Stenotrophomonas and Streptococcus. Although 52 isolates were recovered from dolphin milk samples, no lactobacilli were recovered from these samples; the isolates were predominantly staphylococci. A detailed list of isolates recovered and their specimen of origin is provided in Table 1.
Table 1. Bacterial species recovered from dolphin samples and identified by 16S rDNA sequencing
|Organisma||Rectal swab||Gastric fluid||Oral swab||Milk||Total (%)|
| Achromobacter denitrificans ||1|| || || ||1 (0·3%)|
| Actinobacillus scotiae ||1||5|| || ||6 (2·0%)|
| Alcanivorax dieselolei ||2|| || || ||2 (0·7%)|
| Bacillus thuringiensis || ||1|| || ||1 (0·3%)|
| Citrobacter braakii ||1|| || || ||1 (0·3%)|
| Citrobacter freundii ||4|| || || ||4 (1·3%)|
| Clostridium ghonii ||1|| || || ||1 (0·3%)|
| Clostridium perfringens ||3|| || || ||3 (1·0%)|
| Clostridium sordellii ||2|| || || ||2 (0·7%)|
| Corynebacterium tuberculostearicum || || || ||1||1 (0·3%)|
|Desulfovibrio spp.||1|| || || ||1 (0·3%)|
| Edwardsiella ictaluri ||1|| || || ||1 (0·3%)|
| Edwardsiella tarda ||13|| || || ||13 (4·2%)|
| Enterococcus casseliflavus ||3|| || || ||3 (1·0%)|
| Enterococcus silesiacus || ||2|| || ||2 (0·7%)|
| Enterococcus termitis ||10||3|| || ||13 (4·2%)|
| Eubacterium tenue || ||1|| || ||1 (0·3%)|
| Escherichia/Shigella ||57||4|| || ||61 (19·9%)|
| Lactobacillus salivarius ||4|| || || ||4 (1·3%)|
|Lactobacillus spp.|| ||3|| || ||3 (1·0%)|
| Macrococcus caseolyticus || || || ||1||1 (0·3%)|
| Ochrobactrum tritici ||1|| || || ||1 (0·3%)|
| Peptostreptococcus stomatis ||2|| || || ||2 (0·7%)|
| Photobacterium damselae ||5|| || ||2||7 (2·3%)|
| Pigmentiphaga kullae ||1|| || || ||1 (0·3%)|
| Propionibacterium avidum || || || ||2||2 (0·7%)|
| Pseudomonas otitidis ||1|| || || ||1 (0·3%)|
| Rhodococcus corynebacteroides ||1|| || || ||1 (0·3%)|
| Staphylococcus caprae ||1||1||1||9||12 (3·9%)|
| Staphylococcus cohnii || || || ||1||1 (0·3%)|
| Staphylococcus delphini ||1||8|| || ||9 (2·9%)|
| Staphylococcus epidermidis ||4||6||2||10||22 (7·2%)|
| Staphylococcus hominis || ||2|| ||8||10 (3·3%)|
| Staphylococcus pasteuri ||2||4||1||3||10 (3·3%)|
| Staphylococcus warneri ||3||6|| ||4||13 (4·2%)|
| Stenotrophomonas maltophilia ||1|| || || ||1 (0·3%)|
| Streptococcus australis ||2|| || || ||2 (0·7%)|
| Streptococcus mitis || ||1|| || ||1 (0·3%)|
| Streptococcus parasanguinis ||1|| || ||1||2 (0·7%)|
|Possible yeast (18S rDNA positive)||1||25|| || ||26 (8·5%)|
|Not sequenced or identified||23||25|| ||10||58 (18·9%)|
|Total||154 (50·2%)||97 (31·5%)||4 (1·3%)||52 (16·9%)||307|
Of the 307 bacterial strains isolated from dolphin samples, seven were Lactobacillus spp. Four Lact. salivarius isolates were recovered from rectal swab samples, all from dolphin ‘C’. The isolates had 16S rDNA sequences that were more than 99·8% identical to each other. White, circular, smooth colonies formed on MRS and Brucella agar after 24 h of anaerobic incubation at 37°C, and an alpha-haemolytic phenotype was evident when they were cultured on sheep blood agar. These isolates were nonmotile and did not form spores. They were Gram-positive, catalase-negative, facultative anaerobic bacilli that appeared as single cells and as pairs of cells by microscopy.
In addition, we obtained three novel Lactobacillus spp. isolates from the gastric fluid of dolphin ‘Z’, with 16S rDNA sequences 99·54% identical to each other and with 96·3% 16S rDNA sequence identity to Lactobacillus ceti, a species previously isolated from beaked whales (Vela et al. 2008). The isolates formed small, grey, nonhaemolytic round colonies after 3–7 days of incubation on sheep blood agar at 37°C, under anaerobic conditions. They were Gram-positive, anaerobic, catalase-negative bacilli present as single cells and as pairs of cells. The 16S rDNA sequences obtained from these isolates were 99·8% identical to sequences detected directly in gastric fluid and rectal swab samples from several dolphins studied by whole-community, broad-range 16S rDNA PCR and clone library sequencing (E.M. Bik et al., personal communication). The novel Lactobacillus sp. and the Lact. salivarius isolated in this study had 92·71% 16S rDNA sequence identity to each other. The results of a 16S rDNA-based phylogenetic analysis of lactobacilli found in both studies are provided in Fig. 1. These sequence data have been submitted to the GenBank database under accession numbers JX142127 through JX142133.
Figure 1. Phylogeny of Lactobacillus 16S rDNA sequences found in this study. Lactobacillus sequences found in this study (in bold) were compared with published sequences in a neighbour-joining tree with a Jukes-Cantor correction and a 1323-column filter. Numbers above branches refer to bootstrap values (in percentages out of 1000 trees; numbers below 50% are not shown). The scale bar represents evolutionary distance (1 substitution per 100 nucleotides). Sequence 05283 was obtained in a separate study of bacterial diversity in bottlenose dolphins using broad-range 16S rDNA PCR and clone library sequencing (E.M. Bik et al., personal communication). Refer to Table S1 for the identity matrix of these sequences and to Table 2 for descriptions of MMP strains shown in this figure.
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RepPCR genomic fingerprinting of lactobacilli isolated in this study revealed that, as with ribosomal DNA sequence analysis, the four Lact. salivarius isolates, MMP005, MMP006, MMP007 and MMP077, obtained from dolphin ‘C’, grouped together to form one clade, as did the novel Lactobacillus isolates MMP239, MMP241 and MMP242, obtained from dolphin ‘Z’ (data not shown). Further comparative genomic analyses using microarray or whole-genome sequencing technologies will allow us to refine the phylogenetic relation and genome variability amongst the Lact. salivarius and the novel Lactobacillus sp. isolates, respectively, and determine whether these isolates represent distinct strains of each species (Raftis et al. 2010).
Lactobacillus salivarius isolates were characterized using API 50 CHL strips. These biochemical test kits consist of 50 carbohydrate utilization/fermentation assays used to characterize lactobacilli and related micro-organisms. Substrate utilization results, summarized in Table 2, showed that dolphin-derived Lact. salivarius isolates MMP005, MMP006, MMP007 and MMP077 yielded identical biochemical and carbohydrate fermentation profiles and were positive for d-ribose, d-adonitol, d-galactose, d-glucose, d-fructose, d-mannose, d-mannitol, d-sorbitol, N-acetylglucosamine, d-maltose, d-lactose, d-melibiose, d-saccharose (sucrose), d-trehalose and d-raffinose. These profiles differed slightly from reference strain Lact. salivarius ATCC 11741, which displayed a typical carbohydrate fermentation profile of Lact. salivarius (Jacobsen et al. 1999; Neville and O'Toole 2010), and did not utilize d-ribose or d-adonitol, but was positive for l-rhamnose. The three novel Lactobacillus isolates (MMP239, MMP241 and MMP242) were fastidious and failed to yield sufficient growth for API 50 CHL assays.
Table 2. Summary of characteristics of lactobacilli analysed in this study
|Isolate||Speciesa||Specimen||Dolphin||Enrichment media||Isolation media||Pathogen growth inhibition||TNF modulation||API-CH profileb|
|MMP 005|| Lactobacillus salivarius ||Rectal swab||C||Brucella||MRS||+++||RIB, ADO, GAL, GLU, FRU, MNE, MAN, SOR, NAG, MAL, LAC, MEL, SAC, TRE, RAF.|
|MMP 006|| Lact. salivarius ||Rectal swab||C||Brucella||MRS||+++||RIB, ADO, GAL, GLU, FRU, MNE, MAN, SOR, NAG, MAL, LAC, MEL, SAC, TRE, RAF.|
|MMP 007|| Lact. salivarius ||Rectal swab||C||Brucella||MRS||+++||RIB, ADO, GAL, GLU, FRU, MNE, MAN, SOR, NAG, MAL, LAC, MEL, SAC, TRE, RAF.|
|MMP 077|| Lact. salivarius ||Rectal swab||C||None||MRS||+++||RIB, ADO, GAL, GLU, FRU, MNE, MAN, SOR, NAG, MAL, LAC, MEL, SAC, TRE, RAF.|
|MMP 239||Novel Lactobacillus sp.||Gastric fluid||Z||Brucella||Blood||ND||ND||ND|
|MMP 241||Novel Lactobacillus sp.||Gastric fluid||Z||Brucella||Blood||ND||ND||ND|
|MMP 242||Novel Lactobacillus sp.||Gastric fluid||Z||Brucella||Blood||ND||ND||ND|
|ATCC 11741c|| Lact. salivarius ||−||−||−||−||+++||GAL, GLU, FRU, MNE, RHA, MAN, SOR, NAG, MAL, LAC, MEL, SAC, TRE, RAF|
Pathogen growth inhibition properties
Lactobacillus salivarius candidate probiotic isolates MMP005, MMP006, MMP007 and MMP077, obtained from dolphin ‘C’ rectal swabs, strongly inhibited growth of the marine mammal-derived Salm. enterica serotype Enteritidis (strain MMP-3466467) and human pathogens EHEC-JV.112 and ETEC-JV.3A5 (Fig. 2). Similarly, Lact. reuteri ATCC 55730 and Lact. salivarius ATCC 11741, references established as probiotic strains (Rogosa et al. 1953; Valeur et al. 2004), also inhibited these enteric pathogens. Other marine mammal-derived isolates tested included Staphylococcus sp. MMP123 and strains of Bacillus thuringiensis, Edwardsiella ictaluri, Enterococcus casseliflavus, Escherichia/Shigella, Photobacterium damselae and Staphylococcus spp., all of which yielded inhibition radii smaller than 4 mm and were noninhibitory (not shown). As mentioned earlier, the novel Lactobacillus sp. isolates were not studied further as they were fastidious and yielded insufficient growth. The growth medium alone had no growth inhibitory effect on any of the bacterial pathogens tested (not shown).
Figure 2. Lactobacillus salivarius isolates inhibit growth of selected marine mammal and human pathogens. Candidate probiotic strains were assayed for their ability to inhibit growth of selected marine mammal and human pathogens. Effector strain cultures (Lact. salivarius strains MMP005, MMP006, MMP007 and MMP077, Staphylococcus sp. MMP123 and reference strains Lactobacillus paracasei ATCC 25302, Lact. salivarius ATCC 11741, Lactobacillus reuteri ATCC 55730 and Lact. reuteri ATCC PTA 6475) were spotted onto de Man, Rogosa and Sharpe (MRS) agar and tested with indicator pathogen cell suspensions. Each pathogen growth inhibition zone radius was measured in millimetres (mm). Lactobacillus salivarius isolates obtained in this study from dolphin ‘C’ rectal swabs inhibited growth of marine mammal-derived Salmonella enterica serotype Enteritidis MMP-3466467 and human pathogens EHEC-JV.112 and ETEC-JV.3A5. Reference strains were also pathogen growth inhibitory (anova, P < 0·05). In contrast, Staphylococcus sp. MMP123 and other isolates from dolphin samples tested, including strains of Bacillus thuringiensis, Edwardsiella ictaluri, Enterococcus casseliflavus, Escherichia/Shigella, Photobacterium damselae and Staphylococcus spp. (not shown), were noninhibitory. MRS medium-only control experiments (not shown) confirmed that MRS had no effect on pathogen growth. Bars represent the mean radii of growth inhibition; error bars show standard deviations. () Salmonella; () EHEC and () ETEC.
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