Development of a RAPD fingerprinting method for Lactic Acid Bacteria
To systematically develop a RAPD typing scheme for LAB species, a set of 100 RAPD primers which had proven successful for strain typing other bacterial species [13
] were screened for their ability to amplify multiple polymorphisms from L. acidophilus
. Fifteen primers (Table ) were found to reproducibly amplify 8 or more random DNA fragments from the reference strain L. acidophilus
that ranged in size from 200 to 4000 bp (Fig. ). The complexity of these profiles indicated that discriminatory typing of LAB isolates with these primers was possible.
Specifications of useful RAPD primers for typing Lactic Acid Bacteria
Figure 1 Useful RAPD primers producing diverse polymorphisms from L. acidophilus. The fingerprint patterns generated from strain LMG 9433T are shown for 15 of the primers which were capable of amplifying diverse polymorphisms. The primer number is shown above (more ...)
The primers with the most diverse polymorphisms, 272, 277 and 287 (Table ; Fig. ) were selected for genotyping isolates of further LAB species beyond L. acidophilus
. Primary typing was performed with primer 272 because of its known discriminatory power [13
], and secondary confirmation of strain type was performed with primers 277 and 287.
LAB isolates examined
A collection of 38 LAB isolates was assembled to assess the discriminatory power of the RAPD fingerprinting method (Table ). The collection comprised reference isolates and Type strains of known LAB species obtained from recognised culture collections (14 isolates, 9 species; Table ). In addition, commercially marketed probiotic products were purchased and their constituent LAB isolates cultured and purified (24 isolates, 11 species; Table ). Previous studies have shown that the speciation and labelling of commercially marketed probiotics may often be inaccurate [15
]. Therefore prior to examining the ability of RAPD to differentiate LAB isolates, sequence and phylogenetic analysis of the 16S rRNA gene was used to systematically identify the species of all LAB isolates cultured from commercial samples (Fig. ; Table ). To test the accuracy of this speciation strategy, control sequences from L. brevis
and L. johnsonii
were obtained and found to cluster appropriately with the published sequences from these Type strains (data not shown). The majority of the cultivable bacteria contained within the commercial probiotic products were found to belong to the L. casei
group (L. casei
, L. paracasei
and L. rhamnosus
; 9 isolates) and L. acidophilus
group (L. acidophilus
, L. gallinarum
and L. suntoryeus
species; 6 isolates) (Fig. ; Table ). Other LAB species identified included (Table ): L. gasseri
(3 isolates), L. jensenii
(2 isolates), Enterococcus faecalis
(2 isolates), and L. salivarius
, L. plantarum
, and Pediococcus pentosaceus
(single isolates, respectively).
Reference, probiotic and faecal LAB isolates examined or isolated during the study
Figure 2 Phylogenetic distribution of LAB probiotics and bacteria cultivated during the feeding study. A phylogenetic tree of aligned 16S rRNA genes from representative Lactobacillus reference strains, commercial probiotic strains and dominant isolates recovered (more ...)
Testing the discriminatory power of the RAPD method on other LAB species
The broad collection of systematically identified LAB isolates (Table ) were used to test the efficacy of the RAPD typing scheme. The reproducibility of the RAPD method was excellent, with all 14 reference strains demonstrating identical fingerprint profiles after duplicate analysis. In addition L. acidophilus
was analysed by RAPD at multiple points throughout the study as an internal control; the same fingerprint profile was obtained on each occasion demonstrating that the LAB PCR genotyping scheme demonstrated the same high reproducibility as had been observed with previous RAPD studies on other bacterial species [13
RAPD fingerprinting was able to cluster genetically identical strains as well as differentiate distinct strains within closely related LAB species. For example, multiple isolates of L. acidophilus were found to possess identical RAPD fingerprints (using primer 272) to the type strain for the species, LMG 9433T (Fig. , panel A). These included 4 additional reference isolates that had originally been recovered from diverse sources such as from rat and human faeces, as well as 4 isolates used in the commercial probiotic products (Table ). All L. acidophilus isolates were genotypically indistinguishable even when examined with additional RAPD primers 277 and 287. These data suggested there was little genetic heterogeneity among isolates of L. acidophilus examined in this study. In addition they show that isolates genotypically identical to the L. acidophilus Type strain have been widely adopted for commercial use (Fig. , panel A; Table ). Of the remaining 8 LAB reference isolates examined, 8 distinct RAPD strain types were found that corresponded to each LAB species (Table ).
Figure 3 Discrimination of LAB by RAPD typing. The ability of PCR fingerprinting (with primer 272) to cluster identical isolates (Panel A) and differentiate distinct isolates within the L. casei group (Panel B) is shown. Strains shown in each lane are as follows: (more ...)
RAPD fingerprinting was also able to differentiate genetically unique strain types within very closely related species such as those within the L. casei group (Fig. ); these included L. casei, L. paracasei and L. rhamnosus (Fig. , panel B). From this closely related complex of species (Fig. ), a total of 9 distinct RAPD types (10, 11, 12, 16, 17, 18, 20, 21, and 27; Table ) were identified. Two commercially marketed probiotics were found to contain the same strain of L. rhamnosus (isolates FMD T2 and MW, RAPD type 10; Table ). Another commercial probiotic formulation contained an L. casei strain, designated BF T1, that was identical by RAPD to the L. casei Type strain LMG 6904T (Table ). Overall, the RAPD fingerprinting method was highly effective, working on all 38 LAB isolates examined irrespective of their species and reproducibly defining 26 RAPD types within this diverse collection (Table ).
Application of RAPD fingerprinting to single colonies
To facilitate high throughput typing that could be applied to screening LAB isolates cultivated directly from human faeces, we evaluated if the PCR-fingerprinting method could be adapted for use on single bacteria colonies. Single colonies were picked with a sterile plastic tip and rapid boiling/cooling in a Chelex® resin extraction buffer used to obtain DNA for PCR (see Methods). The RAPD fingerprints obtained from colonies processed in this way were identical to those produced from conventionally extracted high molecular weight DNA (Fig. ). However, it was found that consistent profiles were only obtained if the RAPD PCR was set up immediately after the boiling and chilling cycles of the colony extraction procedure. The amplified PCR fingerprints deteriorated after subsequent frozen storage of the Chelex® resin extracted DNA. To overcome this potential problem, we examined if prolonged frozen storage (-20°C) of the resuspended colony in Chelex® resin prior to full extraction by boiling was possible. This procedure did not affect the quality of the RAPD profiles (Fig. ). The ability to fingerprint from frozen stored colony material provided a high throughput strategy that could be used to systematically screen the multiple colony types isolated from human faeces as part of a Lactobacillus strain feeding study (see below).
Figure 4 Reproducibility of single colony RAPD fingerprints. The polymorphismsamplified by primer 272 from conventionally extracted DNA compared to single colony Chelex® extracted DNA are shown for two LAB strains as follows: lane 1, L. rhamnosus strain (more ...)
Lactobacillus species feeding study design
A small scale proof-of-principle human feeding study was performed to evaluate if the colony-fingerprint strategy could be used to track specific LAB strains from ingestion as capsule recovery from faeces. A capsule for oral administration was formulated to commercial standards which contained two Lactobacillus species isolates: L. salivarius strain NCIMB 30211 (1.8 × 1010 colony forming units [cfu] per capsule) and L. acidophilus strain NCIMB 30156 (5.6 × 109 mean cfu per capsule). Twelve volunteers participated in a feeding study where the capsule was taken daily for 14 days; faecal samples were provided on days before, during and after consumption as described in the Methods. The volunteers were not advised to change their diets in any way other than to take the capsule once a day with some food on each of the trial days. At each faecal sampling point, LAB were plated as described below, enumerated and multiple colonies genotyped by RAPD.
Cultivation of LAB species from human faeces
Although MRS agar is a well established cultivation medium for semi-selective culture of LAB species [17
], we found that several non-LAB species, in particular Gram negative enteric bacteria were frequently encountered as contaminants after plating of human faeces (data not shown). To assist with selection of the Lactobacillus
species in the feeding study, we investigated whether the addition of polymyxin B to MRS medium (MRS-P agar, see Methods) would increase the selectivity of this medium by acting as a counter-selection against coliforms. Addition of polymyxin B at a concentration of 120 units per ml of agar did not inhibit the viability of any of reference LAB species isolates (Table ) or the two Lactobacillus
strains incorporated into the capsule. However, MRS-P was highly effective at reducing the number of contaminating Gram negative enteric colonies seen after plating of human faeces.
To examine the efficacy of the semi-selective MRS-P developed for enrichment of the LAB species within faeces, 29 of the most dominant cultivable isolates recovered from 10 of the volunteers at days -14, 0 and 28 (before and after Lactobacillus feeding) were randomly selected for molecular identification. Using 16S rRNA gene sequence analysis these dominant isolates were identified as (Table ; Fig ): Lactobacillus species (10 isolates), Streptococcus species (7 isolates), Enterococcus species (7 isolates), Weissella species (1 isolate) and Staphylococcus species (4 isolates). The latter Staphylococcus isolates were the only non-LAB species isolated in high numbers on MRS-P agar after faecal plating. These data indicated that the MRS-P agar was effective for selection of LAB species after faecal culture.
Tracking Lactobacillus strains after oral administration
RAPD fingerprinting of the major colony morphotypes appearing after cultivation of each faecal sample was used to determine if the Lactobacillus strains had survived gastric and intestinal passage (Fig. ). The mean faecal LAB count was 8.8 ± 2.7 × 106 cfu per g faeces when all volunteer samples were analysed; consumption of the lactobacilli did not significantly alter the total faecal LAB counts obtained from any of the volunteers (data not shown). Prior to the start of the study, L. salivarius strain NCIMB 30211, was not detected in any of the volunteers, however, strains matching L. acidophilus NCIMB 30156 were cultivated from three of the volunteers at the pre-feeding stage (Table ). The appearance of this L. acidophilus (RAPD strain type 1; Table ) at this point in the study was not unreasonable since it appeared to be a strain commonly found in food/probiotic products which may have been consumed by the volunteers (Table ).
Figure 5 Detection of L. salivarius and L. acidophilus strains after feeding. The colony growth after plating of the day 7 faecal sample from volunteer F are show for the neat and third serial dilutions on MRS-P agar (panels A and B, respectively). Colonies picked (more ...)
Detection of Lactobacillus capsule strains and other faecal bacteria during the feeding study
After consumption of the capsule, the L. salivarius NCIMB 30211 strain was detected on day 2 in three volunteers (B, G and S), on day 7 in two volunteers (F, see Fig. ; S), with only volunteer S remaining faeces positive for this strain on days 21 and 28 (7 and 14 days, respectively, after feeding stopped; Table ). Increased detection of the L. acidophilus NCIMB 30156 strain was also seen with 10 of the volunteers culture positive for this strain at one or more sample points during the feeding period (volunteers A-C, F, G, J, N, P, R and S), and 3 of these (A, N, and S) remained positive on days 21 and 28 (Table ). L. salivarius NCIMB 30211 was never the dominant cultivable LAB strain and was detected at 102 to 104 per g faeces (Fig. ). In contrast, L. acidophilus NCIMB 30156 was the most dominant colony morphotype in volunteers A (day 7 and 28), B (day 2), F (day 7; see Fig. ) and N (day 2, 21 and 28; Table ), where it represented 38% or greater of the total LAB count. The mean LAB count for these volunteers at these time points was 1.8 ± 7.6 × 107 per g faeces indicating that L. acidophilus NCIMB 30156 must have been present at a level of at least 107 per g of faeces.
Statistical evaluation of Lactobacillus feeding in terms of gut colonisation was carried out assuming a null hypothesis that: "Consumption will lead to the subsequent detection by cultivation of the constituent strains within the capsule in the faeces of each subject." Chi Squared analysis demonstrated that the distribution of L. salivarius NCIMB 30211 was significant, with none of the volunteers being positive prior to feeding, and 4 being culture positive (B, F, G and S; Table ) at least once during the feeding period of the trial (Chi square = 4.8; p < 0.05). The distribution of L. acidophilus strain NCIMB 30156 was also significant (3 positive prior to feeding and 10 culture positive during feeding, Table ; Chi square = 8.2, p < 0.01), suggesting that consumption of the organism had led to a significant increase in gut carriage of this L. acidophilus strain. However, limited persistence of the strains was observed in the culture positive volunteers after feeding ceased. For L. acidophilus NCIMB 30156, 10 volunteers were culture positive at least once during the feeding period, this fell to 3 who were still positive on day 21 and 28 (Table ). With L. salivarius NCIMB 30211 only volunteer S retained the strain in faeces at day 21 and 28 after consumption had ceased (Table ).
Specific LAB strains persist in individual humans
Although the persistence of the administered Lactobacillus strains was not substantial after feeding had stopped, other faecal LAB strains were recurrently cultivated at two or more time points from all 12 volunteers (Table ). The RAPD fingerprinting strategy was able to detect the persistence of these strains within the faeces for greater than 28 days in several of the volunteers (Fig. ). Reproducible fingerprints were obtained for Lactobacillus species, Streptococcus species, Enterococcus species, and Weissella species isolates that all persisted in this way (Table and ; Fig. and ). Several strains were also the dominant cultivable isolates recovered from the faeces of certain volunteers, suggesting that they were colonising that individual's gut. For example, the Enterococcus sanguinicola strain (RAPD type 39, representative isolate G-02-a, Table ; Fig. ) recovered from volunteer G was first isolated at 14 days prior to commencing the feeding study and the same strain was also cultivated from their faeces at each subsequent sampling point until day 21 (see Fig. for day 0 and day 21 RAPD fingerprints). At the -14 day sampling point this enterococcal strain was estimated to represent 1% of the cultivable diversity (1.8 × 104 cfu per g faeces), however, within day 0 and day 6 samples it represented 99% of the observed growth (approximately 1.75 × 105 cfu per g faeces); at day 21 it still represented 88% of the cultivable diversity, however, on day 28 it was not detected.
Figure 6 Recurrent LAB strains carried by the human volunteers. Several different strains of LAB were cultivated at several sampling points during the Lactobacillus feeding trial. RAPD fingerprints of these persistent strains are shown for the following in each (more ...)
All the volunteers were colonised with persistent LAB strains (specific to each individual) that represented greater than 1% of their viable faecal growth; at least one of these strains was identified to the species level for each volunteer except J (Table ). Apart from sharing of the L. salivarius NCIMB 30211 and L. acidophilus NCIMB 30156 strains present within the administered feeding capsule, only one other strain was detected in two volunteers, the L. rhamnosus RAPD type 41 strain (Table ). This L. rhamnosus strain was shared by individuals P and T (Table and Table ). Overall, these results demonstrate the ability of the fingerprinting strategy to detect and track the population biology of cultivable faecal strains representative of a broad range of LAB species.