Bacterial strains and isolation

The Lactobacillus strains characterized in this study were isolated from positive blood cultures at Marshfield Laboratories as part of routine diagnostic testing. Marshfield Laboratories uses the BacT/Alert blood culturing system (Organon Teknika Corp., Durham, NC) and performs Gram staining for all positive blood cultures. Positive blood bottles were cultured on trypticase soy–5% sheep blood or chocolate agar at 37°C under the appropriate atmospheric conditions. Blood bottle isolates were initially characterized using the automated VITEK system (bioMérieux Vitek Inc., Hazelwood, MO) and standard biochemical methods. Additional biochemical and molecular analyses described below were performed from isolates propagated in Lactobacillus MRS broth (Difco Laboratories, Detroit, MI).

Anaerobe identification card

The VITEK anaerobe identification card was used in conjunction with the VITEK system for the automated identification of each strain by characterizing the ability to hydrolyze 28 substrates. Following initial isolation and identification of each strain, cultures were propagated through MRS broth and plated on blood agar at 37°C under anaerobic conditions. Identification of isolates was repeated with the anaerobe identification cards as per the manufacturer’s protocol.

Lactobacillus carbohydrate fermentation profile

The API 50 CH test kit and the API CHL medium (bioMérieux Vitek Inc.) were used to determine the species of each Lactobacillus isolate by characterizing the ability to ferment 49 carbohydrates. Strains were grown for 18 hours to stationary phase in MRS broth and 1 mL of culture was harvested by centrifugation at 20,000 x g for 1 minute. The cell pellets were washed with 1 mL sterile saline, suspended in 0.5 mL API CHL medium, and then further diluted 100-fold in API CHL medium. The culture dilutions were then loaded to the API 50 CH test strips as per the manufacturer’s protocol, the capules were covered with mineral oil, and the test strips were incubated at 37°C for up to 48 hours. Test strips information was read and recorded at 16 and 40 hours. Carbohydrate fermentation profiling was performed in duplicate for each strain. Species identification was made by comparison of the results from API 50 CH test strips with known carbohydrate fermentation profiles of lactobacilli as indicated in Bergey’s Manual of Systematic Bacteriology.¹⁹

DNA extraction

DNA was extracted from cultured colonies using a modification of the tissue protocol for the QIAmp DNA Mini Kit (Qiagen, Inc., Valencia, CA). Briefly, 200 μL of a cell suspension in H₂O (containing ~5 to 10 mg cells) was mixed with 200 μL of QIAmp Buffer AL and 10 μL of proteinase K (20 mg/mL). Samples were incubated for 15 minutes at 56°C and then for 15 minutes at 95°C. Two hundred microliters of ethanol was added and mixed by vortexing. The samples were loaded, washed, and eluted in 200 μL from QIAmp spin columns, as indicated in the remainder of the manufacturer’s protocol.

Sequencing of the 16S rRNA gene

The template DNA for sequencing of the 16S rRNA gene was amplified using primers FD1 (5′-AGA GTT TGA TCC TGG CTC AG) and RD1 (5′-AAG GAG GTG ATC CAG CC), resulting in PCR products of ~1500 bp. The reaction mixtures contained 1 x polymerase chain reaction (PCR) buffer, 200 μM each deoxynucleoside triphosphate, 3.0 mM MgCl₂, 0.5 μM each primer species, 1.0 U Taq DNA polymerase, and 1.0 μL chromosomal preparation per 15 μL reaction. The PCR products were used for cycle sequencing reactions without further purification.

Cycle sequence reactions were prepared with BigDye Terminator mix (Applied Biosystems, Foster City, CA) as per supplier’s protocol using primer FD1 (see above), RD1 (see above), 515F (5′-TGC CAG CAG CCG CGG TAA), 806R (5′-GGA CTA CCA GGG TAT CTA AT), 91E (5′-TCA AAK GAA TTG ACG GGG GC), or 13B (5′-AGG CCC GGG AAC GTA TTC AC). Sequence determination was done on an Applied Biosystems model 377XL automated DNA sequencing instrument. DNA sequences were aligned using Lasergene99 software (DNASTAR Inc., Madison, WI). The nucleotide sequences, determined from a minimum of 4-fold coverage, were deposited in GenBank under the accession numbers AY299486, AY299487, and AY299488. Sequence analysis was performed using the BLAST program for database searches at the National Center for Biotechnology Information Web site²⁰ and the sequence match program at the Ribosomal Database Project (RDP-II) Web site.²¹ Multiple sequence alignments were performed using the ClustalW program.²²

PCR primers for determining T-RFLP

Independent reactions with two sets of fluorescent labeled primers targeting the 16S rRNA gene were used for PCR with chromosomal preparations from reference strains and blood isolates. The primers were chosen as described in Christensen et al.¹⁸ based on evaluations of domain specific primers and maximization of matches received from queries using the TAP T-RFLP on-line analysis of RDP-II. As indicated, primers were labeled at the 5′ end with the dyes 6-carboxyfluorescein (6-FAM), 4,7,2′,4′,5′,7′-hexachloro-6-carboxyfluorescein (HEX), or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA).

The 5′ region (encompassing hypervariable regions V1-V3) of the 16S rRNA gene was amplified with S-D-Bact-0045-b-S-20 (5′-6-FAM-GCY TAA CAC ATG CAA GTY GA) and S-D-Bact-0785-a-A-19 (5′-HEX-CTA CCA GGG TAT CTA ATC C). The 3′ region of the 16S rRNA gene was amplified with S-D-Bact-0785-a-S-19 (5′-6-FAM-GGA TTA GAT ACC CTG GTA G) and S-D-Bact-1371-a-A-20 (5′-HEX-AGG CCC GGG AAC GTA TTC AC). PCR products from both sets of primers were predicted to be 600–750 bp, with some variability dependent on the organism from which the template DNA is extracted.

PCR conditions for T-RFLP

Fluorescent dye labeled PCR products for T-RFLP were prepared as described previously.¹⁸ The reaction mixtures for PCR contained 1 x PCR buffer, 200 μM each deoxynucleoside triphosphate, 3.0 mM MgCl₂, 0.5 μM each primer species, 1.0 U Taq DNA polymerase, and 1.0 μL chromosomal preparation per 15 μL reaction. Due to approximately 4-fold higher detection sensitivity of the fluorescence emission from the 6-FAM dye relative to HEX dye, a ratio of 1 part 6-FAM labeled primer (0.125 μM) to 3 parts unlabeled primer (0.375 μM) was used for primers S-D-Bact-0045-b-S-20 and S-D-Bact-0785-a-S-19. DNA was amplified using a model 9600 thermal cycler (Perkin-Elmer, Norwalk, CT) with the following program: 15 minutes at 94°C for denaturation and Taq activation, 35 cycles consisting of denaturation (45 seconds at 94°C), annealing (30 seconds at 52°C), extension (60 seconds at 72°C), and a final extension for 5 minutes at 72°C. All PCR reactions were analyzed by gel electrophoresis and stained with ethidium bromide to assure product formation prior to restriction digest.

Restriction digests of PCR products

PCR products were digested with restriction enzymes (Invitrogen, Carlsbad, CA and New England BioLabs, Beverly, MA) without further purification. Each 10 μL restriction digest contained 1.0 μL of PCR product, 0.5 μL of restriction enzyme, and 8.5 μL of prepared stock containing 2.5 ng/μL each appropriate size/digest control DNA in 1 x buffer. The PCR product of 6-FAM labeled S-D-Bact-0045-b-S-20 and HEX labeled S-D-Bact-0785-a-A-19 (amplifying the 5′ region of the 16S rRNA gene) was digested in separate reactions with AluI, HhaI, MspI, and RsaI. The PCR product of 6-FAM labeled S-D-Bact-0785-aS-19 and HEX labeled S-D-Bact-1371-a-A-20 (amplifying the 3′ region of the 16S rRNA gene) was only digested with HhaI. Restriction digests were performed in a model 9600 Perkin-Elmer thermal cycler programmed for a 1 hour incubation at 37°C followed by 20 minutes at 65°C for enzyme inactivation. The restriction digest products were purified with DyeEx 96-well spin columns (Qiagen) as per manufacturer’s protocol.

Terminal restriction fragment (TRF) profile analysis and organism identification

Samples were prepared for electrophoresis by combining 1.0 μL of restriction digest product, 0.50 μL of X-rhodamine MapMarker 1000 XL (BioVenture, Inc., Muffreesboro, TN), and 14 μL of Hi-Di formamide (Applied Biosystems Instruments). The size standard contains single strands of DNA with a single ROX fluorophore at 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, and 1000 bases. Samples were mixed by pipetting, denatured at 94°C, and immediately cooled to 4°C. The lengths of TRFs derived from amplified DNA products were determined with an Applied Biosystems model 3100 capillary DNA sequencing instrument operating in GeneScan™ mode. The samples were loaded to a 50 cm capillary with an injection voltage of 1 kV for 22 seconds and run at 12.2 kV for 2.2 hours at 50°C. The sizes of control and sample TRFs were calculated by comparison with the internal standard using the local Southern method.²³ Fragment lengths ≥20 but <50 bases were calculated by linear extrapolation from the migration times of the 50 and 75 base standards.

Identification of bacteria was accomplished through the use of a program for calculating terminal fragment sizes and a searchable terminal restriction fragment profile database (TRFPD).¹⁸ Briefly, the maximum area 6-FAM and HEX peak data for each sample are identified and organized according to the respective primer set and restriction digest combination. The program is then used to compare the sample TRF profile with 2435 TRF profiles representing 921 different species of potential relevance to bacteremia in the TRFPD. The results from a Gram stain can also be selected in the data input window to limit the search. The result of a search includes the number of matching fragment lengths (within chosen bp windows), ordering of closest matches, and links to best match TRF profiles. In addition, a sum of differences (SOD) score was determined for each TRF profile relative to the predicted fragments from the database. Specifically, the difference between the size of the experimentally determined TRF and the sequence predicted fragment size was calculated for each fragment/digest combination, and then the sum of these differences was calculated. A smaller SOD score is taken to represent a closer match to a given organism in the database.

Characterization of organisms from positive blood bottles

The three organisms described in this manuscript were isolated from positive anaerobic blood bottles in Marshfield Laboratories. Each culture was determined to contain either Gram positive or Gram variable rod-shaped cells. Isolates grew in 24 hours on trypticase soy–5% sheep blood or chocolate agar at 37°C under anaerobic conditions. Isolates were used for the identification of each organism using the following biochemical tests.

VITEK anaerobe identification

The substrate hydrolysis results from the anaerobe identification card are shown in table 1⇓. The single difference found between the strains was the inability of MCRF-284 to use ρ-nitrophenyl-α-D-mannopyranoside, whereas strains MCRF-271 and MCRF-412 provided a weak reaction. All three strains were identified as L. casei on the basis of the results from Gram reaction, lack of indole production, and substrate hydrolysis on the anaerobic identification card.

Identification of Lactobacillus species by carbohydrate fermentation profile

Table 2⇓ shows the API 50 CH results and the predicted carbohydrate fermentation profiles for L. casei and L. rhamnosus with 22 carbohydrates necessary for species differentiation and identification of most heterofermentative lactobacilli. Strains of L. casei and L. rhamnosus were differentiated by the ability of the latter isolates to ferment arabinose and rhamnose. In addition to the data shown in table 2⇓, all of the blood isolates were positive for fermentation of arbutine, β-gentiobiose, D-tagatose, inositol, N-acetyl-glucosamine, and saccharose. Also, strains MCRF-271 and MCRF-412 were positive for fermentation of L-fucose and L-sorbose, while no reaction was found with MCRF-284. Conversely, MCRF-284 was positive for fermentation of adonitol, D-turanose, and inuline, while no reaction was found with MCRF-271 and MCRF-412.

Identification of species by sequence determination of 16S rRNA gene

The nucleotide sequence was determined for a 1480 base region of the 16S rRNA gene from each of the blood isolates (GenBank accession numbers AY299486, AY299487, and AY299488). The sequences determined for MCRF-271 and MCRF-412 were identical for all 1480 bases. The BLAST analysis of MCRF-271 and MCRF-412 nucleotide sequence indicated the highest identities with a L. casei (99.5%) and L. rhamnosus (99.2%) strain. The similarity scores from the RDP-II sequence match analysis of MCRF-271 and MCRF-412 were determined to be 0.991 for L. casei and 0.986 for L. rhamnosus.

The BLAST analysis of MCRF-284 nucleotide sequence indicated the highest identities with two L. casei and one L. paracasei strains (100% matches). The RDP-II similarity score obtained with the MCRF-284 sequence was determined to be 1.000 with two L. casei and one L. paracasei strains.

Identification of species by TRF profile

The TRF profiles obtained from restriction digest of the 16S rRNA gene PCR products for each of the blood isolates matched closely to those predicted from their respective nucleotide sequences (figure 1⇓ and table 3⇓). The TRF profiles for MCRF-271 and MCRF-412 were closest to those for both L. rhamnosus strains in the sequence predicted TRFPD, matching all 10 fragments within 5 bases and having a SOD score of 14 and 17 bases (table 3⇓). The next closest matches were for three strains of L. plantarum, but the profiles matched only 9 of 10 fragments within 5 bases and had a higher SOD score (26–33 bases). The latter results were primarily due to the significant differences in the 6-FAM labeled AluI restriction digest product (18 base difference).

View larger version:

• In this window
• In a new window

Figure 1.

Representative TRF profile of the PCR products from the 16S rRNA gene of Lactobacillus rhamnosus MCRF-412. Each panel shows a PCR product/restriction digest electropherogram and the applicable range of the standard curve (50–850 bases). The single major 6-FAM (blue peaks) and HEX (green peaks) fragment of each digest are identified by their respective nucleotide size. TRFs from PCR products obtained with (6-FAM)-S-D-Bact-0045-b-S-20 and (HEX)-S-D-Bact-0785-a-A-19 are shown in panels A (AluI), B (HhaI), C (MspI), and D (RsaI). TRFs from PCR products obtained with (6-FAM)-S-D-Bact-0785-aS-19 and (HEX)-S-D-Bact-1371-a-A-20 are shown in panel E (HhaI). The presence of a single peak size >700 bases for both the 6-FAM and HEX fragments in panel D indicates the PCR product was not digested with RsaI.

The TRF profile for MCRF-284 was closest to those for three L. casei strains and one L. zeae strain in the TRFPD, matching all 10 fragments within 5 bases for all four strains and having a SOD score of 13–15 bases. The next closest matches were for three strains of L. paracasei, again matching all 10 fragments within 5 bases, but with a slightly greater SOD score (16 base difference).