Utility of Pyrosequencing in Identifying Bacteria Directly from Positive Blood Culture Bottles

Study hospital setting.The Magee-Women's Hospital of the University of Pittsburgh Medical Center is a 400-bed, full-service women's hospital with a state-of-the-art 75-bed neonatal intensive care unit. The hospital has several specialty surgical services, including orthopedics, bariatrics, and urology. For this study, consecutive positive blood culture bottles were collected during 2008 from both infants and adults.

Bacterial strains.A total of 1,075 bacterial isolates was included in this study for assessing the usefulness of the two proposed 23S rRNA gene targets and consisted of 141 Staphylococcus aureus, 232 coagulase-negative Staphylococcus species (CoNS), 171 Enterococcus species, 107 Streptococcus agalactiae (GBS), 35 Streptococcus pneumoniae, 25 Streptococcus mitis, 74 viridans streptococci, 35 Streptococcus pyogenes, 76 Escherichia coli, 32 Klebsiella oxytoca, two Citrobacter koseri, 23 Enterobacter faecalis, 20 Enterobacter faecium, 10 Citrobacter freundii, five Morganella morganii, 20 Proteus mirabilis, two Proteus vulgaris, 53 Klebsiella pneumoniae, and 12 Serratia marcescens isolates. Most of these bacteria represent well-characterized clinical isolates, but several purchased ATCC reference strains used for quality control testing were also included. The bacterial isolates were identified by standard laboratory protocol using the MicroScan instrument and panels (Siemens Healthcare Diagnostics, Inc., Deerfield, IL) or the RapID STR system (Remel, Lenexa, KS) for identification of streptococci and/or individual biochemical reagents.

Bacterial isolate crude cell lysate preparation.Crude cell lysates were prepared from each of the above-mentioned bacterial isolates for PCR analysis. Using a sterile pipette tip, cells from a well-isolated bacterial colony were transferred to a 1.5-ml Eppendorf tube containing 200 μl of lysis buffer (10 mM Trish-HCl [pH 8.0], 0.5% Brij-35) and 0.24 g of 0.1-mm zirconium beads (catalog no. 11079101z; Biospec Products, Bartlesville, OK). The contents of the tube were vortexed for 5 min, followed by a quick spin to settle the beads out of solution.

Blood culture bottle fluid DNA preparation.Discarded, deidentified Bactec 9240 blood culture bottles, which were resin-containing aerobic bottles, lytic anaerobic bottles, or PedsPlus bottles, were included in this study. The specimen preparation procedure uses a protocol described by Fredricks and Relman (6).

Real-time PCR.Four individual PCR assays were set up with each specimen analyzed. These specimens included the crude cell lysates made from bacterial isolates and purified DNA from positive blood culture bottles. The primers used in each PCR assay are described in Table 1. Each master mix contained 0.5 μl each of 10 μM concentrations of forward and reverse primers (IDT, Coralville, IA), 5 μl of purified DNA prepared from fluid removed from the blood culture bottle or 1 μl of crude cell lysate prepared from isolated bacteria, and 12.5 μl of 2× SYBR TakaRa PreMix Taq polymerase master mix (catalog no. RR039B; Fisher Scientific, Pittsburgh, PA). The PCR cycling conditions consisted of 95°C for 1 min, followed by 40 cycles of 95°C for 20 s, 60°C for 1 min, and 72°C for 15 s. The three positive controls consisted of intact E. coli, S. aureus, and GBS isolates, while the two negative controls consisted of a no-template control and fluid from a discarded, de-identified blood culture bottle containing no growth. All of these controls were included in each PCR run.

Pyrosequencing.All PCR-positive reactions were analyzed by pyrosequencing as previously described (11). The sequencing primer(s) used for each individual pyrosequencing reaction is listed in Table 1. The dispensation order used when sequencing the two 16S rRNA gene target amplicons and the 23S rRNA gene target amplicon (bp 602 to 834 in reference strain GBS AB096754) was 11(ACTG), while the dispensation order used for the 23S rRNA gene target amplicon (bp 1346 to 1625 in reference strain E. coli AJ278710) was AGC12(CTGA).

The PSQ 96 SQA software determined the base sequences and assessed the quality of the pyrograms, which is based on a combination of actual peak height, relative peak height, and background noise. The sequences generated were analyzed using Identifire 1.0.5.0 software that aligns the input sequences with those present in a reference library. The reference library consists of validated sequences generated by us from previously identified clinical isolates, ATCC reference strains, and known sequences within the National Center for Biotechnology Information (NCBI) GenBank using the Basic Local Alignment Search Tool (BLAST) algorithm (http://www.ncbi.nlm.nih.gov/BLAST ).

Assessment of two 23S rRNA gene targets.Our previous work (11) describes two regions within the 16S rRNA gene that are useful for classifying bacteria by PCR and pyrosequencing; one is a universal target (target 1 [Table 1]) that provides broad-based classification of most medically relevant bacteria, while the other is a region that differentiates S. aureus from CoNS (target 2 [Table 1]). However, neither of these 16S rRNA gene targets is helpful for differentiating among enteric gram-negative rods or among Streptococcus species. To that end, we interrogated the 23S rRNA gene and found two regions—one that contained sequence variability among many of the enteric gram-negative rods associated with bloodstream infections (target 4 [Table 1]), and another that contained sequence variability among certain Streptococcus species (target 3 [Table 1]).

Table 2 illustrates the sequence patterns obtained from testing 255 previously identified enteric gram-negative rods with the primers described in Table 1 (target 4). The results illustrated the usefulness of this region for differentiating between E. coli, K. oxytoca, K. pneumoniae, P. mirabilis, P. vulgaris, S. marcescens, M. morganii, and C. koseri. The Enterobacter species and C. freundii isolates had identical sequences and therefore could not be differentiated from each other, but they were distinguishable from the other enteric gram-negative rods analyzed (Table 2). No amplicons were generated when using this primer set for any of the 373 Staphylococcus sp. or 447 Streptococcus sp. isolates tested (data not shown).

Table 3 illustrates the sequence patterns obtain from 447 previously identified streptococcal isolates using the primers described in Table 1 (target 3). The result showed the utility of this region for differentiating between S. pyogenes, GBS, and the Enterococcus spp. The highly diverse group of viridans streptococci demonstrated variability within its group that was distinguishable from the other Streptococcus species analyzed. S. pneumoniae and S. mitis had identical sequences and therefore could not be differentiated from each other but, as a group, were distinguishable from the other Streptococcus species. No amplicons were generated using this primer set for any of the 255 enteric gram-negative rods tested (data not shown). A unique amplicon was generated using this primer set for the 373 staphylococcal isolates tested (5′-CGA CCA AAT ACT CAA CGC CCT-3′) that was distinguishable from those generated for all of the streptococcal isolates.

Positive blood culture bottles with single isolates.A total of 270 bacteria were isolated from these 255 positive blood culture bottles; 242 bottles (94.9%) contained a single bacterial type, while 13 bottles (5.1%) contained polymicrobial growth. Table 4 illustrates the results for 242 blood culture bottles containing a single organism. Compared to culture-based identification, pyrosequencing accurately classified 239/242 (98.8%) of these organisms. The three discordant results were as follows: pyrosequencing reaction repeatedly failed to generate sequencing data for amplicons generated for one viridans streptococcus isolate and two S. pyogenes isolates. As noted above, the sequence generated for the S. pneumoniae could not be distinguished from that of Streptococcus mitis, and neither could the Enterobacter species be distinguished from C. freundii.

Positive blood culture bottles with multiple organisms.Thirteen of 255 (5.1%) positive blood culture bottles contained more than one organism when subcultured. Table 5 compares the results of pyrosequencing with those of culture-based identification of the 28 bacterial isolates from 13 bottles. Pyrosequencing accurately classified 19/28 organisms, while 6/28 bacterial isolates, namely two E. coli, two Enterobacter cloacae, one Enterobacter aerogenes, and one S. mitis isolate, were broadly categorized only as either enteric group 1 (five gram-negative rods) or Streptococcus species (one S. mitis isolate), based solely on the sequences generated from the universal 16S rRNA gene target (target 1 [Table 1]) and not from the 23S rRNA gene target (target 4 [Table 1]). No matches were found for 3/28 organisms, including for two CoNS isolates and one S. mitis isolate.