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

Abstract

Growth in liquid media is the gold standard for detecting microorganisms associated with bloodstream infections. The Gram stain provides the first clue as to the etiology of infection, with phenotypic identification completed 1 or 2 days later. Providing more detailed information than the Gram stain can impart, and in less time than subculturing, would allow the use of more directed empirical therapy and, thus, reduce the patient's exposure to unnecessary or ineffective antibiotics sooner. The study had two objectives, as follows: (i) to identify new targets to improve our ability to differentiate among certain enteric gram-negative rods or among certain Streptococcus species and (ii) to determine whether real-time PCR and pyrosequencing could as accurately identify organisms directly from positive blood culture bottles as culture-based methods. Two hundred and fifty-five consecutive positive blood culture bottles were included. The results showed a high level of agreement between the two approaches; of the 270 bacteria isolated from the 255 blood culture bottles, results for pyrosequencing and culture-based identifications were concordant for 264/270 (97.8%) bacteria with three failed sequences, and three sequences without match. Additionally, compared to the universal 16S rRNA gene target, the new 23S rRNA gene targets greatly improved our ability to differentiate among certain enteric gram-negative rods or among certain Streptococcus species. In conclusion, combining real-time PCR and pyrosequencing provided valuable information beyond that derived from the initial Gram stain and in less time than phenotypic culturebased identification. This strategy, if implemented, could result in a more directed empirical therapy in patients and would promote responsible antibiotic stewardship.Growth in culture is the gold standard for detecting microorganisms present in the bloodstream (7, 24). Although automated blood culture systems have shortened the time needed to detect growth of an organism, we continue to rely heavily on the Gram stain result for the initial information about the organism's identity. This information is then provided to the healthcare team and used to determine the type of empirical therapy that will be ordered for the patient. It is common to start a patient on one or more broad-spectrum antimicrobial drugs while awaiting the culture-based identification and antimicrobial susceptibility test results. Unfortunately, phenotypic identification requires a minimum of 1 to 2 days to complete and another day to perform susceptibility testing. Having a faster way to classify the microorganism(s) present within positive blood culture bottles would allow tailoring of empirical antibiotic therapy and, thus, reduce the patient's exposure to ineffective or unnecessary antibiotic(s) while awaiting susceptibility testing results.

A Sample Extraction Method for Faster, More Sensitive PCR-Based Detection of Pathogens in Blood Culture

“…Of the six different DNA purification methods tested by Fredricks et al, 9 only a benzyl alcoholguanidine hydrochloride organic extraction method generated extracts that were pure enough to be used undiluted in PCR and in pyrosequencing for pathogen identification. 40 Although a direct comparison between the benzyl alcohol extraction method and the Automate Express chemistry was not performed for the present study, both are able to generate extracts that can be directly used in PCR assays. Of lesser importance, Fredricks et al 9 reported that phenol-chloroform extracts require 1000-fold dilution to achieve inhibition-free PCR, whereas in the present study only 10-fold to 100-fold dilutions were required, depending on the experiment.…”

Molecular-based Screening for Perinatal Group B Streptococcal Infection: Implications for Prevention and Therapy

“…Several studies combining a multiplex PCR with different types of identification of amplicons have shown good correlation with cultures [49][50][51]. However, these expensive amplification techniques will probably not be used when starting from positive blood culture bottles.…”

Identification of bacteria directly from positive blood culture samples by DNA pyrosequencing of the 16S rRNA gene

“…In the present study, the rate of concordance between the two techniques was lower. This was partly because a larger number of polymicrobial samples were included in the present study than were present in the study by Jordan et al (2009). Furthermore, the relatively large number of specific strains that went undetected, such as members of the genus Enterobacter, could also decrease the rate of concordance of this study.…”

“…Highly accurate pyrosequencing identification of bacteria from positive blood culture bottles has been reported previously (Jordan et al, 2009). In this report, the 23S rRNA gene was used as the target to improve the accuracy of identification of some specific bacteria, such as members of the family Enterobacteriaceae and the genus Streptococcus, and the agreement between the results of pyrosequencing-and culture-based identification reached 97.8 %.…”

“…Therefore, the rapidity of pyrosequencing-based identification is an attractive advantage for diagnosis. In fact, Jordan et al (2009) reported that the combination of real-time PCR and pyrosequencing methods rapidly identified bacteria from positive blood culture samples and provided highly concordant results with those obtained using phenotypic identification.…”