Beyond the Colony-Forming-Unit: Rapid Bacterial Evaluation in Osteomyelitis
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Curated by eLife
eLife assessment
This useful study addresses discrepancies in determining bacterial burden in osteomyelitis as determined by culture and enumeration using DNA. The authors present compelling data demonstrating the emergence of discrepancies between CFU counts and genome copy numbers detected by PCR in Staphylococcus aureus strains infecting osteocyte-like cells. Whilst the observations may represent a substantial addition to the field of musculoskeletal infection, the broad applicability and clinical benefit are unclear.
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Abstract
Examination of bacteria/host cell interactions is important for understanding the aetiology of many infectious diseases. The colony-forming-unit (CFU) has been the standard for quantifying bacterial burden for the past century, however, this suffers from low sensitivity and is dependent on bacterial culturability in vitro . Our data demonstrate the discrepancy between the CFU and bacterial genome copy number in an osteomyelitis-relevant co-culture system and we confirm diagnosis and quantify bacterial load in clinical bone specimens. This study provides insight into improving the quantification of bacterial burden in such cases.
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eLife assessment
This useful study addresses discrepancies in determining bacterial burden in osteomyelitis as determined by culture and enumeration using DNA. The authors present compelling data demonstrating the emergence of discrepancies between CFU counts and genome copy numbers detected by PCR in Staphylococcus aureus strains infecting osteocyte-like cells. Whilst the observations may represent a substantial addition to the field of musculoskeletal infection, the broad applicability and clinical benefit are unclear.
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Reviewer #1 (Public Review):
Summary:
This work shows, based on basic laboratory investigations of in-vitro-grown bacteria as well as human bone samples, that conventional bacterial culture can substantially underrepresent the quantity of bacteria in infected tissues. This has often been mentioned in the literature, however, relatively limited data has been provided to date. This manuscript compares culture to a digital droplet PCR approach, which consistently showed greater levels of bacteria across the experiments (and for two different strains).Strengths:
Consistency of findings across in vitro experiments and clinical biopsies. There are real-world clinical implications for the findings of this study.Weaknesses:
No major weaknesses. Only three human samples were analyzed, although the results are compelling. -
Reviewer #2 (Public Review):
In this study, the authors address discrepancies in determining the local bacterial burden in osteomyelitis between that determined by culture and enumeration by DNA-directed assay. Discrepancies between culture and other means of bacterial enumeration are long established and highlighted by Staley and Konopka's classic, "The great plate count anomaly" (1985). Here, the authors first present data demonstrating the emergence of discrepancies between CFU counts and genome copy numbers detected by PCR in S. aureus strains infecting osteocyte-like cells. They go on to demonstrate PCR evidence that S. aureus can be detected in bone samples from sites meeting a widely accepted clinicopathological definition of osteomyelitis. They conclude their approach offers advantages in quantifying intracellular bacterial load …
Reviewer #2 (Public Review):
In this study, the authors address discrepancies in determining the local bacterial burden in osteomyelitis between that determined by culture and enumeration by DNA-directed assay. Discrepancies between culture and other means of bacterial enumeration are long established and highlighted by Staley and Konopka's classic, "The great plate count anomaly" (1985). Here, the authors first present data demonstrating the emergence of discrepancies between CFU counts and genome copy numbers detected by PCR in S. aureus strains infecting osteocyte-like cells. They go on to demonstrate PCR evidence that S. aureus can be detected in bone samples from sites meeting a widely accepted clinicopathological definition of osteomyelitis. They conclude their approach offers advantages in quantifying intracellular bacterial load in their in vitro "co-culture" system.
Weaknesses
- My main concern here is the significance of these results outside the model osteocyte system used by this group. Although they carefully avoid over-interpreting their results, there is a strong undercurrent suggesting their approach could enhance aetiologic diagnosis in osteomyelitis and that enumeration of the infecting pathogen might have clinical value. In the first place, molecular diagnostics such as 16S rDNA-directed PCR are well established in identifying pathogens that don't grow. Secondly, it is hard to see how enumeration could have value beyond in vitro and animal model studies since serial samples will rarely be available from clinical cases.- I have further concerns regarding the interpretation of the combined bacterial and host cell-directed PCRs against the CFU results. Significance is attached to the relatively sustained genome counts against CFU declines. On the one hand, it must be clearly recognised that the detection of bacterial genomes does not equate to viable bacterial cells with the potential for further replication or production of pathogenic factors. Of equal importance is the potential contribution of extracellular DNA from lysed bacteria and host cells to these results. The authors must clarify what steps, if any, they have taken to eliminate such contributions for both bacteria and host cells. Even the treatment with lysotaphin may have coated their osteocyte cultures with bacterial DNA, contributing downstream to the ddPCR results presented.
Strengths
- On the positive side, the authors provide clear evidence for the value of the direct buffer extraction system they used as well as confirming the utility of ddPCR for quantification. In addition, the successful application of MinION technology to sequence the EF-Tu amplicons from clinical samples is of interest.- Moreover, the phenomenology of the infection studies indicating greater DNA than CFU persistence and differences between the strains and the different MOI inoculations are interesting and well-described, although I have concerns regarding interpretation.
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