Cutibacterium acnes and the shoulder microbiome
These authors collected samples from the skin, subcutaneous fat, anterior supraspinatus tendon, middle glenohumeral ligament, and humeral head cartilage of 23 patients (14 male and 9 female patients) during primary arthroplasty surgery. Total DNA was extracted and microbial 16S ribosomal RNA sequencing was performed using an Illumina MiSeq system.
After stringent removal of contamination, genomic DNA from various Acinetobacter species (Acinetobacter are widely distributed in hospitals, where up to 27% of hospital sink traps and 20% of hospital floor swabs have yielded isolates of Acinetobacter. The Class Acinetobacter includes the Family Propionibacteriaceae which includes the Genus Cutibacterium) and from the Oxalobacteraceae family (Oxalobacteraceae is a family within the order Burkholderiales, not known to be a common cause of shoulder infections) was identified in 74% of rotator cuff tendon tissue samples. C acnes DNA was detected in the skin of only 1 male patient but not in any other shoulder tissues.
In a thoughtful response to this article Letter to the Editor regarding Qui et al: ‘‘Cutibacterium acnes and the shoulder microbiome’’ the authors call attention to the absence of C acnes in the skin and subcutaneous fat samples which is an unexpected finding and contrasts with many prior studies in which C acnes has consistently been cultured from the dermis of the shoulder despite standard skin antisepsis measures.
The authors of the letter suggest that the likely explanation for the discrepancy is that "the polymerase chain reaction (PCR) primers selected for the study are unable to detect most strains of C acnes . As a result, the presence or absence of this organism as an endogenous element of the shoulder microbiome cannot reliably be evaluated with the methods used. PCR with 16S rRNA provides a highly sensitive method for quantifying the relative abundance of numerous bacterial species in a sample, based on the detection of rRNA sequences in 1 or more variable regions of this gene (V1, V2, V3, V4, and so on) that are unique to various bacterial taxonomic groups. This method uses PCR primers that target conserved nucleotides (occurring between variable regions) that are highly similar across most bacterial organisms and contain only a limited number of species-specific polymorphisms. Unfortunately, the primers used in this experiment (F515 and R806, targeting the V4 region) are particularly inefficient in amplifying the common skin organism C acnes because of mismatches between the critical 3’ ends of the primer sequences and the complementary annealing sites in the C acnes genome. Of all 121 fully sequenced C acnes genomes currently in the Reference Sequence (RefSeq) database, fewer than 1% would be expected to amplify robustly with these primers. This limitation as it relates specifically to C acnes has been previously reported but was most comprehensively described in a recent study comparing the use of V1 to V3 vs. V4 variable regions in the analysis of human skin samples (see Skin Microbiome Surveys Are Strongly Influenced by Experimental Design). This work concluded that V4 amplification using standard primers is almost entirely unable to detect C acnes. As emphasized in a corresponding editorial and letter, alternative primers or target regions should be used in studies of the human skin microbiome because of the inability of this commonly used V4 primer set to detect C acnes . For this reason, subsequent studies, including a recent characterization of the microbiome of the human skin follicle, have used primers targeting the V1 to V3 region. The experiments reported by Qui et al include a series of negative controls (open-air collection blanks) but do not include positive controls, except for 1 PCR–restriction fragment length polymorphism assay for the single sample in which C acnes was detected. As such, the limitations of this specific primer pair with respect to C acnes identification may not have been apparent."
Comment: It is evident that PCR needs to be used and interpreted carefully. It cannot distinguish the DNA from live bacteria from that from dead bacteria. The utility of this technique depends on the appropriate choice of primers and on the analysis of both negative controls (submission of a sample exposed to the air of the operating room) and the use of positive controls (submission of a sample known to contain the organisms of interest).
Similar steps would seem appropriate for next-generation sequencing, as reported for example in
Comparative study of cultures and next-generation sequencing in the diagnosis of shoulder prosthetic joint infections. In that study the inclusion of negative controls may have helped assess the possibility of contaminants. For example, Acinetobacter radioresistens (which was found in 6 of 17 [35.3%] of the NGS-positive cases), is commonly found in laboratory reagents.
In our practice we depend on established culture techniques to identify organisms that we know grow well (Cutibacterium and coagulase-negative Staphylococcus). However, it would be of interest to explore the utility of nucleic acid methods to evaluate so called "culture negative infections".
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