Background
The genus
Corynebacterium is composed of aerobic, non–spore-forming, pleomorphic, Gram-positive bacilli with worldwide distribution. The most well-established and well-described pathogen in this genus,
C. diphtheria, is the main causative agent of diphtheria, the incidence of which has dropped due to effective vaccination programs [
1]. However, in recent years, there has been a considerable increase in reports of non-diphtheriae
Corynebacterium species, which have been linked to multiple hospital outbreaks and nosocomial infections [
2‐
4]. Although these microorganisms are common components of the skin microbiota and mucous membranes, their clinical significance as emerging respiratory pathogens has been demonstrated by various studies [
4‐
6]. Of note, recent reports show that multidrug-resistant strains of the species
C. striatum are emerging rapidly [
7‐
10]. Early detection and identification of
Corynebacterium species are essential to intervention and infection treatment efforts.
Traditionally, these microorganisms have been routinely identified by biochemical tests using the API Coryne system (bioMérieux, Craponne, France) or the RapID CB PLUS system (Thermo Fisher Scientific, Waltham, MA, USA) in clinical microbiology laboratories [
11,
12]. However, these methods have low sensitivity, and are time-consuming and unreliable for species identification, especially in the case of
C. simulans, due to its similarities with
C. striatum [
13,
14]. Identification by 16S ribosomal ribonucleic acid (rRNA) and
rpoB gene sequencing produces more reliable results, but is slow and cost prohibitive in developing countries [
15,
16]. Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS) has been applied to perform accurate species-level identification of
Corynebacterium spp. clinical isolates, but this technology is not yet fully accessible to clinical microbiology laboratories in resource-limited settings [
17]. Also, MALDI-TOF requires pure cultures as starting material, which precludes rapid diagnosis. Therefore, suitable detection assays that are rapid, reliable, and cost-effective are always in demand for effective control and treatment strategies against infections caused by emerging
Corynebacterium species.
The high-resolution melting (HRM) assay, a recently developed technique based on quantitative real-time polymerase chain reaction (qPCR) that detects genetic variation in deoxyribonucleic acid (DNA) sequences, provides a good alternative for molecular diagnosis [
18]. Before HRM analysis can be performed, the region of interest is amplified using PCR in the presence of a fluorescent dye (EvaGreen) that is homogenously intercalated into the double-stranded (ds) DNA. After PCR, the amplicon is gradually heated at increasing temperatures, and the ds PCR product denatures into two single strands, releasing the binding dye and leading to a decrease in fluorescence level. The rate of dissociation of the amplicon mainly depends on GC-content, sequence length, complementarity, and nearest-neighbor thermodynamics [
19]. A specific and characteristic melting profile can be produced for the amplicon by monitoring changes in fluorescence intensity. The HRM assay is an easily implemented, closed-tube method that can simultaneously detect closely related species within approximately 2 h. In addition, it is not restricted to cultured material, but can detect DNA extracted directly from clinical specimens. The HRM technique has been successfully employed in concurrently identifying and differentiating between several pathogens, such as bacteria, viruses, and fungi [
20‐
23].
In this study, we report the development of a qPCR–based HRM assay capable of detecting C. striatum, C. propinquum, and C. simulans, as well as distinguishing between them in pure cultures and clinical specimens with increased specificity and sensitivity.
Discussion
Due to being common components of the skin microbiota, non-diphtheriae
Corynebacterium species are usually thought of as colonizers or contaminants [
31,
32]. Because of this, as well as challenges in identification, they have not received a great deal of attention [
33]. In recent years, the clinical relevance of these microorganisms has been recognized, particularly as a cause of respiratory-tract infections [
5,
6,
34,
35]. A rapid, simple, specific, and sensitive molecular technique for identifying and differentiating between these species is essential for outbreak detection, epidemiological surveillance, and direct patient treatment, as most strains of
C. striatum are resistant to multiple antimicrobials.
However, differentiation between these species remains difficult due to the low genetic variation between them.
C. striatum,
C. propinquum, and
C. simulans have similar colony morphologies and cultural characteristics.
C. simulans and
C. striatum share high genetic homology, and their biochemical reactions are very similar [
36]. Misidentification of
C. simulans or
C. propinquum as
C. striatum by VITEK MS MALDI-TOF MS was observed in our investigation (data not shown), and this has also been reported previously [
8]. Currently, few commercially available assays can rapidly differentiate between these species, and published assays rely on the use of pure cultures.
In this study, we developed a HRM assay that could differentiate between C. striatum, C. propinquum, and C. simulans in cultured samples and clinical specimens within approximately 2 h after DNA extraction. Analysis of the normalized melt curve produced with the universal primers generated species-specific HRM curve profiles. The three species were clearly differentiated by the melting temperature of the dissociation curves, with melting peaks at 88.91 °C for C. striatum, 88.44 °C for C. propinquum, and 87.86 °C for C. simulans. Furthermore, these three strains were successfully identified based on their different plots, which were mutually distinct.
We also evaluated the limit of detection of the newly established HRM assay. This assay could detect quantities as low as 100 fg using DNA from pure cultures as templates. Assay specificity was further evaluated by testing the genomic DNA of 69 corynebacteria clinical isolates and 30 non-target strains. Results showed that the high discriminatory power of the HRM assay developed for C. striatum, C. propinquum, and C. simulans also gave it the ability to specifically distinguish these three species from other related bacteria.
Furthermore, we showed that HRM could be applied to direct tests of clinical sputum samples. Among the 42 culture-positive samples, all were identified by HRM, while one false negative result occurred. This one sample was also found to be negative by the MCDA assay. This result suggested that DNA might have been lost or degraded during the preparation, processing, and storage of the sputum samples.
Furthermore, 22 samples were identified as negative by the culture method, but identified as positive by HRM and MCDA assays. In our study, the culture method was likely to miss one quarter (22/88) of Corynebacterium spp.-positive samples. Putative sensitivity increased from 65.6% using the culture method to 98.4% using the HRM assay. Compared with the culture method, the use of the HRM assay to detect the three Corynebacterium spp. could potentially improve overall sensitivity and reduce turnaround times.
Our HRM assay was also comparable to the MCDA method, showing a high concordance rate (95.5%) [
25]. MCDA technology has revolutionized the detection of pathogens, but the established MCDA method can identify only one
Corynebacterium species,
C. striatum. In addition, this method requires the use of five pairs of primers, which can generate a complex mixture of various DNA products, and it can be difficult to distinguish between specific and non-specific products [
37]. Compared with the MCDA assay, the HRM method we developed based on a pair of universal primers could specifically identify three
Corynebacterium spp. in only one test.
In the present study, 95.3% of pulmonary infections were caused by
C. striatum, 3.1% by
C. propinquum, and 1.6% by
C. simulans, as identified by HRM. Among members of
Corynebacterium spp.,
C. striatum and
C. propinquum have been recognized as pathogens of the respiratory tract, and have been found in cases of pneumonia and chronic obstructive pulmonary disease (COPD) in hospital settings [
4,
38‐
40]. A comparative study noted that
C. striatum was the most prevalent non-diphtheriae
Corynebacterium found in sputum specimens, and found that it caused a large proportion of respiratory infections [
1]. The high incidence of nosocomial outbreaks caused by
C. striatum highlights the importance of routine species-level identification to avoid further spread and outbreaks [
41].
Unlike
C. striatum and
C. propinquum,
C. simulans has not been described as a cause of respiratory infection. Only a few well-documented infections have been reported, including one case of acute pyogenic spondylitis, one prosthetic joint infection, and one case of endocarditis [
42‐
44]. The recovery of
C. simulans from a respiratory specimen here appears to be novel and has clinical relevance, emphasizing its potential role as a causative pathogen of respiratory tract infections.
There were several limitations in our study. i) Sample sizes of
C. propinquum and
C. simulans clinical isolates were small. This was because, despite our efforts, we could not find additional isolates. ii)
C. pseudodiphtheriticum and
C. amycolatum, which other reports have shown to be common non-diphtheriae corynebacteria [
35], were not available for this study; this might have been due to differences in the geographical distribution of non-diphtheriae corynebacteria.
Despite these limitations, our study has some strengths. First, the HRM method can identify and differentiate between
C. striatum,
C. propinquum, and
C. simulans directly from clinical sputum samples without isolating and culturing the pathogens. This meets the need for rapid diagnosis and, more importantly, yields high diagnostic accuracy in culture-negative samples. Second, this assay could potentially be applied to recognize novel or unusual
Corynebacterium spp. in clinical specimens by targeting single loci, with no need to design new assays; this is supported by previous research [
45]. Third, this technique produced results in 2 h with no need for gel electrophoresis of the PCR products, avoiding sample cross-contamination. Finally, HRM analysis is a cost-effective method using common and widely available reagents and equipment, making it especially suitable for resource-limited settings.
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