A case report of native vertebral osteomyelitis caused by Cutibacterium modestum.

BACKGROUND: Cutibacterium modestum was named in 2020. C. modestum was previously called Propionibacterium humerusii. Several implant-associated infections caused by Cutibacterium species have been previously reported, but native vertebral osteomyelitis due to these bacteria has rarely been reported. CASE PRESENTATION: A 72-year-old man, who had previously received several nerve block injections for low back pain, was referred to our hospital for deterioration in back pain in the last 1 month. MRI findings were suggestive of L5-S1 vertebral osteomyelitis. Blood cultures and bone biopsy culture revealed the presence of Gram-positive bacilli. The isolate was identified as C. modestum by 16SrRNA gene sequencing. A diagnosis of vertebral osteomyelitis caused by C. modestum was made. Minocycline followed by oral amoxicillin was administered for 3 months. His symptom improved and did not recur after treatment completion. CONCLUSION: A case of vertebral osteomyelitis caused by C. modestum was encountered. Although C. modestum is very similar to C. acnes, it could be accurately identified by 16SrRNA gene sequencing. This case represents the first documented C. modestum infection in humans.

Common Features and Intra-Species Variation of Cutibacterium modestum Strains, and Emended Description of the Species.

Cutibacterium modestum is a new species coined in 2020 as the fifth species of genus Cutibacterium, which includes Cutibacterium acnes. The species is predicted as a minor but common member of skin microbiome and includes a group tentatively named as "Propionibacterium humerusii". The description of the species has been provided only with a single strain. To establish the characteristics of C. modestum and search for possible disease-related subtypes, we investigated the biochemical characteristics of eight live strains and performed in silico comparison of nine genomes. The common features, which included the morphology of Gram-stain positive short rods, the negativity of phenylalanine arylamidase, and several unique MALDI-TOF MS spectral peaks, were considered useful in laboratory identification. Pairwise comparisons of the genomes by in silico DNA-DNA hybridization showed similarity values of 98.1% or larger, which were far higher than the subspecies cutoff of 79-80%. The 16S rRNA gene sequences of thirteen isolates and genomes were identical. Their recA gene sequences were identical except for two strains, HM-510 (HL037PA2) and Marseille-P5998, which showed unique one-nucleotide polymorphisms. The biochemical features using API kits were slightly different among the isolates but far closer than those of the nearest other species, C. acnes and Cutibacterium namnetense. Spectra of MALDI-TOF mass spectrometry showed slight differences in the presence of m/z 10,512 (10 kD chaperonin GroS) and three other peaks, further clustering the eight isolates into three subtypes. These results indicated that these isolates did not separate to form subspecies-level clusters, but subtyping is possible by using recA gene sequences or MALDI-TOF mass spectrometry spectra. Moreover, this work has confirmed that a group "P. humerusii" is included in C. modestum.

Detection of Bacterial Infection Based on Age-Specific Percentile-Based Reference Curve for Serum Procalcitonin Level in Preterm Infants.

BACKGROUND: Considering the physiological changes in serum procalcitonin (PCT) levels in newborns due to age, we recently established an age-specific percentile-based reference curve for serum PCT level. The present study aimed to determine the best cutoff percentile line using this reference curve for the differentiation between infected and colonized preterm infants. METHODS: A total of 52 preterm infants with positive bacterial culture (9 with bacterial infection, 43 with colonization) were enrolled within the study period. The 97.5th, 95.0th, 92.5th, 90.0th, 80.0th, 70.0th, 60.0th, and 50.0th percentile lines were drawn in the reference curve. PCT levels in infected or colonized infants were used, and sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. The best cutoff percentile line was determined in the receiver operating characteristic curve analysis. RESULTS: Of the 52 preterm infants, 9 were infected (5 and 4 infants with an onset of < 7 days and ≥ 7 days after birth, respectively), whereas 43 were colonized (6 and 37 infants with an onset of < 7 days and ≥ 7 days after birth, respectively). The best cutoff percentile lines were the 90.0th percentile (sensitivity, 0.800; specificity, 0.833; PPV, 0.800; NPV, 0.833) and 97.5th percentile (sensitivity, 1.00; specificity, 0.973; PPV, 0.800; NPV, 1.00) in infants with an onset of < 7 days and ≥ 7 days after birth, respectively. CONCLUSIONS: The age-specific percentile-based reference curve for serum PCT level is clinically applicable as a new tool for diagnosing infections in preterm infants with positive culture results, particularly at ≥ 7 days after birth.

MicroRNA-124 inhibits TNF-α- and IL-6-induced osteoclastogenesis.

Receptor activator for nuclear factor κB ligand (RANKL)-independent osteoclastogenic pathway was reported recently. MicroRNA (miR)-124 has been known to suppress RANKL-dependent osteoclastogenesis by inhibiting NFATc1 expression. However, whether miR-124 regulates a RANKL-independent pathway has not been elucidated. In this study, we examined whether a RANKL-independent pathway is regulated by miR-124 in addition to the RANKL-dependent one. Using osteoclastogenic culture and pit-formation assay, we found that a miR-124 mimic inhibited osteoclastogenesis in mouse bone marrow-derived macrophages stimulated by TNF-α, IL-6, and M-CSF in the presence of osteoprotegerin. We also showed that the expression levels of osteoclast-specific genes and NFATc1 protein were suppressed in the miR-124 mimic-transfected cells by performing quantitative-polymerase chain reaction and western blotting. Our results indicate that miR-124 is important in inhibiting both RANKL-dependent and -independent osteoclast differentiation by suppressing NFATc1-mediated pathway.

Case of Mycobacterium haemophilum misdiagnosed as Mycobacterium intracellulare due to one base insertion in the bacterial genome.

Mycobacterium haemophilum is a slow-growing, non-tuberculous mycobacteria that causes cutaneous infection. We describe a case of cutaneous infection in a 68-year-old Japanese man with polymyositis. This was caused by M. haemophilum harboring one base insertion in gene sequence. At first, the causal microorganism was misidentified as M. intracellulare by COBAS® TaqMan® MAI test. However, poor growth on Ogawa media and growth enhancement on 7H11C agar around a hemin-containing disk prompted us to reinvestigate the causal microorganisms, which were revealed to be M. haemophilum. Amplified polymerase chain reaction products were sequenced, and the 16S rRNA gene, rpoB, hsp65 and internal transcribed spacer region sequences showed a 100%, 100%, 99.66% and 99.7% match, respectively, with the corresponding regions of M. haemophilum, but it harbored a novel gene sequence in hsp65. The sequences determined by gene analysis of the M. haemophilum strain were deposited into the International Nucleotide Sequence Database. Although numerous cases of M. haemophilum infection have been reported in other countries, only six cases have been reported in Japan to date. It could be possible that this novel mutation lead to misdiagnosis. As M. haemophilum prefers a lower growth temperature (30-32°C) and it requires iron in the culture medium, M. haemophilum could be misidentified or overlooked. Accordingly, a M. haemophilum infection should be considered in cases of cutaneous infection of the body sites, of which surface temperature is low.

MicroRNA-124 inhibits the progression of adjuvant-induced arthritis in rats.

OBJECTIVE: MicroRNAs (miRNAs) are small endogenous, non-coding RNAs that act as post-transcriptional regulators. We analysed the in vivo effect of miRNA-124 (miR-124, the rat analogue of human miR-124a) on adjuvant-induced arthritis (AIA) in rats. METHODS: AIA was induced in Lewis rats by injecting incomplete Freund's adjuvant with heat-killed Mycobacterium tuberculosis. Precursor (pre)-miR-124 was injected into the right hind ankle on day 9. Morphological changes in the ankle joint were assessed by micro-CT and histopathology. Cytokine expression was examined by western blotting and real-time RT-PCR. The effect of miR-124 on predicted target messenger RNAs (mRNAs) was examined by luciferase reporter assays. The effect of pre-miR-124 or pre-miR-124a on the differentiation of human osteoclasts was examined by tartrate-resistant acid phosphatase staining. RESULTS: We found that miR-124 suppressed AIA in rats, as demonstrated by decreased synoviocyte proliferation, leucocyte infiltration and cartilage or bone destruction. Osteoclast counts and expression level of receptor activator of the nuclear factor κB ligand (RANKL), integrin ß1 (ITGB1) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) were reduced in AIA rats treated with pre-miR-124. Luciferase analysis showed that miR-124 directly targeted the 3'UTR of the rat NFATc1, ITGB1, specificity protein 1 and CCAAT/enhancer-binding protein α mRNAs. Pre-miR-124 also suppressed NFATc1 expression in RAW264.7 cells. Both miR-124 and miR-124a directly targeted the 3'-UTR of human NFATc1 mRNA, and both pre-miR-124 and pre-miR-124a suppressed the differentiation of human osteoclasts. CONCLUSIONS: We found that miR-124 ameliorated AIA by suppressing critical prerequisites for arthritis development, such as RANKL and NFATc1. Thus, miR-124a is a candidate for therapeutic use for human rheumatoid arthritis.

Rapid detection of B2-ST131 clonal group of extended-spectrum ß-lactamase-producing Escherichia coli by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry: discovery of a peculiar amino acid substitution in B2-ST131 clonal group.

One reason for the spread of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli worldwide is the global pandemic of the B2-ST131 clonal group. We searched for the specific biomarker peaks to distinguish between the B2-ST131 clonal group and other sequence type (ST) clonal groups isolated from clinical specimens obtained in our hospital. Biomarker peaks at m/z 7650 in the B2-ST131 group (sensitivity of 100% and specificity of 89.7%) and m/z 7707 in the other ST clonal groups showed the highest discrimination abilities. We further verified reproducibility against other Japanese clinical isolates obtained in another area of Japan. Differences between the molecular mass at the 7650m/z and 7707m/z peaks indicated an E34A amino acid substitution by proteomic and genomic analysis. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry rapidly and simply identified the B2-ST131 clonal group in routine examinations and will allow for adequate empirical therapy and the possibility to control both hospital infections and the global pandemic.

Sterility Testing of Stem Cell Products by Broad-Range Bacterial 16S Ribosomal DNA Polymerase Chain Reaction.

OBJECTIVE: To evaluate broad-range 16S ribosomal DNA (rDNA) polymerase chain reaction (PCR) as a rapid screening tool to detect bacterial contamination of stem-cell products. METHODS: We performed the evaluation using whole blood spiked with serially diluted bacterial-type strains. Detection sensitivity was defined as the bacterial concentration for which all replicates were positive at each concentration (100% detection). We tested the sterility of 29 bags of autologous peripheral blood stem cell (PBSC) products harvested at our facility using the 16S rDNA PCR method. RESULTS: The detection sensitivity of 16S rDNA PCR in spiked whole blood was 10¹ to 10² colony-forming units (CFU) per mL, depending on the bacterial strain. We detected no amplified 16S rDNA among the PBSCs we used in this study. The BacT/ALERT automated bacterial culture system that we used also showed no positive signals in any of the PBSCs tested. CONCLUSIONS: Our data indicate that bacterial 16S rDNA PCR is a useful alternative for rapid sterility testing, not only for blood products used in transfusion medicine but also for stem-cell products used in regenerative medicine.

[Evaluation of cut-off value for autoantibodies against double-stranded DNA-complexed nucleosomes based on enzyme linked immunosorbent assay].

The new classification criteria for systemic lupus erythematosus (SLE) by Systemic Lupus International Collaborating Clinics (SLICC) published in 2012 have defined positive level titer of antibody against double stranded (ds) DNA as more than double the reference range if tested by enzyme linked immunosorbent assay (ELISA). The aim of this study was to evaluate optimal cut-off value and diagnostic performance of the anti-dsDNA nucleosome-complexed (anti-dsDNA-NcX) ELISA, which uses salmon testis DNA complexed with purified nucleosomes as antigens, for diagnosis of SLE. Titers of antibodies against dsDNA-complexed nucleosome were measured in sera of 76 patients with SLE, 148 with other connective tissue diseases, and 323 healthy volunteers. Sensitivity, specificity, correlation and concordance rate were compared among anti-dsDNA-NcX, conventional ELISA methods (EIA) and radioimmunoassay (RIA). As a results, concordance rates and Spearman's coefficient of rank correlation (r(s)) of anti-dsDNA-NcX with EIA and RIA were 59.2%, r(s) = 0.40 and 53.9%, r(s) = 0.21, respectively. Receiver operating characteristic curve analysis showed that the titer of 44 IU/mL calculated as 99 percentile of 323 healthy volunteers is a better cut-off value for anti-dsDNA-NcX than the 100 IU/mL recommended by the manufacturer. By setting the cut-off value at 44 IU/mL, anti-dsDNA-NcX showed the highest sensitivity (75.0%) and specificity (90.5%) of the 3 assays. With SLICC criterion, positivity rates of anti-dsDNA-NcX, EIA and RIA for SLE patients were 50.0%, 30.3% and 50.0%, respectively. In conclusion, anti-dsDNA-NcX has a good diagnostic performance for SLE with our proposed cut-off value of 44 IU/mL.