Genome sequence of Coxiella burnetii KZQ2 isolated from a clinical strain in the Republic of Korea.

The genome of Coxiella burnetii KZQ2, isolated from clinical patients in Korea, is 2.04 MB long. Multispacer types were ST77, and phylogenetic tree analysis showed that KZQ2 is closely related to the CbuK_Q154 chronic strain isolated from human endocarditis patients in the USA.

Highly efficient graphene terahertz modulator with tunable electromagnetically induced transparency-like transmission.

Graphene-based optical modulators have been extensively studied owing to the high mobility and tunable permittivity of graphene. However, weak graphene-light interactions make it difficult to achieve a high modulation depth with low energy consumption. Here, we propose a high-performance graphene-based optical modulator consisting of a photonic crystal structure and a waveguide with graphene that exhibits an electromagnetically-induced-transparency-like (EIT-like) transmission spectrum at terahertz frequency. The high quality-factor guiding mode to generate the EIT-like transmission enhances light-graphene interaction, and the designed modulator achieves a high modulation depth of 98% with a significantly small Fermi level shift of 0.05 eV. The proposed scheme can be utilized in active optical devices that require low power consumption.

An ECG Stitching Scheme for Driver Arrhythmia Classification Based on Deep Learning.

This study proposes an electrocardiogram (ECG) signal stitching scheme to detect arrhythmias in drivers during driving. When the ECG is measured through the steering wheel during driving, the data are always exposed to noise caused by vehicle vibrations, bumpy road conditions, and the driver's steering wheel gripping force. The proposed scheme extracts stable ECG signals and transforms them into full 10 s ECG signals to classify arrhythmias using convolutional neural networks (CNN). Before the ECG stitching algorithm is applied, data preprocessing is performed. To extract the cycle from the collected ECG data, the R peaks are found and the TP interval segmentation is applied. An abnormal P peak is very difficult to find. Therefore, this study also introduces a P peak estimation method. Finally, 4 × 2.5 s ECG segments are collected. To classify arrhythmias with stitched ECG data, each time series' ECG signal is transformed via the continuous wavelet transform (CWT) and short-time Fourier transform (STFT), and transfer learning is performed for classification using CNNs. Finally, the parameters of the networks that provide the best performance are investigated. According to the classification accuracy, GoogleNet with the CWT image set shows the best results. The classification accuracy is 82.39% for the stitched ECG data, while it is 88.99% for the original ECG data.

Therapeutic effects of boronate ester cross-linked injectable hydrogels for the treatment of hepatocellular carcinoma.

Hepatocellular carcinoma is the most common malignancy with a high incidence rate and is the leading cause of cancer-related deaths. Herein, we developed a thermo-responsive hydrogel comprising poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-lactide (PCLA) that exhibits acidity-accelerated delivery of the tumor-targeting glucuronic acid-bearing doxorubicin (DOX-pH-GA) conjugate into tumor tissues. The PCLA copolymer was post-modified with boronic acid (BA-PCLA) to covalently cross-link with the pH-responsive DOX-pH-GA conjugate. The BA-PCLA copolymer effectively coordinated with the DOX-pH-GA conjugate through the boronate ester formation and showed a lower critical gelation temperature. The DOX conjugated via boronate ester exhibited a sustained release in vitro. Subcutaneous administration of PCLA copolymers formed in situ gels in the subcutaneous layers of Sprague-Dawley rats and degraded after 6 weeks. Similarly, BA-PCLA copolymers coordinated with DOX-pH-GA formed a stable in situ gel in vivo. In vivo imaging studies demonstrated that DOX-pH-GA was released in a sustained manner. The anti-tumor activity of the DOX releasing injectable hydrogel was examined using a HepG2 liver cancer xenograft model. The in vivo antitumor effect demonstrated that the DOX releasing hydrogel depot remarkably suppresses the tumor growth. These results demonstrate that the pH-responsive DOX releasing thermo-responsive hydrogel depot has great potential for application in localized anticancer therapy.

Creation of Fano resonances and bound states in the continuum in metallic metasurface superlattices.

A perfect metal film with a periodic arrangement of cut-through slits, an anisotropic metallic metamaterial film, mimics a dielectric slab and supports guided electromagnetic waves in the direction perpendicular to the slits. Since the guided Bloch modes exist only below the light line, conventional metallic metamaterial films do not exhibit interesting leaky-wave effects, such as bound states in the continuum and Fano resonances. Here, we introduce metallic metasurface superlattices that include multiple slits in a period and demonstrate that the superlattices support the Fano resonances and bound states in the continuum. We show that the number of Fano resonances and bound states depend on the number of slits in a period of superlattices through rigorous finite element method simulations. Experimental results in microwave region also support the creation of Fano resonance and bound states in the continuum by the increment of the number of slits in a period of superlattices.

Metasurfaces with Bound States in the Continuum Enabled by Eliminating First Fourier Harmonic Component in Lattice Parameters.

Conventional photonic lattices, such as metamaterials and photonic crystals, exhibit various interesting physical properties that are attributed to periodic modulations in lattice parameters. In this study, we introduce novel types of photonic lattices, namely Fourier-component-engineered metasurfaces, that do not possess the first Fourier harmonic component in the lattice parameters. We demonstrate that these metasurfaces support the continuous high-Q bound states near second stop bands. The concept of engineering Fourier harmonic components in periodic modulations provides a new method to manipulate electromagnetic waves in artificial periodic structures.

Polarization-differentiated band dynamics of resonant leaky modes at the lattice Γ point.

In the physical description of photonic lattices, leaky-mode resonance and bound states in the continuum are central concepts. Understanding of their existence conditions and dependence on lattice parameters is of fundamental interest. Primary leaky-wave effects are associated with the second stop band at the photonic lattice Γ point. The pertinent band gap is defined by the frequency difference between the leaky-mode band edge and the bound-state edge. This paper address the polarization properties of the band gaps resident in laterally periodic one-dimensional photonic lattices. We show that the band gaps pertinent to TM and TE leaky modes exhibit significantly differentiated evolution as the lattice parameters vary. This is because the TM band gap is governed by a surface effect due to the discontinuity of the dielectric constant at the interfaces of the photonic lattice as well as by a Bragg effect due to the periodic in-plane dielectric constant modulation. We find that when the lattice is thin (thick), the surface (Bragg) effect dominates the Bragg (surface) effect in the formation of the TM band. This leads to complex TM band dynamics with multiple band closures possible under parametric variation. In complete contrast, the TE band gap is governed only by the Bragg effect thus exhibiting simpler band dynamics. This research elucidates the important effect of polarization on resonant leaky-mode band dynamics whose explanation has heretofore not been available.

Albumin affibody-outfitted injectable gel enabling extended release of urate oxidase-albumin conjugates for hyperuricemia treatment.

Therapeutic proteins are attractive candidates for the treatment of human diseases. However, their short half-life often limits their clinical application. To overcome this problem, injectable hydrogels have been developed as depots for controlled release of therapeutic proteins, but these systems have not yet achieved the desired extended, sustained drug release profile. Our strategy herein was to implement selective and strong interactions between the hydrogels and therapeutic proteins. Specifically, we investigated whether strong and specific interactions between human serum albumin (HSA) and albumin-binding peptide (ABP) can be used to achieve extended release of urate oxidase (Uox), a therapeutic protein for hyperuricemia treatment, from pH- and temperature-sensitive injectable hydrogels consisting of poly(ethylene glycol)-poly(ß-amino ester urethane) (PEG-PAEU) copolymer. Thus, HSA was conjugated to Uox (Uox-HSA) and ABP was introduced in PEG-PAEU (PEG-PAEU-ABP). Polymers, conjugates, and hydrogels were extensively characterized for their physicochemical characteristics and in vivo efficacy in a hyperuricemia mouse model. Briefly, the hydrogels exhibited good injectability, in vitro biocompatibility and extended drug release, and in vivo gel formation and degradability. The serum half-life of the Uox-HSA loaded in PEG-PAEU-ABP hydrogels was ~96 h in mice, which was ~88, ~5.5, and ~2 times longer than that of free native Uox, free Uox-HSA, and Uox-HSA loaded in PEG-PAEU hydrogels, respectively. In the hyperuricemia mouse model, Uox-HSA loaded in PEG-PAEU-ABP hydrogels exhibited a substantially extended period of uric acid-lowering efficacy. These results clearly show that by applying ABP-HSA strong interaction to injectable hydrogels and therapeutic protein, the concentration of the therapeutic protein can be maintained for a long period in vivo, prolonging its therapeutic effect. Further, our approach can be tailored to accommodate other therapeutic proteins, which potentially expands the clinical applicability range of these systems.

Wheel Alignment of a Suspension Module Unit Using a Laser Module.

Vehicle wheel alignment inspection is generally carried out using a computer vision-based system. Due to its inspection mechanism using four wheel centers, the computer vision-based system cannot be applied to the wheel alignment inspection of suspension module units. However, when a vehicle suspension module is being developed, there is no complete car ready for wheel alignment inspection even though it is a very important procedure for suspension property tests. This study proposes a novel and efficient way to inspect vehicle wheel alignment for suspension modules. Two laser modules and several mechanical jigs were employed for wheel alignment inspection, allowing the toe and camber angles of the suspension module to be measured. For accurate wheel alignment results, calibration of the laser modules was performed prior to the inspection. This calibration procedure adjusts the yaw and pitch angles of the laser module so that they can be orthogonal to the mounting jig. For the calibration, a novel method of using laser straightness was adopted and, consequently, 0.02 degrees of orthogonality was achieved. The wheel alignment inspection results were determined then verified using a vision system with two cameras. In order to use this vision system, two cameras were used and a new method of modifying the measurement mechanism was developed. According to the verification results, the proposed wheel alignment inspection provided very high measurement accuracy. The wheel alignment inspection mechanism proposed in this study can not only give very reliable results but also provide a cost-efficient method of inspecting the wheel alignment of suspension modules to automakers.

Modularly engineered alginate bioconjugate hydrogel as biocompatible injectable scaffold for in situ biomineralization.

In the present study, a type of bioconjugate was synthesized by post modification of alginate by conjugating temperature-responsive poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-lactide) and O-phosphorylethanolamine as phosphorylation functional groups. Freely flowing bioconjugate sols at low temperature can transform to stable viscoelastic gels at the physiological temperature (37 °C). Subcutaneous administration of temperature-responsive bioconjugate sols into the dorsal region of Sprague-Dawley rats formed in situ hydrogel. in situ formation of bioconjugate gels in stimulated body fluids at 37 °C showed nucleation and hydroxyapatite mineral growth. Furthermore, hydroxyapatite growth was also found in in vivo gels, which suggested the potential of alginate-based bioconjugate gels as a scaffold for bone engineering. Bone morphogenetic protein 2 (BMP-2)-loaded bioconjugate formed stable gel in vivo, and demonstrated sustained release. BMP-2-loaded bioconjugates exhibited in situ biomineralization in vivo. These results imply that the in situ formation of injectable biomimetic materials has potential for bone tissue engineering applications.

Spoligotype Variation of Mycobacterium tuberculosis Strains Prevailing in Korea.

Tuberculosis (TB) is an ongoing global health problem, including in South Korea. To manage TB efficiently, it is necessary to understand the epidemiology, transmission route, and characteristics of prevailing Mycobacterium tuberculosis strains. In this study, we investigated microevolutions over time in the spoligotype patterns of M. tuberculosis isolated from TB patients in Korea. We collected 1,055 clinical M. tuberculosis isolates from 16 provinces in Korea from 1994 to 2006 and analyzed them by spoligotyping. We observed 26 subfamilies, including two large predominant families: a Beijing family (72.7%) and the T family (19.1%). Specifically, the abundance of spoligotype SIT269 from the Beijing-like subfamily significantly increased in the 2000s relative to the 1990s in Korea. This study provides an overview of the M. tuberculosis genotype trends over time in Korea. These data also indicate that we should consider the influence of the newly growing SIT269 subtype identified in the Beijing family.

Degradation-regulated architecture of injectable smart hydrogels enhances humoral immune response and potentiates antitumor activity in human lung carcinoma.

Cancer vaccines that elicit a robust and durable antitumor response show great promise in cancer immunotherapy. Nevertheless, low immunogenicity and weak immune response limit the application of cancer vaccines. To experience next generation cancer vaccines that elicit robust, durable, and anti-tumor T cell response, herein we design injectable smart hydrogels (ISHs) that self-assemble into a cellular microenvironment-like microporous network using a simple hypodermic needle injection, to localize the immune cells and program host cells. ISHs, composed of levodopa- and poly(ε-caprolactone-co-lactide)ester-functionalized hyaluronic acid (HA-PCLA), are loaded with immunomodulatory factor (OVA expressing plasmid, pOVA)-bearing nano-sized polyplexes and granulocyte-macrophage colony-stimulating factor (GM-CSF) as dendritic cell (DC) enhancement factor. Subcutaneous administration of ISHs effectively localized immune cells, and controlled the delivery of immunomodulatory factors to recruit immune cells. The microporous network allowed the recruitment of a substantial number of DCs, which was 6-fold higher than conventional PCLA counterpart. The locally released nano-sized polyplexes effectively internalized to DCs, resulting in the presentation of tumor-specific OVA epitope, and subsequent activation of CD4+ T cells and generation of OVA-specific serum antibody. By the controlled release of nano-sized polyplexes and GM-CSF through a single subcutaneous injection, the ISHs effectively eliminated B16/OVA melanoma tumors in mice. These ISHs can be administered using a minimal invasive technique that could bypass the need for extracorporeal training of cells ex vivo, and provide sustained release of cancer vaccines for immunomodulation. These important findings suggest that ISHs can serve as powerful biomaterials for cancer immunotherapy.

Smart injectable biogels based on hyaluronic acid bioconjugates finely substituted with poly(ß-amino ester urethane) for cancer therapy.

Development of implantable material to control the release of chemotherapeutics in the body is a promising approach to control cancer cell proliferation; however, implantation requires surgical intervention. Herein, we propose the in situ formation of injectable biogels (IBGs) for the programmed delivery of potent chemotherapeutic drugs. IBGs are developed via cohesive molecular assembly of a polysaccharide-polymer network comprised of hyaluronic acid-poly(ß-amino urethane). Biocompatible IBGs could be administered subcutaneously through a hypodermic needle in vivo to subsequently assemble into a microporous network. The hyaluronic acid-shielded network mimics the natural extracellular matrix, avoiding rapid degradation of IBGs, with a soft texture and adhesiveness facilitating integration with dermal tissues after subcutaneous implantation. The natural-mimicking architecture confers the IBG network controlled degradation and bioresorbable properties. Subcutaneous administration of IBGs controlled the delivery of a therapeutic agent in a spatio-temporal manner. Therapeutic agents delivered near the tumors in a sustained manner were effectively infiltrated into the thick solid tumors and provide a durable and enhanced anti-tumor response in the B16/OVA melanoma model in vivo. These results indicate that IBGs could be potential medical interventions for the treatment of cancers.

Modularly engineered injectable hybrid hydrogels based on protein-polymer network as potent immunologic adjuvant in vivo.

Lymphoid organs, which are populated by dendritic cells (DCs), are highly specialized tissues and provide an ideal microenvironment for T-cell priming. However, intramuscular or subcutaneous delivery of vaccine to DCs, a subset of antigen-presenting cells, has failed to stimulate optimal immune response for effective vaccination and need for adjuvants to induce immune response. To address this issue, we developed an in situ-forming injectable hybrid hydrogel that spontaneously assemble into microporous network upon subcutaneous administration, which provide a cellular niche to host immune cells, including DCs. In situ-forming injectable hybrid hydrogelators, composed of protein-polymer conjugates, formed a hydrogel depot at the close proximity to the dermis, resulting in a rapid migration of immune cells to the hydrogel boundary and infiltration to the microporous network. The biocompatibility of the watery microporous network allows recruitment of DCs without a DC enhancement factor, which was significantly higher than that of traditional hydrogel releasing chemoattractants, granulocyte-macrophage colony-stimulating factor. Owing to the sustained degradation of microporous hydrogel network, DNA vaccine release can be sustained, and the recruitment of DCs and their homing to lymph node can be modulated. Furthermore, immunization of a vaccine encoding amyloid-ß fusion proteinbearing microporous network induced a robust antigen-specific immune response in vivo and strong recall immune response was exhibited due to immunogenic memory. These hybrid hydrogels can be administered in a minimally invasive manner using hypodermic needle, bypassing the need for cytokine or DC enhancement factor and provide niche to host immune cells. These findings highlight the potential of hybrid hydrogels that may serve as a simple, yet multifunctional, platform for DNA vaccine delivery to modulate immune response.

Keratinization of Lung Squamous Cell Carcinoma Is Associated with Poor Clinical Outcome.

BACKGROUND: Although the World Health Organization (WHO) classification of lung squamous cell carcinoma (SCC) was revised in 2015, its clinical implications for lung SCC subsets remain unclear. We investigated whether the morphologic characteristics of lung SCC, including keratinization, were associated with clinical parameters and clinical outcome of patients. METHODS: A total of 81 patients who underwent curative surgical resection of diagnosed lung SCC, were enrolled in this study. Attributes such as keratinization, tumor budding, single cell invasion, and nuclear size within the tumor, as well as immunohistochemistry of Bcl-xL and pS6 expressions, were evaluated. RESULTS: The keratinizing and nonkeratinizing subtypes did not differ with respect to age, sex, TNM stage, and morphologic parameters such as nuclear diameter, tumor budding, and single cell invasion at the tumor edge. Most patients with the keratinizing subtype (98.0%) had a history of smoking, whereas the nonkeratinizing group had a relatively higher proportion of never-smokers relative to the keratinizing group (24.0% vs. 2.0%; p=0.008, chi-square test). Expression of pS6 (a surrogate marker of mammalian target of rapamycin complex 1 [mTORC1] signaling that regulates keratinocyte differentiation), and Bcl-xL (a key anti-apoptotic molecule that may inhibit keratinization), did not correlate significantly with the presence of keratinization. Patients with the keratinizing subtype had a significantly shorter overall survival (85.2 months vs. 135.7 months, p=0.010, log-rank test), and a multivariate analysis showed that keratinization was an independent, poor prognostic factor (hazard ratio, 2.389; 95% confidence interval, 1.090-5.233; p=0.030). CONCLUSION: In lung SCC, keratinization is associated with a poor prognosis, and might be associated with smoking.

Birt-Hogg-Dube syndrome prospectively detected by review of chest computed tomography scans.

PURPOSE: Birt-Hogg-Dube syndrome (BHD) is a rare disorder caused by mutations in the gene that encodes folliculin (FLCN) and is inherited in an autosomal dominant manner. BHD is commonly accompanied by fibrofolliculomas, renal tumors, multiple pulmonary cysts, and spontaneous pneumothorax. The aim of this study was to detect BHD prospectively in patients undergoing chest computed tomography (CT) scans and to evaluate further the characteristics of BHD in Korea. METHODS: We prospectively checked and reviewed the chest CT scans obtained for 10,883 patients at Gangnam Severance Hospital, Seoul, Korea, from June 1, 2015 to May 31, 2016. Seventeen patients met the study inclusion criteria and underwent screening for FLCN mutation to confirm BHD. We analyzed the characteristics of the patients confirmed to have BHD and those for a further 6 patients who had previously been described in Korea. RESULTS: Six (0.06%) of the 10,883 patients reviewed were diagnosed with BHD. There was no difference in demographic or clinical features between the patients with BHD (n = 6) and those without BHD (n = 11). Pneumothorax was present in 50% of the patients with BHD but typical skin and renal lesions were absent. The maximum size of the cysts in the BHD group (median 39.4 mm; interquartile range [IQR] 11.4 mm) was significantly larger than that in the non-BHD group (median 15.8 mm; IQR 7.8 mm; P = 0.001). Variable morphology was seen in 100.0% of the cysts in the BHD group but in only 18.2% of the cysts in the non-BHD group (P = 0.002). Nine (95%) of the total of 12 Korean patients with BHD had experienced pneumothorax. Typical skin and renal lesions were present in 20.0% of patients with BHD. CONCLUSIONS: Our findings suggest that BHD can be detected if chest CT scans are read in detail.

Injectable hydrogel-incorporated cancer cell-specific cisplatin releasing nanogels for targeted drug delivery.

Cisplatin (CDDP) is a well-known anticancer agent, and it has been widely used to treat various solid tumors during clinical cancer therapy. Nevertheless, therapeutic applications of CDDP are hampered by its severe side effects. Although CDDP can be encapsulated into nano-scale drug delivery formulations to improve its physicochemical properties, the lack of stability in the formulation and cancer cell-specific targetability have prompted the exploration of novel vectors for the targeted delivery of CDDP. Here, we introduce CDDP-bearing chondroitin sulfate nanogels (CS-nanogels) that are synthesized through a chelating ligand-metal coordination cross-linking reaction, and then incorporated into pH- and temperature-responsive bioresorbable poly(ethylene glycol)-poly(ß-aminoester urethane) (PEG-PAEU) hydrogels for cancer cell-specific delivery of CDDP. The CS-nanogels released from the hydrogels exhibit a pH-dependent release of CDDP. CDDP was released slowly under physiological conditions (pH 7.4), whereas the release of CDDP was triggered under acidic conditions (pH 5.0). Confocal microscopy images demonstrated that fluorescein-5-thiosemicarbazide-labeled CS-nanogels released from the hydrogels selectively bound to the A549 lung carcinoma cell line through the overexpressing CD44 receptor but not to NIH 3T3 cells. An in vitro cytotoxicity test indicated that CS-nanogels released from the hydrogels effectively inhibited the growth of A549 lung carcinoma cells. Subcutaneous injection of CS-nanogel-loaded PEG-PAEU copolymer sols into the dorsal region of Sprague-Dawley rats spontaneously formed a viscoelastic gel without causing noticeable inflammation at the injection site and was found to be bioresorbable in eight weeks. Overall, the injectable hydrogel-incorporated CS-nanogels were demonstrated to be a useful formulation for the targeted delivery of CDDP.

Association of Specific Immunoglobulin E to Staphylococcal Enterotoxin with Airway Hyperresponsiveness in Asthma Patients.

BACKGROUND: Specific immunoglobulin E (IgE) sensitization to staphylococcal enterotoxin (SE) has been recently considered to be related to allergic disease, including asthma. Despite studies on specific IgE (sIgE) to SE and its relationship to asthma diagnosis and severity, the association of sIgE to SE with airway hyperresponsiveness (AHR) remains unclear. METHODS: We enrolled 81 asthma patients admitted to the Severance Hospital in Korea from March 1, 2013, to February 28, 2015 and retrospectively reviewed the electronic medical records of the enrolled subjects. The serum levels of sIgE to SE (A/B) of all subjects was measured using the ImmunoCAP 250 (Phadia) system with SE-sIgE positive defined as >0.10 kU/mL. RESULTS: The SE-sIgE level was not significantly correlated with asthma severity (forced expiratory volume in 1 second [FEV1], FEV1/forced vital capacity, sputum eosinophils, and serum eosinophils), whereas the SE-sIgE level in patients with positive AHR (mean±standard error of the mean, 0.606±0.273 kU/mL) was significantly higher than that in patients with negative AHR (0.062±0.015 kU/mL, p=0.034). In regression analysis, SE sensitization (sIgE to SE ≥0.010 kU/mL) was a significant risk factor for AHR, after adjustment for age, sex, FEV1, and sputum eosinophils (odds ratio, 7.090; 95% confidence interval, 1.180-42.600; p=0.032). Prevalence of SE sensitization was higher in patients with allergic rhinitis and non-atopic asthma patients, as compared to patients without allergic rhinitis and atopic asthma patients, respectively, but without statistical significance. CONCLUSION: SE sensitization is significantly associated with AHR.

Optical activity enhanced by strong inter-molecular coupling in planar chiral metamaterials.

The polarization of light can be rotated in materials with an absence of molecular or structural mirror symmetry. While this rotating ability is normally rather weak in naturally occurring chiral materials, artificial chiral metamaterials have demonstrated extraordinary rotational ability by engineering intra-molecular couplings. However, while in general, chiral metamaterials can exhibit strong rotatory power at or around resonances, they convert linearly polarized waves into elliptically polarized ones. Here, we demonstrate that strong inter-molecular coupling through a small gap between adjacent chiral metamolecules can lead to a broadband enhanced rotating ability with pure rotation of linearly polarized electromagnetic waves. Strong inter-molecular coupling leads to nearly identical behaviour in magnitude, but engenders substantial difference in phase between transmitted left and right-handed waves.

Analysis of species and intra-species associations between the Mycobacterium abscessus complex strains using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST).

PFGE and MLST showed that the strains of M. massiliense hsp65 II-1 were clearly separated from the strains of M. massiliense hsp65 I or II-2 as well as the strains of M. abscessus or M. bolletii; thus, M. massiliense hsp6 5II-1 might represent an additional subspecies of M. massiliense.