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Sarcoptes scabiei (Acari: Sarcoptidae) causes a common contagious skin disease that affects many mammals. Here, the complete mitochondrial genome of a mite, S. scabiei var. nyctereutis, from Japanese wild raccoon dogs was analyzed. The 13,837bp circular genome contained 13 protein-coding genes, two rRNA genes, and 22 tRNA genes. For the first time, two tRNAs (alanine and tyrosine), that were thought to be absent in scabies mites from other animals, were predicted to have short, non-cloverleaf structures by in silico annotation and detected by RT-PCR, sequencing, and northern analysis. The mitochondrial genome structure of S. scabiei is similar to that of Psoroptes cuniculi and Dermatophagoides farinae. While small and unusual tRNA genes seem to be common among acariform mites, further experimental evidence for their presence is needed. Furthermore, through an analysis of the cox1 gene, we have provided new evidence to confirm the transmission of this mite between different animal hosts.

Acinetobacter baumannii is the main causative pathogen of nosocomial infections that causes severe infections in the lungs. In this study, we analyzed the histopathological characteristics of lung infection with two strains of A. baumannii (ATCC 19606 and the clinical isolate TK1090) and Pseudomonas aeruginosa PAO-1 in C3H/HeN mice to evaluate the virulence of A. baumannii. Survival was evaluated over 14 days. At 1, 2, 5, or 14 days postinfection, mice of C3H/HeN were sacrificed, and histopathological analysis of lung specimens was also performed. Histopathological changes and accumulation of neutrophils and macrophages in the lungs after infection with A. baumannii and P. aeruginosa were analyzed. Following intratracheal inoculation, the lethality of ATCC 19606- and TK1090-infected mice was lower than that of PAO-1-infected mice. However, when mice were inoculated with a sub-lethal dose of A. baumannii, the lung bacterial burden remained in the mice until 14 days post-infection. Additionally, histopathological analysis revealed that macrophages infiltrated the lung foci of ATCC 19606-, TK1090-, and PAO-1-infected mice. Although neutrophils infiltrated the lung foci of ATCC 19606- and TK1090-infected mice, they poorly infiltrated the lung foci of PAO-1-infected mice. Accumulation of these cells in the lung foci of ATCC 19606- and TK1090-infected mice, but not PAO-1-infected mice, was observed for 14 days post-infection. These results suggest that A. baumannii is not completely eliminated despite the infiltration of immune cells in the lungs and that inflammation lasts for prolonged periods in the lungs. Further studies are required to understand the mechanism of A. baumannii infection, and novel drugs and vaccines should be developed to prevent A. baumannii infection.

Colistin is used to treat infectious diseases in humans and livestock; it has also been used as a feed additive for livestock for approximately 50 years. Since the mcr-1 plasmid-mediated colistin resistance gene was discovered in China in 2015, it has been detected worldwide, mainly in livestock. In this study, we investigated the prevalence and characteristics of mcr-mediated colistin-resistant Escherichia coli in livestock and farmers in Japan. We collected fecal samples from 295 healthy livestock (202 cattle and 93 swine) and 62 healthy farmers from 72 livestock farms (58 cattle farms and 14 swine farms) between 2013 and 2015. Twenty-eight mcr-1-harboring E. coli strains were isolated from 25 livestock (six cattle and 19 swine) and three farmers (two cattle farmers and one swine farmer). The prevalence rates of mcr-1-harboring E. coli in livestock and farmers were 8.47 and 4.84%, respectively. Of the 28 strains, the resistance genes of three were transferable via the mcr-1-coding plasmids to E. coli J53 at low frequencies (10−7–10−8). Six strains coharbored mcr-1 with CTX-M β-lactamases (CTX-M-14, CTX-M-27, or CTX-M-156). Of the isolates obtained from livestock and farmers in four farms (farms C, I, N, and P), nine strains had the same genotypical characteristics (sequence types and pulsed-field gel electrophoresis band patterns), plasmid characteristics (incompatibility group and plasmid transferability), and minimum inhibitory concentrations. Thus, the findings suggested that clonal strains could spread among livestock and farmers within farms. To our knowledge, this is the first study to detect clonal relatedness of mcr-1-mediated colistin-resistant E. coli in livestock and farmers. It is suggested that farmers are at a higher risk of acquiring mcr-1-harboring strains, calling for our attention based on the One Health concept.

Scabies is a highly contagious skin disease caused by the mite Sarcoptes scabiei that affects many mammals. However, the sensitivity of traditional tests for scabies diagnosis in humans is less than 50%. To simplify the diagnosis of scabies, methods that are simple, sensitive, specific, and cost-effective are required. We developed an immunodiagnostic test based on S. scabiei var. nyctereutis RNA-seq data collected from Japanese raccoon dogs with sarcoptic mange. Three candidate antigens—a highly expressed hypothetical protein “QR98_0091190,” another mite allergen known as “SMIPP-Cc,” and an abundant “vitellogenin-like protein”—were evaluated by western-blot analysis. A lateral flow immunoassay, using specific antibodies against the vitellogenin-like protein, successfully detected scabies in the skin flakes of S. scabiei-infected raccoon dogs. This assay can potentially diagnose scabies more accurately in wildlife, as well as in humans.

Mast cells serve important roles as sentinels against bacterial infection by secreting mediators stored in granules. Much of their effectiveness depends upon recruiting and/or modulating other immune cells. The location of mast cells implies that they recognize pathogens invading tissues or mucosal tissues. Acinetobacter baumannii is a gram-negative bacterium that is considered an emerging nosocomial pathogen and causes a wide range of infections associated with high morbidity and mortality. To date, the interaction of A . baumannii with mast cells remains unclear. In this study, we demonstrated an interaction between human LAD2 mast cells and A. baumannii in vitro . When LAD2 cells were co-cultured with live A. baumannii or Pseudomonas aeruginosa PAO1 in vitro for 4 h, TNF-α and IL-8 were produced in the culture supernatant. These inflammatory cytokines were not detected in the supernatant after the cells were treated with live bacteria without serum. Gene expression analysis showed that TNF-α and IL-8 mRNA expression increased in A. baumannii- and P. aeruginosa -infected LAD2 cells. Scanning electron microscopy showed that A. baumannii was tightly attached to the surface of LAD2 cells and suggested that A. baumannii may bind to FcγRII (CD32) on LAD2 cells. TNF-α in the culture supernatant from A. baumannii -infected LAD2 cells, showed that PMN activation and migration increased in Boyden chamber assays. These results suggest that mast cells recognize and initiate immune responses toward A . baumannii by releasing the preformed mediator TNF-α to activate effector neutrophils.

Neutrophils play a critical role in the innate immune response. Recently, a new neutrophilic biological defense mechanism, termed neutrophil extracellular traps (NETs), has been attracting attention. Neutrophils have been observed to release both lysosomal enzymes and their nuclear contents, including unfolded chromatin, which together trap and inactivate bacteria. The environment in tissues where neutrophils act is thought to be different from that of the blood serum. In this study, we assessed the effect of serum on NET formation. We found that neutrophils spontaneously form NETs in serum-free cultivation conditions at early times. These NETs functioned properly to trap bacteria. Furthermore, we demonstrated that reactive oxygen species play a critical role in the spontaneous formation of NETs. These results suggest that the serum condition must be considered in studies on neutrophils, including the formation and mechanism of action of NETs.

Carbapenem-resistant Acinetobacter baumannii, which are mainly induced by the production of OXA-type β-lactamases, are among the leading causes of nosocomial infections worldwide. Among the β-lactamase genes, the presence of the OXA-51-like gene carrying the upstream insertion sequence, ISAba1, was found to be one of the most prevalent carbapenem resistance mechanisms utilized by these bacteria. Consequently, it is necessary to develop a rapid detection method for ISAba1-blaOXA-51-like sequence for the timely and appropriate antibiotic treatment of A. baumannii infection. In this study, a loop-mediated isothermal amplification (LAMP) assay was optimized for ISAba1-blaOXA-51-like detection. The LAMP primer set was designed to recognize distinct sequences in the ISAba1-blaOXA-51-like gene and could amplify the gene within 25 min at an isothermal temperature of 60°C. This LAMP assay was able to detect the ISAba1-blaOXA-51-like gene with high specificity; in addition, no cross-reactivity was observed for other types of β-lactamase producers (OXA-23-like, OXA-40-like, OXA-58-like, and IMP-1), as indicated by the absence of false positive or false negative results. The detection limit for this assay was found to be 10(0)CFU per tube which was 100-fold more sensitive than a polymerase chain reaction assay for ISAba1-blaOXA-51-like detection. Furthermore, the LAMP assay provided swift detection of the ISAba1-blaOXA-51-like gene, even directly from clinical specimens. In summary, we have described a new, rapid assay for the detection of the ISAba1-blaOXA-51-like gene from A. baumannii that could be useful in a clinical setting. This method might facilitate epidemiological studies and allow monitoring of the emergence of drug resistant strains.

Klebsiella pneumoniae carbapenemases (KPC), which are associated with resistance to carbapenem, have recently spread worldwide and have become a global concern. It is necessary to detect KPC-producing organisms in clinical settings to be able to control the spread of this resistance. We have developed a loop-mediated isothermal amplification (LAMP) method for rapid detection of KPC producers. LAMP primer sets were designed to recognize the homologous regions of blaKPC-2 to blaKPC-17 and could amplify blaKPC rapidly. The specificity and sensitivity of the primers in the LAMP reactions for blaKPC detection were determined. This LAMP assay was able to specifically detect KPC producers at 68 °C, and no cross-reactivity was observed for other types of β-lactamase (class A, B, C, or D) producers. The detection limit for this assay was found to be 10(0) CFU per tube, in 25 min, which was 10-fold more sensitive than a PCR assay for blaKPC detection. Then, the sensitivity of the LAMP reactions for blaKPC detection in human specimens (sputum samples, urine samples, fecal samples and blood samples) was analyzed; it was observed that the LAMP assay had almost the same sensitivity in these samples as when using purified DNA. The LAMP assay is easy to perform and rapid. It may therefore be routinely applied for detection of KPC producers in the clinical laboratory.

Hospital-acquired infections caused by Acinetobacter baumannii have become problematic because of high rates of drug resistance. A. baumannii is usually harmless, but it may cause infectious diseases in an immunocompromised host. Although neutrophils are the key players of the initial immune response against bacterial infection, their interactions with A. baumannii remain largely unknown. A new biological defense mechanism, termed neutrophil extracellular traps (NETs), has been attracting attention. NETs play a critical role in bacterial killing by bacterial trapping and inactivation. Many pathogenic bacteria have been reported to induce NET formation, while an inhibitory effect on NET formation is rarely reported. In the present study, to assess the inhibition of NET formation by A. baumannii, bacteria and human neutrophils were cocultured in the presence of phorbol 12-myristate 13-acetate (PMA), and NET formation was evaluated. NETs were rarely observed during the coculture despite neutrophil PMA stimulation. Furthermore, A. baumannii prolonged the lifespan of neutrophils by inhibiting NET formation. The inhibition of NET formation by other bacteria was also investigated. The inhibitory effect was only apparent with live A. baumannii cells. Finally, to elucidate the mechanism of this inhibition, neutrophil adhesion was examined. A. baumannii suppressed the adhesion ability of neutrophils, thereby inhibiting PMA-induced NET formation. This suppression of cell adhesion was partly due to suppression of the surface expression of CD11a in neutrophils. The current study constitutes the first report on the inhibition of NET formation by a pathogenic bacterium, A. baumannii, and prolonging the neutrophil lifespan. This novel pathogenicity to inhibit NET formation, thereby escaping host immune responses might contribute to a development of new treatment strategies for A. baumannii infections.

Background: We investigated the effects of two antibiotics, erythromycin and rifampicin, on the immunomodulatory gene expression and cellular function of human polymorphonuclear leukocytes (PMNs). Methods: We used real-time quantitative PCR to examine the expression of immunomodulatory genes. The production of reactive oxygen species (ROS) was determined by fluorescence-activated cell sorting. PMN chemotaxis was analyzed using a KK chemotaxis chamber. Results: Stimulation of PMNs with lipopolysaccharide (LPS) resulted in increases in the mRNA levels of immunomodulatory genes. Rifampicin significantly inhibited the overexpression of TLR2, TLR4, CD14 and IL8Rs. However, erythromycin suppressed only the upregulation of TLR2 and TNFA. Neither antibiotic had an effect on the production of ROS. Rifampicin significantly inhibited PMN chemotaxis, but erythromycin had no effect. Conclusions: Erythromycin and rifampicin may play anti-inflammatory roles by affecting the expression levels of immunomodulatory genes or the chemotaxis of PMNs.

Sarcoptes scabiei is a widespread, highly contagious skin disease that affects many mammals including humans. The biological characteristics of S. scabiei remain unclear. Therefore, the ability to collect adequate amount of mites for studies is required to advance our understanding of the parasite. The present study aimed to find a method to collect an adequate amount of live S. scabiei mites within a short time frame. The cornified layer and fur from an infected raccoon dog were inserted into a 50-ml catheter tip-type syringe. A 1.5-ml microtube was attached at the tip of the syringe to collect the mites, which crawled out from the cornified layer and fur. Four conditions were examined, and the following condition was determined to be the best: the syringe and microtube were shaded by aluminum foil, and the microtube was heated using a pet heater (36 °C). In addition, the effectiveness of this method as an alternative method to diagnose S. scabiei infections in animal was evaluated. S. scabiei live mites were not detected in the raccoon dog samples 24 h after the administration of medication (ivermectin or selamectin). The present study revealed that this technique was useful to collect adequate amounts of live mites, and the mites prefer a heated environment and actively move when using the shaded conditions. In addition, this technique was effective as an alternative diagnostic technique to detect live mites on an animal body.

Neutrophils play a pivotal role in immunity against infection by ingesting and killing invading microbes. Neutrophils isolated from human peripheral blood have been used for a number of studies conducted for evaluation of immunomodulating drugs, cytokines, and microbe products. Human promyelocytic leukemia cells, HL-60, have been extensively studied because they can differentiate into neutrophil-like cells by addition of all-trans retinoic acid or dimethyl sulfoxide. For a system that would always allow experimental use of granulocytic cells in a uniformly activated state, we have established HL-60 cell lines with increased migratory activity by transducing the CXC chemokine receptor 1 (CXCR1) gene. When these cell lines were primed with CXC chemokine ligand 8 (IL-8), a slight increase in reactive oxygen species production induced by phorbol myristate acetate (PMA) or zymosan A stimuli was observed. A significance increase in migratory activity was noticed when the HL-60 cells transduced CXCR1 were stimulated with IL-8 in the Boyden chamber method. The gene-transduced HL-60 cell lines may be used as a substitute for neutrophils in screening the effects of various immunomodulating drugs on the migratory activity induced by IL-8.

The aim of the present study was to assess changes of cell membrane antigens on neutrophils in septic patients. Expression levels of neutrophil membrane antigens were measured employing a FACS calibur flow cytometer with several fluorescence-labeled monoclonal antibodies. Expression levels of the CD14 antigen were higher in patients with sepsis than in healthy individuals. In particular, the expression levels of CD14 increased in patients complicated by septic shock. Expression levels of TLR-4 were higher in patients with sepsis or septic shock than in healthy individuals. Expression levels of CD11b and CD16 were lower in patients with sepsis or septic shock than in healthy individuals and were even lower in those complicated by septic shock. Expression levels of neutrophil membrane antigens in patients with sepsis markedly changed in the acute phase. However, these levels tended to return to those of healthy individuals in the convalescing phase. Analyses of the surface antigens on neutrophils strongly involved in biological defense or tissue injury are informative for understanding the pathology of sepsis and for conducting therapy targeting neutrophils in the future.

Hospital-acquired infections as a result of Acinetobacter baumannii have become problematic because of high rates of drug resistance. Although neutrophils play a critical role in early protection against bacterial infection, their interactions with A. baumannii remain largely unknown. To elucidate the interactions between A. baumannii and human neutrophils, we cocultured these cells and analyzed them by microscopy and flow cytometry. We found that A. baumannii adhered to neutrophils. We next examined neutrophil and A. baumannii infiltration into Matrigel basement membranes by an in vitro transmigration assay. Neutrophils were activated by A. baumannii, and invasion was enhanced. More interestingly, A. baumannii was transported together by infiltrating neutrophils. Furthermore, we observed by live cell imaging that A. baumannii and neutrophils moved together. In addition, A. baumannii-activated neutrophils showed increased IL-8 production. The transport of A. baumannii was suppressed by inhibiting neutrophil infiltration by blocking the effect of IL-8. A. baumannii appears to use neutrophils for transport by activating these cells via IL-8. In this study, we revealed a novel bacterial transport mechanism that A. baumannii exploits human neutrophils by adhering to and inducing IL-8 release for bacterial portage. This mechanism might be a new treatment target.

We here present a new method for the expression and purification of recombinant human stem cell factor (rhSCF(164)) in endotoxin-free ClearColi(®) BL21(DE3) cells harboring codon-optimized Profinity eXact™-tagged hSCF cDNA. Previously, we demonstrated that co-expression with thioredoxin increased the solubility of rhSCF in Escherichia coli BL21(DE3), and addition of l-arginine enhanced chromatography performance by removing the endotoxin-masked surface of rhSCF. Initially, we tried to express rhSCF in an endotoxin-free strain using a thioredoxin co-expression system, which resulted in significantly lower expression, possibly due to the stress imposed by overexpressed thioredoxin or antibiotics susceptibility. Therefore, we developed a new expression system without thioredoxin. External redox coupling was tested using persulfides such as glutathione persulfide or cysteine persulfide for the in vivo-folding of hSCF in the cytoplasm. Persulfides improved the protein solubility by accelerating disulfide-exchange reactions for incorrectdisulfides during folding in E. coli. Furthermore, the persulfides enhanced the expression level, likely due to upregulation of the enzymatic activity of T7 RNA polymerase. The recombinant protein was purified via affinity chromatography followed by cleavage with sodium fluoride, resulting in complete proteolytic removal of the N-terminal tag. The endotoxin-free fusion protein from ClearColi(®) BL21(DE3) could bind to the resin in the standard protocol using sodium phosphate (pH 7.2). Furthermore, purified rhSCF enhanced the proliferation and maturation of the human mast cell line LAD2. Thus, we conclude that use of the protein expression system employing E. coli by disulfide shuffling with persulfide addition could be a very useful method for efficient protein production.

Radioprotective 105 (RP105) is a type I transmembrane protein, which associates with a glycoprotein, MD-1. Monoclonal antibody (mAb)-mediated ligation of RP105/MD-1 robustly activates B cells. RP105/MD-1 is structurally similar to Toll-like receptor 4 (TLR4)/MD-2. B-cell responses to TLR2 and TLR4/MD-2 ligands are impaired in the absence of RP105 or MD-1. In addition to RP105/MD-1, MD-1 alone is secreted. The structure of MD-1 shows that MD-1 has a hydrophobic cavity that directly binds to phospholipids. Little is known, however, about a ligand for MD-1 and the role of MD-1 in vivo To study the role of RP105/MD-1 and MD-1 alone, specific mAbs against MD-1 are needed. Here, we report the establishment and characterization of two anti-MD-1 mAbs (JR2G9, JR7G1). JR2G9 detects soluble MD-1, whereas JR7G1 binds both soluble MD-1 and the cell surface RP105/MD-1 complex. With these mAbs, soluble MD-1 was detected in the serum and urine. The MD-1 concentration was altered by infection, diet and reperfusion injury. Serum MD-1 was rapidly elevated by TLR ligand injection in mice. The quantitative PCR and supernatant-precipitated data indicate that macrophages are one of the sources of serum soluble MD-1. These results suggest that soluble MD-1 is a valuable biomarker for inflammatory diseases.

Stem cell factor (SCF) known as the c-kit ligand is a two disulfide bridge-containing cytokine in the regulation of the development and function of hematopoietic cell lineages and other cells such as mast cells, germ cells, and melanocytes. The secreted soluble form of SCF exists as noncovalently associated homodimer and exerts its activity by signaling through the c-Kit receptor. In this report, we present the high level expression of a soluble recombinant human SCF (rhSCF) in Escherichia coli. A codon-optimized Profinity eXact™-tagged hSCF cDNA was cloned into pET3b vector, and transformed into E. coli BL21(DE3) harboring a bacterial thioredoxin coexpression vector. The recombinant protein was purified via an affinity chromatography processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Although almost none of the soluble fusion protein bound to the resin in standard protocol using 0.1M sodium phosphate buffer (pH 7.2), the use of binding buffer containing 0.5M l-arginine for protein stabilization dramatically enhanced binding to resin and recovery of the protein beyond expectation. Also pretreatment by Triton X-114 for removing endotoxin was effective for affinity chromatography. In chromatography performance, l-arginine was more effective than Triton X-114 treatment. Following Mono Q anion exchange chromatography, the target protein was isolated in high purity. The rhSCF protein specifically enhanced the viability of human myeloid leukemia cell line TF-1 and the proliferation and maturation of human mast cell line LAD2 cell. This novel protocol for the production of rhSCF is a simple, suitable, and efficient method.

Acinetobacter baumannii and Pseudomonas aeruginosa are the same aerobic gram-negative bacillus and are usually harmless but cause infectious diseases in compromised hosts. Neutrophils play a critical role in infective protection against the extracellular growth of bacteria. Recently, a new biological defense mechanism called neutrophil extracellular traps (NETs) has been attracting attention. In present study, we investigated the responsiveness of neutrophils to A. baumannii and P. aeruginosa, focusing on NET formation. Neutrophils were co-cultured with A. baumannii or P. aeruginosa, and then DNA, histone and neutrophil elastase were stained, and the formation of NETs was evaluated. Neutrophils stimulated with A. baumannii had spread, but their shapes was maintained, and the nucleus was observed as clearly as that in non-stimulated neutrophils. However, neutrophils stimulated with P. aeruginosa did not maintain their cellular morphology, and the nucleus was disrupted with DNA, histones, and neutrophil elastase released into the extracellular space. These results suggest that A. baumannii does not induce NET formation, in contrast to P. aeruginosa. In addition, we measured expression of myeloperoxidase (MPO), reactive oxygen species (ROS) and superoxide in neutrophils, and we found that these expression in P. aeruginosa-stimulated neutrophils was stronger than that in A. baumannii-stimulated neutrophils. Furthermore, A. baumannii was not killed by neutrophils, in contrast to P. aeruginosa. In this study, we show that the reactivity of neutrophils and their biological defense mechanism are different between A. baumannii and P. aeruginosa, which is important for understanding the pathogenicity of these bacteria.

We developed a PCR-RFLP assay to detect mutations in the quinolone-resistance determining regions of gyrA and parC associated with fluoroquinolone resistance in Enterobacteriaceae. The assay detected mutations associated with reduced susceptibility to fluoroquinolones and may therefore serve as a specific, rapid, inexpensive, and simple testing alternative.

Neutrophils are among the principal effector cells that protect against infectious agents, in part by producing reactive oxygen species (ROS) via the actions of tumor necrosis factor-α (TNF-α). In this study, we investigated whether HL-60 cells that had been differentiated into neutrophil-like cells by all-trans retinoic acid could be primed with TNF-α similar to human neutrophils. Our results showed that when differentiated HL-60 (dHL-60) cells were primed with TNF-α for 10 min, ROS production induced by zymosan A or phorbol myristate acetate (PMA) was enhanced in a TNF-α-dose-dependent manner. In addition, when dHL-60 cells were stimulated with live tachyzoites of Toxoplasma gondii after TNF-α priming, ROS production was also enhanced. Thus, dHL-60, similar to neutrophils, produced ROS after PMA, zymosan A, or T. gondii stimulation. Furthermore, we examined gene expression in dHL-60 cells after TNF-α treatment. The pro-inflammatory cytokine IL-6 was up-regulated more than 1.6-fold by 0.1 ng/mL TNF-α. Endogenous TNF-α was down-regulated by priming. IL-8 receptors genes were not affected by priming with 0.1 ng/mL or 1 ng/mL TNF-α. Complement receptor (CR) 1 and CR3 gene expression was not affected by TNF-α priming for 10 min. However, when the priming period was extended to 1 h, CR1 and CR3 genes were up-regulated 1.3 and 1.4-fold, respectively. Expression of the cell-surface CR3 (CD11b) was not significantly affected by TNF-α for 15 min but was slightly enhanced after priming for 2 h. These results suggest that dHL-60 cells may be used as a substitute for neutrophils when evaluating the effects of cytokines or immunomodulator agents.