Nocardia implantans sp. nov. isolated from a patient with pulmonary infection.

TwoGram-stain-positive and rod-shaped actinomycetes (strains CDC186T and CDC192) were isolated from sputum samples of a patient in Chongqing, PR China, and were investigated to determine their taxonomic status. The results of phylogenetic analysis based on the 16S rRNA gene indicated that CDC186T and CDC192 represented members of the genus Nocardia, and the sequence similarity with Nocardia beijingensis DSM 44636T was the highest, at 99.71 and 99.78 %, respectively. The DNA G+C content of both CDC186T and CDC192 was 69.1 %. Genomic diversity analysis revealed that the average nucleotide identity and in silico DNA‒DNA hybridisation values between the two novel strains and closely related species were significantly below the thresholds of 95-96 and 70 %, respectively, but these values between the two novel strains were 99.96 and 99.90 %, respectively. The phylogenetic relationship based on the dapb1 gene and the single-copy core genes further indicated that the two novel strains were clustered in separate branch adjacent to N. beijingensis DSM 44636T. Growth occurred within the ranges of 20-42 °C, pH 6.0-9.0 and NaCl concentrations of 0.5-4.5 % (w/v). The major fatty acids of CDC186T and CDC192 were C16 : 0 and C18 : 0 10-methyl [tuberculostearic acid (TBSA)]. The predominant respiratory menaquinone was MK-9. The polar lipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannoside, one unidentified glycolipid, one unidentified phospholipid and one unidentified phosphoglycolipid. All the genomes of the studied strains were annotated with virulence factor (VF)-associated genes homologous to those of Mycobacterium tuberculosis, and the results of susceptibility testing indicated that CDC186T and CDC192 were resistant to amoxicillin-clavulanic acid and tigecycline. On the basis of chemotaxonomic characteristics and the results of phylogenetic analyses, strains CDC186T and CDC192 represent a novel species within the genus Nocardia, for which the name Nocardia implantans sp. nov. is proposed. The type strain is CDC186T (=GDMCC 4.206T= JCM 34959T).

Nocardia pulmonis sp. nov., an actinomycete isolated from a patient with pulmonary infection.

A novel bacterial strain, CDC141T, was isolated from sputum samples of a patient with pulmonary infection in Hainan Province, PR China. We performed a polyphasic study to assess the taxonomic position of the new species. Based on the results of 16S rRNA gene sequence analyses, strain CDC141T belonged to the genus Nocardia with the highest sequence similarity to Nocardia nova NBRC 15556T (98.84 %) and Nocardia macrotermitis RB20T (98.54 %). The dapb1 gene sequence-based phylogenetic and phylogenomic trees further showed that the novel strain was clustered in a distinct clade adjacent to Nocardia pseudobrasiliensis DSM 44290T. The DNA G+C content of strain CDC141T was 68.57 mol%. The genomic diversity analysis revealed low average nucleotide identity and in silico DNA‒DNA hybridization values (<84.7 and <28.9 %, respectively) with its closest relative. Growth occurred at 20-40 °C, pH 6.0-9.0 and with NaCl concentrations of 0.5-2.5 % (w/v). The main fatty acids of strain CDC141T were C16 : 0, C18 : 0 10-methyl, TBSA, C16 : 1 ω6c/C16 : 1 ω7c, C18 : 1 ω9c, C18 : 0, C17 : 1 iso I/anteiso B and C17 : 0. The polar lipid profile was dominated by diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, unidentified glycolipids, unidentified phospholipids and unidentified lipids. MK8 (H4ω-cycl) and MK8 (H4) were the major respiratory quinones. These characteristics were consistent with the typical chemotaxonomic properties of members of the genus Nocardia. Based on the results of phenotypic and genetic analyses, strain CDC141T was identified as representing a new species of the genus Nocardia, with the proposed name Nocardia pulmonis sp. nov. (CDC141T=JCM 34955T=GDMCC 4.207T).

Molecular, cellular and neurological consequences of infection by the neglected human pathogen Nocardia.

BACKGROUND: Nocardia is a facultative intracellular pathogen that infects the lungs and brains of immunocompromised patients with consequences that can be fatal. The incidence of such infections is rising, immunocompetent individuals are also being infected, and there is a need to learn more about this neglected bacterial pathogen and the interaction with its human host. RESULTS: We have applied dual RNA-seq to assess the global transcriptome changes that occur simultaneously in Nocardia farcinica (N. farcinica) and infected human epithelial alveolar host cells, and have tested a series of mutants in this in vitro system to identify candidate determinants of virulence. Using a mouse model, we revealed the profiles of inflammation-related factors in the lung after intranasal infection and confirmed that nbtB and nbtS are key virulence genes for Nocardia infection in vivo. Regarding the host response to infection, we found that the expression of many histones was dysregulated during the infection of lung cells, indicating that epigenetic modification might play a crucial role in the host during Nocardia infection. In our mouse model, Nocardia infection led to neurological symptoms and we found that 15 of 22 Nocardia clinical strains tested could cause obvious PD-like symptoms. Further experiments indicated that Nocardia infection could activate microglia and drive M1 microglial polarization, promote iNOS and CXCL-10 production, and cause neuroinflammation in the substantia nigra, all of which may be involved in causing PD-like symptoms. Importantly, the deletion of nbtS in N. farcinica completely attenuated the neurological symptoms. CONCLUSIONS: Our data contribute to an in-depth understanding of the characteristics of both the host and Nocardia during infection and provide valuable clues for future studies of this neglected human pathogen, especially those addressing the underlying causes of infection-related neurological symptoms.

Immediate flap increases patient safety for deep sternal wound infection: A meta-analysis.

Deep sternal wound infection is a severe complication after cardiac surgery. We performed a meta-analysis evaluating the impact of immediate flap and NPWT on mortality and length of hospital stay. The meta-analysis was registered (CRD42022351755). A systematic literature search was conducted from inception to January, 2023, including PubMed, EMBASE, Cochrane Library, ClinicalTrials.gov and EU Clinical Trials Register. The main outcome were in-hospital mortality and late mortality. And additional outcomes were length of stay and ICU stay time. A total of 438 patients (Immediate flap: 229; NPWT: 209) from four studies were included in this study. Immediate flap was associated with lower in-hospital mortality (OR 0.33, 95% CI 0.13-0.81, P = .02) and length of stay (SMD -13.24, 95% CI -20.53 to -5.94, P = .0004). Moreover, pooled analysis demonstrated no significant difference was found in two groups in terms of late mortality (OR 0.64, 95% CI 0.35-1.16, P = .14) and ICU stay time (SMD -1.65, 95% CI -4.13 to 0.83, P = .19). Immediate flap could reduce in-hospital mortality and length of stay for patients with deep sternal wound infection. Flap transplantation as soon as possible may be advised.

rhG-CSF is associated with an increased risk of metastasis in NSCLC patients following postoperative chemotherapy.

BACKGROUND: Recombinant human granulocyte colony-stimulating factor (rhG-CSF) reduces neutropenia events and is widely used in cancer patients receiving chemotherapy. However, the effects of rhG-CSF on distant organ metastasis (DOM) in non-small-cell lung cancer (NSCLC) patients following postoperative chemotherapy are not clear. METHODS: A retrospective cohort study was performed on NSCLC patients who underwent complete surgical resection and postoperative systemic chemotherapy at The First Affiliated Hospital of Nanchang University between 1 January 2012 and 31 December 2017. The effect of rhG-CSF on DOM was assessed with other confounding factors using Cox regression analyses. RESULTS: We identified 307 NSCLC patients who received postoperative systemic chemotherapy (n = 246 in the rhG-CSF group, n = 61 in the No rhG-CSF group). The incidence of DOM in postoperative NSCLC patients with rhG-CSF treatment was observably higher than in patients without rhG-CSF treatment (48.3% vs. 27.9%, p < 0.05). Univariate regression analysis revealed that rhG-CSF and pathological stage were independent risk factors for metastasis-free survival (MFS) (p < 0.05). RhG-CSF users had a higher risk of DOM (adjusted HR: 2.33, 95% CI: 1.31-4.15) than nonusers of rhG-CSF. The association between rhG-CSF and the risk of DOM was significant only in patients presenting with myelosuppression (HR: 3.34, 95% CI: 1.86-6.02) and not in patients without myelosuppression (HR: 0.71, 95% CI: 0.17-2.94, Interaction p-value< 0.01). The risk increased with higher dose density of rhG-CSF compared to rhG-CSF versus no users (p for trend< 0.001). CONCLUSION: These analyses indicate that rhG-CSF use is related to DOM following postoperative chemotherapy in NSCLC.

A CRISPR-based nucleic acid detection platform (CRISPR-CPA): Application for detection of Nocardia farcinica.

AIMS: To establish a CRISPR-based nucleic acid detection platform and apply it to the detection of Nocardia farcinica. METHODS AND RESULTS: A CRISPR-based nucleic acid detection platform, termed CRISPR-CPA (CRISPR/Cas12a combined with PCR amplification), which employed PCR for pre-amplification of target sequences and CRISPR-Cas12a-based detection for decoding of the PCR amplicons, was developed. To demonstrate its feasibility, CRISPR-CPA was applied to the detection of N. farcinica. A pair of PCR primers and a crRNA, which targeting the conservative and specific part of gyrA of N. farcinica reference strain IFM 10152, were designed according to the principle of CRISPR-CPA. The whole detection process of N. farcinica CRISPR-CPA assay, including sample pre-treatment and DNA extraction (~20 min), PCR pre-amplification (60 min), CRISPR-based detection (10 min), can be completed within 90 min. A total of 62 isolates were used to evaluate the specificity of N. farcinica CRISPR-CPA assay. Clinical specimens were employed to determine the feasibility of the method in practical application. The limit of detection of the N. farcinica CRISPR-CPA assay is 1 pg DNA per reaction in pure cultures and 105  CFU/ml in sputum specimens, which is similar with culture but significantly more timesaving. CONCLUSIONS: The N. farcinica CRISPR-CPA assay is an economic and specific method to detect N. farcinica and provides a high-efficiency tool for screening of pathogens especially of some hard-to-culture and slow-growth infectious agents. SIGNIFICANCE AND IMPACT OF THE STUDY: In CRISPR-CPA system, the PCR primers are engineered with a protospacer adjacent motif (PAM) site of Cas12a effector and an additional base A was added at the 5' end of the engineered PCR primer for protecting PAM site, thus the CRISPR-CPA can detect any sequence. Also, we applied CRISPR-CPA to rapidly detect N. farcinica, which is slow-growing bacteria and is firstly detected by a CRISPR-based method.

Decreased Empathy Response to Other's Pain in Parents of Children With Autism Spectrum Disorder.

ABSTRACT: We aimed to investigate pain empathy ability and self-reported empathy among parents of children with autism spectrum disorder (ASD). Twenty-four parents of children with ASD and 26 parents of typically developing children completed the Empathy Quotient (EQ) self-report scale and responded to painful or neutral images during an empathy-for-pain paradigm test. Parents of children with ASD had lower EQ scores, lower accuracy, and longer reaction time (RT) for pain empathy task response (all p < 0.05) compared with controls. There was a negative relationship between cognitive empathy, social skills, total EQ scores, and RT of response in parents of children with ASD. Our findings indicate that self-reported empathy deficits and decreased empathy response to the sight of others' pain in parents of children with ASD are part of a broader autistic phenotype.

Direct detection of Corynebacterium striatum, Corynebacterium propinquum, and Corynebacterium simulans in sputum samples by high-resolution melt curve analysis.

BACKGROUND: Pulmonary infections caused by non-diphtheriae corynebacteria are increasing. However, rapid identification of Corynebacterium species poses a challenge due to the low genetic variation within the genus. METHODS: Three reference strains and 99 clinical isolates were used in this study. A qPCR followed by high-resolution melting (HRM) targeting ssrA was performed to simultaneously identify C. striatum, C. propinquum and C. simulans. To further evaluate this assay's performance, 88 clinical sputum samples were tested by HRM and the detection results were compared with those of the traditional culture method and multiple cross-displacement amplification (MCDA) assay. RESULTS: The melting curve produced by a pair of universal primers generated species-specific HRM curve profiles and could distinguish the three target species from other related bacteria. The limit of detection of HRM assay for DNA from the three purified Corynebacterium species was 100 fg. Compared with the culture method, HRM detected 22 additional positive specimens, representing a 23.9% relative increase in detection rate. The HRM assay had 98.4% (95% confidence interval [CI], 90.5-99.9%) sensitivity and 100% (95% CI, 82.8-100%) specificity. Additionally, 95.5% concordance between HRM and MCDA (κ = 0.89 [95% CI, 0.79-0.99]) was noted. CONCLUSIONS: The HRM assay was a simple, rapid, sensitive, and specific diagnostic tool for detecting C. striatum, C. propinquum, and C. simulans, with the potential to contribute to early diagnosis, epidemiological surveillance, and rapid response to outbreak.

Nfa34810 Facilitates Nocardia farcinica Invasion of Host Cells and Stimulates Tumor Necrosis Factor Alpha Secretion through Activation of the NF-κB and Mitogen-Activated Protein Kinase Pathways via Toll-Like Receptor 4.

The mechanism underlying the pathogenesis of Nocardia is not fully known. The Nfa34810 protein of Nocardia farcinica has been predicted to be a virulence factor. However, relatively little is known regarding the interaction of Nfa34810 with host cells, specifically invasion and innate immune activation. In this study, we aimed to determine the role of recombinant Nfa34810 during infection. We demonstrated that Nfa34810 is an immunodominant protein located in the cell wall. Nfa34810 protein was able to facilitate the uptake and internalization of latex beads coated with Nfa34810 protein into HeLa cells. Furthermore, the deletion of the nfa34810 gene in N. farcinica attenuated the ability of the bacteria to infect both HeLa and A549 cells. Moreover, stimulation with Nfa34810 triggered macrophages to produce tumor necrosis factor alpha (TNF-α), and it also activated mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways by inducing the phosphorylation of ERK1/2, p38, JNK, p65, and AKT in macrophages. Specific inhibitors of ERK1/2, JNK, and NF-κB significantly reduced the expression of TNF-α, which demonstrated that Nfa34810-mediated TNF-α production was dependent upon the activation of these kinases. We further found that neutralizing antibodies against Toll-like receptor 4 (TLR4) significantly inhibited TNF-α secretion. Taken together, our results indicated that Nfa34810 is a virulence factor of N. farcinica and plays an important role during infection. Nfa34810-induced production of TNF-α in macrophages also involves ERK, JNK, and NF-κB via the TLR4 pathway.

Peripheral blood markers predictive of outcome and immune-related adverse events in advanced non-small cell lung cancer treated with PD-1 inhibitors.

BACKGROUND: Selected patients with advanced non-small cell lung cancer (NSCLC) benefit from immunotherapy, especially immune checkpoint inhibitors such as PD-1 (programmed cell death protein 1) inhibitor. Peripheral blood biomarkers would be most convenient to predict treatment outcome and immune-related adverse events (irAEs) in candidate patients. This study explored associations between inflammation-related peripheral blood markers and onset of irAEs and outcome in patients with advanced NSCLC receiving PD-1 inhibitors. METHODS: A retrospective analysis was conducted of 102 patients with advanced NSCLC receiving PD-1 inhibitors from January 2017 to May 2019. Cox regression models were employed to assess the prognostic effect of low/high neutrophil/lymphocyte ratio (NLR), lactate dehydrogenase (LDH), and prognostic nutrition index (PNI) on overall survival (OS) and progression-free survival (PFS). Logistic regression models were used to analyze the correlation between peripheral blood markers and the onset of irAEs. RESULT: NLR < 5, LDH < 240 U/L, or PNI ≥ 45 was favorably associated with significantly better outcomes compared with higher, higher, or lower values, respectively. The multivariate analysis determined that these parameters were independently associated with both better PFS (p = 0.049, 0.046, 0.014, respectively) and longer OS (p = 0.007, 0.031, < 0.001, respectively). Patients with three favorable factors among NLR, LDH, and PNI had better PFS and OS than did those with two, one, or none. PNI and NLR were associated with the onset of irAEs. CONCLUSION: In patients with advanced NSCLC treated with PD-1 inhibitors, pretreatment NLR, LDH, and PNI may be useful predictive markers of clinical outcome and irAEs.

Microbiological profile of distinct virulence of Nocardia cyriacigeorgica strains in vivo and in vitro.

There are significant differences between different Nocardia species regarding geographical distribution, biochemical features, phenotypic characterization, and drug sensitivity. In this study, we explored the differences in virulence and pathogenic mechanisms of two Nocardia cyriacigeorgica strains. We examined the difference in virulence between N. cyriacigeorgica ATCC14759 and N. cyriacigeorgica GUH-2 by measuring cytotoxicity, animal survival after infection, the ability of host cell invasion, and viability in host cells. Western blotting was used to compare the differences in activation of MAPKs, including p38, ERK, and JNK, the NF-κB signaling pathway, and the PI3K/Akt signaling pathway in A549 and RAW264.7 cells. We measured the difference in stimulatory effects on production of the cytokines IL-6, IL-10, and TNF-α by ELISA. We found that N. cyriacigeorgica ATCC14759 causes higher cytotoxicity in cultured cells and higher lethality in mice, and exhibits superior invasion ability and viability in host cells compared with N. cyriacigeorgica GUH-2. Moreover, these two strains show marked differences in activation of the expression of cytokines and signaling pathways. N. cyriacigeorgica ATCC14759 is more virulent than N. cyriacigeorgica GUH-2. Furthermore, there is a significant difference in pathogenesis between the two strains. Our results provide a theoretical basis for the prevention and treatment of Nocardia infection.

Whole-Genome Sequencing Reveals a Prolonged and Persistent Intrahospital Transmission of Corynebacterium striatum, an Emerging Multidrug-Resistant Pathogen.

Corynebacterium striatum is an emerging multidrug-resistant (MDR) pathogen that occurs primarily among immunocompromised and chronically ill patients. However, little is known about the genomic diversity of C. striatum, which contributes to its long-term persistence and transmission in hospitals. In this study, a total of 192 C. striatum isolates obtained from 14 September 2017 to 29 March 2018 in a hospital in Beijing, China, were analyzed by antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing was conducted on 91 isolates. Nearly all isolates (96.3%, 183/190) were MDR. The highest resistance rate was observed for ciprofloxacin (99.0%, 190/192), followed by cefotaxime (90.6%, 174/192) and erythromycin (89.1%, 171/192). PFGE separated the 192 isolates into 79 pulsotypes, and differences in core genome single-nucleotide polymorphisms (SNPs) partitioned the 91 isolates sequenced into four clades. Isolates of the same pulsotype were identical or nearly identical at the genome level, with some exceptions. Two dominant subclones, clade 3a, and clade 4a, were responsible for the hospital-wide dissemination. Genomic analysis further revealed nine resistance genes mobilized by eight unique cassettes. PFGE and whole-genome sequencing revealed that the C. striatum isolates studied were the result mainly of predominant clones spreading in the hospital. C. striatum isolates in the hospital progressively acquired resistance to antimicrobial agents, demonstrating that isolates of C. striatum may adapt rapidly through the acquisition and accumulation of resistance genes and thus evolve into dominant and persistent clones. These insights will be useful for the prevention of C. striatum infection in hospitals.

Correction to: Genomic comparisons of Streptococcus suis serotype 9 strains recovered from diseased pigs in Spain and Canada.

In the original publication of this article [1], the author name 'Pengchen Du' in author list should be 'Pengcheng Du'.

Genomic comparisons of Streptococcus suis serotype 9 strains recovered from diseased pigs in Spain and Canada.

Streptococcus suis is one of the most important bacterial pathogens in the porcine industry and also a zoonotic agent. Serotype 9 is becoming one of the most prevalent serotypes within the S. suis population in certain European countries. In the present study, serotype 9 strains isolated from a country where infection due to this serotype is endemic (Spain), were compared to those recovered from Canada, where this serotype is rarely isolated from diseased pigs. For comparison purposes, strains from Brazil and the only strain isolated from a human case, in Thailand, were also incorporated. Firstly, sequence types (STs) were obtained followed by detection of putative virulence factors. Phylogenetic trees were constructed using the non-recombinant single nucleotide polymorphisms from core genomes of tested strains. Most Spanish strains were either ST123 or ST125, whereas Canadian strains were highly heterogeneous. However, the distribution of putative virulence factors was similar in both groups of strains. The fact that ST16 strains harbored more putative virulence genes and shared greater similarity with the genome of human serotype 2 strains suggests that they present a higher zoonotic and virulence potential than those from Canada and Spain. More than 80% of the strains included in this study carried genes associated with resistance to tetracycline, lincosamides and macrolides. Serotype 9 strains may be nearly 400 years old and have evolved in parallel into 2 lineages. The rapid population expansion of dominant lineage 1 occurred within the last 40 years probably due to the rapid development of the porcine industry.

Genotyping and investigating capsular polysaccharide synthesis gene loci of non-serotypeable Streptococcus suis isolated from diseased pigs in Canada.

Streptococcus suis (S. suis) is an important swine pathogen and an emerging zoonotic agent. Most clinical S. suis strains express capsular polysaccharides (CPS), which can be typed by antisera using the coagglutination test. In this study, 79 S. suis strains recovered from diseased pigs in Canada and which could not be typed using antisera were further characterized by capsular gene typing and sequencing. Four patterns of cps locus were observed: (1) fifteen strains were grouped into previously reported serotypes but presented several mutations in their cps loci, when compared to available data from reference strains; (2) seven strains presented a complete deletion of the cps locus, which would result in an inability to synthesize capsule; (3) forty-seven strains were classified in recently described novel cps loci (NCLs); and (4) ten strains carried novel NCLs not previously described. Different virulence gene profiles (based on the presence of mrp, epf, and/or sly) were observed in these non-serotypeable strains. This study provides further insight in understanding the genetic characteristics of cps loci in non-serotypeable S. suis strains recovered from diseased animals. When using a combination of the previously described 35 serotypes and the complete NCL system, the number of untypeable strains recovered from diseased animals in Canada would be significantly reduced.

Novel Capsular Polysaccharide Loci and New Diagnostic Tools for High-Throughput Capsular Gene Typing in Streptococcus suis.

Streptococcus suis is an important pathogen of pigs and may cause serious disease in humans. Serotyping is an important tool for detection and epidemiological studies of S. suis Thirty-three reference serotypes and nine novel cps loci (NCLs) are recognized in S. suis To gain a better understanding of the prevalence and genetic characteristics of NCLs, we investigated the serotype identity of 486 isolates isolated between 2013 and 2015 in China by capsular gene typing methods. Two hundred seventy-six isolates carried NCLs belonging to 16 groups, 8 of which appear to have not been reported previously. These isolates showed autoagglutination, polyagglutination, or nonagglutination with reference antisera and thus were nonserotypeable. Almost all isolates carrying the unknown NCLs were encapsulated, with various capsular thicknesses, indicating that they are most likely novel serotypes. To simultaneously identify the currently recognized 17 NCLs, an 18-plex detection system using the Luminex xTAG universal array technology was developed. Our data also provide valuable genetic information for monitoring the variations within NCLs by investigating the genetic characteristics of different subtypes within NCLs. IMPORTANCE: Nonserotypeable Streptococcus suis isolates have been reported in many studies, and 9 novel cps loci (NCLs) have already been identified in nonserotypeable isolates. Moreover, novel cps loci are continually being found. The main purpose of this study was to investigate the prevalence and characteristics of NCLs in S. suis isolates recovered between 2013 and 2015 in China. This study provides valuable genetic information for monitoring the variations within NCLs. Meanwhile, a fast and cost-effective 18-plex detection system that can simultaneously identify the currently recognized 17 NCLs was developed in this study. This system will serve as a valuable tool for detecting known and identifying additional novel cps loci among nonserotypeable S. suis isolates.

Capture of armA by a novel ISCR element, ISCR28.

ISCR28 is a fully functional and active member of the IS91-like family of insertion sequences. ISCR28 is 1,708-bp long and contains a 1,293-bp long putative open reading frame that codes a transposase. Sixty ISCR28-containing sequences from GenBank generated 27 non-repeat genetic contexts, all of which represented naturally occurring biological events that had occurred in a wide range of gram-negative organisms. Insertion of ISCR28 into target DNA preferred the presence of a 5'-GXXT-3' sequence at its terIS (replication terminator) end. Loss of the first 4 bp of its oriIS (origin of replication) likely caused ISCR28 to be trapped in ISApl1-based transposons or similar structures. Loss of terIS and fusion with a mobile element upstream likely promoted co-transfer of ISCR28 and the downstream resistance genes. ArmA and its downstream intact ISCR28 can be excised from recombinant pKD46 plasmids forming circular intermediates, further elucidating its activity as a transposase.

Roles of negative pressure wound therapy for scar revision.

The purpose of this study is to review the research progress of negative pressure wound therapy (NPWT) for scar revision and discuss the prospects of its further study and application. The domestic and foreign literatures on NPWT for scar revision were reviewed. The mechanism and application were summarized. NPWT improves microcirculation and lymphatic flow and stimulates the growth of granulation tissues in addition to draining secretions and necrotic tissue. As a significant clinical therapy in scar revision, NPWT reduces tension, fixes graft, and improves wound bed. In the field of scar revision, NPWT has been increasingly used as an innovative and constantly improving technology.

An extraction-free one-step CRISPR-assisted detection platform and a potential Streptococcus pneumoniae at-home self-testing kit.

A rapid, accurate, easy-to-use nucleic acid detection technology is essential for disease diagnosis and control. Herein, we improved CRISPR-top (cluster regularly interspaced short palindromic repeats-mediated testing in one-pot) to develop Extraction-free one-step CRISPR-assistant detection (ExCad), a simple, rapid, accurate gene detection tool for unextracted colonies and samples. We established a pretreatment protocol to rapidly liquify sputum samples and release nucleic acids within 10 min. The ExCad results can be visualised by a real-time fluorescence reader or the naked eye under blue light. We developed an ExCad-Sp assay to detect Streptococcus pneumoniae from unextracted strains and specimens, and optimised the assay conditions. Assay feasibility was evaluated using sputum samples from 32 patients, and it achieved 92.9 % (13/14) sensitivity, 100 % (18/18) specificity, 100 % (13/13) positive predictive value, and 94.7 % (18/19) negative predictive value compared with bacteria culture. The ExCad-Sp assay has potential for developing an at-home self-testing kit for S. pneumoniae.

Accurate, Sensitive, and Rapid Detection of Pseudomonas aeruginosa Based on CRISPR/Cas12b with One Fluid-Handling Step.

Pseudomonas aeruginosa is a major bacterial pathogen causing nosocomial infections and accounts for morbidity and mortality among patients with cystic fibrosis. An accurate, sensitive, and rapid method to detect P. aeruginosa is critical for the early control of infection and patient management. In this study, we established a P. aeruginosa clustered regularly interspaced short palindromic repeats testing in one pot (CRISPR-top) assay which detected P. aeruginosa with one fluid-handling step in one tube. The reaction was performed isothermally within 1 h; thus, specific instruments were not required. The optimal reaction conditions of this assay were determined to be a temperature of 55°C; working concentrations of 1 µM for the forward inner primer and backward inner primer, 0.5 µM for the loop forward primer and loop backward primer, and 0.25 µM for the forward outer primer and backward outer primer; as well as a 2 µM concentration single-stranded DNA reporter molecules. In terms of specificity, our assay showed 100% inclusivity and exclusivity among 48 strains, including 15 P. aeruginosa clinical isolates and 33 non-P. aeruginosa strains. The limit of detection of our method was 10 copies per reaction mixture. Forty-six human sputum specimens from patients with respiratory symptoms were tested. Using the results of quantitative real-time PCR as the gold standard, our method showed 85.3% (29/34) sensitivity, 100% (12/12) specificity, a positive predictive value of 100% (29/29), and a negative predictive value of 70.6% (12/17). In summary, the P. aeruginosa CRISPR-top assay developed in the present study is a high-efficiency alternative tool for the accurate and rapid detection of P. aeruginosa, especially in resource-limited settings. IMPORTANCE This study reports a P. aeruginosa CRISPR-top assay which can precisely identify P. aeruginosa using nucleic acids from pure cultures or clinical samples in one pot with one fluid-handling step. The P. aeruginosa CRISPR-top reaction is suitable for on-site testing, and its diagnostic performance can be compared with that of qPCR.