Phage therapy combats pandrug-resistant Acinetobacter baumannii infection safely and efficiently.

Phage therapy offers a promising approach to combat the growing threat of antimicrobial resistance. Yet, key questions remain regarding dosage, administration routes, combination therapy, and the causes of therapeutic failure. In this study, we focused on a novel lytic phage, ФAb4B, which specifically targeted the A. baumannii strains with KL160 CPS, including the pan-drug resistant A. baumannii YQ4. ФAb4B exhibited the ability to effectively inhibit biofilm formation and eradicate mature biofilms independently of dosage. Additionally, it demonstrated a wide spectrum of antibiotic-phage synergy and did not show any cytotoxic or hemolytic effects. Continuous phage injections, both intraperitoneally and intravenously over 7 days, showed no acute toxicity in vivo. Importantly, phage therapy significantly improved neutrophil counts, outperforming ciprofloxacin (CIP). However, excessive phage injections suppressed neutrophil levels. The combinatorial treatment of phage-CIP rescued 91% of the mice, a superior outcome compared to phage alone (67%). The efficacy of the combinatorial treatment was independent of phage dosage. Notably, prophylactic administration of the combinatorial regimen provided no protection, but even when combined with a delayed therapeutic regimen, it saved all the mice. Bacterial resistance to the phage was not a contributing factor to treatment failure. Our preclinical study systematically describes the lytic phage's effectiveness in both in vitro and in vivo settings, filling in crucial details about phage treatment against bacteriemia caused by A. baumannii, which will provide a robust foundation for the future of phage therapy.

Engineered endolysin of Klebsiella pneumoniae phage is a potent and broad-spectrum bactericidal agent against "ESKAPEE" pathogens.

The rise of antimicrobial resistance in ESKAPEE pathogens poses significant clinical challenges, especially in polymicrobial infections. Bacteriophage-derived endolysins offer promise in combating this crisis, but face practical hurdles. Our study focuses on engineering endolysins from a Klebsiella pneumoniae phage, fusing them with ApoE23 and COG133 peptides. We assessed the resulting chimeric proteins' bactericidal activity against ESKAPEE pathogens in vitro. ApoE23-Kp84B (CHU-1) reduced over 3 log units of CFU for A. baumannii, E. faecalis, K. pneumoniae within 1 h, while COG133-Kp84B (CHU-2) showed significant efficacy against S. aureus. COG133-L1-Kp84B, with a GS linker insertion in CHU-2, exhibited outstanding bactericidal activity against E. cloacae and P. aeruginosa. Scanning electron microscopy revealed alterations in bacterial morphology after treatment with engineered endolysins. Notably, CHU-1 demonstrated promising anti-biofilm and anti-persister cell activity against A. baumannii and E. faecalis but had limited efficacy in a bacteremia mouse model of their coinfection. Our findings advance the field of endolysin engineering, facilitating the customization of these proteins to target specific bacterial pathogens. This approach holds promise for the development of personalized therapies tailored to combat ESKAPEE infections effectively.

ApoE Mimetic Peptide COG1410 Kills Mycobacterium smegmatis via Directly Interfering ClpC's ATPase Activity.

Antimicrobial peptides (AMPs) hold promise as alternatives to combat bacterial infections, addressing the urgent global threat of antibiotic resistance. COG1410, a synthetic peptide derived from apolipoprotein E, has exhibited potent antimicrobial properties against various bacterial strains, including Mycobacterium smegmatis. However, our study reveals a previously unknown resistance mechanism developed by M. smegmatis against COG1410 involving ClpC. Upon subjecting M. smegmatis to serial passages in the presence of sub-MIC COG1410, resistance emerged. The comparative genomic analysis identified a point mutation in ClpC (S437P), situated within its middle domain, which led to high resistance to COG1410 without compromising bacterial fitness. Complementation of ClpC in mutant restored bacterial sensitivity. In-depth analyses, including transcriptomic profiling and in vitro assays, uncovered that COG1410 interferes with ClpC at both transcriptional and functional levels. COG1410 not only stimulated the ATPase activity of ClpC but also enhanced the proteolytic activity of Clp protease. SPR analysis confirmed that COG1410 directly binds with ClpC. Surprisingly, the identified S437P mutation did not impact their binding affinity. This study sheds light on a unique resistance mechanism against AMPs in mycobacteria, highlighting the pivotal role of ClpC in this process. Unraveling the interplay between COG1410 and ClpC enriches our understanding of AMP-bacterial interactions, offering potential insights for developing innovative strategies to combat antibiotic resistance.

[SSR loci information analysis in transcriptome of Andrographis paniculata].

To study the SSR loci information and develop molecular markers, a total of 43 683 Unigenes in transcriptome of Andrographis paniculata were used to explore SSR. The distribution frequency of SSR and the basic characteristics of repeat motifs were analyzed using MicroSAtellite software, SSR primers were designed by Primer 3.0 software and then validated by PCR. Moreover, the gene function analysis of SSR Unigene was obtained by Blast. The results showed that 14 135 SSR loci were found in the transcriptome of A. paniculata, which distributed in 9 973 Unigenes with a distribution frequency of 32.36%. Di-nucleotide and Tri-nucleotide repeat were the main types, accounted for 75.54% of all SSRs. The repeat motifs of AT/AT and CCG/CGG were the predominant repeat types of Di-nucleotide and Tri-nucleotide, respectively. A total of 4 740 pairs of SSR primers with the potential to produce polymorphism were designed for maker development. Ten pairs of primers in 20 pairs of randomly picked primers produced fragments with expected molecular size. The gene function of Unigenes containing SSR were mostly related to the basic metabolism function of A. paniculata. The SSR markers in transcriptome of A. paniculata show rich type, strong specificity and high potential of polymorphism, which will benefit the candidate gene mining and marker-assisted breeding.

An Ime2-like mitogen-activated protein kinase is involved in cellulase expression in the filamentous fungus Trichoderma reesei.

OBJECTIVES: Eukaryotic mitogen-activated protein kinases (MAPKs) play crucial roles in transducing environmental and developmental signals inside the cell and regulating gene expression, however, the roles of MAPKs remain largely unknown in Trichoderma reesei. RESULTS: T. reesei ime2 (TrIme2) encodes an Ime2-like MAPK in T. reesei. The deletion of the TrIme2 gene led to 90% increase in cellulase activity against filter paper during earlier period time of cellulase induction as well as the extracellular protein production. Compared to the parent strain, the transcriptional levels of the three major cellulase genes cbh1,cbh2, egl1 were increased by about 9 times, 4 times, 2 times, respectively, at 8 h after cellulase induction in the ΔTrIme2 mutant. In addition, the disruption of TrIme2 caused over 50% reduction of the transcript levels of cellulase transcriptional regulators cre1 and xyr1. CONCLUSION: TrIme2 functions in regulation of the expression of cellulase gene in T.reesei, and is a good candidate for genetically engineering of T. reesei for higher cellulase production.

A novel major facilitator transporter TrSTR1 is essential for pentose utilization and involved in xylanase induction in Trichoderma reesei.

Trichoderma reesei (teleomorph Hypocrea jecorina) is an industrially important filamentous fungus for glycoside hydrolases production, with its xylanolytic enzymes widely applied in many areas. However, the molecular mechanisms underlying xylanase expression are still insufficiently understood. In particular, the effect of sugar transporter on the induction of xylanase expression is unclear. In this work, we identified a novel major facilitator transporter TrSTR1 that is capable of transporting xylose by using a xylose utilization system in Saccharomyces cerevisiae. In T. reesei, TrSTR1 is essential for the utilization of d-xylose, l-arabinose, and even their downstream metabolites D-xylitol and L-arabitol. TrSTR1 is also involved in the induction of xylanase expression since both the xylanase activity and extracellular protein concentration in the Tu6△str1 strain were decreased, which further confirmed by a qRT-PCR analysis of the transcript levels of the key transcriptional regulators. Our observations provide new insights into connections between pentose utilization and xylanase production in T. reesei.

Characterization and homologous overexpression of an N-acetylglucosaminidase Nag1 from Trichoderma reesei.

Trichoderma reesei is thought to be a promising recombinant host for the production and secretion of complex proteins due to its ability to secrete large amounts of proteins. In this study we identified a functional N-acetyl-ß-glucosaminidase (NAGase) gene Nag1 in T. reesei. Nag1, a putative gene encoding a GH 20 family NAGase in T. reesei, was cloned and homologous overexpressed in the T. reesei RutC30ΔU3 with a strong cellobiohydrolase1 gene (cbh1) promoter. Nag1 was secreted in its active form and the highest expression level was around 499.85IU/ml. Nag1 has a molecular mass of 80kDa. The optimum pH and temperature were 4.0 and 60°C, respectively.

Comparison of anomalous systemic artery to left lower lobe and pulmonary sequestration in left lower lobe by computed tomography.

BACKGROUND: Differentiation of anomalous systemic artery to the left lower lobe (ASALLL) from pulmonary sequestration (PS) is essential, as ASALLL can be corrected by anastomosis, embolization, or ligation of the anomalous artery. PURPOSE: To compare computed tomography (CT) findings of ASALLL and PS in the left lower lobe (LLL). MATERIALS AND METHODS: This study included 16 patients with ASALLL and 25 patients with PS in LLL confirmed by operative and pathologic findings. RESULTS: Cough and sputum were more common in PS (84% and 60%, respectively) than in ASALLL (25% and 12.5%, respectively) (P < 0.05). Hemoptysis was more common in ASALLL (100%) than in PS (24%) (P < 0.05). The frequency of ground glass opacity (GGO), normal bronchial distribution, dilated left inferior pulmonary veins, and absence of the interlobar artery distal to the origin of the superior segmental artery in LLL differed significantly between ASALLL and PS. Mass was less common in ASALLL (0%) than in PS (88%) (P < 0.01). The mean diameter of the anomalous artery (11.88 ± 1.13 mm) in ASALLL was significantly larger than that (5.96 ± 0.98 mm) in PS (P < 0.01). The presence of anomalous artery arising from thoracic aorta was not different between ASALLL (100%) and PS (72%). CONCLUSION: Radiographic indications of ASALLL differ from those of PS in the LLL. Indications that may suggest ASALLL include an enlarged anomalous systemic artery arising from the thoracic aorta, dilated left inferior pulmonary veins, absence of the interlobar artery distal to the origin of the superior segmental artery, normal bronchial distribution, and GGO in the LLL.

Role of multi-detector computed tomography for biliary complications after liver transplantation.

AIM: To investigate the diagnostic performance of multi-detector computed tomography (MDCT) in detecting biliary complications after orthotopic liver transplantation (OLT). METHODS: Eighty-three consecutive OLT recipients, who presented with clinical or biochemical signs of biliary complications, underwent MDCT examination. Two experienced radiologists assessed MDCT images in consensus to determine biliary complications. Final confirmation was based on percutaneous transhepatic cholangiography or endoscopic retrograde cholangiography in 58 patients, surgery in four patients, liver biopsy in 10, and clinical and sonography follow-up in 11 patients. RESULTS: Biliary complications were eventually confirmed in 62 of 83 patients (74.7%), including anastomotic biliary strictures in 32, nonanastomotic biliary strictures in 21, biliary stones in nine (5 with biliary strictures), anastomotic bile leak in five, and biloma in six (all with nonanastomotic strictures, and 2 with biligenic hepatic abscess). Twenty-one patients had no detection of biliary complications. The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of MDCT for detecting biliary strictures were 90.6%, 86.7%, 89.2%, 92.3% and 83.9%, respectively. For detecting biliary stones, anastomotic bile leak and biloma, the sensitivity, specificity, accuracy, positive predictive value and negative predictive value of MDCT were all 100%. CONCLUSION: MDCT is a useful screening tool for detecting biliary complications after OLT.

[Magnetic resonance imaging findings of lesions in limbic system related structures in general paresis of insane].

OBJECTIVE: To analyze the magnetic resonance imaging (MRI) findings of lesions in the limbic system related structures in general paresis of insane (GPI) patients so as to explore its pathogenesis and provide a new MRI diagnostic method. METHODS: The clinical data and MRI findings of lesions in the limbic system related structures were retrospectively analyzed for a total of 31 GPI patients. The parameters were volume and signal abnormality. RESULTS: On MRI, structural abnormalities were found in amygdaloid body (n = 29), hippocampus (n = 28), insular lobe (n = 24), parahippocampal gyrus (n = 23), lenticular nucleus (n = 23), corpus callosum (n = 20), caudate nucleus (n = 11), hypothalamus (n = 10), anterior nucleus of thalamus (n = 10), cingulate gyrus (n = 8) and thalamus (n = 2). The frequent manifestations were atrophy, swelling, T2 hyperintensity in various structures and T2 hypointensity in lenticular nucleus. CONCLUSION: A frequent involvement of limbic system structures in GPI patients may be related with dementia and other psychiatric symptoms. Swelling and T2 hyperintensity of involved structures may be the characteristic MRI manifestations of GPI.

[Initial application of coronary images from 320-slice dynamic volume MDCT].

OBJECTIVE: To evaluate initial application of coronary images from 320-slice dynamic volume MDCT (Toshiba Aquilion One dynamic volume MDCT). METHODS: 180 consecutive patients with suspected coronary artery disease were studied by ECG-gated 320-slice dynamic volume MDCT with non-helical scanning mode in our hospital from November, 2008 to January, 2009. 14 patients also underwent conventional coronary angiography. Images were retrospectively reconstructed under volume rendering technology, maximum intensity projection, multiplanar reconstruction and curved planar reformation. Image quality was analyzed in axial 320-detector row images through the mid-RCA. Sensitivity, specificity, positive and negative predictive value of > or = 50% stenosis were calculated. RESULTS: 145 (80.56%) cases had excellent images at average heart rate of 63 bpm.25 (13.89%) cases had good images at average heart rate of 78 bpm.8 (4.44%) cases had acceptable images at average heart rate of 85 bpm.2 (1.11%) cases had unevaluable images at average heart rate of 96 bpm. No stair-step artifact was found. 40 segments with > or = 50% stenosis were shown in CTA, while 37 segment in CAG. The sensitivity, specificity, positive and negative predictive value of the 320 slice DMCT in detecting coronary artery stenosis > or = 50% were 94.59%, 94.80%, 87.50% and 98.82% respectively. CONCLUSION: Coronary images from 320-slice dynamic volume MDCT with wide area detector and non-helical scanning mode have excellent quality and less artifacts.320-slice coronary CT angiography provides high sensitivity and specificity in detecting coronary artery stenosis.

[Construction and analysis of a metagenomic library from Tengchong hot spring soil in Yunnan Province].

By a combination of freezing/thawing/ proteinase K-based method and SDS/high-salt/heating treatment, the mixed environmental genomic DNA was isolated directly from a hot spring soil in Tengchong, Yunnan, China. With this method, The DNA yield was up to 1 - 2 microg/g soil. After purification with the Wizard DNA clean up system (Promega, Madison, Wis), the mixed genomic DNA was partially digested with restriction enzyme Pst I. Digested DNA fragments of 3 - 8 kb were recovered from agrose gel and ligated to the pSK (+) vector. The ligation mixture was transformed into DH10B strain, resulting in the construction of a metagenomic library with about 2.5 x 10(4) clones. Restriction enzyme analysis revealed that the average insert is about 4.6 kb. Some novel sequences were identified via sequencing and gene annotation analysis of 30 clones randomly chosen from this library.