rss_2.0Polish Journal of Microbiology FeedSciendo RSS Feed for Polish Journal of Microbiology Journal of Microbiology Feed Pressure Level and Effects on Bacterial Growth Kinetics in an Wound Model<abstract> <title style='display:none'>Abstract</title> <p>Negative Pressure Wound Therapy (NPWT) has been widely adopted in wound healing strategies due to its multimodal mechanism of action. While NPWT’s positive impression on wound healing is well-established, its effect on bacterial load reduction remains equivocal. This study investigates NPWT’s efficacy in reducing bioburden using an <italic>in vitro</italic> porcine skin model, focusing on the impact of <italic>Staphylococcus aureus</italic> and <italic>Staphylococcus epidermidis</italic>. Custom-made negative pressure chambers were employed to apply varying negative pressures. Porcine skin was cut into 5 × 5 cm squares and three standardized wounds of 6 mm each were created using a biopsy punch. Then, wounds were infected with <italic>S. aureus</italic> and <italic>S. epidermidis</italic> bacterial suspensions diluted 1:10,000 to obtain a final concentration of 1.5 × 10<sup>4</sup> CFU/ml and were placed in negative pressure chambers. After incubation, bacterial counts were expressed as colony-forming units (CFU) per ml. For <italic>S. aureus</italic> at 120 hours, the median CFU, mean area per colony, and total growth area were notably lower at −80 mmHg when compared to −250 mmHg and −50 mmHg, suggesting an optimal negative pressure for the pressure-dependent inhibition of the bacterial proliferation. While analyzing <italic>S. epidermidis</italic> at 120 hours, the response to the negative pressure was similar but less clear, with the minor CFU at −100 mmHg. The influence of intermittent negative pressure on the <italic>S. epidermidis</italic> growth showed notably lower median CFU with the interval therapy every hour compared to the <italic>S. aureus</italic> control group. This study contributes valuable insights into NPWT’s influence on the bacterial load, emphasizing the need for further research to reformulate its role in managing contaminated wounds.</p> <p><fig id="j_pjm-2024-018_fig_001" position="float" fig-type="figure"> <graphic xmlns:xlink="" xlink:href="graphic/j_pjm-2024-018_fig_001.jpg"/> </fig></p> </abstract>ARTICLEtrue Secondary Metabolites from Higher Plants: Potent Drug Candidates for Chikungunya Using Approaches<abstract> <title style='display:none'>Abstract</title> <p>Chikungunya virus (CHIKV) causes a debilitating fever and joint pain, with no specific antiviral treatment available. Halogenated secondary metabolites from plants are a promising new class of drug candidates against chikungunya, with unique properties that make them effective against the virus. Plants produce these compounds to defend themselves against pests and pathogens, and they are effective against a wide range of viruses, including chikungunya. This study investigated the interactions of halogenated secondary metabolites with nsP2pro, a therapeutic target for CHIKV. A library of sixty-six halogenated plant metabolites screened previously for ADME properties was used. Metabolites without violation of Lipinski’s rule were docked with nsP2pro using AutoDock Vina. To find the stability of the pipoxide chlorohydrin-nsP2pro complex, the GROMACS suite was used for MD simulation. The binding free energy of the ligand-protein complex was computed using MMPBSA. Molecular docking studies revealed that halogenated metabolites interact with nsP2pro, suggesting they are possible inhibitors. Pipoxide chlorohydrin showed the greatest affinity to the target. This was further confirmed by the MD simulations, surface accessible area, and MMPBSA studies. Pipoxide chlorohydrin, a halogenated metabolite, was the most potent against nsP2pro in the survey.<fig id="j_pjm-2024-020_fig_001" position="float" fig-type="figure"> <graphic xmlns:xlink="" xlink:href="graphic/j_pjm-2024-020_fig_001.jpg"/> </fig></p> </abstract>ARTICLEtrue Protein 1 (GBP1) Enhances IFN-α Mediated Antiviral Activity against Hepatitis B Virus Infection<abstract> <title style='display:none'>Abstract</title> <p>Interferon-alpha (IFN-α) is a first-line drug for treating chronic hepatitis B (CHB). Guanylate-binding protein 1 (GBP1) is one of the interferon-stimulating factors, which participates in the innate immunity of the host and plays an antiviral and antibacterial role. In this study, we explored how GBP1 is involved in IFN-α antiviral activity against HBV. Before being gathered, HepG2-NTCP and HepG2 2.15 cells were transfected with the wild-type hGBP1 plasmid or si-GBP1, respectively, and followed by stimulation with Peg-IFNα-2b. We systematically explored the role of GBP1 in regulating HBV infection in cell models. Additionally, we also examined GBP1 levels in CHB patients. GBP1 activity increased, and its half-life was prolonged after HBV infection. Overexpression of GBP1 inhibited the production of HBsAg and HBeAg, as well as HBs protein and HBV total RNA levels, whereas silencing of GBP1 inhibited its ability to block viral infections. Interestingly, overexpressing GBP1 co-treatment with Peg-IFNα-2b further increased the antiviral effect of IFN-α, while GBP1 silencing co-treatment with Peg-IFNα-2b partly restored its inhibitory effect on HBV. Mechanistically, GBP1 mediates the anti-HBV response of Peg-IFNα-2b by targeting HBs. Analysis of clinical samples revealed that GBP1 was elevated in CHB patients and increased with Peg-IFNα-2b treatment, while GBP1 showed good stability in the interferon response group. Our study demonstrates that GBP1 inhibits HBV replication and promotes HBsAg clearance. It is possible to achieve antiviral effects through the regulation of IFN-α induced immune responses in response to HBV.</p> </abstract>ARTICLEtrue of a Nucleic Acid Detection Method for Norovirus GII.2 Genotype Based on RT-RPA and CRISPR/Cas12a-LFS<abstract> <title style='display:none'>Abstract</title> <p>To establish a rapid detection method for norovirus GII.2 genotype, this study employed reverse transcription recombinase polymerase amplification (RT-RPA) combined with CRISPR/Cas12a and lateral flow strip (RT-RPA-Cas12a-LFS). Here, the genome of norovirus GII.2 genotype was compared to identify highly conserved sequences, facilitating the design of RT-RPA primers and crRNA specific to the conserved regions of norovirus GII.2. Subsequently, the reaction parameters of RT-RPA were optimized and evaluated using agar-gel electrophoresis and LFS. The results indicate that the conserved sequences of norovirus GII.2 were successfully amplified through RT-RPA at 37°C for 25 minutes. Additionally, CRISPR/Cas12a-mediated cleavage detection was achieved through LFS at 37°C within 10 minutes using the amplification products as templates. Including the isothermal amplification reaction time, the total time is 35 minutes. The established RT-RPA-Cas12a-LFS method demonstrated specific detection of norovirus GII.2, yielding negative results for other viral genomes, and exhibited an excellent detection limit of 10 copies/μl. The RT-RPA-Cas12a-LFS method was further compared with qRT-PCR by analyzing 60 food-contaminated samples. The positive conformity rate was 100%, the negative conformity rate was 95.45%, and the overall conformity rate reached 98.33%. This detection method for norovirus GII.2 genotype is cost-effective, highly sensitive, specific, and easy to operate, offering a promising technical solution for field-based detection of the norovirus GII.2 genotype.</p> </abstract>ARTICLEtrue Usefulness of IgA for the Early Detection of SARS-CoV-2 Infection: Comparison With IgM<abstract> <title style='display:none'>Abstract</title> <p>Serological testing can be a powerful complementary approach to achieve timely diagnosis of severe acute respiratory coronavirus 2 (SARS-CoV-2) infection, along with nucleic acid detection. Immunoglobulin (Ig) A antibodies are less frequently utilized to detect SARS-CoV-2 infection than IgM and IgG antibodies, even though IgA antibodies play an important role in protective immunity against SARS-CoV-2. This review discusses the differences in kinetics and assay performance between IgA and IgM antibodies and the factors influencing antibody responses. It highlights the potential usefulness of analyzing IgA antibodies for the early detection of SARS-CoV-2 infection. The early appearance of IgA and the high sensitivity of IgA-based immunoassays can aid in diagnosing coronavirus disease 2019. However, because of cross-reactivity, it is important to recognize the only moderate specificity of the early detection of SARS-CoV-2 IgA antibodies against spike antigens. Either the analysis of antibodies targeting the nucleocapsid antigen or a combination of antibodies against the nucleocapsid and spike antigens may strengthen the accuracy of serological evaluation.</p> </abstract>ARTICLEtrue Characteristics of the Mycelium and Optimization of the Culture Medium for<abstract> <title style='display:none'>Abstract</title> <p>This study aimed to elucidate the influence of various culture medium components, including carbon sources, nitrogen sources, inorganic salts, suspension agents, and temperature, on the mycelial growth characteristics of <italic>Phallus dongsun</italic>. Employing single-factor experiments and response surface methodology within glass Petri dishes, the research identified that carrot powder, soybean powder, and ZnSO<sub>4</sub> notably enhanced the proliferation of aerial mycelium, significantly augmenting the growth rate of <italic>P. dongsun</italic> mycelium. The resultant mycelium was observed to be dense, robust, and fluffy in texture. In particular, ZnSO<sub>4</sub> markedly accelerated the mycelium growth rate. Furthermore, xanthan gum was found to effectively modulate the medium’s viscosity, ensuring a stable suspension and facilitating nutrient equilibrium. The optimal cultivation temperature was determined to be 25°C, with mycelial growth ceasing below 5°C and mycelium perishing at temperatures exceeding 35°C. The optimal medium composition was established as follows: wheat starch 5 g/l, carrot powder 5 g/l, soybean powder 7.50 g/l, glucose 10 g/l, ZnSO<sub>4</sub> 0.71 g/l, NH<sub>4</sub>Cl 0.68 g/l, xanthan gum 0.5 g/l, and agar 20 g/l. Under these optimized conditions, the mycelium of <italic>P. dongsun</italic> exhibited a rapid growth rate (1.04 ± 0.14 mm/day), characterized by a thick, dense, and well-developed structure. This investigation provides a theoretical foundation for the conservation, strain selection, and breeding of <italic>P. dongsun</italic>.</p> </abstract>ARTICLEtrue of the Class 1 Integrons, Carbapenemase Genes and Biofilm Formation Genes Occurrence in Clinical Isolates<abstract> <title style='display:none'>Abstract</title> <p><italic>Acinetobacter baumannii</italic> is a non-fermentative Gram-negative bacterium that can cause nosocomial infections in critically ill patients. Carbapenem-resistant <italic>A. baumannii</italic> (CRAB) has spread rapidly in clinical settings and has become a key concern. The main objective of this study was to identify the distribution of integrons and biofilm-formation-related virulence genes in CRAB isolates. A total of 269 <italic>A. baumannii</italic> isolates (219 isolates of CRAB and 50 isolates of carbapenem-sensitive <italic>A. baumannii</italic> (CSAB)) were collected. Carbapenemase genes (<italic>bla</italic><sub>KPC</sub>, <italic>bla</italic><sub>VIM</sub>, <italic>bla</italic><sub>IMP</sub>, <italic>bla</italic><sub>NDM</sub>, and <italic>bla</italic><sub>OXA-23-like</sub>) and biofilm-formation-related virulence genes (<italic>abal</italic>, <italic>bfms</italic>, <italic>bap</italic>, and <italic>cusE</italic>) were screened with PCR. Class 1 integron was screened with PCR, and common promoters and gene cassette arrays were determined with restriction pattern analysis combined with primer walking sequencing. Whole-genome sequencing was conducted, and data were analyzed for a <italic>bla</italic><sub>OXA-23-like</sub>-negative isolate. All 219 CRAB isolates were negative for <italic>bla</italic><sub>KPC</sub>, <italic>bla</italic><sub>VIM</sub>, <italic>bla</italic><sub>IMP</sub>, and <italic>bla</italic><sub>NDM</sub>, while <italic>bla</italic><sub>OXA-23-like</sub> was detected in 218 isolates. The detection rates for <italic>abal</italic>, <italic>bfms</italic>, <italic>bap</italic>, and <italic>cusE</italic> in 219 CRAB were 93.15%, 63.93%, 88.13%, and 77.63%, respectively. Class 1 integron was detected in 75 CRAB (34.25%) and in 3 CSAB. The single gene cassette array <italic>aacA4-catB8-aadA1</italic> with relatively strong PcH2 promoter was detected in class 1 integrons. The <italic>bla</italic><sub>OXA-23-like</sub>-negative CRAB isolate was revealed to be a new sequence type (Oxford 3272, Pasteur 2520) carrying <italic>bla</italic><sub>OXA-72</sub>, <italic>bla</italic><sub>OXA-259</sub>, and <italic>bla</italic><sub>ADC-26</sub>. In conclusion, <italic>bla</italic><sub>OXA-23-like</sub> was the main reason for CRAB’s resistance to carbapenems. A new (Oxford 3272, Pasteur 2520) CRAB sequence type carrying the <italic>bla</italic><sub>OXA-72</sub>, <italic>bla</italic><sub>OXA-259</sub>, and <italic>bla</italic><sub>ADC-26</sub> was reported.</p> </abstract>ARTICLEtrue Characteristics of Shiga Toxin-Producing Responsible for Infections in the Polish Pediatric Population<abstract> <title style='display:none'>Abstract</title> <p>Shiga toxin-producing <italic>Escherichia coli</italic> (STEC) are zoonotic pathogens causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) in children and the elderly. Stool samples were collected from 180 children hospitalized in five pediatric centers in Poland in 2018–2022. Direct <italic>stx1</italic>/<italic>stx2</italic> gene detection by PCR in feces and <italic>E. coli</italic> isolates was performed. Antibiotic susceptibility was tested according to EUCAST v.12. Randomly selected isolates were serotyped with O157 antiserum and genotyped by pulsed-field gel electrophoresis (PFGE). A total of 44 <italic>E. coli</italic> isolates were confirmed as STEC by PCR. Among them, 84.4% were positive for <italic>stx2</italic>, and equally 6,8% for only <italic>stx1</italic> and both <italic>stx1</italic> and <italic>stx2</italic> genes. The <italic>stx1</italic> gene was also found in one <italic>Citrobacter freundii</italic> isolate. <italic>E. coli</italic> serotype O157 was present in 97.6% of the isolates. STEC infections most often occurred between June-October with a peak in July and August (51%). The highest, 77.8% of STEC isolates were found in the 1–5 years old group. No extended-spectrum β-lactamases (ESBL) were found. Resistance only to amoxicillin/clavulanic acid (24.4%), piperacillin/tazobactam (3%), cefotaxime (6%), gentamicin (6%), ciprofloxacin (3%), azithromycin (3%), trimethoprim/sulfamethoxazole (24,2%) was detected. PFGE analysis showed 18 PFGE types with no clonal distribution. Eight isolates with A, B, and C PFGE types showed genetic relatedness in the type with no detection of transmission way of distribution. STEC strains pose a serious threat to human health, therefore demographic and epidemiological characteristics are crucial for their surveillance.</p> </abstract>ARTICLEtrue of Azole Resistance Involving 51A and 51B Genes in Clinical Isolates<abstract> <title style='display:none'>Abstract</title> <p>This study aimed to investigate azole resistance mechanisms in <italic>Aspergillus flavus,</italic> which involve <italic>cyp</italic>51A and <italic>cyp</italic>51B genes. Real-time Reverse Transcriptase qPCR method was applied to determine the overexpression of <italic>cyp</italic>51A and <italic>cyp</italic>51B genes for 34 <italic>A. flavus</italic> isolates. PCR sequencing of these two genes was used to detect the presence of gene mutations. Susceptibility test found sensitivity to voriconazole (VOR) in all strains. 14.7% and 8.8% of isolates were resistant to itraconazole (IT) and posaconazole (POS), respectively, with a cross-resistance in 5.8%. For the double resistant isolates (IT/POS), the expression of <italic>cyp</italic>51A was up to 17-fold higher. PCR sequencing showed the presence of 2 mutations in <italic>cyp</italic>51A: a synonymous point mutation (P61P) in eight isolates, which did not affect the structure of CYP51A protein, and another non synonymous mutation (G206L) for only the TN-33 strain (cross IT/POS resistance) causing an amino acid change in the protein sequence. However, we noted in <italic>cyp</italic>51B the presence of the only non-synonymous mutation (L177G) causing a change in amino acids in the protein sequence for the TN-31 strain, which exhibits IT/POS cross-resistance. A short single intron of 67 bp was identified in the <italic>cyp</italic>51A gene, whereas three short introns of 54, 53, and 160 bp were identified in the <italic>cyp</italic>51B gene. According to the models provided by PatchDock software, the presence of non-synonymous mutations did not affect the interaction of CYP51A and CYP51B proteins with antifungals. In our study, the overexpression of the <italic>cyp</italic>51A and <italic>cyp</italic>51B genes is the primary mechanism responsible for resistance in <italic>A. flavus</italic> collection. Nevertheless, other resistance mechanisms can be involved.</p> </abstract>ARTICLEtrue Carboxylates Facilitate the Counting of Yeasts in Sub-High Temperature Daqu<abstract> <title style='display:none'>Abstract</title> <p>Sub-high temperature Daqu, a traditional solid fermenting agent used in Chinese strong-aroma Baijiu production, is abundant in diverse microorganisms, including bacteria, yeasts, molds, and actinomycetes. Among these, yeasts are pivotal for ethanol production and flavor formation. However, counting yeasts in Daqu is challenging due to interference from molds and bacteria. Antibiotics are employed to inhibit bacterial growth, but there is no practical way to suppress molds without affecting the growth of yeasts. In this study, short-chain carboxylates (C1-C6) were added to the culture medium at various pH conditions to investigate their effects on the growth of molds and yeasts. The results demonstrated distinct inhibitory effects of the short-chain carboxylates, depending on both pH and concentration. Several tested short-chain carboxylates effectively suppressed mold growth on agar plates while leaving yeast growth unaffected. This suggests a simple and feasible method for enhancing the efficiency of yeast isolation and counting in Daqu. Such an approach is valuable for studying yeasts in diverse and complex habitats.</p> </abstract>ARTICLEtrue and Characterization of a Small Thermostable Protease from sp. CNXK100<abstract> <title style='display:none'>Abstract</title> <p>Proteases derived from <italic>Streptomyces</italic> demonstrate numerous commendable properties, rendering it extensively applicable in biotechnology and various industrial sectors. This study focused on the purification and characterization of the thermostable protease obtained from <italic>Streptomyces</italic> sp. CNXK100. The purified protease exhibited an estimated molecular weight of 27 kDa, with optimal activity at 75°C and pH 8.0. Notably, the enzyme remained active even without any metal ions and fully active in the presence of Na<sup>+</sup>, K<sup>+</sup>, Mg<sup>2+</sup>, and Cu<sup>2+</sup>metal ions. The kinetic parameters were determined with a <italic>K<sub>M</sub></italic> value of 3.13 mg/ml and a <italic>V<sub>max</sub></italic> value of 3.28 × 10<sup>6</sup> U/mg. Furthermore, the protease has demonstrated notable stability when subjected to a treatment temperature of up to 65°C for 60 minutes, and across a broad pH range extending from 5.0 to 10.0. This protease also demonstrated resilience against a spectrum of harsh conditions, including exposure to organic solvents, surfactants, bleaching agents, and proteolytic enzymes. Additionally, the enzyme maintained its activity following treatment with commercial detergents, accomplishing complete thrombus lysis at a concentration of 2.50 mg/ml within 4 hours. Remarkably, the protease exhibited stability in terms of activity and protein concentration for 70 days at 4°C. These findings underscore the potential industrial applications of the thermostable protease from <italic>Streptomyces</italic> sp. CNXK100.</p> <p><fig id="j_pjm-2024-014_fig_010" position="float" fig-type="figure"> <graphic xmlns:xlink="" xlink:href="graphic/j_pjm-2024-014_fig_010.jpg"/> </fig></p> </abstract>ARTICLEtrue and Electrochemical Analysis of a Facultative Anaerobic Electrogenic Strain sp. SQ-1<abstract> <title style='display:none'>Abstract</title> <p>Electricigens decompose organic matter and convert stored chemical energy into electrical energy through extracellular electron transfer. They are significant biocatalysts for microbial fuel cells with practical applications in green energy generation, effluent treatment, and bioremediation. A facultative anaerobic electrogenic strain SQ-1 is isolated from sludge in a biotechnology factory. The strain SQ-1 is a close relative of <italic>Klebsiella variicola</italic>. Multilayered biofilms form on the surface of a carbon electrode after the isolated bacteria are inoculated into a microbial fuel cell device. This strain produces high current densities of 625 μA cm<sup>–2</sup> by using acetate as the carbon source in a three-electrode configuration. The electricity generation performance is also analyzed in a dual-chamber microbial fuel cell. It reaches a maximum power density of 560 mW m<sup>–2</sup> when the corresponding output voltage is 0.59 V. The facultative strain SQ-1 utilizes hydrous ferric oxide as an electron acceptor to perform extracellular electricigenic respiration in anaerobic conditions. Since facultative strains possess better properties than anaerobic strains, <italic>Klebsiella</italic> sp. SQ-1 may be a promising exoelectrogenic strain for applications in microbial electrochemistry.</p> <p><fig id="j_pjm-2024-013_fig_006" position="float" fig-type="figure"> <graphic xmlns:xlink="" xlink:href="graphic/j_pjm-2024-013_fig_006.jpg"/> </fig></p> </abstract>ARTICLEtrue of RPA-Cas12a-Based Fluorescence Assay for Rapid Detection of Feline Parvovirus<abstract> <title style='display:none'>Abstract</title> <p>Feline parvovirus (FPV) is highly infectious for cats and other Felidae and often causes severe damage to young kittens. In this study, we incorporated recombinase polymerase amplification (RPA) and Cas12a-mediated detection and developed an RPA-Cas12a-based real-time or end-point fluorescence detection method to identify the NS1 gene of FPV. The total time of RPA-Cas12a-based fluorescence assay is approximately 25 min. The assay presented a limit of detection (LOD) of 1 copies/μl (25 copies/per reaction), with no cross-reactivity with several feline pathogens. The clinical performance of the assay was examined using total genomic DNA purified from 60 clinical specimens and then compared to results obtained with qPCR detection of FPV with 93.3% positive predictive agreement and 100% negative predictive agreement. Together, the rapid reaction, cost-effectiveness, and high sensitivity make the RPA-Cas12a-based fluorescence assay a fascinating diagnostic tool that will help minimize infection spread through instant detection of FPV. <fig id="j_pjm-2024-005_fig_001" position="float" fig-type="figure"> <graphic xmlns:xlink="" xlink:href="graphic/j_pjm-2024-005_fig_001.jpg"/> </fig></p> </abstract>ARTICLEtrue Utilization-Associated Food-Grade Selection Markers in Lactic Acid Bacteria and Yeast<abstract> <title style='display:none'>Abstract</title> <p>This comprehensive review explores the development of food-grade selection markers in lactic acid bacteria and yeast; some of their strains are precisely defined as safe microorganisms and are crucial in the food industry. Lactic acid bacteria, known for their ability to ferment carbohydrates into lactic acid, provide essential nutrients and contribute to immune responses. With its strong fermentation capabilities and rich nutritional profile, yeast finds use in various food products. Genetic engineering in these microorganisms has grown rapidly, enabling the expression of enzymes and secondary products for food production. However, the focus is on ensuring safety, necessitating food-grade selection markers. Traditional antibiotic and heavy metal resistance selection markers pose environmental and health risks, prompting the search for safer alternatives. Complementary selection markers, such as sugar utilization markers, offer a promising solution. These markers use carbohydrates as carbon sources for growth and are associated with the natural metabolism of lactic acid bacteria and yeast. This review discusses the use of specific sugars, such as lactose, melibiose, sucrose, D-xylose, glucosamine, and N-acetylglucosamine, as selection markers, highlighting their advantages and limitations. In summary, this review underscores the importance of food-grade selection markers in genetic engineering and offers insights into their applications, benefits, and challenges, providing valuable information for researchers in the field of food microbiology and biotechnology.</p> </abstract>ARTICLEtrue between Clinical Characteristics and Microbiota in Bronchiectasis Patients Based on Metagenomic Next-Generation Sequencing Technology<abstract> <title style='display:none'>Abstract</title> <p>This study aimed to investigate the disparities between metagenomic next-generation sequencing (mNGS) and conventional culture results in patients with bronchiectasis. Additionally, we sought to investigate the correlation between the clinical characteristics of patients and their microbiome profiles. The overarching goal was to enhance the effective management and treatment of bronchiectasis patients, providing a theoretical foundation for healthcare professionals. A retrospective survey was conducted on 67 bronchiectasis patients admitted to The First Hospital of Jiaxing from October 2019 to March 2023. Clinical baseline information, inflammatory indicators, and pathogen detection reports, including mNGS, conventional blood culture, bronchoalveolar lavage fluid (BALF) culture, and sputum culture results, were collected. By comparing the results of mNGS and conventional culture, the differences in pathogen detection rate and pathogen types were explored, and the diagnostic performance of mNGS compared to conventional culture was evaluated. Based on the various pathogens detected by mNGS, the association between clinical characteristics of bronchiectasis patients and mNGS microbiota results was analyzed. The number and types of pathogens detected by mNGS were significantly larger than those detected by conventional culture. The diagnostic efficacy of mNGS was significantly superior to conventional culture for all types of pathogens, particularly in viral detection (<italic>p</italic> &lt; 0.01). Regarding pathogen detection rate, the bacteria with the highest detection rate were <italic>Pseudomonas aeruginosa</italic> (17/58) and <italic>Haemophilus influenzae</italic> (11/58); the fungus with the highest detection rate was <italic>Aspergillus fumigatus</italic> (10/21), and the virus with the highest detection rate was human herpes virus 4 (4/11). Differences were observed between the positive and negative groups for <italic>P. aeruginosa</italic> in terms of common scoring systems for bronchiectasis and whether the main symptom of bronchiectasis manifested as thick sputum (<italic>p</italic> &lt; 0.05). Significant distinctions were also noted between the positive and negative groups for <italic>A. fumigatus</italic> regarding Reiff score, neutrophil percentage, bronchiectasis etiology, and alterations in treatment plans following mNGS results reporting (<italic>p</italic> &lt; 0.05). Notably, 70% of patients with positive <italic>A. fumigatus</italic> infection opted to change their treatment plans. The correlation study between clinical characteristics of bronchiectasis patients and mNGS microbiological results revealed that bacteria, such as <italic>P. aeruginosa</italic>, and fungi, such as <italic>A. fumigatus</italic>, were associated with specific clinical features of patients. This underscored the significance of mNGS in guiding personalized treatment approaches. mNGS could identify multiple pathogens in different types of bronchiectasis samples and was a rapid and effective diagnostic tool for pathogen identification. Its use was recommended for diagnosing the causes of infections in bronchiectasis patients.</p> </abstract>ARTICLEtrue Proteolytic Activity of Clinical Isolate HU1848 Is Associated with Higher Expression<abstract> <title style='display:none'>Abstract</title> <p><italic>Serratia marcescens</italic> is a global opportunistic pathogen. <italic>In vitro</italic> cytotoxicity of this bacterium is mainly related to metalloprotease serralysin (PrtS) activity. Proteolytic capability varies among the different isolates. Here, we characterized protease production and transcriptional regulators at 37°C of two <italic>S. marcescens</italic> isolates from bronchial expectorations, HU1848 and SmUNAM836. As a reference strain the insect pathogen <italic>S. marcescens</italic> Db10 was included. Zymography of supernatant cultures revealed a single (SmUNAM836) or double proteolytic zones (HU1848 and Db10). Mass spectrometry confirmed the identity of PrtS and the serralysin-like protease SlpB from supernatant samples. Elevated proteolytic activity and <italic>prtS</italic> expression were evidenced in the HU1848 strain through azocasein degradation and qRT-PCR, respectively. Evaluation of transcriptional regulators revealed higher <italic>eepR</italic> expression in HU1848, whereas <italic>cpxR</italic> and <italic>hexS</italic> transcriptional levels were similar between studied strains. Higher <italic>eepR</italic> expression in HU1848 was further confirmed through an <italic>in vivo</italic> transcriptional assay. Moreover, two putative CpxR binding motifs were identified within the <italic>eepR</italic> regulatory region. EMSA validated the interaction of CpxR with both motifs. The evaluation of <italic>eepR</italic> transcription in a <italic>cpxR</italic> deletion strain indicated that CpxR negatively regulates <italic>eepR</italic>. Sequence conservation suggests that regulation of <italic>eepR</italic> by CpxR is common along <italic>S. marcescens</italic> species. Overall, our data incorporates CpxR to the complex regulatory mechanisms governing <italic>eepR</italic> expression and associates the increased proteolytic activity of the HU1848 strain with higher <italic>eepR</italic> transcription. Based on the global impact of EepR in secondary metabolites production, our work contributes to understanding virulence factors variances across <italic>S. marcescens</italic> isolates.</p> </abstract>ARTICLEtrue Detection of a Pathogenic among Symptomatic Children in Eastern Kurdistan of Iraq<abstract> <title style='display:none'>Abstract</title> <p><italic>Entamoeba histolytica</italic> infects the large intestine of humans, causing a spectrum of clinical appearances ranging from asymptomatic colonization to severe intestinal and extra-intestinal disease. The parasite is identical microscopically to commensal nonpathogenic amoeba. To detect the pathogenic <italic>Entamoeba</italic> and estimate the precise prevalence of the parasite among the symptomatic pediatric population using molecular techniques. 323 fecal samples were collected from symptomatic children admitted to Sulaimani Pediatric Teaching Hospital, Sulaimaniyah Province, Iraq, from June to October 2021. A structured, validated questionnaire was prepared and used to report participants’ gender, residency, and drinking water source. Then, stool samples were microscopically examined, and the positive samples were submitted to molecular analysis by amplifying the 18s rRNA gene using nested PCR to differentiate <italic>E. histolytica</italic> from other nonpathogenic <italic>Entamoeba</italic>. Finally, gene sequences were done to confirm the species. Microscopically, 58 positive samples represented <italic>Entamoeba</italic> species infection rate of 18% among symptomatic patients. However, only 18 samples were positive for <italic>E. histolytica</italic> based on molecular methods, which accounts for 31% of the positive by microscopy and 5.6% among the 323 symptomatic populations. NCBI, available in their database, gives the gene sequence and accession number. Patients’ sociodemographic data and water sources were directly related to the infection rate. Classical microscopic examination provides a misleading profile about the prevalence of <italic>E. histolytica</italic> in an endemic region that might lead to unnecessary treatments and a lack of appropriate management for patients.</p> </abstract>ARTICLEtrue and Evaluation of a Rapid GII Norovirus Detection Method Based on CRISPR-Cas12a<abstract> <title style='display:none'>Abstract</title> <p>Norovirus is highly infectious and rapidly transmissible and represents a major pathogen of sporadic cases and outbreaks of acute gastroenteritis worldwide, causing a substantial disease burden. Recent years have witnessed a dramatic increase in norovirus outbreaks in China, significantly higher than in previous years, among which GII norovirus is the predominant prevalent strain. Therefore, rapid norovirus diagnosis is critical for clinical treatment and transmission control. Hence, we developed a molecular assay based on RPA combined with the CRISPER-CAS12a technique targeting the conserved region of the GII norovirus genome, the results of which could be displayed by fluorescence curves and immunochromatographic lateral-flow test strips. The reaction only required approximately 50 min, and the results were visible by the naked eye with a sensitivity reaching 10<sup>2</sup> copies/μl. Also, our method does not cross-react with other common pathogens that cause intestinal diarrhea. Furthermore, this assay was easy to perform and inexpensive, which could be widely applied for detecting norovirus in settings including medical institutions at all levels, particularly township health centers in low-resource areas.</p> </abstract>ARTICLEtrue and Growth Promotion Potential of CTXW 7-6-2 against that Causes Tobacco Target Spot Disease<abstract> <title style='display:none'>Abstract</title> <p>Fungal diseases form perforated disease spots in tobacco plants, resulting in a decline in tobacco yield and quality. The present study investigated the antagonistic effect of <italic>Bacillus subtilis</italic> CTXW 7-6-2 against <italic>Rhizoctonia solani</italic>, its ability to promote the growth of tobacco seedlings, and the expression of disease resistance-related genes for efficient and eco-friendly plant disease control. Our results showed that CTXW 7-6-2 had the most vigorous growth after being cultured for 96 h, and its rate of inhibition of <italic>R. solani</italic> growth <italic>in vitro</italic> was 94.02%. The volatile compounds produced by CTXW 7-6-2 inhibited the growth of <italic>R. solani</italic> significantly (by 96.62%). The fungal growthinhibition rate of the <italic>B. subtilis</italic> CTXW 7-6-2 broth obtained after high-temperature and no-high-temperature sterile fermentation was low, at 50.88% and 54.63%, respectively. The lipopeptides extracted from the <italic>B. subtilis</italic> CTXW 7-6-2 fermentation broth showed a 74.88% fungal growth inhibition rate at a concentration of 100 mg/l. Scanning and transmission electron microscopy showed some organelle structural abnormalities, collapse, shrinkage, blurring, and dissolution in the <italic>R. solani</italic> mycelia. In addition, CTXW 7-6-2 increased tobacco seedling growth and improved leaf and root weight compared to the control. After CTXW 7-6-2 inoculation, tobacco leaves showed the upregulation of the <italic>PDF1.2</italic>, <italic>PPO</italic>, and <italic>PAL</italic> genes, which are closely related to target spot disease resistance. In conclusion, <italic>B. subtilis</italic> CTXW 7-6-2 may be an efficient biological control agent in tobacco agriculture and enhance plant growth potential.</p> </abstract>ARTICLEtrue Resistance and the Prevalence of the Panton-Valentine Leukocidin Gene among Clinical Isolates of in Lithuania<abstract> <title style='display:none'>Abstract</title> <p>This study aimed to determine resistance to antimicrobials of <italic>Staphylococcus aureus</italic> strains isolated from clinical specimens in Lithuanian hospitals and to identify the genes conferring resistance and virulence. The study was carried out from June 2019 to September 2021. <italic>S. aureus</italic> strains were isolated from skin, soft tissues, blood, lower respiratory tract, urine and other specimens. Antibiotic susceptibility testing was performed using the disc diffusion method according to EUCAST guidelines. All isolates were analyzed for detection of the <italic>ermA</italic>, <italic>ermC</italic>, <italic>mecA</italic>, <italic>mecC</italic>, <italic>tetK</italic>, <italic>tetM</italic>, and <italic>lukF-PV</italic> genes by multiplex real-time PCR. The 16S rRNA coding sequence was applied as an internal PCR control. Altogether, 745 <italic>S. aureus</italic> strains were analyzed. Antimicrobial susceptibility testing revealed that all isolates were susceptible to rifampin and vancomycin. Of the 745 strains, 94.8% were susceptible to tetracycline, 94.5% to clindamycin, and 88.3% to erythromycin. The lowest susceptibility rate was found for penicillin (25.8%). Six percent of the tested strains were methicillin-resistant <italic>S. aureus</italic> (MRSA). The majority of methicillin-resistant strains were isolated from skin and soft tissues (73.3%), with a smaller portion isolated from blood (17.8%) and respiratory tract (8.9%). The <italic>ermC</italic> gene was detected in 41.1% of erythromycin-resistant <italic>S. aureus</italic> strains, whereas <italic>ermA</italic> was detected in 32.2% of erythromycin-resistant <italic>S. aureus</italic> strains. 69.2% of tetracycline-resistant <italic>S. aureus</italic> strains had <italic>tetK</italic> gene, and 28.2% had <italic>tetM</italic> gene. 7.3% of <italic>S. aureus</italic> isolates harbored <italic>lukF-PV</italic> gene. The frequency of the <italic>pvl</italic> gene detection was significantly higher in MRSA isolates than in methicillin-susceptible <italic>S. aureus</italic> isolates (<italic>p</italic> &lt; 0.0001).</p> </abstract>ARTICLEtrue