Coli DH5α Research Articles

The removal of antibiotic resistance bacteria and genes and inhibition of RP4 plasmid-mediated conjugative transfer by citric acid chelated Fe(II) catalyzing peroxymonosulfate systems: Performance and mechanisms

The excessive and inappropriate usage of antibiotics accelerated the appearance and proliferation of antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs), posing a considerable threat to human health and ecological systems. The objectives of this study were to construct Fe(II) chelated by citric acid (CA) catalyzing peroxymonosulfate (PMS) systems with different dosages for the elimination of ARB (E. coli DH5α) and ARGs (tetA, tetR, and blaTEM-1), and to reveal the mechanisms on ARGs conjugation transfer. The processes still maintained high ARB/ARGs removal rates in various water matrices (tap water, river water, sewage, and farm wastewater). The results demonstrated Fe(II)-CA/PMS system in high dosages ([Fe(Ⅱ)] = 0.10 mM; [CA] = 0.08 mM; [PMS] = 0.40 mM) significantly enhanced the removal of ARB (8-log) and ARGs (5.11-log tetA, 1.01-log tetR, and 0.62-log blaTEM-1) by promoting the production of reactive oxygen species (ROSs), thereby inhibiting conjugative transfer completely. Interestingly, Fe(II)-CA/PMS treatment at low dosages ([Fe(Ⅱ)] = 0.0025 mM; [CA] = 0.002 mM; [PMS] = 0.02 mM) still inhibited the transfer of ARGs (2.26 × 10−4, 0.65-fold), mainly through downregulation of genes related with ROS synthesis, DNA damage repair (SOS response), ATP synthesis, and transfer-related genes, as well as upregulation of globally regulatory genes. These results presented a hopeful strategy for holding back the dissemination of antibiotic resistance in environments.

MOLECULAR CHARACTERIZATION AND PATHOGENICITY TEST ON CITRUS FRUITS BY GREEN MOLD AND BLUE MOLD

Citrus fruits are essential for preventing various health conditions, including diabetes, neurological diseases, and cancer. Among the postharvest diseases affecting citrus fruits, Penicillium digitatum and Penicillium italicum are particularly significant. This study aimed to characterize Penicillium spp. isolated from three citrus varieties: orange (Citrus sinensis), small orange, and Malta. Pathogenicity tests confirmed that these Penicillium isolates were capable of infecting the tested citrus fruits. For molecular characterization, PCR amplification of the D1/D2 domain of 26S rDNA was performed using universal primers, which target the conserved regions of the nucleotide sequence. The PCR products were inserted into the pGEM-T Easy vector and transformed into E. coli Dh5α. The presence of the D1/D2 domain was verified by endonuclease digestion with EcoR1. Sequencing was conducted using the T7 promoter primer, and the resulting DNA sequences were analyzed with the DNAMAN analysis system. Sequence analysis revealed that the D1/D2 region of 26S rDNA from the orange isolate showed 99% similarity with Penicillium sp., while the D1/D2 region from the small orange isolate had 99.84% similarity with P. digitatum strain CBS 112082. The D1/D2 region from the Malta isolate showed 100% similarity with P. digitatum. Multiple sequence alignments among the three Penicillium isolates revealed a 98.81% identity. This study highlights the use of molecular techniques for understanding the pathogenicity of Penicillium spp. in citrus fruits.

Impact of a random TN5 mutation on endoglucanase secretion in ruminal cellulolytic Escherichia coli

ObjectiveMost protein secretion systems are found in gram-negative bacteria, but the mechanism of endoglucanase (BcsZ) secretion in Escherichia coli (E. coli) remains unclear. MethodsIn this study, we used JBZ-DH5α (which overexpresses BcsZ on the E. coli DH5α genome) as the initial strain. A mutant library was created by randomly inserting the TN5 transposon into the genome, and mutants with reduced transparent circles were identified on Congo red plates. The insertion sites of transposons in the genome were determined through whole-genome sequencing. ResultsThe results revealed that the genes rnc, lon, and suhB, which encode RNC-ribonuclease III (RNC), LON-protease (LON), and SuhB-inositol phosphatase (SuhB), respectively, were disrupted. BcsZ secretion decreased in E. coli DH5α when the lon, rnc, or suhB genes were deleted, but the overexpression of these genes restored their secretion levels. ConclusionThese findings suggest that the lon, rnc, and suhB genes play a role in BcsZ secretion in E. coli, potentially enhancing our knowledge of BcsZ secretion and offering a strategy to increase protein secretion in E. coli as a cell factory.

Preliminary Screening of Phytochemicals and Antibacterial Activity of Medicinal Plant Ber (<i>Ziziphus mauritiana</i> Lamark)

The most important genus in the Rhamnaceae family is Ber (Ziziphus mauritiana Lamk). Phytochemicals present in Ziziphus have antioxidant as well as antimicrobial properties. The present study aimed to detect various phytochemicals and evaluate the antibacterial activity in the Z. mauritiana leaf extracts. Leaves from 14 cultivars/germplasm/varieties of Z. mauritiana were used to explore the presence of various phytoconstituents such as alkaloids, flavonoids, cardiac glycosides, phenols, saponins, protein, steroids, tannins, lignins, and reducing sugar. These phytoconstituents have antioxidant, antibacterial, and hepatoprotective activities. The preliminary antimicrobial activity screening of Ziziphus leaf extract against bacterial strains-Bacillus subtilis (Gram-positive), Staphylococcus aureus (Gram-positive), Salmonella abony (Gram-negative), E. coli DH5α (Gram-negative) were performed by using well diffusion method. The results of various phytochemicals like alkaloids, phenolics, flavonoids, tannins, carbohydrates, proteins, lipids, and amino acids were reported in all selected commercial varieties/cultivars/ germplasm. On the other hand, saponins were found completely absent in methanolic solvents of all varieties/cultivars. The aqueous and methanolic solvent leaf extract were tested for antibacterial activity at different concentrations (25mg/ml, 50mg/ml, and 100mg/ml) against the bacterial strains of B. subtilis, S. aureus, S. abony, and E. coli DH5α, respectively. This study showed that the phytochemicals extracted from Z. mauritiana leaves have potential antimicrobial activities.

Enhancing high-efficient cadmium biosorption of Escherichia coli via cell surface displaying metallothionien CUP1

ABSTRACT Cadmium (Cd) is one of the common heavy metal pollutants in soil, which can induce various diseases and pose a serious threat to human health. Metallothioneins (MTs) are well-known for their excellent metal binding ability due to a high content of cysteine, which has great potential for heavy metal chelation. In this study, we used the Escherichia coli (E. coli) surface display system LPP-OmpA to construct a recombinant plasmid pBSD-LCF encoding LPP-OmpA-CUP1-Flag fusion protein. Then we displayed the metallothionein CUP1 from Saccharomyces cerevisiae on E. coli DH5α surface for Cd removing. The feasibility of surface display of metallothionein CUP1 in recombinant E. coli DH5α (pBSD-LCF) by Lpp-OmpA system was proved by flow cytometry and western blot analysis, and the specificity of the fusion protein in the recombinant strain was also verified. The results showed that the Cd2+ resistance capacity of DH5α (pBSD-LCF) was highly enhanced by about 200%. Fourier-transform infrared spectroscopy showed that sulfhydryl and sulfonyl groups were involved in Cd2+ binding to cell surface of DH5α (pBSD-LCF). Meanwhile, Cd removal rate by DH5α (pBSD-LCF) was promoted to 95.2%. Thus, the recombinant strain E. coli DH5α (pBSD-LCF) can effectively chelate environmental metals, and the cell surface expression of metallothionein on E. coli can provide new ideas and directions for heavy metals remediation.

Role of cspA on the Preparation of Escherichia coliCompetent Cells by Calcium Chloride Method.

One of the fundamental techniques in genetic engineering is the creation of Escherichia colicompetent cells using the CaCl2 method. However, little is known about the mechanism of E. coli competence formation. We have previously found that the cspA gene may play an indispensable role in the preparation of E. coli DH5αcompetent cells through multiomics analysis. In the present study, the cellular localization, physicochemical properties, and function of the protein expressed by the cspA gene were analyzed. To investigate the role of the cspA gene in E. coli transformation, cspA-deficient mutant was constructed by red homologous recombination. The growth, transformation efficiency, and cell morphology of the cspA-deficient strain and E. coli were compared. It was found that there were no noticeable differences in growth and morphology between E. coli and the cspA-deficient strain cultured at 37°C, but the mutant exhibited increased transformation efficiencies compared to E. coli DH5α for plasmids pUC19, pET-32a, and p1304, with enhancements of 2.23, 2.24,and 3.46 times, respectively. It was proved that cspA gene is an important negative regulatory gene in the CaCl2 preparation of competent cells.

Quaternary ammonium biocides promote conjugative transfer of antibiotic resistance gene in structure- and species-dependent manner

The linkage between biocides and antibiotic resistance has been widely suggested in laboratories and various environments. However, the action mechanism of biocides on antibiotic resistance genes (ARGs) spread is still unclear. Thus, 6 quaternary ammonium biocides (QACs) with different bonded substituents or alkyl chain lengths were selected to assess their effects on the conjugation transfer of ARGs in this study. Two conjugation models with the same donor (E. coli DH5α (RP4)) into two receptors, E. coli MG1655 and pathogenic S. sonnei SE6-1, were constructed. All QACs were found to significantly promote intra- and inter-genus conjugative transfer of ARGs, and the frequency was highly impacted by their structure and receptors. At the same environmental exposure level (4 × 10-1 mg/L), didecyl dimethyl ammonium chloride (DDAC (C10)) promoted the most frequency of conjugative transfer, while benzathine chloride (BEC) promoted the least. With the same donor, the enhanced frequency of QACs of intra-transfer is higher than inter-transfer. Then, the acquisition mechanisms of two receptors were further determined using biochemical combined with transcriptome analysis. For the recipient E. coli, the promotion of the intragenus conjugative transfer may be associated with increased cell membrane permeability, reactive oxygen species (ROS) production and proton motive force (PMF)-induced enhancement of flagellar motility. Whereas, the increase of cell membrane permeability and decreased flagellar motility due to PMF disruption but encouraged biofilm formation, maybe the main reasons for promoting intergenus conjugative transfer in the recipient S. sonnei. As one pathogenic bacterium, S. sonnei was first found to acquire ARGs by biocide exposure.

Cloning, sequence analysis, and molecular docking of nuclease B from Bacillus paralicheniformis str. PMp/10

Bacterial biofilms cause serious problems in the industrial sector and human healthcare. Nucleases are characterized as biofilm-degrading enzymes. In this work, the extracellular nuclease nucB gene from a new isolate Bacillus paralicheniformis str. PMp/10 was isolated and cloned successfully into the cloning vector pET 29a+, then transformed into E. coli DH5α. The nucB gene has a molecular weight of 429 bp, its sequence was uploaded to the NCBI GenBank database under the accession number OP712506 which is the first report for B. paralicheniformis. The gene encoded a nuclease B enzyme consisting of 142 amino acids with an estimated molecular weight of 13 kDa. The 3D and 2D structure of nuclease B was predicted and the NucB enzyme's secondary structure prediction showed that it has five β-strands and three α-helices. Finally, the Molecular docking interaction between NucB and deoxyribonucleic acid was performed successfully with a high binding affinity (−7.1 kcal/mol).

Removal of antibiotic resistant bacteria by the coupled system of ferrous ion activated peroxymonosulfate (PMS) and sodium percarbonate (SPC): Performance and mechanisms

Antibiotic resistance has turned into a focus of universal public health concern. Wastewater treatment plants are considered as reservoirs of antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs), which can be transmitted to the environment. In this research, an effective method for inactivating Escherichia coli DH5α (E. coli DH5α) carrying resistance genes was developed utilizing dual oxidant system of peroxymonosulfate (PMS) and sodium percarbonate (SPC) in the presence of ferrous ions (Fe(II)). The results indicated that E. coli DH5α could be inactivated 6.36 log under the conditions of 0.50 mM PMS, 0.50 mM SPC and 0.50 mM Fe(II) after 30 min. Besides, the removal of different types of ARB and pollutants could also be achieved by Fe(II)/PMS/SPC system. The quenching experiments and EPR analysis demonstrated that reactive species (•OH, SO4•−, O2•−, 1O2 and CO3•−) involved in the inactivation of E. coli DH5α. Bacterial damage mechanisms were systematically studied in terms of cell structure and morphology, enzyme activity, malondialdehyde and intracellular reactive oxygen species levels. The inactivation of E. coli DH5α in complex water matrix (including coexisting of anions and natural organic matter) and real wastewater was inhibited. The abundance of intracellular ARGs decreased by 1.15 log, whereas extracellular ARGs increased by 0.32 log. This research supplied a prospective approach for inhibiting the dissemination of antibiotic resistance.

Mechanisms on the removal of gram-negative/positive antibiotic resistant bacteria and inhibition of horizontal gene transfer by ferrate coupled with peroxydisulfate or peroxymonosulfate

The existence of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) has been a global public environment and health issue. Due to the different cell structures, gram-positive/negative ARB exhibit various inactivation mechanisms in water disinfection. In this study, a gram-negative ARB Escherichia coli DH5α (E. coli DH5α) was used as a horizontal gene transfer (HGT) donor, while a gram-positive ARB Bacillus as a recipient. To develop an efficient and engineering applicable method in water disinfection, ARB and ARGs removal efficiency of Fe(VI) coupled peroxydisulfate (PDS) or peroxymonosulfate (PMS) was compared, wherein hydroxylamine (HA) was added as a reducing agent. The results indicated that Fe(VI)/PMS/HA showed higher disinfection efficiency than Fe(VI)/PDS/HA. When the concentration of each Fe(VI), PMS, HA was 0.48 mM, 5.15 log E. coli DH5α and 3.57 log Bacillus lost cultivability, while the proportion of recovered cells was 0.0017 % and 0.0566 %, respectively, and HGT was blocked. Intracellular tetA was reduced by 2.49 log. Fe(IV) and/or Fe(V) were proved to be the decisive reactive species. Due to the superiority of low cost as well as high efficiency and practicality, Fe(VI)/PMS/HA has significant application potential in ARB, ARGs removal and HGT inhibition, offering a new insight for wastewater treatment.

Precision isolation and cultivation of single cells by vortex and flat-top laser ejection.

Single-cell isolation stands as a critical step in single-cell studies, and single-cell ejection technology based on laser induced forward transfer technology (LIFT) is considered one of the most promising methods in this regard for its ability of visible isolating single cell from complex samples. In this study, we improve the LIFT technology and introduce optical vortex laser-induced forward transfer (OV-LIFT) and flat-top laser-induced forward transfer (FT-LIFT) by utilizing spatial light modulator (SLM), aiming to enhance the precision of single-cell sorting and the cell's viability after ejection. Experimental results demonstrate that applying vortex and flat-top beams during the sorting and collection process enables precise retrieval of single cells within diameter ranges of 50 μm and 100 μm, respectively. The recovery rates of Saccharomyces cerevisiae and Escherichia coli DH5α single cell ejected by vortex beam are 89 and 78%, by flat-top beam are 85 and 57%. When employing Gaussian beam sorting, the receiving range extends to 400 μm, with cultivation success rates of S. cerevisiae and E. coli DH5α single cell are 48 and 19%, respectively. This marks the first application of different mode beams in the ejection and cultivation of single cells, providing a novel and effective approach for the precise isolation and improving the viability of single cells.

Innovative DIY drinking water disinfection for underserved communities

Waterborne pathogens threaten 2.2 billion people lacking access to safely managed drinking water services, causing over a million annual diarrheal deaths. Individuals without access to chlorine reagents or filtration devices often resort to do-it-yourself (DIY) methods, such as boiling or solar disinfection (SODIS). However, these methods are not simple to implement. In this study, we introduced an innovative and easily implemented disinfection approach. We discovered that immersing aluminum foil in various alkaline solutions produces alkali-treated aluminum foil (ATA foil) that effectively adsorbs Escherichia coli (E. coli), Salmonella, and Acinetobacter through the generated surface aluminum hydroxide. For example, a 25 cm2 ATA foil efficiently captures all 104E. coli DH5α strains in 100 mL water within 30 min. Using a saturated suspension of magnesium hydroxide, a type of fertilizer, as the alkaline solution, the properties of the saturated suspension eliminate the need for measuring reagents or changing solutions, making it easy for anyone to create ATA foil. ATA foils can be conveniently produced within mesh bags and placed in household water containers, reducing the risk of recontamination. Replacing the ATA foil with a foil improves the adsorption efficiency, and re-immersing the used foil in the production suspension restores its adsorption capacity. Consequently, ATA foil is an accessible and user-friendly alternative DIY method for underserved communities. Verification experiments covering variations in the water quality and climate are crucial for validating the efficacy of the foil. Fortunately, the ATA foil, with DIY characteristics similar to those of boiling and SODIS, is well-suited for testing under diverse global conditions, offering a promising solution for addressing waterborne pathogens worldwide.

Efficient peroxymonosulfate activation by iron-cobalt bimetallic biochar for rapid removal of antibiotic resistant bacteria, antibiotic resistance genes, and ampicillin: The coexisting of free-radical and non-radical pathways

As a typical “One Health” issue, antibiotic resistance has been considered as a worldwide threat to public health. And its spread can be restricted by antibiotics degradation, inactivation of antibiotic resistant bacteria (ARB), and removal of antibiotic resistance genes (ARGs). In this study, iron-cobalt bimetallic biochar (Fe-Co@BC) activated peroxymonosulfate (PMS) system was applied to degrade ampicillin (AMP), inactivate ARB (E. coli DH5α), and remove ARGs (plasmid-encoded blaTEM-1). AMP was degraded efficiently (about 99 %) in the Fe-Co@BC/PMS system within 40 min, and the catalyst still showed good stability after four cycles. Similarly, the Fe-Co@BC/PMS system achieved complete inactivation of ARB (about 107 CFU/mL) within 5 min, and the oxidative damages of ARB were further verified in terms of cell viability, cell membrane integrity, and enzyme activity. The removal efficiency of blaTEM-1 was 3.47 log within 60 min, and the catalyst dose, PMS concentration, initial ARB concentration, pH, and inorganic anions had significant effects on blaTEM-1 removal. The coexistence of non-radical (1O2, electron transfer) and free-radical (SO4−, O2−, and OH) pathways in the Fe-Co@BC/PMS system was demonstrated by electron paramagnetic resonance techniques and free radical quenching experiments, with SO4− and 1O2 being the main reactive species. XPS results showed that redox cycles between Co (III)/Co (II) and Fe (III)/Fe (II) were important to the efficient catalytic performance of Fe-Co@BC. Moreover, possible degradation pathway of AMP was proposed according to LC-MS results and density functional theory analysis. The results obtained in this study provide a novel sight on preventing the propagation of antibiotic resistance in aquatic settings.

Expression and purification of truncated recombinant human angiotensin converting enzyme 2 (trhACE2) capable of binding spike protein of SARS-CoV-2

SARS-CoV-2 infects the host through the interaction be-tween spike protein and the ACE2 (Angiotensin convert-ing enzyme 2) receptor on the host cell surface. There-fore, ACE2 is considered as a key target in drug devel-opment against COVID-19. In this study, we generated the truncated recombinant human ACE2 (trhACE2) expressed on Escherichia coli and evaluated its binding activity to spike protein of SARS-CoV-2. The sequence of ACE2 (18-119 aa) was cloned into pET28a(+) plasmid and transformed into E. coli DH5α strain and finally was transferred to E. coli BL21 (DE3) cells for protein expression. Protein trhACE2 was purified by affinity chromatography using a HisTrap HP column. Protein purity was above 95% and production yield was 75 mg/l. The purified protein was refolded by dialysis method. trhACE2 was evaluated for its ability to bind the spike protein by sandwich ELISA assay and surface plasmon resonance (SPR). The results showed that trhACE2 has the ability to bind spike protein of SARS-CoV-2 with the equilibrium dissociation constant Kd=15.7 nM. With a relatively simple and inexpensive expression and purifi-cation process on E. coli system, there is the potential to develop trhACE2 for preventive and supportive thera-pies against COVID-19.

Induction of proteome changes involved in the cloning of mcr-1 and mcr-2 genes in Escherichia coli DH5-α strain to evaluate colistin resistance

Plasmid genes, termed mobile colistin resistance-1 (mcr-1) and mobile colistin resistance-2 (mcr-2), are associated with resistance to colistin in Escherichia coli (E. coli). These mcr genes result in a range of protein modifications contributing to colistin resistance. This study aims to discern the proteomic characteristics of E. coli-carrying mcr-1 and mcr-2 genes. Furthermore, it evaluates the expression levels of various proteins under different conditions (with and without colistin). Plasmid extraction was performed using an alkaline lysis-based plasmid extraction kit, whereas polymerase chain reaction was used to detect the presence of mcr-1 and mcr-2 plasmids. The E. coli DH5α strain served as the competent cell for accepting and transforming mcr-1 and mcr-2 plasmids. We assessed proteomic alterations in the E. coli DH5α strain both with and without colistin in the growth medium. Proteomic data were analysed using mass spectrometry. The findings revealed significant protein changes in the E. coli DH5α strain following cloning of mcr-1 and mcr-2 plasmids. Of the 20 proteins in the DH5α strain, expression in 8 was suppressed following transformation. In the presence of colistin in the culture medium, 39 new proteins were expressed following transformation with mcr-1 and mcr-2 plasmids. The proteins with altered expression play various roles. The results of this study highlight numerous protein alterations in E. coli resulting from mcr-1 and mcr-2-mediated resistance to colistin. This understanding can shed light on the resistance mechanism. Additionally, the proteomic variations observed in the presence and absence of colistin might indicate potential adverse effects of indiscriminate antibiotic exposure on treatment efficacy and heightened pathogenicity of microorganisms.

Cloning and heterologous expression of Fusarium oxysporum nitrilase gene in Escherichia coli and evaluation in cyanide degradation

Cyanide is widely utilized in the extraction of precious metal extraction even though it has been deemed as the most toxic compound. Fusarium oxysporum has been shown to degrade cyanide through the activity of the Nitrilase enzyme. In this study, the coding sequence of nitrilase gene from F. oxysporum genomic DNA was optimized for cloning and expression in E. coli. The pUC57 containing synthetic optimized nitrilase gene was transferred into E. coli DH5α strain. This nitrilase gene was sub-cloned into pET26b (+) expression vector containing an in-built His-tag at the C-terminal end to facilitate its purification. The recombinant plasmid, pETAM1, was confirmed by PCR, digestion pattern, and sequencing. The recombinant protein was overproduced in E. coli BL21 (DE3). The results of the SDS-PAGE pattern and Western blot analysis confirmed the expression of the expected recombinant protein. For expression optimization of Nitrilase protein, M16 orthogonal experimental design of the Taguchi method was used. The effect of induction time, temperature and IPTG concentration were examined using four levels for each factors. Estimation of the amount of the expressed protein was calculated via densitometry on SDS-PAGE. The enzyme activity and expression in E. coli proved to be successful since there was ammonia production when potassium cyanide and acrylonitrile were used as substrates while the highest enzyme activity of 88% was expressed at 30 °C. The Km and Vm values of the expressed Nitrilase enzyme were determined to be 0.68 mM and 0.48 mM/min respectively.

Effect of Glucose and Methylene Blue in Microbial Fuel Cells Using E. coli

Microbial fuel cells could be used as an alternative for wastewater treatment and electricity generation. Escherichia coli is a representative bacterium that has been widely studied as a model in laboratory assays despite its limited ability to transfer electrons. Although previous studies have employed glucose and methylene blue in electricity production using E. coli, there remains a lack of understanding on how current generation would impact the production of metabolites and what the most appropriate conditions for current production might be. To shed light on those issues, this manuscript used a 32 factorial design to evaluate the effect of the concentration of organic matter (glucose) and the concentration of the mediator methylene blue (MB) using E. coli DH5α as an anodic microorganism. It was found that as the concentration of glucose was increased, the production of electricity increased and at the same time, its degradation percentage decreased. Similarly, a 17-fold increase in current production was observed with an elevation in methylene blue concentration from 0 to 0.3 mM, though inhibition became apparent at higher concentrations. The maximum power generated by the cell was 204.5 µW m−2, achieving a current density of 1.434 mA m−2 at concentrations of 5 g L−1 of glucose and 0.3 mM of MB. Reductions in the production of ethanol, lactate, and acetate were observed due to the deviation of electrons to the anode.

Human pathogenic bacteria on high-touch dry surfaces can be controlled by warming to human-skin temperature under moderate humidity.

Healthcare-associated infections have become a major health issue worldwide. One route of transmission of pathogenic bacteria is through contact with "high-touch" dry surfaces, such as handrails. Regular cleaning of surfaces with disinfectant chemicals is insufficient against pathogenic bacteria and alternative control methods are therefore required. We previously showed that warming to human-skin temperature affected the survival of pathogenic bacteria on dry surfaces, but humidity was not considered in that study. Here, we investigated environmental factors that affect the number of live bacteria on dry surfaces in hospitals by principal component analysis of previously-collected data (n = 576, for CFU counts), and experimentally verified the effect of warming to human-skin temperature on the survival of pathogenic bacteria on dry surfaces under humidity control. The results revealed that PCA divided hospital dry surfaces into four groups (Group 1~4) and hospital dry surfaces at low temperature and low humidity (Group 3) had much higher bacterial counts as compared to the others (Group 1 and 4) (p<0.05). Experimentally, warming to human-skin temperature (37°C with 90% humidity) for 18~72h significantly suppressed the survival of pathogenic bacteria on dry surfaces, such as plastic surfaces [p<0.05 vs. 15°C (Escherichia coli DH5α, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and blaNDM-5 E. coli)] or handrails [p<0.05 vs. 15~25°C (E. coli DH5α, S. aureus, P. aeruginosa, A. baumannii)], under moderate 55% humidity. Furthermore, intermittent heating to human-skin temperature reduced the survival of spore-forming bacteria (Bacillus subtilis) (p<0.01 vs. continuous heating to human-skin temperature). NhaA, an Na+/H+ antiporter, was found to regulate the survival of bacteria on dry surfaces, and the inhibitor 2-aminoperimidine enhanced the effect of warming at human-skin temperature on the survival of pathogenic bacteria (E. coli DH5α, S. aureus, A. baumannii) on dry surfaces. Thus, warming to human-skin temperature under moderate humidity is a useful method for impairing live pathogenic bacteria on high-touch surfaces, thereby helping to prevent the spread of healthcare-associated infections.

Modifying Escherichia coli to mimic Shigella for medical microbiology laboratory teaching: a new strategy to improve biosafety in class.

Laboratory teaching of medical microbiology involves highly pathogenic microorganisms, thus posing potential biosafety risks to the students and the teacher. To address these risks, non/low-pathogenic microorganisms were modified to mimic highly pathogenic ones or highly pathogenic microorganisms were attenuated directly using the CRISPR/Cas9 technology. This study describes the modification of Escherichia coli DH5α to mimic Shigella and its evaluation as a safe alternative for medical laboratory teaching. To generate E. coli DH5α△FliC△tnaA2a, the tnaA and FliC genes in E. coli DH5α were knocked out using CRISPR/Cas9 technology; a plasmid bearing the O-antigen determinant of S. flexneri 2a was then constructed and transformed. Acid tolerance assays and guinea pig eye tests were used to assess the viability and pathogenicity, respectively. Questionnaires were used to analyze teaching effectiveness and the opinions of teachers and students. The survey revealed that most teachers and students were inclined towards real-time laboratory classes than virtual classes or observation of plastic specimens. However, many students did not abide by the safety regulations, and most encountered potential biosafety hazards in the laboratory. E. coli DH5α△FliC△tnaA2a was biochemically and antigenically analogous to S. flexneri 2a and had lower resistance to acid than E. coli. There was no toxicity observed in guinea pigs. Most of teachers and students were unable to distinguish E. coli DH5α△FliC△tnaA2a from pure S. flexneri 2a in class. Students who used E. coli DH5α△FliC△tnaA2a in their practice had similar performance in simulated examinations compared to students who used real S. flexneri 2a, but significantly higher than the virtual experimental group. This approach can be applied to other high-risk pathogenic microorganisms to reduce the potential biosafety risks in medical laboratory-based teaching and provide a new strategy for the development of experimental materials.

Разработка и оценка эффективности ранозаживляющих соединений на основе катионных пептидов и фуллерена

Skin and soft tissue infections following surgical procedures are usually caused by a broad range of bacteria and are the major cause of septic complications and hospital mortality. Treatment of such wounds is a challenge often resulting from the transition from acute to chronic inflammation due to persistence of pathogenic microflora in the wound tissue. The study was aimed to assess the wound-healing activity of the ointment composition based on the dispersion of fullerene C60 (AFD) in the in vivo model of skin wound, to estimate the effects of AFD on the expression of cytokines as markers of regenerative processes, to determine antibacterial activity of the developed cationic peptides. AFD was obtained by tangential ultrafiltration and used to make an ointment composition. The BALB/c mice were used to model the skin injury. The cationic peptides (CPs) were synthesized by the solid-phase method using the Fmoc technology. Antibacterial effects of CPs and AFD were estimated by colony counting. It was found that the AFD-based ointment exerted wound-healing and anti-inflammatory activity. The minimum bactericidal concentrations (MBC) of the CPs most active against the E. coli Dh5α strain, AB-1, AB-2, AB-3, and ST-10, were 1.15, 0.11, 0.74, and 0.74 mM, respectively, while MBC of ampicillin was 0.7 mM. We assume that constructing the hybrid compounds/fullerene C60 conjugates with active CPs will be a promising area of the development of drugs for treatment of wounds complicated by bacterial infection.