Manipulation of genes, including knock-out or knock-in, replacement of gene elements (such as promoters), fusion with a fluorescent protein gene, and construction of in situ gene reporter, is required in most of the biotechnological laboratories. The widely used gene manipulating methods based on two-step allelic exchange are cumbersome in terms of constructing plasmids, transforming and screening. In addition, the efficiency of using this method for long fragment knockout is low. To simplify the process of gene manipulation, we constructed a minimized integrative vector pln2. When a gene needs to be inactivated, an internal fragment of the target gene (non-frameshift) is cloned into the pln2 plasmid. Once the single-crossover recombination between genome and the constructed plasmid occurs, the endogenous gene is segmented by the plasmid backbone and thus inactivated. We developed a toolbox based on pln2 that can be used for different genomic operation mentioned above. With the help of this toolbox, we successfully knocked out large fragments of 20-270 kb.
Genetic Vectors/genetics* ; Pseudomonas aeruginosa/genetics* ; Plasmids/genetics* ; Promoter Regions, Genetic ; Genome

Lysis is a common functional module in synthetic biology and is widely used in genetic circuit design. Lysis could be achieved by inducing expression of lysis cassettes originated from phages. However, detailed characterization of lysis cassettes hasn't been reported yet. Here, we first adopted arabinose- and rhamnose-inducible systems to develop inducible expression of five lysis cassettes (S₁₀₅, A52G, C51S S76C, LKD, LUZ) in Escherichia coli Top10. By measuring OD₆₀₀, we characterized the lysis behavior of strains harboring different lysis cassettes. These strains were harvested at different growth stages, induced with different concentrations of chemical inducers, or contained plasmids with different copy numbers. We found that although all five lysis cassettes could induce bacterial lysis in Top10, lysis behaviors differed a lot at various conditions. We further found that due to the difference in background expression levels between strain Top10 and Pseudomonas aeruginosa PAO1, it was hard to construct inducible lysis systems in strain PAO1. The lysis cassette controlled by rhamnose-inducible system was finally inserted into the chromosome of strain PAO1 to construct lysis strains after careful screen. The results indicated that LUZ and LKD were more effective in strain PAO1 than S₁₀₅, A52G and C51S S76C. At last, we constructed an engineered bacteria Q16 using an optogenetic module BphS and the lysis cassette LUZ. The engineered strain was capable of adhering to target surface and achieving light-induced lysis by tuning the strength of ribosome binding sites (RBSs), showing great potential in surface modification.
Rhamnose/pharmacology* ; Plasmids/genetics* ; Pseudomonas aeruginosa ; Escherichia coli/metabolism*

Objective: To identify the antibiotic resistance, virulence genes, and sequence types of Pseudomonas aeruginosa (P. aeruginosa) strains isolated from blood. Methods: From November 2014 to December 2021, a total of 94 nonrepetitive P. aeruginosa isolates were obtained from blood samples of patients at the First Affiliated Hospital of Shandong First Medical University in Shandong Province, China. The bacteria were identified using matrix-assisted laser desorption ionization time of flight mass spectrometry. Antibiotic resistance of the P. aeruginosa isolates was detected using Vitek 2 Compact system. Polymerase chain reaction (PCR) was conducted for the 18 virulence genes, and multi locus sequence typing (MLST) was performed to identify the sequence types of the P. aeruginosa strains. The resistance rates and distributions of virulence genes between carbapenem resistant pseudomonas aeruginosa (CRPA) and carbapenem susceptible pseudomonas aeruginosa (CSPA) isolates were compared using the Chi-square test. Results: Among 94 P. aeruginosa isolates, 19 (20.2%) isolates were found to be multidrug resistant (MDR) bacteria, of which 17 were CRPA isolates and 2 were CSPA isolates. All strains contained more than 10 virulence genes. Except for exoU gene, the detection rate of other genes was above 83%. MLST analysis revealed a total of 66 different STs, including 59 existing STs and 7 novel STs. Among them, ST244 (n=11, 11.7%) and ST270 (n=7, 7.4%) were the dominant STs. Although these two types of isolates harbored the same virulence genes, the resistance rates to carbapenem were different. 54.5% (6/11) ST244 isolates were CRPA but all 7 ST270 isolates were CSPA. Conclusion: Although the resistance rates of P. aeruginosa strains isolated from blood were at a low level, some MDR and CRPA isolates were detected. As the high virulence gene detection rates and genetic diversity were found for P. aeruginosa strains isolated from blood, close attention should be paid to avoid transmission and outbreaks.
Humans ; Pseudomonas aeruginosa/genetics* ; Multilocus Sequence Typing ; Molecular Epidemiology ; Pseudomonas Infections/microbiology* ; Microbial Sensitivity Tests ; Hospitals ; Carbapenems/pharmacology* ; Drug Resistance, Multiple, Bacterial/genetics* ; Anti-Bacterial Agents/pharmacology* ; beta-Lactamases

Objective: To identify the antibiotic resistance, virulence genes, and sequence types of Pseudomonas aeruginosa (P. aeruginosa) strains isolated from blood. Methods: From November 2014 to December 2021, a total of 94 nonrepetitive P. aeruginosa isolates were obtained from blood samples of patients at the First Affiliated Hospital of Shandong First Medical University in Shandong Province, China. The bacteria were identified using matrix-assisted laser desorption ionization time of flight mass spectrometry. Antibiotic resistance of the P. aeruginosa isolates was detected using Vitek 2 Compact system. Polymerase chain reaction (PCR) was conducted for the 18 virulence genes, and multi locus sequence typing (MLST) was performed to identify the sequence types of the P. aeruginosa strains. The resistance rates and distributions of virulence genes between carbapenem resistant pseudomonas aeruginosa (CRPA) and carbapenem susceptible pseudomonas aeruginosa (CSPA) isolates were compared using the Chi-square test. Results: Among 94 P. aeruginosa isolates, 19 (20.2%) isolates were found to be multidrug resistant (MDR) bacteria, of which 17 were CRPA isolates and 2 were CSPA isolates. All strains contained more than 10 virulence genes. Except for exoU gene, the detection rate of other genes was above 83%. MLST analysis revealed a total of 66 different STs, including 59 existing STs and 7 novel STs. Among them, ST244 (n=11, 11.7%) and ST270 (n=7, 7.4%) were the dominant STs. Although these two types of isolates harbored the same virulence genes, the resistance rates to carbapenem were different. 54.5% (6/11) ST244 isolates were CRPA but all 7 ST270 isolates were CSPA. Conclusion: Although the resistance rates of P. aeruginosa strains isolated from blood were at a low level, some MDR and CRPA isolates were detected. As the high virulence gene detection rates and genetic diversity were found for P. aeruginosa strains isolated from blood, close attention should be paid to avoid transmission and outbreaks.
Humans ; Pseudomonas aeruginosa/genetics* ; Multilocus Sequence Typing ; Molecular Epidemiology ; Pseudomonas Infections/microbiology* ; Microbial Sensitivity Tests ; Hospitals ; Carbapenems/pharmacology* ; Drug Resistance, Multiple, Bacterial/genetics* ; Anti-Bacterial Agents/pharmacology* ; beta-Lactamases

Members of the ferric uptake regulator (Fur) protein family are bacterial transcriptional repressors that control iron uptake and storage in response to iron availability, thereby playing a crucial role in the maintenance of iron homeostasis. The fur null mutants of Pseudomonas aeruginosa could not be obtained because fur is an essential gene. In this study, We constructed a Fur inducibly expression strain Δfur/attB::PBAD-fur in order to study the effect of fur on the growth, biofilm formation, motilities and oxidative stress response of P. aeruginosa. The results showed that a low level of fur expression retarded the growth of P. aeruginosa at an iron-depleted condition, or under high concentration of iron, or in the presence of H2O2. Fur affected the biofilm formation and the motilities (swimming, twitching, and swarming) of strain PAO1. The production of pyoverdine is regulated by Fur. Interestingly, proteins from Magnetospirillum gryphiswaldense MSR-1, which shares homology with Fur, can partially recover the pyoverdine production of strain Δfur/attB::PBAD-fur. This study provides new clues for the prevention and treatment of P. aeruginosa infections.
Bacterial Proteins/genetics* ; Hydrogen Peroxide ; Magnetospirillum ; Pseudomonas aeruginosa/genetics* ; Repressor Proteins/genetics*

OBJECTIVE: To evaluate the in vitro activity of ceftazidime-avibactam (CAZ-AVI) alone or in combination with colistin (COL) against clinically isolated extensively drug-resistant Pseudomonas aeruginosa (XDR-PA). METHODS: Minimum inhibitory concentration (MIC) of 16 clinical XDR-PA isolates was determined by broth dilution method and chessboard design when CAZ-AVI and COL were used alone or in combination, then the combined inhibitory concentration index (FICI) was calculated. Class A [Klebsiella pneumoniae carbapenemase β-lactamase (bla_KPC), Guiana extended-spectrum β-lactamase (bla_GES)], Class B [imipenemase β-lactamase (bla_IMP), Verona-Integronmetallo β-lactamase (bla_VIM), New Delhi metallo β-lactamase (bla_NDM), German imipenemase β-lactamase (bla_GIM), Sao Paulo metallo-β-lactamase (bla_SPM)], Class C [AmpC β-lactamase (bla_AmpC)], Class D [oxacillinase β-lactamase (bla_OXA)] β-lactamase-related resistance genes were detected by polymerase chain reaction. Drug-resistant mutation frequencies of each strain were determined on a drug-containing plate. The time kill curves of three XDR-PA were plotted by colony counting method. A biofilm model was established in vitro, and the synergistic effect of CAZ-AVI and COL on biofilm inhibition was detected by methythiazolyl tetrazolium assay (MTT). RESULTS: The MICs of 16 XDR-PA for CAZ-AVI ranged from 1 mg/L to 128 mg/L, and three of the isolates showed resistance (MIC > 8 mg/L). The FICI range of CAZ-AVI combined with COL was 0.312-1.000. Four isolates were synergistic, while the other 12 isolates were additive. Three isolates resistant to CAZ-AVI contained Class B resistance genes such as bla_IMP and bla_VIM, while 13 susceptible isolates carried resistance genes belonging to Class A, C or D. The logarithm values of mutation frequencies of drug resistance in CAZ-AVI group, COL group and combination group were -4.81±0.88, -7.06±0.69 and -9.70 (-9.78, -9.53), respectively. There were significant differences among the three groups (H = 33.601, P < 0.001), and between every two groups (adjusted P < 0.05). In time kill curves, the phytoplankton load of three XDR-PA decreased more than 6 log CFU/L when these two drugs were used together, and number of PA1819 planktonic bacteria decreased more than 5.1 log CFU/L compared with monotherapy group. Viable quantity in biofilm (A₄₉₀) of normal saline group, CAZ-AVI group, COL group and CAZ-AVI-COL group were 0.665±0.068, 0.540±0.072, 0.494±0.642 and 0.317±0.080, respectively. There was significant difference between the other two groups (all P < 0.001), except for that between CAZ-AVI group and COL group (P = 0.109). CONCLUSIONS CAZ-AVI combined with COL can effectively improve the bactericidal effect of each drug alone on XDR-PA. The regimen can also reduce the production of drug-resistant bacteria and inhibit the formation of biofilm. Therefore, it is a potential treatment for XDR-PA infection.
Anti-Bacterial Agents/therapeutic use* ; Azabicyclo Compounds/therapeutic use* ; Ceftazidime/therapeutic use* ; Colistin/therapeutic use* ; Drug Combinations ; Drug Resistance, Bacterial/genetics* ; Microbial Sensitivity Tests ; Pseudomonas Infections/drug therapy* ; Pseudomonas aeruginosa ; beta-Lactamases

No abstract available.
Anti-Bacterial Agents/*pharmacology ; Azabicyclo Compounds/*pharmacology ; Bacterial Proteins/genetics ; Ceftazidime/*pharmacology ; Cephalosporins/*pharmacology ; DNA, Bacterial/genetics/metabolism ; Drug Resistance, Bacterial/*drug effects ; Gram-Negative Bacteria/drug effects/*isolation & purification ; Humans ; Microbial Sensitivity Tests ; Penicillanic Acid/*analogs & derivatives/pharmacology ; Pseudomonas aeruginosa/drug effects/isolation & purification ; Real-Time Polymerase Chain Reaction

No abstract available.
Anti-Bacterial Agents/pharmacology ; DNA, Bacterial/chemistry/genetics/metabolism ; Drug Resistance, Bacterial ; Integrons/*genetics ; Microbial Sensitivity Tests ; Pseudomonas aeruginosa/drug effects/*enzymology/isolation & purification ; Sequence Analysis, DNA ; beta-Lactamases/*genetics

BACKGROUND: Extensively drug-resistant (XDR) Pseudomonas aeruginosa and Acinetobacter baumannii are a threat to hospitalized patients. We evaluated the effects of antimicrobial combinations on XDR P. aeruginosa and A. baumannii isolates. METHODS: P. aeruginosa and A. baumannii isolates, which were resistant to all antibiotics except colistin (CL), were collected from eight hospitals in Korea. Genes encoding metallo-beta-lactamases (MBLs) and OXA carbapenemases were detected by PCR in eight P. aeruginosa and 30 A. baumannii isolates. In vitro synergy of antimicrobial combinations was tested by using the checkerboard method. RESULTS: Minimum inhibitory concentrations of beta-lactams, aminoglycosides, and fluoroquinolones were very high, while that of CL was low for majority of XDR P. aeruginosa and A. baumannii isolates. Antimicrobial combinations including Imipenem (IPM)-CL, ceftazidime (CAZ)-CL, and rifampin (RIF)-CL exerted only additive/indifferent effects on majority of XDR P. aeruginosa isolates. Proportions of XDR A. baumannii isolates that showed synergistic and additive/indifferent inhibition after treatment with antimicrobial combinations used are as follows: IPM-ampicillin-sulbactam (AMS), 17% and 80% isolates, respectively; IPM-rifampin (RIF), 13% and 81% isolates, respectively; IPM-CL, 13% and 87% isolates, respectively; and RIF-COL, 20% and 73% isolates, respectively. Significant proportion (19%) of XDR P. aeruginosa isolates produced MBLs, and majority (82%) of A. baumannii isolates produced either MBLs or OXA-23. CONCLUSIONS: Our results suggest that combinations of IPM-AMS, IPM-RIF, IPM-CL, and RIF-CL are more useful than individual drugs for treating 13-20% of XDR A. baumannii infections.
Acinetobacter baumannii/*drug effects/genetics/isolation & purification ; Aminoglycosides/pharmacology ; Anti-Infective Agents/*pharmacology ; Bacterial Proteins/genetics/metabolism ; Drug Resistance, Multiple, Bacterial/*drug effects ; Drug Synergism ; Fluoroquinolones/pharmacology ; Imipenem/pharmacology ; Microbial Sensitivity Tests ; Polymerase Chain Reaction ; Pseudomonas aeruginosa/*drug effects/genetics/isolation & purification ; beta-Lactamases/genetics/metabolism

BACKGROUND: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Acinetobacter baumannii (CRAB) are the leading causes of nosocomial infections. A rapid and sensitive test to detect CRPA and CRAB is required for appropriate antibiotic treatment. We optimized a loop-mediated isothermal amplification (LAMP) assay to detect the presence of bla(VIM-2), bla(IMP-1), and bla OXA-23, which are critical components for carbapenem resistance. METHODS: Two sets of primers, inner and outer primers, were manually designed as previously described. The LAMP buffer was optimized (at 2mM MgSO4) by testing different concentrations of MgSO4. The optimal reaction temperature and incubation time were determined by using a gradient thermocycler. Then, the optimized bla(VIM-2), bla(IMP-1), and bla(OXA-23) LAMP reactions were evaluated by using 120 P. aeruginosa and 99 A. baumannii clinical isolates. RESULTS: Only one strain of the 100 CRPA isolates harbored bla(IMP-1), whereas none of them harbored bla(VIM-2). These results indicate that the acquisition of bla(VIM-2) or bla(IMP-1) may not play a major role in carbapenem resistance in Korea. Fifty two strains of the 75 CRAB isolates contained bla(OXA-23), but none contained bla(VIM-2) and bla(IMP-1) alleles. CONCLUSIONS: Our results demonstrate the usefulness of LAMP for the diagnosis of CRPA and CRAB.
Acinetobacter baumannii/genetics/*isolation & purification ; Anti-Bacterial Agents/*pharmacology ; Carbapenems/*pharmacology ; Drug Resistance, Bacterial/*genetics ; *Genes, Bacterial ; Nucleic Acid Amplification Techniques ; Pseudomonas aeruginosa/genetics/*isolation & purification ; Sensitivity and Specificity