The integrase of genomic island GIsul2 mediates the mobilization of GIsul2 and ISCR-related element CR2-sul2 unit through site-specific recombination.

In the worldwide health threat posed by antibiotic-resistant bacterial pathogens, mobile genetic elements (MGEs) play a critical role in favoring the dissemination of resistance genes. Among them, the genomic island GIsul2 and the ISCR-related element CR2-sul2 unit are believed to participate in this dissemination. However, the mobility of the two elements has not yet been demonstrated. Here, we found that the GIsul2 and CR2-sul2 units can excise from the host chromosomal attachment site (attB) in Shigella flexneri. Through establishing a two-plasmid mobilization system composed of a donor plasmid bearing the GIsul2 and a trap plasmid harboring the attB in recA-deficient Escherichia coli, we reveal that the integrase of GIsul2 can perform the excision and integration of GIsul2 and CR2-sul2 unit by site-specific recombination between att core sites. Furthermore, we demonstrate that the integrase and the att sites are required for mobility through knockout experiments. Our findings provide the first experimental characterization of the mobility of GIsul2 and CR2-sul2 units mediated by integrase. They also suggest a potential and unappreciated role of the GIsul2 integrase family in the dissemination of CR2-sul2 units carrying various resistance determinants in between.

Direct antimicrobial susceptibility testing of bloodstream infection on SlipChip.

This paper describes an integrated microfluidic SlipChip device for rapid antimicrobial susceptibility testing (AST) of bloodstream pathogens in positive blood cultures. Unlike conventional AST methods, which rely on an overnight subculture of positive blood cultures to obtain isolated colonies, this device enables direct extraction and enrichment of the bacteria from positive blood cultures by dielectrophoresis. SlipChip technology enables parallel inoculation of the extracted bacteria into nanoliter-scale broth droplets to perform multiplexed ASTs simultaneously. The nanoliter confinement in the droplets increases the effective inoculation amount of the bacteria, shortens the diffusion distance of nutrient elements and gases, and allows faster growth and proliferation rates. Entropy-based image analysis used for the characterization of bacterial susceptibility patterns eliminates the requirement for single-cell morphological analysis and fluorescence labeling. As a proof-of-concept, the susceptibility patterns of Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538p, and a positive blood culture containing Escherichia coli against several broad-spectrum antibiotics were determined by the SlipChip device. The on-chip AST results were well matched with those respectively reported by the broth microdilution method and a BD Phoenix Automated Microbiology System. Reliable AST results can be reported to clinicians within 3-8 h using this simple device after positive blood culture, allowing earlier proper administration of antimicrobial therapy.

Cross-Regulation between the phz1 and phz2 Operons Maintain a Balanced Level of Phenazine Biosynthesis in Pseudomonas aeruginosa PAO1.

Gene duplication often provides selective advantages for the survival of microorganisms in adapting to varying environmental conditions. P. aeruginosa PAO1 possesses two seven-gene operons [phz1 (phzA1B1C1D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2)] that are involved in the biosynthesis of phenazine-1-carboxylic acid and its derivatives. Although the two operons are highly homologous and their functions are well known, it is unclear how the two phz operons coordinate their expressions to maintain the phenazine biosynthesis. By constructing single and double deletion mutants of the two phz operons, we found that the phz1-deletion mutant produced the same or less amount of phenazine-1-carboxylic acid and pyocyanin in GA medium than the phz2-knockout mutant while the phz1-phz2 double knockout mutant did not produce any phenazines. By generating phzA1 and phzA2 translational and transcriptional fusions with a truncated lacZ reporter, we found that the expression of the phz1 operon increased significantly at the post-transcriptional level and did not alter at the transcriptional level in the absence of the phz2 operon. Surprisingly, the expression the phz2 operon increased significantly at the post-transcriptional level and only moderately at the transcriptional level in the absence of the phz1 operon. Our findings suggested that a complex cross-regulation existed between the phz1 and phz2 operons. By mediating the upregulation of one phz operon expression while the other was deleted, this crosstalk would maintain the homeostatic balance of phenazine biosynthesis in P. aeruginosa PAO1.

[Positive regulation in expression of the phenazine-producing operon phz2 mediated by pip in Pseudomonas aeruginosa PAO1].

UNLABELLED: Pseudomonas aeruginosa PAO1, an opportunistic pathogenic bacterium, produces phenazine and its derivatives which play a critical role in pathogen-host interaction during its infection. In a biological control strain P. chlororaphis PCL1391, Pip positively regulates PCN production. OBJECTIVE: Our aim is to identify the function and regulation of an ORF of PA0243 (the homolog of Pip) in Pseudomonas aeruginosa PAO1. METHODS: We first cloned the fragment of the pip gene from the chromosomal DNA of P. aeruginosa PAO1 and constructed the pip-defect mutant PA-PG with the insertion of gentamycin resistance cassette (aacC1). With construction and introduction of pME10P (containing the whole pip gene region) , complementation of the pip was then carried out. With creation of the mutants PA-PD-Z1G and PA-PG-Z2K, phenazine-1-carboxylic acid and pyocyanin were measured in GA medium in relative mutants, respectively. RESULTS: In GA medium, production of phenazine-1-carboxylic acid and pyocyanin in the mutant PA-PG decreased dramatically in comparison with that produced in the wild type strain PAO1. The amounts of phenazine-1-carboxylic acid and pyocyanin, however, were recovered with complementation of the derivative PA-PG bearing pME10P. The production of phenazine-1-carboxylic acid and pyocyanin in mutant PA-PG-Z2K were same to those in parental strain PA-Z2K. Phenazine-1-carboxylic acid and pyocyanin produced by the mutant PA-PD-Z1G were lower than those in the original strain PA-Z1G. CONCLUSION: With these results, it is suggested that Pip exerts positively regulation in phenazine biosynthesis by specifically modulating expression of the phz2 operon, not by mediating expression of the phzl operon in P. aeruginosa PAO1.

[Differential expression of two phenazine-producing loci mediated by deficiency of the global regulator rsmA in Psedomonas aeruginosa PAO1].

UNLABELLED: In many Pseudomonas, RsmA mediates the production of a set of secondary metabolites or virulence factors. OBJECTIVE: Our aim is to evaluate the function and regulation of the rsmA gene on two phenazine-producing operons in Pseudomonas aeruginosa PAO1. METHODS: We first cloned the upstream and downstream fragments of the rsmA gene from the chromosomal DNA. With the insertion of gentamycin resistance cassette (aacC1), the deletion mutant PA-RG was created and verified with PCR. To complement and overexpress the rsmA gene, pME10R and pME32R were also constructed. By constructing the translational fusion plasmids phz1'-'lacZ pMEZ1 and phz2'-'lacZ pMEZ2, we introduced them into the wild type strain PAO1 and the mutant PA-RG, respectively. Activities of beta-galactosidase were determined with Miller method. RESULTS: In glycerol-alanine medium, overexpression of the rsmA gene results in dramatical decrease of pyocyanin production in PA-RG and PAO1 strain. In addition, beta-galactosidase activity of phz1'-'lacZ in the mutant PA-RG was much higher than that in the wild type strain. However, beta-galactosidase activity of phz2'-'lacZ in the wild type strain was 2fold more than that in the mutant PA-RG. CONCLUSION: The regulation mediated by RsmA on two phenazine loci is specific and differential.