The Lrp/AsnC-Type Regulator PA2577 Controls the EamA-like Transporter Gene PA2576 in Pseudomonas aeruginosa.

The regulatory network of gene expression in Pseudomonas aeruginosa, an opportunistic human pathogen, is very complex. In the PAO1 reference strain, about 10% of genes encode transcriptional regulators, many of which have undefined regulons and unknown functions. The aim of this study is the characterization of PA2577 protein, a representative of the Lrp/AsnC family of transcriptional regulators. This family encompasses proteins involved in the amino acid metabolism, regulation of transport processes or cell morphogenesis. The transcriptome profiling of P. aeruginosa cells with mild PA2577 overproduction revealed a decreased expression of the PA2576 gene oriented divergently to PA2577 and encoding an EamA-like transporter. A gene expression analysis showed a higher mRNA level of PA2576 in P. aeruginosa ΔPA2577, indicating that PA2577 acts as a repressor. Concomitantly, ChIP-seq and EMSA assays confirmed strong interactions of PA2577 with the PA2577/PA2576 intergenic region. Additionally, phenotype microarray analyses indicated an impaired metabolism of ΔPA2576 and ΔPA2577 mutants in the presence of polymyxin B, which suggests disturbances of membrane functions in these mutants. We show that PA2576 interacts with two proteins, PA5006 and PA3694, with a predicted role in lipopolysaccharide (LPS) and membrane biogenesis. Overall, our results indicate that PA2577 acts as a repressor of the PA2576 gene coding for the EamA-like transporter and may play a role in the modulation of the cellular response to stress conditions, including antimicrobial peptides, e.g., polymyxin B.

Genome sequence of Pseudomonas aeruginosa PAO1161, a PAO1 derivative with the ICEPae1161 integrative and conjugative element.

BACKGROUND: Pseudomonas aeruginosa is a cause of nosocomial infections, especially in patients with cystic fibrosis and burn wounds. PAO1 strain and its derivatives are widely used to study the biology of this bacterium, however recent studies demonstrated differences in the genomes and phenotypes of derivatives from different laboratories. RESULTS: Here we report the genome sequence of P. aeruginosa PAO1161 laboratory strain, a leu-, RifR, restriction-modification defective PAO1 derivative, described as the host of IncP-8 plasmid FP2, conferring the resistance to mercury. Comparison of PAO1161 genome with PAO1-UW sequence revealed lack of an inversion of a large genome segment between rRNA operons and 100 nucleotide polymorphisms, short insertions and deletions. These included a change in leuA, resulting in E108K substitution, which caused leucine auxotrophy and a mutation in rpoB, likely responsible for the rifampicin resistance. Nonsense mutations were detected in PA2735 and PA1939 encoding a DNA methyltransferase and a putative OLD family endonuclease, respectively. Analysis of revertants in these two genes showed that PA2735 is a component of a restriction-modification system, independent of PA1939. Moreover, a 12 kb RPG42 prophage and a novel 108 kb PAPI-1 like integrative conjugative element (ICE) encompassing a mercury resistance operon were identified. The ICEPae1161 was transferred to Pseudomonas putida cells, where it integrated in the genome and conferred the mercury resistance. CONCLUSIONS: The high-quality P. aeruginosa PAO1161 genome sequence provides a reference for further research including e.g. investigation of horizontal gene transfer or comparative genomics. The strain was found to carry ICEPae1161, a functional PAPI-1 family integrative conjugative element, containing loci conferring mercury resistance, in the past attributed to the FP2 plasmid of IncP-8 incompatibility group. This indicates that the only known member of IncP-8 is in fact an ICE.

Interaction of ArmZ with the DNA-binding domain of MexZ induces expression of mexXY multidrug efflux pump genes and antimicrobial resistance in Pseudomonas aeruginosa.

Multidrug efflux pumps play an important role in antibiotic resistance in bacteria. In Pseudomonas aeruginosa, MexXY pump provides intrinsic resistance to many antimicrobials, including aminoglycosides. The expression of mexXY operon is negatively regulated by MexZ repressor. The repression is alleviated in response to the antibiotic-induced ribosome stress, which results in increased synthesis of anti-repressor ArmZ, interacting with MexZ. The molecular mechanism of MexZ inactivation by ArmZ is not known. Here, we showed that the N-terminal part of MexZ, encompassing the DNA-binding domain, is required for interaction with ArmZ. Using the bacterial two hybrid system based mutant screening and pull-down analyses we identified substitutions in MexZ that diminished (R3S, K6E, R13H) or completely impaired (K53E) the interaction with ArmZ without blocking MexZ activity as a transcriptional repressor. Introduction of corresponding mexZ missense mutations into P aeruginosa PAO1161 chromosome impaired (mexZ K6E, mexZ R13H) or blocked (mexZ K53E) tetracycline mediated induction of mexY expression. Concomitantly, PAO1161 mexZ K53E strain was more susceptible to aminoglycosides. The identified residues are highly conserved in MexZ-like transcriptional regulators found in bacterial genomes encoding both MexX/MexY/MexZ and ArmZ/PA5470 orthologs, suggesting that a similar mechanism may contribute to induction of efflux mediated resistance in other bacterial species. Overall, our data shed light on the molecular mechanism of ArmZ mediated induction of intrinsic antimicrobial resistance in P. aeruginosa.

Drug Susceptibility Profiling and Genetic Determinants of Drug Resistance in Mycobacterium kansasii.

Very few studies have examined drug susceptibility of Mycobacterium kansasii, and they involve a limited number of strains. The purpose of this study was to determine drug susceptibility profiles of M. kansasii isolates representing a spectrum of species genotypes (subtypes) with two different methodologies, i.e., broth microdilution and Etest assays. To confirm drug resistance, drug target genes were sequenced. A collection of 85 M. kansasii isolates, including representatives of eight different subtypes (I to VI, I/II, and IIB) from eight countries, was used. Drug susceptibility against 13 and 8 antimycobacterial agents was tested by using broth microdilution and Etest, respectively. For drug-resistant or high-MIC isolates, eight structural genes (rrl, katG, inhA, embB, rrs, rpsL, gyrA, and gyrB) and one regulatory region (embCA) were PCR amplified and sequenced in the search for resistance-associated mutations. All isolates tested were susceptible to rifampin (RIF), amikacin (AMK), co-trimoxazole (SXT), rifabutin (RFB), moxifloxacin (MXF), and linezolid (LZD) according to the microdilution method. Resistance to ethambutol (EMB), ciprofloxacin (CIP), and clarithromycin (CLR) was found in 83 (97.7%), 17 (20%), and 1 (1.2%) isolate, respectively. The calculated concordance between the Etest and dilution method was 22.6% for AMK, 4.8% for streptomycin (STR), 3.2% for CLR, and 1.6% for RIF. For EMB, INH, and SXT, not even a single MIC value determined by one method equaled that by the second method. The only mutations disclosed were A2266C transversion at the rrl gene (CLR-resistant strain) and A128G transition at the rpsL gene (strain with STR MIC of >64 mg/liter). In conclusion, eight drugs, including RIF, CLR, AMK, SXT, RFB, MXF, LZD, and ethionamide (ETO), showed high in vitro activity against M. kansasii isolates. Discrepancies of the results between the reference microdilution method and Etest preclude the use of the latter for drug susceptibility determination in M. kansasii Drug resistance in M. kansasii may have different genetic determinants than resistance to the same drugs in M. tuberculosis.

The LysR-Type Transcriptional Regulator BsrA (PA2121) Controls Vital Metabolic Pathways in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a facultative human pathogen causing nosocomial infections, has complex regulatory systems involving many transcriptional regulators. LTTR (LysR-Type Transcriptional Regulator) family proteins are involved in the regulation of various processes, including stress responses, motility, virulence, and amino acid metabolism. The aim of this study was to characterize the LysR-type protein BsrA (PA2121), previously described as a negative regulator of biofilm formation in P. aeruginosa. Genome wide identification of BsrA binding sites using chromatin immunoprecipitation and sequencing analysis revealed 765 BsrA-bound regions in the P. aeruginosa PAO1161 genome, including 367 sites in intergenic regions. The motif T-N11-A was identified within sequences bound by BsrA. Transcriptomic analysis showed altered expression of 157 genes in response to BsrA excess; of these, 35 had a BsrA binding site within their promoter regions, suggesting a direct influence of BsrA on the transcription of these genes. BsrA-repressed loci included genes encoding proteins engaged in key metabolic pathways such as the tricarboxylic acid cycle. The panel of loci possibly directly activated by BsrA included genes involved in pilus/fimbria assembly, as well as secretion and transport systems. In addition, DNA pull-down and regulatory analyses showed the involvement of PA2551, PA3398, and PA5189 in regulation of bsrA expression, indicating that this gene is part of an intricate regulatory network. Taken together, these findings reveal the existence of a BsrA regulon, which performs important functions in P. aeruginosa. IMPORTANCE This study shows that BsrA, a LysR-type transcriptional regulator from Pseudomonas aeruginosa, previously identified as a repressor of biofilm synthesis, is part of an intricate global regulatory network. BsrA acts directly and/or indirectly as the repressor and/or activator of genes from vital metabolic pathways (e.g., pyruvate, acetate, and tricarboxylic acid cycle) and is involved in control of transport functions and the formation of surface appendages. Expression of the bsrA gene is increased in the presence of antibiotics, which suggests its induction in response to stress, possibly reflecting the need to redirect metabolism under stressful conditions. This is particularly relevant for the treatment of infections caused by P. aeruginosa. In summary, the findings of this study demonstrate that the BsrA regulator performs important roles in carbon metabolism, biofilm formation, and antibiotic resistance in P. aeruginosa.

Three-year outcomes of mixed astigmatism correction with single-step transepithelial photorefractive keratectomy with a large ablation zone.

PURPOSE: To evaluate refractive and visual outcomes of single-step transepithelial photorefractive keratectomy (transPRK) in the treatment of mixed astigmatism with the use of an aberration-neutral profile and large ablation zone. SETTING: Nicolaus Copernicus University and Oftalmika Eye Hospital, Bydgoszcz, Poland. DESIGN: Retrospective, observational case series. METHODS: This study included patients who underwent transPRK to correct mixed astigmatism and completed the 3-year follow-up. Procedures were performed with an Amaris 750S excimer laser using an aberration-neutral profile and optical zone of 7.2 mm or more. RESULTS: A total 48 eyes of 39 patients were included. Preoperatively, mean spherical manifest refraction was +1.37 ± 0.98 diopter (D) (0.25 to 4.00 D), and astigmatism was -4.00 ± 0.76 D (-2.25 to -6.00 D). Three years postsurgery, it was -0.17 ± 0.26 D and -0.41 ± 0.44 D, respectively. Attempted spherical equivalent correction within ±0.50 D was achieved in 45 eyes (94%) and cylindrical correction in 34 (71%). Preoperative corrected distance visual acuity (CDVA) was 20/20 or better in 38 eyes (79%), and postoperative uncorrected was 20/20 or better in 29 eyes (60.0%). No eye had lost 2 or more Snellen lines of CDVA, whereas 3 eyes (6%) gained 2 or more lines. In 4 eyes (8%), haze of low intensity was observed at the periphery, with scores between 0.5 and 1.0, and only 1 eye getting a score of 2 in 0- to 4-degree scale. CONCLUSIONS: Mixed astigmatism correction with large-ablation-zone transPRK provided good results for efficacy, safety, predictability, and visual outcomes in a 3-year follow-up.

Three-year outcomes after high hyperopia correction using photorefractive keratectomy with a large ablation zone.

AIM: To evaluate refractive and visual outcomes of photorefractive keratectomy (PRK) to treat high hyperopia using an aberration-neutral profile and large ablation zone. METHODS: This was a retrospective, consecutive observational case series at the Oftalmika Eye Hospital, Bydgoszcz, Poland. We included 51 consecutive eyes of 34 patients who underwent alcohol-assisted PRK to correct hyperopia within the range of +3.6 to +6.15 D (mean+4.61±0.67 D). Procedures were performed with an Amaris 750S excimer laser (Schwind eye-tech-solutions GmbH, Kleinostheim, Germany) using an aberration-neutral profile and a 10 mm total ablation zone. Refractive results, predictability, safety and efficacy were evaluated 3 years postoperatively. RESULTS: At 1-year postsurgery, the mean manifest refraction spherical equivalent (MRSE) was -0.002±0.43 D and mean cylinder was -0.181±0.31 D, while the values were +0.09±0.46 D and -0.15±0.26 D, respectively, at 2 years (MRSE p<0.001) and +0.15±0.44 D and -0.15±0.26 D, respectively, at 3 years (MRSE p<0.001). 78% of eyes were within ±0.50 D of the attempted spherical equivalent correction. Three years postoperatively, 22% of eyes lost one line of corrected distance visual acuity and 27% gained a line or two. The change in the mean corneal spherical aberrations for the 6 mm zone was from 0.27±0.07 to 0.08±0.13 µm. CONCLUSIONS: High hyperopia correction with PRK using an aberration-neutral profile and large ablation zone provides good efficacy, safety, predictability and visual outcomes. Relatively low change of corneal spherical aberrations and low increase of hyperopia in the first three postoperative years were observed.

Proposal of a new method for subtyping of Mycobacterium kansasii based upon PCR restriction enzyme analysis of the tuf gene.

Within this study, a new, rapid method for subtyping of Mycobacterium kansasii was developed based on the sequence analysis of the tuf gene coding for the Tu (thermo-unstable) elongation factor (EF-Tu). The method involves PCR amplification of ca. 740-bp tuf gene fragment, followed by digestion with the MvaI restriction endonuclease.