The AE404 antibody recognizes a Pseudomonas aeruginosa PAO1 surface antigen by flow cytometry

The recombinant antibody AE404 detects by flow cytometry P. aeruginosa PAO1 strain; AE409 and AF389 antibodies do not.

Determination of the antimicrobial and antibiofilm effects and ‘Quorum Sensing’ inhibition potentials of Castanea sativa Mill. extracts

The rapid rise of resistance causes existing antibiotics to become dysfunctional. Therefore, search for new antimicrobial active ingredients has increased in recent years. In this study, flower extracts of Castanea sativa were examined for antimicrobial and anti-quorum sensing aspects. The antimicrobial properties of methanol, ethyl acetate, ethanol and hexane extracts of C. sativa against some gram-positive and gram-negative bacterial species, as well as yeasts (Candida albicans and Candida parapsilosis) were investigated by the agar well diffusion method. The minimum inhibition concentration (MIC) and minimum bactericidal concentrations (MBC) of C. sativa extracts were also determined. Chromobacterium violaceum ATCC 12472, C. violaceum 35352, C. violaceum VIR07 and C. violaceum CV026 indicator strains were used for determination of the quorum sensing inhibitions, and the Pseudomonas aeruginosa PAO1 strain was used for the swarming tests. Additionally, biofilm inhibition was detected by the spectrophotometric method using the P. aeruginosa PAO1 strain. Methanol, ethanol and ethyl acetate extracts of C. sativa was found to have high antibacterial and antifungal effects, while the methanol extract also had anti-quorum sensing, anti- swarming and biofilm inhibition effects, but no activity was found in the n- hexane extract. To the best of our knowledge this is the first report revealed that methanol extract obtained from C. sativa flowers induced anti-quorum sensing activities mainly inhibited the violacein production, swarming and biofilm formation. The present investigation provided evidence that the C. sativa flower extract maybe a potential source of antimicrobial agents. Therefore, much attention should be paid to C. sativa flower content, which could be used with high efficacy against microorganisms.

Pseudomonas aeruginosa High-Level Resistance to Polymyxins and Other Antimicrobial Peptides RequirescprA, a Gene That Is Disrupted in the PAO1 Strain

ABSTRACTThearnlocus, found in many Gram-negative bacterial pathogens, mediates resistance to polymyxins and other cationic antimicrobial peptides through 4-amino-l-arabinose modification of the lipid A moiety of lipopolysaccharide. InPseudomonas aeruginosa, several two-component regulatory systems (TCSs) control thearnlocus, which is necessary but not sufficient for these resistance phenotypes. A previous transposon mutagenesis screen to identify additional polymyxin resistance genes that these systems regulate implicated an open reading frame designated PA1559 in the genome of theP. aeruginosaPAO1 strain. Resequencing of this chromosomal region and bioinformatics analysis for a variety ofP. aeruginosastrains revealed that in the sequenced PAO1 strain, a guanine deletion at the end of PA1559 results in a frameshift and truncation of a full-length open reading frame that also encompasses PA1560 in non-PAO1 strains, such asP. aeruginosaPAK. Deletion analysis in the PAK strain showed that this full-length open reading frame, designatedcprA, is necessary for polymyxin resistance conferred by activating mutations in the PhoPQ, PmrAB, and CprRS TCSs. ThecprAgene was also required for PmrAB-mediated resistance to other cationic antimicrobial peptides in the PAK strain. Repair of the mutatedcprAallele in the PAO1 strain restored polymyxin resistance conferred by an activating TCS mutation. The deletion ofcprAdid not affect thearn-mediated lipid A modification, indicating that the CprA protein is necessary for a different aspect of polymyxin resistance. This protein has a domain structure with a strong similarity to the extended short-chain dehydrogenase/reductase family that comprises isomerases, lyases, and oxidoreductases. These results suggest a new avenue through which to pursue targeted inhibition of polymyxin resistance.

Role of Pseudomonas aeruginosa Low-Molecular-Mass Penicillin-Binding Proteins in AmpC Expression, β-Lactam Resistance, and Peptidoglycan Structure

ABSTRACTThis study aimed to characterize the role ofPseudomonas aeruginosalow-molecular-mass penicillin-binding proteins (LMM PBPs), namely, PBP4 (DacB), PBP5 (DacC), and PBP7 (PbpG), in peptidoglycan composition, β-lactam resistance, andampCregulation. For this purpose, we constructed all single and multiple mutants ofdacB,dacC,pbpG, andampCfrom the wild-typeP. aeruginosaPAO1 strain. Peptidoglycan composition was determined by high-performance liquid chromatography (HPLC),ampCexpression by reverse transcription-PCR (RT-PCR), PBP patterns by a Bocillin FL-binding test, and antimicrobial susceptibility by MIC testing for a panel of β-lactams. Microscopy and growth rate analyses revealed no apparent major morphological changes for any of the mutants compared to the wild-type PAO1 strain. Of the single mutants, onlydacCmutation led to significantly increased pentapeptide levels, showing that PBP5 is the majordd-carboxypeptidase inP. aeruginosa. Moreover, our results indicate that PBP4 and PBP7 play a significant role asdd-carboxypeptidase only if PBP5 is absent, and theirdd-endopeptidase activity is also inferred. As expected, the inactivation of PBP4 led to a significant increase inampCexpression (around 50-fold), but, remarkably, the sequential inactivation of the three LMM PBPs produced a much greater increase (1,000-fold), which correlated with peptidoglycan pentapeptide levels. Finally, the β-lactam susceptibility profiles of the LMM PBP mutants correlated well with theampCexpression data. However, the inactivation ofampCin these mutants also evidenced a role of LMM PBPs, especially PBP5, in intrinsic β-lactam resistance. In summary, in addition to assessing the effect ofP. aeruginosaLMM PBPs on peptidoglycan structure for the first time, we obtained results that represent a step forward in understanding the impact of these PBPs on β-lactam resistance, apparently driven by the interplay between their roles in AmpC induction, β-lactam trapping, anddd-carboxypeptidase/β-lactamase activity.

Efficacy of AiiM, anN-Acylhomoserine Lactonase, against Pseudomonas aeruginosa in a Mouse Model of Acute Pneumonia

ABSTRACTQuorum sensing (QS) inPseudomonas aeruginosaregulates the production of many virulence factors and plays an important role in the pathogenesis ofP. aeruginosainfection.N-acyl homoserine lactones (AHL) are major QS signal molecules. Recently, a novel AHL-lactonase enzyme, AiiM, has been identified. The aim of this study was to evaluate the effect of AiiM on the virulence ofP. aeruginosain a mouse model of acute pneumonia. We developed aP. aeruginosaPAO1 strain harboring an AiiM-expressing plasmid. The production of several virulence factors by the AiiM-expressing strain was examined. Mice were intratracheally infected with an AiiM-expressing PAO1 strain. Lung histopathology, bacterial burden, and bronchoalveolar lavage (BAL) fluid were assessed at 24 h postinfection. AiiM expression in PAO1 reduced production of AHL-mediated virulence factors and attenuated cytotoxicity against human lung epithelial cells. In a mouse model of acute pneumonia, AiiM expression reduced lung injury and greatly improved the survival rates. The levels of proinflammatory cytokines and myeloperoxidase activity in BAL fluid were significantly lower in mice infected with AiiM-expressing PAO1. Thus, AiiM can strongly attenuateP. aeruginosavirulence in a mammalian model and is a potential candidate for use as a therapeutic agent againstP. aeruginosainfection.

Analysis of Pseudomonas aeruginosa Conditional Psl Variants Reveals Roles for the Psl Polysaccharide in Adhesion and Maintaining Biofilm Structure Postattachment

ABSTRACT The ability to form biofilms in the airways of people suffering from cystic fibrosis is a critical element of Pseudomonas aeruginosa pathogenesis. The 15-gene psl operon encodes a putative polysaccharide that plays an important role in biofilm initiation in nonmucoid P. aeruginosa strains. Biofilm initiation by a P. aeruginosa PAO1 strain with disruption of pslA and pslB (ΔpslAB) was severely compromised, indicating that psl has a role in cell-surface interactions. In this study, we investigated the adherence properties of this ΔpslAB mutant using biotic surfaces (epithelial cells and mucin-coated surfaces) and abiotic surfaces. Our results showed that psl is required for attachment to a variety of surfaces, independent of the carbon source. To study the potential roles of Psl apart from attachment, we generated a psl-inducible P. aeruginosa strain (Δpsl/p BAD -psl) by replacing the psl promoter region with araC-p BAD , so that expression of psl could be controlled by addition of arabinose. Analysis of biofilms formed by the Δpsl/p BAD -psl strain indicated that expression of the psl operon is required to maintain the biofilm structure at steps postattachment. Overproduction of the Psl polysaccharide led to enhanced cell-surface and intercellular adhesion of P. aeruginosa. This translated into significant changes in the architecture of the biofilm. We propose that Psl has an important role in P. aeruginosa adhesion, which is critical for initiation and maintenance of the biofilm structure.