• Expression of the MexXY aminoglycoside efflux pump and presence of an aminoglycoside modifying enzyme in clinical isolates are highly correlated.

      Seupt, Alexander; Schniederjans, Monika; Tomasch, Jürgen; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (ASM, 2020-10-12)
      The impact of MexXY efflux pump expression on aminoglycoside resistance in clinical Pseudomonas aeruginosa isolates has been debated. In this study, we found that in general, elevated mexXY gene expression levels in clinical P. aeruginosa isolates confer to slight increases in aminoglycoside MIC levels, however those levels rarely lead to clinically relevant resistance phenotypes. The main driver of resistance in the clinical isolates studied here was the acquisition of aminoglycoside modifying enzymes (AMEs). Nevertheless, acquisition of an AME was strongly associated with mexY overexpression. In line with this observation, we demonstrate that the introduction of a gentamicin acetyl-transferase confers to full gentamicin resistance levels in a P. aeruginosa type strain only if the MexXY efflux pump was active. We discuss that increased mexXY activity in clinical AME harboring P. aeruginosa isolates might affect ion fluxes at the bacterial cell membrane and thus might play a role in the establishment of enhanced fitness that extends beyond aminoglycoside resistance.
    • In Vivo Efficacy of Antimicrobials against Biofilm-Producing Pseudomonas aeruginosa.

      Pawar, Vinay; Komor, Uliana; Kasnitz, Nadine; Bielecki, Piotr; Pils, Marina C; Gocht, Benjamin; Moter, Annette; Rohde, Manfred; Weiss, Siegfried; Häussler, Susanne; et al. (2015-08)
      Patients suffering from cystic fibrosis (CF) are commonly affected by chronic Pseudomonas aeruginosa biofilm infections. This is the main cause for the high disease severity. In this study, we demonstrate that P. aeruginosa is able to efficiently colonize murine solid tumors after intravenous injection and to form biofilms in this tissue. Biofilm formation was evident by electron microscopy. Such structures could not be observed with transposon mutants, which were defective in biofilm formation. Comparative transcriptional profiling of P. aeruginosa indicated physiological similarity of the bacteria in the murine tumor model and the CF lung. The efficacy of currently available antibiotics for treatment of P. aeruginosa-infected CF lungs, such as ciprofloxacin, colistin, and tobramycin, could be tested in the tumor model. We found that clinically recommended doses of these antibiotics were unable to eliminate wild-type P. aeruginosa PA14 while being effective against biofilm-defective mutants. However, colistin-tobramycin combination therapy significantly reduced the number of P. aeruginosa PA14 cells in tumors at lower concentrations. Hence, we present a versatile experimental system that is providing a platform to test approved and newly developed antibiofilm compounds.
    • Production of norspermidine contributes to aminoglycoside resistance in pmrAB mutants of Pseudomonas aeruginosa.

      Bolard, Arnaud; Schniederjans, Monika; Haussler, Susanne; Triponney, Pauline; Valot, Benoît; Plesiat, Patrick; Jeannot, Katy; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (American Society of microbiology, 2019-08-05)
      Emergence of resistance to polymyxins in Pseudomonas aeruginosa is mainly due to mutations in two-components systems, that promote addition of 4-amino-4-deoxy-L-arabinose to the lipopolysaccharide (LPS) through upregulation of operon arnBCADTEF-ugd (arn) expression. Here, we demonstrate that mutations occurring in different domains of histidine kinase PmrB or in response regulator PmrA result in coresistance to aminoglycosides and colistin. All seventeen clinical strains tested exhibiting such a cross-resistance phenotype were found to be pmrAB mutants. As shown by gene deletion experiments, the decreased susceptibility of the mutants to aminoglycosides was independent from operon arn but required the efflux system MexXY(OprM) and the products of three genes, PA4773-PA4774-PA4775, that are cotranscribed and activated with genes pmrAB Gene PA4773 (annotated as speD2 in PAO1 genome) and PA4774 (speE2) are predicted to encode enzymes involved in biosynthesis of polyamines. Comparative analysis of cell surface extracts of an in vitro selected pmrAB mutant, called AB16.2, and derivatives lacking PA4773, PA4774 and PA4775, respectively revealed that these genes were needed for norspermidine production via a pathway that likely uses 1,3-diaminoprane, a precursor of polyamines. Altogether, our results suggest that norspermidine decreases the self-promoted uptake pathway of aminoglycosides across the outer membrane and thereby potentiates the activity of efflux pump MexXY(OprM).
    • Pseudomonas aeruginosa ceftolozane-tazobactam resistance development requires multiple mutations leading to overexpression and structural modification of AmpC.

      Cabot, Gabriel; Bruchmann, Sebastian; Mulet, Xavier; Zamorano, Laura; Moyà, Bartolomé; Juan, Carlos; Haussler, Susanne; Oliver, Antonio; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2014-06)
      We compared the dynamics and mechanisms of resistance development to ceftazidime, meropenem, ciprofloxacin, and ceftolozane-tazobactam in wild-type (PAO1) and mutator (PAOMS, ΔmutS) P. aeruginosa. The strains were incubated for 24 h with 0.5 to 64× MICs of each antibiotic in triplicate experiments. The tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64× MIC for 7 consecutive days. The susceptibility profiles and resistance mechanisms were assessed in two isolated colonies from each step, antibiotic, and strain. Ceftolozane-tazobactam-resistant mutants were further characterized by whole-genome analysis through RNA sequencing (RNA-seq). The development of high-level resistance was fastest for ceftazidime, followed by meropenem and ciprofloxacin. None of the mutants selected with these antibiotics showed cross-resistance to ceftolozane-tazobactam. On the other hand, ceftolozane-tazobactam resistance development was much slower, and high-level resistance was observed for the mutator strain only. PAO1 derivatives that were moderately resistant (MICs, 4 to 8 μg/ml) to ceftolozane-tazobactam showed only 2 to 4 mutations, which determined global pleiotropic effects associated with a severe fitness cost. High-level-resistant (MICs, 32 to 128 μg/ml) PAOMS derivatives showed 45 to 53 mutations. Major changes in the global gene expression profiles were detected in all mutants, but only PAOMS mutants showed ampC overexpression, which was caused by dacB or ampR mutations. Moreover, all PAOMS mutants contained 1 to 4 mutations in the conserved residues of AmpC (F147L, Q157R, G183D, E247K, or V356I). Complementation studies revealed that these mutations greatly increased ceftolozane-tazobactam and ceftazidime MICs but reduced those of piperacillin-tazobactam and imipenem, compared to those in wild-type ampC. Therefore, the development of high-level resistance to ceftolozane-tazobactam appears to occur efficiently only in a P. aeruginosa mutator background, in which multiple mutations lead to overexpression and structural modifications of AmpC.
    • Quantitative Contributions of Target Alteration and Decreased Drug Accumulation to Pseudomonas aeruginosa Fluoroquinolone Resistance.

      Bruchmann, Sebastian; Dötsch, Andreas; Nouri, Bianka; Chaberny, Iris F; Häussler, Susanne; Department of Molecular Bacteriology, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2013-03)
      Quinolone antibiotics constitute a clinically successful and widely used class of broad-spectrum antibiotics; however, the emergence and spread of resistance increasingly limits the use of fluoroquinolones in the treatment and management of microbial disease. In this study, we evaluated the quantitative contributions of quinolone target alteration and efflux pump expression to fluoroquinolone resistance in Pseudomonas aeruginosa. We generated isogenic mutations in hot spots of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, and parC and inactivated the efflux regulator genes so as to overexpress the corresponding multidrug resistance (MDR) efflux pumps. We then introduced the respective mutations into the reference strain PA14 singly and in various combinations. Whereas the combined inactivation of two efflux regulator-encoding genes did not lead to resistance levels higher than those obtained by inactivation of only one efflux regulator-encoding gene, the combination of mutations leading to increased efflux and target alteration clearly exhibited an additive effect. This combination of target alteration and overexpression of efflux pumps was commonly observed in clinical P. aeruginosa isolates; however, these two mechanisms were frequently found not to be sufficient to explain the level of fluoroquinolone resistance. Our results suggest that there are additional mechanisms, independent of the expression of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and/or MexXY-OprM efflux pump, that increase ciprofloxacin resistance in isolates with mutations in the QRDRs.
    • Recycling of Peptidyl-tRNAs by Peptidyl-tRNA Hydrolase Counteracts Azithromycin-Mediated Effects on Pseudomonas aeruginosa.

      Gödeke, Julia; Pustelny, Christian; Häussler, Susanne; Gödeke, Julia; Pustelny, Christian; Häussler, Susanne; Department of Molecular Bacteriology, Helmholtz Center for Infection Research, Braunschweig, Germany.; Department of Molecular Bacteriology, Helmholtz Center for Infection Research, Braunschweig, Germany. (2013-04)
      Acute and chronic infections caused by the opportunistic pathogen Pseudomonas aeruginosa pose a serious threat to human health worldwide, and its increasing resistance to antibiotics requires alternative treatments that are more effective than available strategies. Clinical studies have clearly demonstrated that cystic fibrosis (CF) patients with chronic P. aeruginosa infections benefit from long-term low-dose azithromycin (AZM) treatment. Immunomodulating activity, the impact of AZM on the expression of quorum-sensing-dependent virulence factors, type three secretion, and motility in P. aeruginosa seem to contribute to the therapeutic response. However, to date, the molecular mechanisms underlying these AZM effects have remained elusive. Our data indicate that the AZM-mediated phenotype is caused by a depletion of the intracellular pools of tRNAs available for protein synthesis. Overexpression of the P. aeruginosa peptidyl-tRNA hydrolase, which recycles the tRNA from peptidyl-tRNA drop-off during translation, counteracted the effects of AZM on stationary-phase cell killing, cytotoxicity, and the production of rhamnolipids and partially restored swarming motility. Intriguingly, the exchange of a rare for a frequent codon in rhlR also explicitly diminished the AZM-mediated decreased production of rhamnolipids. These results indicate that depletion of the tRNA pools by AZM seems to affect the translation of genes that use rare aminoacyl-tRNA isoacceptors to a great extent and might explain the selective activity of AZM on the P. aeruginosa proteome and possibly also on the protein expression profiles of other bacterial pathogens.