Keith Miller

The role of mutators in the emergence of antibiotic-resistant bacteria

Bacteria contain a number of error prevention and error correction systems that maintain genome stability. However, strains exhibiting elevated mutation frequencies have recently been reported amongst natural populations of pathogenic Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, Neisseria meningitidis, Helicobacter pylori and Streptococcus pneumoniae. The majority of naturally occurring, strong mutators contain defects in the methyl-directed mismatch repair (MMR) system, with mutations in mutS predominating. MMR-deficient strains possess superior genetic backgrounds for the selection of some antibiotic-resistance mutations since mutation frequencies up to 1000-fold higher than normal strains have been reported, and resistance levels achieved in mutators can be greater than those arising in non-mutator hosts. MMR is a major constraint to interspecies recombination events. Removal of this barrier, as in the case of MMR defective mutators, also enhances the frequency of horizontal gene transfer, which is an important mechanism of acquired drug resistance in bacteria. Permanent global mutator status is associated with loss of fitness as mutators accumulate deleterious mutations more frequently than non-mutators. Fitness limitations of mutators may be overcome simply by the high bacterial cell densities that can be achieved during acute infection or by the adoption of transient mutator status. Mutators are a risk factor during the treatment of bacterial infections as they appear to enhance the selection of mutants expressing high- and low-level antibiotic resistance and have the capacity to refine existing plasmid-located resistance determinants.

Treatment of health-care-associated infections caused by Gram-negative bacteria: a consensus statement

This consensus statement presents the conclusions of a group of academic and industrial experts who met in London in September, 2006, to consider the issues associated with the treatment of hospital infections caused by Gram-negative bacteria. The group discussed the severe clinical problems arising from the emergence of antibiotic resistance in these bacteria and the lack of new antibacterial agents to challenge the threat. The discovery of new drugs active against hospital-acquired Gram-negative bacteria is essential to prevent a future medical and social catastrophe. An important strategy to promote drug discovery will be the development of focused cooperations between academic institutions and small pharmaceutical companies.

Increased mutability of Pseudomonas aeruginosa in biofilms

OBJECTIVES Isolates of Pseudomonas aeruginosa from cystic fibrosis (CF) patients are frequently hypermutable due to selection of mutants with defects in DNA repair genes such as mutS. Since P. aeruginosa grows as a biofilm within the infected CF lung, it is possible that this mode of growth enhances the mutability of the organism thereby increasing the opportunity to derive permanent hypermutators through mutation in DNA repair genes. We have now conducted experiments to examine this possibility. METHODS Using established procedures, we examined the mutability of P. aeruginosa PA01 in planktonic cultures and in biofilm cultures generated by growth in a Sorbarod system. Transcriptional profiling by DNA microarray was used to compare gene expression in planktonic and biofilm cells. RESULTS Mutation frequency determinations for resistance to rifampicin and ciprofloxacin demonstrated that biofilm cultures of P. aeruginosa displayed up to a 105-fold increase in mutability compared with planktonic cultures. Several genes (ahpC, katA, sodB and PA3529, a probable peroxidase) that encode enzymes conferring protection against oxidative DNA damage were down-regulated in biofilm cells. In particular, katA, which encodes the major pseudomonal antioxidant catalase, was down-regulated 7.7-fold. CONCLUSIONS Down-regulation of antioxidant enzymes in P. aeruginosa biofilms may enhance the rate of mutagenic events due to the accumulation of DNA damage. Since P. aeruginosa forms biofilms in the CF lung, this mode of growth may enhance the direct selection of antibiotic-resistant organisms in CF patients and also increase the opportunity to derive permanent hypermutators thereby providing a further source of antibiotic-resistant mutants in the CF lung.

Consequences of daptomycin-mediated membrane damage in Staphylococcus aureus

OBJECTIVES The proposed lethal action of daptomycin on Staphylococcus aureus results from the loss of K(+) and membrane depolarization. However, whether these events alone cause cell death has been questioned. We sought to determine whether other consequences of daptomycin-mediated membrane damage may contribute to cell death. METHODS Previously established assays were used to evaluate the membrane damaging activity of daptomycin at a single time-point of 10 min. More detailed time-course experiments were also performed to determine the kinetics of membrane depolarization and leakage of K(+), Mg(2+) and ATP. The kinetics of inhibition of macromolecular synthesis following exposure to daptomycin were also determined by assaying the incorporation of radioactive precursors into macromolecules. RESULTS Daptomycin exhibited no membrane damaging activity in single time-point assays following exposure to the antibiotic for 10 min. Kinetic analysis confirmed these results as leakage of intracellular components did not occur until 20-30 min, membrane depolarization was gradual and cells remained biosynthetically active for at least 30 min after exposure to daptomycin. Viability declined rapidly after exposure to daptomycin and appeared to precede other detectable changes. CONCLUSIONS These data show that daptomycin-induced loss of Mg(2+) and ATP occurs in conjunction with the previously reported leakage of K(+) and membrane depolarization. We propose that the lethal activity of daptomycin is not simply due to loss of K(+) and probably involves more general damage to the membrane.

Antimicrobial peptides from scorpion venoms

Abstract The need for new antimicrobial agents is becoming one of the most urgent requirements in modern medicine. The venoms of many different species are rich sources of biologically active components and various therapeutic agents have been characterized including antimicrobial peptides (AMPs). Due to their potent activity, low resistance rates and unique mode of action, AMPs have recently received much attention. This review focuses on AMPs from the venoms of scorpions and examines all classes of AMPs found to date. It gives details of their biological activities with reference to peptide structure. The review examines the mechanism of action of AMPs and with this information, suggests possible mechanisms of action of less well characterised peptides. Finally, the review examines current and future trends of scorpion AMP research, by discussing recent successes obtained through proteomic and transcriptomic approaches.

XF-70 and XF-73, novel antibacterial agents active against slow-growing and non-dividing cultures of Staphylococcus aureus including biofilms

OBJECTIVES Slow-growing and non-dividing bacteria exhibit tolerance to many antibiotics. However, membrane-active agents may act against bacteria in all growth phases. We sought to examine whether the novel porphyrin antibacterial agents XF-70 and XF-73, which have rapid membrane-perturbing activity against Staphylococcus aureus, retained antistaphylococcal activity against growth-attenuated cells. METHODS The killing kinetics of XF-70, XF-73 and various comparator agents against exponential phase cultures of S. aureus SH1000 were compared with effects on cells held at 4 degrees C, non-growing cultures expressing the stringent response induced by mupirocin and bacteria in the stationary phase. Biofilms of S. aureus SH1000 were generated with the Calgary device to examine the activities of XF-70 and XF-73 under a further system exhibiting diminished bacterial growth. RESULTS Cold culture, stringent response and stationary phase cultures remained susceptible to XF-70 and XF-73, which caused > or =5 log reductions in viability over 2 h. During this period the most active comparator agents (chlorhexidine and cetyltrimethylammonium bromide) only promoted a 3 log drop in viability. XF-70 and XF-73 were also highly active against biofilms, with both agents exhibiting low biofilm MICs (1 mg/L) and minimum biofilm eradication concentrations (2 mg/L). CONCLUSIONS XF-70 and XF-73 remained highly active against various forms of slow-growing or non-dividing S. aureus. The results support the hypothesis that membrane-active agents may be particularly effective in eradicating slow- or non-growing bacteria and suggest that XF-70 and XF-73 could be utilized to treat staphylococcal infections where the organisms are only dividing slowly, such as biofilm-associated infections of prosthetic devices.

Linezolid and Tiamulin Cross-Resistance in Staphylococcus aureus Mediated by Point Mutations in the Peptidyl Transferase Center

ABSTRACT Oxazolidinone and pleuromutilin antibiotics are currently used in the treatment of staphylococcal infections. Although both antibiotics inhibit protein synthesis and have overlapping binding regions on 23S rRNA, the potential for cross-resistance between the two classes through target site mutations has not been thoroughly examined. Mutants of Staphylococcus aureus resistant to linezolid were selected and found to exhibit cross-resistance to tiamulin, a member of the pleuromutilin class of antibiotics. However, resistance was unidirectional because mutants of S. aureus selected for resistance to tiamulin did not exhibit cross-resistance to linezolid. This contrasts with the recently described PhLOPSA phenotype, which confers resistance to both oxazolidinones and pleuromutilins. The genotypes responsible for the phenotypes we observed were examined. Selection with tiamulin resulted in recovery of mutants with changes in the single-copy rplC gene (Gly155Arg, Ser158Leu, or Arg149Ser), whereas selection with linezolid led to recovery of mutants with changes (G2576U in 23S rRNA) in all five copies of the multicopy operon rrn. In contrast, cross-resistance to linezolid was exhibited by tiamulin-resistant mutants generated in a single-copy rrn knockout strains of Escherichia coli, illustrating that the copy number of 23S rRNA is the limiting factor in the selection of 23S rRNA tiamulin-resistant mutants. The interactions of linezolid and tiamulin with the ribosome were modeled to seek explanations for resistance to both classes in the 23S rRNA mutants and the lack of cross-resistance between tiamulin and linezolid following mutation in rplC.

XF-73, a novel antistaphylococcal membrane-active agent with rapid bactericidal activity

OBJECTIVES XF-73 is a novel porphyrin antibacterial agent previously reported to inhibit a range of gram-positive bacterial species, including Staphylococcus aureus. Its mode of action is unknown. Using S. aureus as a model organism we sought to examine the basis of its antibacterial activity. METHODS The effects of XF-73 on the growth and survival of S. aureus SH1000 were investigated by viable count and culture absorbance techniques. Inhibition of macromolecular synthesis and disruption of membrane integrity after exposure to XF-73 were examined by radiolabelling experiments, the BacLight fluorescent dye assay and measurement of K(+) and ATP leakage from the cell. The effect of XF-73 on a staphylococcal coupled transcription-translation system was also investigated. RESULTS XF-73 was rapidly bactericidal against S. aureus SH1000 and demonstrated more rapid killing kinetics than all other comparator agents when tested at an equivalent multiple (4x) of the MIC. Exposure of S. aureus to XF-73 for 10 min completely inhibited DNA, RNA and protein synthesis. XF-73 had no effect on transcription and translation in vitro. Cells exposed to XF-73 gave a positive response in the BacLight assay, which detects membrane damage. The drug also caused substantial loss of K(+) and ATP from the cell, but did not promote bacterial lysis. CONCLUSIONS XF-73 exhibited rapid membrane-perturbing activity, which is likely to be responsible for inhibition of macromolecular synthesis and the death of staphylococci exposed to the drug.

Escherichia coli Mutators Present an Enhanced Risk for Emergence of Antibiotic Resistance during Urinary Tract Infections

ABSTRACT Mutators may present an enhanced risk for the emergence of antibiotic resistance in bacteria during chemotherapy. Using Escherichia coli mutators as a model, we evaluated their ability to develop resistance to antibiotics routinely used for the treatment of urinary tract infections (UTIs). Under conditions that simulate therapeutic drug concentrations in humans, low-level resistance to trimethoprim, gentamicin, and cefotaxime emerged more frequently in mutators than normal strains. Resistance to trimethoprim in both cell types arose from a single point mutation in folA (Ile94→Leu) and cefotaxime resistance resulted from loss of outer membrane porin OmpF. The mechanisms of gentamicin resistance could not be defined, but resistance did not result from mutations in ribosomal protein L6 (rplF). Although similar mechanisms of low-level antibiotic resistance probably arise in these strains, mutators are a risk factor because the increased generation of mutants with low-level resistance enhances the opportunity for subsequent emergence of high-level resistance.

Delayed Development of Linezolid Resistance in Staphylococcus aureus following Exposure to Low Levels of Antimicrobial Agents

ABSTRACT The development of resistance to linezolid (LZD) in gram-positive bacteria depends on the mutation of a single 23S rRNA gene, followed by homologous recombination and gene conversion of the other alleles. We sought to inhibit this process in Staphylococcus aureus using a range of antibacterial agents, including some that suppress recombination. A model for the rapid selection of LZD resistance was developed which allowed the selection of LZD-resistant mutants with G2576T mutations in all five copies of the 23S rRNA gene following only 5 days of subculture. The emergence of LZD-resistant isolates was delayed by exposing cultures to low concentrations of various classes of antibiotics. All antibiotic classes were effective in delaying the selection of LZD-resistant mutants and, with the exception of fusidic acid (FUS) and rifampin (RIF), prolonged the selection window from 5 to ∼15 days. Inhibitors of DNA processing were no more effective than any other class of antibiotics at suppressing resistance development. However, the unrelated antimicrobials FUS and RIF were particularly effective at preventing the emergence of LZD resistance, prolonging the selection window from 5 to 25 days. The enhanced suppressive effect of FUS and RIF on the development of LZD resistance was lost in a recA-deficient host, suggesting that these drugs affect recA-dependent recombination. Furthermore, FUS and RIF were shown to be effective inhibitors of homologous recombination of a plasmid into the staphylococcal chromosome. We suggest that RIF or FUS in combination with LZD may have a role in preventing the emergence of LZD resistance.