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1.  sarA inactivation reduces vancomycin-intermediate and ciprofloxacin resistance expression by Staphylococcus aureus 
International journal of antimicrobial agents  2009;34(2):10.1016/j.ijantimicag.2009.01.018.
It is known that multiple genome-wide transcriptional changes often accompany the development of antimicrobial resistance and occur in response to challenge with antimicrobial agents. We now show that inactivation of the staphylococcal accessory gene regulator sarA, which controls at least tens of genes in Staphylococcus aureus, leads to dramatic reductions in vancomycin and ciprofloxacin resistance in vancomycin-intermediate and ciprofloxacin-resistant strains of S. aureus. This is particularly evident when judged by antimicrobial-gradient plate analysis or population analysis profiles. Whilst the intact sarA cistron is required for full vancomycin resistance expression by vancomycin-intermediate S. aureus (VISA), sarA expression as determined by real-time polymerase chain reaction was found to be VISA strain-dependent. Reductions in vancomycin resistance expression levels following sarA inactivation do not necessarily include an alteration in autolysis. Expression of sarR, the negative regulator of sarA, was downregulated in two VISA mutants, and transcription of the alternative sigma factor sigB was downregulated in one VISA strain. This study contributes to a growing body of evidence demonstrating the importance of loci previously identified to control virulence in the regulation of clinically relevant antibiotic resistance mechanisms.
PMCID: PMC3831611  PMID: 19324528
Staphylococcus aureus; VISA; Ciprofloxacin; Resistance; sarA
2.  Inhibition of Listeria monocytogenes infection by neurological drugs 
To gain insights into the cellular processes required for intracellular bacterial pathogenesis, we previously developed a generalisable screening approach to identify small molecule compounds that alter Listeria monocytogenes infection. In this report, a small molecule library enriched for compounds affecting neurological functions was screened and 68 compounds that disrupted L. monocytogenes infection of macrophages were identified. Many of these compounds were known antimicrobial agents, however 26 compounds were novel inhibitors of intracellular infection. Two of the compounds chosen for further study, the antipsychotic drug thioridazine and the calcium channel blocker bepridil, exhibited dose-dependent inhibition of vacuolar escape and intracellular replication of L. monocytogenes during infection of murine macrophages. These results suggest that clinically approved neurological drugs may provide a novel source of anti-infective agents that are suitable for development as therapeutics against intracellular bacterial infections.
PMCID: PMC2818453  PMID: 20031379
Listeria monocytogenes; Small molecule screen; Intracellular infection; Bepridil; Neurological compounds; Thioridazine
3.  Detection of point mutations associated with antibiotic resistance in Pseudomonas aeruginosa 
Excessive use of broad-spectrum antibiotics in hospitals has led to the emergence of highly resistant strains of Pseudomonas aeruginosa. To reduce the selection pressure for resistance, it is important to determine the antibiotic susceptibility pattern of bacteria so that hospital patients can be treated with more narrow-spectrum and target-specific antibiotics. This study describes the development of a technique for detecting point muations in the fluoroquinolone resistance-determining region of the gyrA and parC genes as well as the efflux regulatory genes mexR, mexZ and mexOZ that are associated with fluoroquinolone and aminoglycoside resistance. The assay is based on a short DNA sequencing method using multiplex-fast polymerase chain reaction (PCR) and Pyrosequencing™ for amplification and sequencing of the selected genes. Fifty-nine clinical isolates of P. aeruginosa were examined for mutations in the abovementioned genes. Mutations related to antibiotic resistance were detected in codons 83 and 87 of gyrA and codon 126 of the mexR regulatory gene. Results of this study suggest Pyrosequencing™ as a substitute for traditional methods as it provides a rapid and reliable technique for determinating the antibiotic resistance pattern of a given bacterial strain in <1 h.
PMCID: PMC2744841  PMID: 19656662
Pyrosequencing; Pseudomonas aeruginosa; Antibiotic resistance
5.  Design and activity of a ‘dual-targeted’ antimicrobial peptide 
Numerous reports have indicated the important role of human normal flora in the prevention of microbial pathogenesis and disease. Evidence suggests that infections at mucosal surfaces result from the outgrowth of subpopulations or clusters within a microbial community and are not linked to one pathogenic organism alone. To preserve the protective normal flora while treating the majority of infective bacteria in the community, a tuneable therapeutic is necessary that can discriminate between benign bystanders and multiple pathogenic organisms. Here we describe the proof-of-principle for such a multitargeted antimicrobial: a multiple-headed specifically-targeted antimicrobial peptide (MH-STAMP). The completed MH-STAMP, M8(KH)-20, displays specific activity against targeted organisms in vitro (Pseudomonas aeruginosa and Streptococcus mutans) and can remove both species from a mixed planktonic culture with little impact against untargeted bacteria. These results demonstrate that a functional, dual-targeted molecule can be constructed from a wide-spectrum antimicrobial peptide precursor.
PMCID: PMC2696886  PMID: 19188046
Antimicrobial peptide; Targeted therapeutic; Streptococcus mutans; Pseudomonas aeruginosa; Peptide synthesis; Novel antibiotic; STAMP; Specifically-targeted antimicrobial peptide; MH-STAMP
6.  Characterisation of a Staphylococcus aureus strain with progressive loss of susceptibility to vancomycin and daptomycin during therapy✰ 
Following an initial response to vancomycin therapy, a patient with meticillin-resistant Staphylococcus aureus (MRSA) bacteraemia developed endocarditis, failed a second course of vancomycin and then failed daptomycin therapy. An increase in the vancomycin minimum inhibitory concentrations of four consecutive MRSA blood isolates from 2 μg/mL to 8 μg/mL was shown by Etest. Population analysis of four successive blood culture isolates recovered over the 10-week period showed that the MRSA strain became progressively less susceptible to both vancomycin and daptomycin. Retrospectively, the macro Etest method using teicoplanin indicated a decrease in vancomycin susceptibility in the second blood isolate. The patient improved after treatment with various courses of trimethoprim/sulphamethoxazole, quinupristin/dalfopristin and linezolid. Early detection of vancomycin-heteroresistant S. aureus isolates, which appeared to have clinical significance in this case, continues to be a challenge for the clinical laboratory. Development of suitable practical methods for this should be given priority. Concurrent development of resistance to vancomycin and daptomycin, whilst rare, must be considered in a patient who is unresponsive to daptomycin following vancomycin therapy.
PMCID: PMC2700752  PMID: 19233622
Staphylococci; Vancomycin; Teicoplanin; Heteroresistance
7.  Green tea inhibits Helicobacter growth in vivo and in vitro 
Helicobacter infection, one of the most common bacterial infections in man worldwide, is a type 1 carcinogen and the most important risk factor for gastric cancer. Helicobacter pylori bacterial factors, components of the host genetics and immune response, dietary cofactors and decreased acid secretion resulting in bacterial overgrowth are all considered important factors for induction of gastric cancer. Components found in green tea have been shown to inhibit bacterial growth, including the growth of Helicobacter spp. In this study, we assessed the bactericidal and/or bacteriostatic effect of green tea against Helicobacter felis and H. pylori in vitro and evaluated the effects of green tea on the development of Helicobacter-induced gastritis in an animal model. Our data clearly demonstrate profound growth effects of green tea against Helicobacter and, importantly, demonstrate that green tea consumption can prevent gastric mucosal inflammation if ingested prior to exposure to Helicobacter infection. Research in the area of natural food compounds and their effects on various disease states has gained increased acceptance in the past several years. Components within natural remedies such as green tea could be further used for prevention and treatment of Helicobacter-induced gastritis in humans.
PMCID: PMC2694061  PMID: 19157800
Helicobacter felis; Helicobacter pylori; Gastric cancer; Green tea; Catechins; Diet
8.  N-(2-hydroxypropyl)methacrylamide–amphotericin B (HPMA–AmB) copolymer conjugates as antileishmanial agents 
Leishmaniasis is a major health problem in many parts of the world, caused by various species of Leishmania. Amastigotes are the clinically relevant form of the parasite in the human host and reside in the parasitophorous vacuole within macrophages. Polymer–drug conjugates have been used for lysosomotropic drug delivery and have already shown potential in anticancer and antileishmanial chemotherapy. We synthesised N-(2-hydroxypropyl)methacrylamide–amphotericin B (HPMA–AmB) copolymer conjugates in which the AmB was attached to the polymer through a degradable GlyPheLeuGly linker. Antileishmanial activity was assessed in vitro against intracellular amastigotes in host macrophages [murine peritoneal exudate macrophages (PEMs), murine bone marrow-derived macrophages (BMMs) and differentiated THP-1 cells]. The most potent copolymers had 50% effective concentration (EC50) values of 0.03 μg/mL AmB equivalent against Leishmania donovani amastigotes in PEMs and BMMs and an EC50 of 0.57 μg/mL AmB equivalent against L. donovani in THP-1 cells. This activity was comparable with free AmB (EC50 = 0.03–0.07 μg/mL against L. donovani in PEMs and BMMs and 0.24–0.42 μg/mL against amastigotes in THP-1 cells) and Fungizone® (EC50 = 0.04–0.07 μg/mL against amastigotes in PEMs). Conjugates also showed potent in vivo activity with ca. 50% inhibition of parasite burden at 1 mg/kg body weight.
PMCID: PMC2669511  PMID: 19097763
Leishmaniasis; Copolymers; Amphotericin B

Results 1-8 (8)