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2.  Pentamidine inhibits Coxiella burnetii growth and 23S rRNA intron splicing in vitro 
Coxiella burnetii is the bacterial agent of Q fever in humans. Acute Q fever generally manifests as a flu-like illness and is typically self-resolving. In contrast, chronic Q fever usually presents with endocarditis and is often life-threatening without appropriate antimicrobial therapy. Unfortunately, available options for the successful treatment of chronic Q fever are both limited and protracted (>18 months). Pentamidine, an RNA splice inhibitor used to treat fungal and protozoal infections, was shown to reduce intracellular growth of Coxiella by ca. 73% at a concentration of 1 μM (ca. 0.6 μg/mL) compared with untreated controls, with no detectable toxic effects on host cells. Bacterial targets of pentamidine include Cbu.L1917 and Cbu.L1951, two group I introns that disrupt the 23S rRNA gene of Coxiella, as demonstrated by the drug's ability to inhibit intron RNA splicing in vitro. Since both introns are highly conserved among all eight genotypes of the pathogen, pentamidine is predicted to be efficacious against numerous strains of C. burnetii. To our knowledge, this is the first report describing antibacterial activity for this antifungal/antiprotozoal agent.
PMCID: PMC2926257  PMID: 20599360
Coxiella; Pentamidine; Group I intron; RNA splicing
3.  Comparison of transformation frequencies among selected Streptococcus pneumoniae serotypes 
Although there are over 90 serotypes of Streptococcus pneumoniae, antimicrobial resistance is predominantly found in a limited number of serotypes/serogroups, namely 6, 9, 14, 19 and 23. There is no compelling mechanism to account for this restriction. We aimed to determine whether serotypes commonly associated with drug resistance have higher transformation frequencies than those that are susceptible to antimicrobial agents. An in vitro investigation of the genetic transformation frequency of drug-resistant serotypes compared with that of susceptible serotypes under the influence of synthetic competence-stimulating peptides was performed. The transforming DNA was genomic DNA carrying a Tn916-like transposon containing the mefE gene that confers resistance to erythromycin. It was observed that serotypes 6, 9, 14, 19 and 23, which are highly associated with drug resistance, do not exhibit a higher degree of transformation efficiency than other serotypes. These findings suggest that the association of serotype with drug resistance is likely due to prolonged exposure to transforming DNA resulting from longer nasopharyngeal carriage and to a greater selective pressure from antimicrobials, particularly in children. This is the first study to compare the transformation frequencies of pneumococcal clinical isolates using genomic DNA that carries the composite Tn916-like element.
PMCID: PMC2902549  PMID: 20472405
Streptococcus pneumoniae; Drug-resistant serotypes/serogroups; Transformation frequency; Tn916 transposon; mefE gene
4.  Candidate vaginal microbicides with activity against Chlamydia trachomatis and Neisseria gonorrhoeae 
Vaginal microbicides with activity towards organisms that cause sexually transmitted infections have been proposed as a strategy to reduce transmission. Small-molecule inhibitors of Chlamydia trachomatis serovar D belonging to the class of salicylidene acylhydrazides (INPs) have been shown to work through a mechanism that involves iron restriction. Expanding on this work, ten INPs were tested against a lymphogranuloma venereum strain of C. trachomatis serovar L2, Neisseria gonorrhoeae, and hydrogen peroxide-producing Lactobacillus crispatus and Lactobacillus jensenii. Seven INPs had minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations of <50 µM towards C. trachomatis L2. Three INPs had an MIC <12.5 µM against N. gonorrhoeae. Inhibition by was reversed by iron, holo-transferrin and holo-lactoferrin but not by the iron-poor forms of these compounds. The compounds exhibited no bactericidal activity toward Lactobacillus. The INPs were not cytotoxic to HeLa 229 cells. When INP 0341 was tested in a mouse model of a Chlamydia vaginal infection there was a significant reduction in the number of mice shedding C. trachomatis up to 4 days after infection (P < 0.01). In summary, select INPs are promising vaginal microbicide candidates as they inhibit the growth of two common sexually transmitted organisms in vitro, are active in a mouse model against C. trachomatis, are not cytotoxic and do not inhibit organisms that compose the normal vaginal flora.
PMCID: PMC2902681  PMID: 20605703
Vaginal microbicide; Sexually transmitted infections; Chlamydia trachomatis; LGV; Neisseria gonorrhoeae
5.  A new-generation 5-nitroimidazole can induce highly metronidazole-resistant Giardia lamblia in vitro 
The 5-nitroimidazole (NI) compound C17, with a side chain carrying a remote phenyl group in the 2-position of the imidazole ring, is at least 14-fold more active against the gut protozoan parasite Giardia lamblia than the 5-NI drug metronidazole (MTR), with a side chain in the 1-position of the imidazole ring, which is the primary drug for the treatment of giardiasis. Over 10 months, lines resistant to C17 were induced in vitro and were at least 12-fold more resistant to C17 than the parent strains. However, these lines had ID90 values (concentration of drug at which 10% of control parasite ATP levels are detected) for MTR of >200 μM, whilst lines induced to be highly resistant to MTR in vitro have maximum ID90 values around 100 μM (MTR-susceptible isolates typically have an ID90 of 5–12.8 μM). The mechanism of MTR activation in Giardia apparently involves reduction to toxic radicals by the activity of pyruvate:ferredoxin oxidoreductase (PFOR) and the electron acceptor ferredoxin. MTR-resistant Giardia have decreased PFOR activity, which is consistent with decreased activation of MTR in these lines, but C17-resistant lines have normal levels of PFOR. Therefore, an alternative mechanism of resistance in Giardia must account for these super-MTR-resistant cells.
PMCID: PMC3103471  PMID: 20456926
Pyruvate:ferredoxin oxidoreductase; Tinidazole; Ronidazole; 5-Nitroimidazole; Cross-resistance
6.  Comparison of two DNA microarrays for detection of plasmid-mediated antimicrobial resistance and virulence factor genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae 
A DNA microarray was developed to detect plasmid-mediated antimicrobial resistance (AR) and virulence factor (VF) genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae. The array was validated with the following bacterial species: Escherichiacoli (n = 17); Klebsiellapneumoniae (n = 3); Enterobacter spp. (n = 6); Acinetobacter genospecies 3 (n = 1); Acinetobacterbaumannii (n = 1); Pseudomonasaeruginosa (n = 2); and Stenotrophomonasmaltophilia (n = 2). The AR gene profiles of these isolates were identified by polymerase chain reaction (PCR). The DNA microarray consisted of 155 and 133 AR and VF gene probes, respectively. Results were compared with the commercially available Identibac AMR-ve Array Tube. Hybridisation results indicated that there was excellent correlation between PCR and array results for AR and VF genes. Genes conferring resistance to each antibiotic class were identified by the DNA array. Unusual resistance genes were also identified, such as blaSHV-5 in a blaOXA-23-positive carbapenem-resistant A. baumannii. The phylogenetic group of each E. coli isolate was verified by the array. These data demonstrate that it is possible to screen simultaneously for all important classes of mobile AR and VF genes in Enterobacteriaceae and non-Enterobacteriaceae whilst also assigning a correct phylogenetic group to E. coli isolates. Therefore, it is feasible to test clinical Gram-negative bacteria for all known AR genes and to provide important information regarding pathogenicity simultaneously.
PMCID: PMC3820026  PMID: 20356716
β-Lactamases; Quinolones; Virulence; Molecular detection

Results 1-6 (6)