Screening a mushroom extract library for activity against Acinetobacter baumannii and Burkholderia cepacia and the identification of a compound with anti-Burkholderia activity

<p>Abstract</p> <p>Background</p> <p><it>Acinetobacter baumannii </it>and species within the <it>Burkholderia cepacia </it>complex (BCC) are significant opportunistic bacterial pathogens of humans. These species exhibit a high degree of antibiotic resistance, and some clinical isolates are resistant to all currently available antimicrobial drugs used for treatment. Thus, new drugs are needed to treat infections by these species. Mushrooms could be a potential source for new drugs to treat <it>A. baumannii </it>and BCC infections.</p> <p>Methods</p> <p>The aim of this study was to screen a library of crude extracts from 330 wild mushrooms by disk diffusion assays for antibacterial activity against <it>A. baumannii </it>and <it>Burkholderia cepacia </it>in the hope of identifying a novel natural drug that could be used to treat infections caused by these species. Once positive hits were identified, the extracts were subjected to bioassay-guided separations to isolate and identify the active drug molecules. MICs were performed to gauge the <it>in vitro </it>activity of the purified compounds.</p> <p>Results</p> <p>Only three crude extracts (0.9%) had activity against <it>A. baumannii </it>and <it>B. cepacia</it>. Compounds from two of these extracts had MICs greater than 128 μg/ml, and further analyses were not performed. From the third extract, prepared from <it>Leucopaxillus albissimus</it>, 2-aminoquinoline (2-AQ) was isolated. This compound exhibited a modest MIC <it>in vitro </it>against strains from nine different BCC species, including multi-drug resistant clinical isolates (MIC = 8-64 μg/ml), and a weak MIC (128 μg/ml) against <it>A baumannii</it>. The IC₅₀against a murine monocyte line was 1.5 mg/ml.</p> <p>Conclusion</p> <p>The small number of positive hits in this study suggests that finding a new drug from mushrooms to treat Gram-negative bacterial infections may be difficult. Although 2-AQ was identified in one mushroom, and it was shown to inhibit the growth of multi-drug resistant BCC isolates, the relatively high MICs (8-128 μg/ml) for both <it>A. baumannii </it>and BCC strains suggests that 2-AQ is not suitable for further drug development in its current form.</p

Use of optical mapping to sort uropathogenic Escherichia coli strains into distinct subgroups

Optical maps were generated for 33 uropathogenic Escherichia coli (UPEC) isolates. For individual genomes, the NcoI restriction fragments aligned into a unique chromosome map for each individual isolate, which was then compared with the in silico restriction maps of all of the sequenced E. coli and Shigella strains. All of the UPEC isolates clustered separately from the Shigella strains as well as the laboratory and enterohaemorrhagic E. coli strains. Moreover, the individual strains appeared to cluster into distinct subgroups based on the dendrogram analyses. Phylogenetic grouping of these 33 strains showed that 32/33 were the B2 subgroup and 1/33 was subgroup A. To further characterize the similarities and differences among the 33 isolates, pathogenicity island (PAI), haemolysin and virulence gene comparisons were performed. A strong correlation was observed between individual subgroups and virulence factor genes as well as haemolysis activity. Furthermore, there was considerable conservation of sequenced-strain PAIs in the specific subgroups. Strains with different antibiotic-resistance patterns also appeared to sort into separate subgroups. Thus, the optical maps distinguished the UPEC strains from other E. coli strains and further subdivided the strains into distinct subgroups. This optical mapping procedure holds promise as an alternative way to subgroup all E. coli strains, including those involved in infections outside of the intestinal tract and epidemic strains with distinct patterns of antibiotic resistance

SK-03-92 Drug Kills Intracellular <i>Mycobacterium tuberculosis</i>

Background: Tuberculosis affects millions of people worldwide. The emergence of drug-resistant Mycobacterium tuberculosis strains has made treatment more difficult. A drug discovery project initiated to screen natural products identified a lead stilbene compound, and structure function analysis of hundreds of analogs led to the discovery of the SK-03-92 stilbene lead compound with activity against several non-tuberculoid mycobacteria. Methods: For this study, an MIC analysis and intracellular killing assay were performed to test SK-03-92 against M. tuberculosis grown in vitro as well as within murine macrophage cells. Results: The MIC analysis showed that SK-03-92 had activity against M. tuberculosis in the range of 0.39 to 6.25 μg/mL, including activity against single-drug-resistant strains. Further, an intracellular kill assay demonstrated that the SK-03-92 lead compound killed M. tuberculosis cells within murine macrophage cells. Conclusion: Together, the data show the SK-03-92 lead compound can kill M. tuberculosis bacteria within mammalian macrophages

Proline Transport and Growth Changes in Proline Transport Mutants of <i>Staphylococcus aureus</i>

Staphylococcus aureus is a major cause of skin/soft tissue infections and more serious infections in humans. The species usually requires the importation of proline to be able to survive. Previous work has shown that single mutations in genes that encode for proline transporters affect the ability of S. aureus to survive in vitro and in vivo. To better understand proline transport in S. aureus, double and triple gene mutant strains were created that targeted the opuD, proP, and putP genes. Single gene mutants had some effect on proline transport, whereas double mutants exhibited significantly lower proline transport. An opuD prop putP triple gene mutant displayed the lowest proline transport under low- and high-affinity conditions. To assess growth differences caused by the mutations, the same mutants were grown in brain heart infusion (BHI) broth and defined staphylococcal medium (DSM) with various concentrations of proline. The triple mutant did not grow in DSM with a low concentration of proline and grew poorly in both DSM with a high proline concentration and BHI broth. These results show that S. aureus has multiple mechanisms to import proline into the cell and knocking out three of the main proline transporters significantly hinders S. aureus growth

Staphylococcus aureus Toxins: Armaments for a Significant Pathogen

Staphylococcus species are common inhabitants of humans and other animals [...

Temporal Regulation of fim Genes in Uropathogenic Escherichia coli during Infection of the Murine Urinary Tract

Uropathogenic Escherichia coli (UPEC) adhere to cells in the human urinary tract via type 1 pili that undergo phase variation where a 314-bp fimS DNA element flips between Phase-ON and Phase-OFF orientations through two site-specific recombinases, FimB and FimE. Three fim-lux operon transcriptional fusions were created and moved into the clinical UPEC isolate NU149 to determine their temporal regulation in UPEC growing in the urinary tract. Within murine urinary tracts, the UPEC strains demonstrated elevated transcription of fimA and fimB early in the infection, but lower transcription by the fifth day in murine kidneys. In contrast, fimE transcription was much lower than either fimA or fimB early, increased markedly at 24 h after inoculation, and then dropped five days after inoculation. Positioning of fimS was primarily in the Phase-ON position over the time span in UPEC infected bladders, whereas in UPEC infected murine kidneys the Phase-OFF orientation was favored by the fifth day after inoculation. Hemagglutination titers with guinea pig erythrocytes remained constant in UPEC growing in infected murine bladders but fell substantially in UPEC infected kidneys over time. Our results show temporal in vivo regulation of fim gene expression in different environmental niches when UPEC infects the murine urinary tract

Transcriptional Activation of the Staphylococcus aureus putP Gene by Low-Proline-High Osmotic Conditions and during Infection of Murine and Human Tissues

Staphylococcus aureus can grow virtually anywhere in the human body but needs to import proline through low- and high-affinity proline transporters to survive. This study examined the regulation of the S. aureus putP gene, which encodes a high-affinity proline permease. putP::lacZ and putP::lux transcriptional fusions were constructed and integrated into the genomes of several S. aureus strains. Enzyme activity was measured after growth in media with various osmolyte concentrations. As osmolarity rose, putP expression increased, with a plateau at 2 M for NaCl in strain LL3-1. Proline concentrations as low as 17.4 μM activated expression of the putP gene. The putP::lux fusion was also integrated into the genomes of S. aureus strains that were either SigB inactive (LL3-1, 8325-4, and SH1003) or SigB active (Newman and SH1000). SigB inactive strains showed increased putP gene expression as NaCl concentrations rose, whereas SigB active strains displayed a dramatic decrease in putP expression, suggesting that the alternative sigma factor B plays a negative role in putP regulation. Mice inoculated with S. aureus strains containing the putP::lux fusion exhibited up to a 715-fold increase in putP expression, although levels in the various murine organs differed. Moreover, urine from human patients infected with S. aureus showed elevated putP levels by use of a PCR procedure, whereas blood and some abscess material had no significant increase. Thus, putP is transcriptionally activated by a low-proline and high osmotic environment both in growth media and in murine or human clinical specimens

The Two-Component Response Regulator RcsB Regulates Type 1 Piliation in Escherichia coli▿

The ability of Escherichia coli cells to produce type 1 pili depends upon the orientation of the fimA promoter. The orientation depends upon the ratios of the FimB and FimE recombinases. Here, we report that the two-component response regulator RcsB influences the piliation state by controlling fimB and fimE transcription