[Show abstract][Hide abstract] ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) infections are a leading cause of death among all fatalities caused by antibiotic-resistant bacteria. With the rise of increasing resistance to current antibiotics, new antimicrobials and treatment strategies are urgently needed. Thiazole compounds have been shown to possess potent antimicrobial activity. A lead thiazole 1 and a potent derivative 2 were synthesized and their activity in combination with glycopeptide antibiotics was determined against an array of MRSA and vancomycin-resistant S. aureus (VRSA) clinical isolates. In addition, the anti-biofilm activity of the novel thiazoles was investigated against S. epidermidis. Compound 2 behaved synergistically with vancomycin against MRSA and was able to resensitize VRSA to vancomycin, reducing its MIC by 512-fold in two strains. In addition, both thiazole compounds were superior to vancomycin in significantly reducing S. epidermidis biofilm mass. Collectively, the results obtained demonstrate that compounds 1 and 2 possess potent antimicrobial activity alone or in combination with vancomycin against multidrug-resistant staphylococci and show potential for use in disrupting staphylococcal biofilm.The Journal of Antibiotics advance online publication, 15 October 2014; doi:10.1038/ja.2014.142.
[Show abstract][Hide abstract] ABSTRACT: The seriousness of microbial resistance combined with the lack of new antimicrobials have increased the interest in the development of antimicrobial peptides (AMPs) as novel therapeutics. In this study, we evaluated the antimicrobial activity of two short synthetic peptides, namely RRIKA and RR. These peptides exhibited potent antimicrobial activity against Staphylococcus aureus and their antimicrobial effects were significantly enhanced by addition of three amino acids in the C terminus, which consequently increased the amphipathicity, hydrophobicity and net charge. Moreover, RRIKA and RR demonstrated a significant and rapid bactericidal effect against clinical and drug-resistant Staphylococcus isolates including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), linezolid-resistant S. aureus and methicillin-resistant S. epidermidis. In contrast to many natural AMPs; RRIKA and RR retained their activity in the presence of physiological concentrations of NaCl and MgCl2. Both RRIKA and RR enhanced the killing of lysostaphin over 1000-fold and eradicated MRSA and VRSA isolates within 20 minutes. Furthermore, the peptides presented were superior in reducing adherent biofilms of S. aureus and S. epidermidis when compared to conventional antibiotics. Our findings indicate that the staphylocidal effects of our peptides were through permeabilization of the bacterial membrane, leading to leakage of cytoplasmic contents and cell death. Furthermore, peptides were not toxic to HeLa cells at 4 to 8 fold their antimicrobial concentrations. The potent and salt-insensitive antimicrobial activities of these peptides present an attractive therapeutic candidate for treatment of multidrug-resistant S. aureus infections.
Antimicrobial Agents and Chemotherapy 05/2014; · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA & VRSA) infections are growing global health concerns. Structure-activity-relationships of phenylthiazoles as a new antimicrobial class have been addressed. We present 10 thiazole derivatives that exhibit strong activity against 18 clinical strains of MRSA and VRSA with acceptable PK profile. Three derivatives revealed an advantage over vancomycin by rapidly eliminating MRSA growth within six hours and no derivatives are toxic to HeLa cells at a concentration of 11 µg/mL.
Journal of Medicinal Chemistry 01/2014; · 5.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inorganic pyrophosphatases are potential targets for the development of novel antibacterial agents. A pyrophosphatase-coupled high-throughput screening assay intended to detect o-succinyl benzoic acid coenzyme A (OSB CoA) synthetase inhibitors led to the unexpected discovery of a new series of novel inorganic pyrophosphatase inhibitors. Lead optimization studies resulted in a series of 3-(3-aryl-pyrrolidin-1-yl)-5-aryl-1,2,4-triazine derivatives that were prepared by an efficient synthetic pathway. One of the tetracyclic triazine analogues 22h displayed promising antibiotic activity against a wide variety of drug-resistant Staphylococcus aureus strains, as well as activity versus Mycobacterium tuberculosis and Bacillus anthracis, at a concentration that was not cytotoxic to mammalian cells.
[Show abstract][Hide abstract] ABSTRACT: De novo design of unnatural proline-rich peptides led to a potent antibacterial peptide that targets both Gram-positive and -negative bacteria. The peptide exerts activity without lysing bacterial membranes or causing hemolysis, and is stable to extended trypsin treatment. Facile entry into macrophages was observed, leading to extensive intracellular clearing of pathogenic bacteria.
Angewandte Chemie International Edition 08/2013; · 11.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Treating intracellular pathogens remains a considerable medical challenge because of the inefficient intracellular delivery of antimicrobials and the frequent emergence of bacterial resistance to therapeutic agents deemed the drugs of last resort. We investigated the capability of antisense peptide nucleic acids (PNAs) conjugated to the (KFF)3K cell penetrating peptide to target RNA polymerase α subunit (rpoA) and RNA polymerase sigma 70 (rpoD) in the intracellular pathogen Listeria monocytogenes. The PNAs tested displayed a concentration dependent inhibition of L. monocytogenes growth in pure culture at the micromolar level and significantly reduced intracellular L. monocytogenes in infected cell culture and Caenorhabditis elegans whole animal model. In vitro, the combined PNAs treatment was synergistic resulting in a clearance of L. monocytogenes at 0.5× the individual PNA concentration. This study demonstrates the potential of anti-rpoA PNA as an antibacterial agent and will provide the basis for improving and developing these PNAs to better target intracellular pathogens like Listeria. This study also establishes C. elegans as a potential model for the screening of PNAs.
[Show abstract][Hide abstract] ABSTRACT: Peptide nucleic acids (PNAs) are single-stranded, synthetic nucleic acid analogues containing a pseudopeptide backbone in place of the phosphodiester sugar-phosphate. When PNAs are covalently linked to cell-penetrating peptides (CPPs) they readily penetrate the bacterial cell envelope, inhibit expression of targeted genes and cause growth inhibition both of Gram-positive and Gram-negative bacteria. However, the effectiveness of PNAs against Brucella, a facultative intracellular bacterial pathogen, was unknown. The susceptibility of a virulent Brucella suis strain to a variety of PNAs was assessed in pure culture as well as in murine macrophages. The studies showed that some of the PNAs targeted to Brucella genes involved in DNA (polA, dnaG, gyrA), RNA (rpoB), cell envelope (asd), fatty acid (kdtA, acpP) and protein (tsf) synthesis inhibit the growth of B. suis in culture and in macrophages after 24h of treatment. PNA treatment inhibited Brucella growth by interfering with gene expression in a sequence-specific and dose-dependent manner at micromolar concentrations. The most effective PNA in broth culture was that targeting polA at ca. 12μM. In contrast, in B. suis-infected macrophages, the most effective PNAs were those targeting asd and dnaG at 30μM; both of these PNAs had little inhibitory effect on Brucella in broth culture. The polA PNA that inhibits wild-type B. suis also inhibits the growth of wild-type Brucella melitensis 16M and Brucella abortus 2308 in culture. This study reveals the potential usefulness of antisense PNA constructs as novel therapeutic agents against intracellular Brucella.
International journal of antimicrobial agents 01/2013; · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the capability of antisense peptide nucleic acids (PNAs) conjugated to the (KFF)3K cell-penetrating peptide to target possible essential genes (ligA, rpoA, rpoD, engA, tsf, and kdtA) in Salmonella enterica serovar Typhimurium and inhibit bacterial growth in vitro and in cell culture. All targeted PNA-based gene inhibition has shown great potency in gene expression inhibition in a sequence-specific and dose-dependent manner at micromolar concentrations. Among tested PNAs, the anti-rpoA and -rpoD PNAs showed the greatest potency.
Antimicrobial Agents and Chemotherapy 12/2012; 56(12). · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intracellular pathogens like Salmonella evade host phagocytic killing by various mechanisms. Classical antimicrobial therapy requires multiple dosages and frequent administration of drugs for a long duration. Intracellular delivery of antimicrobials using nanoparticle may effectively devise therapies for bacterial infections. This review will address the mechanisms used by Salmonella to avoid host pathogenic killing, reasons for therapeutic failure and advances in nanoparticle drug delivery technology for efficient intracellular bacterial clearance.
[Show abstract][Hide abstract] ABSTRACT: Infection by members of the Gram-negative bacterial genus Brucella causes brucellosis in a variety of mammals. Brucellosis in swine remains a challenge, as there is no vaccine in the USA approved for use in swine against brucellosis. Here, we developed an improved recombinant Brucella abortus vaccine strain RB51 that could afford protection against Brucella suis infection by over-expressing genes encoding homologous proteins: L7/L12 ribosomal protein, Cu/Zn superoxide dismutase [SOD] and glycosyl-transferase [WboA]. Using strain RB51leuB as a platform and an antibiotic-resistance marker free plasmid, strains RB51leuB/SOD, RB51leuB/SOD/L7/L12 and RB51leuB/SOD/WboA were constructed to over-express the antigens: SOD alone, SOD and ribosomal protein L7/L12 or SOD and glycosyl-transferase, respectively. The ability of these vaccine candidates to protect against a virulent B. suis challenge were evaluated in a mouse model. All vaccine groups protected mice significantly (P<0.05) when compared to the control group. Within the vaccine groups, the mice vaccinated with strain RB51leuB/SOD/WboA were significantly better protected than those that were vaccinated with either strain RB51leuB/SOD or RB51leuB/SOD/L7/L12. These results suggest that Brucella antigens can be over-expressed in strain RB51leuB and elicit protective immune responses against brucellosis. Since the plasmid over-expressing homologous antigens does not carry an antibiotic resistance gene, it complies with federal regulations and therefore could be used to develop safer multi-species vaccines for prevention of brucellosis caused by other species of Brucella.
[Show abstract][Hide abstract] ABSTRACT: Brucella abortus has been shown to produce two siderophores: 2,3-dihydroxybenzoic acid (2,3-DHBA) and brucebactin. Previous studies on Brucella have shown that 2,3-DHBA is associated with erythritol utilization and virulence in pregnant ruminants. The biosynthetic pathway and role of brucebactin are not known and the only gene shown to be involved so far is entF. Using cre-lox methodology, an entF mutant was created in wild-type B. abortus 2308. Compared with the wild-type strain, the ΔentF strain showed significant growth inhibition in iron minimal media that became exacerbated in the presence of an iron chelator. For the first time, we have demonstrated the death of the ΔentF strain under iron-limiting conditions in the presence of erythritol. Addition of FeCl(3) restored the growth of the ΔentF strain, suggesting a significant role in iron acquisition. Further, complementation of the ΔentF strain using a plasmid containing an entF gene suggested the absence of any polar effects. In contrast, there was no significant difference in survival and growth between the ΔentF and wild-type strains grown in the murine macrophage cell line J774A.1, suggesting that an alternate iron acquisition pathway is present in Brucella when grown intracellulary.
[Show abstract][Hide abstract] ABSTRACT: Brucella abortus strains RB51 and RB51SOD are live attenuated vaccine strains which protect mice against virulent B. abortus strain 2308 intraperitoneal challenge. By comparison, limited information is available on how Brucella vaccines stimulate pulmonary immunity against respiratory infection, another route of exposure in humans. Therefore, in this study, we assessed the ability of intranasally delivered vaccine strains RB51 and RB51SOD to induce innate immunity. Based on parameters assessed, rough strain RB51 induces a better innate immune response in lung versus strain RB51SOD. Additional studies to further delineate strain RB51's ability to stimulate DC and adaptive immunity are warranted.
[Show abstract][Hide abstract] ABSTRACT: Core-shell nanostructures with nonionic amphiphilic shells and ionic cores encapsulating gentamicin were designed for therapy against intracellular pathogens, including Salmonella and Listeria. Flow cytometry and confocal microscopy showed that their uptake into J774A.1 macrophages proceeded mainly by fluid-phase endocytosis and clathrin-mediated pathways. The nanostructures were nontoxic in vitro at doses of 50 to 250 microg/ml, and they significantly reduced the amounts of intracellular Salmonella (0.53 log) and Listeria (3.16 log), thereby suggesting effective transport into the cells.
Antimicrobial Agents and Chemotherapy 08/2010; 54(8):3524-6. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genetic studies of Ochrobactrum anthropi, a bacterial species important in bioremediation and biopesticide degradation, are hindered by the lack of suitably regulated gene expression system. A tightly regulated gene-expression system was developed for O. anthropi using the lacI(q) gene and a re-engineered coliphage T5 promoter containing completely symmetrical DNA segment that binds more efficiently to the lactose repressor. The beta-galactosidase activity was increased 57-fold when the expression of the re-engineered T5 promoter was induced. The degree of induction was controllable by varying the concentration of inducer isopropyl-beta-D: -thiogalactopyranoside.
Current Microbiology 04/2010; 60(4):242-7. · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the capability of biodegradable silica xerogel as a novel carrier of antibiotic and the efficacy of treatment compared to that with the same dose of free drug against murine salmonellosis. The drug molecules (31%) entrapped in the sol-gel matrix remained in biologically active form, and the bactericidal effect was retained upon drug release. The in vitro drug release profiles of the gentamicin from the xerogel and that from the xerogel-polyethylene glycol (PEG) were distinctly different at pH 7.4. A delayed release of gentamicin was observed from the silica xerogel network (57% in 33 h), and with the addition of 2% PEG, the release rate reached 90% in 33 h. Administration of two doses of the silica xerogel significantly reduced the Salmonella enterica serovar Typhimurium load in the spleens and livers of infected AJ 646 mice. The silica xerogel and xerogel-PEG achieved a 0.45-log and a 0.41-log reduction in the spleens, respectively, while for the free drug there was no reduction. On the other hand, silica xerogel and xerogel-PEG achieved statistically significant 1.13-log and 1.15-log reductions in the livers, respectively, while for the free drug the reduction was a nonsignificant value of 0.07 log. This new approach, which utilizes a room-temperature synthetic route for incorporating therapeutic drugs into the silica matrix, should improve the capability for targeting intracellular pathogens.
Antimicrobial Agents and Chemotherapy 09/2009; 53(10):4270-4. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nanostructures encapsulating gentamicin and having either amphiphilic (N1) or hydrophilic (N2) surfaces were designed. Flow cytometry and confocal microscopy studies demonstrated a higher rate of uptake for amphiphilic surfaces. A majority of N1 were localized in the cytoplasm, whereas N2 colocalized with the endosomes/lysosomes. Colocalization was not observed between nanostructures and intracellular Salmonella bacteria. However, significant in vitro reductions in bacterial counts (0.44 log10) were observed after incubation with N1, suggesting that the surface property of the nanostructure influences intracellular bacterial clearance.
Antimicrobial Agents and Chemotherapy 08/2009; 53(9):3985-8. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Six broad-host-range plasmid vectors were developed to study gene expression in Bartonella henselae. The vectors were used to express a beta-galactosidase reporter gene in B. henselae and to generate antisense RNA for gene knockdown. When applied to ompR, a putative transcription response regulator of B. henselae, this antisense RNA gene knockdown strategy reduced bacterial invasion of human endothelial cells by over 60%.
Applied and Environmental Microbiology 07/2009; 75(16):5434-6. · 3.95 Impact Factor