Successful treatment using gentamicin liposomes of Salmonella dublin infections in mice

Fierer, Joshua; Hatlen, L.; Lin, Jan-Ping; Estrella, David Alonso Rivera; Mihalko, Paul J.; Yau-Young, A.

doi:10.1128/aac.34.2.343

Cited by 58 publications

(39 citation statements)

References 15 publications

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“…These dying cells or resulting apoptotic bodies may be phagocytosed by surrounding macrophages, facilitating cell-to-cell spread of the infection. The finding that gentamicin is ineffective in Salmonella infections implies that Salmonella can complete the infectious cycle without being exposed to the extracellular environment (11).…”

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Genetic Requirements for Salmonella -Induced Cytopathology in Human Monocyte-Derived Macrophages

2002

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Infection of human macrophages with Salmonella enterica serovar Typhimurium or Salmonella enterica serovar Dublin produces delayed cytotoxicity characterized by cell detachment and associated apoptosis. Using a site-specific mutant in the SpvB active site, we verify that the ADP-ribosylation activity of SpvB is required for delayed cytotoxicity in human macrophages infected with Salmonella. SipB and the type III protein secretion system (TTSS) encoded by Salmonella pathogenicity island 1 (SPI1) are not involved, whereas the SPI2 TTSS is absolutely required for SpvB-dependent cytotoxicity. Furthermore, we show that infection of macrophage cultures with wild-type or sipB mutant bacteria led to a complete loss of polymerized actin in over half of the cells after 24 h. In contrast, macrophages infected with the spvB or SPI2 (ssaV or ssaJ) mutant strain retained normal F-actin filaments, despite similar numbers of intracellular bacteria. We conclude that SpvB and a functional SPI2 TTSS are essential for Salmonella-induced delayed cytotoxicity of human macrophages.Salmonella enterica subspecies I strains are responsible for enteric fever, gastroenteritis, and bacteremia in humans and a broad array of illnesses in animals, including enteritis and septicemia in livestock. A number of subspecies I serovars carry virulence plasmids encoding the highly conserved spv locus associated with disease syndromes of greater severity (13). The spv genes are found more frequently in human extraintestinal isolates than in environmental or fecal isolates, and most cases of human nontyphoidal bacteremia are caused by serovars that contain the spv genes (12, 13).The plasmid-encoded spv locus consists of the spvR regulatory gene and four structural genes, spvABCD (26). spvR encodes a transcriptional regulator activating its own expression and the spvABCD operon (9, 27). Transcription of the spv operon also requires the stationary-phase alternate factor RpoS (5, 10). Since both rpoS and spv are upregulated after phagocytosis by macrophages (4), expression of the spv genes appears to be strongly induced by the intracellular environment of the host cell. The spv genes appear to promote the macrophage phase of the disease process, avoiding destruction by neutrophils and facilitating proliferation of Salmonella strains at extraintestinal sites of infection (13).In vivo experiments suggest that the spv genes enhance bacterial replication in macrophages (18). In human macrophages, spv gene expression is required for induction of cytotoxicity characterized by cell detachment and eventual apoptosis (29). The spvB gene within the spv operon is essential for the spv virulence phenotype, as shown by mutational analysis (35). The spvB gene encodes a 65.6-kDa protein (recently characterized as an ADP-ribosyl transferase) that modifies actin and blocks polymerization to F-actin filaments (28, 38). Site-directed mutagenesis of the conserved NAD-binding site sequence demonstrates that this ADP-ribosylation activity is essential for virulence in mice (28)...

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Genetic Requirements for Salmonella -Induced Cytopathology in Human Monocyte-Derived Macrophages

2002

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“…Studies with mice have shown that the spv genes are required neither for the invasion and colonization of Peyer's patches nor for the transfer of bacteria to the spleen or liver, but the spv locus is crucial for growth in the liver and spleen (18). The ability to grow inside host cells has been shown to correlate with the virulence phenotype (12,16).…”

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In vitro binding of the Salmonella dublin virulence plasmid regulatory protein SpvR to the promoter regions of spvA and spvR

Grob

Guiney

1996

J Bacteriol

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The spv regulon of Salmonella dublin is essential for virulence in mice. SpvR, a LysR-type regulator, induces the expression of the spvABCD operon and its own expression in the stationary phase of bacterial growth and in macrophages. We constructed fusion proteins to the maltose-binding protein (MBP) and a His tag peptide (His) to overcome the insolubility and to facilitate purification of SpvR. We demonstrated that both fusion proteins, MBP-SpvR and His-SpvR, were able to induce spvA expression in vivo. MBP-SpvR was produced as soluble protein, whereas His-SpvR was only marginally present in the soluble cell fraction. Affinity chromatography resulted in at least 95% pure MBP-SpvR protein and in an enrichment of His-SpvR. Gel mobility shift assay revealed that the SpvR fusion proteins were able to bind to 125-and 147-bp DNA fragments of the spvA and spvR promoter regions, respectively. DNase I footprint experiments showed that the fusion proteins protected DNA regions of 54 and 50 bp within the spvA and spvR promoter regions, respectively.

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“…Because liposomes enhance delivery of drugs to macrophages, liposome-incorporated antibiotics have great promise for treating organisms which survive intracellularly, within the macrophages (15). Examples of such use in experimental animals include treatment of Listeria monocytogenes with ampicillin, murine cryptococcoses with amphotericin B, Mycobacterium avium-M. intracellulare with amikacin, and murine salmonellosis with streptomycin or gentamicin (3,8,9,12,14,26). In general, the liposomeincorporated antibiotics have proven superior to the plain form of the drugs (21).…”

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“…A single inijection of liposome-incorporated ciprofloxacin (LIC) was 10 times more effective than a single inijection of free drug at preventing mortality. When (3,8,9,12,14,26). In general, the liposomeincorporated antibiotics have proven superior to the plain form of the drugs (21).…”

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Liposome-incorporated ciprofloxacin in treatment of murine salmonellosis

Magallanes

Dijkstra

Fierer

1993

Antimicrob Agents Chemother

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We used a dehydration-rehydration procedure in order to efficiently incorporate ciprofloxacin into phospholipid vesicles (liposomes), which we then used to treat BALB/c mice that had been infected per os with SalmoneUla dublin. A single inijection of liposome-incorporated ciprofloxacin (LIC) was 10 times more effective than a single inijection of free drug at preventing mortality. When (3,8,9,12,14,26). In general, the liposomeincorporated antibiotics have proven superior to the plain form of the drugs (21).Ciprofloxacin is a quinolone antibiotic that has excellent in vitro activity against Salmonella species (13). It has been used in the treatment of individuals with Salmonella infections, including those with typhoid fever and chronic typhoid carriers (19,25). In previously published studies of animal models of S. typhimunum infection, both oral and subcutaneous administration of ciprofloxacin resulted in prolongation of survival and reductions in bacterial counts in liver and spleen compared with those in untreated controls and those after treatment with either ampicillin or chloramphenicol (5).In the study described here, we investigated the therapeutic value of liposome-incorporated ciprofloxacin (LIC) in a murine model of fatal S. dublin infection. Untreated, the infection begins in Peyer's patches, spreads to the regional lymph nodes, and then disseminates to the liver and spleen, * Corresponding author.leading to bacteremia and death 10 to 14 days after infection (16). In essence, we attempted to determine whether LIC would prove to be superior to aqueous ciprofloxacin. MATERIALS AND METHODSMice. Virus-free female BALB/cJ mice were purchased from Jackson Laboratory, Bar Harbor, Maine. Animals were housed at four to six per cage and were allowed free access to food and water. Mice weighed approximately 20 g when infected. BALB/c mice carry the itys allele, which makes them susceptible to Salmonella infections (24).Infection. S. dublin Lane pSD6 is a kanamycin-resistant derivative of the parent Lane strain in which TnS is inserted in the cryptic plasmid outside the vir region; it is fully virulent (6). Bacteria were suspended in 0.1 M NaHCO3 solution to 5 x 108 CFU/ml. A total of 0.2 ml of the suspension was delivered into the stomach by gavage. The MIC of ciprofloxacin for S. dublin was 0.03 ,ug/ml.Cultures. Organs and tissues were removed aseptically and homogenized in 2 ml of sterile saline as described previously (16 Preparation of liposomes. The liposomes consisted of dipalmitoyl-phosphatidylcholine (Avanti Polar Lipids, Inc., Alabaster, Ala.), dipalmitoyl-phosphatidylglycerol (Avanti), and cholesterol (type C-8253; Sigma Chemical Co., St. Louis, Mo.) in a molar ratio of 4.1:0.9:4. For the incorporation of ciprofloxacin into phospholipid vesicles, the dehydra-

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Successful treatment using gentamicin liposomes of Salmonella dublin infections in mice

Cited by 58 publications

References 15 publications

Genetic Requirements for Salmonella -Induced Cytopathology in Human Monocyte-Derived Macrophages

Genetic Requirements for Salmonella -Induced Cytopathology in Human Monocyte-Derived Macrophages

In vitro binding of the Salmonella dublin virulence plasmid regulatory protein SpvR to the promoter regions of spvA and spvR

Liposome-incorporated ciprofloxacin in treatment of murine salmonellosis

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