Enhanced contrast of bacteriophage plaques in Salmonella with ferric ammonium citrate and sodium thiosulfate (FACST) and tetrazolium red (TZR)

McLaughlin, M. R.; Balaa, M. F.

doi:10.1016/j.mimet.2005.08.008

Cited by 15 publications

(10 citation statements)

References 5 publications

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“…The addition to the soft agar layer of 2,3,5-triphenyltetrazolium, sodium thiosulfate or ferric ammonium citrate, as it was done in the case of Salmonella enterica subsp. enterica bacteriophages isolated from swine lagoon eﬄuent may be another way to facilitate plaque visualization ( McLaughlin and Balaa, 2006 ).…”

Section: Methods Of Phage Isolationmentioning

confidence: 99%

Bacteriophage Procurement for Therapeutic Purposes

et al. 2016

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Bacteriophages (phages), discovered 100 years ago, are able to infect and destroy only bacterial cells. In the current crisis of antibiotic efficacy, phage therapy is considered as a supplementary or even alternative therapeutic approach. Evolution of multidrug-resistant and pandrug-resistant bacterial strains poses a real threat, so it is extremely important to have the possibility to isolate new phages for therapeutic purposes. Our phage laboratory and therapy center has extensive experience with phage isolation, characterization, and therapeutic application. In this article we present current progress in bacteriophages isolation and use for therapeutic purposes, our experience in this field and its practical implications for phage therapy. We attempt to summarize the state of the art: properties of phages, the methods for their isolation, criteria of phage selection for therapeutic purposes and limitations of their use. Perspectives for the use of genetically engineered phages to specifically target bacterial virulence-associated genes are also briefly presented.

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Section: Methods Of Phage Isolationmentioning

confidence: 99%

Bacteriophage Procurement for Therapeutic Purposes

et al. 2016

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“…Different strategies have been attempted in the past to increase the visualization of tiny plaques formed by different bacteriophages, including S. aureus-specific phages (21,26,27,28,31). Most of the researchers have focused on the use of ␤-lactam antibiotics or quinolone antibiotics for increasing the size of staphylococcal phages (14,17,31).…”

Section: Discussionmentioning

confidence: 99%

Methicillin-Resistant Staphylococcus aureus Phage Plaque Size Enhancement Using Sublethal Concentrations of Antibiotics

Kaur

Harjai

Chhibber

2012

Appl Environ Microbiol

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Phage therapy presents an alternative approach against the emerging methicillin-resistant Staphylococcus aureus (MRSA) threat. Some of the problems encountered during isolation of MRSA phages include the high prevalence of enteric phages in natural sources, nonspecific absorption of viable phage, and the formation of pinpoint or tiny plaques. The phage isolated in this study, MR-5, also formed tiny plaques against its host S. aureus ATCC 43300 (MRSA), making its detection and enumeration difficult. An improved method of increasing the plaque size of MRSA phage by incorporating sublethal concentrations of three different classes of antibiotics (inhibitors of protein synthesis) in the classical double-layer agar (DLA) method was investigated. The ␤-lactam and quinolone antibiotics commonly employed in earlier studies for increasing the plaque size did not show any significant effect on the plaque size of isolated MR-5 phage. Linezolid (oxazolidinone class), tetracycline, and ketolide antibiotics brought significant enhancements (3 times the original size) in the plaque size of MR-5 phage. Prior treatment with these antibiotics resulted in significant reductions in the time of adsorption and the latent period of MR-5 phage. To rule out whether the action of linezolid (which brought the maximum increase in plaque size) was specific for a single phage only, its effect on the plaque size of seven other S. aureus-specific phages was also assessed. Significant enhancements in the plaque size of these phages were observed. These results indicate that this modification can therefore safely be incorporated in the traditional DLA overlay method to search for new MRSA-virulent phages. Staphylococcus aureus is a leading cause of both communityand hospital-acquired infections, including skin and soft tissue infections, wound infections, endocarditis, osteomyelitis, and lifethreatening bacteremia (6, 23). The emergence of high levels of penicillin resistance, followed by the development and spread of strains resistant to the action of nafcillin, oxacillin, methicillin, and now, even vancomycin has made therapy of staphylococcal disease a global challenge (23,24,34). More people in the United States now die from methicillin-resistant S. aureus (MRSA) infection than from AIDS (33). Hence, there is an unmet medical urgency and pressing need to develop novel antibacterials to tackle the staphylococcal menace. Phage therapy can serve as one of the stand-alone approaches for treating staphylococcal infections refractory to the action of commonly deployed antibiotics (6).The first step in investigating the potential of therapy using phage or its products against MRSA infections involves the isolation and selection of a lytic bacteriophage covering a maximum number of MRSA strains, both from community and hospital sources. The difficulty encountered in the isolation of phages specific to staphylococcal strains is partly attributed to the prevalence of enteric phages in sewage samples and partly to the high level of nonspecific adsorpti...

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“…Isolates were not necessarily selected for their association with swine, but rather were chosen to include type strains of S. Typhimurium and Enteritidis, three DT104 isolates, and a small but diverse panel of isolates recommended for use in testing food sanitation processes. Two of the S. Typhimurium isolates (R37 and R47) were unusual in that they were negative for H 2 S production in our laboratory tests (McLaughlin and Balaa, 2006). Freeze‐dried primary cultures were reconstituted in trypticase soy broth (TSB) overnight at 35°C, then stored in 1‐ to 2‐mL aliquots at −70°C.…”

Section: Methodsmentioning

confidence: 97%

Isolation of Salmonella Bacteriophages from Swine Effluent Lagoons

et al. 2006

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Bacteriophages (phages) associated with Salmonella were collected from nine swine manure lagoons in Mississippi. Phages were isolated by an enrichment protocol or directly from effluent. For enrichment, chloroform-treated samples were filtered (0.22 mum) and selectively enriched by adding a cocktail of Salmonella strains in trypticase soy broth. After overnight incubation at 35 degrees C, chloroform was added and samples stored at 5 degrees C. Enriched samples were tested by double agar layer (DAL) plaque assay against individual Salmonella isolates. Phage titers of 2.9 x 10(8) to 2.1 x 10(9) plaque forming units (pfu) per mL were produced, but estimation of phage titers in lagoons was not possible. For direct isolation, effluent was clarified by centrifugation, filtered (0.22 microm), and used in DAL plaque assays to select single-plaque isolates for 15 Salmonella strains. Plaque counts varied among Salmonella strains and lagoons. The most sensitive strain for direct phage recovery was ATCC 13311. Phage titers estimated by direct isolation with ATCC 13311 ranged among lagoons from 12 to 148 pfu per mL. In limited host range tests, 66 isolates recovered by the enrichment protocol produced plaques only on Enteritidis and Typhimurium strains of Salmonella and none produced plaques on lagoon isolates of Citrobacter, Escherichia, Proteus, Providencia, or Serratia. Electron microscopy (EM) showed purified enrichment isolates had Podoviridae morphology (tailless 50-nm icosahedral heads with tail spikes). Electron microscopy of clarified concentrated effluent showed 5.5:1 tailless to tailed phages. The isolated phages have potential as typing reagents, specific indicators, and biocontrol agents of Salmonella.

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Enhanced contrast of bacteriophage plaques in Salmonella with ferric ammonium citrate and sodium thiosulfate (FACST) and tetrazolium red (TZR)

Cited by 15 publications

References 5 publications

Bacteriophage Procurement for Therapeutic Purposes

Bacteriophage Procurement for Therapeutic Purposes

Methicillin-Resistant Staphylococcus aureus Phage Plaque Size Enhancement Using Sublethal Concentrations of Antibiotics

Isolation of Salmonella Bacteriophages from Swine Effluent Lagoons

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