Highly resistant Burkholderia pseudomallei small colony variants isolated in vitro and in experimental melioidosis

AbstractIn response to increasing frequencies of antibiotic-resistant pathogens, there has been a resurrection of interest in the use of bacteriophage to treat bacterial infections: phage therapy. Here we explore the potential of a seemingly ideal phage, PYOSa, for combination phage and antibiotic treatment of Staphylococcus aureus infections. (i) This K-like phage has a broad host range; all 83 tested clinical isolates of S.aureus tested were susceptible to PYOSa. (ii) Because of the mode of action of PYOSaS. aureus is unlikely to generate classical receptor-site mutants resistant to PYOSa; none were observed in the 13 clinical isolates tested. (iii) PYOSa kills S. aureus at high rates. On the downside, the results of our experiments and tests of the joint action of PYOSa and antibiotics raise issues that must be addressed before PYOSa is employed clinically. Despite the maintenance of the phage, PYOSa does not clear the populations of S. aureus. Due to the ascent of a phenotypically diverse array of small colony variants following an initial demise, the bacterial populations return to densities similar to that of phage-free controls. Using a combination of mathematical modeling and in vitro experiments, we postulate and present evidence for a mechanism to account for the demise–resurrection dynamics of PYOSa and S. aureus. Critically for phage therapy, our experimental results suggest that treatment with PYOSa followed by bactericidal antibiotics can clear populations of S. aureus more effectively than the antibiotics alone.Significance StatementThe increasing frequency of antibiotic-resistant pathogens has fostered a quest for alternative means to treat bacterial infections. Prominent in this quest is a therapy that predates antibiotics: bacteriophage. This study explores the potential of a phage, PYOSa, for treating Staphylococcus aureus infections in combination with antibiotics. On first consideration, this phage, isolated from a commercial therapeutic cocktail, seems ideal for this purpose. The results of this population dynamic and genomic analysis study identify a potential liability of using PYOSa for therapy. Due to the production of potentially pathogenic atypical small colony variants, PYOSa alone cannot eliminate S. aureus populations. However, we demonstrate that by following the administration of PYOSa with bactericidal antibiotics, this limitation and potential liability can be addressed.

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Genomic comparison and phenotypic profiling of small colony variants of Burkholderia pseudomallei

PLoS ONE ◽

10.1371/journal.pone.0261382 ◽

2021 ◽

Vol 16 (12) ◽

pp. e0261382

Author(s):

Noorfatin Jihan Zulkefli ◽

Cindy Shuan Ju Teh ◽

Vanitha Mariappan ◽

Soo Tein Ngoi ◽

Jamuna Vadivelu ◽

...

Keyword(s):

Phylogenetic Relationship ◽

Burkholderia Pseudomallei ◽

Whole Genome Sequence ◽

Alkaline Condition ◽

Phenotype Microarray ◽

Growth Responses ◽

Carbon Utilization ◽

Small Colony Variants ◽

Close Phylogenetic Relationship ◽

Small Colony

Burkholderia pseudomallei (B. pseudomallei) is an intracellular pathogen that causes melioidosis, a life-threatening infection in humans. The bacterium is able to form small colony variants (SCVs) as part of the adaptive features in response to environmental stress. In this study, we characterize the genomic characteristics, antimicrobial resistance (AMR), and metabolic phenotypes of B. pseudomallei SCV and wild type (WT) strains. Whole-genome sequence analysis was performed to characterize the genomic features of two SCVs (CS and OS) and their respective parental WT strains (CB and OB). Phylogenetic relationship between the four draft genomes in this study and 19 publicly available genomes from various countries was determined. The four draft genomes showed a close phylogenetic relationship with other genomes from Southeast Asia. Broth microdilution and phenotype microarray were conducted to determine the AMR profiles and metabolic features (carbon utilization, osmolytes sensitivity, and pH conditions) of all strains. The SCV strains exhibited identical AMR phenotype with their parental WT strains. A limited number of AMR-conferring genes were identified in the B. pseudomallei genomes. The SCVs and their respective parental WT strains generally shared similar carbon-utilization profiles, except for D,L-carnitine (CS), g-hydroxybutyric acid (OS), and succinamic acid (OS) which were utilized by the SCVs only. No difference was observed in the osmolytes sensitivity of all strains. In comparison, WT strains were more resistant to alkaline condition, while SCVs showed variable growth responses at higher acidity. Overall, the genomes of the colony morphology variants of B. pseudomallei were largely identical, and the phenotypic variations observed among the different morphotypes were strain-specific.

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Evaluation of the in vitro activity of the gentamicin-collagen sponge against small colony variants of coagulase-negative staphylococci isolated from prosthetic joint infections

10.26226/morressier.56d5ba30d462b80296c95485 ◽

2016 ◽

Author(s):

Alina Olender

Keyword(s):

Collagen Sponge ◽

Vitro Activity ◽

In Vitro Activity ◽

Coagulase Negative Staphylococci ◽

Small Colony Variants ◽

Joint Infections ◽

Prosthetic Joint ◽

Prosthetic Joint Infections ◽

Small Colony

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Hemin-dependent siderophore utilization promotes iron-restricted growth of the Staphylococcus aureus hemB small colony variant

Journal of Bacteriology ◽

10.1128/jb.00458-21 ◽

2021 ◽

Author(s):

Izabela Z. Batko ◽

Ronald S. Flannagan ◽

Veronica Guariglia-Oropeza ◽

Jessica R. Sheldon ◽

David E. Heinrichs

Keyword(s):

Iron Acquisition ◽

Recurrent Infection ◽

Nutritional Requirement ◽

Iron Starvation ◽

Small Colony Variants ◽

Atp Production ◽

Small Colony ◽

Sense And Respond

Respiration deficient S. aureus small colony variants (SCVs) frequently cause persistent infections, which necessitates they acquire iron, yet how SCVs obtain iron remains unknown. To address this, we created a stable hemB mutant in S. aureus USA300 strain LAC. The hemB SCV utilized exogenously supplied hemin but was attenuated for growth under conditions of iron starvation. RNA-seq showed that both WT S. aureus and the hemB mutant sense and respond to iron starvation, however, growth assays show that the hemB mutant is defective for siderophore-mediated iron acquisition. Indeed, the hemB SCV demonstrated limited utilization of endogenous staphyloferrin B or exogenously provided staphyloferrin A, Desferal, and epinephrine. Direct measurement of intracellular ATP in hemB and WT S. aureus revealed that both strains can generate comparable levels of ATP during exponential growth suggesting defects in ATP production cannot account for the inability to efficiently utilize siderophores. Defective siderophore utilization by hemB bacteria was also evident in vivo , as administration of Desferal failed to promote hemB bacterial growth in every organ analyzed except for the kidneys. In support of the hypothesis that S. aureus accesses heme in kidney abscesses, in vitro analyses revealed that increased hemin availability enables hemB bacteria to utilize siderophores for growth when iron availability is restricted. Taken together, our data support the conclusion that hemin is not only used as an iron source itself, but as a nutrient that promotes utilization of siderophore-iron complexes. Importance S. aureus small colony variants (SCVs) are associated with chronic recurrent infection and worsened clinical outcome. SCVs persist within the host despite administration of antibiotics. This study yields insight into how S. aureus SCVs acquire iron which, during infection of a host, is a difficult-to-acquire metal nutrient. Under hemin-limited conditions, hemB S. aureus is impaired for siderophore-dependent growth and, in agreement, murine infection indicates that hemin-deficient SCVs meet their nutritional requirement for iron through utilization of hemin. Importantly, we demonstrate that hemB SCVs rely upon hemin as a nutrient to promote siderophore utilization. Therefore, perturbation of heme biosynthesis and/or utilization represents a viable to strategy to mitigate the ability of SCV bacteria to acquire siderophore-bound iron during infection.

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Highly adherent small-colony variants of Pseudomonas aeruginosa in cystic fibrosis lung infection

Journal of Medical Microbiology ◽

10.1099/jmm.0.05069-0 ◽

2003 ◽

Vol 52 (4) ◽

pp. 295-301 ◽

Cited By ~ 142

Author(s):

Susanne Häußler ◽

Isabell Ziegler ◽

Alexandra Löttel ◽

Franz v. Götz ◽

Manfred Rohde ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Lung Infection ◽

Growth Conditions ◽

Twitching Motility ◽

Small Colony Variants ◽

Environmental Bacterium ◽

Small Colony ◽

Opportunistic Human Pathogen

Pseudomonas aeruginosa, an opportunistic human pathogen and ubiquitous environmental bacterium, is capable of forming specialized bacterial communities, referred to as biofilm. The results of this study demonstrate that the unique environment of the cystic fibrosis (CF) lung seems to select for a subgroup of autoaggregative and hyperpiliated P. aeruginosa small-colony variants (SCVs). These morphotypes showed increased fitness under stationary growth conditions in comparison with clonal wild-types and fast-growing revertants isolated from the SCV population in vitro. In accordance with the SCVs being hyperpiliated, they exhibited increased twitching motility and capacity for biofilm formation. In addition, the SCVs attached strongly to the pneumocytic cell line A549. The emergence of these highly adherent SCVs within the CF lung might play a key role in the pathogenesis of P. aeruginosa lung infection, where a biofilm mode of growth is thought to be responsible for persistent infection.

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Phenotypic diversification in vivo: Pseudomonas aeruginosa gacS− strains generate small colony variants in vivo that are distinct from in vitro variants

Microbiology ◽

10.1099/mic.0.040824-0 ◽

2010 ◽

Vol 156 (12) ◽

pp. 3699-3709 ◽

Cited By ~ 10

Author(s):

Lisa K. Nelson ◽

M. Mark Stanton ◽

Robyn E. A. Elphinstone ◽

Janessa Helwerda ◽

Raymond J. Turner ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Phenotypic Variation ◽

Antimicrobial Agents ◽

Mucosal Surface ◽

Infection Model ◽

Bacterial Survival ◽

Small Colony Variants ◽

Small Colony

Pseudomonas aeruginosa has long been known to produce phenotypic variants during chronic mucosal surface infections. These variants are thought to be generated to ensure bacterial survival against the diverse challenges in the mucosal environment. Studies have begun to elucidate the mechanisms by which these variants emerge in vitro; however, too little information exists on phenotypic variation in vivo to draw any links between variants generated in vitro and in vivo. Consequently, in this study, the P. aeruginosa gacS gene, which has previously been linked to the generation of small colony variants (SCVs) in vitro, was studied in an in vivo mucosal surface infection model. More specifically, the rat prostate served as a model mucosal surface to test for the appearance of SCVs in vivo following infections with P. aeruginosa gacS− strains. As in in vitro studies, deletion of the gacS gene led to SCV production in vivo. The appearance of these in vivo SCVs was important for the sustainability of a chronic infection. In the subset of rats in which P. aeruginosa gacS− did not convert to SCVs, clearance of the bacteria took place and healing of the tissue ensued. When comparing the SCVs that arose at the mucosal surface (MS-SCVs) with in vitro SCVs (IV-SCVs) from the same gacS− parent, some differences between the phenotypic variants were observed. Whereas both MS-SCVs and IV-SCVs formed dense biofilms, MS-SCVs exhibited a less diverse resistance profile to antimicrobial agents than IV-SCVs. Additionally, MS-SCVs were better suited to initiate an infection in the rat model than IV-SCVs. Together, these observations suggest that phenotypic variation in vivo can be important for maintenance of infection, and that in vivo variants may differ from in vitro variants generated from the same genetic parent.

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