Molecular Mechanisms of Staphylococcus and Pseudomonas Interactions in Cystic Fibrosis

Cystic fibrosis (CF) is an autosomal recessive genetic disorder that is characterized by recurrent and chronic infections of the lung predominantly by the opportunistic pathogens, Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa. While S. aureus is the main colonizing bacteria of the CF lungs during infancy and early childhood, its incidence declines thereafter and infections by P. aeruginosa become more prominent with increasing age. The competitive and cooperative interactions exhibited by these two pathogens influence their survival, antibiotic susceptibility, persistence and, consequently the disease progression. For instance, P. aeruginosa secretes small respiratory inhibitors like hydrogen cyanide, pyocyanin and quinoline N-oxides that block the electron transport pathway and suppress the growth of S. aureus. However, S. aureus survives this respiratory attack by adapting to respiration-defective small colony variant (SCV) phenotype. SCVs cause persistent and recurrent infections and are also resistant to antibiotics, especially aminoglycosides, antifolate antibiotics, and to host antimicrobial peptides such as LL-37, human β-defensin (HBD) 2 and HBD3; and lactoferricin B. The interaction between P. aeruginosa and S. aureus is multifaceted. In mucoid P. aeruginosa strains, siderophores and rhamnolipids are downregulated thus enhancing the survival of S. aureus. Conversely, protein A from S. aureus inhibits P. aeruginosa biofilm formation while protecting both P. aeruginosa and S. aureus from phagocytosis by neutrophils. This review attempts to summarize the current understanding of the molecular mechanisms that drive the competitive and cooperative interactions between S. aureus and P. aeruginosa in the CF lungs that could influence the disease outcome.

Involvement of Small Colony Variant-Related Heme Biosynthesis Genes in Staphylococcus aureus Persister Formation in vitro

Background: Persisters are important reasons for persistent infections, and they can lead to antibiotic treatment failure in patients and consequently chronic infection. Staphylococcus aureus small colony variants (SCVs) have been shown to be related to persistent infection. Mutations in the genes of the heme biosynthesis pathway lead to the formation of SCVs. However, the relationship between heme production genes and persister has not been tested.Methods:HemA and hemB were knocked out by allelic replacement from S. aureus strain USA500 separately, and then, the heme deficiency was complemented by overexpression of related genes and the addition of hemin. The stress-related persister assay was conducted. RNA-sequencing was performed to find genes and pathways involved in heme-related persister formation, and relative genes and operons were further knocked out and overexpressed to confirm their role in each process.Results: We found that heme biosynthesis deficiency can lead to decreased persister. After complementing the corresponding genes or hemin, the persister levels could be restored. RNA-seq on knockout strains showed that various metabolic pathways were influenced, such as energy metabolism, amino acid metabolism, carbohydrate metabolism, and membrane transport. Overexpression of epiF and operon asp23 could restore USA500∆hemA persister formation under acid stress. Knocking out operon arc in USA500∆hemA could further reduce USA500∆hemA persister formation under acid and oxidative stress.Conclusion: Heme synthesis has a role in S. aureus persister formation.

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

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.

Characteristics of a Capnophilic Small Colony Variant of Escherichia Coli Co-Isolated With Two Other Strains From a Patient With Bacteremia in China

Small colony variants (SCVs) are a slow-growing subpopulation of bacteria characterized by their atypical colony morphology and distinct biochemical properties, which are known to cause chronic persistent infections. Here, we investigated the characteristics of three phenotypes of Escherichia coli, including a capnophilic SCV, co-isolated from a 64-year-old patient with bacteremia in China. The three strains were identified as a capnophilic strain (EC1), a capnophilic SCV (EC2), and a wild-type strain (EC3). The EC1 and EC2 strains did not grow in the absence of CO2, while the EC2 colonies were pinpoint in appearance and had the ability to revert to the large-form phenotype. The growth of the SCV was slow and not enhanced in the presence of thymidine, hemin, thiamine, and menadione. The results of antimicrobial susceptibility showed similar sensitivity to cefoxitin and imipenem, but resistant to most of the other antimicrobials tested. Whole genome sequencing showed that no genetic mutational variations associated with SCVs were observed, while EC1, EC2 and the revertible strains of EC2 lacked the can gene. Multi-locus sequence typing showed that all strains belonged to ST457 and nucleotide similarity analysis indicated that they had high homology. In conclusion, we report rarely described co-isolated forms of three phenotypes of E. coli that included acapnophilic SCV in a patient with bacteremia. The capnophilic SCV strain had atypical morphology and biochemical characteristics with the absent of can gene. Based on our findings, we have discussed the laboratory identification, characterization, mechanisms, and clinical treatment of capnophilic SCV strains.