Staphylococcus aureus as an intracellular pathogen: the role of small colony variants

doi:10.1016/j.tim.2008.11.004

Trends in Microbiology

Volume 17, Issue 2, February 2009, Pages 54-58

https://doi.org/10.1016/j.tim.2008.11.004 Get rights and content

Increasing evidence indicates that Staphylococcus aureus might be a facultative intracellular pathogen. In particular, certain subpopulations, called small colony variants (SCVs), seem to be well adapted to the intracellular milieu. When compared to ‘normal’ staphylococcal strains, SCVs show increased uptake by host cells, resistance to intracellular defenses and reduced stimulation of host defenses. We propose that the ability to form two subpopulations with different phenotypes might allow S. aureus the option for both extra- cellular and intra-cellular survival in the host.

Section snippets

Small colony variants of Staphylococcus aureus: a link to recurrent infections?

Certain strains of Staphylococcus aureus can produce subpopulations which are phenotypically very different from the parent strain. These naturally occurring subpopulations grow slowly, leading to colonies that are defined as being one-tenth the size of ‘normal’ S. aureus, hence the name ‘small colony variants’ (SCVs) (Figure 1). Prominent features of these SCVs include decreased pigmentation and haemolysis, increased resistance to aminoglycosides and an unstable colony phenotype. The

Intracellular S. aureus: normal colony-size or SCVs?

Recent studies using cell cultures [15] and clinical samples [16] strongly suggest that S. aureus is able to persist intracellularly. Therefore, questions are raised whether all intracellular staphylococci are in fact SCVs, or whether SCVs are an artefact because of the difficulties in culturing intracellular pathogens. No study has fully addressed this question with clinical isolates, most likely because of technical limitations and the ability of SCVs to revert rapidly in the extracellular

SCVs defense strategies to facilitate colonization

In SCVs, a gene coding for an alternate sigma factor, sigB, is upregulated [20]. This gene is recognized as an important regulator of bacterial stress responses and is involved in bacterial strategies to overcome a variety of environmental stress factors, such as pH, temperature and osmolarity [21].

Additionally, SCVs have a thick cell wall [22] and their electrical potential across the cytoplasmic membrane drops immediately when nutrients are expired (e.g. glucose) or do not allow further

Increased uptake and adherence of SCVs?

Bacterial adherence to the host cell is a prerequisite for invasion. The interaction between S. aureus and host cells is believed to occur through a bridging mechanism, in which bacterial adhesion proteins, host proteins (e.g. fibronectin) and cell integrins participate. Integrins mediate communication and adhesion between mammalian cells and the extracellular matrix. S. aureus expresses an array of adhesins, including the ‘microbial surface components recognizing adhesive matrix molecules’

SCVs cause less damage to host cells

In contrast to normal colony-size S. aureus, SCVs have a decreased production of α-toxin and, consequently, do not lyse their host cells [1]. In addition, SCVs produce lower amounts of toxic shock syndrome toxin 1 (TSST-1), which would result in a lower activation of host immune cell-mediated cytotoxicity [31].

Several studies have suggested that normal colony-size intracellular S. aureus mediates apoptosis in epithelial cells [32], endothelial cells [33] and keratinocytes [34], with the

Resistance to intracellular host defenses

As mentioned earlier, a reduced membrane potential can protect SCVs from cationic proteins. Also, because the activity of the tricarboxylic acid cycle is reduced and catabolism of acetate is thus prevented [2], the carboxylate is shunted into the production of polysaccharide intercellular adhesin (PIA), levels of which are elevated in SCVs [45]. PIA is known to contribute to S. aureus resistance against non-oxidative killing mechanisms of neutrophils [46] and thus could enable SCVs to resist

Decreased activation of the host immune system

Bacterial attachment alone is not sufficient to start the signaling process and the inflammatory response. A more intimate connection, such as disruption of normal actin polymerization, is required to set off the cytokine and chemokine alarm system. Infection of endothelial cells with S. aureus leads to induction of cytokines, although different clinical isolates vary greatly in their ability to elicit a proinflammatory response [48], which might reflect their virulence properties. Given that

Proliferation in the cytoplasm of non-professional phagocytes

An assay using human keratinocytes and staphylococci from a patient with Darier's disease revealed that >100-fold more SCVs persisted intracellularly relative to the normal phenotype [18]. Additionally, in a model of endovascular infection used to determine intracellular persistence, >200-fold more SCV (hemB) mutants persisted within cultured endothelial cells relative to the parent strain [47]. Similar results were obtained when endothelial cells were infected with SCVs from cystic fibrosis

Concluding remarks and future directions

Currently, there still is not definitive proof that S. aureus is a (facultative) intracellular pathogen because there are still important questions that need to be addressed (Box 1). Yet, the evidence is accumulating that at least certain strains of S. aureus can persist intracellularly. It is clear that these staphlyococci must adapt their lifestyle to the intracellular milieu. SCVs exhibit many properties that facilitate their attachment to, uptake into and residence and proliferation within

Glossary

Arginine deiminase pathway: a part of the bacteria's metabolism that aims to produce ATP. Arginine is degraded (via citrulline) to ornithine and carbamoyl-phosphate. The latter one serves to generate ATP from ADP by a carbamate kinase.
Auxotrophism: the requirement of specific compounds in order to grow normally. Auxotrophic organisms are unable to synthesize these essential nutrients and are, therefore, dependent on external sources.
Clumping factor: a protein that is excreted by Staphylococcus aureus

References (51)

S. Besier
Molecular analysis of the thymidine-auxotrophic small colony variant phenotype of Staphylococcus aureus
Int. J. Med. Microbiol.
(2007)
F. Schaaff
Mutations are involved in emergence of aminoglycoside-induced small colony variants of Staphylococcus aureus
Int. J. Med. Microbiol.
(2003)
A. Peschel
How do bacteria resist human antimicrobial peptides?
Trends Microbiol.
(2002)
R.I. Lehrer et al.
Antimicrobial peptides in mammalian and insect host defence
Curr. Opin. Immunol.
(1999)
I. Nuzzo
Apoptosis of human keratinocytes after bacterial invasion
FEMS Immunol. Med. Microbiol.
(2000)
W.R. Schwan
Loss of hemolysin expression in Staphylococcus aureus agr mutants correlates with selective survival during mixed infections in murine abscesses and wounds
FEMS Immunol. Med. Microbiol.
(2003)
R.A. Proctor
Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections
Nat. Rev. Microbiol.
(2006)
I. Chatterjee
Enhanced post-stationary-phase survival of a clinical thymidine-dependent small-colony variant of Staphylococcus aureus results from lack of a functional tricarboxylic acid cycle
J. Bacteriol.
(2007)
C. Kohler
Physiological characterization of a heme-deficient mutant of Staphylococcus aureus by a proteomic approach
J. Bacteriol.
(2003)
J. Seggewiss
Reporter metabolite analysis of transcriptional profiles of a Staphylococcus aureus strain with normal phenotype and its isogenic hemB mutant displaying the small-colony-variant phenotype
J. Bacteriol.
(2006)

C. von Eiff

Phenotype microarray profiling of Staphylococcus aureus menD and hemB mutants with the small-colony-variant phenotype

J. Bacteriol.

(2006)

J. Lannergard

Identification of the genetic basis for clinical menadione-auxotrophic small-colony variant isolates of Staphylococcus aureus

Antimicrob. Agents Chemother.

(2008)

I. Chatterjee

In vivo mutations of thymidylate synthase (encoded by thyA) are responsible for thymidine dependency in clinical small-colony variants of Staphylococcus aureus

J. Bacteriol.

(2008)

P. Sendi

Staphylococcus aureus small colony variants in prosthetic joint infection

Clin. Infect. Dis.

(2006)

B. Kahl

Persistent infection with small colony variant strains of Staphylococcus aureus in patients with cystic fibrosis

J. Infect. Dis.

(1998)

A. Schroder

Live cell imaging of phagosome maturation in Staphylococcus aureus infected human endothelial cells: small colony variants are able to survive in lysosomes

Med. Microbiol. Immunol.

(2006)

R.A. Proctor

Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus

Clin. Infect. Dis.

(1995)

Garzoni, C. and Kelley, W.L. (2009) Staphylococcus aureus: new evidence for intracellular persistence Trends Microbiol....

C. Garzoni

A global view of Staphylococcus aureus whole genome expression upon internalization in human epithelial cells

BMC Genomics

(2007)

S. Clement

Evidence of an intracellular reservoir in the nasal mucosa of patients with recurrent Staphylococcus aureus rhinosinusitis

J. Infect. Dis.

(2005)

F. Kipp

Detection of Staphylococcus aureus by 16S rRNA directed in situ hybridisation in a patient with a brain abscess caused by small colony variants

J. Neurol. Neurosurg. Psychiatry

(2003)

C. von Eiff

Intracellular persistence of Staphylococcus aureus small-colony variants within keratinocytes: a cause for antibiotic treatment failure in a patient with darier's disease

Clin. Infect. Dis.

(2001)

C. von Eiff

Recovery of small colony variants of Staphylococcus aureus following gentamicin bead placement for osteomyelitis

Clin. Infect. Dis.

(1997)

H. Moisan

Transcription of virulence factors in Staphylococcus aureus small-colony variants isolated from cystic fibrosis patients is influenced by SigB

J. Bacteriol.

(2006)

M.J. Horsburgh

sigmaB modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325-4

J. Bacteriol.

(2002)

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Staphylococcus aureus (S. aureus) has the ability to invade human cortical bones and cause intracellular infections in osteoblasts, which may lead to a long-term infection that is difficult to eliminate. It is critical to identify the underlying mechanisms of the osteoblast response to the intracellular S. aureus. More recently, multiple circular RNA (circRNA) functions have been identified, including serving as protein scaffolds or miRNA sponges and being translated into polypeptides. The role that circRNAs play in intracellular S. aureus infection of osteoblasts has not, to our knowledge, been investigated. Here, we established an intracellular infection model of S. aureus in osteoblasts and compared the circRNA expression of osteoblasts between the infected and control groups using RNA sequencing technology, by which a significant difference was found. In total, 117 upregulated and 125 down-regulated differentially expressed circRNAs (DEcircRNAs) were identified, and reverse transcription-quantitative PCR was employed to validate the results of RNA sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses demonstrated that DEcircRNAs were enriched in processes associated with macromolecule modification, cellular component organization or biogenesis, and intracellular non-membrane-bound organelles. Finally, a potentially important network of circRNA-miRNA-mRNA based on the DEcircRNAs was constructed. Overall, this study revealed the circRNA expression profile of human osteoblasts infected by intracellular S. aureus for the first time, and identified the circRNAs that may contribute to the pathogenesis of infectious diseases caused by intracellular S. aureus infection in human osteoblasts.
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Small colony variants (SCVs) are biotypes of bacteria that have a size of approximately one-tenth or less of the wild types and has distinct characteristics comparing to the wild type strains. Clinical SCVs are usually associated with persistent infection and require a long-term treatment program with antibiotics. In Saudi Arabia, there are few studies about SCVs Escherichia coli for this reason, this study is aimed to investigate the ability of gentamicin to mutate E. coli ATCC 25922 to produce small SCVs and investigate the genotypes and phenotypes changes and stress tolerance comparing to clinical SCVs E. coli and normal clinical E. coli Isolated from blood samples.
In this investigation, four clinical blood samples were collected ted from patients and the cultivation and isolation were carried out in KFMC between December 2019 and February 2021. The identification of positive blood culture samples was done using phoenix MD. Non-SCV E. coli ATCC25922 were mutated to SCV using exposure to increasing gradual concentrations of gentamicin at 100-generation intervals. Biochemical features and susceptibility to standard antibiotics using automated Phoenix MD 50 and. The survival assays were done using several stresses including heat shock, low pH, high osmotic pressure, and oxidative pressure. Virulence genes screening included the detection of genes that encoded to α-haemolysin, CS12 fimbriae, F17-like fimbrial adhesion, P-related fimbriae, yersiniabactin siderophore system, P-fimbriae, aerobactin, iron-regulated genes using PCR and gel electrophoresis.
The data from the mutating E. coli ATCC 25922 small colony test with gentamicin revealed that the first emergence of the multidrug resistance (MDR) SCV E. coli strain occurred at generation number 250, corresponding to a gentamicin concentration of 57 g/ml. Pathogenicity islands detection revealed that all tested E. coli strains have PAI IV 536 genes on their chromosomes furthermore, mutated SCV E. coli ATCC 25922 acquired PAII CFT₀₇₃ and PAI IV ₅₃₆. Survival tests showed no significant differences changes in tolerance of mutated SCVs comparing to parental strain.
The present work concluded that gentamicin sub-MIC concentration gradual exposure can induce mutation responsible for SCV formation and evolving of MDR E. coli strains. The mutated SCVs evolved high-level aminoglycoside resistance for gentamicin and resistance to amikacin, it also developed resistance to 2 cephalosporin antibiotics cefuroxime, and cephalothin and a resistance to aztreonam.
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Small-colony variants (SCVs) of bacteria are subpopulations with a small colony size, low growth rate, and atypical colony morphology. The purpose of this study was to comprehensively elucidate the characteristics and underlying mechanism of the development of a glutamine-dependent SCV of E. coli, GU-92_SCV, isolated from the blood of a patient with pyelonephritis.
The GU-92_SCV strain was tested for auxotrophy testing for glutamine. DNA mutations in genes related to glutamine synthesis were analysed by sequencing. The isolate's proliferation and antimicrobial susceptibility in Mueller-Hinton II medium supplemented with glutamine were examined.
The colony of the GU-92_SCV strain did not grow on Mueller-Hinton II agar, but growth around the filter paper containing l-glutamine was enhanced on Mueller-Hinton II agar. The GU-92_SCV strain had a single nucleotide substitution in glnA, c.193G>A, corresponding to p.Asp65Asn. Changing c.193G>A to the wild-type sequence in glnA restored these phenotypes. Because GU-92_SCV did not grow in Mueller-Hinton II broth, antimicrobial susceptibility test results were not obtained; however, in the presence of 10 mg mL⁻¹ l-glutamine, the results were consistent with those of the revertant strain GU-92_REV.
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Trends in Microbiology

OpinionStaphylococcus aureus as an intracellular pathogen: the role of small colony variants

Section snippets

Small colony variants of Staphylococcus aureus: a link to recurrent infections?

Intracellular S. aureus: normal colony-size or SCVs?

SCVs defense strategies to facilitate colonization

Increased uptake and adherence of SCVs?

SCVs cause less damage to host cells

Resistance to intracellular host defenses

Decreased activation of the host immune system

Proliferation in the cytoplasm of non-professional phagocytes

Concluding remarks and future directions

Glossary

Int. J. Med. Microbiol.

Int. J. Med. Microbiol.

Trends Microbiol.

Curr. Opin. Immunol.

FEMS Immunol. Med. Microbiol.

FEMS Immunol. Med. Microbiol.

Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections

Nat. Rev. Microbiol.

Enhanced post-stationary-phase survival of a clinical thymidine-dependent small-colony variant of Staphylococcus aureus results from lack of a functional tricarboxylic acid cycle

J. Bacteriol.

Physiological characterization of a heme-deficient mutant of Staphylococcus aureus by a proteomic approach

J. Bacteriol.

Reporter metabolite analysis of transcriptional profiles of a Staphylococcus aureus strain with normal phenotype and its isogenic hemB mutant displaying the small-colony-variant phenotype

J. Bacteriol.

Phenotype microarray profiling of Staphylococcus aureus menD and hemB mutants with the small-colony-variant phenotype

J. Bacteriol.

Identification of the genetic basis for clinical menadione-auxotrophic small-colony variant isolates of Staphylococcus aureus

Antimicrob. Agents Chemother.

In vivo mutations of thymidylate synthase (encoded by thyA) are responsible for thymidine dependency in clinical small-colony variants of Staphylococcus aureus

J. Bacteriol.

Staphylococcus aureus small colony variants in prosthetic joint infection

Clin. Infect. Dis.

Persistent infection with small colony variant strains of Staphylococcus aureus in patients with cystic fibrosis

J. Infect. Dis.

Live cell imaging of phagosome maturation in Staphylococcus aureus infected human endothelial cells: small colony variants are able to survive in lysosomes

Med. Microbiol. Immunol.

Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus

Clin. Infect. Dis.

A global view of Staphylococcus aureus whole genome expression upon internalization in human epithelial cells

BMC Genomics

Evidence of an intracellular reservoir in the nasal mucosa of patients with recurrent Staphylococcus aureus rhinosinusitis

J. Infect. Dis.

Detection of Staphylococcus aureus by 16S rRNA directed in situ hybridisation in a patient with a brain abscess caused by small colony variants

J. Neurol. Neurosurg. Psychiatry

Intracellular persistence of Staphylococcus aureus small-colony variants within keratinocytes: a cause for antibiotic treatment failure in a patient with darier's disease

Clin. Infect. Dis.

Recovery of small colony variants of Staphylococcus aureus following gentamicin bead placement for osteomyelitis

Clin. Infect. Dis.

Transcription of virulence factors in Staphylococcus aureus small-colony variants isolated from cystic fibrosis patients is influenced by SigB

J. Bacteriol.

sigmaB modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325-4

J. Bacteriol.

Opinion
Staphylococcus aureus as an intracellular pathogen: the role of small colony variants