Klebsiella pneumoniae is recognized as an important gram-negative opportunistic pathogen. The ability of bacteria to adhere to host structures is considered essential for the development of infections; however, few studies have examined the influence of adhesion factors on K. pneumoniae virulence. In this study, we cloned and characterized the type 1 fimbria gene cluster of a clinical K. pneumoniae isolate. Although this cluster was not identical to the Escherichia coli type 1 fimbria gene cluster, an overall high degree of structural resemblance was demonstrated. Unique to the K. pneumoniae fim gene cluster is the fimK gene, whose product contains an EAL domain, suggesting that it has a role in regulation of fimbrial expression. Like expression of type 1 fimbriae in E. coli , expression of type 1 fimbriae in K. pneumoniae was found to be phase variable, and an invertible DNA element ( fim switch) was characterized. An isogenic type 1 fimbria mutant was constructed and used to evaluate the influence of type 1 fimbriae in different infection models. Type 1 fimbriae did not influence the ability of K. pneumoniae to colonize the intestine or infect the lungs, but they were determined to be a significant virulence factor in K. pneumoniae urinary tract infection. By use of a PCR-based assay, the orientation of the fim switch during colonization and infection was investigated and was found to be all “off” in the intestine and lungs but all “on” in the urinary tract. Our results suggest that during colonization and infection, there is pronounced selective pressure in different host environments for selection of either the type 1 fimbriated or nonfimbriated phenotype of K. pneumoniae .

Type 3 fimbriae are expressed by most clinical Klebsiella pneumoniae isolates and mediate adhesion to host structures in vitro. However, the role of type 3 fimbriae in K. pneumoniae virulence has not been evaluated by use of in vivo infection models. In this study, the type 3 fimbrial gene cluster ( mrk ) of the clinical isolate C3091 is described in detail. The mrk gene cluster was revealed to be localized in close proximity to the type 1 fimbrial gene cluster. Thus, a 20.4-kb fimbria-encoding region was identified and found to be highly conserved among different K. pneumoniae isolates. Interestingly, a homologue to PecS, known as a global regulator of virulence in Erwinia chrysanthemi , was identified in the fimbria-encoding region. Comparison to the previously characterized plasmid encoded mrk gene cluster revealed significant differences, and it is established here that the putative regulatory gene mrkE is not a part of the chromosomally encoded type 3 fimbrial gene cluster. To evaluate the role of type 3 fimbriae in virulence, a type 3 fimbria mutant and a type 1 and type 3 fimbria double mutant was constructed. Type 3 fimbria expression was found to strongly promote biofilm formation. However, the fimbria mutants were as effective at colonizing the intestine as the wild type, and their virulence was not attenuated in a lung infection model. Also, in a urinary tract infection model, type 3 fimbriae did not influence the virulence, whereas type 1 fimbriae were verified as an essential virulence factor. Thus, type 3 fimbriae were established not to be a virulence factor in uncomplicated K. pneumoniae infections. However, since type 3 fimbriae promote biofilm formation, their role in development of infections in catheterized patients needs to be elucidated.

Methicillin-resistant Staphylococcus aureus (MRSA) continues to be a serious human pathogen, and particularly the spread of community associated (CA)-MRSA strains such as USA300 is a concern, as these strains can cause severe infections in otherwise healthy adults. Recently, we reported that a cyclodepsipeptide termed Solonamide B isolated from the marine bacterium, Photobacterium halotolerans strongly reduces expression of RNAIII, the effector molecule of the agr quorum sensing system. Here we show that Solonamide B interferes with the binding of S. aureus autoinducing peptides (AIPs) to sensor histidine kinase, AgrC, of the agr two-component system. The hypervirulence of USA300 has been linked to increased expression of central virulence factors like α-hemolysin and the phenol soluble modulins (PSMs). Importantly, in strain USA300 Solonamide B dramatically reduced the activity of α-hemolysin and the transcription of psma encoding PSMs with an 80% reduction in toxicity of supernatants towards human neutrophils and rabbit erythrocytes. To our knowledge this is the first report of a compound produced naturally by a Gram-negative marine bacterium that interferes with agr and affects both RNAIII and AgrA controlled virulence gene expression in S. aureus.

Staphylococcus aureus is an opportunistic pathogen related to a variety of life-threatening infections but for which antimicrobial resistance is liming the treatment options. We report here that myricetin, but not its glycosylated form, can remarkably decrease the production of several S. aureus virulence factors, including adhesion, biofilm formation, hemolysis and staphyloxanthin production, without interfering with growth. Myricetin affects both surface proteins and secreted proteins which indicate that its action is unrelated to inhibition of the agr quorum sensing system. Analysis of virulence related gene expression and computational simulations of pivotal proteins involved in pathogenesis demonstrate that myricetin downregulates the saeR global regulator and interacts with sortase A and α-hemolysin. Furthermore, Myr confers a significant degree of protection against staphylococcal infection in the Galleria mellonella model. The present findings reveal the potential of Myr as an alternative multi-target antivirulence candidate to control S. aureus pathogenicity.

Persister cells constitute a small subpopulation of bacteria that display remarkably high antibiotic tolerance and for pathogens such as Staphylococcus aureus are suspected as culprits of chronic and recurrent infections. Persisters formed during exponential growth are characterized by low ATP levels but less is known of cells in stationary phase. By enrichment from a transposon mutant library in S. aureus we identified mutants that in this growth phase displayed enhanced persister cell formation. We found that inactivation of either sucA or sucB, encoding the subunits of the α-ketoglutarate dehydrogenase of the tricarboxylic acid cycle (TCA cycle), increased survival to lethal concentrations of ciprofloxacin by 10–100 fold as did inactivation of other TCA cycle genes or atpA encoding a subunit of the F₁F₀ ATPase. In S. aureus, TCA cycle activity and gene expression are de-repressed in stationary phase but single cells with low expression may be prone to form persisters. While ATP levels were not consistently affected in high persister mutants they commonly displayed reduced membrane potential, and persistence was enhanced by a protein motive force inhibitor. Our results show that persister cell formation in stationary phase does not correlate with ATP levels but is associated with low membrane potential.

Staphylococci are associated with both humans and animals. While most are non-pathogenic colonizers, Staphylococcus aureus is an opportunistic pathogen capable of causing severe infections. S. aureus virulence is controlled by the agr quorum sensing system responding to secreted auto-inducing peptides (AIPs) sensed by AgrC, a two component histidine kinase. agr loci are found also in other staphylococcal species and for S. epidermidis, the encoded AIP represses expression of agr regulated virulence genes in S. aureus. In this study we aimed to determine how other staphylococci affect S. aureus agr, and if such interaction may point to new anti-virulence candidates to target S. aureus infections. Here we show that culture supernatants of 37 out of 52 staphylococcal isolates representing 17 different species inhibit S. aureus agr. The dog pathogen, S. schleiferi, expressed the most potent inhibitory activity and was active against all four agr classes found in S. aureus. By employing a S. aureus strain encoding a constitutively active AIP receptor we show that the activity is mediated via agr. Subsequent cloning and heterologous expression of the S. schleiferi AIP in S. aureus demonstrated that this molecule was likely responsible for the inhibitory activity, and further proof was provided when pure synthetic S. schleiferi AIP was able to completely abolish agr induction of an S. aureus reporter strain. To assess impact on S. aureus virulence, we co-inoculated S. aureus and S. schleiferi in vivo in the Galleria Mellonella wax moth larva, and found that expression of key S. aureus virulence factors was abrogated. Our data show that the S. aureus agr locus is highly responsive to other staphylococcal species suggesting that agr is an inter-species communication system. Based on these results we speculate that interactions between S. aureus and other colonizing staphylococci will significantly influence the ability of S. aureus to cause infection, and we propose that other staphylococci are potential sources of compounds that can be applied as anti-virulence therapy for combating S. aureus infections.