Staphylococcus aureus RN4220, a cloning intermediate, is sometimes used in virulence, resistance, and metabolic studies. Using whole-genome sequencing, we showed that RN4220 differs from NCTC8325 and contains a number of genetic polymorphisms that affect both virulence and general fitness, implying a need for caution in using this strain for such studies.Staphylococcus aureus is a versatile pathogen that is responsible for the majority of nosocomial and community-acquired infections. One of the biggest challenges in treating staphylococcal infections is that many S. aureus strains have developed resistance against various antibiotics. In contrast to clinical isolates, RN4220 is a commonly used laboratory strain that is characterized by a mutation in the sau1 hsdR gene, making it restriction deficient and hence an ideal intermediate cloning host. RN4220 was originally derived from NCTC8325-4 using UV and chemical mutagenesis (10). NCTC8325-4, in turn, was derived from an early clinical isolate, NCTC8325 (also known as PS47 or RN1), cured of three prophages (16). Indeed, RN4220 is not a suitable candidate for the study of antibiotic resistance, because newer methicillin-resistant S. aureus (MRSA) lineages have evolved [e.g., ST239 (hospital), ST80, and ST59 (community)], presumably due to recombination events between lineages. RN4220 is also known to harbor a small deletion in rsbU, a gene within the stress-induced sigB operon, which renders it deficient in B expression. Additionally, RN4220 shows a ⌬agr mutant phenotype and does not produce ␣-hemolysin despite producing small amounts of RNAIII in late log phase (24). Recent studies revealed that a deletion of cvfB (encoding conserved virulence factor B) in RN4220 resulted in diminished agr expression, with a reduction in hla expression, protease production, and virulence, in a silkworm model of systemic infection (14), but the loss of hemolytic activity in the cvfB mutant of RN4220 was found to be due to a defective agr locus and not attributable to the cvfB mutation (14). In another study, SrrAB, a two-component regulatory system, was found to repress the transcription of RNAIII of the agr locus in RN4220 (20,27). However, the interpretation of virulence and of the associated regulatory data in these studies with RN4220 is suspect due to an inherent agr defect in this strain (24). Finally, O'Neill showed by comparative sequencing that NCTC8325-4, which was thought to be identical to its parent NCTC8325 except for the deletion of three prophages, possesses previously undescribed polymorphisms that may influence the virulence and pathogenicity of NCTC8325-4 (18). As a result of these issues, it is extremely important to delineate an accurate picture of the mutations in RN4220, given the polymorphisms in this strain which can have an impact upon virulence and resistance phenotype.Using Illumina Solexa-based whole-genome sequencing (paired end) (P. Mayer, L. Farinelli, and E. Kawashima, U.S. patent application WO98/44151), we obtained the whole-genome sequence of st...

fThe critical role of noncoding small RNAs (sRNAs) in the bacterial response to changing conditions is increasingly recognized. However, a specific role for sRNAs during antibiotic exposure has not been investigated in Staphylococcus aureus. Here, we used Illumina RNA-Seq to examine the sRNA response of multiresistant sequence type 239 (ST239) S. aureus after exposure to four antibiotics (vancomycin, linezolid, ceftobiprole, and tigecycline) representing the major classes of antimicrobials used to treat methicillin-resistant S. aureus (MRSA) infections. We identified 409 potential sRNAs and then compared global sRNA and mRNA expression profiles at 2 and 6 h, without antibiotic exposure and after exposure to each antibiotic, for a vancomycin-susceptible strain (JKD6009) and a vancomycin-intermediate strain (JKD6008). Exploration of this data set by multivariate analysis using a novel implementation of nonnegative matrix factorization (NMF) revealed very different responses for mRNA and sRNA. Where mRNA responses clustered with strain or growth phase conditions, the sRNA responses were predominantly linked to antibiotic exposure, including sRNA responses that were specific for particular antibiotics. A remarkable feature of the antimicrobial response was the prominence of antisense sRNAs to genes encoding proteins involved in protein synthesis and ribosomal function. This study has defined a large sRNA repertoire in epidemic ST239 MRSA and shown for the first time that a subset of sRNAs are part of a coordinated transcriptional response to specific antimicrobial exposures in S. aureus. These data provide a framework for interrogating the role of staphylococcal sRNAs in antimicrobial resistance and exploring new avenues for sRNA-based antimicrobial therapies.

Background: Chronic airway infection by Pseudomonas aeruginosa considerably contributes to lung tissue destruction and impairment of pulmonary function in cystic-fibrosis (CF) patients. Complex interplays between P. aeruginosa and other co-colonizing pathogens including Staphylococcus aureus, Burkholderia sp., and Klebsiella pneumoniae may be crucial for pathogenesis and disease progression.Methods: We generated a library of PA14 transposon insertion mutants to identify P. aeruginosa genes required for exploitative and direct competitions with S. aureus, Burkholderia cenocepacia, and K. pneumoniae.Results: Whereas wild-type PA14 inhibited S. aureus growth, two transposon insertions located in pqsC and carB, resulted in reduced growth inhibition. PqsC is involved in the synthesis of 4-hydroxy-2-alkylquinolines (HAQs), a family of molecules having antibacterial properties, while carB is a key gene in pyrimidine biosynthesis. The carB mutant was also unable to grow in the presence of B. cepacia and K. pneumoniae but not Escherichia coli and S. epidermidis. We further identified a transposon insertion in purF, encoding a key enzyme of purine metabolism. This mutant displayed a severe growth deficiency in the presence of Gram-negative but not of Gram-positive bacteria. We identified a beneficial interaction in a bioA transposon mutant, unable to grow on rich medium. This growth defect could be restored either by addition of biotin or by co-culturing the mutant in the presence of K. pneumoniae or E. coli.Conclusion: Complex interactions take place between the various bacterial species colonizing CF-lungs. This work identified both detrimental and beneficial interactions occurring between P. aeruginosa and three other respiratory pathogens involving several major metabolic pathways. Manipulating these pathways could be used to interfere with bacterial interactions and influence the colonization by respiratory pathogens.

In cystic fibrosis (CF) patients, chronic airway infection by Pseudomonas leads to progressive lung destruction ultimately requiring lung transplantation (LT). Following LT, CF-adapted Pseudomonas strains, potentially originating from the sinuses, may seed the allograft leading to infections and reduced allograft survival. We investigated whether CF-adapted Pseudomonas populations invade the donor microbiota and adapt to the non-CF allograft. We collected sequential Pseudomonas isolates and airway samples from a CF-lung transplant recipient during two years, and followed the dynamics of the microbiota and Pseudomonas populations. We show that Pseudomonas invaded the host microbiota within three days post-LT, in association with a reduction in richness and diversity. A dominant mucoid and hypermutator mutL lineage was replaced after 11 days by non-mucoid strains. Despite antibiotic therapy, Pseudomonas dominated the allograft microbiota until day 95. We observed positive selection of pre-LT variants and the appearance of novel mutations. Phenotypic adaptation resulted in increased biofilm formation and swimming motility capacities. Pseudomonas was replaced after 95 days by a microbiota dominated by Actinobacillus. In conclusion, mucoid Pseudomonas adapted to the CF-lung remained able to invade the allograft. Selection of both pre-existing non-mucoid subpopulations and of novel phenotypic traits suggests rapid adaptation of Pseudomonas to the non-CF allograft.