Characterization of the Prophage Repertoire of African Salmonella Typhimurium ST313 Reveals High Levels of Spontaneous Induction of Novel Phage BTP1

In the past 30 years,Salmonella bloodstream infections have become a significant health problem in sub-Saharan Africa and are responsible for the deaths of anestimated 390,000 people each year. The disease is predominantly caused by a recently described sequence type of SalmonellaTyphimurium: ST313, which has a distinctive set of prophage sequences. We have thoroughly characterized the ST313-associated prophages both genetically and experimentally. ST313 representative strain D23580 contains five full-length prophages: BTP1, Gifsy-2D23580, ST64BD23580, Gifsy-1D23580,and BTP5. We show that commonS.Typhimurium prophages Gifsy-2, Gifsy-1, andST64B are inactivated in ST313 by mutations. Prophage BTP1 was found to be a functional novel phage, and the first isolate of the proposed new species “Salmonellavirus BTP1”, belonging to the P22virusgenus. Surprisingly,∼109BTP1 virus particlesperml were detected in the supernatant of non-induced, stationary-phase culturesof strain D23580, representing the highest spontaneously induced phage titer so farreported for a bacterial prophage. High spontaneous induction is shown to be anintrinsic property of prophage BTP1, and indicates the phage-mediated lysis of around0.2% of the lysogenic population. The fact that BTP1 is highly conserved in ST313 poses interesting questions about the potential fitness costs and benefits of novel prophagesin epidemicS.Typhimurium ST313

Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa

Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch.One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10(-5) on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels.By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system