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Klebsiella pneumoniae is found in the environment and as a harmless commensal, but is also a frequent nosocomial pathogen (causing urinary, respiratory and blood infections) and the agent of specific human infections including Friedländer's pneumonia, rhinoscleroma and the emerging disease pyogenic liver abscess (PLA). The identification and precise definition of virulent clones, i.e. groups of strains with a single ancestor that are associated with particular infections, is critical to understand the evolution of pathogenicity from commensalism and for a better control of infections. We analyzed 235 K. pneumoniae isolates of diverse environmental and clinical origins by multilocus sequence typing, virulence gene content, biochemical and capsular profiling and virulence to mice. Phylogenetic analysis of housekeeping genes clearly defined clones that differ sharply by their clinical source and biological features. First, two clones comprising isolates of capsular type K1, clone CC23K1 and clone CC82K1, were strongly associated with PLA and respiratory infection, respectively. Second, only one of the two major disclosed K2 clones was highly virulent to mice. Third, strains associated with the human infections ozena and rhinoscleroma each corresponded to one monomorphic clone. Therefore, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis should be regarded as virulent clones derived from K. pneumoniae. The lack of strict association of virulent capsular types with clones was explained by horizontal transfer of the cps operon, responsible for the synthesis of the capsular polysaccharide. Finally, the reduction of metabolic versatility observed in clones Rhinoscleromatis, Ozaenae and CC82K1 indicates an evolutionary process of specialization to a pathogenic lifestyle. In contrast, clone CC23K1 remains metabolically versatile, suggesting recent acquisition of invasive potential. In conclusion, our results reveal the existence of important virulent clones associated with specific infections and provide an evolutionary framework for research into the links between clones, virulence and other genomic features in K. pneumoniae.

Staphylococcus aureus is highly adapted to its host and has evolved many strategies to resist opsonization and phagocytosis. Even after uptake by neutrophils, S. aureus shows resistance to killing, which suggests the presence of phagosomal immune evasion molecules. With the aid of secretome phage display, we identified a highly conserved protein that specifically binds and inhibits human myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. We have named this protein "staphylococcal peroxidase inhibitor" (SPIN). To gain insight into inhibition of MPO by SPIN, we solved the cocrystal structure of SPIN bound to a recombinant form of human MPO at 2.4-Å resolution. This structure reveals that SPIN acts as a molecular plug that prevents H 2 O 2 substrate access to the MPO active site. In subsequent experiments, we observed that SPIN expression increases inside the neutrophil phagosome, where MPO is located, compared with outside the neutrophil. Moreover, bacteria with a deleted gene encoding SPIN showed decreased survival compared with WT bacteria after phagocytosis by neutrophils. Taken together, our results demonstrate that S. aureus secretes a unique proteinaceous MPO inhibitor to enhance survival by interfering with MPO-mediated killing.

Local injection of a bacteriophages mix during debridement, antibiotics and implant retention (“DAIR”) was performed to treat a relapsing Staphylococcus aureus chronic prosthetic joint infection (PJI). This salvage treatment was safe and associated with a clinical success. Scientific evaluation of the potential clinical benefit of bacteriophages as antibiofilm treatment in PJI is now feasible and required.

Objectives: To report the management of three consecutive patients with relapsing Staphylococcus aureus prosthetic knee infection (PKI) for whom explantation was not feasible who received a phage therapy during a “Debridement Antibiotics and Implant Retention” (DAIR) procedure followed by suppressive antimicrobial therapy. Methods: Each case was discussed individually in our reference center and with the French National Agency (ANSM). The lytic activity of three phages targeting S. aureus , which was produced with a controlled and reproducible process, was assessed before surgery (phagogram). A hospital pharmacist extemporaneously assembled the phage cocktail (1 ml of 1 × 10 10 PFU/ml for each phage) as “magistral” preparation (final dilution 1 × 10 9 PFU/ml), which was administered by the surgeon directly into the joint, after the DAIR procedure and joint closure (PhagoDAIR procedure). Results: Three elderly patients were treated with the PhagoDAIR procedure. Phagograms revealed a high susceptibility to at least two of the three phages. During surgery, all patients had poor local conditions including pus in contact to the implant. After a prolonged follow-up, mild discharge of synovial fluid persisted in two patients, for whom a subsequent DAIR was performed showing only mild synovial inflammation without bacterial persistence or super-infection. The outcome was finally favorable with a significant and impressive clinical improvement of the function. Conclusions: The PhagoDAIR procedure has the potential to be used as salvage for patients with relapsing S. aureus PKI, in combination with suppressive antibiotics to avoid considerable loss of function. This report provides preliminary data supporting the setup of a prospective multicentric clinical trial.

The diversity and evolution of the class A OXY ␤-lactamase from Klebsiella oxytoca were investigated and compared to housekeeping gene diversity. The entire bla OXY coding region was sequenced in 18 clinical isolates representative of the four K. oxytoca ␤-lactamase gene groups bla OXY-1 to bla OXY-4 and of two new groups identified here, bla OXY-5 (with four isolates with pI 7.2 and one with pI 7.7) and bla OXY-6 (with four isolates with pI 7.75 and three with pI 8.1). Genes bla OXY-5 and bla OXY-6 showed 99.8% within-group nucleotide similarity but differed from each other by 4.2% and from bla OXY-1 , their closest relative, by 2.5% and 2.9%, respectively. Antimicrobial susceptibility to ␤-lactams was similar among OXY groups. Nucleotide sequence diversity of the 16S rRNA (1,454 bp), rpoB (940 bp), gyrA (383 bp), and gapDH (573 bp) genes was in agreement with the ␤-lactamase gene phylogeny. Strains with bla OXY-1 , bla OXY-2 , bla OXY-3 , bla OXY-4 , and bla OXY-6 genes formed five phylogenetic groups, named KoI, KoII, KoIII, KoIV, and KoVI, respectively. Isolates harboring bla OXY-5 appeared to represent an emerging lineage within KoI. We estimated that the bla OXY gene has been evolving within K. oxytoca for approximately 100 million years, using as calibration the 140-million-year estimation of the Escherichia coli-Salmonella enterica split. These results show that the bla OXY gene has diversified along K. oxytoca phylogenetic lines over long periods of time without concomitant evolution of the antimicrobial resistance phenotype.Klebsiella oxytoca is an important opportunistic pathogen causing serious infections in hospitalized patients, including neonates (16,19,28). K. oxytoca is naturally resistant to aminoand carboxy-penicillins (21), a phenotype due to the constitutive expression of a chromosomal class A ␤-lactamase (1), first called K1 (7,20) or KOXY (26) and now OXY (12). Due to the hyperproduction of the chromosomal ␤-lactamase, up to 10 to 20% of K. oxytoca strains (22) can show high-level resistance to certain expanded-spectrum cephalosporins (ceftriaxone and cefotaxime) and aztreonam (8). Up mutations in the promoter sequence of the genes are responsible for this phenotype (9-11). Overproducers of OXY enzymes are commonly resistant to all combinations of ␤-lactams with ␤-lactam inhibitors (21).Sequence diversity of the K. oxytoca chromosomal ␤-lactamase gene and the existence of discrete groups of OXY enzymes have been described. Fournier et al. (12) found a variant (bla OXY-2 ) of the chromosomal ␤-lactamase gene that differed from bla OXY-1 (1) by 12.7% in nucleotide sequence. It was suggested, based on colony hybridization, that the ␤-lactamase genes of K. oxytoca could be classified into two groups (OXY-1 and OXY-2), each representing approximately half of the clinical isolates (12). Recently, Granier et al. (14) identified two additional sequence variants, defined as bla OXY-3 and bla , each found in a single strain so far. Gene bla shares 85 and 84% similarity with bla OXY-1 and bla OXY...

Two bla OKP subgroups were found, diverging by 4.2%. Subgroups bla OKP-A (10 enzyme variants, pIs from 7.1 to 8.3) and bla OKP-B (11 variants, pI 7.1) showed similar antibiotic susceptibilities. Sequencing of rpoB, gyrA, and mdh demonstrated a concordant subdivision of Klebsiella pneumoniae phylogenetic group KpII into two subgroups, KpII-A and KpII-B.Klebsiella pneumoniae clinical isolates are naturally resistant to ampicillin, amoxicillin, carbenicillin, and ticarcillin, but not to extended-spectrum beta-lactams, due to the constitutive expression of a chromosomally encoded beta-lactamase. Three families of the chromosomal beta-lactamase gene, bla SHV , bla OKP , and bla LEN, evolved from a common ancestor over millions of years, in parallel with the diversification of the three K. pneumoniae phylogenetic groups KpI, KpII, and KpIII (3, 6). The bla OKP family was found in group KpII (6) and was more heterogeneous than the SHV and LEN families, with four OKP enzyme variants. These variants appeared to belong to two OKP subgroups, but a higher number of variants was judged necessary to firmly demonstrate the phylogenetic distinctness of these subgroups (6). In addition, because homologous recombination is more frequent among closely related strains than between distant phylogenetic groups (8, 10), the correspondence observed at the level of the three phylogenetic groups between beta-lactamase and housekeeping gene classifications (5, 6) could be disrupted by homologous gene transfer within KpII.Precise identification of bla variants can be critical for epidemiological purposes and infection control. The objectives of the present study were to discover additional OKP enzyme variants, to determine whether strains harboring distinct OKP variants differ in their antibiotic susceptibility, and to determine whether bla OKP gene variants are subdivided into groups that correspond to housekeeping gene sequence groups.In order to discover new OKP variants,

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