c Acinetobacter baumannii, a Gram-negative multidrug-resistant (MDR) bacterium, is now recognized as one of the more common nosocomial pathogens. Because most clinical isolates are found to be multidrug resistant, alternative therapies need to be developed to control this pathogen. We constructed a bacteriophage genomic library based on prophages induced from 13 A. baumannii strains and screened it for genes encoding bacteriolytic activity. Using this approach, we identified 21 distinct lysins with different activities and sequence diversity that were capable of killing A. baumannii. The lysin (PlyF307) displaying the greatest activity was further characterized and was shown to efficiently kill (>5-log-unit decrease) all tested A. baumannii clinical isolates. Treatment with PlyF307 was able to significantly reduce planktonic and biofilm A. baumannii both in vitro and in vivo. Finally, PlyF307 rescued mice from lethal A. baumannii bacteremia and as such represents the first highly active therapeutic lysin specific for Gram-negative organisms in an array of native lysins found in Acinetobacter phage. Members of Acinetobacter are soil bacteria that frequently colonize the human skin without harm (1). However, in environments in which individuals are immunocompromised or suffer from a variety of wounds (e.g., in hospital settings or on battlefields), Acinetobacter baumannii can cause severe life-threatening infections (2-4). Symptoms of A. baumannii infections range from mild skin wounds and urinary tract infections to more severe conditions, including pneumonia, meningitis, and sepsis (5). A. baumannii is now one of the most common causes of hospital-acquired pneumonia (2) and sepsis; while not common (only 1.3% of all sepsis cases), it is associated with mortality rates of up to 58% (6).One of the main threats from A. baumannii is the high rate of resistance to antibiotics commonly used to treat Gram-negative infections. More than 80% of Acinetobacter species are considered to be multidrug resistant (MDR) (i.e., resistant to at least three classes of antibiotics), resulting in infections with poor clinical outcomes, including high rates of morbidity and death, prolonged hospital stays, and substantial health care expenses (3, 7). In addition, several strains of pan-drug-resistant A. baumannii have been isolated, showing resistance to a wide variety of clinically used antibiotics (8). A. baumannii is also capable of surviving treatments with detergents and disinfectants, dehydration, and UV radiation and thus is difficult to eradicate from surfaces in hospital environments (9, 10). The organism not only is intrinsically resistant to many antibiotics (owing to ␤-lactamases, weak membrane permeability, and efficient efflux systems) but also can readily acquire foreign plasmids and is considered to have a high degree of genetic plasticity (11). Outbreaks caused by MDR Acinetobacter have been reported from hospitals worldwide; more recently, they have become a serious problem in military medical facilities (4). One of ...

Propionibacterium acnes is a common and probably underestimated cause of delayed joint prosthesis infection. Bacterial biofilm formation is central in the pathogenesis of infections related to foreign material, and P. acnes has been shown to form biofilm both in vitro and in vivo. Here, biofilm formation by 93 P. acnes isolates, either from invasive infections (n = 45) or from the skin of healthy people (n = 48), was analysed. The majority of isolates from deep infections produced biofilm in a microtitre model of biofilm formation, whereas the skin isolates were poor biofilm producers (p <0.001 for a difference). This indicates a role for biofilm formation in P. acnes virulence. The type distribution, as determined by sequencing of recA, was similar among isolates isolated from skin and from deep infections, demonstrating that P. acnes isolates with different genetic backgrounds have pathogenic potential. The biofilm formed on plastic and on bone cement was analysed by scanning electron microscopy (EM) and by transmission EM. The biofilm was seen as a 10-mum-thick layer covering the bacteria and was composed of filamentous as well as more amorphous structures. Interestingly, the presence of human plasma in solution or at the plastic surface inhibits biofilm formation, which could explain why P. acnes primarily infect plasma-poor environments of, for example, joint prostheses and cerebrospinal shunts. This work underlines the importance of biofilm formation in P. acnes pathogenesis, and shows that biofilm formation should be considered in the diagnosis and treatment of invasive P. acnes infections.

Acinetobacter baumannii is a Gram-negative bacterial pathogen responsible for a range of nosocomial infections. The recent rise and spread of multidrug-resistant A. baumannii clones has fueled a search for alternative therapies, including bacteriophage endolysins with potent antibacterial activities. A common feature of these lysins is the presence of a highly positively charged C-terminal domain with a likely role in promoting outer membrane penetration. In the present study, we show that the C-terminal amino acids 108 to 138 of phage lysin PlyF307, named P307, alone were sufficient to kill A. baumannii (>3 logs). Furthermore, P307 could be engineered for improved activity, the most active derivative being P307 SQ-8C (>5-log kill). Both P307 and P307 SQ-8C showed high in vitro activity against A. baumannii in biofilms. Moreover, P307 SQ-8C exhibited MICs comparable to those of levofloxacin and ceftazidime and acted synergistically with polymyxin B. Although the peptides were shown to kill by disrupting the bacterial cytoplasmic membrane, they did not lyse human red blood cells or B cells; however, serum was found to be inhibitory to lytic activity. In a murine model of A. baumannii skin infection, P307 SQ-8C reduced the bacterial burden by ϳ2 logs in 2 h. This study demonstrates the prospect of using peptide derivatives from bacteriophage lysins to treat topical infections and remove biofilms caused by Gram-negative pathogens.

The role of the skin microbiota in human health is poorly understood. Here, we identified and characterized a novel antioxidant enzyme produced by the skin microbiota, designated RoxP for radical oxygenase of Propionibacterium acnes. RoxP is uniquely produced by the predominant skin bacterium P. acnes, with no homologs in other bacteria; it is highly expressed and strongly secreted into culture supernatants. We show that RoxP binds heme, reduces free radicals, and can protect molecules from oxidation. Strikingly, RoxP is crucial for the survival of P. acnes in oxic conditions and for skin colonization of P. acnes ex vivo. Taken together, our study strongly suggests that RoxP facilitates P. acnes’ survival on human skin, and is an important beneficial factor for the host-commensal interaction. Thus, RoxP is the first described skin microbiota-derived mutualistic factor that potentially can be exploited for human skin protection.

BackgroundPropionibacterium acnes is a Gram positive rod inhabiting the human skin that also infects orthopaedic implants and is associated with acne vulgaris. Previously, one lytic bacteriophage, PA6, from P. acnes has been sequenced and partially characterized. We recently isolated several inducible phages from P. acnes classified as Siphoviruses based on morphology and partial genome sequencing.ResultsIn this study we sequenced the inducible P. acnes phages PAD20 and PAS50, isolated from deep infection and from skin, respectively. The genomes of PAD20 and PAS50 are 29,074 and 29,017 bp, respectively, compared with the 29,739 bp of PA6. The phage genomes have 87.3-88.7% nucleotide sequence identity. The genes are divided into clusters with different levels of similarity between the phages. PAD20 and PAS50 share four genes encoding identical amino acid sequences. Some deletions and insertions in the genomes have occurred, resulting in lack of genes, frame shifts, and possible regulatory differences. No obvious virulence factor gene candidates were found. The phages are inducible, but bacteria can be cured of phages by serial colony isolations and lose their phages during stationary phase, but are still sensitive to new phage infections. Construction of a phylogenetic tree based on more than 459 phage genomes, suggested that P. acnes phages represent a new lineage of Siphoviruses.ConclusionsThe investigated P. acnes Siphovirus genomes share a high degree of homology to other P. acnes phages sequenced, but not to genomes of other phages isolated from Propionibacteria. The phage genomes are not integrated in the bacterial genome, but instead, most likely have a pseudolysogenic life cycle.