Increasing multidrug resistance has led to renewed interest in phage-based therapy. A combination of the bacteriophages and antibiotics presents a promising approach enhancing the phage therapy effectiveness. First, phage candidates for therapy should be deeply characterized. Here we characterize the bacteriophage vB_AbaP_AGC01 that poses antibacterial activity against clinical Acinetobacter baumannii strains. Moreover, besides genomic and phenotypic analysis our study aims to analyze phage–antibiotic combination effectiveness with the use of ex vivo and in vivo models. The phage AGC01 efficiently adsorbs to A. baumannii cells and possesses a bacteriolytic lifecycle resulting in high production of progeny phages (317 ± 20 PFU × cell−1). The broad host range (50.27%, 93 out of 185 strains) against A. baumannii isolates and the inability of AGC01 to infect other bacterial species show its high specificity. Genomic analysis revealed a high similarity of the AGC01 genome sequence with that of the Friunavirus genus from a subfamily of Autographivirinae. The AGC01 is able to significantly reduce the A. baumannii cell count in a human heat-inactivated plasma blood model (HIP-B), both alone and in combination with antibiotics (gentamicin (GEN), ciprofloxacin (CIP), and meropenem (MER)). The synergistic action was observed when a combination of phage treatment with CIP or MER was used. The antimicrobial activity of AGC01 and phage-antibiotic combinations was confirmed using an in vivo larva model. This study shows the greatest increase in survival of G. mellonella larvae when the combination of phage (MOI = 1) and MER was used, which increased larval survival from 35% to 77%. Hence, AGC01 represents a novel candidate for phage therapy. Additionally, our study suggests that phages and antibiotics can act synergistically for greater antimicrobial effect when used as combination therapy.

Staphylococcus aureus is a major cause of skin and soft tissue infections, such as furuncles, carbuncles, and abscesses, but it also frequently colonizes the human skin and mucosa without causing clinical symptoms. Panton-Valentine leukocidin (PVL) is a pore-forming toxin that has been associated with soft tissue infections and necrotizing pneumonia. We have compared the genotypes, virulence gene repertoires, and phage patterns of 74 furunculosis isolates with those of 108 control strains from healthy nasal carriers. The large majority of furunculosis strains were methicillin sensitive. Clonal cluster (CC) 121 (CC121) and CC22 accounted for 70% of the furunculosis strains but for only 8% of the nasal isolates. The PVL-encoding genes luk-PV were detected in 85% of furunculosis strains, while their prevalence among colonizing S. aureus strains was below 1%. luk-PV genes were distributed over several lineages (CCs 5, 8, 22, 30, and 121 and sequence type 59). Even within the same lineages, luk-PV-positive phages characterized furunculosis strains, while their luk-PV-negative variants were frequent among nasal strains. The very tight epidemiological linkage between luk-PV and furunculosis, which could be separated from the genetic background of the S. aureus strain as well as from the gene makeup of the luk-PV-transducing phage, lends support to the notion of an important role for PVL in human furunculosis. These results make a case for the determination of luk-PV in recurrent soft tissue infections with methicillin-sensitive as well as methicillin-resistant S. aureus.Skin and soft tissue infections (SSTIs) are the most frequent type of disease caused by Staphylococcus aureus outside the hospital setting. SSTIs comprise a diverse range of clinical pictures, such as furuncles, carbuncles, subcutaneous abscesses, folliculitis, bullous impetigo, and staphylococcal scalded skin syndrome (23

Autologous vaccines (short: autovaccines) have been used since the beginning of the 20th century to treat chronic staphylococcal infections, but their mechanisms of action are still obscure. This prospective pilot study involved four patients with furunculosis who were vaccinated with autologous formalin-killed Staphylococcus aureus cells. Vaccines were individually prepared from the infecting S. aureus strain and repeatedly injected subcutaneously in increasing doses over several months. We characterized the virulence gene repertoire and spa genotype of the infecting and colonising S. aureus strains. Serum antibody responses to secreted and surface-bound bacterial antigens were determined by two-dimensional immunoblotting and flow-cytometry based assays (Luminex®). All patients reported clinical improvement. Molecular characterization showed that all strains isolated from one patient over time belonged to the same S. aureus clone. Already before treatment, there was robust antibody binding to a broad range of staphylococcal antigens. Autovaccination moderately boosted the IgG response to extracellular antigens in two patients, while the antibody response of the other two patients was not affected. Similarly, vaccination moderately enhanced the antibody response against some staphylococcal surface proteins, e.g. ClfA, ClfB, SdrD and SdrE. In summary, autovaccination only slightly boosted the pre-existing serum antibody response, predominantly to bacterial surface antigens.Electronic supplementary materialThe online version of this article (doi:10.1007/s10096-010-1136-3) contains supplementary material, which is available to authorized users.

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Enterobacteriaceae bacilli are a significant problem in neonatal intensive care units, especially in early-onset infection and for long hospitalized very low birth weight infants. The observed high drug resistance was in large part related to the dominance of epidemic strains as a result of horizontal transmission. The best way to reduce drug resistance would be adequate procedures of isolation and hand hygiene.

Background Pseudomonas aeruginosa is a Gram-negative bacteria responsible for infections in immunocompromised patients and is one of the most common causes of nosocomial infections particularly in intensive care and burn units. We aimed to investigate the population structure of P. aeruginosa strains isolated from patients at different hospital wards. Methods: We analysed the possible presence of P. aeruginosa epidemic or endemic strains in hospitals of the selected region. A genotyping analysis was performed for P. aeruginosa isolates (n = 202) collected from patients of eleven hospitals in north-western Poland. Collections of P. aeruginosa were genotyped using pulsed-field gel electrophoresis (PFGE). Phenotypic screening for antibiotic susceptibility was performed for the common antimicrobial agents. Results Pseudomonas aeruginosa isolates were distributed among 116 different pulsotype groups. We identified 30 groups of clonally related strains, each containing from 2 to 17 isolates and typed the obtained 13 unique patterns, designated as A, D, E, J, K, M, N, Ó, P, T, X, AC, AD, and AH. The two largest clusters, D and E, contained 17 and 13 isolates, respectively. Strains of these groups were continuously isolated from patients at intensive care units and burn units, indicating transmission of these strains. Conclusions In this study, we demonstrate the clonal relatedness of P. aeruginosa strains and their constant exchange in hospitals over a period of 15 months. The obtained results indicate a predominantly non-clonal structure of P. aeruginosa.

Background Atopic dermatitis (AD) is one of the most frequent chronic and inflammatory skin condition. AD is characterized by damaged epidermal barrier, xerosis and pruritus of eczematous skin lesions which tend to flare. The duration and frequency of exacerbation of AD symptoms markedly affects the quality of patient life. AD results from the interplay between host genetics, immunity, and environmental factors, however the detailed pathogenesis of this disease is still not entirely cleared. Furthermore, disturbances of the skin microbiota and skin functional impairment predispose to secondary skin infections. Staphylococcus aureus colonizes skin and mucous membranes of 20 to 80% of healthy individuals and of 90% of patients with AD in whom this bacterium is accounted as an important AD exacerbating factor. It is also proven, that S. aureus nasal carriage significantly increases the risk for self-transmission and endogenous infection. In the current study the presence of S. aureus either in nasal vestibule and on lesioned skin of 64 patients with AD enrolled in 10-year autovaccination program was determined. The genetic relatedness of 86 S. aureus isolated from patients nose and skin using Pulsed Field Gel Electrophoresis (PFGE) and antimicrobial susceptibility of all strains to methicillin, erythromycin, clindamycin, mupirocin, gentamicin, amikacin, tetracycline, chloramphenicol and cotrimoxazole was also evaluated. Results In total 23 PFGE genotypes and 24 unique patterns were distinguished. 34 patients were S. aureus nasal carriers. Simultaneous presence of S. aureus in nose and on affected skin was found in 16 carriers colonized by indistinguishable or potentially related S. aureus vs 2 carriers colonized with non-related S. aureus in nasal vestibule and on skin. 4 isolates were methicillin resistant (MRSA) among which 3 showed constitutive MLSB resistance phenotype and remaining one was resistant to tetracycline and chloramphenicol. In 4 isolates inducible MLSB resistance phenotype was found, one of them was additionally resistant to tetracycline. 7 S. aureus were mupirocin resistant among them 3 - isolated from one patient, were resistant simultaneously to tetracyclines and chloramphenicol. 7 strains demonstrated resistance to chloramphenicol and susceptibility to all tested antimicrobial agents. The susceptibility to gentamicin, amikacin and cotrimoxazole among all examined S. aureus was confirmed. Conclusion The obtained results indicated non-clonal structure of S. aureus circulating in AD patients. PFGE results showed the clonal-structure of vast majority of S. aureus isolated from nose and skin from nasal carriers what may prove the autoinfection in these patients. All examined patients the moderate or strong severity of AD was reported. Susceptibility to most antibiotics among isolated strains was also observed.

Background: Carriers ofStaphylococcus aureus strains can be the source of epidemic infection for patients. Objectives: A molecular epidemiological analysis of an impetigo bullosa outbreak in a neonatal ward was performed in order to determine a potential source of the infection and possible routes of subsequent spreading of the epidemic strain. Methods: The genetic relatedness of S. aureus strains isolated from 6 neonates with epidermal lesions and from 21 staff members was verified by the pulsed field gel electrophoresis (PFGE) method. Additionally, detection of eta and etb genes of S. aureus strains using PCR was performed. Results: None of the infected newborns’ mothers was a carrier. Seven strains, 6 isolated from the newborns and 1 taken from a midwife, showed the same restriction pattern, i.e. type A. In the other 20 health care workers colonized with S. aureus, 3 genetic types could be distinguished, i.e. B (2), C (7) and D (2), as well as 9 strains with unique PFGE patterns. The eta gene detected in 7 strains belonged to the genetic type A; there was no etb gene in any of the 27 S. aureus isolates. Conclusions: The presence of the same genetic type A of S. aureus in the infected newborns is a factor which indicates that the impetigo bullosa was a hospital infection. A probable source of the infection was a midwife who was colonized with the same S. aureus type. She was present at the birth of the first infected newborn. Today, molecular methods are essential for prompt recognition of an epidemic and implementation of appropriate infection control strategies.