Characterization of newly isolated lytic bacteriophages active against Acinetobacter baumannii

Based on genotyping and host range, two newly isolated lytic bacteriophages, myovirus vB_AbaM_Acibel004 and podovirus vB_AbaP_Acibel007, active against Acinetobacter baumannii clinical strains, were selected from a new phage library for further characterization. The complete genomes of the two phages were analyzed. Both phages are characterized by broad host range and essential features of potential therapeutic phages, such as short latent period (27 and 21 min, respectively), high burst size (125 and 145, respectively), stability of activity in liquid culture and low frequency of occurrence of phage-resistant mutant bacterial cells. Genomic analysis showed that while Acibel004 represents a novel bacteriophage with resemblance to some unclassified Pseudomonas aeruginosa phages, Acibel007 belongs to the well-characterized genus of the Phikmvlikevirus. The newly isolated phages can serve as potential candidates for phage cocktails to control A. baumannii infections

Experimental phage therapy of burn wound infection : difficult first steps

Antibiotic resistance has become a major public health problem and the antibiotics pipeline is running dry. Bacteriophages (phages) may offer an ‘innovative’ means of infection treatment, which can be combined or alternated with antibiotic therapy and may enhance our abilities to treat bacterial infections successfully. Today, in the Queen Astrid Military Hospital, phage therapy is increasingly considered as part of a salvage therapy for patients in therapeutic dead end, particularly those with multidrug resistant infections. We describe the application of a well-defined and quality controlled phage cocktail, active against Pseudomonas aeruginosa and Staphylococcus aureus, on colonized burn wounds within a modest clinical trial (nine patients, 10 applications), which was approved by a leading Belgian Medical Ethical Committee. No adverse events, clinical abnormalities or changes in laboratory test results that could be related to the application of phages were observed. Unfortunately, this very prudent ‘clinical trial’ did not allow for an adequate evaluation of the efficacy of the phage cocktail. Nevertheless, this first ‘baby step’ revealed several pitfalls and lessons for future experimental phage therapy and helped overcome the psychological hurdles that existed to the use of viruses in the treatment of patients in our burn unit

Stability of bacteriophages in burn wound care products

Bacteriophages could be used along with burn wound care products to enhance antimicrobial pressure during treatment. However, some of the components of the topical antimicrobials that are traditionally used for the prevention and treatment of burn wound infection might affect the activity of phages. Therefore, it is imperative to determine the counteraction of therapeutic phage preparations by burn wound care products before application in patients. Five phages, representatives of two morphological families (Myoviridae and Podoviridae) and active against 3 common bacterial burn wound pathogens (Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus) were tested against 13 different products commonly used in the treatment of burn wounds. The inactivation of the phages was quite variable for different phages and different products. Majority of the anti-infective products affected phage activity negatively either immediately or in the course of time, although impact was not always significant. Products with high acidity had the most adverse effect on phages. Our findings demonstrate that during combined treatment the choice of phages and wound care products must be carefully defined in advance

Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid `real-time' polymerase chain reaction

INTRODUCTION: Early diagnosis of wound colonisation or prediction of wound sepsis provides an opportunity for therapeutic intervention. There is need for qualitative and quantitative tests that are more rapid than bacterial culture. Pseudomonas aeruginosa results in high morbidity and mortality rates, is inherently resistant to common antibiotics, and is increasingly being isolated as a nosocomial pathogen. We developed three PCR-based methods to detect and quantify P aeruginosa in wound biopsy samples: conventional PCR, enzyme-linked immunosorbent assay (ELISA)-PCR, and RTD-PCR with rapid thermal cycling (LightCycler(™) technology), all based on the amplification of the outer membrane lipoprotein gene oprL. We compared the efficacy of these methods to bacterial culture by quantitatively measuring levels of P aeruginosa in serial dilutions, in reconstituted skin samples and 21 burn wound biopsy samples. MATERIALS AND METHODS: Serial 10-fold dilutions were made from an overnight P aeruginosa culture and plated out onto Luria-Bertani and cetrimide agar plates. The agar plates were incubated overnight at 37°C, and the colonies were counted in order to estimate the number of CFU per dilution tube. A sample was taken from each dilution tube as a template for the three PCR-based methods. Serial P aeruginosa dilutions (see above) were added to uninfected cadaveric skin. The reconstituted biopsy samples were homogenized using a tissue tearer and DNA was extracted using XTRAX DNA buffer. The DNA was resuspended in distilled water. A sample was taken as a template for the PCR-based methods. Twenty-one burn wound biopsy samples were taken from nine patients with suspected P aeruginosa burn wound infection. The biopsy samples were longitudinally divided into two pieces. From one piece, DNA was extracted (using XTRAX DNA buffer) and used as a template for PCR-based techniques (see above). The other piece was homogenized, in physiological water, using a tissue tearer. Serial 10-fold dilutions of the suspension were spread on Luria-Bertani and cetrimide agar plates. Colony counts were performed after overnight incubation at 37°C. The PCR mixture contained sterile distilled water, PCR buffer, deoxynucleotide mixture or digoxigenin labelling mix, MgCl(2), diluted template, primers PAL1 and PAL2, and AmpliTaQ DNA polymerase. The amplification was performed in a GeneAmp(®) PCR System 2400. An aliquot of the reaction mixture was put on an agarose gel for electrophoresis and visualisation of the PCR product. An image of the gel was made using a digital camera. Image analysis software was used to calculate the band mass of the experimental bands. An aliquot of the digoxigenin labelling reaction was denatured and then hybridized with the biotinylated capture probe PrL. Some of the resultant solution was transferred to a well of a streptavidin-coated microtitre plate (MTP) and incubated for 3 h at 45°C. The solution was discarded. Peroxidase conjugated antidigoxigenin was added and the MTP was incubated for 30 min at 37°C. The solution was discarded and ABTS substrate was added. The MTP was incubated for 30 min at 37°C. Absorbance was read at 405 nm. The RTD-PCR mixture contained PCR grade sterile water, diluted template DNA, primers PAL1 and PAL2, 3' fluorescein (FL)-labelled probe oprL-FL, 5' LC Red 640-labelled and 3' phosphorylated probe oprL-LC, MgCl(2), and LC DNA Master Hybridisation Probes, containing Taq DNA polymerase, reaction buffer, dNTP mix with dUTP instead of dTTP, and MgCl(2). The amplification was performed in a LightCycler(™). The fluorescence signal of LC Red 640 was measured during the annealing phase. The measured fluorescence data was processed with analysis software. RESULTS AND DISCUSSION: The three methods showed a good concordance with the culture results. Conventional PCR was at least 100 times less sensitive than bacterial culture and had a low dynamic range (2 logs). With a lower detection limit of 10(3) CFU/g tissue, ELISA-PCR was ten times more sensitive than conventional PCR. The dynamic range, however, did not increase. ELISA-PCR is very time consuming (8 h). The RTD-PCR produced a linear quantitative detection range of 7 logs with a lower detection limit of 10(3) CFU/g tissue. More important, however, was that the time from sample collection to result was less than 1 h. Two biopsy specimens scored significantly higher in ELISA-PCR and RTD-PCR than in bacterial culture. This could indicate that DNA from dead bacteria was amplified. One out of ten culture positive biopsy samples was found negative by all PCR-based methods. Topical antimicrobial agents possibly inhibited PCR. These results show that RTD-PCR has potential for the rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment. Further study is required to assess the reliability of this new technology, and its impact on patient outcome and hospital costs

Microbiological and Molecular Assessment of Bacteriophage ISP for the Control of Staphylococcus aureus

The increasing antibiotic resistance in bacterial populations requires alternatives for classical treatment of infectious diseases and therefore drives the renewed interest in phage therapy. Methicillin resistant Staphylococcus aureus (MRSA) is a major problem in health care settings and live-stock breeding across the world. This research aims at a thorough microbiological, genomic, and proteomic characterization of S. aureus phage ISP, required for therapeutic applications. Host range screening of a large batch of S. aureus isolates and subsequent fingerprint and DNA microarray analysis of the isolates revealed a substantial activity of ISP against 86% of the isolates, including relevant MRSA strains. From a phage therapy perspective, the infection parameters and the frequency of bacterial mutations conferring ISP resistance were determined. Further, ISP was proven to be stable in relevant in vivo conditions and subcutaneous as well as nasal and oral ISP administration to rabbits appeared to cause no adverse effects. ISP encodes 215 gene products on its 138,339 bp genome, 22 of which were confirmed as structural proteins using tandem electrospray ionization-mass spectrometry (ESI-MS/MS), and shares strong sequence homology with the ‘Twort-like viruses’. No toxic or virulence-associated proteins were observed. The microbiological and molecular characterization of ISP supports its application in a phage cocktail for therapeutic purposes

Comparison of the sensitivity of culture, PCR and quantitative real-time PCR for the detection of Pseudomonas aeruginosa in sputum of cystic fibrosis patients

Background: Pseudomonas aeruginosa is the major pathogen involved in the decline of lung function in cystic fibrosis (CF) patients. Early aggressive antibiotic therapy has been shown to be effective in preventing chronic colonization. Therefore, early detection is important and sensitive detection methods are warranted. In this study, we used a dilution series of P. aeruginosa positive sputa, diluted in a pool of P. aeruginosa negative sputa, all from CF patients-to mimick as closely as possible the sputa sent to routine laboratories-to compare the sensitivity of three culture techniques versus that of two conventional PCR formats and four real-time PCR formats, each targeting the P. aeruginosa oprL gene. In addition, we compared five DNA-extraction protocols. Results: In our hands, all three culture methods and the bioMerieux easyMAG Nuclisens protocol Generic 2.0.1, preceded by proteinase K pretreatment and followed by any of the 3 real-time PCR formats with probes were most sensitive and able to detect P. aeruginosa up to 50 cfu/ml, i.e. the theoretical minimum of one cell per PCR mixture, when taking into account the volumes used in this study of sample for DNA-extraction, of DNA-elution and of DNA-extract in the PCR mixture. Conclusion: In this study, no difference in sensitivity could be found for the detection of P. aeruginosa from sputum between microbiological culture and optimized DNA-extraction and real-time PCR. The results also indicate the importance of the optimization of the DNA-extraction protocol and the PCR format

Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa

Artilysins constitute a novel class of efficient enzyme-based antibacterials. Specifically, they covalently combine a bacteriophage-encoded endolysin, which degrades the peptidoglycan, with a targeting peptide that transports the endolysin through the outer membrane of Gram-negative bacteria. Art-085, as well as Art-175, its optimized homolog with increased thermostability, are each composed of the sheep myeloid 29-amino acid (SMAP-29) peptide fused to the KZ144 endolysin. In contrast to KZ144, Art-085 and Art-175 pass the outer membrane and kill Pseudomonas aeruginosa, including multidrug-resistant strains, in a rapid and efficient (similar to 5 log units) manner. Time-lapse microscopy confirms that Art-175 punctures the peptidoglycan layer within 1 min, inducing a bulging membrane and complete lysis. Art-175 is highly refractory to resistance development by naturally occurring mutations. In addition, the resistance mechanisms against 21 therapeutically used antibiotics do not show cross-resistance to Art-175. Since Art-175 does not require an active metabolism for its activity, it has a superior bactericidal effect against P. aeruginosa persisters (up to > 4 log units compared to that of the untreated controls). In summary, Art-175 is a novel antibacterial that is well suited for a broad range of applications in hygiene and veterinary and human medicine, with a unique potential to target persister-driven chronic infections

Pseudomonads from wild free-living sea turtles in Principe Island, Gulf of Guinea

Dissemination of antibiotic resistance is a major concern, especially in aquatic environments, where pollution contributes for resistant bacteria selection. These strains may have serious health implications, especially for endangered species, including the sea turtles' hawksbill Eretmochelys imbricata and green turtles Chelonia mydas. We aimed to evaluate the presence of antibiotic resistant pseudomonads in wild sea turtles from Principe Island, Sao Tome and Principe, Guinea Gulf. Isolates were obtained from oral and cloacal swabs of free-living turtles by conventional techniques. Pseudomonads screening was performed by multiplex-PCR (oprl/oprL) and biochemical identification and antibiotic resistance profiling were achieved using Vitek2. All pseudomonad isolates were genotyped by Rep-PCR. Thirteen isolates were oprl-positive and classified as pseudomonads, eight from the genus Pseudomonas with the species P. aeruginosa, P. stutzeri, and P. mendocina, and five co-isolated Alcaligenes faecalis. The P. aeruginosa isolate was also oprL-positive. Regarding isolates susceptibility profile, 38.5% were susceptible to all antibiotics tested, and multidrug resistant (MDR) strains were not identified. DNA fingerprinting did not show any specific clonal-cluster similarity. Data on the worldwide incidence of antibiotic resistance among wildlife is still very scarce, especially concerning remote tropical areas. Since Pseudomonas genus has emerged as a group of increasingly reported opportunistic microorganisms in human and veterinary medicine with high resistance levels, it could be used as a tool for environmental resistance surveillance, particularly considering their ubiquity.Oceandrio de Lisboa, PortugalMarine Turtle Conservation Act - U.S. Fish & Wildlife Service grantInterdisciplinary Research Centre for Animal Health, Faculty of Veterinary Medicine, University of Lisbon (FMV/UL) [UID/CVT/00276/2013]Laboratory of Molecular and Cellular Technology, Queen Astrid Military Hospital, Brussels, Belgiuminfo:eu-repo/semantics/publishedVersio

Introducing yesterday's phage therapy in today's medicine

The worldwide emergence of 'superbugs' and a dry antibiotic pipeline threaten modern society with a return to the preantibiotic era. Phages - the viruses of bacteria - could help fight antibiotic-resistant bacteria. Phage therapy was first attempted in 1919 by Felix d'Herelle and was commercially developed in the 1930s before being replaced by antibiotics in most of the western world. The current antibiotic crisis fueled a worldwide renaissance of phage therapy. The inherent potential of phages as natural biological bacterium controllers can only be put to use if the potential of the coevolutionary aspect of the couplet phage-bacterium is fully acknowledged and understood, including potential negative consequences. We must learn from past mistakes and set up credible studies to gather the urgently required data with regard to the efficacy of phage therapy and the evolutionary consequences of its (unlimited) use, Unfortunately, our current pharmaceutical economic model, implying costly and time-consuming medicinal product development and marketing, and requiring strong intellectual property protection, is not compatible with traditional sustainable phage therapy. A specific framework with realistic production and documentation requirements, which allows a timely (rapid) supply of safe, tailor-made, natural bacteriophages to patients, should be developed. Ultimately, economic models should be radically reshaped to cater for more sustainable approaches such as phage therapy. This is one of the biggest challenges faced by modern medicine and society as a whole

The Widespread Multidrug-Resistant Serotype O12 <i>Pseudomonas aeruginosa</i> Clone Emerged through Concomitant Horizontal Transfer of Serotype Antigen and Antibiotic Resistance Gene Clusters

The O-specific antigen (OSA) in Pseudomonas aeruginosa lipopolysaccharide is highly varied by sugar identity, side chains, and bond between O-repeats. These differences classified P. aeruginosa into 20 distinct serotypes. In the past few decades, O12 has emerged as the predominant serotype in clinical settings and outbreaks. These serotype O12 isolates exhibit high levels of resistance to various classes of antibiotics. Here, we explore how the P. aeruginosa OSA biosynthesis gene clusters evolve in the population by investigating the association between the phylogenetic relationships among 83 P. aeruginosa strains and their serotypes. While most serotypes were closely linked to the core genome phylogeny, we observed horizontal exchange of OSA biosynthesis genes among phylogenetically distinct P. aeruginosa strains. Specifically, we identified a “serotype island” ranging from 62 kb to 185 kb containing the P. aeruginosa O12 OSA gene cluster, an antibiotic resistance determinant (gyrA(C248T)), and other genes that have been transferred between P. aeruginosa strains with distinct core genome architectures. We showed that these genes were likely acquired from an O12 serotype strain that is closely related to P. aeruginosa PA7. Acquisition and recombination of the “serotype island” resulted in displacement of the native OSA gene cluster and expression of the O12 serotype in the recipients. Serotype switching by recombination has apparently occurred multiple times involving bacteria of various genomic backgrounds. In conclusion, serotype switching in combination with acquisition of an antibiotic resistance determinant most likely contributed to the dissemination of the O12 serotype in clinical settings