Bacteriophage can lyse antibiotic-resistant Pseudomonas aeruginosa isolated from canine diseases.
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ABSTRACT:
SUBMITTER: Furusawa T
PROVIDER: S-EPMC4937139 | biostudies-literature | 2016 Jul
REPOSITORIES: biostudies-literature
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Project description:The rise in multiple-drug-resistant (MDR) phenotypes in Gram-negative pathogens is a major public health crisis. Pseudomonas aeruginosa is one of the leading causes of nosocomial infections in clinics. Treatment options for P. aeruginosa have become increasingly difficult due tdo its remarkable capacity to resist multiple antibiotics. The presence of intrinsic resistance factors and the ability to quickly adapt to antibiotic monotherapy warrant us to look for alternative strategies like combinatorial antibiotic therapy. Here, we report the frequency of P. aeruginosa multidrug-resistant and extensively drug-resistance (XDR) phenotypes in a super-specialty tertiary care hospital in north India. Approximately 60 percent of all isolated P. aeruginosa strains displayed the MDR phenotype. We found highest antibiotic resistance frequency in the emergency department (EMR), as 20 percent of isolates were resistant to 15 antipseudomonal antibiotics. Presence of plasmids with quinolone-resistance determinants were major drivers for resistance against fluoroquinolone. Additionally, we explored the possible combinatorial therapeutic options with four antipseudomonal antibiotics-colistin, ciprofloxacin, tobramycin, and meropenem. We uncovered an association between different antibiotic interactions. Our data show that the combination of colistin and ciprofloxacin could be an effective combinatorial regimen to treat infections caused by MDR and XDR P. aeruginosa.
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Project description:BackgroundPseudomonas aeruginosa is a ubiquitous environmental microorganism and also a common cause of infection. Its ability to survive in many different environments and persistently colonize humans is linked to its presence in biofilms formed on indwelling device surfaces. Biofilm promotes adhesion to, and survival on surfaces, protects from desiccation and the actions of antibiotics and disinfectants.ResultsWe examined the genetic basis for biofilm production on polystyrene at room (22 °C) and body temperature (37 °C) within 280 P. aeruginosa. 193 isolates (69 %) produced more biofilm at 22 °C than at 37 °C. Using GWAS and pan-GWAS, we found a number of accessory genes significantly associated with greater biofilm production at 22 °C. Many of these are present on a 165 kb region containing genes for heavy metal resistance (arsenic, copper, mercury and cadmium), transcriptional regulators and methytransferases. We also discovered multiple core genome SNPs in the A-type flagellin gene and Type II secretion system gene xpsD. Analysis of biofilm production of isolates of the MDR ST111 and ST235 lineages on stainless-steel revealed several accessory genes associated with enhanced biofilm production. These include a putative translocase with homology to a Helicobacter pylori type IV secretion system protein, a TA system II toxin gene and the alginate biosynthesis gene algA, several transcriptional regulators and methytransferases as well as core SNPs in genes involved in quorum sensing and protein translocation.ConclusionsUsing genetic association approaches we discovered a number of accessory genes and core-genome SNPs that were associated with enhanced early biofilm formation at 22 °C compared to 37 °C. These included a 165 kb genomic island containing multiple heavy metal resistance genes, transcriptional regulators and methyltransferases. We hypothesize that this genomic island may be associated with overall genotypes that are environmentally adapted to survive at lower temperatures. Further work to examine their importance in, for example gene-knockout studies, are required to confirm their relevance. GWAS and pan-GWAS approaches have great potential as a first step in examining the genetic basis of novel bacterial phenotypes.
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Project description:Bone and joint infections (BJI) are one of the most difficult-to-treat bacterial infection, especially in the era of antimicrobial resistance. Lytic bacteriophages (phages for short) are natural viruses that can selectively target and kill bacteria. They are considered to have a high therapeutic potential for the treatment of severe bacterial infections and especially BJI, as they also target biofilms. Here we report on the management of a patient with a pandrug-resistant Pseudomonas aeruginosa spinal abscess who was treated with surgery and a personalized combination of phage therapy that was added to antibiotics. As the infecting P. aeruginosa strain was resistant to the phages developed by private companies that were contacted, we set up a unique European academic collaboration to find, produce and administer a personalized phage cocktail to the patient in due time. After two surgeries, despite bacterial persistence with expression of small colony variants, the patient healed with local and intravenous injections of purified phages as adjuvant therapy.
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Project description:Many bacteria use quorum sensing (QS), a bacterial communication system based on the diffusion and perception of small signaling molecules, to synchronize their behavior in a cell-density dependent manner. QS regulates the expression of many genes associated with virulence factor production and biofilm formation. This latter is known to be involved in antibiotic and phage resistance mechanisms. Therefore, disrupting QS, a strategy known as quorum quenching (QQ), appears to be an interesting way to reduce bacterial virulence and increase antibiotic and phage treatment efficiency. In this study, the ability of the QQ enzyme SsoPox-W263I, a lactonase able to degrade acyl-homoserine lactones, was investigated for quenching both virulence and biofilm formation in clinical isolates of Pseudomonas aeruginosa from diabetic foot ulcers, as well as in the PA14 model strain. These strains were further evolved to resist to bacteriophage cocktails. Overall, 10 antibiotics or bacteriophage resistant strains were evaluated and SsoPox-W263I was shown to decrease pyocyanin, protease and elastase production in all strains. Furthermore, a reduction of more than 70% of biofilm formation was achieved in six out of ten strains. This anti-virulence potential was confirmed in vivo using an amoeba infection model, showing enhanced susceptibility toward amoeba of nine out of ten P. aeruginosa isolates upon QQ. This amoeba model was further used to demonstrate the ability of SsoPox-W263I to enhance the susceptibility of sensitive and phage resistant bacteria to bacteriophage and antibiotic.
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Project description:Bacteriophage (phage) therapy is an alternative to traditional antibiotic treatments that is particularly important for multidrug-resistant pathogens, such as Pseudomonas aeruginosa. Unfortunately, phage resistance commonly arises during treatment as bacteria evolve to survive phage predation. During in vitro phage treatment of a P. aeruginosa-type strain, we observed the emergence of phage-resistant mutants with brown pigmentation that was indicative of pyomelanin. As increased pyomelanin (due to hmgA gene mutation) was recently associated with enhanced resistance to hydrogen peroxide and persistence in experimental lung infection, we questioned if therapeutic phage applications could inadvertently select for hypervirulent populations. Pyomelanogenic phage-resistant mutants of P. aeruginosa PAO1 were selected for upon treatment with three distinct phages. Phage-resistant pyomelanogenic mutants did not possess increased survival of pyomelanogenic ΔhmgA in hydrogen peroxide. At the genomic level, large (~300 kb) deletions in the phage-resistant mutants resulted in the loss of ≥227 genes, many of which had roles in survival, virulence, and antibiotic resistance. Phage-resistant pyomelanogenic mutants were hypersusceptible to cationic peptides LL-37 and colistin and were more easily cleared in human whole blood, serum, and a murine infection model. Our findings suggest that hyperpigmented phage-resistant mutants that may arise during phage therapy are markedly less virulent than their predecessors due to large genomic deletions. Thus, their existence does not present a contraindication to using anti-pseudomonal phage therapy, especially considering that these mutants develop drug susceptibility to the familiar FDA-approved antibiotic, colistin.
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Project description:A Pseudomonas aeruginosa (P. aeruginosa) airway infection is one of the predominant causes contributing to the high morbidity and mortality rates in cystic fibrosis (CF) patients. The emergence of antibiotic resistant P. aeruginosa strains has led to an urgent need for new therapeutic approaches. Bacteriophages (phages) are viruses that can infect and lyse specific bacteria, providing a potential alternative approach in targeting antibiotic-resistant strains. We aim to isolate and characterise novel P. aeruginosa phages for combination in a cocktail to kill P. aeruginosa. One particular phage, PA4, could lyse 14/20 clinical isolates as observed through spot assays. This phage could significantly reduce the growth of bacteria in vitro, as determined through planktonic adsorption and inhibition assays as well as crystal violet- and LIVE/DEAD-stained biofilm assays. A morphological and genomic analysis revealed that PA4 belongs to the Myoviridae family and contained 66,450 bp. The broad infectivity profile, good stability in various pH and temperature conditions, lytic ability and the absence of the absences of antibiotic resistance, toxic and lysogenic genes suggest that PA4 is a good candidate for clinical grade use. Overall, phage therapy represents a promising alternative treatment option to antibiotics when treating a P. aeruginosa infection.
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Project description:The Pseudomonas aeruginosa-containing wastewater released by hospitals is treated by wastewater treatment plants (WWTPs), generating sludge, which is used as a fertilizer, and effluent, which is discharged into rivers. We evaluated the risk of dissemination of antibiotic-resistant P. aeruginosa (AR-PA) from the hospital to the environment via the wastewater network. Over a 10-week period, we sampled weekly 11 points (hospital and urban wastewater, untreated and treated water, sludge) of the wastewater network and the river upstream and downstream of the WWTP of a city in eastern France. We quantified the P. aeruginosa load by colony counting. We determined the susceptibility to 16 antibiotics of 225 isolates, which we sorted into three categories (wild-type, antibiotic-resistant and multidrug-resistant). Extended-spectrum ?-lactamases (ESBLs) and metallo-?-lactamases (MBLs) were identified by gene sequencing. All non-wild-type isolates (n?=?56) and a similar number of wild-type isolates (n?=?54) were genotyped by pulsed-field gel electrophoresis and multilocus sequence typing. Almost all the samples (105/110, 95.5%) contained P. aeruginosa, with high loads in hospital wastewater and sludge (?3×10(6) CFU/l or/kg). Most of the multidrug-resistant isolates belonged to ST235, CC111 and ST395. They were found in hospital wastewater and some produced ESBLs such as PER-1 and MBLs such as IMP-29. The WWTP greatly reduced P. aeruginosa counts in effluent, but the P. aeruginosa load in the river was nonetheless higher downstream than upstream from the WWTP. We conclude that the antibiotic-resistant P. aeruginosa released by hospitals is found in the water downstream from the WWTP and in sludge, constituting a potential risk of environmental contamination.
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Project description:UnlabelledPseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates.ImportanceP. aeruginosa is both an antibiotic-refractory pathogen and an important model system for type I CRISPR-Cas bacterial immune systems. By combining the genome sequences of 672 newly and previously sequenced genomes, we were able to provide a global view of the phylogenetic distribution, conservation, and potential targets of these systems. This analysis identified a new and putatively mobile P. aeruginosa CRISPR-Cas subtype, characterized the diverse distribution of known CRISPR-inhibiting genes, and provided a potential new use for CRISPR spacer libraries in accessory genome analysis. Our data demonstrated the importance of CRISPR-Cas systems in modulating the accessory genomes of globally distributed strains while also providing substantial data for subsequent genomic and experimental studies in multiple fields. Understanding why certain genotypes of P. aeruginosa are clinically prevalent and adept at horizontally acquiring virulence and antibiotic resistance elements is of major clinical and economic importance.
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Project description:With the emergence of multidrug-resistant and extensively drug-resistant bacterial pathogens, phage therapy and other alternative or additional therapeutic modalities are receiving resurgent attention. One of the major obstacles in developing effective phage therapies is the evolution of phage resistance in the bacterial host. When Pseudomonas aeruginosa was infected with a phage that uses O-antigen as receptor, phage resistances typically achieved through changing or loss of O-antigen structure. In this study, we showed that dsRNA phage phiYY uses core lipopolysaccharide as receptor and therefore efficiently kills the O-antigen deletion mutants. Furthermore, by phage training, we obtained PaoP5-m1, a derivative of dsDNA phage PaoP5, which is able to infect mutants with truncated O-antigen. We then generated a cocktail by mixing phiYY and PaoP5-m1 with additional three wide host range P. aeruginosa phages. The phage cocktail was effective against a diverse selection of clinical isolates of P. aeruginosa, and in the short-term constrained the appearance of the phage-resistant mutants that had beleaguered the effectiveness of single phage. Resistance to the 5-phage cocktail emerges after several days, and requires mutations in both wzy and migA Thus, this study provides an alternative strategy for designing phage cocktail and phage therapy.
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Project description:Virulent strain Pseudomonas aeruginosa isolated from Mahananda River exhibited the highest hemolytic activity and virulence factors and was pathogenic to fish as clinical signs of hemorrhagic spots, loss of scales, and fin erosions were found. S3 was cytotoxic to the human liver cell line (WRL-68) in the trypan blue dye exclusion assay. Genotype characterization using whole genome analysis showed that S3 was similar to P. aeruginosa PAO1. The draft genome sequence had an estimated length of 62,69,783 bp, a GC content of 66.3%, and contained 5916 coding sequences. Eight genes across the genome were predicted to be related to hemolysin action. Antibiotic resistance genes such as class C and class D beta-lactamases, fosA, APH, and catB were detected, along with the strong presence of multiple efflux system genes. This study shows that river water is contaminated by pathogenic P. aeruginosa harboring an array of virulence and antibiotic resistance genes which warrants periodic monitoring to prevent disease outbreaks.
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