Complete Genomes of Three Pseudomonas aeruginosa Bacteriophages, Kara-mokiny 1, Kara-mokiny 2, and Kara-mokiny 3.

Here, we present the complete genome sequence of Pseudomonas aeruginosa phages Kara-mokiny 1, Kara-mokiny 2, and Kara-mokiny 3. These phages have lytic capabilities against P. aeruginosa and belong to the myovirus morphotype. The genomes of Kara-mokiny 1 and Kara-mokiny 2 are 67,075 bp while that of Kara-mokiny 3 is 66,019 bp long.

Complete Genome Sequences of Four Pseudomonas aeruginosa Bacteriophages: Kara-mokiny 8, Kara-mokiny 13, Kara-mokiny 16, and Boorn-mokiny 1.

Pseudomonas aeruginosa is an opportunistic pathogen. Here, we report the isolation of four bacteriophages from wastewater. All four bacteriophages belong to the Myoviridae family. Kara-mokiny 8, 13, and 16 are of the Pbunavirus genus and have genomes between 65,527 and 66,420-bp. Boorn-mokiny 1 is of the Phikzvirus genus and has a 278,796-bp genome.

Activity of newest generation ß-lactam/ß-lactamase inhibitor combination therapies against multidrug resistant Pseudomonas aeruginosa.

Multidrug resistant (MDR) P. aeruginosa accounts for 35% of all P. aeruginosa isolated from respiratory samples of patients with cystic fibrosis (CF). The usefulness of ß-lactam antibiotics for treating CF, such as carbapenems and later generation cephalosporins, is limited by the development of antibacterial resistance. A proven treatment approach is the combination of a ß-lactam antibiotic with a ß-lactamase inhibitor. New ß-lactam/ß-lactamase inhibitor combinations are available, but data are lacking regarding the susceptibility of MDR CF-associated P. aeruginosa (CFPA) to these new combination therapies. In this study we determined MIC values for three new combinations; imipenem-relebactam (I-R), ceftazidime-avibactam (CZA), and ceftolozane-tazobactam (C/T) against MDR CFPA (n = 20). The MIC90 of I-R, CZA, and C/T was 64/4, 32/4, and 16/8 (all µg/mL), respectively. The susceptibility of isolates to imipenem was not significantly improved with the addition of relebactam (p = 0.68). However, susceptibility to ceftazidime was significantly improved with the addition of avibactam (p < 0.01), and the susceptibility to C/T was improved compared to piperacillin/tazobactam (p < 0.05) These data provide in vitro evidence that I-R may not be any more effective than imipenem monotherapy against MDR CFPA. The pattern of susceptibility observed for CZA and C/T in the current study was similar to data previously reported for non-CF-associated MDR P. aeruginosa.

Pseudomonas aeruginosa Resistance to Bacteriophages and Its Prevention by Strategic Therapeutic Cocktail Formulation.

Antimicrobial resistance poses a significant threat to modern healthcare as it limits treatment options for bacterial infections, particularly impacting those with chronic conditions such as cystic fibrosis (CF). Viscous mucus accumulation in the lungs of individuals genetically predisposed to CF leads to recurrent bacterial infections, necessitating prolonged antimicrobial chemotherapy. Pseudomonas aeruginosa infections are the predominant driver of CF lung disease, and airway isolates are frequently resistant to multiple antimicrobials. Bacteriophages, or phages, are viruses that specifically infect bacteria and are a promising alternative to antimicrobials for CF P. aeruginosa infections. However, the narrow host range of P. aeruginosa-targeting phages and the rapid evolution of phage resistance could limit the clinical efficacy of phage therapy. A promising approach to overcome these issues is the strategic development of mixtures of phages (cocktails). The aim is to combine phages with broad host ranges and target multiple distinct bacterial receptors to prevent the evolution of phage resistance. However, further research is required to identify and characterize phage resistance mechanisms in CF-derived P. aeruginosa, which differ from their non-CF counterparts. In this review, we consider the mechanisms of P. aeruginosa phage resistance and how these could be overcome by an effective future phage therapy formulation.

Overcoming Challenges to Make Bacteriophage Therapy Standard Clinical Treatment Practice for Cystic Fibrosis.

Individuals with cystic fibrosis (CF) are given antimicrobials as prophylaxis against bacterial lung infection, which contributes to the growing emergence of multidrug resistant (MDR) pathogens isolated. Pathogens such as Pseudomonas aeruginosa that are commonly isolated from individuals with CF are armed with an arsenal of protective and virulence mechanisms, complicating eradication and treatment strategies. While translation of phage therapy into standard care for CF has been explored, challenges such as the lack of an appropriate animal model demonstrating safety in vivo exist. In this review, we have discussed and provided some insights in the use of primary airway epithelial cells to represent the mucoenvironment of the CF lungs to demonstrate safety and efficacy of phage therapy. The combination of phage therapy and antimicrobials is gaining attention and has the potential to delay the onset of MDR infections. It is evident that efforts to translate phage therapy into standard clinical practice have gained traction in the past 5 years. Ultimately, collaboration, transparency in data publications and standardized policies are needed for clinical translation.

Correction: Within-host whole genome analysis of an antibiotic resistant Pseudomonas aeruginosa strain sub-type in cystic fibrosis.

[This corrects the article DOI: 10.1371/journal.pone.0172179.].

Whole genome sequencing reveals the emergence of a Pseudomonas aeruginosa shared strain sub-lineage among patients treated within a single cystic fibrosis centre.

BACKGROUND: Chronic lung infections caused by Pseudomonas aeruginosa are a significant cause of morbidity and mortality in people with cystic fibrosis (CF). Shared P. aeruginosa strains, that can be transmitted between patients, are of concern and in Australia the AUST-02 shared strain is predominant in individuals attending CF centres in Queensland and Western Australia. M3L7 is a multidrug resistant sub-type of AUST-02 that was recently identified in a Queensland CF centre and was shown to be associated with poorer clinical outcomes. The main aim of this study was to resolve the relationship of the emergent M3L7 sub-type within the AUST-02 group of strains using whole genome sequencing. RESULTS: A whole genome core phylogeny of 63 isolates indicated that M3L7 is a monophyletic sub-lineage within the context of the broader AUST-02 group. Relatively short branch lengths connected all of the M3L7 isolates. A phylogeny based on nucleotide polymorphisms present across the genome showed that the chronological estimation of the most recent common ancestor was around 2001 (± 3 years). SNP differences between sequential non-hypermutator M3L7 isolates collected 3-4 years apart from five patients suggested both continuous infection of the same strain and cross-infection of some M3L7 variants between patients. The majority of polymorphisms that were characteristic of M3L7 (i.e. acquired after divergence from all other AUST-02 isolates sequenced) were found to produce non-synonymous mutations in virulence and antibiotic resistance genes. CONCLUSIONS: M3L7 has recently diverged from a common ancestor, indicating descent from a single carrier at a CF treatment centre in Australia. Both adaptation to the lung and transmission of M3L7 between adults attending this centre may have contributed to its rapid dissemination. Further genomic investigations are required on multiple intra-sample isolates of this sub-type to decipher potential mechanisms which facilitates its epidemiological success.

Antibiotic perturbation of mixed-strain Pseudomonas aeruginosa infection in patients with cystic fibrosis.

BACKGROUND: Pulmonary exacerbations in cystic fibrosis (CF) remain poorly understood and treatment is usually targeted at Pseudomonas aeruginosa. Within Australia a predominant shared P. aeruginosa strain (AUST-02) is associated with greater treatment needs. This single centre study assessed temporal shared strain population dynamics during and after antibiotic treatment of exacerbations. METHODS: Sputum was collected from 12 adult patients with a history of chronic AUST-02 infection at four time-points during and after treatment of an exacerbation. Forty-eight P. aeruginosa isolates within each sample underwent AUST-02 allele-specific PCR and SNP-based strain genotyping. RESULTS: Various commonly shared Australian strains (AUST-01, 0.1%; AUST-02, 54.3%; AUST-06, 36.6%; AUST-07, 4.6%; AUST-11, 4.3%) and two unique strains (0.1%) were identified from 45 sputum samples (2160 isolates). Based on within-patient relative abundance of strains, a "single-strain infection" (n = 7) or "mixed-strain infection" (n = 5) was assigned to each patient. A significant temporal variation in the P. aeruginosa population composition was found for those with mixed-strain infection (P < 0.001). Patients with mixed-strain infections had more long-term treatment requirements than those with single-strain infection. Moreover, despite both groups having similar lung function at study entry, patients with single-strain infection had greater improvement in FEV1% predicted following their exacerbation treatment (P = 0.02). CONCLUSION: Pulmonary exacerbations may reveal multiple, unrelated P. aeruginosa strains whose relative abundance with one another may change rapidly, in a sustained and unpredictable manner.

Within-host whole genome analysis of an antibiotic resistant Pseudomonas aeruginosa strain sub-type in cystic fibrosis.

A Pseudomonas aeruginosa AUST-02 strain sub-type (M3L7) has been identified in Australia, infects the lungs of some people with cystic fibrosis and is associated with antibiotic resistance. Multiple clonal lineages may emerge during treatment with mutations in chromosomally encoded antibiotic resistance genes commonly observed. Here we describe the within-host diversity and antibiotic resistance of M3L7 during and after antibiotic treatment of an acute pulmonary exacerbation using whole genome sequencing and show both variation and shared mutations in important genes. Eleven isolates from an M3L7 population (n = 134) isolated over 3 months from an individual with cystic fibrosis underwent whole genome sequencing. A phylogeny based on core genome SNPs identified three distinct phylogenetic groups comprising two groups with higher rates of mutation (hypermutators) and one non-hypermutator group. Genomes were screened for acquired antibiotic resistance genes with the result suggesting that M3L7 resistance is principally driven by chromosomal mutations as no acquired mechanisms were detected. Small genetic variations, shared by all 11 isolates, were found in 49 genes associated with antibiotic resistance including frame-shift mutations (mexA, mexT), premature stop codons (oprD, mexB) and mutations in quinolone-resistance determining regions (gyrA, parE). However, whole genome sequencing also revealed mutations in 21 genes that were acquired following divergence of groups, which may also impact the activity of antibiotics and multi-drug efflux pumps. Comparison of mutations with minimum inhibitory concentrations of anti-pseudomonal antibiotics could not easily explain all resistance profiles observed. These data further demonstrate the complexity of chronic and antibiotic resistant P. aeruginosa infection where a multitude of co-existing genotypically diverse sub-lineages might co-exist during and after intravenous antibiotic treatment.

Evaluation of phenotypic screening tests for carbapenemase production in Pseudomonas aeruginosa from patients with cystic fibrosis.

Carbapenemase phenotypic tests (inhibitor-based, modified Hodge and Carba NP) for Pseudomonas aeruginosa isolates from patients with cystic fibrosis (CF) were evaluated with PCR as a reference assay. Except for Carba NP, all methods demonstrated high false positives. Carba NP test presents a promising screening method for CF P. aeruginosa isolates.

Molecular surveillance for carbapenemase genes in carbapenem-resistant Pseudomonas aeruginosa in Australian patients with cystic fibrosis.

The aim of this study was to assess the prevalence of acquired carbapenemase genes amongst carbapenem non-susceptible Pseudomonas aeruginosa isolates in Australian patients with cystic fibrosis (CF). Cross-sectional molecular surveillance for acquired carbapenemase genes was performed on CF P. aeruginosa isolates from two isolate banks comprising: (i) 662 carbapenem resistant P. aeruginosa isolates from 227 patients attending 10 geographically diverse Australian CF centres (2007-2009), and (ii) 519 P. aeruginosa isolates from a cohort of 173 adult patients attending one Queensland CF clinic in 2011. All 1189 P. aeruginosa isolates were tested by polymerase chain reaction (PCR) protocols targeting ten common carbapenemase genes, as well the Class 1 integron intI1 gene and the aadB aminoglycoside resistance gene. No carbapenemase genes were identified among all isolates tested. The intI1 and aadB genes were frequently detected and were significantly associated with the AUST-02 strain (OR 24.6, 95% CI 9.3-65.6; p < 0.0001) predominantly from Queensland patients. Despite the high prevalence of carbapenem resistance in P. aeruginosa in Australian patients with CF, no acquired carbapenemase genes were detected in the study, suggesting chromosomal mutations remain the key resistance mechanism in CF isolates. Systematic surveillance for carbapenemase-producing P. aeruginosa in CF by molecular surveillance is ongoing.