Pseudomonas aeruginosa ventricular assist device infections: findings from ineffective phage therapies in five cases.

Left ventricular assist devices (LVAD) are increasingly used for management of heart failure; infection remains a frequent complication. Phage therapy has been successful in a variety of antibiotic refractory infections and is of interest in treating LVAD infections. We performed a retrospective review of four patients that underwent five separate courses of intravenous (IV) phage therapy with concomitant antibiotic for treatment of endovascular Pseudomonas aeruginosa LVAD infection. We assessed phage susceptibility, bacterial strain sequencing, serum neutralization, biofilm activity, and shelf-life of phage preparations. Five treatments of one to four wild-type virulent phage(s) were administered for 14-51 days after informed consent and regulatory approval. There was no successful outcome. Breakthrough bacteremia occurred in four of five treatments. Two patients died from the underlying infection. We noted a variable decline in phage susceptibility following three of five treatments, four of four tested developed serum neutralization, and prophage presence was confirmed in isolates of two tested patients. Two phage preparations showed an initial titer drop. Phage biofilm activity was confirmed in two. Phage susceptibility alone was not predictive of clinical efficacy in P. aeruginosa endovascular LVAD infection. IV phage was associated with serum neutralization in most cases though lack of clinical effect may be multifactorial including presence of multiple bacterial isolates with varying phage susceptibility, presence of prophages, decline in phage titers, and possible lack of biofilm activity. Breakthrough bacteremia occurred frequently (while the organism remained susceptible to administered phage) and is an important safety consideration.

Correlation of Pseudomonas aeruginosa Phage Resistance with the Numbers and Types of Antiphage Systems.

Phage therapeutics offer a potentially powerful approach for combating multidrug-resistant bacterial infections. However, to be effective, phage therapy must overcome existing and developing phage resistance. While phage cocktails can reduce this risk by targeting multiple receptors in a single therapeutic, bacteria have mechanisms of resistance beyond receptor modification. A rapidly growing body of knowledge describes a broad and varied arsenal of antiphage systems encoded by bacteria to counter phage infection. We sought to understand the types and frequencies of antiphage systems present in a highly diverse panel of Pseudomonas aeruginosa clinical isolates utilized to characterize novel antibacterials. Using the web-server tool PADLOC (prokaryotic antiviral defense locator), putative antiphage systems were identified in these P. aeruginosa clinical isolates based on sequence homology to a validated and curated catalog of known defense systems. Coupling this host bacterium sequence analysis with host range data for 70 phages, we observed a correlation between existing phage resistance and the presence of higher numbers of antiphage systems in bacterial genomes. We were also able to identify antiphage systems that were more prevalent in highly phage-resistant P. aeruginosa strains, suggesting their importance in conferring resistance.

Interlaboratory comparison of Pseudomonas aeruginosa phage susceptibility testing.

Standardized approaches to phage susceptibility testing (PST) are essential to inform selection of phages for study in patients with bacterial infections. There is no reference standard for assessing bacterial susceptibility to phage. We compared agreement between PST performed at three centers: two centers using a liquid assay standardized between the sites with the third, a plaque assay. Four Pseudomonas aeruginosa phages: PaWRA01ø11 (EPa11), PaWRA01ø39 (EPa39), PaWRA02ø83 (EPa83), PaWRA02ø87 (EPa87), and a cocktail of all four phages were tested against 145 P. aeruginosa isolates. Comparisons were made within measurements at the two sites performing the liquid assay and between these two sites. Agreement was assessed based on coverage probability (CP8), total deviation index, concordance correlation coefficient (CCC), measurement accuracy, and precision. For the liquid assay, there was satisfactory agreement among triplicate measurements made on different days at site 1, and high agreement based on accuracy and precision between duplicate measurements made on the same run at site 2. There was fair accuracy between measurements of the two sites performing the liquid assay, with CCCs below 0.6 for all phages tested. When compared to the plaque assay (performed once at site 3), there was less agreement between results of the liquid and plaque assays than between the two sites performing the liquid assay. Similar findings to the larger group were noted in the subset of 46 P. aeruginosa isolates from cystic fibrosis. Results of this study suggest that reproducibility of PST methods needs further development.

Correlation of Host Range Expansion of Therapeutic Bacteriophage Sb-1 with Allele State at a Hypervariable Repeat Locus.

Staphylococci are frequent agents of health care-associated infections and include methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to first-line antibiotic treatments. Bacteriophage (phage) therapy is a promising alternative antibacterial option to treat MRSA infections. S. aureus-specific phage Sb-1 has been widely used in Georgia to treat a variety of human S. aureus infections. Sb-1 has a broad host range within S. aureus, including MRSA strains, and its host range can be further expanded by adaptation to previously resistant clinical isolates. The susceptibilities of a panel of 25 genetically diverse clinical MRSA isolates to Sb-1 phage were tested, and the phage had lytic activity against 23 strains (92%). The adapted phage stock (designated Sb-1A) was tested in comparison with the parental phage (designated Sb-1P). Sb-1P had lytic activity against 78/90 strains (87%) in an expanded panel of diverse global S. aureus isolates, while eight additional strains in this panel were susceptible to Sb-1A (lytic against 86/90 strains [96%]). The Sb-1A stock was shown to be a mixed population of phage clones, including approximately 4% expanded host range mutants, designated Sb-1M. In an effort to better understand the genetic basis for this host range expansion, we sequenced the complete genomes of the parental Sb-1P and two Sb-1M mutants. Comparative genomic analysis revealed a hypervariable complex repeat structure in the Sb-1 genome that had a distinct allele that correlated with the host range expansion. This hypervariable region was previously uncharacterized in Twort-like phages and represents a novel putative host range determinant.IMPORTANCE Because of limited therapeutic options, infections caused by methicillin-resistant Staphylococcus aureus represent a serious problem in both civilian and military health care settings. Phages have potential as alternative antibacterial agents that can be used in combination with antibiotic drugs. For decades, phage Sb-1 has been used in former Soviet Union countries for antistaphylococcal treatment in humans. The therapeutic spectrum of activity of Sb-1 can be increased by selecting mutants of the phage with expanded host ranges. In this work, the host range of phage Sb-1 was expanded in the laboratory, and a hypervariable region in its genome was identified with a distinct allele state that correlated with this host range expansion. These results provide a genetic basis for better understanding the mechanisms of phage host range expansion.

Spatio-temporal patterns of an anthrax outbreak in white-tailed deer, Odocoileus virginanus, and associated genetic diversity of Bacillus anthracis.

BACKGROUND: Anthrax, a soil-borne zoonosis caused by the bacterium Bacillus anthracis, is enzootic in areas of North America with frequent outbreaks in west Texas. Despite a long history of study, pathogen transmission during natural outbreaks remains poorly understood. Here we combined case-level spatio-temporal analysis and high resolution genotyping to investigate anthrax transmission dynamics. Carcass locations from a single white-tailed deer, Odocoileus virginanus, outbreak were analyzed for spatial clustering using K-function analysis and directionality with trend surface analysis and the direction test. RESULTS: The directionalities were compared to results of high resolution genotyping. The results of the spatial clustering analyses, combined with deer movement data, suggest anthrax transmission events occur within limited spatial areas, with carcass locations occurring within the activity space of adjacent cases. The directionality of the outbreak paralleled adjacent dry river beds. Isolates from the outbreak were represented by a single genotype based on multiple locus variable number tandem repeat analysis (MLVA); four sub-genotypes were identified using single nucleotide repeat (SNR) analysis. CONCLUSIONS: Areas of high transmission agreed spatially with areas of higher SNR genetic diversity; however, SNRs did not provide clear evidence of linear transmission. Overlap of case home ranges provides spatial and temporal support for localized transmission, which may include the role of necrophagous or hematophagous flies in outbreaks in this region. These results emphasize the need for active surveillance and prompt cleanup of anthrax carcasses to control anthrax both during outbreaks and between seasons.

Expansion of brucellosis detection in the country of Georgia by screening household members of cases and neighboring community members.

BACKGROUND: Brucellosis is considered as endemic zoonotic disease in the country of Georgia. However, the burden of the disease on a household level is not known. Therefore, this study sought to determine the benefits of active surveillance coupled to serological screening for the early detection of brucellosis among close contacts of brucellosis cases. METHODS: We used an active surveillance approach to estimate the rate of seropositivity among household family members and neighboring community members of brucellosis index cases. All participants were screened using the serum tube agglutination test (SAT). Blood cultures were performed, obtained isolates were identified by a bacteriological algorithm, and confirmed as Brucella spp. using real-time PCR. Further confirmation of Brucella species was done using the AMOS PCR assay. RESULTS: A total of 141 participants enrolled. Of these, 27 were brucellosis index cases, 86 were household family members, and 28 were neighboring community members. The serological evidence of brucellosis in the household member group was 7% and the rate at the household level was 21%. No screened community members were Brucella seropositive. Majority of brucellosis cases were caused by B. melitensis; only one index case was linked to B. abortus. CONCLUSION: We found evidence of brucellosis infection among household family members of brucellosis index cases. B. melitensis was the most common species obtained. Findings of this active surveillance study highlight the importance of screening household family members of brucellosis cases and of the use of culture methods to identify Brucella species in the country of Georgia.

Genome sequence of the Klebsiella quasipneumoniae bacteriophage EKq1 with activity against Klebsiella pneumoniae.

We describe the genome of a lytic phage EKq1 isolated on Klebsiella quasipneumoniae, with activity against Klebsiella pneumoniae. EKq1 is an unclassified representative of the class Caudoviricetes, similar to Klebsiella phages VLCpiS8c, phiKp_7-2, and vB_KleS-HSE3. The 48,244-bp genome has a GC content of 56.43% and 63 predicted protein-coding genes.

Complete genome sequence of the broad host range Acinetobacter baumannii phage EAb13.

We describe the genome of a lytic phage EAb13 isolated from sewage, with broad activity against multidrug-resistant Acinetobacter baumannii. EAb13 is an unclassified siphovirus. Its genome consists of 82,411 bp, with 40.15% GC content, 126 protein-coding sequences, 1 tRNA, and 2,177 bp-long direct terminal repeats.

Genome Sequence of Staphylococcus aureus Phage ESa2.

We describe the genome of a lytic phage, ESa2, isolated from environmental water and specific for Staphylococcus aureus. ESa2 belongs to the family Herelleviridae and genus Kayvirus. Its genome consists of 141,828 bp, with 30.25% GC content, 253 predicted protein-coding sequences, 3 tRNAs, and 10,130-bp-long terminal repeats.

The expanding range of emerging tick-borne viruses in Eastern Europe and the Black Sea Region.

We analysed both pooled and individual tick samples collected from four countries in Eastern Europe and the Black Sea region, using metagenome-based nanopore sequencing (NS) and targeted amplification. Initially, 1337 ticks, belonging to 11 species, were screened in 217 pools. Viruses (21 taxa) and human pathogens were detected in 46.5% and 7.3%, respectively. Tick-borne viral pathogens comprised Tacheng Tick Virus 2 (TTV2, 5.9%), Jingmen Tick Virus (JMTV, 0.9%) and Tacheng Tick Virus 1 (TTV1, 0.4%). An association of tick species with individual virus taxa was observed, with the exception of TTV2, which was observed in both Dermacentor and Haemaphysalis species. Individual ticks from pools with pathogen detection were then further screened by targeted amplification and then NS, which provided extensive genome data and revealed probable pathogen Haseki Tick Virus (HTV, 10.2%). Two distinct TTV2 clades were observed in phylogenetic analysis, one of which included closely related Dermacentor reticulatus Uukuviruses. JMTV detection indicated integrated virus sequences. Overall, we observed an expansion of newly documented pathogenic tick-borne viruses into Europe, with TTV1 being identified on the continent for the first time. These viruses should be included in the diagnostic assessment of symptomatic cases associated with tick bites and vector surveillance efforts. NS is shown as a useful tool for monitoring tick-associated pathogens in pooled or individual samples.

Assays for Identification and Differentiation of Brucella Species: A Review.

Brucellosis is one of the most important and widespread bacterial zoonoses worldwide. Cases are reported annually across the range of known infectious species of the genus Brucella. Globally, Brucella melitensis, primarily hosted by domestic sheep and goats, affects large proportions of livestock herds, and frequently spills over into humans. While some species, such as Brucella abortus, are well controlled in livestock in areas of North America, the Greater Yellowstone Ecosystem supports the species in native wild ungulates with occasional spillover to livestock. Elsewhere in North America, other Brucella species still infect domestic dogs and feral swine, with some associated human cases. Brucella spp. patterns vary across space globally with B. abortus and B. melitensis the most important for livestock control. A myriad of other species within the genus infect a wide range of marine mammals, wildlife, rodents, and even frogs. Infection in humans from these others varies with geography and bacterial species. Control in humans is primarily achieved through livestock vaccination and culling and requires accurate and rapid species confirmation; vaccination is Brucella spp.-specific and typically targets single livestock species for distribution. Traditional bacteriology methods are slow (some media can take up to 21 days for bacterial growth) and often lack the specificity of molecular techniques. Here, we summarize the molecular techniques for confirming and identifying specific Brucella species and provide recommendations for selecting the appropriate methods based on need, sensitivity, and laboratory capabilities/technology. As vaccination/culling approaches are costly and logistically challenging, proper diagnostics and species identification are critical tools for targeting surveillance and control.

Identification and Characterization of vB_PreP_EPr2, a Lytic Bacteriophage of Pan-Drug Resistant Providencia rettgeri.

Providencia rettgeri is an emerging opportunistic Gram-negative pathogen with reports of increasing antibiotic resistance. Pan-drug resistant (PDR) P. rettgeri infections are a growing concern, demonstrating a need for the development of alternative treatment options which is fueling a renewed interest in bacteriophage (phage) therapy. Here, we identify and characterize phage vB_PreP_EPr2 (EPr2) with lytic activity against PDR P. rettgeri MRSN 845308, a clinical isolate that carries multiple antibiotic resistance genes. EPr2 was isolated from an environmental water sample and belongs to the family Autographiviridae, subfamily Studiervirinae and genus Kayfunavirus, with a genome size of 41,261 base pairs. Additional phenotypic characterization showed an optimal MOI of 1 and a burst size of 12.3 ± 3.4 PFU per bacterium. EPr2 was determined to have a narrow host range against a panel of clinical P. rettgeri strains. Despite this fact, EPr2 is a promising lytic phage with potential for use as an alternative therapeutic for treatment of PDR P. rettgeri infections.

Design of a Bacteriophage Cocktail Active against Shigella Species and Testing of Its Therapeutic Potential in Galleria mellonella.

Shigellosis is a leading global cause of diarrheal disease and travelers' diarrhea now being complicated by the dissemination of antibiotic resistance, necessitating the development of alternative antibacterials such as therapeutic bacteriophages (phages). Phages with lytic activity against Shigella strains were isolated from sewage. The genomes of 32 phages were sequenced, and based on genomic comparisons belong to seven taxonomic genera: Teetrevirus, Teseptimavirus, Kayfunavirus, Tequatrovirus, Mooglevirus, Mosigvirus and Hanrivervirus. Phage host ranges were determined with a diverse panel of 95 clinical isolates of Shigella from Southeast Asia and other geographic regions, representing different species and serotypes. Three-phage mixtures were designed, with one possessing lytic activity against 89% of the strain panel. This cocktail exhibited lytic activity against 100% of S. sonnei isolates, 97.2% of S. flexneri (multiple serotypes) and 100% of S. dysenteriae serotypes 1 and 2. Another 3-phage cocktail composed of two myophages and one podophage showed both a broad host range and the ability to completely sterilize liquid culture of a model virulent strain S. flexneri 2457T. In a Galleria mellonella model of lethal infection with S. flexneri 2457T, this 3-phage cocktail provided a significant increase in survival.

Safety and microbiological activity of phage therapy in persons with cystic fibrosis colonized with Pseudomonas aeruginosa: study protocol for a phase 1b/2, multicenter, randomized, double-blind, placebo-controlled trial.

BACKGROUND: Bacteriophages (phages) are a promising anti-infective option for human disease. Major gaps remain in understanding their potential utility. METHODS: This is a randomized, placebo-controlled, double-blind study of a single dose of intravenous phage in approximately 72 clinically stable adult cystic fibrosis volunteers recruited from up to 20 US sites with Pseudomonas aeruginosa airway colonization. The single dose of phage consists of a mixture of four anti-pseudomonal phages. Six sentinel participants will be sequentially enrolled with dose escalation of the phage mixture by one log10 beginning with 4 × 107 plaque-forming units in an unblinded stage 1. If no serious adverse events related to the study product are identified, the trial will proceed to a double-blinded stage 2. In stage 2a, 32 participants will be randomly assigned to one of three phage dosages or placebo in a 1:1:1:1 allocation. An interim analysis will be performed to determine the phage dosage with the most favorable safety and microbiological activity profile to inform phage dosing in stage 2b. During stage 2b, up to 32 additional volunteers will be randomized 1:1 to the phage or placebo arm. Primary outcomes include (1) the number of grade 2 or higher treatment-emergent adverse events, (2) change in log10 P. aeruginosa total colony counts in sputum, and (3) the probability of a randomly selected subject having a more favorable outcome ranking if assigned to receive phage therapy versus placebo. Exploratory outcomes include (1) sputum and serum phage pharmacokinetics, (2) the impact of phage on lung function, (3) the proportion of P. aeruginosa isolates susceptible to the phage mixture before and after study product administration, and (4) changes in quality of life. DISCUSSION: This trial will investigate the activity of phages in reducing P. aeruginosa colony counts and provide insights into the safety profile of phage therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT05453578. Registered on 12 July 2022.

Synergistic Killing and Re-Sensitization of Pseudomonas aeruginosa to Antibiotics by Phage-Antibiotic Combination Treatment.

Multidrug-resistant (MDR) Pseudomonas aeruginosa infections pose a serious health threat. Bacteriophage-antibiotic combination therapy is a promising candidate for combating these infections. A 5-phage P. aeruginosa cocktail, PAM2H, was tested in combination with antibiotics (ceftazidime, ciprofloxacin, gentamicin, meropenem) to determine if PAM2H enhances antibiotic activity. Combination treatment in vitro resulted in a significant increase in susceptibility of MDR strains to antibiotics. Treatment with ceftazidime (CAZ), meropenem, gentamicin, or ciprofloxacin in the presence of the phage increased the number of P. aeruginosa strains susceptible to these antibiotics by 63%, 56%, 31%, and 81%, respectively. Additionally, in a mouse dorsal wound model, seven of eight mice treated with a combination of CAZ and PAM2H for three days had no detectable bacteria remaining in their wounds on day 4, while all mice treated with CAZ or PAM2H alone had ~107 colony forming units (CFU) remaining in their wounds. P. aeruginosa recovered from mouse wounds post-treatment showed decreased virulence in a wax worm model, and DNA sequencing indicated that the combination treatment prevented mutations in genes encoding known phage receptors. Treatment with PAM2H in combination with antibiotics resulted in the re-sensitization of P. aeruginosa to antibiotics in vitro and a synergistic reduction in bacterial burden in vivo.

Genome Sequences of 17 Diverse Pseudomonas aeruginosa Phages.

Here, we describe genome sequences of 17 Pseudomonas aeruginosa phages, including therapeutic candidates. They belong to the families Myoviridae, Podoviridae, and Siphoviridae and six different genera. The genomes ranged in size from 42,788 to 88,805 bp, with G+C contents of 52.5% to 64.3% and numbers of coding sequences from 58 to 179.

Genome sequence of the deep-rooted Yersinia pestis strain Angola reveals new insights into the evolution and pangenome of the plague bacterium.

To gain insights into the origin and genome evolution of the plague bacterium Yersinia pestis, we have sequenced the deep-rooted strain Angola, a virulent Pestoides isolate. Its ancient nature makes this atypical isolate of particular importance in understanding the evolution of plague pathogenicity. Its chromosome features a unique genetic make-up intermediate between modern Y. pestis isolates and its evolutionary ancestor, Y. pseudotuberculosis. Our genotypic and phenotypic analyses led us to conclude that Angola belongs to one of the most ancient Y. pestis lineages thus far sequenced. The mobilome carries the first reported chimeric plasmid combining the two species-specific virulence plasmids. Genomic findings were validated in virulence assays demonstrating that its pathogenic potential is distinct from modern Y. pestis isolates. Human infection with this particular isolate would not be diagnosed by the standard clinical tests, as Angola lacks the plasmid-borne capsule, and a possible emergence of this genotype raises major public health concerns. To assess the genomic plasticity in Y. pestis, we investigated the global gene reservoir and estimated the pangenome at 4,844 unique protein-coding genes. As shown by the genomic analysis of this evolutionary key isolate, we found that the genomic plasticity within Y. pestis clearly was not as limited as previously thought, which is strengthened by the detection of the largest number of isolate-specific single-nucleotide polymorphisms (SNPs) currently reported in the species. This study identified numerous novel genetic signatures, some of which seem to be intimately associated with plague virulence. These markers are valuable in the development of a robust typing system critical for forensic, diagnostic, and epidemiological studies.

HSP-70 mitigates LPS/SKI-induced cell damage by increasing sphingosine kinase 1 (SK1).

Heat shock proteins (HSPs) are potent protectors of cellular integrity against environmental stresses, including toxic microbial products. To investigate the mechanism of HSP-70 cell protection against bacterial lipopolysaccharide (LPS), we established a stable HSP-70 gene-transfected RAW 264.7 murine macrophage model of LPS-induced cell death. Bacterial LPS increases the activity of sphingosine kinase 1 (SK1), which catalyzes formation of sphingosine-1-phosphate (S1P). S1P functions as a critical signal for initiation and maintenance of diverse aspects of immune cell activation and function. When mouse macrophages were incubated with Escherichia coli LPS (1 microg/ml) and sphingosine kinase inhibitor (SKI, 5 microM), 90% of cells died. Neither LPS nor SKI alone at these doses damaged the cells. The LPS/SKI-induced cell death was partially reversed by overexpression of HSP-70 in gene-transfected macrophages. The specificity of HSP-70 in this reversal was demonstrated by transfection of HSP-70-specific siRNA. Down-regulation of HSP-70 expression after transfection of siRNA specific for HSP-70 was associated with increased LPS/SKI-induced cell damage. Overexpression of human or murine HSP-70 (HSPA1A and Hspa1a, respectively) increased both cellular SK1 mRNA and protein levels. Cellular heat shock also increased SK1 protein. These studies confirm the importance of SK1 as a protective moiety in LPS-induced cell injury and demonstrate that HSP-70-mediated protection from cells treated with LPS/SKI is accompanied by upregulating expression of SK1. HSP-70-mediated increases in SK1 and consequent increased levels of S1P may also play a role in protection of cells from other processes that lead to programmed cell death.

Bacteriophage Therapy: Developments and Directions.

In an era of proliferating multidrug resistant bacterial infections that are exhausting the capacity of existing chemical antibiotics and in which the development of new antibiotics is significantly rarer, Western medicine must seek additional therapeutic options that can be employed to treat these infections. Among the potential antibacterial solutions are bacteriophage therapeutics, which possess very different properties from broad spectrum antibiotics that are currently the standard of care, and which can be used in combination with them and often provide synergies. In this review we summarize the state of the development of bacteriophage therapeutics and discuss potential paths to the implementation of phage therapies in contemporary medicine, focused on fixed phage cocktail therapeutics since these are likely to be the first bacteriophage products licensed for broad use in Western countries.

Complete Genome Sequence of Broad-Host-Range Staphylococcus aureus Myophage ESa1.

A potentially therapeutic Twort-like myophage, Esa1, with specificity toward Staphylococcus aureus was isolated from lake water. We report the complete genome sequence of ESa1, assembled using both MinION and Illumina MiSeq reads, consisting of 153,106 bp, with 30.3% GC content, 253 protein coding sequences, 4 tRNAs, and 10,437-bp direct terminal repeats.