An Early History of Phage Therapy in the United States: Is it Time to Reconsider?

Frederick William Twort and Felix d'Hérelle independently discovered bacteriophages in 1915 and 1917, respectively. This led to the early trials of using bacteriophages to treat infectious diseases worldwide. The earliest reported use of bacteriophages therapeutically in the United States was in 1922. With the subsequent discovery of antibiotics in the 1940s, and because of disappointing results of phage therapy in the next decade, use of bacteriophages as therapeutic agents declined in western countries. This paper addresses two questions in the field: what is the historical record of the successes and failures of phage therapy in the United States and, what led to abandoning phage therapy in the United States? We examined the literature from 1915 to 1965, and we present a numerical analysis of the papers published during that period. We report key historical factors leading to a decline in the use of phage therapy in the United States by the 1950s. Since bacteriophages were first used therapeutically, several changes have occurred: increased antimicrobial drug resistance and a better knowledge of the biology of bacteriophages are important examples. Early assessments leading to the rejection of phage therapy in the United States were perhaps appropriate. However, it is time to reconsider the role of bacteriophages in treatment of bacterial infections.

Biochemical adaptation in brain Acetylcholinesterase during acclimation to sub-lethal temperatures in the eurythermal fish Tilapia mossambica.

Tilapia mossambica is a eurythermal tropical fish. We studied the effect of temperature on the kinetics of brain Acetylcholinesterase (AChE) during adaptation to sublethal temperatures by acclimating the fish to 37 °C, and controls to 25 °C. Electrophoresis showed the presence of two AChE bands that did not change in position or intensity with acclimation period or temperature. The apparent Km was 0.23 ± 0.01 mM ATChI and remained relatively constant over the in vitro assay temperature range 10 °C to 40 °C. Biochemical characterization suggested that the enzyme is a 'eurytolerant protein' in its stability of kinetic and thermal properties over a wide temperature range. Thermal stability and arrhenius plots suggested that the AChE was made up of two forms that differed in their thermal properties.The two molecular forms of acetylcholinesterase were purified from the brain of T. mossambica. Molecular weight studies revealed that the two forms were size isomers: a monomer of 59 KDa and a tetramer of 244 KDa. They differed in their Kms, thermal stabilities and energies of activation. We suggest that biochemical adaptation to temperature in the brain acetylcholinerase system of the fish Tilapia mossambica is based on the aggregation-dissociation of these size isomers.

Virulence factor landscape of a Staphylococcus aureus sequence type 45 strain, MCRF184.

BACKGROUND: We describe the virulence factors of a methicillin-sensitive Staphylococcus aureus sequence type (ST) 45 strain, MCRF184, (spa type t917), that caused severe necrotizing fasciitis in a 72-year-old diabetic male. The genome of MCRF184 possesses three genomic islands: a relatively large type III νSaα with 42 open reading frames (ORFs) that includes superantigen- and lipoprotein-like genes, a truncated νSaß that consists mostly of the enterotoxin gene cluster (egc), and a νSaγ island with 18 ORFs including α-toxin. Additionally, the genome has two phage-related regions: phage φSa3 with three genes of the immune evasion cluster (IEC), and an incomplete phage that is distinct from other S. aureus phages. Finally, the region between orfX and orfY harbors a putative efflux pump, acetyltransferase, regulators, and mobilization genes instead of genes of SCCmec. RESULTS: Virulence factors included phenol soluble modulins (PSMs) α1 through α4 and PSMs ß1 and ß2. Ten ORFs identified in MCRF184 had not been reported in previously sequenced S. aureus strains. CONCLUSION: The dire clinical outcome in the patient and the described virulence factors all suggest that MCRF184, a ST45 strain is a highly virulent strain of S. aureus.

Phylogenetic distribution and expression of a penicillin-binding protein homologue, Ear and its significance in virulence of Staphylococcus aureus.

PURPOSE: Staphylococcus aureus is an opportunistic human pathogen that can cause serious infections in humans. A plethora of known and putative virulence factors are produced by staphylococci that collectively orchestrate pathogenesis. Ear protein (Escherichia coli ampicillin resistance) in S. aureus is an exoprotein in COL strain, predicted to be a superantigen, and speculated to play roles in antibiotic resistance and virulence. The goal of this study was to determine if expression of ear is modulated by single nucleotide polymorphisms in its promoter and coding sequences and whether this gene plays roles in antibiotic resistance and virulence. METHODOLOGY: Promoter, coding sequences and expression of the ear gene in clinical and carriage S. aureus strains with distinct genetic backgrounds were analysed. The JE2 strain and its isogenic ear mutant were used in a systemic infection mouse model to determine the competiveness of the ear mutant.Results/Key findings. The ear gene showed a variable expression, with USA300FPR3757 showing a high-level expression compared to many of the other strains tested including some showing negligible expression. Higher expression was associated with agr type 1 but not correlated with phylogenetic relatedness of the ear gene based upon single nucleotide polymorphisms in the promoter or coding regions suggesting a complex regulation. An isogenic JE2 (USA300 background) ear mutant showed no significant difference in its growth, antibiotic susceptibility or virulence in a mouse model. CONCLUSION: Our data suggests that despite being highly expressed in a USA300 genetic background, Ear is not a significant contributor to virulence in that strain.

Complete Genome Sequence of Staphylococcus aureus MCRF184, a Necrotizing Fasciitis-Causing Methicillin-Sensitive Sequence Type 45 Staphylococcus Strain.

We report here the complete genome sequence of a highly virulent methicillin-sensitive Staphylococcus aureus strain, MCRF184, belonging to sequence type 45. This staphylococcal strain was isolated from a surgical biopsy specimen from a patient with necrotizing fasciitis.

Fifty-Three Years after a Pencil Puncture Wound.

A pencil core with an intact pencil tip was excised from the thigh of a 60-year-old male 53 years after a puncture wound. Histologic examination of the excised pencil core and the surrounding tissue revealed a foreign body reaction with abundant entrapped dark black pigment and chronic reparative changes, including dense sclerosis and focal granulation tissue formation.

Characterization of human disease phenotypes associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR, and IFIH1.

Aicardi-Goutières syndrome is an inflammatory disease occurring due to mutations in any of TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR or IFIH1. We report on 374 patients from 299 families with mutations in these seven genes. Most patients conformed to one of two fairly stereotyped clinical profiles; either exhibiting an in utero disease-onset (74 patients; 22.8% of all patients where data were available), or a post-natal presentation, usually within the first year of life (223 patients; 68.6%), characterized by a sub-acute encephalopathy and a loss of previously acquired skills. Other clinically distinct phenotypes were also observed; particularly, bilateral striatal necrosis (13 patients; 3.6%) and non-syndromic spastic paraparesis (12 patients; 3.4%). We recorded 69 deaths (19.3% of patients with follow-up data). Of 285 patients for whom data were available, 210 (73.7%) were profoundly disabled, with no useful motor, speech and intellectual function. Chilblains, glaucoma, hypothyroidism, cardiomyopathy, intracerebral vasculitis, peripheral neuropathy, bowel inflammation and systemic lupus erythematosus were seen frequently enough to be confirmed as real associations with the Aicardi-Goutieres syndrome phenotype. We observed a robust relationship between mutations in all seven genes with increased type I interferon activity in cerebrospinal fluid and serum, and the increased expression of interferon-stimulated gene transcripts in peripheral blood. We recorded a positive correlation between the level of cerebrospinal fluid interferon activity assayed within one year of disease presentation and the degree of subsequent disability. Interferon-stimulated gene transcripts remained high in most patients, indicating an ongoing disease process. On the basis of substantial morbidity and mortality, our data highlight the urgent need to define coherent treatment strategies for the phenotypes associated with mutations in the Aicardi-Goutières syndrome-related genes. Our findings also make it clear that a window of therapeutic opportunity exists relevant to the majority of affected patients and indicate that the assessment of type I interferon activity might serve as a useful biomarker in future clinical trials.

Complete Genome Sequence of a Staphylococcus epidermidis Bacteriophage Isolated from the Anterior Nares of Humans.

We report here the complete genome sequence of a virulent Staphylococcus epidermidis siphophage, phage 6ec, isolated from the anterior nares of a human. This viral genome is 93,794 bp in length, with a 3' overhang cos site of 10 nucleotides, and it codes for 142 putative open reading frames.

A novel case of diabetic muscle necrosis in a patient with cystic fibrosis-related diabetes.

Cystic fibrosis is a recessive autosomal disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. Cystic fibrosis-related diabetes (CFRD) is a common comorbidity of cystic fibrosis. Diabetic myonecrosis is a rare self-limited complication of poorly controlled diabetes mellitus that commonly presents with acute, intense pain and swelling of lower extremities and responds well to conservative management. We report the first case of diabetic myonecrosis in a patient with CFRD.

Staphylococcus epidermidis bacteriophages from the anterior nares of humans.

The role of virulent bacteriophages in staphylococcal colonization of the human anterior nares is not known. This report of lytic bacteriophages against Staphylococcus epidermidis in the anterior nares of 5.5% of human subjects (n = 202) suggests their potential role in modulating staphylococcal colonization in this ecological niche.

Two unusual pediatric cases of fungal infections in farming families.

It is not known whether farming families have more cases of uncommon fungal infections than the general population because of their interdependence on farming environments, including farm animals and other pets. The authors describe here two cases of fungal infections with interesting epidemiology that suggest associations that have been insufficiently described and explored in the literature. The first is a case of otomycosis in a 17-year-old female and is suspected to be linked to hay baling. The second is a case of tinea capitis in a 25-month-old female toddler living on a farm and illustrates that rural farming families with closer association with animals are infected with different species of fungi than are seen in urban areas.

Prevalence of Staphylococcus aureus and lack of its lytic bacteriophages in the anterior nares of patients and healthcare workers at a rural clinic.

BACKGROUND: Nearly 30% of people in the United States are colonized with Staphylococcus aureus and 1% to 2% with methicillin-resistant Staphylococcus aureus (MRSA) in the anterior nares. However, it is not known if lytic bacteriophages against S. aureus are present in the anterior nares, and if they are, what the prevalence rate is, or if they interfere with S. aureus colonization. The aim of this study was to determine the prevalence of nasal carriage of S. aureus and MRSA and to screen for S. aureus lytic bacteriophages in healthcare environment workers and ambulatory patients. METHODS: We enrolled 202 individuals into this study. The anterior nares were swabbed to isolate S. aureus, MRSA, and any lytic S. aureus bacteriophages that may be present. Putative S. aureus colonies on blood agar plates were identified using gram stain, catalase and coagulase tests, and confirmed by S. aureus-specific PCR. Presence of staphylococcal lytic bacteriophages were screened by a plaque assay technique using a methicillin-sensitive reference S. aureus strain ATCC 29213. RESULTS: Of the 49 (24%) individuals who screened positive for S. aureus, two (1%) were positive for MRSA. None of the samples were positive for lytic bacteriophages against S. aureus. Subgroup analysis found no significant difference in the prevalence of S. aureus in the house staff compared to other healthcare environment workers or ambulatory patients of the clinic. Similarly, no significant difference in colonization was noted across the population with respect to age, sex, body mass index, or presence of diabetes mellitus. CONCLUSION: The prevalence of nasal carriage of S. aureus and MRSA in the studied population was 24% and 1%, respectively, similar to the rate of prevalence in the United States. The study also showed that the anterior nares do not seem to harbor lytic bacteriophages against S. aureus.

Contribution of polymorphisms in ankA, gltA, and groESL in defining genetic variants of Anaplasma phagocytophilum.

Analysis of several nucleotide polymorphisms in polymorphic genes (ankA, gltA, and groESL) from 16S rRNA gene-based genetic variants of Anaplasma phagocytophilum from dogs in the western United States defined at least two sets of multigene polymorphisms to further characterize these variants. The multigene polymorphism approach holds promise for development of a genotyping scheme for this important pathogen.

Propionyl-CoA and adenosylcobalamin metabolism in Caenorhabditis elegans: evidence for a role of methylmalonyl-CoA epimerase in intermediary metabolism.

We have utilized Caenorhabditis elegans to study human methylmalonic acidemia. Using bioinformatics, a full complement of mammalian homologues for the conversion of propionyl-CoA to succinyl-CoA in the genome of C. elegans, including propionyl-CoA carboxylase subunits A and B (pcca-1, pccb-1), methylmalonic acidemia cobalamin A complementation group (mmaa-1), co(I)balamin adenosyltransferase (mmab-1), MMACHC (cblc-1), methylmalonyl-CoA epimerase (mce-1) and methylmalonyl-CoA mutase (mmcm-1) were identified. To verify predictions that the entire intracellular adenosylcobalamin metabolic pathway existed and was functional, the kinetic properties of the C. elegans mmcm-1 were examined. RNA interference against mmcm-1, mmab-1, mmaa-1 in the presence of propionic acid revealed a chemical phenotype of increased methylmalonic acid; deletion mutants of mmcm-1, mmab-1 and mce-1 displayed reduced 1-[(14)C]-propionate incorporation into macromolecules. The mutants produced increased amounts of methylmalonic acid in the culture medium, proving that a functional block in the pathway caused metabolite accumulation. Lentiviral delivery of the C. elegans mmcm-1 into fibroblasts derived from a patient with mut(o) class methylmalonic acidemia could partially restore propionate flux. The C. elegans mce-1 deletion mutant demonstrates for the first time that a lesion at the epimerase step of methylmalonyl-CoA metabolism can functionally impair flux through the methylmalonyl-CoA mutase pathway and suggests that malfunction of MCEE may cause methylmalonic acidemia in humans. The C. elegans system we describe represents the first lower metazoan model organism of mammalian propionate spectrum disorders and demonstrates that mass spectrometry can be employed to study a small molecule chemical phenotype in C. elegans RNAi and deletion mutants.