Comparing Methods to Genetically Engineer Bacteriophage and Increase Host Range.

INTRODUCTION: Antibacterial resistance is an emerging problem in military medicine. Disruptions to the health care systems in war-torn countries that result from ongoing conflict can potentially exacerbate this problem and increase the risk to U.S. forces in the deployed environment. Therefore, novel therapies are needed to mitigate the impact of these potentially devastating infections on military operations. Bacteriophages are viruses that infect and kill bacteria. They can be delivered as therapeutic agents and offer a promising alternative to traditional antibiotic chemotherapy. There are several potential benefits to their use, including high specificity and comparative ease of use in the field setting. However, the process of engineering phages for military medical applications can be a laborious and time-consuming endeavor. This review examines available techniques and compares their efficacy. MATERIALS AND METHODS: This review evaluates the scientific literature on the development and application of four methods of bacteriophage genome engineering and their consideration in the context of military applications. Preffered Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed for a systematic review of available literature that met criteria for analysis and inclusion. The research completed for this review article originated from the United States Military Academy's library "Scout" search engine, which compiles results from 254 available databases (including PubMed, Google Scholar, and SciFinder). Particular attention was focused on identifying useful mechanistic insight into the nature of the engineering technique, the ease of use, and the applicability of the technique to countering the problem of antimicrobial resistance in the military setting. RESULTS: A total of 52 studies were identified that met inclusion criteria following PRISMA guidelines. The bioengineering techniques analyzed included homologous recombination (12 articles), in vivo recombineering (9 articles), bacteriophage recombineering of electroporated DNA (7 articles), and the CRISPR-Cas system (10 articles). Rates of success and fidelity varied across each platform, and comparative benefits and drawbacks are considered. CONCLUSIONS: Each of the phage engineering techniques addressed herein varies in amount of effort and overall success rate. CRISPR-Cas-facilitated modification of phage genomes presents a highly efficient method that does not require a lengthy purification and screening process. It therefore appears to be the method best suited for military medical applications.

Genomic Surveillance of Rabies Virus in Georgian Canines.

Rabies is a fatal zoonosis that is considered a re-emerging infectious disease. Although rabies remains endemic in canines throughout much of the world, vaccination programs have essentially eliminated dog rabies in the Americas and much of Europe. However, despite the goal of eliminating dog rabies in the European Union by 2020, sporadic cases of dog rabies still occur in Eastern Europe, including Georgia. To assess the genetic diversity of the strains recently circulating in Georgia, we sequenced seventy-eight RABV-positive samples from the brain tissues of rabid dogs and jackals using Illumina short-read sequencing of total RNA shotgun libraries. Seventy-seven RABV genomes were successfully assembled and annotated, with seventy-four of them reaching the coding-complete status. Phylogenetic analyses of the nucleoprotein (N) and attachment glycoprotein (G) genes placed all the assembled genomes into the Cosmopolitan clade, consistent with the Georgian origin of the samples. An amino acid alignment of the G glycoprotein ectodomain identified twelve different sequences for this domain among the samples. Only one of the ectodomain groups contained a residue change in an antigenic site, an R264H change in the G5 antigenic site. Three isolates were cultured, and these were found to be efficiently neutralized by the human monoclonal antibody A6. Overall, our data show that recently circulating RABV isolates from Georgian canines are predominantly closely related phylogroup I viruses of the Cosmopolitan clade. Current human rabies vaccines should offer protection against infection by Georgian canine RABVs. The genomes have been deposited in GenBank (accessions: OQ603609-OQ603685).

Lessons Learned From the U.S. Military Experience With Hantavirus During the Korean War.

INTRODUCTION: The Korean War (1950-1953) consisted of two phases. The first was a rapid mobile phase, and the second was a slow and stationary phase. During the stationary phase, approximately 3,000 UN troops became infected with a then unknown agent. The resulting illness began with flu-like symptoms and often progressed to a severe hemorrhagic fever leading to kidney failure and death. However, the cause was not to be identified until well over 20 years following the conclusion of the war when Dr. Ho Wang Lee succeeded in isolating Hantavirus from field rodents. The U.S. Military experience with Hantavirus during the Korean War is a case study of the potential impact of war-related environmental change on disease transmission. The lessons learned from this experience should inform future military medical planning and serve as a reminder of the impact that an unknown agent can have on military operations. MATERIALS AND METHODS: A literature review of all available records with respect to the U.S. Military experience with Hantavirus was conducted. PubMed was the primary search engine used for this review. However, primary literature and historical accounts were also evaluated. All records were examined for environmental, epidemiological, and public health data regarding hemorrhagic fever outbreaks among U.S. forces during the Korean War. The quantitative and qualitative data from these sources were analyzed and evaluated within the context of military medical planning and force health protection to derive lessons learned that should be applied to the management and mitigation of viral disease in future wars. RESULTS: Widespread deforestation resulting from war-related efforts most likely played a significant role in the outbreaks of Hantavirus among UN forces during the war. A lack of cultural literacy and an overreliance on erroneous assumptions most likely delayed the identification of the true causative agent. It is conceivable that these delays led to an increased casualty rate and that they had a negative impact on military operations during the war. CONCLUSIONS: A basic understanding of the ecological mechanisms that maintain species diversity in the local environment coupled with an appreciation for the impact of environmental change on this diversity is of paramount importance for the prevention and mitigation of viral disease outbreaks in the deployed setting. Military medical planners should become familiar with the medical literature of the region in which they will be operating as this literature often describes the agents that will most likely be encountered by U.S. forces.

A sustained release cysteamine microsphere/thermoresponsive gel eyedrop for corneal cystinosis improves drug stability.

Cystinosis is a rare, metabolic, recessive genetic disease in which the intralysosomal accumulation of cystine leads to system wide organ and tissue damage. In the eye, cystine accumulates in the cornea as corneal cystine crystals and severely impacts vision. Corneal cystine crystals are treated with cysteamine eyedrops when administrated 6 to 12 times day and used within 1 week. The strict dosing regimen and poor stability are inconvenient and add to the burden of therapy. To reduce the dosing frequency and improve the stability, we present reformulation of cysteamine into a novel controlled release eyedrop. In this work, we characterize and evaluate a topical drug delivery system comprised of encapsulated cysteamine in polymer microspheres with a thermoresponsive gel carrier. Spray-dried encapsulation of cysteamine was performed. In vitro cysteamine release, stability, and ocular irritation and corneal permeation were evaluated. The data suggest that encapsulated cysteamine improves the stability to 7 weeks when compared with 1-week aqueous cysteamine eyedrops. Release studies from one drop of our system show that cysteamine release was present for 24 h and above the minimum cysteamine eyedrop amount (6 drops). Cysteamine from our system also resulted in negligible irritation and enhanced permeation when compared with traditional cysteamine eyedrops. In vivo studies were implemented to support ease of administration, tolerability, and retention for 24 h. These studies suggest that our controlled release delivery system may provide stable cysteamine from a safe, once daily gel eyedrop.

A Longitudinal Evaluation of the Bacterial Pathogens Colonizing Chronic Non-Healing Wound Sites at a United States Military Treatment Facility in the Pacific Region.

PURPOSE: The biology of chronic wounds is complex and many factors act concurrently to impede healing progress. In this study, the dynamics of microflora changes and their antibiotic susceptibility patterns were evaluated longitudinally over 30 days using data from 28 patients with a total of 47 chronic lower extremity wounds. MATERIALS AND METHODS: In this study, colonized wound isolates were characterized using cultural, biochemical, and VITEK 2 methods. Antibiotic susceptibility patterns of the wound isolates were analyzed using various phenotypic assays. Furthermore, antimicrobial resistance patterns and the presence of mutations were evaluated by a genotypic assay, whole-genome sequencing (WGS). RESULTS: Staphylococcus aureus and Pseudomonas aeruginosa were found to be the most common strains at early time points, while members of Enterobacteriaceae were prevalent at later stages of infection. Antimicrobial resistance testing and whole-genome sequencing revealed that the molecular and phenotypic characteristics of the identified wound pathogens remained relatively stable throughout the study period. It was also noted that Enterobacter and Klebsiella species may serve as reservoirs for quinolone resistance in the Pacific region. CONCLUSION: Our observations showed that wounds were colonized with diverse bacteria and interestingly their numbers and/or types were changed over the course of infection. The rapid genetic changes that accompanied the first 4 weeks after presentation did not directly contribute to the development of antibiotic resistance. In addition, standard wound care procedures did not appear to select for resistant bacterial strains. Future efforts should focus on defining those genetic changes associated with the wound colonizing microorganisms that occur beyond 4 weeks.

Escherichia coli as a Potential Reservoir of Antimicrobial Resistance Genes on the Island of O'ahu.

The problem of antimicrobial-resistant bacteria has not been adequately explored in the tropical island environment. To date, there has not been a systematic investigation into the prevalence and distribution of antimicrobial resistance determinants in the Hawaiian Islands. Urinary isolates are the most common bacterial pathogens encountered in the clinical laboratory. Therefore, the antimicrobial resistance determinant profiles of these organisms can serve as a sentinel of the overall antimicrobial resistance situation in a localized patient population. In this study, 82 clinical isolates of Escherichia coli derived from 82 distinct patients were collected at a large medical center on the island of O'ahu. Each isolate was evaluated for the presence of antimicrobial resistance genes using a microarray-based approach. A total of 36 antimicrobial resistance genes covering 10 classes of antimicrobial compounds were identified. Most isolates were found to harbor between 3 and 5 antimicrobial resistance genes. Only a few isolates were found to harbor more than 12 genes. Significantly, a high rate of phenotypic resistance to one of the first-line treatments for uncomplicated urinary tract infection (sulfamethoxazole) was identified. This phenotype was correlated to the presence of sulfonamides and trimethoprim resistance determinants. Since E. coli is one of the most encountered pathogens in the hospital environment, the presence of clinically relevant resistance determinants in isolates of this organism from a clinical setting on O'ahu is a significant finding that warrants further investigation.

Non-Pharmaceutical Interventions and Military Hygiene at the United States Military Academy between 1890 and 1910.

INTRODUCTION: Military installations are at increased risk for the transmission of infectious disease. Personnel who live and train on military installations live and train near one another facilitating disease transmission. An understanding of historical sanitation and hygiene can inform modern practices. This is especially pertinent considering the continuing rise of variants of infectious diseases, such as the recent pandemic of the 2019 severe acute respiratory syndrome coronavirus 2. In this article, we review the rise and decline of infectious disease at the United States Military Academy (USMA) during the period spanning 1890 through 1910, and the public health interventions used to combat disease spread. MATERIALS AND METHODS: Primary data regarding cadet illness were acquired from the historical archives of the USMA. These included annual reports, clinical admission records, casualty ledgers, and sanitation reports. Unpublished documents from the medical history of USMA provide periodic trends of health among cadets because of infectious disease. RESULTS: Between 1890 and 1910, the USMA at West Point was confronted with cases of influenza, measles, mumps, scarlet fever, smallpox, typhus, and malaria. In response, a series of non-pharmaceutical interventions (NPIs) were instituted to curb the spread of infectious disease. These interventions most likely proved effective in suppressing the transmission of communicable diseases. The most common and arguably the most effective NPI was the physical separation of the sick from the well. CONCLUSIONS: The USMA experience mirrored what was occurring in the larger U.S. Army in the early 20th century and may serve as a model for the application of NPIs in response to modern infectious diseases resulting from novel or unknown etiologies.

An evaluation of the impact of clinical bacterial isolates on epithelial cell monolayer integrity by the electric Cell-Substrate Impedance Sensing (ECIS) method.

Virulence is the relative capacity of a pathogenic microorganism to cause damage in susceptible host cells such as those found in airway passages and the gut. In this study, the effect of clinical bacterial isolates on the monolayer integrity of cultured human alveolar basal epithelial cells (A549) was evaluated using the Electric Cell-Substrate Impedance Sensing (ECIS) system. ECIS is a morphological biosensor which records electrical properties of cell-covered microelectrodes in an AC circuit including impedance (ohm), resistance (ohm), and capacitance (µFarad). In the current study, fluctuations in the electrical properties of cell-covered microelectrodes reflect dynamic changes in cell morphology resulting from disrupted cell monolayers following exposure to bacteria. Using the ECIS system, real-time changes of cell morphology and disruption of monolayer integrity of cell-cultures in vitro were revealed for A549 cells infected with either Pseudomonas aeruginosa, ESBL Escherichia coli, Staphylococcus aureus (MRSA), or Enterococcus (VRE). We determined empirically that the optimal signal response was obtained for resistance (ohm) measurements at 4000 hertz. Following infection of A549 cells, the data revealed that Pseudomonas aeruginosa resulted in little change in microelectrode resistance (ohm @4 kHz) as compared to pathogen-free controls within the first 12 h. In contrast, E. coli, MRSA, and VRE caused significant changes in electrode resistance (ohm @4 kHz) values in the infected cells compared to controls over the first 5 h. Resistance (ohm @4 kHz) changes were also observed in cell monolayers infected with different bacterial concentrations for all isolates over 24 h. The highest concentration of bacteria caused the measured resistance (ohm @4 kHz) to drop faster than its' immediate lower concentration, suggesting a dose-dependent effect. Compared to live bacteria, cells exposed to heat-killed bacteria did not show significant changes in resistance (ohm @4 kHz) over 48 h post-exposure. Functionally, cytokine responses were different between cells treated with live and heat-killed bacteria. Of note, live bacteria induced IFNγ, IL-13, and IL-1ß production in A549 cells, whereas heat-killed bacteria induced IL-8 production suggesting a differential interaction with cells that could reveal the underlying causes of resistance (ohm @4 kHz) changes. Our findings indicate that ECIS provides a means to quantify, automate, and measure bacterial virulence, which may have broader implications governing the course of treatment compared to traditional methods alone.

Multivariate relationships between epidemiologic risk factors and zoonotic infections among military personnel in the country of Georgia: A non-linear canonical correlation analysis.

Zoonotic diseases are endemic in the country of Georgia. Using the non-linear canonical correlation (NCC) method, the aim of this study was to examine the relationship between thirteen epidemiological risk factors and seropositivity to five zoonotic infections (anthrax, Q fever, tularemia, leptospirosis, and Crimean-Congo hemorrhagic fever [CCHF]) among Georgian military recruits during 2014-2016. According to this multivariate statistical technique, which is suitable for the analysis of two or more sets of qualitative variables simultaneously, two canonical variables were identified. These variables accounted for 68% of the variation between the two sets of categorical variables ("risk factors" and "zoonotic infections"). For the first canonical variable, there was a relationship among CCHF (canonical loading, which is interpreted in the same way as the Pearson's correlation coefficient, [cl] = 0.715), tick bites (cl = 0.418) and slaughter of animals (cl = 0.351). As for the second canonical variable, Q fever (cl = -0.604) and leptospirosis (cl = -0.486) were related to rodents inside and outside home (cl = -0.346) and sweeping in or around home (cl = -0.317). The NCC method allows researchers to obtain additional insights into the complex relationship between epidemiological risk factors and multiple zoonotic infections.

Latent Lyme Disease Resulting in Chronic Arthritis and Early Career Termination in a United States Army Officer.

Lyme disease is a continuing threat to military personnel operating in arboriferous and mountainous environments. Here we present the case of a 24-year-old Second Lieutenant, a recent graduate from the United States Military Academy, with a history of Lyme disease who developed recurrent knee effusions following surgery to correct a hip impingement. Although gonococcal arthritis was initially suspected from preliminary laboratory results, a comprehensive evaluation contradicted this diagnosis. Despite antibiotic therapy, aspiration of the effusions, and steroid treatment to control inflammation, the condition of the patient deteriorated to the point where he was found to be unfit for duty and subsequently discharged from active military service. This case illustrates the profound effect that latent Lyme disease can have on the quality of life and the career of an active duty military member. It highlights the need for increased surveillance for Borrelia burgdorferi (B. burgdorferi) in military training areas and for the early and aggressive diagnosis and treatment of military personnel who present with the symptoms of acute Lyme disease.

Antibiotic resistance, molecular characterizations, and clinical manifestations of Campylobacteriosis at a military medical center in Hawaii from 2012-2016: a retrospective analysis.

Hawaii has one of the highest incidences of Campylobacteriosis in the United States, but there remains little published data on circulating strains or antimicrobial resistance. We characterized 110 clinical Campylobacter isolates (106 C. jejuni, 4 C. coli) processed at Tripler Army Medical Center in Honolulu, HI from 2012-2016. Twenty-five percent of C. jejuni isolates exhibited fluoroquinolone (FQ) resistance, compared with 16% for tetracycline (TET), and 0% for macrolides. Two of the four C. coli isolates were resistant to FQ, TET, and macrolides. C. jejuni isolates further underwent multilocus sequence typing, pulsed-field gel electrophoresis, and molecular capsular typing. Nineteen capsule types were observed, with two capsule types (HS2 and HS9) being associated with FQ resistance (p < 0.001 and p = 0.006, respectively). HS2 FQ-resistant isolates associated with clonal complex 21, possibly indicating clonal spread in FQ resistance. Macrolides should be considered for treatment of suspect cases due to lack of observed resistance.

Monomer sequence in PLGA microparticles: Effects on acidic microclimates and in vivo inflammatory response.

Controlling the backbone architecture of poly(lactic-co-glycolic acid)s (PLGAs) is demonstrated to have a strong influence on the production and release of acidic degradation by-products in microparticle matrices. Previous efforts for controlling the internal and external accumulation of acidity for PLGA microparticles have focused on the addition of excipients including neutralization and anti-inflammatory agents. In this report, we utilize a sequence-control strategy to tailor the microstructure of PLGA. The internal acidic microclimate distributions within sequence-defined and random PLGA microparticles were monitored in vitro using a non-invasive ratiometric two-photon microscopy (TPM) methodology. Sequence-defined PLGAs were found to have minimal changes in pH distribution and lower amounts of percolating acidic by-products. A parallel scanning electron microscopy study further linked external morphological events to internal degradation-induced structural changes. The properties of the sequenced and random copolymers characterized in vitro translated to differences in in vivo behavior. The sequence alternating copolymer, poly LG, had lower granulomatous foreign-body reactions compared to random racemic PLGA with a 50:50 ratio of lactic to glycolic acid. STATEMENT OF SIGNIFICANCE: This paper demonstrates that changing the monomer sequence in poly(lactic-co-glycolic acid)s (PLGAs) leads to dramatic differences in the rate of degradation and the internal acidic microclimate of microparticles degrading in vitro. We note that the acidic microclimates within these particles were imaged for the first time with two-photon microscopy, which gives an extremely clear and detailed picture of the degradation process. Importantly, we also document that the observed sequence-controlled in vitro processes translate into differences in the in vivo behavior of polymers which have the same L to G composition but differing microstructures. These data, placed in the context of our prior studies on swelling, erosion, and MW loss (Biomaterials2017, 117, 66 and other references cited within the manuscript), provide significant insight not only about sequence effects in PLGAs but into the underlying mechanisms of PLGA degradation in general.

The impact of monomer sequence and stereochemistry on the swelling and erosion of biodegradable poly(lactic-co-glycolic acid) matrices.

Monomer sequence is demonstrated to be a primary factor in determining the hydrolytic degradation profile of poly(lactic-co-glycolic acid)s (PLGAs). Although many approaches have been used to tune the degradation of PLGAs, little effort has been expended in exploring the sequence-control strategy exploited by nature in biopolymers. Cylindrical matrices and films prepared from a series of sequenced and random PLGAs were subjected to hydrolysis in a pH 7.4 buffer at 37 °C. Swelling ranged from 107% for the random racemic PLGA with a 50:50 ratio of lactic (L) to glycolic (G) units to 6% for the sequenced alternating copolymer poly LG. Erosion followed an inverse trend with the random 50:50 PLGA showing an erosion half-life of 3-4 weeks while poly LG required ca. >10 weeks. Stereosequence was found to play a large role in determining swelling and erosion; stereopure analogs swelled less and were slower to lose mass. Molecular weight loss followed similar trends and increases in dispersity correlated with the onset of significant swelling. The relative proportion of rapidly cleavable G-G linkages relative to G-L/L-G (moderate) and L-L (slow) correlates strongly with the degree of swelling observed and the rate of erosion. The dramatic sequence-dependent variation in swelling, in the absence of a parallel hydrophilicity trend, suggest that osmotic pressure, driven by the differential accumulation of degradation products, plays an important role.

An Evaluation of Common Cleaning Methods for the Removal of a Clinical Isolate of Escherichia coli in Personal Hydration System Water Reservoirs.

Waterborne infection is an important cause of morbidity and mortality throughout the world. Personal hydration packs have been used by military personnel since the Gulf War and are now a common issue item. Since military personnel tend to operate under austere conditions and may use a variety of water sources, preventing the acquisition of waterborne infections is extremely important. Further, since hydration pack water reservoir replacements may not be available during combat operations, the development of a reliable cleaning protocol for use in the field is essential. Several methods for cleaning have been described. In the current study, three common cleaning methodologies-bleach treatment, baking soda treatment, and proprietary CAMELBAK Cleaning Tabs™-were evaluated for the ability to remove Escherichia coli contamination from hydration pack water reservoirs. The study results suggest that the use of bleach and proprietary CAMELBAK tablets should be encouraged since they both operate by releasing bactericidal chlorine compounds into solution, which is more effective at reducing post-treatment bacterial burden. It should be noted that no method was 100% effective at completely eliminating bacteria from the reservoirs and that mechanical cleaning was not attempted.

A Case of Wound Infection with Providencia rettgeri and Coincident Gout in a Patient from Guam.

Providencia rettgeri (P. rettgeri) is a ubiquitous organism that is infrequently associated with human disease. Here we report the isolation of this organism from a polymicrobial wound infection resulting from ruptured tophi on a 54-year-old male patient from Guam. We describe the identification and confirmation of this organism, and propose metabolic synergy as a possible mechanism of pathogenesis. To our knowledge, this is the first published report of a wound infection colonized by P. rettgeri from Guam, and the first report to speculate upon the role of bacterial synergy in P. rettgeri pathogenesis.

Effectiveness of a Dental Unit Waterline Treatment Protocol With A-Dec ICX and Citrisil Disinfectants.

This study evaluated the effectiveness of a Dental Unit Waterline disinfection protocol utilizing two waterline disinfectant tablets in a dental treatment clinic. The water effluent from 47 dental treatment units was sampled to determine bacterial load. Four dental treatment units were shocked with the multivalent Sterilex Ultra liquid biocide, followed by a 5-week course of routine disinfection using either the A-dec ICX or Citrisil effervescing tablets. Aseptic samples were taken twice weekly, and bacterial load was determined. No significant difference was found when comparing A-dec ICX with Citrisil, but a significant difference was seen between the use of either tablet and no tablet. In addition, a survey was conducted to evaluate the effect of user compliance on infection control. The results indicate that proper training, coupled with the use of appropriate disinfectants and shock treatment, are important aspects of maintaining low bacterial burden in dental water lines.

A Case of Bordetella brochiseptica at a Military Medical Facility in Hawai'i: Phenotypic and Molecular Testing of an Uncommon Human Pathogen.

Bordetella bronchiseptica (B. bronchiseptica) is rarely implicated in human disease. Human infections typically occur in the context of immunosuppression and while human infection has been sporadically reported in the literature, the majority of these reports are largely descriptive and do not explore the molecular and phenotypic properties of the isolates in question. Here we report the isolation and characterization of a B. bronchiseptica isolate derived from an HIV positive patient at Tripler Army Medical Center on O'ahu. This case represents the first published report of human infection of B. bronchiseptica in the state of Hawai'i and the most detailed description of the biochemical and molecular features of a Hawaiian isolate to date.

Staphylococcus sciuri: An Entomological Case Study and a Brief Review of the Literature.

Staphylococcus sciuri is an emerging gram-positive bacterial pathogen that is infrequently isolated from cases of human disease. This organism is capable of rapid conversion from a state of methicillin sensitivity to a state of methicillin resistance and has been shown to express a set of highly effective virulence factors. The antibiotic-resistance breakpoints of S. sciuri differ significantly from the more common Staphylococcus species. Therefore, the rapid identification of S. sciuri in clinical material is a prerequisite for the proper determination of the antibiotic-resistance profile and the rapid initiation of antimicrobial therapy. Here, we present a brief literature review of S. sciuri and an entomological case study in which we describe the colonization of an American cockroach with this agent. In addition, we discuss potential implications for the distribution and evolution of antibiotic-resistant members of the genus Staphylococcus.