Complete Genome Sequences of Two Bioluminescent Vibrio campbellii Strains Isolated from Biofouling Communities in the Bay of Bengal.

Vibrio campbellii is a pathogen of aquatic animals and has been proposed as a bacterial partner in the formation of bioluminescent milky seas. We present here the complete genome sequences assembled from Illumina and Oxford Nanopore data for two bioluminescent Vibrio campbellii strains (BoB-53 and BoB-90) isolated from biofouled moorings in the Bay of Bengal.

Evaluation of Parameters for High Efficiency Transformation of Acinetobacter baumannii.

Acinetobacter baumannii is an emerging, nosocomial pathogen that is poorly characterized due to a paucity of genetic tools and methods. While whole genome sequence data from several epidemic and environmental strains have recently become available, the functional characterization of genes is significantly lagging. Efficient transformation is one of the first steps to develop molecular tools that can be used to address these shortcomings. Here we report parameters allowing high efficiency transformation of A. baumannii. Using a multi-factorial experimental design we found that growth phase, voltage, and resistance all significantly contribute to transformation efficiency. The highest efficiency (4.3 × 10(8) Transformants/µg DNA) was obtained at the stationary growth phase of the bacterium (OD 6.0) using 25 ng of plasmid DNA under 100 Ohms resistance and 1.7 kV/cm voltage. The optimized electroporation parameters reported here provide a useful tool for genetic manipulation of A. baumannii.

Airworthiness testing of medical maggots.

An investigation was conducted to test and certify medicinal maggots to facilitate rapid healing of traumatic and chronic wound infections in Wounded warriors being transported onboard military aircraft. Our specific aims included (1) to test the ability of medical grade larvae to withstand the rigors of U.S. Army aeromedical certification, including tolerance to change in pressure, temperature, and humidity inside ground-based chambers; (2) to evaluate the efficacy of the medical grade larvae during a high-vibration rotary-wing medical transport flight; and (3) to gain U.S. Army aeromedical certification and U.S. Air Force safe-to-fly approval and begin the steps needed to deploy/implement the use of medicinal maggots in patient care regimes for medical airlift standard operating procedures. This report outlines the ground-based and initial air-based tests performed during the study. Maggot mortality was very low during all tests, with a mortality rate of less than 1%. Maggot growth rates in wound arenas were mixed but generally depended on temperature. Overall, the results of these tests suggest that medicinal maggots can withstand the rigors of aeromedical evacuation flights in simulated flight environments and rotary- or fixed-wing aircraft.

Bacterial Strain Diversity Within Wounds.

Significance: Rare bacterial taxa (taxa of low relative frequency) are numerous and ubiquitous in virtually any sample-including wound samples. In addition, even the high-frequency genera and species contain multiple strains. These strains, individually, are each only a small fraction of the total bacterial population. Against the view that wounds contain relatively few kinds of bacteria, this newly recognized diversity implies a relatively high rate of migration into the wound and the potential for diversification during infection. Understanding the biological and medical importance of these numerous taxa is an important new element of wound microbiology. Recent Advances: Only recently have these numerous strains been discovered; the technology to detect, identify, and characterize them is still in its infancy. Multiple strains of both gram-negative and gram-positive bacteria have been found in a single wound. In the few cases studied, the distribution of the bacteria suggests microhabitats and biological interactions. Critical Issues: The distribution of the strains, their phenotypic diversity, and their interactions are still largely uncharacterized. The technologies to investigate this level of genomic detail are still developing and have not been largely deployed to investigate wounds. Future Directions: As advanced metagenomics, single-cell genomics, and advanced microscopy develop, the study of wound microbiology will better address the complex interplay of numerous individually rare strains with both the host and each other.

Microbial profiling of combat wound infection through detection microarray and next-generation sequencing.

Combat wound healing and resolution are highly affected by the resident microbial flora. We therefore sought to achieve comprehensive detection of microbial populations in wounds using novel genomic technologies and bioinformatics analyses. We employed a microarray capable of detecting all sequenced pathogens for interrogation of 124 wound samples from extremity injuries in combat-injured U.S. service members. A subset of samples was also processed via next-generation sequencing and metagenomic analysis. Array analysis detected microbial targets in 51% of all wound samples, with Acinetobacter baumannii being the most frequently detected species. Multiple Pseudomonas species were also detected in tissue biopsy specimens. Detection of the Acinetobacter plasmid pRAY correlated significantly with wound failure, while detection of enteric-associated bacteria was associated significantly with successful healing. Whole-genome sequencing revealed broad microbial biodiversity between samples. The total wound bioburden did not associate significantly with wound outcome, although temporal shifts were observed over the course of treatment. Given that standard microbiological methods do not detect the full range of microbes in each wound, these data emphasize the importance of supplementation with molecular techniques for thorough characterization of wound-associated microbes. Future application of genomic protocols for assessing microbial content could allow application of specialized care through early and rapid identification and management of critical patterns in wound bioburden.

Proteomic sample preparation for blast wound characterization.

BACKGROUND: Blast wounds often involve diverse tissue types and require substantial time and treatment for appropriate healing. Some of these subsequent wounds become colonized with bacteria requiring a better understanding of how the host responds to these bacteria and what proteomic factors contribute wound healing outcome. In addition, using reliable and effective proteomic sample preparation procedures can lead to novel biomarkers for improved diagnosis and therapy. RESULTS: To address this need, suitable sample preparation for 2-D DIGE proteomic characterization of wound effluent and serum samples from combat-wounded patients was investigated. Initial evaluation of crude effluent and serum proved the necessity of high abundant protein depletion. Subsequently, both samples were successfully depleted using Agilent Multiple Affinity Removal system and showed greatly improved 2-D spot maps, comprising 1,800 and 1,200 protein spots, respectively. CONCLUSION: High abundant protein removal was necessary for both wound effluent and serum. This is the first study to show a successful method for high abundant protein depletion from wound effluent which is compatible with downstream 2-D DIGE analysis. This development allows for improved biomarker discovery in wound effluent and serum samples.

Validation of a novel murine wound model of Acinetobacter baumannii infection.

Patients recovering from traumatic injuries or surgery often require weeks to months of hospitalization, increasing the risk for wound and surgical site infections caused by ESKAPE pathogens, which include A. baumannii (the ESKAPE pathogens are Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). As new therapies are being developed to counter A. baumannii infections, animal models are also needed to evaluate potential treatments. Here, we present an excisional, murine wound model in which a diminutive inoculum of a clinically relevant, multidrug-resistant A. baumannii isolate can proliferate, form biofilms, and be effectively treated with antibiotics. The model requires a temporary, cyclophosphamide-induced neutropenia to establish an infection that can persist. A 6-mm-diameter, full-thickness wound was created in the skin overlying the thoracic spine, and after the wound bed was inoculated, it was covered with a dressing for 7 days. Uninoculated control wounds healed within 13 days, whereas infected, placebo-treated wounds remained unclosed beyond 21 days. Treated and untreated wounds were assessed with multiple quantitative and qualitative techniques that included gross pathology, weight loss and recovery, wound closure, bacterial burden, 16S rRNA community profiling, histopathology, peptide nucleic acid-fluorescence in situ hybridization, and scanning electron microscopy assessment of biofilms. The range of differences that we are able to identify with these measures in antibiotic- versus placebo-treated animals provides a clear window within which novel antimicrobial therapies can be assessed. The model can be used to evaluate antimicrobials for their ability to reduce specific pathogen loads in wounded tissues and clear biofilms. Ultimately, the mouse model approach allows for highly powered studies and serves as an initial multifaceted in vivo assessment prior to testing in larger animals.

Antimicrobial resistance determinants in Acinetobacter baumannii isolates taken from military treatment facilities.

Multidrug-resistant (MDR) Acinetobacter baumannii infections are of particular concern within medical treatment facilities, yet the gene assemblages that give rise to this phenotype remain poorly characterized. In this study, we tested 97 clinical A. baumannii isolates collected from military treatment facilities (MTFs) from 2003 to 2009 by using a molecular epidemiological approach that enabled for the simultaneous screening of 236 antimicrobial resistance genes. Overall, 80% of the isolates were found to be MDR, each strain harbored between one and 17 resistant determinants, and a total of 52 unique resistance determinants or gene families were detected which are known to confer resistance to ß-lactam (e.g., blaGES-11, blaTEM, blaOXA-58), aminoglycoside (e.g., aphA1, aacC1, armA), macrolide (msrA, msrB), tetracycline [e.g., tet(A), tet(B), tet(39)], phenicol (e.g., cmlA4, catA1, cat4), quaternary amine (qacE, qacEΔ1), streptothricin (sat2), sulfonamide (sul1, sul2), and diaminopyrimidine (dfrA1, dfrA7, dfrA19) antimicrobial compounds. Importantly, 91% of the isolates harbored blaOXA-51-like carbapenemase genes (including six new variants), 40% harbored the blaOXA-23 carbapenemase gene, and 89% contained a variety of aminoglycoside resistance determinants with up to six unique determinants identified per strain. Many of the resistance determinants were found in potentially mobile gene cassettes; 45% and 7% of the isolates contained class 1 and class 2 integrons, respectively. Combined, the results demonstrate a facile approach that supports a more complete understanding of the genetic underpinnings of antimicrobial resistance to better assess the load, transmission, and evolution of MDR in MTF-associated A. baumannii.

Wound outcome in combat injuries is associated with a unique set of protein biomarkers.

BACKGROUND: The ability to forecast whether a wound will heal after closure without further debridement(s), would provide substantial benefits to patients with severe extremity trauma. METHODS: Wound effluent is a readily available material which can be collected without disturbing healthy tissue. For analysis of potential host response biomarkers, forty four serial combat wound effluent samples from 19 patients with either healing or failing traumatic- and other combat-related wounds were examined by 2-D DIGE. Spot map patterns were correlated to eventual wound outcome (healed or wound failure) and analyzed using DeCyder 7.0 and differential proteins identified via LC-MS/MS. RESULTS: This approach identified 52 protein spots that were differentially expressed and thus represent candidate biomarkers for this clinical application. Many of these proteins are intimately involved in inflammatory and immune responses. Furthermore, discriminate analysis further refined the 52 differential protein spots to a smaller subset of which successfully differentiate between wounds that will heal and those that will fail and require further surgical intervention with greater than 83% accuracy. CONCLUSION: These results suggest candidates for a panel of protein biomarkers that may aid traumatic wound care prognosis and treatment. We recommend that this strategy be refined, and then externally validated, in future studies of traumatic wounds.

The Hospital Microbiome Project: Meeting Report for the 1st Hospital Microbiome Project Workshop on sampling design and building science measurements, Chicago, USA, June 7th-8th 2012.

This report details the outcome of the 1st Hospital Microbiome Project workshop held on June 7th-8th, 2012 at the University of Chicago, USA. The workshop was arranged to determine the most appropriate sampling strategy and approach to building science measurement to characterize the development of a microbial community within a new hospital pavilion being built at the University of Chicago Medical Center. The workshop made several recommendations and led to the development of a full proposal to the Alfred P. Sloan Foundation as well as to the creation of the Hospital Microbiome Consortium.

Future-generation sequencing and clinical microbiology.

Sequencing technologies are changing the way both laboratory medicine and clinical practice impact patient care. This article focuses on the clinical microbiology laboratory and the potential benefits and limitations of coming generations of sequencing technology. Nucleic acid sequencing technology is rapidly outpacing the infrastructure needed to accurately educate, analyze, and interpret complex massive data sets that are rapidly becoming integrated into clinical practice.

Clustering acinetobacter strains by optical mapping.

Optical mapping is a technique that produces an ordered restriction map of a bacterial or eukaryotic chromosome. We have developed a new method, the BOP method, to compare experimental optical maps with in silico optical maps of complete genomes to infer the presence/absence of short DNA sequences (bops) in each genome. The BOP method, as implemented by the Optical Mapping suite of four programs, circumvents the necessity of whole-genome multiple alignments and permits reliable strain typing and clustering on the basis of optical maps. We have applied the Optical Mapping Suite to 125 strains of Acinetobacter sp., including 11 completely sequenced genomes and 114 Acinetobacter complex from three US military hospitals. We found that optical mapping completely resolves all 125 strains. Signal to noise analysis showed that when the 125 strains were considered together almost 1/3 of the experimental fragments were misidentified. We found that the set of 125 genomes could be divided into three clusters, two of which included sequenced genomes. Signal to noise analysis after clustering showed that only 3.5% of the experimental restriction fragments were misidentified. Minimum spanning trees of the two clusters that included sequenced genomes are presented. The programs we have developed provide a more rigorous approach for analyzing optical map data than previously existed.

Culture-independence for surveillance and epidemiology.

Culture-independent methods in microbiology (quantitative PCR (qPCR), sequencing, microarrays, direct from sample matrix assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS), etc.) are disruptive technology. Rather than providing the same results as culture-based methods more quickly, more cheaply or with improved accuracy, they reveal an unexpected diversity of microbes and illuminate dark corners of undiagnosed disease. At times, they overturn existing definitions of presumably well-understood infections, generating new requirements for clinical diagnosis, surveillance and epidemiology. However, current diagnostic microbiology, infection control and epidemiology rest principally on culture methods elegantly optimized by clinical laboratorians. The clinical significance is interwoven; the new methods are out of context, difficult to interpret and impossible to act upon. Culture-independent diagnostics and surveillance methods will not be deployed unless the reported results can be used to select specific therapeutics or infection control measures. To cut the knots surrounding the adoption of culture-independent methods in medical microbiology, culture-dependent methods should be supported by consistent culture-independent methods providing the microbial context. This will temper existing biases and motivate appropriate scrutiny of the older methods and results.

Maggot debridement therapy in modern army medicine: perceptions and prevalence.

Maggot debridement therapy (MDT), despite its long history and safety profile, finds limited use in the military health care system. Although new methods are continually being investigated to debride wounds more quickly and effectively, MDT remains largely a therapy of last resort. We evaluated the frequency of MDT in the Army sector of the MHS and the decision-making process surrounding its use. A 22 question survey of Army physicians was prepared and distributed through select Medical Corps Consultants in specialties likely to practice debridement. 83% of respondents were familiar with MDT, and of those familiar, 63% were aware of FDA approval for the product and 10% had used the product themselves. The three most frequently cited reasons for not using the therapy were no need (52%), no access (23%), and insufficient experience (19%). Informing the 37% of physicians who are not aware of FDA approval is an obvious target for program improvement. However, as many do not find a need for MDT, targeted improvements to MDT access and education for those physicians who encounter indications for MDT would permit them to apply MDT where there is an unmet need.

Biocompatibility of antimicrobials to maggot debridement therapy: medical maggots Lucilia sericata (Diptera: Calliphoridae) exhibit tolerance to clinical maximum doses of antimicrobials.

The larvae of calliphorid flies are used to debride wounds of patients with severe tissue destruction, often concurrently with antimicrobials. The current study evaluates the effects of ceftazidime, tobramycin, amikacin, gentamicin, polymyxin B, doxycycline, paromomycin, amphotericin B, sodium stibogluconate, and miltefosine at 1, 10, and 100 x the Maximum Clinical Concentration (mg/kg/d) in raw liver assays. Effects on larvae were small and depended on dose and antimicrobial formulation, with hours in assay (24 or 48 h) having a significant effect on larval survival. Sodium stibgluconate had the strongest effect on maggot survival (80.0% at 48 h). These results suggest that the antimicrobials tested here may be used simultaneously with maggot debridement therapy, and may actually increase the effectiveness of maggot debridement therapy in certain applications where >1 x Maximum Clinical Concentration is indicated, such as topical creams for cutaneous leishmaniasis.

Extracellular stress and lipopolysaccharide modulate Acinetobacter baumannii surface-associated motility.

Acinetobacter baumannii is a nosocomial bacterial pathogen, and infections attributed to this species are further complicated by a remarkable ability to acquire antimicrobial resistance genes and to survive in a desiccated state. While the antibiotic resistance and biofilm formation of A. baumannii is well-documented, less is known about the virulence attributes of this organism. Recent studies reported A. baumannii strains display a motility phenotype, which appears to be partially dependent upon Type IV pili, autoinducer molecules, and the response to blue light. In this study, we wanted to determine the prevalence of this trait in genetically diverse clinical isolates, and any additional required factors, and environmental cues that regulate motility. When strains are subjected to a wide array of stress conditions, A. baumannii motility is significantly reduced. In contrast, when extracellular iron is provided or salinity is reduced, motility is significantly enhanced. We further investigated whether the genes required for the production of lipopolysaccharide (lpsB) and K1 capsule (epsA/ptk) are required for motility as demonstrated in other Gram-negative bacteria. Transposon mutagenesis resulted in reduced motility by the insertion derivatives of each of these genes. The presence of the parental allele provided in trans, in the insertion mutant background, could only restore motility in the lpsB mutant. The production of core LPS directly contributes to the motility phenotype, while capsular polysaccharide may have an indirect effect. Further, the data suggest motility is regulated by extracellular conditions, indicating that A. baumannii is actively sensing the environment and responding accordingly.

Optically mapping multiple bacterial genomes simultaneously in a single run.

Optical mapping of bacterial chromosomes provides an unambiguous low-resolution sequence scaffold of the entire chromosome. In comparison to some techniques, such as pulse field gel electrophoresis, cost and throughput limit the application of this technique outside of genome finishing. We have demonstrated the production of multiple bacterial maps using a single set of consumables; this significantly reduces the time and expense of map production.

Rapid whole genome optical mapping of Plasmodium falciparum.

BACKGROUND: Immune evasion and drug resistance in malaria have been linked to chromosomal recombination and gene copy number variation (CNV). These events are ideally studied using comparative genomic analyses; however in malaria these analyses are not as common or thorough as in other infectious diseases, partly due to the difficulty in sequencing and assembling complete genome drafts. Recently, whole genome optical mapping has gained wide use in support of genomic sequence assembly and comparison. Here, a rapid technique for producing whole genome optical maps of Plasmodium falciparum is described and the results of mapping four genomes are presented. METHODS: Four laboratory strains of P. falciparum were analysed using the Argus™ optical mapping system to produce ordered restriction fragment maps of all 14 chromosomes in each genome. Plasmodium falciparum DNA was isolated directly from blood culture, visualized using the Argus™ system and assembled in a manner analogous to next generation sequence assembly into maps (AssemblyViewer™, OpGen Inc.®). Full coverage maps were generated for P. falciparum strains 3D7, FVO, D6 and C235. A reference P. falciparum in silico map was created by the digestion of the genomic sequence of P. falciparum with the restriction enzyme AflII, for comparisons to genomic optical maps. Maps were then compared using the MapSolver™ software. RESULTS: Genomic variation was observed among the mapped strains, as well as between the map of the reference strain and the map derived from the putative sequence of that same strain. Duplications, deletions, insertions, inversions and misassemblies of sizes ranging from 3,500 base pairs up to 78,000 base pairs were observed. Many genomic events occurred in areas of known repetitive sequence or high copy number genes, including var gene clusters and rifin complexes. CONCLUSIONS: This technique for optical mapping of multiple malaria genomes allows for whole genome comparison of multiple strains and can assist in identifying genetic variation and sequence contig assembly. New protocols and technology allowed us to produce high quality contigs spanning four P. falciparum genomes in six weeks for less than $1,000.00 per genome. This relatively low cost and quick turnaround makes the technique valuable compared to other genomic sequencing technologies for studying genetic variation in malaria.