Cell Labeling with Magneto-Endosymbionts and the Dissection of the Subcellular Location, Fate, and Host Cell Interactions.

PURPOSE: The purposes of this study are to characterize magneto-endosymbiont (ME) labeling of mammalian cells and to discern the subcellular fate of these living contrast agents. MEs are novel magnetic resonance imaging (MRI) contrast agents that are being used for cell tracking studies. Understanding the fate of MEs in host cells is valuable for designing in vivo cell tracking experiments. PROCEDURES: The ME's surface epitopes, contrast-producing paramagnetic magnetosomal iron, and genome were studied using immunocytochemistry (ICC), Fe and MRI contrast measurements, and quantitative polymerase chain reaction (qPCR), respectively. These assays, coupled with other common assays, enabled validation of ME cell labeling and dissection of ME subcellular processing. RESULTS: The assays mentioned above provide qualitative and quantitative assessments of cell labeling, the subcellular localization and the fate of MEs. ICC results, with an ME-specific antibody, qualitatively shows homogenous labeling with MEs. The ferrozine assay shows that MEs have an average of 7 fg Fe/ME, ∼30 % of which contributes to MRI contrast and ME-labeled MDA-MB-231 (MDA-231) cells generally have 2.4 pg Fe/cell, implying ∼350 MEs/cell. Adjusting the concentration of Fe in the ME growth media reduces the concentration of non-MRI contrast-producing Fe. Results from the qPCR assay, which quantifies ME genomes in labeled cells, shows that processing of MEs begins within 24 h in MDA-231 cells. ICC results suggest this intracellular digestion of MEs occurs by the lysosomal degradation pathway. MEs coated with listeriolysin O (LLO) are able to escape the primary phagosome, but subsequently co-localize with LC3, an autophagy-associated molecule, and are processed for digestion. In embryos, where autophagy is transiently suppressed, MEs show an increased capacity for survival and even replication. Finally, transmission electron microscopy (TEM) of ME-labeled MDA-231 cells confirms that the magnetosomes (the MRI contrast-producing particles) remain intact and enable in vivo cell tracking. CONCLUSIONS: MEs are used to label mammalian cells for the purpose of cell tracking in vivo, with MRI. Various assays described herein (ICC, ferrozine, and qPCR) allow qualitative and quantitative assessments of labeling efficiency and provide a detailed understanding of subcellular processing of MEs. In some cell types, MEs are digested, but the MRI-producing particles remain. Coating with LLO allows MEs to escape the primary phagosome, enhances retention slightly, and confirms that MEs are ultimately processed by autophagy. Numerous intracellular bacteria and all endosymbiotically derived organelles have evolved molecular mechanisms to avoid intracellular clearance, and identification of the specific processes involved in ME clearance provides a framework on which to develop MEs with enhanced retention in mammalian cells.

Simultaneous Extraction of Viral and Bacterial Nucleic Acids for Molecular Diagnostic Applications.

Molecular detection of microbial pathogens in clinical samples requires the application of efficient sample lysis protocols and subsequent extraction and isolation of their nucleic acids. Here, we describe a simple and time-efficient method for simultaneous extraction of genomic DNA from gram-positive and -negative bacteria, as well as RNA from viral agents present in a sample. This method compared well with existing bacterial- and viral-specialized extraction protocols, worked reliably on clinical samples, and was not pathogen specific. This method may be used to extract DNA and RNA concurrently from viral and bacterial pathogens present in a sample and effectively detect coinfections in routine clinical diagnostics.

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.

Vasopressin decreases pulmonary-to-systemic vascular resistance ratio in a porcine model of severe hemorrhagic shock.

Vasopressors are gaining renewed interest as treatment adjuncts in hemorrhagic shock. The ideal vasoconstrictor will increase systemic blood pressure without increasing pulmonary vascular resistance (PVR), which hinders pulmonary perfusion and exacerbates hypoxemia. However, the selectivity of pressors for pulmonary versus systemic vasoconstriction during hemorrhage has not been characterized. The purpose of this study was to test the hypothesis that vasopressin (VP) has distinct effects on pulmonary versus systemic hemodynamics, unlike the catecholamine vasopressors norepinephrine (NE) and phenylephrine (PE). Anesthetized and ventilated pigs were assigned to resuscitation with saline only (n = 7) or saline with VP (n = 6), NE (n = 6), or PE (n = 6). Animals were hemorrhaged to a target volume of 30 mL/kg and a mean arterial pressure of 35 mmHg. One hour after the start of hemorrhage, animals were resuscitated with saline up to one shed blood volume, followed by either additional saline or a vasopressor. Hemodynamics and oxygenation were measured hourly for 4 h after the start of hemorrhage. Vasopressin increased systemic vascular resistance (SVR) while sparing the pulmonary vasculature, leading to a 45% decrease in the PVR/SVR ratio compared with treatment with PE. Conversely, NE induced pulmonary hypertension and led to an increased PVR/SVR ratio associated with decreased oxygen saturation. Phenylephrine and crystalloid had no significant effect on the PVR/SVR ratio. Sparing of pulmonary vasoconstriction occurs only with VP, not with administration of crystalloid or catecholamine pressors. The ability of VP to maintain blood oxygenation indicates that VP may prevent hypoxemia in the management of hemorrhagic shock.

An experimental comparison of the effects of bacterial colonization on biologic and synthetic meshes.

PURPOSE: Biologic meshes are being used with increasing frequency to repair contaminated abdominal wall defects despite high long-term recurrence and infection rates associated with their use. Recent clinical reports describing the success of lightweight, macroporous synthetic meshes in contaminated ventral hernia repairs have led some surgeons to challenge the belief that synthetics are contraindicated in contaminated fields. We aimed to determine whether a frequently used biologic mesh (Strattice(TM)) is more resistant to bacterial colonization than macroporous synthetic mesh (Parietex(TM) Progrip(TM)) after inoculation with two common pathogens. METHODS: Rats (n = 48) were implanted subcutaneously with Strattice(TM) or Progrip(TM). Meshes were inoculated with sterile saline or a suspension containing 10(6) colony-forming units of Staphylococcus aureus or Escherichia coli prior to wound closure (n = 8 per subgroup). Meshes were explanted at 4 weeks and underwent microbiologic and histologic analyses. RESULTS: Progrip(TM) demonstrated superior bacterial clearance compared to Strattice(TM) (E. coli, 88 vs. 17% clearance, p = 0.03; S. aureus, 75 vs. 50%, p = 0.61; combined bacterial strains, 81 vs. 36%, p = 0.02; respectively). In the Strattice(TM) group, severely degraded meshes were observed in 100% of animals inoculated with E. coli (but 0% inoculated with S. aureus). In contrast, all Progrip(TM) meshes remained intact regardless of inoculum. Scores for neovascularization were higher in the synthetic group irrespective of contamination (p < 0.05). CONCLUSIONS: Biologic meshes may not be more resistant to bacterial colonization than reduced-weight synthetics, and their resistance may differ in response to different pathogens. The routine use of biologics in contaminated ventral hernia repair should be questioned, particularly in the presence of E. coli.

An analysis of Staphylococcus aureus infections at a military medical center using the PLEX-ID combined polymerase chain reaction-mass spectrometry system.

Staphylococcus aureus (S. aureus) is a major cause of morbidity in the military health care setting. Culture-based methods are the most common means of identifying infections caused by this agent. However, culture-based methods lack sensitivity and specificity. The Abbott PLEX-ID instrument uses a combination of the polymerase chain reaction and mass spectrometry for the identification of bacterial isolates. We investigated whether the Abbott PLEX-ID system could identify S. aureus in clinical material and facilitate the epidemiological analysis of individual isolates. The PLEX-ID system positively identified 100% of isolates previously found to be methicillin resistant S. aureus by culture. In addition, analysis using the PLEX-ID software revealed that the majority of S. aureus isolates at Tripler Army Medical Center derive from clonal complex 8 and nearly 100% of these strains express the R-variant of the Panton-Valentine leukocidin virulence factor. These results demonstrate the utility of the PLEX-ID system in identifying clinical isolates and reveal an unexpected level of homogeneity among clinical S. aureus isolates recovered at Tripler Army Medical Center. These results also demonstrate the utility of the PLEX-ID system in identifying the resistance patterns, predicting the virulence properties, and tracking the migration of bacterial pathogens in the clinical setting.