Characterization of Antimicrobial Susceptibility of Bacterial Biofilms on Biological Tissues

abstract: Prosthetic joint infection (PJI) is a devastating complication associated with total joint arthroplasty that results in high cost and patient morbidity. There are approximately 50,000 PJIs per year in the US, imposing a burden of about $5 billion on the healthcare system. PJI is especially difficult to treat because of the presence of bacteria in biofilm, often highly tolerant to antimicrobials. Treatment of PJI requires surgical debridement of infected tissues, and local, sustained delivery of antimicrobials at high concentrations to eradicate residual biofilm bacteria. However, the antimicrobial concentrations required to eradicate biofilm bacteria grown in vivo or on tissue surfaces have not been measured. In this study, an experimental rabbit femur infection model was established by introducing a variety of pathogens representative of those found in PJIs [Staphylococcus Aureus (ATCC 49230, ATCC BAA-1556, ATCC BAA-1680), Staphylococcus Epidermidis (ATCC 35984, ATCC 12228), Enterococcus Faecalis (ATCC 29212), Pseudomonas Aeruginosa (ATCC 27853), Escherichia Coli (ATCC 25922)]. Biofilms of the same pathogens were grown in vitro on biologic surfaces (bone and muscle). The ex vivo and in vitro tissue minimum biofilm eradication concentration (MBEC; the level required to eradicate biofilm bacteria) and minimum inhibitory concentration (MIC; the level required to inhibit planktonic, non-biofilm bacteria) were measured using microbiological susceptibility assays against tobramycin (TOB) and vancomycin (VANC) alone or in 1:1 weight combination of both (TOB+VANC) over three exposure durations (6 hour, 24 hour, 72 hour). MBECs for all treatment combinations (pathogen, antimicrobial used, exposure time, and tissue) were compared against the corresponding MIC values to compare the relative susceptibility increase due to biofilm formation. Our data showed median in vitro MBEC to be 100-1000 times greater than the median MIC demonstrating the administration of local antimicrobial doses at MIC level would not kill the persisting bacteria in biofilm. Also, administering dual agent (TOB+VANC) showed median MBEC values to be comparable or lower than the single agents (TOB or VANC)Dissertation/ThesisMasters Thesis Bioengineering 201

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<p>Hypophosphatasia (HPP) is a rare heritable metabolic bone disease caused by hypomorphic mutations in the ALPL (in human) or Akp2 (in mouse) gene, encoding the tissue-nonspecific alkaline phosphatase (TNAP) enzyme. In addition to skeletal and dental malformations, severe forms of HPP are also characterized by the presence of spontaneous seizures. Initially, these seizures were attributed to an impairment of GABAergic neurotransmission caused by altered vitamin B6 metabolism. However, recent work by our group using knockout mice null for TNAP (TNAP-/-), a well-described model of infantile HPP, has revealed a deregulation of purinergic signaling contributing to the seizure phenotype. In the present study, we report that adult heterozygous (TNAP+/-) transgenic mice with decreased TNAP activity in the brain are more susceptible to adenosine 5′-triphosphate (ATP)-induced seizures. Interestingly, when we analyzed the extracellular levels of ATP in the cerebrospinal fluid, we found that TNAP+/- mice present lower levels than control mice. To elucidate the underlying mechanism, we evaluated the expression levels of other ectonucleotidases, as well as different proteins involved in ATP release, such as pannexin, connexins, and vesicular nucleotide transporter. Among these, Pannexin-1 (Panx1) was the only one showing diminished levels in the brains of TNAP+/- mice. Altogether, these findings suggest that a physiological regulation of extracellular ATP levels and Panx1 changes may compensate for the reduced TNAP activity in this model of HPP.</p

14-3-3ζtg mice are protected against ER stress-induced neuronal death <i>in vivo</i>.

<p>(A) Representative FJB staining of wt and 14-3-3ζtg mouse hippocampus 48 h after i.c.v. injection of tunicamycin (1 µl, 50 µM). Scale bar, 150 µm. Dotted lines depict upper and lower blades of the granule cell layer. str. gr., stratum granulosum. (B) Counts of FJB-positive cells 48 h after tunicamycin in wt and 14-3-3ζtg mice (<i>n</i> = 5 per group; **<i>p</i><0.01 compared to wt). (C, D) Representative photomicrographs showing TUNEL staining in wt and 14-3-3ζtg mice 48 h after tunicamycin injection and graph quantifying the difference (<i>n</i> = 5 per group; ***<i>p</i><0.001 compared to wt. (E, F) Representative western blots (<i>n</i> = 1 per lane) and semi-quantification of UPR and ER-associated protein levels between wt and 14-3-3ζtg mice (<i>n</i> = 3 per group; *<i>p</i><0.05 compared to wt).</p

Reduced levels of ER and UPR-related proteins in 14-3-3ζtg mice.

<p>(A) Representative KDEL (green) immunostaining for wt and 14-3-3ζtg mice hippocampus. Neurons are identified by NeuN counterstaining (red). Merged panels confirm staining is neuronal. Note, lower KDEL immunoreactivity in 14-3-3ζtg mice. Scale bar (top), 700 µm; (bottom), 150 µm. (B) Representative western blots (<i>n</i> = 1 per lane) showing KDEL-containing proteins in microdissected subfields of wt and 14-3-3ζtg mice. (C, D) Basal Grp78 and Grp94 protein levels in wt and 14-3-3ζtg mice (<i>n</i> = 3 per group). (E) Real-time PCR measurement of <i>Grp78</i> levels in wt and 14-3-3ζtg mice in each subfield (<i>n</i> = 3 per group). (F) Western blots (<i>n</i> = 1 per lane) showing levels of various ER-related proteins in the CA3 subfield of 14-3-3ζtg mice compared to wt mice. (G) Graphs showing lower basal levels of p-eIF2α, ATF6 and ATF4 in select hippocampal subfields (<i>n</i> = 3 per group). (H) Gel showing levels of the spiced form of <i>Xbp1</i> from hippocampal lysates of; (lane 2) WT mice subject to seizures levels, (lane 3) Wt mice, (lane 4) 14-3-3ζtg mice. Lane 1 is a ladder. *<i>p</i><0.05 compared to wt.</p

Distribution of the myc-tagged 14-3-3ζ transgene in mouse brain.

<p>(A) Western blot analysis (<i>n</i> = 1 per lane) of microdissected brain regions from wild-type (wt) and 14-3-3ζtg mice immunoblotted with antibodies against the myc tag confirm expression of the transgene in CA subfields, dentate gyrus (DG), cortex (Cx), cerebellum (Cb), striatum (Str) and brain stem (BS). (B) 14-3-3ζ immunostaining in hippocampal tissue sections showing higher immunoreactivity (IR) in 14-3-3ζtg compared to wt mice. Panels to the far right show sections stained for 14-3-3γ revealing normal neuronal distribution and level between wt and 14-3-3ζtg mice. (C) Representative fluorescence immunostaining of the CA1 subfield showing (top) myc (red) with NeuN (green) co-localization and (bottom) myc (green) with GFAP (red) confirming mainly neuronal expression of the transgene. (D) Immunoblots of SJL mouse hippocampal fractions (<i>n</i> = 1 per lane) for the select presence of markers of the nucleus (Nuc), mitochondria (Mito), cytoplasm (Cyto) and microsome-containing ER fraction (Micro). (E) Western blots from pools of 14-3-3ζtg mouse hippocampi (<i>n</i> = 1 per lane) show the presence of various isoforms in different compartments. Note, endogenous 14-3-3ζ and myc-14-3-3ζ are similarly distributed in each fraction. (F) Protein levels of various 14-3-3 isoforms in wt and 14-3-3ζtg mice hippocampal subfields (<i>n</i> = 1 per lane). (G) Real-time PCR measurement of ζ, γ and ε 14-3-3 isoform levels in wt and 14-3-3ζtg mice (<i>n</i> = 3 per group). *<i>p</i><0.05 compared to wt. Scale bars in B, C, 160 µm.</p

Hippocampal morphology and levels of apoptosis- and autophagy-related proteins in 14-3-3ζtg mice.

<p>(A) Nissl-stained sections of wt and 14-3-3ζtg mice. Scale bar, (top) 600 µm; (bottom), 330 µm. (B) Immunohistochemistry showing similar pattern of ZnT3 staining, a protein present in mossy fibers, in wt and 14-3-3ζtg mice. Scale bar, 500 µm. (C) Body weight in wt and 14-3-3ζtg mice at 6 weeks of age (<i>n</i> = 8 per group). (D) Brain weight in wt and 14-3-3ζtg mice at 6 weeks of age (<i>n</i> = 6 per group). *<i>p</i><0.05 compared to wt. (E) NeuN counts in hippocampal subfields from sections of dorsal hippocampus from wt and 14-3-3ζtg mice (<i>n</i> = 6 per group). (F-J) Representative western blots (<i>n</i> = 1 per lane) showing similar levels of (F) NeuN, (G) the astrocyte marker GFAP, (H) microglia marker Iba1, (I) apoptosis-associated 14-3-3 binding proteins and (J) autophagy-related 14-3-3 binding proteins, in adult wt and 14-3-3ζtg mice.</p

Baseline and seizure EEG in 14-3-3ζtg mice.

<p>(A). Protein levels of the kainic acid receptors GluR6/7 and KA2 in microdissected subfields of hippocampus from wt and 14-3-3ζtg mice. (B) Analysis of baseline EEG parameters during 40 min recordings from skull of wt and 14-3-3ζtg mice. No differences were detected between genotypes (<i>n</i> = 6 per group). (C) Representative EEG spectral activity plot of baseline EEG in wt and 14-3-3ζtg mice. (D, E) Representative spectral activity plot of EEG frequency and amplitude, and quantitative analysis of seizure duration (high amplitude and high frequency discharges) for wt and 14-3-3ζtg mice during the 40 min after intra-amygdala microinjection of kainic acid. No differences were detected between genotypes (<i>n</i> = 6-7 per group).</p

14-3-3ζtg mice are protected against seizure-induced neuronal death <i>in vivo</i> and <i>in vitro</i>.

<p>(A) Representative FJB and TUNEL staining for wt and 14-3-3ζtg mice 72 h after status epilepticus for the CA1, CA3 and hilar regions. Scale bar, 120 µm. (B) Semi-quantification of seizure damage and neuron survival (NeuN counts) for wt and 14-3-3ζtg mice (<i>n</i> = 6-10 per group). (C) Primary cultures of hippocampal neurons from wt and 14-3-3ζtg mice were treated with kainic acid and then cell death determined as percentage propidium iodide (PI) positive. (Panels above) Representative photomicrographs of PI-stained neurons 24 h after KA treatment. Scale bar, 25 µm. Graph shows reduced cell death in 14-3-3ζtg mice (<i>n</i> = 3 per group). *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001 compared to wt.</p

Top 12 most abundant miRNA in males and females.

<p>Top 12 most abundant miRNA in males and females.</p

Bar plot showing the number of miRNA detected in samples.

<p>(a) before and (b) after filtering to remove any miRNA not present in 80% of samples.</p