Catheter-Based Medical Device Biofilm Ablation Using Histotripsy: A Parameter Study.

OBJECTIVE: Biofilm formation in medical catheters is a major source of hospital-acquired infections which can produce increased morbidity and mortality for patients. Histotripsy is a non-invasive, non-thermal focused ultrasound therapy and recently has been found to be effective at removal of biofilm from medical catheters. Previously established histotripsy methods for biofilm removal, however, would require several hours of use to effectively treat a full-length medical catheter. Here, we investigate the potential to increase the speed and efficiency with which biofilms can be ablated from catheters using histotripsy. METHODS: Pseudomonas aeruginosa (PA14) biofilms were cultured in in vitro Tygon catheter mimics and treated with histotripsy using a 1 MHz histotripsy transducer and a variety of histotripsy pulsing rates and scanning methods. The improved parameters identified in these studies were then used to explore the bactericidal effect of histotripsy on planktonic PA14 suspended in a catheter mimic. RESULTS: Histotripsy can be used to remove biofilm and kill bacteria at substantially increased speeds compared with previously established methods. Near-complete biofilm removal was achieved at treatment speeds up to 1 cm/s, while a 4.241 log reduction in planktonic bacteria was achieved with 2.4 cm/min treatment. CONCLUSION: These results represent a 500-fold increase in biofilm removal speeds and a 6.2-fold increase in bacterial killing speeds compared with previously published methods. These findings indicate that histotripsy shows promise for the treatment of catheter-associated biofilms and planktonic bacteria in a clinically relevant time frame.

Complete Genome Sequence of Providencia stuartii CMC-4104, Isolated from a Human Splenic Abscess, Containing Multiple Copies of NDM-1 and PER-1 Carbapenem Resistance Genes.

We report the complete genome sequence of a clinical isolate of Providencia stuartii strain CMC-4104, isolated from a splenic abscess. Oxford Nanopore Technologies (ONT) and Illumina sequencing reads were assembled using Geneious to generate a 4,504,925-bp circular chromosome containing multiple copies of the NDM-1 and PER-1 genes in a genomic resistance island.

Particle-Mediated Histotripsy for the Targeted Treatment of Intraluminal Biofilms in Catheter-Based Medical Devices.

Objective. This paper is an initial work towards developing particle-mediated histotripsy (PMH) as a novel method of treating catheter-based medical device (CBMD) intraluminal biofilms. Impact Statement. CBMDs commonly become infected with bacterial biofilms leading to medical device failure, infection, and adverse patient outcomes. Introduction. Histotripsy is a noninvasive focused ultrasound ablation method that was recently proposed as a novel method to remove intraluminal biofilms. Here, we explore the potential of combining histotripsy with acoustically active particles to develop a PMH approach that can noninvasively remove biofilms without the need for high acoustic pressures or real-time image guidance for targeting. Methods. Histotripsy cavitation thresholds in catheters containing either gas-filled microbubbles (MBs) or fluid-filled nanocones (NCs) were determined. The ability of these particles to sustain cavitation over multiple ultrasound pulses was tested after a series of histotripsy exposures. Next, the ability of PMH to generate selective intraluminal cavitation without generating extraluminal cavitation was tested. Finally, the biofilm ablation and bactericidal capabilities of PMH were tested using both MBs and NCs. Results. PMH significantly reduced the histotripsy cavitation threshold, allowing for selective luminal cavitation for both MBs and NCs. Results further showed PMH successfully removed intraluminal biofilms in Tygon catheters. Finally, results from bactericidal experiments showed minimal reduction in bacteria viability. Conclusion. The results of this study demonstrate the potential for PMH to provide a new modality for removing bacterial biofilms from CBMDs and suggest that additional work is warranted to develop histotripsy and PMH for treatment of CBMD intraluminal biofilms.

Complete Genome Sequence of Pseudomonas aeruginosa CMC-097, Isolated from a Ventilator-Associated Pneumonia Patient, Containing a Novel Carbapenem Resistance Class 1 Integron.

We report the complete genome of a clinical strain of Pseudomonas aeruginosa CMC-097, which was isolated from a ventilator-associated pneumonia patient with a chronic infection. Illumina sequence reads were assembled using Geneious to yield a 7,044,064-bp circular chromosome containing a carbapenem resistance integron, In2020.

Focused Ultrasound Biofilm Ablation: Investigation of Histotripsy for the Treatment of Catheter-Associated Urinary Tract Infections (CAUTIs).

Urinary catheters often become contaminated with biofilms, resulting in catheter-associated urinary tract infections (CAUTIs) that adversely affect patient outcomes. Histotripsy is a noninvasive focused ultrasound therapy previously developed for the noninvasive ablation of cancerous tumors and soft tissues. Histotripsy has also previously shown the ability to treat biofilms on glass slides and surgical meshes. Here, we investigate the potential of histotripsy for the treatment of CAUTIs for the first time in vitro. Clinically relevant catheter materials (Tygon, Silicone, and latex catheter mimics) and commonly used clinical catheters were tested to determine the feasibility of producing luminal histotripsy bubble clouds. A Pseudomonas aeruginosa (strain PA14) biofilm model was developed and tested to produce luminal biofilms in an in vitro Tygon catheter mimic. This model was treated with histotripsy to determine the ability to remove a luminal biofilm. Finally, the bactericidal effects of histotripsy were tested by treating PA14 suspended inside the Tygon catheter mimic. Results showed that histotripsy produced precise luminal cavitation within all tested catheter mimics and clinical catheters. Histotripsy treatment of a PA14 biofilm with histotripsy reduced luminal biofilm OD590 signal down to background levels. Further, the treatment of suspended PA14 in Luria-Bertani (LB) showed a 3.45 ± 0.11 log10 reduction in CFU/mL after six histotripsy scans across the catheter mimics. Overall, the results of this study demonstrate the potential of histotripsy to provide a new modality for removing bacterial biofilms from catheter-based medical devices and suggest that additional work is warranted to investigate histotripsy for the treatment of CAUTIs and other biomaterial-associated infections.

Complete Genome Sequence of Pseudomonas aeruginosa CMC-115, a Clinical Strain from an Acute Ventilator-Associated Pneumonia Patient.

We report the complete genome of clinical strain Pseudomonas aeruginosa CMC-115, which was isolated from an acute ventilator-associated pneumonia patient. Illumina sequencing reads were assembled using Geneious to yield a 6,375,262-bp circular chromosome that exhibited an unusual ferrichrome receptor in the pyoverdine synthesis locus and the absence of type 3 secretion system genes.

Tn2008-driven carbapenem resistance in Acinetobacter baumannii isolates from a period of increased incidence of infections in a Southwest Virginia hospital (USA).

OBJECTIVES: The objectives of this study were (i) to determine the genetic basis for carbapenem resistance in multidrug-resistant (MDR) Acinetobacter baumannii strains isolated from patients affected by a sudden increase in the incidence of infections by such organisms in a tertiary care hospital in Virginia, USA, in 2009-2010 and (ii) to examine whether such strains are commonly encountered in the hospital setting. METHODS: The whole genomes of one outbreak strain as well as one carbapenem-resistant and one carbapenem-sensitive strain from sporadic infections in 2010-2012 were sequenced and analysed. Then, 5 outbreak isolates and 57 sporadic isolates (of which 39 were carbapenem-resistant) were screened by PCR for relevant DNA elements identified in the genomics investigation. RESULTS: All three strains for which whole-genome sequences were obtained carried resistance genes linked to MDR phenotypes and a ca. 111-kbp plasmid (pCMCVTAb1) without drug resistance genes. Of these, the two carbapenem-resistant strains possessed a ca. 74-kbp plasmid (pCMCVTAb2) carrying a Tn2008 transposon that provides high-level carbapenem resistance. PCR analysis showed that all of the outbreak isolates carried both plasmids and Tn2008, and of the sporadic isolates 88% carried pCMCVTAb1, 25% contained pCMCVTAb2 and 50% of the latter group carried Tn2008. CONCLUSIONS: Carbapenem resistance in outbreak strains and 12% of sporadic isolates was due to the pCMCVTAb2-borne Tn2008. This is the first report of a Tn2008-driven outbreak of carbapenem-resistant A. baumannii infections in the Commonwealth of Virginia, which followed similar cases in Pennsylvania and Ohio.

A sequence-based approach for prediction of CsrA/RsmA targets in bacteria with experimental validation in Pseudomonas aeruginosa.

CsrA/RsmA homologs are an extensive family of ribonucleic acid (RNA)-binding proteins that function as global post-transcriptional regulators controlling important cellular processes such as secondary metabolism, motility, biofilm formation and the production and secretion of virulence factors in diverse bacterial species. While direct messenger RNA binding by CsrA/RsmA has been studied in detail for some genes, it is anticipated that there are numerous additional, as yet undiscovered, direct targets that mediate its global regulation. To assist in the discovery of these targets, we propose a sequence-based approach to predict genes directly regulated by these regulators. In this work, we develop a computer code (CSRA_TARGET) implementing this approach, which leads to predictions for several novel targets in Escherichia coli and Pseudomonas aeruginosa. The predicted targets in other bacteria, specifically Salmonella enterica serovar Typhimurium, Pectobacterium carotovorum and Legionella pneumophila, also include global regulators that control virulence in these pathogens, unraveling intricate indirect regulatory roles for CsrA/RsmA. We have experimentally validated four predicted RsmA targets in P. aeruginosa. The sequence-based approach developed in this work can thus lead to several testable predictions for direct targets of CsrA homologs, thereby complementing and accelerating efforts to unravel global regulation by this important family of proteins.

Comparisons of Two Proteomic Analyses of Non-Mucoid and Mucoid Pseudomonas aeruginosa Clinical Isolates from a Cystic Fibrosis Patient.

Pseudomonas aeruginosa chronically infects the lungs of cystic fibrosis (CF) patients. The conditions in the CF lung appear to select for P. aeruginosa with advantageous phenotypes for chronic infection. However, the mechanisms that allow the establishment of this chronic infection have not been fully characterized. We have previously reported the transcriptional analysis of two CF isolates strains 383 and 2192. Strain 2192 is a mucoid, alginate overproducing strain whereas strain 383 is non-mucoid. Mucoid strains are associated with chronic infection of the CF lung and non-mucoid strains are the typical initially infecting isolates. To elucidate novel differences between these two strains, we employed two methods of shotgun proteomics: isobaric tags for relative and absolute quantitation (iTRAQ) and two-dimensional gel electrophoresis (2-DE). iTRAQ compares the amount of protein between samples and relies on protein abundance, while 2-DE gel electrophoresis depends on selection of separated protein spots. For both these methods, mass spectrometry was then used to identify proteins differentially expressed between the two strains. The compilation of these two proteomic methods along with Western blot analysis revealed proteins of the HSI-I operon of the type 6 secretion system, showed increased expression in 383 compared to 2192, confirming the our previous transcriptional analysis. Proteomic analysis of other proteins did not fully correlate with the transcriptome but other differentially expressed proteins are discussed. Also, differences were noted between the results obtained for the two proteomic techniques. These shotgun proteomic analyses identified proteins that had been predicted only through gene identification; we now refer to these as "proteins of unknown functions" since their existence has now been established however their functional characterization remains to be elucidated.

RahU: an inducible and functionally pleiotropic protein in Pseudomonas aeruginosa modulates innate immunity and inflammation in host cells.

The aim of this study was to define the functional role of a recently identified RahU protein from Pseudomonas aeruginosa in macrophages and its role in bacterial defense. Recombinant (r)-RahU had no significant effect on cell apoptosis or cell viability in human monocytic THP-1 cells. Gene expression array of murine macrophage cells (RAW 264.7) stimulated with LPS showed modulation of common transcripts (by r-RahU and predisone) involved in inflammation. Functional cellular analysis showed RAW cells incubated with r-RahU at 1.0-10 µg/ml (0.06-0.6 µM) inhibited accumulation of nitric oxide (NO) in the presence of LPS by 10-50%. The IC(50) of r-RahU (0.6 µM) was distinct from the known inhibitors of NO production: prednisone (50 µM) and L-NMMA (100 µM). r-RahU also significantly inhibited chemotactic activity of THP-1 cells toward CCL2 or chemotactic supernatants from apoptotic T-cells. These reports show previously unknown pleiotropic properties of RahU in modulating both microbial physiology and host innate immunity.

Host derived inflammatory phospholipids regulate rahU (PA0122) gene, protein, and biofilm formation in Pseudomonas aeruginosa.

This study describes the role of "inflammatory" oxidized (Ox) phospholipids in regulation of rahU (PA0122) expression and biofilm formation in Pseudomonas aeruginosa (383) wild type (rahU(+)) and rahU mutant (rahU(-)) strains. Functional analysis of RahU protein from P. aeruginosa in presence of Ox-phospholipids show: (a) LysoPC modulates RahU gene/and protein expression in rahU(+) cells; (b) rahU promoter activity is increased by lysoPC and inhibited by PAPC, Ox-PAPC and arachidonic acid; the latter inhibitory effect can be reversed by lysoPC, which was enzymatically derived from PAPC; (c) biofilm formation increased in rahU(-) cells as compared to rahU(+); and (d) inhibition of rahU promoter activity by PAPC and AA (but not lysoPC) showed significantly augmented biofilm formation in rahU(+) but not in rahU(-) cells. This study shows that host derived Ox-phospholipids affect P. aeruginosa-rahU gene and protein expression, which in turn modulates biofilm formation. The accompanying paper describes the role of RahU protein in eukaryotic-host cells.

A novel oxidized low-density lipoprotein-binding protein from Pseudomonas aeruginosa.

A novel protein, PA0122, has been identified in Pseudomonas aeruginosa and shown to bind to oxidized low-density lipoprotein (Ox-LDL). The PA0122 gene was recognized based on gene expression pattern differences between two strains of P. aeruginosa isolated from the sputum of an individual with cystic fibrosis (CF). There was an approximately eightfold increase in PA0122 expression in the non-mucoid strain 383, compared to that in the mucoid strain 2192. Quantitative real-time RT-PCR (qRT-PCR) supported PA0122 transcript expression differences between strains 383 and 2192 and revealed growth-phase dependence, with the highest level of expression at early stationary phase (OD(600) 1.5). PA0122 encodes a 136 aa 'conserved hypothetical' protein that has similarity to Aspergillus fumigatus Asp-haemolysin, which is an Ox-LDL-binding protein, and possessed a motif that is homologous to the fungal aegerolysin family of proteins. Antibodies produced to purified recombinant PA0122 recognized a 16 kDa protein band in cell lysates as well as in the supernatant fractions of strain 383. The PA0122 protein expression pattern was growth phase-dependent, with maximal production observed at OD(600) 1.5 that was consistent with the PA0122 transcript expression profile. Subcellular fractionation studies revealed differences in the localization of PA0122 between strains 383 and 2192. In 383, PA0122 was observed in the cytoplasm and in membrane fractions. In 2192, PA0122 was found in the cytoplasm but was not detected in membrane fractions. Surface plasmon resonance revealed that recombinant PA0122 binds with high affinity to Ox-LDL and to its major subcomponent, lysophosphatidylcholine, but not to non-oxidized LDL.

Novel phosphorylcholine-containing protein of Pseudomonas aeruginosa chronic infection isolates interacts with airway epithelial cells.

Pseudomonas aeruginosa undergoes phase variation in the expression of the phosphorylcholine (ChoP) epitope, a structure crucial for the virulence of several respiratory pathogens. In this study, ChoP expression analysis comparing organisms from acute and chronic infections revealed that expression of ChoP at 37 degrees C was higher among strains from chronic infections. Coimmunoprecipitation experiments and mass spectrometry analysis demonstrated that ChoP was on the protein elongation factor Tu. The presence of ChoP at the surface was confirmed by immunofluorescence and flow cytometry analysis of intact bacteria. Pretreatment of bronchial epithelial cells or mice with a platelet-activating factor receptor (PAFR) antagonist reduced adhesion and invasion of the ChoP-positive P. aeruginosa isolates. Results of this study suggest that ChoP expression may represent a novel phenotype expressed by the chronic infection isolates that could mediate P. aeruginosa colonization of the epithelial airway by means of the interaction with the PAFR.

Intranasal immunization with heterologously expressed polysaccharide protects against multiple Pseudomonas aeruginosa infections.

Surface-expressed bacterial polysaccharides are often immunodominant, protective antigens. However, these antigens are chemically and serologically highly heterogeneous, and conjugation to protein carriers is often necessary to enhance their immunogenicity. Here we show the efficacy of intranasal immunization of mice with attenuated Salmonella enterica serovar Typhimurium expressing the O antigen portion of Pseudomonas aeruginosa lipopolysaccharide. P. aeruginosa is an ideal model system because it can cause a myriad of localized and systemic infections. In particular, this bacterium is a leading cause of hospital-acquired pneumonia and is responsible for infections after burns and after eye injury. In addition, there are mouse models of infection that mimic the clinical manifestations of P. aeruginosa infections. Immunized mice were highly protected against infection, with long-lasting immunity to acute P. aeruginosa pneumonia, whereas mice immunized with Salmonella containing only the cloning vector or PBS were not. Prophylactic and therapeutic administration of sera from vaccinated animals protected naive mice. Intranasal vaccination also provided complete protection from infections after burns and reduced pathology after corneal abrasions. These results indicate that intranasal delivery of heterologously expressed polysaccharide antigens provides protection at distinct sites of infection. This approach for the expression and delivery of polysaccharide antigens as recombinant immunogens could be easily adapted to develop vaccines for many infectious agents, without the need for complicated purification and conjugation procedures.

Purification and characterization of an esterase isozyme involved in hydrolysis of organophosphorus compounds from an insecticide resistant pest, Helicoverpa armigera (Lepidoptera: Noctüidae).

An esterase isozyme was purified from the insecticide resistant pest, Helicoverpa armigera collected from field crops. Purification involved ammonium sulfate precipitation, hydrophobic interaction and ion exchange chromatography followed by gel filtration chromatography. The purification was 212-fold with 1% yield of the enzyme. The optimum pH of the isozyme was found to be 10.5 and 8.5 for p-nitrophenyl phosphate and paraoxon, respectively. The enzyme was unstable at temperature >50 degrees C. The molecular mass determined by SDS-PAGE was 66 kDa. Cations such as Hg(+2), Ag(+2), Cd(+2) inhibited the activity while Zn(+2) stimulated it. Kinetic studies indicated that the enzyme had low K(m) values of 0.238 and 0.348 mM for p-nitrophenyl phosphate and paraoxon, respectively. The enzyme had broad substrate specificity with high K(m) values for ATP, ADP and beta-glycerophosphate. This enzyme was partially sequenced and identified as an alkaline phosphatase.

Oral vaccination of BALB/c mice with Salmonella enterica serovar Typhimurium expressing Pseudomonas aeruginosa O antigen promotes increased survival in an acute fatal pneumonia model.

Pseudomonas aeruginosa is a leading cause of nosocomial pneumonia. We compared the efficacies of oral and intraperitoneal (i.p.) vaccinations of BALB/c mice with attenuated Salmonella enterica serovar Typhimurium SL3261 expressing P. aeruginosa serogroup O11 O antigen to protect against P. aeruginosa infection in an acute fatal pneumonia model. Oral and i.p. vaccines elicited O11-specific serum immunoglobulin G (IgG) antibodies, but IgA was observed only after oral immunization. Challenge of orally vaccinated mice with an O11 strain (9882-80) at 6 and 12 times the 50% lethal dose showed increased survival in mice that received the vaccine compared to phosphate-buffered saline (PBS)- and vector-treated controls; no difference in survival was seen with a heterologous strain, 6294 (serogroup O6). In addition, significant protection against 9882-80 was not observed in i.p. vaccinated animals. Bronchoalveolar lavage fluid taken from immunized mice harbored O11-specific IgA and IgG in orally immunized mice but only modest levels of IgG in i.p. vaccinated mice. To correlate protection, opsonophagocytosis assays were performed with pooled sera from orally immunized animals. Efficient killing of five O11 clinical isolates was observed, while no killing was noted with 6294, indicating that the recombinant SL3261 oral vaccine induces an O11-specific reaction. We next determined the ability of orally vaccinated animals to clear bacteria from their lungs. Following P. aeruginosa challenge, the numbers of viable bacteria were significantly fewer in orally vaccinated animals than in PBS- and vector-treated controls. Our results suggest that oral immunization with recombinant SL3261 is efficacious in protection against pneumonia caused by P. aeruginosa.

Cloning and characterization of a novel sperm-associated isoantigen (E-3) with defensin- and lectin-like motifs expressed in rat epididymis.

In the present study we report the identification of a novel epididymis-specific secretory glycoprotein, E-3, which is a sperm-associated isoantigen containing defensin- and lectin-like motifs. E-3 was detected in rat epididymal fluid and in sperm extracts by two-dimensional (2-D) Western blotting using rat hyperimmune sera raised against rat sperm. The immunoreactive spot of approximately 28 kDa with an isoelectric point (pI) of 3.5 was cored from silver-stained gels. Microsequencing by tandem mass spectrometry and database searches revealed several peptides to be novel sequences. Degenerate deoxyinosine-containing primers corresponding to the novel peptides were used in rapid amplification of cDNA ends and polymerase chain reaction to clone E-3 from a rat epididymal cDNA library. A 449-base pair nucleotide sequence was subsequently obtained consisting of a complete open reading frame (ORF) of 111 amino acids, which showed similarity to the defensin and lectin families. The first 21 amino acids constituted a putative signal peptide, suggesting that E-3 is a secretory protein. Mature E-3 protein corresponding to amino acids 22-111 was expressed in E. coli, and chickens were immunized with recombinant E-3 (rE-3). The resulting anti-rE-3 antisera recognized the recombinant immunogen as well as a "native" protein of 28 kDa, pI 2.5-3.5 in both epididymal fluid and in sperm extracts on 2-D Western blots. Northern hybridization indicated that E-3 mRNA was present in the epididymis but not in testis or other tissues, and that E-3 mRNA was predominantly expressed in the corpus and cauda of the epididymis, but not in the initial segment or caput. Similarly, Western blots detected the E-3 protein only in the epididymal fluid and sperm from the corpus and caudal regions. Finally, indirect immunofluorescence localized E-3 on the entire tail, and with less intensity on the head of the sperm. These observations indicate that E-3 is a secreted epididymal protein that becomes associated with the sperm as it transits through the corpus and cauda. The presence of a defensin-like motif suggests that E-3 may play a role in protecting the sperm from microbial infections in the epididymis and in the female reproductive tract.