Publications by authors named "Nancy Hanson"

80 Publications

OmpC regulation differs between ST131 and non-ST131 Escherichia coli clinical isolates and involves differential expression of the small RNA MicC.

J Antimicrob Chemother 2020 05;75(5):1151-1158

Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE, USA.

Background: Virulence genes and the expression of resistance mechanisms undoubtedly play a role in the successful spread of the pandemic clone Escherichia coli ST131. Porin down-regulation is a chromosomal mechanism associated with antibiotic resistance. Translation of porin proteins can be impacted by modifications in mRNA half-life and the interaction among small RNAs (sRNAs), the porin transcript and the sRNA chaperone Hfq. Modifications in the translatability of porin proteins could impact the fitness and therefore the success of E. coli ST131 isolates in the presence of antibiotic.

Objectives: To identify differences in the translatability of OmpC and OmpF porins for different STs of E. coli by comparing steady-state RNA levels, mRNA half-life, regulatory sRNA expression and protein production.

Methods: RNA expression was evaluated using real-time RT-PCR and OmpC mRNA half-life by northern blotting. OmpC, OmpF and Hfq protein levels were evaluated by immunoblotting.

Results: Differences between ST131 and non-ST131 isolates included: (i) the level of OmpC RNA and protein produced with mRNA expression higher for ST131 but OmpC protein levels lower compared with non-ST131 isolates; (ii) OmpC mRNA half-life (21-30 min for ST131 isolates compared with <2-23 min for non-ST131 isolates); and (iii) levels of the sRNA MicC (2- to 120-fold for ST131 isolates compared with -4- to 70-fold for non-ST131 isolates).

Conclusions: Mechanisms involved in the translatability of porin proteins differed among different STs of E. coli. These differences could provide a selective advantage to ST131 E. coli when confronted with an antibiotic-rich environment.
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http://dx.doi.org/10.1093/jac/dkz566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177473PMC
May 2020

A Standard Numbering Scheme for Class C β-Lactamases.

Antimicrob Agents Chemother 2020 02 21;64(3). Epub 2020 Feb 21.

Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA

Unlike for classes A and B, a standardized amino acid numbering scheme has not been proposed for the class C (AmpC) β-lactamases, which complicates communication in the field. Here, we propose a scheme developed through a collaborative approach that considers both sequence and structure, preserves traditional numbering of catalytically important residues (Ser, Lys, Tyr, and Lys), is adaptable to new variants or enzymes yet to be discovered and includes a variation for genetic and epidemiological applications.
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http://dx.doi.org/10.1128/AAC.01841-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038296PMC
February 2020

lptG contributes to changes in membrane permeability and the emergence of multidrug hypersusceptibility in a cystic fibrosis isolate of Pseudomonas aeruginosa.

Microbiologyopen 2019 11 12;8(11):e844. Epub 2019 Apr 12.

Department of Medical Microbiology and Immunology, Creighton University, Omaha, Nebraska.

Purpose: In the lungs of cystic fibrosis patients, Pseudomonas aeruginosa is exposed to a myriad of antibiotics leading to alterations in antibiotic susceptibility. This study identifies mutations resulting in hypersusceptibility in isogenic mutants of a P. aeruginosa clinical isolate, PA34.

Methods: PA34 was exposed to subinhibitory concentrations of doripenem or meropenem during growth to mid-log phase. Antibiotic susceptibility of surviving colonies was determined by agar dilution. Two carbapenem-resistant colonies hypersusceptible to non-carbapenem antibiotics were selected for further analysis. Antibiotic resistance gene expression was evaluated by RT-rtPCR and OprD production by SDS-PAGE. PA34 and isogenic mutants were evaluated with whole genome sequencing. Sequence variants were confirmed by Sanger sequencing, and cognate genes in eight carbapenem-resistant clinical isolates hypersusceptible to non-carbapenem antibiotics were sequenced. Lipopolysaccharide preparations of PA34 and hypersusceptible mutants were evaluated with ProQ-Emerald stain.

Results: Isogenic mutants showed 4- to 8-fold MIC increase for imipenem, meropenem, and doripenem. However, they were hypersusceptible (≥4-fold MIC decrease) to aminoglycosides, fluoroquinolones, and non-carbapenem β-lactams. Expression of ampC or mex-opr efflux pumps was unchanged, but OprD production was decreased. Mutations causing Q86H AlgU and G77C LptG amino acid substitutions and nonsense mutations within OprD were observed in both mutants. Lipopolysaccharide modifications were observed between isogenic mutants and PA34. Non-synonymous mutations in LptF or LptG were observed in 6/8 hypersusceptible clinical isolates resistant to carbapenem antibiotics.

Conclusion: Evaluation of hypersusceptible mutants identified the association between lptG and a hypersusceptible phenotype. Modifications in lipopolysaccharide profiles suggests LptG modification interferes with lipopolysaccharide transport and contributes to hypersusceptibility.
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http://dx.doi.org/10.1002/mbo3.844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854846PMC
November 2019

Draft Genome Assemblies of Clinical Isolates of Klebsiella pneumoniae V9011662 and Enterobacter hormaechei Entb306.

Microbiol Resour Announc 2019 Apr 11;8(15). Epub 2019 Apr 11.

Department of Medical Microbiology and Immunology, Creighton University, Omaha, Nebraska, USA

and are pathogenic that have been associated with the spread of antibiotic resistance. Here, we report draft genome assemblies of an clinical isolate and a multidrug-resistant clinical isolate of .
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http://dx.doi.org/10.1128/MRA.00091-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6460023PMC
April 2019

Draft Genome Sequence of the Mucoid Clinical Isolate PA34.

Genome Announc 2017 Nov 16;5(46). Epub 2017 Nov 16.

Department of Medical Microbiology and Immunology, Creighton University, Omaha, Nebraska, USA

is a serious threat to patients suffering from cystic fibrosis. These organisms are exposed to a unique set of selective pressures within the lung. Here, we report the draft genome sequence of a mucoid clinical isolate obtained from a cystic fibrosis patient colonized with .
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http://dx.doi.org/10.1128/genomeA.01307-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5690347PMC
November 2017

Impact of CLSI and EUCAST Cefepime breakpoint changes on the susceptibility reporting for Enterobacteriaceae.

Diagn Microbiol Infect Dis 2017 Dec 12;89(4):328-333. Epub 2017 Oct 12.

University of Maryland School of Medicine, Department of Pathology, Baltimore, MD.

Objective: We analyzed the effects of different cefepime MIC breakpoints on Enterobacteriaceae cefepime susceptibility and the presence of AmpC and extended-spectrum β-lactamase (ESBL) genes within the cefepime MIC interpretative categories.

Methods: Using Enterobacteriaceae susceptibility data from 2013 comparisons of MIC breakpoints were performed using Pearson's chi-squared test. Molecular testing on a subset of isolates was done.

Results: Among 3784 non-duplicate clinical isolates, cefepime susceptibility decreased from 97.6% to 96.1% to 93.7% for CLSI 2013, CLSI 2014, and EUCAST 2011, respectively. In ceftriaxone non-susceptible isolates, cefepime susceptibility decreased from 79% to 66% (P<0.0001) using CLSI 2013 and 2014, respectively, which was greater and statistically significant for Escherichia coli and Klebsiella spp. but not for Enterobacter spp. (P=0.06). Isolates with MIC ≤1μg/mL more often harbored AmpC (77%) than ESBL (18%) genes.

Conclusions: Lower cefepime MIC breakpoints decrease cefepime susceptibility for isolates harboring ESBLs, while sparing the majority of those with AmpCs.
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http://dx.doi.org/10.1016/j.diagmicrobio.2017.08.020DOI Listing
December 2017

High-Resolution Melting Analysis for Rapid Detection of Sequence Type 131 Escherichia coli.

Antimicrob Agents Chemother 2017 06 24;61(6). Epub 2017 May 24.

Department of Medical Microbiology and Immunology, Creighton University, Omaha, Nebraska, USA

isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and β-lactam resistance. Whole-genome sequencing and multilocus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using high-resolution melting (HRM) analysis to differentiate ST131 from non-ST131 in large sample populations in the absence of sequence analysis. The method was optimized using DNA from 12 isolates. Singleplex PCR was performed using 10 ng of DNA, Type-it HRM buffer, and multilocus sequence typing primers and was followed by multiplex PCR. The amplicon sizes ranged from 630 to 737 bp. Melt temperature peaks were determined by performing HRM analysis at 0.1°C resolution from 50 to 95°C on a Rotor-Gene Q 5-plex HRM system. Derivative melt curves were compared between sequence types and analyzed by principal component analysis. A blinded study of 191 isolates of ST131 and unknown sequence types validated this methodology. This methodology returned 99.2% specificity (124 true negatives and 1 false positive) and 100% sensitivity (66 true positives and 0 false negatives). This HRM methodology distinguishes ST131 from non-ST131 without sequence analysis. The analysis can be accomplished in about 3 h in any laboratory with an HRM-capable instrument and principal component analysis software. Therefore, this assay is a fast and cost-effective alternative to sequencing-based ST131 identification.
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http://dx.doi.org/10.1128/AAC.00265-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444143PMC
June 2017

IMP-27, a Unique Metallo-β-Lactamase Identified in Geographically Distinct Isolates of Proteus mirabilis.

Antimicrob Agents Chemother 2016 10 23;60(10):6418-21. Epub 2016 Sep 23.

Creighton University School of Medicine, Department of Medical Microbiology and Immunology, Center for Research in Anti-Infectives and Biotechnology, Omaha, Nebraska, USA

A novel metallo-β-lactamase gene, blaIMP-27, was identified in unrelated Proteus mirabilis isolates from two geographically distinct locations in the United States. Both isolates harbor blaIMP-27 as part of the first gene cassette in a class 2 integron. Antimicrobial susceptibility testing indicated susceptibility to aztreonam, piperacillin-tazobactam, and ceftazidime but resistance to ertapenem. However, hydrolysis assays indicated that ceftazidime was a substrate for IMP-27.
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http://dx.doi.org/10.1128/AAC.02945-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5038328PMC
October 2016

The effect of referral for genetic counseling on genetic testing and surgical prevention in women at high risk for ovarian cancer: Results from a randomized controlled trial.

Cancer 2016 Nov 22;122(22):3509-3518. Epub 2016 Jul 22.

Translational Outcomes Research, Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington.

Background: Guidelines recommend genetic counseling and testing for women who have a pedigree suggestive of an inherited susceptibility for ovarian cancer. The authors evaluated the effect of referral to genetic counseling on genetic testing and prophylactic oophorectomy in a randomized controlled trial.

Methods: Data from an electronic mammography reporting system identified 12,919 women with a pedigree that included breast cancer, of whom 625 were identified who had a high risk for inherited susceptibility to ovarian cancer using a risk-assessment questionnaire. Of these, 458 women provided informed consent and were randomized 1:1 to intervention consisting of a genetic counseling referral (n = 228) or standard clinical care (n = 230).

Results: Participants were predominantly aged 45 to 65 years, and 30% and 20% reported a personal history of breast cancer or a family history of ovarian cancer, respectively. Eighty-five percent of women in the intervention group participated in a genetic counseling session. Genetic testing was reported by 74 (33%) and 20 (9%) women in the intervention and control arms (P < .005), respectively. Five women in the intervention arm and 2 in the control arm were identified as germline mutation carriers. Ten women in the intervention arm and 3 in the control arm underwent prophylactic bilateral salpingo-oophorectomy (P < .05).

Conclusions: Routine referral of women at high risk for ovarian cancer to genetic counseling promotes genetic testing and prophylactic surgery. The findings from the current randomized controlled trial demonstrate the value of implementing strategies that target women at high risk for ovarian cancer to ensure they are offered access to recommended care. CA Cancer J Clin 2016. © 2016 American Cancer Society, Inc. Cancer 2016;122:3509-3518. © 2016 American Cancer Society.
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http://dx.doi.org/10.1002/cncr.30190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5253334PMC
November 2016

Structural and Mutagenic Analysis of Metallo-β-Lactamase IMP-18.

Antimicrob Agents Chemother 2016 09 22;60(9):5521-6. Epub 2016 Aug 22.

Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Yamagata, Japan Faculty of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Akiha-ku, Niigata, Japan

IMP-type metallo-β-lactamases (MBLs) are exogenous zinc metalloenzymes that hydrolyze a broad range of β-lactams, including carbapenems. Here we report the crystal structure of IMP-18, an MBL cloned from Pseudomonas aeruginosa, at 2.0-Å resolution. The overall structure of IMP-18 resembles that of IMP-1, with an αβ/βα "folded sandwich" configuration, but the loop that covers the active site has a distinct conformation. The relationship between IMP-18's loop conformation and its kinetic properties was investigated by replacing the amino acid residues that can affect the loop conformation (Lys44, Thr50, and Ile69) in IMP-18 with those occupying the corresponding positions in the well-described enzyme IMP-1. The replacement of Thr50 with Pro considerably modified IMP-18's kinetic properties, specifically those pertaining to meropenem, with the kcat/Km value increased by an order of magnitude. The results indicate that this is a key residue that defines the kinetic properties of IMP-type β-lactamases.
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http://dx.doi.org/10.1128/AAC.00985-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4997881PMC
September 2016

Evaluation of CTX-M steady-state mRNA, mRNA half-life and protein production in various STs of Escherichia coli.

J Antimicrob Chemother 2016 Mar 26;71(3):607-16. Epub 2015 Nov 26.

Department of Medical Microbiology and Immunology, Center for Research in Anti-Infectives and Biotechnology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA

Objectives: High levels of β-lactamase production can impact treatment with a β-lactam/β-lactamase inhibitor combination. Goals of this study were to: (i) compare the mRNA and protein levels of CTX-M-15- and CTX-M-14-producing Escherichia coli from 18 different STs and 10 different phylotypes; (ii) evaluate the mRNA half-lives and establish a role for chromosomal- and/or plasmid-encoded factors; and (iii) evaluate the zones of inhibition for piperacillin/tazobactam and ceftolozane/tazobactam.

Methods: Disc diffusion was used to establish zone size. RNA analysis was accomplished using real-time RT-PCR and CTX-M protein levels were evaluated by immunoblotting. Clinical isolates, transformants and transconjugants were used to evaluate mRNA half-lives.

Results: mRNA levels of CTX-M-15 were up to 165-fold higher compared with CTX-M-14. CTX-M-15 protein levels were 2-48-fold less than their respective transcript levels, while CTX-M-14 protein production was comparable to the observed transcript levels. Nineteen of 25 E. coli (76%) had extended CTX-M-15 mRNA half-lives of 5-15 min and 16 (100%) CTX-M-14 isolates had mRNA half-lives of <2-3 min. Transformants had mRNA half-lives of <2 min for both CTX-M-type transcripts, while transconjugant mRNA half-lives corresponded to the half-life of the donor. Ceftolozane/tazobactam zone sizes were ≥19 mm, while piperacillin/tazobactam zone sizes were ≥17 mm.

Conclusions: CTX-M-15 mRNA and protein production did not correlate. Neither E. coli ST nor phylotype influenced the variability observed for CTX-M-15 mRNA or protein produced. mRNA half-life is controlled by a plasmid-encoded factor and may influence mRNA transcript levels, but not protein levels.
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http://dx.doi.org/10.1093/jac/dkv388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4743699PMC
March 2016

The OpdQ porin of Pseudomonas aeruginosa is regulated by environmental signals associated with cystic fibrosis including nitrate-induced regulation involving the NarXL two-component system.

Microbiologyopen 2015 Dec 12;4(6):967-82. Epub 2015 Oct 12.

Department of Medical Microbiology and Immunology, Center for Research in Anti-Infectives and Biotechnology, Creighton University School of Medicine, 2500 California Plaza, Omaha, Nebraska, 68178.

Pseudomonas aeruginosa is a versatile opportunistic pathogen that causes chronic infections in immunocompromised hosts. Multiple porins modulate outer membrane permeability under various environmental conditions. The lung environment of cystic fibrosis (CF) patients is unique with changes occurring in nutrient availability, osmolarity, and oxygen content. Although P. aeruginosa gene expression is modified under these conditions, little is known about how they influence porin regulation. In this study, we evaluated the regulation of the outer membrane porin OpdQ, a member of the OprD family of porins, with regard to oxygen, nitrate, and/or NaCl levels. We demonstrated using promoter::fusion clones of P. aeruginosa PAO1 and clinical strains collected from CF patients that OpdQ was transcriptionally repressed under low oxygen but increased in the presence of nitrate. The nitrate-induced regulation of OpdQ was found to be dependent on the transcription factor NarL via the NarXL two-component system. In addition, NaCl-induced osmotic stress increased OpdQ production among most of the clinical strains evaluated. In conclusion, these data identify for the first time that specific environmental cues associated with the CF microenvironment influence porin regulation, and that the nitrate-induced regulation of OpdQ is associated with nitrate metabolism via the NarXL two-component system of P. aeruginosa.
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http://dx.doi.org/10.1002/mbo3.305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4694141PMC
December 2015

Identification of Gram-Negative Bacteria and Genetic Resistance Determinants from Positive Blood Culture Broths by Use of the Verigene Gram-Negative Blood Culture Multiplex Microarray-Based Molecular Assay.

J Clin Microbiol 2015 Aug 20;53(8):2460-72. Epub 2015 May 20.

The Medical College of Wisconsin, Milwaukee, Wisconsin, USA

Bloodstream infection is a serious condition associated with significant morbidity and mortality. The outcome of these infections can be positively affected by the early implementation of effective antibiotic therapy based on the identification of the infecting organism and genetic markers associated with antibiotic resistance. In this study, we evaluated the microarray-based Verigene Gram-negative blood culture (BC-GN) assay in the identification of 8 genus or species targets and 6 genetic resistance determinants in positive blood culture broths. A total of 1,847 blood cultures containing Gram-negative organisms were tested using the BC-GN assay. This comprised 729 prospective fresh, 781 prospective or retrospective frozen, and 337 simulated cultures representing 7 types of aerobic culture media. The results were compared to those with standard bacterial culture and biochemical identification with nucleic acid sequence confirmation of the resistance determinants. Among monomicrobial cultures, the positive percent agreement (PPA) of the BC-GN assay with the reference method was as follows; Escherichia coli, 100%; Klebsiella pneumoniae, 92.9%; Klebsiella oxytoca, 95.5%; Enterobacter spp., 99.3%; Pseudomonas aeruginosa, 98.9%; Proteus spp., 100%; Acinetobacter spp., 98.4%; and Citrobacter spp., 100%. All organism identification targets demonstrated >99.5% negative percent agreement (NPA) with the reference method. Of note, 25/26 cultures containing K. pneumoniae that were reported as not detected by the BC-GN assay were subsequently identified as Klebsiella variicola. The PPA for identification of resistance determinants was as follows; blaCTX-M, 98.9%; blaKPC, 100%; blaNDM, 96.2%; blaOXA, 94.3%; blaVIM, 100%; and blaIMP, 100%. All resistance determinant targets demonstrated >99.9% NPA. Among polymicrobial specimens, the BC-GN assay correctly identified at least one organism in 95.4% of the broths and correctly identified all organisms present in 54.5% of the broths. The sample-to-result processing and automated reading of the detection microarray results enables results within 2 h of culture positivity.
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http://dx.doi.org/10.1128/JCM.00581-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4508435PMC
August 2015

Assessing Community-Based Injury Prevention Services in U.S. Children's Hospitals.

AIMS Public Health 2014 31;1(4):199-210. Epub 2014 Oct 31.

Children's Hospital Association, Washington, DC 20005, USA.

Objective: Not-for-profit hospitals are required to meet federal reporting requirements detailing their community benefit activities, which support their tax-exempt status. Children's hospitals have long provided community injury prevention (IP) programming and thus can inform public health outreach work in other areas. This work describes IP programming as a community service offered by children's hospitals in the U.S.

Methods: The IP specialist at 232 US-based member institutions of the Children's Hospital Association were invited to complete an assessment of their hospital's IP outreach programming.

Results: 47.7 percent of hospitals request financial data from IP programming for tax reporting purposes. Almost all offer injury prevention (IP) services; the majority are in the community (60.3%) and 34.5% are hospital-based. Most IP units are independent (60.3%) and 71.8% are responsible for their own budgets.

Conclusions: By integrating dissemination and implementation sciences and community health needs assessments, these findings can help advance community services provided by hospitals to impact public health.
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http://dx.doi.org/10.3934/publichealth.2014.4.199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5690253PMC
October 2014

Cancer Risk Awareness and Concern among Women with a Family History of Breast or Ovarian Cancer.

Behav Med 2016 3;42(1):18-28. Epub 2014 Nov 3.

a Fred Hutchinson Cancer Research Center and University of Washington.

Women with a documented deleterious mutation in BRCA1 or BRCA2 are at substantially elevated risk for ovarian cancer. To understand what percentage of women with high-risk family histories know their risk is elevated we surveyed 1,885 women with a high- or moderate-risk family history and no personal history of breast or ovarian cancer, and asked about their perceived risk of breast and ovarian cancer. Among high-risk women, fewer than 20% reported use of genetic counseling, and knowledge of elevated risk of ovarian cancer was low. Prior genetic counseling was associated with greater perceived risk for ovarian cancer. Results suggest that most high-risk women (>75%) do not know their risk for ovarian cancer. Identification of potentially high-risk women for referral to genetic counseling may improve informed ovarian cancer risk management.
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http://dx.doi.org/10.1080/08964289.2014.947234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4469617PMC
October 2016

Whole genome mapping of the first reported case of KPC-2-positive Klebsiella pneumoniae ST258 in Nebraska.

Diagn Microbiol Infect Dis 2014 Jul 12;79(3):384-6. Epub 2014 Apr 12.

Center for Research in Anti-Infectives and Biotechnology, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178. Electronic address:

Three ertapenem-resistant Klebsiella pneumoniae carrying bla(KPC-2) were isolated from a single patient in Nebraska over a span of 5 months. A comparative analysis of the genetic relatedness of these isolates was investigated using pulsed-field gel electrophoresis, multilocus sequence typing, and whole genome mapping.
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http://dx.doi.org/10.1016/j.diagmicrobio.2014.03.023DOI Listing
July 2014

Emergence of carbapenem resistance due to the novel insertion sequence ISPa8 in Pseudomonas aeruginosa.

PLoS One 2014 10;9(3):e91299. Epub 2014 Mar 10.

Creighton University School of Medicine, Department of Medical Microbiology and Immunology, Center for Research in Anti-Infectives and Biotechnology, Omaha, Nebraska, United States of America.

Chronic lung infections due to the persistence of Pseudomonas aeruginosa in cystic fibrosis patients are typically associated with the emergence of antibiotic resistance. The purpose of this study was to investigate the mechanisms responsible for the emergence of carbapenem resistance when a clinical isolate of P. aeruginosa collected from a patient with cystic fibrosis was challenged with meropenem. Nine carbapenem-resistant mutants were selected with subinhibitory concentrations of meropenem from a clinical isolate of P. aeruginosa and characterized for carbapenem resistance. Increased carbapenem MICs were associated with the identification of the novel insertion sequence ISPa8 within oprD or its promoter region in all the mutants. The position of ISPa8 was different for each of the mutants evaluated. In addition, Southern blot analyses identified multiple copies of ISPa8 within the genomes of the mutants and their parent isolate. These data demonstrate that transposition of IS elements within the Pseudomonas genome can influence antibiotic susceptibility. Understanding the selective pressures associated with the emergence of antibiotic resistance is critical for the judicious use of antimicrobial chemotherapy and the successful treatment of bacterial infections.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0091299PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3948848PMC
March 2016

A clinical scoring system to identify patients with sebaceous neoplasms at risk for the Muir-Torre variant of Lynch syndrome.

Genet Med 2014 Sep 6;16(9):711-6. Epub 2014 Mar 6.

Department of Dermatology, Mayo Clinic, Jacksonville, Florida, USA.

Purpose: The Muir-Torre syndrome variant of Lynch syndrome is characterized by the presence of sebaceous neoplasms (adenoma, epithelioma/sebaceoma, carcinoma) and Lynch syndrome-associated cancers (colon, endometrial, and others). Several clinical scoring systems have been developed to identify patients with colon cancer at high risk of Lynch syndrome. However, no such system has been described for patients presenting with sebaceous neoplasms.

Methods: Based on logistic regression analysis, a scoring system was developed for patients with sebaceous neoplasm to identify those with the highest likelihood of having Muir-Torre syndrome. The final version of the scoring system included variables such as age at presentation of initial sebaceous neoplasm, total number of sebaceous neoplasms, personal history of a Lynch-related cancer, and family history of Lynch-related cancers.

Results: Patients with a score of 3 or more were more likely to have Muir-Torre syndrome (28 of 29 patients), those with a score of 2 had intermediate likelihood (12 of 20 patients), and no patient with a score of 0 or 1 was diagnosed with Muir-Torre syndrome.

Conclusion: The Mayo Muir-Torre syndrome risk scoring system appears to identify whether patients who present with sebaceous neoplasms are in need of further Lynch syndrome evaluation using easily ascertained clinical information. Abnormal mismatch repair gene immunohistochemistry of a sebaceous neoplasm is a poor predictor in regard to diagnosing Lynch syndrome.
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http://dx.doi.org/10.1038/gim.2014.19DOI Listing
September 2014

Multiplex high-resolution melting analysis as a diagnostic tool for detection of plasmid-mediated AmpC β-lactamase genes.

J Clin Microbiol 2014 Apr 29;52(4):1262-5. Epub 2014 Jan 29.

Creighton University School of Medicine, Department of Medical Microbiology and Immunology, Center for Research in Anti-Infectives and Biotechnology, Omaha, Nebraska, USA.

High-resolution melting (HRM) analysis can be a diagnostic tool to evaluate the presence of resistance genes with the added bonus of discriminating sequence modifications. A real-time, multiplex PCR assay using HRM was designed for the detection of plasmid-mediated ampC genes. The specificity and sensitivity of this assay were 96% and 100%, respectively.
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http://dx.doi.org/10.1128/JCM.00214-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3993522PMC
April 2014

Purification, crystallization and preliminary X-ray analysis of IMP-18, a class B carbapenemase from Pseudomonas aeruginosa.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2013 Dec 29;69(Pt 12):1397-400. Epub 2013 Nov 29.

Department of Material and Biological Chemistry, Graduate School of Science and Engineering, Yamagata University, 1-4-12 Kojirakawa, Yamagata 990-8560, Japan.

Class B β-lactamases are known as metallo-β-lactamases (MBLs) and they hydrolyze most β-lactams, including carbapenems. IMP-18, an MBL cloned from Pseudomonas aeruginosa, was overexpressed, purified and crystallized by vapour diffusion for X-ray crystallographic analysis. Preliminary X-ray analysis showed that the crystal diffracted to 2.4 Å resolution and belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 120.77, c = 96.54 Å, α = β = γ = 90°, suggesting the presence of two molecules in the asymmetric unit.
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http://dx.doi.org/10.1107/S1744309113030480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3855729PMC
December 2013

Rapid PCR amplification protocols decrease the turn-around time for detection of antibiotic resistance genes in Gram-negative pathogens.

Diagn Microbiol Infect Dis 2013 Oct 23;77(2):113-7. Epub 2013 Jul 23.

Creighton University School of Medicine, Department of Medical Microbiology and Immunology, Center for Research in Anti-Infectives and Biotechnology (C.R.A.B), 2500 California Plaza, Omaha, NE 68178, USA.

A previously designed end-point multiplex PCR assay and singleplex assays used to detect β-lactamase genes were evaluated using rapid PCR amplification methodology. Amplification times were 16-18 minutes with an overall detection time of 1.5 hours. Rapid PCR amplifications could decrease the time required to identify resistance mechanisms in Gram-negative organisms.
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http://dx.doi.org/10.1016/j.diagmicrobio.2013.06.015DOI Listing
October 2013

Effect of drug treatment options on the mobility and expression of blaKPC.

J Antimicrob Chemother 2013 Dec 16;68(12):2779-85. Epub 2013 Jul 16.

Center for Research in Anti-Infectives and Biotechnology, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA.

Objectives: Both transposition and increases in gene expression have been implicated in the success of KPC-producing pathogens, but the stimulus required for these phenomena are unknown. It is possible that exposure to antimicrobials during patient treatment increases bla(KPC) expression or induces Tn4401 transposition. The purpose of this study was to determine if exposure to carbapenems or other antimicrobial drug classes could stimulate expression of bla(KPC) or the in vitro transposition of Tn4401.

Methods: Five KPC-producing clinical isolates were evaluated in this study. Gene expression of RNA from each isolate exposed to subinhibitory, MIC or suprainhibitory levels of antibiotics was evaluated using real-time RT-PCR. Southern blots were performed on plasmids from isolates exposed to subinhibitory levels of antibiotics.

Results: There were subtle changes in bla(KPC) RNA expression following antibiotic exposure that were both strain and drug dependent. Multiple plasmids ranging from ~8 to >200 kb were observed for the Enterobacteriaceae isolates, whereas the Pseudomonas aeruginosa isolate had one ~55 kb plasmid. No changes in hybridization patterns or binding intensity for the bla(KPC) probe were observed after antibiotic exposure.

Conclusions: While the changes in bla(KPC) RNA expression are subtle, the different responses observed suggest both strain- and genera-specific variations in response to different antibiotic treatments.
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http://dx.doi.org/10.1093/jac/dkt280DOI Listing
December 2013

Release of luminal exosomes contributes to TLR4-mediated epithelial antimicrobial defense.

PLoS Pathog 2013 4;9(4):e1003261. Epub 2013 Apr 4.

Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska, United States of America.

Exosomes are membranous nanovesicles released by most cell types from multi-vesicular endosomes. They are speculated to transfer molecules to neighboring or distant cells and modulate many physiological and pathological procedures. Exosomes released from the gastrointestinal epithelium to the basolateral side have been implicated in antigen presentation. Here, we report that luminal release of exosomes from the biliary and intestinal epithelium is increased following infection by the protozoan parasite Cryptosporidium parvum. Release of exosomes involves activation of TLR4/IKK2 signaling through promoting the SNAP23-associated vesicular exocytotic process. Downregulation of let-7 family miRNAs by activation of TLR4 signaling increases SNAP23 expression, coordinating exosome release in response to C. parvum infection. Intriguingly, exosomes carry antimicrobial peptides of epithelial cell origin, including cathelicidin-37 and beta-defensin 2. Activation of TLR4 signaling enhances exosomal shuttle of epithelial antimicrobial peptides. Exposure of C. parvum sporozoites to released exosomes decreases their viability and infectivity both in vitro and ex vivo. Direct binding to the C. parvum sporozoite surface is required for the anti-C. parvum activity of released exosomes. Biliary epithelial cells also increase exosomal release and display exosome-associated anti-C. parvum activity following LPS stimulation. Our data indicate that TLR4 signaling regulates luminal exosome release and shuttling of antimicrobial peptides from the gastrointestinal epithelium, revealing a new arm of mucosal immunity relevant to antimicrobial defense.
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http://dx.doi.org/10.1371/journal.ppat.1003261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3617097PMC
January 2014

Rapid detection and statistical differentiation of KPC gene variants in Gram-negative pathogens by use of high-resolution melting and ScreenClust analyses.

J Clin Microbiol 2013 Jan 17;51(1):61-5. Epub 2012 Oct 17.

Center for Research in Anti-Infectives and Biotechnology, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska, USA.

In the United States, the production of the Klebsiella pneumoniae carbapenemase (KPC) is an important mechanism of carbapenem resistance in Gram-negative pathogens. Infections with KPC-producing organisms are associated with increased morbidity and mortality; therefore, the rapid detection of KPC-producing pathogens is critical in patient care and infection control. We developed a real-time PCR assay complemented with traditional high-resolution melting (HRM) analysis, as well as statistically based genotyping, using the Rotor-Gene ScreenClust HRM software to both detect the presence of bla(KPC) and differentiate between KPC-2-like and KPC-3-like alleles. A total of 166 clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii with various β-lactamase susceptibility patterns were tested in the validation of this assay; 66 of these organisms were known to produce the KPC β-lactamase. The real-time PCR assay was able to detect the presence of bla(KPC) in all 66 of these clinical isolates (100% sensitivity and specificity). HRM analysis demonstrated that 26 had KPC-2-like melting peak temperatures, while 40 had KPC-3-like melting peak temperatures. Sequencing of 21 amplified products confirmed the melting peak results, with 9 isolates carrying bla(KPC-2) and 12 isolates carrying bla(KPC-3). This PCR/HRM assay can identify KPC-producing Gram-negative pathogens in as little as 3 h after isolation of pure colonies and does not require post-PCR sample manipulation for HRM analysis, and ScreenClust analysis easily distinguishes bla(KPC-2-like) and bla(KPC-3-like) alleles. Therefore, this assay is a rapid method to identify the presence of bla(KPC) enzymes in Gram-negative pathogens that can be easily integrated into busy clinical microbiology laboratories.
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http://dx.doi.org/10.1128/JCM.02193-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536237PMC
January 2013

Production of KPC-2 alone does not always result in β-lactam MICs representing resistance in gram-negative pathogens.

J Clin Microbiol 2012 Dec 12;50(12):4183-4. Epub 2012 Sep 12.

Department of Medical Microbiology and Immunology Creighton University Omaha, Nebraska, USA.

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http://dx.doi.org/10.1128/JCM.02194-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3502953PMC
December 2012

Editorial: Resistance in gram-negative pathogens: a threat to global health.

Authors:
Nancy D Hanson

Curr Pharm Des 2013 ;19(2):163

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July 2013

Development of a TaqMan multiplex PCR assay for detection of plasmid-mediated ampC β-lactamase genes.

J Clin Microbiol 2012 Nov 15;50(11):3722-5. Epub 2012 Aug 15.

Creighton University School of Medicine Department of Medical Microbiology and Immunology Center for Research in Anti-Infectives and Biotechnology, Omaha, Nebraska, USA.

A multiplex, real-time TaqMan assay was designed to identify clinical isolates carrying plasmid-mediated ampC genes. The specificity and sensitivity of this assay were 100% when testing characterized AmpC/non-AmpC-producing isolates and randomly selected clinical isolates. This is a rapid assay that can be performed in a clinical microbiology laboratory.
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http://dx.doi.org/10.1128/JCM.02038-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486237PMC
November 2012

Prophylactic oophorectomy rates in relation to a guideline update on referral to genetic counseling.

Gynecol Oncol 2012 Aug 4;126(2):229-35. Epub 2012 May 4.

Group Health Research Institute, 1730 Minor Ave, Suite 1600, Seattle, WA 98101-1448, USA.

Objective: We sought to determine whether prophylactic oophorectomy rates changed after the introduction of a 2007 health plan clinical guideline recommending systematic referral to a genetic counselor for women with a personal or family history suggestive of an inherited susceptibility to breast/ovarian cancer.

Methods: We conducted a retrospective cohort study of female members of Group Health, an integrated delivery system in Washington State. Subjects were women aged ≥ 35 years during 2004-2009 who reported a personal or family history consistent with an inherited susceptibility to breast/ovarian cancer. Personal and family history information was collected on a questionnaire completed when the women had a mammogram. We ascertained oophorectomies from automated claims data and determined whether surgeries were prophylactic by medical chart review. Rates were age-adjusted and age-adjusted incidence rate ratios (IRR) and 95% confidence intervals (CI) were computed using Poisson regression.

Results: Prophylactic oophorectomy rates were relatively unchanged after compared to before the guideline change, 1.0 versus 0.8/1000 person-years, (IRR=1.2; 95% CI: 0.7-2.0), whereas bilateral oophorectomy rates for other indications decreased. Genetic counseling receipt rates doubled after the guideline change (95% CI: 1.7-2.4) from 5.1 to 10.2/1000 person-years. During the study, bilateral oophorectomy rates were appreciably greater in women who saw a genetic counselor compared to those who did not regardless of whether they received genetic testing as part of their counseling.

Conclusion: A doubling in genetic counseling receipt rates lends support to the idea that the guideline issuance contributed to sustained rates of prophylactic oophorectomies in more recent years.
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http://dx.doi.org/10.1016/j.ygyno.2012.04.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383401PMC
August 2012

Molecular epidemiological analysis of Escherichia coli sequence type ST131 (O25:H4) and blaCTX-M-15 among extended-spectrum-β-lactamase-producing E. coli from the United States, 2000 to 2009.

Antimicrob Agents Chemother 2012 May 21;56(5):2364-70. Epub 2012 Feb 21.

Veterans Affairs Medical Center, Minneapolis, Minnesota, USA.

Escherichia coli sequence type ST131 (from phylogenetic group B2), often carrying the extended-spectrum-β-lactamase (ESBL) gene bla(CTX-M-15), is an emerging globally disseminated pathogen that has received comparatively little attention in the United States. Accordingly, a convenience sample of 351 ESBL-producing E. coli isolates from 15 U.S. centers (collected in 2000 to 2009) underwent PCR-based phylotyping and detection of ST131 and bla(CTX-M-15). A total of 200 isolates, comprising 4 groups of 50 isolates each that were (i) bla(CTX-M-15) negative non-ST131, (ii) bla(CTX-M-15) positive non-ST131, (iii) bla(CTX-M-15) negative ST131, or (iv) bla(CTX-M-15) positive ST131, also underwent virulence genotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). Overall, 201 (57%) isolates exhibited bla(CTX-M-15), whereas 165 (47%) were ST131. ST131 accounted for 56% of bla(CTX-M-15)-positive- versus 35% of bla(CTX-M-15)-negative isolates (P < 0.001). Whereas ST131 accounted for 94% of the 175 total group B2 isolates, non-ST131 isolates were phylogenetically distributed by bla(CTX-M-15) status, with groups A (bla(CTX-M-15)-positive isolates) and D (bla(CTX-M-15)-negative isolates) predominating. Both bla(CTX-M-15) and ST131 occurred at all participating centers, were recovered from children and adults, increased significantly in prevalence post-2003, and were associated with molecularly inferred virulence. Compared with non-ST131 isolates, ST131 isolates had higher virulence scores, distinctive virulence profiles, and more-homogeneous PFGE profiles. bla(CTX-M-15) was associated with extensive antimicrobial resistance and ST131 with fluoroquinolone resistance. Thus, E. coli ST131 and bla(CTX-M-15) are emergent, widely distributed, and predominant among ESBL-positive E. coli strains in the United States, among children and adults alike. Enhanced virulence and antimicrobial resistance have likely promoted the epidemiological success of these emerging public health threats.
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http://dx.doi.org/10.1128/AAC.05824-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3346636PMC
May 2012

Point mutations in the inc antisense RNA gene are associated with increased plasmid copy number, expression of blaCMY-2 and resistance to piperacillin/tazobactam in Escherichia coli.

J Antimicrob Chemother 2012 Feb 24;67(2):339-45. Epub 2011 Nov 24.

Center for Research in Anti-Infectives and Biotechnology, Creighton University School of Medicine, Omaha, NE 668178, USA.

Background: High-level expression of AmpC β-lactamase genes is associated with increased resistance to β-lactam antibiotics. bla(CMY-2) is the most prevalent plasmid-encoded AmpC gene found in Escherichia coli worldwide, and the gene is often found on plasmids of the IncI1 replicon type. Replication of IncI1 plasmids is controlled by antisense RNA transcribed from the gene inc, and nucleotide changes in the hairpin loop region of inc have been associated with increased plasmid copy number of IncI1 mini-plasmid constructs. The objective of this study was to determine the mechanism(s) responsible for increased bla(CMY-2) expression in three piperacillin/tazobactam-selected E. coli mutant strains with bla(CMY-2) encoded on a 100 kb IncI1 plasmid.

Methods: Mutants were selected from a clinical E. coli strain by exposure to superinhibitory concentrations of piperacillin/tazobactam. β-Lactam susceptibilities were measured by agar dilution. Relative bla(CMY-2) transcript levels, gene copy number and IncI1 plasmid copy number were measured by real-time PCR. The inc gene of all strains was sequenced.

Results: Piperacillin/tazobactam MICs were 16- to 128-fold higher for mutant strains than for their parent strain. This increase in MICs correlated with 3- to 13-fold increases in bla(CMY-2) gene expression, bla(CMY-2) copy number and IncI1 plasmid copy number. Two mutants with 8- and 13-fold increases in IncI1 copy number had single point mutations located within the hairpin loop region of inc.

Conclusions: These findings demonstrate that inc point mutations can be associated with increased copy number of a 100 kb IncI1 plasmid, and lead to increased bla(CMY-2) expression and piperacillin/tazobactam resistance.
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http://dx.doi.org/10.1093/jac/dkr479DOI Listing
February 2012