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1.  Nanoparticulate iron(III) oxo-hydroxide delivers safe iron that is well absorbed and utilised in humans 
Nanomedicine  2014;10(8):1877-1886.
Iron deficiency is the most common nutritional disorder worldwide with substantial impact on health and economy. Current treatments predominantly rely on soluble iron which adversely affects the gastrointestinal tract. We have developed organic acid-modified Fe(III) oxo-hydroxide nanomaterials, here termed nano Fe(III), as alternative safe iron delivery agents. Nano Fe(III) absorption in humans correlated with serum iron increase (P < 0.0001) and direct in vitro cellular uptake (P = 0.001), but not with gastric solubility. The most promising preparation (iron hydroxide adipate tartrate: IHAT) showed ~80% relative bioavailability to Fe(II) sulfate in humans and, in a rodent model, IHAT was equivalent to Fe(II) sulfate at repleting haemoglobin. Furthermore, IHAT did not accumulate in the intestinal mucosa and, unlike Fe(II) sulfate, promoted a beneficial microbiota. In cellular models, IHAT was 14-fold less toxic than Fe(II) sulfate/ascorbate. Nano Fe(III) manifests minimal acute intestinal toxicity in cellular and murine models and shows efficacy at treating iron deficiency anaemia.
From the Clinical Editor
This paper reports the development of novel nano-Fe(III) formulations, with the goal of achieving a magnitude less intestinal toxicity and excellent bioavailability in the treatment of iron deficiency anemia. Out of the tested preparations, iron hydroxide adipate tartrate met the above criteria, and may become an important tool in addressing this common condition.
Graphical Abstract
Current oral treatment of iron deficiency anaemia is based upon soluble iron compounds, all of which have undesirable gastrointestinal effects. However, natural dietary iron (III) may be nano-formed ligand-modified iron hydroxide particles that are acquired endocytically and thus not soluble or available for adverse effects. Here, a series of five different nano Fe(III) hydroxide particles was developed: absorption in humans matched endocytic uptake by gut epithelial cells but not solubility in (stomach) acid. In murine and cellular models nano iron(III) treated iron deficiency anaemia as well as ferrous sulfate but, unlike the latter, was safe to the gut environment.
PMCID: PMC4228177  PMID: 24983890
Ligand-modified Fe(III) poly oxo-hydroxide; Iron supplementation; Bioavailability; Microbiota; Redox damage
2.  Nanopore film based enrichment and quantification of low abundance hepcidin from human bodily fluids 
Endogenous peptides that represent biological and pathological information of disease have attracted interest for diagnosis. However, the extraction of those low abundance peptides is still a challenge because of the complexity of human bodily fluids (HBF). Hepcidin, a peptide hormone, has been recognized as a biomarker for iron-related diseases. There is no rapid and reliable way to enrich them from HBF. Here we describe a peptides extraction approach based on nanoporous silica thin films to successfully detect hepcidin from HBF. Cooperative functions of nanopore to biomolecule, including capillary adsorption, size-exclusion and electrostatic interaction, were systematically investigated to immobilize the target peptide. To promote this new approach to clinical practices, we further applied it to successfully assay the hepcidin levels in HBF provided by healthy volunteers and patients suffering from inflammation. Our finding provides a high-throughput, rapid, label-free and cost-effective detection method for capturing and quantifying low abundance peptides from HBF.
PMCID: PMC4077980  PMID: 24566273
Biomarker discovery; Nanoporous silica film; Peptide; MALDI-TOF MS; Hepcidin
3.  The Multicopper Ferroxidase Hephaestin Enhances Intestinal Iron Absorption in Mice 
PLoS ONE  2014;9(6):e98792.
Hephaestin is a vertebrate multicopper ferroxidase important for the transfer of dietary iron from intestinal cells to the blood. Hephaestin is mutated in the sex-linked anemia mouse, resulting in iron deficiency. However, sex-linked anemia mice still retain some hephaestin ferroxidase activity. They survive, breed, and their anemia improves with age. To gain a better understanding of the role of hephaestin in iron homeostasis, we used the Cre-lox system to generate knockout mouse models with whole body or intestine-specific (Villin promoter) ablation of hephaestin. Both types of mice were viable, indicating that hephaestin is not essential and that other mechanisms, multicopper ferroxidase-dependent or not, must compensate for hephaestin deficiency. The knockout strains, however, both developed a microcytic, hypochromic anemia, suggesting severe iron deficiency and confirming that hephaestin plays an important role in body iron acquisition. Consistent with this, the knockout mice accumulated iron in duodenal enterocytes and had reduced intestinal iron absorption. In addition, the similarities of the phenotypes of the whole body and intestine-specific hephaestin knockout mice clarify the important role of hephaestin specifically in intestinal enterocytes in maintaining whole body iron homeostasis. These mouse models will serve as valuable tools to study the role of hephaestin and associated proteins in iron transport in the small intestine and other tissues.
PMCID: PMC4045767  PMID: 24896847
4.  Quinolone Antimicrobial Agents, 3rd Edition 
Emerging Infectious Diseases  2004;10(6):1177.
PMCID: PMC3323177
Quinolone Antimicrobial Agents
6.  HFE C282Y homozygotes are at increased risk of breast and colorectal cancer 
Hepatology (Baltimore, Md.)  2010;51(4):10.1002/hep.23448.
The evidence that mutations in the HFE gene are associated with increased cancer risk is inconsistent.
The Melbourne Collaborative Cohort Study is a prospective cohort study that commenced recruitment in 1990. Participants of born in Australia, New Zealand, the United Kingdom or Ireland (n = 28,509) were genotyped for the HFE C282Y variant. Incident cancers were ascertained from Australian cancer registries during an average of 14 years follow up. Hazard ratios, confidence intervals and p-values were obtained from separate Cox regression analyses for colorectal, breast and prostate cancers, all other solid cancers and all cancers.
Compared with those with no C282Y variant, C282Y homozygotes were at increased risk of colorectal cancer (HR 2·28; 95% CI 1·22, 4·25; p = 0.01) and female C282Y homozygotes were at increased risk of developing breast cancer (HR 2·39; 95% CI 1·24, 4·61; p = 0.01) but male C282Y homozygotes were not at increased risk for prostate cancer (HR 0·96; 95% CI 0·43, 2·15; p = 0.92). C282Y/H63D compound heterozygotes were not at increased risk for colorectal cancer, (HR 1 27; 95% CI 0·80, 2·01); breast cancer, (HR 1·16; 95% CI 0·74, 1·84); or prostate cancer (HR 1·08; 95% CI 0·68, 1·70).
HFE C282Y homozygotes have twice the risk of colorectal and breast cancer compared with those without the C282Y variant.
PMCID: PMC3815603  PMID: 20099304
Haemochromatosis; neoplasm; prospective cohort; iron
7.  HFE C282Y/H63D Compound Heterozygotes Are at Low Risk of Hemochromatosis-Related Morbidity 
Hepatology (Baltimore, Md.)  2009;50(1):94-101.
The risk of hemochromatosis-related morbidity is unknown among HFE compound heterozygotes (C282Y/H63D). We used a prospective population-based cohort study to estimate the prevalence of elevated iron indices and hemochromatosis-related morbidity for compound heterozygotes. In all, 31,192 subjects of northern European descent were genotyped for HFE C282Y and H63D. An HFE-genotype stratified random sample of 1,438 subjects, followed for an average of 12 years to a mean age of 65 years, completed questionnaires and gave blood. Clinical examinations were blinded to HFE genotype. A total of 180 (84 males) clinically examined C282Y/H63D participants were compared with 330 (149 males) controls with neither HFE mutation; 132 (65 males) and 270 (122 males), respectively, had serum iron measures at both timepoints. Mean serum ferritin (SF) and transferrin saturation (TS) were significantly greater for male and female compound heterozygotes than for wild-types at baseline and follow-up (all P < 0.02) except for females who were pre-menopausal at baseline, where SF was similar in both genotype groups. For subjects with serum measures from both baseline and follow-up, mean SF and TS levels did not change significantly for men or for postmenopausal women, but for premenopausal women SF levels increased from 43 to 109 μg/L for compound heterozygotes and from 35 to 64 μg/L for wild-types (both P < 0.001). Male and female compound heterozygotes had a similar prevalence of hemochromatosis-related morbidity to wild-types. One of 82 males and zero of 95 females had documented iron overload-related disease.
For male compound heterozygotes, mean iron indices do not change during middle age but for female compound heterozygotes menopause results in increased mean SF. Although compound heterozygotes might maintain elevated iron indices during middle age, documented iron overload-related disease is rare.
PMCID: PMC3763940  PMID: 19554541
8.  Effects of gestational age and surface modification on materno-fetal transfer of nanoparticles in murine pregnancy 
Scientific Reports  2012;2:847.
Nanoparticle exposure in pregnancy may result in placental damage and fetotoxicity; however, the factors that determine fetal nanoparticle exposure are unclear. Here we have assessed the effect of gestational age and nanoparticle composition on fetal accumulation of maternally-administered nanomaterials in mice. We determined the placental and fetal uptake of 13 nm gold nanoparticles with different surface modifications (ferritin, PEG and citrate) following intravenous administration at E5.5-15.5. We showed that prior to E11.5, all tested nanoparticles could be visualized and detected in fetal tissues in significant amounts; however, fetal gold levels declined dramatically post-E11.5. In contrast, Au-nanoparticle accumulation in the extraembryonic tissues (EET) increased 6–15 fold with gestational age. Fetal and EET accumulation of ferritin- and PEG-modified nanoparticles was considerably greater than citrate-capped nanoparticles. No signs of toxicity were observed. Fetal exposure to nanoparticles in murine pregnancy is, therefore, influenced by both stage of embryonic/placental maturation and nanoparticle surface composition.
PMCID: PMC3496197  PMID: 23150793
9.  Gastrins, Iron Homeostasis and Colorectal Cancer 
Biochimica et biophysica acta  2011;1813(5):889-895.
The peptide hormone gastrin has been identified as a major regulator of acid secretion and a potent mitogen for normal and malignant gastrointestinal cells. The importance of gastric acid in the absorption of dietary iron first became evident 50 years ago when iron-deficiency anemia was recognised as a long-term consequence of partial gastrectomy. This review summarises the connections between circulating gastrins, iron status and colorectal cancer. Gastrins bind two ferric ions with micromolar affinity and, in the case of non-amidated forms of the hormone, iron-binding is essential for biological activity in vitro and in vivo. The demonstration of an interaction between gastrin and transferrin by biochemical techniques led to the proposal that gastrins catalyse the loading of transferrin with iron. Several lines of evidence, including the facts that the concentrations of circulating gastrins are increased in mice and humans with the iron-overload disease hemochromatosis and that transferrin saturation positively correlates with circulating gastrin concentration, suggest the potential involvement of gastrins in iron homeostasis. Conversely, recognition that ferric ions play an unexpected role in the biological activity of gastrins may assist in the development of useful therapies for colorectal carcinoma and other disorders of mucosal proliferation in the gastrointestinal tract.
PMCID: PMC3078979  PMID: 21320535
10.  HFE C282Y homozygotes with serum ferritin concentrations below 1000 μg/L are at low risk of hemochromatosis 
Hepatology (Baltimore, Md.)  2010;52(3):925-933.
HFE-associated hereditary hemochromatosis (HH) is a genetic predisposition to iron overload and subsequent signs and symptoms of disease potentially affecting around 80,000 in Australia and almost one million people in the USA. Most clinical cases are homozygous for the C282Y mutation in the HFE gene, with serum ferritin (SF) concentration >1000 μg/L the strongest predictor of cirrhosis. The optimal treatment regimen for those with SF concentrations above the normal range but <1000 μg/L is unknown. We assessed HFE mutations in a prospective cohort of 31,192 participants of northern European descent, aged 40–69 years. An HFE-stratified random sample of 1438 participants including all C282Y homozygotes with iron studies 12 years apart were examined by physicians blinded to HFE genotype. All previously undiagnosed C282Y homozygotes (35 male, 67 female) and all HFE wild-types (131 male, 160 female) with baseline and follow-up SF concentrations <1000 μg/L were assessed for HH-associated signs and symptoms including abnormal second/third metacarpophalangeal joints (MCP 2/3), raised liver enzymes, hepatomegaly, and self-reported liver disease, fatigue, diabetes mellitus, and use of arthritis medication. The prevalence of HH-associated signs and symptoms was similar for C282Y homozygotes and HFE wild-types for both normal and moderately elevated SF concentrations. The maximum prevalence difference between HFE genotype groups with moderately elevated SF was 11% (MCP 2/3 95% CI (−6%, 29%), p=0.22) and for normal SF was 6% (arthritis medicine use, 95% CI (−3, 16), p=0.11).
Previously undiagnosed C282Y homozygotes with SF concentrations that remain below 1000 μg/L are at low risk of developing HH-associated signs and symptoms at an age when disease would be expected to have developed. These observations have implications for the management of C282Y homozygotes.
PMCID: PMC2932765  PMID: 20583211
hemochromatosis; iron overload; C282Y homozygotes; serum ferritin
11.  A genome-wide screen for modifiers of transgene variegation identifies genes with critical roles in development 
Genome Biology  2008;9(12):R182.
An extended ENU screen for modifiers of transgene variegation identified four new modifiers, MommeD7-D10.
Some years ago we established an N-ethyl-N-nitrosourea screen for modifiers of transgene variegation in the mouse and a preliminary description of the first six mutant lines, named MommeD1-D6, has been published. We have reported the underlying genes in three cases: MommeD1 is a mutation in SMC hinge domain containing 1 (Smchd1), a novel modifier of epigenetic gene silencing; MommeD2 is a mutation in DNA methyltransferase 1 (Dnmt1); and MommeD4 is a mutation in Smarca 5 (Snf2h), a known chromatin remodeler. The identification of Dnmt1 and Smarca5 attest to the effectiveness of the screen design.
We have now extended the screen and have identified four new modifiers, MommeD7-D10. Here we show that all ten MommeDs link to unique sites in the genome, that homozygosity for the mutations is associated with severe developmental abnormalities and that heterozygosity results in phenotypic abnormalities and reduced reproductive fitness in some cases. In addition, we have now identified the underlying genes for MommeD5 and MommeD10. MommeD5 is a mutation in Hdac1, which encodes histone deacetylase 1, and MommeD10 is a mutation in Baz1b (also known as Williams syndrome transcription factor), which encodes a transcription factor containing a PHD-type zinc finger and a bromodomain. We show that reduction in the level of Baz1b in the mouse results in craniofacial features reminiscent of Williams syndrome.
These results demonstrate the importance of dosage-dependent epigenetic reprogramming in the development of the embryo and the power of the screen to provide mouse models to study this process.
PMCID: PMC2646286  PMID: 19099580
13.  SNP selection for genes of iron metabolism in a study of genetic modifiers of hemochromatosis 
BMC Medical Genetics  2008;9:18.
We report our experience of selecting tag SNPs in 35 genes involved in iron metabolism in a cohort study seeking to discover genetic modifiers of hereditary hemochromatosis.
We combined our own and publicly available resequencing data with HapMap to maximise our coverage to select 384 SNPs in candidate genes suitable for typing on the Illumina platform.
Validation/design scores above 0.6 were not strongly correlated with SNP performance as estimated by Gentrain score. We contrasted results from two tag SNP selection algorithms, LDselect and Tagger. Varying r2 from 0.5 to 1.0 produced a near linear correlation with the number of tag SNPs required. We examined the pattern of linkage disequilibrium of three levels of resequencing coverage for the transferrin gene and found HapMap phase 1 tag SNPs capture 45% of the ≥ 3% MAF SNPs found in SeattleSNPs where there is nearly complete resequencing. Resequencing can reveal adjacent SNPs (within 60 bp) which may affect assay performance. We report the number of SNPs present within the region of six of our larger candidate genes, for different versions of stock genotyping assays.
A candidate gene approach should seek to maximise coverage, and this can be improved by adding to HapMap data any available sequencing data. Tag SNP software must be fast and flexible to data changes, since tag SNP selection involves iteration as investigators seek to satisfy the competing demands of coverage within and between populations, and typability on the technology platform chosen.
PMCID: PMC2289803  PMID: 18366708
14.  Circulating Gastrin is Increased in Hemochromatosis. 
FEBS letters  2006;580(26):6195-6198.
Gastric acid production is important in intestinal iron absorption. The peptide hormone gastrin exists in both amidated and non-amidated forms, which stimulate and potentiate gastric acid secretion, respectively. Since non-amidated gastrins require ferric ions for biological activity in vitro, this study investigated the connection between iron status and gastrin by measurement of circulating gastrin concentrations in mice and humans with hemochromatosis. Gastrin concentrations are increased in the plasma and gastric mucosa of Hfe-/- mice, and in the sera of humans with HFE-related hemochromatosis. The discovery of a relationship between iron status and circulating gastrin concentrations opens a new perspective on the mechanisms of iron homeostasis.
PMCID: PMC1780041  PMID: 17064691
ferric; gastrin; hemochromatosis; iron
15.  Obesity and pre-hypertension in family medicine: Implications for quality improvement 
Prevention of pre-hypertension is an important goal for primary care patients. Obesity is a risk factor for hypertension, but has not been addressed for pre-hypertension in primary care populations. The objective of this study was to assess the degree to which obesity independently is associated with risk for pre-hypertension in family medicine patients.
This study was a retrospective analysis of information abstracted from medical records of 707 adult patients. Multivariable logistic regression was used to test the relationship between body mass index (BMI) and pre-hypertension, after adjustment for comorbidity and demographic characteristics. Pre-hypertension was defined as systolic pressure between 120 and 139 mm Hg or diastolic pressure between 80 and 89 mm Hg.
In our sample, 42.9% of patients were pre-hypertensive. Logistic regression analysis revealed that, in comparison to patients with normal body mass, patients with BMI > 35 had higher adjusted odds of being pre-hypertensive (OR = 4.5, CI 2.55–8.11, p < .01). BMI between 30 and 35 also was significant (OR = 2.7, CI 1.61–4.63, p < 0.01) as was overweight (OR = 1.8, CI 1.14–2.92, p = 0.01).
In our sample of family medicine patients, elevated BMI is a risk factor for pre-hypertension, especially BMI > 35. This relationship appears to be independent of age, gender, marital status and comorbidity. Weight loss intervention for obese patients, including patient education or referral to weight loss programs, might be effective for prevention of pre-hypertension and thus should be considered as a potential quality indicator.
PMCID: PMC2222627  PMID: 18154676
16.  Does the ‘Old Bag’ Make a Good ‘Wind Bag’?: Comparison of Four Fabrics Commonly Used as Exclusion Bags in Studies of Pollination and Reproductive Biology 
Annals of Botany  2004;93(5):603-607.
• Background and Aims Fabrics used in pollination bags may exclude pollen carried by biotic vectors, but have varying degrees of permeability to wind‐borne pollen. The permeability of bags to wind‐borne pollen may have important consequences in studies of pollination and reproductive biology. The permeability of four fabrics commonly used in the construction of pollination bags was examined.
• Methods Deposition of wind‐borne pollen on horizontally and vertically oriented microscope slides was assessed on slides enclosed in pollination bags, as well as on control slides.
• Key Results It was found that the permeability of fabrics to wind‐borne pollen, as measured by deposition on both horizontally and vertically oriented slides, decreased with pore size. However, deposition on horizontal slides was always greater than on vertical slides for a given fabric; this could manifest itself as differential success of pollination of flowers in bags—dependent on flower orientation.
• Conclusions Obviously, bags with mesh size smaller than most pollen grains are impermeable to pollen. However, material for such bags is very expensive. In addition, it was also observed that bags with even moderately small pore size, such as pores (approx. 200 µm) in twisted fibre cotton muslin, offered highly significant barriers to passage of wind‐borne pollen. Such bags are sufficiently effective in most large‐sample‐size reproductive biology studies.
PMCID: PMC4242310  PMID: 15037446
Amphiboly; anemophily; pollen exclusion bag; pollination; pollination bag; pollinator exclusion bag; technique; wind pollination
17.  Potential of essential fatty acid deficiency with extremely low fat diet in lipoprotein lipase deficiency during pregnancy: A case report 
Pregnancy in patients with lipoprotein lipase deficiency is associated with high risk of maternal pancreatitis and fetal death. A very low fat diet (< 10% of calories) is the primary treatment modality for the prevention of acute pancreatitis, a rare but potentially serious complication of severe hypertriglyceridemia. Since pregnancy can exacerbate hypertriglyceridemia in the genetic absence of lipoprotein lipase, a further reduction of dietary fat intake to < 1–2% of total caloric intake may be required during the pregnancy, along with the administration of a fibrate. It is uncertain if essential fatty acid deficiency will develop in the mother and fetus with this extremely low fat diet, or whether fibrates will cross the placenta and concentrate in the fetus.
Case presentation
A 23 year-old gravida 1 woman with primary lipoprotein lipase deficiency was seen at 7 weeks of gestation in the Lipid Clinic for management of severe hypertriglyceridemia that had worsened with pregnancy. While on her habitual fat intake of 10% of total calories, her pregnancy resulted in an exacerbation of the hypertriglyceridemia, which prompted further restriction of fat intake to < 2% of total calories, as well as administration of gemfibrozil at a lower than average dose. The level of gemfibrozil, as the active metabolite, in the venous and arterial fetal cord blood was within the expected therapeutic range for adults. The clinical signs and a biomarker of essential fatty acid deficiency, namely the ratio of 20:3 [n-9] to 20:4 [n-6] fatty acids, were closely monitored throughout her pregnancy. Despite her extremely low fat diet, the levels of essential fatty acids measured in the mother and in the fetal blood immediately postpartum were normal. Normal essential fatty acid levels may have been achieved by the topical application of sunflower oil.
An extremely low fat diet in combination with topical sunflower oil and gemfibrozil administration was safely implemented in pregnancy associated with the severe hypertriglyceridemia of lipoprotein lipase deficiency.
PMCID: PMC544881  PMID: 15610556
18.  Carbapenem Resistance in a Clinical Isolate of Enterobacter aerogenes Is Associated with Decreased Expression of OmpF and OmpC Porin Analogs 
Antimicrobial Agents and Chemotherapy  2002;46(12):3817-3822.
We investigated the mechanism of imipenem resistance in Enterobacter aerogenes strain 810, a clinical isolate from the United States for which the imipenem MIC was 16 μg/ml and the meropenem MIC was 8 μg/ml. An imipenem-susceptible revertant, strain 810-REV, was obtained after multiple passages of the strain on nonselective media. For the revertant, the imipenem MIC was ≤1 μg/ml and the meropenem MIC was ≤0.25 μg/ml. Cefepime MICs also decreased from 8 to 1 μg/ml; however, the MICs of ceftazidime (≥128 μg/ml), cefoxitin (≥32 μg/ml), and cefotaxime (≥64 μg/ml) remained the same. The β-lactamase and porin profiles of the parent, the revertant, and carbapenem-susceptible type strain E. aerogenes ATCC 13048 were determined. Strains 810 and 810-REV each produced two β-lactamases with pIs of 8.2 and 5.4. The β-lactamase activities of the parent and revertant were similar, even after induction with subinhibitory concentrations of imipenem. While 810-REV produced two major outer membrane proteins of 42 and 39 kDa that corresponded to Escherichia coli porins OmpC and OmpF, respectively, the parent strain appeared to produce similar quantities of the 39-kDa protein (OmpF) but decreased amounts of the 42-kDa protein (OmpC). When the parent strain was grown in the presence of imipenem, the 42-kDa protein was not detectable by gel electrophoresis. However, Western blot analysis of the outer membrane proteins of the parent and revertant with polyclonal antisera raised to the OmpC and OmpF analogs of Klebsiella pneumoniae (anti-OmpK36 and anti-OmpK35, respectively) showed that strain 810 expressed only the 42-kDa OmpC analog in the absence of imipenem (the 39-kDa protein was not recognized by the anti-OmpF antisera) and neither the OmpC nor the OmpF analog in the presence of imipenem. The OmpC analog is apparently down-regulated in the presence of imipenem; however, 810-REV expressed both OmpC and OmpF analogs. These data suggest that imipenem resistance in E. aerogenes 810 is primarily associated with the lack of expression of the analogs of the OmpC (42-kDa) and OmpF (39-kDa) outer membrane proteins, which also results in decreased susceptibility to meropenem and cefepime.
PMCID: PMC132746  PMID: 12435682
19.  TEM-71, a Novel Plasmid-Encoded, Extended-Spectrum β-Lactamase Produced by a Clinical Isolate of Klebsiella pneumoniae 
TEM-71, a novel extended-spectrum β-lactamase from a Klebsiella pneumoniae clinical isolate, had an isoelectric point of 6.0 and a substrate profile showing preferential hydrolysis of cefotaxime over ceftazidime. It differed from TEM-1 by two substitutions, Gly238Ser and Glu240Lys, and was under the control of the strong P4 promoter.
PMCID: PMC127224  PMID: 12019125
20.  Characterization of Clinical Isolates of Klebsiella pneumoniae from 19 Laboratories Using the National Committee for Clinical Laboratory Standards Extended-Spectrum β-Lactamase Detection Methods 
Journal of Clinical Microbiology  2001;39(8):2864-2872.
Extended-spectrum β-lactamases (ESBLs) are enzymes found in gram-negative bacilli that mediate resistance to extended-spectrum cephalosporins and aztreonam. In 1999, the National Committee for Clinical Laboratory Standards (NCCLS) published methods for screening and confirming the presence of ESBLs in Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli. To evaluate the confirmation protocol, we tested 139 isolates of K. pneumoniae that were sent to Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) from 19 hospitals in 11 U.S. states. Each isolate met the NCCLS screening criteria for potential ESBL producers (ceftazidime [CAZ] or cefotaxime [CTX] MICs were ≥2 μg/ml for all isolates). Initially, 117 (84%) isolates demonstrated a clavulanic acid (CA) effect by disk diffusion (i.e., an increase in CAZ or CTX zone diameters of ≥5 mm in the presence of CA), and 114 (82%) demonstrated a CA effect by broth microdilution (reduction of CAZ or CTX MICs by ≥3 dilutions). For five isolates, a CA effect could not be determined initially by broth microdilution because of off-scale CAZ results. However, a CA effect was observed in two of these isolates by testing cefepime and cefepime plus CA. The cefoxitin MICs for 23 isolates that failed to show a CA effect by broth microdilution were ≥32 μg/ml, suggesting either the presence of an AmpC-type β-lactamase or porin changes that could mask a CA effect. By isoelectric focusing (IEF), 7 of the 23 isolates contained a β-lactamase with a pI of ≥8.3 suggestive of an AmpC-type β-lactamase; 6 of the 7 isolates were shown by PCR to contain both ampC-type and blaOXA genes. The IEF profiles of the remaining 16 isolates showed a variety of β-lactamase bands, all of which had pIs of ≤7.5. All 16 isolates were negative by PCR with multiple primer sets for ampC-type, blaOXA, and blaCTX-M genes. In summary, 83.5% of the K. pneumoniae isolates that were identified initially as presumptive ESBL producers were positive for a CA effect, while 5.0% contained β-lactamases that likely masked the CA effect. The remaining 11.5% of the isolates studied contained β-lactamases that did not demonstrate a CA effect. An algorithm based on phenotypic analyses is suggested for evaluation of such isolates.
PMCID: PMC88252  PMID: 11474005
21.  Novel Carbapenem-Hydrolyzing β-Lactamase, KPC-1, from a Carbapenem-Resistant Strain of Klebsiella pneumoniae 
A Klebsiella pneumoniae isolate showing moderate to high-level imipenem and meropenem resistance was investigated. The MICs of both drugs were 16 μg/ml. The β-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. The strain was also resistant to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing studies demonstrated three β-lactamases, with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). The presence of blaSHV and blaTEM genes was confirmed by specific PCRs and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the β-lactamase with a pI of 6.7, KPC-1 (K. pneumoniae carbapenemase-1), was encoded on an approximately 50-kb nonconjugative plasmid. The gene, blaKPC-1, was cloned in E. coli and shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of the novel carbapenem-hydrolyzing β-lactamase, KPC-1, showed 45% identity to the pI 9.7 carbapenem-hydrolyzing β-lactamase, Sme-1, from Serratia marcescens S6. Hydrolysis studies showed that purified KPC-1 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and monobactams. KPC-1 had the highest affinity for meropenem. The kinetic studies also revealed that clavulanic acid and tazobactam inhibited KPC-1. An examination of the outer membrane proteins of the parent K. pneumoniae strain demonstrated that the strain does not express detectable levels of OmpK35 and OmpK37, although OmpK36 is present. We concluded that carbapenem resistance in K. pneumoniae strain 1534 is mainly due to production of a novel Bush group 2f, class A, carbapenem-hydrolyzing β-lactamase, KPC-1, although alterations in porin expression may also play a role.
PMCID: PMC90438  PMID: 11257029
22.  Possible Horizontal Transfer of the vanB2 Gene among Genetically Diverse Strains of Vancomycin-Resistant Enterococcus faecium in a Korean Hospital 
Journal of Clinical Microbiology  2001;39(3):1165-1168.
A total of 25 isolates of vanB-containing Enterococcus faecium were recovered from patients in a single Korean hospital over a 20-month period. There were two distinct vanB2 patterns among the 11 pulsed-field gel electrophoresis types; 17 contained the prototype vanB2 and 8 contained a novel vanB2 with a 177-bp deletion in vanYB. Both vanB2 genes were transmissible in vitro at a mean frequency of 1.1 × 10−8 transconjugants/donor. These results suggest the horizontal spread of vanB2 is occurring among genetically diverse strains of E. faecium in Korean hospitals.
PMCID: PMC87896  PMID: 11230450
23.  Characterization of the Extended-Spectrum β-Lactamase Reference Strain, Klebsiella pneumoniae K6 (ATCC 700603), Which Produces the Novel Enzyme SHV-18 
Klebsiella pneumoniae K6 (ATCC 700603), a clinical isolate, is resistant to ceftazidime and other oxyimino-β-lactams. A consistent reduction in the MICs of oxyimino-β-lactams by at least 3 twofold dilutions in the presence of clavulanic acid confirmed the utility of K. pneumoniae K6 as a quality control strain for extended-spectrum β-lactamase (ESBL) detection. Isoelectric-focusing analysis of crude lysates of K6 demonstrated a single β-lactamase with a pI of 7.8 and a substrate profile showing preferential hydrolysis of cefotaxime compared to ceftazidime. PCR analysis of total bacterial DNA from K6 identified the presence of a blaSHV gene. K6 contained two large plasmids with molecular sizes of approximately 160 and 80 kb. Hybridization of plasmid DNA with a blaSHV-specific probe indicated that a blaSHV gene was encoded on the 80-kb plasmid, which was shown to transfer resistance to ceftazidime in conjugal mating experiments with Escherichia coli HB101. DNA sequencing of this blaSHV-related gene revealed that it differs from blaSHV-1 at nine nucleotides, five of which resulted in amino acid substitutions: Ile to Phe at position 8, Arg to Ser at position 43, Gly to Ala at position 238, and Glu to Lys at position 240. In addition to the production of this novel ESBL, designated SHV-18, analysis of the outer membrane proteins of K6 revealed the loss of the OmpK35 and OmpK37 porins.
PMCID: PMC90073  PMID: 10952583

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