Clostridium difficile: Investigating Transmission Patterns Between Infected and Colonized Patients Using Whole Genome Sequencing.

Background: Whole genome sequencing (WGS) studies can enhance our understanding of the role of patients with asymptomatic Clostridium difficile colonization in transmission. Methods: Isolates obtained from patients with Clostridium difficile infection (CDI) and colonization identified in a study conducted during 2006-2007 at 6 Canadian hospitals underwent typing by pulsed-field gel electrophoresis, multilocus sequence typing, and WGS. Isolates from incident CDI cases not in the initial study were also sequenced where possible. Ward movement and typing data were combined to identify plausible donors for each CDI case, as defined by shared time and space within predefined limits. Proportions of plausible donors for CDI cases that were colonized, infected, or both were examined. Results: Five hundred fifty-four isolates were sequenced successfully, 353 from colonized patients and 201 from CDI cases. The NAP1/027/ST1 strain was the most common strain, found in 124 (62%) of infected and 92 (26%) of colonized patients. A donor with a plausible ward link was found for 81 CDI cases (40%) using WGS with a threshold of ≤2 single nucleotide polymorphisms to determine relatedness. Sixty-five (32%) CDI cases could be linked to both infected and colonized donors. Exclusive linkages to infected and colonized donors were found for 28 (14%) and 12 (6%) CDI cases, respectively. Conclusions: Colonized patients contribute to transmission, but CDI cases are more likely linked to other infected patients than colonized patients in this cohort with high rates of the NAP1/027/ST1 strain, highlighting the importance of local prevalence of virulent strains in determining transmission dynamics.

Host and pathogen factors for Clostridium difficile infection and colonization.

BACKGROUND: Clostridium difficile infection is the leading cause of health care-associated diarrhea, and the bacterium can also be carried asymptomatically. The objective of this study was to identify host and bacterial factors associated with health care-associated acquisition of C. difficile infection and colonization. METHODS: We conducted a 15-month prospective study in six Canadian hospitals in Quebec and Ontario. Demographic information, known risk factors, potential confounding factors, and weekly stool samples or rectal swabs were collected. Pulsed-field gel electrophoresis (PFGE) was performed on C. difficile isolates to determine the genotype. Levels of serum antibodies against C. difficile toxins A and B were measured. RESULTS: A total of 4143 patients were included in the study; 117 (2.8%) and 123 (3.0%) had health care-associated C. difficile infection and colonization, respectively. Older age and use of antibiotics and proton-pump inhibitors were significantly associated with health care-associated C. difficile infection. Hospitalization in the previous 2 months; use of chemotherapy, proton-pump inhibitors, and H(2) blockers; and antibodies against toxin B were associated with health care-associated C. difficile colonization. Among patients with health care-associated C. difficile infection and those with colonization, 62.7% and 36.1%, respectively, had the North American PFGE type 1 (NAP1) strain. CONCLUSIONS: In this study, health care-associated C. difficile infection and colonization were differentially associated with defined host and pathogen variables. The NAP1 strain was predominant among patients with C. difficile infection, whereas asymptomatic patients were more likely to be colonized with other strains. (Funded by the Consortium de Recherche sur le Clostridium difficile.).

Use of amplified-fragment length polymorphism to study the ecology of Campylobacter jejuni in environmental water and to predict multilocus sequence typing clonal complexes.

We determined the genetic variability among water isolates of Campylobacter jejuni by using amplified-fragment length polymorphism (AFLP) and multilocus sequence typing (MLST). Across a highly diverse collection of isolates, AFLP clusters did not correlate with MLST clonal complexes, suggesting that AFLP is not reliable for deciphering population genetic relationships and may be problematic for larger epidemiologic analyses.

Staphylococcus aureus sigma B-dependent emergence of small-colony variants and biofilm production following exposure to Pseudomonas aeruginosa 4-hydroxy-2-heptylquinoline-N-oxide.

BACKGROUND: Staphylococcus aureus and Pseudomonas aeruginosa are often found together in the airways of cystic fibrosis (CF) patients. It was previously shown that the P. aeruginosa exoproduct 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) suppresses the growth of S. aureus and provokes the emergence of small-colony variants (SCVs). The presence of S. aureus SCVs as well as biofilms have both been associated with chronic infections in CF. RESULTS: We demonstrated that HQNO stimulates S. aureus to form a biofilm in association with the formation of SCVs. The emergence of SCVs and biofilm production under HQNO exposure was shown to be dependent on the activity of the stress- and colonization-related alternative sigma factor B (SigB). Analysis of gene expression revealed that exposure of a prototypical S. aureus strain to HQNO activates SigB, which was leading to an increase in the expression of the fibronectin-binding protein A and the biofilm-associated sarA genes. Conversely, the quorum sensing accessory gene regulator (agr) system and the alpha-hemolysin gene were repressed by HQNO. Experiments using culture supernatants from P. aeruginosa PAO1 and a double chamber co-culture model confirmed that P. aeruginosa stimulates biofilm formation and activates SigB in a S. aureus strain isolated from a CF patient. Furthermore, the supernatant from P. aeruginosa mutants unable to produce HQNO induced the production of biofilms by S. aureus to a lesser extent than the wild-type strain only in a S. aureus SigB-functional background. CONCLUSIONS: These results suggest that S. aureus responds to HQNO from P. aeruginosa by forming SCVs and biofilms through SigB activation, a phenomenon that may contribute to the establishment of chronic infections in CF patients.

Nox2-containing NADPH oxidase deficiency confers protection from hindlimb ischemia in conditions of increased oxidative stress.

OBJECTIVE: Because Nox2-containing NADPH oxidase is a major source of ROS in the vasculature, we investigated its potential role for the modulation of ischemia-induced neovascularization in conditions of increased oxidative stress. METHODS AND RESULTS: To mimic a clinical situation of increased oxidative stress, mice were exposed to cigarette smoke before and after the surgical induction of hindlimb ischemia. Nox2 expression and oxidative stress in ischemic tissues were significantly increased in wild-type mice, but not in mice deficient for the Nox2-containing NADPH oxidase (Nox2(-/-)). Nox2(-/-) mice demonstrated faster blood flow recovery, increased capillary density in ischemic muscles, and improved endothelial progenitor cell functional activities compared to Nox2(+/+) mice. In addition, Nox2 deficiency was associated with increased antioxidant and nitrite concentrations in plasma, together with a preserved expression of eNOS in ischemic tissues. In vitro, Nox2(-/-) endothelial cells exhibit resistance against superoxide induction and improved VEGF-dependent angiogenic activities compared to Nox2(+/+) endothelial cells. Importantly, the beneficial effects of Nox2 deficiency on neovascularization in vitro and in vivo were lost after treatment with the NO inhibitor L-NAME. CONCLUSIONS: Nox2-containing NADPH oxidase deficiency protects against ischemia in conditions of increased oxidative stress. The mechanism involves improved neovascularization through a reduction of ROS formation, preserved activation of the VEGF/NO angiogenic pathway, and improved functional activities of endothelial progenitor cells.

Thermotolerant coliforms are not a good surrogate for Campylobacter spp. in environmental water.

This study aimed to assess the importance of quantitatively detecting Campylobacter spp. in environmental surface water. The prevalence and the quantity of Campylobacter spp., thermotolerant coliforms, and Escherichia coli in 2,471 samples collected weekly, over a 2-year period, from 13 rivers and 12 streams in the Eastern Townships, Québec, Canada, were determined. Overall, 1,071 (43%), 1,481 (60%), and 1,463 (59%) samples were positive for Campylobacter spp., thermotolerant coliforms, and E. coli, respectively. There were weak correlations between the weekly distributions of Campylobacter spp. and thermotolerant coliforms (Spearman's rho coefficient = 0.27; P = 0.008) and between the quantitative levels of the two classes of organisms (Kendall tau-b correlation coefficient = 0.233; P < 0.0001). Well water samples from the Eastern Townships were also tested. Five (10%) of 53 samples from private surface wells were positive for Campylobacter jejuni, of which only 2 were positive for thermotolerant coliforms. These findings suggest that microbial monitoring of raw water by using only fecal indicator organisms is not sufficient for assessing the occurrence or the load of thermophilic Campylobacter spp. Insights into the role of environmental water as sources for sporadic Campylobacter infection will require genus-specific monitoring techniques.

A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality.

BACKGROUND: In March 2003, several hospitals in Quebec, Canada, noted a marked increase in the incidence of Clostridium difficile-associated diarrhea. METHODS: In 2004 we conducted a prospective study at 12 Quebec hospitals to determine the incidence of nosocomial C. difficile-associated diarrhea and its complications and a case-control study to identify risk factors for the disease. Isolates of C. difficile were typed by pulsed-field gel electrophoresis and analyzed for binary toxin genes and partial deletions in the toxin A and B repressor gene tcdC. Antimicrobial susceptibility was evaluated in a subgroup of isolates. RESULTS: A total of 1703 patients with 1719 episodes of nosocomial C. difficile-associated diarrhea were identified. The incidence was 22.5 per 1000 admissions. The 30-day attributable mortality rate was 6.9 percent. Case patients were more likely than matched controls to have received fluoroquinolones (odds ratio, 3.9; 95 percent confidence interval, 2.3 to 6.6) or cephalosporins (odds ratio, 3.8; 95 percent confidence interval, 2.2 to 6.6). A predominant strain, resistant to fluoroquinolones, was found in 129 of 157 isolates (82.2 percent), and the binary toxin genes and partial deletions in the tcdC gene were present in 132 isolates (84.1 percent). CONCLUSIONS: A strain of C. difficile that was resistant to fluoroquinolones and had binary toxin and a partial deletion of the tcdC gene was responsible for this outbreak of C. difficile-associated diarrhea. Exposure to fluoroquinolones or cephalosporins was a risk factor.

Multilocus sequence typing of Campylobacter jejuni isolates from humans, chickens, raw milk, and environmental water in Quebec, Canada.

Molecular strain typing is essential for deciphering the epidemiology of Campylobacter jejuni infections. We applied two different methods, multilocus sequence typing (MLST) and analysis of the flaA short variable repeat (SVR), to 289 isolates (163 human, 56 chicken, 34 raw milk, and 36 environmental water isolates) collected in the province of Québec, Canada, over 3 years; in addition, the analysis included the pulsed-field gel electrophoresis (PFGE) typing results for a subset of 131 isolates studied previously. MLST defined 96 sequence types (STs) and 20 clonal complexes (CCs), including 49 STs (73 isolates, 25%) and 39 alleles not previously documented in an international database. The frequency of new STs was significantly higher among water isolates than among isolates from other sources (18/36 [50%] and 55/253 [22%], respectively; P < 0.001). Nine of the 10 most prevalent CCs included isolates from humans and at least one other source; five CCs comprised exclusively or mostly human and chicken isolates. However, water and milk were the predominant nonhuman sources among the remaining CCs, suggesting that sporadic C. jejuni infections in humans may frequently arise from sources other than chickens. All three typing systems were discriminatory (discriminatory index > 0.9). Among 131 isolates analyzed by PFGE, each of the 20 types represented by two or more isolates corresponded to a single CC. In contrast, among the 14 most prevalent types detected by analysis of the flaA SVR (5 to 27 isolates each), 8 (57%) included isolates that represented multiple different CCs. The basis for these discordant results was uncertain. Antimicrobial resistance was randomly distributed among the CCs and appeared to be more closely related to the source of an isolate than its genotype. Although MLST is labor-intensive and expensive, it remains the single best method for the genotyping of C. jejuni isolates and deciphering the epidemiologic relationships among isolates.

Moderate consumption of red wine (cabernet sauvignon) improves ischemia-induced neovascularization in ApoE-deficient mice: effect on endothelial progenitor cells and nitric oxide.

Moderate consumption of red wine is associated with a decreased incidence of cardiovascular diseases in populations with relatively high amount of fat in the diet. However, the mechanisms involved in this protective effect are not completely understood. Here we show that moderate consumption of red wine (equivalent to 2 glasses/day in humans) but not ethanol only, improves blood flow recovery by 32% after hindlimb ischemia in hypercholesterolemic ApoE-deficient mice. In ischemic tissues, red wine consumption reduces oxidative stress and increases capillary density by 46%. Endothelial progenitor cells (EPCs) have been shown to have an important role in postnatal neovascularization. We found that the number of EPCs is increased by 60% in ApoE mice exposed to red wine. Moreover, the migratory capacity of EPCs is significantly improved in red wine-drinking mice. The wine used in our study is a cabernet sauvignon from Languedoc-Roussillon, France, which contains a relatively high concentration (4-6 mg/L) of the polyphenolic antioxidant resveratrol. We demonstrate that resveratrol can rescue oxidized low-density lipoprotein (oxLDL)-induced impairment of in vitro angiogenic activities in human umbilical vein endothelial cells (HUVECs). Resveratrol exposure is also associated with increased activation of Akt/eNOS together with a restoration of nitric oxide production in HUVECs exposed to oxLDL. Our study suggests that moderate consumption of red wine improves ischemia-induced neovascularization in high-cholesterol conditions by increasing the number and the functional activities of EPCs and by restoring the Akt-eNOS-NO pathway.

Plesiobrachytherapy for chest wall recurrences of breast cancer after mastectomy and radiotherapy for breast cancer.

PURPOSE: The purpose of the study was to evaluate the results of high-dose-rate plesiobrachytherapy for local relapse after mastectomy and radiotherapy in terms of both local control and survival. METHODS: We reviewed retrospectively 43 patients who experienced a chest wall relapse of breast cancer after local excision (22 patients) or not (21 patients). Patients were treated with an individually designed mold with four to six fractions of 3-6 Gy high-dose-rate brachytherapy, two fractions per week. Mean total dose was 24 Gy. RESULTS: After surgical resection, the 3- and 5-year local control rates were 80% and 73%, respectively. For nonresectable patients, the overall response rate was 86%, and the 3-year infield local control and chest wall local control were 51% and 26%, respectively. The 5-year survival rate was 50.5% for the whole population, 62% after surgery, and 45.4% for irresectable patients. Acute Grade 2 or 3 toxicity occurred in 43% of the patients, resolving in a few days. Two patients had a local necrosis lasting 3 to 7 months. Late toxicity was observed in 5 patients. CONCLUSIONS: High-dose-rate plesiobrachytherapy is a simple outpatient technique to treat chest wall local relapse of breast cancer. As a reirradiation technique, its tolerance is acceptable. This technique may obtain long-term local control after incomplete surgery; in case of nonresectable disease, a high response rate was observed, which might improve the quality of life of these patients.

Comparison of antimicrobial resistance of Campylobacter jejuni isolated from humans, chickens, raw milk, and environmental water in Québec.

This study compares the occurrence of antimicrobial resistance to erythromycin, ciprofloxacin, and tetracycline among 384 Campylobacter jejuni isolates from humans (245), fresh whole retail chickens (56), raw milk (33), and environmental water (41) collected between 2000 and 2003 in Québec, Canada. Resistance to ciprofloxacin was significantly more frequent in human isolates acquired abroad than in those acquired locally (50 versus 5.9%; P < 0.001); ciprofloxacin resistance was almost absent in water, chicken, and raw milk isolates. In contrast, resistance to erythromycin was significantly more common in chicken than in locally acquired human isolates (16 versus 3.0%, respectively; P < 0.001); no erythromycin resistance was found among water, raw milk, and human isolates acquired abroad. Resistance to tetracycline was significantly more common in chicken and human isolates acquired locally (58.9 and 45.8%, respectively) than in raw milk and water isolates (9.1 and 7.3%, respectively, P < 0.001). Tetracycline resistance was also observed in 44.4% of human isolates acquired abroad. No human isolate was resistant to both ciprofloxacin and erythromycin, but one chicken isolate was resistant to all three antimicrobial agents. Our results suggest that from 2000 to 2003 in Québec, antimicrobial resistance remained stable among locally acquired C. jejuni human clinical isolates and might even have decreased. However, the high erythromycin resistance rate observed among chicken isolates is concerning because of the risk of transmission of such isolates to humans. Additional studies are needed to monitor trends in antimicrobial resistance among food, environment, and human C. jejuni isolates as well as antibiotic use in animals.

10-Year follow-up of 621 patients treated using high-dose rate brachytherapy as ambulatory boost technique in conservative breast cancer treatment.

PURPOSE: Breast conserving treatment, consisting of lumpectomy followed by whole-breast irradiation, is considered the standard of care in early-stage breast cancer. Randomized studies have reported that delivering boost doses to tumor bed improves local control rates, particularly in young women. This study sought to evaluate local control and cosmetic results of delivering boost doses using a high-dose-rate (HDR) brachytherapy (HDRBT) in breast cancer conservative treatment. METHODS: We included 621 T1-T2, N0-N1 breast cancer patients who underwent lumpectomy, external irradiation (44Gy over 5weeks), and a boost dose of two fractions of 5Gy to the tumor bed by means of HDR iridium brachytherapy. Implantation was performed during the lumpectomy or 2-3weeks after external irradiation. Population characteristics were as follows: pTis=11.6%; pT1=63.4%; pT2=25.0%; median tumor size=1.5cm; histology: ductal carcinoma in situ (DCIS): 72 (11.6%); infiltrative ductal carcinoma (IDC): 471 (75.8%); other: 78 (12.6%). For IDCs, the surgical margins were positive in 38cases (6.2%) and an extensive intraductal component was present in 254 cases. RESULTS: With a median follow-up of 10.3years, 47 local relapses were observed (10-year local relapse rate: 7.4%). Small-volume implantation (V100<45cc) and ductal carcinoma in situ histology both significantly correlated with local relapse. The 10-year overall survival was 91%. Cosmetic results were evaluated in 264patients, proving excellent in 58 (22%), good in 153 (58%), fair in 40 (15%), and poor in 13 (5%). CONCLUSIONS: Small implant volume and ductal carcinoma in situ histology significantly correlated with local relapse following HDR brachytherapy dose boost in breast cancer conservative treatment. Modern image-guided breast brachytherapy techniques using surgical clips as a guide may decrease potential treatment targeting errors, consequently improving local control without increasing toxicity.

Circulating endothelial progenitor cells from healthy smokers exhibit impaired functional activities.

OBJECTIVE: Endothelial dysfunction is one of the earliest pathological effects of cigarette smoking. It has recently been suggested that endothelial progenitor cells (EPCs) could contribute to ongoing endothelial maintenance and repair. Accordingly, we tested the hypothesis that cigarette smoking is associated with EPC dysfunction. METHODS AND RESULTS: EPCs were isolated from the peripheral venous blood of 15 healthy smokers and 11 age-matched nonsmokers. The number of EPCs was significantly reduced in smokers versus control subjects (51.6+/-1.9 versus 120.3+/-10.0 per power field, p<0.001). Moreover, the functional activities of EPCs isolated from smokers were severely compromised. First, the proliferative and migratory response of EPCs isolated from smokers were reduced by 75% and 19%, respectively (p<0.05). Second, EPCs from smokers showed an important decreased adherence to HUVECs that had been previously activated with tumor necrosis factor-alpha (TNF-alpha) (p<0.01). Finally, the participation of EPCs to tube formation in a matrigel assay was reduced by 38% in smokers versus control subjects (p<0.001). We found that EPCs from smokers had a significant reduction in the expression of the endothelial cell-specific markers (VE-cadherin, KDR, and vWF). Moreover, ROS formation was significantly increased in EPCs from smokers, whereas the serum antioxidant and nitrite levels of smokers were reduced and correlated with impaired EPC number and functional activity. CONCLUSIONS: Cigarette smoking is associated with a reduced number of EPCs together with an important impairment of EPC differentiation and functional activities. Our results suggest that EPC dysfunction could contribute to impair blood vessel healing and growth in smokers.

First reported case of Campylobacter lanienae enteritis in a human.

INTRODUCTION: Campylobacters are the most frequently identified bacteria causing diarrhoea in humans worldwide. Campylobacter lanienae was isolated for the first time in 2000 from faecal samples of two asymptomatic abattoir workers in Switzerland during a routine hygiene screen, but has never been associated with human disease. CASE PRESENTATION: At hospital admission, the patient reported diarrhoea, lower abdominal cramps, nausea, one episode of bilious vomiting and low-grade fever of 38 °C. The patient was having 10 or more diarrheic stools per day as well as during the night, and had noticed blood mixed with the stools on several occasions. Stool cultures were negative for species of Salmonella and Shigella, Escherichia coli O157:H7 and Yersinia enterocolitica, but were positive for C. lanienae. Identification was made by classical biochemical testing, as well as 16S rRNA gene and cpn60 sequencing. The patient slowly improved without antibiotic treatment and was discharged nine days after admission with complete resolution of symptoms. CONCLUSION: On the whole it seems very likely that C. lanienae was the causative agent. Clinical microbiologists should be aware of this micro-organism which can be identified by phenotypic and molecular methods. The real burden of C. lanienae infection in humans might be underestimated and should be further investigated as a potential cause of human diarrhoea disease.

Inhibition of vascular smooth muscle cell proliferation and neointimal formation in injured arteries by a novel, oral mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor.

BACKGROUND: Mitogen-activated protein kinases (MAPKs) are rapidly induced after arterial injury in different animal models. However, their precise role in vascular smooth muscle cell (VSMC) proliferation and neointimal formation in vivo remains to be determined. METHODS AND RESULTS: We investigated the properties of a novel, selective inhibitor of the upstream kinase, MAPK/extracellular signal-regulated kinase, that is orally active (PD0185625). In vitro, PD0185625 was shown to abrogate p44/p42 MAPK activation in VSMCs after serum stimulation. This was associated with a dose-dependent inhibition of VSMC proliferation. In vivo, PD0185625 was administered orally to rats (200 mg x kg(-1) x d(-1)) beginning 2 days before balloon injury of the left carotid artery and for 2 weeks thereafter. Treatment with PD0185625 led to nearly complete inhibition of p44/p42 MAPK activation after balloon injury. This resulted in a significant decrease in VSMC proliferation (BrdU incorporation) at day 7 after injury. Moreover, neointimal formation was significantly reduced in PD0185625-treated animals at 14 and 28 days after arterial injury. We found that PD0185625 did not increase the rate of apoptotic cell death but prevented cell cycle progression and induced a G1 block. CONCLUSIONS: PD0185625 reduced neointimal formation after arterial injury. The mechanism involved inhibition of VSMC proliferation via a G1 block of the cell cycle. Orally active selective MAPK inhibitors could represent a novel therapeutic approach for vascular diseases.

Age-dependent impairment of reendothelialization after arterial injury: role of vascular endothelial growth factor.

BACKGROUND: The mechanisms responsible for the association between advanced age and atherosclerotic diseases are not clear. Because atherosclerosis develops in response to local endothelial injuries, we investigated the effect of aging on vascular healing and reendothelialization. METHODS AND RESULTS: Endothelium denudation was performed by balloon angioplasty of the iliac arteries in young and old New Zealand White rabbits. Planimetric analysis after Evans Blue staining at 28 days after injury showed a significant decrease in reendothelialization in old versus young animals, which was associated with an important increase in neointimal formation in old rabbits. Vascular endothelial growth factor (VEGF) was rapidly induced after balloon injury. However, arterial VEGF expression was significantly reduced in old versus young animals. To confirm the role of VEGF in the age-dependent impairment of reendothelialization, an adenoviral vector encoding for VEGF(165) (adeno-VEGF) was locally delivered at the time of iliac artery angioplasty. Compared with animals treated with the control vector (adeno-betaGal), reendothelialization was significantly improved and neointimal formation reduced in old rabbits treated with adeno-VEGF. CONCLUSIONS: These results document for the first time an age-dependent impairment of reendothelialization after arterial injury. Our study indicates that VEGF supplementation may represent a useful strategy to accelerate reendothelialization and improve vascular healing in the context of aging.

Inhibition of hypoxia-induced angiogenesis by cigarette smoke exposure: impairment of the HIF-1alpha/VEGF pathway.

Smoking is a major risk factor for atherosclerotic diseases. However, the impact of cigarette smoke exposure on neovascularization that develops in response to tissue ischemia is unknown. Here we demonstrate that cigarette smoke extracts inhibit hypoxia-induced in vitro angiogenesis (matrigel assay) in human umbilical vascular endothelial cells. In vivo, mice exposed to cigarette smoke (MES) were shown to have a significant impairment of angiogenesis following surgically induced hindlimb ischemia. The reduced angiogenic response in MES was documented by Laser Doppler flow perfusion studies and capillary density analyses in ischemic hindlimbs. Inhibition of angiogenesis by cigarette smoke in vitro and in vivo was associated with a reduced expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in hypoxic conditions. Administration of an adenoviral vector encoding for HIF-1alpha/VP16, a hybrid transcription factor that is stable in hypoxic and normoxic conditions, restored VEGF expression and completely reversed the cigarette smoke inhibition of angiogenesis in hypoxic conditions. Taken together, these results suggest that cigarette smoke exposure impairs angiogenesis by inhibiting VEGF through decreased expression of HIF-1alpha in hypoxic conditions.

Role of p44/p42 MAP kinase in the age-dependent increase in vascular smooth muscle cell proliferation and neointimal formation.

OBJECTIVE: Age-dependent increase in vascular smooth muscle cell (VSMC) proliferation is thought to contribute to the pathology of atherosclerotic diseases. In this study, we investigated the role of mitogen-activated protein kinases (MAPKs) on VSMC proliferation and neointimal formation in the context of aging. METHODS AND RESULTS: VSMCs were isolated from the aorta of young and old rabbits. The proliferative index after serum stimulation was significantly increased in old versus young VSMCs. This was associated with a significant and specific age-dependent increase in p44/p42 MAPK activation. Treatment with MEK inhibitor PD98059 successfully inhibited p44/p42 MAPK activities and VSMC proliferation. These results were confirmed in vivo using a model of balloon injury in rabbit iliac arteries. p44/p42 MAPK activities were rapidly induced by angioplasty in young and old animals. However, the levels of p44/p42 MAPK activities achieved in arteries of old rabbits were significantly higher than those of young rabbits. This was associated with a higher cellular proliferative index and a significant increase in neointimal formation in old animals. Local delivery of PD98059 in old rabbits successfully inhibited p44/p42 MAPK activities after angioplasty, which led to a significant reduction in cellular proliferation and neointimal formation in treated animals. CONCLUSIONS: Our study suggests for the first time that increased p44/p42 MAPK activation contributes to augmented VSMC proliferation and neointimal formation with aging. p44/p42 MAPK inhibition could represent a novel therapeutic avenue against atherosclerotic diseases.

Antibiotic resistance and hypermutability of Escherichia coli O157 from feedlot cattle treated with growth-promoting agents.

In a longitudinal study (165 days), we investigated the effect of growth-promoting agents (monensin and trenbolone acetate-estradiol) and an antibiotic (oxytetracycline) on the incidence in feedlot steers of Escherichia coli O157, including antibiotic-resistant and hypermutable isolates. Eighty steers in 16 pens were treated with eight combinations of promoters, and each treatment was duplicated. Fecal samples were collected at nine different sampling times for detection of E. coli O157. Overall, 50 E. coli O157 isolates were detected in treated animals, and none were found in untreated animals. Compared with untreated controls, there was a significant association between the utilization of growth-promoting agents or antibiotics and the shedding of E. coli O157 at day 137 (P = 0.03), when a prevalence peak was observed and 50% of the isolates were detected. Multiplex PCR assays were conducted for some virulence genes. PCR results indicated that all except one isolate possessed at least the Shiga toxin gene stx2. MICs for 12 antibiotics were determined, and eight oxytetracycline-resistant E. coli O157 strains were identified. Antibiotic-resistant strains were considered a distinct subpopulation of E. coli O157 by pulsed-field gel electrophoresis typing. Seven of these antibiotic-resistant strains were isolated early in the study (on or before day 25), and among them two were also hypermutable as determined by rifampin mutation frequencies. The proportion of hypermutable strains among E. coli O157 isolates remained relatively constant throughout the study period. These results indicate that the use of growth-promoting agents and antibiotics in beef production may increase the risk of environmental contamination by E. coli O157.

Predictors of asymptomatic Clostridium difficile colonization on hospital admission.

BACKGROUND: Clostridium difficile (CD) is the leading cause of health care-associated diarrhea and can result in asymptomatic carriage. Rates of asymptomatic CD colonization on hospital admission range from 1.4%-21%. The objective of this study was to evaluate host and bacterial factors associated with colonization on admission. METHODS: The Consortium de recherche québécois sur le Clostridium difficile study provided data for analysis, including demographic information, known risk factors, and potential confounding factors, prospectively collected for 5,232 patients from 6 hospitals in Quebec and Ontario over 15 months from 2006-2007. Stool or rectal swabs were obtained for culture on admission. Pulsed-field gel electrophoresis was performed on the isolates. The presence of antibody against CD toxins A and B was measured. RESULTS: There were 212 (4.05%) patients colonized with CD on admission, and 5,020 patients were not colonized with CD. Multivariate logistic regression analysis showed that hospitalization within the last 12 months, use of corticosteroids, prior CD infection, and presence of antibody against toxin B were associated with colonization on admission. Of patients colonized on admission, 79.4% had non-NAP1, non-NAP2 strains. CONCLUSION: There are identifiable risk factors among asymptomatic CD carriers that could serve in their detection and provide a basis for targeted screening.