Diet alters age-related remodeling of aortic collagen in mice susceptible to atherosclerosis.

Vascular cells restructure extracellular matrix in response to aging or changes in mechanical loading. Here, we characterized collagen architecture during age-related aortic remodeling in atherosclerosis-prone mice. We hypothesized that changes in collagen fiber orientation reflect an altered balance between passive and active forces acting on the arterial wall. We examined two factors that can alter this balance, endothelial dysfunction and reduced smooth muscle cell (SMC) contractility. Collagen fiber organization was visualized by second-harmonic generation microscopy in aortic adventitia of apolipoprotein E (apoE) knockout (KO) mice at 6 wk and 6 mo of age on a chow diet and at 7.5 mo of age on a Western diet (WD), using image analysis to yield mean fiber orientation. Adventitial collagen fibers became significantly more longitudinally oriented with aging in apoE knockout mice on chow diet. Conversely, fibers became more circumferentially oriented with aging in mice on WD. Total collagen content increased significantly with age in mice fed WD. We compared expression of endothelial nitric oxide synthase and acetylcholine-mediated nitric oxide release but found no evidence of endothelial dysfunction in older mice. Time-averaged volumetric blood flow in all groups showed no significant changes. Wire myography of aortic rings revealed decreases in active stress generation with age that were significantly exacerbated in WD mice. We conclude that the aorta displays a distinct remodeling response to atherogenic stimuli, indicated by altered collagen organization. Collagen reorganization can occur in the absence of altered hemodynamics and may represent an adaptive response to reduced active stress generation by vascular SMCs.NEW & NOTEWORTHY The following major observations were made in this study: 1) aortic adventitial collagen fibers become more longitudinally oriented with aging in apolipoprotein E knockout mice fed a chow diet; 2) conversely, adventitial collagen fibers become more circumferentially oriented with aging in apoE knockout mice fed a high-fat diet; 3) adventitial collagen content increases significantly with age in mice on a high-fat diet; 4) these alterations in collagen organization occur largely in the absence of hemodynamic changes; and 5) circumferential reorientation of collagen is associated with decreased active force generation (contractility) in aged mice on a high-fat diet.

Hepatic Histology in Treatment-naïve Children With Chronic Hepatitis B Infection Living in the United States and Canada.

OBJECTIVES: Chronic hepatitis B virus infection is a major cause of morbidity and mortality. The aim of the study is to describe the hepatic histology in children chronically infected with hepatitis B virus living in the United States and Canada. METHODS: Liver biopsies of 134 treatment-naïve children with chronic hepatitis B virus infection were scored for inflammation, fibrosis, and other histological features, and correlated with clinical and laboratory data. RESULTS: Sixty percentage of subjects acquired the infection vertically, 51% were male, and 69% were hepatitis B e antigen-positive at the time of the biopsy. Hepatitis B DNA levels were generally high (mean 7.70 log IU/mL), as was serum alanine aminotransferase (median 120 U/L). Using the Ishak-modified histology activity index scoring system, interface hepatitis was mild in 31%, moderate in 61%, and severe in 6%. Lobular inflammation was mild in 54%, moderate in 29%, and marked in 7%. Portal inflammation was mild in 38% and moderate in 62% of subjects. Eighteen percentage had no fibrosis, 59% had portal expansion without bridging fibrosis, 19% had bridging fibrosis, and 4% had cirrhosis. Alanine aminotransferase positively correlated with inflammation and fibrosis. Neither age, duration of infection, nor Hepatitis B virus DNA levels correlated with fibrosis. Fibrosis-4 index did not correlate with fibrosis but correlated with inflammation. Aspartate aminotransferase/platelet ratio index correlated with both inflammation and fibrosis. CONCLUSIONS: Chronic hepatitis B virus infection results in significant inflammation and fibrosis during childhood. Serum alanine aminotransferase is a strong indicator of the severity and extent of hepatic inflammation and fibrosis.

Hepatic Venous Pressure Gradient Measurements in Children: Correlation With Hepatic Histology and Clinical Indicators of Portal Hypertension.

OBJECTIVES: In adults, elevated hepatic venous pressure gradients (HVPGs) are correlated with the degree of liver fibrosis on histopathology and predict worse outcomes including variceal bleeding and death. We aimed to examine the association between HVPG measurements, histopathologic findings, and clinical indicators of portal hypertension in children. METHODS: Utilizing retrospective data from 2 pediatric centers between 2006 and 2015, we identified children who underwent simultaneous HVPG measurement and transjugular liver biopsy. Medical charts were reviewed for histopathology, imaging, endoscopic, and clinical data. RESULTS: Forty-one children (median age 11 years) were included in the analysis with diagnoses of acute hepatitis (n = 15), chronic liver disease (n = 12), hepatic noncirrhotic portal hypertension (n = 4), acute liver failure (n = 3), and nonhepatic causes of portal hypertension (n = 7). Elevated mean HVPG measurements were found in children with acute liver failure (10 mmHg, range 4-12) and chronic liver disease (7 mmHg, range 1-12). HVPG measurements did not correlate with the histological severity of fibrosis (ρ = 0.23, P = 0.14) or portal inflammation (ρ = 0.24, P = 0.29), and no difference was found in HVPG when comparing children with and without a history of variceal bleeding (P = 0.43). CONCLUSIONS: HVPG measurements do not correlate significantly with the degree of hepatic fibrosis on biopsy. Furthermore, HVPG measurements are not associated with the presence of varices or history of variceal bleeding, suggesting the possibility of intrahepatic shunting in children with advanced liver disease. Therefore, unlike in adults, HVPG measurements may not accurately predict children who are at risk of complications from portal hypertension.

Suitability of the molecular subtyping methods intergenic spacer region, direct genome restriction analysis, and pulsed-field gel electrophoresis for clinical and environmental Vibrio parahaemolyticus isolates.

Vibrio parahaemolyticus is the leading cause of infectious illness associated with seafood consumption in the United States. Molecular fingerprinting of strains has become a valuable research tool for understanding this pathogen. However, there are many subtyping methods available and little information on how they compare to one another. For this study, a collection of 67 oyster and 77 clinical V. parahaemolyticus isolates were analyzed by three subtyping methods--intergenic spacer region (ISR-1), direct genome restriction analysis (DGREA), and pulsed-field gel electrophoresis (PFGE)--to determine the utility of these methods for discriminatory subtyping. ISR-1 analysis, run as previously described, provided the lowest discrimination of all the methods (discriminatory index [DI]=0.8665). However, using a broader analytical range than previously reported, ISR-1 clustered isolates based on origin (oyster versus clinical) and had a DI=0.9986. DGREA provided a DI=0.9993-0.9995, but did not consistently cluster the isolates by any identifiable characteristics (origin, serotype, or virulence genotype) and ∼ 15% of isolates were untypeable by this method. PFGE provided a DI=0.9998 when using the combined pattern analysis of both restriction enzymes, SfiI and NotI. This analysis was more discriminatory than using either enzyme pattern alone and primarily grouped isolates by serotype, regardless of strain origin (clinical or oyster) or presence of currently accepted virulence markers. These results indicate that PFGE and ISR-1 are more reliable methods for subtyping V. parahemolyticus, rather than DGREA. Additionally, ISR-1 may provide an indication of pathogenic potential; however, more detailed studies are needed. These data highlight the diversity within V. parahaemolyticus and the need for appropriate selection of subtyping methods depending on the study objectives.

Development and validation of a PulseNet standardized protocol for subtyping isolates of Cronobacter species.

Cronobacter (formerly known as Enterobacter sakazakii) is a genus comprising seven species regarded as opportunistic pathogens that can be found in a wide variety of environments and foods, including powdered infant formula (PIF). Cronobacter sakazakii, the major species of this genus, has been epidemiologically linked to cases of bacteremia, meningitis in neonates, and necrotizing enterocolitis, and contaminated PIF has been identified as an important source of infection. Robust and reproducible subtyping methods are required to aid in the detection and investigation, of foodborne outbreaks. In this study, a pulsed-field gel electrophoresis (PFGE) protocol was developed and validated for subtyping Cronobacter species. It was derived from an existing modified PulseNet protocol, wherein XbaI and SpeI were the primary and secondary restriction enzymes used, generating an average of 14.7 and 20.3 bands, respectively. The PFGE method developed was both reproducible and discriminatory for subtyping Cronobacter species.

Standardization and international multicenter validation of a PulseNet pulsed-field gel electrophoresis protocol for subtyping Shigella flexneri isolates.

Shigella flexneri is one of the agents most frequently linked to diarrheal illness in developing countries and often causes outbreaks in settings with poor hygiene or sanitary conditions. Travel is one of the means by which S. flexneri can be imported into developed countries, where this pathogen is not commonly seen. A robust and discriminatory subtyping method is needed for the surveillance of S. flexneri locally and regionally, and to aid in the detection and investigation of outbreaks. The PulseNet International network utilizes standardized pulsed-field gel electrophoresis (PFGE) protocols to carry out laboratory-based surveillance of foodborne pathogens in combination with epidemiologic data. A multicenter validation was carried out in nine PulseNet laboratories located in North and South America, Europe, and Asia, and it demonstrated that a new protocol is highly robust and reproducible for subtyping of S. flexneri. This protocol, already approved for PulseNet laboratories, applies NotI and XbaI as primary and secondary restriction enzymes, respectively, under electrophoresis conditions of initial switch time of 5 s to final switch time of 35 s, at 6 volts/cm.

Global Distribution of Shigella sonnei Clones.

To investigate global epidemiology of Shigella sonnei, we performed multilocus variable number tandem repeat analysis of 1,672 isolates obtained since 1943 from 50 countries on 5 continents and the Pacific region. Three major clonal groups were identified; 2 were globally spread. Type 18 and its derivatives have circulated worldwide in recent decades.

Re-evaluation, optimization, and multilaboratory validation of the PulseNet-standardized pulsed-field gel electrophoresis protocol for Listeria monocytogenes.

The PulseNet Methods Development and Validation Laboratory began a re-evaluation of the standardized pulsed-field gel electrophoresis (PFGE) protocols with the goal of optimizing their overall performance and robustness. Herein, we describe a stepwise evaluation of the PulseNet-standardized PFGE protocol for Listeria monocytogenes that led to the modification of several steps which significantly improved the overall appearance and reproducibility of the resulting PFGE data. These improvements included the following: (1) reducing the cell suspension concentration, (2) increasing lysozyme incubation temperature from 37 degrees C to 56 degrees C, and (3) decreasing the number of units of restriction enzymes AscI and ApaI. These changes were incorporated into a proposed protocol that was evaluated by 16 PulseNet participating laboratories, including 2 international participants. Results from the validation study indicated that the updated L. monocytogenes protocol is more robust than the original PulseNet-standardized protocol established in 1998 and this resulted in the official adoption of the new protocol into the PulseNet system in the spring of 2008. The modifications not only represent an improvement to the protocol but also describe procedural improvements that could be potentially applied to the PFGE analysis of other Gram-positive organisms.

Severe diarrhea caused by cholera toxin-producing vibrio cholerae serogroup O75 infections acquired in the southeastern United States.

BACKGROUND: From 2003 through 2007, Vibrio cholerae serogroup O75 strains possessing the cholera toxin gene were isolated from 6 patients with severe diarrhea, including 3 in Georgia, 2 in Alabama, and 1 in South Carolina. These reports represent the first identification of V. cholerae O75 as a cause of illness in the United States. V. cholerae O75 was isolated from a water sample collected from a pond in Louisiana in 2004. Subsequently, 3 V. cholerae isolates from Louisiana (2 from patients with diarrhea in 2000 and 1 from a water sample collected in 1978) that had been previously reported as serogroup O141 were also discovered to be serogroup O75. RESULTS: All 8 patients who were infected with V. cholerae O75 were adults who became ill after consuming seafood; 2 had eaten raw oysters traced back to the Gulf Coast of the United States. All 10 isolates possessed the cholera toxin gene and were susceptible to 10 antimicrobials. One clinical isolate and 1 environmental (water) isolate had the same pulsed-field gel electrophoresis pattern; 4 clinical isolates shared a common pulsed-field gel electrophoresis pattern. CONCLUSIONS: The occurrence of these cases over many years and the concurrent identification of V. cholerae O75 in water from a Gulf Coast state suggest that these strains may survive for long periods in this environment. The patients' exposure histories suggest that infection can be acquired from consumption of raw oysters from the Gulf Coast. Clinicians and public health authorities should be vigilant for the occurrence of new toxigenic serogroups of V. cholerae that are capable of causing severe diarrhea.

Evaluation and validation of a PulseNet standardized pulsed-field gel electrophoresis protocol for subtyping Vibrio parahaemolyticus: an international multicenter collaborative study.

The pandemic spread of Vibrio parahaemolyticus is an international public health issue. Because of the outbreak potential of the organism, it is critical to establish an internationally recognized molecular subtyping protocol for V. parahaemolyticus that is both rapid and robust as a means to monitor its further spread and to guide control measures in combination with epidemiologic data. Here we describe the results of a multicenter, multicountry validation of a new PulseNet International standardized V. parahaemolyticus pulsed-field gel electrophoresis (PFGE) protocol. The results are from a composite analysis of 36 well-characterized V. parahaemolyticus isolates from six participating laboratories, and the isolates represent predominant serotypes and various genotypes isolated from different geographic regions and time periods. The discriminatory power is very high, as 34 out of 36 sporadic V. parahaemolyticus strains tested fell into 34 distinguishable PFGE groups when the data obtained with two restriction enzymes (SfiI and NotI) were combined. PFGE was further able to cluster members of known pandemic serogroups. The study also identified quality measures which may affect the performance of the protocol. Nonadherence to the recommended procedure may lead to high background in the PFGE gel patterns, partial digestion, and poor fragment resolution. When these quality measures were implemented, the PulseNet V. parahaemolyticus protocol was found to be both robust and reproducible among the collaborating laboratories.

Laboratory-based surveillance of paratyphoid fever in the United States: travel and antimicrobial resistance.

BACKGROUND: The incidence of paratyphoid fever, including paratyphoid fever caused by antimicrobial-resistant strains, is increasing globally. However, the epidemiologic and laboratory characteristics of paratyphoid fever in the United States have never been studied. METHODS: We attempted to interview all patients who had been infected with laboratory-confirmed Salmonella serotypes Paratyphi A, Paratyphi B, or Paratyphi C in the United States with specimens collected from 1 April 2005 through 31 March 2006. At the Centers for Disease Control and Prevention (CDC), isolates underwent serotype confirmation, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis typing. RESULTS: Of 149 patients infected with Salmonella Paratyphi A, we obtained epidemiologic information for 89 (60%); 55 (62%) of 86 were hospitalized. Eighty-five patients (96%) reported having travel internationally, and 80 (90%) had traveled to South Asia. Of the 146 isolates received at the CDC, 127 (87%) were nalidixic acid resistant; nalidixic acid resistance was associated with travel to South Asia (odds ratio, 17.0; 95% confidence interval, 3.8-75.9). All nalidixic acid-resistant isolates showed decreased susceptibility to ciprofloxacin (minimum inhibitory concentration, > or = 0.12 microg/mL). Of 49 patients infected with Salmonella Paratyphi B, only 12 (24%) were confirmed to have Paratyphi B when tested at the CDC. Four (67%) of 6 patients were hospitalized, and 5 (83%) reported travel (4 to the Andean region of South America). One case of Salmonella Paratyphi C infection was reported in a traveler to West Africa with a urinary tract infection. CONCLUSIONS: Physicians should be aware of the increasing incidence of infection due to Salmonella Paratyphi A and treatment options given its widespread antimicrobial resistance. A paratyphoid fever vaccine is urgently needed. Continued surveillance for paratyphoid fever will help guide future prevention and treatment recommendations.

Recent developments and future prospects in subtyping of foodborne bacterial pathogens.

Infections caused by foodborne bacterial pathogens continue to be a major public health issue around the world. During the past decade, pulsed-field gel electrophoresis (PFGE) has become the gold standard for molecular subtyping and source tracking of most foodborne bacteria. Owing to problems inherent in PFGE technology, new methods have been developed focusing on DNA sequence-based subtyping. This review discusses the feasibility of using multilocus sequence typing, multiple-locus variable-number tandem repeat analysis, single nucleotide polymorphisms, microarrays, whole genome sequencing and mass spectrometry for subtyping foodborne bacterial pathogens.

Human Salmonella and concurrent decreased susceptibility to quinolones and extended-spectrum cephalosporins.

The National Antimicrobial Resistance Monitoring System monitors susceptibility among Enterobacteriaceae in humans in the United States. We studied isolates exhibiting decreased susceptibility to quinolones (nalidixic acid MIC >32 microg/mL or ciprofloxacin MIC > or =0.12 microg/mL) and extended-spectrum cephalosporins (ceftiofur or ceftriaxone MIC > or =2 microg/mL) during 1996-2004. Of non-Typhi Salmonella, 0.19% (27/14,043) met these criteria: 11 Senftenberg; 6 Typhimurium; 3 Newport; 2 Enteridis; and 1 each Agona, Haifa, Mbandaka, Saintpaul, and Uganda. Twenty-six isolates had gyrA mutations (11 at codon 83 only, 3 at codon 87 only, 12 at both). All Senftenberg isolates had parC mutations (S801 and T57S); 6 others had the T57S mutation. The Mbandaka isolate contained qnrB2. Eight isolates contained bla(CMY-2); 1 Senftenberg contained bla(CMY-23). One Senftenberg and 1 Typhimurium isolate contained bla(SHV-12); the Mbandaka isolate contained bla(SHV-30). Nine Senftenberg isolates contained bla(OXA-1) contained bla(OXA-9). Further studies should address patient outcomes, risk factors, and resistance dissemination prevention strategies.

Development of a universal probe for electronic microarray and its application in characterization of the Staphylococcus aureus polC gene.

Electronic microarray technology is an exceptionally accurate and effective technique for detecting and defining single nucleotide polymorphisms (SNPs) in DNA sequences. Target oligonucleotides are electronically addressed to a gel matrix containing streptavidin to which biotinylated polymerase chain reaction (PCR) amplicons are bound. Typically, a fluorescent-labeled reporter oligonucleotide specific for each locus of interest is hybridized and reported. We detail the development of a universal reporter system to replace the standard method that is used to detect many different sequences accurately. The universal reporter eliminates the need to synthesize specific labeled reporters for each SNP sequence thereby dramatically reducing the cost and time required for assay development. The feasibility of this approach was demonstrated by successfully analyzing eight SNPs distributed within a highly variable 1-kb region of the polC gene from six isolates of Staphylococcus aureus.