Functionally Distinct Bacterial Cytochrome c Peroxidases Proceed through a Common (Electro)catalytic Intermediate.

The diheme cytochrome c peroxidase from Shewanella oneidensis (So CcP) requires a single electron reduction to convert the oxidized, as-isolated enzyme to an active conformation. We employ protein film voltammetry to investigate the mechanism of hydrogen peroxide turnover by So CcP. When the enzyme is poised in the active state by incubation with sodium l-ascorbate, the graphite electrode specifically captures a highly active state that turns over peroxide in a high potential regime. This is the first example of an on-pathway catalytic intermediate observed for a bacterial diheme cytochrome c peroxidase that requires reductive activation, consistent with the observed voltammetric response from the diheme cytochrome c peroxidase from Nitrosomonas europaea (Ne), which is constitutively active and does not require the same one electron activation. Mutational analysis at the active site of So CcP confirms that the rate-limiting step involves a proton-coupled single electron reduction of a high valent iron species centered on the low-potential heme, consistent with the same mutation in Ne CcP. The pH dependence of catalysis for wild-type So CcP suggests that reduction shifts the pK(a)'s of at least two amino acids. Mutation of His81 in "loop 1", a surface exposed loop thought to shift conformation during the reductive activation process, eliminated one of the pH dependent features, confirming that the loop 1 shifts, changing the environment of His81 during the rate-limiting step. The observed catalytic intermediate has the same electron stoichiometry and similar pH dependence to that previously reported for Ne CcP, which is constitutively active and therefore hypothesized to follow a different catalytic mechanism. The prominent similarities between the rate-limiting steps of differing mechanistic classes of bCcPs suggest unexpected similarities in the intermediates formed.

Methionine ligand lability of homologous monoheme cytochromes c.

Direct electrochemical analysis of adsorbed bacterial monoheme cytochromes c has revealed a phenomenological loss of the axial methionine when examined using pyrolytic "edge-plane" graphite (EPG) electrodes. While prior findings have reported that the Met-loss state may be quantitatively understood using the cytochrome c from Hydrogenobacter thermophilus as a model system, here we demonstrate that the formation of the Met-loss state upon EPG electrodes can be observed for a range of cytochrome orthologs. Through an electrochemical comparison of the wild-type proteins from organisms of varying growth temperature optima, we establish that Met-ligand losses at graphite surfaces have similar energetics to the "foldons" for known protein folding pathways. Furthermore, a downward shift in reduction potential to approximately -100 mV vs standard hydrogen electrode was observed, similar to that of the alkaline transition found in mitochondrial cytochromes c. Pourbaix diagrams for the Met-loss forms of each cytochrome, considered here in comparison to mutants where the Met-ligand has been substituted to His or Ala, suggest that the nature of the Met-loss state is distinct from either a His-/aquo- or a bis-His-ligated heme center, yet more closely matches the pKa values found for bis-His-ligated hemes., We find the propensity for adoption of the Met-loss state in bacterial monoheme cytochromes c scales with their overall thermal stability, though not with the specific stability of the Fe-Met bond.

Outbreaks associated with cantaloupe, watermelon, and honeydew in the United States, 1973-2011.

Fresh fruits and vegetables are an important part of a healthy diet. Melons have been associated with enteric infections. We reviewed outbreaks reported to the Centers for Disease Control and Prevention's Foodborne Disease Outbreak Surveillance System during 1973-2011 in which the implicated food was a single melon type. We also reviewed published literature and records obtained from investigating agencies. During 1973-2011, 34 outbreaks caused by a single melon type were reported, resulting in 3602 illnesses, 322 hospitalizations, 46 deaths, and 3 fetal losses. Cantaloupes accounted for 19 outbreaks (56%), followed by watermelons (13, 38%) and honeydew (2, 6%). Melon-associated outbreaks increased from 0.5 outbreaks per year during 1973-1991 to 1.3 during 1992-2011. Salmonella was the most common etiology reported (19, 56%), followed by norovirus (5, 15%). Among 13 outbreaks with information available, melons imported from Mexico and Central America were implicated in 9 outbreaks (69%) and domestically grown melons were implicated in 4 outbreaks (31%). The point of contamination was known for 20 outbreaks; contamination occurred most commonly during growth, harvesting, processing, or packaging (13, 65%). Preventive measures focused on reducing bacterial contamination of melons both domestically and internationally could decrease the number and severity of melon-associated outbreaks.

Foodborne disease outbreaks caused by Bacillus cereus, Clostridium perfringens, and Staphylococcus aureus--United States, 1998-2008.

From 1998 to 2008, 1229 foodborne outbreaks caused by Bacillus cereus, Clostridium perfringens, and Staphylococcus aureus were reported in the United States; 39% were reported with a confirmed etiology. Vomiting was commonly reported in B. cereus (median, 75% of cases) and S. aureus outbreaks (median, 87%), but rarely in C. perfringens outbreaks (median, 9%). Meat or poultry dishes were commonly implicated in C. perfringens (63%) and S. aureus (55%) outbreaks, and rice dishes were commonly implicated in B. cereus outbreaks (50%). Errors in food processing and preparation were commonly reported (93%), regardless of etiology; contamination by a food worker was only common in S. aureus outbreaks (55%). Public health interventions should focus on these commonly reported errors to reduce the occurrence of outbreaks caused by B. cereus, C. perfringens, and S. aureus in the United States.

Outbreak-associated Salmonella enterica serotypes and food Commodities, United States, 1998-2008.

Salmonella enterica infections are transmitted not only by animal-derived foods but also by vegetables, fruits, and other plant products. To clarify links between Salmonella serotypes and specific foods, we examined the diversity and predominance of food commodities implicated in outbreaks of salmonellosis during 1998-2008. More than 80% of outbreaks caused by serotypes Enteritidis, Heidelberg, and Hadar were attributed to eggs or poultry, whereas >50% of outbreaks caused by serotypes Javiana, Litchfield, Mbandaka, Muenchen, Poona, and Senftenberg were attributed to plant commodities. Serotypes Typhimurium and Newport were associated with a wide variety of food commodities. Knowledge about these associations can help guide outbreak investigations and control measures.

Risk factors for venous thromboembolism in patients with chronic liver disease.

BACKGROUND: Pharmacologic prophylaxis for venous thromboembolism (VTE) in patients with chronic liver disease (CLD) presents a unique challenge because of coagulopathies associated with the disease. When evaluating whether these patients require VTE prophylaxis upon hospitalization, it would be advantageous if risk factors for the development of VTE in this population were known. OBJECTIVE: To evaluate risk factors associated with the development of VTE in patients with CLD. METHODS: A retrospective case-control study was conducted. Patients admitted to the University of Kentucky Chandler Hospital from October 2006 to July 2010 with a diagnosis of CLD and VTE were matched in a 1:3 fashion with CLD patients without VTE. The primary objective was to determine whether there were significant differences in laboratory values between the 2 groups. RESULTS: During this time, 27 patients with CLD (1.0%) were diagnosed with VTE. These patients had significantly lower median aspartate aminotransferase (AST) (47 vs 70 U/L, p = 0.04), alanine transaminase (ALT) (24.5 vs 36 U/L, p = 0.02), albumin (2.1 vs 2.4 g/dL, p = 0.02) and hematocrit (Hct) (28.3% vs 32%, p = 0.03) values compared to the control patients. Patients with albumin lower than 1.9 g/dL had a 5.1 times greater risk of VTE compared to patients with albumin of 2.8 g/dL and higher (OR 5.14, 95% CI 1.05-25.2). CONCLUSIONS: Patients with CLD who developed VTE had significantly lower AST, ALT, albumin, and Hct compared to those of control patients. Studies are necessary to further examine the significance of this finding.

Outbreaks of Acute Gastroenteritis Transmitted by Person-to-Person Contact, Environmental Contamination, and Unknown Modes of Transmission--United States, 2009-2013.

PROBLEM/CONDITION: Acute gastroenteritis (AGE) is a major cause of illness in the United States, with an estimated 179 million episodes annually. AGE outbreaks propagated through direct person-to-person contact, contaminated environmental surfaces, and unknown modes of transmission were not systematically captured at the national level before 2009 and thus were not well characterized. REPORTING PERIOD: 2009-2013. DESCRIPTION OF SYSTEM: The National Outbreak Reporting System (NORS) is a voluntary national reporting system that supports reporting of all waterborne and foodborne disease outbreaks and all AGE outbreaks resulting from transmission by contact with contaminated environmental sources, infected persons or animals, or unknown modes. Local, state, and territorial public health agencies within the 50 U.S. states, the District of Columbia (DC), five U.S. territories, and three Freely Associated States report outbreaks to CDC via NORS using a standard online data entry system. RESULTS: A total of 10,756 AGE outbreaks occurred during 2009-2013, for which the primary mode of transmission occurred through person-to-person contact, environmental contamination, and unknown modes of transmission. NORS received reports from public health agencies in 50 U.S. states, DC, and Puerto Rico. These outbreaks resulted in 356,532 reported illnesses, 5,394 hospitalizations, and 459 deaths. The median outbreak reporting rate for all sites in a given year increased from 2.7 outbreaks per million population in 2009 to 11.8 outbreaks in 2013. The etiology was unknown in 31% (N = 3,326) of outbreaks. Of the 7,430 outbreaks with a suspected or confirmed etiology reported, norovirus was the most common, reported in 6,223 (84%) of these outbreaks. Other reported suspected or confirmed etiologies included Shigella (n = 332) and Salmonella (n = 320). Outbreaks were more frequent during the winter, with 5,716 (53%) outbreaks occurring during December-February, and 70% of the 7,001 outbreaks with a reported setting of exposure occurred in long-term-care facilities (n = 4,894). In contrast, 59% (n = 143) of shigellosis outbreaks, 36% (n = 30) of salmonellosis outbreaks, and 32% (n = 84) of other or multiple etiology outbreaks were identified in child care facilities. INTERPRETATION: NORS is the first U.S. surveillance system that provides national data on AGE outbreaks spread through person-to-person contact, environmental contamination, and unknown modes of transmission. The increase in reporting rates during 2009-2013 indicates that reporting to NORS improved notably in the 5 years since its inception. Norovirus is the most commonly reported cause of these outbreaks and, on the basis of epidemiologic data, might account for a substantial proportion of outbreaks without a reported etiology. During 2009-2013, norovirus accounted for most deaths and health care visits in AGE outbreaks spread through person-to-person contact, environmental contamination, and unknown modes of transmission. PUBLIC HEALTH ACTION: Recommendations for prevention and control of AGE outbreaks transmitted through person-to-person contact, environmental contamination, and unknown modes of transmission depend primarily on appropriate hand hygiene, environmental disinfection, and isolation of ill persons. NORS surveillance data can help identify priority targets for the development of future control strategies, including hygiene interventions and vaccines, and help monitor the frequency and severity of AGE outbreaks in the United States. Ongoing study of these AGE outbreaks can provide a better understanding of certain pathogens and their modes of transmission. For example, certain reported outbreak etiologies (e.g., Salmonella) are considered primarily foodborne pathogens but can be transmitted through multiple routes. Similarly, further examination of outbreaks of unknown etiology could help identify barriers to making an etiologic determination, to analyze clinical and epidemiologic clues suggestive of a probable etiology, and to discover new and emerging etiologic agents. Outbreak reporting to NORS has improved substantially since its inception, and further outreach efforts and system improvements might facilitate additional increases in the number and completeness of reports to NORS.

Surveillance for foodborne disease outbreaks - United States, 1998-2008.

PROBLEM/CONDITION: Foodborne diseases cause an estimated 48 million illnesses each year in the United States, including 9.4 million caused by known pathogens. Foodborne disease outbreak surveillance provides valuable insights into the agents and foods that cause illness and the settings in which transmission occurs. CDC maintains a surveillance program for collection and periodic reporting of data on the occurrence and causes of foodborne disease outbreaks in the United States. This surveillance system is the primary source of national data describing the numbers of illnesses, hospitalizations, and deaths; etiologic agents; implicated foods; contributing factors; and settings of food preparation and consumption associated with recognized foodborne disease outbreaks in the United States. REPORTING PERIOD: 1998-2008. DESCRIPTION OF THE SYSTEM: The Foodborne Disease Outbreak Surveillance System collects data on foodborne disease outbreaks, defined as the occurrence of two or more cases of a similar illness resulting from the ingestion of a common food. Public health agencies in all 50 states, the District of Columbia, U.S. territories, and Freely Associated States have primary responsibility for identifying and investigating outbreaks and use a standard form to report outbreaks voluntarily to CDC. During 1998-2008, reporting was made through the electronic Foodborne Outbreak Reporting System (eFORS). RESULTS: During 1998-2008, CDC received reports of 13,405 foodborne disease outbreaks, which resulted in 273,120 reported cases of illness, 9,109 hospitalizations, and 200 deaths. Of the 7,998 outbreaks with a known etiology, 3,633 (45%) were caused by viruses, 3,613 (45%) were caused by bacteria, 685 (5%) were caused by chemical and toxic agents, and 67 (1%) were caused by parasites. Among the 7,724 (58%) outbreaks with an implicated food or contaminated ingredient reported, 3,264 (42%) could be assigned to one of 17 predefined commodity categories: fish, crustaceans, mollusks, dairy, eggs, beef, game, pork, poultry, grains/beans, oils/sugars, fruits/nuts, fungi, leafy vegetables, root vegetables, sprouts, and vegetables from a vine or stalk. The commodities implicated most commonly were poultry (18.9%; 95% confidence interval [CI] = 17.4-20.3) and fish (18.6%; CI = 17.2-20), followed by beef (11.9%; CI = 10.8-13.1). The pathogen-commodity pairs most commonly responsible for outbreaks were scombroid toxin/histamine and fish (317 outbreaks), ciguatoxin and fish (172 outbreaks), Salmonella and poultry (145 outbreaks), and norovirus and leafy vegetables (141 outbreaks). The pathogen-commodity pairs most commonly responsible for outbreak-related illnesses were norovirus and leafy vegetables (4,011 illnesses), Clostridium perfringens and poultry (3,452 illnesses), Salmonella and vine-stalk vegetables (3,216 illnesses), and Clostridium perfringens and beef (2,963 illnesses). Compared with the first 2 years of the study (1998-1999), the percentage of outbreaks associated with leafy vegetables and dairy increased substantially during 2006-2008, while the percentage of outbreaks associated with eggs decreased. INTERPRETATION: Outbreak reporting rates and implicated foods varied by state and year, respectively; analysis of surveillance data for this 11-year period provides important information regarding changes in sources of illness over time. A substantial percentage of foodborne disease outbreaks were associated with poultry, fish, and beef, whereas many outbreak-related illnesses were associated with poultry, leafy vegetables, beef, and fruits/nuts. The percentage of outbreaks associated with leafy vegetables and dairy increased during the surveillance period, while the percentage associated with eggs decreased. PUBLIC HEALTH ACTIONS: Outbreak surveillance data highlight the etiologic agents, foods, and settings involved most often in foodborne disease outbreaks and can help to identify food commodities and preparation settings in which interventions might be most effective. Analysis of data collected over several years of surveillance provides a means to assess changes in the food commodities associated most frequently with outbreaks that might occur following improvements in food safety or changes in consumption patterns or food preparation practices. Prevention of foodborne disease depends on targeted interventions at appropriate points from food production to food preparation. Efforts to reduce foodborne illness should focus on the pathogens and food commodities causing the most outbreaks and outbreak-associated illnesses, including beef, poultry, fish, and produce.