Glutathione S-transferase alpha 4 induction by activator protein 1 in colorectal cancer.

Glutathione S-transferase alpha 4 (GSTA4) is a phase II detoxifying enzyme that metabolizes electrophiles and carcinogens including 4-hydroxy-2-nonenal (4-HNE), an endogenous carcinogen that contributes to colorectal carcinogenesis. In this study, we investigated GSTA4 expression and regulation in murine primary colonic epithelial cells, microbiome-driven murine colitis and human carcinomas. Exposure of YAMC cells to 4-HNE induced Gsta4 expression. Using an inflammation-associated model of colorectal cancer (CRC), Gsta4 expression increased in vivo in colon macrophages and serum after 2 weeks of colonization of IL-10 deficient (Il10-/-) mice with Enterococcus faecalis. Increased expression was noted after 9 months of colonization in colon macrophages and epithelia in areas of inflammation. In human colon biopsies, immunohistochemistry showed no GSTA4 expression in normal epithelial cells, whereas GSTA4 was strongly expressed in the neoplastic epithelia of invasive carcinomas. For tubular adenomas, increased expression was primarily noted in stromal macrophages. Increased GSTA4 was confirmed in established human CRC cell lines and associated with 4-HNE-protein adducts in human colon adenomas and CRC. Next, we showed that 4-HNE induced activation of c-Jun and Nrf2, two components of the oncogenic transcription factor AP-1. AP-1 inhibitors and gene-specific small interfering RNAs partially suppressed GSTA4 expression. Co-immunoprecipitation confirmed interactions between c-Jun and Nrf2 supporting a role for AP-1 in regulating 4-HNE-induced GSTA4 expression. These findings demonstrate GSTA4 activation during 4-HNE-induced neoplastic transformation in colorectal carcinogenesis. GSTA4 is a potential surrogate biomarker for CRC screening and should provide novel approaches for chemoprevention.

Extracellular superoxide production by Enterococcus faecalis requires demethylmenaquinone and is attenuated by functional terminal quinol oxidases.

The intestinal commensal bacterium, Enterococcus faecalis, is unusual among prokaryotic organisms in its ability to produce substantial extracellular superoxide. Transposon mutagenesis, allelic replacement, and electron spin resonance (ESR)-spin trapping showed that superoxide production and generation of derivative hydroxyl radical were dependent on membrane-associated demethylmenaquinone. Extracellular superoxide was generated through univalent reduction of oxygen by reduced demethylmenaquinone. Moreover, extracellular superoxide production was inhibited by exogenous haematin, an essential cofactor for cytochrome bd, and by fumarate, a substrate for fumarate reductase. As integral membrane quinol oxidases, cytochrome bd and fumarate reductase redox cycle demethylmenaquinone, and are necessary for aerobic and anaerobic respiration respectively. A rat model of intestinal colonization demonstrated that conditions exist in the mammalian intestinal tract that permit a mode of respiration for E. faecalis that results in the formation of hydroxyl radical. These results identify and characterize the mechanism by which E. faecalis generates extracellular free radicals.

Identification of a p28 gene in Ehrlichia ewingii: evaluation of gene for use as a target for a species-specific PCR diagnostic assay.

PCR was used to amplify a 537-bp region of an Ehrlichia ewingii gene encoding a homologue of the 28-kDa major antigenic protein (P28) of Ehrlichia chaffeensis. The E. ewingii p28 gene homologue was amplified from DNA extracted from whole blood obtained from four humans and one canine with confirmed cases of infection. Sequencing of the PCR products (505 bp) revealed a partial gene with homology to outer membrane protein genes from Ehrlichia and Cowdria spp.: p30 of Ehrlichia canis (< or =71.3%), p28 of E. chaffeensis (< or =68.3%), and map1 of Cowdria ruminantium (67.3%). The peptide sequence of the E. ewingii partial gene product was deduced (168 amino acids) and the antigenicity profile was analyzed, revealing a hydrophilic protein with < or =69.1% identity to P28 of E. chaffeensis, < or =67.3% identity to P30 of E. canis, and < or =63.1% identity to MAP1 of C. ruminantium. Primers were selected from the E. ewingii p28 sequence and used to develop a species-specific PCR diagnostic assay. The p28 PCR assay amplified the expected 215-bp product from DNA that was extracted from EDTA-treated blood from each of the confirmed E. ewingii infections that were available. The assay did not produce PCR products with DNA extracted from E. chaffeensis-, E. canis-, or E. phagocytophila-infected samples, confirming the specificity of the p28 assay for E. ewingii. The sensitivity of the E. ewingii-specific PCR assay was evaluated and determined to detect as few as 38 copies of the p28 gene.

Infections with Ehrlichia chaffeensis and Ehrlichia ewingii in persons coinfected with human immunodeficiency virus.

The clinical course and laboratory evaluation of 21 patients coinfected with human immunodeficiency virus (HIV) and Ehrlichia chaffeensis or Ehrlichia ewingii are reviewed and summarized, including 13 cases of ehrlichiosis caused by E. chaffeensis, 4 caused by E. ewingii, and 4 caused by either E. chaffeensis or E. ewingii. Twenty patients were male, and the median CD4(+) T lymphocyte count was 137 cells/microL. Exposures to infecting ticks were linked to recreational pursuits, occupations, and peridomestic activities. For 8 patients, a diagnosis of ehrlichiosis was not considered until > or =4 days after presentation. Severe manifestations occurred more frequently among patients infected with E. chaffeensis than they did among patients infected with E. ewingii, and all 6 deaths were caused by E. chaffeensis. Ehrlichiosis may be a life-threatening illness in HIV-infected persons, and the influence of multiple factors, including recent changes in the epidemiology and medical management of HIV infection, may increase the frequency with which ehrlichioses occur in this patient cohort.

A double-blind placebo-controlled crossover trial of intravenous magnesium sulfate for foscarnet-induced ionized hypocalcemia and hypomagnesemia in patients with AIDS and cytomegalovirus infection.

Foscarnet (trisodium phosphonoformate hexahydrate) is an antiviral agent used to treat cytomegalovirus disease in immunocompromised patients. One common side effect is acute ionized hypocalcemia and hypomagnesemia following intravenous administration. Foscarnet-induced ionized hypomagnesemia might contribute to ionized hypocalcemia by impairing excretion of preformed parathyroid hormone (PTH) or by producing target organ resistance. Prevention of ionized hypomagnesemia following foscarnet administration could blunt the development of ionized hypocalcemia. To determine whether intravenous magnesium ameliorates the decline in ionized calcium and/or magnesium following foscarnet infusions, MgSO(4) at doses of 1, 2, and 3 g was administered in a double-blind, placebo-controlled, randomized, crossover trial to 12 patients with AIDS and cytomegalovirus disease. Overall, increasing doses of MgSO(4) reduced or eliminated foscarnet-induced acute ionized hypomagnesemia. Supplementation, however, had no discernible effect on foscarnet-induced ionized hypocalcemia despite significant increases in serum PTH levels. No dose-related, clinically significant adverse events were found, suggesting that intravenous supplementation with up to 3 g of MgSO(4) was safe in this chronically ill population. Since parenteral MgSO(4) did not alter foscarnet-induced ionized hypocalcemia or symptoms associated with foscarnet, routine intravenous supplementation for patients with normal serum magnesium levels is not recommended during treatment with foscarnet.

Outcomes of an antimicrobial control program in a teaching hospital.

The clinical outcomes and cost-effectiveness of an antimicrobial control program (ACP) were studied. The impact of an ACP in a teaching hospital was analyzed by comparing clinical outcomes and intravenous antimicrobial costs over two two-year periods, the two years before the program and the first two years after the program's inception. Admission baseline data, length of stay, mortality, and readmission rates were gathered for each patient. Patients were identified by using the International Classification of Diseases. Multivariate logistic regression models were constructed for mortality and for lengths of stay of 12 or more days. The acquisition costs of intravenous antimicrobial agents for the second baseline year and the entire program period were tabulated and compared. The average daily inpatient census was determined. The ACP was associated with a 2.4-day decrease in length of stay and a reduction in mortality from 8.28% to 6.61%. Rates of readmission for infection within 30 days of discharge remained about the same. Inpatient pharmacy costs other than intravenous antimicrobials decreased an average of only 5.7% over the two program years, but the acquisition cost of intravenous antimicrobials for both program years yielded a total cost saving of $291,885, a reduction of 30.8%. The institution's average daily census fell 19% between the second baseline year and the second program year. An ACP directed by a clinical pharmacist trained in infectious diseases was associated with improvements in inpatient length of stay and mortality. The ACP decreased intravenous antimicrobial costs and facilitated the approval process for restricted and nonformulary antimicrobial agents.

Infection-derived Enterococcus faecalis strains are enriched in esp, a gene encoding a novel surface protein.

We report the identification of a new cell wall-associated protein of Enterococcus faecalis. Studies on the distribution of the gene encoding this novel surface protein, Esp, reveal a significant (P < 0.001) enrichment in infection-derived E. faecalis isolates. Interestingly, the esp gene was not identified in any of 34 clinical E. faecium isolates or in 4 other less pathogenic enterococcal species tested. Analysis of the structural gene among various E. faecalis isolates reveals the existence of alternate forms of expression of the Esp protein. The deduced primary structure of the Esp protein from strain MMH594, inferred to be 1,873 amino acids (aa) with a predicted mass of approximately 202 kDa, reveals a core region consisting of repeat units that make up 50% of the protein. Esp bears global organizational similarity to the Rib and C alpha proteins of group B streptococci. Identity among Esp, Rib, and C alpha proteins is strikingly localized to a stretch of 13 aa within repeats of similar length. The high degree of conservation of this 13-residue sequence suggests that it plays an important role in the natural selection for this trait among infection-derived E. faecalis and group B streptococcal isolates.

Prospective case-cohort study of intestinal colonization with enterococci that produce extracellular superoxide and the risk for colorectal adenomas or cancer.

OBJECTIVE: The aim of this study was to determine whether intestinal colonization with enterococci that produce extracellular superoxide (O2*-), a free radical implicated in the development of colorectal cancer, is associated with these lesions or their precursors. METHODS: A prospective case-cohort study was performed by isolating enterococci from stools of consecutive patients undergoing colonoscopy who had no prior history of colonoscopy or colorectal cancer. A food frequency questionnaire was also administered to control for dietary factors known to affect the risk for these lesions. RESULTS: Among 159 evaluable participants were 77 with no precancerous or cancerous pathology, 61 with adenomas <2 cm, 10 with adenomas > or =2 cm, and 11 with cancer. Regression analyses found no associations for those subjects with adenomas of any size or with cancer and colonization with O2*--producing enterococci, any nutrient, or age. For those patients with large adenomas > or = 2 cm or cancer, however, significant associations were noted for age (OR 1.94 per decade, 95% CI 1.2-3.5), beta-carotene (OR 0.44 per 500 microg/1000 kcal/day, 95% CI 0.2-0.8), vitamin A (OR 3.20 per 500 microg/1000 kcal/day, 95% CI 1.2-8.9), and vitamin E (OR 0.09 per 10 mg/ 1000 kcal/day, 95% CI 0.006-0.9), but not colonization with O2*--producing enterococci. Second stools collected 1 yr later, however, often contained dissimilar enterococcal flora, undermining an important study assumption. CONCLUSIONS: Significant associations were found for those with large adenomas or cancer (but not small adenomas), with age, and with foods enriched for vitamin A, vitamin E, and beta-carotene. An association between colonization with O2*--producing enterococci and colorectal adenomas or cancer, however, could not be ascertained, possibly because intestinal enterococcal flora changes over time, leaving a potentially cohesive hypothesis of colon cancer and risk factors as yet unanswered.

A hospital-based pharmacy intervention program for pneumococcal vaccination.

BACKGROUND: Current pneumococcal vaccination rates are well below national goals. OBJECTIVE: To determine whether pneumococcal vaccination rates could be increased with a hospital pharmacy-based program using simple chart reminders. METHODS: On a daily basis, inpatient records on general medicine and cardiology services at an academic medical center were reviewed to determine which patients were eligible to receive pneumococcal vaccine. Eligible inpatients were interviewed, and the percentage of nonvaccinated inpatients given vaccine during hospitalization was determined. During an intervention period, reminders were placed on charts after the interview requesting a vaccine when indicated. RESULTS: Of 447 inpatients, 224 (50.1%) had 1 or more indications for receiving pneumococcal vaccine. Only 64 (28.6%) had been previously vaccinated. One hundred fifty-eight (70.5%) of 224 vaccine-eligible patients had a prior hospitalization within the previous 5 years. Previous hospitalization was not significantly associated with having (48 [30.4%] of 158) or not having (16 [24.2%] of 66; P=.35) been vaccinated prior to admission. During the observational period, 0 of 80 vaccine-eligible, nonvaccinated inpatients were vaccinated before discharge. In comparison, 23 (28.8%) of 80 inpatients were vaccinated after a chart reminder (P<.001). During the intervention period, vaccination rates were 10-fold higher on general medicine services than on cardiology services. CONCLUSIONS: A hospital-based pharmacy vaccination program that relied on simple chart reminders was significantly associated with increased vaccination rates among inpatients at risk for invasive pneumococcal disease.

Multiple-drug resistant enterococci: the nature of the problem and an agenda for the future.

Enterococci, leading causes of nosocomial bacteremia, surgical wound infection, and urinary tract infection, are becoming resistant to many and sometimes all standard therapies. New rapid surveillance methods are highlighting the importance of examining enterococcal isolates at the species level. Most enterococcal infections are caused by Enterococcus faecalis, which are more likely to express traits related to overt virulence but--for the moment--also more likely to retain sensitivity to at least one effective antibiotic. The remaining infections are mostly caused by E. faecium, a species virtually devoid of known overt pathogenic traits but more likely to be resistant to even antibiotics of last resort. Effective control of multiple-drug resistant enterococci will require 1) better understanding of the interaction between enterococci, the hospital environment, and humans, 2) prudent antibiotic use, 3) better contact isolation in hospitals and other patient care environments, and 4) improved surveillance. Equally important is renewed vigor in the search for additional drugs, accompanied by the evolution of new therapeutic paradigms less vulnerable to the cycle of drug introduction and drug resistance.

Surgical Infection Society position on vancomycin-resistant Enterococcus.

The risk of transfer of vancomycin resistance to staphylococci is a real possibility and has been achieved in the laboratory. Prolonged colonization occurs with vancomycin-resistant Enterococcus (VRE), and many more patients are colonized than infected. The failure to identify, isolate, and adhere to infection control measures when caring for VRE-colonized patients dooms to failure any means to control its spread. Control of vancomycin use alone is unlikely to greatly affect the number of patients at risk for VRE colonization. The global spread of VRE may be impossible to stop, but infection control measures are the most important line of defense inside hospitals.

Augmented production of extracellular superoxide by blood isolates of Enterococcus faecalis.

To assess the frequency of extracellular superoxide (O-2) production by enterococci, multiple species were surveyed in a whole organism assay for their ability to reduce ferricytochrome c in a superoxide dismutase-inhibitable fashion. For stool and clinical enterococcal isolates and 12 type strains, only Enterococcus faecalis (87/91 isolates and ATCC 19433), Enterococcus faecium (5/13 isolates), Enterococcus casseliflavus (ATCC 25778), and Enterococcus gallinarum (ATCC 35038) produced extracellular O-2. Among 106 strains comprising 13 species of enteric gram-negative bacilli and gram-positive cocci, only Lactococcus lactis subspecies lactis produced extracellular O-2. Mean (+/-SE) rates of extracellular O-2 production in vitro by E . faecalis for isolates associated with bacteremia and endocarditis and for isolates from stool were 2.4+/-0.2, 1.9+/-0.2, 1.5+/-0.3 nmol of O-2 min(-1) 10(9) cfu(-1), respectively (P=.025, analysis of variance), suggesting an association between invasiveness and extracellular O-2 production.

Frequency of aggregation substance and cytolysin genes among enterococcal endocarditis isolates.

Enterococcus faecalis blood isolates were probed for the serine protease activator of cytolysin (cylA) and aggregation substance (asa1), traits that generally reside on pheromone-responsive plasmids, to determine how commonly these genotypes were associated with disease. In dot blot assays, no significant difference was found in the frequency of asa1 for blood isolates [55 of 103 (54%)] and isolates recovered from stool [9 of 14 (60%); P > 0.1, chi 2 test]. In contrast, cylA occurred more frequently among bacteremia isolates [34 of 68 (50%)] than endocarditis [4 of 35 (11%)] or stool isolates (0 of 14; P < 0.001; chi 2 test). However, when the clonality of isolates was accounted for, the frequency of asa1 and cylA among unrelated strains was not significantly different among the three sets of strains (P > 0.2, chi 2 test). The lack of enrichment for asa1 or cylA among clonally unrelated E. faecalis bloodstream isolates fails to support a role for plasmid-encoded aggregation substance or cytolysin in the transition from bacteremia to endocarditis. Clonally related, cytolytic strains, however, demonstrated an increased propensity to cause bloodstream infection.

Apophysomyces elegans infection in a renal transplant recipient.

A 50-year-old cadaveric renal transplant recipient on immunosuppressive therapy is described with post-traumatic cutaneous infection caused by Apophysomyces elegans. He showed no evidence of hematogenous dissemination and recovered fully after therapy with extensive local debridement and amphotericin B lipid complex. An apparent drug-drug interaction between amphotericin B lipid complex and cyclosporine was encountered. The course of A elegans infection in transplant recipients may be similar to that described in immunocompetent hosts. A elegans infection should be considered in evaluation of post-traumatic cutaneous infection not readily responsive to antibacterial therapy.

Enterococcus faecalis cytolysin without effect on the intestinal growth of susceptible enterococci in mice.

A murine model was developed to determine whether the Enterococcus faecalis cytolysin, through its bacteriolytic action on gram-positive bacteria, could promote intestinal overgrowth of cytolytic strains. Sets of E. faecalis strains with varying cytolytic production and susceptibility to cytolytic activity were mixed 1:1 and allowed to compete in vitro in broth or in vivo after orogastric administration in mice pretreated with antibiotics. In general, cytolytic strains outgrew, by as much as 2000-fold, competing cytolysin-susceptible or -hypersusceptible strains in vitro. In contrast, no growth advantage was observed in vivo, despite similar transient colonization of the murine intestinal tract by both cytolytic and cytolysin-susceptible strains. These data suggest that cytolysin plays little role in promoting intestinal overgrowth of enterococci through bacteriolytic activity.

Virulence of enterococci.

Enterococci are commensal organisms well suited to survival in intestinal and vaginal tracts and the oral cavity. However, as for most bacteria described as causing human disease, enterococci also possess properties that can be ascribed roles in pathogenesis. The natural ability of enterococci to readily acquire, accumulate, and share extrachromosomal elements encoding virulence traits or antibiotic resistance genes lends advantages to their survival under unusual environmental stresses and in part explains their increasing importance as nosocomial pathogens. This review discusses the current understanding of enterococcal virulence relating to (i) adherence to host tissues, (ii) invasion and abscess formation, (iii) factors potentially relevant to modulation of host inflammatory responses, and (iv) potentially toxic secreted products. Aggregation substance, surface carbohydrates, or fibronectin-binding moieties may facilitate adherence to host tissues. Enterococcus faecalis appears to have the capacity to translocate across intact intestinal mucosa in models of antibiotic-induced superinfection. Extracellular toxins such as cytolysin can induce tissue damage as shown in an endophthalmitis model, increase mortality in combination with aggregation substance in an endocarditis model, and cause systemic toxicity in a murine peritonitis model. Finally, lipoteichoic acid, superoxide production, or pheromones and corresponding peptide inhibitors each may modulate local inflammatory reactions.