Staphylococcus aureus infections among children receiving a solid organ transplant: clinical features, epidemiology, and antimicrobial susceptibility.

BACKGROUND: Staphylococcus aureus is among the most common causes of healthcare-associated infection (HAI) in the United States. Patients who have received a solid organ transplant (SOT) represent a unique population for the acquisition of HAIs, given their preoperative organ failure, immunosuppression, and need for invasive procedures. However, limited literature is published on S. aureus infections among children with SOT. We describe the epidemiology, antimicrobial susceptibility, and clinical features of S. aureus infections among pediatric SOT recipients. DESIGN: An ongoing prospective S. aureus surveillance database from 2001 to 2012 was searched for infections in patients with a history of SOT at Texas Children's Hospital. Medical records and antibiotic susceptibility profiles were reviewed; specific attention was applied to the time since transplantation to infection. RESULTS: Out of the total of 696 transplants performed during the study period, 38 pediatric SOT recipients developed 41 S. aureus infections; the highest incidence of infection was among heart recipients. Overall, the most common infectious diagnoses were skin-and-soft-tissue infections (66.1%), followed by bacteremia (15.3%). Among isolates in SOT patients, 47.5%, 16.9%, and 6.7% were resistant to methicillin, clindamycin, or mupirocin, respectively. Three infections (7.3%) occurred in the early post-transplant period (<1 month), all of which were bacteremia (P = 0.007) and all caused by methicillin-susceptible S. aureus (MSSA). The majority of infections (90.2%) occurred in the late post-transplant period (>6 months). In 10 cases (16.9%), S. aureus infection was associated with graft rejection during the same admission. CONCLUSIONS: S. aureus represents an important cause of morbidity in pediatric SOT recipients. While the majority of infections occurred late after transplant (>6 months), those acquired in the early post-transplant period were more often invasive and caused by MSSA in our hospital. Physicians caring for SOT recipients should be aware of the risks posed by this pathogen and the potential concomitant morbidity including graft rejection.

Successful treatment of Candida albicans osteomyelitis of the spine with fluconazole and surgical debridement: case report.

A 65-year old diabetic male presented with progressive bone destruction of thoracic spine (T-11&12) with cord compression. Candida albicans was isolated from aspirated materials pre-and intra-operative. Two weeks of fluconazole was given prior to surgical debridement, and fixation of the lesion. C. albicans isolated pre-and 2-weeks after fluconazole treatment were DNA-typed using AP-PCR. MIC was 2-4 mg/l in all isolates tested. The pre-and post treatment isolates had two DNA patterns, indicating the existence of two different strains. Surgical treatment was necessary for patient recovery.

DNA sequence analysis of rdxA and frxA from 12 pairs of metronidazole-sensitive and -resistant clinical Helicobacter pylori isolates.

We previously reported that inactivation of rdxA and/or frxA converted Helicobacter pylori from metronidazole sensitive to metronidazole resistant. To examine the individual roles of rdxA and frxA in the development of metronidazole resistance in H. pylori, we examined the status of rdxA and frxA from 12 pairs of metronidazole-sensitive and -resistant H. pylori isolates obtained following unsuccessful therapy containing metronidazole. Arbitrary primed fingerprinting analyses revealed that the genotypes of 11 sensitive and resistant pairs of strains were essentially identical. Amino acid sequence identities of RdxA and FrxA from the 14 metronidazole-sensitive isolates ranged from 92 to 98% and 95 to 98%, respectively, compared to that of H. pylori J99 (MIC, 1 microg/ml). All strains with high-level metronidazole resistance (MICs, 128 microg/ml) contained premature truncation of both RdxA and FrxA caused by nonsense and/or frameshift mutations. Strains with intermediate resistance to metronidazole (MICs, 32 to 64 microg/ml) contained a single premature truncation and/or altered RdxA and FrxA caused by nonsense, frameshift, and unique missense mutations. The low-level metronidazole-resistant strains (MICs, 8 microg/ml) contained unique missense mutations in FrxA but no specific changes in RdxA. The results demonstrate that alterations in both the rdxA and frxA genes are required for moderate and high-level metronidazole resistance and that metronidazole resistance that develops during anti-H. pylori therapy containing metronidazole is most likely to involve a single sensitive strain infection rather than a coinfection with a metronidazole-resistant strain.

Carotenoids, alpha-tocopherols, and retinol in plasma and breast cancer risk in northern Sweden.

OBJECTIVE: Using a nested case-referent design we evaluated the relationship between plasma levels of six carotenoids, alpha-tocopherol, and retinol, sampled before diagnosis, and later breast cancer risk. METHODS: In total, 201 cases and 290 referents were selected from three population-based cohorts in northern Sweden, where all subjects donated blood samples at enrolment. All blood samples were stored at -80 degrees C. Cases and referents were matched for age, age of blood sample, and sampling centre. Breast cancer cases were identified through the regional and national cancer registries. RESULTS: Plasma concentrations of carotenoids were positively intercorrelated. In analysis of three cohorts as a group none of the carotenoids was found to be significantly related to the risk of developing breast cancer. Similarly, no significant associations between breast cancer risk and plasma levels of alpha-tocopherol or retinol were found. However, in postmenopausal women from a mammography cohort with a high number of prevalent cases, lycopene was significantly associated with a decreased risk of breast cancer. A significant trend of an inverse association between lutein and breast cancer risk was seen in premenopausal women from two combined population-based cohorts with only incident cases. A non-significant reduced risk with higher plasma alpha-carotene was apparent throughout all the sub-analyses. CONCLUSION: In conclusion, no significant associations were found between plasma levels of carotenoids, alpha-tocopherol or retinol and breast cancer risk in analysis of three combined cohorts. However, results from stratified analysis by cohort membership and menopausal status suggest that lycopene and other plasma-carotenoids may reduce the risk of developing breast cancer and that menopausal status has an impact on the mechanisms involved.

Detection of Mycobacterium avium subspecies paratuberculosis in Crohn's diseased tissues by in situ hybridization.

OBJECTIVES: Reports about the association between Crohn's disease (CD) and cell wall-deficient (CWD) forms of Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) are controversial. This may be due to the heterogeneous nature of CD where only about 50% of the patients show granulomatous inflammation. Detection of CWD forms of M. paratuberculosis in tissues from patients with CD would support its association with the disease. To help identify these forms in inflamed tissues, a previously developed and optimized nonradioactive in situ hybridization method was applied on well-defined tissue materials obtained from patients with CD, ulcerative colitis (UC), and controls. METHODS: Specimens from 37 patients with CD (15 with epitheloid cell granulomas and 22 without granulomas), 21 UC, and 22 noninflammatory bowel disease (IBD) patients were analyzed by the in situ hybridization method based on the digoxigenin-labeled M. paratuberculosis IS900 fragment, previously shown to be species specific. Samples were counterstained with hematoxylin and eosin to show the location of the positive signal. Positive controls made of beef cubes injected with CWD and acid-fast M. paratuberculosis and negative controls were included in each experiment to monitor for nonspecific hybridization or staining. RESULTS: Six of 15 (40%) patients with CD and granulomas showed positive signals in myofibroblasts and macrophages. Interestingly, no positive signals were observed within granulomas. Only 4.5% of 22 CD samples from patients with nongranulomatous disease, 9.5% of 21 UC, and remarkably, none of the 22 non-IBD patients were M. paratuberculosis positive. CONCLUSION: The demonstration of DNA from CWD forms of M. paratuberculosis in this limited number of CD tissues further supports and confirms previous reports of its association with the granulomatous type of the disease.

Review article: Mycobacterium avium subsp. paratuberculosis as one cause of Crohn's disease.

A number of theories regarding the aetiology of Crohn's disease have been proposed. Diet, infections, other unidentified environmental factors and immune disregulation, all working under the influence of a genetic predisposition, have been viewed with suspicion. Many now believe that Crohn's disease is a syndrome caused by several aetiologies. The two leading theories are the infectious and autoimmune theories. The leading infectious candidate is Mycobacterium avium subspecies paratuberculosis (Mycobacterium paratuberculosis), the causative agent of Johne's disease, an inflammatory bowel disease in a variety of mammals including cattle, sheep, deer, bison, monkeys and chimpanzees. The evidence to support M. paratuberculosis infection as a cause of Crohn's disease is mounting rapidly. Technical advances have allowed the identification and/or isolation of M. paratuberculosis from a significantly higher proportion of Crohn's disease tissues than from controls. These methodologies include: (i) improved culture techniques; (ii) development of M. paratuberculosis-specific polymerase chain reaction assays; (iii) development of a novel in situ hybridization method; (iv) efficacy of macrolide and anti-mycobacterial drug therapies; and (v) discovery of Crohn's disease-specific seroreactivity against two specific M. paratuberculosis recombinant antigens. The causal role for M. paratuberculosis in Crohn's disease and correlation of infection with specific stratification(s) of the disorder need to be investigated. The data implicating Crohn's as an autoimmune disorder may be viewed in a manner that supports the mycobacterial theory. The mycobacterial theory and the autoimmune theory are complementary; the first deals with the aetiology of the disorder, the second deals with its pathogenesis. Combined therapies directed against a mycobacterial aetiology and inflammation may be the optimal treatment of the disease.

Specific seroreactivity of Crohn's disease patients against p35 and p36 antigens of M. avium subsp. paratuberculosis.

Crohn's disease (CD) is a chronic inflammatory bowel disease that is similar to Johne's disease in ruminants. Recent data have strengthened the association of M. avium subsp. paratuberculosis (M. paratuberculosis) with CD. To provide more evidence of an etiological association, antibody reactivities from CD patients were tested by immunoblotting against recombinant antigens that were identified previously from our M. paratuberculosis genomic library. Two clones (designated pMptb#40 (3.2-kb insert) and #48 (1.4-kb insert) expressing a 35K (p35)- and 36K(p36)-antigens showed specific reactivities with serum samples from CD patients. Serum samples from 75% of 53 CD patients, 14% of 35 normal individuals and 10% of 10 ulcerative colitis patients reacted to p35 antigen. Reactivities were also observed with serum samples from 89% of 89 CD patients, 14% of 50 normal controls and 15% of 29 ulcerative colitis patients reacted with p36 antigen. When the reactivity results from p35 and p36 were combined, the background from the controls was eliminated, i.e. only the CD patients reacted to both p35 and p36. The positive predictive value was 98% with specificity of 98% and the negative predictive value was 76% with sensitivity of 74% (39 positive out of 53). A statistical significance (p<0.0001) was observed when the results from CD serum samples reacting with either or both antigens were compared to the controls. The reactivity of anti-M. paratuberculosis (specifically against p35 and p36 antigens) antibodies in a significant proportion of CD patients would suggest a causal role for the organism in CD.

In situ hybridization method for studies of cell wall deficient M. paratuberculosis in tissue samples.

Cell wall deficient forms of mycobacteria may be important in the pathogenesis of Crohn's disease and sarcoidosis. However, no method has been available to localize this type of organisms in tissue sections. We developed an in situ hybridization method for the demonstration of Mycobacterium paratuberculosis spheroplasts (cell wall deficient forms) in paraffin embedded tissue sections.M. paratuberculosis spheroplasts were prepared by treatment with glycine and lysozyme. Pieces of beef were injected with the prepared spheroplasts. The samples were fixed in buffered formalin and paraffin embedded. A M. paratuberculosis-specific probe derived from the IS900 gene was used. Specificity was controlled by using an irrelevant probe and by hybridizing sections with spheroplasts from other bacteria. Beef samples injected with M. paratuberculosis spheroplasts were the only samples that hybridized with the probe. Beef samples containing acid-fast or spheroplast forms of M. smegmatis and M. tuberculosis as well as the acid-fast forms of M. paratuberculosis did not hybridize with the probe. Unrelated bacterial controls, i.e. Helicobacter pylori and Escherichia coli were also negative in the assay. In situ hybridization with the IS900 probe provides a specific way to localize M. paratuberculosis spheroplasts in tissue sections and may be useful for studies of the connection between M. paratuberculosis and Crohn's disease and sarcoidosis. The assay may also be valuable for studies on Johne's diseased animals.

Identification of cell wall deficient forms of M. avium subsp. paratuberculosis in paraffin embedded tissues from animals with Johne's disease by in situ hybridization.

M. avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease (JD) in ruminants leading to enormous economical losses in dairy and meat industries worldwide. During the subclinical stage of the disease, the infected animals are difficult if not impossible to detect by the available diagnostic tests including the PCR based ones. Although only considered an animal pathogen, cell wall deficient (CWD) forms of M. paratuberculosis have been isolated from patients with sarcoidosis and Crohn's disease (idiopathic diseases) in humans. Hence, the CWD form of this organism has been suspected to play a role in the pathogenesis of these diseases by persisting in the affected tissues and triggering a localized immune response and pathology. Differentiating between the CWD and acid-fast forms of this organism may lead to the determination of whether the CWD form is the pathogenic form in the subclinical cases of JD in animals and/or the etiologic agent for the above human diseases. To localize such organisms in tissue sections, CWD forms of mycobacteria were prepared in vitro and injected into beef cubes which were then formalin fixed and paraffin embedded. An in situ hybridization (ISH) technique, combined with the IS900 M. paratuberculosis-specific probe labeled with digoxigenin, was developed for the detection of nucleic acids specifically from the CWD forms but not their acid-fast forms in tissue sections. Specificity was confirmed by the negative finding with an irrelevant probe and with control tissue preparations containing CWD cells of related mycobacteria and unrelated organisms. This ISH procedure provides a way to distinguish between the acid-fast and CWD forms of M. paratuberculosis and to localize them in tissue sections. ISH may prove useful to evaluate the significance of CWD forms of M. paratuberculosis in the pathogenesis of JD, Crohn's disease and sarcoidosis.

Helicobacter pylori entry into human gastric epithelial cells: A potential determinant of virulence, persistence, and treatment failures.

BACKGROUND AND OBJECTIVES: Intracellular location of Helicobacter pylori in human gastric epithelial cells has been observed in biopsies. Whether this reflects an ability to invade host cells and establish an intracellular niche remains to be determined. METHODS: The interactions between a clinical isolate of H. pylori and primary cell cultures from human gastric epithelium or the human epithelial cell line HEp-2 were monitored using time-lapse photography. This technique allows studies of the dynamics of host-microbial interactions. RESULTS: H. pylori cells readily approached and established close contacts with epithelial cells followed by uptake of the bacteria into the cellular cytoplasm. Entry into epithelial cells was achieved through an active process of bacterial motility and penetration of the cell membranes. In conventional invasion assays using HEp-2 cells, an increased internalization in a strain producing the vacuolating cytotoxin was observed, compared to the isogenic VacA knockout mutant. CONCLUSION: Invasion of gastric epithelium represents a hitherto unappreciated trait of H. pylori that could contribute to the bacterium's ability to establish persistent infection that evades the mucosal immune defense and sometimes also antimicrobial therapy. A small number of bacterial cells with a transient intracellular habitat could serve as a seeder population, providing a backup for a constantly challenged and fluctuating luminal population.

Antibacterial therapy for Crohn's disease: a review emphasizing therapy directed against mycobacteria.

The most commonly used antibiotics in Crohn's disease are nitroimidazoles and macrolides often combined with corticosteroids or sulfasalazine. There has been interest in a mycobacterial involvement in Crohn's disease since its earliest description. It is not recognized that Mycobacterium avium subspecies paratuberculosis, a proven but uncommon cause of human disease, is widespread in the human food chain especially in dairy products and beef. M. paratuberculosis has been identified in tissues from a higher proportion of Crohn's disease patients than controls, suggesting that it may be one of the causes of Crohn's disease. We review the large number of antibiotic trials in Crohn's disease. Although studies have been performed with many different protocols and variations in the definition of success, preliminary reports of multiple drug therapies are encouraging. Nevertheless, large-well designed preferably placebo-controlled studies are needed before one could recommend such therapy.

Antibiotics as primary therapy for Crohn's disease.

Crohn's disease, an inflammatory bowel disease of unknown cause, was initially considered to have an infectious etiology. As the infectious agent could not be identified, it was grouped among the immune disorders. As a result, research and clinical trials were directed towards the autoimmune theory and patients were treated with steroids, immunomodulators, aminosalicylates and, most recently, anti-tumor necrosis factor-alpha. Because of the inconsistency of treatment success and the failure to cure Crohn's disease, many physicians turned to antibiotics in search for alternative solutions, especially when other regimens failed. Attention has recently been directed toward possible infectious causes of Crohn's disease. Although it is still unknown whether microbial agents are etiologically involved in the pathogenesis of Crohn's disease, there has been a growing interest in trying antibiotics in the management of Crohn's disease. This review summarizes the data available regarding antibiotic treatment of Crohn's disease in relation to a possible mycobacterial etiology. Multidrug therapies are in clinical trials and the results of these randomized, controlled, double-blind studies are needed before guidelines about whether to include antibiotics as part of the treatment of Crohn's disease management can be made.

Fatty-acid composition in serum phospholipids and risk of breast cancer: an incident case-control study in Sweden.

The study of the relationship between dietary intake of fatty acids and the risk of breast cancer has not yielded definite conclusions with respect to causality, possibly because of methodological issues inherent to nutritional epidemiology. To evaluate the hypothesis of possible protection of n-3 polyunsaturated fatty acids (PUFA) against breast cancer in women, we examined the fatty-acid composition of phospholipids in pre-diagnostic sera of 196 women who developed breast cancer, and of 388 controls matched for age at recruitment and duration of follow-up, in a prospective cohort study in Umeâ, northern Sweden. Individual fatty acids were measured as a percentage of total fatty acids, using capillary gas chromatography. Conditional logistic-regression models showed no significant association between n-3 PUFA and breast-cancer risk. In contrast, women in the highest quartile of stearic acid had a relative risk of 0.49 (95% confidence interval, 0.22-1.08) compared with women in the lowest quartile (trend p = 0.047), suggesting a protective role of stearic acid in breast-cancer risk. Besides stearic acid, women in the highest quartile of the 18:0/18:1 n-9c ratio had a relative risk of 0.50 (95% confidence interval, 0.23-1.10) compared with women in the lowest quartile (trend p = 0.064), suggesting a decrease in breast-cancer risk in women with low activity of the enzyme delta 9-desaturase (stearoyl CoA desaturase), which may reflect an underlying metabolic profile characterized by insulin resistance and chronic hyper-insulinemia.

Characterization of Mycobacterium paratuberculosis p36 antigen and its seroreactivities in Crohn's disease.

Recent data using improved cultural, molecular, and serological techniques have strengthened the association of Mycobacterium paratuberculosis with Crohn's disease, an inflammatory bowel disease (IBD) with unknown etiology. To provide more evidence of an etiological association, antibody reactivities of Crohn's disease patients were tested by immunoblotting against M. paratuberculosis-recombinant antigens. A clone containing a 1,402-bp insert and expressing a 36K-antigen (p36) was analyzed. No homology was found between the deduced amino acid sequence of p36 and any protein sequences compiled in the GenBank indicating that p36 is a novel mycobacterial protein. The reactivity of 199 serum samples was tested against the p36 by immunoblotting technique. Sera from 77 of 89 (86.5%) Crohn's disease patients and 16 of 18 (89%) sera from patients with tuberculosis and leprosy reacted with p36 compared to 5 of 42 (12%) ulcerative colitis and non-IBD control sera (p < 0.0001). In addition, p36 reacted to all sera from 10 normal controls that were Bacillus Calmette-Guerin (BCG)-immunized and only to 10% of 40 normal controls that were not BCG-immunized. The fact that sera from Crohn's disease patients reacted to p36 with the same high frequency as the sera from patients that were exposed to mycobacterial antigens further supports the hypothesis of the mycobacterial etiology in Crohn's disease.

Studies of the regulation of the mouse carboxyl ester lipase gene in mammary gland.

The lactating mammary gland and pancreas of mouse constitute the main tissues for synthesis and secretion of a bile-salt-stimulated lipase called carboxyl ester lipase (CEL). In this paper we have analysed the endogenous CEL gene expression in mammary gland. It is shown that the gene is expressed at day 14 of pregnancy, which is synchronous with that of the whey acidic protein (WAP) gene. Even though the CEL and WAP genes are induced at the same time during mammary gland differentiation, their regulation is different with respect to dependence on lactogenic hormones. The high induction of the WAP gene expression due to the activation of signal transducer and activator of transcription (STAT)5 by prolactin has not been observed for the CEL gene, even though it has been demonstrated that both STAT5 isoforms interact with one of the gamma-interferon activation sequence sites in the promoter of the CEL gene. Hence we have demonstrated that the prolactin/STAT5 signal is not involved in a general and significant activation of 'milk genes'. Instead of a direct effect of the lactogenic hormones, the up-regulation of the CEL gene is correlated with an increase in the number of differentiated epithelial cells. Furthermore, promoter studies using the mammary-gland-derived cell line, HC11, show that a major positive element in the CEL gene promoter interacts with a member(s) of the CCAAT-binding transcription factor/nuclear factor 1 family, binding to a palindromic site. Binding of this factor(s) is important for the tissue-specific activation of the CEL gene in the mammary gland, because no activation by this factor(s) was seen in cells of pancreatic origin.

Presence of Helicobacter species DNA in Swedish water.

Municipal water, treated waste-water and well-water from all 25 counties in Sweden were analysed for the presence of Helicobacter spp. DNA. Bacteria were concentration by immunomagnetic separation. Culture, Gram staining and urease tests were performed before lysis of bacteria. Two polymerase chain reaction (PCR) assay with high sensitivity (adhesin and 16S rRNA) were followed by Helicobacter spp. specific hybridization. Nine of 24 private wells, three of 25 municipal tapwater and three of 25 wastewater samples were positive for both PCR assays. Positive municipal and waste-water samples were positive for counties. Non-specificity of PCR methods due to the presence of unknown bacteria within the genus helicobacter cannot be totally ruled out. Thus, the clinical significance of findings Helicobater spp. DNA in drinking water needs to be further evaluated.

Effects of suramin on polyamine metabolism in B16 murine melanoma cells.

Polyamines and their biosynthetic enzymes, such as ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are crucial for normal and neoplastic cell growth and differentiation. Suramin inhibits the growth of several tumor cells by affecting various intracellular targets, but its effects on polyamines are not known. In this study, the effects of suramin on some parameters of polyamine metabolism in B16 melanoma cells were investigated in vitro. Suramin increased cellular ODC activity and ODC mRNA levels, whereas the drug was directly inhibitory to the enzyme. AdoMetDC was not affected. Cellular putrescine levels were enhanced by suramin, whereas spermidine and spermine pools were unaltered. Cells cultured in the presence of suramin showed decreased cellular polyamine transport, but no direct inhibitory effect on the polyamine transporter could be found. Fluorescence spectroscopy demonstrated a direct interaction between suramin and spermine. It may be concluded that suramin affects polyamine metabolism, and that its effects in some respects are opposite to those of alpha-difluoromethylomithine (DFMO), a specific inhibitor of ODC.