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Inducible Metronidazole Resistance in nim–Positive and nim–Negative Bacteroides fragilis Group Strains after Several Passages Metronidazole Containing Columbia Agar Plates

Abstract Background: Recent data show an emergence of resistance in the Bacteroides fragilis group against several antimicrobial agents and inducible resistance against metronidazole in nim–positive strains. The aim of the present study was to investigate inducible metronidazole resistance in nim–positive as well as in nim–negative B. fragilis group strains. Materials and Methods: Of 18 B. fragilis strains (including four nim–positive reference strains and one ATCC strain), two Bacteroides ovatus strains, and one Bacteroides thetaiotaomicron DSM strain minimum inhibitory concentration (MIC) values for metronidazole were determined by Etest^? and analyzed for nim genes (nimA to –G) by PCR. For this purpose bacterial suspensions were incubated on supplemented Columbia agar plates containing metronidazole at twice the MIC value of the specific strain and incubated under anaerobic conditions for 48 hours. After incubation, growing bacteria were harvested and thereafter incubated at four times the original MIC. This procedure was repeated with increasing antibiotic concentrations. The resulting MIC values were confirmed by Etest^?. Results: The MIC values for metronidazole of the four nim–positive reference strains ranged from 3 to 8 mg/l. The B. fragilis ATCC 25285 strain and the B. thetaiotaomicron DSM 2255 strain were nim negative with MIC values of 0.19 mg/l and 0.75 mg/l, respectively. Three clinical isolates of B. fragilis strains showed MIC values of > 256 mg/l. In all three strains, nim genes were detected by PCR. The other clinical isolates were nim negative. In these strains, MIC values ranged from 0.19 to 0.75 mg/l. After several passages on metronidazole containing agar, all B. fragilis group strains exhibited MIC values of > 256 mg/l determined by Etest^?. Conclusion: Metronidazole resistance can be selected not only in nim–positive strains but also in nim–negative strains, suggesting that mechanisms other than nim genes are involved. These findings and the emerging resistance of the B. fragilis group against several antimicrobial agents underscore the importance of susceptibility testing of anaerobes even in routine laboratories. This paper is dedicated to the founders of the Walter Marget Foundation, D. Adam and F. Daschner, in gratitude for their support of the training in infectious diseases.     

A low-flow adaptation phase improves shear-stress resistance of artificially seeded endothelial cells

INTRODUCTION: The purpose of this study was to evaluate the effect of different adaptation phases on the shear-stress resistance of endothelial cells seeded artificially onto vascular prostheses and biological heart valves. MATERIAL AND METHODS: Human endothelial cells (EC), fibroblasts (FB), and smooth muscle cells (SMC) were isolated from vena saphena magna pieces and expanded in culture. Group A: 15 polyurethane vascular grafts (20 mm diameter) were seeded with FB and SMC (53 +/- 1.2 million cells), followed by EC seeding (39 +/- 0.9 million cells). Group B: eight stentless porcine valves (Freestyle, Medtronic, USA) were seeded with FB (68 +/- 1.5 million cells) and EC (42 +/- 1.1 million cells). Shear-stress testing was done under pulsatile flow (pulse rate: 80 pulses/min.). Adaptation phase: flow was set to 0.9 +/- 0.3 l/min (systolic pressure: 40 - 50 mm Hg). High flow was 3.2 +/- 0.6 l/min. (systolic pressure: 140 - 160 mm Hg) and lasted over four hours in all groups. The vascular grafts were divided into three groups (n = 5 each): group 1 (high flow immediately), group 2 (adaptation phase of 15 minutes), and group 3 (adaptation phase of 30 minutes). The valves either were given high flow immediately (n = 4) or had an adaptation phase of 30 minutes (n = 4). Specimens were obtained after cell seeding, before, and after perfusion. RESULTS: A confluent EC layer was achieved on all grafts. After perfusion without adaptation, large defects within the cell layer were found. No FB and SMC were seen at the bottom of these defects. In group B, the defects were largest on the ventricular surface of the leaflets. After an adaptation phase of 15 minutes in group A, only a few defects within the EC layer were detected with a still confluent FB and SMC. After a 30-minute adaptation phase defects within the EC layer were very rare and no interruption of the underlying FB and SMC layer was seen. Immunohistochemical staining for factor VIII and CD31 proved the EC to be viable and staining for collagen IV and laminin revealed the formation of a basement membrane. After perfusion, the specimen also stained positive for eNOS. CONCLUSION: An adaptation phase of 30 minutes proved to be sufficient to allow artificially seeded endothelial cells to adapt to shear stress. The formation of a basement membrane was of great importance for the maintenance of a confluent EC layer.     

The triple A syndrome is due to mutations in ALADIN, a novel member of the nuclear pore complex

The triple A syndrome (MIM#231550) is a rare autosomal recessive disorder characterized by adrenocorticotropic hormone (ACTH) resistant adrenal failure, achalasia, alacrima, and a variety of neurological and dermatological features. The triple A syndrome is caused by mutations in the AAAS gene, which encodes a protein known as ALADIN (ALacrima Achalasia aDrenal Insufficiency Neurologic disorder). ALADIN is a new WD-repeat protein that has no significant homology to any previously identified WD-repeat protein. It has been shown that it colocalizes with nuclear pore complexes (NPCs), a finding that strongly suggests an involvement of ALADIN in nucleocytoplasmic transport. An investigation of 110 families with triple A syndrome disclosed mutation hot spots including Q15K (exon 1), and S293P (exon 8), which occur in 17 and 21 families from different geographical regions, respectively. The variable phenotype of all patients cannot be correlated with the localization and the nature of the ALADIN mutations. Thus, modifying genes/factors may be involved in the progression of this neurodegenerative disease. The lack of AAAS mutations in eight patients and negative linkage to chromosome 12q13 in three families are suggestive of genetic heterogeneity. To examine the cellular localization of ALADIN mutants causing triple A syndrome, we investigated nine different ALADIN-mutants: 2 nonsense (W84X, Q456X), 2 frameshift (F157fsX171, G397fsX414) and 5 point mutations (Q15K, L25P, H160R, S263P, L381R) by transfection experiments with green fluorescence protein. Mutants were predominantly localized in the cytoplasm, but also found in the nucleus indicating that ALADIN is essential for NPC targeting. To investigate physiological functions of ALADIN in vivo, we generated and analysed Aaas-/- knockout mice by homologous recombination in embryonic stem cells. Surprisingly, required animals lack any gross abnormality in adrenal and nervous system function. Further studies have to investigate the role of ALADIN at NPCs and to identify interacting proteins. Functional analyses of ALADIN may permit further understanding of its role for adrenocortical function and neurodevelopment.     

Comparison of moxalactam and cefazolin as prophylactic antibiotics during cesarean section.

Prophylactic antibiotics have been shown to be effective in decreasing the incidence of febrile morbidity associated with cesarean section after labor. However, the relative effectiveness of different single antibiotics has been studied infrequently, and these investigations have been limited by small patient samples. Several new, broad-spectrum antibiotics are now available, and any further benefit from more traditional antibiotics for surgical prophylaxis remains untested. A randomized prospective double-blind therapeutic trial was therefore undertaken to compare the value of a first-generation cephalosporin (cefazolin) with a new third-generation cephalosporin (moxalactam). Between July 1981 and June 1983, 254 qualifying women who underwent primary cesarean section after labor were randomly chosen for either of the two treatment groups. Although not statistically significant, the rates of febrile morbidity, wound infection, and endometritis were less for those treated with cefazolin (4.0, 3.2, and 0.8%, respectively) than for those treated with moxalactam (9.2, 7.7, and 1.6%, respectively). No serious adverse effects were apparent in the mother and newborn infant from short-term exposure to either drug. Although the newer, more expensive, and broader-spectrum cephalosporin, moxalactam, was associated with a low postoperative febrile morbidity rate and short postpartum hospitalization, it was no more beneficial than cefazolin.     

Neutrophil alkaline phosphatase: comparison of enzymes from normal subjects and patients with polycythemia vera and chronic myelogenous leukemia.

To determine whether decreased alkaline phosphatase activity in the granules from neutrophils of patients with chronic myelogenous leukemia (CML) was due to an absence of enzyme or the production of defective enzyme, we compared the immunologic properties of granule alkaline phosphatase derived from patients with CML with that of normal subjects and patients with polycythemia vera (PRV). Antisera prepared in rabbits against granule alkaline phosphatase purified from the neutrophils of a patient with PRV produced a single precipitin line of antigenic identity when reacted with extracts of normal, PRV, and CML neutrophil granules. A histochemical stain for alkaline phosphatase activity (alpha-naphthyl acid phosphate coupled with Fast Blue RR) specifically stained the precipitin line. A variety of quantitative precipitin techniques failed to produce satisfactory precipitation of alkaline phosphatase activity. Comparative analyses were therefore performed by affinity chromatography using goat antirabbit-gammaglobulin linked to Sepharose 4B to adsorb alkaline phosphatase complexed with rabbit gamma globulin. With this method, 100% of CML, normal, and PRV alkaline phosphatase could be adsorbed. Using limiting concentrations of antibody, a proportionally smaller fraction of enzyme activity was absorbed as the concentration of PRV alkaline phosphatase or normal alkaline phosphatase was increased. Extracts of CML granules containing comparable amounts of protein but 200-fold less alkaline phosphatase activity per milligram did not specifically reduce adsorption. Thus, in CML, we found no evidence that the granulocytes contained a large amount of antigenically normal but enzymatically defective alkaline phosphatase. Examination of electron micrographs revealed no significant differences in the number or distribution of granules in the granulocytes of normal subjects or patients with PRV or CML. This suggests that the low level of neutrophil alkaline phosphatase in CML granulocytes is the result of decreased enzyme content and not a consequence of synthesis of catalytically defective enzyme.