Human isolates of Staphylococcus caprae: association with bone and joint infections.

Staphylococcus caprae is a coagulase-negative, DNase-positive member of the genus Staphylococcus usually associated with goats, but since 1991 a few laboratories have reported isolating the organism from human clinical specimens. We report on the isolation of 14 strains from human specimens and note that 10 strains were obtained from patients with bone and joint infections. Nine of the 10 infections started with traumatic fractures, and the other was a case of mastoiditis. Seven of these 10 infections were in patients with orthopedic prostheses, which appears to be a risk factor. Three of the 14 strains were from transplant patients. For three of the patients, S. caprae was the only organism isolated. S. caprae may be misidentified because it is not represented in the current MicroScan or Vitek identification systems which are in use in many laboratories, but the organism can be differentiated by a few biochemical tests. S. caprae produces positive results for DNase, pyrrolidonyl aminopeptidase, and acid production from mannitol and maltose; it produces negative results for ornithine decarboxylase and tube coagulase.

Fatty acid characterization of rapidly growing pathogenic aerobic actinomycetes as a means of identification.

The fatty acid compositions of 39 type strains and 529 clinical or reference strains of pathogenic aerobic actinomycetes were analyzed after standardized culture by using the Microbial Identification System (MIS). Library entries for each type strain were created by using the MIS Library Generation Software, and the fatty acid profiles of clinical and reference strains were compared to these library entries. The bacteria separated into two large groups based upon major amounts of branched-chain or of saturated or monounsaturated straight-chain fatty acids. Identification of isolates was possible by using only the type strains for comparison, but fatty acid heterogeneity occurred within most species.

Identification of staphylococci with a self-educating system using fatty acid analysis and biochemical tests.

We characterized all of the 35 aerobic taxa of the genus Staphylococcus by using an objective, self-learning system combining both whole-cell fatty acid (FA) analysis and the results of 35 biochemical tests. Isolates were compared with the type strain for each taxon to generate an FA profile library and a biochemical table of test responses. Isolates were accepted into the system if they had a similarity index of > or = 0.6 for a taxon within the FA profile library and if they were identified as the same taxon by a computer program using a probability matrix constructed from the biochemical data. These stringent criteria led to acceptance of 1,117 strains assigned to legitimate taxa. Additional FA groups were assembled from selected strains that did not meet the inclusion criteria based on the type strains and were added to the system as separate entries. Currently, 1,512 isolates have bee accepted into the system. This approach has resulted in a comprehensive table of biochemical test results and a FA profile library, which together provide a practical system for valid identifications.

Influence of macrophage depletion on bacterial translocation and rejection in small bowel transplantation.

Rejection and sepsis can be intimately related following small bowel transplantation when rejection compromises normal intestinal barrier mechanisms and bacterial translocation results. Macrophages play a role in controlling the egress of intestinal luminal bacteria--and they have also been implicated in allograft rejection. In this study, the role of macrophages in rejection and bacterial translocation was evaluated by depleting macrophages in donors and/or recipients of rat small bowel allografts with injection of liposome-encapsulated dichloromethylene diphosphonate (CL2MDP). In preliminary studies, we demonstrated that a single intraperitoneal injection of liposome-encapsulated CL2MDP (350 mg/kg) depleted ED2-positive macrophages by > 90% in the liver mesenteric lymph nodes and proximal and distal small bowel, and by approximately 50% in the spleen. ED1-positive macrophages were depleted by > 90% in the liver and by approximately 50% at the other sites. ED3-positive macrophages were completely depleted. Dendritic cells were > 90% depleted in the spleen and mesenteric lymph nodes, but were not depleted in the small bowel. Macrophage depletion in the donor resulted in increased translocation of bacteria to the peritoneal cavity (P = 0.03) if recipient macrophages were present. With histopathologic analysis, a significantly milder rejection with less arteritis was seen in the allografts of the recipient macrophage-depleted group compared with nondepleted controls (P = 0.045). This suggests that recipient macrophages play an important role in rejection. With macrophage depletion in both the donor and the recipient, graft survival was prolonged significantly (13.2 +/- 1.9 days) compared with non-macrophage-depleted controls (9.2 +/- 1.3 days) (P = 0.003). These studies suggest that strategies targeting recipient macrophages may be useful in controlling small bowel allograft rejection without increasing bacterial translocation.

Diversity of methicillin-resistant Staphylococcus aureus isolated in a Canadian hospital.

Three neonates and three other patients located elsewhere in the hospital became infected with Staphylococcus aureus. Initial automated microdilution susceptibility testing with oxacillin and disk diffusion testing with amoxicillin-clavulanic acid indicated the isolates had borderline oxacillin resistance (MICs 4 micrograms/ml), presumably due to hyperproduction of beta-lactamase. Chromosomal DNA restriction fingerprinting and phage typing revealed the neonatal isolates to be identical; whereas, the other patients were infected with three different strains. Further analysis of the four strains by Southern hybridization with a mecA specific oligoprobe and a quantitative beta-lactamase assay demonstrated that two strains carried the mecA gene (coding for low affinity penicillin-binding protein 2a), and two strains were hyperproducers of beta-lactamase, including one which was mecA gene positive. One strain neither carried the mecA gene nor hyperproduced beta-lactamase. The two mecA gene positive strains displayed oxacillin MICs of 16 micrograms/ml on dilution susceptibility testing in 4% NaCl supplemented Mueller-Hinton agar. Hence, they were considered intrinsically methicillin-resistant Staphylococcus aureus. Both oxacillin and amoxicillin-clavulanic acid MICs were increased on NaCl supplementation. Results of amoxicillin-clavulanic acid disk diffusion susceptibility testing did not correlate with quantitative beta-lactamase production. It is recommended that clinical laboratories do not use amoxicillin-clavulanic acid disk diffusion assays to differentiate suspected borderline resistance due to beta-lactamase hyperproduction from mecA gene expression of PBP-2a since additional mechanisms may account for resistance.

Treatment with FK506 prevents rejection of rat colon allografts.

Colon transplantation has been proposed as a method to improve the function of an intestinal allograft. The present study examined the risk of colon rejection and the effect of FK506 on colon rejection in BN-->LEW rats with orthotopic bowel transplants. The first 4 groups included rats with untreated allografts (group 1), rats with isografts treated with 0.6 mg/kg FK506 (group 2), rats with allografts treated with 0.6 mg/kg FK506 (group 3), and rats with allografts treated with 0.4 mg/kg FK506 (group 4). In each of these groups (10-12 rats), half of the animals received a small bowel graft only (SB), while the other half received a small bowel, ascending colon, and cecum graft (SBC). The animals were followed daily until they died or were killed at 4 weeks. In group 5, an additional 18 untreated rats with SBC allografts were randomly killed on the third, fifth, seventh, and tenth postoperative days to study the sequential histopathologic and immunopathologic changes of colon rejection. There was no difference in survival, body weight, nutritional parameters, or bacterial contamination after SB and SBC transplantation. Intestinal transit was slower after SBC than SB transplantation (P < 0.05). Sequential histopathologic studies revealed that (1) the severity and time course of colon rejection was similar to small intestine rejection, and (2) the features of colon rejection were similar to ulcerative colitis. There was no evidence of graft-versus-host disease after SBC transplantation. In summary, adding a segment of large bowel to a small bowel allograft does not increase the risk of rejection or surgical complications. The transplanted colon slows intestinal transit. Treatment with FK506 effectively prevents colon rejection. These data suggest that adding a colon graft may improve the outcome of clinical small bowel transplantation.

Transplantation of segmental rat intestinal grafts including the ileocecal value and the ascending colon.

Extrinsic denervation and lymphatic disruption impair nutrient absorption after small bowel transplantation. The present study was undertaken to determine whether adding the ileocecal valve with or without the ascending colon would improve the function of a segmental intestinal graft. Five groups of Lewis rats (n = 10/group) were studied. Group I had a sham laparotomy. Groups II, III, IV, and V had the native jejunum, ileum, and cecum replaced with a graft. Inbred Lewis rats were used as isogeneic donors for the transplants to avoid the confounding effect of graft rejection. Group II had the entire jejunum and ileum transplanted. Group III had 20 cm of terminal ileum transplanted. Group IV had 20 cm of the terminal ileum including the ileocecal valve transplanted. Group V had 20 cm of the terminal ileum, the ileocecal valve, and the ascending colon transplanted. The terminal ileum-transplanted and terminal ileum/ileocecal valve-transplanted groups lost more than 25% of their preoperative weight by the end of the second postoperative week; most of these animals were killed because of inanition. In contrast, the sham laparotomy, jejunum/ileum-transplanted, and ascending colon-transplanted groups remained healthy until completion of the study on the 28th postoperative day. The ascending colon-transplanted group had slower intestinal transit and less bacterial contamination of the terminal ileum compared with the terminal ileum-transplanted and terminal ileum/ileocecal valve-transplanted groups (P < 0.05). Transplantation of the ascending colon and the ileocecal valve significantly improves the function of segmental small bowel isografts in rats. These data suggest that adding a colonic segment may be a simple method to improve the function of short-segment cadaveric and living-related intestinal grafts in humans.

Synergistic effects of tumour necrosis factor and morphine on gut barrier function.

OBJECTIVE: To study the effects of tumour necrosis factor (TNF) and morphine on intestinal permeability, intestinal transit and bacterial translocation in the rat. DESIGN: A randomized interventional controlled experiment. SETTING: University surgery and microbiology research laboratory. PARTICIPANTS: Forty-four rats in five groups as follows: control (n = 9); treated with morphine every 2 hours for 8 hours (n = 9); treated with TNF for 5 minutes (n = 10); treated with TNF plus morphine every 2 hours for 8 hours (n = 6); and treated with TNF plus morphine every 3 hours for 24 hours (n = 10). MAIN OUTCOME MEASURES: Intestinal permeability as measured by the uptake of chromium-51 ethylenediaminetetraacetate (51Cr-EDTA) over 8 hours, intestinal transit as measured by the amount of 51Cr-EDTA remaining in the gastrointestinal tract at the time of animal sacrifice, intestinal bacteria counts and translocation of bacteria as measured from bacterial counts of mesenteric lymph nodes, spleen and liver at the time of sacrifice. RESULTS: Morphine increased intestinal transit time and ileal bacteria counts (p < 0.05). TNF alone did not increase intestinal permeability or bacterial translocation. TNF plus morphine increased intestinal transit time, intestinal permeability, bacterial counts and bacterial translocation (p < 0.05). CONCLUSIONS: Morphine or increased intestinal transit time, or both, increases the concentration of intestinal bacteria. Morphine plus TNF increases intestinal bacteria counts, intestinal permeability and bacterial translocation. Morphine alone does not increase intestinal permeability or bacterial translocation.

Heat of fusion measurement of a low melting polymorph of carbamazepine that undergoes multiple-phase changes during differential scanning calorimetry analysis.

Two polymorphs of carbamazepine that melt at 176 and 189 degrees C and are known to be enantiotropic have been characterized more fully than in previous reports. For the first time, a value for the heat of fusion (29.3 kJ/mol) of the lower melting form is estimated, even though single melting DSC endotherms were not obtained. A method is described for summing the heat flows of intermediate transitions that occur when low melting carbamazepine is heated to a temperature where it will remain melted. The method should have general applicability in studies of polymorphs. Other characteristics of the carbamazepine enantiotropic pair are reported, including the transition temperature (71 degrees C).

Intestinal permeability and bacterial translocation following small bowel transplantation in the rat.

In addition to its role in absorbing nutrients, the intestinal mucosa provides an important barrier against toxins and bacteria in the bowel lumen. The present study evaluated gut barrier function following orthotopic (in continuity) intestinal grafting in rats. Graft histology, intestinal permeability, and bacterial translocation to the grafted mesenteric lymph nodes, the host's liver, and the host's spleen were assessed on the 3rd, 5th, and 7th postoperative days. The study group received no immunosuppression after allotransplantation. The two control groups included rats with isografts and rats with cyclosporine-treated allografts. On the 7th POD, the study animals had moderate transmural inflammation due to rejection, with normal histology in the isografts and CsA-treated allografts; increased intestinal permeability, measured by urinary excretion of oral 51Cr-EDTA (P less than 0.01); and increased number of bacteria in the MLN and spleen (P less than 0.05). The number of bacteria in the MLN and spleen of the study group positively correlated with the changes in intestinal permeability (P less than 0.05). Rejection of the orthotopic intestinal graft leads to increased intestinal permeability and bacterial translocation from the lumen of the graft to the host's reticuloendothelial system. Measures to improve gut barrier function and antibiotic therapy during rejection episodes may help reduce the incidence of septic complications after intestinal grafting.

Laboratory investigation of outbreak of hemorrhagic colitis caused by Escherichia coli O157:H7.

A severe outbreak of hemorrhagic colitis occurred in London, Ontario, during the month of September 1985. A total of 55 residents and 18 employees of a nursing home developed diarrhea, and 17 residents (age range, 78 to 99 years) died. Specimens from 38 patients, 37 employees and contacts, and 10 autopsies were investigated for all enteric pathogens. Specimens were also planted on MacConkey-sorbitol agar. Fecal extracts were tested on Vero cells for cytotoxin (FVT). Escherichia coli isolates were serotyped and tested for verotoxin and beta-glucuronidase production. Of the 38 symptomatic patients, 26 were positive for FVT, verotoxin-producing E. coli (VTEC), or both. Of the 105 specimens that were examined from these 38 patients, FVT and VTEC were both positive in 30 specimens, FVT only was positive in 13 specimens, and VTEC only was positive in 4 specimens. None of the 27 specimens from 10 autopsies was positive for FVT or VTEC. No other enteric pathogen was found in any of the cases. All asymptomatic individuals were negative for both FVT and VTEC. Of 19 VTEC strains that were isolated, 18 belonged to serotype O157:H7. These 18 strains and 2 more strains that were obtained from sporadic cases that had occurred within the 2 previous months were found to give similar biochemical reactions in a 36-test identification system. All isolates of serotype O157:H7 were beta-glucuronidase negative and susceptible to the antimicrobial agents that are used to treat E. coli infections. Testing for FVT and VTEC was found to be the most sensitive and specific technique for the laboratory diagnosis of this disease. Negative sorbitol, positive raffinose, and negative beta-glucuronidase tests appeared to be consistent markers for aiding in the detection of E. coli O157:H7.

Enterobacter meningitis--treatment complicated by emergence of mutants resistant to cefotaxime.

A case of Enterobacter cloacae meningitis in a postoperative patient is reported. A slow response to cefotaxime necessitated the use of gentamicin and trimethoprim-sulfamethoxazole for cure. Two types of resistance in the strain of E. cloacae isolated to cefotaxime were demonstrated: an inducible beta-lactamase that likely was the cause of the poor response to cefotaxime and a constitutive beta-lactamase in a mutant strain detected by a disc susceptibility test.

Characterization of polymorphism of gepirone hydrochloride.

Gepirone hydrochloride, an investigational anxiolytic drug, was found to have at least three polymorphic forms which melted at 180 degrees C (I), 212 degrees C (II), and 200 degrees C (III). Thermal analytical studies showed that forms I and II were an enantiotropic pair, as were forms I and III. Form III was monotropic with form II and there was no temperature at which III was the most stable polymorph. Solubility data from powder dissolution studies were used to estimate a transition temperature of 74 degrees C for the enantiotropic pair of I and II. The difference in enthalpy was 4.5 kcal/mol at 74 degrees C and 2.54 kcal/mol at 25 degrees C. Form I was the most physically stable below 74 degrees C, whereas form II was the most stable above 74 degrees C. Essentially pure samples of I and II could be obtained easily, but pure III could be developed only transiently on the differential scanning calorimeter heating block. Video taping of hot-stage microscopic observations for review was helpful for detecting seed crystals of II or III in samples of I. The information developed is presented in a hypothetical free energy-temperature diagram.

High-performance liquid chromatographic analysis of cyclophosphamide.

A reversed-phase high-performance liquid chromatographic technique is described for the analysis of cyclophosphamide in the presence of its hydrolysis products. The drug was quantified using a UV detector at a low wavelength. A single band was observed for the intact drug, which was well separated from tis hydrolysis product(s). Quantificaiton was obtained with adequate precision by the use of an injector loop or a suitable internal standard (hydrocortisone). The technique requires no extraction of the drug from aqueous solution or derivatization for analysis. The method was applied to partially hydrolyzed and to known solutions of cyclophosphamide. With suitable modification, the method may be useful for analysis of dosage forms but probably lacks the sensitivity necessary for analysis of the drug in biological samples.

Transfer of multiple antibiotic resistance from Bacteroides fragilis to Escherichia coli.

Multiple antibiotic resistance was transferred from a clinical isolate of Bacteroides fragilis to a strain of Escherichia coli K12. Resistance to ampicillin, amoxicillin, cephalothin, tetracycline, minocycline, and chloramphenicol was transferred as a unit, but the resistance markers became segregated during storage of the recipient strains.