Identification, Phylogenetic Analysis, and Biological Characterization of Serratia marcescens Strains Causing Cucurbit Yellow Vine Disease.

ABSTRACT A serious vine decline of cucurbits known as cucurbit yellow vine disease (CYVD) is caused by rod-shaped bacteria that colonize the phloem elements. Sequence analysis of a CYVD-specific polymerase chain reaction (PCR)-amplified 16S rDNA product showed the microbe to be a gamma-proteobacterium related to the genus Serratia. To identify and characterize the bacteria, one strain each from watermelon and zucchini and several noncucurbit-derived reference strains were subjected to sequence analysis and biological function assays. Taxonomic and phylogenetic placement was investigated by analysis of the groE and 16S rDNA regions, which were amplified by PCR and directly sequenced. For comparison, eight other bacterial strains identified by others as Serratia spp. also were sequenced. These sequences clearly identified the CYVD strains as Serratia marcescens. However, evaluation of metabolic and biochemical features revealed that cucurbit-derived strains of S. marcescens differ substantially from strains of the same species isolated from other environmental niches. Cucurbit strains formed a distinct cluster, separate from other strains, when their fatty acid methyl ester profiles were analyzed. In substrate utilization assays (BIOLOG, Vitek, and API 20E), the CYVD strains lacked a number of metabolic functions characteristic for S. marcescens, failing to catabolize 25 to 30 compounds that were utilized by S. marcescens reference strains. These biological differences may reflect gene loss or repression that occurred as the bacterium adapted to life as an intracellular parasite and plant pathogen.

Genotyping of Serratia marcescens Strains Associated with Cucurbit Yellow Vine Disease by Repetitive Elements-Based Polymerase Chain Reaction and DNA-DNA Hybridization.

ABSTRACT The bacterium that causes cucurbit yellow vine disease (CYVD) has been placed in the species Serratia marcescens based on 16S rDNA and groE sequence analysis. However, phenotypic comparison of the organism with S. marcescens strains isolated from a variety of ecological niches showed significant heterogeneity. In this study, we compared the genomic DNA of S. marcescens strains from different niches as well as type strains of other Serratia spp. through repetitive elements-based polymerase chain reaction (rep-PCR) and DNA-DNA hybridization. With the former, CYVD strains showed identical banding patterns despite the fact that they were from different cucurbit hosts, geographic locations, and years of isolation. In the phylogenetic trees generated from rep-PCR banding patterns, CYVD strains clearly were differentiated from other strains but formed a loosely related group with S. marcescens strains from other niches. The homogeneity of CYVD strains was supported further by the DNA relatedness study, in that labeled DNA from the cantaloupe isolate, C01-A, showed an average relative binding ratio (RBR) of 99%, and 0.33% divergence to other CYVD strains. Used as a representative strain of CYVD, the labeled C01-A had a RBR of 76%, and a 4.5% divergence to the S. marcescens type strain. These data confirm the previous placement of CYVD strains in S. marcescens. Our investigations, including rep-PCR, DNA-DNA hybridization, and previous phenotyping experiments, have demonstrated that CYVD-associated strains of S. marcescens cluster together in a group significantly different from other strains of the species.

The effect of trauma program registry on reported mortality rates.

BACKGROUND: This study assesses the relationship that the brand of trauma program registry (TPR) has on mortality rate (MR) in the reports prepared by the American College of Surgeons Committee on Trauma (ACSCOT) trauma center (TC) site surveyors. METHODS: Data from 242 ACSCOT adult TC survey reports (88 Level I, 115 Level II, and 39 Level III) were analyzed for annual trauma volume, injury severity score (ISS), MR, and TPR. Six TPR (A through F) were identified; group F was a composite of several infrequently used TPRs. This report focuses on the ISS range 16-24 because of the likelihood that the mean for each TC would be near 20 and MR is high enough so that a difference, if present, could be statistically documented. RESULTS: For the total group, MR showed no correlation with TC volume or TC level for ISS 16-24. MR was significantly different according to which TPR was used by the TCs. The MR is less (4.8%) for 14 high volume TCs (over 1200 admits) using TPR A compared with 33 low volume TCs (below 800 admits) using TPR A (6.34%). CONCLUSION: The MR for ISS 16-24 in ACSCOT-surveyed TCs differs within subgroups based on type of TPR utilized. This may reflect improper use of the software programs. Enhanced skill in the application of software programs designed to generate ISS scores is essential if meaningful studies on the effects of improved trauma care on MR are to be conducted. Hand scored ISS by trained personnel may circumvent this problem.

Yellow Vine of Watermelon and Pumpkin in Tennesse.

Yellow vine (YV) is a recently recognized decline of cucurbits expressed as plant yellowing, phloem discoloration, and death of vines as fruit approach maturity. In severely affected fields, YV incidence can range from 50 to 100% with similar yield loss. The disease has been associated with a phloem-limited, walled bacterium belonging to the gamma-3-proteobacteria (1), for which specific polymerase chain reaction (PCR) primers have been developed and used in diagnosis (2). First observed in 1988 in Oklahoma and Texas squash and pumpkin, YV was not detected in watermelon and cantaloupe until 1991. The disease has never been detected in cucumber. Efforts to date have been unsuccessful in transmitting the disease with dodder, grafting, or selected insects. Initially, the geographic range of the disease appeared to be generally confined to central and northeastern Oklahoma and north central Texas, an area known as the Cross Timbers Region. In 1997 to 1998, YV was diagnosed in commercial fields of watermelon and muskmelon from east Texas (Post Oak Savannah) and all cucurbit-growing areas of Oklahoma. In late summer 1998, symptoms similar to those of YV were observed in one watermelon (Hardeman County) and three pumpkin (Rhea and Morgan counties) fields in Tennessee where the leaves turned yellow and chlorotic and affected plants exhibited phloem discoloration. Estimated incidence of YV ranged from less than 1 to 20% of the plants in affected fields. PCR, with the YV-specific primers (2), amplified a band of the expected size (409 bp) from all watermelon and pumpkin plants exhibiting phloem discoloration. In contrast, no bands were amplified from asymptomatic (no phloem discoloration) watermelon or pumpkin. The nucleotide sequence of the DNA fragment amplified from a Tennessee watermelon and pumpkin plant was identical to that of the YV bacterium. The occurrence of YV outside of the Cross Timbers Region, and in a location as distant as Tennessee, suggests that the disease may be much more widespread than previously recognized. Diagnosis and monitoring of YV in all cucurbit-growing areas is critical for determining the geographic distribution and losses caused by this emerging disease. References: (1) F. J. Avila et al. Phytopathology 88:428, 1998. (2) U. Melcher et al. (Abstr.) Phytopathology. 89(suppl.):S95, 1999.

Analysis of American College of Surgeons trauma consultation program.

OBJECTIVE: To identify the criteria deficiencies found during peer consultation of hospitals and the relationship to subsequent verification. METHODS: Between September 1987 and December 1992, 52 hospitals had consultation visits using American College of Surgeons criteria. Each report was studied for deficiencies, frequency of deficiencies, and relationship to verification. RESULTS: There are 108 American College of Surgeons criteria. Thirty-five different criteria deficiencies were found. The number of deficiencies per hospital ranged from zero to 12. The more frequent deficiencies included a lack of the following: quality improvement, 35 (67%); trauma service, 20 (38%); trauma surgeon in emergency department, 20 (38%); 24-hour operating room availability, 17 (33%); trauma registry, 17 (33%); trauma continuing medical education, 16 (31%); trauma director, 15 (29%); computed tomography technician in hospital, 15 (29%); research, 14 (27%); trauma coordinator, 14 (27%); and neurosurgeon availability, 13 (25%). No hospital that lacked commitment of surgeons (n = 12) or hospital (n = 3) requested a verification visit. Twenty-four hospitals (46%) achieved verification by February 1994. Twenty-eight hospitals had six or fewer deficiencies, with 19 (68%) verified. Twenty-four hospitals had seven or more deficiencies, with only five (21%) subsequently verified. Verification visits followed consultation by 3 to 52 months. Two hospitals with nine deficiencies were verified after 30 and 48 months, although one failed its first verification visit. CONCLUSIONS: American College of Surgeons consultation assists hospitals to identify their trauma center capability and appears to improve their ability to pass subsequent trauma center verification. Most criteria deficiencies are correctable. Lack of commitment by the surgeons or hospital is difficult to correct. There is an inverse relationship between the number of deficiencies and subsequent verification.

American College of Surgeons Verification/Consultation Program: analysis of unsuccessful verification reviews.

UNLABELLED: This study was designed to document the reasons hospitals have been unsuccessfully peer reviewed as potential trauma centers. METHOD: 120 trauma center reviews were performed by a peer review program between September 1987 and December 1992 using the American College of Surgeons (ACS) criteria. Fifty-four hospitals had criteria deficiencies. These reviews were studied for criteria deficiencies for each hospital with documentation of frequency and relationship to re-review outcome. RESULTS: There are 108 ACS criteria. The 54 hospitals had various combinations of 28 different criteria deficiencies. Deficiencies ranged from 1 to 15 per hospital. Thirty-one hospitals underwent a second review. Twenty-five hospitals had corrected the deficiencies and were verified. No hospital with over 8 deficiencies was subsequently verified. The Quality Improvement program was the most common deficiency (74%) and was correctable (50%). Other frequent deficiencies were no trauma service (46%), no surgeons in ED (41%), inadequate neurosurgeon response (35%), no trauma coordinator (31%), no trauma registry (28%), lack of surgical commitment (26%), and lack of 24 hour OR availability (24%). The lack of surgeon or hospital commitment accounted for most of the 28 criteria deficiencies. Subsequent verification was notably poorer for hospitals seeking verification for the purpose of designation versus verification only (29% versus 75%). Only 1 hospital with a prior ACS consultation visit failed the first verification review. CONCLUSIONS: A limited but critical set of criteria enable a hospital to function as a trauma center. Trauma quality improvement is a poorly understood but a correctable issue. Surgical and hospital commitment are essential for verification. Prior consultation may be of benefit.

Computed tomographic evaluation to exclude traumatic aortic disruption.

Traumatic aortic disruption from blunt trauma remains a lethal injury. The role of computed tomographic (CT) scanning in the diagnosis of traumatic aortic disruption (TAD) has been debated and varying results have been reported. We reviewed our experience with 133 consecutive cases of blunt trauma with abnormal findings on chest x-ray films of sufficient concern to require further evaluation for TAD. Of the 105 patients who underwent CT scanning as the initial evaluation, 11 (10%) required aortography (Ao) for diagnosis; seven had TAD. Twenty-eight patients with highly suggestive signs of TAD underwent Ao as the initial diagnostic test; five had TAD. Ten of the 12 patients (83%) undergoing surgical repair had good results; one died of exsanguination at surgery and the other suffered a profound neurologic injury. Follow-up by phone or chart review at 6 months to more than 5 years after injury revealed no late mortalities from unrecognized TAD. We conclude that high quality CT evaluation of patients with worrisome chest x-ray films following blunt trauma can be used to exclude TAD in the majority of cases. Aortography is reserved for cases in which there is a high clinical suspicion of TAD and for those patients in whom TAD cannot be confidently excluded by CT scanning.

Total parathyroidectomy and autotransplantation in hyperplasia of the parathyroid gland.

Hyperparathyroidism caused by multiple-gland hyperplasia has traditionally been treated by subtotal parathyroidectomy. Excellent results have been reported by some, particularly in primary hyperparathyroidism, but other have reported a significant incidence of recurrent hyperparathyroidism. Since 1979, we have chosen to avoid the possibility of remedial exploration of the neck and its attendant risks by treating all patients with primary and secondary hyperplasia with total parathyroidectomy and heterotopic autotransplantation. A total of 20 patients were studied. There were no failures of grafts and no operative complications. We conclude that this procedure is a reliable and safe alternative in the treatment of primary or secondary hyperplasia of the parathyroid gland.

Results of venous interposition grafts in arterial injuries.

Controversy continues regarding the use of PTFE versus autogenous vein grafts in the repair of arterial injuries. This study was designed to evaluate the results of a large series of autogenous interposition vein grafts used for arterial trauma. The charts of 191 patients with 192 arterial injuries repaired with an autogenous vein graft were reviewed. Specific areas of interest included graft-related complications such as thrombosis, infection, rupture, incidence of amputation, and mortality. Seventy-six per cent of the injuries were due to penetrating trauma. Forty-five per cent involved the upper and 51% the lower extremity. Shock (B. P. less than 80) occurred in one third of the patients. There were 23 (12%) graft-related complications. Sixteen (8.3%) of the grafts thrombosed. Three of these patients required an amputation and one a nephrectomy. Seven grafts (3.6%) became infected; all seven eventually ruptured. Five of these patients required an amputation. Eighteen patients (9.4%) required amputation; however, only eight (4.2%) of these cases were graft related. One patient died from non-graft-related multiple organ failure, establishing a mortality rate of 0.5%. Based on the data reported in this series, it is concluded that autogenous grafts continue to provide a safe, readily accessible, and effective means by which selected arterial injuries can be repaired.

Trichloroethylene: an update.

The toxicity of tricholoroethylene (TCE) has been summarized in a number of reviews. In this particular update, only the more recent studies that deal with metabolism and carcinogenicity have been examined. In reviewing the more recent publications on metabolism of TCE, we determined that differences exist in its metabolism if low doses are compared with high doses in animals. There may also be a difference in the metabolism of TCE between different species--namely mice, rats, and humans. TCE has not been shown to be a potent carcinogen in rats and it only seems to be a potent carcinogen in one specific strain of mice, namely the B6C3F1 mouse. Epidemiology studies have been rather limited. The number of persons examined so far for chronic toxic effects is small, compared with the enormous size of the work force that is exposed to TCE over prolonged periods. On an empirical basis, the occupational experience with TCE does not suggest that this compound is a potent carcinogen. The risk associated with exposure to trace amount (ppb) concentrations of TCE in water appear to be minimal or perhaps negligible. Because there are differences in metabolism of TCE, it is important that theoretical risks attributed to TCE in the past be reexamined. It is highly possible that in humans, the metabolic pathway leading to the formation of the proximate carcinogen is not activated at low doses, where TCE is excreted by first-order kinetics.

Fibrinolytic response to trauma.

The fibrinolytic response to trauma was investigated in 23 patients. Patients were triaged upon arrival in the emergency center into three groups; group I-patients with significant trauma who maintained normal vital signs, had a good prognosis, and tolerated the trauma well (mean injury severity score 8, range 4 to 12); group II--patients with significant trauma and transient episodes of hypotension, hypoxia, or acidosis who recovered (mean injury severity score 22, range 9 to 38); and group III--patients with profound or continued hypoxia and hypotension who eventually died of the trauma (mean injury severity score 41, range 30 to 50). Serial measurements of prothrombin time, activated partial thromboplastin time, and platelet count; concentrations of fibrinogen, plasminogen, and fibrin degradation products; and assays of euglobulin fraction fibrinolytic activity on plasminogen-free and plasminogen-rich fibrin plates were obtained on all patients. Coagulation studies documented a trauma-related coagulopathy that correlated with the degree of trauma. Plasminogen concentrations were initially depressed in all three groups; however by 24 hours group III patients were noted to have significantly elevated plasminogen concentrations while group I and group II patients had normal plasminogen concentrations. Fibrinolytic activity measured on plasminogen-free and plasminogen-rich fibrin plates was initially increased in all three groups with group III patients demonstrating the greatest increase. Over the succeeding 14 hours fibrinolytic activity returned to baseline values in group I and group II patients while group III patients demonstrated no detectable fibrinolytic activity for the remainder of the study period. This absence of fibrinolytic activity and increase in plasminogen concentrations in group III patients is thought to be caused by depletion of the intravascular plasminogen activator with the subsequent development of a hypofibrinolytic state.

Stability of four steroids in lyophilised human serum.

The stability of a preparation of lyophilised serum and its suitability for use as a reference material for routine assays of cortisol, oestradiol, and progesterone have been studied in four laboratories with a variety of assay systems. Cortisol and oestradiol were also measured by gas chromatography-mass spectrometry. The lyophilised serum was suitable for use in all routine and reference assay methods examined, with between-method variability no greater than that for frozen serum pools. The concentrations of cortisol, oestradiol, and progesterone were predicted by accelerated degradation studies to decline by 0.01% per annum if the preparations were to be stored at --20 degrees C. The testosterone content of the preparation, determined in one laboratory, provided no evidence for degradation. The preparation can be shipped for use at ambient temperature without deleterious effect.

Metabolic and drug profiling.

Over recent years, advances in analytical technology have greatly improved our ability to study the metabolism of compounds from either endogenous or erogenous sources. The application of gas-liquid chromatography, mass spectrometry, high-performance liquid chromatography and immunological approaches are discussed in relation to the analysis of steroids, bile acids, organic acids, prostaglandins, porphyrins and bile pigments, amino acids, proteins, nucleotides, catecholamines, vitamins and drugs.