Silage review: Using molecular approaches to define the microbial ecology of silage.

Ensiling of forages was recognized as a microbial-driven process as early as the late 1800s, when it was associated with the production of "sweet" or "sour" silage. Classical microbiological plating techniques defined the epiphytic microbial populations associated with fresh forage, the pivotal role of lactic acid-producing bacteria in the ensiling process, and the contribution of clostridia, bacilli, yeast, and molds to the spoilage of silage. Many of these classical studies focused on the enumeration and characterization of a limited number of microbial species that could be readily isolated on selective media. Evidence suggested that many of the members of these microbial populations were viable but unculturable, resulting in classical studies underestimating the true microbial diversity associated with ensiling. Polymerase chain reaction-based techniques, including length heterogeneity PCR, terminal RFLP, denaturing gradient gel electrophoresis, and automated ribosomal intergenic spacer analysis, were the first molecular methods used to study silage microbial communities. Further advancements in whole comparative genomic, metagenomic, and metatranscriptomic sequencing have or are in the process of superseding these methods, enabling microbial communities during ensiling to be defined with a degree of detail that is impossible using classical microbiology. These methods have identified new microbial species in silage, as well as characterized shifts in microbial communities with forage type and composition, ensiling method, and in response to aerobic exposure. Strain- and species-specific primers have been used to track the persistence and contribution of silage inoculants to the ensiling process and the role of specific species of yeast and fungi in silage spoilage. Sampling and the methods used to isolate genetic materials for further molecular analysis can have a profound effect on results. Primer selection for PCR amplification and the presence of inhibitors can also lead to biases in the interpretation of sequence data. Bioinformatic analyses are reliant on the integrity and presence of sequence data within established databases and can be subject to low taxonomic resolution. Despite these limitations, advancements in molecular biology are poised to revolutionize our current understanding of the microbial ecology of silage.

Differing populations of endemic bacteriophages in cattle shedding high and low numbers of Escherichia coli O157:H7 bacteria in feces.

The objectives of this study were to identify endemic bacteriophages (phages) in the feedlot environment and determine relationships of these phages to Escherichia coli O157:H7 from cattle shedding high and low numbers of naturally occurring E. coli O157:H7. Angus crossbred steers were purchased from a southern Alberta (Canada) feedlot where cattle excreting ≥ 10(4) CFU · g(-1) of E. coli O157:H7 in feces at a single time point were identified as supershedders (SS; n = 6), and cattle excreting <10(4) CFU · g(-1) of feces were identified as low shedders (LS; n = 5). Fecal pats or fecal grabs were collected daily from individual cattle for 5 weeks. E. coli O157:H7 in feces was detected by immunomagnetic separation and enumerated by direct plating, and phages were isolated using short- and overnight-enrichment methods. The total prevalence of E. coli O157:H7 isolated from feces was 14.4% and did not differ between LS and SS (P = 0.972). The total prevalence of phages was higher in the LS group (20.9%) than in the SS group (8.3%; P = 0.01). Based on genome size estimated by pulsed-field gel electrophoresis and morphology determined by transmission electron microscopy, T4- and O1-like phages of Myoviridae and T1-like phage of Siphoviridae were isolated. Compared to T1- and O1-like phages, T4-like phages exhibited a broad host range and strong lytic capability when targeting E. coli O157:H7. Moreover, the T4-like phages were more frequently isolated from feces of LS than SS, suggesting that endemic phages may impact the shedding dynamics of E. coli O157:H7 in cattle.

Prevalence and diversity of class 1 integrons and resistance genes in antimicrobial-resistant Escherichia coli originating from beef cattle administered subtherapeutic antimicrobials.

AIMS: To characterize class 1 integrons and resistance genes in tetracycline-resistant Escherichia coli originating from beef cattle subtherapeutically administered chlortetracycline (A44), chlortetracycline and sulfamethazine (AS700), or no antimicrobials (control). METHODS AND RESULTS: Tetracycline-resistant E. coli (control, n = 111; AS700, n = 53; A44, n = 40) were studied. Class 1 integrons, inserted gene cassettes and the presence of other antimicrobial resistance genes, as well as phylogenetic analysis, were performed by PCR, restriction enzyme analysis and sequencing. Susceptibilities to 11 antimicrobials were conducted on all isolates. Prevalence of class 1 integrase was higher (P < 0·001) in isolates from AS700 (33%) and A44 (28%) steers as compared to control (7%). Most integron gene cassettes belonged to the aad or dfr families. Correlations were found between the tet(A) gene and the genetic elements sul1 (r = 0·44), aadA1 (r = 0·61), cat (r = 0·58) and intI1(r = 0·37). Both closely and distantly related isolates harboured integrons with identical gene cassette arrays. CONCLUSIONS: Subtherapeutic administration of chlorotetracycline alone or in combination with sulfamethazine may select for class 1 integrons in bovine tetracycline-resistant E. coli isolates. Vertical spread and horizontal transfer are responsible for the dissemination of a particular type of class 1 integron, but this study could not differentiate if this phenomenon occurred within or outside of the feedlot. Tetracycline-resistant E. coli strains with sul1 and tet(A) genes were more likely to harbour class 1 integrons. SIGNIFICANCE AND IMPACT OF THE STUDY: Subtherapeutic use of chlortetracycline and sulfamethazine may promote the presence of class 1 integrons in tetracycline-resistant E. coli isolated from feedlot cattle.

Influence of preservation time on outcome and metabolic function of bladder-drained pancreas transplants.

The influence of cold storage preservation time on graft survival and metabolic function of pancreatic transplants was studied in 130 recipients of bladder-drained grafts (47 simultaneous with, 33 after, and 50 without a kidney transplant) between October 1, 1984 and May 1, 1989. The recipients were divided into four groups according to the preservation time: less than 6 hr (n = 11), 6-12 hr (n = 24), 12-24 hr (n = 75), and greater than 24 hr (n = 20). Twenty-six grafts were procured by other transplant teams and sent to us. Silica gel fractionated plasma was used for preservation in 104 cases and the University of Wisconsin solution in 25 (1 in the less than 6 hr, 2 in the 6-12 hr, 16 in the 12-24 hr, and 6 in the greater than 24 hr groups). The technical failure rate at 1 month was 13% (17 grafts), 1 (9%) in the less than 6 hr, 5 (21%) in the 6-12 hr, 9 (12%) in the 12-24 hr, and 2 (10%) in the greater than 24 hr groups. At 1 month, 107 (82%) of the grafts were functioning, 10 (91%) in the less than 6 hr, 18 (75%) in the 6-12 hr, 62 (83%) in the 12-24 hr and 17 (85%) in the greater than 24 hr groups, the longest preserved for 30 hr. The respective 1-year graft survival rates were 51%, 50%, 57%, and 70%. Ninety patients (10 in the less than 6 hr, 16 in the 6-12 hr, 51 in the 12-24 hr, and 13 in the greater than 24 hr groups) had metabolic studies between 2 and 6 weeks postransplant. The results of 24-hour profiles (14 blood glucose determinations) were similar in each preservation time group; the means of the mean (+/- SD) profile glucose (mg/dl) values were 130 +/- 19, 126 +/- 31, 130 +/- 24, and 129 +/- 30, respectively (P greater than 0.6). Mean plasma glucose levels at 2 hr during OGTT were 141 +/- 32, 145 +/- 43. 163 +/- 49, and 184 +/- 100 in the respective preservation groups (P greater than or equal to 0.064). According to the National Diabetes Data Group classification, 75% of recipients in the less than 6 hr, 50% in the 6-12 hr, 44% in the 12-24 hr, and 33% in the greater than 24 hr groups had normal OGTT results.(ABSTRACT TRUNCATED AT 400 WORDS)

Pancreas retransplants compared with primary transplants.

The improved results with pancreas transplantation in general, and the emerging evidence that the procedure favorably influences the course of secondary diabetic complications, given an impetus to retransplant patients whose initial graft has failed. In order to determine whether a pancreas retransplant policy is justified, we analyzed the results at our own institution. From 1978 through 1989, 327 pancreas transplants were performed in 261 patients, including 259 primary (79%) and 68 retransplants (21%) after a previous one failed (including 2 primary transplants performed elsewhere), with 48 second (15%), 18 third (5%), and 2 fourth (1%) transplants. The surgical techniques used in the 261 primary PxTxs were open-duct free drainage into the peritoneal cavity in 15 recipients, of whom 3 (20%) were retransplanted: duct occlusion in 34, of whom 9 (26%) were retransplanted intestinal drainage in 78, of whom 23 (29%) were retransplanted; and bladder drainage in 134, of whom 13 (10%) were retransplanted. The surgical techniques used for the 68 pancreas retransplants were duct occlusion in 11 (10 second, 1 third), intestinal drainage in 12 (9 second, 3 third), and bladder drainage in 45 (29 second, 14 third, and 2 forth); bladder drainage has been used nearly exclusively for the most recent pancreas retransplants. The recipient categories in the 261 primary transplants were pancreas alone in 115, of whom 29 (25%) were retransplanted, pancreas after kidney in 81, of whom 17 (21%) were retransplanted, and simultaneous pancreas and kidney transplants in 63, of whom 2 (3%) were retransplanted. Of the 68 pancreas retransplants, 32 (47%) were pancreas alone (26 second, 6 third), 24 (35%) were pancreas after kidney (17 second, 6 third, 1 fourth), and 12 (18%) were simultaneous pancreas and kidney (5 second, 6 third, 1 fourth). Overall patient survival rates were similar (P = 0.48), at 1 month (actual [98% after primary and 94% after retransplantation]) and at 1 year (actuarial [91% vs. 89%]). Overall graft functional rates were also similar, at 1 month (actual [76% for all primary and 79% for all retransplants - P = 0.9]), and at 1 year (actuarial [46% vs. 43% - P = 0.9]). Causes of graft losses at 1 months were similar for primary (18% were technical failures, 6% were rejected) and retransplant (16% were technical failures, 3% were rejected) cases.(ABSTRACT TRUNCATED AT 400 WORDS)

Long-term metabolic function of pancreas transplants and influence of rejection episodes.

Pancreas grafts, when not rejected, can sustain an insulin-independent state in type I diabetic recipients for indefinite periods. To what extent the metabolic control achieved approaches that of normal individuals, the relationships between graft endocrine and exocrine function, the effect of reversible rejection episodes on subsequent graft function, and the correlation between the results of serial tests of graft function were determined by studies at 1 month, 1 year, and 2 years in a cohort of 39 recipients (29 females, 10 males; mean age (+/- SD), 33 +/- 5 years; mean duration of diabetes, 22 +/- 6 years) of bladder-drained pancreas transplants performed between November 1984 and December 1988. Fifteen patients received a pancreas transplant alone, 8 a pancreas after a kidney, and 16 a simultaneous pancreas/kidney transplant. Graft endocrine function was tested by a 24-hr metabolic profile of blood glucose levels before meals, at 1 and 2 hr after meals, and during the night (14 values in all), by intravenous and oral blood glucose tolerance tests, and by glycosylated hemoglobin levels (HA1 and HA1c). Graft exocrine function was assessed by urine amylase activity (U/hr). The results of the tests in the recipients were subjected to paired comparisons between timepoints and at each timepoint to the results of the same tests in 55 normal nondiabetic control individuals. The means of the mean 24-hr profile glucose (mg/dl) values were significantly lower (P less than 0.05) at 1 and 2 years posttransplant (116 +/- 27 and 115 +/- 15, respectively) than at 1 month (128 +/- 31) in the recipients, but the mean of the mean values in the normal controls (100 +/- 7) was even lower (P less than 0.05). Mean values of individual timepoints during the profile were significantly lower for 6 of the 14 values in the controls than in the recipients. The mean IVGTT K value of the normal controls (-1.9 +/- 0.4%) was significantly lower than the 1-month and 2-year values of the recipients (-1.5 +/- 0.5% and -1.3 +/- 0.6%, respectively), but the comparison with the 1-year value (-1.6 +/- 0.6%) was not significant. The mean glucose levels at zero minutes and between 120 and 300 min of the OGTTs were significantly lower at both 1 and 2 years than at 1 month in the recipients, and the values in the control group were also significantly lower than in the recipients at 1 month but not at 1 and 2 years.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence that combined procurement of pancreas and liver grafts does not affect transplant outcome.

We compared outcome after pancreas and liver transplantation when both organs were retrieved from the same donor to outcome when only one or the other organ was retrieved. A total of 166 cadaver pancreata were transplanted at our institution between November 1984 and August 1989; 64 were obtained from donors in whom the liver was also donated (LD), and 102 were retrieved from non-liver donors (non-LD). Of the 64 LD pancreata, 53 were the entire organ with a segment of duodenum and 11 were segmental. Both the superior mesenteric artery (SMA) and celiac axis (CA) were retained with the pancreas in 13, while in 40 pancreata the CA was retrieved with the liver and the blood supply to the pancreas was reconstructed [end-to-side anastomosis of splenic artery (SA) to SMA in 11 and a Y-graft of donor iliac bifurcation to SA and SMA in 29]; a graft of common iliac vein was used to extend the portal vein in 10. The technical failure rate was 8/64 (12%) in LD pancreata, and 13/102 (13%) in non-LD pancreata (P greater than 0.1). The overall pancreas allograft survival rate at 1 year was 76% for pancreata obtained from LD (n = 64) and 64% for technically successful transplanted pancreata obtained from non-LD (n = 102, P greater than 0.1). One-year actuarial patient survival was 95% in the LD group and 90% in the non-LD group (P greater than 0.1). Among the 64 livers from pancreas donors (PD), 20 were transplanted at our hospital, 42 were transported to other institutions, and 2 were not transplanted. Follow-up information regarding 47 primary orthotopic adult, whole liver PD recipients (18 at our hospital, 29 at other institutions) was available for analysis and was compared with information concerning 62 adult recipients of primary orthotopic whole livers from non-PD transplanted during the same period at our institution. The total PNF rate among 47 PD liver allografts was 2/47 (4%), compared with 1/62 (1%) for the livers from non-PD (P greater than 0.1). The technical failure rate for the PD group was 1/47 (2%) versus 5/62 (8%) in the non-PD group (P greater than 0.1). The overall liver allograft survival rate at 1 year was 75% for livers obtained from PD (n = 47) and 81% for livers obtained from non-PD (n = 62, P greater than 0.1). One-year actuarial patient survival was 88% in the PD group and 81% in the non-PD group (P greater than 0.1). We concluded that simultaneous procurement of liver and pancreas grafts had no significant detrimental effect on the rate of technical failure, or on allograft or patient survival after either pancreas or liver transplantation.

International Pancreas Transplant Registry report.

In summary, BD pancreas graft survival rates in SPK recipients were similar to those for other solid organs in both the United and non-United States. In this category more than 90% of the patients were alive at 1 year, more than 80% had functioning kidney grafts, and more than 70% were insulin independent. Most solitary pancreas transplants (PAK and PTA categories) have been performed in the United States, and approximately one half of the grafts have functioned for 1 or more years. The potential for further improvement in results is greatest in the latter two groups.

Discordant graft loss from rejection of organs from the same donor in simultaneous pancreas-kidney recipients.

Either organ (pancreas or kidney) from a single donor in a single recipient can be discordantly lost from rejection. One can be totally rejected with the other totally normal, or the other may function within the context of chronic rejection. The pancreas is more likely to function with the kidney being rejected than vice versa.