Risk factors for severe morbidity and definitive stoma after elective surgery for sigmoid diverticulitis: a multicenter national cohort study.

BACKGROUND: In the decision to perform elective surgery, it is of great interest to have data about the outcomes of surgery to individualize patients who could safely undergo sigmoid resection. The aim of this study was to provide information on the outcomes of elective sigmoid resection for sigmoid diverticular disease (SDD) at a national level. METHODS: All consecutive patients who had elective surgery for SDD (2010-2021) were included in this retrospective, multicenter, cohort study. Patients were identified from institutional review board-approved databases in French member centers of the French Surgical Association. The endpoints of the study were the early and the long-term postoperative outcomes and an evaluation of the risk factors for 90-day severe postoperative morbidity and a definitive stoma after an elective sigmoidectomy for SDD. RESULTS: In total, 4617 patients were included. The median [IQR] age was 61 [18.0;100] years, the mean ± SD body mass index (BMI) was 26.8 ± 4 kg/m2, and 2310 (50%) were men. The indications for surgery were complicated diverticulitis in 50% and smoldering diverticulitis in 47.4%. The procedures were performed laparoscopically for 88% and with an anastomosis for 83.8%. The severe complication rate on postoperative day 90 was 11.7%, with a risk of anastomotic leakage of 4.7%. The independent risk factors in multivariate analysis were an American Society of Anesthesiologists (ASA) score ≥ 3, an open approach, and perioperative blood transfusion. Age, perioperative blood transfusion, and Hartmann's procedure were the three independent risk factors for a permanent stoma. CONCLUSIONS: This series provides a real-life picture of elective sigmoidectomy for SDD at a national level. TRIAL REGISTRATION: Comité National Information et Liberté (CNIL) (n°920361).

Robotic-assisted right colectomy. Official expert recommendations delivered under the aegis of the French Association of Surgery (AFC).

Twenty-seven experts under the aegis of the French Association of Surgery (AFC) offer this reference system with formalized recommendations concerning the performance of right colectomy by robotic approach (RRC). For RRC, experts suggest patient installation in the so-called "classic" or "suprapubic" setup. For patients undergoing right colectomy for a benign pathology or cancer, RRC provides no significant benefit in terms of intra-operative blood loss, intra-operative complications or conversion rate to laparotomy compared to laparoscopy. At the same time, RRC is associated with significantly longer operating times. Data from the literature are insufficient to define whether the robot facilitates the performance of an intra-abdominal anastomosis, but the robotic approach is more frequently associated with an intra-abdominal anastomosis than the laparoscopic approach. Experts also suggest that RRC offers a benefit in terms of post-operative morbidity compared to right colectomy by laparotomy. No benefit is retained in terms of mortality, duration of hospital stay, histological results, overall survival or disease-free survival in RRC performed for cancer. In addition, RRC should not be performed based on the cost/benefit ratio, since RRC is associated with significantly higher costs than laparoscopy and laparotomy. Future research in the field of RRC should consider the evaluation of patient-targeted parameters such as pain or quality of life and the technical advantages of the robot for complex procedural steps, as well as surgical and oncological results.

Adult appendicitis: Clinical practice guidelines from the French Society of Digestive Surgery and the Society of Abdominal and Digestive Imaging.

INTRODUCTION: The French Society of Digestive Surgery (SFCD) and the Society of Abdominal and Digestive Imaging (SIAD) have collaborated to propose recommendations for clinical practice in the management of adult appendicitis. METHODS: An analysis of the literature was carried out according to the methodology of the French National Authority for Health (HAS). A selection was performed from collected references and then a manual review of the references listed in the selected articles was made in search of additional relevant articles. The research was limited to articles whose language of publication was English or French. Articles focusing on the pediatric population were excluded. Based on the literature review, the working group proposed recommendations whenever possible. These recommendations were reviewed and approved by a committee of experts. RESULTS: Recommendations about appendicitis in adult patients were proposed with regard to clinical, laboratory and radiological diagnostic modalities, treatment strategy for uncomplicated and complicated appendicitis, surgical technique, and specificities in the case of macroscopically healthy appendix, terminal ileitis and appendicitis in the elderly and in pregnant women. CONCLUSION: These recommendations for clinical practice may be useful to the surgeon in optimizing the management of acute appendicitis in adults.

Colon sparing resection versus extended colectomy for left-sided obstructing colon cancer with caecal ischaemia or perforation: a nationwide study from the French Surgical Association.

AIM: It is not known whether patients with obstructive left colon cancer (OLCC) with caecal ischaemia or diastatic perforation (defined as a blowout of the caecal wall related to colonic overdistension) should undergo a (sub)total colectomy (STC) or an ileo-caecal resection with double-barrelled ileo-colostomy. We aimed to compare the results of these two strategies. METHOD: From 2000 to 2015, 1220 patients with OLCC underwent surgery by clinicians who were members of the French Surgical Association. Of these cases, 201 (16%) were found to have caecal ischaemia or diastatic perforation intra-operatively: 174 patients (87%) underwent a STC (extended colectomy group) and 27 (13%) an ileo-caecal resection with double-end stoma (colon-sparing group). Outcomes were compared retrospectively. RESULTS: In the extended colectomy group, 95 patients (55%) had primary anastomosis and 79 (45%) had a STC with an end ileostomy. In the colon-sparing group, 10 patients (37%) had simultaneous resection of their primary tumour with segmental colectomy and an anastomosis which was protected by a double-barrelled ileo-colostomy. The demographic data for the two groups were comparable. Median operative time was longer in the STC group (P = 0.0044). There was a decrease in postoperative mortality (7% vs 12%, P = 0.75) and overall morbidity (56% vs 67%, P = 0.37) including surgical (30% vs 40%, P = 0.29) and severe complications (17% vs 27%, P = 0.29) in the colon-sparing group, although these differences did not reach statistical significance. Cumulative morbidity included all surgical stages and the rate of permanent stoma was 66% and 37%, respectively, with no significant difference between the two groups. Overall survival and disease-free survival were similar between the two groups. CONCLUSION: The colon-sparing strategy may represent a valid and safe alternative to STC in OLCC patients with caecal ischaemia or diastatic perforation.

Antibiotics alone as an alternative to appendectomy for uncomplicated acute appendicitis in adults: Changes in treatment modalities related to the COVID-19 health crisis.

The massive inflow of patients with COVID-19 requiring urgent care has overloaded hospitals in France and impacts the management of other patients. Deferring hospitalization and non-urgent surgeries has become a priority for surgeons today in order to relieve the health care system. It is obviously not simple to reduce emergency surgery without altering the quality of care or leading to a loss of chance for the patient. Acute appendicitis is a very specific situation and the prevalence of this disease leads us to reconsider this particular disease in the context of the COVID-19 crisis. Indeed, while the currently recommended treatment for uncomplicated acute appendicitis is surgical appendectomy, the non-surgical alternative of medical management by antibiotic therapy alone has been widely evaluated by high-quality studies in the literature. Insofar as the main limitation of exclusively medical treatment of uncomplicated acute appendicitis is the risk of recurrent appendicitis, this treatment option represents an alternative of choice to reduce the intra-hospital overload in this context of health crisis. The aim of this work is therefore to provide physicians and surgeons with a practical guide based on a review of the literature on the medical treatment of uncomplicated acute appendicitis in adults, to offer this alternative treatment to the right patients and under good conditions, especially when access to the operating room is limited or impossible.

[Antibiotics alone as an alternative to appendectomy for uncomplicated acute appendicitis in adults: Changes in treatment modalities related to the COVID-19 health crisis]. / Crise sanitaire liée au COVID-19 : modalités du traitement de l'appendicite aiguë non compliquée de l'adulte par antibiothérapie seule comme alternative à l'appendicectomie.

The massive inflow of patients with COVID-19 requiring urgent care has overloaded hospitals in France and impacts the management of other patients. Deferring hospitalization and non-urgent surgeries has become a priority for surgeons today in order to relieve the health care system. It is obviously not simple to reduce emergency surgery without altering the quality of care or leading to a loss of chance for the patient. Acute appendicitis is a very specific situation and the prevalence of this disease leads us to reconsider this particular disease in the context of the COVID-19 crisis. Indeed, while the currently recommended treatment for uncomplicated acute appendicitis is surgical appendectomy, the non-surgical alternative of medical management by antibiotic therapy alone has been widely evaluated by high-quality studies in the literature. Insofar as the main limitation of exclusively medical treatment of uncomplicated acute appendicitis is the risk of recurrent appendicitis, this treatment option represents an alternative of choice to reduce the intra-hospital overload in this context of health crisis. The aim of this work is therefore to provide physicians and surgeons with a practical guide based on a review of the literature on the medical treatment of uncomplicated acute appendicitis in adults, to offer this alternative treatment to the right patients and under good conditions, especially when access to the operating room is limited or impossible.

Intra-operative fluorescence angiography is reproducible and reduces the rate of anastomotic leak after colorectal resection for cancer: a prospective case-matched study.

AIM: Intra-operative fluorescence angiography (IOFA) with indocyanine green provides information on tissue perfusion that may help prevent an anastomotic leak (AL). The aim of this study was to assess the impact of IOFA on outcomes after left-sided colonic or low anterior resection with anastomosis for colorectal cancer. METHODS: All patients with left-sided colonic or rectal cancer, operated between June 2017 and December 2018, were prospectively included. IOFA has been routinely implemented since May 2018. Reproducibility of IOFA, after a 1:1 matching for relevant clinical risk factors of AL, was studied in patients with IOFA (IOFA+) and without IOFA (IOFA-). Outcomes were compared in terms of postoperative events such as clinically relevant AL as the primary end-point. RESULTS: In the IOFA+ group, changing of the initially planned colon transection due to inadequate perfusion occurred in five out of 46 patients (10.9%). Agreement between intra-operative assessment and postoperative blind review of IOFA was deemed strong (Cohen's kappa index 0.893, 95% CI 0.788-0.998, P < 0.001). Among 111 patients, 42 matched patients were included in each group. There was significantly more clinically relevant AL in the IOFA- group compared to the IOFA+ group (16.7% vs 2.4%, P = 0.026) involving significantly more anastomotic dehiscence which required re-intervention (19% vs 2.4%, P = 0.014). Additionally, more descending colon ischaemia/necrosis was observed in the IOFA- group compared with the IOFA+ group (9.5% vs 0%, P = 0.040). CONCLUSION: In this prospective case-matched study, IOFA decreased the occurrence of clinically relevant AL due to necrosis of the descending colon or anastomosis. Upon blind review, perfusion assessment using IOFA was reproducible.

Operating room hygiene: Clinical practice recommendations.

INTRODUCTION: The French Society of Gastro-Intestinal Surgery (SociétéFrançaisedeChirurgieDigestive) and the Association of hepato-bilio-pancreatic and transplantation surgery (AssociationdeChirurgieHépato-Bilio-PancréatiqueetTransplantation) requested that clinical practice recommendations be established with regard to operating room hygiene. METHODS: The literature was analyzed according to the High Authority of Health (HauteAutoritédesanté [HAS]) methodology and after consultation of the Cochrane and Medline databases. Pertinent references were selected, and supplementary references were hand-picked from the reference lists. Only English or French language papers were retained. The recommendations of learned societies and the World Health Organization were also considered. RESULTS: Recommendations were proposed with regard to pre-operative patient preparation, skin preparation, draping, wound edge protectors, surgeon hygiene, wound closure, and operating room environment. CONCLUSION: These clinical practice recommendations should guide and improve the daily practice of gastro-intestinal surgeons.

Morbidity and oncological outcomes of rectal cancer impaired by previous prostate malignancy.

BACKGROUND: Specific surgical and oncological outcomes in patients with rectal cancer surgery after a previous diagnosis of prostate cancer have not been well described. The aim of this study was to compare surgical outcomes in patients with rectal cancer with or without a history of prostate cancer. METHODS: Patients who had surgery for rectal cancer with (PC group) or without (no-PC group) previous curative treatment for prostate cancer were enrolled between January 2001 and December 2015. Comparisons between the two groups were performed by multivariable Cox analysis, and after propensity score matching in a 3 : 1 ratio for demographic and tumour characteristics, and surgical and oncological outcomes. RESULTS: A total of 944 patients with rectal cancer were enrolled, of whom 10·8 per cent had a history of prostate cancer. After matching, 83 patients who had received treatment for prostate cancer were compared with 249 who had not. The PC and no-PC groups were similar regarding patient characteristics. Extended total mesorectal excision, conversion to open surgery, transfusion and tumour perforation were more frequent in the PC group than in the no-PC group. Major surgical morbidity (28 versus 17·2 per cent; P = 0·036), anastomotic leakage (25 versus 13·7 per cent; P = 0·019) and permanent stoma (41 versus 12·4 per cent; P < 0·001) occurred more frequently in the PC group. Local recurrence was increased significantly in the PC group (17 versus 8·0 per cent; P = 0·019), and resulted in a significant decrease in disease-free and overall survival. CONCLUSION: Prostate cancer treatment increases short- and long-term surgical morbidity in patients with rectal cancer, and impairs oncological outcomes.

Surgical management of obstructive left colon cancer at a national level: Results of a multicentre study of the French Surgical Association in 1500 patients.

PURPOSE: Surgical management of obstructive left colon cancer (OLCC) is controversial. The objective is to report on postoperative and oncological outcomes of the different surgical options in patients operated on for OLCC. METHODS: From 2000-2015, 1500 patients were treated for OLCC in centers members of the French Surgical Association. Colonic stent (n=271), supportive care (n=5), palliative derivation (n=4) were excluded. Among 1220 remaining patients, 456 had primary diverting colostomy (PDC), 329 a segmental colectomy (SC), 246 a Hartmann's procedure (HP) and 189 a subtotal colectomy (STC) as first-stage surgery. Perioperative data and oncological outcomes were compared retrospectively. RESULTS: There was no difference between the 4 groups regarding gender, age, BMI and comorbidities. Postoperative mortality and morbidity were 4-27% (PDC), 6-47% (SC), 9-55% (HP), 13-60% (STC), respectively (P=0.005). Among the 431 living patients after PDC, 321 (70%) patients had their primary tumour removed. Cumulative mortality and morbidity favoured PDC (7-39%) and SC (6-40%) compared to HP (1-47%) and STC (13-50%) (P=0.04). At the end of follow-up definitive stoma rates were 39% (HP), 24% (PDC), 10% (SC), and 8% (STC) (P<0.0001). Five-year overall and disease-free survival was: SC (67-55%), PDC (54-48%), HP (54-37%) and STC (48-49%). After multivariate analysis, SC and PDC were associated with better prognosis compared to HP and STC. CONCLUSION: In OLCC, SC and PDC are the two preferred options in patients with good medical conditions. For patients with severe comorbidities PDC should be recommended, reserving HP and STC for patients with colonic ischaemia or perforation complicating malignant obstruction.

Thirty-day mortality after emergency surgery for obstructing colon cancer: survey and dedicated score from the French Surgical Association.

AIM: The aim was to define risk factors for postoperative mortality in patients undergoing emergency surgery for obstructing colon cancer (OCC) and to propose a dedicated score. METHOD: From 2000 to 2015, 2325 patients were treated for OCC in French surgical centres by members of the French National Surgical Association. A multivariate analysis was performed for variables with P value ≤ 0.20 in the univariate analysis for 30-day mortality. Predictive performance was assessed by the area under the receiver operating characteristic curve. RESULTS: A total of 1983 patients were included. Thirty-day postoperative mortality was 7%. Multivariate analysis found five significant independent risk factors: age ≥ 75 (P = 0.013), American Society of Anesthesiologists (ASA) score ≥ III (P = 0.027), pulmonary comorbidity (P = 0.0002), right-sided cancer (P = 0.047) and haemodynamic failure (P < 0.0001). The odds ratio for risk of postoperative death was 3.42 with one factor, 5.80 with two factors, 15.73 with three factors, 29.23 with four factors and 77.25 with five factors. The discriminating capacity in predicting 30-day postoperative mortality was 0.80. CONCLUSION: Thirty-day postoperative mortality after emergency surgery for OCC is correlated with age, ASA score, pulmonary comorbidity, site of tumour and haemodynamic failure, with a specific score ranging from 0 to 5.

Long-term outcome and management of right colonic diverticulitis in western countries: Multicentric Retrospective Study.

AIM OF THE STUDY: Right colonic Diverticulitis (RD) is rare in Europe; few studies have focused on it and its management is not standardised. The aim of this study was to analyse the clinical presentation (complicated, uncomplicated), acute phase management and long-term outcome of RD in western countries. PATIENTS AND METHODS: From 2003 to 2017, 93 consecutive patients who presented with RD were retrospectively included at 11 French Hospital Centres. RESULTS: The study population consisted of two groups: Uncomplicated Right Diverticulitis (URD) group (63.5%, (n=59)) and Complicated Right Diverticulitis (CRD) group (36.5%, [n=34]). 84.7% (n=50/59) of URD were treated conservatively. 41.2% (n=14/34) of patients with CRD had emergency surgery (mostly laparotomy) for Hinchey III peritonitis, clinical intolerance or hemodynamic instability. Altogether 5.2% (n=2/34) patients with CRD had surgery after a cooling off period (initially abscess). The overall rate of severe postoperative complications was low (8%). Recurrence rate was low and comparable in both groups: 6.8% (n=4/59) for URD and 8.8% (n=3/34) for CRD, all recurrences occurred in the same locations with an uncomplicated form, 42.9% (n=3/7) of them had elective laparoscopic surgery and the rest were conservatively treated. Median follow up was 33.2 months. CONCLUSION: Conservative treatment can be proposed safely and efficiently for URD and for selected patients with CRD. Surgery should be reserved for unstable patients or patients with severe forms of complicated diverticulitis in emergency.

In situ delivery of allogeneic natural killer cell (NK) combined with Cetuximab in liver metastases of gastrointestinal carcinoma: A phase I clinical trial.

Despite successful introduction of NK-based cellular therapy in the treatment of myeloid leukemia, the potential use of NK alloreactivity in solid malignancies is still elusive. We performed a phase I clinical trial to assess the safety and efficacy of in situ delivery of allogeneic NK cells combined with cetuximab in liver metastasis of gastrointestinal origin. The conditioning chemotherapy was administrated before the allogeneic NK cells injection via hepatic artery. Three escalating doses were tested (3.106, 8.106 and 12.106 NK cells/kg) following by a high-dose interleukin-2 (IL-2). Cetuximab was administered intravenously every week for 7 weeks. Nine patients with liver metastases of colorectal or pancreatic cancers were included, three per dose level. Hepatic artery injection was successfully performed in all patients with no report of dose-limiting toxicity. Two patients had febrile aplasia requiring a short-term antibiotherapy. Grade 3/4 anemia and thrombopenia were also observed related to the chemotherapy. Objective clinical responses were documented in 3 patients and among them 2 occurred in patients injected with cell products harboring two KIR ligand mismatches and one in a patient with one KIR ligand mismatch. Immune monitoring revealed that most patients presented an increase but transient of IL-15 and IL-7 cytokines levels one week after chemotherapy. Furthermore, a high expansion of FoxP3+regulatory T cells and PD-1+ T cells was observed in all patients, related to IL-2 administration. Our results demonstrated that combining allogeneic NK cells transfer via intra-hepatic artery, cetuximab and a high-dose IL-2 is feasible, well tolerated and may result in clinical responses.

Is trans-anal total mesorectal excision really safe and better than laparoscopic total mesorectal excision with a perineal approach first in patients with low rectal cancer? A learning curve with case-matched study in 68 patients.

AIM: To compare the learning curve for trans-anal total mesorectal excision (TATME) with laparoscopic TME started by a perineal approach (LTME). METHOD: The first 34 consecutive patients who underwent TATME for low rectal cancer were matched with LTME (performed by the same surgeon) for gender, body mass index and chemoradiation. RESULTS: Thirty-four patients undergoing TATME (23 men; 58 ± 14 years) were matched with 34 undergoing LTME (23 men; 59 ± 13 years). Intra-operative complications occurred more frequently during TATME (21%) than LTME (6%), but this difference was not significant (P = 0.07). The complications of TATME included rectal (n = 4), bladder (n = 1) and vaginal (n = 1) injury and bleeding (n = 1). Length of stay and postoperative overall and major morbidities were similar between groups. Early symptomatic anastomotic leakage (AL) occurred in 1/34 TATME and 5/34 LTME (15%; P = 0.02) procedures. Asymptomatic AL occurred in four TATME (12%) and four LTME (12%, P = 1). Thus, the overall rate of AL was 5/34 (15%) for TATME vs 9/34 (26%) for LTME (P = 0.4). No significant difference between the two groups was noted with regard to tumour, number of harvested and positive lymph nodes, R1 resection rate or completeness of the mesorectum. Metastatic recurrence was similar between groups (15% vs 18%, P = 0.7), but follow-up was shorter after TATME (13 ± 6 months) than after LTME (25 ± 14 months; P < 0.0001). CONCLUSION: The TATME learning curve seems to be associated with a significant rate of intra-operative complications. Because no significant benefit has been reported to date, more evidence is needed before TATME can be considered as a better approach than laparoscopic TME with a perineal approach first in patients with low rectal cancer.

Perioperative docetaxel, cisplatin, and 5-fluorouracil compared to standard chemotherapy for resectable gastroesophageal adenocarcinoma.

BACKGROUND: Even though the perioperative chemotherapy improves the overall survival (OS) compared to surgery alone in patients with a resectable gastroesophageal adenocarcinoma (GEA), prognosis of these patients remains poor. Docetaxel (D), cisplatin (C), and 5-fluorouracil (F) regimen improves OS compared to CF among patients with advanced GEA. We evaluated the potential interest of a perioperative DCF regimen, compared to standard (S) regimens, in resectable GEA patients. METHODS: We identified 459 patients treated with preoperative DCF or S regimens. The primary endpoint was OS. Propensity scores were estimated with a logistic regression model in which all baseline covariates were included. We then used two methods to take PS into account and thus make DCF and S patients comparable. OS analyses were performed with Kaplan-Meier and Cox models in propensity score matched samples, and inverse probability of treatment weighted (IPTW) samples. RESULTS: In the propensity score matched sample, the p-value from the log rank test for OS was 0.0961, and the 3-year OS rate was 73% and 55% in DCF and S groups, respectively. The multivariate Cox regression underlined a Hazard Ratio of 0.55 (95% CI 0.27-1.13) for DCF patients compared to S patients. The results from IPTW analyses showed that DCF was significantly and independently associated with OS (HR = 0.52; 95% CI 0.40-0.69). CONCLUSIONS: In this retrospective multicenter, hypothesis-generating study, the propensity score analyses underlined encouraging results in favor of DCF compared to S regimens regarding OS. This promising result should be validated in a phase-3 trial.

Management of rectal cancer: the 2016 French guidelines.

AIM: Rectal cancer is a malignant disease requiring multidisciplinary management. In view of the increasing number of studies published over the past decade, a comprehensive update is required to draw recommendations for clinical practice mandated by the French Research Group of Rectal Cancer Surgery and the French National Coloproctology Society. METHOD: Seven questions summarizing the treatment of rectal cancer were selected. A search for evidence in the literature from January 2004 to December 2015 was performed. A drafting committee and a large group of expert reviewers contributed to validate the statements. RESULTS: Recommendations include the indications for neoadjuvant therapy, the quality criteria for surgical resection, the management of postoperative disordered function, the role of local excision in early rectal cancer, the place of conservative strategies after neoadjuvant treatment, the management of synchronous liver metastases and the indications for adjuvant therapy. A level of evidence was assigned to each statement. CONCLUSION: The current clinical practice guidelines are useful for the treatment of rectal cancer. Some statements require a higher level of evidence due to a lack of studies.

Delay before implanting a port-a-cath after removing the previous one because of infection.

AIMS: We had for objective to assess the risk of a new Port-a-Cath (PAC) infection after re-implantation when the first one had been removed because of infection in the previous month. METHODS: We conducted a retrospective bicentric study including all patients having undergone PAC removal because of infection, followed by re-implantation of a new one in the next month, between 2004 and 2012. RESULT: One hundred and forty-nine PAC removals were followed by re-implantation in the next month. The cause of removal was infection for 63 patients: 34 presented with bacteremia (13 PAC removals and simultaneous re-implantation, and 21 with delayed re-implantation for a mean 14days) and 29 presented with local signs of infection (20 PAC removals and simultaneous re-implantation, and with delayed re-implantation for a mean 13days). Two out of 13 PACs were re-infected in the bacteremia group, (15.4%) when the removal and re-implantation were performed simultaneously, and 1 out of 21 (4.8%) when re-implantation of the second PAC was delayed. The 3 re-infected PACs were successfully treated with antibiotics. CONCLUSION: The early re-implantation of a PAC after removal of the previous one because of infection is a relatively safe procedure. The risk of infection can be higher when PAC removal and re-implantation are performed simultaneously in patients presenting with bacteremia; nevertheless, all reinfections were successfully treated with antibiotics.

Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans.

The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30 degrees C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity.