Crucial role of Salmonella genomic island 1 master activator in the parasitism of IncC plasmids.

IncC conjugative plasmids and the multiple variants of Salmonella Genomic Island 1 (SGI1) are two functionally interacting families of mobile genetic elements commonly associated with multidrug resistance in the Gammaproteobacteria. SGI1 and its siblings are specifically mobilised in trans by IncC conjugative plasmids. Conjugative transfer of IncC plasmids is activated by the plasmid-encoded master activator AcaCD. SGI1 carries five AcaCD-responsive promoters that drive the expression of genes involved in its excision, replication, and mobilisation. SGI1 encodes an AcaCD homologue, the transcriptional activator complex SgaCD (also known as FlhDCSGI1) that seems to recognise and activate the same SGI1 promoters. Here, we investigated the relevance of SgaCD in SGI1's lifecycle. Mating assays revealed the requirement for SgaCD and its IncC-encoded counterpart AcaCD in the mobilisation of SGI1. An integrative approach combining ChIP-exo, Cappable-seq, and RNA-seq confirmed that SgaCD activates each of the 18 AcaCD-responsive promoters driving the expression of the plasmid transfer functions. A comprehensive analysis of the activity of the complete set of AcaCD-responsive promoters of SGI1 and the helper IncC plasmid was performed through reporter assays. qPCR and flow cytometry assays revealed that SgaCD is essential to elicit the excision and replication of SGI1 and destabilise the helper IncC plasmid.

The Florida Pancreas Collaborative Next-Generation Biobank: Infrastructure to Reduce Disparities and Improve Survival for a Diverse Cohort of Patients with Pancreatic Cancer.

Background: Well-annotated, high-quality biorepositories provide a valuable platform to support translational research. However, most biorepositories have poor representation of minority groups, limiting the ability to address health disparities. Methods: We describe the establishment of the Florida Pancreas Collaborative (FPC), the first state-wide prospective cohort study and biorepository designed to address the higher burden of pancreatic cancer (PaCa) in African Americans (AA) compared to Non-Hispanic Whites (NHW) and Hispanic/Latinx (H/L). We provide an overview of stakeholders; study eligibility and design; recruitment strategies; standard operating procedures to collect, process, store, and transfer biospecimens, medical images, and data; our cloud-based data management platform; and progress regarding recruitment and biobanking. Results: The FPC consists of multidisciplinary teams from fifteen Florida medical institutions. From March 2019 through August 2020, 350 patients were assessed for eligibility, 323 met inclusion/exclusion criteria, and 305 (94%) enrolled, including 228 NHW, 30 AA, and 47 H/L, with 94%, 100%, and 94% participation rates, respectively. A high percentage of participants have donated blood (87%), pancreatic tumor tissue (41%), computed tomography scans (76%), and questionnaires (62%). Conclusions: This biorepository addresses a critical gap in PaCa research and has potential to advance translational studies intended to minimize disparities and reduce PaCa-related morbidity and mortality.

Replication of the Salmonella Genomic Island 1 (SGI1) triggered by helper IncC conjugative plasmids promotes incompatibility and plasmid loss.

The mobilizable resistance island Salmonella genomic island 1 (SGI1) is specifically mobilized by IncA and IncC conjugative plasmids. SGI1, its variants and IncC plasmids propagate multidrug resistance in pathogenic enterobacteria such as Salmonella enterica serovars and Proteus mirabilis. SGI1 modifies and uses the conjugation apparatus encoded by the helper IncC plasmid, thus enhancing its own propagation. Remarkably, although SGI1 needs a coresident IncC plasmid to excise from the chromosome and transfer to a new host, these elements have been reported to be incompatible. Here, the stability of SGI1 and its helper IncC plasmid, each expressing a different fluorescent reporter protein, was monitored using fluorescence-activated cell sorting (FACS). Without selective pressure, 95% of the cells segregated into two subpopulations containing either SGI1 or the helper plasmid. Furthermore, FACS analysis revealed a high level of SGI1-specific fluorescence in IncC+ cells, suggesting that SGI1 undergoes active replication in the presence of the helper plasmid. SGI1 replication was confirmed by quantitative PCR assays, and extraction and restriction of its plasmid form. Deletion of genes involved in SGI1 excision from the chromosome allowed a stable coexistence of SGI1 with its helper plasmid without selective pressure. In addition, deletion of S003 (rep) or of a downstream putative iteron-based origin of replication, while allowing SGI1 excision, abolished its replication, alleviated the incompatibility with the helper plasmid and enabled its cotransfer to a new host. Like SGI1 excision functions, rep expression was found to be controlled by AcaCD, the master activator of IncC plasmid transfer. Transient SGI1 replication seems to be a key feature of the life cycle of this family of genomic islands. Sequence database analysis revealed that SGI1 variants encode either a replication initiator protein with a RepA_C domain, or an alternative replication protein with N-terminal replicase and primase C terminal 1 domains.

IncC conjugative plasmids and SXT/R391 elements repair double-strand breaks caused by CRISPR-Cas during conjugation.

Bacteria have evolved defence mechanisms against bacteriophages. Restriction-modification systems provide innate immunity by degrading invading DNAs that lack proper methylation. CRISPR-Cas systems provide adaptive immunity by sampling the genome of past invaders and cutting the DNA of closely related DNA molecules. These barriers also restrict horizontal gene transfer mediated by conjugative plasmids. IncC conjugative plasmids are important contributors to the global dissemination of multidrug resistance among pathogenic bacteria infecting animals and humans. Here, we show that IncC conjugative plasmids are highly resilient to host defence systems during entry into a new host by conjugation. Using a TnSeq strategy, we uncover a conserved operon containing five genes (vcrx089-vcrx093) that confer a novel host defence evasion (hde) phenotype. We show that vcrx089-vcrx090 promote resistance against type I restriction-modification, whereas vcrx091-vcxr093 promote CRISPR-Cas evasion by repairing double-strand DNA breaks via recombination between short sequence repeats. vcrx091, vcrx092 and vcrx093 encode a single-strand binding protein, and a single-strand annealing recombinase and double-strand exonuclease related to Redß and λExo of bacteriophage λ, respectively. Homologous genes of the integrative and conjugative element R391 also provide CRISPR-Cas evasion. Hence, the conserved hde operon considerably broadens the host range of large families of mobile elements spreading multidrug resistance.

Entry Exclusion of Conjugative Plasmids of the IncA, IncC, and Related Untyped Incompatibility Groups.

Conjugative plasmids of incompatibility group C (IncC), formerly known as A/C2, disseminate antibiotic resistance genes globally in diverse pathogenic species of Gammaproteobacteria. Salmonella genomic island 1 (SGI1) can be mobilized by IncC plasmids and was recently shown to reshape the conjugative type IV secretion system (T4SS) encoded by these plasmids to evade entry exclusion. Entry exclusion blocks DNA translocation between cells containing identical or highly similar plasmids. Here, we report that the protein encoded by the entry exclusion gene of IncC plasmids (eexC) mediates entry exclusion in recipient cells through recognition of the IncC-encoded TraGC protein in donor cells. Phylogenetic analyses based on EexC and TraGC homologs predicted the existence of at least three different exclusion groups among IncC-related conjugative plasmids. Mating assays using Eex proteins encoded by representative IncC and IncA (former A/C1) and related untyped plasmids confirmed these predictions and showed that the IncC and IncA plasmids belong to the C exclusion group, thereby explaining their apparent incompatibility despite their compatible replicons. Representatives of the two other exclusion groups (D and E) are untyped conjugative plasmids found in Aeromonas sp. Finally, we determined through domain swapping that the carboxyl terminus of the EexC and EexE proteins controls the specificity of these exclusion groups. Together, these results unravel the role of entry exclusion in the apparent incompatibility between IncA and IncC plasmids while shedding light on the importance of the TraG subunit substitution used by SGI1 to evade entry exclusion.IMPORTANCE IncA and IncC conjugative plasmids drive antibiotic resistance dissemination among several pathogenic species of Gammaproteobacteria due to the diversity of drug resistance genes that they carry and their ability to mobilize antibiotic resistance-conferring genomic islands such as SGI1 of Salmonella enterica While historically grouped as "IncA/C," IncA and IncC replicons were recently confirmed to be compatible and to abolish each other's entry into the cell in which they reside during conjugative transfer. The significance of our study is in identifying an entry exclusion system that is shared by IncA and IncC plasmids. It impedes DNA transfer to recipient cells bearing a plasmid of either incompatibility group. The entry exclusion protein of this system is unrelated to any other known entry exclusion proteins.

Genome Sequence of a Canadian Vibrio parahaemolyticus Isolate with Unique Mobilizing Capacity.

Vibrio parahaemolyticus is a clinically significant marine bacterium implicated in gastroenteritis among consumers of raw or undercooked seafood. This report presents the whole-genome sequence of a unique strain of V. parahaemolyticus isolated from oysters harvested in Canada.

A toxin antitoxin system promotes the maintenance of the IncA/C-mobilizable Salmonella Genomic Island 1.

The multidrug resistance Salmonella Genomic Island 1 (SGI1) is an integrative mobilizable element identified in several enterobacterial pathogens. This chromosomal island requires a conjugative IncA/C plasmid to be excised as a circular extrachromosomal form and conjugally mobilized in trans. Preliminary observations suggest stable maintenance of SGI1 in the host chromosome but paradoxically also incompatibility between SGI1 and IncA/C plasmids. Here, using a Salmonella enterica serovar Agona clonal bacterial population as model, we demonstrate that a Toxin-Antitoxin (TA) system encoded by SGI1 plays a critical role in its stable host maintenance when an IncA/C plasmid is concomitantly present. This system, designated sgiAT for Salmonella genomic island 1 Antitoxin and Toxin respectively, thus seems to play a stabilizing role in a situation where SGI1 is susceptible to be lost through plasmid IncA/C-mediated excision. Moreover and for the first time, the incompatibility between SGI1 and IncA/C plasmids was experimentally confirmed.

Whipple's procedure in a renal transplant recipient with polycystic liver disease.

CONTEXT: Polycystic disease is a rare disorder, which most commonly manifests in the kidney and liver. Recently an increased risk for pancreatic malignancies in subsets of patients with polycystic disease has been reported. CASE REPORT: We report a patient with polycystic liver and kidney disease who successfully underwent a Whipple's procedure for pancreatic adenocarcinoma. CONCLUSION: Although technical difficulty may increase, pancreaticoduodenectomy can be safely performed in patients with polycystic liver disease.

Laparoscopic gastric gastrointestinal stromal tumor resection: the mayo clinic experience.

HYPOTHESIS: Laparoscopic resection of gastric gastrointestinal stromal tumors (GISTs) is safe and effective. DESIGN: Retrospective medical record review. SETTING: Tertiary referral center. PATIENTS: Patients undergoing laparoscopic resection of gastric GISTs from April 1, 2000, to April 1, 2006. MAIN OUTCOME MEASURES: Demographic data, diagnostic workup, operative technique, tumor characteristics, morbidity, mortality, and follow-up. RESULTS: Thirty-three patients underwent attempted laparoscopic resection of gastric GISTs, with 31 operations completed laparoscopically. The mean patient age was 68 years (age range, 35-86 years). The female to male ratio was 18:15. Sixteen patients (49%) were asymptomatic, and their tumors were found incidentally. Of 24 patients (73%) who underwent preoperative endoscopic ultrasonography, the results of fine-needle aspiration verified the diagnosis in 13 patients (54%). The mean operative time was 124 minutes (range, 30-253 minutes). A combined endoscopic-laparoscopic approach was used in 11 patients (33%). The mean tumor size was 3.9 cm (range, 0.5-10.5 cm). Two patients (6%) underwent conversion to an open procedure. The median hospital stay duration was 3 days. The mean follow-up was 13 months (range, 3-64 months). There were no local recurrences. Three patients (9%) experienced complications, including 1 wound infection and 2 episodes of upper gastrointestinal tract bleeding. There were no mortalities. CONCLUSION: Although technically demanding, the laparoscopic approach to gastric GISTs is safe and effective, resulting in a short hospital stay duration and low morbidity.

Late gastric perforations after laparoscopic fundoplication.

BACKGROUND: Late complications are rarely encountered after laparoscopic Nissen fundoplication. These complications include acute gastric herniation through the esophageal hiatus, port-site herniation, recurrent reflux, and anatomic failure of the fundoplication. Only three cases of late gastric perforation after laparoscopic Nissen fundoplication have been reported, all associated with intrathoracic wrap herniation. METHODS: We retrospectively reviewed all cases of gastric perforation after laparoscopic antireflux procedures performed between July 1991 and March 2002 by a single surgeon. RESULTS: In this series of 1,600 laparoscopic antireflux procedures, we found six delayed gastric fundal perforations occurring in three patients at 1, 41, 48, 51, 68, and 72 months after surgery. All the perforations were on the anterior wall of the fundus of the stomach and were distant from the stitches of the fundoplication. None of the perforations was associated with severe peritoneal contamination. CONCLUSIONS: This series of late gastric fundal perforations in 0.2% of our patients after laparoscopic fundoplication may have been caused by medications, gastric stasis, ischemia, or a foreign body such as a stitch or Teflon pledget.

Colorectal lymphangioma.

Lymphangiomas of the colon are historically rare benign tumors. Only 331 cases have been reported in the world medical literature between 1931 and 2004. With widespread use of colonoscopy, however, they are being found more frequently. We report the case of a 74-year-old woman in whom a colonoscopy revealed a 3 x 4-cm submucosal lesion in the cecum that was eventually diagnosed as a lymphangioma. A CT of the abdomen showed a soft-tissue mass in the cecum and a low-density hepatic lesion. An endoscopic ultrasound of the colon showed a 3 x 4-cm hypoechoic lesion with internal septa arising from the submucosal layer of the cecum. This lesion resembled a vascular malformation; therefore a biopsy specimen was not taken. Pathologic findings of a specimen taken after a subsequent right hemicolectomy identified a submucosal lymphangioma. Published reports indicate that colonoscopy cures most lesions smaller than 2.5 cm in diameter. Resection should be reserved for larger lesions or those in patients exhibiting protein-losing enteropathy.

Gallbladder carcinosarcoma: a case report and literature review.

Carcinosarcoma of the gallbladder is a rare malignancy characterized by both malignant epithelial and mesenchymal components. The clinical behavior of this tumor is extremely aggressive. Only 26 cases have been reported in the world literature to date. We report the case of a 64-year-old woman who had fever associated with a right upper quadrant mass. An exploratory laparotomy through a right upper quadrant incision was performed at another institution, and the patient was thought to have severe acute cholecystitis that would require additional antibiotic therapy before attempted resection. She was referred to our center, where abdominal CT showed a 6.4 x 8.2 cm pericholecystic mass involving the hepatic flexure of the colon. The patient underwent cholecystectomy and hepatic wedge resection, pancreaticoduodenectomy, and right hemicolectomy. Pathologic examination of the surgical specimen revealed two histologic components consisting of squamous cell carcinoma and spindle cell sarcoma of gallbladder origin, consistent with carcinosarcoma. All seven lymph nodes in the pancreaticoduodenectomy specimen were negative for tumor. We present this case and a review of the literature and current treatment recommendations.

Occult fifth gland intrathyroid parathyroid adenoma identified by gamma probe.

We report the case of a 54-year-old woman undergoing radioguided parathyroidectomy in whom four normal glands were identified on cervical exploration. The gamma probe was instrumental in guiding us to the adenoma in a fifth gland that was not visible and was localized to the parenchyma of the left lobe of the thyroid gland. Many surgeons do not advocate use of the gamma probe in minimally invasive parathyroidectomy and rely solely on focused exploration on the basis of a preoperative sestamibi scan and intraoperative rapid parathyroid hormone assays. This case, however, illustrates the utility of the gamma probe in parathyroid adenoma localization.