Characterization of vB_Kpn_F48, a Newly Discovered Lytic Bacteriophage for Klebsiella pneumoniae of Sequence Type 101

Resistance to carbapenems in Enterobacteriaceae, including Klebsiella pneumoniae, represents a major clinical problem given the lack of effective alternative antibiotics. Bacteriophages could provide a valuable tool to control the dissemination of antibiotic resistant isolates, for the decolonization of colonized individuals and for treatment purposes. In this work, we have characterized a lytic bacteriophage, named vB_Kpn_F48, specific for K. pneumoniae isolates belonging to clonal group 101. Phage vB_Kpn_F48 was classified as a member of Myoviridae, order Caudovirales, on the basis of transmission electron microscopy analysis. Physiological characterization demonstrated that vB_Kpn_F48 showed a narrow host range, a short latent period, a low burst size and it is highly stable to both temperature and pH variations. High throughput sequencing and bioinformatics analysis revealed that the phage is characterized by a 171 Kb dsDNA genome that lacks genes undesirable for a therapeutic perspective such integrases, antibiotic resistance genes and toxin encoding genes. Phylogenetic analysis suggests that vB_Kpn_F48 is a T4-like bacteriophage which belongs to a novel genus within the Tevenvirinae subfamily, which we tentatively named “F48virus”. Considering the narrow host range, the genomic features and overall physiological parameters phage vB_Kpn_F48 could be a promising candidate to be used alone or in cocktails for phage therapy applications

KPC-Kp strains of clinical origin from the Italian nationwide survey investigated for the nature of the <i>cps</i> gene cluster by the modified multiplex PCR developed in this work.

<p>The first two characters of each strain ID identify the center from which the isolate was obtained. Identifiers are as reported in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096827#pone-0096827-g002" target="_blank">Fig. 2</a>.</p

Map showing the distribution of Italian centers from which the 46 KPC-Kp strains of ST258 or ST512 investigated for CPS typing by the modified multiplex PCR were originated, and distribution of the different types of <i>cps</i> gene clusters.

<p>Centers were as follows: 01, Milan; 02, Varese; 03, Lecco; 04, Torino; 05, Novara; 06, Genoa; 07, Sanremo; 08, Verona; 09, Bolzano; 10, Modena; 11, Modena; 15, Ancona; 16, Rome; 18, Foggia; 19, Lecce; 20, Naples; 22, Cosenza; 23, Palermo; 24, Catania.</p

Comparison of the CPS gene clusters from <i>K. pneumoniae</i> strains KKBO-4 (<i>cps</i>_BO-4), HS11286 (<i>cps</i>_HS11286), KK207-2 (<i>cps</i>_207-2), and 1996/49 (K-type 22, <i>cps</i>_K22).

<p>Sequence accession numbers and STs for the respective strains are also indicated (the ST of strain 1996/49 was deduced from ref. 30). The CPS gene cluster of strain 8238 (K-type 37) (accession number AB819894), differing from <i>cps</i>_K22 by a single nucleotide deletion resulting in a frameshift mutation located in a putative acetyltransferase downstream <i>gnd</i>, is not included for simplicity. Homologous regions are connected by areas of different colors reflecting the degree of nucleotide identity (from 67% to 100%). Open reading frames encoding transposases are colored in red, while those encoding hypothetical glycosyltransferases are colored in yellow. The locations of synonymous, non-synonymous and intergenic single nucleotide variations (SNVs) occurring between the CPS gene clusters of KKBO-4 and Kp13 are indicated by green, red and black stars, respectively. The <i>cps</i>_207-2 gene cluster exhibited regions of similarity to <i>cps</i>_BO-4 including the conserved <i>galF</i>-<i>wzc</i> region (83.2% of nucleotide identity), and the conserved <i>gnd</i> and <i>ugd</i> genes (95.5% and 96.8% nucleotide identity, respectively).</p

Differences between the CPS gene clusters of strains KKBO-4 (<i>cps</i>_BO-4, 26,587 bp) or KK207-2 (<i>cps</i>_207-2, 23,994 bp) and closely related CPS gene clusters detected in other sequenced genomes.

<p>The dash indicates 100% identity. The cut-off values used for the inclusion in the analysis were ≥99% nucleotide identity and ≥99% of query coverage, based on results from a BLAST search performed at the NCBI website (<a href="http://blast.ncbi.nlm.nih.gov/" target="_blank">http://blast.ncbi.nlm.nih.gov/</a>) using either nr or wgs databases, using default values but without the low complexity filter option.</p>a<p>KPNIH21 was chosen as a representative of the outbreak clone described in reference 24.</p>b<p>strains ST258 K26BO and ST258 K28BO, both described in reference 26, were characterized by identical CPS gene clusters.</p>c<p>strains UHKPC02 and UHKPC06 were representatives of those included in the <i>Klebsiella pneumoniae</i> Genome Sequencing Center Project (<a href="http://gsc.jcvi.org/projects/gsc/klebsiella_pneumoniae/index.php" target="_blank">http://gsc.jcvi.org/projects/gsc/klebsiella_pneumoniae/index.php</a>).</p>d<p>2 out of 5 SNVs are located in intergenic regions.</p

Regret affects the choice between neoadjuvant therapy and upfront surgery for potentially resectable pancreatic cancer

Background: When treating potentially resectable pancreatic adenocarcinoma, therapeutic decisions are left to the sensibility of treating clinicians who, faced with a decision that post hoc can be proven wrong, may feel a sense of regret that they want to avoid. A regret-based decision model was applied to evaluate attitudes toward neoadjuvant therapy versus upfront surgery for potentially resectable pancreatic adenocarcinoma. Methods: Three clinical scenarios describing high-, intermediate-, and low-risk disease-specific mortality after upfront surgery were presented to 60 respondents (20 oncologists, 20 gastroenterologists, and 20 surgeons). Respondents were asked to report their regret of omission and commission regarding neoadjuvant chemotherapy on a scale between 0 (no regret) and 100 (maximum regret). The threshold model and a multilevel mixed regression were applied to analyze respondents' attitudes toward neoadjuvant therapy. Results: The lowest regret of omission was elicited in the low-risk scenario, and the highest regret in the high-risk scenario (P &lt; .001). The regret of the commission was diametrically opposite to the regret of omission (P ≤ .001). The disease-specific threshold mortality at which upfront surgery is favored over the neoadjuvant therapy progressively decreased from the low-risk to the high-risk scenarios (P ≤ .001). The nonsurgeons working in or with lower surgical volume centers (P&nbsp;= .010) and surgeons (P&nbsp;= .018) accepted higher disease-specific mortality after upfront surgery, which resulted in the lower likelihood of adopting neoadjuvant therapy. Conclusion: Regret drives decision making in the management of pancreatic adenocarcinoma. Being a surgeon or a specialist working in surgical centers with lower patient volumes reduces the likelihood of recommending neoadjuvant therapy

Risk factors for anastomotic leakage after anterior resection for rectal cancer (RALAR study): A nationwide retrospective study of the Italian Society of Surgical Oncology Colorectal Cancer Network Collaborative Group

Aim: Anastomotic leakage after restorative surgery for rectal cancer shows high morbidity and related mortality. Identification of risk factors could change operative planning, with indications for stoma construction. This retrospective multicentre study aims to assess the anastomotic leak rate, identify the independent risk factors and develop a clinical prediction model to calculate the probability of leakage. Methods: The study used data from 24 Italian referral centres of the Colorectal Cancer Network of the Italian Society of Surgical Oncology. Patients were classified into two groups, AL (anastomotic leak) or NoAL (no anastomotic leak). The effect of patient-, disease-, treatment- and postoperative outcome-related factors on anastomotic leak after univariable and multivariable analysis was measured. Results: A total of 5398 patients were included, 552 in group AL and 4846 in group NoAL. The overall incidence of leaks was 10.2%, with a mean time interval of 6.8 days. The 30-day leak-related mortality was 2.6%. Sex, body mass index, tumour location, type of approach, number of cartridges employed, weight loss, clinical T stage and combined multiorgan resection were identified as independent risk factors. The stoma did not reduce the leak rate but significantly decreased leak severity and reoperation rate. A nomogram with a risk score (RALAR score) was developed to predict anastomotic leak risk at the end of resection. Conclusions: While a defunctioning stoma did not affect the leak risk, it significantly reduced its severity. Surgeons should recognize independent risk factors for leaks at the end of rectal resection and could calculate a risk score to select high-risk patients eligible for protective stoma construction

Implementation of the ERAS (Enhanced Recovery after Surgery) protocol for colorectal cancer surgery in the Piemonte Region with an Audit and Feedback approach: Study protocol for a stepped wedge cluster randomised trial: A study of the EASY-NET project

Introduction The ERAS protocol (Enhanced Recovery After Surgery) is a multimodal pathway aimed to reduce surgical stress and to allow a rapid postoperative recovery. Application of the ERAS protocol to colorectal cancer surgery has been limited to a minority of hospitals in Italy. To promote the systematic adoption of ERAS in the entire regional hospital network in Piemonte an Audit and Feedback approach (A&F) has been adopted together with a cluster randomised trial to estimate the true impact of the protocol on a large, unselected population. Methods A multicentre stepped wedge cluster randomised trial is designed for comparison between standard perioperative management and the management according to the ERAS protocol. The primary outcome is the length of hospital stay (LOS). Secondary outcomes are: incidence of postoperative complications, time to patients' recovery, control of pain and patients' satisfaction. With an A&F approach the adherence to the ERAS items is monitored through a dedicated area in the study web site. The study includes 28 surgical centres, stratified by activity volume and randomly divided into four groups. Each group is randomly assigned to a different activation period of the ERAS protocol. There are four activation periods, one every 3 months. However, the planned calendar and the total duration of the study have been extended by 6 months due to the COVID-19 pandemic. The expected sample size of about 2200 patients has a high statistical power (98%) to detect a reduction of LOS of 1 day and to estimate clinically meaningful changes in the other endpoints. Ethics and dissemination The study protocol has been approved by the Ethical Committee of the coordinating centre and by all participating centres. Study results will be timely circulated within the hospital network and published in peer-reviewed journals. Trial registration number NCT04037787

SICE national survey: current status on the adoption of laparoscopic approach to the treatment of colorectal disease in italy

The real di usion of laparoscopy for the treatment of colorectal diseases in Italy is largely unknown. The main purpose of the present study is to investigate among surgeons dedicated to minimally invasive surgery, the volume of laparoscopic colorectal procedures, the type of operation performed in comparison to traditional approach, the indication for surgery (benign and malignant) and to evaluate the di erent types of technologies used. A structured questionnaire was developed in collaboration with an international market research institute and the survey was published online; invitation to participate to the survey was issued among the members of the Italian Society of Endoscopic Surgery (SICE). 211 surgeons working in 57 surgical departments in Italy ful lled and answered the online survey. A total of 6357 colorectal procedures were recorded during the year 2015 of which 4104 (64.1%) were performed using a minimally invasive approach. Colon and rectal cancer were the most common indications for laparoscopic approach (83.1%). Left colectomy was the operation most commonly performed (41.8%), while rectal resection accounted for 23.5% of the cases. Overall conversion rate was 5.9% (242/4104). Full HD standard technology was available and routinely used in all the responders’ centers. The proportion of colorectal resec- tions that are carried out laparoscopically in dedicated centers has now reached valuable levels with a low conversion ra