Double copies of blaKPC-3::Tn4401a on an IncX3 plasmid in Klebsiella pneumoniae successful clone ST512 from Italy

A carbapenem-resistant sequence type 512 (ST512) Klebsiella pneumoniae carbapenemase 3 (KPC-3)-producing K. pneumoniae strain showing a novel variant plasmid content was isolated in Palermo, Italy, in 2014. ST512 is a worldwide successful clone associated with the spread of blaKPC genes located on the IncFIIk pKpQIL plasmid. In our ST512 strain, the blaKPC-3 gene was unusually located on an IncX3 plasmid, whose complete sequence was determined. Two copies of blaKPC-3::Tn4401a caused by intramolecular transposition events were detected in the plasmid

Detection of SHV β-lactamases in Gram-negative bacilli using fluorescein-labeled antibodies

<p>Abstract</p> <p>Background</p> <p>β-lactam resistance in Gram-negative bacteria is a significant clinical problem in the community, long-term care facilities, and hospitals. In these organisms, β-lactam resistance most commonly results from the production of β-lactamases. In Gram-negative bacilli, TEM-, SHV-, and CTX-M-type β-lactamases predominate. Therefore, new and accurate detection methods for these β-lactamase producing isolates are needed.</p> <p>Results</p> <p><it>E. coli </it>DH10B cells producing SHV-1 β-lactamase and a clinical isolate of <it>K. pneumoniae </it>producing SHV-5 β-lactamase were rendered membrane permeable, fixed and adhered to poly-L-lysine coated slides, and stained with purified polyclonal anti-SHV antibodies that were fluorescein labeled. <it>E. coli </it>DH10B cells without a <it>bla</it>_SHVgene were used as a negative control. The procedure generated a fluorescence signal from those slides containing cells expressing SHV β-lactamase that was sufficient for direct imaging.</p> <p>Conclusion</p> <p>We developed a rapid and accurate method of visualizing the SHV family of enzymes in clinical samples containing Gram-negative bacilli using a fluorescein-labeled polyclonal antibody.</p

Acinetobacter baumannii isolates from pets and horses in Switzerland: molecular characterization and clinical data

Objectives We investigated whether Acinetobacter baumannii isolates of veterinary origin shared common molecular characteristics with those described in humans. Methods Nineteen A. baumannii isolates collected in pets and horses were analysed. Clonality was studied using repetitive extragenic palindromic PCR (rep-PCR) and multilocus sequence typing (MLST). PCR and DNA sequencing for various β-lactamase, aminoglycoside-modifying enzyme, gyrA and parC, ISAba1 and IS1133, adeR and adeS of the AdeABC efflux pump, carO porin and class 1/2/3 integron genes were performed. Results Two main clones [A (n = 8) and B (n = 9)] were observed by rep-PCR. MLST indicated that clone A contained isolates of sequence type (ST) ST12 (international clone II) and clone B contained isolates of ST15 (international clone I). Two isolates of ST10 and ST20 were also noted. Seventeen isolates were resistant to gentamicin, 12 to ciprofloxacin and 3 to carbapenems. Isolates of ST12 carried blaOXA-66, blaADC-25, blaTEM-1, aacC2 and IS1133. Strains of ST15 possessed blaOXA-69, blaADC-11, blaTEM-1 and a class 1 integron carrying aacC1 and aadA1. ISAba1 was found upstream of blaADC (one ST10 and one ST12) and/or blaOXA-66 (seven ST12). Twelve isolates of different STs contained the substitutions Ser83Leu in GyrA and Ser80Leu or Glu84Lys in ParC. Significant disruptions of CarO porin and overexpressed efflux pumps were not observed. The majority of infections were hospital acquired and in animals with predisposing conditions for infection. Conclusions STs and the molecular background of resistance observed in our collection have been frequently described in A. baumannii detected in human patients. Animals should be considered as a potential reservoir of multidrug-resistant A. baumanni

Characterization and genomic analyses of two newly isolated Morganella phages define distant members among Tevenvirinae and Autographivirinae subfamilies

Morganella morganii is a common but frequent neglected environmental opportunistic pathogen which can cause deadly nosocomial infections. The increased number of multidrug-resistant M. morganii isolates motivates the search for alternative and effective antibacterials. We have isolated two novel obligatorily lytic M. morganii bacteriophages (vB_MmoM_MP1, vB_MmoP_MP2) and characterized them with respect to specificity, morphology, genome organization and phylogenetic relationships. MP1s dsDNA genome consists of 163,095bp and encodes 271 proteins, exhibiting low DNA (10kb chromosomal inversion that encompass the baseplate assembly and head outer capsid synthesis genes when compared to other T-even bacteriophages. MP2 has a dsDNA molecule with 39,394bp and encodes 55 proteins, presenting significant genomic (70%) and proteomic identity (86%) but only to Morganella bacteriophage MmP1. MP1 and MP2 are then novel members of Tevenvirinae and Autographivirinae, respectively, but differ significantly from other tailed bacteriophages of these subfamilies to warrant proposing new genera. Both bacteriophages together could propagate in 23 of 27M. morganii clinical isolates of different origin and antibiotic resistance profiles, making them suitable for further studies on a development of bacteriophage cocktail for potential therapeutic applications.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684) and the Project PTDC/BBB-BSS/6471/2014 (POCI-01-0145-FEDER-016678). RL contributed to the genome sequencing analysis, supported by the KU Leuven GOA Grant ‘Phage Biosystems’. JP acknowledges the project R-3986 of the Herculesstichting.info:eu-repo/semantics/publishedVersio

Whole-genome characterization of a Shewanella algae strain coharboring blaCTX-M-15 and armA genes on a novel IncC plasmid

no abstract per pubblicazione letter

Environmental dissemination of carbapenemase-producing Enterobacteriaceae in rivers in Switzerland

The aquatic environment takes on a key role in the dissemination of antimicrobial-resistant Enterobacteriaceae. This study assesses the occurrence of carbapenemase-producing Enterobacteriaceae (CPE) in freshwater samples from rivers, inland canals, and streams throughout Switzerland, and characterizes the isolated strains using phenotypic and NGS-based genotypic methods. CPE producing KPC-2 (n = 2), KPC-3 (n = 1), NDM-5 (n = 3), OXA-48 (n = 3), OXA-181 (n = 6), and VIM-1 (n = 2) were detected in 17/164 of the water samples. Seven Escherichia coli had sequence types (STs) that belonged to extra-intestinal pathogenic clonal lineages ST38, ST73, ST167, ST410, and ST648. The majority (16/17) of the carbapenemase genes were located on plasmids, including the widespread IncC (n = 1), IncFIIA (n = 1), and IncFIIB plasmids (n = 4), the epidemic IncL (n = 1) and IncX3 (n = 5) plasmids, a rare Col156 plasmid (n = 1), and the mosaic IncFIB, IncR, and IncQ plasmids (n = 3). Plasmids were composed of elements that were identical to those of resistance plasmids retrieved from clinical and veterinary isolates locally and worldwide. Our data show environmental dissemination of high-risk CPE clones in Switzerland. Epidemic and mosaic-like plasmids carrying clinically relevant carbapenemase genes are replicating and evolving pollutants of river ecosystems, representing a threat to public health and environmental integrity

Antimicrobial resistance prediction and phylogenetic analysis of Neisseria gonorrhoeae isolates using the Oxford Nanopore MinION sequencer

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is common, compromising gonorrhoea treatment internationally. Rapid characterisation of AMR strains could ensure appropriate and personalised treatment, and support identification and investigation of gonorrhoea outbreaks in nearly real-time. Whole-genome sequencing is ideal for investigation of emergence and dissemination of AMR determinants, predicting AMR, in the gonococcal population and spread of AMR strains in the human population. The novel, rapid and revolutionary long-read sequencer MinION is a small hand-held device that generates bacterial genomes within one day. However, accuracy of MinION reads has been suboptimal for many objectives and the MinION has not been evaluated for gonococci. In this first MinION study for gonococci, we show that MinION-derived sequences analysed with existing open-access, web-based sequence analysis tools are not sufficiently accurate to identify key gonococcal AMR determinants. Nevertheless, using an in house-developed CLC Genomics Workbench including de novo assembly and optimised BLAST algorithms, we show that 2D ONT-derived sequences can be used for accurate prediction of decreased susceptibility or resistance to recommended antimicrobials in gonococcal isolates. We also show that the 2D ONT-derived sequences are useful for rapid phylogenomic-based molecular epidemiological investigations, and, in hybrid assemblies with Illumina sequences, for producing contiguous assemblies and finished reference genomes

Treatment and outcomes in carbapenem-resistant Klebsiella pneumoniae bloodstream infections

Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) is an emerging multidrug-resistant nosocomial pathogen. This is a retrospective chart review describing the outcomes and treatment of 60 cases of CR-Kp bloodstream infections. All CR-Kp isolated from blood cultures were identified retrospectively from the microbiology laboratory from January 2007 to May 2009. Clinical information was collected from the electronic medical record. Patients with 14-day hospital mortality were compared to those who survived 14 days. The all-cause in-hospital and 14-day mortality for all 60 CR-Kp bloodstream infections were 58.3% and 41.7%, respectively. In this collection, 98% of tested isolates were susceptible in vitro to tigecycline compared to 86% to colistimethate, 45% to amikacin, and 22% to gentamicin. Nine patients died before cultures were finalized and received no therapy active against CR-Kp. In the remaining 51 patients, those who survived to day 14 (n = 35) were compared to nonsurvivors at day 14 (n = 16). These patients were characterized by both chronic disease and acute illness. The 90-day readmission rate for hospital survivors was 72%. Time to active therapy was not significantly different between survivors and nonsurvivors, and hospital mortality was also similar regardless of therapy chosen. Pitt bacteremia score was the only significant factor associated with mortality in Cox regression analysis. In summary, CR-Kp bloodstream infections occur in patients who are chronically and acutely ill. They are associated with high 14-day mortality and poor outcomes regardless of tigecycline or other treatment regimens selected

Effects of type and level of training on variation in physician knowledge in the use and acquisition of blood cultures: a cross sectional survey

BACKGROUND: Blood culture (BCX) use is often sub-optimal, and is a user-dependent diagnostic test. Little is known about physician training and BCX-related knowledge. We sought to assess variations in caregiver BCX-related knowledge, and their relation to medical training. METHODS: We developed and piloted a self-administered BCX-related knowledge survey instrument. Expert opinion, literature review, focus groups, and mini-pilots reduced > 100 questions in multiple formats to a final questionnaire with 15 scored content items and 4 covariate identifiers. This questionnaire was used in a cross-sectional survey of physicians, fellows, residents and medical students at a large urban public teaching hospital. The responses were stratified by years/level of training, type of specialty training, self-reported practical and theoretical BCX-related instruction. Summary scores were derived from participant responses compared to a 95% consensus opinion of infectious diseases specialists that matched an evidence based reference standard. RESULTS: There were 291 respondents (Attendings = 72, Post-Graduate Year (PGY) = 3 = 84, PGY2 = 42, PGY1 = 41, medical students = 52). Mean scores differed by training level (Attending = 85.0, PGY3 = 81.1, PGY2 = 78.4, PGY1 = 75.4, students = 67.7) [p ≤ 0.001], and training type (Infectious Diseases = 96.1, Medicine = 81.7, Emergency Medicine = 79.6, Surgery = 78.5, Family Practice = 76.5, Obstetrics-Gynecology = 74.4, Pediatrics = 74.0) [p ≤ 0.001]. Higher summary scores were associated with self-reported theoretical [p ≤ 0.001] and practical [p = 0.001] BCX-related training. Linear regression showed level and type of training accounted for most of the score variation. CONCLUSION: Higher mean scores were associated with advancing level of training and greater subject-related training. Notably, house staff and medical students, who are most likely to order and/or obtain BCXs, lack key BCX-related knowledge. Targeted education may improve utilization of this important diagnostic tool

Failure of levofloxacin treatment in community-acquired pneumococcal pneumonia

BACKGROUND: Streptococcus pneumoniae is the leading cause of community-acquired pneumonia (CAP). High global incidence of macrolide and penicillin resistance has been reported, whereas fluoroquinolone resistance is uncommon. Current guidelines for suspected CAP in patients with co-morbidity factors and recent antibiotic therapy recommend initial empiric therapy using one fluoroquinolone or one macrolide associated to other drugs (amoxicillin, amoxicillin/clavulanate, broad-spectrum cephalosporins). Resistance to fluoroquinolones is determined by efflux mechanisms and/or mutations in the parC and parE genes coding for topoisomerase IV and/or gyrA and gyrB genes coding for DNA gyrase. No clinical cases due to fluoroquinolone-resistant S. pneumoniae strains have been yet reported from Italy. CASE PRESENTATION: A 72-year-old patient with long history of chronic obstructive pulmonary disease and multiple fluoroquinolone treatments for recurrent lower respiratory tract infections developed fever, increased sputum production, and dyspnea. He was treated with oral levofloxacin (500 mg bid). Three days later, because of acute respiratory insufficiency, the patient was hospitalized. Levofloxacin treatment was supplemented with piperacillin/tazobactam. Microbiological tests detected a S. pneumoniae strain intermediate to penicillin (MIC, 1 mg/L) and resistant to macrolides (MIC >256 mg/L) and fluoroquinolones (MIC >32 mg/L). Point mutations were detected in gyrA (Ser81-Phe), parE (Ile460-Val), and parC gene (Ser79-Phe; Lys137-Asn). Complete clinical response followed treatment with piperacillin/tazobactam. CONCLUSION: This is the first Italian case of community-acquired pneumonia due to a fluoroquinolone-resistant S. pneumoniae isolate where treatment failure of levofloxacin was documented. Molecular analysis showed a group of mutations that have not yet been reported from Italy and has been detected only twice in Europe. Treatment with piperacillin/tazobactam appears an effective means to inhibit fluoroquinolone-resistant strains of S. pneumoniae causing community-acquired pneumonia in seriously ill patients