Pamela Kibsey

Carbapenem-resistant Gram-negative bacilli in Canada 2009–10: results from the Canadian Nosocomial Infection Surveillance Program (CNISP)

OBJECTIVES To investigate the occurrence and molecular mechanisms associated with carbapenemases in carbapenem-resistant Gram-negative isolates from Canadian cases. METHODS Twenty hospital sites across Canada submitted isolates for a 1 year period starting 1 September 2009. All Enterobacteriaceae with MICs ≥ 2 mg/L and Acinetobacter baumannii and Pseudomonas aeruginosa with MICs ≥ 16 mg/L of carbapenems were submitted to the National Microbiology Laboratory (NML) where carbapenem MICs were confirmed by Etest and isolates were characterized by PCR for carbapenemase genes, antimicrobial susceptibilities, PFGE and plasmid isolation. RESULTS A total of 444 isolates (298 P. aeruginosa, 134 Enterobacteriaceae and 12 A. baumannii) were submitted to the NML of which 274 (61.7%; 206 P. aeruginosa, 59 Enterobacteriaceae and 9 A. baumannii) met the inclusion criteria as determined by Etest. Carbapenemase genes were identified in 30 isolates: bla(GES-5) (n = 3; P. aeruginosa), bla(KPC-3) (n = 7; Enterobacteriaceae), bla(NDM-1) (n = 2; Enterobacteriaceae), bla(VIM-2) and bla(VIM-4) (n = 8; P. aeruginosa) bla(SME-2) (n = 1; Enterobacteriaceae) and bla(OXA-23) (n = m9; A. baumannii). PFGE identified a cluster in each of Enterobacteriaceae, P. aeruginosa and A. baumannii corresponding to isolates harbouring carbapenemase genes. Three KPC plasmid patterns (IncN and FllA) were identified where indistinguishable plasmid patterns were identified in unrelated clinical isolates. CONCLUSIONS Carbapenemases were rare at the time of this study. Dissemination of carbapenemases was due to both dominant clones and common plasmid backbones.

Clinical presentation, diagnosis and management of Cryptococcus gattii cases: Lessons learned from British Columbia.

The environmental fungus Cryptococcus gattii emerged on Vancouver Island, British Columbia (BC), in 1999. By the end of 2006, it led to 176 cases and eight deaths - one of the highest burdens of C gattii disease worldwide. The present paper describes three cases, and the BC experience in the diagnosis and management of this infection. All three cases presented with pulmonary findings, including cryptococcomas and infiltrates. One also presented with brain cryptococcomas. Cases were diagnosed by chest and brain imaging, and laboratory evidence including serum or cerebrospinal fluid cryptococcal antigen detection and culture of respiratory or cerebrospinal fluid specimens. Genotyping of fungal isolates confirmed infection with C gattii VGIIa. Pulmonary cases were treated with fluconazole. One patient with central nervous system disease was treated with amphotericin B followed by fluconazole. Although this infection remains rare, clinicians should be aware of it in patients with a compatible clinical presentation who are either living in or returning from a trip to BC.

Characterization of a stable, metronidazole-resistant Clostridium difficile clinical isolate.

Background Clostridium difficile are Gram-positive, spore forming anaerobic bacteria that are the leading cause of healthcare-associated diarrhea, usually associated with antibiotic usage. Metronidazole is currently the first-line treatment for mild to moderate C. difficile diarrhea however recurrence occurs at rates of 15–35%. There are few reports of C. difficile metronidazole resistance in the literature, and when observed, the phenotype has been transient and lost after storage or exposure of the bacteria to freeze/thaw cycles. Owing to the unstable nature of the resistance phenotype in the laboratory, clinical significance and understanding of the resistance mechanisms is lacking. Methodology/Principal Findings Genotypic and phenotypic characterization was performed on a metronidazole resistant clinical isolate of C. difficile. Whole-genome sequencing was used to identify potential genetic contributions to the phenotypic variation observed with molecular and bacteriological techniques. Phenotypic observations of the metronidazole resistant strain revealed aberrant growth in broth and elongated cell morphology relative to a metronidazole-susceptible, wild type NAP1 strain. Comparative genomic analysis revealed single nucleotide polymorphism (SNP) level variation within genes affecting core metabolic pathways such as electron transport, iron utilization and energy production. Conclusions/Significance This is the first characterization of stable, metronidazole resistance in a C. difficile isolate. The study provides an in-depth genomic and phenotypic analysis of this strain and provides a foundation for future studies to elucidate mechanisms conferring metronidazole resistance in C. difficile that have not been previously described.

Proteomic Analysis of a NAP1 Clostridium difficile Clinical Isolate Resistant to Metronidazole

Background Clostridium difficile is an anaerobic, Gram-positive bacterium that has been implicated as the leading cause of antibiotic-associated diarrhea. Metronidazole is currently the first-line treatment for mild to moderate C. difficile infections. Our laboratory isolated a strain of C. difficile with a stable resistance phenotype to metronidazole. A shotgun proteomics approach was used to compare differences in the proteomes of metronidazole-resistant and -susceptible isolates. Methodology/Principal Findings NAP1 C. difficile strains CD26A54_R (Met-resistant), CD26A54_S (reduced- susceptibility), and VLOO13 (Met-susceptible) were grown to mid-log phase, and spiked with metronidazole at concentrations 2 doubling dilutions below the MIC. Peptides from each sample were labeled with iTRAQ and subjected to 2D-LC-MS/MS analysis. In the absence of metronidazole, higher expression was observed of some proteins in C. difficile strains CD26A54_S and CD26A54_R that may be involved with reduced susceptibility or resistance to metronidazole, including DNA repair proteins, putative nitroreductases, and the ferric uptake regulator (Fur). After treatment with metronidazole, moderate increases were seen in the expression of stress-related proteins in all strains. A moderate increase was also observed in the expression of the DNA repair protein RecA in CD26A54_R. Conclusions/Significance This study provided an in-depth proteomic analysis of a stable, metronidazole-resistant C. difficile isolate. The results suggested that a multi-factorial response may be associated with high level metronidazole-resistance in C. difficile, including the possible roles of altered iron metabolism and/or DNA repair.

Complete sequences of a novel blaNDM-1-harbouring plasmid from Providencia rettgeri and an FII-type plasmid from Klebsiella pneumoniae identified in Canada

OBJECTIVES Emergence of plasmids harbouring bla(NDM-1) is a major public health concern due to their association with multidrug resistance and their potential mobility. METHODS PCR was used to detect bla(NDM-1) from clinical isolates of Providencia rettgeri (PR) and Klebsiella pneumoniae (KP). Antimicrobial susceptibilities were determined using Vitek 2. The complete DNA sequence of two bla(NDM-1) plasmids (pPrY2001 and pKp11-42) was obtained using a 454-Genome Sequencer FLX. Contig assembly and gap closures were confirmed by PCR-based sequencing. Comparative analysis was done using BLASTn and BLASTp algorithms. RESULTS Both clinical isolates were resistant to all β-lactams, carbapenems, aminoglycosides, ciprofloxacin and trimethoprim/sulfamethoxazole, and susceptible to tigecycline. Plasmid pPrY2001 (113 295 bp) was isolated from PR. It did not show significant homology to any known plasmid backbone and contained a truncated repA and novel repB. Two bla(NDM-1)-harbouring plasmids from Acinetobacter lwoffii (JQ001791 and JQ060896) shared 100% similarity to a 15 kb region that contained bla(NDM-1). pPrY2001 also contained a type II toxin/antitoxin system. pKp11-42 (146 695 bp) was isolated from KP. It contained multiple repA genes. The plasmid backbone had the highest homology to the IncFIIk plasmid type (51% coverage, 100% nucleotide identity). The bla(NDM-1) region was unique in that it was flanked upstream by IS3000 and downstream by a novel transposon designated Tn6229. pKp11-42 also contained a number of mutagenesis and plasmid stability proteins. CONCLUSIONS pPrY2001 differed from all known plasmids due to its novel backbone and repB. pKp11-42 was similar to IncFIIk plasmids and contained a number of genes that aid in plasmid persistence.

Rationale for and protocol of a multi-national population-based bacteremia surveillance collaborative

BackgroundBloodstream infections are frequent causes of human illness and cause major morbidity and death. In order to best define the epidemiology of these infections and to track changes in occurrence, adverse outcome, and resistance rates over time, population based methodologies are optimal. However, few population-based surveillance systems exist worldwide, and because of differences in methodology inter-regional comparisons are limited. In this report we describe the rationale and propose first practical steps for developing an international collaborative approach to the epidemiologic study and surveillance for bacteremia.FindingsThe founding collaborative participants represent six regions in four countries in three continents with a combined annual surveillance population of more than 8 million residents.ConclusionFuture studies from this collaborative should lead to a better understanding of the epidemiology of bloodstream infections.

Results from the Canadian Nosocomial Infection Surveillance Program on Carbapenemase-Producing Enterobacteriaceae, 2010 to 2014

ABSTRACT Carbapenemase-producing Enterobacteriaceae (CPE) are increasing globally; here we report on the investigation of CPE in Canada over a 5-year period. Participating acute care facilities across Canada submitted carbapenem-nonsusceptible Enterobacteriaceae from 1 January 2010 to 31 December 2014 to the National Microbiology Laboratory. All CPE were characterized by antimicrobial susceptibilities, pulsed-field gel electrophoresis, multilocus sequence typing, and plasmid restriction fragment length polymorphism analysis and had patient data collected using a standard questionnaire. The 5-year incidence rate of CPE was 0.09 per 10,000 patient days and 0.07 per 1,000 admissions. There were a total of 261 CPE isolated from 238 patients in 58 hospitals during the study period. blaKPC-3 (64.8%) and blaNDM-1 (17.6%) represented the highest proportion of carbapenemase genes detected in Canadian isolates. Patients who had a history of medical attention during international travel accounted for 21% of CPE cases. The hospital 30-day all-cause mortality rate for the 5-year surveillance period was 17.1 per 100 CPE cases. No significant increase in the occurrence of CPE was observed from 2010 to 2014. Nosocomial transmission of CPE, as well as international health care, is driving its persistence within Canada.

Containing Cefoxitin Costs through a Program to Curtail Use in Surgical Prophylaxis

OBJECTIVE To reduce drug costs attributable to anti-anaerobic cephalosporins - specifically to reduce cefoxitin use in surgical prophylaxis. DESIGN Before and after intervention cefoxitin use comparison. SETTING Tertiary care hospital. PARTICIPANTS Hospitalized patients. INTERVENTIONS Chart review of patients identified through pharmacy records as cefoxitin recipients was carried out to determine which physicians were the principal users of cefoxitin and the purpose for such use. These data were used to direct cost containment strategies. MAIN OUTCOME MEASURES Hospital quarterly pharmacy acquisition costs and grams of cefoxitin used. RESULTS The departments of surgery (49%) and obstetrics/gynecology (37%) were the principal users of cefoxitin, and surgical prophylaxis was found to be the principal indication for use (63%). These departments were invited by the Antibiotic Utilization Subcommittee of the hospitals Pharmacy and Therapeutics Committee to draft surgical prophylaxis guidelines in keeping with published recommendations. Such guidelines were written and distributed to medical staff and substituted cefazolin for most forms of prophylaxis, gentamicin/metronidazole for colorectal prophylaxis and cefoxitin only for appendectomies. Over the following 21 months, hospital-wide cefoxitin use fell from 6093 g,

Characterization of Clostridium difficile Strains in British Columbia, Canada: A Shift from NAP1 Majority (2008) to Novel Strain Types (2013) in One Region

70,076 per quarter, to 1316 g,

Epidemiologic and Genotypic Review of Carbapenemase-Producing Organisms in British Columbia, Canada, between 2008 and 2014

11,515 per quarter (partially offset by a 2595 g,