The extent to which clonal spread contributes to emerging antimicrobial resistance in Escherichia coli is incompletely defined. To address this question within a recent, nationally representative strain collection, three established drug-resistant E. coli clonal groups (i.e., clonal group A, E. coli O15:K52:H1, and sequence type 131 [ST131]) were sought among 199 E. coli urine isolates recovered from across Canada from 2002 to 2004, with stratification by resistance to trimethoprim-sulfamethoxazole (TS) and fluoroquinolones (FQs). The isolates' clonal backgrounds, virulence genotypes, and macrorestriction profiles were assessed. The three clonal groups were found to account for 37. 2% of isolates overall, but accounted for 0% of TS-susceptible (TS-S) and FQ-susceptible (FQ-S) isolates, 20% of TS-resistant (TS-R) and FQ-S isolates, 60% of TS-S and FQ-R isolates, and 68% of TS-R and FQ-R isolates (P < 0.001). E. coli ST131, the most prevalent clonal group, accounted for 23.1% of isolates overall and for 44% of the FQ-R isolates.Nearly all ST131 isolates were FQ-R (96%) but, notably, cephalosporin susceptible (98%). Although the distinctive virulence profiles of the FQ-R clonal group isolates were less extensive than those of the susceptible isolates, they were significantly more extensive than those of the other FQ-R isolates. These findings indicate that among the E. coli urine isolates studied, resistance to TS and FQs has a prominent clonal component, with the O15:K52:H1 clonal group and especially E. coli ST131 being the major contributors. These clonal groups appear to be more virulent than comparably resistant isolates, possibly contributing to their success as emerging multi-drug-resistant pathogens.

, 19 medical centers collected 4,180 isolates recovered from clinical specimens from patients in intensive care units (ICUs) in Canada. The 4,180 isolates were collected from 2,292 respiratory specimens (54.8%), 738 blood specimens (17.7%), 581 wound/tissue specimens (13.9%), and 569 urinary specimens (13.6%). The 10 most common organisms isolated from 79.5% of all clinical specimens were methicillin-susceptible Staphylococcus aureus (MSSA) (16.4%), Escherichia coli (12.8%), Pseudomonas aeruginosa (10.0%), Haemophilus influenzae (7.9%), coagulase-negative staphylococci/Staphylococcus epidermidis (6.5%), Enterococcus spp. (6.1%), Streptococcus pneumoniae (5.8%), Klebsiella pneumoniae (5.8%), methicillin-resistant Staphylococcus aureus (MRSA) (4.7%), and Enterobacter cloacae (3.9%). MRSA made up 22.3% (197/884) of all S. aureus isolates (90.9% of MRSA were health care-associated MRSA, and 9.1% were community-associated MRSA), while vancomycin-resistant enterococci (VRE) made up 6.7% (11/255) of all enterococcal isolates (88.2% of VRE had the vanA genotype). Extended-spectrum ␤-lactamase (ESBL)-producing E. coli and K. pneumoniae occurred in 3.5% (19/536) and 1.8% (4/224) of isolates, respectively. All 19 ESBL-producing E. coli isolates were PCR positive for CTX-M, with bla CTX-M-15 occurring in 74% (14/19) of isolates. For MRSA, no resistance against daptomycin, linezolid, tigecycline, and vancomycin was observed, while the resistance rates to other agents were as follows: clarithromycin, 89.9%; clindamycin, 76.1%; fluoroquinolones, 90.1 to 91.8%; and trimethoprim-sulfamethoxazole, 11.7%. For E. coli, no resistance to amikacin, meropenem, and tigecycline was observed, while resistance rates to other agents were as follows: cefazolin, 20.1%; cefepime, 0.7%; ceftriaxone, 3.7%; gentamicin, 3.0%; fluoroquinolones, 21.1%; piperacillin-tazobactam, 1.9%; and trimethoprim-sulfamethoxazole, 24.8%. Resistance rates for P. aeruginosa were as follows: amikacin, 2.6%; cefepime, 10.2%; gentamicin, 15.2%; fluoroquinolones, 23.8 to 25.5%; meropenem, 13.6%; and piperacillin-tazobactam, 9.3%. A multidrug-resistant (MDR) phenotype (resistance to three or more of the following drugs: cefepime, piperacillin-tazobactam, meropenem, amikacin or gentamicin, and ciprofloxacin) occurred frequently in P. aeruginosa (12.6%) but uncommonly in E. coli (0.2%), E. cloacae (0.6%), or K. pneumoniae (0%). In conclusion, S. aureus (MSSA and MRSA), E. coli, P. aeruginosa, H. influenzae, Enterococcus spp., S. pneumoniae, and K. pneumoniae are the most common isolates recovered from clinical specimens in Canadian ICUs. A MDR phenotype is common for P. aeruginosa isolates in Canadian ICUs.

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M atrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has been routinely used for the identification of bacteria and fungi from agar culture in many centers in Europe and is increasingly used in North America and elsewhere for primary identification of microorganisms (1, 2, 25). MALDI-TOF instruments use an ionizing laser to vaporize the abundant structural elements (primarily ribosomal proteins) of bacteria and yeasts and analyze the weight and relative abundance of each particle to generate a spectrum. Spectra are compared to a computer database of reference or user-defined organism spectra, and identification is obtained by matching the most similar spectrum in the database to the unknown organism. Performance of the Bruker MALDI BioTyper has been extensively studied in multiple centers and confirms that reliable identification can be obtained for Ͼ95% of the isolates grown on solid media routinely encountered in the clinical laboratory (1,4,5,8,19).More recently, protocols for the direct identification of pathogens from positive blood culture broths have been developed (3,12,16,21,22). Although blood culture broths are usually monobacterial (or monofungal) cultures, the presence of proteins from red cells, white blood cells, and serum interferes with the analysis by adding spectral peaks not found in the organism database. Furthermore, interfering substances such as charcoal (when present) and low organism numbers (as might be encountered with slow-growing or contaminating bacteria) present additional challenges in the use and interpretation of MALDI-TOF spectra for identification pathogens directly from positive blood cultures (24). As a result, many centers have developed in-house methods for preprocessing of blood cultures to optimize recovery of the bacterial proteome. More recently, a commercial kit (Bruker Sepsityper) has been released to simplify the processing steps required for the purification and extraction of the bacterial proteome from positive blood cultures. The system serves to facilitate preprocessing and minimize the impact of the interfering human proteome on the MALDI-TOF analysis. Here we report the performance of the Sepsityper system on the Bruker MALDI BioTyper for the direct identification of pathogens from blood cultures and a cost and turnaround time analysis of the results. MATERIALS AND METHODS Blood cultures.Blood was collected at the bedside and directly inoculated into BacT/Alert SA (aerobic culture) and/or SN (anaerobic) (bioMérieux, Marcy l'Etoile, France). Both bottle types are charcoal free. Bottles were loaded onto the a BacT/Alert instrument (bioMérieux, Marcy l'Etoile, France) and incubated. Bottles were incubated for up to 5 days, and when the operator was notified of a positive blood culture, a Gram stain was performed. All positive bottles were subjected to subculture and routine