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 ß-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.

Population-based laboratory assessment of the burden of community-onset bloodstream infection in Victoria, Canada.

Although community-onset bloodstream infection (BSI) is recognized as a major cause of morbidity and mortality, its epidemiology has not been well defined in non-selected populations. We conducted population-based laboratory surveillance in the Victoria area, Canada during 1998-2005 in order to determine the burden associated with community-onset BSI. A total of 2785 episodes were identified for an overall annual incidence of 101·2/100,000. Males and the very young and the elderly were at highest risk. Overall 1980 (71%) episodes resulted in hospital admission for a median length of stay of 8 days; the total days of acute hospitalization associated with community-onset BSI was 28 442 days or 1034 days/100,000 population per year. The in-hospital case-fatality rate was 13%. Community-onset BSI is associated with a major burden of illness. These data support ongoing and future preventative and research efforts aimed at reducing the major impact of these infections.

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.

Cryptococcus gattii infections on Vancouver Island, British Columbia, Canada: emergence of a tropical fungus in a temperate environment.

BACKGROUND: Cryptococcus gattii causes disease among immunocompetent individuals in the tropics and subtropics. We document the appearance of C. gattii infections on Vancouver Island (VI), a temperate region, and discuss reasons for this emergence. METHODS: Data on Cryptococcus hospitalizations for the calendar years 1995 through 2004 were reviewed. Viable historic isolates stored at the provincial public health laboratory between 1987 and 2000 were serotyped. Human cases were mapped by place of residence. RESULTS: Cryptococcosis among HIV negative individuals diagnosed on VI increased sharply after 1999. C. gattii was not detected in stored isolates prior to 1999. C. gattii cases lived in a specific biogeoclimatic zone on VI. Higher rates of illness were associated with exposure to the central region of VI. CONCLUSIONS: The emergence of C. gattii in a temperate region is unprecedented. Clinicians should consider C. gattii in the differential diagnosis of individuals who travelled to certain areas in British Columbia.

Outbreak of invasive Streptococcus pneumoniae among an inner-city population in Victoria, British Columbia, 2016-2017.

BACKGROUND: Invasive pneumococcal disease (IPD) is a significant cause of morbidity and mortality; however, outbreaks of IPD are relatively rare. Homelessness and substance use are known risk factors for IPD and have been associated with several outbreaks in Canada, despite national recommendations for routine childhood and targeted adult pneumococcal vaccination. OBJECTIVES: To describe the epidemiology and public health challenges related to an outbreak of novel serotype 4 IPD in a homeless and unstably housed population in Victoria, British Columbia during the autumn and winter of 2016-2017. RESULTS: Prospective, enhanced surveillance was initiated for laboratory confirmed cases reported to public health, including variables recording housing status and substance use. Thirty-three cases of serotype 4 IPD within the Victoria area were reported to public health between August 1, 2016 and September 1, 2017. Compared with other serotypes, these cases were more likely to be middle-aged, homeless or unstably housed, and to have a recent history of substance use. A targeted pneumococcal vaccination campaign was initiated in collaboration with external community organizations; however, these initiatives were challenged by incomplete data and staffing constraints. CONCLUSION: This report illustrates an outbreak of serotype 4 IPD among an inner-city population with multiple risk factors, including homelessness, unstable housing and substance use. Given the challenges controlling the outbreak, outreach capacity and pneumococcal vaccination coverage is needed among this marginalized population.

Predominance of staphylococcal organisms in infections occurring in a burns intensive care unit.

To assess the sites, incidence, and bacteriology of infections in intensive care burn patients, a prospective survey of all admissions to a tertiary care institution burn unit was carried out over a 12-month period. One hundred and sixteen patients were admitted, 106 with a diagnosis of thermal burns. Forty patients developed 90 infections. Only two deaths occurred, one in a patient with sepsis. In order of frequency, pneumonia, burn infection, UTI and primary bacteraemia were most common. Staphylococcal species accounted for a majority of infections at all body sites except UTI (47 per cent of all infections, including 11 of 14 bacteraemic infections). Staph. aureus sepsis was more common in those carrying the organism on admission. Strain typing of paired admission and subsequent clinical isolates in 19 patients with Staph. aureus sepsis indicated that eight (42 per cent) became infected with a strain they carried on admission. Further reductions in septic complications of burns in our center would be best directed at staphylococcal species, particularly Staph. aureus. Both eradication of carrier state, and prevention of acquisition of Staph. aureus strains could be explored.

Epidemiological studies of nosocomial infections with Pseudomonas aeruginosa using a DNA probe.

A DNA probe encoding the Pseudomonas aeruginosa pilin gene has been developed in the authors' laboratory and has been shown to be a useful epidemiological tool. In the present study this technology, together with other typing methods, has been used to define relatedness and possible transmission routes of P aeruginosa strains isolated in several hospital wards. Clusters of P aeruginosa infections, suspected to be the result of nosocomial transmission, developed in a general intensive care unit (ICU) and a neurosurgical ward/ICU, as well as in a burn unit, were studied using antibiograms, lipopolysaccharide-serotyping, and gene probe analysis. Results of these studies demonstrated that each of the general and neurosurgical ICU isolates were different, making nosocomial transmission very unlikely. However, within the burn unit, patient isolates had identical profiles, suggesting that spread between patients was occurring or that a common source of infection was present. Changes in infection control measures within the unit were introduced and may have contributed to eradication of the outbreak. DNA probe studies were valuable in clarifying epidemiological relatedness of isolates that was not evident with the other typing strategies and identified a possible burn-associated strain.

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 hospital's 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, $70,076 per quarter, to 1316 g, $11,515 per quarter (partially offset by a 2595 g, $9,131 per quarter increase in cefazolin use). CONCLUSION: As a first step in reducing hospital costs of anti-anaerobic cephalosporins, rationalization of cefoxitin use may be preferable to formulary interchange with alternatives such as ceftizoxime or cefotetan.

Disk diffusion versus broth microdilution susceptibility testing of Haemophilus species and Moraxella catarrhalis using seven oral antimicrobial agents: application of updated susceptibility guidelines of the National Committee for Clinical Laboratory Standards.

Susceptibility testing of Haemophilus species and Moraxella catarrhalis is medium and inoculum dependent. Seven oral agents, ampicillin, amoxicillin-clavulanic acid, cefaclor, loracarbef, cefuroxime-axetil, cefixime, and erythromycin, were tested against 400 beta-lactamase-positive and -negative clinically significant respiratory strains of Haemophilus species and 100 strains of M. catarrhalis. Sources of the strains included teaching and regional hospitals and a private laboratory. All strains were tested by broth microdilution and disk diffusion in haemophilus test medium for Haemophilus species and Mueller-Hinton broth and agar for M. catarrhalis. Appropriate National Committee for Clinical Laboratory Standards (NCCLS) standards were followed. For Haemophilus species, by disk diffusion and broth microdilution, respectively, 27 and 27% of strains were resistant to ampicillin, 37 and 5% were resistant to erythromycin, 3 and 0.5% were resistant to cefaclor, 2 and 0.5% were resistant to loracarbef, and 0% were resistant to cefuroxime-axetil, cefixime, and amoxicillin-clavulanic acid. beta-Lactamase-negative ampicillin-resistant strains were not observed. Of M. catarrhalis strains, 56% were resistant to ampicillin by disk diffusion and 95% were resistant by broth microdilution. This species was susceptible to all other agents tested by either method. The disagreements between disk diffusion results and MICs for cefaclor, ampicillin, cefuroxime, and loracarbef that occurred with use of the 1990 NCCLS tables were resolved when the 1992 NCCLS tables were used.

Relapsing Clostridium difficile colitis and Reiter's syndrome.

We describe a patient with recurrent Clostridium difficile-associated colitis who suffered severe arthritis and urethritis with each of three episodes of diarrhea. Although immune complex formation was demonstrated in synovial fluid, neutralizing antibodies to C. difficile cytotoxin A and B were not found in either serum or synovial fluid. Cholestyramine did not prevent a third episode of colitis which followed the use of amikacin. This patient was HLA-B27 positive; she developed sacroiliitis, tenosynovitis, oligoarthritis, culture negative urethritis, and cervicitis. With the successful treatment of each episode of diarrhea, these additional symptoms resolved. The close temporal correlation between recurrences of C. difficile-associated colitis and these other symptoms strengthens evidence from other reports which suggests that colonic infections with C. difficile may precipitate the Reiter's syndrome. Evidence from this case does not support the contention that antibodies to C. difficile toxins are implicated in producing joint inflammation.

Laboratory and clinical evaluations of media for the primary isolation of Haemophilus species.

There has not previously been an objective comparison of medium formulations for the primary isolation of Haemophilus species. This study was undertaken to evaluate the components required for the optimal growth of large, easily identifiable colonies of these bacteria. We compared six medium bases and seven supplements for their ability to support the growth of 86 strains of Haemophilus influenzae and 17 strains of other species of Haemophilus. By using a growth index that combines colony size and the dilution factor, a formulation of GC agar base with 1% yeast autolysate and 5% sheep blood (chocolated) promoted the growth of large, easily recognizable colonies of H. influenzae and other Haemophilus species. This medium was designated GCYSB. The addition of hematin to supplements that supplied NAD (or factor V) to the medium was inhibitory to the growth of all of the Haemophilus species tested. In a clinical comparison of GCYSB with routinely used chocolate agar medium in two laboratories for the primary isolation of Haemophilus species, overall GCYSB promoted better growth of 124 strains of H. influenzae and H. parainfluenzae. GCYSB is easy to prepare and inexpensive compared with the ease of preparation and expense of other Haemophilus isolation media.

The effects of ischemia and ischemia-reperfusion on bacterial translocation, lipid peroxidation, and gut histology: studies on hemorrhagic shock in pigs.

The bacterial translocation hypothesis was tested in two studies (acute and subacute) in a porcine model of hemorrhagic shock. Male pigs (30-40 kg each) under general anesthesia had their femoral vein, femoral artery, and portal vein catheterized. After stabilization (1 hour) they were bled (40% of blood volume) over 30 minutes, then maintained in the hypotensive state (MAP = 30-40 mm Hg) for 2 hours, following which, according to randomization, they entered the control group or were resuscitated with whole blood (WB group) or with lactated Ringer's solution (LR group). In the acute study, the mesenteric efferent lymphatic was also cannulated, the control group was not resuscitated, and the animals remained under general anesthesia to the end of the experiment (8.5 hours), when gut tissue was obtained for histologic study and measurement of lipid peroxidation. In the subacute study, the control group was not bled, the animals were awakened at 6.5 hours, and the portal vein catheter remained in situ until 48 hours. In both studies, samples of portal blood were obtained for culture at regular intervals and on completion, samples from mesenteric lymph nodes (MLNs) for culture were taken in the acute study, and in the subacute study samples from MLNs, spleen, and liver were obtained. In the acute study significant bacterial translocation to the MLNs and portal blood did not occur among the controls (n = 3), the LR group (n = 5), and the WB group (n = 6). Significant evidence of lipid peroxidation was found in both the LR and WB groups. Histologic assessment showed no difference among the groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of a DNA probe with culture for detecting Chlamydia trachomatis directly from genital specimens.

A study was conducted to compare results between culture methods and the Gen-Probe (Gen-Probe Inc. San Diego, California) chemiluminescent technique of nucleic acid hybridization to identify Chlamydia trachomatis from genital specimens from 117 females and 70 males. Specimens collected from sexually transmitted diseases (STD) and infertility clinics were randomized as to whether probe or culture swabs were collected first. The Gen-Probe demonstrated a sensitivity of 83% and a specificity of 75% in the female population and a sensitivity of 68% and a specificity of 75% in the male population when compared to the reference culture method using cycloheximide-treated McCoy cells. Gen-Probe had an overall sensitivity of 74% and specificity of 75% when the two groups were combined. Chlamydiazyme (Abbott Labs) results were obtained on 135 specimens; 90 of which correlated with probe and culture. The remaining 45 specimens had varying combinations of probe, culture and Chlamydiazyme results. MicroTrak (Syva) was done on 49 specimens; 35 of which correlated with probe and culture. The remaining 14 specimens had varying combinations of probe, culture and MicroTrak results. The apparent lack of sensitivity of the DNA probe is a major drawback of this system.

Hemorrhagic shock and bacterial translocation in a swine model.

Bacterial translocation is proposed as an explanation for sepsis associated with hemorrhagic shock. This study attempted to document these events in a large animal model. Male swine were randomly assigned to control (n = 10) or experimental (n = 10) groups. Animals were anaesthetized, and the bladder, portal vein, and a mesenteric lymphatic vessel cannulated. Experimental animals were bled 40% of blood volume. Over the next six hours maintenance fluids were given, and cultures of portal blood and mesenteric lymph taken. Before the swine were killed, cultures were taken from portal and systemic blood, mesenteric lymph, and lymph nodes, and a portion of terminal ileum was resected for histologic study. Experimental animals experienced significant shock as demonstrated by changes in hemodynamic and biochemical variables. Cultures and histologic examination of the terminal ileum showed no significant difference between control and experimental animals. In an unresuscitated swine model, significant bacterial translocation was not demonstrated within six hours of hemorrhagic shock.

Comparison of BACTEC Plus 26 and 27 media with and without fastidious organism supplement with conventional methods for culture of sterile body fluids.

We compared the BACTEC Plus 26/27 culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) with and without fastidious organism supplement with conventional centrifugation preparation and plating for the recovery and speed of detection of microorganisms. A total of 1,101 sterile body fluid specimens were collected and processed at five hospital laboratories, yielding 234 (21%) positive cultures. Of the 176 isolates considered clinically significant, 133 (76%) were recovered by both the BACTEC system and conventional culture, while 28 (16% [P < 0.005]) were recovered by BACTEC only and 11 (6%) were recovered by conventional culture alone. There were no statistically significant differences in the speed of detection of microbial growth. It was found that BACTEC, with or without the addition of fastidious organism supplement, exhibited improved sensitivity for the recovery of microorganisms, including fastidious bacteria.

Survey of Bacteroides fragilis group susceptibility patterns in Canada.

The in vitro activities of penicillin, clindamycin, chloramphenicol, metronidazole, piperacillin, piperacillin-tazobactam, ticarcillin, ticarcillin-clavulanate, ampicillin-sulbactam, cefoxitin, ceftizoxime, cefotetan, moxalactam, and imipenem against 348 Bacteroides fragilis group isolates collected from six Canadian cities during 1990 were determined by the National Committee for Clinical Laboratory Standards (NCCLS) agar dilution technique. All isolates were susceptible to chloramphenicol, metronidazole, piperacillin-tazobactam, and imipenem. For the other antibiotics tested, the following resistance rates were observed: penicillin, 97%; clindamycin, 9%; piperacillin, 19%; ticarcillin, 31%; ticarcillin-clavulanate, 0.28%; ampicillin-sulbactam, 0.85%; cefoxitin, 26%; ceftizoxime, 15%; cefotetan, 53%; and moxalactam, 17%. Susceptibility profiles to beta-lactam antibiotics varied among the different species tested: B. fragilis and Bacteroides vulgatus demonstrated lower resistance rates than Bacteroides distasonis and indole-positive Bacteroides thetaiotaomicron and Bacteroides ovatus. Ceftizoxime results should be interpreted cautiously, because the MICs obtained with the recommended NCCLS control strain were lower than expected.

Epidemiology of infections with Pseudomonas aeruginosa in burn patients: the role of hydrotherapy.

Pseudomonas aeruginosa remains a cause of serious wound infection and mortality in burn patients. By means of restriction fragment length polymorphism analysis and a DNA probe for the pilin gene of Pseudomonas, a lethal strain of nosocomial P. aeruginosa was identified as the cause of an outbreak of wound infections among burn patients. Environmental surveys suggested an association of the outbreak with hydrotherapy provided to many patients in a common facility. In a trial of burn wound care without hydrotherapy, overall mortality was reduced significantly, mortality associated with pseudomonas sepsis was eliminated, and the strain of P. aeruginosa associated with earlier mortality was eradicated. Moreover, fewer nosocomial pseudomonas infections, lower levels of pseudomonas resistance to aminoglycoside antibiotics, significantly fewer pseudomonas infections of skin graft donor sites, and later appearance of Pseodomonas species in burn patients were found during the period when hydrotherapy was not used.