Biomedical subjects

Johann D D Pitout

Publications and source records attributed to Johann D D Pitout.

16 recordsLinked to original sources

PubMed2006

Molecular epidemiology of metallo-beta-lactamase-producing Pseudomonas aeruginosa in the Calgary Health Region: emergence of VIM-2-producing isolates.

A study was designed to describe the molecular epidemiology of carbapenem-resistant (CR) Pseudomonas aeruginosa in a large well-defined geographical region with a centralized laboratory system serving one pediatric and three large adult hospitals (acute care centers I, II, and III). Molecular characterization was done using PCR with sequencing of the integron-associated gene cassettes. Pulsed-field gel electrophoresis (PFGE) using a modified combined Stenotrophomas maltophilia and Streptococcus pneumoniae protocol with SpeI was performed on CR P. aeruginosa strains isolated in the Calgary Health Region during 2002-2006. The majority (96%) of metallo-beta-lactamase (MBL)-producing isolates produced VIM-2 with gene cassettes aacC1 and aacA4, while 4% produced IMP-7 with gene cassettes aacC4 and aacC1. Eighty-six percent of VIM-2 producers belonged to a cluster (MBLV) that was responsible for nosocomial outbreaks during 2003 (intensive care unit) and 2004 (bone marrow transplant unit) at acute care center I. Environmental isolates from these units also belonged to MBLV. The majority of strains from cluster MBLVR (related to MBLV) were present in acute care center III. Isolates producing IMP-7 belonged to a different cluster (MBLI) and were related to strains described during the 1990 s. PFGE of the MBL-negative CR strains showed that 37% belonged to a closely related cluster, NMBL, whose members were predominantly isolated from acute care center II. Our findings suggest that CR and dissemination of MBL clusters among P. aeruginosa populations in large geographic healthcare regions are dynamic processes that require continuous molecular surveillance.

Alberta↗

PubMed2006

Population-based laboratory surveillance of Hafnia alvei isolates in a large Canadian health region.

BACKGROUND: Hospital-based series have characterized Hafnia alvei primarily as an infrequent agent of polymicrobial nosocomial infections in males with underlying illness. METHODS: We conducted population-based laboratory surveillance in the Calgary Health Region during 2000-2005 to define the incidence, demographic risk factors for acquisition, and anti-microbial susceptibilities of Hafnia alvei isolates. RESULTS: A total of 138 patients with Hafnia alvei isolates were identified (2.1/100,000/year) and two-thirds were of community onset. Older age and female gender were important risk factors for acquisition. The most common focus of isolation was urine in 112 (81%), followed by lower respiratory tract in 10 (7%), and soft tissue in 5 (4%), and the majority (94; 68%) were mono-microbial. Most isolates were resistant to ampicillin (111;80%), cephalothin (106; 77%), amoxicillin/clavulanate (98; 71%), and cefazolin (95; 69%) but none to imipenem or ciprofloxacin. CONCLUSION: Hafnia alvei was most commonly isolated as a mono-microbial etiology from the urinary tract in women from the community. This study highlights the importance of population-based studies in accurately defining the epidemiology of an infectious disease.

Age Distribution↗

PubMed2006

In vivo selection of fluoroquinolone-resistant Escherichia coli isolates expressing plasmid-mediated quinolone resistance and expanded-spectrum beta-lactamase.

A ciprofloxacin-resistant Escherichia coli isolate, isolate 1B, was obtained from a urinary specimen of a Canadian patient treated with norfloxacin for infection due to a ciprofloxacin-susceptible isolate, isolate 1A. Both isolates harbored a plasmid-encoded sul1-type integron with qnrA1 and blaVEB-1 genes. Isolate 1B had amino acid substitutions in gyrase and topoisomerase.

Anti-Infective Agents↗

PubMed2005

Population-based epidemiological study of infections caused by carbapenem-resistant Pseudomonas aeruginosa in the Calgary Health Region: importance of metallo-beta-lactamase (MBL)-producing strains.

BACKGROUND: A study was conducted in the Calgary Health Region between May 2002 and April 2004 to define the population-based epidemiological characteristics of infections caused by imipenem-resistant Pseudomonas aeruginosa and to explore the clinical outcomes due to metallo- beta -lactamase (MBL)-producing and non-MBL-producing strains. METHODS: Detailed clinical information was obtained by chart review, and phenotypic and molecular characterizations were performed using the MBL E-test, polymerase chain reaction with sequencing, and pulsed-field gel electrophoresis. RESULTS: A total of 228 patients with infections caused by imipenem-resistant P. aeruginosa were identified (annual incidence, 10.5 cases/100,000 population), with the highest incidence rate in those >or=75 years old. MBL-producing strains (98/228) were associated with higher rates of multidrug resistance and bacteremia. Ninety MBL-producing strains also produced VIM-2, 4 produced IMP-7, and 4 were unclassified. A cluster of VIM-2-producing strains was responsible for a nosocomial outbreak during 2003. The case-fatality rate was significantly higher for infections caused by MBL-producing strains than for those caused by non-MBL-producing strains (25% vs. 13%; relative risk, 1.98 [95% confidence interval, 1.00-3.90]; P=.05). CONCLUSION: MBL-producing P. aeruginosa strains were associated with a higher case-fatality rate and invasive disease. Our study highlights the potential importance of molecular laboratory techniques in infection control and patient care.

Adolescent↗

PubMed2005

Emergence of Enterobacteriaceae producing extended-spectrum beta-lactamases (ESBLs) in the community.

Enterobacteriaceae, especially Klebsiella spp. producing extended-spectrum beta-lactamases (ESBLs) such as SHV and TEM types, have been established since the 1980s as a major cause of hospital-acquired infections. Appropriate infection control practices have largely prevented the dissemination of these bacteria within many hospitals, although outbreaks have been reported. However, during the late 1990s and 2000s, Enterobacteriaceae (mostly Escherichia coli) producing novel ESBLs, the CTX-M enzymes, have been identified predominantly from the community as a cause of urinary tract infections. Resistance to other classes of antibiotics, especially the fluoroquinolones, is often associated with ESBL-producing organisms. Many clinical laboratories are still not aware of the importance of screening for ESBL-producing Enterobacteriaceae originating from the community. A heightened awareness of these organisms by clinicians and enhanced testing by laboratories, including molecular surveillance studies, is required to reduce treatment failures, to limit their introduction into hospitals and to prevent the spread of these emerging pathogens within the community.

Community-Acquired Infections↗

PubMed2005

Virulence factors of Escherichia coli isolates that produce CTX-M-type extended-spectrum beta-lactamases.

This study determined the phylogenetic groups and virulence factors of 37 Escherichia coli isolates producing types of CTX-M compared with those of 19 isolates producing different types of extended-spectrum beta-lactamases (ESBLs) in a well-defined North American population. Most CTX-M-14 producers (97%) were from phylogenic group D; 67% of the CTX-M-15 producers were from group B2. A single CTX-M-14-producing strain belonged to clonal group A. There were significant prevalence differences for individual virulence factors among CTX-M producers and nonproducers; however, aggregate virulence factor scores were similar. CTX-M producers more commonly caused repeat urinary tract infections. Our results indicate that CTX-M type predicts phylogenetic background, and the virulence potential of ESBL-producing E. coli isolates is a complex issue, requiring further study and ongoing surveillance.

Escherichia coli↗

PubMed2005

Community-wide outbreaks of clonally related CTX-M-14 beta-lactamase-producing Escherichia coli strains in the Calgary health region.

Enterobacteriaceae producing extended-spectrum beta-lactamases (ESBLs) typically cause nosocomial infections. Previous surveillance in the Calgary Health Region showed that Escherichia coli strains producing ESBLs were common among community patients. During the period (2000 to 2002): 23 of 157 (15%) of the strains were positive for blaCTX-M genes from the CTX-M-I group (CTX-M-1-like) and 87 of 157 (55%) of the strains were positive for blaCTX-M genes from the CTX-M-III group (CTX-M-14-like). The objective of this study was to investigate the molecular epidemiology of these strains. The beta-lactamases were characterized, and the genetic relatedness of the isolates was analyzed by digesting genomic DNA with the restriction endonuclease XbaI and by performing pulse-field gel electrophoresis (PFGE). PFGE revealed two closely related restriction patterns (clusters CTXM14A and CTXM14AR) among 67 (77%) CTX-M-14 producers. These strains from CTXM14A had nearly identical susceptibility patterns and were isolated most often from urine samples obtained at community sites during 2000 and 2001. Strains from the CTX-M-1-like and CTX-M-negative groups were unrelated to clusters CTXM14, CTXM14AR, and CTXM14NR. We conclude that clonally related strains of E. coli producing CTX-M-14 beta-lactamases were responsible for a predominantly community-wide outbreak. Further studies are warranted to investigate whether community-onset diseases caused by ESBL-producing E. coli are related to a point source or transmission within the community.

Anti-Bacterial Agents↗

PubMed2005

Detection of Pseudomonas aeruginosa producing metallo-beta-lactamases in a large centralized laboratory.

Metallo-beta-lactamases (MBLs) have been increasingly recognized from clinical isolates worldwide, but the laboratory detection of these strains is not well defined. We report a study that developed an EDTA disk screen test and a molecular diagnostic assay for the detection of MBL-producing Pseudomonas aeruginosa. Using NCCLS disk methodology, inhibition zone diameters were determined in tests with imipenem (IPM) and meropenem (MEM) disks alone and in combination with 930 microg of EDTA. This test was compared with the MBL Etest. The duplex PCR assay showed 100% sensitivity and specificity for detecting MBL-producing control strains. Of the 241 clinical strains of IPM-nonsusceptible P. aeruginosa from the Calgary Health Region isolated from 2002 to 2004, 110/241 (46%) were MBL positive using phenotypic methods while 107/241 (45%) were PCR positive for MBL genes: 103/241 (43%) for bla(VIM) and 4/241 (2%) for bla(IMP). The EDTA disk screen test using MEM showed 100% sensitivity and 97% specificity for detecting MBLs in control and clinical strains. The EDTA disk screen test is simple to perform and to interpret and can easily be introduced into the workflow of a clinical laboratory. We recommend that all IPM-nonsusceptible P. aeruginosa isolates be routinely screened for MBL production using the EDTA disk screen test and that PCR confirmation be performed at a regional laboratory.

Alberta↗

PubMed2005

Evaluation of beta-lactamase inhibitors in disk tests for detection of plasmid-mediated AmpC beta-lactamases in well-characterized clinical strains of Klebsiella spp.

The diagnostic utility of the AmpC beta-lactamase inhibitors LN-2-128, 48-1220, and Syn 2190 in combination with cefotetan (CTT) or cefoxitin in a disk test for the detection of clinical isolates of Klebsiella spp. producing plasmid-mediated AmpC beta-lactamases (pAmpCs) was evaluated. The combination of Syn 2190 and CTT had a sensitivity of 91%, a specificity of 100%, and a reproducibility of 100% and showed the best potential of using an inhibitor for detection of Klebsiella spp. producing pAmpCs.

Bacterial Proteins↗

PubMed2004

Population-based laboratory surveillance for Escherichia coli-producing extended-spectrum beta-lactamases: importance of community isolates with blaCTX-M genes.

A prospective, population-based laboratory surveillance study was conducted to define the epidemiology of extended-spectrum beta -lactamase-producing Escherichia coli infections in the Calgary Health Region during the years 2000-2002. The incidence was 5.5 cases per 100,000 population per year. The annualized incidence of 3.9 cases per 100,000 population for January through March was significantly lower than the incidence for the other quarters of the year (6.0 per 100,000 population; P<.01). Seventy-one percent of subjects had community-onset disease. Patients aged > or =65 years (22.0 vs. 3.8 cases per 100,000 population per year; P<.0001) and women (9.2 vs. 1.7 cases per 100,000 population per year; P<.0001) had significantly higher rates of infection. Polymerase chain reaction identified 23 (15%) of 157 isolates as positive for blaCTX-M genes from the CTX-M-I subgroup and 87 (55%) from the CTX-M-III subgroup. Ciprofloxacin resistance was independently associated with CTX-M- beta -lactamases (odds ratio, 14.2; 95% confidence interval, 3.69-54.84). Strains of E. coli with blaCTX-M genes commonly cause community-onset infections, and women and older patients are at highest risk.

Adolescent↗

PubMed2004

Emerging gram-negative enteric infections.

This is an overview describing the microbiology, epidemiology, clinical presentations, laboratory diagnosis, and treatment of emerging gram-negative enteric infections. This review illustrates the shifts in epidemiology of infections caused by new and reemerging bacteria and highlights the role of the clinical microbiology laboratory in detecting these pathogens. It concludes with suggestions for the implementation of molecular techniques to ensure rapid surveillance and enhanced patient care.

Communicable Diseases, Emerging↗

PubMed2004

Community-onset disease caused by Citrobacter freundii producing a novel CTX-M beta-lactamase, CTX-M-30, in Canada.

Strains of Citrobacter freundii intermediate to cefotaxime but sensitive to ceftazidime were isolated from four different patients in Canada. Sequencing of PCR products by use of CTX-M-specific primers revealed a new combination of four amino acid substitutions. This new gene was designated bla(CTX-M-30) and was encoded on a 3-kb plasmid. The pI of CTX-M-30 was 8.0.

Blotting, Southern↗

PubMed2004

Phenotypic and molecular detection of CTX-M-beta-lactamases produced by Escherichia coli and Klebsiella spp.

Organisms producing CTX-M-beta-lactamases are emerging around the world as a source of resistance to oxyiminocephalosporins such as cefotaxime (CTX). However, the laboratory detection of these strains is not well defined. In this study, a molecular detection assay for the identification of CTX-M-beta-lactamase genes was developed and used to investigate the prevalence of these enzymes among clinical isolates of Escherichia coli and Klebsiella species in the Calgary Health Region during 2000 to 2002. In addition, National Committee for Clinical Laboratory Standards (NCCLS) recommendations were evaluated for the ability to detect isolates with CTX-M extended-spectrum beta-lactamases (ESBLs). The PCR assay consisted of four primer sets and demonstrated 100% specificity and sensitivity for detecting different groups of CTX-M-beta-lactamases in control strains producing well-characterized ESBLs. Using these primer sets, 175 clinical strains producing ESBLs were examined for the presence of CTX-M enzymes; 24 (14%) were positive for bla(CTX-M-1-like) genes, 95 (54%) were positive for bla(CTX-M-14-like) genes, and the remaining 56 (32%) were negative for bla(CTX-M) genes. Following the NCCLS recommendations for ESBL testing, all of the control and clinical strains were detected when screened with cefpodoxime and when both cefotaxime and ceftazidime with clavulanate were used as confirmation tests.

Anti-Bacterial Agents↗

PubMed2004

Use of beta-lactamase inhibitors in disk tests to detect plasmid-mediated AmpC beta-lactamases.

Seeking a simple disk test for detection of organisms producing plasmid-mediated AmpC beta-lactamases, we evaluated the diagnostic utility of the beta-lactamase inhibitors 48-1220 (Ro 48-1220) and LN-2-128. Using NCCLS disk methodology, inhibition zone diameters were determined for five beta-lactam antibiotics tested alone and in combination with 20 microg of either 48-1220 or LN-2-128. Using an increase of > or =4 mm in zone diameter in the presence of an inhibitor as a positive test, cefotetan with LN-2-128 and 48-1220 was adequate for the detection of organisms producing plasmid-mediated AmpCs (specificity of 90% and sensitivity of 100%).

Bacterial Proteins↗

PubMed2003

Association between handling of pet treats and infection with Salmonella enterica serotype newport expressing the AmpC beta-lactamase, CMY-2.

Resistance to the extended-spectrum cephalosporins can occur in Salmonella species via the production of extended-spectrum and AmpC beta-lactamases. We describe human infections with Salmonella enterica serotype Newport phage type 14 strains resistant to ceftazidime (CAZ) and cefoxitin (FOX) related to the handling of pet treats containing dried beef. These strains were isolated from five patients in Calgary, Alberta, Canada, during 2002 and were compared to a strain cultured from a commercial pet treat present at the property of one of the patients. The strains were resistant to FOX, CAZ, cefpodoxime, ampicillin, and chloramphenicol; intermediate resistant to ceftriaxone and cefotaxime; and sensitive to the aminoglycosides, ciprofloxacin, cefepime, and imipenem. Isoelectric focusing, multiplex PCR, and sequencing of the amplicons showed that all strains produced the plasmid-encoded AmpC beta-lactamase, CMY-2. Restriction analysis of plasmid DNA following transformation demonstrated that bla(CMY-2) was encoded on an approximately 140-kb plasmid. Pulsed-field gel electrophoresis showed the human and pet treat Salmonella strains to be highly related. This study is the first to implicate the transfer of multidrug-resistant Salmonella species through the handling of commercial pet treats containing animal products. In addition to documenting the first cases of human infection caused by CMY-2-producing S. enterica serotype Newport strains in Canada, this study illustrates the necessity of rapid and accurate laboratory-based surveillance in the identification of novel types of antimicrobial resistance.

Adult↗

PubMed2003

Development and validation of a molecular beacon probe-based real-time polymerase chain reaction assay for rapid detection of methicillin resistance in Staphylococcus aureus.

CONTEXT: A rapid, real-time, duplex, fluorescent molecular beacon probe-based polymerase chain reaction (PCR) assay was recently developed for the detection of methicillin-resistant Staphylococcus aureus. OBJECTIVE: To describe the development and validation of this unique assay. DESIGN: Prospective laboratory analysis. SETTING: Urban health region/centralized diagnostic microbiology laboratory. BACTERIAL STRAINS: One hundred eighty-one previously characterized clinical and American Type Culture Collection isolates, including 50 strains each of methicillin-resistant and methicillin-sensitive S aureus, plus 50 strains of coagulase-negative staphylococci and 31 nonstaphylococcal isolates to ensure assay specificity. INTERVENTION: Assays were performed on purified genomic DNA extracted from growing bacterial colonies. Two sets of oligonucleotide primers were used to specifically amplify the mecA and nuc genes, followed by detection of amplicons using fluorophore-labeled molecular beacon probes. Assays were performed on the Mx4000 Multiplex Quantitative PCR System (Stratagene Inc, La Jolla, Calif). MAIN OUTCOME MEASURES: (1) Assay sensitivity and specificity, and (2) analytical sensitivity. RESULTS: The assay demonstrated 100% sensitivity and 100% specificity, and accurately characterized isolates as methicillin-resistant S aureus, methicillin-sensitive S aureus, or methicillin-resistant coagulase-negative staphylococci, with test results available in 2.5 hours. The analytical sensitivity of the assay was determined to be between 6 and 60 genomic equivalents. CONCLUSIONS: This assay is rapid, accurate, easy to perform, and is compatible with other real-time PCR instruments, making it a suitable alternative to conventional PCR methodologies.

Bacterial Proteins↗