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In Canada, most notifiable disease case definitions use only traditional non-molecular tests, such as culture or direct examination for pathogens and serological tests, as evidence of disease. Because nucleic acid tests are generally superior to traditional tests in terms of sensitivity and turnaround time, these newer assays are highly appealing approaches for diagnosing infectious diseases. However, interpretation of molecular assays is not straightforward and requires caution and a firm understanding of the technology to optimize adoption for public health purposes. Accepting nucleic acid testing as evidence for “probable cases” is a prudent approach, enabling the integration of these methodologies into existing public health notifiable disease case definitions.

Salmonella is one of the most common enteric pathogens related to foodborne illness. Alberta's Provincial Laboratory for Public Health (ProvLab) provides Outbreak and Surveillance support by performing serotyping. The Check&Trace Salmonella™ (CTS) assay (Check-Points, Netherlands), a commercial DNA microarray system, can determine the serotype designation of a Salmonella isolate with automated interpretation. Here we evaluate 1028 Salmonella isolates of human clinical or environmental sources in Alberta, Canada with the CTS assay. CTS was able to assign a serovar to 98.7% of the most frequently occurring human clinical strains in Alberta (82.5% overall), and 71.7% of isolates which were inconclusive by conventional methods. There was 99.7% concordance in environmental isolates. The CTS database has potential to expand to identify rare serovars. With the anticipated shift to molecular methods for identification, CTS provides an easy transition and demonstrates ease-of-use and reduces the turn-around-time of a reported result significantly compared to classical serotyping.

Background Antibiotic administration to individuals with Shiga toxin-producing Escherichia coli (STEC) infection remains controversial. We assessed if antibiotic administration to individuals with STEC infection is associated with development of hemolytic uremic syndrome (HUS). Methods The analysis included studies published up to 29 April 2015, that provided data from patients (1) with STEC infection, (2) who received antibiotics, (3) who developed HUS, and (4) for whom data reported timing of antibiotic administration in relation to HUS. Risk of bias was assessed; strength of evidence was adjudicated. HUS was the primary outcome. Secondary outcomes restricted the analysis to low-risk-of-bias studies employing commonly used HUS criteria. Pooled estimates of the odds ratio (OR) were obtained using random-effects models. Results Seventeen reports and 1896 patients met eligibility; 8 (47%) studies were retrospective, 5 (29%) were prospective cohort, 3 (18%) were case-control, and 1 was a trial. The pooled OR, including all studies, associating antibiotic administration and development of HUS was 1.33 (95% confidence interval [CI], .89-1.99; I(2) = 42%). The repeat analysis including only studies with a low risk of bias and those employing an appropriate definition of HUS yielded an OR of 2.24 (95% CI, 1.45-3.46; I(2) = 0%). Conclusions Overall, use of antibiotics was not associated with an increased risk of developing HUS; however, after excluding studies at high risk of bias and those that did not employ an acceptable definition of HUS, there was a significant association. Consequently, the use of antibiotics in individuals with STEC infections is not recommended.

Human exposure to antimicrobial-resistant bacteria may result in the transfer of resistance to commensal or pathogenic microbes present in the gastrointestinal tract, which may lead to severe health consequences and difficulties in treatment of future bacterial infections. It was hypothesized that the recreational waters from beaches represent a source of antimicrobial-resistant Escherichia coli for people engaging in water activities.To describe the occurrence of antimicrobial-resistant E coli in the recreational waters of beaches in southern Quebec.Sampling occurred over two summers; in 2004, 674 water samples were taken from 201 beaches, and in 2005, 628 water samples were taken from 177 beaches. The minimum inhibitory concentrations of the antimicrobial-resistant E coli isolates against a panel of 16 antimicrobials were determined using microbroth dilution.For 2004 and 2005, respectively, 28% and 38% of beaches sampled had at least one water sample contaminated by E coli resistant to one or more antimicrobials, and more than 10% of the resistant isolates were resistant to at least one antimicrobial of clinical importance for human medicine. The three antimicrobials with the highest frequency of resistance were tetracycline, ampicillin and sulfamethoxazole.The recreational waters of these beaches represent a potential source of antimicrobial-resistant bacteria for people engaging in water activities. Investigations relating the significance of these findings to public health should be pursued.L’exposition humaine à des bactéries résistant aux antimicrobiens peut provoquer le transfert de la résistance à des microbes commensaux ou pathogènes présents dans le tube digestif, ce qui peut avoir de graves conséquences sur la santé et compliquer le traitement de futures infections bactériennes. On a soulevé l’hypothèse que les eaux de baignade des plages représentent une source d’infection à l’Les chercheurs ont procédé à l’échantillonnage sur deux étés. En 2004, ils ont prélevé 674 échantillons d’eau sur 201 plages, et en 2005, 628 échantillons d’eau sur 177 plages. Ils ont établi les concentrations inhibitrices minimales des isolats d’En 2004 et en 2005, respectivement, 28 % et 38 % des plages échantillonnées comptaient au moins un échantillon d’eau contaminée par l’Les eaux de baignade de ces plages représentent une source potentielle de bactéries résistant aux antimicrobiens pour les personnes qui s’adonnent à des activités aquatiques. Il faudrait poursuivre les recherches sur la signification de ces observations en matière de santé publique.

The isolation of Shiga toxin-producing Escherichia coli (STEC) other than serogroup O157 from clinical stool samples is problematic due to the lack of differential phenotypic characteristics from non-pathogenic E. coli. The development of molecular reagents capable of identifying both toxin and serogroup-specific genetic determinants holds promise for a more comprehensive characterization of stool samples and isolation of STEC strains. In this study, 876 stool samples from paediatric patients with gastroenteritis were screened for STEC using a cytotoxicity assay, commercial immunoassay and a conventional PCR targeting Shiga-toxin determinants. In addition, routine culture methods for isolating O157 STEC were also performed. The screening assays identified 45 stools presumptively containing STEC, and using non-differential culture techniques a total of 20 O157 and 22 non-O157 strains were isolated. These included STEC serotypes O157 : H7, O26 : H11, O121 : H19, O26 : NM, O103 : H2, O111 : NM, O115 : H18, O121 : NM, O145 : NM, O177 : NM and O5 : NM. Notably, multiple STEC serotypes were isolated from two clinical stool samples (yielding O157 : H7 and O26 : H11, or O157 : H7 and O103 : H2 isolates). These data were compared to molecular serogroup profiles determined directly from the stool enrichment cultures using a LUX real-time PCR assay targeting the O157 fimbrial gene lpfA, a microsphere suspension array targeting allelic variants of espZ and a gnd-based molecular O-antigen serogrouping method. The genetic profile of individual stool cultures indicated that the espZ microsphere array and lpfA real-time PCR assay could accurately predict the presence and provide preliminary typing for the STEC strains present in clinical samples. The gnd-based molecular serogrouping method provided additional corroborative evidence of serogroup identities. This toolbox of molecular methods provided robust detection capabilities for STEC in clinical stool samples, including co-infection of multiple serogroups.

Coagulase-negative staphylococci (CNS), frequently associated with both community-acquired and nosocomial bloodstream infections, must be distinguished from Staphylococcus aureus for clinical purposes. Conventional methods are too laborious and time-consuming and often lack sensitivity to CNS. Fourier transform infrared (FTIR) spectroscopy combined with the use of a universal growth medium (Que-Bact® Universal Medium No. 2) and chemometrics was evaluated for its potential as a rapid and simple clinical tool for making this distinction. FTIR spectra of 11 methicillin-sensitive and 11 methicillin-resistant CNS isolates as well as 25 methicillin-sensitive, 47 methicillin-resistant, 34 borderline oxacillin-resistant and 35 glycopeptide intermediate S. aureus isolates were obtained from dried films of stationary-phase cells grown on the universal medium. Principal component analysis (PCA), self-organizing maps, and the K-nearest neighbor algorithm were employed to cluster the different phenotypes based on similarity of their FTIR spectra. PCA of the first-derivative normalized spectral data from a single narrow region (2888–2868 cm− 1) yielded complete differentiation of CNS from both methicillin-sensitive and methicillin-resistant S. aureus. The rate of correct classification was somewhat reduced, from 100% to 90%, after inclusion of borderline oxacillin-resistant and glycopeptide intermediate S. aureus strains in the data set. Differentiation based on the data in broader spectral regions was much less reliable. The results of this study indicate that with proper spectral region selection, FTIR spectroscopy and cluster analysis may provide a simple and accurate means of CNS species identification.

BACKGROUND: In 2004, an outbreak of the USA300 strain of methicillin-resistantStaphylococcus aureus(MRSA) was identified in persons with histories of homelessness, illicit drug use or incarceration in the Calgary Health Region (Calgary, Alberta). A prevalence study was conducted to test the hypotheses for factors associated with USA300 colonization or infection.METHODS: Participants were recruited at sites accessed by this marginalized population. Health care staff administered a questionnaire and collected crack pipes and nasal, axillary and skin infection swabs. Pipes and swabs were cultured according to standard techniques. MRSA isolates were further characterized by polymerase chain reaction (mecA, Panton-Valentine leukocidin and Staphylococcal cassette chromosomemec) and typing methods (pulsed-field gel electrophoresis, staphylococcal protein A typing and multilocus sequence typing). Colonization or infection was determined by having any one of nasal, axillary, skin infection or pipe swabs positive for USA300. Colonized participants had one or more nasal, axillary or pipe swab positive for USA300; infected participants had one or more skin infection swab positive for USA300.RESULTS: The prevalence of USA300 colonization or infection among 271 participants was 5.5% (range 3.1% to 9.0%). USA300 cases were more likely to report manipulation of skin infections (OR 9.55; 95% CI 2.74 to 33.26); use of crack pipes was not significant despite identification of the USA300 strain on two of four crack pipes tested. USA300 cases were more likely to report drug use between sex trade workers and clients (OR 5.86; 95% CI 1.63 to 21.00), and with casual sex partners (OR 5.40; 95% CI 1.64 to 17.78).CONCLUSION: Ongoing efforts to promote the appropriate treatment of skin infections in this population are warranted. The association of USA300 colonization or infection and drug use with sexual partners suggest a role for sexual transmission of the USA300 strain of MRSA.

Three commercially available real-time reverse transcriptase PCR assays (the Artus RealArt HPA coronavirus LightCycler, the Artus RealArt HPA coronavirus Rotor-Gene, and the EraGen severe acute respiratory syndrome coronavirus POL assay) and three RNA extraction methodologies were evaluated for the detection of severe acute respiratory syndrome coronavirus RNA from 91 stool specimens. The assays' sensitivities were highest (58% to 75%) for specimens obtained 8 to 21 days after symptom onset. The assays were less sensitive when specimens were obtained less than 8 days or more than 21 days after the onset of symptoms. All assays were 100% specific.

OBJECTIVE: How to eradicate methicillin-resistantStaphylo-coccus aureus(MRSA) colonization in hospitalized patients is uncertain. We reviewed our experience with MRSA decolonization therapy in hospitalized patients.SETTING: An 1100-bed, university-affiliated tertiary care teaching hospital in Toronto, Ontario.DESIGN: Retrospective chart review of 207 adult inpatients with MRSA colonization hospitalized between February 1996 and March 1999.INTERVENTIONS: All patients with MRSA colonization were assessed for possible decolonization therapy with a combination of 4% chlorhexidine soap for bathing and washing, 2% mupirocin ointment applied to the anterior nares three times/day, rifampin (300 mg twice daily) and either trimethoprim/sulfamethoxazole (160 mg/800 mg twice daily) or doxycycline (100 mg twice daily). This treatment was given for seven days.RESULTS: A total of 207 hospitalized patients with MRSA colonization were identified and 103 (50%) received decolonization therapy. Patients who received decolonization therapy were less likely than untreated patientsto have intravenous (P=0.004) or urinary catheters (PCONCLUSIONS: Combined topical and oral antimicrobial therapy was found to be effective in eradicating MRSA colonization in selected hospitalized patients, especially those without indwelling medical devices or extranasal sites of colonization.

Pandemic (H1N1) 2009 virus-positive specimens were collected from autopsy patients and matched to pandemic (H1N1) 2009 virus-positive nasopharyngeal specimens from community control patients and pandemic (H1N1) 2009 virus-positive specimens from intensive-care unit (ICU) patients. Specimens were analysed for polymorphisms at amino acid 222 of the haemagglutinin (HA) glycoprotein. Whereas some specimens from autopsy patients were positive for D222N, none was positive for D222G. All control patient specimens were wild-type D222. D222G polymorphisms were also identified in a subset of ICU patients with admixtures of D222G and D222 and of D222N, D222G and D222 present. The relevance of D222N and D222G to influenza pathogenesis and transmissibility currently remains unclear.

Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most significant pathogens affecting global public health and health care systems. In Canada and the United States, the spread of MRSA is primarily attributed to a single dominant epidemic clone: CMRSA10/USA300. Despite this, the CMRSA7/USA400 epidemic clone has been reported to be the predominate epidemic clone in several Canadian provinces and some parts of the United States. This study examined the epidemiology of CMRSA7/USA400 MRSA in Alberta, Canada, from June 2005 to December 2012. Molecular characterization of CMRSA7/USA400 isolates was done using spa , SCC mec , PVL, and PFGE typing and identified two predominant spa types in Alberta: t128 and t1787. Although closely related, these spa types have distinct geographic distributions. From 2010 to 2012, the number of t128 infections has remained stable while there has been a nearly 3-fold increase in the number of provincial t1787 infections, accompanied by 10-fold increases in t1787 infection rates in some communities. Most t128 and t1787 patients were First Nations or Inuit people, and isolates were usually from skin and soft tissue infections in outpatients. t128 patients were significantly older than t1787 patients. Antimicrobial susceptibility testing showed higher mupirocin resistance in t1787 than in t128 MRSA. Improved strategies to reduce or stabilize t1787 infections in Alberta are needed.

Genotypic-based methods hold promise for the rapid and accurate detection or confirmation of antimicrobial resistance; however, phenotypic methods will continue to have an advantage when resistance to the same antimicrobial agent may be caused by several different mechanisms. The diversity of genetic mechanisms may exceed the capabilities of current molecular technology. Genotypic assays have the ability to detect resistance but not susceptibility. Although resutls can be obtained rapidly, many molecular methods are labor-intensive, expensive, and lack standardization. Clinical studies will be required to validate the genotypic approach to detection of antimicrobial resistance. Molecular assays are also at risk for false-positive results because of contamination of specimens by other specimens that carry the DNA targeted for the assay, or carryover of amplified target DNA (amplicons) from a previous PCR assay during sample preparation. Detection of certain genetic resistance loci in clinical specimens must be interpreted with caution, because organisms in normal flora may also harbor the same loci. All these factors must be taken into consideration when introducing a genotypic method in the clinical laboratory. Other considerations include cost, turnaround time, and assay performance. It must be emphasized that the bedside assessment of the patient should always be considered in addition to the results of antimicrobial susceptibility tests (whether phenotypic or genotypic) so that the best outcome is assured for the patient.

We evaluated the BacT/Alert Microbial Detection System (Organon Teknika Corporation, Durham, NC, USA) by using FAN bottles compared to conventional culture methods for the recovery of microorganisms from normally sterile body fluids other than blood and dialysates. Clinically significant pathogens were isolated from 116 (11%) of 1, 099 consecutive specimens (80 from both conventional media and FAN bottles; 23 from FAN bottles only; 13 from conventional media only). Gram-positive cocci were more likely to be recovered from FAN bottles than from conventional media (p = 0.04). Contaminants were also more likely to have grown in FAN bottles (3%) than on conventional media (1%) (p = 0.04). The mean time to detection of significant pathogens was 20.9 h using FAN bottles as compared to 30. 9 h using conventional media (p = 0.0001). These results indicate that the BacT/Alert Microbial Detection System using FAN blood culture bottles improves the yield of clinically significant Gram-positive isolates from normally sterile body fluids with a reduced time to detection.

OBJECTIVE: To evaluate parenteral antibiotic utilization and bacterial resistance patterns in a critical care unit (CrCU).DESIGN: Descriptive, prospective audit of infection site, culture and antimicrobial susceptibility test results, parenteral antibiotic usage and duration, total antibiotic acquisition costs, and length of stay.SETTING: A 17-bed medical-surgical CrCU in a tertiary care teaching hospital in Metropolitan Toronto.PATIENTS: Two hundred and fifty-eight patients admitted to the CrCU between May 1995 and April 1996 who received antimicrobial therapy.RESULTS: The most frequently prescribed antibiotics were cefazolin (47%, 1098 g), gentamicin (33%,141 g) and ceftriaxone (20%, 255 g). The most common indications for antimicrobial therapy included surgical prophylaxis (34%) and pneumonia (35%). The following organisms were isolated from patients treated with antibiotics:Staphylococcus aureus(26%),Pseudomonas aeruginosa(13%), enterococci (12%),Haemophilus influenzae(11%),Escherichia coli(11%),Enterobacter cloacae(8%) and other Gram-negative bacilli (19%). Only 9% of Gram-negative bacilli were resistant to aminoglycosides, 3% were resistant to ciprofloxacin and no extended-spectrum beta-lactamases or imipenem-resistance were detected. No vancomycin-resistant enterococci and only two methicillin-resistantStaphylococcus aureusisolates were identified.CONCLUSIONS: Antibiotic use during the audit appeared appropriate for the specific clinical indications. Low levels of bacterial resistance were detected during the audit.

Objectives Over the last decade, a marked increase in Salmonella enterica serotype 4,[5],12:i:- with a core resistance to ampicillin, streptomycin, sulphonamides and tetracycline (ASSuT) has been observed in Europe. This study describes the emergence and characterization of isolates of multidrug-resistant Salmonella 4,[5],12:i:- in Canada. Methods Human clinical isolates of Salmonella 4,[5],12:i:- were identified by provincial laboratories from 2003 to 2010. Serotyping and phage typing were performed by standardized methodologies. MIC values were determined using broth microdilution. PCR was used to determine the presence of resistance genes. Multilocus sequence typing was performed on a selected number of isolates. Results A total of 26 251 Salmonella were submitted as part of the Canadian Integrated Program on Antibiotic Resistance Surveillance (CIPARS). Of these, Salmonella 4,[5],12:i:- accounted for a total of 766 isolates (2.9%), and the number increased significantly from 42 (1.4%) in 2003 to 164 (4.8%) in 2010. The ASSuT+ phenotype was observed in 11.9% (n = 91) of Salmonella 4,[5],12:i:- isolates and increased from two isolates in 2003 to 35 isolates in 2010. Two sequence types (STs) were observed. ST34 was mainly associated with the ASSuT isolates (n = 24; 38%), which contained blaTEM, strA-strB, tet(B) and sul2. ST19 was more likely to be associated with the ACSSuT phenotype and contained blaTEM, floR, strA-strB, sul2 and tet(A) or blaPSE-1, floR, aadA2, sul1 and tet(G). Conclusions The prevalence of Salmonella 4,[5],12:i:- has significantly increased from 2003 to 2010 and it is now the fifth most common serotype reported in Canada causing human disease. Similar antimicrobial resistance patterns, phage types and STs have been observed in Europe.

Objective: To determine the prevalence of resistance ofStreptococcus pneumoniaeto penicillin and other antimicrobial agents in metropolitan Toronto.Design: Consecutive pneumococcal isolates from different patients were obtained from two private community-based laboratories and from patients assessed in the emergency department of a tertiary-care teaching hospital in Toronto, Ontario between June and December 1993, and between March and October 1994. In vitro susceptibility testing was done by broth microdilution in accordance with National Committee for Clinical Laboratory Standards guidelines.Results: Twenty (7.3±3.1%) of 274 pneumococcal isolates were resistant to penicillin; six (30%) isolates had high-level resistance (minimal inhibitory concentration [mic] 2.0 μg/mL or greater); and 14 isolates had intermediate resistance (mic0.1 to 1.0 μg/mL). Penicillin-resistant strains were also frequently resistant to tetracycline (55%), cotrimoxazole (50%), erythromycin (40%) and cefuroxime (35%). Resistant strains comprised several serotypes: 19F (six isolates), 9V (three), 23F (three), and one each of 6A, 6B, 14, and 19A; four isolates were nontypeable.Conclusions: There has been a recent emergence of penicillin-resistantS pneumoniaein southern Ontario. National and regional surveillance is warranted to determine the extent of the problem elsewhere in Canada.

A total of 244 CMY-2 plasmids from 5 separate studies involving Escherichia coli and Salmonella human clinical cases as well as E. coli from feedlots and water sources were examined. Genetically similar CMY-2 plasmids isolated from either E. coli or Salmonella from human, animal, and environmental sources are widely distributed across Canada and cluster into replicon types I1, A/C, and K/B and an unidentified group.

Five methods for the rapid identification and differentiation of Branhamella catarrhalis from other Neisseria species in 86 respiratory specimens were compared. These tests included the 4-methylumbelliferyl butyrate (MUB), API quadFerm, B.CAT.Confirm, Gonochek II, and the Tributyrin disc. All five tests reliably and accurately identified 31 B. catarrhalis isolates. However, the MUB test was the least expensive, least labor intensive, and did not require overnight purity plates for performance. The MUB test provided same-day identification of B. catarrhalis isolates from the initial primary isolation culture.

Borderline oxacillin-resistant Staphylococcus aureus (BORSA) exhibit oxacillin MIC values of 1–8 μg ml−1, but lack mecA, which encodes the low-affinity penicillin-binding protein (PBP)2a. The relationship of the BORSA phenotype with specific genetic backgrounds was assessed, as well as amino acid sequence variation in the normal PBP2. Among 38 BORSA, 26 had a common PFGE profile of genomic DNA, and were multilocus sequence type (ST)25. The other isolates were genetically diverse. Complete pbp2 sequences were determined for three BORSA, corresponding to ST25, ST1 and ST47, which were selected on the basis of lacking blaZ-encoded β-lactamase. The essential transpeptidase-domain-encoding segment of pbp2 was also sequenced from seven additional ST25 isolates. Amino acid substitutions occurred in the transpeptidase domain of all BORSA, irrespective of clonal type. A Gln629→Pro substitution was common to all ST25 BORSA, but most could be distinguished from one another by additional unique substitutions in the transpeptidase domain. The ST1 and ST47 isolates also possessed unique substitutions in the transpeptidase domain. Plasmid-mediated expression of pbp2 from an ST25 or ST1 isolate in S. aureus RN6390 increased its oxacillin MIC from 0.25 to 4 μg ml−1, while pbp2 from a susceptible strain, ATCC 25923, had no effect. Therefore, different amino acid substitutions in PBP2 of diverse BORSA lineages contribute to borderline resistance. The predominant ST25 lineage was not related to any of the five clonal complexes that contain meticillin-resistant S. aureus (MRSA), suggesting that ST25 cannot readily acquire mecA-mediated resistance.

OBJECTIVE:To describe rates of antimicrobial resistance (AMR) amongSalmonellaandShigellaisolates reported in five Canadian provinces, focusing on clinically important antimicrobials.METHODS:The authors retrospectively investigated AMR rates among 6219Salmonellaand 1673Shigellaisolates submitted to provincial public health laboratories in Alberta, Newfoundland and Labrador, Ontario, Prince Edward Island and Saskatchewan from 1997 to 2000; these isolates were estimated to represent 41% ofSalmonellacases and 72% ofShigellacases reported by the study provinces.RESULTS:AmongSalmonellaisolates, 27% (1704 of 6215) were resistant to ampicillin, 2.2% (135 of 6122) to trimethoprim/sulfamethoxazole, 1.5% (14 of 938) to nalidixic acid, 1.2% (one of 84) to lomafloxacin and 0.08% (five of 6163) to ciprofloxacin. AmongShigellaisolates, 70% (1144 of 1643) were resistant to trimethoprim/sulfamethoxazole, 65% (1079 of 1672) to ampicillin, 3.1% (eight of 262) to nalidixic acid, 0.49% (eight of 1636) to ciprofloxacin, 0.14% (one of 700) to ceftriaxone and 0.08% (one of 1292) to ceftazidime.CONCLUSIONS:Higher rates of resistance to clinically important antimicrobials (including ciprofloxacin) were observed among bothSalmonellaandShigellaisolates than has previously been reported. Current Canadian data on rates of AMR for these pathogens are required.

Resistance to the extended-spectrum cephalosporins can occur in Salmonella species via the production of extended-spectrum and AmpC β-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 β-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.

We compared the results of Gram staining and culture of cerebrospinal fluid to results obtained with a rapid PCR assay for the diagnosis of meningococcal meningitis in 281 cases of suspected bacterial meningitis. PCR had a sensitivity of 97% compared to a sensitivity of 55% for culture, and the PCR specificity was 99.6%. PCR results were available within 2 h of the start of the assay.

Rapid detection and accurate identification of methicillin-resistant staphylococci are critical for the effective management of infections caused by these organisms. We describe a multiplex PCR-based assay for the direct detection of methicillin-resistant staphylococci from blood culture bottles (BacT/Alert; Organon-Teknika, Durham, N.C.). A simple lysis method followed by a multiplex PCR assay designed to detect the nuc , mecA , and bacterial 16S rRNA genes was performed. A total of 306 blood culture specimens were collected over a period of 10 months from June 1998 to April 1999, consisting of 236 blood cultures growing staphylococci (including 124 methicillin-resistant Staphylococcus spp.), 50 positive blood cultures which grew organisms other than staphylococci, and 20 blood cultures that were negative for bacterial and fungal pathogens after 5 days of incubation and terminal subculture. DNA extraction, PCR, and detection could be completed in 2.5 h. Of the positive blood cultures with staphylococci, the multiplex PCR assay had a sensitivity and specificity of 99.2% and 100%, respectively. Our results show that rapid, direct detection of methicillin-resistant staphylococci is possible, allowing clinicians to make prompt and effective decisions for the management of patients with staphylococcal bacteremia.

Objectives:To describe the investigation and management of an outbreak due to multiresistantAcinetobacter baumanniiand to determine risk factors for acquisition of the organism.Setting:A 14-bed regional burn unit in a Canadian tertiary-care teaching hospital.Design:Case-control study with multivariate analysis of potential risk factors using logistic regression analysis. Surveillance cultures were obtained from the hospital environment, from noninfected patients, and from healthcare providers.Results:A total of 31 (13%) of 247 patients with acute burn injuries acquired multiresistantA. baumanniibetween December 1998 and March 2000; 18 (58%) of the patients were infected. The organism was recovered from the hospital environment and the hands of healthcare providers. Significant risk factors for acquisition of multiresistantA. baumanniiwere receipt of blood products (odds ratio [OR], 10.8; 95% confidence interval [CI95], 3.4 to 34.4;P< .001); procedures performed in the hydrotherapy room (OR, 4.1; CI95, 1.3 to 13.1;P= .02); and increased duration of mechanical ventilation (OR, 1.1 per day; CI95, 1.0 to 1.1;P=. 02).Interventions:Improved compliance with hand hygiene, strict patient isolation, meticulous environmental cleaning, and temporary closure of the unit to new admissions.Conclusions:Acquisition of multiresistantA. baumanniiwas likely multifactorial, related to environmental contamination and contact with transiently colonized healthcare providers. Control measures addressing these potential sources of multiresistantA. baumanniiwere successful in terminating the outbreak. Ongoing surveillance and continued attention to hand hygiene and adequate environmental cleaning are essential to prevent recurrent outbreaks due to antibiotic-resistant bacteria in burn units.

The MRSA-Screen (Denka-Seiken, Tokyo, Japan) latex agglutination test was evaluated for its ability to detect PBP 2a from 200 clinical isolates of coagulase-negative staphylococci (CoNS; 84 mecA -positive strains and 116 mecA -negative strains) consisting of 108 Staphylococcus epidermidis , 37 S. saprophyticus , 15 S. haemolyticus , 11 S. hominis , 10 S. capitis , 10 S. warneri , and 3 S. lugdunensis species as well as 6 other species of CoNS. The assay was compared with susceptibility testing with an agar screen plate with oxacillin at 6 μg/ml (OXA6), by oxacillin disk diffusion (DD), by broth microdilution (BMDIL), by the E test, and with Vitek GPS-SV and Vitek GPS-107 susceptibility cards. PCR for the detection of the mecA gene was used as the “gold standard.” The sensitivities and specificities for the methods evaluated were as follows: MRSA-Screen, 100 and 100%, respectively; OXA6, 100 and 99%, respectively; DD, 98 and 62%, respectively; BMDIL, 100 and 60%, respectively; E test, 100 and 51%, respectively; Vitek GPS-SV susceptibility card, 98 and 87%, respectively; and Vitek GPS-107 susceptibility card, 100 and 61%, respectively. The MRSA-Screen test accurately and rapidly detected oxacillin resistance in CoNS.

The incidence of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) continues to increase in many countries worldwide. Rapid identification of MRSA from clinical specimens and screening of high-risk patients for MRSA colonization have been found to be cost-effective measures for

Extended-spectrum β-lactamase (ESBL) activity in Escherichia coli, Klebsiella pneumoniae, or Klebsiella oxytoca can be detected by using a standard disk diffusion susceptibility test method with cefotaxime (30-μg), ceftriaxone (30-μg), ceftazidime (30-μg), cefpodoxime (10-μg), or aztreonam (30-μg) disks (2, 3). The presence of an ESBL can then be confirmed by a phenotypic confirmatory disk diffusion test recommended by the National Committee for Clinical Laboratory Standards (3) or by the double disk synergy test described by Jarlier et al. (1). The phenotypic confirmatory disk diffusion test is relatively simple to perform and easy to interpret. It requires testing of both cefotaxime (30-μg) and ceftazidime (30-μg) disks each alone and in combination with clavulanic acid (10 μg). An increase in the zone diameter of ≥5 mm for either antimicrobial agent tested in combination with clavulanic acid over that when tested alone indicates that the isolate is an ESBL producer. However, as clavulanic acid is labile, it is recommended that the disks be used immediately after preparation and that they not be stored (3). This may be an inconvenience for many clinical laboratories. Therefore, we evaluated the stability of disks containing clavulanic acid stored at −20°C for 14 consecutive days. Clavulanic acid powder was obtained from Smith Kline Beecham Pharma (Oakville, Ontario, Canada). A stock solution of clavulanic acid (1,000 μg/ml) was freshly prepared. A 10-μl aliquot was added to each of the cefotaxime (30-μg) and ceftazidime (30-μg) disks, which were then allowed to air dry at room temperature for 30 min. The cefotaxime-clavulanic acid and ceftazidime-clavulanic acid disks were placed in separate screw-capped glass vials containing dessicators (Dricap 1.0 silica gel; Multi-Sorb Technology Inc., Buffalo, N.Y.) and placed in a −20°C freezer. Each day for 14 consecutive days, the containers were removed from the freezer and brought to room temperature. The disks were removed, and the containers were closed and immediately returned to the freezer. The disks were used for phenotypic confirmatory disk diffusion testing (3) to detect ESBLs using E. coli ATCC 25922 (ESBL-negative) and a well-characterized ESBL-producing strain of E. coli (LPTP 9711). Linear regression analysis was used to determine if there was a significant change in the zone diameters over time. There was no significant change in the zone diameters obtained with any of the four disks tested with the two organisms over the 14 days of evaluation (Table ​(Table1).1). For each of the disks tested, the maximum variability in the measured zone diameters was 3 mm. There were no statistically significant changes in the zone diameters with the β-lactam disks alone in comparison to those containing clavulanic acid over the 14 days. These results suggest that disks with clavulanic acid prepared for ESBL phenotypic confirmatory disk diffusion testing (3) are sufficiently stable for use up to 14 days after preparation, provided that they are stored at −20°C. TABLE 1 Phenotypic confirmatory disk diffusion test results using cefotaxime and ceftazidime disks, with and without clavulanic acid, that were stored frozen for up to 14 daysa

The probe-based Velogene Rapid MRSA Identification Assay (ID Biomedical Corp., Vancouver, British Columbia, Canada) and the latex agglutination MRSA-Screen (Denka Seiken Co., Tokyo, Japan) were evaluated for their ability to identify methicillin-resistant Staphylococcus aureus (MRSA) and to distinguish strains of MRSA from borderline oxacillin-resistant S. aureus (BORSA; mecA -negative, oxacillin MICs of 2 to 8 μg/ml). The Velogene is a 90-min assay using a chimeric probe to detect the mecA gene. MRSA-Screen is a 15-min latex agglutination test with penicillin-binding protein 2a antibody-sensitized latex particles. We compared these assays with the BBL Crystal MRSA ID System (Becton Dickinson, Cockeysville, Md.) and with PCR for mecA gene detection. A total of 397 clinical isolates of S. aureus were tested, consisting of 164 methicillin-susceptible strains, 197 MRSA strains, and 37 BORSA strains. All assays performed well for the identification of MRSA with sensitivities and specificities for Velogene, MRSA-Screen, and BBL Crystal MRSA ID of 98.5 and 100%, 98.5 and 100%, and 98.5 and 98%, respectively. Three MRSA strains were not correctly identified by each of the Velogene and MRSA-Screen assays, but repeat testing with a larger inoculum resolved the discrepancies. The BBL Crystal MRSA ID test misclassified four BORSA strains as MRSA. Both the Velogene and the MRSA-Screen assays are easy to perform, can accurately differentiate BORSA isolates from MRSA isolates, and provide a rapid alternative for the detection of methicillin resistance in S. aureus in clinical laboratories, especially when mecA PCR gene detection is unavailable.

Objectives:To determine the cost-effectiveness of a policy of screening high-risk patients for methicillin-resistantStaphylococcus aureus(MRSA) colonization on admission to hospital.Setting:980-bed university-affiliated tertiary-care hospital.Patients:Between June 1996 and May 1997, patients directly transferred from another hospital or nursing home, or who had been hospitalized in the previous 3 months, were screened for MRSA within 72 hours of hospital admission.Design:Nasal, perineal, and wound swabs were obtained for MRSA screening using standard laboratory methods. Laboratory and nursing costs associated with screening patients for MRSA on admission to hospital were calculated. The costs associated with the implementation of recommended infection control measures for patients with MRSA also were determined.Results:3,673 specimens were obtained from 1,743 patients. MRSA was found on admission in 23 patients (1.3%), representing 36% of the 64 patients with MRSA identified in the hospital during the year. MRSA-colonized patients were more likely to have been transferred from a nursing home (odds ratio [OR], 6.4;P=.04) or to have had a previous history of MRSA colonization (OR, 13.1;P=.05). Laboratory and nursing costs were found to be $8.34 per specimen, for a total cost of $30,632 during the year. The average cost of implementing recommended infection control measures for patients colonized with MRSA was approximately $5,235 per patient.Conclusion:If early identification of MRSA in colonized patients prevents nosocomial transmission of the organism to as few as six new patients, the screening program would save money.

This study evaluated a PCR assay for detection of the streptococcal pyrogenic exotoxin B ( speB ) gene from tissue biopsy specimens of patients with necrotizing fasciitis. speB was detected in specimens from all 10 patients with necrotizing fasciitis due to group A streptococcus. The assay was negative for all 11 patients without culture or serologic evidence of streptococcal infection. These results suggest that the detection of speB by PCR may be useful for confirming group A streptococcal infection when cultures are negative or not available.

The in-vitro susceptibilities of 1658 blood culture isolates to ciprofloxacin and 13 other antimicrobial agents were determined, and compared with the results for isolates obtained before and after the availability of ciprofloxacin in 1989. Only six (0.6%) of 995 Enterobacteriaceae were resistant to the fluoroquinolones tested; all of the ciprofloxacin-resistant strains were isolated after 1989 (P = 0.04). No significant increase in ciprofloxacin resistance was found in Pseudomonas aeruginosa or Acinetobacter spp. No resistance to ciprofloxacin was found in 124 Staphylococcus aureus isolates prior to 1989, but five (2.4%) of 208 S. aureus strains recovered after 1989 were ciprofloxacin-resistant (P = 0.16). Rates of resistance to ciprofloxacin and other antimicrobial agents commonly used to treat bacteraemic infections have remained relatively low in this Canadian teaching hospital over the past 16 years.

The Helisal test is a quantitative enzyme immunoassay for the measurement of Helicobacter pylori-specific immunoglobulin G antibodies in saliva. This test was evaluated in comparison with culture and histopathologic examination of gastric biopsy specimens obtained from 195 patients who underwent 200 endoscopic procedures for the investigation of gastrointestinal symptoms. Forty-one (21%) patients were found to have peptic ulcer disease, and one other patient had a gastric carcinoma. H. pylori was detected in gastric biopsy specimens obtained from 98 (49%) of the procedures. The sensitivity, specificity, and positive and negative predictive values of the Helisal test were 81, 75, 76, and 80%, respectively. The test was negative for 16 (38%) of the 42 patients with peptic ulcer disease or a gastric malignancy diagnosed at endoscopy. These results suggest that the Helisal assay is only moderately accurate for the detection of H. pylori infection in symptomatic patients.

Background Feedlot cattle in North America are routinely fed subtherapeutic levels of antimicrobials to prevent disease and improve the efficiency of growth. This practice has been shown to promote antimicrobial resistance (AMR) in subpopulations of intestinal microflora including Escherichia coli. To date, studies of AMR in feedlot production settings have rarely employed selective isolation, therefore yielding too few AMR isolates to enable characterization of the emergence and nature of AMR in E. coli as an indicator bacterium. E. coli isolates (n = 531) were recovered from 140 cattle that were housed (10 animals/pen) in 14 pens and received no dietary antimicrobials (control - 5 pens, CON), or were intermittently administered subtherapeutic levels of chlortetracycline (5 pens-T), chlortetracycline + sulfamethazine (4 pens-TS), or virginiamycin (5 pens-V) for two separate periods over a 9-month feeding period. Phenotype and genotype of the isolates were determined by susceptibility testing and pulsed field gel electrophoresis and distribution of characterized isolates among housed cattle reported. It was hypothesized that the feeding of subtherapeutic antibiotics would increase the isolation of distinct genotypes of AMR E. coli from cattle. Results Overall, patterns of antimicrobial resistance expressed by E. coli isolates did not change among diet groups (CON vs. antibiotic treatments), however; isolates obtained on selective plates (i.e., MA,MT), exhibited multi-resistance to sulfamethoxazole and chloramphenicol more frequently when obtained from TS-fed steers than from other treatments. Antibiograms and PFGE patterns suggested that AMR E. coli were readily transferred among steers within pens. Most MT isolates possessed the tet(B) efflux gene (58.2, 53.5, 40.8, and 50.6% of isolates from CON, T, TS, and V steers, respectively) whereas among the MA (ampicillin-resistant) isolates, the tem1-like determinant was predominant (occurring in 50, 66.7, 80.3, and 100% of isolates from CON, T, TS, and V steers, respectively). Conclusions Factors other than, or in addition to subtherapeutic administration of antibiotics influence the establishment and transmission of AMR E. coli among feedlot cattle.

We compared the MicroScan Pos MIC Type 6 panel and AMS-Vitek Gram Positive Susceptibility Card (GPS-TA) to agar dilution screen plates for the detection of high-level aminoglycoside resistance in 182 enterococcal isolates. The specificity of the two commercial systems was 100%, with the exception of one susceptible isolate found to be streptomycin resistant by the Vitek system. The MicroScan and Vitek systems had comparable sensitivities for the detection of gentamicin resistance (90 and 95% respectively) and streptomycin resistance (85 and 78%, respectively). These results suggest that screening tests such as agar dilution screen plates, broth dilution, or disk diffusion should continue to be used to detect high-level gentamicin and streptomycin resistance.