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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.

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.

The objective of this study was to investigate tetracycline and ampicillin resistance in Escherichia coli isolated from the feces of 50 crossbred steers housed in 5 feedlot pens. The steers were not administered antibiotics over a 246-day feeding period. A total of 216 isolates were selected for further characterization. The E. coli isolates were selected on MacConkey agar or on MacConkey agar amended with ampicillin (50 µg/mL) or tetracycline (4 µg/mL). Pulsed-field gel electrophoresis (PFGE) typing (XbaI digestion), screening against 11 antibiotics, and multiplex PCR for 14 tet and 3 β-lactamase genes were conducted. Prevalence of antimicrobial resistance in E. coli at each sampling day was related both temporally and by pen. Multiplex PCR revealed that tet(B) was most prevalent among tetracycline-resistant isolates, whereas β-lactamase tem1-like was detected mainly in ampicillin-resistant isolates. Our results suggest that antimicrobial resistance in E. coli populations persists over the duration of the feeding period, even in the absence of in-feed antibiotics. Many of the isolates with the same antibiograms had indistinguishable PFGE patterns. Characterization of the factors that influence the nature of this nonselective resistance could provide important information for consideration in the regulation of in-feed antimicrobials for feedlot cattle.

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.

A multi-province outbreak of listeriosis occurred in Canada from June to November 2008. Fifty-seven persons were infected with 1 of 3 similar outbreak strains defined by pulsed-field gel electrophoresis, and 24 (42%) individuals died. Forty-one (72%) of 57 individuals were residents of long-term care facilities or hospital inpatients during their exposure period. Descriptive epidemiology, product traceback, and detection of the outbreak strains of Listeria monocytogenes in food samples and the plant environment confirmed delicatessen meat manufactured by one establishment and purchased primarily by institutions was the source of the outbreak. The food safety investigation identified a plant environment conducive to the introduction and proliferation of L. monocytogenes and persistently contaminated with Listeria spp. This outbreak demonstrated the need for improved listeriosis surveillance, strict control of L. monocytogenes in establishments producing ready-to-eat foods, and advice to vulnerable populations and institutions serving these populations regarding which high-risk foods to avoid.

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.

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.

Potential relationships between virulence factor expression and transmissibility were assessed in epidemic methicillin-resistant Staphylococcus aureus (MRSA) clones CMRSA-1 and CMRSA-3. A major subtype of CMRSA-1 exhibited normal transcription of RNAIII, which facilitates the induction of secreted virulence factors and repression of colonization factor expression at high cell density. However, these isolates characteristically did not express alphatoxin or protease and displayed a limited profile of secreted proteins. CMRSA-1 also expressed a novel cell surface glycoprotein and exhibited a unique polymorphism within the accessory gene regulator (agr) locus. CMRSA-3 displayed attenuated activation of RNAIII transcription, which was consistent with its higher fibronectin-binding and coagulase activity relative to sporadic MRSA or CMRSA-1 ( ), low protease activity, and limited profile of secreted P= .05 proteins. Thus, the balance of virulence factor expression in CMRSA-1 and CMRSA-3 favors the colonization phase of infection, and CMRSA-1 possesses unique genotypic and phenotypic traits.

Pulsed-field gel electrophoresis (PFGE) is a valuable molecular typing assay used for methicillin-resistant Staphylococcus aureus (MRSA) surveillance and genotyping. However, there are several limitations associated with PFGE. In Alberta, Canada, the significant increase in the number of MRSA isolates submitted to the Provincial Laboratory for Public Health (ProvLab) for PFGE typing led to the need for an alternative genotyping method. In this study, we describe the transition from PFGE to Staphylococcus protein A (spa), Staphylococcal cassette chromosome (SCCmec), and Panton-Valentine leukocidin (PVL) typing. A total of 1915 clinical MRSA isolates collected from 2005 to 2009 were used to develop and validate an algorithm for assigning PFGE epidemic types using spa, SCCmec, and PVL typing and the resulting data was used to populate a new Alberta MRSA typing database. An additional 12620 clinical MRSA isolates collected from 2010 to 2012 as part of ongoing routine molecular testing at ProvLab were characterized using the new typing algorithm and the Alberta MRSA typing database. Switching to spa, SCCmec, and PVL from PFGE typing substantially reduced hands-on and turn-around times while maintaining historical PFGE epidemic type designations. This led to an approximate $77,000 reduction in costs from 2010 to 2012. PFGE typing is still required for a small subset of MRSA isolates that have spa types that are rare, novel, or associated with more than one PFGE epidemic type.

A model biofilm, formed of multiple species from environmental drinking water, including opportunistic pathogens, was created to explore the tolerance of multi-species biofilms to chlorine levels typical of water-distribution systems. All species, when grown planktonically, were killed by concentrations of chlorine within the World Health Organization guidelines (0.2–5.0 mg l−1). Higher concentrations (1.6–40-fold) of chlorine were required to eradicate biofilm populations of these strains, ∼70% of biofilms tested were not eradicated by 5.0 mg l−1 chlorine. Pathogenic bacteria within the model multi-species biofilms had an even more substantial increase in chlorine tolerance; on average ∼700–1100 mg l−1 chlorine was required to eliminate pathogens from the biofilm, 50–300-fold higher than for biofilms comprising single species. Confocal laser scanning microscopy of biofilms showed distinct 3D structures and multiple cell morphologies and arrangements. Overall, this study showed a substantial increase in the chlorine tolerance of individual species with co-colonization in a multi-species biofilm that was far beyond that expected as a result of biofilm growth on its own.

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.

Consumption of foods containing Staphylococcus aureus can cause severe gastro-intestinal illness. Given the fact that over the past decade, Canada has seen increasing rates of methicillin-resistant S. aureus (MRSA) carriage and infection, the objective of this study was to investigate the impact of methicillin-susceptible S. aureus (MSSA) and MRSA on foodborne illness in Alberta, Canada. Between January 2007 and December 2010, there were 693 food samples associated with foodborne investigations submitted to the Alberta Provincial Laboratory for Public Health (ProvLab). These foods were screened for: Bacillus cereus, Clostridium perfringens, S. aureus, Aeromonas spp., Campylobacter spp., Escherichia coli O157:H7, Salmonella, Shigella spp., and Yersinia spp. S. aureus was identified in 10.5% (73/693) of samples, and of these, 59% (43/73) were co-contaminated with at least one other organism on the screening panel. The S. aureus positive samples included 29 meat, 20 prepared foods containing meat, 11 prepared foods not containing meat, 10 dairy, and three produce. Methicillin-resistance was not detected in any isolates tested. These findings indicate that the presence of S. aureus in food associated with foodborne investigations is a cause for concern, and although MRSA was not found, the potential for outbreaks exists, and ongoing surveillance should be sustained.

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.

A total of 142 cefoxitin-resistant Escherichia coli isolates from water sources were collected across Canada. Multidrug resistance was observed in 65/142 (45.8%) isolates. The bla CMY-2 gene was identified in 110/142 (77.5%) isolates. Sequencing of the chromosomal ampC promoter region showed mutations from the wild type, previously shown to hyperproduce AmpC. CMY-2-producing plasmids predominantly belonged to replicon groups I1-Iγ, A/C, and K/B. The majority of the E. coli isolates belonged to the nonvirulent phylogenetic groups A and B1.

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.

It is not understood how immune inflammation influences the pathogenesis of severe acute respiratory syndrome (SARS). One area of strong controversy is the role of interferon (IFN) responses in the natural history of SARS. The fact that the majority of SARS patients recover after relatively moderate illness suggests that the prevailing notion of deficient type I IFN-mediated immunity, with hypercytokinemia driving a poor clinical course, is oversimplified. We used proteomic and genomic technology to systematically analyze host innate and adaptive immune responses of 40 clinically well-described patients with SARS during discrete phases of illness from the onset of symptoms to discharge or a fatal outcome. A novel signature of high IFN-α, IFN-γ, and IFN-stimulated chemokine levels, plus robust antiviral IFN-stimulated gene (ISG) expression, accompanied early SARS sequelae. As acute illness progressed, SARS patients entered a crisis phase linked to oxygen saturation profiles. The majority of SARS patients resolved IFN responses at crisis and expressed adaptive immune genes. In contrast, patients with poor outcomes showed deviated ISG and immunoglobulin gene expression levels, persistent chemokine levels, and deficient anti-SARS spike antibody production. We contend that unregulated IFN responses during acute-phase SARS may culminate in a malfunction of the switch from innate immunity to adaptive immunity. The potential for the use of the gene signatures we describe in this study to better assess the immunopathology and clinical management of severe viral infections, such as SARS and avian influenza (H5N1), is therefore worth careful examination.

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.

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.

Using checkerboard and time-kill assays, we evaluated the in vitro activity of quinupristin-dalfopristin (RP 59500) alone and in combination with five other antimicrobial agents against 12 clinical strains of vancomycin-resistant Enterococcus faecium (VREF). In time-kill studies, six VREF strains exhibited synergism with the combination of quinupristin-dalfopristin and doxycycline and three exhibited synergism with quinupristin-dalfopristin plus ampicillin-sulbactam. Combinations of quinupristin-dalfopristin with these and other agents warrant further clinical evaluation for the treatment of serious VREF infections.

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.

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.

The eae gene has recently been shown to be necessary for the attaching and effacing (AE) activity of enteropathogenic Escherichia coli (EPEC) on intestinal epithelial cells. In this paper we report the cloning and nucleotide sequence of a similar gene from a strain of enterohemorrhagic E. coli (EHEC) serotype O157:H7. An EHEC eae sequence was identified which was 97% homologous to the EPEC eae gene for the first 2200 bp and 59% homologous over the last 800 bp. Both eae sequences show 50% homology to the central region of the Yersinia pseudotuberculosis inv gene. The receptor-binding domain of the inv gene product lies near the carboxyl terminus. This suggests that the predicted amino acid sequence divergence in the carboxyl termini of the eae gene products might result in different antigenic and receptor specificity of these putative adhesins.

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.

The aim of this study was to examine the prevalence and distribution of Escherichia coli O157:H7 lineage-specific polymorphism assay (LSPA) 6 genotypes from cattle (n = 313) and clinical human (n = 203) isolates from northern and southern Alberta, Canada, to understand possible associations of genotypes with host and geographic location. The majority of cattle isolates (feedlot and dairy) typed as LSPA-6 111111 (72.2%), with proportionately higher LSPA-6 222222 (19.4%) than other LSPA-6 genotypes (10.7%). Clinical human isolates also typed primarily as LSPA-6 111111 (90.1%), but a higher percentage of genotypes (6.8%) other than LSPA-6 222222 (3.1%) was observed. A significantly higher frequency of LSPA-6 111111 in southern Alberta cattle (P < 0.0001) and a significant difference in LSPA-6 genotypes between human versus feedlot cattle from northern Alberta (P < 0.0001) were detected. LSPA-6 211111 genotype was third and second most common in cattle and humans, respectively, and several new LSPA-6 genotypes (n = 19) were also discovered. Despite avoiding over-representation of isolates from specific farms or outbreaks, higher strain diversity among cattle by pulsed-field gel electrophoresis (PFGE; 50 genotypes) in contrast to human (9 PFGE genotypes) isolates was observed. The majority of cattle (74.4%) and human (90.6%) isolates were susceptible to the antimicrobials tested. Within resistant cattle isolates, sulfisoxazole-tetracycline resistance was common (62.5%) and was accounted for by the presence of sul1 and sul2, and tet(A) and tet(B) determinants. An association between LSPA-6 and PFGE genotypes but not between geographic location and PFGE genotype for both hosts was evident.

Verotoxin-producing Escherichia coli strains of the serotype O157:H7 belong to a class of gastrointestinal pathogens that adhere to epithelial cells in a characteristic pattern known as attaching and effacing. Recent insight into the nature of E. coli O157:H7 adhesion was provided by the cloning and sequencing of the chromosomal eaeA (for E. coli attaching and effacing) gene homolog (G. Beebakhee, M. Louie, J. De Azavedo, and J. Brunton, FEMS Microbiol. Lett. 91:63-68, 1992, and J. Yu and J. B. Kaper, Mol. Microbiol. 6:411-417, 1992) and isolation of a 60-MDa plasmid referred to as pO157 (I. Toth, M. L. Cohen, H. S. Rumschlag, L. W. Riley, E. H. White, J. H. Carr, W. W. Bond, and I. K. Wachsmuth, Infect. Immun. 58:1223-1231, 1990, and S. Tzipori, H. Karch, K. I. Wachsmuth, R. M. Robins-Browne, A. D. O'Brien, H. Lior, M. L. Cohen, J. Smithers, and M. M. Levine, Infect. Immun. 55:3117-3125, 1987) and an approximately 94-kDa outer membrane protein (94-kDa OMP; P. Sherman, F. Cockerill III, R. Soni, and J. Brunton, Infect. Immun. 59:890-899, 1991). In this study, we examined the gene products of both eaeA and pO157 in relation to the 94-kDa OMP and as candidate effectors for O157:H7 attachment-effacement. Peptide sequencing and immunoassay demonstrated that the C. coli O157:H7 eaeA gene product is distinct from the 94-kDa OMP. Using ultrastructural analyses, we found that both parent and pO157 plasmid-cured O157:H7 strains demonstrated attaching and effacing adhesion to host epithelial cells and reacted equally well to rabbit antiserum raised against the 94-kDa OMP. By both transmission electron microscopy and light microscopy, E. coli HB101 transformed separately with the cloned eaeA gene and the pO157 plasmid did not form attaching and effacing lesions on cultured epithelial cells in vitro and rabbit intestinal tissues in vivo. Since additional determinants may mediate the attaching and effacing phenotype, we examined transposon TnphoA mutants constructed from E. coli O157:H7 strain CL8. Two TnphoA mutants were found deficient in bacterial factors that are necessary for O157:H7 attachment-effacement and likely distinct from the eaeA gene product.