Detection of OXA-181 Carbapenemase in Shigella flexneri.

We report the detection of OXA-181 carbapenemase in an azithromycin-resistant Shigella spp. bacteria in an immunocompromised patient. The emergence of OXA-181 in Shigella spp. bacteria raises concerns about the global dissemination of carbapenem resistance in Enterobacterales and its implications for the treatment of infections caused by Shigella bacteria.

A Practical Workflow for the Identification of Aspergillus, Fusarium, Mucorales by MALDI-TOF MS: Database, Medium, and Incubation Optimization.

There is an increasing body of literature on the utility of MALDI-TOF MS in the identification of filamentous fungi. However, the process still lacks standardization. In this study, we attempted to establish a practical workflow for the identification of three clinically important molds: Aspergillus, Fusarium, and Mucorales using MALDI-TOF MS. We evaluated the performance of Bruker Filamentous Fungi database v3.0 for the identification of these fungi, highlighting when there would be a benefit of using an additional database, the MSI-2 for further identification. We also examined two other variables, namely, medium effect and incubation time on the accuracy of fungal identification. The Bruker database achieved correct species level identification in 85.7% of Aspergillus and 90% of Mucorales, and correct species-complex level in 94.4% of Fusarium. Analysis of spectra using the MSI-2 database would also offer additional value for species identification of Aspergillus species, especially when suspecting species with known identification limits within the Bruker database. This issue would only be of importance in selected cases where species-level identification would impact therapeutic options. Id-Fungi plates (IDFP) had almost equivalent performance to Sabouraud dextrose agar (SDA) for species-level identification of isolates and enabled an easier harvest of the isolates with occasional faster identification. Our study showed accurate identification at 24 h for Fusarium and Mucorales species, but not for Aspergillus species, which generally required 48 h.

Assessment of panfungal PCR performance with formalin-fixed paraffin-embedded tissue specimens†.

We reviewed the performance of a panfungal ITS-2 PCR and Sanger sequencing assay performed on 88 FFPE specimens at The Hospital for Sick Children (Toronto, Canada) in 2019. A potential fungal pathogen was identified by ITS PCR in 62.7 and 2.9% of positive and negative direct slide examination of tissue specimens, respectively. ITS amplicons were detected in 87/88 specimens, with 53/88 (60.2%) considered as 'positive-contaminants' and 34/88 (38.6%) as 'positive-potential pathogen' upon sequencing. Potential pathogens included Blastomyces dermatitidis (17.1%), Cryptococcus neoformans (17.1%), Histoplasma capsulatum (14.3%) and Mucormycetes (11.4%). Laboratories should only perform ITS PCR on FFPE tissues if fungal elements have been confirmed on histopathology slides. LAY SUMMARY: In this study, we examined how well a DNA-based test could detect DNA from fungi in archived human biopsy tissues. The best performance was achieved if fungi were seen in the tissue under a microscope before being tested. Our results indicate that we should only use this test if these conditions are met.

Burkholderia cenocepacia ET12 transmission in adults with cystic fibrosis.

This report describes transmission of a Burkholderia cenocepacia ET12 strain (ET12-Bc) at the Toronto Adult Cystic Fibrosis (CF) Centre occurring from 2008 to 2017. Epidemiological and genomic data from 11 patients with CF were evaluated. Isolates were analysed using whole genome sequencing (WGS). Epidemiological investigation and WGS analysis suggested nosocomial transmission, despite enhanced infection control precautions. This was associated with subsequent deaths in 10 patients. ET12-Bc positive patients are no longer cared for on the same unit as ET12-Bc negative patients.

Epidemiologic and Genotypic Review of Carbapenemase-Producing Organisms in British Columbia, Canada, between 2008 and 2014.

Carbapenemase-producing organisms (CPOs) are a serious emerging problem for health care facilities worldwide. Owing to their resistance to most antimicrobial therapies, CPOs are difficult to treat and pose a challenge for infection prevention and control. Since 2010, lab-based surveillance for CPOs and PCR-based testing were implemented in British Columbia (BC), Canada. A review of CPOs in BC from 2008 to March 2014 was done to characterize the resistance mechanisms and possible clonal strain transmission and to compare pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and plasmid restriction fragment length polymorphism (RFLP) as molecular typing tools. During this study period, a total of 177 CPO cases were identified. Patient demographics and travel history were reviewed, and a descriptive analysis was carried out. PFGE profiles, MLST, and plasmid RFLP analysis for a subset of Escherichia coli, Klebsiella pneumoniae, and Enterobacter species isolates were obtained and analyzed. Our findings demonstrate that CPOs have been increasing in number in BC over time, from 1 isolate/year retrospectively identified in 2008 and 2009 to 82 isolates in 2013 and 30 isolates in the first quarter of 2014. Overall, K. pneumoniae isolates lack clonality, although some seemingly related clusters have been found. Plasmid analysis showed evidence of the spread of plasmids carrying carbapenemase-encoding genes between the examined isolates. Analysis of Enterobacter cloacae isolates revealed a more clonal nature of these CPOs in BC. The presence of related clusters provides evidence of interpatient organism transmission both within and between institutions. Although in our study, NDM-harboring E. cloacae isolates appeared to spread clonally, the spread of carbapenem resistance in K. pneumoniae seems to be plasmid mediated.

Evaluation of MALDI-TOF mass spectrometry and Sepsityper Kit™ for the direct identification of organisms from sterile body fluids in a Canadian pediatric hospital.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) can be used to identify bacteria directly from positive blood and sterile fluid cultures. The authors evaluated a commercially available kit - the Sepsityper Kit (Bruker Daltonik, Germany) - and MALDI-TOF MS for the rapid identification of organisms from 80 flagged positive blood culture broths, of which 73 (91.2%) were blood culture specimens and seven (8.7%) were cerebrospinal fluid specimens, in comparison with conventional identification methods. Correct identification to the genus and species levels was obtained in 75 of 80 (93.8%) and 39 of 50 (78%) blood culture broths, respectively. Applying the blood culture analysis module, a newly developed software tool, improved the species identification of Gram-negative organisms from 94.7% to 100% and of Gram-positive organisms from 66.7% to 70%. MALDI-TOF MS is a promising tool for the direct identification of organisms cultured from sterile sites.

La spectrométrie de masse à temps de vol par désorption-ionisation par impact laser assisté par matrice (MALDI-TOF MS) peut être utilisée pour identifier les bactéries directement dans le sang et les cultures positives de liquide stérile. Les auteurs ont évalué une trousse commerciale, la Sepsityper Kit (Bruker Daltonik, Allemagne), et la MALDI-TOF MS pour identifier rapidement des organismes dans 80 bouillons d'hémoculture signalés comme positifs, dont 73 (91,2 %) étaient des échantillons d'hémoculture et sept (8,7 %), des échantillons de liquide céphalorachidien, par rapport aux méthodes d'identification classiques. Les chercheurs ont obtenu la bonne identification du genre et de l'espèce dans 75 des 80 (93,8 %) et 39 des 50 (78 %) bouillons d'hémoculture, respectivement. La mise en application du module d'analyse d'hémoculture, un nouvel outil informatique, a fait passer l'identification des espèces d'organismes Gram négatif de 94,7 % à 100 % et des organismes Gram positif de 66,7 % à 70 %. La MALDI-TOF MS est un outil prometteur pour l'identification directe d'organismes cultivés à partir de foyers stériles.

Post-traumatic Fungal Keratitis and Endophthalmitis Caused by Coniochaeta Hoffmannii with Late Recurrence following Therapeutic Full-thickness Penetrating Keratoplasty.

BACKGROUND: To report a rare case of fungal keratitis and endophthalmitis due to Coniochaeta hoffmannii. METHODS: Case report. RESULTS: A 71-year-old immunocompetent male sustained a corneal laceration, traumatic cataract, and retinal detachment due to penetrating injury from a nail pulled from a wooden deck. The patient's postoperative course was complicated by infectious keratitis. Fungal cultures, DNA sequencing and analysis of the internal transcribed spacer sequence confirmed Coniochaeta hoffmannii. Topical and oral voriconazole treatments were initiated; however, due to impending perforation, a therapeutic corneal transplant was required. One year later, the patient developed a new corneal infiltrate at the graft-host junction: Corneal scrapings were culture positive for Coniochaeta hoffmannii. This was treated with topical and intrastromal voriconazole along with oral itraconazole 200 mg once daily for 8 months. CONCLUSIONS: Coniochaeta hoffmannii may cause recalcitrant keratitis and endophthalmitis, which required longstanding antifungal treatment.

New Delhi metallo-ß-lactamase-1: local acquisition in Ontario, Canada, and challenges in detection.

New Delhi metallo-ß-lactamase-1 (NDM-1) is a recently identified metallo-ß-lactamase that confers resistance to carbapenems and all other ß-lactam antibiotics, with the exception of aztreonam. NDM-1 is also associated with resistance to many other classes of antibiotics. The enzyme was first identified in organisms isolated from a patient in Sweden who had previously received medical treatment in India, but it is now recognized as endemic throughout India and Pakistan and has spread worldwide. The gene encoding NDM-1 has been found predominantly in Escherichia coli and Klebsiella pneumoniae. We describe the isolation NDM-1-producing organisms from two patients in Toronto, Ontario. To the best of our knowledge, this is the first report of an organism producing NDM-1 that was locally acquired in Canada. We also discuss the evidence that NDM-1 can affect bacterial species other than E. coli and K. pneumoniae, the limited options for treatment and the difficulty laboratories face in detecting organisms that produce NDM-1.

Fourier Transform Infrared Spectroscopy for Typing Burkholderia cenocepacia ET12 Isolates.

The IR Biotyper and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using ClinProTools software (MALDI-TOF MS-ClinProTools) are two novel typing methods that rely on the analysis of carbohydrate and peptide residues in intact bacterial cells. These two methods have shown promising results in the rapid and accurate typing of bacteria. In this study, we evaluated these novel typing methods in comparison with genotypic typing for cluster analysis of Burkholderia cenocepacia epidemic strain ET12, isolated from adult cystic fibrosis patients. Sixty-six isolates of B. cenocepacia were used in this study, 35 of which were identified as the ET12 strain and 31 as non-ET12 strains by repetitive-element PCR (rep-PCR). Twelve isolates were used for the creation of typing models using IR Biotyper and MALDI-TOF MS-ClinProTools, and 54 isolates were used for external validation of the typing models. The IR Biotyper linear discriminant analysis (LDA) model had a diagnostic sensitivity of 84.6% for typing the epidemic strain, ET12. At a cutoff of 70%, MALDI-TOF MS-ClinProTools had 87.5% diagnostic sensitivity in detecting the ET12 strain (P = 1.00). Both methods had a diagnostic specificity of ≥80% for detecting the ET12 strain. In conclusion, IR Biotyper and MALDI-TOF MS-ClinProTools offer rapid typing using proteomics and analysis of small cellular molecules with a low running cost. Our pilot study showed suboptimal accuracy of both methods for typing outbreak strains of B. cenocepacia. Extending the spectral region analyzed by the IR Biotyper can improve the accuracy and has the potential of improving the generalizability of this technique for typing other organisms. IMPORTANCE Respiratory infections due to Burkholderia cenocepacia, particularly the ET12 epidemic strain, are considered sentinel events for persons with cystic fibrosis, as they are often associated with person-to-person transmission and accelerated decline in lung function and early mortality. Current typing methods are generally only available at reference centers, with long turn-around-times, which can affect the identification of outbreaks and critical patient triage. This pilot study aims to add to the growing literature illustrating the potential utility of Fourier transform infrared spectroscopy (FTIR), a novel rapid method, for the successful typing of clinically significant bacteria. In this study, we evaluated its utility to discriminate between the ET12 clone and non-ET12 isolates of B. cenocepacia and compared it to proteomics cluster analysis using MALDI-TOF MS and ClinProTools software. Both methods had encouraging but suboptimal accuracy (≥85% sensitivity and ≥83% specificity), which will likely be improved by extending the spectral region analyzed by the IR Biotyper with updated software.

Evaluation of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry and ClinPro Tools as a Rapid Tool for Typing Streptococcus pyogenes.

BACKGROUND: Timely strain typing of group A Streptococci (GAS) is necessary to guide outbreak recognition and investigation. We evaluated the use of (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) combined with cluster analysis software to rapidly distinguish between related and unrelated GAS isolates in real-time. METHODS: We developed and validated a typing model using 177 GAS isolates with known emm types. The typing model was created using 43 isolates, which included 8 different emm types, and then validated using 134 GAS isolates of known emm types that were not included in model generation. RESULTS: Twelve spectra were generated from each isolate during validation. The overall accuracy of the model was 74% at a cutoff value of 80%. The model performed well with emm types 4, 59, and 74 but showed poor accuracy for emm types 1, 3, 12, 28, and 101. To evaluate the ability of this tool to perform typing in an outbreak situation, we evaluated a virtual outbreak model using a "virtual outbreak strain; emm74" compared with a non-outbreak group or an "outgroup " of other emm types. External validation of this model showed an accuracy of 91.4%. CONCLUSIONS: This approach has the potential to provide meaningful information that can be used in real time to identify and manage GAS outbreaks. Choosing isolates characterized by whole genome sequencing rather than emm typing for model generation should improve the accuracy of this approach in rapidly identifying related and unrelated GAS strains.

Mandibular osteomyelitis due to Raoultella species.

INTRODUCTION: Raoultella is a genus of aerobic Gram-negative bacilli belonging to the family Enterobacteriaceae that are commonly found in water, soil and aquatic environments. With improved bacterial identification techniques, Raoultella species (namely R. planticola and R. ornithinolytica) have been an increasingly reported cause of infections in humans. CASE PRESENTATION: An 85-year-old man presented to hospital with a several-week history of left jaw pain and trismus. His medical history was significant for left mandibular osteomyelitis treated 1 year previously with amoxicillin-clavulanate. On admission, a computed tomography scan demonstrated a 2.6×1.7×1.6 cm peripherally enhancing collection surrounding the left posterior mandibular body. Two aspirates of the abscess grew a bacterium belonging to the genus Raoultella, with discordant species identification (R. ornithinolytica versus R. planticola) using two different techniques. A potential source of infection included a left lower molar tooth which was extracted months preceding the original diagnosis of osteomyelitis. CONCLUSION: This is the first case of mandibular osteomyelitis caused by Raoultella species reported in the literature. In contrast to other forms of osteomyelitis, the pathogenesis of mandibular osteomyelitis involves contiguous spread from an odontogenic focus. Risk factors for mandibular osteomyelitis include a history of fracture, irradiation, diabetes and steroid therapy. This report adds to the growing literature of infections caused by this genus of bacteria, and raises the possibility of this organism's role in odontogenic infections.

Rapid detection of resistance to carbapenems and cephalosporins in Enterobacteriaceae using liquid chromatography tandem mass spectrometry.

Carbapenemase producing Enterobacteriaceae (CPE) are becoming a global healthcare concern. Current laboratory methods for the detection of CPE include screening followed by confirmatory phenotypic and genotypic tests. These processes would generally take ≥72 hours, which could negatively impact patient care and Infection Control practices. To this end, we developed a protocol for rapid resistance testing (RRT) to detect hydrolysis in a panel of beta lactam antibiotics consisting of ampicillin, cefazolin, cefotaxime and imipenem, using liquid chromatography tandem mass spectrometry. Ninety-nine beta lactamase producing Enterobacteriaceae isolates were used to evaluate the RRT method, 54 isolates were CPE and 45 isolates were Class A or AmpC beta lactamase producing Enterobacteriaceae but not carbapenemase producers. We also tested 10 E.coli isolates that were susceptible to ampicillin, cefazolin, cefotaxime and imipenem. Receiver Operating Characteristic (ROC) Curves analysis showed that imipenem had a sensitivity and a specificity of 100% for crabapenemase detection at hydrolysis cut off values that are greater than 50% and less than or equal to 80%. The RRT protocol can be conducted in a time frame of less than 2 hours. This preliminary study shows that the rapid resistance testing protocol might have utility for the rapid detection of CPE. Additional work with a greater number and variety of beta- lactamase producing Enterobacteriaceae isolates is required to validate these preliminary findings.

Risk factors for Staphylococcus aureus surgical site infection during an outbreak in patients undergoing cardiovascular surgery.

BACKGROUND: This study examined the epidemiology of an outbreak of Staphylococcus aureus surgical site infections (SSI) after cardiovascular surgery, and analyzed risk factors for S aureus SSIs. METHODS: This was a retrospective case-control study to determine risk factors for S aureus SSI in 38 patients who developed S aureus SSI during the outbreak period, compared with age-, sex-, and procedure-matched controls. S aureus strains were typed by pulsed-field gel electrophoresis. RESULTS: A total of 38 patients had S aureus SSI. Pulsed-field gel electrophoresis identified transmission of 3 S aureus clones (2 MSSA clones and 1 MRSA clone). Twenty-one health care workers were carriers of outbreak strains. In multivariate analysis, the significant risk factors for S aureus SSI were previous cardiac surgery (odds ratio, 7.41; 95% confidence interval, 1.05-52.16) and long procedure duration (odds ratio, 1.49; 95% confidence interval, 1.00-2.21). CONCLUSIONS: This outbreak demonstrates evidence of nosocomial transmission of 3 clones of S aureus in the setting of incomplete compliance with recommended standard perioperative infection control measures, associated with a high prevalence of staff carriage of the predominant outbreak strains.

Epidemiology and outcome of pneumonia caused by methicillin-resistant Staphylococcus aureus (MRSA) in Canadian hospitals.

BACKGROUND: MRSA remains a leading cause of hospital-acquired (HAP) and healthcare-associated pneumonia (HCAP). We describe the epidemiology and outcome of MRSA pneumonia in Canadian hospitals, and identify factors contributing to mortality. METHODS: Prospective surveillance for MRSA pneumonia in adults was done for one year (2011) in 11 Canadian hospitals. Standard criteria for MRSA HAP, HCAP, ventilator-associated pneumonia (VAP), and community-acquired pneumonia (CAP) were used to identify cases. MRSA isolates underwent antimicrobial susceptibility testing, and were characterized by pulsed-field gel electrophoresis (PFGE) and Panton-Valentine leukocidin (PVL) gene detection. The primary outcome was all-cause mortality at 30 days. A multivariable analysis was done to examine the association between various host and microbial factors and mortality. RESULTS: A total of 161 patients with MRSA pneumonia were identified: 90 (56%) with HAP, 26 (16%) HCAP, and 45 (28%) CAP; 23 (14%) patients had VAP. The mean (± SD) incidence of MRSA HAP was 0.32 (± 0.26) per 10,000 patient-days, and of MRSA VAP was 0.30 (± 0.5) per 1,000 ventilator-days. The 30-day all-cause mortality was 28.0%. In multivariable analysis, variables associated with mortality were the presence of multiorgan failure (OR 8.1; 95% CI 2.5-26.0), and infection with an isolate with reduced susceptibility to vancomycin (OR 2.5, 95% CI 1.0-6.3). CONCLUSIONS: MRSA pneumonia is associated with significant mortality. Severity of disease at presentation, and infection caused by an isolate with elevated MIC to vancomcyin are associated with increased mortality. Additional studies are required to better understand the impact of host and microbial variables on outcome.