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Background Pneumonia from SARS-CoV-2 is difficult to distinguish from other viral and bacterial etiologies. Broad-spectrum antimicrobials are frequently prescribed to patients hospitalized with COVID-19 which potentially acts as a catalyst for the development of antimicrobial resistance (AMR). Objectives We conducted a systematic review and meta-analysis during the first 18 months of the pandemic to quantify the prevalence and types of resistant co-infecting organisms in patients with COVID-19 and explore differences across hospital and geographic settings. Methods We searched MEDLINE, Embase, Web of Science (BioSIS), and Scopus from November 1, 2019 to May 28, 2021 to identify relevant articles pertaining to resistant co-infections in patients with laboratory confirmed SARS-CoV-2. Patient- and study-level analyses were conducted. We calculated pooled prevalence estimates of co-infection with resistant bacterial or fungal organisms using random effects models. Stratified meta-analysis by hospital and geographic setting was also performed to elucidate any differences. Results Of 1331 articles identified, 38 met inclusion criteria. A total of 1959 unique isolates were identified with 29% (569) resistant organisms identified. Co-infection with resistant bacterial or fungal organisms ranged from 0.2 to 100% among included studies. Pooled prevalence of co-infection with resistant bacterial and fungal organisms was 24% (95% CI 8–40%; n = 25 studies: I2 = 99%) and 0.3% (95% CI 0.1–0.6%; n = 8 studies: I2 = 78%), respectively. Among multi-drug resistant organisms, methicillin-resistant Staphylococcus aureus, carbapenem-resistant Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and multi-drug resistant Candida auris were most commonly reported. Stratified analyses found higher proportions of AMR outside of Europe and in ICU settings, though these results were not statistically significant. Patient-level analysis demonstrated > 50% (n = 58) mortality, whereby all but 6 patients were infected with a resistant organism. Conclusions During the first 18 months of the pandemic, AMR prevalence was high in COVID-19 patients and varied by hospital and geography although there was substantial heterogeneity. Given the variation in patient populations within these studies, clinical settings, practice patterns, and definitions of AMR, further research is warranted to quantify AMR in COVID-19 patients to improve surveillance programs, infection prevention and control practices and antimicrobial stewardship programs globally.

KEY POINTS Clinical presentation in persons infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ranges from asymptomatic to the life-threatening respiratory distress that can occur with coronavirus disease 2019 (COVID-19).[1][1] Diagnosis of acute or new cases of SARS-CoV-2

From 2014–2019, invasive pulmonary aspergillosis complicated 7.2% (0–23.1% in different influenza seasons) of cases of influenza-associated respiratory failure in Edmonton, Alberta. Disease outcomes ranged from survival without therapy to death despite antifungals. Clinician vigilance, longitudinal local surveillance, and refined criteria to identify patients requiring therapy are needed.

Background: Pneumonia from SARS-CoV-2 is difficult to distinguish from other viral and bacterial etiologies. Broad-spectrum antimicrobials are frequently prescribed to patients hospitalized with COVID-19 which potentially acts as a catalyst for the development of antimicrobial resistance (AMR). Objectives: We conducted a systematic review and meta-analysis during the first 18 months of the pandemic to quantify the prevalence and types of resistant co-infecting organisms in patients with COVID-19 and explore differences across hospital and geographic settings. Methods: We searched MEDLINE, Embase, Web of Science (BioSIS), and Scopus from November 1, 2019 to May 28, 2021 to identify relevant articles pertaining to resistant co-infections in patients with laboratory confirmed SARS-CoV-2. Patient- and study-level analyses were conducted. We calculated pooled prevalence estimates of co-infection with resistant bacterial or fungal organisms using random effects models. Stratified meta-analysis by hospital and geographic setting was also performed to elucidate any differences. Results: Of 1331 articles identified, 38 met inclusion criteria. A total of 1959 unique isolates were identified with 29% (569) resistant organisms identified. Co-infection with resistant bacterial or fungal organisms ranged from 0·2 to 100% among included studies. Pooled prevalence of co-infection with resistant bacterial and fungal organisms was 24% (95% CI: 8-40%; n=25 studies: I 2 =99%) and 0·3% (95% CI: 0·1-0·6%; n=8 studies: I 2 =78%), respectively. Among multi-drug resistant organisms, methicillin-resistant Staphylococcus aureus, carbapenem-resistant Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Candida auris were most commonly reported. Stratified analyses found higher proportions of AMR outside of Europe and in ICU settings, though these results were not statistically significant. Patient-level analysis demonstrated >50% (n=58) mortality, whereby all but 6 patients were infected with a resistant organism. Conclusions: During the first 18 months of the pandemic, AMR was moderately prevalent in COVID-19 patients and varied by hospital and geography although there was substantial heterogeneity. Given the variation in patient populations within these studies, clinical settings, practice patterns, and definitions of AMR, further research is warranted to quantify AMR in COVID-19 patients to improve surveillance programs, infection prevention and control practices and antimicrobial stewardship programs globally. Funding: The Antimicrobial Resistance - One Health Consortium is funded through the Major Innovation Fund Program of the Ministry of Jobs, Economy, and Innovation (JEI), Government of Alberta, Canada. Declaration of Interest: We declare no competing interests.

Candida auris is an emerging yeast that is associated with antifungal resistance and healthcare-associated outbreaks. From 2012 to 2019, there were 24 known cases of C. auris colonization or infection in Canada. Isolates were from axilla/groin (n = 6), ear (n = 5), blood (n = 4), toe (n = 2), and a variety of other sites (n = 7). Canadian isolates belonged to the four main genomic clades: Clade I (formerly called South Asian clade, n = 12), Clade II (East Asian, n = 3), Clade III (African, n = 4), and Clade IV (South American, n = 5). Isolates within each clade were clonal; however, whole genome sequencing may be helpful in identifying clusters within healthcare facilities. Lay summary The fungal pathogen Candida auris has caused many hospital outbreaks and is often multidrug resistant. All four major strains of C. auris were identified in Canada from 2012 to 2019. Genomic epidemiology may be useful for identifying and reducing transmission of C. auris within hospitals.

Pulmonary aspergillosis may complicate coronavirus disease 2019 (COVID-19) and contribute to excess mortality in intensive care unit (ICU) patients. The disease is poorly understood, in part due to discordant definitions across studies.We sought to review the prevalence, diagnosis, treatment, and outcomes of COVID-19-associated pulmonary aspergillosis (CAPA) and compare research definitions.PubMed, Embase, Web of Science, and MedRxiv were searched from inception to October 12, 2021.ICU cohort studies and CAPA case series including ≥3 patients were included.Adult patients in ICUs with COVID-19.Patients were reclassified according to four research definitions. We assessed risk of bias with an adaptation of the Joanna Briggs Institute cohort checklist tool for systematic reviews.We calculated CAPA prevalence using the Freeman-Tukey random effects method. Correlations between definitions were assessed with Spearman's rank test. Associations between antifungals and outcome were assessed with random effects meta-analysis.Fifty-one studies were included. Among 3297 COVID-19 patients in ICU cohort studies, 313 were diagnosed with CAPA (prevalence 10%; 95% CI 8%-13%). Two hundred seventy-seven patients had patient-level data allowing reclassification. Definitions had limited correlation with one another (ρ = 0.268-0.447; p 0.001), with the exception of Koehler and Verweij (ρ = 0.893; p 0.001); 33.9% of patients reported to have CAPA did not fulfill any research definitions. Patients were diagnosed after a median of 8 days (interquartile range 5-14) in ICUs. Tracheobronchitis occurred in 3% of patients examined with bronchoscopy. The mortality rate was high (59.2%). Applying CAPA research definitions did not strengthen the association between mould-active antifungals and survival.The reported prevalence of CAPA is significant but may be exaggerated by nonstandard definitions.

RationalePulmonary aspergillosis may complicate COVID-19 and contribute to excess mortality in intensive care unit (ICU) patients. The incidence is unclear because of discordant definitions across studies.ObjectiveWe sought to review the incidence, diagnosis, treatment, and outcomes of COVID-19-associated pulmonary aspergillosis (CAPA), and compare research definitions.MethodsWe systematically reviewed the literature for ICU cohort studies and case series including ≥ patients with CAPA. We calculated pooled incidence. Patients with sufficient clinical details were reclassified according to 4 standardized definitions (Verweij, White, Koehler, and Bassetti).MeasurementsCorrelations between definitions were assessed with Spearman’s rank test. Associations between antifungals and outcome were assessed with Fisher’s Exact test.Main Results38 studies (35 cohort studies and 3 case series) were included. Among 3,297 COVID-19 patients in ICU cohort studies, 313 were diagnosed with CAPA (pooled incidence 9.5%). 197 patients had patient-level data allowing reclassification. Definitions had limited correlation with one another (ρ=0.330 to 0.621, pConclusionsThe reported incidence of CAPA is exaggerated by use of non-standard definitions. Further research should focus on identifying patients likely to benefit from antifungals.

ObjectivesUnderstanding the epidemiology of invasive Candida infections is essential to patient management decisions and antifungal stewardship practices. This study characterized the species distribution and antifungal susceptibilities of prospectively collected isolates of Candida species causing bloodstream infections (BSIs) in patients admitted to tertiary care hospitals located in 14 cities across 8 of the 10 Canadian provinces between 2011 and 2016.MethodsAntifungal susceptibility testing was performed by broth microdilution using CLSI methods, breakpoints and epidemiological cut-off values. DNA sequencing of fks loci was performed on all echinocandin-non-susceptible isolates.ResultsCandida albicans (49.6%), Candida glabrata (20.8%) and Candida parapsilosis complex (12.0%) were the most common species out of 1882 isolates associated with BSIs. Candida tropicalis (5.2%), Candida krusei (4.3%), Candida dubliniensis (4.1%), Candida lusitaniae (1.4%) and Candida guilliermondii (1.1%) were less frequently isolated. Between 2011 and 2016, the proportion of C. albicans significantly decreased from 60.9% to 42.1% (P ConclusionsAntifungal resistance in contemporary isolates of Candida causing BSIs in Canada is uncommon. However, the proportion of C. glabrata isolates has increased and echinocandin resistance in this species has emerged. Ongoing surveillance of local hospital epidemiology and appropriate antifungal stewardship practices are necessary to preserve the utility of available antifungal agents.

Background Staphylococcus aureus bacteremia (SAB) is associated with high morbidity and mortality. Infectious disease consultation (IDC) is associated with increased adherence to guideline management and improved patient outcomes. We describe the IDC rate over time and impact of IDC on the management and outcomes of patients with SAB. Methods This retrospective chart review includes adult patients (≥ 18 years) hospitalized at the University of Alberta Hospital, Edmonton, Canada who had at least 1 blood culture growing Staphylococcus aureus during two time periods (A: Jan 2010 to Dec 2012; B: Jan to Oct 2020). Patients who died or were made palliative within 48hrs following bacteremia were excluded. Descriptive statistics were used to compare appropriateness of SAB management and outcomes in patients receiving IDC and those who did not (NIDC). Results 325 patients in period A and 129 in period B were included. Baseline demographics were similar. IDC rate increased from 63% to 88% (p< 0.001) between the study periods. IDC was associated with increased odds of receiving an echocardiogram (OR=3.56, 95% CI 2.22 – 5.57; OR=20.4, 95% 4.13 – 110.6, p< 0.001) and appropriate duration of antimicrobial therapy (OR=6.74, 95% 3.93 – 11.54; OR=43.2, 95% 5.72 – 529.5, p< 0.001) between study periods. Mean length of stay decreased in patients receiving IDC (44.8 vs 28.1 days, p=0.005) and increased in NIDC patients (19.9 vs 28.7 days, p=0.216). IDC was associated with lower 30-day mortality in period A (OR=3.53, 95% 1.95 – 6.36), however this association was not observed in period B (OR=1.43, 95% 0.40 – 5.56). There was a trend towards decreased odds of mortality in patients receiving early IDC (≤2 days from bacteremia, n=65) compared to late IDC (≥3 days from bacteremia, n=45) (OR=2.59, 95% 0.95 – 7.10, p=0.077). Conclusion Our centre’s IDC rate for SAB increased over time without specific intervention. IDC increased the odds of appropriate SAB management and was associated with decreased length of stay in period B. IDC was associated with lower 30-day mortality in period A and trended towards lower mortality in period B. Specifically, early IDC decreased odds of 30-day mortality compared to late IDC. These results suggest that routine early IDC be part of SAB management. Disclosures All Authors: No reported disclosures

Antimicrobial resistance (AMR) is a top priority for several national and international organizations, including the World Health Organization (WHO), the European Centre for Disease Prevention and Control (ECDC), the National Institutes of Health (NIH), and the Centers for Disease Control and Prevention (CDC). All the initiatives describe a multifaceted approach involving multiple disciplines to combat this problem. A part of this approach is to gain an understanding of what genes are responsible for antimicrobial resistance and how we can use this knowledge to develop rapid diagnostic assays and new antimicrobial agents. Here, we describe the current knowledge of the genetic markers that confer resistance in Gram-negative organisms and provide some insights into how this knowledge can be used in the ongoing battle against antimicrobial resistance.

Summary Background The classic geographical range of histoplasmosis in North America primarily includes the states and provinces adjacent to the Ohio, Mississippi, and St Lawrence riverways. Although Alberta, Canada is not typically considered a region of risk for histoplasmosis, cases with suspected local acquisition have been reported. We aimed to investigate the epidemiology and geographical distribution of cases of histoplasmosis in Alberta to assess evidence for local acquisition of infections, using genomic analysis for corroboration. Methods We did an epidemiological and genomic investigation, in which laboratory-confirmed cases of histoplasmosis were reviewed in Alberta from 2011, when the disease became reportable, until 2018. We used data attained from Alberta Health. Travel and exposure histories and clinical features were reviewed. Definite local acquisition was defined as a case without previous travel outside Alberta or associated with a common-source outbreak within the province, whereas probable local acquisition was a sporadic case with travel outside Alberta but compelling local exposures. Genomes of selected case isolates were analysed, including those from cases suspected to have been locally acquired and imported. Findings Between Jan 1, 2011, and June 30, 2018, 45 cases of histoplasmosis were identified. Participants had a median age of 53 years (range 17–77) and 32 [71%] were male. Among 34 patients with documented travel histories, ten (29%) had never left the province. 11 cases were of definite local acquisition, including eight cases from three common-source outbreaks and three sporadic cases in patients who had never travelled outside Alberta. The common-source outbreaks all involved exposure to bats or their droppings in chimneys or attics of private dwellings or churches. Four patients had travelled outside Alberta but compelling evidence was seen for local exposure to bat guano. Genome sequencing showed that isolates from cases of definite and probable local acquisition clustered together and were genetically distinct from isolates from suspected imported cases and other published isolates. Interpretation Using epidemiological and genomic analyses, we established that cases of histoplasmosis have been acquired in Alberta, thus expanding the geographical range of Histoplasma spp much further northwest than was previously appreciated. Histoplasmosis should be considered in patients with compatible symptoms outside areas of classic geographical risk. Funding None.

Whole-genome sequencing (WGS) of Staphylococcus aureus is increasingly used as part of infection prevention practices. In this study, we established a long-read technology-based WGS screening program of all first-episode methicillin-resistant Staphylococcus aureus (MRSA) blood infections at a major urban hospital., Whole-genome sequencing (WGS) of Staphylococcus aureus is increasingly used as part of infection prevention practices. In this study, we established a long-read technology-based WGS screening program of all first-episode methicillin-resistant Staphylococcus aureus (MRSA) blood infections at a major urban hospital. A survey of 132 MRSA genomes assembled from long reads enabled detailed characterization of an outbreak lasting several months of a CC5/ST105/USA100 clone among 18 infants in a neonatal intensive care unit (NICU). Available hospital-wide genome surveillance data traced the origins of the outbreak to three patients admitted to adult wards during a 4-month period preceding the NICU outbreak. The pattern of changes among complete outbreak genomes provided full spatiotemporal resolution of its progression, which was characterized by multiple subtransmissions and likely precipitated by equipment sharing between adults and infants. Compared to other hospital strains, the outbreak strain carried distinct mutations and accessory genetic elements that impacted genes with roles in metabolism, resistance, and persistence. This included a DNA recognition domain recombination in the hsdS gene of a type I restriction modification system that altered DNA methylation. Transcriptome sequencing (RNA-Seq) profiling showed that the (epi)genetic changes in the outbreak clone attenuated agr gene expression and upregulated genes involved in stress response and biofilm formation. Overall, our findings demonstrate the utility of long-read sequencing for hospital surveillance and for characterizing accessory genomic elements that may impact MRSA virulence and persistence.

The goal of this document was to provide Canadian laboratories with a framework for consistent reporting and monitoring of multidrug resistant organisms (MDRO) and extensively drug resistant organisms (XDRO) for common gram-negative pathogens. This is the final edition of the interim recommendations, which were modified after one year of broad consultative review. This edition represents a consensus of peer-reviewed information and was co-authored by the Canadian Public Health Laboratory Network and the Canadian Association of Clinical Microbiology and Infectious Diseases. There are two main recommendations. The first recommendation provides standardized definitions for MDRO and XDRO for gram-negative organisms in clinical specimens. These definitions were limited to antibiotics that are commonly tested clinically and, to reduce ambiguity, resistance (rather than non-susceptibility) was used to calculate drug resistance status. The second recommendation identifies the use of standardized laboratory reporting of organisms identified as MDRO or XDRO. Through the broad consultation, which included public health and infection prevention and control colleagues, these definitions are ready to be applied for policy development. Both authoring organizations intend to review these recommendations regularly as antibiotic resistance testing evolves in Canada.

We report three cases of hospital-acquired mucormycosis in heart and lung transplant patients over a 6-month period. Traditional epidemiological investigation tools were used to look for a common link between patients to explain the outbreak. Genome sequencing of each fungal strain was used to supplement the investigation. By disproving a close genetic link between infecting strains of mucormycosis, we were able to conclude the outbreak investigation. Genome sequencing is a novel tool that can be used in addition to traditional epidemiologic investigations to help determine linkage of patients during outbreak investigations.

Objectives To identify the β-lactamase responsible for the positive detection of carbapenemase production in four clinical isolates of Pseudomonas aeruginosa that were negative by PCR for KPC, OXA-48, NDM, VIM, IMP, GES and NMC/IMI carbapenemase genes. Methods WGS using short-read and long-read methods was used to characterize the isolates. Bioinformatic analysis was used to identify the potential gene encoding a carbapenemase. Cloning, antimicrobial susceptibility testing and biochemical and phenotypic characterization were used to determine metallo-enzyme activity. Single-nucleotide variant (SNV) typing was used to determine strain relatedness. Conjugation experiments were used to determine transmissibility of the novel carbapenemase-encoding gene. Results WGS analysis revealed a novel class B β-lactamase gene, blaCAM-1 (Central Alberta Metallo-β-lactamase), located in a 73 kb integrative element, named IMEPaCAM-1, in the chromosome of four clinical isolates of P. aeruginosa. The cloned blaCAM-1 gene conferred carbapenem resistance to Escherichia coli TOP10. The four isolates, which were all closely related, were from three patients, all of whom spent time in the same hospital in 2008 and/or 2009. IMEPaCAM-1 could not be transferred by conjugation. Conclusions A novel metallo-enzyme, CAM-1, is encoded on an integrative element, IMEPaCAM-1, located in the chromosome of clinical isolates of P. aeruginosa. No additional isolates harbouring CAM-1 have been identified in Alberta since 2007.

We thank Dr. Yeung for his interest in our primer for Canadian clinicians on Candida auris .[1][1],[2][2] Given word-limit constraints, we were unable to elaborate further in our article, and so we welcome the opportunity to provide clarification regarding limitations in the ability of diagnostic

Background Histoplasmosis is a serious fungal infection caused by the geographically restricted, dimorphic fungus Histoplasma capsulatum. In Canada, the geographic range of H. capsulatum is classically thought to be restricted to southern parts of Ontario and Quebec. Over the past decade, histoplasmosis has occasionally been diagnosed in patients in Alberta without travel to areas of known geographic risk (Figure 1). We studied the epidemiology and geographic distribution of histoplasmosis in Alberta to assess evidence for locally-acquired infections. Methods We retrospectively reviewed all laboratory-confirmed (culture, antigen and/or immunodiffusion positive) cases of histoplasmosis diagnosed from January 1, 2011 to June 30, 2018. Data collected by public health and clinical charts were reviewed for clinical presentation, exposure and travel histories, and geographic distribution of cases. Cases of histoplasmosis in patients who had not left Alberta or associated with a local point source were classified as definite local acquisition; cases in patients with remote travel but with local exposures and appropriate timing of disease onset were deemed “probable” cases of local infection. University of Alberta’s Research Ethics Board approved this study. Results We identified 45 laboratory-confirmed cases of histoplasmosis, including 17 cases that were locally acquired. Among these, there were 12 cases of definite local acquisition, including 8 patients from 3 point-source outbreaks—all involving exposure to bats and/or their droppings in chimneys or attics of private dwellings or churches—and 4 sporadic cases in patients who had never traveled. Of the other 5 cases probably acquired in Alberta, patients had previously traveled (n = 4) or travel history was incomplete (n = 1) but local exposures preceding infection were considered compelling. The mean incidence rate of locally acquired infection was 0.062/100,000 population with incidence increasing since 2014. Table 1 shows features of locally acquired cases. Conclusion This study, for the first time, establishes Alberta as a region of geographic risk for histoplasmosis. The diagnosis should be considered in patients with compatible symptoms and exposure history, even in the absence of travel. Disclosures All authors: No reported disclosures.

To assess the impact of MALDI-TOF MS coupled with antimicrobial stewardship on clinical outcomes for pediatric inpatients with bloodstream infections.Outcomes of pediatric inpatients were compared before and after MALDI-TOF MS implementation. Outcomes measured included time until organism identification and susceptibility, duration of antibiotics, patient length of stay (LOS), mortality and hospital costs.210 and 135 patient events were compared pre- and post-intervention. Average time to organism identification decreased from 41 to 11 hours (P = 0.0001). Time to i) susceptibilities decreased from 50.8 to 37.7 hours (P = 0.0001), ii) de-escalation of antibiotics decreased from 58 to 23 hours (P = 0.0001), iii) discontinuation of unnecessary antibiotics decreased from 49 to 20 hours (P = 0.0001). Infection-related LOS decreased from 10.5 to 8.37 days (P = 0.006). No significant differences were seen for other outcomes.MALDI-TOF MS identification of bacteria from blood culture broth improves time to appropriate antibiotic treatment for pediatric inpatients.

We report the first documented isolation of Wohlfahrtiimonas chitiniclastica from a human in the United States. Initially misidentified as Acinetobacter lwoffii by Vitek-2, the isolate was subsequently identified as W . chitiniclastica by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing. While the clinical significance of the isolate in this case is unclear, it highlights the superior performance of MALDI-TOF MS for bacterial identification.

Background Antimicrobial resistance has emerged as a major threat to global public health. Thus, the surveillance of changes in antimicrobial resistance in local and global settings is a paramount necessity. While many studies have tracked antimicrobial resistance, only a small percentage surveyed ocular isolates. The purpose of this study was to report the in vitro susceptibility of bacterial pathogens isolated from ocular samples in New York, NY from 2010 to 2015. Methods A retrospective review of ocular isolates was conducted. All organisms were collected by 25 separate inpatient wards and outpatient clinics, and were analyzed by the clinical microbiology laboratory at Mount Sinai Hospital. Clinical Laboratory and Standards Institute (CLSI) guidelines were followed for susceptibility testing and breakpoint interpretations. Results A total of 549 bacterial organisms were isolated from 1664 cultures (33%) during the 6-year study period. Of these, 358 isolates (65.2%) underwent susceptibility testing. 182 (50.8%) isolates were Gram-positive. The most common Gram-positive bacterium was Staphylococcus aureus (62.1%). Methicillin-resistance decreased in S. aureus isolates (31.3% in 2010, 14.1% in 2015) but was without significant change (p = 0.25). When analyzing all S. aureus isolates recovered during the study period, there were significantly more methicillin-resistant S. aureus (MRSA) isolates resistant to fluoroquinolones (p

The occurrence and significance of Haemophilus spp. isolated from the genitourinary tract are not well known. Herein, we describe the clinical significance and characteristics of Haemophilus influenzae type b genogroup strains isolated from genitourinary tract specimens from an adult male veteran patient population and, in particular, their associations with prostatitis and epididymitis.

Although toxins A and B are known to be important contributors to the acute phase of Clostridium difficile infection, the role of colonization and adherence to host tissues in the overall pathogenesis of these organisms remains unclear. Consequently, we used the recently introduced intron-based ClosTron gene interruption system to eliminate the expression of two reported C. difficile colonization factors, the major flagellar structural subunit (FliC) and the flagellar cap protein (FliD), to gain greater insight into how flagella and motility contribute to C. difficile 's pathogenic strategy. The results demonstrate that interrupting either the fliC or the fliD gene results in a complete loss of flagella, as well as motility, in C. difficile . However, both the fliC and fliD mutant strains adhered better than the wild-type 630Δerm strain to human intestine-derived Caco-2 cells, suggesting that flagella and motility do not contribute to, or may even interfere with, C. difficile adherence to epithelial cell surfaces in vitro . Moreover, we found that the mutant strains were more virulent in hamsters, indicating either that flagella are unnecessary for virulence or that repression of motility may be a pathogenic strategy employed by C. difficile in hamsters.

Herein we present evidence for the therapeutic potential of colonization factor (CF)-specific egg yolk antibodies (IgY) for potentially treating acute and recurring Clostridium difficile infection (CDI) in humans. The study involved cloning, expressing as 6×His-tagged proteins in Escherichia coli, and Ni-affinity purifying three previously identified CFs (FliC, FliD and Cwp84) from C. difficile. The recombinant CF antigens were then used to immunize Leghorn chickens and CF-specific IgY antibodies were prepared from their eggs. The specificity and titre of the resulting C. difficile CF-specific IgY antibodies were assessed by ELISA and Western immunoblotting techniques. The antibodies were also screened for their ability to inhibit C. difficile adherence to human colon-derived T84 cells, and, based on these findings, one of them (FliD-specific IgY) was evaluated for its potential to prevent C. difficile-mediated morbidity and mortality in Syrian hamsters. The results revealed that purified FliD-specific IgY significantly protected hamsters from C. difficile strain 630 infection relative to control animals treated with carbonate buffer alone or IgY produced from unimmunized chicken eggs. The results suggest that egg yolk preparations obtained from chickens immunized with recombinant C. difficile CFs may represent another safe and cost-effective treatment option in humans suffering from acute or recurring CDI.

Herein we describe a real-time quantitative PCR assay for evaluating the adherence of Clostridium difficile to differentiated human intestinal Caco-2 cells. Our investigations demonstrated that the method, employing the C. difficile-specific triose-phosphate isomerase gene, is as reliable but less time-consuming than counting c.f.u. We conclude that the method will be useful for evaluating the role of host cell adherence in the pathogenesis of C. difficile infection.

A systematic investigation into the assembly and stability of native and modified subunits of the Shiga toxins (Stx) in vitro is described. Analysis of the assembly of native and modified B subunits of Stx1 and Stx2 in solution, carried out using electrospray ionization mass spectrometry (ES-MS), suggests that the lower thermodynamic stability of the B subunit homopentamer of Stx2, compared to that of Stx1, is due to the presence of a repulsive interaction involving Asp70 of the Stx2 B subunit. In Stx1 B, the corresponding (spatially) residue is Arg. Using temperature-controlled ES-MS, it is shown that the Stx1 and Stx2 holotoxins exhibit differences in their resistance to temperature- and acid-induced dissociation. However, both Stx1 and Stx2 are fully assembled at pH3.5 and 37 degrees C. This finding has several important biological implications. First, it argues against the likelihood that the difference in Stx1 and Stx2 toxicity arises from differential dissociation of the toxins during the intracellular trafficking steps of the cellular intoxication process. Second, it implies that the activation of the A subunits of Stx1 and Stx2 by enzymatic cleavage must occur while the A subunit is assembled with the B subunit homopentamer. It is, therefore, proposed that the differential toxicities of Stx1 and Stx2 reflect the relative efficiencies of intracellular activation of the A subunits.

The biological and ligand-binding properties of recombinant C-terminal cell-binding domains (CBDs) and subdomains of the two large exotoxins, Toxin A (TcdA) and Toxin B (TcdB) expressed by Clostridium difficile were examined in the hemagglutination and Verocytotoxicity neutralization assays and by qualitative affinity chromatography using Sepharose-linked alpha Gal(1,3)betaGal(1,4)beta Glc as well as the direct electrospray ionization mass spectrometry (ES-MS) assay. These studies revealed that, whereas the full-length TcdA CBD agglutinated rabbit erythrocytes, neutralized TcdA-mediated Vero cell death and bound to alpha Gal(1,3)betaGal(1,4)beta Glc-derivatized Sepharose, the TcdB CBD was inactive in these functional assays. Moreover, retention by alpha Gal(1,3)betaGal(1,4)beta Glc-derivatized Sepharose corresponded to the number of available TcdA subdomain ligand-binding sites. By contrast, the ES-MS assays revealed that both the TcdA and TcdB CBD bind to 8-methoxycarbonyloctyl-alpha Gal(1,3)betaGal(1,4)beta Glc sequences with similar avidities. Additional ES-MS experiments using chemically altered alpha Gal(1,3)betaGal(1,4)beta Glc sequences also revealed that the TcdA and TcdB CBD will tolerate a fair amount of structural variation in their complementary glycan ligands. Although the studies are consistent with the known ligand-binding properties of the TcdA and TcdB holotoxins, they also revealed subtle heretofore unrecognized functional differences in their receptor recognition properties.

At the turn of the century, a shift in incidence of Clostridium difficile infection occurred with the emergence of a novel, more virulent strain of C. difficile . It became apparent that strain typing was critical to our understanding of the epidemiology and transmission of the disease. Over the past 25 years, C. difficile strain typing methods have developed from phenotypic to genotypic techniques, each with their own advantages and disadvantages. Unfortunately, with the advent of polymerase chain reaction for C. difficile diagnosis, many clinical laboratories no longer perform anaerobic culture of the organism, and therefore routine strain typing for surveillance or detection of hospital outbreaks is rarely performed. Even among reference laboratories that do perform strain typing of C. difficile , standardisation of methods and inter-laboratory exchange of data continues to be problematic. Herein, an overview of molecular strain typing methods for C. difficile is provided, along with specific applications and limitations of each technique. In addition, current efforts to standardise laboratory protocols and a review of recent studies are discussed.

Background Guidelines currently provide conflicting recommendations regarding the diagnosis of group A streptococcal (GAS) pharyngitis in adults. Clinical guidelines state that negative rapid antigen detection tests (RADTs) do not require confirmation by a backup method in adults, whereas laboratory-based guidelines mandate confirmation of a negative RADT in patients of all ages. The objective of this study was to assess the utility of reflexive culture following a negative RADT in adolescents and adults with suspected GAS pharyngitis. Methods A retrospective analysis of 726 patients, aged ≥13 years, with negative RADTs and positive GAS throat cultures, was performed between 1 January 2000 and 31 December 2011 at 2 academic medical centers in Seattle, Washington. Complication rates, treatment, modified Centor score, and bacterial burden in patients with negative RADTs and positive GAS throat cultures were assessed. Results Modified Centor scores ≥2 were observed in 55% of patients with a negative RADT and positive GAS culture. Of these, 77% of patients had a moderate or heavy bacterial burden (≥2+). RADTs failed to detect some patients who presented with serious complications of GAS pharyngitis: 29 (4.0%) had peritonsillar abscesses and 2 (0.28%) were diagnosed with acute rheumatic fever. Providers found culture results to be useful for initiating antibiotic therapy or confirming a clinical diagnosis. Antibiotic treatment was prescribed in 68.7% of patients, with culture-directed initiation of therapy documented in 43.5%. Conclusions Reflexive GAS culture is clinically useful when RADTs are negative. RADTs fail to detect a substantial number of adult patients with clinically significant pharyngitis who can benefit from treatment.

Clostridium difficile , a major cause of hospital-acquired diarrhea, triggers disease through the release of two toxins, toxin A (TcdA) and toxin B (TcdB). These toxins disrupt the cytoskeleton of the intestinal epithelial cell, increasing intestinal permeability and triggering the release of inflammatory mediators resulting in intestinal injury and inflammation. The most prevalent animal model to study TcdA/TcdB-induced intestinal injury involves injecting toxin into the lumen of a surgically generated “ileal loop.” This model is time-consuming and exhibits variability depending on the expertise of the surgeon. Furthermore, the target organ of C. difficile infection (CDI) in humans is the colon, not the ileum. In the current study, we describe a new model of CDI that involves intrarectal instillation of TcdA/TcdB into the mouse colon. The administration of TcdA/TcdB triggered colonic inflammation and neutrophil and macrophage infiltration as well as increased epithelial barrier permeability and intestinal epithelial cell death. The damage and inflammation triggered by TcdA/TcdB isolates from the VPI and 630 strains correlated with the concentration of TcdA and TcdB produced. TcdA/TcdB exposure increased the expression of a number of inflammatory mediators associated with human CDI, including interleukin-6 (IL-6), gamma interferon (IFN-γ), and IL-1β. Finally, we were able to demonstrate that TcdA was much more potent at inducing colonic injury than was TcdB but TcdB could act synergistically with TcdA to exacerbate injury. Taken together, our data indicate that the intrarectal murine model provides a robust and efficient system to examine the effects of TcdA/TcdB on the induction of inflammation and colonic tissue damage in the context of human CDI.