675. Evaluation of PLST for Clostridioides difficile Sequence-based Strain Typing

Abstract Background Clostridioides difficile is a leading cause of healthcare-associated infection (HAI), most often following antibiotic therapy. The source of these infections may be endogenous or nosocomial; effective intervention requires distinquishing between these, which in turn requires strain typing. Numerous methods have been developed for C. difficile typing, ranging from length-based ribotyping and MLVA to whole genome sequencing. However, none are routinely used in clinical settings due to low resolution, high cost, technical complexity, or requirement for cultured isolates. The application of polymorphic locus sequence typing (PLST) to epidemiological analysis of HAI and foodborne infections has recently been described; here this approach is extended to C. difficile. Methods Tandem repeats were bioinformatically identified in the genome sequence of ribotype 027 strain R20291. These were screened by BLASTN of GenBank databases for the most polymorphic locus, which identified CdMT1 (Mbp 3.149). DNA was purified from colonies or environmental (Banana Broth) cultures; bead-beating and PCR inhibitor removal steps were required for consistent results. Results CdMT1 encompassed MLVA repeat C6cd which, based on length alone, yielded the highest diversity index (DI) of 0.96. In contrast, CdMT1 sequence analysis yielded DI of >0.99. Comparison to ribotype further illustrated high level resolution; e.g., 9 ribotype 027 strains were resolved into 8 CdMT1 alleles. For initial laboratory evalulation, veterinary C. difficile isolates (44 canine, 4 bovine) were CdMT1 typed. Bioinformatic analysis of the 48 sequences resolved 24 CdMT1 alleles, including 8 clusters of 2 to 6 canine strains. Six of these clusters represented isolates from individual puppies in the same litter, or from different litters but the same household, while the bovine isolates formed a phylogenetically distinct group. Using the same DNA purification protocol, CdMT1 typing demonstrated compatibility with C. difficile-spiked stool samples and Banana Broth environmental cultures. Conclusion CdMT1 typing represents a potentially useful tool for outbreak detection and investigation in healthcare facilities, particularly in light of its compatibility with both stool and environmental samples. Disclosures Tom Edlind, PhD, MicrobiType LLC (Employee, Scientific Research Study Investigator)

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678. Polymorphic Locus Sequence Typing for Pseudomonas aeruginosa Outbreak Detection and Investigation

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.870 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S392-S392

Author(s):

Tom Edlind

Keyword(s):

Pseudomonas Aeruginosa ◽

Tandem Repeats ◽

Diversity Index ◽

Polymorphic Locus ◽

Hybrid Approach ◽

Healthcare Facility ◽

Outbreak Detection ◽

Snp Analysis ◽

Healthcare Associated Infection ◽

Genome Sequences

Abstract Background Pseudomonas aeruginosa is a common agent of healthcare-associated infection (HAI), which can be challenging to treat due to intrinsic or acquired antibiotic resistance. In contrast to most HAI, the source of P. aeruginosa infection is typically environmental; e.g., from the faucets, drains, and showers in patient rooms. To unambiguously identify the source, strain typing of patient and environmental isolates is required. Typing methods include sequence-based MLST and WGS which resolve strains based on relatively rare SNPs, and length-based MLVA which resolves strains based on relatively frequent insertions/deletions within tandem repeats. These and other methods previously applied to P. aeruginosa have one or more limitations relating to cost, technical complexity, reproducibility, and turnaround time that preclude their routine use. Polymorphic locus sequence typing (PLST) is a hybrid approach that employs sequence analysis of the one or two most phylogenetically informative tandem repeat-containing loci within the genome of a microbial species. Strain resolution equals or exceeds MLST and MLVA and approaches that of WGS. Here, the identification and evaluation of candidate PLST loci for P. aeruginosa epidemiology are described. Methods Tandem repeats were bioinformatically identified in representative genome sequences, and screened by BLASTN queries of GenBank genome databases followed by clustal alignments and phylogenetic analysis. Candidate loci were amplified from heat-treated colony lysates and Sanger sequenced. Results PLST locus PaMT1 (Mbp 2.568-2.569 in the PA01 genome) resolved 99 alleles from 144 epidemiologically unrelated isolates with GenBank genome sequences, yielding diversity index = 0.991. Nineteen sets of isolates shared healthcare facility and PaMT1 allele, consistent with nosocomial transmission. Examples, confirmed by WGS-SNP analysis, include isolates from 8 VAP patients, 2 sets of cancer patient isolates, and a pair of isolates from patient and hospital sewage. In the laboratory, locus PaMT1 was readily amplified and sequenced from colony lysates, yielding typing data with 1-2 day turnaround. Conclusion PaMT1 PLST provides an affordable, user-friendly, and timely tool for P. aeruginosa outbreak detection and investigaton. Disclosures Tom Edlind, PhD, MicrobiType LLC (Employee, Scientific Research Study Investigator)

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Evaluation of the bioMérieux EPISEQ-CS Software for wgMLST-Based Bacterial Strain Typing

Infection Control and Hospital Epidemiology ◽

10.1017/ice.2020.780 ◽

2020 ◽

Vol 41 (S1) ◽

pp. s231-s231

Author(s):

Amorce Lima ◽

Steven Bruzek ◽

Amanda Lasher ◽

Grant Vestal ◽

Suzane Silbert

Keyword(s):

Bacterial Strain ◽

Diversity Index ◽

Bacterial Species ◽

Outbreak Detection ◽

Epidemiological Surveillance ◽

Strain Typing ◽

Sequencing Data ◽

Hospital Acquired Infections ◽

Main Challenge ◽

Hospital Acquired

Background: Whole-genome sequencing (WGS) is becoming the method of choice for outbreak analysis of microbial pathogens. However, the main challenge with WGS for microbial strain typing is the conversion of raw sequencing data to actionable results for epidemiology and surveillance analysis. We evaluated the bioMrieux EPISEQ-CS, a cloud-based WGS data analysis software for outbreak detection to compare the results for 4 groups of different species previously characterized by strain typing and commonly isolated in hospital-acquired infections. Methods: In total, 30 methicillin-resistant Staphylococcus aureus (MRSA), 15 Clostridioides difficile (CDIFF), 17 Pseudomonas aeruginosa (PSA), and 10 Acinetobacter baumannii (ACB) isolates were included in this study. All isolates had been previously characterized by rep-PCR using the DiversiLab system (bioMrieux, France) and saved at 70C. Before testing, samples were thawed and plated, and DNA extraction was performed on the QIAcube (Qiagen, Hilden, Germany) using the DNEasy Ultra Clean Microbial kit extraction protocol. DNA libraries were prepared using the Nextera DNA Flex Kit and sequenced on the Illumina iSeq100 platform according to manufacturer’s recommendations (Illumina, San Diego, CA). Generated sequences were uploaded into EPISEQ-CS, and wgMLST-based analysis was performed. We compared clusters generated by the DiversiLab system and EPISEQ-CS. Results: DiversiLab identified 9 MRSA clusters among 30 isolates. EPISEQ-CS reclassified 14 of 30 isolates into 5 MRSA clusters and the remaining 16 isolates were unrelated. DiversiLab identified 2 CDIFF clusters among 15 isolates. EPISEQ-CS reclassified 3 isolates into 1 CDIFF cluster and determined the remaining 12 to be unrelated. DiversiLab identified 5 PSA clusters among 17 isolates, whereas EPISEQ-CS reclassified all 17 isolates as unrelated. DiversiLab identified 2 ACB clusters among 10 isolates, whereas EPISEQ-CS reclassified 2 ACB isolates into 1 cluster and determined 8 to be unrelated. Analysis using Simpson’s diversity index (D) suggested that the EPISEQ-CS showed increased diversity when compared to DiversiLab clustering across all bacterial species analyzed in this study. Conclusions: EPISEQ-CS enabled a comprehensive wgMLST analysis, including quality control and comparative epidemiological analysis, thereby providing a more reliable method for bacterial strain typing. As WGS becomes more affordable and applicable to routine epidemiological surveillance, EPISEQ-CS provides an informative tool in the monitoring of hospital-acquired infections.Funding: NoneDisclosures: None

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Targeted Assessment for Prevention: A Statewide Collaborative

Infection Control and Hospital Epidemiology ◽

10.1017/ice.2020.1047 ◽

2020 ◽

Vol 41 (S1) ◽

pp. s401-s401

Author(s):

Cindy Hou ◽

Shannon Davila ◽

Mary Miller ◽

Ashlee Hiester ◽

Katherine Hosmer ◽

...

Keyword(s):

Quality Improvement ◽

Infection Prevention ◽

Local Level ◽

Central Line ◽

Interdisciplinary Teams ◽

System Level ◽

Healthcare Associated Infection ◽

Hospital System ◽

Healthcare Facilities ◽

Clostridioides Difficile

Background: Infection preventionists (IPs) are the backbone of the quality and safety matrix of their organizations. Tools to help locate potential gaps can provide unique viewpoints from frontline staff. The CDC provides a Targeted Assessment for Prevention (TAP) strategy that identifies vulnerabilities in the prevention of healthcare-associated infection (HAIs). Methods: A statewide quality improvement organization, partnering with the CDC TAP team, administered TAP facility assessments for catheter-associated urinary tract infection (CAUTI), central-line–associated bloodstream infection (CLABSI), and Clostridioides difficile infection (CDI) to a collaborative of 15 acute-care and 2 long-term acute hospitals. More than 800 respondents filled out surveys based on their individualized perceptions of infection prevention practices. Results: The survey results yielded the following lagging indicators: lack of awareness of nursing and physician champions, need for competency-based training of clinical equipment, and feedback on device utilization. At the hospital system level, one improvement team focused on CDI, uncovered leading and lagging areas in general infrastructure, antibiotic stewardship, early detection and appropriate testing, contact precautions, and environmental cleaning. To culminate the TAP collaborative, the cohort of organizations, supported by interdisciplinary teams, participated in a full-day TAP workshop in which they reviewed detailed analyses of their HAI data and assessment results, shared best practices for infection prevention and planned for specific improvement projects using the plan-do-study-act model. Conclusions: Results of a statewide analysis of HAI prevention data and opportunities at a local level were reviewed. The TAP strategy can be used to target opportunities for improvement, to assess gaps in practice, and to develop and implement interventions for improving outcomes. Healthcare facilities and quality improvement organizations can drive infection prevention actions.Funding: NoneDisclosures: None

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Molecular characterization of Clostridioides difficile ribotype 027 in a major Chinese hospital

Journal of Microbiology Immunology and Infection ◽

10.1016/j.jmii.2021.01.003 ◽

2021 ◽

Author(s):

Ren-feng Zhang ◽

Yu-xia Man ◽

Yuan-yuan Bai ◽

Chun-hong Shao ◽

Chun-mei Liu ◽

...

Keyword(s):

Molecular Characterization ◽

Ribotype 027 ◽

Clostridioides Difficile

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Homologous Recombination in Clostridioides difficile Mediates Diversification of Cell Surface Features and Transport Systems

mSphere ◽

10.1128/msphere.00799-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Hannah D. Steinberg ◽

Evan S. Snitkin

Keyword(s):

Cell Wall ◽

Homologous Recombination ◽

Sugar Transport ◽

The United States ◽

Strain Typing ◽

Cell Wall Proteins ◽

Genetic Loci ◽

Content Type ◽

Clostridioides Difficile ◽

Host Pathogen

ABSTRACT Illness caused by the pathogen Clostridioides difficile is widespread and can range in severity from mild diarrhea to sepsis and death. Strains of C. difficile isolated from human infections exhibit great genetic diversity, leading to the hypothesis that the genetic background of the infecting strain at least partially determines a patient’s clinical course. However, although certain strains of C. difficile have been suggested to be associated with increased severity, strain typing alone has proved insufficient to explain infection severity. The limited explanatory power of strain typing has been hypothesized to be due to genetic variation within strain types, as well as genetic elements shared between strain types. Homologous recombination is an evolutionary mechanism that can result in large genetic differences between two otherwise clonal isolates, and also lead to convergent genotypes in distantly related strains. More than 400 C. difficile genomes were analyzed here to assess the effect of homologous recombination within and between C. difficile clades. Almost three-quarters of single nucleotide variants in the C. difficile phylogeny are predicted to be due to homologous recombination events. Furthermore, recombination events were enriched in genes previously reported to be important to virulence and host-pathogen interactions, such as flagella, cell wall proteins, and sugar transport and metabolism. Thus, by exploring the landscape of homologous recombination in C. difficile, we identified genetic loci whose elevated rates of recombination mediated diversification, making them strong candidates for being mediators of host-pathogen interaction in diverse strains of C. difficile. IMPORTANCE Infections with C. difficile result in up to half a million illnesses and tens of thousands of deaths annually in the United States. The severity of C. difficile illness is dependent on both host and bacterial factors. Studying the evolutionary history of C. difficile pathogens is important for understanding the variation in pathogenicity of these bacteria. This study examines the extent and targets of homologous recombination, a mechanism by which distant strains of bacteria can share genetic material, in hundreds of C. difficile strains and identifies hot spots of realized recombination events. The results of this analysis reveal the importance of homologous recombination in the diversification of genetic loci in C. difficile that are significant in its pathogenicity and host interactions, such as flagellar construction, cell wall proteins, and sugar transport and metabolism.

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Comparison of Multilocus Variable-Number Tandem-Repeat Analysis with Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis for Enterococcus faecalis

Polish Journal of Microbiology ◽

10.33073/pjm-2011-048 ◽

2011 ◽

Vol 60 (4) ◽

pp. 335-339 ◽

Cited By ~ 2

Author(s):

EWA SADOWY ◽

ALEKSANDRA SIEŃKO ◽

WALERIA HRYNIEWICZ

Keyword(s):

Enterococcus Faecalis ◽

Gel Electrophoresis ◽

Tandem Repeats ◽

Diversity Index ◽

Variable Number Tandem Repeat ◽

Variable Number ◽

Pulsed Field Gel Electrophoresis ◽

Discriminatory Power ◽

Pulsed Field ◽

High Discriminatory Power

Enterococcus faecalis represents recently an important etiological agent of health care-associated infections (HAIs) and there is a need for evaluation and comparison of typing methods available for this microorganism. We tested multilocus VNTR (variable-number tandem repeats) analysis (MLVA) on a well-characterized collection of 153 clinical isolates of E. faecalis, corresponding to 52 multilocus sequence types and 67 pulsed-field gel electrophoresis (PFGE) profiles. MLVA showed high discriminatory power, discerning 111 different types (diversity index equal 98.9%). The concordance MLVA/MLST and MLVA/PFGE was 0.95 and 0.74, respectively. High discriminatory power of MLVA indicates its utility for local epidemiology such as outbreak investigation, and for differentiation of clones defined by other methods.

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Clinical impact of a pharmacist-led antimicrobial stewardship initiative evaluating patients with Clostridioides difficile colitis

Journal of Investigative Medicine ◽

10.1136/jim-2019-001173 ◽

2020 ◽

Vol 68 (4) ◽

pp. 888-892 ◽

Cited By ~ 1

Author(s):

Paige A Bishop ◽

Carmen Isache ◽

Yvette S McCarter ◽

Carmen Smotherman ◽

Shiva Gautam ◽

...

Keyword(s):

Clinical Outcomes ◽

Antimicrobial Stewardship ◽

Treatment Guidelines ◽

Clinical Success ◽

Audit And Feedback ◽

Healthcare Associated Infection ◽

Clostridioides Difficile ◽

Stewardship Program ◽

The Usa ◽

The Impact

Clostridioides difficile is the most common cause of healthcare-associated infection and gastroenteritis-associated death in the USA. Adherence to guideline recommendations for treatment of severe C. difficile infection (CDI) is associated with improved clinical success and reduced mortality. The purpose of this study was to determine whether implementation of a pharmacist-led antimicrobial stewardship program (ASP) CDI initiative improved adherence to CDI treatment guidelines and clinical outcomes. This was a single-center, retrospective, quasi-experimental study evaluating patients with CDI before and after implementation of an ASP initiative involving prospective audit and feedback in which guideline-driven treatment recommendations were communicated to treatment teams and documented in the electronic health record via pharmacy progress notes for all patients diagnosed with CDI. The primary endpoint was the proportion of patients treated with guideline adherent definitive regimens within 72 hours of CDI diagnosis. Secondary objectives were to evaluate the impact on clinical outcomes, including length of stay (LOS), infection-related LOS, 30-day readmission rates, and all-cause, in-hospital mortality. A total of 233 patients were evaluated. The proportion of patients on guideline adherent definitive CDI treatment regimen within 72 hours of diagnosis was significantly higher in the post-interventional group (pre: 42% vs post: 58%, p=0.02). No differences were observed in clinical outcomes or proportions of patients receiving laxatives, promotility agents, or proton pump inhibitors within 72 hours of diagnosis. Our findings demonstrate that a pharmacist-led stewardship initiative improved adherence to evidence-based practice guidelines for CDI treatment.

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Automated outbreak detection of hospital-associated pathogens: Value to infection prevention programs

Infection Control and Hospital Epidemiology ◽

10.1017/ice.2020.233 ◽

2020 ◽

Vol 41 (9) ◽

pp. 1016-1021

Author(s):

Meghan A. Baker ◽

Deborah S. Yokoe ◽

John Stelling ◽

Ken Kleinman ◽

Rebecca E. Kaganov ◽

...

Keyword(s):

Infection Prevention ◽

Detection System ◽

Statistical Significance ◽

Prevention Programs ◽

Multidrug Resistant ◽

Outbreak Detection ◽

Convenience Sample ◽

Clostridioides Difficile ◽

Surveillance Programs ◽

Infection Preventionists

AbstractObjective:To assess the utility of an automated, statistically-based outbreak detection system to identify clusters of hospital-acquired microorganisms.Design:Multicenter retrospective cohort study.Setting:The study included 43 hospitals using a common infection prevention surveillance system.Methods:A space–time permutation scan statistic was applied to hospital microbiology, admission, discharge, and transfer data to identify clustering of microorganisms within hospital locations and services. Infection preventionists were asked to rate the importance of each cluster. A convenience sample of 10 hospitals also provided information about clusters previously identified through their usual surveillance methods.Results:We identified 230 clusters in 43 hospitals involving Gram-positive and -negative bacteria and fungi. Half of the clusters progressed after initial detection, suggesting that early detection could trigger interventions to curtail further spread. Infection preventionists reported that they would have wanted to be alerted about 81% of these clusters. Factors associated with clusters judged to be moderately or highly concerning included high statistical significance, large size, and clusters involving Clostridioides difficile or multidrug-resistant organisms. Based on comparison data provided by the convenience sample of hospitals, only 9 (18%) of 51 clusters detected by usual surveillance met statistical significance, and of the 70 clusters not previously detected, 58 (83%) involved organisms not routinely targeted by the hospitals’ surveillance programs. All infection prevention programs felt that an automated outbreak detection tool would improve their ability to detect outbreaks and streamline their work.Conclusions:Automated, statistically-based outbreak detection can increase the consistency, scope, and comprehensiveness of detecting hospital-associated transmission.

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Antimicrobial susceptibility of Clostridioides difficile isolated from diarrhoeal stool specimens of Canadian patients: summary of results from the Canadian Clostridioides difficile (CAN-DIFF) surveillance study from 2013 to 2017

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa118 ◽

2020 ◽

Vol 75 (7) ◽

pp. 1824-1832

Author(s):

James A Karlowsky ◽

Heather J Adam ◽

Melanie R Baxter ◽

Christopher W Dutka ◽

Kim A Nichol ◽

...

Keyword(s):

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Surveillance Study ◽

Dilution Method ◽

Agar Dilution Method ◽

In Vitro Susceptibility ◽

Ribotype 027 ◽

Clostridioides Difficile ◽

Vancomycin Mics ◽

Stool Specimens

Abstract Objectives To summarize data generated by the Canadian Clostridioides difficile (CAN-DIFF) surveillance study from 2013 to 2017. Methods Isolates of C. difficile (n = 2158) were cultured from toxin-positive diarrhoeal stool specimens submitted by eight hospital laboratories to a coordinating laboratory. Antimicrobial susceptibility testing was performed according to the CLSI agar dilution method (M11, 2018). Isolate ribotypes were determined using an international, standardized, high-resolution capillary gel-based electrophoresis protocol. Results Of the 2158 isolates of C. difficile, 2133 (98.8%) had vancomycin MICs ≤2 mg/L [i.e. were vancomycin susceptible (EUCAST breakpoint tables, v 9.0, 2019) or WT (CLSI M100, 29th edition, 2019)]. Fidaxomicin MICs were lower than those of all other agents tested (MIC90, 0.5 mg/L); however, one isolate with a fidaxomicin MIC of >8 mg/L was identified. Metronidazole MICs ranged from 0.12 to 4 mg/L; all isolates were metronidazole susceptible by the CLSI breakpoint (≤8 mg/L) compared with 96.8% susceptible by the EUCAST breakpoint (≤2 mg/L). In total, 182 different ribotypes were identified from 2013 to 2017. The most common ribotypes identified were 027 (19.3% of isolates) and 106 (8.2%). Ribotype 027 isolates were frequently moxifloxacin resistant (87.3% of isolates) and MDR (48.6%), associated with vancomycin (10/25, 40.0%) and metronidazole (58/69, 84.1%) resistance and from patients aged ≥80 years. The prevalence of ribotype 027 decreased significantly (P < 0.0001) from 2013 (27.5%) to 2017 (9.0%) and was replaced by increases in ribotype 106 (P = 0.0003) and multiple less common ribotypes. Conclusions Periodic surveillance is required to monitor clinical isolates of C. difficile for changes to in vitro susceptibility testing profiles and ribotype evolution.

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Evaluation of variable numbers of tandem repeat as molecular epidemiological markers of Mycobacterium tuberculosis in Japan

Journal of Medical Microbiology ◽

10.1099/jmm.0.46990-0 ◽

2007 ◽

Vol 56 (8) ◽

pp. 1052-1057 ◽

Cited By ~ 26

Author(s):

Takayuki Wada ◽

Shinji Maeda ◽

Atsushi Hase ◽

Kazuo Kobayashi

Keyword(s):

Mycobacterium Tuberculosis ◽

Tandem Repeat ◽

Tandem Repeats ◽

Allelic Diversity ◽

Rflp Analysis ◽

Strain Typing ◽

Beijing Family ◽

Discrimination Power ◽

Family Based ◽

Vntr Analysis

Using 243 Mycobacterium tuberculosis isolates obtained in 2001 in Osaka City, Japan, the discriminatory power of variable numbers of tandem repeats (VNTRs) of 12 standard mycobacterial interspersed repetitive units (MIRUs) was assessed. The biggest cluster defined by MIRU-VNTRs consisted of 57 (23.5 %) isolates and they belonged to the Beijing family based on spoligotyping. When additional VNTR loci were included in the MIRU-VNTR analysis, the 57 originally clustered strains were further differentiated by the addition of Queen's University Belfast (QUB)-VNTRs, but not exact tandem repeat-VNTR. The allelic diversity of additional VNTR loci such as VNTR 3232 (QUB-3232), VNTR 2163a (QUB-11a), VNTR 2163b (QUB-11b) and VNTR 1982 (QUB-18) was high in the 57 strains. When the 243 M. tuberculosis isolates were analysed using 16-locus VNTR (the 12 standard MIRUs and the 4 QUB loci) and IS6110 RFLP, the respective Hunter–Gaston discriminatory indexes were 0.9966 and 0.9971. The discrimination power of 16-locus VNTR was equal to that of IS6110 RFLP analysis. If appropriate loci are added to the standard MIRU analysis, VNTR genotyping could be a valuable tool for strain typing and epidemiological research of M. tuberculosis in Japan.

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